M E T H O D S IN E N Z Y M O L O G Y EDITORS-IN-CHIEF
John N. Abelson
Melvin I. Simon
DIVISION OF BIOLOGY CALIFORNIA...
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M E T H O D S IN E N Z Y M O L O G Y EDITORS-IN-CHIEF
John N. Abelson
Melvin I. Simon
DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
Methods in Enzymology Volume 234
Oxygen Radicals in Biological Systems Part D E D I T E D BY
Lester Packer DEPARTMENT OF MOLECULAR AND CELL BIOLOGY UNIVERSITY OF CALIFORNIA, BERKELEY BERKELEY, CALIFORNIA
Editorial Advisory Board Bruce Ames Kelvin Davies Barry HaUiwell
Etsuo Niki William Pryor Helmut Sies
® ACADEMIC PRESS San Diego
A Division of Harcourt Brace & Company New York Boston London Sydney Tokyo
Toronto
Contributors to V o l u m e 2 3 4 Article numbers are in parentheses following the names o f contributors. Affiliations listed are current.
ELIAS N. ABOUJAOUDE (2), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720
MICHAEL BOCKSTETTE (13), Department of lmmunochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany WOLF BORS (41), Institutfiir Strahlenbiologie, GSF-Forschungszentrum fiir Umwelt und Gesundheit Neuherberg, D-85758 Oberschleissheim, Germany KARLIS BRIVIBA (37), Institut fiir Physiologische Chemie I, Heinrich-Heine-Universitgit, D-40001 Diisseldorf, Germany GARRY W. BUCHKO (8), Department of Radiobiology, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2 MARK J. BURKITT (7), Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland, United Kingdom LARRY G. BUTLER (42), Department of BiDchemistry, Purdue University, West Lafayette, Indiana 47907 JEAN CADET (8), D~partement de Recherche Fondamentale sur la Matidre Condens6e, SESAM/LAN, CEA - Centre d'Etudes Nacldaires de Grenoble, F-38054 Grenoble, France JOHN M. CARNEY (53), Department of BiDchemistry and Molecular Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 RAJAGOPAL CHATTOPADHYAYA (5), Bose Institute, 700054 Calcutta, India QIN CHEN (2), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 JOSIANE CILLARD (43), Laboratoire de Biologie Cellulaire et V~gdtale, Facult~ de Pharmacie, 35043 Rennes, France PIERRE CILLARD (43), Laboratoire de Biologie CeUulaire et V6gdtale, Facult~ de Pharmacie, 35043 Rennes, France
SHOSHY ALTUVIA (17), Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 BRUCE N. AMES (2, 8, 23), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 MARY E. ANDERSON (49, 50), Department of Biochemistry, Cornell University Medical College, New York, New York 10021 MIGUEL ASENSI (35), Departamento de Fisiolo~ia, Universidad de Valencia, 46010 Valencia, Spain LAURA AUGERI (10), Departments of Biochemistry and Radiation Oncology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 PATRICK A. BAEUERLE (14), Institute of Biochemistry, University of Freiburg, D79104 Freiburg, Germany G. BARJA (31), Department of Animal Biology-H (Animal Physiology), Complutense University, 28040 Madrid, Spain SHARMILA BAsu-MODAK (18), Swiss Institute for Experimental Cancer Research, Physical Carcinogenesis Unit, CH-1066 Epalinges, Switzerland CHRISTA BAUMSTARK-KHAN (9), Radiologische Universitgitsklinik, Experimentelle Radiologie und Strahlenbiologie, Universiti~t Bonn, D-53105 Bonn, Germany JOHN S. BERTRAM (19), Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii 96813 xi
xii
CONTRIBUTORS TO VOLUME 2 3 4
IAN A. COTGREAVE (48), Institute of Envi-
CHERYL A. EDBAUER-NECHAMEN (16), De-
ronmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm 171 77, Sweden DANA R. CRAWFORD (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208 CARROLL E. CROSS (21), Department of Medicine and Physiology, PulmonaryCritical Care Medicine, University of California, Davis Medical Center, Sacramento, California 95817 TOM CURRAN (15), Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110 DIPAK K. DAS (40), Cardiovascular Division, Department of Surgery, Surgical Research Center, University of Connecticut School of Medicine, Farmington, Connecticut 06030 JOANNA M. S. DAVIES (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208 KELVIN J. A. DAVIES (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208 MiRAL DIZDAROGLU (1), Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 PAUL W. DOETSCH (3, 10), Departments of Biochemistry and Radiation Oncology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 WULF DROGE (13), Department oflmmunochemistry, Deutsehes Krebsforschungszentrum, D-69120 Heidelberg, Germany MARIA-THERESE DROY-LEFAIX (46), Department of Pharmacology, IPSEN Institute, 75016 Paris, France TIMOTHY R. DUVALL (22), California Regional Primate Research Center, University of California at Davis, Davis, California 95616
partment of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208 BERND EPE (12), Institute of Pharmacology
and Toxicology, University of Wiirzburg, D-97078 Wiirzburg, Germany Jose M. ESTRELA (35), Departamento de Fi-
siologia, Universidad de Valencia, 46010 Valencia, Spain ROBERT A. FLOYD (6, 53), Department of
Biochemistry and Molecular Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 BALZ FREI (23), Whitaker Cardiovascular
Institute, Boston University Medical Center, Boston, Massachusetts 02118 HANS-JOACHIM FREISLEBEN (36), Depart-
ment of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 T. G. GANTCHEV (63), MRC Group in
the Radiation Sciences, Department of Nuclear Medicine and Radiobiology, University of Sherbrooke, Sherbrooke, Qudbec, Canada J1H 5N4 MONIQUE GARDgS-ALBERT (46), Rend Des-
cartes University, Physical Chemistry Laboratory, URA 400 CNRS , 75006 Paris, France ERNST GLINZ (26), Pharma Research, F.
Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland DAVID V. GOEDDEL (20), Department of
Immunology and Molecular Biology, Genentech, Inc., South San Francisco, California 94080 MATTHEW B. GRISHAM (57), Department of
Physiology and Biophysics, Louisiana State University Medical Cener, Shreveport, Louisiana 71130 ANN E. HAGERMAN (42), Department of Chemistry, Miami University, Oxford, Ohio 45056 EDWARD D. HALL (56), CNS Diseases Re-
search Unit, Upjohn Company, Kalamazoo, Michigan 49001
. o °
CONTRIBUTORS TO VOLUME 234
Xlll
BARRY HALLIWELL (21), Pharmacology
KAr~KI KOMIYAMA (29), Research Division,
Group, King's College, University of London, London SW3 6LX, United Kingdom KRISTA K. HAMILTON (3), Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 KRISTA K. HAMILTON (10), Departments of Biochemistry and Radiation Oncology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 JUTTA HEGLER (12), Institute of Pharmacology and Toxicology, University of Wiirzburg, D-97078 Wiirzburg, Germany ERNST S. HENLE (5), Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 A. HERNANZ (31) Department of Biochemistry, lnsalud La Paz Hospital, 28046 Madrid, Spain LUBICA HOR~KOV.g. (58), Department of Neuropharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, 842 16 Bratislava, Slovakia J. R. S. HOULT (44), Department of Pharmacology, King's College London, London SW3 6LX, United Kingdom MASAYASU INOUE (32), Department of BiDchemistry, Osaka City University Medical School, Abeno, Osaka 545, Japan RICHARD L. JACKSON (51), Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania 19101 RUCHENG JIN (5), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 VALERIAN E. KAGAN (28, 33, 36, 63), Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15238 S. KHWAIA (33), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 YONG K. KIM (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208
The Kitasato Institute, Minato-ku, Tokyo 108, Japan ALFRED W. KORMANN (26), Vitamins and Fine Chemicals Research, Research and Technology Development, F. HoffmannLa Roche Ltd., CH-4002 Basel, Switzerland KE1KO KOYAMA (32), Department of Biochemistry, Osaka City University Medical School, Abeno, Osaka 545, Japan MURALI C. KRISHNA (59), Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 JEROLD A. LAST (22), California Regional Primate Research Center, University of California at Davis, Davis, California 95616 DIANA M. LEE (52), Free Radical Biology and Aging Research Program, Oklahoma Medical Resarch Foundation, Oklahoma City, Oklahoma 73104 KEUNMYOUNG LEE (3), Department of Botany and Plant Sciences, University of California at Riverside, Riverside, California 92521 GI~RARD LESCOAT (43), Laboratoire de Biologie Cellulaire et Vdg(tale, Facultd de Pharmacie, 35043 Rennes, France ELLEN J. LEVY (49, 50), Department of Anatomy and Cell Biology, State University of New York Health Science Center at Brooklyn, New York, New York 11203 DANIEL C. LIEBLER (27), Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, Arizona 85721 STUART LINN (5), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 MARIA A. LIVREA (39), lstituto de Chimica Biologica, Universit& di Palermo, Policlinico, 90127 Palermo, Italy CHARLES V. LOWRY (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208
xiv
CONTRIBUTORS TO VOLUME 2 3 4
YONGZHANG LUO (5), Department of Bio-
NICHOLAS J. MILLER (24), Free Radical Re-
logical Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 I. TONG MAK (62), Departments of Medicine and Physiology, George Washington University Medical Center, Washington, D.C. 20037 MASANOBU MANABE (36), Department of Anesthesiology and Resuscitology, Kochi Medical School, Nankoku-shi, Kochi 783, Japan SIMON J. T. MAO (51), Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215 LUCIA MARCOCCI (46, 54), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 AMY M. MARTIN (10), Departments of Biochemistry and Radiation Oncology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 JOHN M. MCCALL (56), Discovery Research, Upjohn Company, Kalamazoo, Michigan 49001 ALTON MEISTER (49, 50), Department of Biochemistry, Cornell University Medical College, New York, New York 10021
search Group, UMDS-dash Guy's Hospital, London SE1 9RT, United Kingdom PETER MOLDI~US (48), Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm 171 77, Sweden OLIVIER H. MORAND (61), Pharma Division, Preclinical Research, F. HoffmanLaRoche Ltd., CH-4002 Basel, Switzerland ISABELLE MOREL (43), Laboratoire de Biologie Cellulaire et V~g~tale, Facult~ de Pharmacie, 35043 Rennes, France MICHELE A. MORONEY (44), Department of Pharmacology, King's College London, London SW3 6LX, United Kingdom PAUL A. MOTCHNIK (23), Xoma Corporation, Berkeley, California 94710 MAIK S. W. OBENDORF (4), Institutfiir Physiologische Chemie 1, Heinrich-HeineUniversitdt, D-40001 Diisseldorf, Germany
CARLOS FREDERICO MARTINS MENCK (11),
Department of Biology, Institute of Biosciences, University of Sao Paulo, CEP 05422-970 Sao Paulo, Brazil CI-IRISTA MICHEL (41), Institutfiir Strahlenbiologie, GSF Forschungszentrum fiir Umwelt und Gesundheit Neuherberg, D85758 Oberschleissheim, Germany SABINE MIHM (13), Department of Gastroenterology and Endocrinology, University of G6ttingen, D-37075 GiSttingen, Germany MASAYUKO MIKI (55), Department of Pediatrics, Osaka Medical College, Takatsuki, Osaka 569, Japan ALLEN M. MILES (57), Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, Louisiana 71130
LESTER PACKER (28, 33, 34, 36, 45, 46, 54),
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 FEDERICO V. PALLARDO (35), Departamento de Fisiologia, Universidad de Valencia, 46010 Valencia, Spain MIGUEL PAYA (44), Departamento de Fisiologla, Universidad de Valencia, 46100 Valencia, Spain ORS B. RANALDER (26), Pharma Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland JEAN-Luc RAVANAT (8), Ddpartement de Recherche Fondamentale sur la Matidre Condensde, SESAM/LAN, CEA - Centre d'Etudes Nucldaires de Grenoble, F38054 Grenoble, France ROSEMARIE RETTENMAIER (25), Vitamins and Fine Chemicals Research, Research and Technology Development, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland CATHERINE RICE-EVANS (24), Free Radical Research Group, UMDS-Guy's Hospital, London SE1 9RT, United Kingdom
CONTRIBUTORS TO VOLUME 234
XV
GEORGES RISS (26), Vitamins and Fine
STEEN STEENKEN (58), Max-Planck-lnstitut
Chemicals Research, Research and Technology Development, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland STEFFEN ROTH (13), Mannheimer Strasse 129, 68309 Mannheim, Germany SHARON L. SALMON (16), Department of Biochemistry and Molecular Biology, The Albany Medical College, Albany, New York 12208 AMRAM SAMUNI (59), Department of Molecular Biology, Hebrew University Medical School, 91010 Jerusalem, Israel MANFRED SARAN (41), Institutfiir Strahlenbiologie, GSF Forschungszentrum fiir Umwelt und Gesundheit Neuherberg, D85758 Oberschleissheirn, Germany JUAN SASTRE (35), Departamento de Fisiologia, Universidad de Valencia, 46010 Valencia, Spain RALF SCHRECK (14), Fred Hutchinson Cancer Center, Seattle, Washington 98104 WILLY SCHLrEP (25), Vitamins and Fine Chemicals Research, Research and Technology Development, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland WOLFGANG A. SCHULZ (4), Institutfiir Physiologische Chemie 1, Heinrich-HeineUniversit?it, D-40001 Diisseldorf, Germany ABDELHAFID SEKAKI (46), Rend Descartes University, Physical Chemistry Laboratory, URA 400 CNRS , 75006 Paris, France ELENA A. SERBINOVA (33, 34), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 MARK K. SHIGENAGA (2), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 HELMUT SIES (4, 37, 38, 47, 58), Institutffw Physiologische Chemie I, HeinrichHeine-Universitiit, D-40001 Diisseldorf, Germany WILHELM STAHL (38), Institutfi2r Physiologische Chemie I, Heinrich-Heine-Universiti~t, D-40001 Diisseldorf, Germany
far Strahlenchemie, W-4330 Miilheim, Germany GISELA STORZ (17), Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 O. A. STOYANOVSKY (33, 63), Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15238 ALFRED R. SUNDQUIST (37), Department of Chemistry, University of California at San Diego, San Diego, California 92093 YUICHIRO JUSTIN SUZUKI (45, 54), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 BRIAN K. TARKINGTON (22), California Regional Primate Research Center, University of California at Davis, Davis, California 95616 LUISA TESORIERE (39), Istituto de Chimica Biologica, Universitft di Palermo, Policlinico, 90127 Palermo, Italy MASAHIKO TSUCHIYA (36, 45, 54), Department of Anesthesiology and Resuscitology, Kochi Medical School, Nankokushi, Kochi 783, Japan REX M. TYRRELL (18), Swiss Institute for Experimental Cancer Research, Physical Carcinogenesis Unit, CH-I066 Epalinges, Switzerland J. E. VAN LIER (63), MRC Group in the Radiation Sciences, Department of Nuclear Medicine and Radiobiology, University of Sherbrooke, Sherbrooke, Qudbec, Canada J1H 5N4 GOVlND T. VATASSERY (30), Research Service, and Geriatric Research Education and Clinical Center, V.A. Medical Center, Minneapolis, Minnesota 55417 JOSE VIIZ4A (35), Departamento de Fisiologia, Universidad de Valencia, 46010 Valencia, Spain WILLI WALTHER (26), Pharma Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland
xvi
CONTRIBUTORS TO VOLUME 2 3 4
WILLIAM B. WEGLICKI (62), Departments
of Medicine and Physiology, George Washington University Medical Center, Washington, D.C. 20037 HELEN WISEMAN (60), Departments of Pharmacology and Biochemistry, Royal Free Hopsital School of Medicine, London NW3 2PF, United Kingdom GRACE H. W. WONG (20), Cardiovascular Department, Genetech, Inc., South San Francisco, California 94080 PETER K. WONG (6), Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 STEVEN XANTHOUDAKIS (15), Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 J. C. YALOWICH (63), Department of Pharmacology, University of Pittsburgh, and
Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15238 MASAKAZU YAMAOKA (29), Applied Microbiology Division, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305, Japan MARK T. YATES (51), Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215 HELEN C. YEO (8), Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 PAULOS G. YOHANNES (10), Science Division, DeKalb College, Decatur, Georgia 30034 L1-XIN ZHANG (19), Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Preface The importance of oxygen-derived radicals, reactive oxygen species, and antioxidants in health and disease is now recognized by every branch of medicine and biological science. Overwhelming evidence indicates that free radicals play a role in most major health problems of the industrialized world, including cardiovascular diseases, cancer, neurological disease, and aging, and that antioxidants play a critical role in wellness, health maintenance, and the prevention of chronic and degenerative diseases. Oxidants also play a role in some aspects of health, as in the oxidative burst of neutrophils and macrophages which allows them to kill foreign organisms. The discovery that endothelial relaxing factor is nitric oxide has provided further evidence of the role of reactive oxygen species in transcellular signaling pathways; the inducible nitric oxide synthetase in macrophages produces large amounts of nitric oxide which are cytotoxic. Transcellular signaling and cytotoxicity have generated enormous interest, not only in nitric oxide, but also in hydrogen peroxide, carbon monoxide, and other oxygen-containing compounds as modulators of cell proliferation and differentiation. Recently, a new branch of these studies has emerged. It is becoming increasingly evident that oxygen radicals and antioxidants have roles in modulating gene expression; e.g., reactive oxygen species affect transcription factors (NFK-B, AP-1) and early growth response genes (c-los, c-jun, etc.). These effects can be important both in normal growth as well as in pathological conditions. The discovery and continued exploration of such actions, as well as clarification of the subtle interactions between oxidants and antioxidants and between various antioxidants themselves, have been the result of new, more sensitive techniques for the detection and quantitation of oxygen radicals in biological systems and the merging of these techniques with the explosive and ever-changing fields of molecular biology and molecular genetics. The enormous array of technologies and new developments has required two new Methods in Enzymology volumes, Oxygen Radicals in Biological Systems (Part C, Volume 233, and Part D, Volume 234), to contain some of the best and most recent technical improvements in the field of oxidants in biological systems. The contributions to these volumes describe methods for the generation and determination of various radical species and antioxidant actions and for the study of the products of their attack on cellular components. xvii
. ° °
XVIII
PREFACE
We express great appreciation to the editorial advisory board--Bruce Ames, Kelvin J. A. Davies, Barry Halliwell, Etsuo Niki, William Pryor, and Helmut Sies--whose advice, suggestions, and contributions have helped these volumes represent the state of the art in new techniques and methods. LESTER PACKER
METHODS IN ENZYMOLOGY VOLUME I. Preparation and Assay of Enzymes
Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes
Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates
Edited by SIDNEY P, COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist
Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes
Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index
Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates
Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism
Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation
Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure
Edited by C. H. W. HIas VOLUME XII. Nucleic Acids (Parts A and B)
Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle
Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids
Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids
Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions
Edited by KENNETH KUSTIN xix
XX
METHODS IN ENZYMOLOGY
VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)
Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)
Edited by DONALD B. McCORMICK AND LEMUEL n . WRIGHT VOLUME X l X . Proteolytic Enzymes
Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND
VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)
Edited by KIVlE MOLDAVEAND LAWRENCEGROSSMAN VOLUME XXI. Nucleic Acids (Part D)
Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques
Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A)
Edited by ANTHONY SAN PIETRO VOLUME XXlV. Photosynthesis and Nitrogen Fixation (Part B)
Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B)
Edited by VICTORGINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)
Edited by LAWRENCEGROSSMANAND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)
Edited by KIVIE MOLDAVEAND LAWRENCEGROSSMAN VOLUME XXXI. Biomembranes (Part A)
Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME XXXII. Biomembranes (Part B)
Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX
Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)
Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B)
Edited by JOHN M. LOWENSTEIN
METHODS iN ENZYMOLOGY
xxi
VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)
Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)
Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)
Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)
Edited by W. A. WOOD VOLUME XLIII. Antibiotics
Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes
Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B)
Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling
Edited by WILLIAM B. JAKOBY AND MEIR WiLCHEK VOLUME XLVII. Enzyme Structure (Part E)
Edited by C. H. W. HiRS AND SERGE N. TIMASHEEE VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME XLIX. Enzyme Structure (Part G)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME L. Complex Carbohydrates (Part C)
Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism
Edited by PATRICIA A . HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes, (Part C" Biological Oxidations)
Edited by SIDNEY FLEISCHER~AND LESTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations)
Edited by SIDNEY FLEISCHER' ~ND LESTER PACKER VOLUME LIV. Biomembranes (~?art E: Biological Oxidations)
Edited by SIDNEY FLEISCHER, AND LESTER PACKER
xxii
METHODS IN ENZYMOLOGY
VOLUME LV. Biomembranes (Part F: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence
Edited by MARLENE A. DELuCA VOLUME LVIII. Cell Culture
Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)
Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)
Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME 62. Vitamins and Coenzymes (Part D)
Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I)
Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E)
Edited by DONALD B. McCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F)
Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA
Edited by RAY Wu VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)
Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A)
Edited by HELEN VAN VUNAKIS AND JortN J. LANGONE VOLUME 71. Lipids (Part C)
Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D)
Edited by JOHN M. LOWENSTEIN
METHODS IN ENZYMOLOGY
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VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE
VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton)
Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies)
Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLIS A. WOOD
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METHODS IN ENZYMOLOGY
VOLUME 90. Carbohydrate Metabolism (Part E)
Edited by WILLIS A. WOOD VOLUME 91. Enzyme Structure (Part I)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines
Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80
Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)
Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B)
Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C)
Edited by RAY Wu, LAWRENCEGROSSMAN,AND KIVlE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins)
Edited by ANTHONY R. MEANS AND BERT W. O'MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)
Edited by P. MICHAELCONN VOLUME 104. Enzyme Purification and Related Techniques (Part C)
Edited by WILLIAMB. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems
Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)
Edited by FINN WOLD AND KIVIE MOLDAVE
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VOLUME 107. Posttranslational Modifications (Part B)
Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS
VOLUME 109. Hormone Action (Part I: Peptide Hormones)
Edited by LUTZ BIRNBAUMER AND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A)
Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B)
Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A)
Edited by KENNETH J. WIDDERAND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds
Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)
Edited by HAROLDW. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B)
Edited by HAROLDW. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNIDI SABATO,JOHN J. LANGONE, AND HELEN VAN VUNAKIS
VOLUME 117. Enzyme Structure (Part J)
Edited by C. H. W. Hms AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology
Edited by ARTHUR WEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C)
Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)
Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G)
Edited by FRANK CHYTIL AND DONALD B. McCoRMICK VOLUME 123. Vitamins and Coenzymes (Part H)
Edited by FRANK CHYTIL AND DONALD B. McCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)
Edited by P. MICHAELCONN
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VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGRESTAND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERSAND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and CellMediated Cytotoxicity) Edited by GIOVANNIDI SABATOAND JOHANNESEVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B)
Edited by MARLENEDELUCA AND WILLIAMD. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARDB. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B)
Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C)
Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D)
Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E)
Edited by VICTOR GINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONYR. MEANS AND P. MICHAELCONN VOLUME 140. Cumulative Subject Index Volumes 102-119, 121-134
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VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids)
Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOUR KAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids
Edited by WILLIAM B. JAKOBY AND OWEN GRIFEITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix)
Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A)
Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B)
Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes
Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B)
Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques
Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D) Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E)
Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) Edited by RAY Wu VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na,K-Pump) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A) Edited by JAMES F. RIORDAN AND BERT L. VALLEE
.°. XXVIII
METHODS IN ENZYMOLOGY
VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action
Edited by JACKIE D. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and HemiceUulose)
Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin)
Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 164. Ribosomes
Edited by HARRY F. NOLLER, JR., AND KIVlE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology
Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids
Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria
Edited by LUSTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides)
Edited by P. MICHAELCONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A)
Edited by JACEK HAWlGER VOLUME 170. Nucleosomes
Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135-139, 141-167
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VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics)
Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism)
Edited by NORMANJ. OPPENHEIMERAND THOMASL. JAMES VOLUME 178. Antibodies, Antigens, and Molecular Mimicry
Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F)
Edited by VICTOR GINSBURG VOLUME 180. RNA Processing (Part A: General Methods)
Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 181. RNA Processing (Part B: Specific Methods)
Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification
Edited by MURRAY P. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELL F. DOOLITTLE VOLUME 184. Avidin-Biotin Technology
Edited by MEIR WILCHEK AND EDWARD A. BAYER VOLUME 185. Gene Expression Technology
Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants)
Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators
Edited by ROBERT C. MURPHY AND FRANKA. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy
Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects)
Edited by LESTERPACKER VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications)
Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells)
Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER
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METHODS IN ENZYMOLOGY
VOLUME 193. Mass Spectrometry
Edited by JAMES A. McCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology
Edited by CHRISTINEGUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN VOLUME 196. Molecular Motors and the Cytoskeleton
Edited by RICHARDB. VALLEE VOLUME 197. Phospholipases
Edited by EDWARD A. DENNIS VOLUME 198. Peptide Growth Factors (Part C)
Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168-174, 176-194 (in preparation) VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by TONY HUNTER AND BARTHOLOMEWM. SEFTON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTER AND BARTHOLOMEWM. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems
Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME 206. Cytochrome P450
Edited by MICHAELR. WATERMANAND ERIC F. JOHNSON VOLUME 207. Ion Channels Edited by BERNARDO RUDY AND LINDA E. IVERSON VOLUME 208. P r o t e i n - D N A
Interactions
Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis Edited by EDWARD A. DENNIS AND DENNIS E. VANCE
METHODS IN ENZYMOLOGY
xxxi
VOLUME 210. Numerical Computer Methods Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA)
Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA)
Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by JACEK J. HAWIGER VOLUME 216. Recombinant DNA (Part G) Edited by RAY Wu VOLUME 217. Recombinant DNA (Part H) Edited by RAY Wu VOLUME 218. Recombinant DNA (Part I) Edited by RAY Wu VOLUME 219. Reconstitution of Intracellular Transport Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A) Edited by NEJAT DOZGUNE~ VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT Dt2ZGONE~ VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitots) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 224. Molecular Evolution: Producing the Biochemical Data Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON
VOLUME 225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMANAND MELVIN L. DEPAMPHILIS
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VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME 228. Aqueous Two-Phase Systems Edited by HARRY WALTERAND GOTE JOHANSSON VOLUME 229. Cumulative Subject Index Volumes 195-198, 200-227 (in preparation) VOLUME 230. Guide to Techniques in Glycobiology
Edited by WILLIAMJ. LENNARZAND GERALD W. HART VOLUME 231. Hemoglobins (Part B: Biochemical and Analytical Methods)
Edited by JOHANNES EVERSE, KIM D. VANDEGRIFFAND ROBERT M. WINSLOW VOLUME 232. Hemoglobins (Part C: Biophysical Methods)
Edited by JOHANNES EVERSE, KIM D. VANDEGRIFFAND ROBERT M. WINSLOW VOLUME 233. Oxygen Radicals in Biological Systems (Part C)
Edited by LESTER PACKER VOLUME 234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTER PACKER VOLUME 235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIAL. CLARKAND PATRIKM. BAVOIL VOLUME 236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by VIRGINIAL. CLARKAND PATRIKM. BAVOIL VOLUME 237. Heterotrimeric G Proteins
Edited by RAVI IYENGAR VOLUME 238. Heterotrimeric G-Protein Effectors (in preparation)
Edited by RAVI IYENGAR VOLUME 239. Nuclear Magnetic Resonance (Part C) (in preparation) Edited by THOMAS L. JAMES AND NORMANJ. OPPENHEIMER VOLUME 240. Numerical Computer Methods (Part B) (in preparation)
Edited by MICHAELL. JOHNSON AND LUDWIG BRAND VOLUME 241. Retroviral Proteases (in preparation)
Edited by LAWRENCEC. Kuo AND JULES A. SHAFER VOLUME 242. Neoglycoconjugates (Part A: Synthesis) (in preparation) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 243. Inorganic Microbial Sulfur Metabolism (in preparation)
Edited by HARRY D. PECK, JR., AND JEAN LEGALL
METHODS
IN
ENZYMOLOGY
..° XXXIII
VOLUME 244. Proteolytic Enzymes: Serine and Cysteine Peptidases (in preparation) Edited by ALAN J. BARRETT VOLUME 245. Extracellular Matrix Components (in preparation) Edited by ERICKIRUOSLAHTI AND EVA ENGVALL VOLUME 246. Biochemical Spectroscopy (in preparation) Edited by KENNETH SAUER VOLUME 247. Neoglycoconjugates (Part B, Biomedical Applications) (in preparation) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases (in preparation) Edited by ALAN J. BARRETT VOLUME 249. Enzyme Kinetics and Mechanism (Part D) (in preparation) Edited by DANIEL L. PURICH
[1]
DETERMINING OXIDATIVEDNA DAMAGEBY GC/MS
3
[1] Chemical D e t e r m i n a t i o n o f Oxidative D N A D a m a g e by Gas C h r o m a t o g r a p h y - M a s s Spectrometry B y MIRAL DIZDAROGLU
Introduction Oxidative DNA damage produced by free radicals or other DNAdamaging agents has been implicated to play a role in mutagenesis, carcinogenesis, reproductive cell death, and aging.l Oxygen-derived species such as superoxide radical (O2-) and H202 are generated in all aerobic cells. 1,2 Excess generation of these species by endogenous sources or exogenous sources (e.g., redox-cyclic drugs, ionizing radiation) may cause damage to cellular DNA by a variety of mechanisms): The toxicity of these species in vivo, however, is thought to result from their metal ion-catalyzed conversion to the highly reactive hydroxyl radical (-OH). l Ionizing radiation can also produce .OH in cells and tissues by interacting with cellular water. 3 Hydroxyl radical causes formation of a number of modified bases and sugars in DNA, and DNA-protein cross-links in nucleoprotein) -7 A number of these lesions are also produced by nonradical mechanisms.8-15 For understanding of the role of oxidative DNA damage in carcinogenesis i B. Halliwell and J. M. C. Gutteridge, " F r e e Radicals in Biology and Medicine," 2nd ed. Oxford Univ. Press (Clarendon), Oxford, 1989. 2 I. Fridovich, Arch. Biochem. Biophys. 247, 1 (1986). 3 C. von Sonntag, "The Chemical Basis of Radiation Biology," pp. 116-166, 221-294. Taylor & Francis, London, 1987. 4 B. Halliwell and O. I. Aruoma, FEBS Lett. 281, 9 (1991). 5 N. L. Oleinick, S. Chiu, N. Ramakrishnan, and L. Xue, Br. J. Cancer 55, Suppl. 8, 135 (1987). 6 M. Dizdaroglu, Free Radical Biol. Med. 10, 225 (1991). 7 K. Frenkel, Pharmacol. Ther. 53, 127 (1992). 8 M. Dizdaroglu, E. Holwitt, M. P. Hagan, and W. F. Blakely, Biochem. J. 235, 531 (1986). 9 R. A. Floyd, M. S. West, K. L. Eneff, and J. E. Schneider, Arch. Biochem. Biophys. 273, 106 (1989). l0 S. Steenken, Chem. Rev. 89, 503 (1989). ii S. A. Akman, J. H. Doroshow, and M. Dizdaroglu, Arch. Biochem. Biophys. 282, 202 (1990). 12 D. J. Deeble, M. N. Schuchmann, S. Steenken, and C. von Sonntag, J. Phys. Chem. 94, 8186 (1990). J3 D. Angelov, M. Berger, J. Cadet, N. Getoff, E. Keskinova, and S. Solar, Radiat. Phys. Chem. 37, 717 (1991). 14 S. Boiteux, E. Gajewski, J. Laval, and M. Dizdaroglu, Biochemistry 31, 106 (1992). i5 H. Sies and C. F. M. Menck, Mutat. Res. 275, 367 (1992).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[l]
and other biological processes, it is essential to chemically characterize and quantify DNA lesions. In this chapter, we describe characterization and quantification of oxidative DNA damage by the technique of gas chromatography-mass spectrometry (GC/MS). Materials and Methods Reagents and enzymes used in this methodology are available commercially from a number of suppliers.16 Reference Compounds. Isobarbituric acid (5-hydroxyuracil), 5,6-dihydrothymine, 6-azathymine, 8-azaadenine, 5,6-dihydrouracil, alloxan, 5-(hydroxymethyl)uracil, 4,6-diamino-5-formamidopyrimidine, 8-bromoadenine, isoguanine (2-hydroxyadenine), 2,5,6-triamino-4-hydroxypyrimidine, and xanthine-l,3-15N2 are available from Sigma Chemical Company (St. Louis, MO), and 8-hydroxyguanine from Schweizerhall Inc. (formerly Chemical Dynamics Corporation) (Piscataway, N J). Thyminec~,ot,ot,6-2H4 is available from Merck & Co. Inc./Isotopes (St. Louis, MO). 8-Hydroxyadenine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine are synthesized by treatment of 8-bromoadenine and 2,5,6-triamino4-hydroxypyrimidine with formic acid, respectively. 17,18cis-Thymine glycol is obtained by treatment of thymine with osmium tetroxide. 8 5-Hydroxy-5-methylhydantoin is obtained by the reaction between pyruvic acid and u r e a . 19 Isodialuric acid (5,6-dihydroxyuracil) is synthesized by oxidation of isobarbituric acid with bromine. E°5-Hydroxyhydantoin is obtained by treatment of alloxan with formic a c i d . E1 These and other reference compounds, and their stable isotope-labeled analogs, which are dealt with in this chapter, are available on a custom-synthesis basis from the Chemical Synthesis and Analysis Laboratory of Program Resources Inc./Dyncorp, National Cancer Institute-FCRD (Frederick, MD). The following stable isotope-containing analogs of modified DNA bases have also become available: 5,6-dihydrothymine-l,3JSNE-2J3C, 5,6-
dihydrouracil-l,3JSNE-EJ3C, 5-hydroxy-5-methylhydantoin-l,3JSNE-EJ3C, 16 Certain commercial equipment or materials are identified in this chapter in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. 17 M. Dizdaroglu and D. S. Bergtold, Anal. Biochem. 156, 182 (1986). 18 L. F. Cavalieri and A. Bendich, J. Am. Chem. Soc. 72, 2587 (1950). ~9 S. Murahashi, H. Yuki, K. Kosai, and F. Doura, Bull. Chem. Soc. Jpn. 39, 1559 (1966). 2o R. Behrend and O. Roosen, Justus Liebig's Ann. Chem. 251, 235 (1889). 21 M. Dizdaroglu, FEBS Lett. 315, 1 (1993).
[1]
DETERMINING OXIDATIVE D N A DAMAGE BY G C / M S
5
alloxan-1,3-15N2-2,4-13C2, 5-hydroxyhydantoin-1,3A5N2-2,433C 2, 5-hydroxyuracil-l,3JSN2-2J3C , 5-(hydroxymethyl)uracil-2,433C2-a,a-2H 2 , 5-hydroxycytosine-l,3A5N2-2A3C, cis-thymine glycol-a,a,a,6-2H4, 5,6dihydroxyuracil-I,3J5N2-2J3C (isodialuric acid-l,3A5N2-233C), 4,6-diamino-5-formamidop yrimidine-l ,3-15N2-2-~3C-(5-formamidoA5 N,2 H ) , 8-hydroxyadenine-l,3,7-tSN3-2,8J3C2, 2,6-diamino-4-hydroxy-5-formamidopyrimidine-l,3J5N2-(5-amino-15N)-2A3C, and 8-hydroxyguanine-l,3JSN 2(2-aminoA5N)-233C. The synthesis of 5-(hydroxymethyl)uracil-2-13C-5 2H2-6-2H has also been reported elsewhere. 22 Most compounds listed above are soluble in water. 8-Hydroxyguanine and 8-hydroxyguanine-l,3-t5N2-(2-amino35N)-233C are not completely soluble. However, complete solubility can be obtained by increasing the pH of the solutions to 9.5 with dilute NaOH and then stirring the solutions for several hours at room temperature.
Hydrolysis The preparation of DNA or nucleoprotein samples for analysis by GC/MS involves hydrolysis followed by derivatization. DNA can be hydrolyzed by either acidic hydrolysis or enzymatic hydrolysis. Acidic Hydrolysis. Acidic hydrolysis cleaves the glycosidic bonds between bases and sugar moieties in DNA, releasing intact and modified bases. Formic acid is well suited for hydrolysis of DNA. z3-25 In the case of DNA-protein cross-links, nucleoprotein is hydrolyzed by the standard method of protein hydrolysis using 6 M HC1. By this procedure, peptide bonds in proteins as well as glycosidic bonds in DNA are cleaved to release DNA base-amino acid c r o s s - l i n k s . 26'27 The stability of numerous pyrimidine- and purine-derived modified DNA bases and their release from DNA have been studied under various conditions of formic acid hydrolysis, because this information is important for the assessment of the accuracy of the DNA damage measurement. 25,28 It was found that most of the modified bases are stable under all studied conditions of hydrolysis, and only a few undergo partial destruction, depending on the concentration of formic acid. 25Furthermore, the possibility that some of the modified bases may be formed by acidic treatment in 22 Z. Djuric, D. A. Luongo, and D. A. Harper, Chem. Res. Toxicol. 4, 687 (1991). 23 G. R. Wyatt and S. S. Cohen, Biochem. J. 55, 774 (1953). 24 M. Dizdaroglu, Anal. Biochem. 144, 593 (1985). z5 Z. Nackerdien, R. Olinski, and M. Dizdaroglu, Free Radical Res. Commun. 16, 259 (1992). 26 E. Gajewski, A. F. Fuciarelli, and M. Dizdaroglu, Int. J. Radiat. Biol. 54, 445 (1988). 27 M. Dizdaroglu, E. Gajewski, P. Reddy, and S. A. Margolis, Biochemistry 28, 3625 (1989). 28 A. F. Fuciarelli, B. J. Wegher, E. Gajewski, M. Dizdaroglu, and W. F. Blakely, Radiat. Res. 119, 219 (1989).
6
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[1]
DNA from corresponding intact bases has been investigated. The results indicated that formic acid caused no significant formation of modified bases under the conditions used. As a conclusion of these studies, formic acid at a concentration of 60% (v/v) has been found to be optimal for DNA hydrolysis. 25 The following procedure is used for DNA hydrolysis. An aliquot of DNA (10 to 100/zg) is treated with 0.5 ml of formic acid (60%) in evacuated and sealed tubes at 140° for 30 min. The sample is then transferred to a vial, frozen in liquid nitrogen, and lyophilized. If chromatin instead of pure DNA is to be hydrolyzed for detection of modified bases, the same hydrolysis procedure is followed. It should be pointed out that formic acid hydrolysis causes deamination and dehydration of cytosine-derived products as follows: cytosine glycol yields a mixture of 5-hydroxycytosine and 5-hydroxyuracil, the former by dehydration and the latter by dehydration and deamination. 8 5,6-Dihydrocytosine, 5-hydroxy-6-hydrocytosine and 5,6-dihydroxycytosine deaminate to give 5,6-dihydrouracil, 5-hydroxy-6-hydrouracil and 5,6-dihydroxyuracil, respectively. 5-Hydroxyhydantoin, which has been identified in the past as a product of cytosine, 29 results from acid-induced decarboxylation of alloxan. 21 For detection of DNA-protein cross-links, nucleoprotein containing 100 ~g DNA is hydrolyzed with 0.5 ml of 6 M HCI in evacuated and sealed tubes at 120° for 6 hr. Subsequently, the sample is transferred into a vial, frozen in liquid nitrogen and lyophilized. Enzymatic Hydrolysis. This type of hydrolysis has been discussed elsewhere in detail. 3° The following procedure can be used to hydrolyze DNA to nucleosides. An aliquot (0.1 mg) of DNA is incubated in 0.5 ml of 10 mM Tris-HCl buffer, pH 8.5 (containing 2 mM MgC12), with deoxyribonuclease I (100 units), spleen exonuclease (0.01 unit), snake venom exonuclease (0.5 units), and alkaline phosphatase (10 units) at 37 ° for 24 hr. The sample is then transferred to a vial, frozen in liquid nitrogen, and lyophilized. The drawback of enzymatic hydrolysis is that the products of the 2'-deoxycytidine moiety in DNA cannot be readily analyzed by GC/MS because of the poor gas chromatographic properties of cytidine derivatives. 31 On the other hand, generally less volatile trimethylsilyl [(CH3)3Si] derivatives of modified purine 2'-deoxynucleosides can be analyzed successfully by GC/MS.31'32 Deamination of 2'-deoxyadenosine products may 29 M. Polverelli and R. Troule, Z. Naturforsch., C: Biosci. 29C, 12 (1974). 3o p. F. Crain, this series, Vol. 193, p. 782. 3z M. Dizdaroglu, J. Chromatogr. 367, 357 0986). 32 M.-L. Dirksen, W. F. Blakely, E. Holwitt, and M. Dizdaroglu, Int. J. Radiat. Biol. 54, 195 (1988).
[1]
DETERMINING OXIDATIVEDNA DAMAGEBY GC/MS
7
occur during enzymatic hydrolysis owing to contaminating deaminase activity in the enzymes. Removal of excess salt from the hydrolyzates and removal of deaminases from the enzymes may prevent problems associated with analysis of 2'-deoxycytidine products and deamination of 2'-deoxyadenosine products. 3°'33
Derivatization DNA bases, nucleosides, and DNA base-amino acid cross-links are not sufficiently volatile for gas chromatography, and thus must be converted to volatile derivatives. For this purpose, trimethylsilylation is the mode of derivatization most frequently used. 34 (tert)-Butyldimethylsilylation is also u s e d . 33'35'36 The following procedure can be used for trimethylsilylation. Lyophilized hydrolyzates of DNA or nucleoprotein samples containing 0.01-0.1 mg of DNA are heated with 0.1 ml of a mixture of bis(trimethylsilyl)trifluoroacetamide (containing I% trimethylchlorosilane) and acetonitrile (4 : 1, v/v) at 140° for 30 rain in polytetrafluoroethylene-capped vials (sealed under dry nitrogen). The amounts of the reagents can be modified according to the amount of DNA. After derivatization, samples are cooled to room temperature. Without any further treatment, an aliquot (e.g., l tzl) of each derivatized sample is injected into the injection port of the gas chromatograph.
Instrumentation
A GC/MS instrument equipped with a capillary inlet system and a computer data system can be used. Data presented and reviewed here were obtained on commercial quadrupole mass spectrometers. Fusedsilica capillary columns are used for separation of derivatized hydrolyzates of DNA or nucleoprotein. These types of columns provide high inertness, excellent separation efficiency, and measurement of high sensitivity. Fused-silica capillary columns coated with cross-linked 5% phenyl methylsilicone gum phase appear to be best for the p u r p o s e . 24'37 Column length may vary depending on the type of analysis. A column 12.5 m in length (0.2 mm internal diameter, 0.33 tzm film thickness) is generally used for analysis of derivatized bases. A shorter column (e.g., 8 m, 0.2 mm internal 33 p. F. Crain, Mass Spectrom. Rev, 9, 505 (1990). 34 K. H. Schram, this series, Vol. 193, p. 791. 35 M. A. Quilliam and J. B. Westmore, Anal. Chem. 50, 59 (1978). 36 M. Dizdaroglu, BioTechniques 4, 536 (1986). 37 M. Dizdaroglu, J. Chromatogr. 295, 103 (1984).
8
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[1]
diameter, 0.11 /~m film thickness) serves best for analysis of derivatized DNA base-amino acid cross-links. Helium (ultrahigh purity) is used as the carrier gas. The split mode of injection is used to avoid overloading the column. The split ratio (i.e., ratio of the carrier gas flow through the splitter vent to carrier gas flow through the column) is adjusted according to the DNA amount in samples. An aliquot of DNA hydrolyzates corresponding to approximately 0.1 to 0.4 ~g of DNA on the GC column after splitting of the injected sample is generally sufficient. The injection port of the gas chromatograph and the GC/MS interface are kept at 250°. The temperature of the ion source of the mass spectrometer is usually kept at around 200° . Some instruments may permit variation of this temperature zone. The glass liner in the injection port of the gas chromatograph is filled with silanized glass wool, which allows homogeneous vaporization of injected samples. Electronionization (El) mode at 70 eV has been used to record mass spectra and to perform selected-ion monitoring in studies presented or reviewed here.
Gas Chromatography-Mass Spectrometry Free Bases. Gas chromatography on a fused-silica capillary column (usually 12.5 m long) permits separation of (CH3)3Si derivatives of a large number of modified bases from one another and from the four intact DNA bases in a single analysis. 24 Compounds eluting from the GC column are ionized in the ion source and then analyzed by the mass analyzer of the mass spectrometer) 8 Electron-ionization mass spectra of (CH3)3Si derivatives of modified DNA bases provide considerable structural detail that can be used for unequivocal identification. These mass spectra are characterized by an intense molecular ion (M "+ion), an intense (M - 15) ÷ ion, and other characteristic i o n s , 24'37 as are those of the intact bases, a9'4° The (M - 15) + ion results from the loss of a methyl radical from the M .+ ion. 39'4° In some instances, an intense (M - l) ÷ ion resulting from loss of an H atom from the M .+ ion also appears in the m a s s spectra. 24'37 Nucleosides. Trimethylsilyl derivatives of modified 2'-deoxynucleosides follow the same fragmentation patterns as those of other nucle-
38 j. T. Watson, "Introduction to Mass Spectrometry," Chapters 1 and 3. Raven, New York, 1985. 39 E. White, V. P. M. Krueger, and J. A. McCIoskey, J. Org. Chem. 37, 430 (1972). 40 j. A. McCloskey, in "Basic Principles in Nucleic Acid Chemistry" (P. O. P. Ts'o, ed.), Vol. l, p. 209. Academic Press, New York, 1974.
[1]
DETERMINING OXIDATIVE D N A DAMAGE BY G C / M S
9
osides/1,42 Prominent ions are the (base + H) + ion [(B + 1) + ion] and the (base + H - CH3) + ion, whereas the M .+ and the (M - 15) + ions are of low intensity. 31For example, in the mass spectra of (CH3)3Si derivatives of 8-hydroxy-2'-deoxyguanosine and 8-hydroxy-2'-deoxyadenosine, the (B + 1) + ion appears as the most prominent ion (100% relative intensity) owing to stabilization through an electron-donating substituent at C-8 of the purine ring. Trimethylsilyl derivatives of 8,5'-cyclopurine 2'-deoxynucleosides provide partly different fragmentation patterns from those of other nucleosides. 32'43These spectra are characterized by prominent ions containing the base plus portions of the sugar moiety and by an intense M "+ ion, most likely due to stabilization by the increased number of rings in the molecule. 44 DNA Base-Amino Acid Cross-links. Mass spectra of (CH3)3Si derivatives of DNA base-amino acid cross-links contain M .+ and (M - 15) + ions and other characteristic ions resulting from typical fragmentations of base and amino acid moieties, z6'~7'37 For example, the most prominent ion (m/z 448) in the mass spectrum of the (CH3)3Si derivatives of the thymine-tyrosine cross-link results from cleavage of the bond between the a and/3 carbons of the tyrosine moiety accompanied by an H atom transfer [(M - 218 + 1) + ion]. The high abundance of this ion is due to resonance stabilization through the aromatic ring. This cleavage, which is typical of (CH3)3Si derivatives of amino acids, also accounts for the intense m/z 218 ion when the charge is retained on the a carbon without an H atom transfer. In the case of DNA base-aliphatic amino acid crosslinks, these fragmentations also occur. However, an ion arising from loss of "CO~Si(CH3)3 from the M "+ ion generally appears as one of the most prominent ions in the mass spectra, whereas the abundance of the (M 218 + 1) + ions depends on the aliphatic amino acid residue. Figures 1 and 2 illustrate the structures of modified DNA bases and nucleosides and some DNA base-amino acid cross-links that can be measured by the use of the GC/MS technique. These compounds are formed in DNA or nucleoprotein by free radicals or other agents causing oxidative damage. 6,45 DNA base-amino acid cross-links involving thymine and the amino acids glycine, alanine, valine, leucine, isoleucine, and threonine, 41 H. Pang, K. H. Schram, D. L. Smith, S. P. Gupta, L. B. Towsend, and J. A. McCloskey, J. Org. Chem. 47, 3923 (1982). 42 j. A. McCloskey, this series, Vol. 193, p. 825. 43 M. Dizdaroglu, Biochem. J. 238, 247 (1986). 44 F. W. McLafferty, "Interpretation of Mass Spectra." Univ. Sci. Books, Mill Valley, California, 1980. 45 M. Dizdaroglu, Mutat. Res. 275, 331 (1992).
10
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
o
,~= •It"
z
o
~
-,-
o ~"
~
}
,~o
==
/ / ~
=
0
t.
Eta
== =
.=. ~;
"o
0
z: :ooz
"
:
= .d==
~ - o o ~-
.:
~
~.
_
~
~
~95% supercoiled form; the plasmid, available from Stratagene (Heidelberg, Germany), is purified from Escherichia coli strains DHFa or XLl-blu by CsCI double banding 3 or by chromatography on affinity columns (Diagen, Dtisseldorf, Germany) TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0
Agarose Gel Electrophoresis Loading buffer (agarose gel): 20 mM EDTA, 40% glycerol, pH 7.4, 0.4 mg/ml bromphenol blue TBE buffer: 45 mM Tris, 45 mM boric acid, 10 mM EDTA, pH 8.3 Agarose gel: 1% in TBE, boil for 2 min, let cool to 65°, pour Molecular weight marker: h phage DNA digested with BstEII Ethidium bromide: 10 mg/ml stock solution
End-Labeling of Damaged DNA Buffers and nucleotides may have to be changed when enzymes other than KpnI and XhoI are to be used. 10 × Restriction buffer: 100 mM Tris-HC1, pH 7.5, 100 mM MgCI2, I0 mM dithiothreitol (DTT) Restriction enzymes: KpnI and XhoI at - 10 U//.d; restriction enzymes must be devoid of endonuclease activity, which is assayed by incubating overnight 1/xg of supercoiled plasma DNA that does not contain recognition sites for the enzymes used (e.g., pBR322) with 10 U enzyme each in restriction buffer and checking for loss of supercoiled form as described below 3 H. Miller, this series, Vol. 152, p. 145.
48
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[4]
Adaptation buffer: 10 mM Tris-HCl, I0 mM MgCIz, 200 mM NaC1, 1 mM DTT, pH 7.5 Dilution buffer: 30 mM Tris, 14 mM MgCl 2 , 14 mM DTT, pH 7.5, 25 p.M dTTP, 0.5/.tCi//zl [a-3Zp]dCTP (3000 Ci/mmol) Sequenase 2.0: Available from United States Biochemical (Bad Homburg, Germany) Loading buffer (polyacrylamide gel): Included in Sequenase kit, see below
Preparation of Sequence Marker Lanes Sequenase 2.0 kit: Available from United States Biochemical KS primer: Available from Stratagene
Sequencing Gel A 5% polyacrylamide denaturing gel is prepared and run under standard conditions. 4 Procedure
Treatment of Plasmid DNA with Activated Oxygen It is essential to start with a plasmid preparation containing as high a fraction of the intact supercoiled form as possible (usually >95%). Two micrograms DNA is incubated with activated oxygen, the reaction is terminated, and the source of activated oxygen is removed. The DNA is dialyzed and/or purified by ethanol precipitation 5 to remove contaminants from the reaction. Enzymes such as glucose oxidase or xanthine oxidase used to generate activated oxygen species may be removed by proteinase K digestion followed by extraction with high-quality phenol. 6 Finally, the DNA is redissolved in 10 mM Tris, 1 mM EDTA, pH 8.0, at 0.1 mg/ml.
Determining Amount of Damage Before proceeding to localization of the strand breaks, the amount and type of DNA damage are determined by agarose gel electrophoresis. This is useful for several reasons. The method described below will not distinguish between double- and single-strand breaks. Also, among multiple 4 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning," 2nd Ed., Chapter 13. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 5 D. M. Wallace, this series, Vol. 152, p. 41. 6 D. M. Wallace, this series, Vol. 152, p. 33.
[4]
LOCALIZING STRAND BREAKS IN PLASMID D N A
C
1
2
3
4
5
6
7
8
9
49
L
FIc. 3. Estimation of DNA damage by agarose gel electrophoresis. One microgram of pBluescript plasmid DNA was treated for 10 min with different concentrations of DNase I in the presence of Mg2÷ ; the reaction was terminated by addition of EDTA and heating at 65° for 10 min. The products were analyzed on an agarose gel as described in the text. C, Untreated plasmid; L, h phage DNA digested with BstElI; lanes 1-9, plasmid DNA treated with 10-is-10 -7 U DNase I.
single-strand breaks in the same molecule only the one closest to the restriction sites will be seen. Spontaneous strand breaks will also be detected, and their frequency limits the sensitivity of the assay. Therefore, a level of damage with a maximum of single-strand breaks and few doublestrand breaks is optimal. Moreover, with some reagents the level of damage may be difficult to reproduce. In that case it is advisable to carry several samples differing in exposure to the damaging agent through the procedure in addition to appropriate controls. Aliquots containing approximately 1 p.g plasmid DNA treated with activated oxygen species are mixed with agarose gel loading buffer and electrophoresed on a 1% agarose gel alongside 1 k~g untreated pBluescript DNA. 7 A molecular weight marker, for example, ?~-phage DNA digested with BstEII, is included on the gel. Figure 3 shows a model experiment with DNase I using conditions where the enzyme introduces mostly singlestrand breaks. The control plasmid still contains greater than 90% supercoiled form after incubation in buffer alone, but also a fraction of relaxed circular plasmid form with single-strand breaks that runs more slowly. With increasing concentration of DNase I the proportion of plasmid in the relaxed form increases, and eventually double-strand breaks appear 7 R. C. Ogden and D. A. Adams, this series, Vol. 152, p. 61.
50
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[4]
as indicated by a fragment at 3.0 kb, the size expected for linearized plasmid. Further increases in DNase I concentration cause degradation of DNA detectable as a smear of fragments smaller than 3 kb. In this experiment, samples 4-7 in addition to the control plasmid would have been chosen for further investigation.
End-Labeling Analysis For end-labeling analysis, 7.2 ~1 of each sample in a small reaction tube is mixed with 0.8/zl of 10 × restriction buffer. Eight units KpnI in a maximum volume of 0.8/.d is added (or another chosen enzyme yielding 3'-protruding ends). Following thorough mixing by hand, the reaction mixture is incubated at 37° for 1 hr. Then 4/zl is removed and checked on an agarose minigel for complete digestion. Meanwhile, 4/zl of adaptation buffer and 5 U of XhoI are added to the remainder of the sample, and incubation is continued for at least I hr. After the minigel has been analyzed and digestion by KpnI has been found to be complete, 8/xl of dilution buffer containing the 32p-labeled deoxynucleotide triphosphate and 2.5 U of Sequenase 2.0 are added. Following incubation for another 30 min, 4 ttl of loading buffer is added; the samples are denatured for 2 min at 95 ° and put rapidly on ice, where they may be stored until loading. Approximately 4-5 /zl of each sample is loaded on a standard 5% polyacrylamide sequencing gel 4 alongside the 35S-labeled marker DNA. The four marker lanes should be next to one another, but it is useful to alternate empty ones with the sample lanes, since/3 radiation from 32p may obscure the signal from the markers and from weaker samples and the control. Marker DNA is prepared by running standard sequencing reactions 4 with pBluescript plasmid DNA using an appropriate primer and a-35Slabeled deoxynucleotide triphosphates. With the above enzymes, KS primer is most convenient to use. The marker DNA can be prepared in advance and may be stored for several weeks at - 20°. Following fixation and drying, the gel is exposed to X-ray film. 8 An amplifying screen is usually not required.
Interpretation of Data The location of single-strand breaks is readily determined from the autoradiograph (Fig. 2). Note, however, that the strand investigated for strand breaks is opposite to the one synthesized in the marker DNA. Thus, a band in the C-marker lane corresponds to a strand break at G in 8 W. M. Bonner, this series, Vol. 152, p. 55.
[S]
Fe/H202
DNA
DAMAGE PRODUCTS
51
the sample, and so on. Also, marker DNA and sample fragments do not start at the same position. Depending on which enzyme is used, which nucleotides are filled in, and which primer is employed, marker and samples are off by several bases. For instance, when cutting with XhoI, filling in with dTTP and labeled dCTP, and using KS primer for marker synthesis, the fragments differ in length by only a single base (Fig. 1). To facilitate interpretation of complex damage patterns, it has proved useful to load marker DNAs on both sides of the samples on the sequencing gel. To compare the frequency of single-strand breaks at individual sites, suitable autoradiographs are analyzed using one of several commercially available densitometer systems (e.g., GSXL, LKB, Piscataway, N J).
[5] D e t e c t i n g D N A D a m a g e C a u s e d b y I r o n a n d Hydrogen Peroxide B y YONGZHANG LUO, ERNST S. HENLE, RAJAGOPAL CHATOPADHYAYA, RUCHENG JIN, and STUART LINN
Introduction
This chapter describes methods to assess DNA damage mediated by Fenton reactions via hydroxyl radicals (.OH): Fe 2+ + H202 + H+---~ Fe 3+ + H20 + "OH
Depending on the state of chelation of the iron, including DNA chelation, other oxygen radicals such as ferryl radicals 1 are possible, and these may also be reactive toward DNA. Additionally superoxide radical (02 ~) is formed by the following scavenging reaction: •OH
+
H202 ---> H20 + O2: + H ÷
Identification of the stable products derived from DNA after exposure to Fenton reactions is difficult owing to the multitude of products. Therefore, we have devised methods for observing the damage spectra of the four deoxynucleosides, nucleotides, oligonucleotides, homopolymers, and finally duplex DNA. After enzymatic conversion to nucleosides, products are identified by high-performance liquid chromatography (HPLC) retention times, radiolabeling, mass spectrometry, nuclear magnetic resonance, i j. D. Rush and W. H. Koppenol, J. Biol. Chem. 261, 6730 (1986).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[S]
Fe/H202
DNA
DAMAGE PRODUCTS
51
the sample, and so on. Also, marker DNA and sample fragments do not start at the same position. Depending on which enzyme is used, which nucleotides are filled in, and which primer is employed, marker and samples are off by several bases. For instance, when cutting with XhoI, filling in with dTTP and labeled dCTP, and using KS primer for marker synthesis, the fragments differ in length by only a single base (Fig. 1). To facilitate interpretation of complex damage patterns, it has proved useful to load marker DNAs on both sides of the samples on the sequencing gel. To compare the frequency of single-strand breaks at individual sites, suitable autoradiographs are analyzed using one of several commercially available densitometer systems (e.g., GSXL, LKB, Piscataway, N J).
[5] D e t e c t i n g D N A D a m a g e C a u s e d b y I r o n a n d Hydrogen Peroxide B y YONGZHANG LUO, ERNST S. HENLE, RAJAGOPAL CHATOPADHYAYA, RUCHENG JIN, and STUART LINN
Introduction
This chapter describes methods to assess DNA damage mediated by Fenton reactions via hydroxyl radicals (.OH): Fe 2+ + H202 + H+---~ Fe 3+ + H20 + "OH
Depending on the state of chelation of the iron, including DNA chelation, other oxygen radicals such as ferryl radicals 1 are possible, and these may also be reactive toward DNA. Additionally superoxide radical (02 ~) is formed by the following scavenging reaction: •OH
+
H202 ---> H20 + O2: + H ÷
Identification of the stable products derived from DNA after exposure to Fenton reactions is difficult owing to the multitude of products. Therefore, we have devised methods for observing the damage spectra of the four deoxynucleosides, nucleotides, oligonucleotides, homopolymers, and finally duplex DNA. After enzymatic conversion to nucleosides, products are identified by high-performance liquid chromatography (HPLC) retention times, radiolabeling, mass spectrometry, nuclear magnetic resonance, i j. D. Rush and W. H. Koppenol, J. Biol. Chem. 261, 6730 (1986).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
52
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[5]
and UV absorption spectra. Quantitation is achieved either by UV absorption or radiolabeling. Materials Chromatography Columns. Analytical silica-based C18 reversed-phase (RP) columns (250 × 4.6 mm, 5/zm) and semipreparative normal-phase and RP columns are from Alltech (Deerfield, IL). Polystyrene reversedphase (PRP) columns are from Hamilton (Reno, NV). Saturator columns are constructed from empty analytical columns which are gravity-filled with chromatographic grade 230-400 mesh silica gel from Sigma (St. Louis, MO). Enzymes. DNase I is from Sigma, and units are defined by Kunitz. 2 P1 nuclease is from Boehringer Mannheim (Indianapolis, IN) with units as defined by Fujimoto et al. 3 Snake venom phosphodiesterase (SVP) is from Worthington (Freehold, N J) with units defined by Razell and Khorana. 4 Bacterial alkaline phosphatase (BAP) is from Worthington with units defined by Garen and Levinthal. 5Enzymes are dialyzed extensively versus 25 mM triethylammonium bicarbonate (TEAB), pH 7.5, to remove contaminants which may be mistaken for products and to remove nonvolatile salts which interfere with subsequent analyses by mass spectrometry (MS) or nuclear magnetic resonance (NMR). Substrates. Substrates are repurified by HPLC where possible. When unavailable, radiolabeled deoxynucleoside monophosphates (dNMPs) can be produced from commercially available deoxynucleoside triphosphates (dNTPs) by treatment with S V P 6 and then purified by ion-pairing C~8 HPLC (mobile phase TEAB, pH 7, 0-30% methanol), followed by C18 RP-HPLC (mobile phase water) with a 100-fold molar excess of NaHCO3 injected with the sample. Treatment of nucleotides with BAP will yield the corresponding nucleosides, which are purified by RP-HPLC (mobile phase water). Suitable dNpNs and dNpNpNs are purified by ion-pairing HPLC (mobile phase TEAB, pH 9, 0-30% methanol) on a PRP column. The sample is then further purified on a C18 RP-HPLC column (mobile phase water for 10 min followed by a 0-50% methanol gradient), with a 100-fold molar excess of NaHCO3 injected with the sample. The dNpN and dNpNpN structures are confirmed by SVP digestion, to dNp and dN, and quantitation after HPLC. 2 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). s M. Fujimoto, A. Kuninaka, and H. Yoshino, Agric. Biol. Chem. 38, 777 (1974). 4 W. E. RazeU and H. G. Khorana, J. Biol. Chem. 234, 2105 (1959). 5 A. Garen and C. Levinthal, Biochim. Biophys. Acta 38, 470 (1960). 6 S. E. Pollack and D. S. Auld, Anal. Biochem. 127, 81 (1982).
[5]
Fe/HzO2 DNA DAMAGEPRODUCTS
53
DNA is best obtained from purified virus particles. Covalently closed and supercoiled DNA is preferred, since its integrity is easily tested. Most eukaryotic DNAs contain 5-methylcytosine which adds to the product spectrum, so we use supercoiled phage PM2 DNA which lacks modified bases and which can be radiolabeled in vivo 7 or by nick-translation. The DNA is extensively dialyzed against EDTA then against 25 mM NaCI. It can also be used to monitor nicking by the Fenton reagents. 8 Concentrations of H202 are measured by UV absorbance, assuming an extinction coefficient (e240) of 39.4 M - 1 cm- 1.9 Ferrous concentrations are determined either with Ferrozine, assuming that Fe 2+(Ferrozine)3 has an 8562 value of 28,000 M - 1 cm- 1,10 or with o-phenanthroline, assuming that Fe2+(o-phenanthroline)3 has an e510 value of l 1,100 M -~ cm -1. Methods Fenton Reaction Conditions. Substrates can be subjected to H202induced damage under a variety of conditions using Fe z+ or Fe 3+. NADH
and/or ethanol can be present, and the reaction can be made anaerobic by flushing with N 2 . Two millimolar H202 is used because this concentration gives maximal killing of Escherichia coli. 8 In most cases, damage is proportional to Fe z+ concentrations up to 1.2 mM. For 1 mM substrate, ! mM iron is used to get 5-50% damage, and 2 mM NADH is used where desired. To avoid secondary attack on the highly reactive compounds dC and dCMP, 0.4 mM iron and 0.8 mM NADH are used. Damage caused by freely diffusible .OH can be eliminated in the presence of 100 mM ethanol, an .OH scavenger. When purging O2 with N2, for a 2-ml volume the Oz concentration drops from 8 to less than or equal to 0.1 ppm by 5 min. Prior to H 2 0 2 addition, the pH of reaction mixtures is adjusted with NaOH to between pH 6.5 and 6.8. The order of reactant addition is substrate-iron-(NADH)-(ethanol)-H202 so that the iron can associate with the substrate before it reacts with HzO z or is chelated by NADH. The reaction is started by adding a 10 mM H2Oz stock solution while vortexing and then is maintained at 25° for 30 min. Vortexing during rapid addition of HzO2 is necessary to transient differences in concentrations. Postreaction Digestion to Nucleosides. For most products, separation and purification are best attained by enzymatic digestion to nucleosides followed by C18 RP-HPLC. DNase I digests DNA to short oligomers which 7 U. Kuhnlein, E. E. Penhoet, and S. Linn, Proc. Natl. Acad. Sci. U.S.A. 73, 1169 (1976). 8 j. A. Imlay and S. Linn, Science 240, 1302 (1988). 9 D. P. Nelson and L. A. Kiesow, Anal. Biochem. 49, 474 (1972). I0 T. J. Giovanniello and T. Peters, Jr., in "Standard Methods of Clinical Chemistry" (D. Seligson, ed.), Vol. 4, p. 139. Academic Press, New York, 1963.
54
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[5]
are substrates for P1 nuclease and SVP. Both P1 nuclease and SVP should be used to ensure complete digestion of oligonucleotides to mononucleotides.l~'12 Because deaminases may contaminate SVP preparations, excessive use of the enzyme should be avoided. Mononucleotides are dephosphorylated to nucleosides by BAP. To our knowledge, no damage products are refractory to digestion or altered by these procedures with the exception that we did not observe dephosphorylation of 2-deoxy-D-ribono-8lactone-5-phosphate by BAP. For DNA, reactions contain 1 mM substrate (nucleotide residues), 20 mM Tris-HCl, pH 7.5, 2.25 mM MgC12 , 50/xM CaC12, and 20-100 units/ ml DNase I and are incubated for 30 min at 25 °. Twenty microliters of 1 M sodium acetate, pH 5.2, and 30 units of P1 nuclease are then added per milliliter, and incubation is continued for 30 min. Twenty-five microliters of 1 M Tris-HC1, pH 8.0, 20 units SVP, and 20 units BAP are then added per milliliter, and incubation is continued for 30 min. For homopolymers, the substrate is suspended in 20 mM sodium acetate, pH 5.5, 3 mM MgC12, and digestion with P1 nuclease, SVP, and BAP is carried out as above. For mononucleotides, the substrate is suspended in 20 mM TrisHCI, pH 7.5, and digested with BAP as above. Digests are centrifuged for 1 min at 5000 g, then the supernatants are filtered through 0.22-/xm Millipore (Bedford, MA) filters and finally brought to 10 mM potassium phosphate, pH 6.5. For large-scale reactions of dNs or dNMPs for NMR or MS analysis 1 M N H 4 H C O 3 o r TEAB is added to a 500-ml Fenton reaction mixture to bring the pH to 6-7. After centrifugation for 1 min at 5000 g, the supernatant is passed through a 0.22-/.~m Millipore filter and then dried under vacuum. TEAB is generally preferred as a buffer for desiccation since it is not so volatile as N H 4 H C O 3 . It does not interfere with fast atom bombardment MS in the negative ion mode [FAB-MS(- )], but will interfere in the positive ion mode [FAB-MS(+)]. N H 4 H C O 3 interferes with both modes of FAB, but since it is so volatile, it can be adequately removed from sample preparations destined for FAB-MS(+). The dried sample is redissolved in the mobile phase before HPLC. High-Performance Liquid Chromatography. We use a binary Perkin Elmer (Norwalk, CT) LC-250 pump and a saturator column upstream of the injector to prolong the life of silica-based columns. Mobile phases are purged with helium to minimize bubble formation and UV absorption by oxygen below 210 nm. The pH of the mobile phase is important for obtaining symmetric peaks. When the pH approaches the PKa of an eluate, its II M. Liuzzi, M. Weinfeld, and M. C. Paterson, J. Biol. Chem. 264, 6355 (1989). 12 M. Weinfeld, M. Liuzzi, and M. C. Paterson, Nucleic Acids Res. 17, 3735 (1989).
[5]
Fe/H202 D N A DAMAGE PRODUCTS 4000
-
55
4 dG
3000
o. 0
2000
2
1000
1
67
3
5 8
0
~ 0
5
9
----'-"~ 10 15 Time
1
11
"~"
~ 20
r. 25
-'--I 30
(min)
FIG. 1. Radiochromatogram of products from [I',2'-3H]dG (dashed line) and [8-14C]dG (solid line) after exposure to FeZ÷/H202 under aerobic conditions after RP-HPLC. The reaction mixture contained 1 mM" dG, 1 mM FeSO4, and 2 mM H202 and was processed as described in the text. Approximately 105 cpm of each isotope was injected. Peaks: (1) unknown; (2) 2-deoxy-o-ribono-~-lactone and unknown 247-Da compound; (3) unknown 179-Da compound; (4) unknown 273-Da compound; (5) unknown; (6) 5',8-cyclodeoxyguanosine; (7) guanine; (8) 9-(2'-deoxy-fl-D-erythro-pentopyranosyl)guanine; (9) 9-(2'-deoxy-fl-oerythro-pento-l,5-dialdo-l,4-furanosyl)guanine; (10) 9-(2'-deoxy-a-D-erythro-pentofuranosyl)guanine; (dG) 2'-deoxyguanosine; (11) 5',8-cyclo-2',5'-dideoxyguanosine.
peak may be distorted. We have found that pH 6.5 is well-suited for the separations. Potassium phosphate buffer is used because it can chelate residual iron that would otherwise damage the column. For nucleoside mixtures, a C]8 RP-HPLC column is eluted with 2% methanol v/v in 10 mM potassium phosphate, pH 6.5, at 1 ml/min for 10 min followed by a gradient to 30% methanol v/v 70% 10 mM potassium phosphate, pH 6.5, for 40 min. For dC products, all products elute before the gradient is applied. For resolution of nucleotides by C]8 RP-HPLC for FAB-MS( - ), elution is with 10 mM TEAB, pH 7, for 60 min followed by 100% methanol. Alternatively, for resolution by PRP columns, the TEAB is adjusted to pH 9. Samples for FAB-MS(+) or NMR spectroscopy are purified by RP-HPLC with water at 1 ml/min for 60 min followed by 100% methanol. The dC products are not well-resolved by RP-HPLC because of their greater polarity. Accordingly they are separated first by normal-phase silica HPLC 13 followed by RP-HPLC with 2% methanol in 10 mM potassium phosphate, pH 6.5, before RP-HPLC with water. The HPLC eluates are monitored by a diode array detector. We use a Perkin Elmer LC-480 detector which continuously records UV spectra t3 j. R. Wagner, J. E. van Lier, C. Decarroz, M. Berger, and J. Cadet, this series, Vol. 186, p. 502.
56
OXIDATIVE DAMAGETO DNA AND DNA REPAIR 400 -
10-15 1 -.__55 8 -
a.
dC
16-18
/
300'
0
[5]
200" 21-22 100"
19-20 ~
23
A_
0 0
2
4
6 Time
;
'0
1 '2
1'4
(min)
FIG. 2. Radiochromatogram of products from [UJ4C]dCMP after exposure to Fe2+/H202 under aerobic conditions resolved on RP-HPLC prior to final resolution on normal-phase HPLC. The reaction mixture contained 1 mM dCMP, 0.4 mM FeSO4, and 2 mM H202 and was processed as described in the text. Peaks: (1-5) 2-deoxy-D-ribono-8-1actone-5phosphate, 2'-deoxyribosyl-NLformyl-N2-glyoxylurea,4-amino-l-formyl-5-hydroxy-2-oxoA3-imidazoline, parabanic acid, alloxan; (6-7) 2'-deoxyribosylurea, 5,6-dihydro-5,6-dihydroxydeoxycytidine; (8-9) 2'-deoxyribosylformamide, trans-l-carbamoylimidazolidone-4, 5-diol; (10-15) cis/trans-dihydroxyuracil, cis-dihydroxyuracil, cis/trans-dihydroxydeoxyuridine, cis/trans-dihydroxyuracil, 2,4,5-trihydroxydeoxypyrimidine, 2'-deoxyribosyl-aminol-formyl-5-hydroxy-2-oxo-A3-imidazoline;(16-18) cytosine, 5-hydroxyhydantoin, cis/transdihydroxyuracil; (19-20) 2'-deoxyribosyl-trans-1-carbamoylimidazolidone-4,5-diol,oxaluric acid; (21-22) 2'-deoxyribosyl-5-hydroxyhydantoin, 2'-deoxyribosylbiouret; (23) 1-carbamoyl-l-carboxy-4-(2-deoxy-fl-D-erythro-pentofuranosyl) glycinamide; (dC) 2'-deoxycytidine. for storage on a computer. A diode array detector allows immediate UV spectra for identification of products, quantitation by integration of UV profiles at the maximum absorbance for each product, a useful UV trace without test runs, and determination of peak purity by assessing absorption ratios across a peak. Radiochromatograms have the advantages of reliable quantitation, reduction of the number of observable products to avoid overcrowding, and confirmation of structure by which labeled atoms remain. Tritiated water from 3H abstraction b y . O H or 14CO2 from decarboxylation can be r e m o v e d by desiccation. Substrate radiolabeling is essential for identifying degradation products from iron/NADH/H202 systems, since UV-absorbing N A D H and its degradation products coelute with products of interest. Characterization o f Eluates. Eluates may be characterized by their H P L C retention times, UV absorbance spectra, and/or the use of specifically radiolabeled substrates. If standard compounds are unavailable, MS
[5]
Fe/H202 DNA DAMAGEPRODUCTS
dA
dT
L
0.050 ~D
57
5
A
0.0
-
i
0
i
i
lo
i
I
11
I
I
i
i
5
i
I
i
l
10 Time
i
i
15
i
i
i
i
20
(rain)
FIG. 3. Ultraviolet absorbance profile of products from poly(dA) after exposure of poly (dA) : oligo(dT) to Fe 2+/H202 under aerobic conditions resolved by RP-HPLC. The reaction mixture contained 0.52 mM (dA)2000 and 0.48 mM (dT)z0 (nucleotide residues), 50 mM NaCl, 1 mM FeSO4, and 2 mM H202. After reaction, 8 M was added to disrupt the duplex structure, and the mixture was centrifuged in a Centricon concentrator 30 (Amicon, Danvers, MA) at 4000 rpm. Small molecules (e.g., released bases) and thymine oligomers not associated with the poly(dA) passed through the filter. The filter was washed 7 times with water to remove urea, and the retentate was removed from the filter by reverse centrifugation and then digested to nucleosides and processed as described in the text. Peaks: (1) unknown; (2) 2'-deoxyribosyl-4,6-diamino-5-formamidopyrimidine; (3) unknown from thymine family; (4) unknown; (5) unknown from thymine family; (6, 7) unknown; (8) 2'-deoxyribosyl-5formyluracil; (9) 2'-deoxyribosylisoguanosine; (10) 5',8-cyclo-dA (R and S); (dT) 2'-deoxythymidine; (11) adenine; (12) unknown; (dA) 2'-deoxyadenosine; (13) 8-oxo-7,8dihydrodeoxyadenosine.
and NMR analysis can be used to determine a structure. Samples for NMR are desiccated twice in D20 and redisolved in D20 or deuterated dimethyl sulfoxide (DMSO). We have chosen FAB-MS over other forms of MS since the molecular ion is less likely to be fragmented, and polar samlbles are more easily volatilized. 14 FAB-MS(+) is used for bases and nucleosides, since these generally bear no charge but can associate with a proton under these conditions. F A B - M S ( - ) is sensitive for anionic dNMP and dNpN residues. A glycerol matrix is used as the internal marker. Figures 1-4 are representative chromatograms, with preliminary peak identifications, from a member of the guanine, cytosine, adenine, and thymine families, respectively. 14 A. G. Harrison and R. J. Cotter, this series, Vol. 193, p. 3.
58
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[5]
dT
2501
2001
3
15° I
8
O lOOoi O
J
0
.
.
.
.
i
5
.
.
.
.
i
10
.
.
.
.
i
•
5
20 25 Time (rain)
-
i
30
•
•
,~,
i
35
.
.
.
.
i
40
FIG. 4. Radiochromatogram of products from [methyl, l',2'-3H]dT-labeled PM2 DNA under aerobic conditions resolved by RP-HPLC. The reaction mixture contained 1 mM DNA (nucleotide residues), 10 mM NaC1, 1 mM FeSO4, and 2 mM H202. After 30 min at 25°, the reaction mixture was subjected to enzymatic digestion as described in the text. Peaks: (1) 2'-deoxyribosyl-5-hydroxylmethyluracil; (2, 3, 4, 5) 2'-deoxyribosyithymine glycol (four isomers); (X) contaminant from undamaged DNA substrate; (6) 2'-deoxyribosylpyruvylurea; (7) 2'-deoxyribosyl-5-hydroxy-5-methylhydantoin; (8) thymine; (Y) contaminant from undamaged DNA substrate; (dT) 2'-deoxythymidine.
Discussion The above procedures have been applied to characterize DNA damage at the nucleoside, nucleotide, and polynucleotide levels. Enzymatic conversion to nucleosides avoids harsh chemical hydrolysis conditions which can destroy (or form) products. To our knowledge no products are lost owing to their resistance to enzymatic conversion. However, these procedures are not necessarily recommended as a replacement for chemical hydrolysis before analysis, but rather as a complement to those techniques. An isotope effect is noted with dG (Fig. 1). Singly or multiply labeled [14C]dG coelutes with its nonradioactive counterpart, but [ I ' , 2 ' - 3 H ] d G elutes 2.2% faster under isocratic conditions. The [I',2'-3H]dGTP, from which the nucleoside was prepared, was tested for its authenticity by incorporation into DNA with DNA polymerase I and digesting the purified DNA to nucleosides. The recovered 3H-labeled material behaves in the manner described above. Similar 3H isotope effects have been previously reported. 15,16 15 K. Frenkel, M. S. Goldstein, and G. W. Teebor, Biochemistry 20, 7566 (1981). 16 p. D. Klein, in "Advances in Chromatography" (J. C. Giddings and R. A. Keller, eds.), pp. 3-65. Dekker, New York, 1966.
[6]
PHOTOCHEMICAL
SYNTHESIS
OF 8-HYDROXYGUANINE
59
Acknowledgments This research was supported by U.S. Public Health Service Grants R37 GM19020, T32 ES07075, and P30 ES01896 and a grant from the Chevron Corporation Risk Assessment Program. We are indebted to Drs. Richard Wagner and Clinton Ballou for helpful advice, to Sherry Ogden for MS analyses, and to Richard Mazzarisi and Graham Ball for NMR analyses.
[6] P h o t o c h e m i c a l 8-Hydroxyguanine
Synthesis
of
Nucleosides
B y PETER K. WONG and ROBERT A. FLOYD
Introduction O x y g e n free radicals have been implicated in the etiology of processes associated with m a n y diseases and pathological states. The molecular basis of the action of oxygen free radicals is probably manifold but most likely involves oxidative damage to macromolecules including proteins and nucleic acids. Oxygen free radical-mediated damage to nucleic acids to form base adducts represents an attractive notion to explain their effect in cancer development. Kasai and Nishimura I first noted that reducing agents, involving the action of hydroxyl free radicals, mediated formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) when these agents acted on either isolated D N A or the free nucleoside. Since these first observations, n u m e r o u s biological models have been investigated to determine if 8-oxo-dG can be implicated in carcinogenesis. The status of research in this area has been summarized, and it is clear that in models where oxidative damage has been implicated, there is a close association between the increased presence of 8-oxo-dG and the d e v e l o p m e n t of cancer, z In addition, there have been extensive studies devoted to the use of 8-oxodG excretion in the urine as an index of oxidative stress and as a p a r a m e t e r to assess the role of oxygen free radicals in aging.3 Singlet oxygen produced by the action of light on methylene blue or by chemical systems has been shown to form 8-oxo-dG in D N A . 4,5 The use of high-performance liquid l H. Kasai and S. Nishimura, Nucleic Acids Res. 12, 2137 (1984). z R. A. Floyd, Carcinogenesis (London) 11, 1447 (1990). 3 j. McCann, V. Simmon, D. Streitwieser, and B. N. Ames, Proe. Natl. Acad. Sci. U.S.A. 72, 3190 (1975). 4 R. A. Floyd, M. S. West, K. L. Eneff, and J. E. Schneider, Arch. Biochem. Biophys. 273, 106 (1989). 5 T. P. A. Devasagayam, S. Steenken, M. S. W. Obendorf, W. A. Schulz, and H. Sies, Biochemistry 30, 6283 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[6]
PHOTOCHEMICAL
SYNTHESIS
OF 8-HYDROXYGUANINE
59
Acknowledgments This research was supported by U.S. Public Health Service Grants R37 GM19020, T32 ES07075, and P30 ES01896 and a grant from the Chevron Corporation Risk Assessment Program. We are indebted to Drs. Richard Wagner and Clinton Ballou for helpful advice, to Sherry Ogden for MS analyses, and to Richard Mazzarisi and Graham Ball for NMR analyses.
[6] P h o t o c h e m i c a l 8-Hydroxyguanine
Synthesis
of
Nucleosides
B y PETER K. WONG and ROBERT A. FLOYD
Introduction O x y g e n free radicals have been implicated in the etiology of processes associated with m a n y diseases and pathological states. The molecular basis of the action of oxygen free radicals is probably manifold but most likely involves oxidative damage to macromolecules including proteins and nucleic acids. Oxygen free radical-mediated damage to nucleic acids to form base adducts represents an attractive notion to explain their effect in cancer development. Kasai and Nishimura I first noted that reducing agents, involving the action of hydroxyl free radicals, mediated formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) when these agents acted on either isolated D N A or the free nucleoside. Since these first observations, n u m e r o u s biological models have been investigated to determine if 8-oxo-dG can be implicated in carcinogenesis. The status of research in this area has been summarized, and it is clear that in models where oxidative damage has been implicated, there is a close association between the increased presence of 8-oxo-dG and the d e v e l o p m e n t of cancer, z In addition, there have been extensive studies devoted to the use of 8-oxodG excretion in the urine as an index of oxidative stress and as a p a r a m e t e r to assess the role of oxygen free radicals in aging.3 Singlet oxygen produced by the action of light on methylene blue or by chemical systems has been shown to form 8-oxo-dG in D N A . 4,5 The use of high-performance liquid l H. Kasai and S. Nishimura, Nucleic Acids Res. 12, 2137 (1984). z R. A. Floyd, Carcinogenesis (London) 11, 1447 (1990). 3 j. McCann, V. Simmon, D. Streitwieser, and B. N. Ames, Proe. Natl. Acad. Sci. U.S.A. 72, 3190 (1975). 4 R. A. Floyd, M. S. West, K. L. Eneff, and J. E. Schneider, Arch. Biochem. Biophys. 273, 106 (1989). 5 T. P. A. Devasagayam, S. Steenken, M. S. W. Obendorf, W. A. Schulz, and H. Sies, Biochemistry 30, 6283 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
60
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[6]
2ram ID
FIG. 1. Glass coil used to carry out the irradiation reaction in the synthesis of 8-OH derivatives of guanosine or deoxyguanosine by the action of methylene blue and light.
chromatography with electrochemical detection (HPLC/ED) as an extremely sensitive method to measure 8-oxoguanine in DNA and RNA 6 has prompted us to develop newer and easier methods to prepare the 8-oxo-dG and 8-hydroxyguanosine (8-oxo-G). Here we present methods based on the use of methylene blue plus light. The ease of synthesis by these newer methods is valuable for researchers in the field.
Materials and Methods
Reagents Guanosine, 2'-deoxyguanosine, and 8-hydroxyguanine; Sigma Chemical Company (St. Louis, MO) Methylene blue; Aldrich Chemical Company (Milwaukee, WI) AG 50W-X2 cation-exchange resin (200-400 mesh, H + form); BioRad Laboratories (Richmond, CA) Sephadex G- 15; Pharmacia Fine Chemicals (Piscataway, N J)
Synthesis of 8-Hydroxyguanosine by Methylene Blue and Light Prepare a reaction mixture consisting of 450 ml of 2.2 mM guanosine in water and 50 ml of 1.0 mM methylene blue in water. The above mixture is stored in a reservoir and allowed to slowly pass through a glass coil having dimensions as shown in Fig. 1. The flow rate of the mixture is adjusted so that a portion of the mixture is exposed to a 100-W flood light at a distance of 15 cm for approximately 30 min. The liquid, which contains unreacted guanosine, 8-oxo-G, and a small amount of by-products as well 6 R. A. Floyd, J. J. Watson, P. K. Wong, D. H. Altmiller, and R. C. Rickard, Free Radical Res. C o m m u n . 1, 163 (1986),
[6]
PHOTOCHEMICAL SYNTHESIS OF 8-HYDROXYGUANINE
61
as methylene blue, is collected as it exits from the coil and is stored at 4° in the dark. The reaction mixture is then first passed through an AG 50W-X2 cation-exchange column, 38 x 50 mm, at a flow rate of 1.5 ml/ min. Methylene blue and unreacted guanosine are strongly retained on the column while 8-oxo-G and some by-products are eluted more quickly. After all the liquid containing the reaction products has passed through the column, 190 ml of deionized water is added to the column to wash out the remaining 8-oxo-G. The first 160 ml of eluate does not contain 8-oxo-G and is discarded, and the remaining clear eluate is collected and evaporated under vacuum to about 150 ml. The solution containing 8-oxo-G is then loaded on to a second AG 50W-X2 column, 38 x 120 mm, and allowed to run through at a rate of 1.5 ml/min. Deionized water is added to continue the flow. The eluate is collected in 20-ml fractions. Fractions 14 through 25 are rich in 8-oxo-G and are pooled together for a total of 220 ml. Because the elution profile changes as the chromatographic conditions vary, it is necessary to determine if 8-oxo-G is present by injecting each fraction into a HPLC/ED system. The pooled fractions are then evaporated under vacuum to a volume of about 2 ml and stored at 4° for crystallization. The yield of product is about 3%.
Synthesis of 8-Hydroxyguanosine by Udenfriend System The method of 8-oxo-G synthesis is similar to that described by Kasai and Nishimura ~ for 8-oxo-dG synthesis with some modifications. The reaction mixture consists of the following: 0.25 g guanosine in 390 ml of 0.1 M phosphate buffer (pH 6.8), 32 ml of 0.1 M EDTA, 8 ml of 0.1 M FeSO4, and 70 ml of 0.1 M ascorbic acid. The reaction is allowed to proceed at 37° with constant bubbling of oxygen for 3 hr in the dark. The removal of iron, ascorbate, and buffer is accomplished by adding about 100 ml of the reaction mixture onto a Sephadex G-15 gel filtration column, 38 x 200 mm. This is followed by addition of deionized water. The eluate is collected in 20-ml fractions. The first 300 ml of liquid is discarded. At this point, all the yellow bands should have passed out of the column. The remaining fractions, about 180 ml, are collected and pooled. The elution profile is monitored for products using HPLC/ED. The maximum load of the column is about 100 ml, so the remaining reaction mixture has to be run in 100-ml batches following the same procedure. The eluates containing 8-oxo-G collected from each run are pooled together and vacuum evaporated to a final volume of about 150 ml. Guanosine and other by-products are removed by the procedure described previously for the purification of 8-oxo-G formed by the action of methylene blue and light. Thus, 150 ml of the pooled eluate, which
62
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[6]
consists mainly of unreacted guanosine and 8-oxo-G, are loaded onto an AG 50W-X2 cation-exchange column, 38 × 120 mm, and allowed to run through at a rate of 1.5 ml/min. Deionized water is added to continue the elution. The eluate is collected in 20-ml fractions. Fractions rich in products (14 through 25) are pooled and then evaporated under vacuum to a volume of about 2 ml, followed by storage at about 4° for crystallization. The yield is about 4%.
Synthesis of 8-Hydroxy-2'-deoxyguanosine by Methylene Blue and Light The reaction mixture consists of 450 ml of 3.8 mM 2'-deoxyguanosine and 50 ml of 1.0 mM methylene blue in water. The above mixture is stored in a reservoir and slowly passed through a glass coil as described before. Experimental conditions are similar to those used for the 8-oxo-G synthesis. After exposure to light the reaction mixture, consisting of dG, 8-oxo-dG, methylene blue, and a small amount of by-products, is fractionated, collected, and stored in the dark at about 4° for further treatment as described before. About 500 ml of the reaction mixture is first passed through an AG 50W-X2 cation-exchange column, 38 × 50 mm, at a flow rate of 1.5 ml/min. Methylene blue and unreacted deoxyguanosine are strongly absorbed on the column, while the product, 8-oxo-dG, and small amount of by-products are eluted faster. After all the liquid containing the reaction product has passed through the column, about 250 ml of deionized water is added to wash out the remaining product. After discarding the first 180 ml of liquid, the rest of the eluate is collected and evaporated under vacuum to about 150 ml. This is then loaded onto a second AG 50W-X2 cation-exchange column, 38 × 120 mm, as described before, and allowed to run through at a rate of 1.5 ml/min. The first 420 ml is discarded, and fractions 22 through 36, a total of 280 ml rich in product, are pooled, evaporated under vacuum to about 2 ml, and stored at 4 ° for crystallization. The yield is about 1%.
Characterization of 8-Hydroxy-2'-deoxyguanosine and 8-Hydroxyguanosine The UV spectra of 8-oxo-dG and 8-oxo-G were obtained, and the spectral parameters agree with previously obtained v a l u e s , 6 a s listed below. Ultraviolet Spectra. Spectral parameters for 8-oxo-dG and 8-oxo-G are as follows: 8-oxo-dG, max (e) 248 nm (12,034), 295 nm (10,440), 206 nm (26,170); 8-oxo-G, max (e) 248 nm (12,880), 295 nm (10,940), 206 nm (25,640).
[6]
PHOTOCHEMICAL
SYNTHESIS OF 8-HYDROXYGUANINE
63
High-Performance Liquid Chromatography with Electrochemical Detection Figure 2 s h o w s the H P L C / E D traces w h e n 8 - o x o - G and 8 - o x o - d G p r e p a r e d by the K a s a i and N i s h i m u r a p r o c e d u r e o r the m e t h y l e n e blue plus light p r o c e d u r e w e r e injected and run. It is clear that the p r o d u c t s p r e p a r e d b y either p r o c e d u r e are identical. Figure 3 s h o w s the h y d r o d y n a m i c v o l t a m m o g r a m s o f 8 - o x o - G p r e p a r e d by the K a s a i and N i s h i m u r a ~ p r o c e d u r e as c o m p a r e d to the m e t h y l e n e blue plus light p r o c e d u r e . T h e plots are identical. T h e same is true f o r
T C
® t'O
B
O~ nr'~ ¢9 LU
1
A
I
I
I
I
0
10
20
30
(Min)
Fro. 2. HPLC chromatograms of 8-oxo-O (peak 1) and 8-oxo-dO (peak 2). (A) Ten microliters of a solution containing 10 mM 8-oxo-G and 10 mM 8-oxo-dG prepared by the method described by Kasai and Nishimura 1was analyzed. (B) Ten microliters of a solution consisting of 10 mM 8-oxo-G and 10 mM of 8-oxo-dG prepared by the action of methylene blue plus light was analyzed. In (C) 10 t~l each of the solutions used in (A) and (B), for 20 ~l total, was injected onto the HPLC/ED system. Chromatographic conditions: mobile phase; acetate/citrate buffer, pH 5.1, with 4% methanol; flow rate, 0.8 ml/min; column, Hibar Cl8 column, 10 ram, 4 x 250 ram; electrochemical detector, set at +0.750 V versus Ag/AgC1; UV detector, 0.05 AUFS, 254 nm.
64
OXIDATIVEDAMAGETODNA ANDDNA REPAIR
[6]
1.0
0.8
u) C o CL
0.6
n" C]
o
0.4
0.2 ~ 0
0.~
|
0'.6
i
!
0.8
|
110
E vs Ag/AgCI (volt)
FIG. 3. Hydrodynamic voltammograms of (e) 8-oxo-G prepared by the method described by Kasai and NishimuraI and ((3) 8-oxo-G prepared by the action of methylene blue plus light.
1.00.8ta
c
O O nO I.U
0.6
02/
S
0.4
0
|
o.,
0'.6
o18
11o
E vs Ag/AgCI (volt)
FIG. 4. Hydrodynamic voltammograms of(e) 8-oxo-dG prepared by the method described by Kasai and NishimuraI and (©) 8-oxo-dG prepared by the action of methylene blue plus light.
[6]
PHOTOCHEMICAL SYNTHESIS OF 8-HYDROXYGUANINE
65
the hydrodynamic voltammograms of 8-oxo-dG prepared by the Kasai and Nishimura I procedure as compared to the methylene blue plus light procedure (Fig. 4). Discussion The Udenfriend system for synthesis of 8-oxo-dG or 8-oxo-G requires that reaction components and buffer present in the incubation mixture be removed before the evaporation step. Incomplete removal of these components could result in the formation of large amount of an unknown white precipitate which interferes with the isolation of the product. In the Kasai and Nishimura I method, active charcoal was used to accomplish this goal. Because properties of charcoal vary, some strongly absorbing the product and thus causing problems in the desorption step, it was found that the use of the gel filtration material Sephadex G-15 can better achieve separation of product from the starting materials and buffer. By using the methylene blue system for 8-hydroxyguanine nucleoside synthesis, the isolation step is simplified because only a few components are present in the system, and the use of the AG 50W-X2 cation-exchange resin can then effectively separate the product from the mother compounds. Guanosine or deoxyguanosine were strongly retained on the AG 50W-X2 cation exchanger. The use of stronger acid may then be required to elute the compound. Alternatively, the resin can be regenerated by stirring in ascorbic acid solution. Ascorbate reduces methylene blue, a cation, to a colorless neutral leuco form of methylene blue which can then be washed from the resin. It was found that deoxyguanosine or guanosine dissolved in water reacts with methylene blue to produce the 8-OH derivative in the highest yield. Phosphate buffer ranging from pH 5.2 to 7.4 was tested, and all systems gave less product than the reaction mixture without any buffer present. If the reaction mixture was placed inside an open beaker and irradiated with light, the product generated was susceptible to further changes; therefore, the timing of irradiation is important. Thus, when a glass coil was used as described above, the product was stable and fewer by-products were formed.
66
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7] C o p p e r - D N A
[7]
Adducts
By MARK J. BURKITT Introduction Redox active metal ions such as iron and copper are believed to play a central role in the formation of reactive oxygen species in biological systemsJ ,2 Although iron has received the most attention in this capacity (perhaps because of its greater cellular abundance), the role of copper may be particularly crucial because copper occurs in the mammalian cell nucleus where it may be involved in the condensation of DNA-histone fibers into higher order chromatin structures. 3-5 Consequently, to those concerned with the role of oxygen radicals in biomolecular damage, interest in copper-DNA adducts stems from the possibility that endogenous, DNA-associated copper may be able to promote oxidative damage to DNA. 4-7 As with iron, copper serves to convert superoxide (02"-3 and hydrogen peroxide, formed during the metabolism of oxygen 8,9 (particularly in the presence of redox-cycling xenobiotics l°'H), to the highly reactive hydroxyl radical (.OH), which is generally considered to be the oxidizing species l B. HaUiwell and J. M. C. Gutteridge, this series, Vol. 186, p. 1. 2 S. D. Aust, L. A. Morehouse, and C. Thomas, J. Free Radicals Biol. Med. 1, 3 (1985). 3 C. D. Lewis and U. K. Laemmli, Cell (Cambridge, Mass.) 29, 171 0982). 4 A. M. George, S. A. Sabovljev, L. E. Hart, W. A. Cramp, G. Harris, and S. H o r n s e y , Br. J. Cancer 55, Suppl. 8, 141 (1987). 5 W. A. Cramp, A. M. George, H. Khan, and M. B. Yatvin, in "Free Radicals, Metal Ions and Biopolymers" (P. C. Beaumont, D. J. Deeble, B. J. Parsons, and C. Rice-Evans, eds.), p. 127. Richelieu Press, London, 1989. 6 W. A. Cramp, A. M. George, J. C. Edwards, S. A. Sabovljev, G. Harris, L. E. Hart, H. Lambert, and M. B. Yatvin, in "Prostaglandin and Lipid Metabolism in Radiation Injury" (T. L. Walden, Jr. and H. N. Hughes, eds.), p. 59. Plenum, New York, 1987. 7 R. Stoewe and W. Prtitz, Free Radical Biol. Med. 3, 97 (1987). s A. Boveris and E. Cadenas, in "Superoxide Dismutase" (L. Oberley, ed.), Vol. 2, p. 15. CRC Press, Boca Raton, Florida, 1982. 9 N. Oshino, B. Chance, H. Sies, and T. Biicher, Arch. Biochem. Biophys. 154, 117 (1973). i0 R. P. Mason, Environ. Health Perspect. 87, 237 (1990). 11 G. V. Rumyantseva and L. M. Weiner, FEBS Lett. 234, 459 (1988).
METHODS IN ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress, Inc. All fights of reproductionin any form reserved.
[7]
COPPER-DNA ADDUCTS
67
responsible for the induction of biomolecular damage [reactions ( 1 ) - ( 3 ) ] . 1'2'7A2 The ability of DNA-bound copper ions to participate in O f + Cu 2 + ~ 02 + Cu + 202-7+ 2H + ~ H202 + 02 Cu ÷ + H202--~ Cu 2+ + .OH + O H -
(1) (2) (3)
the catalysis of hydroxyl radical formation has been demonstrated by numerous investigators in well-defined chemical s y s t e m s . 7,13-16 Evidence for the participation of endogenous copper in such reactions in intact cells is, however, less extensive) 7 In this chapter, methods are described for the investigation of DNA oxidation by bound copper ions in well-defined systems, and approaches are suggested for demonstrating the (possible) participation of endogenous copper ions in the mediation of damage to DNA in intact cells exposed to oxidants. The oxidative lesions induced in DNA following exposure to copper ions and reduced oxygen species, which include strand breaks and hydroxylated base products, are expected to be similar in nature (though not distribution) to those induced by oxygen radicals generated via other means (e.g., the Fe2+-H202 system). ~5Therefore, any of the conventional methods of determining oxidative damage to DNA may be applied to systems employing copper. These methods, which are described in detail elsewhere, include alkaline elution, DNA unwinding, and plasmid nicking techniques for the determination of strand breaks and gas chromatography-mass spectrometry (GC-MS) techniques for the determination of oxidized base products (e.g., 8-hydroxyguanine). ~3-~5'18-2~In this chapter, full experimental details are given only for techniques that are particularly applicable to copper systems.
12 M. J. Burkitt and B. C. Gilbert, Free Radical Res. Commun. 14, 107 (1991). 13 J.-L. Sagripanti and K. H. Kraemer, J. Biol. Chem. 264, 1729 (1989). 14 C. J. Reed and K. T. Douglas, Biochem. J. 275, 601 (1991). 15 O. I. Aruoma, B. Halliwell, E. Gajewski, and M. Dizdaroglu, Biochem. J. 273, 601 (1991). 16 L. Milne, P. Nicotera, S. Orrenius, and M. J. Burkitt, Arch. Biochem. Biophys. 304, 102 (1993). ~7 H. C. Birnboim, Arch. Biochem. Biophys. 294, 17 (1992). 18 G. Ahnstr6m and K. Erixon, in " D N A Repair: A Laboratory Manual of Research Procedures" (E. C. Friedberg and P. C. Hanawalt, eds.), Vol. 1, Part B, p. 403. Dekker, New York, 1981. ~9C. von Sonntag and H.-P. Schuchmann, this series, Vol. 186, p. 511. 2o M. K. Shigenaga, J.-W. Park, K. C. Cundy, C. J. Gimeno, and B. N. Ames, this series, Vol. 186, p. 521. 21 M. Dizdaroglu and E. Gajewski, this series, Vol. 186, p. 530.
68
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7]
Physical Properties of Copper-DNA Complexes
Copper(II) In 1968, Eichhorn and Shin 22 described the effects of various divalent metal ions, including those of the first transition series, on DNA melting and annealing. Metal ions believed to bind to DNA primarily at the phosphate moiety [e.g., Mg(II)] were found to stabilize DNA, whereas those which bind primarily at the base residues [such as Cd(II) and Cu(II)] were found to destabilize the double helix. Copper(II) was shown to bind to DNA with a higher affinity than any of the other divalent cations studied, which is believed to be one reason why copper is particularly effective in promoting the oxidation of DNA: oxidation by H 2 0 2 with copper as a catalyst has been reported to be 50 times faster than that with iron. 7 Kagawa et al.23 have resolved the crystal structure (to 1.2 A resolution) of CuCIE-soaked duplex DNA and suggested that Cu(II) forms a regular octahedral complex, with four water ligands in the equatorial plane and a fifth water along with the N-7 of a guanine residue in the axial positions. This appears to be the prototypical structure, of which all other complexes with guanine are a variation. 23 Although studies employing Cu(II)-GMP complexes have indicated that the Y-phosphate oxygen atoms from neighboring GMP molecules can also act as equatorial ligands, 24 this has not been observed in duplex DNA. 23 Regular octahedral binding is not observed at adenine residues, but an axially distorted trigonal bipyrimidal complex may occur when both adenine and guanine residues are able to interact and share a single copper(II) center. 25
Copper(l) Whereas Cu(II) binding causes destabilization of the double helix, 22 Cu(I) binding causes stabilization, 26 and it may involve the conversion of certain DNA regions from the B conformation to the Z conformation, z7 Indeed, Minchenkova and Ivanov suggested that the oxidation state of copper may play a role in the control of DNA synthesis by determining 22 G. L. Eichhorn and Y. Ae Shin, J. Am. Chem. Soc. 90, 7323 (1968). 23 Z. F. Kagawa, B. H. Geierstanger, A. H.-J. Wang, and P. Shing Ho, J. Biol. Chem. 266, 20175 (1991). 24 K. Aoki, G. R. Clark, and J. D. Orbell, Biochim. Biophys. Acta 425, 369 (1976). 25 B. H. Geierstanger, T. F. Kagawa, S.-L. Chen, G. J. Quigley, and P. Shing Ho, J. Biol. Chem. 266, 20185 (1991). 26 S. J. Atherton, in " F r e e Radicals, Metal Ions and Biopolymers" (P. C. Beaumont, D. J. Deeble, B. J. Parsons, and C. Rice-Evans, eds.), p. 93. Richelieu Press, London, 1989. 27 W. A. Prfitz, J. Butler, and E. J. Land, Int. J. Radiat. Biol. 58, 215 (1990).
[7]
COPPER-DNA ADDUCTS
69
the conformation of the double helix. 28 Compared with Cu(II), there have been considerably fewer studies on the binding of copper(I) to DNA. This is presumably a reflection of the instability of the copper(I) oxidation state. Copper(I) ions in aqueous solution undergo rapid disproportionation, z9 Although stable stock solutions of copper(l) can be prepared using nonprotic solvents, such as acetonitrile, 3° the interaction of Cu(I) with DNA has been studied most successfully following its generation in situ via the addition of a reducing agent (e.g., ascorbate) to DNA in the presence of Cu(II). 7'28 Copper(I) may also be formed via the radiolytic reduction of Cu(II). 27 This involves the y-irradiation (0.33 Gy sec -1) of air-saturated formate solutions. The radiolysis products from water are rapidly converted to the reducing superoxide radical [reactions (4)-(8)]. Binding of the Cu(I) to DNA competes with its loss via disproportionation. H20 _1~ -OH(0.28), eaq(0.28), H'(0.06) •OH + HCO2- ~ H20 + CO27 C O 2 "7"q- 0 2 ~
CO 2 + 027
e~-q(H-) + 02--~ (H ÷) + O Z 027 + Cu 2+ ~ 02 + Cu +
(4) (5) (6) (7) (8)
Using this method of Cu(I) generation, Prtitz et al. 27 have obtained UV absorbance difference spectra of the DNA-Cu(I) complex. Spectra are characterized by a minimun at 250-255 nm, a maximum at 295 nm, and an isobestic point around 270 nm. These features are believed to be due primarily to the fixation of Cu(I) to the guanine and cytosine bases, accompanied by enolization, proton transfer from the N-l-guanine to the N-3-cytosine, and conformational changes, z7'28 Similar spectra have been obtained using ascorbate as the reductant of Cu(II). 28 Detection of Copper-Dependent Hydroxyl Radical Formation Copper-dependent hydroxyl radical formation has been demonstrated in many model systems, generally via the application of standard methods of .OH determination. For example, van Steveninck et al. 31 have used salicylate hydroxylation to detect the hydroxyl radical in systems containing HzO2, Cu(II), and a reducing agent. However, the most direct method of hydroxyl radical detection is electron spin resonance (ESR) spectros2s L. E. Minchenkova and V. I. Ivanov, Biopolymers 5, 615 (1967). 29 F. A. Cotton and G. Wilkinson, "Advanced Inorganic Chemistry," 5th Ed. Wiley (Interscience), New York, 1988. 3o p. M. Hanna and R. P. Mason, Arch. Biochem. Biophys. 295, 205 (1992). 31 j. van Steveninck, J. van der Zee, and T. M. A. R. Dubbelman, Biochem. J. 232, 309 (1985).
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OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7]
copy in conjunction with a spin trapping reagent. 32'33 ESR spin-trapping evidence for .OH formation via reaction (3) has been obtained by Hanna and Mason, 3° who used Cu(I) prepared in deoxygenated acetonitrile. The •OH radical was detected as its adduct to the spin trap 5,5-dimethyll-pyrroline N-oxide (DMPO). The DMPO/.OH adduct can also arise, however, via a nonradical mechanism involving the Cu(II)-stimulated nucleophilic addition of water to DMPO. 3°'34 Although this can lead to problems in the detection of copper-dependent. OH formation, the contribution of the nucleophilic route to DMPO/.OH formation can be minimized by the careful use of copper chelating agents (e.g., by using a citrate-based buffer. 34,35 A further problem is that the DMPO/.OH adduct is prone to reduction to an ESR-silent hydroxylamine in the presence of Cu(I)) 5 Since the radical adducts resulting from the trapping of carbon-centered radicals are generally more stable than those derived from oxygen-centered radicals, 36 a more reliable indication of .OH formation is obtained if the radical is first reacted with a suitable scavenger molecule to form a carbon-centered radical before trapping (see below and Refs. 16, 35, and 36). Electron Spin Resonance Detection of Copper-DNA Adduct-Dependent Hydroxyl Radical Formation The pattern of hydroxylated base products detected following the incubation of DNA with copper(II), H202, and a reducing agent indicates that DNA-bound copper ions can support hydroxyl radical formation.15 Indeed, Yamamoto and Kawanishi 37 have described possible mechanisms by which DNA-bound copper ions may interact with hydrogen peroxide to form oxidizing species. These workers suggested that a peroxide bridge is formed between two copper centers prior to electron transfer. In support of this hypothesis, Kagawa and co-workers have described models which indicate that DNA can accommodate peroxide-bridged copper(II) centers at adjacent guanine residues. 23 ESR spin-trapping techniques have been applied to the detection of hydroxyl radical formation following the interaction of DNA-bound copper(II) ions with ascorbate and H202 [reactions (9) and (10), in which 32 G. R. Buettner and R. P. Mason, this series, Vol. 186, p. 127. 33 R. P. Mason, P. M. Hanna, M. J. Burkitt, and M. B. Kadiiska, Environ. Health Perspect. (in press). 34 p. M. Hanna, W. Chamulitrat, and R. P. Mason, Arch. Biochem. Biophys. 296, 640 (1992). 35 M. J. Burkitt, L. Milne, S. Y. Tsang, and S. C. Tam, Arch. Biochem. Biophys., (in press). 36 M. J. Burkitt, Free Radical Res. Commun. 18, 43 (1993). 37 K. Yamamoto and S. Kawanishi, J. Biol. Chem. 264, 15435 (1989).
[7]
COPPER-DNA ADDUCTS
71
Asc 2- and Asc: represent the dianion of ascorbic acid and the ascorbyl radical, respectively]. 16 Because oxygen-radical adducts of DMPO (the D N A - C u 2+ + ASC 2- ~ D N A - C u + + Asc-:D N A - C u + + H202----~ D N A - C u z+ + OH- + .OH
(9) (10)
spin trap used most commonly to detect .OH) can undergo conversion to ESR-silent hydroxylamines in the presence of reductants (e.g., superoxide), 32 and because of the instability of the DMPO/-OH adduct in the presence of metal ions, 36 a secondary trapping technique has been employed in which the .OH radical is first converted to the methyl radical ('CH3) via scavenging with dimethyl sulfoxide (DMSO). The methyl radical is then trapped and detected as its relatively stable adduct to the spin trap N-tert-butyl-et-phenylnitrone (PB N), PBN/. CH 3 [reactions (11) and (12)].38 (CH3)280 + "OH---> CHaSO2H + "CH3 PBN + -CH 3~ PBN/'CH 3
(11) (12)
Method The standard 2-ml reaction mixture contains 25 mM potassium phosphate buffer (pH 7), 2 M DMSO, 100 mM PBN, 2 mg/ml DNA, 1 mM CuCI2, 1 mM H202, and 1 mM ascorbate. The phosphate buffer is added from a 100 mM stock solution treated with chelating resin (from Sigma, St. Louis, MO) to remove contaminating metal i o n s . 39 The DNA (sodium salt, from Sigma, St. Louis, MO) is added from a concentrated stock solution prepared daily (8 mg/ml, in water). The CuCI: and H202 (prepared daily) are added from concentrated stock solutions prepared in water (100 and 200 mM, respectively), and ascorbic acid (added last) is taken from a 25 mM stock solution prepared daily in the 100 mM phosphate buffer and adjusted to pH 7. Following the initiation of reactions with ascorbate, mixtures are transferred to a quartz fiat cell, either manually or using a rapid sampling device. 4° Spectra are then recorded using a Bruker E 109 time constant, 41 ms; spectrometer (Bruker Spectrospin Ltd., Coventry, England) with the following instrument settings: sweep width, 60 G (1 G = 10 -4 T ) , modulation frequency, 100 kHz; modulation amplitude, 1 G; sweep time, 84 sec; power, 20 mW. Hydroxyl radical formation is indicated by the observation of the six-line signal (a N = 16.3 G, a~ = 3.6 G) from the 38 M. J. Burkitt and R. P. Mason, Proc. Natl. Acad. Sci. U.S.A. 88, 8440 (1991). 39 G. R. Buettner, J. Biochem. Biophys. Methods 16, 27 (1988). 4o R. P. Mason, this series, Vol. 105, p. 416.
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OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7]
PBN/'CH3 adduct (Fig. IA). The weaker six-line signal (a TM= 14.8 G, aft = 3.5 G), also present in the spectrum in Fig. 1A, is believed to arise from the PBN-methoxyl adduct, PBN/.OCH3, formed following the reaction of methyl radicals with oxygen prior to trapping with PBN. 38 The dependence of hydroxyl radical formation on copper, hydrogen peroxide, and ascorbate is demonstrated in Fig. 1B-D, respectively. The weak doublet signal in Fig. 1C is from the ascorbyl radical, the formation of
IO.OG X/ Cu 2+ DNA
/~
sscoF'os|e
minus Cu 2+
B
minus H202
C
minus ascorbate
D ,....--,..
FIG. 1. Demonstration of copper-DNA adduct-dependent hydroxyl radical formation. Reaction mixtures contained 2 mg/ml DNA, 1 mM CuC12, 1 mM H202, 0.1 M PBN, 2 M DMSO, and 1 mM ascorbate in 25 mM KHPO4 buffer, pH 7. (A) Complete system. (B) As in (A), but without CuC12. (C) As in (A), but without H20:. (D) As in (A), but without ascorbate. Hydroxyl radical formation is indicated by the detection of the six-line ESR signal from the PBN-methyl radical adduct.
[7]
COPPER-DNA ADDUCTS
73
which is not H202-dependent [see reaction (9)]. Hydroxyl radical formation can also be demonstrated to ocur when DNA is omitted from the above reaction mixture. Indeed, the signal from the PBN/.CH3 adduct detected in the absence of DNA is more intense than that detected in the presence of DNA (data not shown). This may reflect differences in the reactivities of the free and bound metal ions: the rate constant for the reaction of the aquacopper(I) ion with H20 2 is 4 x 103 M -1 sec -1, whereas the corresponding value for the slower reaction of the DNA-bound Cu(I) ion is less than 1.3 M -1 sec-l. ~6 The intensity of the PBN/.CH 3 signal from reactions containing DNA increases with time, whereas that from incubations not containing DNA is stable (not shown). The steady increase in the production of hydroxyl radicals in the presence of DNA may reflect the redox cycling of copper ions following the generation of reducing radicals on the DNA. 41 The spin-trapping technique described above can be used to examine the effects of a variety of agents on hydroxyl radical formation catalyzed by DNA-bound copper ions. For example, glutathione, which occurs in cell nuclei at a relatively high concentration (-19.2 mM),4z has been shown to suppress -OH formation when included in reaction mixtures containing DNA, copper(II), hydrogen peroxide, and ascorbateJ 6 Glutathione is believed to suppress -OH formation by stabilizing copper in the + 1 oxidation state, thereby preventing its participation in reaction (3)J 6 Measurement of Copper-Dependent DNA Oxidation Many of the standard, well-documented techniques for measuring DNA oxidation have been applied to copper systems. 13-15,17In the author's laboratory, use has been made of the ethidium-binding assay,16 developed initially by Prtitz for studies on radiation damage to DNA. 43 The assay, which has been applied subsequently to copper systems,7 provides a quantitative determination of copper-dependent oxidative damage to DNA under well-defined reaction conditions. The assay is based on the fact that a highly fluorescent complex is formed between native DNA and the intercalating agent ethidium bromide. When DNA is modified following exposure to hydroxyl radicals, intercalation by ethidium bromide is disrupted and the fluorescence from the ethidium bromide-DNA complex 41M. J. Burkitt, M. Fitchett, and B. C. Gilbert, in "Medical, Biochemicaland Chemical Aspects of Free Radicals" (O. Hayaishi,E. Niki, M. Kondo, and T. Yoshikawa,eds.), p. 63. Elsevier, Amsterdam, 1989. 42G. Bellomo, M. Vairetti, L. Stivala, F. Mirabelli, P. Richelmi, and S. Orrenius, Proc. Natl. Acad. Sci. U.S.A. 89, 4412 (1992). 43W. A. Prfitz, Radiat. Environ. Biophys. 23, 1 (1984).
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OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7]
is compromised. Several forms of DNA lesions, including strand scission, base oxidation, and base liberation, are believed to contribute to the loss of fluorescence. Hence, the assay is not specific for any single lesion. The basic assay, essentially as used by Stoewe and Priitz, 7 is described below, along with modifications which permit its application to the investigation of the effects of chelating agents on damage. 16 Method The 2-ml standard reaction mixture contains (in order of addition): 20 mM phosphate buffer (chelating resin-treated, pH 7), 100 /zg/ml DNA (sodium salt, Sigma), 50/zM CuClz, 2 mM H202, and 2 mM ascorbate. DNA is added from a concentrated stock solution prepared freshly (400 /~g/ml in water). The CuCI2 and H202 (prepared daily) are added from concentrated aqueous stock solutions (10 and 80 mM, respectively). Reactions are initiated via the addition ofascorbate from a 40 mM stock solution prepared daily in phosphate buffer and adjusted to pH 7. Reactions are carried out at 25° in open tubes. In the original method described by Stoewe and Prtitz, 7 reactions are terminated via the addition of 10 mM EDTA, to form a redox-inactive complex with the metal ion. Although EDTA can be used successfully to terminate reactions in the simple incubation described above, the reagent may not be able to stop reactions in more complex incubations containing other copper chelating agents. For example, in the presence of 1,10phenanthroline (OP), which promotes the degradation of DNA by copper (involving the formation of a reactive ternary complex), 16 EDTA would not be expected to remove and inactivate the metal ion. To broaden the application of the ethidium bromide binding assay to a greater variety of reaction conditions, catalase can be used to terminate reactions~6: following the addition of 400 units catalase (in 5/zl water), 50 /zM ethidium bromide is added to tubes and the fluorescence intensity measured with excitation at 510 nm and emission at 590 nm. Because the enhancement in the fluorescence of ethidium bromide measured following intercalation with DNA is a measure of the integrity of the DNA, the 100% value on the instrument is set using a solution containing the same reagents as the reaction under study with the exception of H202 , and catalase is added before the CuCI 2 and ascorbate. The zero reading on the machine is set using a solution prepared the same as the 100% reference solution except that DNA is omitted. Using these reference solutions, 100% fluorescence intensity refers to a 100% enhancement in the fluorescence ofethidium bromide following intercalation with (undamaged) DNA. Damage is then indicated as a loss in the enhancement of fluorescence.
[7]
cOPPER-DNA ADDUCTS
75
Figure 2 shows a time course of copper-dependent DNA oxidation in which reactions were terminated using either catalase or EDTA. Clearly, catalase is as effective as EDTA in the termination of DNA oxidation. The findings from ESR spin-trapping measurements of .OH formation carried out as described above indicate, however, that although both EDTA and catalase prevent the formation of .OH in the absence of 1,10phenanthroline, only catalase is able to inhibit .OH formation in the presence of the copper chelator (data not shown). A time course of DNA oxidation by copper in the presence of OP, terminated using catalase, is shown in Fig. 3; at the low concentration of copper used (5 ~M), no oxidation is detected in the absence of OP. Intact Cells: Catalysis by Endogenous Copper The oxidation of DNA following incubation with exogenous copper has been demonstrated in a variety of in vitro model systems. ~3-16 However, considerably fewer studies have addressed the possibility that the endogenous, DNA-bound metal ion may serve to promote the oxidation of DNA under conditions of oxidative stress. DNA single-strand breaks are often
100
...
80 60 40
0
20
0
i
i
i
i
5
10
15
20
i
25
i
30
Time ( m i n ) FIG. 2. Time course of copper-dependent DNA oxidation. DNA (100/xg/ml) was incubated with 50 ~M CuCI~, 2 mM H202, and 2 mM ascorbate in 20 mM KHPO4 buffer, pH 7. Reactions were terminated at the times indicated via the addition of either 10 mM EDTA (O) or 400 units catalase (©). Ethidium bromide (50 ~M) was then added and damage indicated as the failure of the DNA to cause a 100% enhancement in the fluorescence of the dye (compared with undamaged DNA). Values represent means with standard deviation values no greater than 2.6 (n = 2). (From Milne e t al., 16 with permission.)
76
OXIDATIVEDAMAGETO DNA AND DNA REPAIR
[7]
100- ,~:
80
60'
40-
20
. 0
5
.
. 10
. 15
20
J
i
25
30
Time (min)
Fie. 3. Effect of 1,10-phenanthroline (OP) on copper-dependent DNA oxidation. DNA (100/xg/ml) was incubated with 5 p~MCuCIz, 200 p.M H202, and 200 p.M ascorbate, in either the presence (O) or absence (0) of 15 ~M OP, in 20 mM KHPO4 buffer, pH 7. Reactions were terminated at the times indicated via the addition of 400 units catalase. Ethidium bromide (50 p.M) was then added and damage indicated as the failure of the DNA to cause a 100% enhancement in the fluorescence of the dye (compared with undamaged DNA). Values represent means with S.D. values no greater than 0.9 (n = 3). (From Milne e t a l . , 16 with permission.)
detected following the exposure of cells to either hydrogen peroxide or c o m p o u n d s that promote the cellular formation of superoxide and hydrogen peroxide.44 Although a significant proportion of the D N A strand breaks detected in cells following exposure to reactive oxygen species may be brought about by the action o f calcium-dependent nucleases, 44,45 the fact that oxidized purine and pyrimidine bases have been detected in cells following such treatment indicates that direct radical damage to D N A must also occur.44 Lipophilic metal ion-chelating agents have been used to probe for the participation o f metals of the induction of strand breaks in cells exposed to reduced o x y g e n species, such as superoxide and hydrogen peroxide. A key issue under investigation by several groups is the elucidation o f the identity o f the metal ion(s) responsible for the promotion of hydroxyl radical formation, and hence biomolecular damage, in cells exposed to 44B. Halliwell and O. I. Aruoma, F E B S Lett. 281, 9 (1991). 45S. Orrenius, M. J. Burkitt, G. E. N. Kass, J. M. Dypbukt, and P. Nicotera, Ann. Neurol. 32, $33 (1992).
[7]
COPPER-DNA ADDUCTS
77
o x i d a n t s . 17,35,46-49 Although
low molecular weight iron chelates may support •OH formation, the possibility that DNA-associated copper may promote formation in the nucleus is particularly interesting and highly relevant to the mechanism(s) of DNA damage.
Use of 1,10-Phenanthroline and Neocuproine as Probes for the Involvement of Copper and Iron in DNA Oxidation Although OP and 2,9-dimethyl- 1,10-phenanthroline (neocuproine, NC) will each chelate both copper and iron, their effects on the redox chemistry of the two metal ions are distinct: Cu(I) chelated to OP reacts readily with HzOz to form .OH [see reaction (3)]: 0 whereas reaction of the Fe(II) complex is inhibited46; NC inhibits the reaction of Cu(I) with HzO25~ but appears to have little effect o n F e ( I I ) . 46 Therefore, by determining the effects of OP and NC on DNA oxidation in cells exposed to oxidants, it is possible, in principle, to determine whether iron or copper is the catalytic metal responsible for -OH formation and damage. 35 From the data shown in Fig. 4, 5z demonstrating that H202-induced DNA fragmentation in HeLa cells is inhibited by OP and not NC, it appears that iron rather than copper is responsible for the catalysis of .OH formation: had copper been responsible, NC would have been expected to inhibit fragmentation and OP would have had, if anything, a stimulatory effect (see Fig. 3). OP does not remove copper from DNA, but it participates in the formation of a reactive ternary complex with both copper and DNA. 53-55 It is considered unlikely, therefore, that the protection afforded by OP could be due to its removal of copper, and hence -OH formation, to a site distant to the target molecule. Lipophilic chelating agents can be used as probes for the participation of copper and iron in DNA oxidation in experiments employing most of the standard techniques for the determination of end points of damage. 46 A. C. Mello-Fihlo and R. Meneghini, Mutat. Res. 251, 109 (1991). 47 A. C. Mello-Filho and R. Meneghini, Biochim. Biophys. Acta 781, 56 (1984). 48 I. Schraufstatter, P. A. Hyslop, J. H. Jackson, and C. G. Cochrane, J. Clin. Invest. 82, 1040 (1988). 49 O. Cantoni, P. Sestili, F. Cattabeni, G. Bellomo, S. Pou, M. Cohen, and P. Cerutti, Eur. J. Biochem. 182, 209 (1989). 50 S. Goldstein and G. Czapski, J. Am. Chem. Soc. 108, 2244 (1986). 51 G. Czapski and S. Goldstein, Free Radical Res. Commun. 1, 157 (1986). 52 C. M. Gedik and A. R. Collins, Nucleic Acids Res. 18, 1007 (1990). 53 S. Goldstein and G. Czapski, J. Free Radicals Biol. Med. 2, 3 (1986). 54 F. Liu, K. A. Meadows, and D. R. McMillin, J. Am. Chem. Soc. 115, 6699 (1993). 55 D. S. Sigman, T. W. Bruice, A. Mazumder, and C. L. Sutton, Acc. Chem. Res. 26, 98 (1993).
78
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[7]
Control
h
100 .aM H202
100 gM H202 + 1 mM OP
100 p.M H202 + 1 mM NC i
0
i
20
40
h
i
60
% ss breaks
FIG. 4. Effects of 1,10-phenanthroline (OP) and neocuproine (NC) on the level of DNA single-strand breaks detected in HeLa cells following incubation with H20 2. Cells were grown and DNA labeled with [3H]thymidine according to Gedik and Collins.52After a wash in phosphate-buffered saline (PBS), and following preincubation (4°) for 5 min with either 1 mM OP or 1 mM NC (each added from 15 mM aqueous stock solutions), cells were incubated with 100 p.M H20 2, and additions as indicated, for 15 min (4°). Cells not exposed to OP or NC were also preincubated for 5 min. No additions were made to the control incubations. Strand breaks were then determined by alkaline denaturation and hydroxyapatite chromatography/8 Values represent means -+1 S.D. (n = 3). (Data from Burkett et al., 35 with permission). H o w e v e r , w i t h t h e e x c e p t i o n o f t h e findings f r o m s o m e r a d i a t i o n s t u d ies,4-6,56 t h e r e is little e v i d e n c e f o r t h e p a r t i c i p a t i o n o f e n d o g e n o u s c o p p e r in D N A o x i d a t i o n d u r i n g o x i d a t i v e s t r e s s . F o r e x a m p l e , B i r n b o i m 17 h a s s h o w n t h a t O P e n h a n c e s D N A s t r a n d b r e a k a g e in p h o r b o l e s t e r - t r e a t e d l e u k o c y t e s . It r e m a i n s to d e m o n s t r a t e w h e t h e r t h e c e l l u l a r p o o l o f c o p p e r r e s p o n s i b l e f o r this is i n d e e d t h a t w h i c h e x i s t s in t h e n u c l e u s b o u n d to D N A . Concluding Remarks It h a s b e c o m e i n c r e a s i n g l y a p p a r e n t t h a t c o p p e r o c c u r s n a t u r a l l y in c h r o m o s o m e s , w h e r e it is b e l i e v e d to p l a y a r o l e in t h e a t t a c h m e n t o f D N A to s c a f f o r d p r o t e i n s v i a t h e f o r m a t i o n o f m e t a l l o p r o t e i n b r i d g e s ) I n 56 S.-M. Chiu, L.-Y. Xue, L. R. Friedman, and N. L. Oleinick, Biochemistry 32, 6214 (1993).
[8]
HPLC AND MS ANALYSISOF DNA DAMAGEPRODUCTS
79
view of the increasing awareness of the role played by redox active metal ions and reactive oxygen species in the inducement of biomolecular damage and disease, it seems particularly remarkable that the interconversion of bound Cu(I) and Cu(II) ions should play a role in the regulation of the conformation of such a critical cellular target molecule as D N A . 4-6 It is anticipated that this interesting dilemma will continue to stimulate research from which an appreciation of the true significance of D N A oxidation by the bound metal ion will emerge. Acknowledgments The author thanks Mrs. L. Milne for technical assistance and Scottish Office Agriculture and Fisheries Department for support.
[8] S i n g l e t O x y g e n D N A D a m a g e : C h r o m a t o g r a p h i c and Mass Spectrometric Analysis of Damage Products B y JEAN CADET, JEAN-LUC RAVANAT, GARRY W. BUCHKO,
HELEN C. YEO, and BRUCE N. AMES Introduction Singlet oxygen (~O2), the lowest excited state of molecular oxygen (lAg, 94.2 kJ/mol), m a y be generated through energy transfer involving a type II photosensitized reaction L2 and by thermal decomposition of endoperoxides 3 and dioxetanes.4 In addition, the formation of 10 2 in biological systems m a y be mediated by several enzymatic reactions 5 and by chemiexcitation during lipid peroxidation. 6 Like the highly reactive hydroxyl radical (-OH), but in a more specific manner, IO 2 is capable of inducing genotoxic, carcinogenic, and mutagenic effects 7-9 and is likely involved in aging. ~° 1c. s. Foote, in "Free Radicals in Biology" (W. A. Pryor, ed.), Vol. 2, p. 85. Academic Press, New York, 1976. 2 E. Sage, T. Le Doan, V. Boyer, D. E. Helland, L. Kittler, C. Hfl~ne, and E. Moustacchi, J. Mol. Biol. 209, 297 (1989). 3 p. di Mascio and H. Sies, J. Am. Chem. Soc. 111, 2909 (1989). 4 W. Adam and C. Cilento, Angew. Chem., Int. Ed. Engl. 22, 529 (1983). 5 E. Cadenas and H. Sies, this series, Vol. 105, p. 221. 6 E. Cadenas and H. Sies, Eur. J. Biochem. 124, 349 (1982). 7 j. Piette, J. Photochem. Photobiol., B 4, 335 (1990). 8 R. A. Floyd, Carcinogenesis (London) 11, 1447 (1990). 9 B. Epe, Chem.-Biol. Interact. 83, 239 (1991). 10B. N. Ames, Science 221, 1258 (1983).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[8]
HPLC AND MS ANALYSISOF DNA DAMAGEPRODUCTS
79
view of the increasing awareness of the role played by redox active metal ions and reactive oxygen species in the inducement of biomolecular damage and disease, it seems particularly remarkable that the interconversion of bound Cu(I) and Cu(II) ions should play a role in the regulation of the conformation of such a critical cellular target molecule as D N A . 4-6 It is anticipated that this interesting dilemma will continue to stimulate research from which an appreciation of the true significance of D N A oxidation by the bound metal ion will emerge. Acknowledgments The author thanks Mrs. L. Milne for technical assistance and Scottish Office Agriculture and Fisheries Department for support.
[8] S i n g l e t O x y g e n D N A D a m a g e : C h r o m a t o g r a p h i c and Mass Spectrometric Analysis of Damage Products B y JEAN CADET, JEAN-LUC RAVANAT, GARRY W. BUCHKO,
HELEN C. YEO, and BRUCE N. AMES Introduction Singlet oxygen (~O2), the lowest excited state of molecular oxygen (lAg, 94.2 kJ/mol), m a y be generated through energy transfer involving a type II photosensitized reaction L2 and by thermal decomposition of endoperoxides 3 and dioxetanes.4 In addition, the formation of 10 2 in biological systems m a y be mediated by several enzymatic reactions 5 and by chemiexcitation during lipid peroxidation. 6 Like the highly reactive hydroxyl radical (-OH), but in a more specific manner, IO 2 is capable of inducing genotoxic, carcinogenic, and mutagenic effects 7-9 and is likely involved in aging. ~° 1c. s. Foote, in "Free Radicals in Biology" (W. A. Pryor, ed.), Vol. 2, p. 85. Academic Press, New York, 1976. 2 E. Sage, T. Le Doan, V. Boyer, D. E. Helland, L. Kittler, C. Hfl~ne, and E. Moustacchi, J. Mol. Biol. 209, 297 (1989). 3 p. di Mascio and H. Sies, J. Am. Chem. Soc. 111, 2909 (1989). 4 W. Adam and C. Cilento, Angew. Chem., Int. Ed. Engl. 22, 529 (1983). 5 E. Cadenas and H. Sies, this series, Vol. 105, p. 221. 6 E. Cadenas and H. Sies, Eur. J. Biochem. 124, 349 (1982). 7 j. Piette, J. Photochem. Photobiol., B 4, 335 (1990). 8 R. A. Floyd, Carcinogenesis (London) 11, 1447 (1990). 9 B. Epe, Chem.-Biol. Interact. 83, 239 (1991). 10B. N. Ames, Science 221, 1258 (1983).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
80
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[8]
One major biological target of 10 2 is DNA. It has been shown that D N A synthesis blocking lesions induced by a chemical source of singlet oxygen are targeted to guanine residues in single-stranded DNA. 11 This may be accounted for by the specific oxidation of the guanine moiety of DNA, which is the most reactive nucleobase toward electrophilic addition of IOz .12 One of the main resulting oxidation products, at least in DNA, has been identified as 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). 13-15 Various studies involving site-specific incorporation of 8-oxo-dG into oligonucleotides have shown that this oxidized nucleoside is potentially mutagenic 16and is a substrate for the formamidopyrimidine glycosylase (FPG) D N A repair protein. 17It should be noted that a sensitive high-performance liquid chromatography (HPLC)-electrochemical detection assay 18 and postlabeling techniques 19 are now available to monitor the formation of 8-oxo-dG in both isolated and cellular DNA. In addition, a monoclonal antibody column has been developed for the prepurification of 8-oxo-dG in urine. 2° However, the formation of 8-oxo-dG cannot be used as a probe for 10 z reactions in cellular D N A since this modified nucleoside is also produced through the reaction of .OH (and related reactive species of the Fenton reaction) with the guanine moiety. 21 In addition, it has been observed that the hydration reaction of the guanine radical cation within D N A leads to the predominant formation of 8 - o x o - d G . z2 On the other hand, the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxoH D. T. Ribeiro, F. Bourre, A. Sarasin, P. Di Mascio, and C. F. M. Menck, Nucleic Acids Res. 211, 2465 (1992). 12j. Cadet and P. Vigny, in "Bioorganic Photochemistry" (H. Morrison, ed.), Vol. 1, p. 1. Wiley, New York, 1990. ~3R. A. Floyd, M. S. West, K. L. Eneff, and J. E. Schneider, Free Radical Biol. Med. 8, 327 (1990). 14 j. E. Schneider, S. Price, L. Maidt, J. M. C. Gutteridge, and R. A. Floyd, Nucleic Acids Res. 18, 631 (1990). 15 T. P. A. Devasagayam, S. Steenken, M. S. W. Obendorf, W. A. Schultz, and H. Sies, Biochemistry 311, 6283 (1991). 16 S. Shibutani, M. Takeshida, and A. P. Grollman, Nature (London) 349, 431 (1991). 17 j. Tchou, H. Kasai, S. Shibutani, M.-H. Chung, J. Laval, A. P. Grollman, and S. Nishimura, Proc. Natl. Acad. Sci. U.S.A. 88, 4690 (1991). ~8 R. A. Floyd, J. J. Watson, P. T. Wong, D. H. Altmiller, and R. C. Rickard, Free Radical Res. Commun. 1, 163 (1986). 19 j. Cadet, F. Odin, J.-F. Mouret, M. Polverelli, A. Audic, P. Giacomoni, A. Favier, and M.-J. Richard, Mutat. Res. 275, 343 (1992). 20 E.-M. Park, M. K. Shigenaga, P. Degan, T. S. Korn, J. W. Kitzler, C. M. Wehr, P. Kolachana, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 89, 3375 (1992). 2~ K. Kasai and S. Nishimura, in "Oxidative Stress, Oxidants and Antioxidants" (H. Sies, ed.), p. 99. Academic Press, San Diego, 1991. 22 H. Kasai, Z. Yamaizumi, M. Berger, and J. Cadet, J. Am. Chem. Soc. 114, 9692 (1992).
[8]
HPLC AND MS ANALYSISOF DNA DAMAGEPRODUCTS
81
2'-deoxyguanosine (4-hydroxy-8-oxo-dG), 23-25 whose formation arises from initial [2 ÷ 4] Diels-Alder cycloaddition of 10 2 across the 4,8-purine carbons, are specific for 102-mediated DNA oxidation. 12Emphasis in this chapter is placed on the chromatographic behavior and mass spectroscopic (MS) features of the diastereoisomers of 4-hydroxy-8-oxo-dG and the corresponding base. Materials and Methods 2'-Deoxyguanosine is obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Acetonitrile (HPLC grade) and ammonium formate are purchased from Carlo Erba (Farmitalia, Carlo Erba, Milano, Italy) and Kodak (Eastman Kodak Co., Rochester, NY), respectively. Phthalocyanine complexed with zinc (ZnPcS2), prepared as described by Langlois et a1.,26 is a gift from Prof. J. E. van Lier (University of Sherbrooke, Qu6bec, Canada). The two diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine are prepared by dye photosensitization of 2'-deoxyguanosine. Typically, aerated Milli-Q deionized aqueous solutions of 1.0 mM 2'deoxyguanosine containing ZnPcS2 (OD6732.0) are exposed to the visible light generated from a 100-W halogen lamp equipped with a Kodak Model 23A (590 nm) cutoff filter. The cooling of the irradiation system is achieved by interfacing a heat filter (10 mm, circulating water) between the lamp and the photolyzed solution. The 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'deoxyguanosine are separated by HPLC on an analytical (250 x 4.6 mm i.d.) amino-substituted silica gel (mean particle size 5 /zm) Lichrocart column (Merck, Darmstadt, Germany) under isocratic conditions using an 8 : 2 (v/v) mixture of acetonitrile and 50 mM ammonium formate at a flow rate of 1.0 ml/min. 24 The HPLC system consists of two Model 302 Gilson dual pumps (Middleton, WI) equipped with a sil-9A Shimadzu automatic injector (Touzart & Matignon, Paris, France) and a L-4000 UV spectrophotometer (Hitachi, Tokyo, Japan) set at 230 nm. The pump is interfaced to an Apple II microcomputer that controls the eluent and to a Model 621 Gilson Data Master for quantitative analysis. 23 J.-L. Ravanat, M. Berger, F. Benard, R. Langlois, R. Ouellet, J. E. van Lier, and J. Cadet, Photochem. Photobiol. 55, 809 (1992). 24 J.-L. Ravanat, Ph.D. Thesis, University of Grenoble (1992). 25 G. W. Buchko, J. Cadet, M. Berger, and J.-L. Ravanat, Nucleic Acids Res. 20, 4847 (1992). 26 R. Langlois, H. Ali, N. Brasseur, J. R. Wagner, and J. E. van Lier, Photochem. Photobiol. 44, 117 (1986).
82
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[8]
Fast atom bombardment (FAB) mass spectra (glycerol matrix, 35-keV cesium atoms) in the positive and negative ion modes are obtained on a VG ZAB 2-EQ spectrometer (Fisons-V.G., Manchester, UK). A Hewlett Packard (HP) 5890 Series II gas chromatograph interfaced with a HP 5971 mass-selective detector is used for gas chromatography-mass spectrometry (GC-MS) analysis of the 4R* and 4S* diastereoisomers of 4-hydroxy-8-oxo-dG and the corresponding purine base. The GC apparatus is equipped with a fused-silica capillary column (12 m, 0.2 mm i.d., 0.33/zm film thickness) coated with cross-linked 5% phenylmethylsilicone (w/w). The GC oven is held at 150° for 1 min and programmed at 10°/min to a final temperature of 280 °. The temperature of both the injector and the detector is 280 °. Prior to GC-MS analysis, the 4R* and 4S* diastereoisomers of 4hydroxy-8-oxo-dG are subjected to acid hydrolysis in evacuated tubes with either 88% formic acid (v/v) for 30 min at 140°27 or 48% aqueous hydrofluoric acid (v/v) for 30 min at 0°28 in evacuated tubes. The samples are then lyophilized to dryness and subsequently derivatized with a mixture of bis(trimethylsilyl)trifluoroacetamide and acetonitrile (2 : 1, v/v) at 130° for 30 min. Photosensitized Reactions of 2'-Deoxyguanosine One of the problems associated with using phthalocyanine or other dyes, such as methylene blue, to generate type II photooxidation products is that they usually also give rise to a significant amount of type I photoproducts. Consequently it is necessary to be able to distinguish between the two main classes of photooxidation damage. One approach is to use a dye, such as benzophenone, which produces predominantly type I photooxidation lesions. The main type I photosensitization products of 2'-deoxyguanosine are 2,2-diamino-4-[2-deoxy-fl-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone, its unstable precursor 2-amino-5-[(2-deoxy~-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one, 29 and (2S)-2,5'anhydro- 1-(2-deoxy-fl-D-erythro-pentofuranosyl)-5-guanidinylidene-2hydroxy-4-oxoimidazolidine.30 The formation of these photoproducts may be rationalized in terms of quantitative initial hydrogen or electron abstraction from the guanine ring by photoexcited benzophenone. It is interesting 27 S. Boiteux, E. Gajewski, J. Laval, and M. Dizdaroglu, Biochemistry 167, 347 (1992). 28 j. Catania, B. C. Keenan, G. P. Margison, and D. S. Fairweather, Anal. Biochem. 167, 347 (1987). 29 j. Cadet, M. Berger, C. Decarroz, J.-F. Mouret, J. E. van Lier, and J. R. Wagner, J. Chim. Phys. 88, 1021 (1991). 30 G. W. Buchko, J. Cadet, J.-L. Ravanat, and P. Labataille, Int. J. Radiat. Biol. 63, 669 (1993).
[8]
HPLC AND MS ANALYSISOF DNA DAMAGEPRODUCTS
83
also to note that these compounds have also been shown to be produced from initial .OH addition at the C-4 position of the guanine ring followed by a fast dehydration reaction, 29'3~giving rise to a strong oxidizing neutral radical. Another method of distinguishing between type I and type II photooxidation products is to identify singlet oxygen-mediated lesions on the basis of the significant D20 enhancement effect or by using the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate as a clean source of singlet oxygen effect. 23 Using these methods the two main 2'-deoxyguanosine type II photooxidation products are characterized as the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine on the basis of extensive nuclear magnetic resonance (NMR) measurements and mass spectrometry analysisfl 4'25 The formation of the two modified nucleosides is likely to be explained by the initial [2 + 4] cycloaddition of 10 2 to the 4,8-carbons leading to unstable endoperoxides (Fig. l) which, subsequently, undergo thermal decomposition. In addition, 7,8-dihydro-8-oxo-2'-deoxyguanosine has also been characterized as a relatively minor ~O2 oxidation product of 2'-deoxyguanosine. 23Note that 8-oxo-dG becomes a competitive substrate with respect to 2'-deoxyguanosine as soon as its yield of formation reaches a value of 0.75%. The two 4-hydroxy-8-oxo-dG diastereoisomers are the main singlet oxygen secondary oxidation products of 8 - o x o - d G . 23 It is likely that the formation of 8-oxo-dG involves the reduction of the transient 2'-deoxyguanosine 4,8-endoperoxides as inferred from the observation that the presence of Fe 2+ in the photooxidation reaction leads to a significant decrease in the yield of diastereoisomeric 4-hydroxy-8oxo-dG with a concomitant increase in the formation of 8-oxo-dG. This effect, which was also shown to decrease the yield of the oxazolone compound, is likely to explain the decrease in the ratio of 4-hydroxy8-oxo-dG to 8-oxo-dG in singlet oxygen-mediated oxidation of doublestranded DNA. Chromatographic Separation of the 4R* and 4S* Diastereoisomers of 4,8-Dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine Separation of the ZnPcS2-mediated photooxidation products of 2'deoxyguanosine on the amino-substituted column is illustrated in Fig. 2. Note that the two diastereoisomers of 4-hydroxy-8-oxo-dG are well separated from 2'-deoxyguanosine and 2,2-diamino-4-[(2-deoxy-fl-Derythro-pentofuranosyl)amino]-5(2H)-oxazolone (the main stable type I photooxidation product). A baseline separation of the 4R* and 4S* diaster31 S. Steenken, Chem. Rev. 89, 503 (1989).
0
"0
Z
Z
"0
=
Z
TZ
Z
i\
Z
0
0
0
0
"0
[
1 Z
_= "0
_= cJ
0
-~
o 0 -r L
I (M
0 Q
"lZ
0
o~-,
=p 0"0
"~r"N
o
No
0 1-
._~ "0
[8]
H P L C AND M S ANALYSIS OF D N A DAMAGE PRODUCTS
85
dGuo
2 -
O E C
1 -
i
0.00
i
i
i
=
5.00
i
i
I
i
i
10.00
i
i
I
i
i
15.00
|
i
I
i
!
20.00
Time (min)
Fl•. 2. HPLC elution profile of 2'-deoxyguanosine (dGuo), 2,2-diamino-4-[(2-deoxy-fl-
o-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (type I), and the S* and R* diastereoisomers of 7,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine(type II) on a Lichrocart aminosubstituted analytical column (250 x 4.6 mm i.d.). The eluent consisted of an 8 : 2 mixture of acetonitrile and 50 mM ammonium formate at a flow rate of 1 ml/min. Detection of the compounds was achieved by a variable wavelength spectrophotometer set at 230 nm.
eoisomers (the early eluting c o m p o u n d has been assigned as the 4S diastere o i s o m e r 25) is obtained. The purity of each of the diastereoisomers, as determined by 1H N M R spectroscopy, is greater than 95%. Hence, the amino-substituted silica gel column constitutes an appropriate analytical s y s t e m to separate these two specific products of singlet-mediated oxidation of 2'-deoxyguanosine. Mass Spectrometry of the 4R* and 4S* Diastereoisomers of 4,8-Dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine A quasi-molecular ion at m/z 322 corresponding to (M + Na) ÷ was o b s e r v e d in the positive mode F A B - m a s s spectrum of both diastereoisomers of 4-hydroxy-8-oxo-dG. In addition, the negative m o d e F A B - m a s s spectrum of both oxidized nucleosides exhibited a parent ion at m/z 298 corresponding to the pseudomolecular ion (M - H ) - . 25
86
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[8]
73
6,000,000:
43o
5,000,000 ~
4,000,000
.1~
-
i--
_z
5
125 10075 ~
50-
g 25 o 1
2
3
4
5
6
7
8
9
10
CELL NUMBERS ( x l O s)
FIG. 4. Fluorescence enhancement of bisbenzamide by cell DNA. Varying numbers of CHO cells were treated with unwinding solution (T and B samples) and mixed afterward with bisbenzamide-containing buffer. (o) B, Single-stranded DNA; (e) T, double-stranded DNA.
The basis for DNA unwinding is a time-dependent transformation of duplex DNA to single-stranded DNA under moderate alkaline conditions. 9-15 Quantification of the remaining double-stranded DNA can be performed either by scintillation counting of [3H]thymidine in fractions of double- and single-stranded DNA after hydroxyapatite chromatography 9 or by fluorescence measurements, thus omitting radioactive labeling. The fluorochrome bisbenzamide reacts specifically with DNA but not with RNA and protein. This results in an enhanced fluorescence, the extent of which depends on the adenine-thymine content of the DNA. 2° The degree of fluorescence of DNA-bound bisbenzamide is a function of the amount and state of the DNA (double- or single-stranded form). Data in Fig. 4 show the fluorescence of different numbers of CHO cells which were subjected to unwinding conditions (T and B samples). There are linear relationships between cell numbers and fluorescence, showing that the degree of fluorescence is dependent on the amount (cell numbers) and the state of DNA. The fluorescence of double-stranded DNA is about twice as high as that of single-stranded DNA for cell numbers up to 1 × 106. For higher cell numbers, the intensity of fluorescence of doublestranded DNA becomes saturated (data not shown), but not that for singlestranded DNA because binding affinities of bisbenzamide to duplex and 20 C. F. Ceasrone, C. Bolognesias, and L. Santi, Anal. Biochem. 100, 188 (1979).
[9]
FLUORESCENCE ANALYSIS OF D N A STRAND BREAKS
97
single-stranded DNA are different. The difference in fluorescence (T minus B) for high cell numbers (> 106) is smaller than for low cell numbers (< 106) owing to unsaturated binding between the dye and duplex DNA. Figure 5 shows the dependence of fluorescence intensity on dye concentration up to 2.5/xM for T and B samples prepared from 2 x 10 6 CHO cells. For higher dye concentrations the fluorescence intensity becomes saturated. It appears that the optimal values for cell numbers and dye concentrations in the FADU assay are 5 x 105 to 1 x 106 cells and 1.25 /xM bisbenzamide, respectively. The FADU technique is based on time-dependent alkaline denaturation of DNA under moderate denaturing conditions (Fig. 6). Aliquots of cells (5 x 105) were subjected to the alkaline unwinding treatment for different periods of time. Unwinding starts at the strand ends or other unwinding units. The fluorescence intensities for B samples are low and stay constant during the unwinding period. Because of extensive DNA fragmentation, conversion of the native DNA to single-stranded DNA comes to a completion during the first seconds of the treatment (F = 0). The DNA in T samples remain in duplex form and show no decrease in fluorescence intensity (F = I). The P samples contain DNA in a partially denatured form; accordingly, the amount of double-stranded DNA is a function of unwinding time and dose (0 < F < 1). As shown in Fig. 6 in a log-log plot, DNA unwinding proceeds linearly with time. The initial unwinding 300 275
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[1 1]
SINGLET OXYGEN AND SHUTTLE VECTORS
115
3'-terminal a,fl-unsaturated aldehyde (4-hydroxy-trans-2-pentenal). Such DNA scission products have electrophoretic mobilities different from those produced by hot alkali cleavage (a, 3'-phosphoryl, Fig. 4B) or endonuclease IV cleavage (c, 3'-hydroxyl, Fig. 4B) of the same A P - D N A substrates. Yeast redoxyendonuclease generates DNA scission products identical to those produced by other AP lyases, placing this enzyme into the same category of DNA base excision repair enzymes as endo III with respect to the N-glycosylase/AP lyase mode of action (Fig. 5). Acknowledgments We thank the followingindividuals for gifts of enzymes: R. Cunningham (endonuclease III), R. S. Lloyd (T4 endonuclease V), and B. Demple (endonuclease IV). This work was supported by Grants CA42607,CA01441,and Training Grant T32GM08367from the National Institutes of Health and Grant NP-806from the American Cancer Society.
[1 1] S h u t t l e V e c t o r b e t w e e n P r o k a r y o t e s a n d E u k a r y o t e s for A s s a y i n g S i n g l e t O x y g e n - I n d u c e d D N A D a m a g e and Mutagenicity
By
CARLOS FREDERICO MARTINS M E N C K
Introduction Investigations on the in oioo consequences of the interactions of singlet oxygen (102) with genetic material face some difficulties owing to the high reactivity and short lifetime of singlet oxygen. To overcome these difficulties methods are needed in which DNA is treated in vitro with 102 and then introduced into living cells in order to analyze how the cells handle this modified genetic molecule and the subsequent outcomes. For mammalian cells, special plasmids, the shuttle vectors, can be used as exogenous probes to obtain data on the mutagenicity of lO2-induced DNA damage. Shuttle vectors are plasmid molecules which can replicate in both bacteria and mammalian cells.l The basic idea is to submit the vector to IO2 treatment, to analyze the damaging effects, and to introduce it into mammalian cells. The DNA lesions are processed, that is, replicated and repaired in the eukaryotic environment, causing mutations. Finally, the 1 A. Sarasin, J. Photochem. Photobiol., B 3, 143 (1989).
METHODS IN ENZYMOLOGY,VOL. 234
Copyright © 1994by Academic Press, Inc. All rights of reproductionin any form reserved.
[1 1]
SINGLET OXYGEN AND SHUTTLE VECTORS
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3'-terminal a,fl-unsaturated aldehyde (4-hydroxy-trans-2-pentenal). Such DNA scission products have electrophoretic mobilities different from those produced by hot alkali cleavage (a, 3'-phosphoryl, Fig. 4B) or endonuclease IV cleavage (c, 3'-hydroxyl, Fig. 4B) of the same A P - D N A substrates. Yeast redoxyendonuclease generates DNA scission products identical to those produced by other AP lyases, placing this enzyme into the same category of DNA base excision repair enzymes as endo III with respect to the N-glycosylase/AP lyase mode of action (Fig. 5). Acknowledgments We thank the followingindividuals for gifts of enzymes: R. Cunningham (endonuclease III), R. S. Lloyd (T4 endonuclease V), and B. Demple (endonuclease IV). This work was supported by Grants CA42607,CA01441,and Training Grant T32GM08367from the National Institutes of Health and Grant NP-806from the American Cancer Society.
[1 1] S h u t t l e V e c t o r b e t w e e n P r o k a r y o t e s a n d E u k a r y o t e s for A s s a y i n g S i n g l e t O x y g e n - I n d u c e d D N A D a m a g e and Mutagenicity
By
CARLOS FREDERICO MARTINS M E N C K
Introduction Investigations on the in oioo consequences of the interactions of singlet oxygen (102) with genetic material face some difficulties owing to the high reactivity and short lifetime of singlet oxygen. To overcome these difficulties methods are needed in which DNA is treated in vitro with 102 and then introduced into living cells in order to analyze how the cells handle this modified genetic molecule and the subsequent outcomes. For mammalian cells, special plasmids, the shuttle vectors, can be used as exogenous probes to obtain data on the mutagenicity of lO2-induced DNA damage. Shuttle vectors are plasmid molecules which can replicate in both bacteria and mammalian cells.l The basic idea is to submit the vector to IO2 treatment, to analyze the damaging effects, and to introduce it into mammalian cells. The DNA lesions are processed, that is, replicated and repaired in the eukaryotic environment, causing mutations. Finally, the 1 A. Sarasin, J. Photochem. Photobiol., B 3, 143 (1989).
METHODS IN ENZYMOLOGY,VOL. 234
Copyright © 1994by Academic Press, Inc. All rights of reproductionin any form reserved.
116
OXIDATIVE DAMAGETO DNA AND DNA REPAIR
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vectors are rescued back into E s c h e r i c h i a coli, where mutations in a target gene can be screened and examined at the sequence level. Specific shuttle vectors have been used for the investigation of 10 2 effects on genetic material. 2'3 These vectors contain the origin of replication of pBR322 and the c a t (chloramphenicol acetyltransferase) gene for amplification and selection (chloramphenicol resistance) in E. coli. They carry the SV40 origin of replication, so when introduced in monkey COS7 cells, which supply the T-antigen in trans, 4 the plasmid replicates episomally. They also have the SV40 late genes, responsible for production of capsid proteins, and thus the plasmids are packaged and amplified as pseudoviruses. As the target gene for mutation studies, the vectors have the tRNA s u p F gene of E. coli. One of the vectors used has a particular and interesting feature. It also carries the replication origin from the bacteriophage fl, which allows the plasmid to enter the phage replication mode in permissive bacteria after infection with a helper phage, generating single-stranded DNA (ssDNA). Therefore, the use of these shuttle vectors also enables the comparison of IO2 action on ssDNA and double-stranded DNA (dsDNA) structures. The shuttle vector approach is straightforward (Fig. 1). Plasmid Treatment and DNA Damage Assays Shuttle vectors are exposed to IO2generated by thermal decomposition of the water-soluble endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). This chemical method is ideally suited for these studies because it is simple and produces IO2 as the only reactive molecule in solution. 5 Other methods, such as the use of photosensitizers, may generate other reactive products under some conditions, making interpretation of results uncertain. DNA samples (2/zg/200/A) are incubated with NDPO2, at a concentration up to 100 mM, in 50 mM sodium phosphate buffer in D20, pD 7.4. The reaction proceeds for 90 min at 37°, yielding 3,3'-(1,4-naphthylidene) dipropionate (NDP) and molecular oxygen, half in the ground state and half in the excited singlet state, which reacts with DNA. During the first 30 min of incubation, the samples are vortexed every 2 min in order to homogenize the IO2 produced in the solution. After treatment, DNA may 2 p. Di Mascio, C. F. M. Menck, R. G. Nigro, A. Sarasin, and H. Sies, Photochem. Photobiol. 51, 293 (1990). 3D. T. Ribeiro, C. Madzak, A. Sarasin, P. Di Mascio, H. Sies, and C. F. M. Menck, Photochem. Photobiol. 55, 39 (1992). 4 y. Gluzman, Cell (Cambridge, Mass.) 23, 175 (1981). s p. Di Mascio and H. Sies, J. Am. Chem. Soc. 111, 2909 (1989).
[11]
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Virus
Sitesthat b l o c k DNAbreaks DNAsynthesis DNAdaiageassays ssDNA ~.~ dsDNA
NDPO2 treatment
Mutant sequencing ~
~nfection
q~:~o:~3 extraction E. co/i
~ transformation ~ i "~ ~,°~(~ supfmutants screening
FIG. 1. Schematicrepresentation of the general use o£ shuttle vectors for assayingthe DNA-damaging and mutageni¢effects of IO2 in mammaliancells. be purified by the addition of 0.6 volume of a solution of 20% polyethylene glycol (PEG) and 2.5 M NaCI to the samples, which are kept for 1 hr in ice and then centrifuged (13,000 rpm, 20 min). The precipitated DNA is resuspended for further analysis. The DNA can be checked for detectable lesions using any of the procedures described in the literature, such as chromatographic6and enzymatic assays.7 For 102-treated shuttle vectors, single- and double-stranded breaks can be detected by conventional electrophoresis in 0.7% agarose gels, in Tris-borate buffer, pH 7.5. 8 After migration the DNA is stained by ethidium bromide (0.5 /~g/ml) and visualized by fluorescence in an UV (330 nm) transilluminator. This methodology allows discrimination between unbroken plasmid molecules (supercoiled structure) and those having single-stranded (open relaxed circles) or double-stranded (linear) breaks, and the relative number of any of the three forms can be quantified by scanning the gel pictures with a densitometer. For ssDNA, agarose 6 M. K. Shigenaga, J. W. Park, K. C. Cundy, C. J. Gimeno, and B. N. Ames, this series, Vol. 186, p. 521. 7 S. Boiteux, E. Gajewski, J. Laval, and M. Dizdaroglu, Biochemistry 31, 106 (1992). 8 j. Sambrook, E. F. Fritsch, and T. Maniatis, 2nd ed. "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
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gel electrophoresis also separates covalently closed and whole linear molecules (resulting from a single break in the DNA). In this case, degradation of the ssDNA can also be observed, as a result of at least two nonspecific breaks in the same molecule. This generates single-stranded linear fragments with different DNA sizes which migrate to various positions and, consequently, are dispersed in the gel. The relative number of covalently closed and whole linear molecules can also be obtained as for dsDNA. In both cases, the number of breaks can be calculated based on the Poisson distribution. The results obtained after treatment of shuttle vectors with NDPO2 indicate that 102 induces single- and double-strand breaks in dsDNA. 2 It also reacts with ssDNA, yielding breaks in this molecule. Quantification of such lesions revealed that more breaks are induced in ssDNA than in dsDNA. 3 A second approach was employed in order to examine DNA damage in 102-treated shuttle vectors, namely, the analysis of sites that block in vitro DNA synthesis by DNA polymerases. This method was first described by Moore and Strauss, 9 who found that DNA lesions induced by UV-irradiation block DNA synthesis by the DNA polymerase I from E. coli. The method is simple and uses conventional sequencing techniques. 102-treated and denatured DNA (1/zg) is annealed with a specific primer (30 ng), which is complementary to the vector in a position next to the sequence to be investigated. Annealing is performed in 10/zl of 40 mM Tris-HCl, pH 7.5, 20 mM MgCI2,and 50 mM NaC1, at 65° for 5 min, followed by gentle cooling to room temperature. To 5/~1 of annealed DNA are added standard buffer [26.5 mM Tris-HCl, pH 7.5, 13.3 mM MgCI2, 16.6 mM NaCI, and 6.7 mM dithiothreitol (DTT)] and 0.3 /zM of each [35S]dATP (1000 Ci/mmol), dGTP, dCTP, and dTTPs and DNA polymerase (final volume of 7.5/zl). A mixture (1/~1) of 83/zM of the four deoxynucleoside triphosphates (dNTPs) is added for DNA elongation. Both time and temperature vary for labeling and elongation reactions, based on the DNA polymerase used. The reactions are terminated by the addition of 0.6 volume of stop solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The products of the polymerization reactions are heat denatured and then loaded and electrophoresed on standard high-resolution denaturing sequencing gels (8% polyacrylamide, 7 M urea). After migration the gels are dried and subjected to autoradiography. Sites on the template DNA containing lesions that block DNA synthesis appear in the autoradiogram as bands, with the specific location being identified by comparison with the regular DNA sequence in the same gel. 9 p. D. Moore and B. S. Strauss, Nature (London) 278, 664 (1979).
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Although the nature of the blocking lesions cannot be determined by this kind of experiment, at least two important results are obtained: (i) the identified lesions correspond to biologically significant damages that interrupt DNA synthesis, and (ii) the final result is a DNA damage spectrum induced by 102 in a specific DNA sequence. In the case of shuttle vectors, the DNA sequence investigated corresponds to the supF gene, which is also used to obtain the mutation spectrum. Thus, one can correlate the mutagenicity of a specific region of the supF gene with the presence of lesions in the same position. The data obtained 1° show that, depending on the enzyme used, DNA synthesis is interrupted either opposite or one nucleotide 3' to the deoxyguanosine positions on the template. This suggests that the blocking lesions induced by IO2 are specifically located at deoxyguanosine residues, confirming the high and specific reactivity of 102 with guanine. Moreover, there are marked variations among the different deoxyguanosines of the supF gene in the efficiency of blocking DNA synthesis, which may reflect the distribution of blocking lesions in the sequence. In general, no particular sequence context showed specific susceptibility to the attack of 1 0 2 .
Shuttling DNA into Mammalian Cells The IO2-damaged vectors can be introduced in monkey COS7 cells by any of a number of established transfection procedures. The DEAE-dextran technique n works well for this cell line. Basically, a sterile solution is prepared in Dulbecco's modified Eagle's medium (without serum or sodium bicarbonate) containing 2/.~g/ml of DNA, 50 mM Tris-HCl, pH 7.4, and 0.75 mg/ml of DEAE-dextran. A 90-mm cell culture dish with approximately 10 6 cells is rinsed twice with phosphate-buffered saline (PBS), and 0.5 ml of the DNA solution is carefully spread over the cells. The cells are incubated for 30 min at room temperature and then rinsed twice with PBS. Culture proceeds for 3 to 7 days in order to amplify the vector. The cells are harvested, and low molecular weight DNA is extracted by the small-scale alkaline lysis procedure. An alkaline sodium dodecyl sulfate (SDS) solution (450/zl of 2.7% sucrose, 1.7% Triton X-100, 16 mM EDTA, 16 mM Tris-HCl, 0.67 N NaOH, and 0.7% SDS) is applied over the cell monolayer. The culture dish is rocked gently, and the cell lysate is scraped into an Eppendorf tube. Ammonium acetate (7.5 M, pH 7.8, ~0 D. T. Ribeiro, F. Bourre, A. Sarasin, P. Di Mascio, and C. F. M. Menck, Nucleic Acids Res. 20, 2465 (1992). u j. H. Wilson, Virology 91, 380 (1978).
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225/~1) is mixed into the lysate; the tube is then cooled on ice for 10 min and centrifuged (13,000 rpm, 30 min). The clear supernatant is transferred to a fresh tube and incubated with RNase I (50/zg/ml) for 15 min, at 45 °. DNA is purified by two phenol-chloroform extractions and sedimented after addition of 0.6 volume of 2-propanol by centrifugation (13,000 rpm, 15 min). The pellet (almost invisible) is rinsed with 70% ethanol and resuspended in 100 /zl of 10 mM Tris-HCl pH 8.0, 1 mM EDTA. The plasmids are ready to transform bacteria in order to check for relative survival and to screen for mutants in the supF gene induced by ~O2 treatment.
Shuttling Back to Escherichia coli and Screening for Mutations Plasmid DNA is rescued in E. coli made competent by the method of Hanahan.12 The transformants are plated on LB medium containing chloramphenicol (34/~g/ml), X-Gal (5-bromo-4-chloroindolyl-fl-D-galactoside, 0.08 mg/ml), and IPTG (isopropyl-fl-D-thiogalactoside, 0.1 raM). The number of colonies recovered reflects the amount of DNA molecules replicated in the monkey cells, and thus plasmid survival can be defined as the ratio of colonies transformed with DNA from cells transfected with ~O2-treated vector to those obtained with untreated vector. Such analysis indicated that lesions induced by 102 in ssDNA strongly inhibit its conversion to the double-stranded replicative form after transfection in mammalian cells. This is in contrast with the high recovery of plasmids from cells transfected with damaged dsDNA. 3 The E. coil strain MBM7070 is used for screening of plasmids containing mutations that inactivate the tRNA supF gene. The product of the supF gene suppresses an amber mutation in the lacZ gene of the bacterial chromosome, leading to the production of an active fl-galactosidase and, thus, metabolization of the indicator dye X-Gal. Consequently, colonies bearing plasmids with a functional supF gene have a bright blue phenotype. Mutations that inactivate the suppressor tRNA lead to the formation of white or light blue colonies. These colonies are screened in a background of blue colonies and restreaked three times for confirmation of the mutant phenotype. The mutation frequency is defined as the ratio of white or light blue colonies to total colonies examined. The experiments with '02treated shuttle vectors have shown that ~O2-induced lesions are highly mutagenic in mammalian cells. The mutagenicity of ~O2 is higher for ssDNA than for dsDNA. However, when comparison of the data obtained 12 D. Hanahan, J. Mol. Biol. 166, 557 (1983).
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for both DNA structures is based on the number of lesions induced on each molecule, it seems that damages on the dsDNA have a higher mutability, t3 Mutation Analysis The mutated plasmids are amplifed in bacteria and extracted by conventional procedure. 8 The supF locus is then sequenced by the Sanger chain-termination method, using specific primers complementary to the plasmid DNA in positions adjacent to the supF gene. This locus is particularly interesting as a mutation target, since extensive studies have demonstrated that base substitutions at almost any site in the 85 structural base pairs of the tRNA inactivate the supF function, with few silent mutations. 14 For double-stranded shuttle vectors, mostly single and multiple base substitutions were found among the ~O2-induced mutants. The great majority of these point mutations involve G : C base pairs, resulting mainly in G : C to T : A followed by G : C to C : G transversions) s Consistent with these data, the sequence of mutants obtained in experiments employing singlestranded vectors indicated that transversions involving G (G to T and G to C) are the most frequent mutations induced by ~O2 (unpublished results). Similar results were also obtained for the M13 lacZ phage system replicated in E. co[i. 16 Therefore, mutagenesis, mediated by 102-induced DNA damage, is targeted selectively at guanine residues in both prokaryotic and eukaryotic cells. Concluding Remarks Shuttle vector systems, in which DNA is exposed to damaging agents outside of the cell, are being used in order to understand the biological consequences of the interaction of ~O2with DNA. These and other studies provided information showing that lO2-induced DNA damages may interfere with DNA replication, are repaired, and are mutagenic in both prokaryotic and eukaryotic cells. The mechanisms and the enzymology of these processes are just beginning to be understood, especially in mammalian cells. The use of shuttle vectors may continue to contribute to solving these questions by shuttling ~O2-damaged plasmids into cells impaired in the metabolism of damaged DNA, such as those derived from persons 13 H. Sies and C. F. M. Menck, Mutat. 14 K. H. Kraemer and M. M. Seidman, ~5R. Costa de Oliveira, D. T. Ribeiro, Nucleic Acids Res. 20, 4319 (1992). 16 D. Decuyper-Debergh, J. Piette, and
Res. 275, 367 (1992). Mutat. Res. 220, 61 (1989). R. G. Nigro, P. Di Mascio, and C. F. M. Menck,
A. Van de Vorst, EMBO J. 10, 3155 (1987).
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suffering from syndromes like xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, Cockayne, and Bloom syndromes. If any of the genes related to these syndromes is involved in the processing of DNA damages, the replication or the mutagenicity of the ~O2-treated vectors may be affected. Not only will such information help in understanding the syndrome itself, but it may also give clues regarding the deleterious role of ~O2 over the genetic material in oivo and how the cells deal with DNA damage induced by this excited molecule. Acknowledgments I a m grateful to Dr. H e l m u t Sies and Dr. Paolo Di Mascio for continuing e n c o u r a g e m e n t during the course of this research. This work was supported by grants from Fundaqfm de A m p a r o ~ P e s q u i s a do E s t a d o de S~o Paulo (FAPESP) and C o n s e l h o Nacional de Desenvolvim e n t o Cientffico e Tecnol6gico (CNPQ), Brazil.
[12] O x i d a t i v e D N A D a m a g e : E n d o n u c l e a s e F i n g e r p r i n t i n g By BERND EPE and JUTTA HEGLER
Introduction Most cells contain a number of repair endonucleases which specifically recognize types of DNA modifications that are induced by reactive oxygen species (hydroxyl radicals, singlet oxygen) and some other agents, for example, UV-irradiation and methylating agents, that apparently constitute a natural hazard for the DNA. These specific repair endonucleases work independently from and in addition to the nonspecific nucleotide excision repair of the cells, which is represented in Escherichia coli by the uvrABC endonucleases. Several specific repair endonucleases have been cloned and well characterized; the substrate specificities of some of them, according to the available data, are summarized in Table I. Several types of both base modifications and AP sites (apurinic/apyrimidinic sites, sites of base loss) are recognized. The spectrum of substrate modifications comprises most of the modifications known to be generated by reactive oxygen species. All the enzymes shown in Table I incise the DNA at the site of a modification, that is, they generate a DNA single-strand break. The enzymes recognizing base modifications have combined glycosylase and AP endonuclease activity: they first remove the modified base from the deoxyribose moiety and then incise the DNA backbone at the AP site generated. Therefore, regular AP sites (i.e., those not oxidized in the sugar moiety) are substrates for all the enzymes. METHODS 1N ENZYMOLOGY, VOL. 234
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
122
OXIDATIVE DAMAGE TO D N A AND D N A REPAIR
[12]
suffering from syndromes like xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, Cockayne, and Bloom syndromes. If any of the genes related to these syndromes is involved in the processing of DNA damages, the replication or the mutagenicity of the ~O2-treated vectors may be affected. Not only will such information help in understanding the syndrome itself, but it may also give clues regarding the deleterious role of ~O2 over the genetic material in oivo and how the cells deal with DNA damage induced by this excited molecule. Acknowledgments I a m grateful to Dr. H e l m u t Sies and Dr. Paolo Di Mascio for continuing e n c o u r a g e m e n t during the course of this research. This work was supported by grants from Fundaqfm de A m p a r o ~ P e s q u i s a do E s t a d o de S~o Paulo (FAPESP) and C o n s e l h o Nacional de Desenvolvim e n t o Cientffico e Tecnol6gico (CNPQ), Brazil.
[12] O x i d a t i v e D N A D a m a g e : E n d o n u c l e a s e F i n g e r p r i n t i n g By BERND EPE and JUTTA HEGLER
Introduction Most cells contain a number of repair endonucleases which specifically recognize types of DNA modifications that are induced by reactive oxygen species (hydroxyl radicals, singlet oxygen) and some other agents, for example, UV-irradiation and methylating agents, that apparently constitute a natural hazard for the DNA. These specific repair endonucleases work independently from and in addition to the nonspecific nucleotide excision repair of the cells, which is represented in Escherichia coli by the uvrABC endonucleases. Several specific repair endonucleases have been cloned and well characterized; the substrate specificities of some of them, according to the available data, are summarized in Table I. Several types of both base modifications and AP sites (apurinic/apyrimidinic sites, sites of base loss) are recognized. The spectrum of substrate modifications comprises most of the modifications known to be generated by reactive oxygen species. All the enzymes shown in Table I incise the DNA at the site of a modification, that is, they generate a DNA single-strand break. The enzymes recognizing base modifications have combined glycosylase and AP endonuclease activity: they first remove the modified base from the deoxyribose moiety and then incise the DNA backbone at the AP site generated. Therefore, regular AP sites (i.e., those not oxidized in the sugar moiety) are substrates for all the enzymes. METHODS 1N ENZYMOLOGY, VOL. 234
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[12]
ENDONUCLEASE FINGERPRINTING
+ .~,.~
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100 mM DTT.
DNA Mobility Shift The ability of a protein to bind to DNA can most simply be examined by gel retardation assays. Numerous gel conditions for the retardation assays have been described; however, we have found that low ionic strength gel conditions give significantly better shifts with the OxyR protein. The protein sample, either purified OxyR protein (0.2 to 5 ng) or a soluble extract from cells lysed by sonication ( - 1 ~g), is diluted in 1 x TM [50 mM Tris-C1, pH 7.9, 12.5 mM MgC12,20% glycerol, 1 mM EDTA, pH 8, 0.1% Nonidet P-40 (NP-40), and 100 mM KCI]. The labeled DNA sample (5000-10,000 cpm, 1-5 fmol), either plasmid restriction fragments or complementary 5'-end-labeled oligonucleotides annealed in 0.1 MNaC1 at 65 °, is diluted in water. Then the protein and the binding sites are mixed to give a final concentration of 0.5 × TM and a final volume of 25/zl. The binding reaction is incubated at room temperature for 10 to 15 min and subsequently loaded directly onto a low ionic strength gel prepared exactly as described) ° Nonspecific poly(dI-dC) (0.1 to 0.5 ~g) can be added if less pure samples of OxyR are assayed. To assay the ability of OxyR to bind a DNA sequence under reducing conditions we add DTT (---100 mM final concentration) to the binding reactions. The state of OxyR binding may change once the binding reaction has been loaded on the polyacrylamide gel; however, DTT can also be added to the running buffer. (DTT cannot be added to the gel since it prevents polymerization.)
~0F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., "Current Protocols in Molecular Biology." Wiley (Interscience), New York, 1987.
222
ASSAY OF STRESS GENES/PROTEINS
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DNase I Footprinting DNase I footprinting assays can be used to map the site of OxyR binding precisely. For the footprinting reactions, a DNA fragment that is labeled only on one end is required. All of the in vivo OxyR binding sites described to date map at or near the - 35 promoter consensus sequence, and we generally label a restriction fragment of 300 to 400 base pairs (bp) carrying the promoter as follows. DNA (20/zg) is digested with a restriction enzyme giving a 5' overhang (the restriction enzymes EcoRI, BamHI, and HindlII are ideal) between 100 and 200 bp from the putative binding site. The restriction enzymes are removed by phenol and chloroform extractions and ethanol precipitation. Phosphatase is added to remove the 5'-phosphate groups and then removed by thorough phenol and chloroform extractions. The 5' ends are then labeled by treating 5/~g of the cut and phosphatase-treated DNA (in 20/zl) with kinase in the presence of 200-300 /zCi [32p]ATP. The volume of the reaction is then adjusted to 50/xl, and the DNA is digested with a restriction enzyme that cuts 100 to 200 bp from the other side of the putative binding site. To purify the fragment, we load the entire labeled and digested sample on a 6%, 0.8-mm-thick acrylamide gel and cut out the fragment of interest after a 1- to 2-rain exposure to film. The fragment is eluted from the gel slice in 200 to 400 tzl TE at 37° for greater than 8 hr. After elution, the fragment is extracted with phenol and chloroform and precipitated. The DNase I required for the footprinting reactions is obtained as a lyophilized powder and then dissolved in cold water at a concentration of 2.5 mg/ml. Aliquots (10/xl) of the stock solution can be frozen in liquid nitrogen and stored at - 70°. The binding reactions are carried out almost exactly as for the mobility shift assay. Less than 30 ng of OxyR protein in I × TM is mixed with 2000 to 6000 cpm (3 fmol) 5'-end-labeled DNA in water to give a total volume of 25 /zl at 0.5 z TM and then incubated for I0 min at room temperature. Again DTT (-> 100 mM) can be added to examine the binding under reducing conditions. During the 10-min incubation, the DNase I stock is diluted to 1 to 3/xg/ml in cold water (use lower concentrations for larger fragments to obtain a more complete set of partially digested fragments). After the 10-min incubation, 25 ~1 of a Mg 2+,Ca2+-containing solution (10 mM MgCI2 and 5 mM CaCI2) is added to all tubes. Immediately thereafter, 2/xl diluted DNase I is added to each tube at room temperature at 10- to 15-sec intervals. Then each DNase I digestion is stopped after exactly 1 min by adding 200/~1 of stop solution (20 mM EDTA, pH 8.0, 1% SDS, 0.2 M NaCI, and 250/~g/ml total yeast RNA which is purified by abundant phenol and chloroform extractions). The digested fragments are subsequently extracted with phenol and chloroform and precipitated
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with 600 txl of ethanol (no additional salt is needed for the precipitation), The pellet can be washed with 70% ethanol, dried, resuspended in 2-4 tzl sequencing formamide dye mix, heated at 90° to I00 ° for 2 to 3 min, and then loaded on an 8% sequencing gel. A sample corresponding to the G and A sequence can be generated as follows. Five micrograms of yeast total RNA and 80,000 cpm of the labeled DNA fragment are dried, resuspended in 6/~1 of fresh 2% (v/v) formic acid (J. T. Baker Chemical Co., Phillipsburg, NJ), and incubated at 37° for 5 min (use shorter times if too much cleavage is observed). The formic acid is removed by evaporation for 2 hr in a Speed Vac concentrator. The dried pellet is resuspended in 100/xl of 1 M piperidine (Fisher Scientific, Fair Lawn, N J) and heated at 90 ° for 40 min. The piperidine is removed by evaporation overnight. Then the dried DNA is resuspended in 50 /xl of water and the water removed by evaporation. Finally the cleaved DNA is resuspended in 100 /xl of water, extracted twice with chloroform, and then precipitated on the addition of salt and ethanol. An amount equivalent to only one-third of the radioactivity in the footprinting lanes should be loaded. In Vitro Transcription
The ability of OxyR to activate transcription can be assayed in vitro as follows. Purified OxyR (-0.5 mg/ml) with 15 /zg of bovine serum albumin in a total volume of 50 tzl is exchanged into transcription buffer (40 mM Tris-Cl, pH 7.9, 0.1 M KCI, 10 mM MgCI2) containing 1 mM DTT, 5% glycerol, and 0.1% NP-40 by centrifugation through 800 ~1 of Sephadex. An aliquot (5/zl) together with 2.5 /xl water is the incubated with a supercoiled template DNA (0.2/zg in 36.5/zl of transcription buffer) for 10 min at 37°. RNA polymerase holoenzyme (0.5/zg in 5/zl of transcription buffer) is then added to the bound template, and the reaction is incubated for an additional 10 min at 37 °. After the addition of 1 /~1 of a 25 mM mixture of dNTPs, the reactions are incubated another 5 min at 37°. The transcription reactions are terminated by the addition of phenol, and the mRNA is extracted with phenol and chloroform several times and then assayed by primer extension.
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[18] T r a n s i e n t E n h a n c e m e n t o f H e m e O x y g e n a s e 1 m R N A A c c u m u l a t i o n : A M a r k e r o f O x i d a t i v e S t r e s s to E u k a r y o t i c Cells By REX M. TYRRELL and SHARMILA BASU-MODAK Introduction
Bacteria respond to oxidative stress by the rapid and transient expression of a large number of genes. Two major regulatory pathways have been recognized to date, namely, the oxyR and soxR systems. The oxyR system regulates nine of the genes induced by hydrogen peroxide in both Escherichia coli and Salmonella typhimurium. 1 Several of the oxyR-controlled genes (e.g., catalase, glutathione reductase, alkyl hydroperoxide reductase) clearly play a role in defense against oxidative stress. OxyR codes for a regulatory protein which appears to act as an oxygen sensor by being oxidized directly. 2 Another 40 or so genes are turned on by redox cycling agents in E. coli, and 9 of these have been shown to have a common regulatory pathway and have been grouped together as the soxR regulon. 3 The detection of expression of the oxyR regulon and/or the soxR regulon should provide an early marker of oxidative stress in prokaryotes. The binding of several eukaryotic transcription factors such as NFKB and possibly the fos-jun heterodimer (AP-1) may be redox regulated. 4,5 Expression of the c-jun oncogene is stimulated by either UVC radiation (254 nm) or hydrogen peroxide. 6 On the other hand, expression of the human heme oxygenase 1 (HO-1) gene is not induced by UVC radiation, whereas it is strongly induced by oxidizing agents such as UVA (320-380 nm) radiation or hydrogen peroxide. 7 In addition to oxidative stress, the gene is induced by other agents such as phorbol esters, heavy metals, and sodium arsenite. 7 Although this phenomenon was originally observed in fibroblasts cultured from human skin, induction occurs in most human l L. A. Tartaglia, G. Storz, S. B. Farr, and B. N. Ames, in "Oxidative Stress: Oxidants and Antioxidants" (H. Sies, ed.), p. 155. Academic Press, London, 1991. 2 G. Storz, L. A. Tartaglia, and B. N. Ames, Science 248, 189 (1990). 3 B. Demple and J. D. Levin, in "Oxidative Stress: Oxidants and Antioxidants" (H. Sies, ed.), p. ll9. Academic Press, London, 1991. 4 C. Abate, L. Patel, F. J. Rauscher III, and T. Curran, Science 249, 1157 (1990). R. Schreck, P. Rieber, and P. A. Baeuede EMBO J. 10, 2247 (1991). 6 y . Devary, R. A. Gottlieb, L. F. Lau, and M. Karin, Mol. Cell. Biol. 11, 2804 (1991). 7 S. M. Keyse and R. M. Tyrrell, Proc. Natl. Acad. Sci. U.S.A. 86, 99 (1989).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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cell types and all mammalian cell types so far tested. 8 The induction is clearly related to the cellular redox state since lowering cellular glutathione levels strongly enhances HO-1 mRNA accumulation) For these reasons, altered expression of the HO-1 gene appears to be a fairly sensitive marker of oxidative stress. Indeed, enhanced expression of the HO-1 gene is now being used in several laboratories as a positive control when testing other genes suspected of being oxidant-inducible. Choice of Assay
Because oxidants induce expression of the HO-1 gene by enhancing the transcription rate, 1° altered expression can be monitored at a variety of levels. Direct measurement of altered transcription rates is probably the most sensitive method, but the run-off transcription assays usually employed for this measurement are more labor-intensive and subject to greater interexperimental variation than assays that measure a later step. One-dimensional sodium dodecyl sulfate (SDS)-poylacrylamide gels are normally sensitive enough to detect induction of de novo synthesis of the 32-kDa protein corresponding to HO-l,n but background levels are high due to the large number of constitutive proteins in this molecular size range. Induction of HO-1 enzyme activity several hours after the initial treatment is also fairly simple to measure by a spectrophotometric assay,~Z although the biliverdin reductase required for the coupled assay is not available commercially. However, a resolution problem now arises because HO-1 cannot be distinguished enzymatically from the constitutive HO-2 form which is present in various amounts according to cell t y p e ) TM The two forms can be distinguished by Western blot analysis, but antibodies to the proteins are not yet commercially available. With these considerations in mind, the current method of choice is measurement of the accumulation of HO-1 mRNA using a specific cDNA probe. The most commonly used techniques for measuring such accumulation are the dot-blot and Northern blot procedures, the latter being preferred given the high level of nonspecific background that can be associated with the dot-blot procedure. RNA extraction and Northern blot methods 8 L. A. Applegate, P. Luscher, and R. M. Tyrrell, Cancer Res. 51, 974 (1991). 9 D. Lautier, P. Luscher, and R. M. Tyrrell, Carcinogenesis (London) 13, 227 (1992). l0 S. M. Keyse, L. A. Applegate, Y. Tromvoukis, and R. M. Tyrrell, Mol. Cell. Biol. 10, 4967 (1990). ii S. M. Keyse and R. M. Tyrrell, J. Biol. Chem. 262, 14821 (1987). 12 S. Shibahara, T. Yoshida, and G. Kikuchi, Arch. Biochem. Biophys. 188, 243 (1978). 13 M. D. Maines, G. M. Trakshel, and R. K. Kutty, J. Biol. Chem. 261, 411 (1986). 14 G. M. Trakshel, R. K. Kutty, and M. D. Maines, J. Biol. Chem. 261, 11131 (1986).
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are commonly described in laboratory manuals for molecular biology, 15,16 but we describe here the sequence of procedures that works best for us for our work with HO-1 mRNA. The precise conditions of cell culture and preparation before and after treatment will depend on the cell type employed. We describe the procedure for human fibroblasts cultured as attached monolayers and treated with either UVA radiation or hydrogen peroxide.
Methods Treatment Procedure Irradiation with UVA. A broad-spectrum UVA lamp with a large beam area (e.g., we use the UVASUN 3000 lamp supplied by Mutzhas, Munich Germany) is a convenient source of UVA radiation for the processing of many culture dishes simultaneously.
1. Human fibroblast cells (FEK 417) are seeded in 10-cm dishes at a density of 5 × 105 cells in 10 ml Earle's minimal essential medium [supplemented with 50 U/ml penicillin, 50/zg/ml streptomycin, 0.2% (w/v) sodium bicarbonate, and 15% fetal calf serum (FCS, v/v) (heat-inactivated at 56°)] per dish and cultured for 3 days at 37° in 5% CO2, at which time they reach 60-80% confluency. 2. Remove the conditioned medium and keep aside at 37°. Rinse the cell monolayers with 10-15 ml phosphate-buffered saline (PBS) at room temperature. 3. Add 5 ml of PBS to each 10-cm dish. The PBS should be supplemented with CaZ'-/Mg2+ salts (each 0.01% final concentration)just prior to use. 4. Irradiate the culture dishes for the time required at a distance from the UVA source that avoids a temperature rise during irradiation. A fluence range of 0-1 MJ m -2 is generally used for the HO-1 gene, and we find that the HO-1 mRNA levels peak at fluences between 400 and 500 kJ m -2. The fluence rate at 30 cm from the UVASUN 3000 lamp source is approximately 300 W m-2 as measured by an IL 1700 radiometer (International Light Inc., Newburyport, MA). 15 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. ~6 L. G. Davis, M. D. Dibner, and J. F. Battey, "Basic Methods in Molecular Biology." Elsevier, New York, 1986. 17 R. M. Tyrrell and M. Pidoux, Cancer Res. 46, 2665 (1986).
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5. After irradiation, aspirate the buffer and rinse the monolayer with 10-15 ml of PBS. Add back the original medium and incubate the cells for 3 hr before extracting total RNA, Addition of fresh medium instead of the conditioned medium induces the gene and gives incorrect estimates of the basal levels of the HO-1 mRNA.
Treatment with Oxidant 1. Cells are grown to 60-80% confluency and prepared for chemical treatment as described above for irradiation with UVA. 2. Add 5 ml of Ca2+/Mg2+-containing PBS to each culture dish and then hydrogen peroxide to a final concentration of 100 /xM. A dilute solution of hydrogen peroxide is prepared in sterile water just prior to use. At 240 nm the molar extinction coefficient of hydrogen peroxide is 43.6 M 1cm-l. 18The appropriate (i.e., nontoxic but effective) concentration of hydrogen perioxide is highly dependent on the cell number and needs to be determined for each given set of experimental conditions. 3. Incubate the cell monolayers with the oxidant for 30 min at 37° in the CO2 incubator. After chemical treatment, aspirate the buffer containing the oxidant, rinse the monolayers with PBS, and add back the original medium. Incubate cultures for 3 hr at 37° before extracting total RNA.
Isolation of Total Cellular RNA We use the acid-guanidinum thiocyanate-phenol-chloroform (AGPC) extraction method ~9 for isolation of total cellular RNA as it is rapid and a large number of samples can be processed simultaneously. All solutions (except Tris and EDTA) are prepared in water treated with DEPC (diethyl pyrocarbonate). 15 RNase-free glassware and plasticware is used for all manipulations. Use of gloves during all procedures is obligatory for RNA work.
Solutions Guanidinium thiocyanate stock: 4 M guanidinium thiocyanate (250 g plus 293 ml of water), 25 mM sodium citrate (pH 7.0) (17.6 ml of 0.75 M stock), 0.5% sarkosyl (26.4 ml of 10% stock); guanidinium thiocyanate is dissolved at 65 ° directly in the manufacturer's bottle; the stock solution is stable at room temperature for 3 months ~s A. Claiborne, in "CRC Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), p. 283. CRC Press, Boca Raton, Florida, 1985. 19 p. Chomczynski and N. Sacchi, Anal, Biochem. 162, 156 (1987).
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Solution D: 0.36 ml of 2-mercaptoethanol in 50 ml of guanidinium thiocyanate stock; the solution is stable at room temperature for 1 month Phenol equilibrated with Tris-Cl (pH 8.0) 15 Chloroform-isoamyl alcohol mixture (49:1, v/v) 75% Ethanol (v/v) 2 M Sodium acetate (pH 4.0)
Procedure 1. Rinse the cell monolayers with 10-15 ml PBS and add 2 ml solution D per 10-cm dish. Cells are lysed directly in the culture dishes for 5 min at room temperature, after which the dishes are left in an inclined position for 2 min to allow the viscous lysate to accumulate on one side of the dish. This allows the collection of 95% of the lysate and is especially useful when a large number of dishes are processed simultaneously. 2. Transfer the lysate to polypropylene tubes with caps and add 0.2 ml of 2 M sodium acetate (pH 4.0), 2.5 ml phenol, and 0.4 ml chloroform-isoamyl mixture sequentially. Mix well by rapidly inverting the tube for at least 30 sec after each addition. The mixing steps should be thorough but gentle. 3. Cool suspensions on ice for 15 min and centrifuge at 10,000 g for 20 min at 4 ° to separate the phases. 4. Collect the aqueous phase in a fresh tube and precipitate the RNA with an equal volume of 2-propanol at - 2 0 ° for at least 1 hr. When a large number of samples are being processed, it may be more convenient to leave the samples overnight at - 2 0 ° at this step. 5. Pellet the RNA by centrifuging at 10,000 g for 30 min and redissolve (room temperature) the pellet in 0.3 ml of solution D. At this step, samples are transferred to 1.5-ml Eppendorf tubes and reprecipitated with an equal volume of 2-propanol (0.3 ml) at - 2 0 ° for I hr. 6. Pellet the RNA by centrifuging in a microcentrifuge at 4 °. Wash the RNA pellet twice with 75% ethanol as follows. Add 500/xl of 75% ethanol to each tube and release the RNA pellet by tapping the tube gently against the laboratory bench. Microcentrifuge for I0 min at 4 ° and then aspirate the supernatant. 7. Vacuum-dry the pellet for 5 min. If RNA is dried for too long, then it does not go into solution easily. 8. Dissolve the RNA in 25 /~1 of DEPC-treated water by heating to 65 ° for 10 min followed by quick cooling on ice. Determine the RNA concentration by measuring the absorbance of an aliquot at 260 and 280 nm. The A26o/A2so ratios obtained should be between 1.95 and 2.0. Store aliquots containing 12-15/~g of total RNA at - 2 0 ° until further use.
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Northern Analysis of HO-1 mRNA Electrophoresis of RNA. The gel casting trays, combs, and buffer tanks should be used routinely only for RNA work and should be soaked in 1% SDS (w/v) and rinsed well with ultrapure deionized water prior to each use. If RNase is also used in the laboratory, then extra attention should be given to cleaning the gel electrophoresis accessories. 15 We use formaldehyde/agarose gels for separation of RNA. Solutions 10× MOPS [3-(N-morpholino)propanesulfonic acid] buffer (final pH between 5.5 and 7.0)*: 0.2 M MOPS [3-(N-morpholino)propanesulfonic acid], 50 mM sodium acetate, 10 mM EDTA Loading buffer (10 ml): 4.8 ml deionized formamide, 1.07 ml of 10× MOPS buffer, 1.73 ml (37%) formaldehyde, 0.533 ml glycerol, 1.033 ml bromphenol blue (saturated solution), 0.834 ml water Rinse solution*: 75 mM NaOH, 100 mM NaC1 100 mM Tris-Cl (pH 7.5)
Procedure 1. Melt 1.3 g agarose (Bio-Rad, Richmond, CA) in 74 ml of sterile water and add 10 ml of 10× MOPS buffer. Cool to 50° and add 16.2 ml of 37% formaldehyde solution (2.2 M final), then pour the contents into a 10 by 15 cm gel casting tray with the comb in position. Formaldehyde gels are cast in a fume hood and allowed to set for 30-45 min. We do not use ethidium bromide in gels which are subjected to Northern transfer. 2. Remove the comb gently and cover the gel with 1 × MOPS buffer. 3. To prepare RNA samples for electrophoresis, vacuum-dry aliquots containing 12-15/~g of total RNA to decrease the volume to 5-10/~1. Do not dry completely. Add 20/~1 of loading buffer to each sample and heat to 65° for 10 min followed by quick cooling on ice. 4. Load samples and electrophorese at 50 V until the bromphenol blue migrates a distance of approximately 7 cm from the well. 5. After electrophoresis, rinse the gel with deionized water and soak in the rinse solution for 40 min at room temperature on a rocking platform. 6. Neutralize the gel with 100 mM Tris-Cl (pH 7.5) for 45-60 min on a rocking platform with one change of buffer and set up the Northern transfer as described below. * Sterile ultrapure deionizedwater can be used to make these solutions instead of DEPCtreated water.
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It is usually not necessary to electrophorese samples in ethidium bromide-containing gels since RNA prepared by the AGPC method is usually undegraded, provided that proper care has been taken during isolation and electrophoresis. However, if RNA isolation is carried out for the first time, an aliquot of 2/zg of total RNA can be electrophoresed in 1% agarose gels containing ethidium bromide using 0.5 × TBE bufferJ 5 Northern Transfer: Capillary Blot Procedure. We use Gene Screen nylon membranes (NEN Du Pont, Dreieich, Germany) for Northern transfer and use the manufacturer's conditions for transfer and hybridization. These are described briefly here.
Solutions Transfer buffer (pH 6.5)*: 25 mM Na2HPO4, 25 mM NaH2PO4
Procedure 1. Place a sponge (small pore, 2 × 15 × 19 cm) in a RNase-free plastic container and add enough transfer buffer to soak the sponge. D o n o t submerge the sponge in buffer. Air bubbles can be easily removed by poking the sponge with an RNase-free pipette or glass rod. 2. Place a Whatman (Clifton, N J) paper 3MM (prewet in transfer buffer), cut to a size intermediate between the gel size and the sponge size, on the sponge. Remove entrapped air bubbles by rolling a pipette or glass rod over the paper. Overlay with two more wet pieces of Whatman 3MM paper. Care should be taken to remove air bubbles at each overlay step. 3. Place the gel with the well bottom facing away from the sponge and overlay with a wet piece of Gene Screen membrane cut to the same size as the gel. The nylon membrane should be wetted in transfer buffer for at least 15 min prior to use. 4. Overlay the nylon membrane with one piece of wet and two pieces of dry Whatman 3MM paper cut to the same size as the gel. Place Parafilm strips along the edges of the gel to prevent short-circuiting during transfer. 5. Place a stack of paper towels (8-10 cm high) cut to the same size as the gel and a 1-kg lead weight on top of the stack and leave for at least 16 hr for transfer. 6. Rinse the membrane with transfer buffer to remove residual agarose, place the blot on a Whatman 3MM paper with the transferred RNA side facing upward, and air dry. Bake the blot at 80° for 2-4 hr. After this step the nylon membrane can be stored at room temperature until use.
Hybridization of RNA. We probe the Northern blot for the HO-1 mRNA with the large EcoRI fragment (1000 bp) of a full-length cDNA
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clone (clone 2/107). The full-length cDNA is inserted into the EcoRI sites of the phagemid pBluescript SK (MI3-), and we maintain it in the Stratagene (LaJolla, CA) E. coli host XL1-Blue {endA1, hsdR17 (rk-, mk+), supE44, thi-1, k-, recA1, gyrA96, relA1, lac- [F', proAB, laclqZ A M15, TnlO (TetR)]}. This plasmid DNA (15-20 /xg) is digested to completion with EcoRI and electrophoresed in a 0.6% low melt agarose gel. The 1000-bp fragment is cut out from the gel and used directly for labeling. This cDNA clone is available from our laboratory. To control for the variation between RNA samples in the same gel, we reprobe each membrane for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, 2° levels of which are not affected by UVA or other oxidants. Probes for other constitutive mRNA species can also be used as loading controls. The cDNA probes are labeled with [a-32p]dCTP or [ot-32p]dATP using a Random Primed labeling kit supplied by Boehringer Mannheim (Mannheim, Germany). We purify the 3Zp-labeled fragment on a Elutip-d column (Schleicher and Schuell, Dassel, Germany) according to the procedure recommended by the manufacturer.
Solutions Prehybridization buffer (recommended for Gene Screen nylon membranes by manufacturer): 50% formamide, v/v (deionized), 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin (BSA), 0.2% Ficoll, 50 mM Tris-HCl (pH 7.5), 1.0 M NaC1, 0.1% sodium pyrophosphate, 1.0% SDS (w/v), 10% dextran sulfate (optional), 100 /zg/ml denatured salmon sperm DNA 20 x SSC*: 3 M sodium chloride, 0.3 M sodium citrate Washing solution 1": 2× SSC, 0.1% SDS Washing solution 2*: 0.1x SSC, 0.1% SDS Procedure 1. For prehybridization, prewet the blot in 6x SSC and place in a plastic bag. Remove as much liquid as possible. Add 12 ml ofprehybridization buffer at 42° to the bag, remove air bubbles, and seal the bag. Leave the bag in a 42 ° water bath with constant agitation for at least 4 hr (can be left for 24 hr). Two filters can be placed in each bag, with the sides containing the RNA facing away from one another. 2. For denaturing the probe, boil in a water bath for 10 rain and quick cool on ice for 10 rain. Add the denatured probe (specific activity 108 cpm/ mg) to the prehybridization bag (2-3 x 10 6 cpm/ml, 10-50 ng/ml), remove 20 p. Fort, L, Marty, M. Piechaczyk, S. E. Sabrouty, C. Dani, P. Jeanteur, and J. M. Blanchard, Nucleic Acids Res. 13, 1431 (1985).
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air bubbles, seal the bag, and leave for hybridization at 42 ° for 16--24 hr. The probe purified on an Elutip-d column is in a volume of 500 ~1, and we add this directly to the prehybridization solution. 3. Collect the hybridization solution and store at - 2 0 °. The solution can be reused once; to do so, remove all buffer from the bag after prehybridization and add the hybridization solution which has been boiled in a water bath for 10 min. 4. After hybridization, wash the membrane with constant agitation as follows: (a) once in 100 ml of 2× SSC for 5 min at room temperature; (b) once in 100 ml of washing solution 1 for 30 min at 50°; and (c) once in 100 ml of washing solution 2 for 30 rain at 65 °. For the HO-I cDNA probe, the third washing step (c) is usually not necessary. 5. Air dry the membrane, wrap it in Saran wrap and expose to a preflashed film at - 7 0 °. 15Because the membranes are usually rehybridized with the GAPDH probe, they should be left slightly damp. 6. Prior to rehybridization, the labeled probe is stripped from the membrane by boiling in washing solution 2 for 40 min with one solution change. It is recommended that the labeled probe be stripped off from the membrane soon after autoradiography if rehybridization is to be carried out. The stripped Northern blot can be air dried and stored between sheets of Whatman 3MM paper at room temperature. A typical Northern blot analysis of total cellular RNA isolated by the AGPC method and probed for the HO-1 and GAPDH mRNA species is shown in Fig. 1. For this experiment cell cultures were exposed to a range of fluences of UVA radiation in the presence of 5 mM N-acetylcysteine. Quantification of Northern Analysis. Because there is a fluence-dependent change in HO-1 mRNA levels, it is necessary to carry out autoradiography for different periods of time. Autoradiographs in which the control sample (see Fig. 1) which has the lowest signal intensity is clearly visible and the band with the highest signal intensity is still in the linear range MJm N-AC
-2
0 -
0.1 +
0.2 +
-
0.3 +
0.4 +
-
0.5 +
-
+
HO
GAPDH
FIG. 1. Human skin fibroblasts (FEK 4) were irradiated with increasing Iluences of UVA in the presence (+) or absence ( - ) of 5 mM N-acetylcysteine (N-AC). Total RNA was subjected to Northern blotting and probed first for HO- 1 mRNA and then for GAPDH mRNA.
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should be used for densitometric analysis. A detailed densitometric analysis of autoradiographs with different exposure times needs to be done for one experiment, after which it is possible to judge the signal intensity visually. Radioactive signals are quantified in our laboratory by densitometry using an Elscript 400 (Hirschmann) densitometer with evaluation software. We usually scan vertically down each track rather than scanning the band of interest horizontally along the gel. The area under the curve of the band of interest is integrated with the evaluation software supplied with the densitometer. Areas determined by densitometry of autoradiographs in Fig. 1 are shown in Table I. Calculation o f Increase in HO m R N A The HO-1 mRNA signal intensity increases severalfold over basal levels in response to oxidative stress, and this response can be modulated by chemical agents such as N-acetylcysteine (see Fig. 1). The first step in the quantification is to normalize for the variation in loading between samples on the same gel using the GAPDH mRNA signal (or any other constitutive mRNA) on autoradiographs. A GAPDH ratio is calculated as follows: 1. Calculate the mean areas under the curves of all the GAPDH bands on a gel from the autoradiograph.
TABLE I AREAS DETERMINED BY DENSITOMETRY FOR HO-1 m R N A AND G A P D H m R N A
Lane number a 1 2 3 4 5 6 7 8 9 10 11 12
HO-1 area
GAPDH area
GAPDH ratio
GAPDH ratio x HO-1 area
3.21 8.01 8.86 6.19 15.57 13.23 53.22 30.13 109.17 55.04 93.16 73.72
33.89 26.42 23.82 30.63 18.03 19.41 20.52 23.51 48.72 33.52 33.65 43.21
0.87 1.12 1.24 0.97 1.64 1.53 1.44 1.26 0.61 0.88 0.88 0.69
2.79 8.97 10.99 6.00 25.53 20.24 76.64 37.96 66.59 48.44 81.98 50.87
a See Fig. 1; lanes are n u m b e r e d from left to right.
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2. Divide the mean by the GAPDH area of each sample to obtain a GAPDH ratio for each lane. If equal amounts of total RNA were loaded in all tracks, then the signal intensity would be the same in all the tracks. This is usually not the case and thus the need for this normalization step. 3. Multiply the area under the curve of each HO-1 band by the corresponding GAPDH ratio. The numbers thus obtained are normalized for the variation between samples on the same gel. 4. The calculated area of the control sample (Table I, lane 1) is used as a unit to calculate the increase(-fold) in HO-1 mRNA above basal levels. A plot of relative increase in HO- 1 mRNA levels as a function of fluence is shown in Fig. 2A.
30 t "--
u) 25
"
t
°
15
OT 2.0
.
,
•
,
•
,
•
,
•
,
"
B 0 1.5
-,-~ 1.0 o~ o
0.0 0.0
0.1
0.2
Fluence
0.3
0.4
0.5
(MJm -2 )
FIG. 2. The HO-I and GAPDH mRNA signals were quantified by densitometry. The GAPDH signal was used as an internal control to normalize for the variation in loading between samples. (A) Normalized HO-1 mRNA signals are expressed as the relative increase above basal levels and plotted as a function of fluence. (©) Control cells, (0) cells treated with N-acetylcysteine. (B) The ratios of the normalized HO-1 mRNA signal areas in samples treated with 5 mM N-acetylcysteine (N-AC) and control samples are plotted as a function of fluence.
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The increase(-fold) of HO-1 mRNA varies between experiments and thus error bars are large; however, the pattern is consistent for any agent. To quantify the effect of a test agent it is useful to calculate the ratios of the HO-1 mRNA areas (normalized) in untreated and treated samples of at least three experiments. Such ratios with their standard deviations are plotted against fluence in Fig. 2B. Transient enhancement of HO- 1 mRNA accumulation appears to be an extremely sensitive marker of oxidative stress, and it has been employed in assays in several laboratories. In this chapter we have described detailed methodology based on the Northern blot procedure for the comparative measurement of HO-1 mRNA levels and provide an example in which increased accumulation of HO- I mRNA after a mild oxidative stress (UVA radiation) is suppressed by a free radical scavenging antioxidant (N-acetylcysteine). Acknowledgments The studiesdescribedherein have been supportedby the SwissNational ScienceFoundation (FN 31-30880-91)and the Swiss League against Cancer. N-Acetylcysteinewas a kind gift of Imphazarrn S.A. (Cadempino, Switzerland).
[ 1 9] A s s a y s for R e g u l a t i o n o f G a p J u n c t i o n a l C o m m u n i c a t i o n and Connexin Expression by Carotenoids
By
JOHN S. BERTRAM and LI-XIN
ZHANG
Introduction Compelling epidemiologic evidence has shown that certain carotenoids have cancer chemopreventive activities.1 However, their mode of action in this respect is unclear. Carotenoids as a group are considered to possess antioxidant properties,2 and a limited number act as provitamin A sources in mammals.3 In view of the postulated role of oxidative damage in carcinogenesis 4'5 and the known activity ofretinoids as cancer chemopreventives,6 I j. S. Bertram, L. N. Kolonel, and F. L. M e y s k e n s , Cancer Res. 47, 3012 (1987). 2 N. I. Krinsky, Free Radical Biol. Med. 7, 617 (1989). 3 j. A. Olson, J. Nutr. 119, 105 (1989). 4 p. A. Cerutti, Science 227, 375 (1985). 5 L. H. Breimer, Mol. Carcinog. 3, 188 (1990). 6 R. C. Moon, J. Nutr. 119, 127 (1989).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
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The increase(-fold) of HO-1 mRNA varies between experiments and thus error bars are large; however, the pattern is consistent for any agent. To quantify the effect of a test agent it is useful to calculate the ratios of the HO-1 mRNA areas (normalized) in untreated and treated samples of at least three experiments. Such ratios with their standard deviations are plotted against fluence in Fig. 2B. Transient enhancement of HO- 1 mRNA accumulation appears to be an extremely sensitive marker of oxidative stress, and it has been employed in assays in several laboratories. In this chapter we have described detailed methodology based on the Northern blot procedure for the comparative measurement of HO-1 mRNA levels and provide an example in which increased accumulation of HO- I mRNA after a mild oxidative stress (UVA radiation) is suppressed by a free radical scavenging antioxidant (N-acetylcysteine). Acknowledgments The studiesdescribedherein have been supportedby the SwissNational ScienceFoundation (FN 31-30880-91)and the Swiss League against Cancer. N-Acetylcysteinewas a kind gift of Imphazarrn S.A. (Cadempino, Switzerland).
[ 1 9] A s s a y s for R e g u l a t i o n o f G a p J u n c t i o n a l C o m m u n i c a t i o n and Connexin Expression by Carotenoids
By
JOHN S. BERTRAM and LI-XIN
ZHANG
Introduction Compelling epidemiologic evidence has shown that certain carotenoids have cancer chemopreventive activities.1 However, their mode of action in this respect is unclear. Carotenoids as a group are considered to possess antioxidant properties,2 and a limited number act as provitamin A sources in mammals.3 In view of the postulated role of oxidative damage in carcinogenesis 4'5 and the known activity ofretinoids as cancer chemopreventives,6 I j. S. Bertram, L. N. Kolonel, and F. L. M e y s k e n s , Cancer Res. 47, 3012 (1987). 2 N. I. Krinsky, Free Radical Biol. Med. 7, 617 (1989). 3 j. A. Olson, J. Nutr. 119, 105 (1989). 4 p. A. Cerutti, Science 227, 375 (1985). 5 L. H. Breimer, Mol. Carcinog. 3, 188 (1990). 6 R. C. Moon, J. Nutr. 119, 127 (1989).
METHODS IN ENZYMOLOGY, VOL. 234
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both activities could contribute to the observed epidemiologic findings. In studies conducted with a highly characterized system of in vitro carcinogenesis, 7 we have demonstrated a novel carotenoid action: that of upregulation of gap junctional communication (GJC). 8 This action is highly correlated with the chemopreventive properties but not with the provitamin A status or lipid-phase antioxidant potency of a series of dietary carotenoids. 8 We have further demonstrated that this induction of GJC by carotenoids is achieved by increasing the expression of connexin 43 (Cx43), 9 o n e of a family of genes coding for a structural subunit of the gap junction. 10 Here we describe the measurement of gap junctional communication at the functional level by means of assays of dye transfer. Because junctions may be in the open or closed state, any observed increased dye transfer can be the result of an increased number of junctions and/or an increased number of open junctions. To distinguish between these possibilities we have included assays for the amount and cellular location of connexin 43. Gap junctions are clusters of hydrophilic channels traversing the plasma membranes of coupled cells. The channels when open allow the passive diffusion of inorganic ions and small water-soluble compounds up to about 1000 Da, such as small metabolites, and second messengers. 11:2 Each gap junctional channel is made of two tightly joined hemichannels, each donated by one of the two participating cells. Each hemichannel is termed a hemiconnexon, and two connexons in register are paired to form a continuous aqueous channel by end-to-end joining. Each hemiconnexon is composed of six transmembrane protein subunits termed connexins. A family of connexins has been described with tissue and cell type specificity) 3 Connexin 43 (Cx43), one member of the connexin family, is a major gap junctional protein expressed in many cell types. In the mouse C3H/ 10T1/2 cell culture system (10T1/2) used here, Cx43 appears to be the only connexin expressed) 4 This cell line has been extensively used for studies of carcinogenesis, growth control, and differentiation. 7 7 j. S. Bertram, IARC Sci. Publ. 67, 77 (1985). 8 L.-X. Zhang, R. V. Cooney, and J. S. Bertram, Carcinogenesis (London) 12, 2109 (1991). 9 L.-X. Zhang, R. V. Cooney, and J. S. Bertram, Cancer Res. 52, 5707 (1992). l0 E. C. Beyer, D. L. Paul, and D. A. Goodenough, J. Membr. Biol. 116, 187 (1990). ii W. R. Loewenstein, Cell (Cambridge, Mass.) 48, 725 (1987). lz j. C. Sfiez, J. A. Connor, D. C. Spray, and M. V. L. Bennett, Proc. Natl. Acad. Sci. U.S.A. 86, 2708 (1989). 13 M. Tornomura, K. Kadomatsu, S. Matsubara, and T. Muramatsu, J. Biol. Chem. 265, 10765 (1990). i4 M. Rogers, J. M. Berestecky, M. Z. Hossain, H. Guo, R. Kadle, B. J. Nicholson, and J. S. Bertram, Mol. Carcinog. 3, 335 (1990).
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Methods
Cell Cultures 10T1/2 cells, which can be obtained from the ATCC (Rockville, MD), are grown in Eagle's basal medium supplemented with 5% serum. 15 We have found that iron-supplemented calf serum (HyClone, Logan, UT) can substitute for the increasingly expensive fetal serum previously used for growth of this cell line. Substitution results in an approximate 6-fold reduction of costs and the opportunity to purchase larger lots of serum which reduces the need for frequent screening of new lots of serum. Cells are used between passages 5 and 15 and are cultured at 37° in 5% CO2 in air. Because of the requirement for low passage cells it is essential that multiple vials of cells be frozen in liquid N2 as soon as possible. We have found that 10% (v/v) dimethyl sulfoxide (DMSO) in complete medium is a superior cryopreservative to glycerol. For communication assays cells are normally seeded at a density of 104 cells/60-mm culture dish; under these conditions cells should be confluent within 7 days.
Drug Delivery Extensive research in vitro with carotenoids was previously obstructed by difficulty in delivering these highly lipophilic compounds to target cells. We have overcome this problem by the use of tetrahydrofuran (THF) as a delivery vehicle. This allows the formation of pseudosolutions of carotenoids in cell culture medium at high concentrations. 16,17Carotenoids are dissolved in T H F [99.9% pure containing 0.025% (w/v) butylated hydroxytoluene (BHT) as preservative, Aldrich Chemical Co., Milwaukee, WI]. Solutions must be kept in dark bottles, sealed with a neoprene septum, under inert gas at - 20°. Solutions can be withdrawn by hypodermic glass syringe without addition of atmosphere, as described in detail previously. 17The final concentration of THF in the culture medium should not be above 0.5%. Routinely, 2 mM stock solutions of carotenoids are prepared. Under these conditions, solutions of canthaxanthin can be kept for at least 1 month, and most carotenoids can be stored for up to 2 weeks without degradation. Lycopene should be kept for only 1 week under these conditions. 15 C. A. Reznikoff, J. S. Bertram, D. W. Brankow, and C. Heidelberger, Cancer Res. 33, 2339 (1973). ~6j. S. Bertram, A. Pung, M. Churley, T. J. I. Kappock, L. R. Wilkins, and R. V. Cooney, Carcinogenesis (London) 12, 671 (1991). 17 R. V. Cooney and J. S. Bertram, this series, Vol. 214, p. 55.
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Cultures should be mildly agitated after addition of the drugs. Readers are strongly recommended to consult Cooney and Bertram 17 for details of the preparation and handling of carotenoid solutions.
Measurement of Gap Junctional Communication by Dye Microinjection To minimize interexperimental variations cells should be confluent prior to treatment with carotenoids and should have an identical history of plating and refeeding. In particular, increasing age of the cultures results in progressively increasing communication, whereas recent (within 24-48 hr) refeeding causes decreases in communication. Cells are injected with the fluorescent dye Lucifer Yellow CH (LH) (Sigma, St. Louis, MO), which has been widely utilized for the measurement of gap junctional communication. LH is a substituted naphthalimide with two sulfonate groups (Mr 472.2). It has found wide usage in the measurement of gap junctional communication because (1) it is water soluble and sufficiently small to be able to travel through gap junctions but, since it is charged, cannot diffuse through the surface of cell membranes; (2) it does not become protein-bound and thus remains mobile; (3) its excitation and fluorescence spectra are similar to fluorescein and thus can be visualized by the usual fluorescence optics; and (4) it is nontoxic at useful concentrations. Junctional permeability is assayed by microinjection of LH into a single cell with the aid of an Eppendorf microinjector. Micropipettes are prepared from glass capillary tubes with a Flaming-Brown micropipette puller, Model P-80/PC (Sutter Instrument Co., San Rafael, CA). The correct parameters for pulling a good micropipette needle must be determined empirically, since each heating filament and each batch of glass tubes require individual settings. Once determined the parameters can be stored in the electronic memory of the instrument. The puller allows for up to 10 programs to be stored. As a guide we use tubes of borosilicate glass (outer diameter 1 mm, inner diameter 0.78 mm) from Sutter Instruments. The tip after pulling should have a diameter of about 0.2/~m with an acute taper. These parameters result in a robust, fairly rigid pipette which causes a rapidly sealing puncture in the cell membrane. The micropipette is backfilled with a few microliters of 10% Lucifer Yellow solution in 0.33 M LiC1 through a 10-/~1glass syringe, then attached to a Zeiss micromanipulator connected to an Eppendorf microinjector. The fluorescent dye is microinjected under consistent pressure of Nz into the perinuclear region a single cell identified under phase-contrast or Nomarsky optics. A final magnification of at least 200 × is required. Injection pressures must be determined empirically.
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We normally inject 15 to 20 cells in a line across a dish over a 10-rain time period; at the end of this time the first injected cell is relocated under fluorescence optics and the image recorded on videotape. All cells are similarly recorded in sequence over a 10-min interval. The total number of fluorescent cells adjacent to each dye-injected cell is then determined and serves as an index of gap junctional communication. Measurements of gap junctional communication are performed in duplicate cultures for each treatment. Figures 1 and 2A,B show the induction of gap junctional communication by carotenoids using this method.
Immunofluorescent Detection of Gap Junctional Plaques Cells are seeded on Permanox plastic slides (Nunc Inc., Naperville, 1L) for fluorescent microscopy. Conventional plastic dishes will themselves fluoresce and interfere with the fluorescein isothiocyanate (FITC) signal, whereas cells grown on glass have a different morphology from those grown on plastic. Treatment with carotenoids is carried out as described above. After treatment, the slides are briefly washed in 37° PBS + [phosphate-buffered 40-
-~ C)
321
t--
h-~
24-
O c-
E E
o o
16 g
0
0 0
7
14
21
28
Days of T r e a t m e n t FIG. 1. Carotenoid effects on gap junctional communication in 10T1/2 cells. The cells were seeded at 1000 cells/60-mm dish and treated with 10 -s M carotenoids dissolved in THF when confluent on the seventh day and weekly thereafter. Junctional communication was indexed as the number of cells to which Lucifer Yellow was transferred within 10 min of injection into a test cell. Data points are the means -+ SE of 15 microinjections performed in two dishes each with the following carotenoids: Q, 0.5% THF control; V, fl-carotene; ¢,, canthaxanthin; II, lutein; A, lycopene. (From Zhang e t al., s with permission.)
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saline (PBS) as defined below, plus 0.01% MgSO4 and 0.01% CaCI 2] and fixed with cold methanol ( - 20°). If slides are to be stored they should be first transferred to room temperature methanol before drying. If they are to be immediately processed they should be hydrated for 10 min in cold PBS (0.8% NaC1, 0.115% Na2HPO 4, 0.02% KH2PO4, and 0.02% KC1), followed by blocking for 1 hr in PBS containing 5% bovine serum albumin (BSA) and 0.2% sodium azide at 4 °. The primary antibody is then applied diluted 1 : 30 with the blocking solution. We use a polyclonal antiserum raised in rabbits by immunization against a synthetic polypeptide corresponding to the C-terminal region (residues 368-382) of the predicted sequence of connexin 43.14 Slides are incubated at 4 ° for 60 min in a humidified chamber, then rinsed with cold PBS and washed for I hr in high salt PBS (0.5 M NaC1 in PBS) followed by a final wash in PBS. FITC-conjugated second antibody (goat anti-rabbit IgG F(ab)z fragment (Sigma) is diluted 1 : 40 in blocking solution, placed on the slides, and incubated for 60 min at 4 °. Slides are washed as above and mounted under coverslips using 0.233 g 1,4-diazobicyclo[2.2.2]octane (DABCO), 800/zl water, 200 ~1 1 M Tris-HC1, pH 8.0, and 9.0 ml glycerol to retard photobleaching (P. Lichter, personal communication, 1992). The slides are finally sealed with clear nail polish. Immunofluorescent microscopy of the slides is performed using a Zeiss Axioplan microscope (Thornwood, NY). Gap junctions are characterized as fluorescent plaques in junctional regions of the cell membrane (Fig. 2).
Western Blot Analysis of Connexin Proteins Isolation of Membrane Proteins. Cultures of 10T1/2 cells are grown in 150-mm dishes and treated with carotenoids as described above. After removal of culture medium dishes are washed with ice-cold PBS containing I mM NaF and 1 mM phenylmethylsulfonyl fluoride (PMSF, dissolved in ethanol), and then cells are scraped from the dish with a rubber policeman into 10 ml of the same solution. After centrifugation at 3000 rpm for 10
FIG. 2. Increased gap junctional communication is accompanied by an increased number and size of immunofluorescent plaques in regions of cell-cell contact. Confluent cultures were treated with 10 -5 M canthaxanthin for 7 days. (A, B) Photomicrographs of dye transfer. (A) Solvent control; (B) canthaxanthin. Junctional permeability was assayed by microinjection of the fluorescent dye Lucifer Yellow CH (10% in 0.33 M LiC1) into a single cell as described. 21(C-F) Immunofluorescent detection of junctional plaques induced by canthaxanthin. (C, E) Control, fluorescent and respective phase-contrast image; (D, F) canthaxanthintreated cells, fluorescent and respective phase-contrast image. 10T1/2 cells were seeded on Permanox plastic slides and treated as in Fig. 3A,B. Arrows indicate junctional plaques surrounding a single cell. (From Zhang et al., 9 with permission.)
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min, cell pellets are either frozen at - 7 0 ° (up to 2 weeks) or lysed in 30-50/xl of lysis buffer [1% Tergitol NP-40, 50 mM iodoacetamide, 10 mM PMSF, 1 mM EDTA, 1 p.M leupeptin, 2/zg/ml aprotinin, 0.7 ~g/ml pepstatin in borate buffer] for 1 hr at 4 °. Lysates are then centrifuged at 10,000 g for 15 min at 4 ° and the supernatant assayed by Western blotting for connexin 43. Prior to electrophoresis, the protein content is assayed using the BCA Protein Assay Reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Protein Electrophoresis and Western Blotting. Protein samples are separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli. ~8 Cell lysates containing equal amounts of protein (30-60 ~g/lane) are mixed well with 2 x SDS sample buffer containing 10% of 2-mercaptoethanol. After at least 15 min of reaction at room temperature without boiling (the samples must be reduced with 2-mercaptoethanol but not heated~4), proteins are electrophoresed on a 10% SDS-polyacrylamide minigel (Bio-Rad, Richmond, CA) at 200 V. Prestained SDS-PAGE protein standards (Bio-Rad) are also loaded for the indication of both molecular weight and transference efficiency. After electrophoresis, the proteins in the gel are transferred to an Immobilon membrane (Millipore, Bedford, MA) at 100 V and 4 ° with stirring for 35-40 min. After transfer, the gel can be stained with 0.25% Coomassie blue R solution [methanol-glacial acetic acid-water, 50 : 10 : 40 (v/v)] and destained with 5% methanol/9% acetic acid for checking protein transference. Detection of Connexin 43. After Western blotting, the membrane is blocked with blotto for 1 hr or overnight at 4° and then reacted with the same primary anti-Cx43 antibody as used in the immunofluorescence studies diluted 1 : 200 in blotto-borate buffer (1 : 2, v/v) with gentle shaking. (blotto is 5% nonfat dry milk in PBS; borate buffer contains 1.03% boric acid, 0.785% NaCI, and 0.11% NaC1, pH 8.0). After incubation, the blot is washed with excess borate buffer for 1 hr with 3-4 changes of the buffer. ~25I-labeled protein A is diluted 1:2 in blotto-borate buffer to achieve a concentration of 106 cpm/I 0 ml of solution and the blot incubated with this solution for 1 hr at room temperature with shaking, followed by a 1-hr wash in borate buffer with three changes of washing buffer. The blot is then gently pressed against absorbent paper to remove excess liquid, wrapped in plastic film, and exposed to Kodak (Rochester, NY) X-Omat AR film with double intensifying screens at - 7 0 ° for 2-3 days. Figure 3 demonstrates the use of this method to detect induction of Cx43 by carotenoids. I8 U. K. Laemmli, Nature (London) 227, 680 (1970).
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R E G U L A T I O N OF GAP J U N C T I O N A L C O M M U N I C A T I O N
A
1
234
0
1
5
6
4
7
243
78
Cx43 B
2
3
Cx43 treatment (days)
Cx43 0 0.31.03.010 canthaxanthin (uM) FIG. 3. Induction of Cx43 in 10T1/2 cells by carotenoids and the synthetic retinoid tetrahydrotetramethylnapthalenylpropenylbenzoic acid Hoffmann-La Roche (Nutley, NJ) (TTNPB) but not ~-tocopherol. (A) Structure-activity studies. Cultures were treated with 10-8 M TTNPB for 3 days, 10-5 M fl-carotene or canthaxanthin for 7 days, or 10-5 M lycopene or a-tocopherol for 7 days with retreatment every 3 days. Lane 1, Solvent control; 2, a-tocopherol; 3, canthaxanthin; 4, lycopene; 5, fl-carotene; 6, TTNPB. Lanes 7 and 8 show results of a separate experiment in which cultures received solvent control (lane 7) or 10-5 M methylbixin for 7 days (lane 8). (B) Time course. Cultures were treated with 10-5 M canthaxanthin and analyzed at the indicated times. (C) Dose-response. Cultures were treated with the stated concentrations of canthaxanthin for 7 days then analyzed. (From Zhang e t al., 9 with permission.)
Chemiluminescent Detection. As an alternative detection m e t h o d , we have f o u n d that a c o m m e r c i a l n o n i s o t o p i c kit, W e s t e r n - L i g h t Chemilumin e s c e n t D e t e c t i o n S y s t e m ( T R O P I X , B e d f o r d , MA), is m o r e sensitive and c o n v e n i e n t than the a b o v e m e t h o d o f radioactive detection. Using the kit, only a few minutes is required to identify i m m u n o r e a c t i v e proteins immobilized on m e m b r a n e s , in c o n t r a s t to the 2 or 3 d a y s n e e d e d for the isotopic detection m e t h o d . A f t e r processing, the m e m b r a n e should be stained for a b o u t 10 min with 0.25% C o o m a s s i e R solution as used for gel staining, destained with 50% m e t h a n o l / 2 5 % acetic acid, and p h o t o g r a p h e d . This serves as an internal c o n t r o l for normalizing for e a c h sample the a m o u n t o f total proteins loaded and blotted.
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Analysis and Quantitation. Analysis and quantification can best be carried out using digital image analysis which allows the subtraction of background and the direct comparison of treated and control bands. We use the National Institutes of Health Image program, a public domain program written for the Macintosh.
[20] O n e - D a y N o r t h e r n Blotting for D e t e c t i o n of m R N A : N D G A Inhibits t h e I n d u c t i o n of M n S O D m R N A b y Agonists of T y p e 1 T N F R e c e p t o r
By GRACE H. W. WONG and DAVm V. GOEDDEL Introduction Production of tumor necrosis factor (TNF) by cells is transient, tightly regulated, and can be triggered by infection and by oxidative stress. 1 In addition to its originally defined tumoricidal activity, TNF has been shown to act on many cell types and mediate pleiotropic activities2 including protection against various types of oxidative stress. 3 TNF also induces the expression of manganous superoxide dismutase (MnSOD), an enzyme that scavenges superoxide radical (02 ~) in mitochondria.4 The induction of MnSOD expression occurs at the transcriptional level and requires very low levels of TNF (0.1 ng/ml). The induction of MnSOD mRNA is rapid (less than 1 hr), substantial (more than 10-fold), and direct because it can be blocked by actinomycin D but not by cycloheximide.5 This chapter describes a 1-day Northern blotting method to examine the regulation of MnSOD mRNA. B. Beutler, ed., "Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine." Raven, New York, 1992. 2 W. Fiers, F E B S Lett. 285, 199 (1991). 3 G. H. W. Wong, A. Kamb, L. A. Tartaglia, and D. V. Goeddel, "Molecular Biology of Free Radical Scavenging Systems," pp. 69-96. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1992. 4 G. H. W. Wong and D. V. Goeddel, Science 242, 941 (1988). 5 G. H. W. Wong, A. Kamb, J. H. Elwell, L. W. Oberley, and D. V. Goeddel, in "Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine" (B. Beutler, ed.), p. 473. Raven, New York, 1992.
METHODSIN ENZYMOLOGY,VOL. 234
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[20]
Analysis and Quantitation. Analysis and quantification can best be carried out using digital image analysis which allows the subtraction of background and the direct comparison of treated and control bands. We use the National Institutes of Health Image program, a public domain program written for the Macintosh.
[20] O n e - D a y N o r t h e r n Blotting for D e t e c t i o n o f m R N A : N D G A Inhibits t h e I n d u c t i o n of M n S O D m R N A b y Agonists of T y p e 1 T N F Receptor
By GRACE H. W. WONG and DAVID V. GOEDD~L Introduction Production of tumor necrosis factor (TNF) by cells is transient, tightly regulated, and can be triggered by infection and by oxidative stress. ~ In addition to its originally defined tumoricidal activity, TNF has been shown to act on many cell types and mediate pleiotropic activities2 including protection against various types of oxidative stress. 3 TNF also induces the expression of manganous superoxide dismutase (MnSOD), an enzyme that scavenges superoxide radical (02:) in mitochondria.4 The induction of MnSOD expression occurs at the transcriptional level and requires very low levels of TNF (0.1 ng/ml). The induction of MnSOD mRNA is rapid (less than 1 hr), substantial (more than 10-fold), and direct because it can be blocked by actinomycin D but not by cycloheximide.5 This chapter describes a 1-day Northern blotting method to examine the regulation of MnSOD mRNA. B. Beutler, ed., "Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine." Raven, New York, 1992. 2 W. Fiers, FEBS Lett. 285, 199 (1991). 3 G. H. W. Wong, A. Kamb, L. A. Tartaglia, and D. V. Goeddel, "Molecular Biology of Free Radical Scavenging Systems," pp. 69-96. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1992. 4 G. H. W. Wong and D. V. Goeddel, Science 242, 941 (1988). 5 G. H. W. Wong, A. Kamb, J. H. Elwell, L. W. Oberley, and D. V. Goeddel, in "Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine" (B. Beutler, ed.), p. 473. Raven, New York, 1992.
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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NORTHERN BLOTTING FOR DETECTION OF m R N A
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One-Day Northern Blotting A. Isolation of Total Cytoplasmic RNA 1. Harvest adherent cultured cells either by trypsinization or with a cell scraper. Transfer cells (106-108) to an autoclaved siliconized l-ml Eppendorf tube and wash once with cold phosphate-buffered saline (PBS). Autoclave tubes, tips, water, and all buffers before use. Wear plastic gloves throughout. 2. Fast centrifuge the cells at room temperature (12,000 rpm, 5 sec). To avoid cell death, never centrifuge for longer than 20 sec. 3. Remove the supernatant and keep the cell pellet on ice. 4. Add 300/zl ice-cold lysis buffer A [10 mM Tris-HC1, pH 7.5, 0.1 M NaC1, 5 mM MgCI2, 0.5% Nonidet P-40 (NP-40)] containing 10 mM of the RNase inhibitor vanadyl-ribonucleoside complexes (VRC, from BioRad, Richmond, CA). 5. Vortex (maximum speed on a bench-top mixer) and incubate on ice for 30 sec (never more than 2 min; otherwise the buffer will lyse the nuclear membrane and the cytoplasmic RNA will be contaminated with DNA and nuclear RNA). 6. Centrifuge for 1 min to pellet nuclei (save intact nuclei for DNA extraction); RNA is in the supernatant. 7. Add 100/zl sodium dodecyl sulfate (SDS) buffer B (10 mM TrisHCI, pH 7.5, 0.1 M NaC1, 5 mM EDTA, 0.1% SDS) containing fresh 0.1% 2-mercaptoethanol (2-ME) and 0.1% diethyl pyrocarbonate (DEPC, Sigma, St. Louis, MO) to the RNA-containing supernatant. Add 700/zl phenol-chloroform (50:50, v/v) and mix thoroughly. (The RNA can be stored in this mixture at - 2 0 ° without degradation for several years.) Precaution: When samples are removed from storage at - 2 0 °, open the cap immediately to avoid explosion during warming and add 10/zl of a mixture of 2-ME and DEPC to prevent RNA degradation. Work in a fume hood and wear protective clothing. 8. Extract the RNA with 700/zl phenol-chloroform at least twice. [Phenol is equilibrated with water, 8-hydroxyquinoline (0.1%), and 2-ME (0. I%) to avoid oxidation and also provide yellow color indication.] If no aqueous phase can be recovered, remove the organic phase (yellow layer) from the bottom and extract with chloroform alone to obtain a clear aqueous phase and proceed. 9. Precipitate the RNA with 800/zl of 95% ethanol and 30/zl of 3 M sodium acetate (pH 7.5) at - 2 0 ° for 20 rain. 10. Collect the RNA pellet by microcentrifugation (15 rain, 14,000 rpm, cold room).
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1 I. Rinse the pellet with cold 75% ethanol and drain dry. 12. Redissolve the pellet in 100 /zl water containing 0.1% sarkosyl (sodium N-lauroylsarcosinate, 30%, from Pfalz and Bauer Inc., Waterbury, CT). 13. Dilute 5/~1 in 500/~1 water to determine A260 and A280. Check the quality of the RNA by analytical electrophoresis on a vertical 1.2% agarose formaldehyde gel before preparation of poly(A) + RNA. Store the RNA at - 2 0 ° until use.
B. Monitoring Quality of RNA by Vertical Agarose Formaldehyde Gel Electrophoresis 1. Prepare running gel buffer 1 × MEN, pH 7.5 [10 x MEN is 200 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM sodium acetate, 10 mM EDTA; the 10 x MEN buffer will be yellow after autoclaving]. 2. Prepare the 1.2% agarose gel (1.8 g SeaKern HGT agarose (FMC BioProducts, Rockland, ME), 15 ml of 10 × MEN, 110 ml water). 3. Microwave for 3 rain to melt the agarose. 4. Heat the taped vertical gel plates in an oven (80°) for 5 min (because agarose solidifies quickly on cold plates). 5. Add 25 ml formaldehyde to the melted agarose (the temperature drops to approximately 70 °) and prepare to pour the gel (do not wait for it to cool). 6. Transfer the agarose mix into a 50-ml blue-cap tube and pour into the vertical gel plates carefully (without bubbles). 7. Insert a gel comb into the agarose and let it solidify for 30 min inside a fume hood. 8. Denature 5 ~g RNA at 65 ° for 10 rain using 15 p~lof RNA denaturing buffer per sample (stock contains 1000/A formamide, 200/A of 10 × MEN, 320/~1 formaldehyde, and 480/A water). The RNA in the denaturing buffer can be stored a t - 20° without degradation for at least I year, and the denaturing buffer is good for at least 6 months. 9. Remove the gel comb very carefully or the wells may break. Remove the comb as slowly as possible and constantly apply water into each well and make sure the binder clips are off before bending the comb forward and backward. 10. Always prerun the gel to ensure that the buffer and the gel are working before loading the RNA samples. 11. Add 3 /~1 of loading dye [50% glycerol, 1 mM EDTA (pH 8.0), 0.25% xylene cyanol FF] to each sample (15/~1); spin, mix, and carefully load the sample into the vertical gel. 12. Run the gel at a constant voltage of 150 V for 30 min to complete.
['90]
NORTHERN BLOTTING FOR DETECTION OF m R N A
247
13. Detect the RNA by UV shadowing without staining the gel. Wrap the gel with Saran wrap and place the gel on a fluorescence precoated silica gel thin-layer chromatography (TLC) plate (Plate 60 F254 from Merck, Darmstadt, Germany, or E. M. Science, Cherry Hill, N J). Hold the UV lamp over the wrapped gel, and two sharp bands should appear, the 28 S and 18 S ribosomal RNA. I f R N A is intact, then proceed to make poly(A) ÷ RNA. If a smear of degraded RNA appears, stop and discard the RNA samples. The quality of RNA samples can also be checked even before the phenol-chloroform extraction. Spin the mixture of RNA and phenol-chloroform from Step A.7 (14,000 rpm, 3 min, room temperature (RT)), and mix 5/zl of RNA-containing supernatant (clear top phase; phenol is in the yellow bottom layer) with 10/xl denaturing buffer (from Step B.8) to denature the RNA at 65 ° for I0 min. Run samples on the gel and check the RNA by UV shadowing as described in steps B.8 to B. 13. C. Isolation o f Poly(A) ÷ R N A Preparation of Oligo(dT)-Cellulose 1. Dissolve 1 g of oligo(dT)-cellulose (type III, Collaborative Research, Bedford, MA) in 50 ml water and let swell for 3 min in a 50 ml screw-cap tube. 2. Spin (tabletop centrifuge; 1000 rpm for 10 sec at RT) and remove the water. 3. Clean the oligo(dT)-cellulose with 0.1 M NaOH (50 ml) by inverting and mixing thoroughly for 10 sec. 4. Centrifuge (1000 krpm, 10 sec at RT) to remove the supernatant. 5. Wash once with sterile autoclaved water and centrifuge to remove the supernatant. 6. Wash once with 50 ml of 10 x oligo(dT) buffer (4 M NaC1, 0.1 M Tris, pH 7.5, 10 mM EDTA). 7. Centrifuge (1000 rpm, 10 sec, RT); remove the supernatant. 8. Wash with 50 ml of 1 x oligo(dT) buffer until the pH is neutral (approximately pH 7.5), testing with pH paper. 9. Centrifuge (1000 rpm, 10 sec, RT) to remove the supernatant. 10. Store the oligo(dT)-cellulose pellet at - 2 0 ° until use. Enrichment ofPoly(A) ÷RNA. Always check the quality of RNA before the preparation of poly(A) ÷ RNA by analyzing 5/xl of total RNA on a vertical 1.2% agarose formaldehyde gel as described in Section B. 1. Dissolve the RNA in 300/zl water and incubate at 65 ° for 10 min in order to denature. Transfer to ice. 2. Add 30/zl sarkosyl (sarkosyl is preferable to SDS because sarkosyl does not precipitate at 0 °) to inactivate RNase.
248
ASSAY OF STRESS GENES/PROTEINS
[20]
3. Add 30 ~I of 10 x oligo(dT) buffer. 4. Dissolve the RNase-free oligo(dT)-cellulose pellet in 15 ml of I x oligo(dT) buffer. 5. Take 300 tzl of this mixture and add to the RNA samples (from Step 3). 6. Shake at room temperature in a capped Eppendorf tube for at least 10 min to ensure that the poly(A) ÷ RNA has bound to the oligo(dT)-cellulose. 7. Centrifuge (14,000 rpm, 1 min, RT) to separate the poly(A)- RNA in the supernatant. Save the supernatant until certain that the poly(A) ÷ RNA has been recovered. 8. Rinse the pellet with 1 x oligo(dT) buffer. 9. Elute the poly(A) + RNA by adding 300/zl water containing 0.1% sarkosyl to the pellet; mix thoroughly and incubate at 65 ° for 30 sec. 10. Mix, invert, and centrifuge (14,000 rpm, 1 min, RT). 11. Precipitate the poly(A) ÷ RNA from the supernatant with 800/zl ethanol (95%) and 30/zl sodium acetate (3 M, pH 7.5) at - 20° for 20 min. 12. Centrifuge (14,000 rpm, 10 min, 4 °) and carefully remove the supernatant. 13. Wash the pellet with cold 75% ethanol. 14. Dry and redissolve the poly(A)+ RNA in 20/xl water containing 0.1% sarkosyl and store the poly(A) + RNA at - 2 0 ° until use.
D. Hybridization with Radioactive D N A Probes I. Check the quality of the poly(A) + RNA by UV shadowing as described in Step B. 13. Two sharp bands should be seen indicating that 28 S and 18 S ribosomal RNA remaining in the poly(A) + RNA preparation. The ribosomal RNAs are coprecipitated and usually a sign that goodquality poly(A) + RNA has been made. 2. Transfer the RNA from the thin gel onto a nitrocellulose membrane filter using 20 x SSC. Complete transfer takes 20 rain. The 28 S and 18 S ribosomal RNAs will disappear from the gel after transfer (check with the UV shadowing technique). 3. Bake the filter in a vacuum oven at 80° for 10 min. 4. Prehybridize the filter at 42° for 10 rain in a hybridization box containing 50 ml prehybridization mix (see below). 5. Add the denatured 3ZP-labeled probe (I00 ° for 5 rain, then ice for 1 min) to the prehybridization mix (see below), add dextran sufate to 10%, and further hybridize for at least 3 hr at 42°. 6. Rinse the filter 3 times in 0.2 x SSC plus 0.1% SDS at room temperature for 1-2 min.
[20]
249
NORTHERN BLOTTING FOR DETECTION OF m R N A
7. Wash in 0.2 x SSC plus 0.1% SDS at 42° with constant shaking for 20 min. 8. Transfer the filter onto the blotting paper and wrap the filter with Saran wrap immediately (never let the filter completely dry). 9. Expose the filter to X-ray film at - 70° (normally, 10 min is sufficient to detect/3-actin signal and 1-3 hr is required to detect MnSOD mRNA signal if 3/zg of poly(A) + RNA is used per lane).
Prehybridization Mix 500ml 250ml 50ml 50 ml 10 ml I0 ml 10 ml 120 ml 1000ml
Formamide 20 x SSC (3 M NaC1, 0.3 M sodium citrate, pH 7.0) 100 x Denhardt's solution [2% (w/v) each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin (BSA)] 1 M Sodium phosphate, pH 6.5 3 M Tris-HC1, pH 8.5 SDS (10%) Salmon sperm DNA (Sigma) (10 mg/ml, boiled for 5 min then chilled in ice) Water (pH8.0-8.5)
E. Labeling Overlapping Oligonucleotides by Filling in with 32P-Labeled Nucleotides I. Add 1 ~1 of each oligonucleotide (concentration of 2.5OD260/ml) to 35 ~lwater.
,
A ,
3,111/I/3' B
Sequence of MnSOD 66-mer A: 5'-GGG GAG TTG CTG GAA GCC ATC AAA CGT GAC TTT GGT TCC TTT GAC AAG TTT AAG GAG AAG CTG ACG-3' Sequence of MnSOD 63-mer B: 5'-GAA ACC AAG CCA ACC CCA ACC TGA GCC TTG GAC ACC AAC AGA TGC AGC CGT CAG CTT CTC CTT-3' /3-Actin sequence (45-mer) A: 5'-GCC TCT GGC CGT ACC ACT GGC ATC GTG TAG GAC TCC GGT GAC GGG-3'
250
ASSAY OF STRESS GENES/PROTEINS
[20]
fl-Actin sequence (45-mer) B: 5'-CCC CTC GTA GAT GGG CAC AGT GTG GGT GAC CCC GTC ACC GGA GTC-3' The underlined regions of sequences A and B are complementary. 2. Add 5/zl of 10x Klenow polymerase buffer (0.5 M Tris-Cl, 0.1 M MgCI2, pH 7.5). 3. Incubate at 55 ° for 10 min. 4. Further incubate at 25 ° for 10 rain, then put on ice. 5. Add 3/zl of dGTP (2 mM) and 3/zl of dTTP (2 mM). 6. Add 10/xl of a-a2p-labeled dATP (Amersham, Arlington Heights, IL, 10204, 10 mCi/ml) and 10/zl of ot-32p-labeled dCTP (Amersham 10205, 10 mCi/ml). 7. Add 2 units of Klenow polymerase. 8. Incubate for 15 min at 25°. 9. Further incubate for 15 min at 37°. 10. Add 200/zl water and ethanol precipitate with 30/xl sodium acetate (3 M), 700/zl ethanol (95%), and 1 ~1 of carrier tRNA or DNA (10 mg/ml), or purify the probe with a Bio-Spin chromatography column (Bio-Rad). 11. Redissolve the DNA probe in 50 ~1 water. The probe should contain at least about 108 cpm and is ready for use (use the entire probe per 50 ml in a hybridization box). Store the labeled probe at 4°; it may be reused for at least 3-4 weeks (by reheating to 70 ° for 5 min and chilling in ice for 3 min). Example
Figure 1 shows an example of Northern blot analysis using the 1-day procedure. 6-9 Both MnSOD and fl-actin probes were used. Agonist antibodies against the type 1 TNF receptor (TNF-R1) but not the type 2 TNF receptor (TNF-R2) were found to induce high levels of MnSOD mRNA, whereas/3-actin mRNA remained unchanged. Thus, TNF induces MnSOD expression by activation of TNF-R1 and is not involved in intracellular events per se. The intracellular mediators of the activation signal are unknown. Surprisingly, induction of MnSOD by both TNF and the antiTNF-R1 antibodies can be blocked by nordihydroguaiaretic acid (NDGA), 6 y . Beck, R. Oren, B. Arnit, A. Levanon, M. Gorecki, and J. R. Hartman, Nucleic Acids Res. 15, 9076 (1987). 7 p. Ponte, S. Y. Ng, J. Engel, P. Gunning, and L. Kedes, Nucleic Acids Res. 12, 1687 (1984). 8 G. H. W. Wong, L. A. Tartaglia, M. S. Lee, and D. V. Goeddel, J. Immunol. 149, 3350 (1992). 9 L. A. Tartaglia and D. V. Goeddel, J. Biol. Chem. 267, 4304 (1992).
[20]
NORTHERN BLOTTING FOR DETECTION OF m R N A
251
fl I
8
II
I
.,,, -
- M n S O D (4kb)
I~-Actin-- m - M n S O D (lkb) 1 2 3 4 5 6 7 8 FIG. 1. Nordihyguaiaretic acid (NDGA) but not indomethacin (INDO) inhibits the induction of MnSOD mRNA by TNF and by anti-TNF-Rl antibodies. Confluent cultures of A549 human lung carcinoma cells were treated with TNF-a (0.1 /xg/ml) or with a 1/100 dilution of polyclonal antibodies against TNF-R1 or TNF-R2 for 1 hr at 4°, washed three times with medium, and further incubated with or without 10 mM of either NDGA or INDO for 12 hr. Poly(A) ÷ RNA (3/zg/lane) was hybridized to both 32p-labeled MnSOD and/3-actin probes. 6-8 The specific activity of human TNF-~ is 4 × 107 units/rag, and the rabbit antisera against human TNF-R1 or TNF-R2 have been described previously. 9-j°
an inhibitor of lipooxygenase. Indomethacin (INDO), an inhibitor of cyclooxygenase, does not block the induction (Fig. 1). These results suggest that leukotrienes or some other product(s) of the lipoxygenase pathway may mediate the induction of MnSOD mRNA by TNF. The 1-day Northern blotting method can be used to isolate pure and undegraded poly(A) + RNA from a large number of samples for hybridization with various DNA probes. The method is simple, inexpensive, rapid, and reliable and is suitable for gene regulation studies using cultured cells. Enrichment of poly(A) + RNA by the batch method in an Eppendorf tube allows one to enrich a large sample of poly(A) + RNA within a short time. This I-day Northern blotting method is not suitable for isolating RNA from tissues. The intact nuclei isolated during the preparation of cyto-
252
ASSAY OF STRESS G E N E S / P R O T E I N S
[21]
plasmic R N A can also be used for nuclear runon/runoff experiments and for isolating D N A . The U V shadowing m e t h o d can be conveniently used to detect and p h o t o g r a p h 28 S and 18 S R N A without staining or other manipulation of the gel. Acknowledgments We thank the manufacturing group at Genentech (San Francisco, CA) for providing pure recombinant human TNF-a; Greg Bennett for preparation of rabbit anti-TNF-R1 and antiTNF-R2 antibodies; Louis Tamayo for art work; Tracey Rivas and Sunita Sohrabji for help in preparing the manuscript; and Drs. Alan Harris, Susanne Baumhueter, and Louis Tartaglia for critical comments. G.H.W.W. thanks Dr. Linus Pauling, Linda Pauling-Kamb, and Alexander David Kamb for support.
[21] E v a l u a t i o n
of Biomolecular
Damage
by Ozone
B y CARROLL E. CROSS and BARRY HALLIWELL
Introduction O z o n e (O3), an important toxic c o m p o n e n t of photochemical air pollution, is believed to exert its toxic effects via its strong oxidizing capacity. 1-4 Although its pathophysiological effects on the respiratory tract have been extensively studied, 1,5 and m u c h is k n o w n concerning its chemical reactivity with a variety of biological molecules studied in i s o l a t i o n , 2-4,6-8 the precise biomolecular m e c h a n i s m s of inhaled 03 toxicity are not fully understood. The respiratory tract lining fluids ( R T L F s ) represent the first biological substances coming into contact with inhaled 03. As such, constituents of the R T L F s m a y well represent an important first line of respiratory tract defense, p r e s u m a b l y b y absorbing, reacting with, and detoxifying 03. Indeed, it has been suggested that, at concentrations expected to be present in polluted air, inhaled 03 and NO2 will react p r i m a r i l y with the oxidizable biomolecules present in the R T L F s and that these inhaled I D. B. Menzel, 13, 183 (1984). 2 M. A. Mehlman and C. Borek, Environ. Res. 42, 36 (1987). 3 M. G. Mustafa, Free Radical Biol. Med. 9, 245 (1990). 4 W. A. Pryor, Am. J. Clin. Nutr. 53, 702 (1991). 5 M. Lippmann, J. Air Pollut. Control Assoc. 39, 692 (1989). 6 j. B. Mudd, R. Leavitt, A. Ongun, and T. T. McManus, Atmos. Environ. 3, 669 (1969). 7 R. S. Oosting, M. M. J. Van Greevenbroek, J. Verheof, L. M. G. Van Golde, and H. P. Haagsman, Am. J. Physiol. 261, L77 (1991). 8 W. A. Pryor, B. Das, and D. F. Church, Chem. Res. Toaicol. 4, 341 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
252
ASSAY OF STRESS G E N E S / P R O T E I N S
[21]
plasmic R N A can also be used for nuclear runon/runoff experiments and for isolating D N A . The U V shadowing m e t h o d can be conveniently used to detect and p h o t o g r a p h 28 S and 18 S R N A without staining or other manipulation of the gel. Acknowledgments We thank the manufacturing group at Genentech (San Francisco, CA) for providing pure recombinant human TNF-a; Greg Bennett for preparation of rabbit anti-TNF-R1 and antiTNF-R2 antibodies; Louis Tamayo for art work; Tracey Rivas and Sunita Sohrabji for help in preparing the manuscript; and Drs. Alan Harris, Susanne Baumhueter, and Louis Tartaglia for critical comments. G.H.W.W. thanks Dr. Linus Pauling, Linda Pauling-Kamb, and Alexander David Kamb for support.
[21] E v a l u a t i o n
of Biomolecular
Damage
by Ozone
B y CARROLL E. CROSS and BARRY HALLIWELL
Introduction O z o n e (O3), an important toxic c o m p o n e n t of photochemical air pollution, is believed to exert its toxic effects via its strong oxidizing capacity. 1-4 Although its pathophysiological effects on the respiratory tract have been extensively studied, 1,5 and m u c h is k n o w n concerning its chemical reactivity with a variety of biological molecules studied in i s o l a t i o n , 2-4,6-8 the precise biomolecular m e c h a n i s m s of inhaled 03 toxicity are not fully understood. The respiratory tract lining fluids ( R T L F s ) represent the first biological substances coming into contact with inhaled 03. As such, constituents of the R T L F s m a y well represent an important first line of respiratory tract defense, p r e s u m a b l y b y absorbing, reacting with, and detoxifying 03. Indeed, it has been suggested that, at concentrations expected to be present in polluted air, inhaled 03 and NO2 will react p r i m a r i l y with the oxidizable biomolecules present in the R T L F s and that these inhaled I D. B. Menzel, 13, 183 (1984). 2 M. A. Mehlman and C. Borek, Environ. Res. 42, 36 (1987). 3 M. G. Mustafa, Free Radical Biol. Med. 9, 245 (1990). 4 W. A. Pryor, Am. J. Clin. Nutr. 53, 702 (1991). 5 M. Lippmann, J. Air Pollut. Control Assoc. 39, 692 (1989). 6 j. B. Mudd, R. Leavitt, A. Ongun, and T. T. McManus, Atmos. Environ. 3, 669 (1969). 7 R. S. Oosting, M. M. J. Van Greevenbroek, J. Verheof, L. M. G. Van Golde, and H. P. Haagsman, Am. J. Physiol. 261, L77 (1991). 8 W. A. Pryor, B. Das, and D. F. Church, Chem. Res. Toaicol. 4, 341 (1991).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
[21]
EVALUATION OF BIOMOLECULAR DAMAGE BY OZONE
253
gases are unlikely to reach the underlying respiratory tract epithelial cells (RTECs). 9-1° It follows that the toxicity of O3 to R T E C s may not be mediated by direct O 3 attack, but by cytotoxic products generated when 03 interacts with the R T L F s . Respiratory tract lining fluids are difficult to obtain and variable in composition. To examine the spectrum of reactions that can be expected to occur when complex biological fluids (such as R T L F s ) are exposed to 03, we have used plasma as a target. 11 Plasma is easier to obtain than R T L F s and contains a wide range of antioxidants (both aqueous and in the lipid phase). 12-14 Of course, it is not an ideal model (e.g., mucin and surfactant are missing). Injury to the lung, for example, by high concentrations of oxidants, however, can be expected to cause transudation of plasma constituents into the R T L F s , 15 that is, they become closer to plasma in composition, Indeed, it has been proposed 12 that this transudation, often used as a marker of injury, is also beneficial in that it will increase the total antioxidant capacity of the R T L F s . Hence, exposure of plasma to 03, followed by measurements of consumption of antioxidants in relation to oxidative protein modification and the appearance of lipid hydroperoxides,11 provides important clues in understanding 03 reactions with other complex biological fluids (such as RTLFs) and cell culture media. Often cells in culture are exposed to 03 without considering the reactions of 03 with constituents of the culture medium. Experimental Design Fresh heparinized human plasma (or other biological fluid or culture medium) is obtained, and aliquots are placed into open plastic Falcon dishes contained within a closed fully humidified chamber which is exposed to a constantly monitored and maintained level of O3 in 5% CO2/ 9 E. M. Postlethwait, S. D. Langford, and A. Bidani, Toxicol. Appl. Pharamcol. 109, 464 (1991). 9a E. M. Postlethwait, S. D. Langford, and A. Bidani, Toxicol. Appl. Pharamcol. 125, 77 (1994). i0 W. A. Pryor, Free Radical Biol. Med. 12, 83 (1992). 11C. E. C r o s s , P. A. Motchnik, B. A. Bruener, D. A. Jones, H. Kauf, B. N. Ames, and B. Halliwell, FEBS Lett. 298, 269 (1992). 12B. Halliwell, Biochem. Pharmacol. 34, 569 (1988). 13B. Halliwell and J. M. C. Gutteridge, Arch. Biochem. Biophys. 2~0, 1 (1990). 14B. Frei and B. N. Ames, in "Molecular Biology of Free Radical Scavenging Systems" (J. Scandalios, ed.), p. 23. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1992. 15C. G. A. Persson, I. Erjefalt, N, Alkner, C. Baumgarden, L. Greiff, B. Gustafsson, A. Luts, U. Pipkorn, F. Sundler, C. Svensson, and P. Wollmer, Clin. Exp. Allergy 21, 17 (1991).
254
ASSAY OF STRESS GENES/PROTEINS
[2 II
95% air at 37°, control plasma being exposed to an identical gas stream without O3.11'16'17 Our system is designed to maintain an ambient 03 concentration at a designated level by maintaining a flow to volume ratio of approximately 10/min, varying the 03 flow to maintain a constant 03 level in the chamber. However, the exposure system can be modified to monitor 03 "uptake" into the exposure solution by precise determinations of both intake and outflow 0 3 concentrations. ~6'17 The overall research strategy is to determine the oxidative changes in the medium of interest in relation to levels of 0 3 , time of 03 exposure, and depletions of antioxidants. Antioxidants that can be measured include those in the aqueous phase [ascorbic acid, uric acid, reduced glutathione (GSH), cysteine] and lipid-soluble antioxidants (ubiquinol, a-tocopherol, and fl-carotene and other carotenoids). Oxidative damage to proteins can be measured by several methods, including the carbonyl assay 18,19 and formation of fluorescent products from tyrosine, 2° including bityrosine. 21 Damage to lipids can be measured by a multiplicity of methods,22 especially useful being the high-performance liquid chromatography (HPLC)-based measurement of hydroperoxides with chemiluminescence detection. 23'24 In addition, products of free radical attack on antioxidants can be measured [e.g., dehydroascorbic acid, oxidized glutathione (GSSG), ubiquinone, allantoin and other degradation products of uric acid, 25 and a-tocopherol quinone]. • Figure 1 illustrates a typical experiment showing the loss of antioxidants and the occurrence of oxidative damage when fresh human plasma is exposed to 03.11.26 There is a rapid depletion of both uric acid and ascorbic acid, important aqueous antioxidants, whereas protein SH thiol groups are lost more slowly. By contrast, there is little change in the 16 D. C. Bolton, B. K. Tarkington, Y. C. Zhee, and J. W. Osebold, Environ. Res. 27, 466 (1982). 17 B. K. Tarkington, T. R. Duvall, and J. A. Last, this volume [22]. 18 R. L. Levine, D. Garland, C. N. Oliver, A. Amici, I. Climent, A. G. Lenz, B. W. Ahn, S. Shaltiel, and E. R. Stadtman, this series, Vol. 186, p. 464. 19 A. Z. Reznick, and L. Packer, this series, Vol. 233 [38]. 2o C. A. O'Neill, A. van der Vliet, M. L. Hu, H. Kaur, C. E. Cross, S. Louie and B. HaUiwell, J. Lab. Clin. Med. 122, 497 (1993). 21 C. Giulivi and K. J. A. Davies, J. Biol. Chem. 268, 8752 (1993). 22 B. Halliwell and S. Chirico, Am. J. Clin. Nutr. 57, 7155 (1993). 23 B. Frei, R. Stocker, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9748 (1988)• 24 C. E. Cross, T. Forte, R. Stocker, S. Louie, Y. Yamamoto, B. N. Ames, and B. Frei, J. Lab. Clin. Med. 115, 396 (1990). 25 H. Kaur and B. HaUiwell, Chem.-Biol. Interact. 73, 235 (1991). 26 C. E. Cross, A. Z. Reznick, L. Packer, P. A. Davis, Y. J. Suzuki, and B. Halliwell, Free Radical Res. Commun. 15, 347 (1991).
[21]
EVALUATION OF BIOMOLECULAR DAMAGE BY OZONE
255
"
o
".~_~
1 8o
O
~.)
~
60 4o
~e..,
20
•
0
0
1
2 3 Time (hours)
FIG. 1. Depletion of selected antioxidants and protein thiol groups in human plasma exposed to 16 ppm O3 .II Results are expressed as the percentage of antioxidants initially present corrected for loss in air-exposed controls. A, a-Tocopherol; II, protein thiols; ©, ascorbic acid; [2, uric acid.
major lipid-soluble antioxidant a-tocopherol. Unlike what is found with 03 exposures of isolated lipids, little lipid hydroperoxide formation could be demonstrated (< 1 /zM), and no shifts in lipoprotein electrophoretic mobility could be demonstrated. On the other hand, it was readily possible to demonstrate protein oxidation, as revealed by the protein thiol oxidation and by the increase in protein carbonyls (Fig. 2). 1.75 ¢X,
e~o
1.50
o
1.25'
r-.,
1.00" O
(..)
/
0.75 O
r.)
0.50 0
2
4
Time (Hours) FIo. 2. Oxidative damage to human plasma proteins exposed to 16 Plasma + air; II, plasma + ozone.
ppm
0 3 .26 A ,
256
ASSAY OF STRESS GENES/PROTEINS
[21]
Comments Experimental approaches designed to characterize the mechanisms of 03 reactions with constituents of the respiratory tract should ideally include a systematic evaluation of the entire spectrum of 03 reactions occurring in the RTLFs. The described methodology provides a strategy for evaluating the bioreactivity of 03 in an extracellular fluid that has many, but not all, of the constituents present in RTLFs. Extrapolation of our results to the in vivo situation should bear the following in mind: (1) the role of"reactive absorption" at the fluid surface9; (2) the packaging and antioxidant potentials of RTLF mucin 27-29and surfactant and its constituents3°-32; (3) the turnover of RTLF constituents; (4) the reactions of 0 3 to give other reactive oxygen species33,34; (5) the role of transudative/inflammatory processes, including production of reactive oxygen species by activated phagocytic cellslS; (6) the role of metal chelators such as ceruloplasmin, transferrin, and lactoferrin, 13,35known to be present in RTLFs36'37; (7) the role of antioxidant enzymes known to be present in RTLFs and presumably active in vivo, such as catalaseaS; and (8) the cytopathological effects of products of 03 reaction in the RTLFs on the underlying epithelial cells (e.g., such as effects on cytokine and eicosanoid production).
27 C. E. Cross, B. Halliwell, and A. Allen, Lancet 1, 1328 (1984). 28 M. B. Grisham, C. yon Ritter, B. F. Smith, J. T. LaMont, and D. N. Granger, Am. J. Physiol. 253, G93 (1991). 29 H. Hiraishi, A. Terano, S. Ota, H. Mutoh, T. Sugimoto, T. Harada, M. Razandi, and K. J. Ivey, J. Lab. Clin. Med. 121, 570 (1993). 30 S. Matalon, B. A. Holm, R. R. Baker, M. K. Whitfield, and B. A. Freeman, Biochim. Biophys. Acta 1035, 121 (1990). 31 B. Rustow, R. Haupt, P. A. Stevens, and D. Kunze, Am. J. Physiol. 265, L133 (1993). 32 G. E. Hatch, in "Treatise on Pulmonary Toxicology" (R. A. Parent, ed.), Vol. 1, p. 617. CRC Press, Boca Raton, Florida, 1992. 33 W. H. Glaze, Environ. Health Perspect. 69, 151 (1986). 34 j. R. Kanofsky and P. Sima, J. Biol. Chem. 266, 9039 (1991). 35 j. M. C. Gutteridge and B. HaUiwell, in "Atmospheric Oxidation and Antioxidants" (G. Scott, ed.), Vol. 3, pp. 71-99. Elsevier, Amsterdam, 1993. 36 E. R. Pacht and W. B. Davis, J. Appl. Physiol. 64, 2093 (1988). 37 W. B. Davis and E. R. Pacht, in "Lung Injury" (R. G. Crystal and J. B. West, eds.), p. 61. Raven Press, New York, 1992. 38 A. M. Cantin, G. A. Fells, R. C. Hubbard, and R. G. Crystal, J. Clin. Invest. 86, 962 (1990).
[22]
OZONE EXPOSURE OF CULTURED CELLS AND TISSUES
257
[22] O z o n e E x p o s u r e o f C u l t u r e d Cells a n d T i s s u e s By BRIAN K. TARKINGTON, TIMOTHY R. D U V A L L , a n d JEROLD A . LAST
Introduction The effects of oxidant air pollutants such as ozone (03) and nitrogen dioxide (NO2) on the respiratory tract have been studied using in vivo inhalation exposures, ~'2 organ culture or explant systems, 3,4 and cell culture systems. 5'6 With in vitro systems, a major problem is design of the exposure system to deliver constant and reproducible concentrations of ozone to replicate culture dishes, since ozone is so highly reactive chemically. The other challenge is to mimic in vivo exposure conditions where the luminal surfaces of respiratory epithelial cells are exposed almost directly to the inspired air, except for a mucous or surfactant layer of variable thickness. Exposure of organ or cell cultures through a stationary liquid layer is undesirable because relatively insoluble gases have little effect except at high concentrations. 7'8 In addition, the mechanism of action may be different if an oxidant first reacts with the components of a relatively thick liquid layer rather than reacting directly with the cell or its surface layer. 9 For the latter problem, a biphasic cell culture system of respiratory epithelial cells has recently been developed. 10In this biphasic culture, epithelial cells are maintained between air and the liquid medium so that a direct exposure of epithelial cells to ozone is feasible. In vitro systems for exposure of respiratory epithelial cells or explants to ozone have been developed and used successfully by several groups at the Air Pollution Exposure Facility of the California Regional Primate D. W. Wilson, C. G. Plopper, and D. L. Dungworth, Am. J. Pathol. 116, 193 (1984). 2 j. A. Last, in "Air Pollution, the Automobile and Public Health," (A. Y. Watson, R. R. Bates, and D. Kennedy, eds.), p. 415. National Academy Press, Washington, DC, 1988. 3 K. J. Nikula, D. W. Wilson, D. L. Dungworth, and C. G. Plopper, Toxicol. Appl. Pharmacol. 93, 394 (1988). 4 W. C. Eisenberg, K. Tyler, and L. J. Schiff, Experientia 40, 514 (1984). 5 R. E. Rasmussen, J. Toxicol. Environ. Health 13, 397 (1984). 6 D. C. Bolton, B. K. Tarkington, Y. C. Zee, and J. W. Osebold, Environ. Res. 27, 466 (1982). 7 D. M. Pace, P. A. LandoR, and B. T. Aftonomes, Arch. Environ. Health 18, 165 (1969). s W. L. Hagar, W. E. Sweet, and F. Sweet, J. Air Pollut. Control Assoc. Notebook 31, 993 (1981). 9 D. G. Wenzel and D. L. Morgan, Drug Chem. Toxicol. 5, 201 (1982). i0 j. M. Whitcutt, K. B. Adler, and R. Wu, In Vitro Cell. Dev. Biol. 24, 420 (1988).
METHODS IN ENZYMOLOGY,VOL. 234
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ASSAY OF STRESS GENES/PROTEINS
i
Oxyoe. ~v, I
A
B c,
-
H
r- I N
~
v6 D
[22]
~
II I
~
r~
N
L
L Carbon dioxide I FIG. 1. Schematic diagram of small in vitro exposure system. A, Ozonizer; B, ozone bypass valve; C, incubator; D, humidificationbottles; E, exposure vessel; F, control vessel; G, pressure gauges; H, ozone analyzer; I, thermometer; J, heating tape; K, thermometer; L, thermal insulation. (From Tarkington et al?2).
Research Center (University of California, Davis). 3,11-13 The systems are designed to generate and monitor consistent, reproducible levels of ozone o v e r a range of concentrations in a humidified atmosphere, allowing exposure times greater than those previously used by others .4,5 Several replicate culture vials or plates can be equally exposed to the experimental atmosphere, and the epithelial cells are exposed directly with only a thin aqueous film o v e r the epithelial surface. Control cells or explants may be simultaneously exposed to the experimental atmosphere without ozone.
Materials and Methods Small in Vitro Exposure System The atmospheres used are filtered air with 5% by volume carbon dioxide added, all saturated with water vapor and held at 37.5 °. A schematic diagram of the system is shown in Fig. 1. One vessel for ozone exposure and one control vessel are used. Most of the components of the system H K. J. Nikula and D. W. Wilson, Fundam. Appl. Toxicol. 15, 121 (1990). lz B. K. Tarkington, R. Wu, W. Sun, K. J. Nikula, D. W. Wilson, and J. A. Last, Toxicology 88, 112 (1994). 53 j. M. Cheek, A. R. Buckpitt, C. Li, B. K. Tarkington, and C. G. Plopper, Toxicol. Appl. Pharmacol. 125, 59 (1994).
[22]
OZONE EXPOSURE OF C U L T U R E D CELLS A N D TISSUES
259
with which the ozone comes in contact are made from Teflon. A few items are glass or type 316 stainless steel. To ensure very stable flows, three stages of pressure regulation are used on all gases. Oxygen, air, or carbon dioxide are introduced to the associated control valves at a constant pressure of 0.21 kg/cm 2. Valve V1 and rotameter R1 are used to deliver a flow of medical grade oxygen of 5 liters/min through the electrical discharge ozonizer (Model IV, Erwin Sander Elektroapparatebau G.m.b.H., Uetze-Eltze, Germany). This flow rate is used to provide adequate cooling of the ozonizer. With the unit set to maximum output, as much as 700 ppm ozone can be produced in the oxygen. After closure of the ozone bypass valve slightly, valve V2 and rotameter R2 are used to meter 50, 100, or 150 ml/min of the ozone in oxygen stream into the exposure portion of the system. Flow rate selection is dependent on the maximum ozone concentration desired. Finer adjustments to ozone production are electrical. Flow rates of 2.8, 2.75, or 2.7 liters/min of filtered air are added through valve V3 and rotameter R3. Dry 99.8% carbon dioxide is introduced through valve V4 and rotameter R4. The carbon dioxide flow rate is 150 ml/min. By adjusting the total flow rate of the air and oxygen streams to 2.85 liters/min, a 5% by volume carbon dioxide atmosphere is maintained in a total flow rate of 3 liters/min through the exposure vessel. This provides excess flow for the ozone analyzer (Model 1003-AH, Dasibi Environmental Corporation, Glendale, CA), which samples at 2 liters/rain and prevents recirculation of the sampled gas. Except for the lack of provisions for ozone introduction, the atmosphere stream for the control vessel is produced in the same way with the air flow rate of 2.85 liters/min controlled by valve V5 and rotameter R5, and the carbon dioxide flow rate of 150 ml/min controlled by valve V6 and rotameter R6. Lines 6.4 mm (1/4 inch) in diameter pass through a port in the incubator and convey the gas streams to the exposure vessel and control vessel. The gas streams are humidified by bubbling through 1000-ml bottles containing sterile distilled water. Production of complete saturation necessitates slight heating on the outside of the bottles. Heating mats with input controlled by ac power supplies are used, and the outside of the bottles are wrapped with thermal insulation. The humidified gases are then conveyed to 320ml (or l-liter)jars (Savillex Corporation, Minnetonka, MN) that serve as the exposure and control vessels. The jars can be mounted on a rocking platform if needed. The platform is used for tracheal explants so that they are regularly bathed with culture medium. Each vessel (Fig. 2) can contain five glass culture vials. Inside each vessel the gases enter at the top through a jet oriented so that the atmosphere is injected tangentially to the wall and swirls across the tops of the culture vials. This is to promote mixing
260
ASSAY OF STRESS GENES/PROTEINS EXHAUST ATMOSPHERE
III
[22]
INLET ATMOSPHERE
--
GLASS VIAL CONTAINING MEDIUM AND TRACHEA
FIG. 2. Side view of exposure vessell for small in vitro exposure system. Details are given in the text.
and even exposure among each of the five vials. Exhaust from each vessel is taken from the bottom in the center well below the tops of the vials. Humidification of the atmospheres is adjusted so that the 2 ml of medium contained in each culture vial neither loses nor gains volume during a 24hr period. Exhaust lines 9.5 mm (3/8 inch) in diameter exit from the vessels. Near each vessel a T is provided to connect a pressure line and a remotely mounted gauge so that the pressure in each vessel is monitored. The vessels are operated at a pressure of 1.5 cm (water gauge) above that of the laboratory to prevent any influx of biological contaminants in the event of a leak. Changes in pressure are also used to monitor the system for leaks. The exposure vessel exhaust line passes through a port in the incubator and connects to the ozone analyzer inlet and exhaust so that 2 liters/min of this atmosphere is circulated through and sampled by the analyzer. Because the gases exiting the incubator are saturated with moisture and at 37.5 ° , all exhaust lines, pressure lines, sample lines, gauges, and valves are wrapped with heating tape and thermal insulation. Electrical power to the heating tape is controlled so that the humid gases are held at about 40 ° until they are vented to a fume hood. This prevents the considerable condensation in the system that would occur if the gases were allowed to cool to the laboratory temperature of 22°. In addition, condensation on the optics of the ozone analyzer is avoided by partially blocking off the cooling air intake to increase the internal temperature of the instrument from the normal 34° to about 42° to 43 °. This is below the maximum recommended ambient operating temperature of 45 °. The internal temperature of the analzyer is monitored with a remote thermistor probe thermometer affixed to the absorption chamber near the sample exit.
[22]
OZONE EXPOSURE OF CULTURED CELLS AND TISSUES
261
Large in Vitro Exposure System A larger scale in vitro exposure system enables the simultaneous exposure of cells to three ozone or nitrogen dioxide levels with a filtered air control. Pollutant levels in the three exposure vessels are independently adjustable. This is the preferred system for performing dose-response studies. Also, more material can be exposed in each vessel. The arrangement of components is similar to that used for the small in vitro exposure system except for the addition of two more flow channels for exposure vessels, larger tubing diameters to accommodate higher flow rates, and solenoid valves to control which vessel is sampled by the ozone analyzer. Standard 86 by 127 mm culture plates, available in a wide variety of well configurations, containing the cells on inserts, are exposed to ozone, nitrogen dioxide, or filtered air in specially designed cylindrical glass vessels 3.66 liters in volume (Fig. 3). Atmospheres saturated with water vapor at 37.5 ° and containing 95% air and 5% carbon dioxide by volume flow through each vessel at a total rate of 15 liters/rain. Incorporated in the lid of each vessel is a tangential mixing dome followed by a diffuser plate with 19 symmetrically located holes, each 1.6 mm in diameter, to distribute the flow evenly. Exhaust is taken from a central point below a perforated desiccator plate on which the culture plate is placed. Vessel
FIG. 3. Exposure vessel for large in vitro exposure system. Details are given in the text.
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ASSAY OF STRESS GENES/PROTEINS
[22]
geometry and flow patterns are critical in assuring that a homogeneous pollutant concentration is maintained within and that each culture receives the same exposure.
Ozone Monitoring Ozone concentrations in each vessel are continuously monitored during exposures with an ultraviolet ozone analyzer. In the large system with three exposure vessels and a control vessel, the analyzer is connected to computer-controlled valves that cycle from vessel to vessel. Typically, each is sampled for 2 min during a total cycle of 8 min. All materials in contact with the sampled ozone are Teflon or glass. Concentration data from the ozone analyzer are collected with a computer-based data acquisition system (PC-AT, IBM Corporation, Boca Raton, FL, and System 4000, ADAC Corporation, Woburn, MA) that is also used to produce statistical reports on the exposure conditions. Calibration of the analyzer is performed according to the national reference method 14and is traceable to a National Institute of Standards and Technology absolute ozone photometer. It is necessary to prevent condensation from the atmospheres saturated with moisture at 37.5 ° on the optics of the analyzer by raising the internal temperature to about 42° to 43°. Since the response of the instrument to a given ozone concentration is inversely proportional to the absolute temperature of the sample, ~5 calibrations are performed at the elevated temperature to eliminate this temperature difference as a small source of error. During exposure, stable ozone concentrations are easily maintained with few adjustments. Table I shows a portion of the computerized data report for a typical exposure. The desired concentration of 0.5 ppm in this case was achieved with a mean level for the 24-hr period of 0.49 ppm and a standard deviation of about 2% of the mean. High peak excursions in concentration, which could in theory influence any biological effects independently of the mean level, did not occur.
Testing Uniformity of Exposure To ensure that each of the culture vials in the exposure apparatus receive the same amount of ozone, 4 ml of 2% neutral buffered potassium iodide solution ~6is pipetted into each. In a typical series of tests, the vial 14 U.S. Code of Federal Regulations, Prot. Environ. 40, 667 (1988). 15 W. B. DeMore, J. C. Romanovsky, M. Feldstein, W. J. Haming, and P. K. Mueller, in "Calibration in Air Monitoring," p. 131. Am. Soc. Test. Mater., Philadelphia, 1976. 16 B. E. Saltzman, in "Selected Methods for the Measurement of Air Pollutants" (M. Storlazzi and S. Hochheiser, eds.), p. D-1. Public Health Service, Durham, North Carolina, 1965.
[22]
OZONE EXPOSURE OF C U L T U R E D CELLS A N D TISSUES
7 O N
0
0
z
0
Toc3. Through these processes, Toc3 and Toc may show different inhibition when reacting with peroxide within the membrane.
[30] D e t e r m i n a t i o n o f T o c o p h e r o l s a n d T o c o p h e r o l q u i n o n e in H u m a n R e d B l o o d Cell a n d P l a t e l e t S a m p l e s B y G O V I N D T . VATASSERY
Introduction Prolonged vitamin E deficiency in the rat results in a variety of hematological problems that include higher platelet and reticulocyte counts, increased platelet aggregation in vitro, and normocytic anemia.l'2 The susceptibility of red cells to hemolysis in the presence of dialuric acid was proposed as a laboratory test for the assessment of vitamin E nutritional status by Gyorgy and Rose. 3 Even though this test is rarely used, the importance of vitamin E for maintaining the biological integrity of red cells is well established. Platelets have been employed in investigations dealing with assessment of nutritional status in humans. 4'5 One of the oxidation products of vitamin E is tocopherolquinone which exhibits interesting biological activities. Tocopherolquinone has been reported to be an antisterility factor in the vitamin E-deficient male hamster and rat. 6 Mackenzie et al. 7 have shown that a-tocopherolquinone is active in alleviating muscular dystrophy in vitamin E-deficient rabbits. Tocopherol as well as its oxidation products have also been found to inhibit platelet aggregation under in vitro conditions. 8'9 We have attempted to use the concentrations of tocopherol and tocopherolquinone as an index of the oxidative status of membranes with respect 1 L. Machlin, R. F. Filipski, A. L. Willis, D. C. Kuhn, and M. Brin, Proc. Soc. Exp. Biol. Med. 149, 275 (1975). 2 G. F. Combs, Proc. Nutr. Soc. 40, 187 (1981). 3 p. Gyorgy and C. S. Rose, Ann. N.Y. Acad. Sci. 52, 231 (1949). 4 j. Lehmann, Am. J. Clin. Nutr. 34, 2104 (1981). 5 G. T. Vatassery, A. M. Krezowski, and J. H. Eckfeldt, Am. J. Clin. Nutr. 37, 1020 (1983). 6 S. I. Mauer and K. E. Mason, J. Nutr. 105, 491 (1975). 7 j. B. Mackenzie, H. Rosenkrantz, S. Ulick, and A. T. Milhorak, J. Biol. Chem. 183, 655 (1950). 8 A. C. Cox, G. H. R. Rao, J. M. Gerrard, and J. G. White, Blood 55, 907 (1980). 9 R. Mower and M. Steiner, Prostaglandins 24, 137 (1982).
METHODS IN ENZYMOLOGY.VOL. 234
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[30]
to vitamin E. l°'ll This involves determination of the two compounds in the same sample. A few publications dealing with the analysis of tocopherolquinone can be found in the literature. 12-15 The method recommended in this report is the latest modification of our earlier procedure. 16
Assay Method Samples of red cells or platelets are saponified, and the tocopherol derivatives are extracted with hexane. The isolated compounds are then separated and quantitated using high-performance liquid chromatography (HPLC). Reagents. All chemicals and solvents used are reagent or chromatography grade from standard suppliers. Absolute ethanol is redistilled prior to use. Standard tocopherols and quinone are from Eastman Organic Chemicals (Rochester, NY). Solutions. The following solutions are used for the assay: butylated hydroxytoluene (BHT), 0.025% (w/v) in redistilled ethanol; ascorbic acid, 30% (w/v), and pyrogallol, 25% (w/v), in deionized water. Stock solutions (100 mg/100 ml) of a-, Y-, and 8-tocopherols, tocopherolquinone, and tocol are made in redistilled ethanol. The stock standards are stable for months if kept in the freezer at - 40°. Dilute working solutions of standards can be made in ethanol containing 0.025% BHT, and the diluted stock solutions are stable in the refrigerator for several weeks. Collection o f Blood and Isolation o f Red Cells and Platelets. It should be kept in mind that platelet function and biochemical properties are very susceptible to a number of drugs. Therefore special care has to be taken to ascertain that blood is obtained in a drug-flee state. Factors that influence the properties of platelets such as drug treatment, the method of drawing blood, and storage conditions have been discussed in an earlier volume in this series.17 Blood samples for the isolation of platelets are drawn into tubes containing sodium citrate (1.25 ml of 3.8% sodium citrate per 12 ml of blood). The blood is mixed gently for a few minutes and transferred into polypropylene tubes (16 × 100 ram) and centrifuged at 250 g for 15 min. The l0 G. T. Vatassery, Biochim. Biophys. Acta 926, 160 (1987). 11 G. T. Vatassery, Lipids 24, 299 (1989). i2 S. K. Howeland and Y. M. Wang, J. Chromatogr. 227, 174 (1982).
13D. D. Stump, E. E. Roth, Jr., and H. S. Gilbert,J. Chromatogr. 306, 371 (1984). 14G. A. Pascoe, C. T. Duda, and D. Reed, J. Chromatogr. 414, 440 (1987). 15M. E. Murphy and J. P. Kehrer, J. Chromatogr. 421, 71 (1987). 16G. T. Vatassery and W. E. Smith, Anal. Biochem. 167, 411 (1987). 17M. B. Zucker, this series, VoL 169, p. 117.
[30]
VITAMIN E COMPOUNDS IN PLATELETS AND RED CELLS
329
platelet-rich plasma (PRP) is then drawn out with a Pasteur pipette, mixed with EDTA (1 ml of 0.2 M disodium EDTA per 10 ml of PRP), and centrifuged at 2000 g for 15 min. The platelet pellet is then washed twice with phosphate-buffered saline (PBS) at pH 7.4 containing 4 mM EDTA. Platelets are resuspended in the appropriate medium required for the specific oxidation experiments in vitro. Blood samples for collection of red blood cells are drawn into Vacutainers (purple top, 16 x 100 mm; evacuated blood collection tubes made by Becton Dickinson and Company, Rutherford, NJ) containing disodium EDTA as anticoagulant and centrifuged at 2600 g for 15 min. Plasma and the buffy coat on top of the red cells are removed. The cells are washed twice with PBS, pH 7.4. The washed red cells can then be used for analysis. Saponification and Sample Treatment. Two milliliters ethanol containing 0.025% BHT, 0.1 ml of 30% ascorbic acid, and 0.2 ml of 25% pyrogallol are pipetted into saponification tubes (16 x 125 mm Teflon-capped culture tubes). The red cell or platelet samples are then added. With red cell samples it is preferable to add the samples to tubes containing the saponification medium while being vortexed. One milliliter of 10% (w/v) potassium hydroxide solution is added, and the mixture is saponified at 60° for 30 min. The tubes are cooled and 2 ml of water is added. Two milliliters of hexane containing 0.025% BHT is then added. Tocopherols and quinone are extracted into the hexane phase by vortexing the mixture for 1 min. The hexane phase is separated out and evaporated under a stream of nitrogen. The residue is redissolved in mobile phase and injected on the chromatographic column. Chromatography of Tocopherol and Tocopherolquinone. Instruments used for HPLC consist of the following components: Model 126 pump, Model 507 autosampler, and System Gold software (Beckman Instruments, San Ramon, CA) supported by an IBM Model 80-386 computer. Tocopherol compounds are detected electrochemically with a Coulochem 5100 A detector (ESA Inc., Bedford, MA) using the various cells and potentials as follows: 5011 analytical cell with detector 1 at -0.25 V and detector 2 at +0.55 V and 5021 conditioning cell at -0.75 V. These electrochemical conditions are modified from the recommendations of Murphy and Kehrer ~5and involve reduction of tocopherolquinone at one electrode followed by oxidation of the resultant species at the second electrode. Chromatographic conditions are as follows: column, ultrasphere ODS, 5 tzm, 4.6 x 250 mm from Beckman Instruments; mobile phase, 6% buffer, 12% acetonitrile, and 82% methanol with the mixture containing 7.5 mM NaH2PO 4 . H20 (final concentration); flow rate, 3 ml/ min. Representative retention times of the various tocopherol derivatives under these conditions are the following: ~-tocopherol, 15.6 min; y-tocoph-
330
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[30]
erol, 12.5 min; 8-tocopherol, 10.4 min; tocopherolquinone, 9.4 rain; and tocol (internal standard), 7.5 min. A typical chromatogram obtained during analysis of a sample of platelets is shown in Fig. 1. General Comments
All operations involving platelets are performed at room temperature (20 °) using siliconized glassware or plastics. Resuspension of platelets is done gently, employing plastic Pasteur pipettes. It is better not to subject the platelet samples to vortex mixing. Red cells may be processed at 4 °. Detection limits for the assay depend largely on the chromatographic detector used. Electrochemical detectors are the most sensitive (detection limit 25 pg injected on the column). Tocopherols can also be estimated using fluorescence (295 nm excitation and 340 nm emission) or UV absorption at 295 nm and tocopherolquinone by UV absorption at 265 nm.
A
3.6-
E i
(~ ¢-)
E
3.4
~ 3.2
i r~ 2.8
. . . .
;
. . . .
. . . .
1;'
'
'
Time (minutes) FIG. l. Chromatogram obtained during the assay of human platelets. A sample of blood was obtained from a human volunteer, and the platelets were isolated and assayed for tocopherols and tocopherolquinone using the recommended procedure. Peak A, Tocol; B, a-tocopherolquinone; C, 8-tocopherol; D, y-tocopherol; E, a-tocopherol.
[31]
URIC AND ASCORBATE/DEHYDROASCORBATERATIO
331
Tocopherols in red cell samples tend to be very labile, and a mixture of the three antioxidants is necessary to avoid losses during sample processing. With platelet samples a mixture of BHT and ascorbate is sufficient. Interestingly, ascorbate is necessary to avoid losses of tocopherols as well as quinone during assays. Use of 8-tocopherol as an internal standard is recommended in some publications. However, 8-tocopherol may occur naturally in small amounts in many samples. For this reason tocol is the preferred internal standard. Finally, Burton et al. z8 have recommended an extraction procedure using sodium dodecyl sulfate (SDS) as a detergent solubilizer of the biological samples. When the proposed method involving saponification was compared with the SDS extraction procedure, the results obtained by both methods were not significantly different with either platelet or red cell samples. is G. W. Burton, A. Webb, and K. U. Ingold, Lipids 20, 29 (1985).
[31] V i t a m i n C, D e h y d r o a s c o r b a t e , a n d U r i c A c i d in T i s s u e s and Serum: High-Performance Liquid Chromatography B y G. BARJA and A. HERNANZ
Introduction Ascorbic acid and uric acid 1 are low molecular weight water-soluble antioxidants present in significant amounts in the tissues and body fluids of humans and other mammals. Their measurement in biological samples is of current interest in biomedical research since they can provide protection against many pathologies implicating free radicals at various stages of their progression. High-performance liquid chromatography (HPLC) offers reliable methods for the measurement of ascorbic and uric acid that should replace less specific colorimetric or electroanalytic ,3 techniques. Two HPLC methods specifically designed for the measurement of these
1 B. N. Ames, R. Cathart, E. Schwiers, and P. Hochstein, Proc. Natl. Acad. Sci. U.S.A. 78, 6858 (1981). 2 p. j. Garry, G. M. Owen, D. W. Lashley, and P. C. Ford, Clin. Biochem. 7, 131 (1974). 3 E. L. McGowen, M. G. Rusnack, M. Lewis, and J. A. Tillotson, Anal. Biochem. 119, 55 (1982).
METHODS IN ENZYMOLOGY, VOL. 234
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[31]
URIC AND ASCORBATE/DEHYDROASCORBATERATIO
331
Tocopherols in red cell samples tend to be very labile, and a mixture of the three antioxidants is necessary to avoid losses during sample processing. With platelet samples a mixture of BHT and ascorbate is sufficient. Interestingly, ascorbate is necessary to avoid losses of tocopherols as well as quinone during assays. Use of 8-tocopherol as an internal standard is recommended in some publications. However, 8-tocopherol may occur naturally in small amounts in many samples. For this reason tocol is the preferred internal standard. Finally, Burton et al. z8 have recommended an extraction procedure using sodium dodecyl sulfate (SDS) as a detergent solubilizer of the biological samples. When the proposed method involving saponification was compared with the SDS extraction procedure, the results obtained by both methods were not significantly different with either platelet or red cell samples. is G. W. Burton, A. Webb, and K. U. Ingold, Lipids 20, 29 (1985).
[31] V i t a m i n C, D e h y d r o a s c o r b a t e , a n d U r i c A c i d in T i s s u e s and Serum: High-Performance Liquid Chromatography B y G. BARJA and A. HERNANZ
Introduction Ascorbic acid and uric acid 1 are low molecular weight water-soluble antioxidants present in significant amounts in the tissues and body fluids of humans and other mammals. Their measurement in biological samples is of current interest in biomedical research since they can provide protection against many pathologies implicating free radicals at various stages of their progression. High-performance liquid chromatography (HPLC) offers reliable methods for the measurement of ascorbic and uric acid that should replace less specific colorimetric or electroanalytic ,3 techniques. Two HPLC methods specifically designed for the measurement of these
1 B. N. Ames, R. Cathart, E. Schwiers, and P. Hochstein, Proc. Natl. Acad. Sci. U.S.A. 78, 6858 (1981). 2 p. j. Garry, G. M. Owen, D. W. Lashley, and P. C. Ford, Clin. Biochem. 7, 131 (1974). 3 E. L. McGowen, M. G. Rusnack, M. Lewis, and J. A. Tillotson, Anal. Biochem. 119, 55 (1982).
METHODS IN ENZYMOLOGY, VOL. 234
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ANTIOXIDANT CHARACTERIZATION AND ASSAY
[31]
substances in tissues 4 or blood 5 are described. They can be performed with basic HPLC equipment, since elution is isocratic and detection is based in ultraviolet absorption in both cases. The tissue method 4 is adapted here to the simultaneous measurement of ascorbate, dehydroascorbate, and uric acid in the same sample in two consecutive HPLC runs. Measurement of Ascorbic Acid, Uric Acid, 4 and Dehydroascorbate in Tissues
Principle. Ascorbic and uric acid can be efficiently and rapidly separated on standard reversed-phase columns with isocratic elution after addition of appropriate concentrations of a counterion to a buffered waterbased mobile phase. This solves many problems associated with ionexchange chromatography. As maximums of absorption are found around 267 nm for ascorbic acid and around 292 nm for uric acid, the choice of an intermediate wavelength (280 nm) allows the simultaneous measurement of both antioxidants in the same chromatogram with enough sensitivity for most mammalian tissues using a conventional UV detector. 4 Standards. Prepare a solution containing 0.4 mM ascorbic acid and 89 /zM uric acid in 50 mM perchloric acid. The solution is maintained in the cold (5°), protected from light, and promptly injected in the HPLC system every day that samples are processed. Sample Preparation. Tissue samples (100 mg/ml) are homogenized in cold 50 mM perchloric acid, centrifuged at 3000 g for 10 min at 5 °, and filtered through 0.5/xm pore diameter membranes, and 20/zl is directly injected in the HPLC system. The use of other acids with higher acidity and ionic strength for extraction and stabilization of ascorbic and uric acid produces strong reductions of retention times on successive injections and will greatly shorten the useful life of reversed-phase columns. This problem is absent with perchloric acid at the concentration cited without compromising extraction efficiency from the tissue or the stability of the substrates. Chromatography Conditions. The mobile phase is acetonitrile/water (12.5 : 87.5, v/v), pH 5.5, containing 4.3 mM disodium hydrogen phosphate and 1.07 mM myristyltrimethylammonium bromide as counterion. When the mobile phase is isocratically pumped at 0.75 ml/min through a C~8 reversed-phase Nucleosil 7/zm (4.6 × 100 mm) column (Machery-Nagel, Oensingen, Switzerland), retention times for uric and ascorbic acid are around 5.5 and 9 min, respectively (column temperature 25°). Peaks are 4 G. Barja de Quiroga, M. L6pez-Torres, R. P6rez-Campo, and C. Rojas, Anal. Biochem. 199, 81 (1991). 5 R. E. Hughes, Biochem. J. 64, 203 (1956).
[3 II
URIC AND ASCORBATE/DEHYDROASCORBATE RATIO
333
detected at 280 nm for both substances. I f a programmable UV detector is available, further sensitivity can be gained setting the wavelength at 292 nm during the first part of the chromatogram and automatically changing to 267 nm after uric acid has eluted. A total of 25-35 min between injection of two consecutive samples is convenient for ,many tissues tested. It is essential to control carefully the pH of the mobile phase since, as is shown in Table I, an increase in this parameter strongly reduces the retention time of ascorbic acid without affecting that of uric acid. This characteristic of the mobile phase affects more intensely the differential retention of both acids than the acetonitrile or counterion concentration. Thus, pH should be the first parameter to vary when direct application of the method described here to a different sample does not initially allow good resolution from interfering peaks. Comments. The method described here is widely applicable to tissue samples; good, fast chromatographic resolutions have been obtained using rat brain, lung, and liver; pigeon liver; mouse liver; guinea pig liver and lung; and trout liver. A detailed study performed in mouse live# showed 93.9-96.2% stability of both substances in the perchloric acid extract during 8 hr at 5° in the dark, 94.3-96.5% recovery, good reproducibility, and linearity of response in homogenates containing from 1.5 to 30 mg protein/ml. In addition to retention times, peak purity can be assessed with a conventional UV detector after incubating the homogenates with specific degradative enzymes. Thus, total disappearance of the ascorbic acid peak is obtained after 5 min of incubation of a freshly prepared mouse liver homogenate (pH 6.4 at 25°C) with 500 IU/ml of ascorbate oxidase prior to the addition of perchloric acid. Similarly, incubation of a mouse TABLE I EFFECT OF pH OF MOBILE PHASE ON DIFFERENTIAL CHROMATOGRAPHIC SEPARATION OF ASCORBIC AND URIC ACIDa Retention time (min) Compound
pH 6.4
pH 5.9
pH 5.5
Uric acid Ascorbic acid
5.4 5.5
5.4 7.0
5.4 9.1
a HPLC conditions: mobile phase, acetonitrile/water (12.5 : 87.5, v/v) containing 4.3 mM disodium hydrogen phosphate and 1.07 mM myristyltrimethylammonium bromide pumped at 0.75 ml/min; column, Cm Nucleosil 7/xm Macherey-Nagel (4.6 x 100 mm); temperature, 25° -+ 1°.
334
A N T I O X I D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
[31]
liver homogenate (pH 7.4, 25 °) with uricase (5 IU/ml) leads to total disappearance of the uric acid peak. a Dehydroascorbate Analysis. Information about the dehydroascorbate content of the tissues can be obtained by performing the method of Hughes, 5 adapted to HPLC of liver tissue as described here. For this purpose the centrifuged and filtered perchloric acid extract is divided into two aliquots. The first is diluted by a factor of 10 with 50 mM perchloric acid and injected in the chromatograph to measure the reduced form of ascorbate as described above. The second aliquot is carefully neutralized to pH 7.0 using a pH micrometer by adding around 100/xl of 45% K2HPO 4 per milliliter of sample. A portion of the neutralized sample is immediately added to a vial containing a previously weighed amount of DL-homocysteine (e.g., 4 mg). The amount of sample added is that needed to get a final DL-homocysteine concentration of I% (e.g., add 0.4 ml of sample to 4 mg of DL-homocysteine). Shake and incubate for 15 min at 25°. This procedure fully reduces the dehydroascorbate present in the sample to ascorbate (DL-homocysteine is fully effective only at neutral pH). At the end of the 15-min period the sample is diluted by a factor of 10 with 50 mM perchloric acid and is immediately injected in the chromatograph (direct injection of the undiluted samples containing 1% homocysteine causes strong reductions in retention times and shortens the column half-life; these problems are eliminated by decreasing the homocysteine concentration to 0.1%). Now the ascorbate peak represents total ascorbate (reduced plus oxidized forms). Dehydroascorbic acid is calculated by subtracting the amount of reduced ascorbate from total ascorbic acid, and the ratio dehydroascorbic/ascorbic can thus be calculated. To obtain optimal results the reduced ascorbate sample must also be diluted by a factor of 10 with 50 mM perchloric acid before injection in the chromatograph. This is especially important in tissues such as liver where 90% or more of the ascorbic acid is present in the reduced form. We have obtained good, reproducible results with this method using guinea pig liver. Incubation of the "total ascorbate" sample with ascorbate oxidase (see "Comments" above) at the optimum pH of the enzyme leads to total disappearance of the "ascorbate plus dehydroascorbate" peak. This demonstrates that all the increase in peak area from the untreated to the treated (homocysteine) sample is due to reduction of dehydroascorbate and not to reduction of other substances present in the tissue. Thus, this method allows the simultaneous measurement of the three substances, ascorbate, dehydroascorbate, and uric acid, in the same tissue sample in two consecutive HPLC runs. The sensitivity of a conventional UV detector is much higher than needed for accurate detection of these substances in many tissues using the described method.
[31]
URIC AND ASCORBATE/DEHYDROASCORBATE RATIO
335
Measurement of Ascorbic and Uric Acids in Serum 6
Principle. Different high-performance liquid chromatography methods have been used to determine ascorbic acid in serum from humans. 7-9 However, few HPLC methods using UV spectrophotometric detection have been shown to be simple and sensitive. Here, a simple, rapid, and sensitive reversed-phase HPLC procedure for ascorbic and uric acid determination in 0.5 ml serum (or less) using paired-ion chromatography with UV spectrophotometric determination is described. The effects of solvent pH and ascorbic acid oxidation are also examined. With this method analysis of clinical samples in order to investigate presymptomatic decreases in the ascorbic acid as well as antioxidant status can be carried out without difficulty. Standards. Prepare stock solutions of ascorbic acid (6 raM) and uric acid (1 mM) in 50 ml of 0.3 mM trifluoroacetic acid in the presence of 10 mM 1,4-dithioerythritol (DTE) as antioxidant. This solution is stable for 1 week when frozen at - 2 5 °. Ascorbic and uric acid working standards are prepared flesh daily by diluting the stock solutions (1 : 10) with the mobile phase. Samples. Serum samples are prepared from blood collected from the antecubital vein in the presence of 1 mM DTE as antioxidant. A 0.5-ml serum sample is mixed with 0.1 ml of 1.2 M trifluoroacetic acid to obtain a protein-flee extract after centrifugation. To eliminate trifluoroacetic acid, supernatants are desiccated under vacuum with centrifugation, then stored at - 2 5 ° under N2 until analysis. Immediately before being analyzed the serum extracts are reconstituted with 0.2 ml of mobile phase and passed through a 0.45-nm HV filter (Millipore, Bedford, MA); 5-20/zl is injected in the HPLC system. Chromatography Conditions. The mobile phase is 5 mM ammonium formate buffer, pH 6.0, containing 5 mM tetrahexylammonium chloride (Fluka, Ronkonkoma, NY) as a paired-ion reagent in water/methanol (65 : 35, v/v). The addition of tetrahexylammonium chloride to the mobile phase allows ionic compounds, such as ascorbic acid, to be separated on C~8 reversed-phase columns, eliminating problems of precise pH, temperature control, reproducibility, and short column life associated with ion 6 A. Hernanz, J. Clin. Chem. Clin. Biochem. 26, 459 (1988). 7 W. Lee, P. Hamernyik, M. Hutchinson, V. A. Raisys, and R. F. Labb6, Clin. Chem. (Winston-Salem, N.C.) 28, 2165 (1982). 8 T. Iwata, M. Yamaguchi, S. Hara, and M. Nakamura, J. Chromatogr. Biomed. Appl. 344, 351 (1985). 9 A. J. Speek, J. Schrijver, and W. H. P. Schreurs, J. Chromatogr. Biomed. Appl. 305, 53 (1984).
336
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[31]
T A B L E II EFFECT OF 1,4-DITHIOERYTHRITOL ON ASCORBIC ACID LEVELS IN SERUM a Ascorbic acid (t~mol/liter) Sample
With DTE
Without DTE
Serum Aqueous standard
63.4 --- 12.2 55.7 4- 3.5
14.5 -4- 12.2 18.6 - 1.2
Determination in duplicate, with and without 1,4-dithioerythritol (DTE), of ascorbic acid levels (txmol/liter, mean -+- SD) in serum from fifteen healthy adults and in ten different aliquots of an aqueous standard of 58/xmol/ liter ascorbic acid was performed. HPLC conditions: mobile phase, methanol/water (35:65, v/v) containing 5 m M ammonium formiate buffer, pH 6.0, and 5 m M tetrahexylammonium chloride pumped at 0.7 ml/min; column, C18 Novapak 4 /zm Waters (3.6 x 150 mm); temperature, 25 -4- 1°.
exchange. Further filtration with a 0.5-nm FHUP filter (Millipore) was performed. The flow rate of the mobile phase is adjusted to 0.7 ml/min. When the mobile phase is isocratically pumped through a C18 reversedphase Novapak (15 cm × 3.6 mm i.d.) column (Waters, Milford, MA), retention times for ascorbic and uric acids are around 8 and 10 min, respectively (column temperature 25°). Peaks are detected at 265 nm for both substances, or at 254 nm if a filter photometer is available. It is essential to control carefully the pH of the mobile phase because a decrease in mobile phase pH significantly reduces the absorption at 265 nm of ascorbic acid. Comments. The method described here has wide application to serum samples if ascorbic acid oxidation is avoided. With this method it is possible to measure without any risk of oxidation serum ascorbic acid amounts as low as 10 pmol (2 ng), which corresponds to 0.5 txmol/liter if 10/xl of serum extract is injected. Correlation between peak responses and injected ascorbic acid concentrations was found to be linear from 10 to 600 pmol (2 to 110 ng). The day-to-day reproducibility of the total HPLC procedure was determined by measuring in duplicate separate portions of a serum pool on 10 consecutive days. The coefficient of variation obtained was 7. I% for the serum pool having a mean concentration of 50 tzmol/liter. Precision was calculated by measuring a serum pool 10 times in a single run. The coefficient of variation was 5.4%. Analytical recovery was carried out by adding 50/xl of 30/zmol/liter ascorbic acid to 0.5 ml of a serum
[31]
URIC AND ASCORBATE/DEHYDROASCORBATE RATIO
337
pool and measuring the total ascorbic acid on 10 different days. The recovery obtained was 94.5 -+ 6.0 (mean % m SD). In addition to the retention times, peak purity can be assessed by incubating the serum extracts with specific degradative enzymes. Total disappearance of the ascorbic acid peak is obtained after incubation with ascorbate oxidase or H20: . Similarly, incubation with uricase leads to total disappearance of the uric acid peak. 4 The reliable measure of dehydroascorbic content in serum samples is controversial. Whereas some authors 1° have shown that when 1,4-dithiothreitol is added to urine samples the amount of ascorbic acid increased, owing to reduction of dehydroascorbic acid to ascorbic acid, others ll also using 1,4-dithiothreitol have shown minimum amounts of dehydroascorbic acid (only 3% of total ascorbic acid) in human plasma. In agreement with these last authors, Levine et al.~2 have also demonstrated the absence of dehydroascorbic acid in human blood. Schmidt et al. 13have indicated that dehydroascorbic acid is unstable at neutral pH and in the presence of mild biological oxidants, as occurs in many vital biological processes in the living organism, and it is rapidly metabolized. As noted in Table II serum samples, obtained from blood collected without 1,4-dithioerythritol as the antioxidant, as well as standard samples stored without 1,4-dithioerythritol, present lower values of ascorbic acid after being subjected to the entire HPLC method on the same day as blood collection. This sample oxidation has not been described when UV spectrophotometric9 or electrochemical7 detection was used.
10 L. W. Donner and K. B. Hicks, Anal. Biochem. 115, 225 (1981). 11 M. Okamura, Clin. Chim. Acta 103, 259 (1980). 12 M. Levine, K. R. Dhariwal, P. W. Washko, J. DeB Butter, R. W. Welch, Y. Wang, and P. Bergsten, Am. J. Clin. Nutr. 54, 11575 (1991). 13 K. Schmidt, H. Oberritter, G. Bruchelt, V. Hagmaier, and O. Hornig, Int. J. Vitam. Nutr. Res. 53, 77 (1983).
338
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[32]
[32] I n V i v o D e t e r m i n a t i o n o f S u p e r o x i d e a n d V i t a m i n C Radicals Using Cytochrome c and Superoxide Dismutase Derivatives By MASAYASU INOUE and KEIKO KOYAMA
Introduction Most reactive oxygen species rapidly react with various molecules and interfere with cellular functions. 1 Based on experiments using antioxidant enzymes and scavengers for reactive oxygen species and free radicals, such as Cu/Zn-type superoxide dismutase (SOD), glutathione, ascorbic acid, and dimethyl sulfoxide (DMSO), critical roles of these reactive species in the pathogenesis of various diseases have been postulated. 2 However, these antioxidants react with a wide variety of compounds and are involved in various metabolic pathways. Thus, even if these scavengers inhibited tissue injury, the corresponding reactive oxygen species may not always be involved in their pathogenesis. To get direct evidence for the involvement of reactive oxygen species in the pathogenesis of various diseases, these species should be determined quantitatively in vivo.
Cytochrome c (Cyt c) has been used for determining superoxide radicals in vitro. 3 Because cytochrome c reacts not only with superoxide radicals but also with other compounds with reducing activity and serves as a substrate for cytochrome c-reductase and cytochrome c-oxidase, acetylated Cyt c (AC) has been used for the detection of superoxide radicals, particularly in complex biological systems. 4 However, both Cyt c and AC rapidly undergo glomerular filtration and disappear from circulation with a half-life of 2-3 min. Thus, in practice it is difficult to use Cyt c and AC for in vivo detection of superoxide radicals and related metabolites. To overcome such frustrating situations, an acetylated Cyt c derivative that circulates bound to albumin with a prolonged in vivo half-life was synthesized. i H. Sies, ed., " O x i d a t i v e S t r e s s . " A c a d e m i c Press, Orlando, Florida, 1985. 2 I. Emerit, L. Packer, and C. Auclair, eds., " A n t i o x i d a n t s in T h e r a p y and Preventive M e d i c i n e . " P l e n u m , N e w York, 1990. 3 j. M. M c C o r d and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 4 S. Minakami, K. Titani, and H. Ishikawa, J. Biochem. (Tokyo) 45, 341 (1958).
METHODS 1N ENZYMOLOGY,VOL. 234
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[32]
LONG-ACTING CYTOCHROME C AND S O D DERIVATIVES
339
SM NH2 H2N~
NH2
H?-- CO0-
H2N~NH2
NH, NH2
HC--C~ 0 ~'0 HC--C~o
Cyt.c S
HCH
NH2~ NH2
C~-cH 0 II
H E="l
H2N ~'F~• J~ H SM-Cyt.c NH2
~
NH2
NH2
mm' 2-3
I
CH~-C~
CH3--CO ~ 1 ~
I
NH
°
'
L
=
0 ..~-~'-""~...~
~ NH2
/
II ~
^,. ..... ~,
. NH I
CH3--CO
NH I
CO
SMAC
I
CH3 FIG. I. Synthesis of a cytochrome c derivative that circulates bound to albumin with prolonged in vivo half-life. Cytochrome c was incubated with the anhydride of half-butylesterified poly(styrene-co-maleic acid) (SM). The SM-Cyt c thus formed was further reacted with acetic anhydride. The resulting SMAC forms a dissociable complex with albumin and escapes from being filtered by the glomerulus.
Synthesis of Long-Acting Cytochrome c Derivative We previously reported that superoxide dismutase covalently linked with half-butyl-esterified poly(styrene-co-maleic acid) (SM, molecular weight 1600) circulates bound to albumin and has a prolonged in vivo half-
340
ANTIOXIDANT CHARACTERIZATION AND ASSAY
REACTIVITY
OF CYTOCHROME
TABLE I SMAC
C AND
WITH
VARIOUS
[32]
COMPOUNDS
a
Rate b Antioxidant
Concentration (/~M)
Cytochrome c
SMAC
30 60 10 400 30 500
0.036 0.065 nd nd nd nd
0.003 0.005 nd nd nd nd
Ascorbic acid Reduced glutathione Uric acid Bilirubin Bilirubin + albumin
Incubation mixtures contained, in a final volume of 1 ml, 0.1 M phosphate buffer, pH 7.4, varying concentrations of low molecular weight compounds, and 20/xM cytochrome c or SMAC. The reaction was started by adding the reducing agents at 25°. The change in absorbance at 550 nm was monitored. b Change in absorbance at 550 nm/min, nd, Below detectable levels (I-¢,/) Z ILl I.-Z UJ (..) Z UJ ¢..) ¢t) UJ n-
O :ZD _J LL
100
8 ~mNM~
~ ' - ~
6 4 0 i-
100 p.M l cis-
~PARINARIC
0~ 0
I
160 p . ~ . . ~ et-TOCOPHEROL / ¢x-TOCOTRIENOL
/I 4
EX 304 nm, EM 421
I 8
I,
I 12
I
I 16
I
I 20
nm I 24
TIME IN MINUTES FIG. 2. Comparison of antioxidant activity between tx-tocopheroI and tx-tocotrienol in hexane.
Incubation Conditions. The reaction mixture (3 ml) contains AMVN (100 mM) and cis-parinaric acid (30/zM) in hexane, a-Tocopherol and a-tocotrienol (160/zM each) have to be initially dissolved in chloroform and added to the incubation medium during the course of AMVN-induced fluorescence loss of cis-parinaric acid. Radical Scavenging Activity of a-Tocopherol and a-Tocotrienol in Liposomes: Azo Initiator-Based Assay Addition of A M V N to a suspension of diolcoylphosphatidylcholinc (DOPC) liposomes in the presence of luminol produces a characteristic chcmilumincsccnt response. This response is not observed in the absence of liposomes, indicating that the recorded chemiluminescence represents the reaction of AMVN-dcrivcd pcroxyl radicals with luminol in D O P C liposomal membranes. Liposomcs with incorporated a-tocophcrol or o~-tocotricnol inhibit AMVN-induced luminol-sensitized chemiluminescence in a concentration-dependent fashion22 (Fig. 3). The antioxidant efficiency of a-tocopherol and a-tocotricnol is different. The concentrations of a-tocophcrol and a-tocotricnol producing 50% inhibition of AMVN-induccd chemiluminescence arc 7.5 and 5/~M, respectively. Thus in liposomcs a-tocotricnol is 1.5 times more efficientscavenger ofpcroxyl radicals than o~-tocopherol. These data are in agreement with the results
360
[34]
ANTIOXlDANT CHARACTERIZATION AND ASSAY A DOSE DEPENDENCY
B REACTION CURVE 200
lOOq
A
s=
80
mTOCOPHEROL
160
.._1
i,.,--
0 re, Z
60
120
O O
3 p.M c~ TOCOTRIENOL
ii
O
4
'I,-" z u.I O
80
ua o :z
4
2 ~-TOCOTRIENOL
"._..= -' ,~= O
0 0
I
2
CONCENTRATION(gM)
0
4
8
TIME IN MINUTES
FIG. 3. Radical scavenging activity of a-tocopherol and a-tocotrienol incorporated in DOPC liposomes.
reported by Yamaoka and Komiyama24 on the antioxidant activity of a-tocopherol and a-tocotrienol in the 2.2'-azobis(2-aminopropane) dihydrochloride (AAPH)-initiated oxidation of dilinoleoylphosphatidylcholine (DLPC) liposomes, a-Tocotrienol added after liposome formation shows higher antioxidant activity than a-tocopherol. Incubation Conditions. Incubation medium (2 ml) contains DOPC liposomes (2.5 mM), luminol (150/zM), and a-tocopherol (or a-tocotrienol) in various concentrations in Tris-HCl buffer, pH 7.4. The reaction starts at 40° by the addition of AMVN (2.5 mM). Chromanols and DOPC must be initially dissolved in chloroform, dried under nitrogen, and resuspended in Tris buffer by sonicating for 10 rain at 22 kHz, 4°.
Antioxidant Activity of a-Tocopherol and a-Tocotrienol in Membranes Comparison of the antioxidative properties of different tocopherols and tocotrienols in preventing the oxidation of lard showed that tocotrienols are more active than the corresponding tocopherols. 25The antioxidative efficiency of tocotrienol isomers measured at 1I0 ° in the dark increases in the following order: c~- >/3- > y- > 8-tocotrienol. 24 M. Yamaoka and K. Komiyama, J. Jpn. Oil Chem. Soc. 38, 478 (1989). 25 V. A. Seher and S. A. Ivanov, Fette, Seifen, Anstrichm. 7S, 606-9 (1973).
[34]
o~-TOCOPHEROL AND Ot-TOCOTRIENOL
361
TABLE I CONSTANTS FOR 50% INHIBITION OF LIPID PEROXIDATION BY ot-ToCOPHEROL AND ot-ToCOTRIENOL IN RAT HEART MICROSOMES AND MITOCHONDRIA a
Constants (M) Microsomes Antioxidant a-Tocopherol a-Tocotrienol
Fe(II) + N A D P H 3.8 × 10 -5 0.2 × 10 -5
Fe(II) + a s c o r b a t e
Mitochondria Fe(lI) + ascorbate
7.1 × 10 _6 0 . 4 × 10 _6
2.8 × 10 -6 0.3 × 10 -6
a Timeof lipid peroxidation: 5 min. Incubationmediumcontained0.5 mg protein/ml, 10 mM F e S O 4 • 7H20, and 0.5 mM NADPH or ascorbate in 0.1 M potassium, sodium phosphate buffer, pH 7.4, at 37°. Quantitative comparison of the antioxidant potencies of c~-tocopherol and a-tocotrienol in a more physiological system [Fe(II) plus ascorbateor Fe(II) plus NADPH-induced lipid peroxidation in rat liver microsomes] has shown that a-tocotrienol exerts much higher antioxidant activity than a-tocopherol. The concentrations of a-tocopherol producing 50% inhibition (Ks0) are 40 and 60 times higher than those for a-tocotrienol for Fe E+ plus NADPH- and Fe E+ plus ascorbate-dependent lipid peroxidation, respectively. 26 Similar results are obtained for rat heart mitochondria and microsomes (Table I). I n c u b a t i o n C o n d it io n s . Incubation medium contains NADPH or ascorbate (0.5 mM), FeSO4" 7 H20 (10/zM), and protein (0.3 mg/ml) in 0.1 M potassium, sodium phosphate buffer, pH 7.4 at 37°. Secondary lipid peroxidation products, interacting with 2-thiobarbituric acid, are determined spectrophotometrically. Chromanols must first be dissolved in ethanol. To prevent the effect of ethanol on the accumulation of lipid peroxidation products the final ethanol concentration in the reaction mixture has to be less than 0.5%. Methods for Generation of Chromanoxyl Radicals and Recycling Efficiency of a-Tocopherol and a-Tocotrienol The steady-state concentrations of vitamin E in membranes are determined by (1) the efficiency of incorporation into membranes following transfer from blood lipoproteins and (2) its metabolism in membranes. The main intramembrane metabolic pathway of vitamin E is believed to 26E. A. Serbinova, V. E. Kagan, D. Han, and L. Packer, Free Radical Biol. Med. 10, 263 (1991).
362
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[34]
be scavenging of lipid radicals in the course of initiation and propagation of lipid peroxidation. 3'8'27 The vitamin E free radical is formed during reaction (1). It has been generally believed that great mobility of vitamin E in the lateral plane of the membrane and its exact positioning in the membrane are extremely important for this reaction. Vitamin E is anchored in the hydrocarbon part of membrane bilayer by the phytol tail which is 13 carbons long, just the right length to position the chromanol nucleus, which possesses the antioxidant activity, at the membrane interface. There, vitamin E through its phenolic hydroxyl group quenches free radicals, in the process becoming the (phenoxyl or chromanoxyl) tocopheroxyl radical. Tocopheroxyl free radicals are less reactive than other lipid radicals (peroxyl or alkoxyl radicals) generated in membranes and thus serve to break the chain of free radical reactions in lipid peroxidation. However, the free radical, tocopheroxyl form of vitamin E is susceptible to oxidation or to destruction by reacting with itself or by other reactions that cause it to decompose as a result of radical-initiated reactions. Unless reduced (regenerated) to its original antioxidant form, it will be lost before prooxidant reactions occur. This type of vitamin E action has been suspected for a long time. In fact it was proposed in 1966 by Mellors and Tappel that the ubiquinone components of membranes may serve to protect vitamin E against loss by interacting with it. 2s It is also known from in vitro studies that ascorbate (vitamin C) regenerates vitamin E from its free radical form, but whether this was an important activity of membranes was not known. Studies in the laboratory of Burke revealed that reduced glutathione (GSH), the primary preventative water-soluble antioxidant in most aerobic cells, protects against lipid perodixation of microsomal membranes in vitro. However, glutathione does not exhibit this activity if membranes are prepared from vitamin E-deficient animals. From this finding Bast, McCay, and others have suggested the existence of a vitamin E free radical reductase activity, that is, some enzyme or enzyme systems capable of specifically regenerating vitamin E, and that glutathione may be one of the substrates for this type of activity.29'3° However no direct experimental data on enzymatic regeneration of vitamin E was obtained. The only way to elucidate these types of membrane reactions of vitamin E is to follow 27 G. 28 A. 29 A. 30 p.
W. Burton and K. U. Ingold, Acc. Chem. Res. 19, 194 (1986). Mellors and A. L. Tappel, J. Biol. Chem. 214, 4353 (1966). Bast and G. R. M. M. Haenen, Biochim. Biophys. 574, 537 (1988). B. McCay, Annu. Reo. Nutr. 5, 323 (1985).
[34]
t~-TOCOPHEROL AND ot-TOCOTRIENOL
363
dynamically the reactions of the vitamin E tocopheroxyl radicals in natural membranes using highly sensitive electron spin resonance (ESR) techniques.26, 31-33
Generation of Chromanoxyl Radicals Generation of Chromanoxyl Radicals in Microsomes, Mitochondria, and Low Density Lipoproteins. Chromanoxyl radicals from a-tocotrienol and a-tocopherol are generated using (1) an enzymatic oxidation system (soybean 15-1ipoxygenase plus linolenic acid), (2) a hydrophobic azo-initiator of peroxyl radicals, AMVN, and (3) UV i r r a d i a t i o n . 26'31-33 When the enzymatic oxidation system is used the incubation medium (100/~1) contains low density lipoprotein (LDL) (9-13 mg protein/ml), microsomes or mitochondria (30-50 mg protein/ml), or liposomes (30 mg lipids/ml) in 50 mM phosphate buffer, pH 7.4 at 25°. The concentration of exogenously added chromanols is 80 nmol/mg protein. Linolenic acid (1.4 mM) plus lipoxygenase (10 U//A) and chromanols are subsequently added to the LDL suspension. Chromanols are added in ethanolic solution. With the azo-initiator the incubation medium is essentially the same, but AMVN (5.0 mM) is added instead of lipoxygenase plus linolenic acid and the reaction carried out at 40 °. Generation of Chromanoxyl Radicals in Erythrocyte Ghosts. Chromanoxyl radicals from a-tocopherol and homologs are generated in erythrocyte ghosts using an enzymatic oxidation system consisting lipoxygenase and arachidonic a c i d . 34 The reaction medium (50/zl) contains erythrocyte membrane suspension (5 mg/ml protein), arachidonic acid (1.97 mM), lipoxygenase (4.5 U//xl), and chromanols (9 mM) in 50 mM phosphate buffer, pH 7.4 at 20°. The reaction is started with lipoxygenase addition. Incorporation of Chromanols in Membranes. Membranes are preincubated with chromanols (added from ice-cold ethanolic solution) for 20 min at 25 ° (for microsomal and mitochondrial membranes) or at 20° (for erythrocyte ghosts). The suspensions are centrifuged for 60 min at 105,000 g at 4 ° and for 20 min at 30,000 g at 4° for microsomal and mitochondrial membranes or erythrocyte ghosts, respectively. Under the above conditions the amount of incorporated a-tocopherol/a-tocotrienol in all types of membranes is 84-90%. 31 L. Packer, J. J. Maguire, R. J. Mehlhorn, E. A. Serbinova, and V. E. Kagan, Biochem. Biophys. Res. Commun. 159, 229 (1989). 32 V. Kagan, E. Serbinova, T. Forte, G. Scita, and L. Packer, J. Lipid Res. 33, 385 (1992). 33 V. Kagan, E. Serbinova, and L. Packer, Arch. Biochem. Biophys. 280, 33 (1990).
364
ANTIOXIDANT CHARACTERIZATION AND ASSAY
40°C
CONTROL
40°C
+AMVN
40°C
+UV
40°C
+UV + AMVN
25°C t,.,~
~
[34]
+LIPOXYGENASE+ LINOLENICACID
g = 2.00 FIG. 4. Electron spin resonance spectra of chromanoxyl radicals generated in LDL from endogenous vitamin E.
Irradiation
Irradiation is achieved by a solar simulator (Solar Light Co., Model 14S), whose output closely matches the solar spectrum in the wavelengths 290-400 nm. The samples are illuminated directly in the ESR resonator cavity; the distance between the light source and the sample is
~~x~ MICROSOMES
~ ~ ~ LIPOSOMES
FIG. 5. Electron spin resonance spectra of chromanoxyl radicals generated from atocopherol and a-tocotrienol by an enzymatic oxidation system (lipoxygenase plus linolenic acid) in microsomes or liposomes.
[34]
O~-TOCOPHEROL AND O~-TOCOTRIENOL
365
T A B L E II RECYCLING EFFICIENCY AND DELAY TIME FOR REAPPEARANCE OF CHROMANOXYL RADICALS FROM ot-TocOPHEROL AND ot-ToCOTRIENOL IN RAT LIVER MICROSOMES Antioxidant
Delay time (min) a
Recycling efficiency b
(+)-~-Tocopherol (+)-a-Tocotrienol
1.0 - 0.2 ¢ 3.0 -+ 0.3 c
0.23 - 0.02 c 0.37 - 0.04 c
a The delay time was measured after addition of ascorbyl palmitate. b The recycling efficiency was measured after addition of NADPH. c Values were averaged from 5 data points.
30 cm. The power density of the light at the sample surface in the spectral region 310-400 nm is 1.5 mW/cm 2 and drops to 10% of this value at 290 nm.
Electron Spin Resonance Measurements The ESR measurements are made on a Varian E 109E or Brucker IBM ER 200 D-SRC spectrometer in gas-permeable Teflon tubings (0.8 mm internal diameter, 0.013 mm thickness) obtained from Zeus Industrial Products (Raritan, NJ). The gas-permeable tube ( - 8 cm in length) is filled with 60 ~1 of a mixed sample, folded into quarters, and placed in an open 3.0 mm internal diameter ESR quartz tube such that all of the sample is within the effective microwave irradiation area. ESR spectra are recorded either in the dark or under continuous UVAB-irradiation by the solar simulator in the ESR cavity. Spectra are recorded at 100 mW power and 2.5 gauss modulation, and 25 gauss/min scan time. Spectra are recorded at room temperature under aerobic conditions by flowing oxygen through the ESR cavity. Chromanoxyl and ascorbyl radical ESR signals are recorded at 3245 gauss magnetic field strength, scan range 100 gauss, and time constant 0.064 sec.
Efficiency of Chromanoxyl Radical Reduction To evaluate quantitatively the efficiency of chromanoxyl radical reduction the recycling efficiency coefficient (Re) can be calculated:
Re
=
(A_re d -
A+red)/Are d
where A_red and A +red are the magnitudes of ESR signals of chromanoxyl radicals in the absence and presence of a reductant, respectively. The
366
ANTIOXlDANT CHARACTERIZATION AND ASSAY
[34]
values of recycling efficiency vary from 1 to 0, which correspond to 100% reduction (complete transient disappearance of ESR signal) and 0% reduction (no effect on ESR signal), respectively. 34 Under the conditions described chromanoxyl radicals from vitamin E and its homologs are generated using all three methods (Fig. 4). 33 Both a-tocopherol and a-tocotrienol produce characteristic pentameric chromanoxyl radical signals with component g values of 2.0122 2.0092, 2.0061, 2.0028, and 1.9993 both in microsomes and in liposomes (Fig. 5). 26'35'36 It has been reported that ~-tocotrienol radical ESR signals are significantly higher than those of ~-tocopherol in the presence of either microsomes or liposomes. 26 Addition of NADPH to the microsomal suspension results in a decrease of the magnitude (but not complete disappearance) of the ESR signals of a-tocopherol (or a-tocotrienol). The NADPH-dependent decrease of ESR signals is much more pronounced for a-tocotrienol than for a-tocopherol. The data presented on Table II show that in microsomes NADPH-supported recycling efficiency (Re) for o~-tocotrienol is higher than for a-tocopherol. Also, the delay time of chromanoxyl radical ESR signal reappearance after addition of ascorbyl palmitate is greater for o~-tocotrienol than for a-tocopherol (Table II). These results show that o~-tocotrienol has a higher recycling efficiency than a-tocopherol. The higher recycling efficiency of a-tocotrienol must be contributing to its higher antioxidant activity compared to a-tocopherol. However, whereas the recycling efficiency and the delay time for a-tocopherol are only about 1.6 and 2.5-3 times less than those for o~-tocotrienol, the concentrations exerting 50% inhibition of lipid peroxidation differ by 40-60 times. This indicates that the higher antioxidant activity of a-tocotrienol in vitro must result from the contribution of other factors in addition to its higher recycling efficiency, such as more uniform distribution in membrane bilayer and stronger disordering of membrane lipids compared to c~-tocopherol.26 Conclusion It is recognized that differences in vivo in the antioxidant activity of tocopherols and tocotrienols may depend greatly on their pharmacokinetics. However ot-tocotrienol may have higher antioxidant activity in oioo under conditions of oxidative stress owing to its more effective antioxidant potency in membranes. 34 A. Constantinescu, D. Han, and L. Packer, J. Biol. Chem. 268(15), 10906 (1993). 35 K. Mukai, K. Takamatsu, and K. Ishizu, Bull. Chem. Soc. Jpn. 57, 3507 (1984). 36 K. Mukai, N. Tsuzuki, S. Ouchi, and K. Fukuzawa, Chem. Phys. Lipids 30, 337 (1982).
[35]
HPLC DETERMINATIONOF GSSG IN BLOOD
367
[35] D e t e r m i n a t i o n o f O x i d i z e d G l u t a t h i o n e in B l o o d : High-Performance Liquid Chromatography B y M I G U E L ASENSI, JUAN SASTRE, FEDERICO V . PALLARDO,
JOSE M. ESTRELA, and JOSE Vlr~A Introduction The measurement of glutathione status 1 is important in determining oxidative stress 2 in tissues and biological fluids. The ratio of reduced to oxidized glutathione (GSH/GSSG) is thus a good indicator of the oxidative stress that may occur under physiological and pathological conditions. 3'4 Changes in GSSG levels have been considered as intracellular signals able to modulate enzyme activity. 5,6 Thus, it is important to have accurate methods to determine GSSG in biological fluids and in cells. In many cases, it is possible to use tissues such as liver, muscle, or brain to determine GSH/GSSG. However, especially in human studies, samples from these tissues are not readily available, and measurement of blood samples is required. A major problem is the measurement of GSSG in the presence of GSH because spontaneous or catalyzed GSH oxidation must be prevented. Indeed, assuming that glutathione reductase (GR) is at equilibrium, we calculated7 that the ratio of GSH to GSSG must be about 105. Even if this is not the case, that is, if GR is not at equilibrium and if the GSH/GSSG ratio is about 100, a 2% oxidation of GSH will cause a 100% change in GSSG. Thus, oxidation of GSH must be kept to a minimum if measurements of GSH/GSSG are to be meaningful. Several methods have been devised to measure GSH and GSSG. 8-11 l N. S. Kosower and E. M. Kosower, Int. Rev. Cytol. 54, 109 (1978). 2 H. Sies, Angew. Chem. 25, 1058 (1986). 3 j. Vifia, ed., "Glutathione: Metabolism and Physiological Functions." CRC Press, Boca Raton, Florida, 1990. 4 A. Meister, in "Metabolism and Functions of Glutathione" (D. Dolphin, R. Poulson, and O. Avramovic, eds.), pp. 367-474. Wiley, New York, 1989. 5 H. F. Gilbert, J. Biol. Chem. 257, 12086 (1982). 6 H. F. Gilbert, this series, Vol. 107, p. 330. 7 j. Vifia, R. Hems, and H. A. Krebs, Biochem. J. 170, 627 (1978). 8 M. W. Fariss and D. J. Reed, this series, Vol. 143, p. 101. 9 T. P. M. Akerboom and H. Sies, this series, Vol. 77, p. 373. 10 F. A. M. Redegeld, A. S. Koster, and W. P. van Bennekom, in "Glutathione: Metabolism and Physiological Functions" (J. Vifia, ed.), pp. 11-20. CRC Press, Boca Raton, Florida, 1990. ti R. C. Fahey and G. L. Newton, this series, Vol. 143, p. 85.
METHODS IN ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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ANTIOXlDANT CHARACTERIZATION AND ASSAY
[35]
Most methods rely on chelating thiols in order to prevent GSH oxidation. An excellent, probably the best, thiol chelator is N-ethylmaleimide (NEM).12 However, it must be removed before GSSG measurements because it inactivates the enzymes used for such measurements .9 Thus, highperformance liquid chromatography (HPLC) methods are preferred to measure glutathione. One of the most widely used HPLC methods was des.cribed by Reed and colleagues in the early 1980s. a We have used this method and found oxidation of internal standards of GSH added to extracts of liver or kidney ranging from about 3 to 4%. When blood was used, however, oxidation was up to 30%. This appears to have been the case in other studies in which GSSG was measured in blood using this method. 13 In this chapter, an HPLC method to determine GSSG in blood is described which minimizes GSH oxidation to about 0.25%. Assay Procedure
Principle. The accurate measurement of GSSG in the presence of GSH relics on rapid and effective GSH quenching. To prevent GSH oxidation during sample preparation, several quenching agents, such as N-ethylmaleimide (NEM), 2-vinylpyridine, and iodoacetic acid, have been used to alkylate thiol groups. Among them, NEM is preferred because of its rapid reaction rate (completion within I min), in contrast to 2-vinylpyridine (20-50 rain) or iodoacetic acid (5-15 rain)) ° Furthermore, treatment with NEM can be achieved on ice and in acidic medium, which minimize GSH oxidation, whereas quenching by 2-vinylpyridine or iodoacetic acid occurs at room temperature and in a neutral or basic medium. Thus, GSH quenching by NEM under acidic conditions is the most convenient because it prevents GSH autoxidation that may occur in a neutral or basic medium. According to our method, blood samples are treated with pcrchloric acid (5% final concentration) containing NEM (20 mM final concentration) and bathophenanthrolinedisulfonic acid (1 mM final concentration) as metal chelator. Then, blood samples are derivatized and analyzed by HPLC to determine GSSG. Reagents 12% Perchloric acid (PCA) containing 40 mM N-ethylmaleimide (NEM) and 2 mM bathophenanthrolinedisulfonic acid (BPDS) 1 mM 3,-Glutamylglutamate (Glu-Glu) prepared in 0.3% perchloric acid 12j. p. Richie and C. A. Lang, Anal. Biochem. 163, 9 (1987). 13 K. Gohii, C. Viguie, W. C. Stanley, G. A. Brooks, and L. Packer, J. Appl. Physiol, 64, 115 (1988).
[35]
HPLC DETERMINATIONOF GSSG IN BLOOD
369
2 M Potassium hydroxide (KOH) containing 0.3 M 3-(N-morpholino)propanesulfonic acid (MOPS) 1% 1-Fluoro-2,4-dinitrobenzene (FDNB) dissolved in ethanol. Mobile phase A: 80% methanol (HPLC grade), 20% water (HPLC grade) Mobile phase B: Prepared by adding 800 ml of a stock sodium acetate solution to 3.2 liters of solvent A; the stock sodium acetate solution is prepared by adding 1 kg sodium acetate (HPLC grade) and 448 ml of water (HPLC grade) to 1.39 liter of glacial acetic acid (HPLC grade) 8
Sample Preparation 1. Add 0.5 ml of whole blood to 0.5 ml of ice-cold 12% PCA containing 40 mM NEM and 2 mM BPDS. Blood samples must be treated with PCA immediately after extraction from the animal or subject. Mix thoroughly. 2. Centrifuge at 15,000 g for 5 min at 4°. 3. Take 0.5 ml of the acidic supernatant and keep it on ice until derivatization. Samples can also be stored frozen at - 2 0 ° for 1 week.
Derivatization 1. Add 50 tzl of 1 mM glutamylglutamate and 10/xl of a pH indicator solution to 500 tzl of acidic supernatant. 2. Adjust to pH 8.0-8.5 with 2 M KOH containing 0.3 M MOPS to prevent excessive alkalinization. Check the pH after neutralization with a pH meter. 3. Centrifuge samples at 15,000 g for 5 min. 4. Add an aliquot of 25/zl of each supernatant to 50/zl of 1% 1-fluoro2,4-dinitrobenzene in a small glass tube. After 45 min of incubation in the dark at room temperature, the derivatized samples are desiccated under vacuum and stored at - 20° in the dark until injection. Samples processed in this way are stable for several weeks.
Analysis by Chromatography. Samples processed as mentioned above are dissolved in 50/~1 of 80% methanol (mobile phase A) and injected onto the HPLC system. A Spherisorb NH2 column (20 x 0.4 cm, 5 tzm particles) is used. An NH2-~Bondapak column is also suitable for this method. The flow rate is 1.0 ml/min during the procedure. The mobile phases and the gradient are as followsS: solvent A is 80% methanol, and solvent B is 0.5 M sodium acetate in 64% methanol, prepared as described by Fariss and Reed. 8 After injection of 25 ~1 of derivatized solution, the mobile phase is held at 80% A, 20% B for 5 min followed by a 10-min linear gradient up to 1% A, 99% B. 8 The mobile phase is held
370
[35]
A N T I O X I D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
8.0
6°0
.1 0
4.0
2.0 i
0o0
'
0.0
'
I 2.0
'
'
' 4.0
'
I
'
'
i
'
6.0
I 8.0
'
'
' lO.O
I
'
I 12.0
'
~
I 14.0
MINUTES
FIo. 1. Chromatogram of the N-dinitrophenyl derivatives of a blood sample processed and analyzed as described in the text. The retention time for the N-dinitrophenyl derivative of GSSG was 11.78 rain. The G S H - N E M adduct decomposes and appears as three peaks for the corresponding N-dinitrophenyl derivatives (retention times: 3.16, 5.39, 7.02 rain). Ten volts on the y axis is equivalent to 0.05 AOD units at 365 nm.
at 99% B until GSSG has eluted. Using this method, chromatograms such as that shown in Fig. 1 are obtained. The G S H - N E M adduct decomposes and appears as three peaks (see Fig. 1). Thus, GSH must be measured by an enzymatic method, such as the one using glyoxalase7 or glutathione transferasefl 4 in an aliquot to which no NEM has been added. 14 R. Brigelius, C. Muckel, T. P. M. Akerboom, and H. Sies, Biochem. Pharmacol. 32, 2529 (1983).
'
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID
371
Applications When we measured GSH and GSSG levels in blood samples following the method described by Fariss and Reed, 8 we obtained remarkably high GSSG levels. Indeed, GSH/GSSG ratios were 10 or lower for all blood samples assayed. When we added 0.5 ml of 12% PCA containing 2 mM BPDS and 1/zM GSH to 0.5 ml of whole blood and assayed the samples following the method by Fariss and Reed, 8 we found that 23 -- 6% (n = 5) of the GSH present originally in the standard solution was oxidized. When our method was used, the percent of oxidation of an standard GSH solution (1084 nmol/ml) was 0.22 - 0.23% (n = 4). High GSSG levels can be erroneously obtained owing to oxidation of GSH during sample preparation, especially with blood samples. This may lead to erroneous conclusions concerning the pathophysiological changes of glutathione status. For instance, Gohil et al.13 reported GSH/GSSG ratios in blood of about 1. These artifactual increases in GSSG may be important when trying to assess oxidative status. For instance, Gohil et al. 13reported that in humans changes in GSSG in blood during exhaustive exercise were not related to changes in blood lactate. When we repeated these experiments, but using the present method for GSSG determination, we observed an excellent linear relationship between GSSG/GSH and lactate/pyruvate ratios.~5 15 j. Sastre, M. Asensi, E. Gasc6, F. V. Pallard6, J. A. Ferrero, T. Furukawa, and J. Villa, Am. J. Physiol. 32, R992 (1992).
[36] A n t i o x i d a n t A c t i v i t y o f a - T o c o p h e r o l , f l - C a r o t e n e , a n d U b i q u i n o l in M e m b r a n e s : c i s - P a r i n a r i c Acid-Incorporated Liposomes By MASAHIKO TSUCHIYA, VALERIAN E. K A G A N , HANS-JOACHIM FREISLEBEN, MASANOBU MANABE, and LESTER PACKER
Introduction Owing to their high reactivities, oxygen free radicals, which are generated by various biological and chemical processes in vivo, are potentially dangerous to living cells. These radicals can induce oxidative destruction of the polyunsaturated fatty acyl chains of membrane lipids by the processes known as lipid peroxidation. The resultant loss of membrane integMETHODS IN ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress.Inc. All rightsof reproductionin any formreserved.
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID
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Applications When we measured GSH and GSSG levels in blood samples following the method described by Fariss and Reed, 8 we obtained remarkably high GSSG levels. Indeed, GSH/GSSG ratios were 10 or lower for all blood samples assayed. When we added 0.5 ml of 12% PCA containing 2 mM BPDS and 1/zM GSH to 0.5 ml of whole blood and assayed the samples following the method by Fariss and Reed, 8 we found that 23 -- 6% (n = 5) of the GSH present originally in the standard solution was oxidized. When our method was used, the percent of oxidation of an standard GSH solution (1084 nmol/ml) was 0.22 - 0.23% (n = 4). High GSSG levels can be erroneously obtained owing to oxidation of GSH during sample preparation, especially with blood samples. This may lead to erroneous conclusions concerning the pathophysiological changes of glutathione status. For instance, Gohil et al.13 reported GSH/GSSG ratios in blood of about 1. These artifactual increases in GSSG may be important when trying to assess oxidative status. For instance, Gohil et al. 13reported that in humans changes in GSSG in blood during exhaustive exercise were not related to changes in blood lactate. When we repeated these experiments, but using the present method for GSSG determination, we observed an excellent linear relationship between GSSG/GSH and lactate/pyruvate ratios.~5 15 j. Sastre, M. Asensi, E. Gasc6, F. V. Pallard6, J. A. Ferrero, T. Furukawa, and J. Villa, Am. J. Physiol. 32, R992 (1992).
[36] A n t i o x i d a n t A c t i v i t y o f a - T o c o p h e r o l , f l - C a r o t e n e , a n d U b i q u i n o l in M e m b r a n e s : c i s - P a r i n a r i c Acid-Incorporated Liposomes By MASAHIKO TSUCHIYA, VALERIAN E. K A G A N , HANS-JOACHIM FREISLEBEN, MASANOBU MANABE, and LESTER PACKER
Introduction Owing to their high reactivities, oxygen free radicals, which are generated by various biological and chemical processes in vivo, are potentially dangerous to living cells. These radicals can induce oxidative destruction of the polyunsaturated fatty acyl chains of membrane lipids by the processes known as lipid peroxidation. The resultant loss of membrane integMETHODS IN ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress.Inc. All rightsof reproductionin any formreserved.
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ANTIOXIDANT CHARACTERIZATION AND ASSAY
[36]
rity and function is implicated in such pathological conditions as inflammation, diabetes, reperfusion injury, radiation damage, cancer, aging, and neurological diseases. However, in healthy living cells, oxidative processes can be ingeniously intercepted by a network of interacting antioxidants. a-Tocopherol, a lipid-soluble membrane constituent, has been demonstrated to be an essential factor in the cellular antioxidant defense system.2 By donating a hydrogen atom, it functions as an efficient chain-breaking antioxidant that blocks lipid peroxidation. 3 Other biological compounds, including carotenoids and ubiquinones, appear also to play a role in protection of biological membranes against oxygen free radicals. Carotenoids inhibit peroxidation of methyl linoleate and microsomal lipids,4'5 as well as peroxyl radical-initiated fatty acid peroxidation in hexane. 6 Mitochondrial membranes depleted of ubiquinones appear to be more sensitive to oxidative damage by lipid peroxidation inducers,7 and, functioning in its reduced or semireduced form, ubiquinone prevents human low density lipoprotein (LDL) oxidation. 8 Although these natural antioxidants may react directly with oxygen free radicals, it has been suggested that they eliminate radicals indirectly via a recycling process that conserves ct-tocopherol stores. 9 This process may be a very important antioxidant defense system in vivo. The fundamental chemistry of these reactions has been investigated in detail, mainly in homogeneous solutions and/or aqueous dispersions, and is now believed to be fairly well understood. 3A°Such chemical studies, however, are not sufficient to understand antioxidant reactions in vivo, since vital antioxidants function in connection with biomembranes, which compartmentalize the reactions. We have previously demonstrated that antioxidant activity is highly dependent on the environment. 6 The effects of membranes on antioxidant reactions must therefore be taken into consideration. Approaches to this problem include biochemical studies with natural materials such as membranous fractions, whole cells, or tissues,
1 B. Halliwell and J. M. C. Gutteridge, "Free Radicals in Biology and Medicine," 2nd Ed. Oxford Univ. Press (Clarendon), Oxford, 1989. 2 L. Packer, Am. J. Clin. Nutr. 53, 1050S (1991). 3 G. W. Burton and K. U. Ingold, J. Am. Chem. Soc. 103, 6472 (1981). 4 j. Terao, Lipids 24, 659 (1989). 5 p. Palozza and N. I. Krinsky, Free Radical Biol. Med. U , 407 (1991). 6 M. Tsuchiya, G. Scita, H. J. Freisleben, V. E. Kagan, and L. Packer, this series, Vol. 213, p. 460. 7 V. C. Joshi, J. Jayaraman and T. Ranasarma, Biochem. J. 88, 25 (1963). 8 R. Stocker, V. W. Bowry, and B. Frei, Proc. Natl. Acad. Sci. U.S.A. 88, 1646 (1991). 9 V. E. Kagan, E. A. Serbinova, and L. Packer, Arch. Biochem. Biophys. 282, 221 (1990). l0 E. Niki, T. Saito, A. Kawakami, and Y. Kamiya, J. Biol. Chem. 259, 4177 (1984).
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID
373
that is, systems that mimic well in vivo reactions, u,12 Even these methods, however, cannot fully clarify the details of the reactions owing to the lack of reliable methodology under these complex conditions, and there is still an experimental gap between fundamental chemical studies and biochemical studies aimed at understanding the reactions of vital antioxidants in the presence of membranes. One technique that has helped to close this gap is electron spin resonance (ESR) spectroscopy, which can sensitively detect and elucidate short-duration antioxidant reactions with free radicals in the presence of membranes. A drawback is that ESR spectroscopy requires special large-scale equipment. 9,13 Recently, it was demonstrated that a fluorescent polyunsaturated fatty acid, cis-parinaric acid, can be used as a sensitive and reliable reporting molecule for peroxidation in membranes.14 It shows potent fluorescence, as well as high susceptibility to peroxidation by various oxygen free radicals. Attenuation of its fluorescence is a good index of the oxidative stress at the site where cis-parinaric acid is present. In addition, this lipid is readily and easily incorporated into membranes and causes little unfavorable disturbance of the lipid bilayer. 14 Thus, it is considered to be a sensitive and almost ideal probe that allows direct continuous monitoring of oxidative stress in membranes. Moreover, cis-parinaric acid-incorporated liposomal membranes provide a good model for in vivo reactions, although this is a quite simple system. In the present study, we have used this model to investigate the antioxidant activity of a-tocopherol and other natural compounds against peroxyl radicals, major oxygen free radicals that damage biomembranes. Materials and Methods
Chemicals 2,2'-Azobis(2,4-dimethylvaleronitrile) (AMVN) is purchased from Polysciences, Inc. (Warrington, PA) and cis-parinaric acid from Molecular Probes (Junction City, OR). Butylated hydroxytoluene (BHT), fl-carotene, dioleoylphosphatidylcholine (DOPC), isoluminol, and methyl linoleate ubiquinol 10 are from Sigma Chemical Company (St. Louis, MO). 11 R. Ferrari, O. Vesioli, C. Guarnieri, and M. Caldarera, Acta Vitaminol. Enzymol. 5, 11 (1983). 12 C. Michielis, M. Race, and J. Remacle, Arch. Int. Physiol. Biochim. 94, S13 0986). 13 V. E. Kagan, E. A. Serbinova, T. Forte, G. Scita, and L. Packer, J. Lipid Res. 33, 385 0992). 14 F. A. Kuypers, J. J. M. van den Berg, C. Schalkwijk, B. Roelofsen, and J. A. F. Op den Kamp, Biochim. Biophys. Acta 921, 266 (1987).
374
[36]
ANTIOXIDANT CHARACTERIZATION AND ASSAY
a-Tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC) are gifts from Henkel Corp. (LaGrange, IL) and Eisai Co. (Tokyo, Japan), respectively. Other reagents are commercial products of analytical grade.
Rate of Radical Generation by Azo Initiator AMVN, [CN(CH3)z--CH2--C(CH3)CN--N=N--C(CH3)CN-CHz--CH(CH3)z], a diazo compound, is thermally decomposed without enzymes or biotransformation to yield peroxyl radicals as follows15: (I
-
R - - N ~ N - - R - - * R. + N2 + R. --~ 2eR-
e)R--R
+ N2
R. + 02--~ ROO. where e is the efficiency of free radical production. The rate constant, K~, of peroxyl radical generation from AMVN is obtained by the method of Barclay and Ingold 16 and Niki et al. 17 In brief, a known concentration of a chain-breaking phenolic antioxidant, BHT or PMC (both of which have been shown to react with exactly two peroxyl radicals and terminate oxidation chains) is added to a system where oxidation is induced only by the decomposition of AMVN, and the induction period, t~nh, is measured during which oxidation is suppressed. The rate constant kl is given by Eq. (1)16'17: k~ = 2[IN]/tinh[AMVN]
(1)
where [IN] and [AMVN] are the concentrations of PMC (or BHT) and AMVN, respectively, in the system. The oxidation of a sample is measured by monitoring the oxygen consumption of the reaction mixture with an oxygen electrode. The reaction mixture contains 1.67 mM AMVN, 100 mM methyl linoleate, and various concentrations of PMC (or BHT) in 10 mM Triton X-100 solution at 45 °. Concentrations of PMC and BHT are confirmed by spectrophotometric measurement at 292 nm [log(extinction coefficient) = 3.54] and 277 nm [log(extinction coefficient) = 3.34], respectively. 18
Measurement of Fluorescence of cis-Parinaric Acid in Liposomes The DOPC liposomes with incorporated cis-parinaric acid and antioxidants are prepared by sonication of 6 txM cis-parinaric acid, various antiox~5 E. Niki, this series, Vol. 186, p. 100. 16 L. R. C. Barclay and K. U. Ingold, J. Am. Chem. Soc. 103, 6478 (1981). 17 E. Niki, M. Saito, Y. Yoshikawa, Y. Yamamoto, and Y. Kamiya, Bull. Chem. Soc. Jpn. 59, 471 (1986). is R. C. Weast, ed., "Handbook of Chemistry and Physics," 52nd Ed. Chem. Rubber Publ. Co., Cleveland, OH, 1971.
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID
375
idants, and 1.3 mM DOPC dispersion in 20 mM phosphate buffer (pH 7.4) under nitrogen gas at 4°. The oxidation is started by quickly raising the sample temperature to 45 ° just after the incorporation of 300/xM AMVN into the DOPC liposomes by further sonication. The fluorescence intensity is then monitored at an excitation wavelength of 324 nm with a 5 nm slit and an emission wavelength of 413 nm with a 5 nm slit at 45 °, using a Perkin-Elmer (Norwalk, CT) MPF-44A spectrofluorometer, a-Tocopherol and/3-carotene concentrations are determined by high-performance liquid chromatography (HPLC). Ubiquinol 10 is obtained by the reduction of ubiquinone 10 with NaBH 4 in hexane/ethanol solution, and the concentration of the reduced form is spectrophotometrically determined r~-I% ~ L ' I cm = 46.4 at 290 nm). ~9 Concentrations of ascorbate and cis-parinaric acid are determined using the extinction coefficients of 1.45 × 10 3 M - l c m -1 at 265 nm z° and 80 x 10 3 M -~ c m - l at 303 nm (or 74 x 103 M -1 at 318 nm), 2~ respectively.
Measurement of Lipid Hydroperoxides Resulting from Peroxidation of cis-Parinaric Acid with Chemiluminescence Chromatography System A hexane extract of the above DOPC liposomes after 60 min of reaction with AMVN is analyzed by a chemiluminescence HPLC system that can sensitively detect lipid hydroperoxide products. The measurement is performed according to the method developed by Ames and others. 2z The HPLC system consists of a chemiluminescence detector S-3400 (Soma Optic Ltd., Japan), a reversed-phase octadecylsily column No. 235329 (Beckman, San Ramon, CA), a pulseless low pressure pump LPP297 (Lazar Research Laboratories, California) for isoluminol, an HPLC pump 114 M (Beckman) for mobile phase, and an HPLC injector 7125 (Rheodyne, Coati, CA). Samples are chromatographed using methanol as the mobile phase; eluted lipid hydroperoxides are detected by the chemiluminescence reaction with isoluminol. Results
Rate of Radical Generation by Azo Initiator The rate constant of radical generation by AMVN, ki, at 45 ° was obtained from Fig. 1B by using Eq. (I). The experimental value of ~9F. 20 G. 21 L. 22 y .
L. Crane and R. Barr, this series, Vol. 18C, p. 137. R. Buettner, Free Radical Res. Comrnun. 10, 5 (1990). A. Sklar, B. S. Hudson, and R. D. Simoni, J. Supramol. Struct. 4, 449 (1976). Yamamoto, M. H. Brodsky, J. C. Baker, and B. N. Ames, Anal. Biochem. 160, 7 (1987).
376
ANTIOXIDANT CHARACTERIZATION AND ASSAY A
120
[36]
B
+AMVN
60
-
80
40
~"
+AMVN +BHT
0
40
20
.~,oo //-~.VN-I[ ~ 0
0
~
I ~
/
7
"PMC l OBHT
~
I
20 40 60 80 0 2 4 6 8 Time (min) 10 3[IN]/[AMVN]
0
FIG. 1. (A) Rate of oxygen uptake induced by AMVN and induction period by BHT during the oxidation of 100 mM methyl linoleate dispersion at 45° in the presence of l0 mM Triton X-100, 1.67 mM AMVN. (B) Plot of induction period as a function of the ratio of PMC (or BHT) concentration [IN] to AMVN concentration [AMVN]. 4.41 × 10 -6 sec -~ is in agreement with that previously reported by B raughler and Pregenzer at 37 °.23 Thus, the rate of peroxyl radical production by A M V N in this experiment is 1.32 × 10 - 9 M sec -].
Effects of Dioleoylphosphatidylcholine and cis-Parinaric Concentration on Fluorescence The relation between the fluorescence intensity of cis-parinaric acid and the concentration of DOPC is shown in Fig. 2A. The intensity reached a plateau at concentrations greater than 0.8 m M DOPC, indicating incorporation o f whole fractions of probe and minimal collisional interactions of the probe throughout this range. The fluorescence intensity increased linearly with increasing cis-parinaric acid concentration from 0.5 to 6.5 M when the DOPC concentration was in the plateau range (Fig. 2B). This enables estimation of the probe concentration under these conditions.
Time Course and Spectra of Fluorescence Decay of cis-Parinaric Acid Induced by Azo Initiator and Concomitant Lipid Hydroperoxide Production The fluorescence of cis-parinaric acid (excitation at 324 nm and emission at 413 nm) was stable in the absence of A M V N while the measurement 23 j. M. Braughler and J. F. Pregenzer, Free Radical Biol. Med. 7, 125 (1989).
[36]
ASSAY ANTIOXIDANTS WITH
A
ciS-PARINARIC ACID
377
B
S 1oo 80
60 t'-
40
3
zo
w_ 0
I
0.0
I
1.0
2.0
DOPC(rr~)
I
3.0
i
O..0
2.0
i
4.0
6).0
cis-Padnaric Acid (/aM)
FIG. 2. Fluorescence intensity of cis-parinaric acid in DOPC liposomes as a function of DOPC (A) and cis-parinaric acid concentration (B). The reaction mixture contained 6/xM cis-parinaric acid and DOPC as indicated (A), or 1.3 mM DOPC and cis-parinaric acid as indicated (B) in 20 mM phosphate buffer (pH 7.4).
was performed (Fig. 3). With AMVN-incorporated liposomes, the fluorescence decreased to about 20% of maximal intensity in 60 min. The analogous decrease in the fluorescence spectra indicates that the decrease in fluorescence intensity is a result of the actual decomposition and disappearance of cis-parinaric acid. The peroxidation of cis-parinaric acid was further confirmed by chemiluminescence HPLC (Fig. 4). Lipid hydroperoxide with a retention time of 8.9 min was eluted from samples of the fluorescence experiment during the 60-min reaction. It was completely undetected in the absence of AMVN.
Effects of Antioxidants on Azo Initiator-Induced Fluorescence Decrease of cis-Parinaric Acid Various natural antioxidants, such as a-tocopherol,/3-carotene, and ubiquinol 10 (reduced form of ubiquinone 10), that had previously and individually been incorporated into DOPC liposomes prevented cis-parinaric acid fluorescence decay by AMVN in a concentration-dependent
378
A N T I O X I D A NCHARACTERIZATION T AND ASSAY A
B I
I
C ~
I
I
I
I
"~ 100
I
I
/ I
"~ D "~
! ~
I
O0 "~
nin
"~ D
0 min ")
80
..0 80
.*2_
i
CO 60 E nin
C.I +AMVN ~ m 40 C +{x_t ocopherol/X Q) 0to 20 0
--~ U_
[36]
t
/
cco
.*2_ 60 rm
0 40 Q) C GO 20 CO o G :3
+AMVN o
o
0
I
I
20 40 Time (rain)
60
I
270
I
I
I
I
I
I
310 350 380 440 Wavelength (nm)
I
Ii -
-
500
FIG. 3. (A) Time course of cis-parinaric acid fluorescence decay induced by A M V N at 45° (excitation at 324 nm and emission at 413 nm). The reaction mixture consisted of 1.3 mM DOPC liposomes containing 6 i~M cis-parinaric acid and 300/zM AMVN (and 1.3 ~M a-tocopherol if needed) in 20 mM phosphate buffer (pH 7.4). (B) Fluorescence excitation and (C) emission spectra of cis-parinaric acid corresponding to each point of the time course recording. Excitation spectra were scanned with 413 nm emission wavelength, and emission spectra with 324 nm excitation wavelength.
manner (Fig. 3). The protective effect was further confirmed by measuring the generated lipid hydroperoxides by chemiluminescence HPLC (Fig. 4). A lipid hydroperoxides peak (retention time 8.9 min) from cis-parinaric acid peroxidation was progressively inhibited by increasing doses of atocopherol. A large negative peak (retention time 10.8 min) was identified as ot-tocopherol remaining in the sample. Thus, the decrease in the hydroperoxide peak was not due to direct quenching of the chemiluminescence HPLC reaction by residual free ot-tocopherol. The ratio of the initial decay rate of cis-parinaric acid fluorescence in the absence of an antioxidant to that in the presence of an antioxidant, V/VA, is related to the rate constant for peroxyl radical quenching by the antioxidant and to the concentration of the antioxidant by Eq. (2).24: 24y. Kono, M. Takahashi, and K. Asada, Arch. Biochem. Biophys. 174, 454 (1976).
[36]
ASSAY
ANTIOXIDANTS
WITH
¢i$-PARINARIC
4-300/./M AMVN I
,~ ~
ACID
379
+300//MAMVN . +0.3WM~-tocopneroI +300pMAMVN +?pM ~-tocopherol
~
-AMVN I
I
I
I
I
I
I
I
0
2
4
6
8
10
12
14
Time (min) FIG. 4. Chemiluminescencechromatograms of the samplesof Fig. 3 in a 60-rain reaction that contained 6 l.tM cis-parinaric acid, various concentrations of a-tocopherol as indicated, 300/~M AMVN, and 1.3 mM DOPC liposomes in 20 mM phosphatebuffer (pH 7.4). Detailed conditions were described in the Materials and Methods section.
V/V A = 1 + (kA/kpnA[PnA])[A]
(2)
where kA, kpnA, [PnA], and [A] are, respectively, the second-order rate constant for the reaction between the antioxidant and peroxyl radicals, the rate constant for the reaction between cis-parinaric acid and peroxyl radicals from AMVN, the concentration of cis-parinaric acid, and the concentration of the antioxidant. Thus, the slope of the line of V/VA against antioxidant concentration indicates the scavenging potency of the incorporated antioxidant for peroxyl radicals, with cis-parinaric acid as a standard. Of the three antioxidants tested, a-tocopherol was the most effective peroxyl radical scavenger (Fig. 5), which supports previous reports that a-tocopherol is a major and potent natural antioxidant in membranes. 2 From the slopes of the lines, the rate constant for quenching of peroxyl radicals by a-tocopherol was calculated to be 4.4 times larger than that for quenching by fl-carotene, and 4.8 times larger than that for quenching by ubiquinol 10.
380
A N T I O X I D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
[36]
4.0
>
3.0
2.0
1.0
, 0.0
, 1.0
, 2.0
3.0
Concentration (~M) FIG. 5. Relationship between V~VA and antioxidant concentration. V is the rate of initial fluorescence decay of cis-parinaric acid in the absence of antioxidants; VA, the rate of initial fluorescence decay of cis-parinaric acid in tile presence of the antioxidants indicated. For experimental conditions, see text. ([]) ~-Tocopherol; (A) fl-carotene; and (11) Q10 (ubiquinol 10, the reduced form of ubiquinone 10).
Interaction of a-Tocopherol with Other Antioxidants Because this system is simple and allows easy estimation of kinetics in the presence of membranous components, it was used to investigate the interaction of ct-tocopherol with other natural antioxidants, which is still a major question in in vivo antioxidant reactions. The addition of ascorbate or ubiquinol 10 to the a-tocopherol-incorporated liposomes increased the slope of V/VA, indicating that the apparent rate constant of ot-tocopherol for quenching peroxyl radicals became larger, whereas the addition of/3-carotene did not change the slope, indicating that/3-carotene did not alter the apparent rate constant of o~-tocopherol (Fig. 6). Discussion
Use of DOPC liposomes possessing cis-parinaric acid as a probe for oxidative stress, AMVN as a lipid-soluble radical initiator, and an antioxidant has provided a good model for the reaction of antioxidants with oxygen free radicals in biological membranes. This assay system, which enables continuous reaction monitoring with simple equipment, has potentially wide applications for investigation of fundamental antioxidant reactions.
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID A
C
B I
I
I
i
+1.3 uM I}-carotene
3.0-
381
T Jj/
Q1O
/J.
2.0
1.0~ 0 I0
'
'
015
1.0
I
0.0
0.5
I
I
1.0
0.0
0.S
I
1.0
1.5
c~-Tocopherol (HM) FIo. 6. Effect of (A) ascorbate, (B)/3-carotene, and (C) Ql0 (ubiquinol 10, the reduced form of ubiquinone 10) on the plot of V~VA against a-tocopherol concentration. Experimental conditions are described in the text.
The natural lipid-soluble compounds a-tocopherol, r-carotene, and ubiquinol 10 are expected to function differently as antioxidants in vivo, according to the differences in molecular structure (Fig. 7). Because it possesses a phenol group, ct-tocopherol scavenges free radicals by donating a hydrogen atom to them. 3 Ubiquinol 10 is expected to function in a similar manner. 25 The scavenging mechanism of/3-carotene is still unclear. 26 Besides, several fragmented products that may be harmful to membranes have been reported after the reaction of/3-carotene with free radicals. 27 Tsuchiya et al. 6 have reported that the conjugated double bond structure between rings is important for scavenging activity. The results presented here confirm that/3-carotene and ubiquinol, as well as a-tocopherol, are able to prevent oxidation of membranous components. However, the effects of r-carotene and ubiquinol are approximately 5 times weaker than those of a-tocopherol. It is surprising that a-tocopherol efficiently protects membranes against oxidation despite its low concentration in membranes. 28 There have been 25 V. Kagan, E. Serbinova, and L. Packer, Biochem. Biophys. Res. Commun. 169, 851 (1990). 26 N. I. Krinsky, Free Radical Biol. Med. 7, 617 (1989). 27 G. J. Handelman, F. J. G. M. van Kuijk, A. Chatterjee, and N. I. Krinsky, Free Radical Biol. Med. 10, 427 (1991). z8 j. j. Maguire, V. Kagan, B. A. C. Ackrell, E. Serbinova, and L. Packer, Arch. Biochem. Biophys. 292, 47 (1992).
382
[36]
ANTIOXIDANT CHARACTERIZATION AND ASSAY
o~-Tocopherol CH 3 ]
H~
HO"
~
_ CH3
CH 3
CH3
.CH3
v
CH 3
I~-Carotene H3C
H3c\/cH3 I
/
CH 3
/
CH3
11
II
__ I
__ L
~.,,.,,,,';~CH 3
H3C
H3C
1
n3c cn3
Ubiquinol 10 OH
CH30~CH3.~
°.30- y
o.
Iv[
_ I'"
~.3j, °
FIG. 7. Molecular structures of a-tocopherul,/3-carotene, and ubiquinol 10.
several reports suggesting that some antioxidant such as ascorbate or ubiquinol can regenerate tocopherol that has been oxidized by reacting with free radicals and thereby maintain the apparent concentration of tocopherol (recycling tocopherol). 9A°'25'29 The potent protective effect of tocopherol as an antioxidant in biomembranes may be partly explained by such a recycling mechanism. The apparent increase in the rate constant of a-tocopherol by ascorbate or ubiquinol 10 provides evidence for atocopherol recycling. Ubiquinol 10 affected V/VA even at the absence of t~-tocopherol but ascorbate did not, which suggests that ubiquinol 10 scavenges radicals, as well as recycles a-tocopherol in membranes, but ascorbate only recycles a-tocopherol. On the other hand, E-carotene did not change the apparent rate constant of a-tocopherol, which suggests 29 j. j. M. van den Berg, F. A. Kuypers, B. Roelofsen, and J. A. F. Op den Kamp, Chem. Phys. Lipids 53, 309 (1990).
[36]
ASSAY ANTIOXIDANTS WITH ciS-PARINARIC ACID
383
the absence of interaction. These two antioxidants probably function independently in membranes. It can be supposed that the production of peroxyl radicals in the presence of excess cis-parinaric acid leads to the first-order decrease in fluorescence in the initial stage of the reaction, under the assumption that the chain reaction is negligible at this stage since interaction of each molecule is highly restricted by incorporation into the membranes. 3° Based on this assumption, the apparent first-order rate constant was obtained from the inverse half-time of fluorescence decay, and the second-order rate constant for the reaction, calculated from the dependence of the first-order rate constants on the concentration of cis-parinaric acid, was estimated to be 1.32 × 103 M -1 s e c -1. Using Eq. (2) to describe the competition of cis-parinaric acid and antioxidants for peroxyl radicals (Fig. 5), secondorder rate constants for the reaction of the antioxidants a-tocopherol,/3carotene, and ubiquinol I0 with peroxyl radicals were estimated to be 8.76 × 103, 1.97 × 103, and 1.84 × 103 M -1 sec -1, respectively. These values are smaller than those previously reported in the literature (e.g., ranging from 0.8 to 230 × 105 M -1 sec -~ for a-tocopherol), which were chemically determined in solution or suspension. 31 Niki et al. 32 reported a similar phenomenon, namely, that the rate constant of a-tocopherol in liposomal membranes was approximately 50 to I00 times less than that in homogeneous solution. They report that a possible reason for this difference is the restriction of antioxidant mobility resulting from the incorporation of tocopherol into membranes. 32 Using analogous compounds, we also found that mobility has a significant impact on the activity of antioxidants in membranes. 33Thus, the effect of membranes on antioxidant behavior appears to be another important factor for evaluating antioxidant activity, although the pure chemical reactivity of an antioxidant with radicals is certainly of great importance. Acknowledgments This work was supported by a grant from the National Institutes of Health (CA 47597) and by the Foundation for Total Health Promotion (1992). The authors thank Dr. Kozo Utsumi (Kochi Medical School) for valuable suggestions.
3o M. Takahashi and K. Asada, J. Biochem. (Tokyo) 91, 889 (1982). 3t E. Niki, Chem. Phys. Lipids 44, 227 (1987). 32 E. Niki, M. Takahashi, and E. Komuro, Chem. Lett. p. 1573 (1986). 33 E. Serbinova, V. Kagan, D. Han, and L. Packer, Free Radical Biol. Med. 10, 263 (1991).
384
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[37]
[37] S i n g l e t O x y g e n Q u e n c h i n g b y C a r o t e n o i d s By ALFRED
R.
SUNDQUIST, KARLIS BRIVIBA,
and
H E L M U T SIES
Introduction The quenching of singlet molecular oxygen [Oz(1Ag),abbreviated below as IO2] by carotenoids was first demonstrated by Foote and Denny ~using a chemical competition technique which measured the inhibition by/3carotene of the 102-dependent photooxygenation of 2-methyl-2-pentene. Since then considerable attention has been given to the antioxidant activities of carotenoids and oxycarotenoids (xanthophylls), 2,3 and a variety of additional chemical and physical techniques to evaluate 102 quenching have been described. Other chemical competition techniques determine the degree to which the test carotenoid inhibits the autosensitized 4'5 or methylene blue-sensitized 6 photooxidation of rubrene, or the chlorophyll-sensitized photooxidation of soybean oil. 7 Bleaching of the quencher has been used to measure the 10 2 quenching activity of the pigments crocin8'9 and bixin) ° Physical techniques are based on spectral properties of the excited species involved in the quenching process. For example, quenching can be measured as a decrease in the level or lifetime of 102 photoemission [reaction (I)] using 102 ~ 02 + hv (1270 nm)
(1)
sensitive infrared detectors. 11With some methods, 102 is produced continously either with a chemicaP 2 or photochemicaP 3 source, and the effect I C. S. Foote and R. W. Denny, J. Am. Chem. Soc. 9t), 6233 (1968). 2 N. I. Krinsky, Free Radical Biol. Med. 7, 617 (1989). 3 p. Di Mascio, M. E. Murphy, and H. Sies, Am. J. Nutr. 53, 194S (1991). 4 S. R. Fahrenholtz, F. H. Doleiden, A. M. Trozzolo, and A. A. Lamola, Photochem. Photobiol. 20, 505 (1974). s D. J. Carlsson, T. Suprunchuk, and D. M. Wiles, J. Polym. Sci. Part B U , 61 (1973). 6 M. M. Mathews-Roth, T. Wilson, E. Fujimori, and N. I. Krinsky, Photochem. Photobiol. 19, 217 (1974). 7 S.-H. Lee and D. B. Min, J. Agric. Food Chem. 38, 1630 (1990). 8 W. Bors, C. Michel, and M. Saran, Biochim. Biophys. Acta 796, 312 (1984). 9 p. Manitto, G. Speranza, D. Monti, and P. Gramatica, Tetrahedron Lett. 28, 4221 (1987). l0 G. Speranza, P. Manitto, and D. Monti, J. Photochem. Photobiol., B. 8, 51 (1990). II A. A. Gorman and M. A. J. Rodgers, J. Photochem. Photobiol,, B 14, 159 (1992). 12 p. Di Mascio, S. Kaiser, and H. Sies, Arch. Biochem. Biophys. 274, 532 (1989). 13 E. Oliveros, P. Murasecco-Suardi, A. M. Braun, and H.-J. Hansen, this series, Vol. 213, p. 420.
METHODS IN ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[37]
SINGLET OXYGEN QUENCHING BY CAROTENOIDS
385
of the carotenoid on the steady-state level of photoemission is determined. In another method, the carotenoid-dependent decrease in the lifetime of a pulse of photochemically generated 10 2 is measured by time-resolved luminescence spectroscopy. 14 Rate constants have been also determined by spectrophotometrically monitoring the triplet excited carotenoid (3C) formed during the quenching process [reaction (2)]. 15-17 1 0 2 q- C ~
(2)
0 2 q- 3C
In this chapter we describe in more detail the technique 12which makes use of a germanium photodiode to monitor 10 2 photoemission and a thermodissociable endoperoxide to generate a steady-state level of 10 2.18
Method
Reagents The endoperoxide of 3,3'-(l,4-naphthylidene) dipropionate (NDPO2) decomposes at moderate temperatures to yield the parent compound, NDP, and molecular oxygen, a portion of which (-50%) is in the singlet excited state [reaction (3)]. 19,20A convenient method to synthesize NDPOz in high yield is to incubate a solution of NDP 2°'21with HEOz in the presence
2No00C~ooc~ ~~ "~ 37°Ci ~aO0~ No NaOOC NDP02
'-'1"- 02 + 102 (3)
NDP
of sodium molybdate, 19,2° afterward precipitating the NDPO 2 with acid. Samples of carotenoids have been generously provided by Dr. J. Bausch (F. Hoffmann-La Roche, Basel, Switzerland). 14 p. F. Conn, W. Schalch, and T. G. Truscott, J. Photochem. Photobiol., B 11, 41 (1991). 15 A. Farmilo and F. Wilkinson, Photochem. Photobiol. 18, 447 (1973). 16 F. Wilkinson and W.-T. Ho, Spectrosc. Lett. 11, 455 (1978). i7 M. A. J. Rodgers and A. L. Bates, Photochem. Photobiol. 31, 533 (1980). 18 p. Di Mascio, A. R. Sundquist, T. P. A. Devasagayam, and H. Sies, this series, Vol. 213, p. 429. 19 j. M. Aubry, J. Am. Chem. Soc. 107, 5844 (1985). 2o p. Di Mascio and H. Sies, J. Am. Chem. Soc. 111, 2909 (1989). 21 R. Saint-Jean and P. Cannone, Bull. Soc. Chim. Ft., p. 3330 (1971).
386
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[37]
Assay Carotenoid solutions are freshly prepared in amber-colored vials with Nz-purged chloroform and kept on ice. Stock solutions of NDPO2 are made by neutralizing the free acid (see above) with NaOH in DzO18; the solution is stored at - 7 0 ° as small aliquots and thawed as needed. For reasons of solubility a mixed assay solvent [chloroform-ethanol, 1 : 1 (v/v)] is preferred. The 10 z quenching assays are conducted with a liquid nitrogen-cooled germanium diode (North Coast Scientific Co. Model EO-817L, Santa Rosa, CA) attached to a sample chamber.18 The assays are carried out in a cuvette (35 × 6 × 55 mm) placed in a thermostatted (37 °) holder. The signal is processed with a lock-in amplifier and monitored continuously with a chart recorder. After recording the baseline with the assay solvent, NDPO2 is added to the cuvette and the photoemission followed until a maximum (So) is reached; immediately thereafter the carotenoid is added, and the resulting level of photemission (S) is recorded. The assays are repeated over a range of carotenoid concentrations (e.g., 0.2-10 tzM).
Calculations Quenching rate constants are determined graphically by plotting the degree of quenching (i.e., the ratio So~S)versus the carotenoid concentration. The slope of this plot (Stern-Volmer) is equivalent to (kq + kr) ~', namely, the sum of the rate constants for physical quenching and chemical reaction multiplied by the lifetime of l o 2 (33 /zsec in the present assay solvent). The contribution of chemical reaction to the quenching of 10 2 by carotenoids is minor ( retinoid 5,6-epoxide + LO-
(11)
This can also be considered an autoxidative pathway for retinoid oxidation, because it does not lead to a net radical consumption. The antioxidant activity of retinoids is seen as the net effect of all these competing reactions. 25 V. M. Samokyszyn and L. J. Marnett, J. Biol. Chem. 262, 14119 (1987). 26 V. M. Samokyszyn and L. J. Marnett, this series, Vol. 190, p. 282. 27 G. W. Burton and K. U. Ingold, Science 224, 569 (1984). R. Stocker, Y. Yamamoto, A. F. McDonagh, A. N. Glazer, and B. N. Ames, Science 235, 1043 (1987) V. M. Samokyszyn and L. J. Marnett, Free Radical Biol. Med. 8, 491 (1990). 30 R. Yamauchi, N. Miyake, K. Kato, and Y. Ueno, Biosci. Biotechnol. Biochem. 56, 1529 (1992).
[39]
ANTIOXIDANT
ACTIVITY OF VITAMIN
25"
.......
407
A
-100
I I. . . . . . . . . . .
-"J
I I I
#1 II II I
12.5"
Z
-50
AB
I1','
30
O m
4b
s'o
"T 0
60
.,A g
15-
,..........
100
O Z
u._
-r
? o p
7.5-
i¢ //i
fl
,
1
,50
I" IM
X
~E
In
el_ u
0 15-
_
I Jl 30
40
.. o
50 ,- . . . . . . . . . .
60
Z Ill U
100
C
ZS-
0
30 FRACTION
,50
40
5'0
0 60
NUMBER
FIG. 1. Reversed-phase HPLC separation of all-trans-[3H]retinol (solid bar) and metabolites (open bars) generated during the reaction of 16 i x M all-trans-[3H]retinol with linoleic acid-derived peroxyl radicals. The elution system consisted of a linear methanol-water gradient from 30 to 90% methanol over 30 min, followed by isocratic elution with 90% methanol for 20 min, then with 100% methanol for additional 10 min, at a flow rate of 1.0 ml/min. Fractions of 1.0 mi are collected and the radioactivity measured. (a) Zero time; (b) 10 min incubation; (c) 20 min incubation.
408
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[39]
Partitioning of the retinoid carbon-centered intermediate between competing antioxidant and autoxidative pathways may also govern the prooxidant and antioxidant activity of all-trans-retinol, since the shift of the equilibrium of reaction (10) in favor of the formation of retinoid-derived peroxyl radical will actually increase the steady-state concentrations of peroxyl radicals. Under the experimental conditions described, autoxidation reactions of all-trans-retinol with Oz do not seem to occur nor to affect the antioxidant efficacy of all-trans-retinol. Given the autocatalytic nature of the peroxidative chain reactions that would be set in motion by the retinoid-derived peroxyl radical, compared to the 1 : 1 stoichiometry of the inhibition reaction [Eq. (9)], the occurrence and linear dependence of lag times on antioxidant concentrations (Table I) could not be observed. On the other hand, since the measured stoichiometric factor is less than 1, it appears evident that even when lag times are linearly dependent on the retinol concentration, some retinol is consumed without a net radical consumption, possibly via epoxide formation. Kinetics of Peroxyl Radical-Scavenging Activity of Natural and Synthetic Retinoids Oxidation of linoleic acid methyl ester in methanol may be employed to measure the lipoperoxyl radical-scavenging activity of vitamin A analogs. Table II shows the inhibition kinetic parameters obtained with some natural and synthetic retinoids. Retinoids are chosen and assayed on the basis of the solubility in methanol, all-trans-Retinyl palmitate and all-trans-
TABLE II INHIBITION KINETIC PARAMETERSOF OXIDATION OF LINOLEIC ACID METHYL ESTER IN METHANOL BY VITAMIN A ANALOGS
Inhibitor
all-trans-Retinyl palmitate all-trans-Retinoic acid 13-cis-Retinoic acid Acitretin Etretinate Temarotene Ro 15-1570
Concentration (/~M)
LAME (raM)
AMVN (mM)
10 2 2 10 10 10 10
315 315 315 315 315 315 315
3.0 3.0 3.0 3.0 3.0 3.0 3.0
tinh (sec)
780 1320
108 Rinh (M sec -I)
10 -5 kinh (M-Isec -I) n
No inhibition* 5.5 6.3 0.7 28.8 No inhibition No inhibition No inhibition No inhibition
2.7 4.0
* Since the Sigma compound contains 1% butylated hydroxytoluene (BHT), reference assays must be performed with the same amount of BHT. When corrected for the effect of BHT itself, no antioxidant activity of all-trans retinyl palmitate was evident.
[39]
A N T I O X I D A N T ACTIVITY OF VITAMIN A
409
"13
¢5 e-
0.)
:.=
).,
e'~
,.d "e
z
R - - . C H - - O H + H20
(2) (3)
Owing to the high reactivity of .OH radicals, such reactions occur quite randomly, 25,66,67 and it is understandable that the subsequent addition of O2 to these alkyl radicals by a diffusion-controlled reaction 68'69 leads to various isomeric peroxyl radicals: R - - . C H - - R ' - - C O O H + O2--->R - - C ( H ) O O - - - R ' - - C O O H R--.CH---OH + O2 ~ R - - C ( H ) O O . - - O H
(4) (5)
Aside from being scavenged by antioxidants, peroxyl radicals can undergo competing decay reactions, if concentration and/or reactivity of the antioxidant are low. Fatty acid peroxyl radicals have been calculated to decay in bimolecular reactions with rate constants of 2-4 x 108 M -~ sec-~. 25 The fate of isopropyl peroxyl radicals is more complicated and depends on pH and on the radical concentration (i.e., pulse dose). Both a spontaneous first-order decay and a base-catalyzed reaction lead to the liberation of 02- (and acetone): (CH3)aC(OH)OO" ~ 02- + H + + ( C H 3 ) 2 C = O + O H - --~ O2~ + H 2 0 + ( C H 3 ) 2 C - ~ - O
(CH3)zC(OH)OO"
(6) (7)
albeit with widely differing rate constants, k6 being 6.5-7.0 x 102 sec -~ and k7 being 5 x l0 9 M -I s e c - l . 70,71 The same products are formed in 66 M. G. J. Heijman, A. J. P. Heitzman, H. Nauta, and Y. K. Levine, Radiat. Phys. Chem. 26, 83 (1985). 67 K. D. Asmus, H. M6ckel, and A. Henglein, J. Phys. Chem. 77, 1218 (1973). 68 G. E. Adams and R. L. Willson, Trans. Faraday Soc. 65, 2981 (1969). 69 A. Marchaj, D. G. Kelley, A. Bakac, and J. H. Espenson, J. Phys. Chem. 95, 4440 (1991). 70 y . Ilan, J. Rabani, and A. Henglein, J. Phys. Chem. 80, 1558 (1976). 71 E. Bothe, G. Behrens, and D. Schulte-Frohlinde, Z. Naturforsch. B: Anorg. Chem., Org. Chem. 32B, 886 (1977).
[41]
SCAVENGING OF PEROXYL RADICALS BY FLAVONOIDS
a reaction catalyzed by phosphate buffer (Ilan M-I
et
427
a/.7°; k8 = 1.1 x 107
sec-1):
(CH3)zC(OH)OO" + HPO42- ~ 027 + H2PO 4- + (CH3)2C=O
(8)
In addition, second-order decay may also occurT°: (CH3)2C(OH)OO-
0 2 q"- 2 ( C H 3 ) z C ( O H ) O . O 2 + (CH3)zC(OH)--OO--C(OH)--(CH3)
2
(9) Because peroxyl radicals lack a distinctive absorption spectrum, the scavenging reaction by flavonoids can only be determined from the buildup of the absorption of the flavonoid aroxyl radical. 25 Using this approach, absolute rate constants of flavonoids with isopropyl peroxyl radicals 65 and with linoleyl peroxyl radical isomers 25 have been determined (Table I). An alternative generation system for peroxyl radicals has been established to determine whether the various peroxyl radical isomers, produced by the random attack of .OH radicals at linoleic acid [reactions (2) and (4)], are kinetically equivalent, z5 In NzO-saturated solutions containing
TABLE I RATE CONSTANTS OF SELECTED FLAVONOIDS WITH PEROXYL RADICALSa
Rate constant (x 108 M -1 sec -I) Substrate b Flavonol Kaempferol (3,5,7,4') Quercetin (3,5,7,3',4') Flavone Apigenin (5,7,4') Luteolin (5,7,3' ,4')
R(OH)OO.
n-LOO.
13-LOO-
19.5 2.1
0.34 0.18
0.42 0.15
n.d. c n.d.
n.d. n.d.
0.77 1.85
a Kinetic evaluation of aroxyl radical formation after pulse-radiolytic generation of peroxyl radicals: R(OH)OO., isopropylperoxyl radicals at pH 8.5-9.065, n-LOO., linoleic acid peroxyl radical isomers in N20/O2 system, pH 11.5; 13-LOO., 13-hydroperoxyl linoleic acid radical in N20/N 3- system, pH 11.5. 25 b Numbers in parentheses denote positions of hydroxyl groups. c n.d., Not determined.
428
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[41]
sodium azide, all .OH radicals obtained in reaction (1) are converted to azide radicals: •O H + N 3- ~
0o)
"N 3 + O H -
Azide radicals are highly electrophilic but are more restricted in their reactivity than .OH radicals. Thus, they are unable to abstract aliphatic H atoms from fatty acids at an appreciable rate 72 and react preferentially by electron transfer. 73 In the specific case of enzymatically produced fatty acid hydroperoxides, the dissociation of the hydroperoxy group with a pK value above 12TM facilitates a direct oxidation of this group by .N 3 radicals in strongly alkaline solutions25: R--C(H)OO---CH2--COO-
+ "N 3 R--C(H)OO'--CH2--COO-
+ N 3-
(11)
These conditions furthermore allow the use of millimolar concentrations of fatty acids in homogeneous solutions, as at lower pH micelle formation and consequently lower solubility prevails. 72It turned out that these selectively generated 13-hydroperoxyllinoleic acid radicals were kinetically quite similar to the random mixture of peroxyl radical isomers, obtained in reactions (2) and (4). 25 This obviates the need of using the latter, more complicated and more limited, system.
Conclusions The determination of the reactivities of flavonoid antioxidants as well as the identification of the corresponding radical structures 24,75are worthwhile endeavors as several open questions beckon to be answered. First, do flavonoids also function as antioxidants in plant tissue itself? Second, should secondary reactions of the flavonoid aroxyl radicals occur with lipid moieties (on time scales too slow for pulse-radiolytic observation), such an initiation of chain reactions would diminish the apparent antioxidao tive potential of these polyphenols. A third area of potential surprises concerns the unresolved interactions of flavonoids or their aroxyl radicals 72 M. Erben-Russ, W. Bors, R. Winter, and M. Saran, Radiat. Phys. Chem. 27, 419 (1986). 73 Z. B. Alfassi and R. H. Schuler, J. Phys. Chem. 89, 3359 (1985). 74 L. S. Silbert, in "Organic Peroxides" (D. Swern, ed.), Vol. 2, p. 637. Wiley (Interscience), New York, 1970. 75 W. Bors, W. Heller, C. Michel, and K. Stettmaier, in "Free Radicals: From Basic Science to Medicine" (G. Poli, M. Albano, M. U. Gianzani, eds.), p. 374. Birkhaeuser, Basel (1993).
[42]
ASSAYS FOR C O N D E N S E D T A N N I N S
429
with other biological cofactors, such a s ascorbate 76,77 or glutathione. A reminder of the complexities of flavonoid structure-activity studies is the fact that similar structural requirements seem to exist for various functions of flavonoids, some of which may not involve radical intermediates at all. 65 76 A. Bentsath, S. Rusznyak, and A. Szent-Gy6rgyi, Nature (London) 139, 326 (1937). 77 A. Negre-Salvayre, A. Affany, C. Hariton, and R. Salvayre, Pharmacology 42, 262 (1991).
[42] A s s a y o f C o n d e n s e d T a n n i n s or F l a v o n o i d O l i g o m e r s a n d R e l a t e d F l a v o n o i d s in P l a n t s
By ANN E. HAGERMAN and LARRY G. BUTLER Introduction The flavonoids are a diverse group of plant phenolics based on a 15carbon skeleton. The chemistry of the simple flavonoids is well known, and reviews of their structures, reactions, and distribution in higher plants are available. 1 The discussion in this chapter is restricted to the flavonoid oligomers and polymers known as condensed tannins, and a few closely related flavonoids. The condensed tannins (I) are formally polymers of flavan-3-ols such as catechin (II). The probable biosynthetic precursors of the condensed tannins are flavan-3,4-diols, or leucoanthocyanidins, such as catechin4fl-ol (111).2 Oxidative degradation of condensed tannins yields the corresponding anthocyanidins, such as cyanidin (IV), so that the condensed tannins are sometimes called proanthocyanidins. The pattern of hydroxyl-
oLO. vOH v ~OH I
8
6~
°"
O H OH 4
II
I R. J. Grayer, Methods Plant Biochem. 1, 283 (1989). 2 H. A. Stafford, "Flavonoid Metabolism." CRC Press, Boca Raton, Florida, 1990.
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
[42]
ASSAYS FOR C O N D E N S E D T A N N I N S
429
with other biological cofactors, such a s ascorbate 76,77 or glutathione. A reminder of the complexities of flavonoid structure-activity studies is the fact that similar structural requirements seem to exist for various functions of flavonoids, some of which may not involve radical intermediates at all. 65 76 A. Bentsath, S. Rusznyak, and A. Szent-Gy6rgyi, Nature (London) 139, 326 (1937). 77 A. Negre-Salvayre, A. Affany, C. Hariton, and R. Salvayre, Pharmacology 42, 262 (1991).
[42] A s s a y o f C o n d e n s e d T a n n i n s or F l a v o n o i d O l i g o m e r s a n d R e l a t e d F l a v o n o i d s in P l a n t s
By ANN E. HAGERMAN and LARRY G. BUTLER Introduction The flavonoids are a diverse group of plant phenolics based on a 15carbon skeleton. The chemistry of the simple flavonoids is well known, and reviews of their structures, reactions, and distribution in higher plants are available. 1 The discussion in this chapter is restricted to the flavonoid oligomers and polymers known as condensed tannins, and a few closely related flavonoids. The condensed tannins (I) are formally polymers of flavan-3-ols such as catechin (II). The probable biosynthetic precursors of the condensed tannins are flavan-3,4-diols, or leucoanthocyanidins, such as catechin4fl-ol (111).2 Oxidative degradation of condensed tannins yields the corresponding anthocyanidins, such as cyanidin (IV), so that the condensed tannins are sometimes called proanthocyanidins. The pattern of hydroxyl-
oLO. vOH v ~OH I
8
6~
°"
O H OH 4
II
I R. J. Grayer, Methods Plant Biochem. 1, 283 (1989). 2 H. A. Stafford, "Flavonoid Metabolism." CRC Press, Boca Raton, Florida, 1990.
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All fights of reproduction in any form reserved.
430
[42]
ANTIOXIDANT CHARACTERIZATION AND ASSAY OH
HO
HO
"~H T
"OH
0 OH
OH
III
IV
ation on the A and B rings of the flavonoids is variable, yielding structural diversity among the condensed tannins) Structural complexity also results from the diversity of positions for polymerization of the parent flavan-3,4-diols, and from the possible stereochemical variations in the interflavan bonds. For example, simple linear condensed tannins such as the 4 ~ 8-1inked procyanidin B-1 (I) are common in some plants, but species rich in 5-deoxyflavan-3-ols produce branched chain polymers such as the profisetinidins (e.g., V). Some compounds with multiple interflavan bonds, such as proanthocyanidin A-2 (VI), have been isolated. Stereochemical diversity is illustrated by the series of procyanidin dimers B-1 (I), B-2 (VII), B-3 (VIII) and B-4 (IX). The condensed tannins commonly isolated from plant tissues and used in most studies of the biological effects of tannins contain not only oligomers like those depicted here but also less well-characterized polymers. Degrees of polymerization as high as 50 have been claimed for some condensed tannins.4 OH
O
OH
OH
o.
0~ ]
0H
OH
HO
V
VI
3 R. W. Hemingway, in "Chemistry and Significance of Condensed Tannins" (R. W. Hemingway and J. J. Karchesy, eds.), p. 83. Plenum, New York, 1989. 4 V. M. Williams, L. J. Porter, and R. W. Hemingway, Phytochemistry 22, 569 (1983).
[42]
ASSAYS FOR CONDENSED TANNINS
431
r~"~OH
HOe"IT OH VII
OH
H
O
~
~
OH
OH
OH
VIil
IX
Condensed tannins can be isolated from a wide variety of plant species and tissues. Among the most common sources of tannins used in laboratory studies are grain from Sorghum bicolor and extracts from the bark of quebracho (Schinopsis spp.). The tannin from Sorghum grain is largely comprised of 4 --~ 8-1inked procyanidin polymers similar to B-1 (I), and that from quebracho is a complex mixture of profisetinidins (V). The condensed tannins are structurally and chemically distinct from the hydrolyzable tannins (gallotannins and ellagitannins),5 so the commercial gallotannin known as tannic acid is an inappropriate material to use as a standard in studies of condensed tannins.
Extraction and Purification of Condensed Tannins Fresh, frozen, or lyophilized tissue can be extracted, although in general fresh material yields the best results. Sample treatment does affect phenolic extractability, so all samples should be treated similarly. 6 To obtain sufficient quantities of tannin for routine characterization, about 2 g dry weight of the tissue is needed. The method can be scaled down for analytical work. The ground or homogenized plant tissue is extracted with 10 volumes of 70% acetone (acetone/water, 70:30, v/v) and is sonicated for 30 min at 4°. The sample is centrifuged and the supernatant is stored at 4 °. The extraction is repeated three more times, and the extracts are pooled to obtain an overall recovery of about 90% of the total extractible phenolics in the tissue. The first two extracts usually contain about 75% of the total extractable phenolics. Tannins are usually purified by taking advantage of their sorption by Sephadex LH-20. 7 The acetone is removed from the crude plant extract 5 E. Haslam, "Plant Polyphenols: Vegetable Tannins Revisited." Cambridge Univ. Press, Cambridge, 1989. 6 A. E. Hagerman, J. Chem. Ecol. 14, 453 0988). 7 D. H. Strumeyer and M. J. Malin, J. Agric. Food Chem. 23, 909 (1975).
432
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[42]
and replaced by a smaller volume of ethanol before applying the sample to the Sephadex, which is equilibrated in ethanol. Nontannin phenolics are eluted with ethanol while monitoring absorbance at 280 nm. The tannin is then recovered by elution with aqueous acetone. The adsorption can be done either batchwise, for purification of large quantities of tannin, or in a column, for separation of individual tannin polymers. This method can be used either to purify tannin from the plant source of interest, or to purify well-characterized condensed tannins that are to be used as analytical standards. Quebracho tannin is commercially available as a crude powder which contains 30-50% nontannin materials. The material can be obtained in bulk from Trask Chemical Corporation [3200 W. Somerset Court, Marietta, GA 30067; (404) 955-9190] or from Tannin Corporation [60 Pulaski Street, P.O. Box 606, Peabody, MA 01960; (617) 532-4010]. Small amounts of quebracho tannin to be used for analytical standards can be obtained from Dr. Ann Hagerman (Department of Chemistry, Miami University, Oxford OH 45056). Sorghum tannin is often a more useful standard than quebracho tannin, because it gives higher color yields in several of the methods used to determine tannin. Quebracho tannin and Sorghum tannin can be purified by a modification of the Sephadex LH-20 method described above. 8,9
General Phenolic Assays M a n y methods for determining total phenolics have been described. These methods rely on the chemistry of the phenolic group and are thus not specific for tannins, but they provide insight into the levels of total (tannin plus nontannin) phenolics in plant extracts. A m o n g the most comm o n general phenolic methods are a variety of spectrophotometric redox assays, which detect the easily oxidized phenolic group. Redox methods are subject to interference by many compounds; of particular importance are the antioxidants often added to plant extracts, such as ascorbic acid. One widely used rcdox method is the Folin (Lowry, Folin-Denis) method) ° W e prefer the Prussian blue method, H since it is less subject to interference from nonphenolic compounds than is the Folin method.~2 For routine analysis using the Prussian blue method, dispense 0.10ml samples into 125-mi Erlenmeyer flasks and add 50.0 ml distilledwater. To each sample, add 3.0 rnl of 0.10 M FeNH4(SO4)2 in 0. I M HCI and s T. N. Asquith and L. G. Butler, J. Chem. Ecol. 11, 1535 (1985). 9 A. E. Hagerman and L. G. Butler, J. Agric. Food Chem. 28, 947 (1980). 10T. Swain and W. E. Hillis, J. Sci. Food Agric. 10, 63 (1959). 11 M. P. Price and L. G. Butler, J. Agric. Food Chem. 25, 1268 (1977). t2 A. E. Hagerman and L. G. Butler, J. Chem. Ecol. 15, 1795 (1989).
[42]
ASSAYS FOR CONDENSED TANNINS
433
swirl. Additions should be timed; 1-min intervals are convenient. Exactly 20 min after the addition of the FeNH4(SO4) 2, start timed additions (1min intervals) of 3.0 ml of 8 mM K3Fe(CN)6 , mixing after each addition. Exactly 20 min after the addition of the K3Fe(CN) 6 , read the absorbance at 720 nm, making readings at 1-min intervals. The spectrophotometric blank should undergo the same timed reaction but substituting sample solvent for the sample; the solvent can influence the reaction progress, so standards should be dissolved in the same solvent as the unknowns. Any phenolic compound can be used as a standard for the Prussian blue assay; gallic acid or catechin are convenient. When interpreting the results obtained with any redox assay, including the Prussian blue assay, it must be recognized that color yield is dependent on phenolic structure and the redox potential of the phenolic groups. F u n c t i o n a l G r o u p Assays
Anthocyanidin Formation A characteristic reaction of condensed tannins is oxidative cleavage of the interflavan bond to produce anthocyanidin pigments such as cyanidin (IV). This reaction has long been used as the basis for determining condensed tannins, but the chemistry of the reaction has only recently been elucidated. 13The method in which color yield and reproducibility of the assay have been optimized is described here. 13 In a screw-capped culture tube add 6.0 ml of acid butanol reagent (950 ml of n-butanol with 50 ml concentrated HCI) to a 1.0-ml aliquot of the sample. Add 0.2 ml of iron reagent [2% FeNH4(SO4) 2 in 2 N HCI] and vortex the sample. Cap the tube loosely and put it in a boiling water bath for 50 min. Cool the tube and read the absorbance at 550 nm. The spectrophotometric blank should contain solvent, acid butanol reagent, and iron reagent. The color yield in the assay is dependent on the stability of the interflavan bond. Bond stability is determined by tannin structure. For example, 5-deoxyproanthocyanidins such as quebracho tannin give substantially less color than simple procyanidins such as Sorghum tannin) Furthermore, the presence of water in the reaction decreases color formation substantially. Samples dissolved in nonaqueous solvents will give the greatest color, but the assay is sensitive enough to give acceptable results even when substantial amounts of water are present if similar amounts of water are included in the standards. 13L. J. Porter, L. N. Hrstich, and B. C. Chan, Phytochemistry 25, 223 (1986).
434
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[42]
If the samples are colored before heating (owing to flower pigments or chlorophyll) the absorbance obtained before heating can be subtracted from the absorbance obtained after heating. For chlorophyll-containing samples this correction is only approximate. Because chlorophyll is partially destroyed during heating, the absorbance obtained before heating is larger than the absorbance due to chlorophyll in the heated sample. Better results can be obtained by adsorbing the polyphenols on polyvinylpyrrolidone before developing the color. 14 If flavan-3,4-diols are present, they can be assayed by initially developing the color at room temperature. 14
Reaction with Substituted Benzaldehydes In the vanillin reaction an aromatic aldehyde such as vanillin reacts with the meta-substituted ring of flavanols to yield a red adduct. Although the vanillin reaction has been widely used to determine condensed tannin, the reaction is not specific for condensed tannins. Any appropriately substituted flavanol reacts in the assay. Thus flavan-3-ols such as catechin and epicatechin also react with vanillin to yield a red-colored adduct. Furthermore, because the vanillin reacts only with meta-substituted flavonoids, condensed tannins composed principally of 5-deoxyflavonoids, such as quebracho tannin, produce little color in the vanillin reaction. The vanillin method described here was devised to eliminate problems with reproducibility. 15 The vanillin reagent must be prepared daily by mixing 1 part of 1% (w/v) vanillin in methanol with 1 part of 8% HC1 in methanol (8 ml concentrated HC1 brought to 100 ml with absolute methanol). A solution of 4% HC1 in methanol is also prepared (4 ml concentrated HC1 brought to 100 ml with absolute methanol). The vanillin reagent and the 4% HC1 solution are brought to 30°, and 1.0-ml aliquots of the samples are dispensed into culture tubes and brought to 30°. Each sample must be run in duplicate, with one of the pair used for the reaction and the other for the background. At exactly 1-min intervals 5.0 ml of the vanillin reagent is added to one set of samples, and 5.0 ml of the 4% HCI solution is added to the second set of samples. The samples are left in the water bath for exactly 20 min, and the absorbance at 500 nm is read. The spectrophotometric blank contains vanillin reagent with sample solvent. The absorbance of each background sample is subtracted from the absorbance of the corresponding reaction tube. The background absorbance is substantial if the samples contain pigments. The vanillin reaction is very sensitive to the presence of water. Even a small amount of water in the reaction mixture will substantially quench i4 j. j. Watterson and L. G. Butler, J. Agric. Food Chem. 31, 41 (1983). t5 M. L. Price, S. Van Scoyoc, and L. G. Butler, J. Agric. Food Chem. 26, 1214 (1978).
[42]
ASSAYS FOR CONDENSED TANNINS
435
color yield. All standards should be prepared in anhydrous organic solvents (usually methanol). If water must be present in the samples to be analyzed, the same amount of water should be added to the standards. Catechin is commonly used to standardize the vanillin reaction, but interpretation of the results ("catechin equivalents") obtained with this standard is difficult. Under the normal conditions for the vanillin assay (methanol solvent), condensed tannins and catechin both react with vanillin, but the rates of reaction of the polymer and the monomer are quite different.~5 In general, the absorbances obtained with catechin are lower than the absorbances obtained with similar amounts of condensed tannin. Use of an alternate solvent to overcome these problems has been described. 16
Protein Precipitation Methods The characteristic reaction of tannins is their ability to precipitate protein. Many of the biological effects of tannins have been attributed to their ability to precipitate protein, but it is not possible at this time to predict biological activity reliably based on any of the many methods available for measuring protein precipitation. Among the factors to be considered when evaluating protein precipitation are the effects of pH, ~7 protein structure, TM tannin structure, ~9 the tannin to protein ratio, 2° and nonprotein modifiers in the reaction mixture (e.g., detergents2~). Of the many methods that have been developed for measuring protein precipitation, we summarize two of the simplest and most generally applicable methods here. Other methods have been compared in a more extensive review.12
Protein Precipitable Phenolics In the following assay, a standard protein is used to precipitate tannin, and the amount of tannin precipitated is assessed using a spectrophotometric method based on the formation of colored iron-phenolate complexes.~7 The method can be combined with methods for determining precipitated protein 2z to evaluate both components of the precipitate. 16 L. G. Butler, M. L. Price, and J. E. Brotherton, J. Agric. Food Chem. 30, 1087 (1982). 17 A. E. Hagerman and L. G. Butler, J. Agric. Food Chem. 26, 809 (1978). 18 A. E. Hagerman and L. G. Butler, J. Biol. Chem. 256, 4494 (1981). 19 T. N. Asquith and L. G. Butler, Phytochemistry 25, 1591 (1986). 2o A. E. Hagerman and C. T. Robbins, J. Chem. Ecol. 13, 1243 (1987). 21 M. M. Martin, D. C. Rockholm, and J. S. Martin, J. Chem. Ecol. 11, 485 (1985). 22 A. E. Hagerman and L. G. Butler, J. Agric. Food Chem. 28, 944 (1980).
436
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[42]
Dispense 2.0 ml of 1 mg/ml bovine serum albumin (BSA) dissolved in buffer (0.20 M acetic acid, 0.17 M NaC1, pH 4.9) into tubes. Up to 1 ml of the tannin-containing sample is added with mixing, and the mixtures are incubated for 15 min at room temperature (purified tannin) or for 24 hr at 4° (plant extracts).2° The samples are centrifuged and the supernatants discarded. The pellets are redissolved by vigorous agitation in 4.0 ml SDS/ TEA [ 1% (w/v) sodium lauryl sulfate, 5% (v/v) triethanolamine]. The color is developed by adding 1.0 ml of 10 mM FeCI 3 in I0 mM HCI and vortexing immediately. After about 15 min the absorbance is recorded at 510 nm. The spectrophotometric blank is a mixture containing reagents but no sample. Even traces of acetone inhibit precipitation of protein by phenolics, 2° so all acetone must be removed from plant extracts before attempting the method.
Radial Diffusion Assay In the radial diffusion assay, the tannin-containing sample diffuses through a protein-containing agar gel, and an opaque ring forms as the tannin precipitates protein in the gel. The area (or diameter squared) of the ring is proportional to the amount of tannin used. The response of the method is dependent in part on the molecular size of the tannin, since diffusion is a function of molecular size. For samples containing chemically similar tannins, the method provides a rapid, simple way to determine tannins, z3 To make eight diffusion plates (suitable for analysis of 32 samples), dissolve 1.0 g agarose [Type I, low EEO, gel point 36° (e.g,, Sigma, St. Louis, MO, A-6013)] in 100 ml of buffer (50 mM acetate containing 60 /zM ascorbic acid, pH 5.0) by heating with continuous stirring. Allow the agarose solution to cool, with occasional stirring, to 45 °, and then add 0.10 g BSA while gently stirring. The protein should be completely dissolved without allowing the solution to cool further. Using a serological pipette with a large opening at the tip, dispense 9.5 ml of solution into 10-cm plastic petri dishes. Allow the solution to gel while the dishes are on a level surface, and then cover each dish and seal with a strip of Parafilm. The plates can be stored at 4° for up to a week before use. Wells are punched in the plates using a 4.0-mm punch (e.g., Bio-Rad, Richmond, CA, 170-4029), with the wells arranged so there are four evenly spaced wells per plate. The plugs of agarose are removed from the wells with gentle suction. Up to 8/xl of sample is then added to each well, and the dishes are resealed and incubated on a level surface at 30° for 96 hr. 23 A. E. H a g e r m a n , J. Chem. Ecol. 13, 437 (1987).
[43]
ROLE OF FLAVONOIDS AND IRON CHELATION
437
A ruler is then used to record the diameter of the ring that has formed. The plates can be stored at 4° for several weeks after development of the rings. The sample can be dissolved in any solvent that is convenient, but we find that aqueous organic mixtures (50% methanol or 70% acetone) are most convenient. If the tannin-containing extract is very dilute, 8/xl may not be sufficient for a response. Larger volumes can be applied to a well by dispensing repetitive 8-/xl samples, but the well must not become completely dry between successive aliquots that are to be added.
[43] R o l e o f F l a v o n o i d s a n d I r o n C h e l a t i o n in A n t i o x i d a n t A c t i o n
By
ISABELLE MOREL, GI~RARD LESCOAT, PIERRE CILLARD,
and JOSIANE CILLARD Introduction The antioxidant effect of flavonoids has been of interest for a considerable time) -4 The potential of flavonoids to inhibit lipid peroxidation in biological models is supposed to reside mainly in their free radical scavenging capacity rather than in their iron chelating activity. 5,6This last property has often been considered as a minor mechanism in the antioxidant action, since it has not been clearly established in biological systems. The assessment of a relationship between the antioxidant effect and the iron chelating capacity of flavonoids is subsequently of interest. 7 For this purpose, we used rat hepatocyte cultures as a biological model where lipid peroxidation was induced by iron [Fe(III)] in its complexed form with nitrilotriacetic
T. F. Slater and N. N. Eakins, in " N e w Trends in the Therapy of Liver Diseases" (A. Bertelli, ed.), p. 84. Karger, Basel, 1975. 2 W. Bors and M. Saran, Free Radical Res. Cornmun. 2, 289 (1987). 3 W. Bors, W. Heller, C. Michel, and M. Saran, this series, Vol. 186, p. 343. 4 j. Torel, J. Cillard, and P. Cillard, Phytochemistry 2,5, 383 (1986). 5 C. G. Fraga, V. S. Martino, G. E. Ferraro, J. F. Coussio, and A. Boveris, Biochem. Pharmacol. 36, 717 (1987). 6 A. K. Ratty and N. P. Das, Biochem. Med. Metab. Biol. 39, 69 (1988). 7 I. Morel, G. Lescoat, J. Cillard, P. Cogrel, O. Sergent, N. Pasdeloup, P. Brissot, and P. Cillard, Biochem. Pharmacol. 45, 13 (1993).
METHODS IN ENZYMOLOGY,VOL.234
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[43]
ROLE OF FLAVONOIDS AND IRON CHELATION
437
A ruler is then used to record the diameter of the ring that has formed. The plates can be stored at 4° for several weeks after development of the rings. The sample can be dissolved in any solvent that is convenient, but we find that aqueous organic mixtures (50% methanol or 70% acetone) are most convenient. If the tannin-containing extract is very dilute, 8/xl may not be sufficient for a response. Larger volumes can be applied to a well by dispensing repetitive 8-/xl samples, but the well must not become completely dry between successive aliquots that are to be added.
[43] R o l e o f F l a v o n o i d s a n d I r o n C h e l a t i o n in A n t i o x i d a n t A c t i o n
By
ISABELLE MOREL, GI~RARD LESCOAT, PIERRE CILLARD,
and JOSIANE CILLARD Introduction The antioxidant effect of flavonoids has been of interest for a considerable time) -4 The potential of flavonoids to inhibit lipid peroxidation in biological models is supposed to reside mainly in their free radical scavenging capacity rather than in their iron chelating activity. 5,6This last property has often been considered as a minor mechanism in the antioxidant action, since it has not been clearly established in biological systems. The assessment of a relationship between the antioxidant effect and the iron chelating capacity of flavonoids is subsequently of interest. 7 For this purpose, we used rat hepatocyte cultures as a biological model where lipid peroxidation was induced by iron [Fe(III)] in its complexed form with nitrilotriacetic
T. F. Slater and N. N. Eakins, in " N e w Trends in the Therapy of Liver Diseases" (A. Bertelli, ed.), p. 84. Karger, Basel, 1975. 2 W. Bors and M. Saran, Free Radical Res. Cornmun. 2, 289 (1987). 3 W. Bors, W. Heller, C. Michel, and M. Saran, this series, Vol. 186, p. 343. 4 j. Torel, J. Cillard, and P. Cillard, Phytochemistry 2,5, 383 (1986). 5 C. G. Fraga, V. S. Martino, G. E. Ferraro, J. F. Coussio, and A. Boveris, Biochem. Pharmacol. 36, 717 (1987). 6 A. K. Ratty and N. P. Das, Biochem. Med. Metab. Biol. 39, 69 (1988). 7 I. Morel, G. Lescoat, J. Cillard, P. Cogrel, O. Sergent, N. Pasdeloup, P. Brissot, and P. Cillard, Biochem. Pharmacol. 45, 13 (1993).
METHODS IN ENZYMOLOGY,VOL.234
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
438
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[43]
acid (NTA). The F e - N T A complex is known to induce a rapid accumulation of iron inside the cells. 8-11 Materials and Methods Ferric Nitrilotriacetate Solution
Nitrilotriacetic acid (NTA) is used to maintain ferric iron in a soluble state; it is a low-affinity iron chelator. The ferric nitrilotriacetate solution is prepared according to the following method12: to 47 mg of nitrilotriacetic acid disodium salt (Sigma Chimie, St. Quentin Fallavier, France), dissolved in 10 ml of sterile water, is added 20 mg of ferric ammonium citrate (Merck, Darmstadt, Germany). The final concentration of ferric ion is 10 mM, and the molar ratio ferric iron to NTA is 1 : 2. The solution is sterilized before use by filtration through a 0.22-/xm filter. Flavonoids
Based on solubility, the flavonoid to be tested is dissolved in either water or dimethyl sulfoxide (DMSO). A constant concentration of DMSO is maintained in control samples (2%), a dose that does not affect control rates of lipid peroxidation or iron mobilization. Cell Isolation and Culture
Adult rat hepatocytes are isolated from 2-month-old Sprague-Dawley male rats by cannulating the portal vein and perfusing the liver with a coUagenase solution. 13 The cells are collected in Leibovitz medium containing, per milliliter, 1 mg bovine serum albumin (BSA) and 5/zg bovine insulin. The cell suspension is filtered through gauze and allowed to sediment for 20 rain in order to eliminate cell debris, blood, and sinusoidal cells. The cells are then washed three times by centrifugation at 50 g for 1 min at 4 °, tested for viability, and counted. The hepatocytes are then suspended in a mixture of 75% (v/v) Eagle's minimum essential medium 8 B. R. Bacon, A. S. Tavill, G. M. Brittenham, C. H. Park, and R. O. Recknagel, J. Clin. Invest. 71, 429 (1983), 9 B. Desvergne, G. Baffet, P. Loyer, M. Y. Rissel, G. Lescoat, C. Guguen-Guillouzo, and P. Brissot, Eur. J. Cell Biol. 49, 162 (1989). i0 p. Brissot, J. Farjanel, D. Bourel, J. P. Campion, A. Guillouzo, A. Rattner, Y. Deugnier, B. Desvergne, B. Ferrand, M. Simon, and M. Bourel, Dig. Dis. Sci. 32, 620 (1987). i1 S. Shedlofsky, H. L. Bonkowsky, P. R. Sinclair, W. J. Bement, and J. J. Pomeroy, Biochem. J. 212, 321 (1983). 12 G. P. White and A. Jacobs, Biochim. Biophys. Acta 543, 217 (1978). 13 C. Guguen, A. Guillouzo, M. Boisnard, A. Le Cam, and M. Bourel, Biol. Gastroenterol. 8, 223 (1975).
[43]
ROLE OF FLAVONOIDS AND IRON CHELATION
439
and 25% medium 199, supplemented with 10% fetal calf serum (FCS) and containing, per milliliter, the following: streptomycin (50/~g), penicillin (7.5 IU), bovine insulin (5/~g), bovine serum albumin (1 mg), and NaHCO3 (2.2 mg). Usually, according to the experimental procedure, either 2.5 x 10 6 hepatocytes are plated in 25-cm 2 Nunclon flasks (Roskilde, Denmark), or 1.5 x 105 hepatocytes are suspended in I ml of medium in multiwell tissue culture plates. The medium is changed 3-4 hr later and renewed the day after with the same medium as above but without serum and supplemented with 10 -7 M dexamethasone.
Lipid Peroxidation Chromatography Procedure. Free malondialdehyde (MDA) quantification ~4 is performed using a high-performance liquid chromatography (HPLC) system [Laboratory Data Control (LDC) Finnigan, Orsay, France] which is equipped with a Spherogel-TSK G 1000 PW size-exclusion column (7.5 mm i.d. x 30 cm, Cluzeau, Ste. Foy, France). This sizeexclusion column separates compounds using a gel-permeation technique which allows a diffusion of the smallest molecules such as MDA inside the gel and then permits their separation from larger compounds which are eluted earlier. The retention time (56 min for MDA) is, however, greatly increased in comparison with classic C~8 columns. The use of this size-exclusion column is nevertheless highly recommended, especially in biological systems where many interferences may occur. The eluant is composed of 0.1 M disodium phosphate buffer, pH 8, at a flow rate of 1 ml/min. The absorbance of free MDA is monitored at 267 nm, using the maximal sensitivity (0.001 absorbance unit full scale). The injections (250 ~1) are performed by an autosampling injector (Promis, LDC) and the data are recorded and treated using a chromatography software (Thermochrom, LDC). Preparation of Free MDA Standard. Five microliters of 1,1,3,3-tetramethoxypropane (Sigma) are hydrolyzed in 5 ml of 0.1 N HC1 during 5 min in boiling water. This is performed in a closed vial in order to prevent evaporation. The solution is then diluted 1/ 1000 in 10 mM Na2HPO4 buffer, pH 7.45, which corresponds to a final concentration of 6/~M of MDA. Because the stability of diluted solutions of MDA is very weak, a new solution must be prepared daily. The concentration of MDA in samples is calculated using a standard curve of free MDA. Preparation of Samples for Analysis. During the first day of experimentation (day 1), the cultures are maintained in the presence of ferric 14 A. S. Csallany, D. M. Guan, J. D. Manwaring, and P. B. Addis, Anal. Biochem. 142, 277 (1984).
440
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[43]
iron nitrilotriacetate (Fe-NTA) in order to obtain final iron concentrations of I00/.,M. More precisely, each sample with F e - N T A is compared to control cultures without any supplementation and to cultures supplemented with 200/zM NTA, which is the amount of NTA added to the cultures in the experimental procedure. After 24 hr of incubation, the medium is renewed with the same medium supplemented or not with iron with or without DMSO, and the flavonoids are added. After another 24-hr period of incubation (day 2), culture media are collected and hepatocytes are washed twice with 10 mM phosphate buffer, pH 7.45. They are resuspended in 1 ml of the same buffer. The cells are lysed using an ultrasonic homogenizer during 1 min in ice. An aliquot was stored at - 1 7 ° until protein content is estimated. The samples (culture media or cell homogenates) are filtered through a 500-Da membrane ultrafilter (Amicon, Danvers, MA) in a 10-ml Amicon cell pressurized at 4 bars with nitrogen gas. The filtrate is used for the HPLC procedure. Free MDA is quantified separately in culture media and in cell homogenates (see Fig. 1).
MDA
MDA
A
0
i6
B
6
i6
FIG. 1. Chromatograms of free MDA in hepatocytes (A) and in the medium (B) of cultures which have been supplemented with F e - N T A (100/zM) for 48 hr. The size-exclusion HPLC system allowed good separation of free MDA (retention time 56 min) from other biological components.
[43]
ROLE OF FLAVONOIDS AND IRON CHELATION
441
The results may be expressed in nanograms per milligram of protein for free MDA in the cells and in nanograms per milliliter of culture medium for extracellular free MDA. For convenience, they can also be expressed as total free MDA (nanomolar) present in each culture sample (MDA in the cells plus MDA in the culture medium).~5 However the absolute level of free MDA in biological models may vary from one experiment to another, especially when the cells are provided by different animals. Moreover, variations in culture medium composition or in incubation times before treatment of the cells may induce considerable modifications in MDA levels. Thus, the standardization of culture conditions is of major importance to obtain reproducible values. In the hepatocyte culture model where all precautions have been taken, absolute values for total free MDA levels are 1724 --- 32 nM in cultures supplemented only with 100 ~M F e - N T A , whereas the basal level of free MDA in hepatocyte cultures without supplementation is 37.3 - 3.6 nM. To avoid experimental variations, total free MDA may also be expressed as a percentage of control values. In our experiments, where the antioxidant activity was investigated, 100% of MDA recovery corresponded to MDA level in cultures supplemented with iron only. 16 Another source of variation in experimental values is the weak stability of free MDA, which results in a loss of MDA in the samples. Stability experiments have shown that the samples must be subjected to ultrafiltration as soon as possible to stop lipid peroxidation and to avoid MDA linkage to biological substrates such as proteins or DNA, and that MDA level in the ultrafiltrates has to be evaluated within 24 hr, unless samples are frozen in liquid nitrogen. When working with frozen samples, two main indications should be kept in mind: first, ultrafiltration is necessary before freezing since MDA levels decrease in nonultrafiltrated frozen samples; second, a standard solution of MDA must be frozen simultaneously to prevent any variations resulting from a small loss in the samples. However, direct analysis of MDA on fresh ultrafiltrates remains the best and the simplest way to obtain accurate results (Fig. 2). Protein content is determined on thawed cell homogenates according to the Bradford reaction, 17performed on a Cobas-Bio automatic analyzer (Roche, Neuilly/Seine, France) using the Bio-Rad protein assay reagent diluted with phosphate-buffered saline (1 : 4, v/v) and using bovine serum albumin as a standard (0-700/xg/ml). 15 I. Morel, G. Lescoat, J. Cillard, N. Pasdeloup, P. Brissot, and P. CiUard, Biochem. Pharmacol. 39, 1647 (1990). 16 I. Morel, J. Cillard, G. Lescoat, O. Sergent, N. Pasdeloup, A. Z. Ocaktan, M. A. Abdallah, P. Brissot, and P. Cillard, Free Radical Biol. Med. 13, 499 (1992). iv M. M. Bradford, Anal. Biochem. 72, 248 (1976).
442
[43]
ANTIOXIDANT CHARACTERIZATION AND ASSAY
-N-CH2CH s
"N=~
,N-C.~C..
CH2 CH3
0~
•
x HCI
FIG. 1. Chemicalstructures of the glucocorticoidmethylprednisolone,the nonglucorticoid steroid U-72099E, and the 21-aminosteroids U-74006FandU-74500A. The first compound in the 21-aminosteroid or lazaroid series was synthesized in 1985. U-74006F (Fig. 1; generic name tirilazad mesylate, trade name Freedox) has been selected for clinical development for the acute treatment of brain and spinal injury, subarachnoid hemorrhage, and stroke, and is currently involved in Phase III clinical trials in each of those disorders. In the present discussion, a main focus is on the known antioxidant mechanisms of U-74006F. Another 21-aminosteroid, U-74500A (Fig. 1), is actually a more potent inhibitor of iron-catalyzed lipid peroxidation than U-74006F, but it has not been chosen for development owing to pharmaceutical instability and rapid elimination in vivo.
Mechanisms of Lipid Peroxidation Inhibition U-74006F is a very lipophiliccompound (the log of the calculated octanol/water partitioncoefficientequals 8) that distributespreferentially
550
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[56]
to the lipid bilayer of cells. It appears that the compound exerts its effects through cooperative effects: a radical scavenging action (i.e., chemical antioxidant effect) and a physicochemical interaction with the cell membrane that serves to decrease membrane fluidity (i.e., membrane stabilization). Cytoprotection is governed partly by intrinsic reactivity toward free radicals, partly by location and orientation of U-74006F within the membrane, and partly by the ability of the compound to modify the physical properties of the lipid bilayer of the membrane. Activity in different in oitro and in vivo models probably reflect different balances between these actions. Antioxidant Effects in Membrane Systems
The 21-aminosteroids are potent inhibitors of lipid peroxidation in vitro. Using rat brain homogenates or purified rat brain synaptosomes as the lipid source, U-74006F and U-74500A potently inhibit iron-dependent lipid peroxidation, with an efficacy greatly surpassing that of the glucocorticoid steroid methylprednisolone. In a model that uses synaptic membranes prepared from rat brain as a lipid source and 200/zM ferrous chloride to initiate and catalyze the lipid peroxidation reactions, U-74006F inhibited lipid peroxidation with IC50 values ranging from 10 to 60/zM. 7 U-74006F also protects isolated liver microsomes from oxidative injury that is initiated by ferrous ammonium sulfate. The IC50 value is 3.8/.~M when the U-74006F is added in ethanol to a suspension of microsomes in Krebs buffer. Interestingly, when U-74006F is added as a lipid emulsion (triglyceride, phosphatidylcholine, drug, and water) rather than in ethanol, the IC50 value drops to below 0. I/zM. This illustrates one of the problems in testing very lipophilic compounds like U-74006F. When such compounds are added in organic solution to physiological buffers, they microprecipitate. Emulsion delivery is often a superior delivery technique for compounds of this class. Since many of the lipid peroxidation models involve initiation of oxidative injury by iron, the 21-aminosteroids have been mistakenly described as inhibitors that affect exclusively iron-dependent lipid peroxidation. However, we have also studied iron-free systems and shown lipid antioxidant effects of U-74006F and U-74500A. In addition, U-74006F has been shown effective in a model of lipid peroxidation that involved rat liver microsomes with initiation by cumene hydroperoxide. Free iron was removed with a chelation column, thus ensuring that the system was truly 7 j. M. Braughler, J. F. Pregenzer, R. L. Chase, L. A. Duncan, J. M. McCall, and E. J. Jacobsen, J. Biol. Chem. 262, 10438 (1987).
[56]
LAZAROIDS
551
iron-free. 8 U-74006F has further been demonstrated to inhibit diquatinduced lipid peroxidation in liver microsomes. 9 U-74006F and U-74500A have been reported to scavenge lipid peroxyl and phenoxy radicals in a methanol solution of linoleic acid in the presence of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) which induces peroxidation of the polyunsaturated linoleic acid, although U-74006F and U-74500A both possess slower rate constants in this environment than the prototypical peroxyl radical scavenger vitamin E. However, both compounds act to slow the oxidation of vitamin E during linoleic acid peroxidation and potentiate the antioxidant efficacy of vitamin E. ~° We further studied the lipid radical scavenging properties of the 21aminosteroids in three different models. H Model 1 involves a homogeneous methanolic solution of linoleic acid as the peroxidizable lipid substrate in methanol, with AMVN as the free radical initiator as previously described. 9 Model 2 involves multilamellar vesicles of dilinoleyllecithin with 2,2'-azobis(2-amidinoaminopropane), ABAP, as the water-soluble initiator. In Model 2, U-74006F or U-74500A was incorporated in the multilamellar vesicle as it was prepared. Both initiators are thermally activated and produce lipid free radicals at a constant and readily reproducible rate, thereby creating a steady-state kinetic system. Hydroperoxide formation was measured by high-performance liquid chromatography (HPLC) in Model I and by a xylenol orange color test for lipid hydroperoxides in Model 2; hydroperoxide LOOH formation was found to be linear for the time periods measured. The rates of hydroperoxide formation were proportional to the square root of the concentration of initiator and to the concentration of substrate. When U-74500A was added as the inhibitor, a transient decrease in the hydroperoxide production was observed, and during the same time period the compound was degraded in a first-order manner. Thus, in the homogeneous system, the inhibitor acts by scavenging lipid radicals, and its reactivity is about 30 times greater than that of linoleic acid. In Model 3, rat liver microsomes were treated with ferrous ammonium sulfate. This initiates an iron-mediated lipid peroxidation that is empirically described by the measure of malonyldialdehyde (MDA) that is formed. U-74006F was effective in all of these models. However, it was most effective when it was in the ordered environment of the lipid vesicle (Model 2) or the microsome (Model 3). U-74500A is actually a better 8 C. L. Bryan, R. A. Lawrence, E. D. Hall, and S. G. Jenkinson, FASEB J. 4, A630 (1990). 9 G. H. I. Wolfgang, R. A. Jolly, and T. W. Petry, Free Radical Biol. Med. 10, 403 (1991). 10j. M. Braughler and J. F. Pregenzer, Free Radical Biol. Med. 7, 125 (1989). ~! K. L. Linseman, B. S. Lutzke, J. M. McCall, and D. E. Epps, Toxicologist 13, 337 (1993).
552
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[56]
antioxidant than U-74006F. It has a lower oxidation potential, and this makes it a superior in vitro lipid peroxidation inhibitor. In addition to scavenging o f lipid peroxyl radicals, U-74006F also reacts with reactive oxygen species such as hydroxyl radicals generated during in vitro F e n t o n reactions (i.e., Fe 2+ + H202----> Fe 3+ + O H - + .OH). 12 In vivo studies employing the salicylate trapping method for measurement of hydroxyl radical have demonstrated that U-74006F administration decreases brain hydroxyl radical levels in models of concussive head injury in mice, 13 as well as global cerebral ischemia-reperfusion injury in gerbils. 12 This may be due to either direct scavenging of hydroxyl radical or a decrease in its injury-induced formation. A n t i o x i d a n t E f f e c t s in Whole Cells
The 21-aminosteroids have also been shown to inhibit lipid peroxidation in whole cells. F o r example, U-74500A inhibits copper-induced red cell lipid peroxidation. The compound is effective at concentrations as low as 1/xM. At 1/zM, it significantly reduces copper-induced and H202induced erythrocyte lipid peroxidation by 76.5 and 27.6%, respectively. The inhibition of erythrocyte lipid peroxidation was accompanied by an inhibition of hemolysis.14 U-74006F (5 ~M) has been shown to protect murine neocortical cell cultures that were exposed to 5 0 / x M ferric iron and 50/xM ferrous iron for 24 hr from neuronal degeneration. 15 The compound has also been reported to protect cultured murine spinal neurons from damage by 200/.tM ferrous iron. 16 U-74006F is also effective in an in vitro model for predicting the ability of a compound to prevent cell damage during periods of energy failure. Iodoacetic acid (IAA) was administered to cultured human astroglial cells (UC-11MG) at a concentration of 50 ~ M for 4 hr. This agent shuts down glycolysis and leads to subsequent irreversible breakdown of cellular membranes, and ultimately to cell death. During the first hours after addition, IAA rapidly depleted cellular levels of ATP and decreased active uptake of tritiated aminoisobutyric acid. Subsequent irreversible cellular injuries were characterized by the release of large amounts of free arachidonic acid into the extracellular medium, massive calcium influx, and leakage 12j. s. Althaus, C. W. Williams, P. K. Andrus, P. F. von Voigtlander, and E. D. Hall, Soc. Neurosci. Abstr. 17, 164 (1991). 13E. D. Hall, P. K. Andrus, and P. A. Yonkers, J. Neurochem. 60, 588 (1993). 14A. C. Fernandes, P. M. Filipe, and C. F. Manso, Eur. J. Pharmacol. 220, 211 (1992). 15H. Monyer, D. M. Hartley, and D. W. Choi, Neuron 5, 121 (1990). 16E. D. Hall, J. M. Braughler, P. A. Yonkers, S. L. Smith, K. L. Linseman, E. D. Means, H. M. Scherch, P. F. von Voigtlander, R. A. Lahti, and E. J. Jacobsen, J. Pharmacol. Exp. Ther. 258, 688 (1991).
[56]
LAzARoIos
553
of cytoplasmic contents (51Cr release). The appearance of 15-hydroxyeicosatetraenoic acid in membrane phospholipids and loss of cellular thiol groups indicated that the cell constituents were being assaulted by oxidative species. These manifestations oflAA-induced cell damage were inhibited by U-74006F. The IAA-induced release of tritiated arachidonic acid was inhibited with an IC50 value of 6/.~M. U-74006F was effective even when it was administered up to 1 hr after the onset of the metabolic insult.~7 In other work, U-74006F was also shown to decrease the release of arachidonic acid from cultured AtT-20 pituitary tumor cells triggered by exposure to either IAA or ferrous iron.~8 Physicochemical Effects on Membranes The 21-aminosteroids U-74006F and U-74500A also have potent stabilizing effects on cell membranes. As noted above, the compounds have a high affinity for the lipid bilayer because of their lipophilicity. Reflecting its membrane interaction, U-74006F has been shown to exert physicochemical effects on endothelial cell membranes. Bovine brain microvessel endothelial cells (BMECs) were labeled with diphenylhexatriene (DPH) fluorophores. Interactions with cell membranes were characterized by fluorescence anisotropy and fluorescence lifetimes. U-74500A and U-74006F preferentially altered the fluorescence anisotropy and lifetime parameters of the fluorescent DPH probe that distributed into the membranes throughout the BMECs. Little or no effect of the compounds were observed on the fluorescence parameters of the probe trimethylammonium-phenyl-DPH) that localized on the surface of BMEC plasma membranes. In contrast, cholesterol, used as a positive control, substantially altered the fluorescence parameters of BMECs labeled with either surface or membrane core probes. These experiments suggest that the 21-aminosteroids induce changes in the molecular packing order in membrane hydrophobic domains throughout the BMEC.19 Other research has also demonstrated physicochemical effects of the 21-aminosteroids on membranes. U-74006F and vitamin E were studied in bilayer lipid membranes with time-resolved fluorescence depolarization and angle-resolved fluorescence depolarization techniques, and by electron paramagnetic resonance utilizing probe molecules. 2° Lipid peroxida17 F. Sun, B. M. Taylor, and W. E. Fleming, FASEB J. 7, A658 (1992). 18 j. M. Braughler, R. L. Chase, G. L. Neff, P. A. Yonkers, J. S. Day, E. D. Hall, V. H. Sethy, and R. A. Lahti, J. Pharmacol. Exp. Ther. 44, 423 (1988). 19 K. L. Audus, F. L. Guillot, and J. M. Braughler, Free RadicaIBiol. Med. 11, 361 (1991). 2o G. Van Ginkel, J. M. Muller, F. Siemsen, A. A. van't Veld, L. J. Korstanje, M. A. M. van Zandvoort, M. L. Wratten, and A. Sevanian, J. Chem. Soc., Faraday Trans. 88, 1901 (1992).
554
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[56]
H~OIH /0 H
+1
/N~N CH2 V
O~'~"CH3
N -Ni~ Tk,Y
I~ N
©
FIG. 2. Membrane orientation of the 21-aminosteroid U-74006F.
tion products (oxidized fatty acids) strongly disorder the unsaturated lipid membranes they inhabit, but they do not affect the lipid dynamics. This is compatible with a model where the lipid hydroperoxy or hydroxy moieties reside closer to the polar head group region of the membrane lipids. U-74006F also has a disordering effect in the lipid systems, although the effects on dynamics vary depending on the surrounding lipids. Generally, U-74006F decreased dynamics (increased head group order). The decrease for U-74006F is consistent with the observations from additional work showing that the 21-aminosteroids are incorporated into the lipid bilayer where they occupy strictly defined positions and orientations. 2l As shown in Fig. 2, we hypothesize that U-74006F resides in the cell membrane, and that the piperazine nitrogen, which is largely protonated (i.e., positively charged) at physiologic pH, should orient with the head groups of the membrane bilayer by ionic interaction to the negatively charged phosphate-containing head groups. The steroid moiety, on the other hand, should localize within the hydrophobic core of the membrane. The pyrimidine amine of the molecule should help compress membrane phospholipid head groups. Indeed, head group viscosity in a lipid monolayer increases significantly with as little as 1.0 mol% (relative to lipid) of U-74006F (F. Kezdy et al., personal communication). 2[ j. S. Hinzmann, R. L. McKenna, T. S. Pierson, F. Han, F. Kezdy, and D. E. Epps, Chem. Phys. Lipids 62, 123 (1992).
[57]
ANTIOXIDANT
PROPERTIES
OF AMINOSALICYLATES
555
In addition to the chemical antioxidant properties of U-74006F (and U-74500A) described above, this "membrane stabilizing" action may help to inhibit the propagation of lipid peroxidation by restricting the movement of lipid peroxyl and alkoxyl radicals within the membrane. Thus, the 21aminosteroids block oxygen radical-induced lipid peroxidation apparently via a combination of chemical antioxidant (i.e., radical scavenging) and membrane stabilizing effects.
[57] A n t i o x i d a n t P r o p e r t i e s o f A m i n o s a l i c y l a t e s
By ALLEN M. MXLES and MATTHEW B. GRISHAM Introduction Ulcerative colitis (UC) is a recurrent inflammation of the colon and rectum that is characterized by rectal bleeding, diarrhea, fever, pain, anorexia, and weight loss. Active episodes of the disease are characterized by the extravasation and infiltration of large numbers of phagocytic leukocytes (neutrophils, monocytes, and macrophages) into the colonic mucosa. 1 This enhanced inflammatory infiltrate is accompanied by extensive mucosal injury including edema, crypt abscesses, loss of goblet cells, decreased production of mucus, erosions, and mucosal ulcerations. The apparent association between leukocyte infiltration and mucosal injury has led to the proposition that phagocytic leukocytes may mediate much of the pathophysiology associated with active disease. 2 There is a growing body of experimental data to suggest that the chronically inflamed intestine and/or colon may be subjected to considerable oxidative stress. 3'4 The most probable source of these oxidants are the phagocytic leukocytes since these cells are known to be present in large numbers in the inflamed
I R. H. Riddell, in "Inflammatory Bowel Disease" (J. Kirsner and R. G. Shorter, eds.), pp. 329-350. Lea & Febiger, Philadelphia, 1988. 2 M. B. Grisham and D. N. Granger, in "Current Topics in Gastroenterology" (R. MacDermott and W. Stenson, eds.), pp. 225-239. Elsevier, Amsterdam, 1992. 3 N. J. Simmonds, R. E. Allen, T. R. J. Stevens, R. N. M. Van Someren, D. R. Blake, and D. S. Rampton, Gastroenterology 103, 186 (1992). 4 A. Keshavarzian, S. Sedghi, J. Kanofsky, T. List, C. Robinson, C. Ibrahim, and D. Winship, Gastroenterology 103, 177 (1992).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[57]
ANTIOXIDANT
PROPERTIES
OF AMINOSALICYLATES
555
In addition to the chemical antioxidant properties of U-74006F (and U-74500A) described above, this "membrane stabilizing" action may help to inhibit the propagation of lipid peroxidation by restricting the movement of lipid peroxyl and alkoxyl radicals within the membrane. Thus, the 21aminosteroids block oxygen radical-induced lipid peroxidation apparently via a combination of chemical antioxidant (i.e., radical scavenging) and membrane stabilizing effects.
[57] A n t i o x i d a n t P r o p e r t i e s o f A m i n o s a l i c y l a t e s
By ALLEN M. MXLES and MATTHEW B. GRISHAM Introduction Ulcerative colitis (UC) is a recurrent inflammation of the colon and rectum that is characterized by rectal bleeding, diarrhea, fever, pain, anorexia, and weight loss. Active episodes of the disease are characterized by the extravasation and infiltration of large numbers of phagocytic leukocytes (neutrophils, monocytes, and macrophages) into the colonic mucosa. 1 This enhanced inflammatory infiltrate is accompanied by extensive mucosal injury including edema, crypt abscesses, loss of goblet cells, decreased production of mucus, erosions, and mucosal ulcerations. The apparent association between leukocyte infiltration and mucosal injury has led to the proposition that phagocytic leukocytes may mediate much of the pathophysiology associated with active disease. 2 There is a growing body of experimental data to suggest that the chronically inflamed intestine and/or colon may be subjected to considerable oxidative stress. 3'4 The most probable source of these oxidants are the phagocytic leukocytes since these cells are known to be present in large numbers in the inflamed
I R. H. Riddell, in "Inflammatory Bowel Disease" (J. Kirsner and R. G. Shorter, eds.), pp. 329-350. Lea & Febiger, Philadelphia, 1988. 2 M. B. Grisham and D. N. Granger, in "Current Topics in Gastroenterology" (R. MacDermott and W. Stenson, eds.), pp. 225-239. Elsevier, Amsterdam, 1992. 3 N. J. Simmonds, R. E. Allen, T. R. J. Stevens, R. N. M. Van Someren, D. R. Blake, and D. S. Rampton, Gastroenterology 103, 186 (1992). 4 A. Keshavarzian, S. Sedghi, J. Kanofsky, T. List, C. Robinson, C. Ibrahim, and D. Winship, Gastroenterology 103, 177 (1992).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
556
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[57]
-i:~
\""
H202 + C l -
Fe+3
• HOCI
:
0=+ OH-+OH"
(.. o-,/ o, NADPH oxidase
FIG. 1. Release of reactive oxygen metabolites by activated human neutrophils. Interaction of certain proinflammatory mediators (e.g., leukotriene B4, platelet-activating factor, or bacterial products) with specific receptors on the neutrophilic plasma membrane activates the membrane-associated enzyme NADPH oxidase. This oxidase reduces molecular oxygen (02) by one electron to yield the superoxide anion radical (027) which rapidly and spontaneously dismutates to yield hydrogen peroxide (H202). Superoxide may interact with H202 in the presence of trace amounts of iron (Fe 3+) to yield the highly reactive hydroxyl radical (.OH). Activated neutrophils also secrete the hemoprotein myeloperoxidase (MPO) into the extracellular space where it catalyzes the H202-dependent, two-electron oxidation of chloride (C1-) to yield the potent oxidizing and chlorinating agent hypochlorous acid (HOCI).
mucosa and are known to produce large amounts of reactive oxygen species in response to certain inflammatory stimuli (Fig. 1). 5,6 Oral administration of the drug sulfasalazine (SAZ) has proved effective in attenuating the inflammation and mucosal injury associated with 5 T. Yamada and M. B. Grisham, in "Inflammatory Bowel Disease: From Bench to Bedside" (F. Shanahan and S. Targan, eds.), pp. 133-150. Williams & Wilkens, Baltimore, 1994. 6 S. J. Klebanoff, in "Inflammatory--Basic Principles and Clinical Correlates" (J. I. Gallen, I. M. Goldstein, and R. Synderman, eds.), pp. 391-444. Raven, New York, 1985.
[57]
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
557
ulcerative colitis. 7-9 Pharmacokinetic studies have demonstrated that SAZ passes unmodified through the upper gastrointestinal (GI) tract until it reaches the colon where it is metabolized by endogenous bacteria to yield sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA). Several clinical studies have shown that 5-ASA is the pharmacologically active moiety of SAZ; however, its mechanism of action remains only speculative. 7-9 It has been suggested that 5-ASA may exert its anti-inflammatory activity in vivo by inhibiting prostaglandin synthase and/or lipoxygenase activities as well as by interfering with antibody synthesis. ~0-~4Although these proposed mechanisms of action of 5-ASA can be readily demonstrated in vitro, the concentrations required for significant inhibition are at least 10to 50-fold higher than those achieved in the colonic mucosal interstitium of a normal bowel perfused with clinically relevant concentrations of 5-ASA.~5 Our laboratory, as well as others, have demonstrated that 5-ASA is a very potent antioxidant and free radical scavenger at concentrations similar to those found within the colonic mucosal interstitium. 16-z4 Thus, we have proposed that the antioxidant and metal binding properties of 5-ASA are important mechanisms by which 5-ASA exerts its anti-inflammatory activity in rio0. 23'24 In this chapter, we have assembled a variety 7 A. H. Azad-Khan, J. Piris, and S. C. Truelove, Lancet 2, 892 (1977). 8 p. A. M. van Hees, J. H. Bakker, and J. H. M. van Tongeren, Gut 21, 632 (1980). 9 U. Klotz, K. Maier, C. Fischer, and K. Heinkel, N. Engl. J. Med. 303, 1499 (1980). 10 j. R. S. Hoult and P. K. Moore, Br. J. Pharmacol. 68, 719 (1980). 11 C. J. Hawley and S. C. Truelove, Gut 24, 213 (1983). 12 W. F. Stenson and E. Lobos, J. Clin. lnoest. 69, 494 (1982). 13 H. Allgayer and W. F. Stenson, Immunopharmacology 15, 39 (1988). t4 R. P. MacDermott, S. R. Schloemann, M. J. Bertovich, G. S. Nash, M. Peters, and W. F. Syenson, Gastroenterology 96, 442 (1989). 15 M. B. Grisham and D. N. Granger, Dig. Dis. Sci. 34, 573 (1989). 16 G. Carlin, R. Djursater, G. Smedegard, and B. Gerdin, Agents Actions 16, 377 (1985). 17 W. H. Betts, M. W. Whitehouse, L. G. Cleland, and B. Vernon-Roberts, J. Free Radical Biol. Med. 1, 273 0985). 18 O. I. Aruoma, M. Wasil, B. Halliwell, B. M. Hoey, and J. Butler, Biochem. Pharmacol. 36, 3739 (1987). 19 p. A. Carven, J. Pfanstiel, R. Saito, and F. R. DeRubertis, Gastroenterology 92, 1998 (1987). 2o I. Ahnfelt-Ronne and O. H. Nielson, Agents Actions 21, 1991 (1987). 21 B. J. Dull, K. Salata, A. Van Langenhove, and P. Goldman, Biochem. Pharmacol. 36, 2467 (1987). 22 I. Ahnfelt-Ronne, O. H. Nielson, A. Christensen, E. Langholz, and P. Riis, Gastroenterology 98, 1162 (1990). 23 M. B. Grisham, in "Inflammatory Bowel Disease: Current Status and Future Approach" (R. P. MacDermott, ed.), pp. 261-266. Elsevier, Amsterdam, 1988. 24 T. Yamada, C. Volkmer, and M. B. Grisham, Can. J. Gastroenterol. 4(7), 295 (1990).
558
ANTIOXlDANT CHARACTERIZATION AND ASSAY
[57]
of techniques that may be used to assess the antioxidant properties of several different anti-inflammatory drugs including the aminosalicylates. Antioxidant Properties of Aminosalicylates
Superoxide The most popular method for measuring the superoxide (02 :) scavenging properties of various compounds is the inhibition of O2--mediated reduction of cytochrome. 25In most cases this assay is entirely appropriate; however, it should be noted that some low molecular weight, easily oxidizable compounds will reduce hemoproteins like cytochrome c in a superoxide-independent mechanism, thereby introducing an artifactually high background rate of reduction. One such compound is 5-ASA (A. M. Miles and M. Grisham, unpublished observations, 1993). To assess the superoxide dismutase (SOD)-like activity of aminosalicylates and eliminate potentially interfering artifacts associated with the cytochrome c assay we have used a modification of the spectrophotometric assay of superoxide dismutase originally described by Marklund. 26 The assay directly measures the rate of spontaneous dismutation of potassium superoxide at 250 nm. 26 At alkaline pH and low 02: concentrations, this radical is stable enough to be studied using a laboratory spectrophotometer. The rate of dismutation of 02: can be monitored using the broad absorbance maximum (molar extinction coefficient = 2000) of 02: at 250 nm. 26 Using these conditions it is possible to observe the 02: scavenging properties of a variety of compounds including SOD and 5-ASA directly. Prior to the experiments all glassware is cleaned with potassium dichromic acid and rinsed extensively with deionized water (i.e., double-distilled and deionized by reversed osmosis). Three milliliters of filtered and degassed 50 mM 2-amino-2-methyl-l-propanol (AMP-HC1; pH 9.5; 25°) buffer containing 0.2 mM desferrioxamine and 20/zg/ml of catalase is added to a 1-cm quartz cuvette positioned in the sample holder of a recording spectrophotometer. Pulverized potassium superoxide (100 mg; KO2), prepared immediately before use, is then dissolved in 25 ml of icecold 50 mM NaOH containing 0.5 mM desferrioxamine. The flask is rapidly swirled and at exactly 10 sec after adding the KO2 to the flask, an aliquot (10/xl) of the KO2 solution is transferred to the tip of a Teflon-coated 25 I. Fridovich, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), pp. 121-122. CRC Press, Boca Raton, Florida, 1985. 26 S. S. Marklund, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), pp. 249-255. CRC Press, Boca Raton, Florida, 1985.
[57]
ANTIOXIDANT
PROPERTIES
559
OF AMINOSALICYLATES
spatula. The strip chart recorder is then started (300 mm/min) and the tip of the spatula containing the KO 2 solution is immediately inserted into the cuvette containing the AMP-HCI buffer and the contents mixed rapidly. The decrease in absorbance at 250 nm is then continuously recorded. We have found that 5-ASA directly interacts with 02 ~, causing the rapid decomposition of the radical (Fig. 2). The disappearance of O2" may occur by two possible pathways. One pathway would require that O2" is reduced by one electron by 5-ASA to give H202 . The other pathway could involve the one-electron oxidation of 02- to yield molecular oxygen. Because 5-ASA would acquire or lose one electron by either pathway, it would become, by definition, a free radical itself. We are currently investigating the mechanism of 02- decomposition by 5-ASA.
Hydrogen Peroxide There are a variety of methods available to measure the hydrogen decomposing activity of various substances. Virtually all these methods are based on the ability of a hemoprotein peroxidase (usually horseradish peroxidase) to catalyze the HzOz-dependent oxidation of an electron-donating detector molecule. In many cases the detector substrate is a leuko dye which, when oxidized, produces a chromogenic p e r o x i d e (H202)
~_o "~ C
°
1O0 80
\
u cO
oe
•
60
lo
•
27
20
& + 5-ASA
0
5
10
15 Time
20
25
30
(seconds)
FIG. 2, Decomposition of superoxide anion radical by 5-aminosalicylic acid (5-ASA) and SOD. Decomposition of superoxide (potassium superoxide) was assessed by measuring the decrease in absorbance at 250 nm (hmax for Oz ~) at pH 9.0 in the absence or presence of SOD (20 t~g/ml) or 5-ASA (0.25 mM).
560
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[57]
or fluorometric product that can easily be quantified. Unfortunately, we have found that certain aminosalicylates (including 5-ASA) are excellent substrates for peroxidase-catalyzed reactions and thus will compete with the detector compound for oxidation by the peroxidase (see section on Hemoprotein-Mediated Oxidative Reactions). These types of interfering reactions render this assay unsuitable for measuring the H202-decomposing activity of 5-ASA. To circumvent this problem we have used a modification of a method originally described by Heath and Tappe127 in which H 2 0 2 is detected using glutathione peroxidase-catalyzed oxidation of reduced glutathione (GSH). It is well known that the selenoprotein glutathione peroxidase has H20 2 +
2GSH--> H20 + GSSG
an absolute specificity for GSH as its electron donating substrate. 2a We have found that a variety of antioxidants will not interfere with the detection of H202 using this assay, unless of course it decomposes the oxidant! Briefly, the assay involves an initial incubation period in which H202 (50-200 ~M) is allowed to interact with the compound in question (e.g., 5-ASA) in l0 mM potassium phosphate buffer (pH 7.4) for 15-60 min at 37°. Following the incubation period, 1 mM GSH and 1 unit/ml of GSH peroxidase (bovine erythrocyte; Sigma, St. Louis, MO) is added to the reaction volume and incubated for an additional 5 min at 37 °. The tubes are then diluted 10-fold using 0.2 M Tris-HCl (pH 8.5), 1 mM 5,5'-dithiobis(2nitrobenzoic acid) (DTNB) added, the tubes incubated for 5 rain at 37 °, and the absorbances determined at 412 nm. DTNB is used to detect GSH via its interaction with GSH to yield 5-thio-2-nitrobenzoic acid (EM 13,600 at 412 nm). Because 2 mol of GSH are oxidized for every mole of H202 present, one may calculate the concentration of H202remaining following its interaction with potential H2OE-Scavenging compounds. Therefore, any compound capable of decomposing H202 will attenuate the oxidation of GSH. We have found that 5-ASA is unable to decompose significant amounts of H202 even when the ratio of 5-ASA to H 2 0 2 is 20 : 1.29
Hydroxyl Radicals It has been demonstrated that activated polymorphonuclear lymphocytes (PMNs) may produce relatively large amounts of hydroxyl radicals (. OH) in the presence of a metal catalyst such as iron (Fe 3÷) (Fig. 1). The source of the Fe is not known; however, it has been suggested to be 27 R. L. Heath and A. L. Tappel, Anal. Biochem. 76, 184 (1976). 28 A. Wendel, this series, Vol. 77, p. 325. 29 M. B. Grisham, Biochem. Pharm. 39, 2060 (1990).
[57]
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
561
fenitin since it is well known that 0 2" will reductively release Fe from the protein core of ferritin. 3° More recent evidence suggests that activated PMNs may produce small amounts of • OH in the absence of a metal catalyst via the interaction between 02- and the hypochlorous acid (HOCI) generated from the myeloperoxidase-catalyzed oxidation of chloride (C1-). 31 In either case it is apparent that activated PMNs have the potential to produce significant quantities of the very reactive • OH. The hydroxyl radical is an extremely reactive species, reacting immediately with virtually all known biomolecules at diffusion-limited rates of reactions (-107-1010 M -1 s e c - l ) . Therefore, it is very short-lived and will react at the site where it is formed, that is, in a site-specific fashion. This reactivity dictates that the radical will be injurious to cells only if the metal catalyst is localized on biomolecules essential for survival. For example, DNA, membrane phospholipids, or proteins required for essential metabolic processes could be potential targets for such a metabolism. If, on the other hand, • OH is generated on biomolecules present in high concentrations (e.g., albumin, glucose) the physiological consequences of such degradative reactions may be minimal. 32 The iron-catalyzed, site-specific generation o f . OH may be measured using ferrous sulfate-mediated degradation of deoxyribose to yield thiobarbituric acid-reactive substances (TBARS) as described by Gutteridge. 33 It has been demonstrated that Fe 2÷, in the absence of an exogenous chelator, will associate with deoxyribose (DOR) where it autoxidizes to generate • OH on the surface of the carbohydrate (i.e., in a site-specific manner) 32,33Only those compounds capable of removing Fe 2÷ from deoxyDOR + Fe 2+ ~ DOR-Fe 2+ DOR-Fe 2+ + 02--> DOR-Fe 3+ + 02" 20 2v + 2H + --~ 2H202 + 0 2 DOR-Fe 2+ + H202 --* DOR-Fe 3+ + OH- + • OH ribose and rendering it poorly redox active or those compounds with H202 decomposing activity will be effective inhibitors of this system. 29'32'33 To generate • OH site specifically, each reaction volume (0.5 ml) contains the following compounds which are added in the following order: 2 mM deoxyribose, 0.1 mM ferrous sulfate, various concentrations of drug, and 10 mM potassium phosphate buffer (pH 7.4). Following a 30-min 30 p. Biemond, H. G. Van Eijk, A. J. G. Swaak, and J. F. Koster, J. Clin. Invest. 73, 1576 (1984). 31 C. L. Ramos, S. Pou, B. E. Britigan, M. S. Cohen, and G. M. Rosen, J. Biol. Chem. 267, 8307 (1992). 32 B. Halliwell and J. M. C. Gutteridge, Arch. Biochem. Biophys. 246, 501 0986). 33 j. M. C. Gutteridge, Biochem. J. 243, 709 (1987).
562
[57]
ANTIOXIDANT CHARACTERIZATION AND ASSAY
incubation period at 37°, reactions are terminated by the addition of catalase (20/xg/ml). The TBARS are then quantified by the sequential addition of 0.5 ml trichloroacetic acid (2.8%) and 0.5 ml thiobarbituric acid (TBA) (1% in 0.05 N NaOH), and the tubes are heated at 100° for 15 min in a boiling water bath. The tubes are then cooled and the absorbance determined at 532 nm. 33Each absorbance value is corrected for nonspecific development that may occur in the absence of iron. Using this method to generate • OH we have found that 5-ASA was much more effective at inhibiting the formation of TBARS than was N-acetyl-ASA (NASA), SAZ, or SP (Fig. 3). Because we had already determined that 5-ASA did not decompose H202, we concluded that the mechanism by which 5-ASA inhibited this reaction was due to its ability to bind Fe and render it poorly redox active. Indeed, we subsequently demonstrated that 5-ASA but not NASA, SAZ, or SP chelates Fe 2+ or Fe3+. 29 To directly assess the ability of 5-ASA to chelate Fe 2+, ferrous sulfate (0.2 mM) was added to a solution of 5-ASA (1 mM) in 0.1 M NaCI (pH 7.4), and the absorbance spectrum was determined. The interaction
125[ 100 , ~ r
-m SP
•~
I'--.. 75
m
50
-&
SAZ
5 -ASA
!
25 0
1 O0
200
300
400
500
Drug Concentrotion (pJvI) FIG. 3. Effects of 5-aminosalicylic acid (5-ASA), N-acetyl-5-ASA (NASA), sulfasalazine (SAZ), and sulfapyridine (SP) on ferrous sulfate-mediated degradation of deoxyribose to yield thiobarbituric acid-reactive substances (TBARS). Each data point is the mean from at least four determinations and varied by less than ---5%. A mean absorbance value of 0.218 was achieved in the absence of drug and was designated as 100%. (Data derived from Grisham. 29)
[57]
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
563
b e t w e e n Fe 2+ and 5-ASA produced a purple chromophore indicative of a charge transfer complex (Fig. 4). 29
Hydroxyl radicals may also be generated in free solution by exposing a detector molecule such as deoxyribose to a ferrous iron (Fe z+) chelate in the presence of H202: F e 2+ + H 2 0 2 ~
F e 3+ + O H -
+ • OH
In these experiments Fe 2+ is chelated to diethylenetriaminepentaacetic acid (DTPA), thereby preventing the metal from associating with deoxyribose. Thus, any" OH generated from the interaction between Fe2+-DTPA and HzOzwill have equal access to all components of the reaction volume including deoxyribose. Hydroxyl radical production is measured by quantifying the oxidative degradation of deoxyribose to yield TBARS as described by Gutteridge. 33
0,20-
0,15-
U C
0.1-
0.05-
b 0
400
s~o
e~o
7~o
Wavelength (nm)
FIG. 4. Absorbance spectrum of the Fe2+-5-ASA chelate in the absence or presence of deoxyribose. Ferrous sulfate (0.2 mM) was added to a solution containing 1 mM 5-ASA and 0.1 M NaC1, pH 7.4, in the absence or presence of 4 mM deoxyribose and the absorbance spectrum determined. (a) Fe 2÷, (b) 5-ASA, (c) Fe 2÷ plus 5-ASA, and (d) Fe 2÷ plus 5-ASA in the presence of deoxyribose. (Data derived from Grisham. 29)
564
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[57]
1001 g
60
~ ' ~ ~
NASA SP SAZ
,o
5-ASA
20 I 0
, 100
, 200
i 300
Drug C o n c e n t r a t i o n
i 400
i 500
OzM)
FIG. 5. Effects of 5-aminosalicylic acid (5-ASA), N-acetyl-5-ASA (NASA), sulfasalazine (SAZ), and sulfapyridine (SP) on the Fe2+-DPTA-mediated degradation of deoxyribose to yield thiobarbituric acid-reactive substances (TBARS). Each data point is the mean from four determinations and varied by less than ---5%. A mean absorbance value of 0.426 ~vas achieved in the absence of drug and was designated as 100%. (Data derived from Grisham. 29)
The FeZ+-DTPA complex is prepared by the method of Cohen 34 in which 1 mM ferrous sulfate is added to a solution containing 2 mM DTPA and 50 mM potassium phosphate buffer. 34 Each reaction (volume 0.5 ml) contains the following compounds which are added in the following order: 2 mM deoxyribose, 0.1 mM FeZ+-DTPA (0.1 mM Fe2+-0.2 mM DTPA), various concentrations of the aminosalicylate, and 10 mM potassium phosphate buffer (pH 7.4). Addition of 0.2 m M H202 is used to initiate the reaction. Following a 10-min incubation period at 37° reactions are terminated by the addition of 20/~g/ml catalase. The TBARS are then quantified as described above using the sequential addition of 0.5 ml trichloroacetic acid (2.8%) and 0.5 ml TBA (1% in 0.05 N NaOH). We have found that SAZ as well as 5-ASA, NASA, and SP are equally effective at inhibiting the • OH-mediated degradation of deoxyribose when • OH is generated in free solution (Fig. 5). 29 This is not at all surprising since virtually all phenolic compounds have been shown react with -OH at diffusion-limited rates. The data also illustrate two important points. First, the mechanism of action of 5-ASA cannot be due to its ability to G. Cohen, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), pp. 55-64. CRC Press, Boca Raton, Florida, 1985.
[57]
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
565
scavenge selectively the. OH since the pharmacology inactive metabolites NASA and SP are equally effective in this assay. Second, the data dispel the erroneous suggestion that certain compounds may act as specific "hydroxyl radical scavengers" since virtually all compounds react rapidly with • OH.
Peroxyl Radicals Hydroxyl radicals interact with certain carbohydrates, proteins, nucleotide bases, and lipids to produce peroxyl radicals (ROO .) as intermediates.35 Peroxyl radicals are slightly less reactive than. OH and thus possess "extended" half-lives of seconds instead of nanoseconds. These radicals would then be expected to react at site distant from those of. OH generation, thereby promoting the toxicity of" OH. Indeed, peroxyl radical intermediates have been shown to damage biomolecules. The best characterized example of peroxyl radical-mediated reactions is the peroxidation of polyunsaturated fatty acids (PUFA) initiated by • OH: •OH+LH--~HOH+L. L" + 02---> L 0 0 " L 0 0 " + LH --~ L 0 0 t t + L"
where • OH, LH, L., L O 0 . , and LOOH represent the hydroxyl radical, polyunsaturated lipid, lipid alkyl radical, lipid hydroperoxyl radical, and lipid hydroperoxide, respectively. Peroxyl radicals will also oxidize proteins, carbohydrates, and sulfhydryl components and hemolyze erythrocytes.3~ Because most free radical generators require the addition of potentially interfering cofactors and transition metals such as iron (or copper) to produce the free radicals we chose to use the thermolabile, peroxyl radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). AAPH is a free radical initiator that decomposes unimolecularly without requirement for enzymes or biotransformation to yield nitrogen and carbon radicals. 37,3s The carbon-centered radicals react rapidly with molecular oxygen (02) to yield peroxyl radicals. The rate of decomposition of AAPtt ~5 R. B. Wilson, in "Oxidative Stress" (H. Sies, ed.), pp. 41-72. Academic Press, London and New York, 1985. 36 I. S. Sandu, K. Ware, and M. B. Grisham, Free Radical Res. Commun. 16, 111 (1992). ~7 E, Niki, this series, Vol. 186, p. 100. 38 E. Niki, E. Komuro, M. Takahashi, S. Urano, E. Ito, and K. Terao, J. Biol. Chem. 263, 19809 (1988).
566
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[57]
is determined primarily by temperature, and the rate of generation of radicals is virtually constant for the first few hours. The reaction sequence is as follows: A - - N ~ N - - A ~ A. + N 2 + A" A- + O 2 ~ A O O " AOO. + L H ~ AOOH + L . L . + 02 ~ LOO" LOO. + L H ~ LOOH + L . where A - - N ~ - - N - - A , A., AOO., and AOOH represent AAPH, alkyl radical, peroxyl radical, and hydroperoxide, respectively. Because peroxyl radical-mediated oxidative reactions are not initiated by Fe-catalyzed hydroxyl radical formation, an inhibitory effect by an antioxidant is due solely to its ability to scavenge the peroxyl radical. We have found that peroxyl radical-mediated lipid peroxidation represents a sensitive and simple method to assess the antioxidant activity of a variety of compounds such as 5-ASA. Lipid peroxidation is quantified by measuring the peroxyl radical-mediated formation of TBARS from a phospholipid substrate as described by Buege and Aust. 39 Folch fraction III phospholipid (brain extract; 80-85% phophatidyl serine) is obtained from Sigma. The phospholipid preparation is stored at - 2 0 ° as a 10% (w/v) solution in chloroform which contains 140 mM phospholipid phosphate. Aqueous solutions ofphospholipid (liposomes) are prepared by evaporating the chloroform from 100-/zl aliquots of the lipid preparation using oxygen-free nitrogen, adding 2 ml saline, and then sonicating the suspension for 10 sec under nitrogen. Briefly, AAPH (50 mM) is incubated in phosphate-buffered saline (PBS) containing phospholipid (brain extract; 0.7 mM phospholipid phosphate) for varying lengths of time at 37°. At 15 min intervals aliquots (0.5 ml) are removed from each tube and added to 1 ml of a solution containing 15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, and 0.25 N HC1. To prevent spurious lipid peroxidation during subsequent steps, 0.02% (w/v) butylated hydroxytoluene is added. Each mixture is then heated in a boiling water bath for 15 min; the tubes are allowed to cool and then are centrifuged at 8000 g for 5 rain to remove precipitate. The absorbance of each sample is then determined at 532 nm. We have found that 5-ASA but not SAZ, SP, NASA is capable of completely inhibiting peroxyl radical-mediated lipid peroxidation such that the concentration necessary for 5-ASA to inhibit peroxidation by 50% (ICs0) is equal to approximately 10/xM (Fig. 6). 24,36 39j. A. Buege and S. D. Aust, this series, Vol. 52, p. 303.
[57]
567
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
^^ L _
tO .m
_,,._._.~v_
_-
SAZ
v
60
o
E
o
40
(/}
20
0
0
!
a
10
20
__
I,
30
40
5"0
Drug Concentrotion (/u,M) FIG. 6. Effects of 5-aminosalicylic acid (5-ASA), sulfasalazine (SAZ), N-acetyl-5-ASA (NASA), and sulfapyridine (SP) on peroxyl radical-mediated lipid peroxidation. Lipid (80% phosphatidylcholine) was incubated with the thermolabile free radical initiator 2,2'-azobis(2amidinopropane) dihydrochloride for 30 min at 37° in the absence of presence of SAZ or its metabolites. Lipid peroxidation was determined by measuring the formation of thiobarbituric acid-reactived substances (TBARS). (Data derived from Sandhu et al) 6)
We have also used the ability of aminosalicylates to inhibit peroxyl radical-mediated hemolysis of human erythrocytes to assess their antioxidant activity. Briefly, blood is collected from human volunteers by venipuncture. The erythrocytes are separated from leukocytes and platelets by dextran sedimentation, and the erythrocyte pellet is washed by centrifugation three times with PBS. The cells (2 × 109/ml) are incubated with 100 t~Ci sodium [SlCr] chromate for I hr at 37°. The cells are then washed three times with PBS and resuspended to 2 × l 0 9 cells/ml with Dulbecco's phosphate-buffered saline (DPBS). The erythrocytes are then diluted to 2 × 108/ml and incubated with varying concentrations of AAPH in DPBS for varying lengths of time at 37° with occasional swirling. At 15-rain intervals aliquots of cell suspension are removed and microcentrifuged, and the supernatant and pellet are counted for the presence of 51Cr to determine the extent of hemolysis. Interestingly, both 5-ASA and NASA are equally effective at inhibiting peroxyl radical-mediated hemolysis, whereas SAZ and SP are either much less effective or inactive (Fig. 7). 36 The data suggest that the mechanism by which peroxyl radicals mediated hemolysis may not be due solely to lipid peroxidation. In fact, we find very little evidence for lipid peroxidation in this system. 36
568
ANTIOXIDANT CHARACTERIZATION AND ASSAY
100~
[57]
_
90 t
70
'i
0
10
20
30 Drug
40
50
60
Concentration
70
80
90
100
(/~M)
FIG. 7. Effect of sulfasalazine and its metabolites on AAPH-induced hemolysis. Erythrocytes (2 × 108 cells/ml) were incubated with 50 mM AAPH in the absence or presence of anti-inflammatory drugs for 120 min at 37°. (@) 5-ASA, (A) 4-ASA, (11) N-acetyl-5-ASA, 01') sulfapyridine, and (T) sulfasalazine. Each data point represents the mean - SEM of duplicate determinations from at least three different donors. (Data derived from Sandu et al. 36)
Hemoprotein-Mediated Oxidative Reactions Work from our laboratory as well as that of others have demonstrated that hemoglobin (Hb) will interact with H2Oz or lipid hydroperoxides to yield a hemoprotein-associated oxidant that is capable of peroxidizing polyunsaturated fatty acids (PUFA), phospholipids, and cellular membranes. 4°-45 The interaction between methemoglobin (MetHb; Hb III) and H202 results in the two-electron oxidation of the hemoprotein to yield the radical form of ferrylhemoglobin (ferryltb; • Hb ~v) which is a potent oxidant. Inasmuch as active episodes of ulcerative colitis are accompanied 40 M. B. Grisham, J. Free Radical Biol. Med. 1, 227 (1985). 41 T. I. Yamada, C. Volkmer, and M. B. Grisham, Free Radical Biol. Med. 10, 41 (1991). 42 S. M. H. Sadrzadeh, E. Graf, S. S. Panter, P. E. Hallaway, and J. W. Eaton, J. Biol. Chem. 259, 14354 (1984). 43 j. Kanner and S. Harel, Arch. Biochem. Biophys. 237, 314 (1985). 44 M. B. Grisham and D. N. Granger, Dig. Dis. Sci. 33, Suppl., 6S (1988). 45 S. M. H. Sadrzadeh, D. K. Anderson, S. S. Panter, P. E. Hallaway, and J. W. Eaton, J. Clin. Invest. 79, 662 (1987).
[57]
569
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES I
,?
=~&'
'&"--
SAZ
4o _
0/ 0
,
,
,
l
20
40
60
80
•
1O0
Drug Concentration (b~M) FIG. 8. Effects of 5-ASA, NASA, SP, and SAZ on hemoglobin-catalyzed lipid peroxidation. Lipid was incubated in the absence or presence of varying amounts of SAZ or its metabolites with hemoglobin (0.02 mmol/liter) and HzO2 (0.1 mmol/liter) for 20 min at 37°. Lipid peroxidation was determined by measuring the formation of TBARS. (Data derived from Yamada et al. 41)
by subepithelial hemorrhage and hemolysis as well as mucosal lipid peroxidation, 2z'44 we have suggested that Hb-catalyzed reactions may play an important role in mediating and/or exacerbating inflammatory tissue injury. 41'44Indeed, the presence of Hb has been proposed to mediate and/or exacerbate oxidative injury in tissues such as the central nervous system, retina, kidney, and s y n o v i u m . 45-47 U s i n g Hb-catalyzed peroxidation of phospholipid as a model of oxidative degradation of membrane lipids, we assessed the ability of SAZ and its metabolites 5-ASA, NASA, and SP to inhibit the oxidative reaction. Hemoglobin-catalyzed lipid peroxidation is quantified using the method of Buege and Aust 39 in which TBARS are determined by measuring the absorbance at 532 nm. Human hemoglobin (predominantly HbIII; Sigma) is determined by the method of Tentori and Salvati 48in which cyanomethemoglobin is quantified. Briefly, each reaction (volume 0.5 ml) contains 20 mM potassium phosphate buffer (pH 7.4), 0.7 mM phospholipid phos46 S. Yoshino, D. R. Blake, S. Hewitt, C. Morris, and P. A. Bacon, Ann. Rheum. Dis. 44, 485 (1985). 47 G. J. Handelman and E. A. Dratz, Ado. Free Radical Biol. Med. 2, 1 (1986). 48 L. Tentori and A. M. Salvati, this series, Vol. 76, p. 707.
570
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[57]
120 10o
8O
o
~
A
~
a
SP
20 0
0
20
~
60
80
100
Drug Concentration (j~l)
FIG. 9. Effectsof 5-ASA, 4-ASA, NASA, and SP on myeloperoxidase(MPO) activity. HumanMPO activitywas assessed by measuringthe HzO2-dependentoxidationof 3,3',5,5'tetramethylbenzidinein the absence or presence of varyingconcentrationsof metabolites. (Data derived from von Ritter eta/. 49)
phate, Hb (12.5 tzM; 50/zM heme), varying concentrations of sulfasalazine or its metabolites, and 100/xM H202. Following a 15-min incubation at 37°, reactions are terminated by the addition of 1 ml of a solution containing 15% (w/v) trichloroacetic acid, 0.375% (v/v) thiobarbituric acid, and 0.25 N H C I . 39 To prevent spurious lipid peroxidation during subsequent steps, 0.02% butylated hydroxytoluene is added at this point. Each reaction mixture is then heated in a boiling water bath for 15 min; the tubes are removed and allowed to cool and then centrifuged at 8000 g for 5 rain to remove precipitate. The absorbance of each sample is then determined at 532 nm. 39 We found that 5-ASA and to a lesser extent NASA is effective at inhibiting Hb-catalyzed lipid peroxidation such that 5-ASA and NASA exhibited IC50 values of 50 and 125/~M, respectively (Fig. 8). 41 Neither SAZ nor SP were effective in this system. We have also found that 5-ASA is oxidized to a golden brown chromophore during its interaction with Hb and H202, suggesting that it is acting as an alternative substrate for the ferryl hemoprotein-mediated oxidation. 4'1 These are interesting data in view of a report demonstrating the presence of several different uncharacterized oxidation products of 5-ASA ob,tained from rectal dialyzates of patients with active ulcerative colitis being treated with 5-ASA. 22
[57]
ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES
571
100"
C
o
5
O
Dapsone 0
0
• 50
N-AcelyI-S~ASA m 100pM
Drug Concenlration [..M]
FIG. 10. Hypochlorous acid (HOC1)-scavenging properties of sulfapyridine (SP), 5-ASA, 4-ASA, N-acetyl-5-ASA, and dapsone. All substances demonstrated a similar potential for scavenging HOC1. Each data point represents the mean for triplicate samples and did not vary by more than -+7%. (Data derived from von Ritter eta/. 49)
Another hemoprotein of interest in inflammation is myeloperoxidase (MPO), the enzyme released by activated PMNs at sites of inflammation. It is known that this enzyme combines with H202 to yield a potent hemoprotein-associated free radical (known as compound I) which oxidizes chloride by two electrons to yield the potent cytotoxic oxidant hypochlorous acid (HOCI) (Fig. 1). Using the H202-dependent oxidation of 3,3',5,5'tetramethylbenzidine at 655 nm to quantify MPO activity in the absence or presence of SAZ or its metabolites, we found that 5-ASA and its analog 4-ASA were very effective inhibitors of the catalytic activity of MPO (Fig. 9). 49 These aminosalicylates might act as alternative substrates for MPO in much the same way as they do with Hb-catalyzed oxidation reactions. In addition to showing inhibition of the catalytic activity of MPO, we, as well as others, have also demonstrated that 5-ASA is an exceedingly good scavenger of HOCI (Fig. 10). 18,49The concentration of HOC1 is determined by the method of Thomas e t al. 5° in which HOC1 is trapped by taurine to form taurine monochloramine. Taurine monochloramine is then quantified 49 C. von Ritter, M. B. Gfisham, and D. N. Granger, Gastroenterology 96, 811 (1989). 50 E. L. Thomas, M. B. Grisham, and M. M. Jefferson, J. Clin. Invest. 72, 444 (1983).
572
A N T I O X l D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
[58]
by its ability to oxidize 2 mol of the yellow chromophore 5-thio-2-nitrobenzoic acid to 1 mol of the colorless 5,5'-dithiobis(2-nitrobenzoic acid). Conclusion Taken together the experimental data demonstrate that 5-ASA possesses potent antioxidant activity with the ability to scavenge a variety of free radicals and the ability to decompose neutrophilic oxidants (e.g., HOC1) and detoxify hemoprotein-associated oxidizing agents. Furthermore, 5-ASA has the additional property of being able to chelate iron and render it poorly redox active. Thus, the reason that 5-ASA may be so effective as an antiinflammatory agent in vivo may be due to its multitude of antioxidant properties. Acknowledgments Some of the work reported in this chapter was supported by a grant from the National Institutes of Health (DK43785; Project 6).
[58] A n t i o x i d a n t A c t i o n o f S t o b a d i n e By LUBICA
HORAKovA, H E L M U T SIES, and STEEN STEENKEN
Introduction Stobadine is a pyridoindole derivative synthesized in a search of new antiarrhythmic drugs) '2 It has been demonstrated that besides this effect it also reveals a-adrenolytic, antihistaminic, local anesthetic, and sedative effects. In comparison to its (+)-cis isomer it is less toxic and more powerful in diminishing epinephrine-induced arrhythmia. A protective, antihypoxic effect on myocardium and brain tissue has also been observed. The pharmacological effects of stobadine have been summarized. 3 The synthesis of stobadine is shown in Scheme 1. Treatment of ptolylhydrazine (1) with N-methyl-4-piperidone (II) yields 2,8-dimethyl2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indole (liD. After catalytic hydrogenation of Ill, the (-+)-cis isomer (IV) is obtained. This isomer is recrystallized several times with (+)-dibenzoyltartaric acid to obtain the optically S. ~tolc, V. Bauer, L. Bene~, and M. Tich~, Czech. Patent 229,067 (1983). 2 S. ~tolc, V. Bauer, L. Bench, and M. Tich~, Swiss Patent 651,754 (1985). 3 L. Bene~ and S. ~tolc, Drugs Future 14, 135 (1989).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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A N T I O X l D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
[58]
by its ability to oxidize 2 mol of the yellow chromophore 5-thio-2-nitrobenzoic acid to 1 mol of the colorless 5,5'-dithiobis(2-nitrobenzoic acid). Conclusion Taken together the experimental data demonstrate that 5-ASA possesses potent antioxidant activity with the ability to scavenge a variety of free radicals and the ability to decompose neutrophilic oxidants (e.g., HOC1) and detoxify hemoprotein-associated oxidizing agents. Furthermore, 5-ASA has the additional property of being able to chelate iron and render it poorly redox active. Thus, the reason that 5-ASA may be so effective as an antiinflammatory agent in vivo may be due to its multitude of antioxidant properties. Acknowledgments Some of the work reported in this chapter was supported by a grant from the National Institutes of Health (DK43785; Project 6).
[58] A n t i o x i d a n t A c t i o n o f S t o b a d i n e By LUBICA
HORAKovA, H E L M U T SIES, and STEEN STEENKEN
Introduction Stobadine is a pyridoindole derivative synthesized in a search of new antiarrhythmic drugs) '2 It has been demonstrated that besides this effect it also reveals a-adrenolytic, antihistaminic, local anesthetic, and sedative effects. In comparison to its (+)-cis isomer it is less toxic and more powerful in diminishing epinephrine-induced arrhythmia. A protective, antihypoxic effect on myocardium and brain tissue has also been observed. The pharmacological effects of stobadine have been summarized. 3 The synthesis of stobadine is shown in Scheme 1. Treatment of ptolylhydrazine (1) with N-methyl-4-piperidone (II) yields 2,8-dimethyl2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indole (liD. After catalytic hydrogenation of Ill, the (-+)-cis isomer (IV) is obtained. This isomer is recrystallized several times with (+)-dibenzoyltartaric acid to obtain the optically S. ~tolc, V. Bauer, L. Bene~, and M. Tich~, Czech. Patent 229,067 (1983). 2 S. ~tolc, V. Bauer, L. Bench, and M. Tich~, Swiss Patent 651,754 (1985). 3 L. Bene~ and S. ~tolc, Drugs Future 14, 135 (1989).
METHODS IN ENZYMOLOGY, VOL. 234
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[58]
573
ANTIOXIDANT ACTION OF STOBADINE
H3C~ + C?/CH3 ~t'~N~'NH2 O" v H
~.H 3 C ~ N ~ ' C H 3 H
(m)
H3C~N/CH3 (iv)
STOBADINE SCHEME 1
pure ( - ) - c i s enantiomer. The reaction with corresponding acids yields stobadine dihydrochloride or dipalmitate. Intensive studies of stobadine as an antioxidant were initiated after the finding of its antiarrhythmic cardioprotective and antihypoxic effects on myocardium.] As the participation of free radicals in the pathobiology of hypoxia (ischemia)-reperfusion injury has received considerable experimental support, the possibility of stobadine forming stable free radicals was investigated. Some Characteristics of Stobadine
For stobadine dihydrochloride, the pK a values for indole nitrogen and methyl nitrogen deprotonation are 3.2 and 8.7 as determined by spectrophotometric and potentiometric titration: The one-electron oxidation potential of stobadine at pH 7 (0.58 V/NHE) studied by pulse radiolysis 5 is lower than the potentials determined for biological target molecules such as purines and pyrimidines 6 as well as amino acids, 7,8 which indicates that stobadine can repair oxidized bases and amino acids by electron donation in biological systems. 4 M. ~tefek, L. Bene~, and V. Zelnik, Xenobiotica 19, 143 (1989). 5 S. Steenken, A. R. Sundquist, S. V. Jovanovi6, R. Crockett, and H. Sies, Chem. Res. Toxicol. 5, 355 (1992). 6 S. V. Jovanovi~ and M. G. Simic, Biochim. Biophys. Acta 1008, 39 (1989). 7 S. V. Jovanovi~, S. Steenken, and M. G. Simic, J. Phys. Chem. 95, 684 (1991). 8 M. R. De Felippis, C. P. Murthy, M. Faraggi, and M. H. Klapper, Biochemistry 28, 4847 (1989).
574
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[58]
Assay for Stobadine Several assay methods for stobadine in various biological matrices have been described: spectrofluorometric analysis of extracts from serum and urine,9 gas-liquid chromatography to study stobadine and metabolites in liver microsomes, 1° radiometric methods using radioactively labeled stobadine dihydrochloride to measure the unchanged drug by liquid scintillation counting, H and high-performance liquid chromatography (HPLC) to assess stobadine pharmacokinetics in dogs and humans ~2and to determine the concentration of stobadine in microsomes and liposomes.13 Antioxidant Properties of Stobadine
Radical Formation The ability of stobadine to form stable free radicals was first established in 1989.14 In the presence of oxygen, stobadine dissolved in ethanol generated free radicals by the action of y radiation. The electron spin resonance (ESR) spectrum reflected the presence of an NO. radical on the indole nitrogen. 14These findings were confirmed in a study where an ESR-observable radical of stobadine with a well-resolved hyperfine structure was detected on oxidation of stobadine with PbO 2 and tert-butyl hydroperoxide (tBuOOH). 15 Because no hyperfine splittings from the proton in the/3 position to the nitroxyl group could be detected, it was proposed that the intermediate radical II was converted by oxidation to nitrone III. After trapping a radical (possibly tBuOO, or tBuO-), adduct IV was formed (Scheme 2). It might be supposed that these steps of radical formation reflect one way by which stobadine scavenges reactive radicals. An alternative scavenging mechanism, involving electron transfer, was evaluated by using pulse radiolysis with optical detection, 5 showing the importance of the indole nitrogen. One-electron oxidation of stobadine with radicals such as C6H50., CC1302- , Br2 v, and HO. (reaction rate constants - 5 × 108-101°M - 1 sec-~) led to formation of the radical cation (absorbance maxima at 280 and 445 nm) which deprotonates from the indolic nitrogen (pKa 5.0) to give a nitrogen-centered radical (absorbance 9 V.Marko, Pharmazie 40, 192 (1985). l0 M. ~tefek and L. Bene~, J. Chromatogr. 415, 163 (1987). 11 V. ~ a s n ~ and M. ~tefek, Radioanal. Nucl. Chem. 111, 117 (1987). 12 L. ~olt6s, Z. K~llay, ~. Bezek, and V. Fedele~ovtt, BiopharmacoL Drug Dispos. 12, 29 (1991). 13 L'. Horfikovfi, K. Briviba, and H. Sies, Chem.-Biol. Interact. 83, 85 (1992). 14 H. Sz6csov~i and L. Bene~, Csl. Pharmazie 37, 121 (1989). 15 A. Sta~ko, K. Ondria~, V. Mi~fk, H. SzOcsovfi, and D. Gergel', Chem. Pap. 44, 493 (1990).
[58]
ANTIOXIDANT ACTION OF STOBADINE 9
H
I
H3C- .~S/ "A~ ~ r ~ b I A I -N-'CH3 ter¢-BuOOH H3Cv/~x ~ 7 ~ N ~ 3 PbO2 6 -i~I H 4
H
HY 'C ~N--CH3
H
~
575
N"-OH3
tert-
BuOOH
PbO 2
H
tert ~lauOO" or X
H 3 C ~ ~ x
N--CH3
O" IV
i
0 |1| SCHEME 2
maxima at 275, 335, and 410 nm), probably bearing a positive charge at the pyrido-nitrogen (Fig. 1).
Singlet Oxygen Quenching Stobadine is an effective quencher of ~O2 with an overall quenching rate constant of 1,3 × 108 M-1 sec-~, determined with the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) as 10 2 source and by monitoring ~Ozphotoemission with a germanium diode.5 This rate constant is comparable to the constants for different tocopherol homologs (108 M sec-~) and is intermediate between the range of values determined for carotenoids (109 M -1 sec-1) 16 and thiols (106 M -1 sec-l), t7 Stobadine represents one of the most efficient water-soluble ~O2 quenchers reported to date.
Hydroxyl Radical Scavenging The ability of stobadine to scavenge the hydroxyl radical generated by a Fenton-type reaction was demonstrated using ESR spin trapping with 5,5-dimethyl-l-pyrroline N-oxide (DMPO). t8 The signal intensity of the DMPO-OH adduct reflected the relative O H radical concentration in the system. In the presence of 12 mM stobadine the height of the DMPO-OH signal was markedly decreased. i~p. Di Mascio, S. Kaiser, and H. Sies, Arch. Biochem. Biophys. 2"/4,532 (1989). 17T. P. A. Devasagayam,A. R. Sundquist, P. Di Mascio, S. Kaiser, and I-t. Sies, J. Photochem. Photobiol., B 9, 105 (1991). is K. Ondria~,V. Mi~fk,D. Gergel', and A. Stagko,Biochim. Biophys. Acza 111113,238 (1989).
ANTIOXlDANT CHARACTERIZATIONAND ASSAY
576
[58]
Me
Me
~
~N÷I H
-
/
I
-
I
I-I '
+H ÷ I
H
pKa
5.0
FIG. 1. Radical formation from stobadine.
The rate constant for the stobadine-'OH radical interaction was determined 19using two chemical methods for detecting "OH radicals: (I) deoxyribose oxidation to thiobarbituric acid (TBA)-reactive products and (2) ethylene production from 2-keto-4-methylthiobutyric acid (KMBA). Stobadine was found to be an efficient scavenger of "OH characterized by a second-order rate constant of approximately 1 × 101°M - 1 sec- ~ in both assays, in agreement with the directly measured value of 7 × 109 M - l sec-1.5 The potency of stobadine to prevent ethylene production from KMBA, characterized by the IC50 value, was compared for three different "OH generating systems, namely, a chemical, an enzymatic, and a membrane-bound enzymatic system. Similar IC50 values (0.74-0.93 mM) were obtained for the three systems studied, ~9 suggesting that stobadine is an efficient scavenger of "OH radicals produced not only free in solution but also in biological membranes.
Superoxide Radical Scavenging In the concentration range of 10 to 100/zM stobadine did not significantly scavenge superoxide anions generated in an enzymatic (xanthine/ xanthine oxidase) system, where superoxide anions were determined in vitro by spectrophotometric measurement of the reduction of ferricytochrome. 2° The rate constant for interaction of stobadine with superoxide was determined as approximately 103 M -1 s e c - l . 21 The IC50 value for inhibition ofpyrogallol autoxidation by stobadine was l0 mM, also indicating a low affinity of stobadine to superoxide; for comparison, the same ICs0 value for ascorbate is about 0.04 mM. 21 19M. ~tefek and L. Beneg, FEBS Lett. 294, 264 (1991). 20L'. Horfikov~, V. Uraz, O. Ondreji~kov~i,L'. LukoviC and I. Jur~nek, Biomed. Biochim. Acta 50, 1019 (1991). 21j. Humplov~f,M. ~tefek, L. Bene~, and L. Kri~anov~i, Cesk. Fysiol. 41, 80 (1992).
[58]
ANTIOXIDANT ACTION OF STOBADINE
577
As measured by superoxide-induced lucigenin-amplified chemiluminescence, the second-order rate constant for the reaction of stobadine with superoxide was estimated to be 7.5 x 102 M -1 sec-1. 22
Antioxidative Effect in Liposomes Stobadine exhibited its antioxidative effect in phosphatidylcholine liposomes, incubated under air at 50°, where lipid peroxidation was detected spectroscopically for conjugated diene and thiobarbituric acid product formation. 18 Several drugs were tested in different liposomal/drug molar ratios, and stobadine was found to be more effective than butylated hydroxytoluene (BHT). Stobadine reactivity with peroxyl radicals in liposomes was examined using a lipid-soluble azo initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Stobadine exerted scavenging as evidenced by the inhibition of (1) cis-parinaric acid fluorescence decay (half-maximal effect at 20/zM) and (2) luminol-sensitized chemiluminescence (half-maximal effect at 33/zM). 22
Mechanisms of Antioxidant Action in Microsomes Stobadine effectively inhibited thiobarbituric acid-reactive product formation in a concentration-dependent manner, with an ICs0 value of 56 t~M in cumene hydroperoxide-induced lipid peroxidation in microsomes, and it also inhibited oxygen consumption. 23Nonenzymatic lipid peroxidation induced by ascorbate (0.5 mM)/ferrous ion (50/xM) in heat-denatured microsomes was also inhibited, with an ICs0 of 25/zM. Because stobadine did not prevent cumene hydroperoxide bioactivation 23and because superoxide anion radicals and hydroxyl radicals are not involved in cumene hydroperoxide-dependent lipid peroxidation, 24'25 the inhibitory effect of stobadine may be at least partially explained by its scavenging of alkoxyl (LO') and peroxyl (LOO') radicals. Stobadine was equally efficient in inhibiting microsomal lipid peroxidation induced by the lipid-soluble azo initiator of peroxyl radicals AMVN or the water-soluble 2,2'-azobis(2-aminopropane) hydrochloride (AAPH), azo initiators of peroxyl radicals with half-maximal effect at 17/zm. 22The stobadine octanol-water partition coefficient (log P of 0.57) explains its ability to quench peroxyl radicals in both lipid and aqueous phases. 22 V. E. Kagan, M. Tsuchiya, E. Serbinova, L. Packer, and H. Sies, Biochem. Pharmacol. 45, 393 (1993). 23 M. ~tefek, M., Masarykov~i, and L. Bene~, Pharmacol. Toxicol. 711, 407 (1992). 24 R. H. Weiss and R. W. Estabrook, Arch. Biochem. Biophys. 251, 348 (1986). 25 j. A. Thompson and N. P. Yumibe, Drug Metab. Rev. 20, 365 (1989).
578
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[58]
The antioxidant effect of stobadine was also examined with iron/ADP/ N A D P H as prooxidant in normal and vitamin E-deficient rat liver microsomes. 13 L o w level chemiluminescence was used as a sensitive method of monitoring lipid peroxidation. The duration of the lag phase is a measure of susceptibility to peroxidation, and stobadine at 5 /zM was found to double the lag phase. Stobadine at 5 / z M was without effect on vitamin E-deficient microsomes. Addition of N A D P H to microsomes activated the c y t o c h r o m e P-450 system to metabolize stobadine, z6 When liposomal peroxidation was started by adding F e S O 4 with the reductants N A D P H or ascorbate, stobadine was not consumed. It thus appears that in microsomes the antioxidant effect of stobadine depends on vitamin E, and stobadine is metabolized in the presence of N A D P H . In liposomes, stobadine reacted with lipid radicals, and N A D P H prevented the loss of stobadine. Antioxidant Effect in Mitochondria
The inhibitory effect of stobadine on FeZ+/ascorbate-induced lipid peroxidation was studied in brain mitochondria in comparison to the effect of B H T and nifedipine, z7 The ICs0 values for B H T , stobadine, and nifedipide were about 1,200, and 800/zM, respectively. Preventive Effect in lschemia and Reperfusion
Stobadine markedly delayed the onset of epinephrine-induced arrhythmia in laboratory animals, 28 decreased functional damage, and reduced the infarct size of isolated rat hearts subjected to a period of ischemia followed by reperfusion. 3'29 Isoproterenol could be a trigger of heart injury caused by free radicals.3° Administration of stobadine prevented a decrease in the content o f protein thiol groups and glutathione in hearts treated with high doses ofisoproterenol (30 mg/kg). 31 Moreover, stobadine also attenuated the increase in the content of malondialdehyde and decreased the activities of catalase and glutathione reductase as well as the ratio of reduced to oxidized glutathione 26M. ~tefek, L. Bene~, M. Jergelov~i, V. S~asmir, and L. Turi-Nagy, Xenobiotica 17, 1067 (1987). 27L'. Hor,'ikovfi,I. Jurfmek, and B. Bokn~ov~i, Biologia (Bratislava) 45, 313 (1990). 28A. Babulovfi, L. Buran, and L. Bene~, Farm. Obz. 54, 15 (1985). 29I. Gabauer, J. Styk, J. Slezfik,J. Okoli~finy,V. Holec, and L'. Bene~,Bratisl. Lek. Listy 85, 265 (1986). 30S. L. Jewet, L. J. Eddy, and P. Hochstein, Free Radical Biol. Med. 6, 185 (1989). 3l O. Ondreji~kov~, A. D~urba, J. Sedlfik, J. Tokfirov~i,T. Ma~i~kovfi, and L. Bene~, Biomed. Biochim. Acta 50, 1251 (1991).
[58]
ANTIOXIDANT ACTION OF STOBADINE
579
(GSH/GSSG) observed in heart mitochondria isolated from isoproterenoltreated animals. 31 A protective effect of stobadine was also demonstrated in several models of brain hypoxia followed by reoxygenation. 2°'a2'33 The dose employed in these experiments (2 mg/kg) is within the dose range typically used in testing the pharmacodynamic effects of stobadine in preclinical studies and is in the upper range of expected therapeutic doses. Stobadine decreased the level of conjugated dienes and TBA-reactive substances and maintained the level of total thiol groups after complete brain ischemia followed by incubation of brain cortex homogenates in a stream of wet nitrogen (hypoxia) and wet air (reoxygenation). 3z The ability of stobadine to prevent lipid peroxidation was tested also in vivo in incomplete rat cerebral ischemia models induced by 4 hr of ligation of the common carotid arteries with a subsequent 10 min of reperfusion. 2° The concentration of conjugated dienes and TBA-reactive substances significantly decreased in animals treated with therapeutic doses of stobadine (2 mg/kg), administered intravenously immediately before reperfusion or 10 rain after the onset of reperfusion. Stobadine was more effective than vitamin E, given in a dose of 30 mg/kg per day intramuscularly over 3 consecutive days prior to ischemia. Significant changes were found in the activities of antioxidative enzymes, namely, an increase in superoxide dismutase and a decrease in glutathione reductase activities in brain cortex samples. Stobadine prevented these changes. The density and affinity of a-adrenergic binding sites was investigated in the same experiments. Compared to the group of sham-operated animals, decreased density and increased affinity of [3H]dihydroergocryptine binding sites were found in cerebrocortical membranes of rats subjected to 4 hr of incomplete ischemia and 1 hr of reperfusion. Stobadine and vitamin E prevented these changes, indicating that oxygen free radicals might play a role in these processes. 34 The beneficial effect of stobadine was also demonstrated by higher survival of rats subjected to brain ischemia followed by reperfusion. 2° In the group of animals subjected to ischemia and reperfusion, 61% of the animals survived for 1 hr after reperfusion, 11.8% had seizures. After treatment with vitamin E or stobadine, 82 and 90% of the animals survived, respectively. Animals treated with either vitamin E or stobadine before ischemia and reperfusion were without seizures. 32 S. ~tolc and L. Horfikovfi, in " N e w Trends in Clinical Neuropharmacology" (D. Bartko, P. Tur~finy, and G. Stern, eds.), pp. 59-63. John Libbey, London, 1988. 33 L'. Horftkovfi, O. Ondreji6kovfi, V. Uraz, L'. Lukovi6, and I. Jur~nek, Experientia 48, 872 (1992). 34 Z. Kvaltfnovfi, L'. Lukovi~, and S. ~tolc, Neuropharmacology 38, 785 (1993).
580
ANTIOXlDANT CHARACTERIZATION AND ASSAY
[59]
Summary
The above-mentioned physicochemical, chemical, as well as pharmacological properties including successful results of Phase I and II clinical testing of stobadine as an antianginal agent permit us to consider this compound as potential drug in prevention and/or treatment of tissue injuries caused by oxidative stress.
[59] Nitroxides as A n t i o x i d a n t s
By MURALI C. KRISHNA and AMRAM SAMUN! Introduction Low molecular weight nitroxides are nonimmunogenic, cell-permeable, nontoxic 1stable radicals that readily partition among various cellular compartments. As paramagnetic species, detectable by electron paramagnetic resonance (EPR), nitroxides report on subtle changes in their chemical environment. 2 Consequently they have been predominantly used as biophysical markers to probe cellular metabolism, intracellular pH, oxygen level, molecular mobility of proteins and lipids, and membrane structure. 3-6 Additionally, nitroxides were proposed as contrast agents for nuclear magnetic resonance (NMR) imaging. 7,8These capacities of nitroxides have been extensively investigated, reported, and previously reviewed. 9 Being fully substituted in the ortho position, low molecular weight nitroxides are subject to steric hindrance that inhibits their dismutation, thus rendering them stable. However, nitroxides undergo chemical and cellular reduction to the corresponding one-electron reduced hydroxylamine, l° The hydroxylamine, on the other hand, is oxidized back to the nitroxide by various oxidants (chemical and cellular). The cellular reducI E. G. Ankel, C. S. Lai, L. E. Hopwood, and Z. Zivkovic, Life Sci. 40, 495 (1987). 2 L. J. Berliner, ed., "Spin Labeling," Vols. 1 and 2. Academic Press, New York, 1979. 3 H. M. Swartz, M. Sentjurc, and P. D. Morse II, Biochim. Biophys. Acta 888, 82 (1986). 4 j. Fuchs, W. H. Nitschmann, L. Packer, O. H. Hankovszky, and K. Hideg, Free Radical Res. Commun. 10, 315 (1990). 5 W. Froncisz, C. S. Lai, and J. S, Hyde, Proc. Natl. Acad. Sci. U.S.A. 82, 411 (1985). 6 j. F. Glockner, H. C. Chan, and H. M. Swartz, Magn. Reson. Med. 20, 123 (1991). 7 R. C. Brasch, Radiology 147, 781 (1983). 8 j. F. Keana and N. F. Van, Physiol. Chem. Phys. Med. N M R 16, 477 (1984). 9 A, Ionnone and A. Tomasi, Acta Pharm. Jugosl. 41, 277 (1991). l0 S. Belkin, R. J. Mehlhorn, K. Hideg, O. Hankovsky, and L. Packer, Arch. Biochem. Biophys. 256, 232 (1987).
METHODS 1N ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress, Inc. All fightsof reproductionin any formreserved.
580
ANTIOXlDANT CHARACTERIZATION AND ASSAY
[59]
Summary
The above-mentioned physicochemical, chemical, as well as pharmacological properties including successful results of Phase I and II clinical testing of stobadine as an antianginal agent permit us to consider this compound as potential drug in prevention and/or treatment of tissue injuries caused by oxidative stress.
[59] Nitroxides as A n t i o x i d a n t s
By MURALI C. KRISHNA and AMRAM SAMUN! Introduction Low molecular weight nitroxides are nonimmunogenic, cell-permeable, nontoxic 1stable radicals that readily partition among various cellular compartments. As paramagnetic species, detectable by electron paramagnetic resonance (EPR), nitroxides report on subtle changes in their chemical environment. 2 Consequently they have been predominantly used as biophysical markers to probe cellular metabolism, intracellular pH, oxygen level, molecular mobility of proteins and lipids, and membrane structure. 3-6 Additionally, nitroxides were proposed as contrast agents for nuclear magnetic resonance (NMR) imaging. 7,8These capacities of nitroxides have been extensively investigated, reported, and previously reviewed. 9 Being fully substituted in the ortho position, low molecular weight nitroxides are subject to steric hindrance that inhibits their dismutation, thus rendering them stable. However, nitroxides undergo chemical and cellular reduction to the corresponding one-electron reduced hydroxylamine, l° The hydroxylamine, on the other hand, is oxidized back to the nitroxide by various oxidants (chemical and cellular). The cellular reducI E. G. Ankel, C. S. Lai, L. E. Hopwood, and Z. Zivkovic, Life Sci. 40, 495 (1987). 2 L. J. Berliner, ed., "Spin Labeling," Vols. 1 and 2. Academic Press, New York, 1979. 3 H. M. Swartz, M. Sentjurc, and P. D. Morse II, Biochim. Biophys. Acta 888, 82 (1986). 4 j. Fuchs, W. H. Nitschmann, L. Packer, O. H. Hankovszky, and K. Hideg, Free Radical Res. Commun. 10, 315 (1990). 5 W. Froncisz, C. S. Lai, and J. S, Hyde, Proc. Natl. Acad. Sci. U.S.A. 82, 411 (1985). 6 j. F. Glockner, H. C. Chan, and H. M. Swartz, Magn. Reson. Med. 20, 123 (1991). 7 R. C. Brasch, Radiology 147, 781 (1983). 8 j. F. Keana and N. F. Van, Physiol. Chem. Phys. Med. N M R 16, 477 (1984). 9 A, Ionnone and A. Tomasi, Acta Pharm. Jugosl. 41, 277 (1991). l0 S. Belkin, R. J. Mehlhorn, K. Hideg, O. Hankovsky, and L. Packer, Arch. Biochem. Biophys. 256, 232 (1987).
METHODS 1N ENZYMOLOGY,VOL. 234
Copyright© 1994by AcademicPress, Inc. All fightsof reproductionin any formreserved.
[59]
581
NITROXIDES AS ANTIOXIDANTS TABLE I OXIDATION/REDUCTION MIDPOINT POTENTIALS OF NITROXIDE RADICALS
Nitroxide TEMPO TEMPOL TEMPAMINE TEMPONE
3-Carboxyproxyl 3-Aminomethylproxyl 3-Carbamoylproxyl 3-Cyanoproxyl 3-Carbamoyl-3-pyrroline OXANO C y c l o h e x a n e doxyl
Midpoint potential (mV versus normal hydrogen electrode)
Reversibility
722 810 826 913 792 853 861 976 966 960 900
+ + + + + + + + + -
tion of the nitroxide and the oxidation of the hydroxylamine depend on many factors which include the redox status of the cells and oxygen tension. The chemical reduction of nitroxides has also been shown to be determined by the substituent inductive effects, n Nitroxides can be classified into two types based on electrochemical behavior in cyclic voltammetric experiments. 12(1) The oxazolidine derivatives exhibit irreversible redox behavior in the regions of positive and negative potentials. (2) The piperidine and proxyl derivatives form a redox couple with their oxidized intermediate and exhibit a reversible behavior in the regions of positive potential. The midpoint redox potentials are listed in Table I. Less common has been the use of nitroxides as antioxidants. Initially, the participation of nitroxide radicals in one-electron redox reactions attracted research interest because such reactions affected their stability in cellular systems) ° Only a few attempts have been made to investigate their toxic effects 13 or potential role as antioxidants. 14'~ The protection of various biological systems by nitroxides, however, and particularly 11 S. Morris, G. S o s n o v s k y , B. Hui, C. O. Huber, N. U. Rao, and H. M. Swartz, J. Pharm. Sci. 80, 149 (1991). 12 M. C. Krishna, D. A. G r a h a m , A. Samuni, J. B. Mitchell, and A. Russo, Proc. Natl. Acad. Sci. U.S.A. 89, 5537 (1992). 13 H. Sies and R. Mehlhorn, Arch. Biochem. Biophys. 251, 393 (1986). 14 I. T. Brownlie and K. U. Ingold, Can. J. Chem. 45, 2427 (1967). 15 T. J. Weil, J. Van der Veen, and H. S. Olcot, Nature (London) 219, 168 (1968).
582
ANTIOXIDANT CHARACTERIZATIONAND ASSAY
[59]
their antioxidative activity have been rapidly established. 16-23In this chapter, the procedures adopted for applying and assaying the antioxidative activity o f nitroxides are presented.
Assay of Superoxide Dismutase Mimetic Activity Because the redox potential o f the nitroxide/hydroxylamine couple of oxazolidine derivatives, such as O X A N O , is about - 0 . 3 4 V , the system can oxidize superoxide anion ( 0 2 0 to O2.24 The hydroxylamine in turn can react with another 027 reducing it to H202. By this cyclic reaction, O X A N O is continuously restored and hence effectively dismutates O2 ~ yielding 02 and H202.
N - - O + 0 2-
O--~
/Z•
+
H+ ~ & ,
OH + 027 + H +
o-V/--OH
k2) 0 7 - - 0
+ 02
(1)
+ H20 2
(2)
Such superoxide dismutase (SOD) mimic activity of nitroxides is demonstrable by EPR, UV-visible spectrophotometry, and chemiluminescence techniques. 24 Unlike the case for native SOD, both k 1 and k2 depend on the p H and so does the ratio kl/k2. The individual reaction rate constants, however, cannot be derived from EPR measurements and should be determined by some independent technique such as the SOD-inhibitable 16U. A. Nilsson, L. I. Olsson, G. Carlin, and A. C. Bylund-Fellenius, J. Biol. Chem. 2,64, 11131 (1989). 17j. B. Mitchell, A. Samuni, M. C. Krishna, W. G. DeGraff, M. S. Ahn, U. Samuni, and A. Russo, Biochemistry 29, 2802 (1990). 18R. I. Zhdanov and P. G. Komarov, Free Radical Res. Commun. 9, 367 (1990). 19M. V. Bilenko, P. G. Komarov, A. A. Morgunov, and R. I. Zhdanov, Byull. Eksp. Biol. Med. 111, 500 (1991). 20j. An and A. W. Hsie, Mutat. Res. 270, 167 (1992). 21N. P. Konovalova, R. F. Diatchkovskaya, L. M. Volkova, and V. N. Varfolomeev, Anticancer Drugs 2, 591 (1991). = A. Samuni, D. Winkelsberg, A. Pinson, S. M. Hahn, J. B. Mitchell, and A. Russo, J. Clin. Invest. 87, 1526(1991). 23y. Miura, H. Utsumi, and A. Hamada, Arch. Biochem. Biophys. 300, 148(1993). 24A. Samuni, C. M. Krishna, P. Riesz, E. Finkeistein, and A. Russo, J. Biol. Chem. 263, 17921 (1988).
[59]
NITROXIDES AS ANTIOXIDANTS
583
reduction of ferricytochrome c (Cyt cnI). Piperidine nitroxide derivatives such as TEMPO, TEMPOL, or TEMPAMINE are oxidized by superoxide, but the oxidized intermediate, oxo-ammonium cation, is rapidly reduced by another O2 ~ as demonstrated for TEMPOL~2:
HO
- - O + 027 + 2H ÷
HO~~N~O
+ 027
~4 )
) HO
HOCN'--O
=O
+ H202
+ 0 2
(3)
(4)
Because k 3 is much less than k4, the nitroxide concentration is practically unaffected, although the net reaction is the same and the nitroxide is regenerated while 02 ~ is catalytically removed.12 Electron Paramagnetic Resonance The EPR experiment is performed by exposing approximately 10/zM nitroxide to 02- flux and following the residual nitroxide signal. A prolonged continuous flux of 02 ~ is achieved using 0.1 U/ml xanthine oxidase (EC 1.1.3.22, xanthine : oxygen oxidoreductase) and 1 mM hypoxanthine in 10 mM phosphate buffer at pH 7 containing 0.1 mM diethylenetriaminepentaacetic acid (DTPA). To ensure a steady rate of 027 formation, the reaction is carried out in a gas-permeable Teflon capillary (Zeus Industries, Raritan, NJ) of 0.8 mm inner diameter and 0.038 mm wall thickness. The tube containing the sample is folded twice, inserted in a quartz EPR tube opened at both ends, and placed in the EPR cavity. A constant oxygen concentration is maintained by flowing gas of the desired composition and temperature (measured by a thermocoupled probe set within the cavity) around the sample. Nitroxides do not react directly with H202, yet, in order to regenerate 02 from any accumulated H202, it is beneficial to add 100 U/ml catalase (SOD-free preparation from Boehringer, Mannheim, Germany). The intensity of the middle line of the nitroxide EPR spectra is monitored as a function of time. Spin loss occurring by reaction (1) is noted by a decrease of the initial signal (/initial) which subsequently reaches an intermediate equilibrium intensity. This decrease is attributed to the formation of the one-electron reduction product from the nitroxide, namely, the hydroxylamine. Later,
584
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[59]
following an accumulation of the hydroxylamine, reactions (1) and (2) balance one another, and the intensity (I) of the residual EPR signal becomes time-invariant. Since O2: is continuously removed through reactions (1) and (2) while H202 and molecular oxygen are generated, the reaction mechanism does not represent a genuine chemical equilibrium but rather a steady state where three ongoing processes affect the nitroxide concentration:
d[nitroxide]/dt = kE[hydroxylamine][O2-] + k_l[OE][hydroxylamine ] -
kl[nitroxide][O2:]
(5)
Therefore, in the presence of both oxygen and 02", a steady state is achievable reflected by a time-invariant ratio of [nitroxide]/[hydroxylamine]: [nitroxide]/[hydroxylamine] = kz/kl + k_l[OE]/kl[02-]
(6)
Under conditions where k_ 1[O2]
>
!
o
> > > >
> > > / >
FIG. 1. Decomposition of plasmenylethanolamine (1-alk-l'-enyl-2-acyl-sn-glycero-3phosphoethanolamine): formation of a dioxetane intermediate with singlet oxygen (type II chemistry 29'3°) and breakdown products. 1, Plasmenylethanolamine; 2, dioxetane intermediate; 3, 2-monoacylglycerophosphoethanolamine; 4, formic acid; 5, pentadecanal.
chromatography (HPLC) quantification and identification of fatty aldehyde moieties after dinitrophenylhydrazine (DNP) hydrazine derivatization.
Methods Method 1: Photosensit&ed Oxidation of Cultured Cells Principle. The method uses 12-(1'-pyrene)dodecanoic acid, a fluorescent fatty acid analog and also a photosensitizer. 21'27'35'36which is covalently incorporated into phospholipids and neutral lipids of cultured cells.21,37-39 Excitation of the pyrene moiety with long-wavelength UV light 35 S. T. Mosley, J. L. Goldstein, M. S. Brown, J. R. Falck, and R. G. W. Anderson, Proc. Natl. Acad. Sci. U.S.A. 78, 5717 (1981). 36 E. Fibach, O. Morand, and S. Gatt, J. Cell Sci. 85, 149 (1986). 37 O. H. Morand, E. Fibach, A. Dagan, and S. Gatt, Biochim. Biophys. Acta 711, 539 (1982). 38 j. Radom, R. Salvayre, T. Levade, and L. Douste-Blazy, Biochem. J. 269, 107 (1990). 39 j. Kasurinen and P. Somerharju, J. Biol. Chem. 267, 6563 (1992).
[61]
PLASMALOGENS
607 R'
'o
0% )(CH2)2NH3+
y_o_
.o.../o
R\
102
Oxidation
(
~. H
H
or
Radical
6
R"
> > > X
7 R"~ H-Abstraction
i
H k~ H
%. 10
H
R"
FIG. 2. Decomposition of plasmenylethanolamine: formation of allylic hydroperoxide and alkoxyl radical intermediates (type I chemistry29,3°) and breakdown products. R' is the remainder of the phospholipid moiety, and R" the remainder of the alkyl chain. 1, Plasmenylethanolamine; 6, allylic hydroperoxide; 7, alkoxyl radical; 8, diacylglycerophosphoethanolamine; 9, AZ-hexadecenal; 10, 2-monoacylglycerophosphoethanolamine. The allylic 1'-hydroperoxide intermediate 6 can be also converted to products 9 and 10 by a peroxidase.
(>300 nm) generates reactive oxygen species, including singlet oxygen and radicals. 4° Reagents and Materials 12-(l'-Pyrene)dodecanoic acid (Molecular Probes, Eugene, OR), 20 mM stock solution in dimethyl sulfoxide (DMSO), stored under nitrogen, in the dark, and at -20% diluted into growth medium Transilluminator (Ultraviolet Products Inc., San Gabriel, CA) equipped with two bulbs (15-W Sylvania F1ST8 Blacklight Blue) UV Intensity meter (Blak-Ray, Ultraviolet Products Inc.) Procedure. Cells are seeded, in the appropriate growth medium, in sterile plastic tissue culture dishes or in sterile glass tubes. Cells are incubated in the presence of 12-(1'-pyrene)dodecanoic acid together with 40 j. D. Spikes, in "The Science of Photobiology" (K. C. Smith, ed.), p. 87. Plenum, New York, 1977.
608
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[61]
a radioactive precursor for labeling phospholipids. 27 Prior to photosensitization, medium is aspirated and cells are washed with phosphate-buffered saline, pH 7.4 (PBS). Tissue culture dishes or glass tubes containing cells in PBS are placed above a Transilluminator equipped with UV light bulbs (>300 nm) and exposed to UV light for the appropriate amount of time. Cells must be placed at a distance of more than 2 cm from the UV source to assure homogeneous exposure of all samples, and exposure is calibrated with the UV intensity meter. Standard conditions of exposure are as follows: 10-20 W/m 2 at 365 nm for 5-30 rain. A glass plate must be intercalated between the tissue culture dish and the UV source to block any UV light below 300 nm.
Method 2: Plasmalogen Breakdown in [32p]Pi-Labeled Cells Principle. The following method is used to characterize the disappearance of 32p-plasmalogens in cultured Chinese hamster ovary (CHO-K1) cells under conditions of photooxidative stress. 27 Cells are preincubated with [32p]Pi to label all phospholipids, and with 12-(1 '-pyrene)dodecanoic acid to incorporate the photosensitizer into cell membrane lipids. After UV-irradiation, phospholipids are extracted and separated by two-dimensional thin-layer chromatography (TLC). Reagents and Materials Phosphorus-32 (orthophosphoric acid), approximately 9 Ci//zmol (Du P o n t - N e w England Nuclear, MA) 12-(1'-Pyrene)dodecanoic acid, 20 mM stock solution in DMSO, diluted at 10-I00/zM into growth medium Mercuric chloride, 10 mM in glacial acetic acid (heating helps solubilization) Silica gel 60 TLC plates (10 × 10 cm, E. Merck, Darmstadt, Germany) Solvent system A: chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) Solvent system B: chloroform/methanol/formic acid (65:25 : 10, v/v) Container to dry TLC plates in a stream of nitrogen Procedure. The CHO-KI cells are seeded in 1 ml of growth medium in sterile glass tubes ( - 2 x 104 cells/tube). One day after seeding, the medium is replaced with 0.9 ml of fresh medium containing [32p]Pi (200 /zCi/ml). The [32p]Pi can be also added at the time of seeding. After another day, each tube receives 0.1 ml of medium containing 12-(1'-pyrene)dodecanoic acid to provide a final concentration of the pyrene fatty acid of 1-10 t~M, and the tube is incubated for another 12-24 hr. Then, the medium is removed by aspiration, and cells are washed twice with PBS and placed in 1 ml of PBS.
[61]
PLASMALOGENS
609
Immediately after UV-irradiation (see Method 1) the reaction is quenched by addition of 2.5 ml of methanol and 1 ml of chloroform. After heating at 60°, samples receive 1.25 ml of PBS and 1.5 ml of chloroform to obtain a two-phase solvent system. 41 After vortexing and centrifuging, the lower organic phase is collected, and lipids are dried under nitrogen. Lipids are analyzed by two-dimensional thin-layer chromatography42 using I0 by 10 cm silica gel plates. Lipids solubilized in a small volume of chloroform/methanol (1 : 1, v/v) are spotted at the comer of the plate at a distance of 1.5 cm from both edges. Lipids are chromatographed in the first dimension in solvent system A to the top of the plate. Next, the plate must be carefully dried in a stream of nitrogen for 10 min. The plate is sprayed with 10 mM mercuric chloride in glacial acetic acid and allowed to dry thoroughly for 20 min in a stream of nitrogen. Treatment with mercuric chloride in acid will cleave the vinyl ether linkage of plasmalogens, 5 allowing for the separation of diacylphosphatidylcholine and diacylphosphatidylethanolamine from the plasmalogen-derived lysophospholipids (i.e., 2-monoacylglycerophosphocholine and 2-monoacylglycerophosphoethanolamine, respectively) in the second dimension.16'27 Ether phospholipids (e.g., plasmanylcholine and plasmanylethanolamine) are not hydrolyzed under these conditions. The plate is chromatographed in the second dimension in solvent system B to the top of the plate. After drying the plate, radioactive phospholipids are visualized by autoradiography, scraped off, and counted by liquid scintillation spectrometry. The percent distribution of each radioactive phospholipid is calculated. Comments. It is important that the photosensitization reaction is quenched rapidly. If cells cultured in suspension are used, they should be prepared in buffer (PBS, Tris, or HEPES) in glass tubes just before applying oxidative stress. Then the reaction is rapidly stopped by addition of methanol and chloroform. If cells usually growing on plastic are used (e.g., CHO-K1 cells) one possibility consists of seeding them instead in glass tubes, and replacing the growth medium with buffer just before applying oxidative stress and quenching with chloroform and methanol. Alternatively, the cells are seeded in conventional polystyrene tissue culture dishes. Then, the growth medium is replaced by buffer before applying oxidative stress, and reaction stopped with methanol alone, and cells scraped off and transferred to glass tubes prior to addition of chloroform. Figure 3 shows a typical distribution of phospholipids extracted from control and photosensitized 32p-labeled CHO-K1 cells. In control cells, ethanolamine-containing phospholipids were separated between diacyl41 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 42 j. D. Esko and C. R. H. Raetz, J. Biol. Chem. 255, 4474 (1980).
610
Control cells
E
G) >, (n 4)
>
[61]
A N T I O X I D A N T C H A R A C T E R I Z A T I O N A N D ASSAY
5 A
4e
Photosensitized cells
3
2
O (n A o4
6
....
/
....
4e
2
'lj,
11
1) Solvent system A
FIG. 3. Schematic representation of the separation of [32p]Pi-labeledphospholipids by two-dimensional thin-layer chromatography showing the disappearance of plasmalogens in photosensitized CHO-K1 cells.27O, origin; 1 and 2, sphingomyelin;3, phosphatidylcholine; 4, phosphatidylinositol; 5, phosphatidylserine; 6, lysophosphatidylethanolamine (derived from acidic cleavage of plasmenylethanolamine); 7, phosphatidylethanolamine.
phosphatidylethanolamine (spot 7, Fig. 3) and plasmenylethanolaminederived lysophosphatidylethanolamine (spot 6, Fig. 3). CHO-K1 cells contain little or no plasmenylcholine as evidenced by the absence of a second radioactive spot below diacylphosphatidylcholine (spot 3, Fig. 3). Photosensitization induces the selective disappearance of plasmenylethanolamine. No radioactive breakdown product derived from plasmenylethanolamine was found in this two-dimensional thin-layer chromatography, and the method described next can be used to identify the product that might have migrated with another phospholipid.
Method 3: Plasmenylethanolamine Breakdown in [2J4 C]Ethanolamine-Labeled Cells Principle. The following method is used to characterize (1) the disappearance of plasmenylethanolamine and (2) the formation of lysophosphatidylethanolamine in cultured cells under conditions of photooxidative stress. 27Cells are preincubated with [2-14C]ethanolamine to label plasmenylethanolamine, and with 12-(l'-pyrene)dodecanoic acid to incorporate the photosensitizer into cell membrane lipids. After UV-irradiation, radioactive phospholipids are extracted and separated by two-step one-dimensional thin-layer chromatography.
[61]
PLASMALOGENS
611
Reagents and Materials [2-14C]Ethanolamine, 50-60 mCi/mmol (Amersham Corp., Amersham, UK) 12-(1 '-Pyrene) dodecanoic acid, 20 mM stock solution in DMSO, then diluted at 10-100/xM into growth medium Silica gel 60 TLC plates (20 x 20 cm, E. Merck) Mercuric chloride, 10 mM in glacial acetic acid Solvent system A: chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) Container to dry TLC plates in a stream of nitrogen Procedure. The CHO-K1 cells are grown at the bottom of sterile glass tubes ( - 1 × 105 cells/tube) in 0.9 ml of medium containing approximately 0.2/~Ci/ml [2-14C]ethanolamine. After 1 day, each tube receives 0.1 ml of growth medium containing 12-(1'-pyrene)dodecanoic acid to provide a final concentration of the pyrene fatty acid of 1-10/zM and is incubated for another 12-24 hr. After the medium is removed by aspiration, the cells are washed twice with PBS and incubated in 1 ml of PBS. Immediately after UV-irradiation (see Method 1), the reaction is quenched by the addition of 2.5 ml of methanol and 1 ml of chloroform. After heating at 60°, samples receive 1.25 ml of PBS and 1.5 ml of chloroform to obtain a two-phase solvent system. After vortexing and centrifuging, the lower organic phase is collected, and lipids are evaporated to dryness under nitrogen. Lipids are analyzed by two-step one-dimensional thin-layer chromatography using 20 by 20 cm silica gel plates. Lipids solubilized in a small volume of chloroform/methanol (1 : 1, v/v) are spotted in 1-cm bands at a distance of 2 cm from the bottom edge of the plate and first chromatographed in solvent system A to a distance of 5 cm. Next, the plate must be carefully dried in a stream of nitrogen for 10 min. The plate is sprayed with 10 mM mercuric chloride in glacial acetic acid 5 and allowed to dry thoroughly for 20 min in a stream of nitrogen. The plate is rechromatographed in the same dimension using the same solvent system A to a distance of 15 cm. After drying the plate, the radioactive phospholipids are visualized by autoradiography, scraped off, and counted by liquid scintillation spectrometry. This two-step one-dimensional TLC procedure allows for the separation of diacylphosphatidyl[2-14C]ethanolamine (Rf -0.67), 2-monoacylglycerophospho[2-14C]ethanolamine derived from plasmenyl[2J4C]etha nolamine after acidic cleavage on the plate (Rf -0.48), and 2-monoacylglycerophospho[2-14C]ethanolamine generated by photooxidation and originally present in the extract (Rf -0.39). 27 The radioactive breakdown
612
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[61]
product 2-monoacylglycerophosphoethanolamine can be also characterized directly by one-step one-dimensional TLC using solvent systems such as chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) or chloroform/acetone/methanol/acetic acid/water (6 : 8 : 2 : 2 : 1, v/v) with a standard lysophospholipid running in parallel. Comments. This method can be partially transposed to characterize the decomposition of plasmenylcholine. CHO-K 1 cells contain little or no plasmenylcholine, but other cell types may have higher levels. In this case, [methylJ4C]choline will be used to label plasmenylcholine and diacylphosphatidylcholine. For identification the two-step two-dimensional system of Method 2 is preferable because 2-monoacylglycerophosphocholine, diacylphosphatidylcholine, and sphingomyelin will separate well. Lysophosphatidylcholine and sphingomyelin have low Rf values, and they separate rather poorly in conventional one-dimensional TLC systems.
Method 4: Formation of Radioactive Formic Acid in [1J4C]Hexadecanol-Labeled Cells Principle. [1-14C]Hexadecanol is used to label the vinyl ether-linked fatty chain of plasmalogens. The long-chain fatty alcohol will condense with acyI-DHAP to form alkyI-DHAP prior to acylation and subsequent polar head group transfer. 43 Carbon atom 1 of hexadecanol will be the source of radioactive formic acid produced from photooxidized plasmalogens (Fig. 1). After photosensitization, radioactive formic acid is characterized by its specific conversion to CO2 by formate dehydrogenase (EC 1.2.1.2) in the presence of NAD+. 44'45 Because the water-soluble formate can rapidly diffuse out of cells, both cell extracts and extracellular media must be analyzed. 27 Reagents and Materials [1J4C]Hexadecanol is synthesized46 from [1-14C]palmitic acid, 40-60 mCi/mmol (Du Pont-New England Nuclear) [14C]Formate, sodium salt, 40-60 mCi/mmol (Du Pont-New England Nuclear) 12-(1 '-Pyrene)dodecanoic acid, 20 mM stock solution in DMSO, then diluted at 9-90/zM into growth medium Formate dehydrogenase (from Pseudomonas oxalaticus, Sigma, St. Louis, MO), 7 units/ml in water 43 F. Paltauf, in " E t h e r Lipids, Biochemical and Biomedical Aspects" (H. K. Mangold and F. Paltaulf, eds.), p. 107. Academic Press, New York, 1983. J. R. Quayle, this series, Vol. 9, p. 360. 45 j. S. Blanchard and W. W. Cleland, Biochemistry 19, 3543 (1980). 46 p. A. Davis and A. K. Hajra, J. Biol. Chem. 254, 4760 (1979).
[61]
PLASMALOGENS
613
Formic acid, 0.4 mM in water NAD + (Sigma), 40 mM in water Methylbenzethonium hydroxide (Aldrich, Milwaukee, WI) Trichloroacetic acid (TCA), 80% (w/v) in water Glass vials with rubber stoppers and small plastic wells (insert) like those used for radioactive fatty acid/3-oxidation measurements Filter paper Disposable syringes (1-ml) Procedure. Dry radioactive [1-14C]hexadecanol (50/zCi) is dissolved in 0.2 ml of ethanol, added to 5 ml of growth medium, sonicated twice for 30 sec, and diluted to 25 ml with growth medium. CHO-K1 cells are seeded in 3 ml of medium at a density of 5 × 104 cells in 35-mm tissue culture dishes, and supplemented with 1 ml of the radioactive hexadecanol stock to get a final activity of 0.5 /zCi/ml ( - 9 /xM). After 2 days of incubation, cells receive 0.5 ml of medium containing 12-(l'-pyrene)dodecanoic acid to provide a final concentration of the pyrene fatty acid of 1-10/zM and are incubated further for 12-24 hr. The medium is removed by aspiration, and the cells are washed twice with PBS and incubated in 1.5 ml of PBS. Immediately after UV-irradiation (see Method 1), the buffer is collected, cells are washed twice with 1.5 ml of cold PBS, and the three PBS fractions are pooled. The pooled material defined as the extracellular medium is kept on ice. Cells are scraped off using a rubber policeman and transferred to glass tubes, subjected to three cycles of freezing and thawing, and kept on ice. Sonication can be also used to disrupt the cells. Both extracellular media and cells are centrifuged at 130,000 g for 60 min in a Beckman 50Ti rotor to pellet cell debris, and supernatants are kept on ice. For the formate dehydrogenase assay, 400/~1 of centrifuged extracellular medium or cell supernatant, 50/zl of 0.4 mM nonradioactive formate as carrier, 50/zl of 40 mM NAD +, and 400/xl of water are mixed in small glass vials on ice. Vials are closed with a rubber stopper, equipped with a small plastic well containing a filter paper soaked with 40/zl of methylbenzethonium hydroxide as the CO2 trapping agent. The mixture is warmed to 37° for 1 min, and the reaction is initiated by injecting 0.1 ml of formate dehydrogenase (7 units/ml) with a syringe. Final conditions are as follows: 20/~M cold formate, 2 mM NAD +, 0.7 units formate dehydrogenase/ml in 1 ml of 2.5-fold diluted PBS. The mixture is incubated at 37° for 30 min, and the reaction is stopped by injecting 0.1 ml of 80% TCA. The vials are kept in the water bath for another 1 hr to allow for the trapping of CO2. Next, filter papers are removed and counted by liquid scintillation spectrometry. [~4C]Formate diluted with cold formate is assayed in parallel
614
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[61]
for calibration. Between 50 and 90% of the radioactive formic acid produced from plasmalogens of photooxidized CHO-K1 cells is found in the extracellular medium. Method 5: Formation of Fatty Aldehydes in [UJ4C]Hexadecanol Labeled Cells Principle. Fatty aldehydes are breakdown products of photooxidized plasmalogens (Fig. 1 and 2). In the following method [U-14C]hexadecanol is used to label the vinyl ether-linked fatty chain of plasmalogens. 43 This vinyl ether-linked fatty chain will be the source of fatty aldehyde produced from photooxidized plasmalogens; it is extracted and identified according to its retention factor after separation by thin-layer chromatography. Its conversion to fatty acid in the presence of chromium trioxide 47 will also confirm its nature. Reagents and Materials [U-14C]Hexadecanol is synthesized46from [U-i4C]palmitic acid, >500 mCi/mmol (Du Pont-New England Nuclear) 12-(1'-Pyrene)dodecanoic acid, 20 mM stock solution in DMSO, then diluted at 10-100 ~ M into growth medium Silica gel 60 TLC plates (20 × 20 cm, E. Merck) Solvent system C: n-hexane/diethyl ether/acetic acid (80 : 20 : 1, v/v) Solvent system D: n-hexane/diethyl ether/acetic acid (90 : 10 : 1, v/v) Solvent system E: n-heptane/isopropyl ether/acetic acid (60:40:4, v/v) cis-Hexadecenal (Sigma or Aldrich) or another long-chain fatty aldehyde standard Chromium trioxide (Sigma), saturated solution in glacial acetic acid Sodium hydroxide, 3 N in water Concentrated HCI Procedure. [U-14C]Hexadecanol (10/zCi) is dissolved in 0.2 ml of ethanol, added to 5 ml of growth medium, and sonicated twice for 30 sec. CHO-K1 cells are seeded in 6 ml of medium in 100-mm plastic culture dishes at a density of 5 x 106 cells/dish. Each dish receives also [U-14C]hexadecanol in 1.2 ml of medium and 12-(l'-pyrene)dodecanoic acid at 10-100/xM in 0.8 ml of medium. Final conditions are as follows: 0.3/xM [U-14C]hexadecanol (0.3/xCi/ml) and 1-10/zM 12-(1 '-pyrene)dodecanoic acid. Cells are incubated for 12-24 hr, washed twice with PBS, and UV-irradiated in 4.5 ml of PBS for 5-30 min (see Method 1). The 47 G. M. Gray, in "Lipid Chromatographic Analysis" (G. V. Marinetti, ed.), Vol. 1, p. 401. Dekker, New York, 1967.
[61]
PLASMALOGENS
615
reaction is immediately quenched with 5 ml of methanol, and the cells are scraped off the dishes with a rubber policeman and transferred to glass tubes. After heating at 60 °, each tube receives 5 ml of chloroform prior to mixing and centrifuging. The lower organic phase is collected and evaporated to dryness under nitrogen, and the lipids are further analyzed. Lipids are separated first by thin-layer chromatography using solvent system C with standard lipids running in parallel. In this system the Rf values for fatty alcohol, fatty acid, triacyglycerol, fatty aldehyde, and cholesteryl ester are 0.09, 0.14, 0.35, 0.44, and 0.62, respectively. Phospholipids remain at the origin. Other minor lipids are usually seen. Radioactive lipids are visualized by autoradiography, and the band corresponding to long-chain fatty aldehyde is scraped off the plate and counted by liquid scintillation spectrometry. The lipids from the band can be also reextracted from the silica gel three times with 2 ml of chloroform/methanol (2: 1, v/v). After drying under nitrogen, the lipid is analyzed again by thin-layer chromatography in two different solvent systems D and E for comparison to a standard such as cis-hexadecenal running in parallel, cis-Hexadecenal is visualized on the plate with iodine vapors. For complete identification, the extracted lipid is tested for its ability to be oxidized to fatty acid in the presence of chromium trioxide. 47 The dry lipid is mixed with 1.5 ml of glacial acetic acid and heated at 40 ° prior to the addition of 0.5 ml of chromium trioxide solution. After 10 min at 40°, 3 ml of water is added, and free fatty acids are extracted twice with 2 ml of benzene. After evaporation under nitrogen the extract is mixed into 4 ml of methanol, 0.5 ml of 3 N NaOH, and impurities are extracted twice with 2 ml of n-hexane. The lower phase is acidified with 0.25 ml of concentrated HC1, and fatty acids are extracted three times with 2 ml of n-hexane. The extract is dried under nitrogen and analyzed by thin-layer chromatography using solvent system E with fatty aldehyde, fatty alcohol, and fatty acid standards running in parallel. Radioactive lipids are visualized by autoradiography. Comments. Long-chain fatty alcohols given to cells are for a good part converted to long-chain fatty acids by a fatty alcohol : NAD ÷ oxidoreductase (EC 1.1.1.192, long-chain-alcohol dehydrogenase).48 As a consequence, [UJ4C]palmitic acid can be recovered from [U-14C]hexadecanollabeled cells in its free form and esterified into complex liquids. In addition, long-chain fatty aldehydes can be readily converted to long-chain fatty acids by a fatty aldehyde dehydrogenase;49 this was evidenced by the 48 W. B. Rizzo, D. A. Craft, A. L. Dammann, and M. W. Phillips, J. Biol. Chem. 17412 (1987). 49 R. Lindhal and S. Evces, J. Biol. Chem. 259, 11991 (1984).
262,
616
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[61]
finding of pentadecanoic acid in photooxidized CHO-K1 cells, 27 supposedly derived from pentadecanal, a breakdown product of plasmalogens.
Method 6: High-Performance Liquid Chromatography of Fatty Aldehyde Moieties after Dinitrophenylhydrazine Derivatization Principle. The previous method to identify the formation of radioactive long-chain fatty aldehydes in photosensitized [U-14C]hexadecanol-labeled cells is simple and broad. It is, however, incomplete and does not provide accurate data on the nature of the fatty aldehydes produced from plasmalogen breakdown. It is possible to analyze the fatty acids derived from chromium trioxide treatment after another conversion to the corresponding phenacyl derivatives for characterization by HPLC, but unstable fatty aldehydes could be lost in this procedure. A more direct and reliable method consists of the derivatization of fatty aldehydes and plasmalogens with 2,4-dinitrophenylhydrazine (DNP hydrazine) producing the corresponding DNP hydrazine hydrazones.5° Plasmalogens and preexisting fatty aldehydes extracted from cells or tissues are first separated by silica gel chromatography, then derivatized with DNP hydrazine under acidic conditions, and finally subjected to HPLC for identification and quantification. The following procedure is adapted from published methods5°-5z with some modifications. Reagents and Materials 2,4-Dinitrophenylhydrazine (DNP hydrazine, Sigma) must be first repurified. A 4 mM DNP hydrazine solution is prepared in 1 N HCI, and kept at 70° for 30 min. After cooling, carbonyl impurities are extracted twice with two 3-ml portions of n-hexane; the nhexane remaining on top of the aqueous phase is evaporated under nitrogen. The DNP hydrazine reagent in 1 N HC1 is stored under nitrogen, in the dark, and at room temperature to be used within 24 hr. 12-(1 '-Pyrene)dodecanoic acid, 20 mM stock solution in DMSO, then diluted at 1-10/zM into growth medium Silica gel (200-400 mesh, Bio-Rad, Richmond, CA) HPLC 250 × 4.6 mm 5-~m Econosil-Cl8 column (Alltech) with precolumn 5o T. Huque, J. G. Brand, J. L. Rabinowitz, and F. F. Gavarron, Comp. Biochem. Physiol. B 86B, 135 (1987). 51 C. Pries and C. J. F. B6ttcher, Biochim. Biophys. Acta 98, 329 (1965). 52 H. Esterbauer, K. H. Cheeseman, M. U. Dianzani, G. Poli, and T. F. Slater, Biochem. J. 208, 129 (1982).
[61 ]
PLASMALOGENS
617
Mobile phase for HPLC: acetonitrile/water (95 : 5, v/v) Methanol, chloroform, and n-hexane (all carbonyl-free) cis-Hexadecenal (Sigma) Saturated and unsaturated long-chain fatty aldehyde standards, which can be synthesized by selective oxidation of the sulfanate esters of corresponding fatty acids 53 Procedure. The CHO-K1 cells are seeded in medium in 60-mm tissue culture dishes at a density of approximately 1 × 105 cells/well and incubated for 1 day. The next day, medium is replaced by 3 ml of medium containing 1-10 /zM of P12. After 12-24 hr of incubation the medium is removed, and cells are washed twice with PBS and UV-irradiated in 1 ml of PBS (see Method 1). The reaction is immediately quenched with 2.5 ml of methanol. Cells are scraped off the dishes with a rubber policeman, and the methanol/water suspension is transferred to glass tubes containing 1 ml of chloroform, mixed, and heated at 60 ° for 10 min. Next, samples receive 1.25 ml PBS and 1.5 ml of chloroform to obtain a two-phase solvent system. After vortexing and centrifuging, the lower organic phase is collected and lipids dried under nitrogen. Silica gel chromatography is used to separate phospholipids, including the plasmalogens, from the neutral lipids, including long-chain fatty aldehydes released during photooxidation. Small columns, containing about 0.2 g of silica gel in Pasteur pipettes, are first washed with three 2-ml portions of methanol and then with four 2-ml portions of chloroform. Commercial small silica gel columns (e.g., Supelco, Bellafonte, PA) can also be used. Lipids in 0.2 ml of chloroform are applied to the columns; neutral lipids are eluted with three 1-ml portions of chloroform, and polar lipids with four 1-ml portions of methanol. All fractions are evaporated to dryness under nitrogen in glass tubes with Teflon screw caps prior to derivatization with DNP hydrazine. Polar lipid fractions and neutral lipid fractions are redissolved in 0.5 ml of methanol, and 0.5 ml of DNP hydrazine reagent is added. Samples are vortexed and incubated at 70° for 30 min. After cooling, the DNP hydrazones are extracted twice with 1.5 ml of n-hexane. The pooled extracts are dried under nitrogen and redissolved in 20-50/~1 of acetonitrile/water (95 : 5, v/v) for HPLC analysis. Reversed-phase HPLC analyses are performed under isocratic conditions with acetonitrile/water (95 : 5, v/v) as the mobile phase at a flow rate of 2 ml/min with detection at 395 nm. Standard fatty aldehydes are derivatized and analyzed in parallel for identification and calibration. 53 V. Mahadevan, F. Phillips, and W. O. Lundberg, Lipids 1, 183 (1966).
618
ANTIOXIDANT CHARACTERIZATION AND ASSAY
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Comments. This method does not require radiolabeling, and it can be used with cells and tissues exposed to a variety of oxidative stress conditions. The DNP hydrazine derivatization of plasmalogens must be performed under acidic conditions, and it will be used to quantify the disappearance of plasmalogens. Depending on the type of cell or tissue, plasmalogens contain mostly C16 and C~8 vinyl ether-linked fatty chains, and possible long-chain fatty aldehyde breakdown products include pentadecanal and A2-hexadecenal, and heptadecanal and A2-0ctadecenal, respectively. The distribution of the fatty aldehydes produced at room temperature during photooxidation might be different from that obtained at 4°; instability of some of the fatty aldehydes could explain this difference. A higher sensitivity of detection will be obtained using dansyl hydrazine 54 for derivatization in place of DNP hydrazine; dansylhydrazones separated by HPLC are then detected by spectrofluorometry.
General Discussion Whether plasmalogens are a class of natural membrane antioxidants remains an open question. The analytical methods described in this chapter can be extended to other biological systems involving oxidative stress to examine the hypothetical decomposition of plasmalogens. Plasmalogen breakdown in tissues might escape detection when total fatty aldehydes are measured by acidic DNP hydrazine derivatization. Thus, it is important that neutral lipids are first separated from phospholipids as in Method 6, to allow for the detection of preexisting fatty aldehydes originating from oxidative decomposition ofplasmalogens. The identification ofpentadecanal and heptadecanal (Method 6), and of formic acid (Method 4) will point to the occurrence of singlet oxygen. The reactivity of plasmalogens to singlet oxygen and radicals is the basis for an antioxidant, scavenger function, provided that breakdown products are not cytotoxic and/or are rapidly metabolized. Interestingly, CHO-K1 cells can lose up to 30% of plasmenylethanolamine after photosensitization and remain viable,2~'27 pointing to plasmalogens as first-line membrane antioxidants. The cytotoxicity of photosensitization in plasmalogen-deficit cells supports the notion that other biochemical entities essential to cell integrity and functions are indeed protected from activated oxygen species when vinyl ether phospholipids are restored. If plasmalogens are scavengers of singlet oxygen it remains to be seen whether singlet oxygen is produced in defined biological systems causing plasmalogen decomposition. Natural photosensitization is limited to a few 54 j. M. Anderson, Anal. Biochem. 152, 146 0986)°
[61]
PLASMALOGENS
619
tissues such as the skin and eye, and it does not apply to tissues rich in plasmalogens such as heart, brain, muscle, and white cells. Although evidence has accumulated to show the occurrence of activated oxygen species such as superoxide anion in biological systems 55 the production of singlet oxygen in such systems needs further investigation. Kanofsky 56 reviewed several singlet oxygen-generating enzyme systems such as the peroxidase-hydrogen peroxide-halide system. One must be very careful when it comes to extrapolating these observations to living systems, and emphasize that most model enzyme systems producing singlet oxygen are using high, nonphysiological concentrations of hydroperoxides, hydrogen peroxide, and halides. The measurement of 1268 nm oxygen chemiluminescence 57'58 is an unequivocal method for the detection of singlet oxygen in biological systems and should help in sorting out these questions. It has already permitted the characterization of the production of singlet oxygen by stimulated intact human eosinophils 59via the myeloperoxidase-hydrogen peroxide-halide system. Whether stimulated eosinophils lose plasmalogens is not known. Stimulated polymorphonuclear leukocytes do produce fatty aldehydes, although these are not fully identified yet. 6° The oxidation of plasmalogens by radicals is more likely to occur in vivo because radicals are known to play a significant role in a number of biological situations. 55 These situations include ischemia-reperfusion injury in myocardial infarction and stroke, hyperoxygenation syndromes, oxidative-burst inflammatory disorders, lipoprotein oxidation, ionizing radiations, and others. Yavin and Gatt 23'24 have already demonstrated the oxygen-dependent cleavage of plasmenylcholine by rat brain homogenates, together with the production of lysophosphatidylcholine and longchain aldehydes. Loss of plasmalogens has been shown in a situation of ischemia-reperfusion injury in the traumatized cat spinal cord. 61 Older observations have confirmed the disappearance of plasmalogens in the infarcted area of human heart following lethal myocardial infarction. 62 It may well reflect ischemia-reperfusion injury of the myocardium63 causing the oxidative decomposition of plasmalogens; enzymatic hydrolysis of 55 C. E. Cross, B. Halliwell, E. T. Borish, W. A. Pryor, B. N. Ames, R. L. Saul, J. M. McCord, and D. Harman, Ann. Intern. Med. 107, 526 (1987). 56 j. R. Kanofsky, Chem.-Biol. Interact. 70, 1 (1989). 57 A. U. Kahn, J. Am. Chem. Soc. 105, 7195 (1983). 58 j. R. Kanofsky, J. Biol. Chem. 259, 5596 (1984). 59 j. R. Kanofsky, H. Hoogland, R. Wever, and S. J. Weiss, J. Biol. Chem. 263, 9692 (1988). 6o O. H. Morand and C. R. H. Raetz, unpublished results (1989). 61 p. Demediuk, R. D. Saunders, D. K. Anderson, E. D. Means, and L. A. Horrocks, Proc. Natl. Acad. Sci. U.S.A. 82, 7071 (1985). 62 K. A. Oster and P. Hope-Ross, J. Cardiol. 17, 83 (1966). 63 M. S. Sussman and G. B. Bukley, this series, Vol. 186, 711.
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ANTIOXlDANT CHARACTERIZATION AND ASSAY
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plasmalogens by a plasmalogen-selective phospholipase A 2 is also possible. 64 In conclusion, biological systems involving oxidative stress need to be reexamined to establish whether oxidative decomposition of plasmalogens takes place under physiological conditions. 64 D. A. Ford, S. L. Hazen, J. E. Saffitz, and R. W. Gross, J. Clin. Invest. 88, 331 (1991).
[62] A n t i o x i d a n t A c t i v i t y o f C a l c i u m C h a n n e l Blocking Drugs
By I. TONG MAK and WILLIAM B. WEGLICKI Introduction In common with most cardiovascular agents, the clinically used calcium channel blockers (nicardipine, nifedipine, verapamil, diltiazem) are amphiphilic in nature. Thus, in addition to their specific binding to protein receptors, these agents may readily partition into the phospholipid domain of cardiovascular membranes to various degrees according to their lipophilicity. Efforts from our laboratory have focused on the effects of such agents on the sensitivities of cardiac membranes and vascular cells to free radical injury. 1-7 At the membrane level, we have chosen the highly purified sarcolemmal membranes of ventricular myocytes as model membranes. Compared to other subcellular membranes, the sarcolemmal membranes were much more sensitive to free radical-mediated damage, 8 probably owing to the highly enriched phospholipid content. 8-1° To assess the extent of membrane lipid peroxidation, we chose to use the thiobarbituric acid (TBA) method because of its sensitivity and convenience. 1 I. T. Mak and W. B. Weglicki, Circ. Res. 63, 262 (1988). 2 I. T. Mak and W. B. Weglicki, Circ. Res. 66, 1449 (1990). 3 I. T. Mak, C. M. Arroyo, and W. B. Weglicki, Circ. Res. 65, 1151 (1989). 4 W. B. Weglicki, I. T. Mak, and M. G. Simic, J. Mol. Cell. Cardiol. 22, 1199 (1990). 5 I. T. Mak, A. M. Freedman, B. F. Dickens, and W. B. Weglicki, Biochem. Pharmacol. 40, 2169 (1990). 6 I. T. Mak, P. Boehme, and W. B. Weglicki, Circ. Res. 70, 1099 (1992). 7 I. T. Mak, J. H. Kramer, and W. B. Weglicki, Coronary Artery Dis. 3, 1095 (1992). s j. H. Kramer, I. T. Mak, and W. B. Weglicki, Circ. Res. 55, 120 (1984). 9 W. B. Weglicki, K. Owens, F. F. Kennett, A. Kessner, L. Harris, R. M. Wise, and G. V. Vahoouny, J. Biol. Chem. 255, 3605 (1980). l0 W. B. Weglicki, J. H. Kramer, I. T. Mak, B. F. Dickens, and T. M. Phillips, in "Isolated Adult Cardiomyocytes" (H. Piper and G. Isenberg, eds.), Vol. 1, p. 1. CRC Press, Boca Raton, Florida, 1988.
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plasmalogens by a plasmalogen-selective phospholipase A 2 is also possible. 64 In conclusion, biological systems involving oxidative stress need to be reexamined to establish whether oxidative decomposition of plasmalogens takes place under physiological conditions. 64 D. A. Ford, S. L. Hazen, J. E. Saffitz, and R. W. Gross, J. Clin. Invest. 88, 331 (1991).
[62] A n t i o x i d a n t A c t i v i t y o f C a l c i u m C h a n n e l Blocking Drugs
By I. TONG MAK and WILLIAM B. WEGLICKI Introduction In common with most cardiovascular agents, the clinically used calcium channel blockers (nicardipine, nifedipine, verapamil, diltiazem) are amphiphilic in nature. Thus, in addition to their specific binding to protein receptors, these agents may readily partition into the phospholipid domain of cardiovascular membranes to various degrees according to their lipophilicity. Efforts from our laboratory have focused on the effects of such agents on the sensitivities of cardiac membranes and vascular cells to free radical injury. 1-7 At the membrane level, we have chosen the highly purified sarcolemmal membranes of ventricular myocytes as model membranes. Compared to other subcellular membranes, the sarcolemmal membranes were much more sensitive to free radical-mediated damage, 8 probably owing to the highly enriched phospholipid content. 8-1° To assess the extent of membrane lipid peroxidation, we chose to use the thiobarbituric acid (TBA) method because of its sensitivity and convenience. 1 I. T. Mak and W. B. Weglicki, Circ. Res. 63, 262 (1988). 2 I. T. Mak and W. B. Weglicki, Circ. Res. 66, 1449 (1990). 3 I. T. Mak, C. M. Arroyo, and W. B. Weglicki, Circ. Res. 65, 1151 (1989). 4 W. B. Weglicki, I. T. Mak, and M. G. Simic, J. Mol. Cell. Cardiol. 22, 1199 (1990). 5 I. T. Mak, A. M. Freedman, B. F. Dickens, and W. B. Weglicki, Biochem. Pharmacol. 40, 2169 (1990). 6 I. T. Mak, P. Boehme, and W. B. Weglicki, Circ. Res. 70, 1099 (1992). 7 I. T. Mak, J. H. Kramer, and W. B. Weglicki, Coronary Artery Dis. 3, 1095 (1992). s j. H. Kramer, I. T. Mak, and W. B. Weglicki, Circ. Res. 55, 120 (1984). 9 W. B. Weglicki, K. Owens, F. F. Kennett, A. Kessner, L. Harris, R. M. Wise, and G. V. Vahoouny, J. Biol. Chem. 255, 3605 (1980). l0 W. B. Weglicki, J. H. Kramer, I. T. Mak, B. F. Dickens, and T. M. Phillips, in "Isolated Adult Cardiomyocytes" (H. Piper and G. Isenberg, eds.), Vol. 1, p. 1. CRC Press, Boca Raton, Florida, 1988.
METHODS IN ENZYMOLOGY,VOL. 234
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ANTIOXlDANT PROPERTIES OF CALCIUM BLOCKERS
621
Increasing evidence has accumulated to suggest that lipid peroxidative processes are involved in the pathogenesis of vascular diseases.ll,12 Among those processes, oxidative injury of endothelial cells may represent a critical event in propagating atherogenesis. 11 The endothelial cells are potential targets of reactive oxygen radicals released from activated blood cells (e.g., neutrophils, macrophages, platelets) and oxidizable drugs and chemicals. Therefore, at the cellular level, we have used cultured endothelial cells to assess the cytoprotective effects of the calcium channel blockers against free radical-induced loss of glutathione and increased membrane permeability. Both parameters were relatively sensitive to the oxidative stress generated from a chemical oxygen-radical system. Cardiac Sarcolemmal Membrane Model
Sarcolemmal Preparation. Sarcolemmal membranes are isolated from adult canine ventricular myocytes. The isolation procedure has been described in detail elsewhere, s-l° Briefly, adult canine myocytes are isolated from ventricular tissue by enzymatic digestion with 0.05% collagenase. Following disruption of the myocytes by nitrogen cavitation (1000 psi, 30 min), the sarcolemmal membranes are enriched by differential and sucrose gradient centrifugation. The sarcolemmal fractions, which band between 21 and 26% sucrose, are about 80-fold enriched in the specific activity of the marker enzyme Na ÷, K+-ATPase over that of the myocyte homogenate.9'l° Generation of Free Radicals. The chemical system we use to generate oxygen-radicals consists of dihydroxyfumarate (DHF) and FeCI3-ADP. 13 Oxidation of D H F (Sigma Chemical Co., St. Louis, MO) in solution generates sustained levels of superoxide anions14'15; the rate of production is further promoted by the presence of metal chelates such as Fe-ADP. Hydroxyl radicals (.OH) are generated according to the following chemical reactions: DHF + 02--> .DHF + 02 ~ •DHF + 02 ~ diketosuccinate + O2 ~ 202: + 2H ÷ ~ H202 + 02 Fe3+-ADP + 02 ~ ~ Fe2+-ADP + 02 Fe2+-ADP + H202 --> -OH + Fe3+-ADP + O H 11 B. Hennig and C. K. Chow, Free Radical Biol. Med. 4, 99 (1988). 12 B. Halliwell, Br. J. Exp. Pathol. 70, 737 (1989). 13 I. T. Mak, H. P. Misra, and W. B. Weglicki, J. Biol. Chem. 258, 13733 (1983). t4 S. A. Goscin and I. Fridovich, Arch. Biochem. Biophys. 153, 778 (1972). t5 B. Halliwell, Biochem. J. 163, 441 (1977).
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ANTIOXIDANT CHARACTERIZATION AND ASSAY
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Presumably, other iron-oxygen species (such as hypervalent iron complexes) may also be formed in Fenton-type reactionsl6; these species could be just as deleterious as .OH radicals. In our system, a concentrated solution of D H F is prepared in the incubation buffer; a brief period ( verapamil > diltiazem; this appears to follow their membrane antiperoxidative activities.
Comparison of Pharmacologically Active and Inactive Enantiomers of Nicardipine. Of all the calcium blockers tested, nicardipine (racemic form) is the most effective cytoprotective agent. In an effort to further TABLE III ANTIOXIDANT PROPERTIES OF ISOMERS OF NICARDIPINE AND BUTYLATED HYDROXYTOLUENE IN ENDOTHELIAL CELLSa
Conditions Buffer control R. (DHF + Fe-ADP) R' plus agent: ( + )-Nicardipine ( - )-Nicardipine BHT
Concentration (~M)
20 5 20 5 5
GSH loss (%)
Cell death (%)
2 47
11 59
8 24 11 26 7
19 28 22 31 16
Incubation conditions were as described in Table II. Values are means of 4-9 determination. Results adapted from Mak et al., 7 with permission.
630
ANTIOXIDANT CHARACTERIZATION AND ASSAY
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distinguish the nature of this cytoprotection, we have examined the effects of the pharmacologically active ( + ) and inactive ( - ) isomers of nicardipine. Owing to an asymmetric carbon at position 4 of the dihydropyridine ring, nicardipine has two optical isomers, namely, (+)- and (-)-nicardipine; the ( + ) isomer is about 5-fold more potent than the ( - ) isomer as a vasodilator or calcium channel blocker. 24 Both (+)- and (-)-nicardipine are supplied by Syntex Research Laboratory (Palo Alto, CA). When the membrane antioxidant activities of these two compounds are studied using the sarcolemmal membranes, both isomers display identical dose-dependent inhibitory activities against lipid peroxidation; the estimated ECs0 values are 20.6/xM for ( + )-nicardipine and 22.8/xM for ( - )-nicardipine. For comparison, the ECs0 value for BHT is 5.2/xM. Therefore the antioxidant potency of either nicardipine isomer was about one-fourth that of BHT. Using the endothelial cell system, the protection of the nicardipine isomers against free radical-induced losses of GSH and viability is evaluated. As summarized in Table III, both nicardipine isomers (5 and 20 /zM) provide comparable, concentration-dependent protection against the peroxidative depletion of GSH and loss of cellular viability. These results indicate that the cytoprotective effects of the agents are due to intrinsic chemical antioxidant activity rather than calcium channel blocking ability. Comments. The initial rate of endothelial GSH loss is proportional to the concentration of the free radicals generated; doubling the concentration of the free radical components leads to a 50% loss of GSH in 15 min of incubation. Linear regression analysis indicates that the loss of cellular viability is correlated significantly with the decrease of GSH (r = 0.89, p < 0.001). Lipid peroxidation at the cell level cannot be assessed accurately (compared to the isolated membranes); however, we believe that the loss of endothelial GSH is due to its oxidation by increased levels of lipid peroxides. The calcium blocker-mediated effects appear to be secondary to their inhibition of peroxide formation in the membranes. Our results indicate that the dihydropyridine calcium blockers exhibit the greatest antioxidant potency, which is partly due to their higher membrane partitioning and, perhaps, to their more active redox chemistry. It remains to be determined whether such antioxidant properties contribute to their established anti-atherogenic effects, z5 Acknowledgments This research was supported by National Institutes of Health Grants PO 1-HL-38079 and ROI-HL-36418. 24 K. Iwatsuli, F. Iijima,and S. Chiba, Clin. Exp. PharmacoL Physiol. 11, 1 (1984). 25 p. D. Henry, J. Cardiovasc. Pharmacol. 15~ $6 (1991).
[63]
ESR OF ETOPOSIDE(VP-16) PHENOXYLRADICAL
631
[63] I n t e r a c t i o n s o f P h e n o x y l R a d i c a l o f A n t i t u m o r D r u g , E t o p o s i d e , w i t h R e d u c t a n t s in S o l u t i o n a n d in Cell a n d Nuclear H o m o g e n a t e s : Electron Spin R e s o n a n c e and High-Performance Liquid Chromatography
By T. G.
G A N T C H E V , J. E. VAN LIER, D. A. STOYANOVSKY, J. C . Y A L O W I C H , and VALERIAN E . K A G A N
Introduction Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers, including testicular and small lung cancers and lymphoma.1 Mechanisms of cytotoxicity of VP-16 may involve DNA strand cleavage and/or topoisomerase II inhibition accompanied by formation of DNA-topoisomerase cross-links and DNA strand breaks. 2,3 The VP-16 molecule contains a hindered phenolic group (Fig. 1) which is crucial for its antitumor activity. It has been reported that several enzymatic systems (peroxidase, tyrosinase, prostaglandin synthase, cytochrome P-450), as well as exogenous sources of peroxyl radicals (e.g., azo initiators) activate VP-16 via one-electron oxidation of the phenolic group to yield reactive metabolites (quinones) capable of irreversible binding to macromolecular targets (DNA and proteins) and/or generation of hydroxyl radicals in the presence of metal catalysts. 4-6 An essential step in the process of VP-16 activation is the formation of its phenoxyl radical which can be further either converted to oxidation products or reduced by intracellular reductants to the initial phenolic form. 7'8 Obviously, this may be critical for enhancing or suppressing the cytotoxic effects of VP16 in tumor cells'or surrounding tissues. The formation of the transient VP-16 phenoxyl radical in the course of its oxidative/reductive conversions can be directly followed by electron spin resonance (ESR) spectroscopy. Together with simultaneous highI M. L. Slevin, Cancer (Philadelphia) 67, 319 (1991). 2 N. Osheroff, Pharmacol. Ther. 41, 223 (1989). 3 L. A. Zwelling, Cancer Metastasis Rev. 4, 263 (1985). 4 B. Sinha and M. Trush, Biochem. Pharrnacol. 32, 3495 (1983). 5 j. M. S. Van Maanen, J. Retel, H. M. J. de Vries, and H. M. Pinedo, J. Natl. Cancer Inst. 80, 1526 (1988). 6 N. Haim, J. Nemec, J. Roman, and B. Sinha, Cancer Res. 47, 5835 (1987). 7 N. Usui and B. K. Sinha, Free Radical Res. Commun. 10, 287 (1990). 8 j. C. Yalowich, D. A. Stoyanovsky, W. P. Allan, B. W. Day, and V. E. Kagan, Proc. Am. Assoc. Cancer Res. 34, 1779 (1993).
METHODS IN ENZYMOLOGY, VOL. 234
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H H3C"~ 0 HO
0 0
o
OH Fro. 1. Structural formula of etoposide (VP-16).
performance liquid chromatography (HPLC) measurements of the concentrations of VP-16 and its oxidation product(s), this may give detailed information on the interaction of VP-16, its intermediates, and oxidation product(s) with metabolizing enzymes and other intracellular and exogenous oxidants as well as with physiologically relevant reductants in model chemical and biological systems. Several examples below illustrate ESR detection of VP-16 phenoxyl radicals generated by different enzymatic and nonenzymatic systems as well by light-activated reactions.
Methods
Generation of VP-16 Phenoxyl Radicals by Enzymatic and Nonenzymatic Systems and Electron Spin Resonance Detection Generation of VP-16 Phenoxyl Radical by Tyrosinase and Peroxidase. Tyrosinase, a copper-containing enzyme widely distributed in biological systems, can hydroxylate phenols to form catechols and o-quinones. 9 Peroxidative attack on the phenolic moiety of VP-16 by tyrosinase in airsaturated phosphate-buffered aqueous solution (pH 7.4) results in generation of a typical phenoxyl free radical as detected by ESR spectroscopy 7,1° 9 S. H. Pomerantz, J. Biol. Chem. 241, 161 (1966). 10 B. Kalyanaraman, J. Nemec, and B. K. Sinha, Biochemistry 28, 4839 (1989).
[63]
ESR OF ETOPOSIDE(VP-16) PHENOXYLRADICAL
633
(Fig. 2A). The oxygen-centered phenoxyl radical exhibits a hyperfine structure similar to semiquinone radicals, but is relatively more stable and does not require additional metal ion or alkaline stabilization. In aqueous solution the twenty-line ESR spectrum is characterized by the following magnetic resonance parameters: g = 2.0048 -+ 0.0002 and hyperfine splitting arising from six identical methoxy protons (aHcH3 -----0.14 mT), two phenoxyl ring protons (ar~ng = 0.14 mT), one fl-proton (a H = 0.45 mT), and a y-proton (a H = 0.06 mT). Horseradish peroxidase generates the VP-16 phenoxyl radical of the same structure, but requires the presence of hydrogen peroxide 1° (Fig. 2B). Generation of VP-16 Phenoxyl Radical by Azo Initiators of Peroxyl Radicals. Azo compounds are known to decompose unimolecularly and nonezymatically to yield N2 and two identical carbon-centered radicals, which further react with molecular oxygen to form corresponding peroxyl radicals, ll Both hydrophilic [e.g., 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)] and lipophilic [e.g., 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN)] azo initiators of peroxyl radicals can be used to induce oxidation in the aqueous phase or in the membranous phases, respectively.11 The practical advantage of these compounds as peroxyl radical initiators follows from their constant spontaneous decomposition rate (radical yield) at physiological conditions. Peroxyl (alkoxyl) radicals readily abstract the hydrogen atom from phenols to produce phenoxyl radicals.~2 Oxidation rates of VP-16 by AAPH in aqueous solution or by AMVN in dioleoylphosphatidylcholine liposomes at 37° are high enough to produce steady-state concentrations of the VP-16 phenoxyl radical easily detectable by ESR (Fig. 2C,D). AAPH and AMVN decomposition can be induced by UV-irradiation, causing decomposition of the azo bond (h -> 320 nm). Thus UV-irradiation of VP-16 in the presence of an azo initiator can also be used for generating ESR-detectable steady-state concentrations of the VP-16 phenoxyl radicals (not shown). Photosensitized Generation of VP-16 Phenoxyl Radicals. Metallophthalocyanines (MePc) and their sulfonated water-soluble derivatives [e.g., aluminum tetrasulfophthalocyanine (A1PcS4) or zinc tetrasulfophthalocyanine (ZnPcS4)] are red-light-absorbing photosensitizers with a promising application in the so-called photodynamic therapy of tumors.~3 Triplet-state excited phthalocyanines initiate photooxidation of biological substrates either via direct electron/hydrogen atom abstraction from subII E. Niki, this series, Vol. 186, p. 100. 12 G. W. Burton and K. U. Ingold, Acc. Chem. Res. 19, 194 (1986). is j. E. Van Lier and J. D. Spikes, in "Photosensitizing Compounds: Their Chemistry, Biology and Chemical Use" (G. Bock and S. Harnett, eds.), p. 17. Wiley, Chichester, 1989.
g=2.0048 Gain
lx10 2
A '
~
mT
B
lx103
, 1.1
J,I
C
D
E
z4
FIG. 2. ESR spectra of the VP-16 phenoxyl radical generated by enzymatic and nonenzymatic systems. Tyrosinase-induced VP-16 oxidation: 50 mM phosphate buffer (pH 7.4) containing 0.5 mM VP-16 and 160 U mushroom tyrosinase (A); peroxidase-induced VP-16 oxidation: horseradish peroxidase (8/zg/ml), H2O2 (2 mM), and VP-16 (0.2 mM) in acetate buffer (pH 5.0) (B); VP-16 oxidation induced by a water-soluble azo initiator 2,2'-azobis(2amidinopropane) dihydrochloride (AAPH, 250 raM) (C); VP-16 oxidation induced by a lipidsoluble azo initiator 2,2'-azobis(2,4-dimethylvaleronitrile)(AMVN, 60 mM) incorporated into dioleoylphosphatidylcholinetiposomes (10 mg/ml) (D); metallophthalocyanine-photosensitized oxidation of VP-16: aluminum tetrasulfophthalocyanine (A1PcS4, 0.1 mM), continuous irradiation with visible-light(~. -> 400 nm; P = 15 mW/cm2) of 1 mMVP-16 in phosphate buffer (pH 7.4) containing 2 mM sodium dodecyl sulfate (SDS) (E).
[63]
ESR OF ETOPOSIDE(VP-16) PHENOXYLRADICAL
,/
~1 t
635
0 min 5 min
~----- 30 min
~
45 min
'75 mirr~n '90 min 0
7 Retention time
i 14
FIG. 3. HPLC tracings of VP-16 and its oxidation product formed in the course of incubation with tyrosinase in phosphate buffer. Incubation conditions: VP-16 (0.5 raM) and tyrosinase (160 U) in 0.1 M phosphate buffer (pH 7.4 at 25°).
strate molecules or via generation of activated oxygen species: singlet oxygen (102) , superoxide anion (O20 and hydroxyl radicals (-OH). 14,15 Irradiation of air-saturated aqueous solutions containing AIPcS4 (or ZnPcS4) and VP-16 with visible light (h -> 400 nm) results in generation of the characteristic VP-16 phenoxyl radical as followed by the direct photoESR measurements (Fig. 2E). The reactive intermediate(s) involved in VP-16 oxidation by PcS4 remain unknown (possibilities include activated oxygen species and/or direct triplet state dye-ground state VP-16 interaction). 14 R. Langlois, H. Ali, N. Brasseur, R. J. Wagner, and J. E. van Lier, Photochem. Photobiol. 44, 117 (1986). 15 T. G. Gantchev, M. G. Kaltchev, and G. P. Gotchev, Int. J. Radiat. Biol. 60, 597 (1991).
636
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[63]
A
Gain
t(min)
4
_._._z
28
lx100 ' t 32
FIG. 4. ESR spectra of the tyrosinase-induced radicals in the presence of VP-16 and reductants (A) ascorbate and (B) reduced glutathione (GSH). Incubation conditions: 50 mM phosphate buffer (pH 7.4) containing 0.5 mM VP-16, 160 U mushroom tyrosinase, and 0.8 mM ascorbate (A) or 0.1 mM GSH (B). The prescan incubation time is shown in the right column; the receiver gain is shown in the left column.
(63]
E S R OF ETOPOSIDE (VP-16) PI-IENOXYL RADICAL
B Gain
t(min)
lx100
I mT -
-
-
-
-
. . . . . . . . .
.
,
12
2O
FIG. 4.
(continued)
637
638
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[63]
Reduction of VP-16 Phenoxyl Radical Studied by Electron Spin Resonance and Chromatography in Aqueous Solution Physiologically important reductants with appropriate redox potentials, such as reduced glutathione (GSH) and ascorbate are capable of donating electrons (hydrogen atoms) to phenoxyl radicals, thus regenerating phenols at the expense of their own oxidation. For hindered phenols, this regeneration process can be followed directly by ESR spectroscopy. In particular, reduction ofphenoxyl radicals of vitamin E and its homologs, the food preservatives butylated hydroxytoluene (BHT) and hydroxyanisole (BHA), and some phenolic drugs (e.g., probucol, acetaminophen) has been demonstrated by E S R . 16'17 Reduction of the VP-16 phenoxyl radical by intracellular reductants may prevent its activation to a cytotoxic species. We have used ESR spectroscopy to study interactions of the VP-16 phenoxyl radicals with some physiologically relevant reductants (reduced glutathione, ascorbate)/8 In these experiments, we use tyrosinase as the VP-16 phenoxyl radical generating system, which permits persistent detection of the characteristic ESR signal for 50-60 min. HPLC measurements show that incubation with tyrosinase resulted in a timedependent consumption of VP-16 and accumulation of its oxidation product(s) (Fig. 3). Thus, the effects of reductants should be conveniently evaluated both by ESR and HPLC measurements. Tyrosinase is not likely to cause secondry reactions, affecting the stability of the phenoxyl radical, as has been reported for horseradish peroxidase. 10In addition, tyrosinase is possibly one of the major VP-16 metabolizing enzymes in a number of malignant cells, especially in melanomas. 6'~9 When ascorbate is added to the incubation medium, the characteristic doublet of the semidehydroascorbyl radical could be observed in the ESR spectra unless ascorbate is consumed. This is followed by a rapid appearance and growth of the VP-16 phenoxyl radical ESR signal (Fig. 4A). Replacing ascorbate by reduced glutathione (100 /zM GSH) under the same conditions results in several "blank" ESR scans, followed by a less steep (relative to the case of ascorbate) growth of the VP-16 phenoxyl radical ESR signal (Fig. 4B). In both cases, in the presence of ascorbate or GSH, the buildup of the VP-16 phenoxyl radical ESR signal after the lag period reaches a saturation intensity level nearly the same as in the 16 V. E. Kagan, E. A. Serbinova, and L. Packer, Arch. Biochem. Biophys. 280, 33 (1990). 17 R. D. N. Rao, V. Fischer, and R. P. Mason, J. Biol. Chem. 265, 844 (1990). ~s D. Stoyanovsky, J. Yalowich, T. Gantchev, and V. Kagan, Biochem. Pharmacol., submitted for publication. 19 D. H. Kern, R. H. Shoemaker, S. U. Hildebrand-Zanki, and J. S. Driscoll, Cancer Res. 48, 5178 (1988).
[63]
ESR OF ETOPOSIDE(VP-16) PHENOXYL RADICAL
639
A [Asc],pM
t-.m c o~ £E CO UJ
j/
t(min)
I
I
0
I
10
20 [GSH],pM
(D
E UJ
~ 0
1,
210
t(min) I 30
FIG. 5. Direct ESR measurements of the kinetics of the VP-16 phenoxyl radical buildup in the presence of different concentrations of (A) ascorbate and (B) reduced glutathione. Conditions: Magnetic field lock at 336.3 mT; modulation amplitude 0.32 mT; and scan time 64 min. For more details, see text. absence of reductants. Quenching of the VP-16 phenoxyl radical E S R signal by reductants and its reconstitution after reductant depletion are reversible by m e a n s of repetitive addition of reductants to the solution after their consumption. In their oxidized form, neither d e h y d r o a s c o r b a t e nor G S S G causes a lag period in the a p p e a r a n c e of the VP-16 phenoxyl radical E S R signal (not shown). The lag periods produced by reductants
640
[63]
ANTIOXIDANT CHARACTERIZATION AND ASSAY 0.6-
VP-16 (control) z
.-
~
"r
I
~
x
T
~
~
+ Tyrosinase
¢.,
o
0.3.
~¢~
012'
tO
> 0.1 " 0.0
0
+ Tyrosinase 1~, ~ I
20
+Tyrosinase + GSH
10
8b
Time, rain FIG. 6. Effect of ascorbate and GSH on the kinetics of the tyrosinase-induced VP-16 oxidation as measured by HPLC. Concentrations of ascorbate and GSH were 0.8 and 0.15 mM, respectively.
during VP-16 oxidation can be conveniently recorded by kinetic measurements of the VP-16 phenoxyl radical ESR signal by locking the magnetic field. Typical kinetic scans obtained in the presence of different concentrations of ascorbate or GSH are presented in Fig. 5. The lag periods induced by GSH are longer than those induced by ascorbate under equivalent conditions. This is most likely due to a tyrosinase-catalyzed slow oxidation of ascorbate in the absence of VP-16. The HPLC tracings demonstrate that no decay of VP-16 is observed until ascorbate is completely consumed. After depletion of ascorbate the rate of VP-16 oxidation is the same as in the absence of ascorbate (Fig. 6). Similarly, in the presence of GSH, VP-16 oxidation proceeds with a lag period during which the concentration of VP-16 does not change (Fig. 6). The durations of lag periods for the appearance of the VP-16 phenoxyl radical signal in the ESR spectra in the presence of ascorbate or GSH are exactly the same as the lag periods observed for the onset of VP-16 oxidation in HPLC tracings. Neither ascorbate nor GSSG affects the time course of VP-16 oxidation by tyrosinase (data not shown).
Interactions of Endogenous Reductants with VP-16 Phenoxyl Radical in Presence of Cell and Nuclear Homogenates A delicate balance between oxidants (including oxidative enzymes) and reductants may predetermine the prevalence of oxidative over reductive
[63]
ESR OF ETOPOSIDE(VP-16) PHENOXYLRADICAL
~ 0i
.-~"" 2 , 10
3
"3
2b
641
t(min) l 30
4~
0
FIG. 7. ESR spectra of tyrosinase-induced radicals in the presence of VP-16 and K-562 cell nuclear homogenates. K-562 cell homogenates were prepared from (1) 0.3 x 106, (2) 0.6 x 106, and (3) 0.9 × 106, cells isolated from a culture at the mid-exponential growth; nuclear homogenates were prepared from I × 106 nuclei (4). ESR conditions were as in Fig. 5.
metabolism of VP-16 in specific intracellular compartments. Shifts in this balance may be at least partially responsible for the drug resistance of tumor cells via mechanism(s) involving reduction of the VP-16 phenoxyl radical. The redox balance toward the VP-16 phenoxyl radical can be evaluated by kinetic measurements of the tyrosinase-induced ESR signal of the VP-16 radical in the presence of cell homogenates. When tyrosinase and VP-16 are incubated in the presence of homogenates prepared from different numbers of K-562 human leukemia cells (Fig. 7, curves 1-3) or from nuclei isolated from these cells (curve 4, Fig. 7), the phenoxyl radical buildup to an ESR-detectable concentration follows a lag period with a subsequent relatively steep increase in the magnitude of the ESR signal. In nuclear homogenates the lag period is
10 tiM GSH
.__>,
~
.E
nuJ
I
k
I
I
0
10
20
30
r
FIG. 8. Effect of exogenous GSH added to K-562 cell homogenate after depletion of endogenous antioxidants on kinetics of the VP-16 phenoxyl radical ESR signal. K-562 cell homogenates were prepared from 0.5 × 106 cells; the GSH concentration was 10/.tM.
642
ANTIOXIDANT CHARACTERIZATION AND ASSAY
[63]
shorter as compared to the cell homogenate prepared from an equivalent number of cells. In K-562 cell and nuclear homogenates no semidehydroascorbyl radical ESR signals are detected during the lag period (preceding the appearance of the VP-16 phenoxyl radical ESR signal) as is observed in a model system on addition of ascorbate; hence, ascorbate is not likely to be the major intracellular reductant responsible for the VP-16 regeneration. The slow kinetics of the VP-16 phenoxyl radical ESR signal after a lag period are similar to those observed in a model system in the presence of GSH. Exogenously added GSH causes a rapid quenching and a slow reappearance of the VP-16 phenoxyl radical ESR signal if added to K-562 cell homogenates after the initial lag period is over (Fig. 8). Thus, it seems likely that reduced thiols may primarily contribute to the VP-16 regeneration in K-562 cell homogenates. Addition of ascorbate to K-562 cell or nuclear homogenates after completion of the lag period results in the same kinetics of quenching and abrupt reappearance of the VP-16 phenoxyl radical ESR signal after ascorbate oxidation as is observed in a model system (not shown). HPLC measurements reveal a lag period in a tyrosinase-catalyzed VP16 oxidation in the presence of K-562 cell or nuclear homogenates. They correspond to the lag periods for appearance of the VP- 16 phenoxyl radical ESR signal: the onset of the VP-16 oxidation coincides in time with the appearance of the VP-16 radical ESR signal. In conclusion, the procedure we have developed for measuring the duration of the lag periods for the VP-16 phenoxyl radical appearance in cell homogenates can be used for convenient evaluation of cell reductive capacity. Intracellular reductants may eliminate activation of VP-16 by oxidative metabolism via mechanisms involving reduction of the VP-16 phenoxyl radical. Deliberate depletion of intracellular reductants (e.g., by photosensitizers generating reactive oxygen species, azo initiators of peroxyl radicals, overexpression of oxidative enzymes) may be used in the future to enhance the cytotoxic effects of VP-16 in tumor cells.
AUTHOR INDEX
643
Author Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.
A Aakvaag, A., 591 Abate, C., 164, 165(1, 2), 166(2, 7), 167, 167(2), 168(2, 16), 174(7), 224 Abbas, A. K., 141 Abbott, W. A., 493 Abdallah, M., 443 Abdallah, M. A., 441 Abe, K., 274 Aberg, F., 344 Aboujaoude, E. N., 16 Abrams, J., 491 Aburada, M., 415,416(31) Ackrell, B.A.C., 317, 354, 381 Adam, W., 79, 129 Adams, D. A., 49 Adams, G. E., 426 Adams, M., 592 Addis, P. B., 439 Adler, K. B., 257 Ae Shin, Y., 68 Afanas'ev, I. B., 421 Affany, A., 429 Aftonomes, B. T., 257 Aggarwal, B. B., 461 Aggarwar, B. B., 156 Ahn, B. W., 254 Ahn, M. S., 582, 587(17), 589(17) Ahnadi, C., 591 Ahnfelt-Ronne, I., 557, 569(22), 570(22) Ahnstrfm, G., 67, 78(18), 89, 94(9, 14), 96(9, 14), 100(9, 14), 102 Aho, P., 527 Ahokas, J., 477, 478(13) Akatsuka, N., 491 Akerboom, T.P.M., 367, 368(9), 370 Akeson, A. L., 507 Akman, S. A., 3 Alaupovic, P., 515 Albermann, K., 152, 154(4), 156(4)
Albiero, R., 401 Albroscheit, G., 415,416(28) Alcaraz, M. J., 421, 446, 449(15), 450(15), 453,453(14, 15), 454(14, 21) Alessio, H., 24, 31(27) Alfassi, Z. B., 428 Ali, H., 81,635 Alkner, N., 253,256(15) Allan, W. P., 631 Allen, A., 256 Allen, L. A., 604 Allen, R. E., 555 Allgayer, H., 557 Almeda, S., 411 Alperin, P. E., 24 Althaus, J. S., 552 Altmann, A., 149 Altmiller, D. H., 60, 62(6), 80 Altuvia, S., 217, 219 Ames, B. N., 16-17, 22, 24, 24(9), 25(25), 26(25), 27(9), 32(9), 59, 67, 79-80, 88, 117, 151, 156(1), 175(3), 176, 181(3), 187, 214, 216(18), 220,224, 253-254, 254(11), 255(11), 269, 273, 331, 344, 348(7), 375,406, 619 Amici, A., 254 Amstad, P., 191, 192(22) Amstad, P. A., 187, 191(19), 203(19) An, J., 582 Andersen, L., 421 Anderson, D. K., 568, 569(45), 619 Anderson, J. M., 618 Anderson, M. E., 492-493, 493(2, 3), 494, 494(16), 496(2, 21), 497, 497(2, 21), 498(21, 29), 499, 499(29), 500, 501(1), 502,502(1), 503,503(1, 3), 504, 504(3, 5), 627 Anderson, R.G.W., 606 Anderson, V. C., 604 Andersson, C. M., 479 Ando, Y., 341
644
AUTHOR INDEX
Andreef, M., 592 Andrus, P. K., 552 Andus, K. L., 553 Angel, P., 170 Angelov, D., 3 Ankel, E. G., 580 Antus, S., 422, 425(26) Aoki, K., 68 Applegate, L. A., 225 Arashima, S., 547 Arcioni, A., 601 Arichi, S., 446 Ariga, T., 421,423(22) Armstrong, D., 404 Armstrong, D. W., 394 Armstrong, R. N., 505 Arnit, B., 250, 251(6) Arnstein, H.R.V., 591-592, 592(32, 33), 594(33, 37, 39), 595(32, 37, 39), 596(33, 37, 39), 597(33, 39), 598(47, 48), 599(48), 600(47), 601(33, 37, 39, 47, 48) Aronovitch, J., 586, 589(27) Arroyo, C. M., 620, 625, 625(3) Arshad, M.A.Q., 283 Artz, W. E., 397 Arudi, R. L., 421 Aruoma, O. I., 3, 17, 67, 70(15), 73(15), 75(15), 76, 76(15), 489, 557, 571(18) Arvidsson, S., 528 Asada, K., 378, 383,459 Asahara, H., 123 Ascarelli, I., 424 Ascherio, A., 269 Asensi, M., 367, 371 Asmus, K. D., 422,426, 479 Asquith, T. N., 432,435 Astengo, M., 490 Astor, M. B., 494 Atherton, S. J., 68 Attaway, J. A., 416 Aubert, M. C., 397 Aubry, J. M., 385 Auclair, C., 530 Audic, A., 17, 80 Aufderheide, E., 89 Augeri, L., 35, 102, 110 Auld, D. S., 52 Auld, P.A.M., 492(9), 493, 494(9) Aust, S. D., 66, 67(2), 566, 569(39), 570(39) Ausubel, F. M., 221
Avron, M., 400 Awai, M., 341 Azad-Khan, A. H., 557
B Babulovfi, A., 578 Backer, U., 491 Backstr6m, R., 527 Bacon, B. R., 438 Bacon, K. B., 452 Bacon, P. A., 569 Badley, J. E., 194 Baeuerle, P. A., 151-152, 153(3), 154, 154(4), 155(3, 10), 156(4, 5), 157(3, 5, 10), 158(5), 159-160, 161(15), 162(3, 10), 163, 163(10), 217, 224 Baffet, G., 438 Baggen, R.G.A., 627 Baggio, G., 511 Bagnasco, M., 490 Bailey, J. M., 514 Bailly, V., 111, 113, 113(12) Baiocchi, M. R., 511 Bakac, A., 426 Baker, J. C., 375 Baker, P. F., 311,312(8), 313(8), 314(8) Baker, R. R., 256 Bakerian, R. H., 123 Bakker, J. H., 557 Balser, L., 514 Baltimore, D., 153, 159, 162(9), 163, 172 Bank, J. F., 123 Bannai, S., 137, 139(14, 15), 148 Banoun, H., 530 Baranes, L., 462 Barber, A. A., 280 Barclay, L.R.C., 269, 374,423,506, 533,543 Barden, H. B., 590 Barenholz, Y., 324 Barindelli, E., 487 Barja, G., 331 Barja da Quiroga, G., 332, 333(4), 334(4), 337(4) Barklay, J.R.C., 403 Barlow, D. J., 592,598(47), 600(47), 601(47) Bamhart, J. W., 505 Barnhart, R. L., 505, 506(4) Barr, R., 375
AUTHOR INDEX Bartels, K., 478 Barth, S. A., 462 Bartlett, P. D., 605 Barton, D., 414 Basset, P., 602 Bassett, J. M., 603 Bast, A., 362, 455,478,489 Bastide, P., 421 Basu-Modak, S., 224 Bates, A. L., 385 Battey, J. F., 226 Bauer, V., 572, 573(1) Bauernfeind, J. C., 388 Bauernfidd, J. C , 355 Baum, M., 590-591 Baumann, J., 411,421 Baumgarden, C., 253, 256(15) Baumstark-Khan, C., 88-89, 90(12), 91(12), 94(12), 96(12), I00, 100(12) Beaune, P., 592 Beck, Y., 250, 251(6) Becker, J. T., 524 Beckman, J. S., 475 Beckman, K. B., 22 Beecher, G. R., 396, 398 Beehler, B. C., 24, 36, 38(19) Behrend, R., 4 Behrens, G., 426 Behrens, W. A., 270, 271(12) Beier, H., 185 Beli, J., 590 Belkin, S., 580 Bell, S. D., 165 Bellocq, J. P., 602 Bellomo, G., 73, 77, 628 Bement, W. J., 438 Ben Amotz, A., 400 Benard, F., 81, 83(23) Ben Aziz, A., 424 Bendich, A., 4, 270 Beneg, L., 572-573, 573(1), 574, 576-578, 578(3), 579(31) Bennett, M.V.L., 236 Bennicelli, C., 490 Benninghoff, B., 135, 140(5), 142(5), 144(5) Be'nsasson, R. V., 480 Bentsath, A., 429 Berestecky, J. M., 236, 241(14) Berger, M., 3, 55, 80-82, 83(23, 25, 29), 85(25), 87, 87(25)
645
Berggren, M., 479, 487-489, 489(8) Bergsten, P., 337 Bergtold, D. S., 4, 24, 31(27), 131 Berliner, L. J., 580 Berman, E., 592 Bernard, G. R., 491 Bernhard, K., 390, 394(14) Bernier, J. L., 421,423(13), 425(13) Bernstein, L., 590 Berstock, D., 590 Berthou, F., 592 Bertovich, M. J., 557 Bertram, J. S., 235-237, 238(17), 239(8), 241(9, 14), 243(9) Bertrand, M., 423 Bessho, T., 24 Betts, W. H., 557 Beutler, B., 244 Beyer, E. C., 236 Bezek, 5., 574 Bhadra, S., 283 Biasin, M. R., 100 Biber, T.U.L., 458 Bidani, A., 253,256(9) Bieber, L. L., 536 Biedermann, J., 477 Bielski, B.H.J., 421,622 Biemond, P., 561,566(30) Bieri, J. G., 397 Bignon, E., 591 Bindoli, A., 425 Bing, D. H., 411 Birnboim, H. C., 67, 73(17), 77(17), 78(17), 89, 90(10, 13), 91(10, 13), 94(10, 13), 96(10, 13) Bischoff, F., 189 Bishop, G. A., 194 Bishop, J. E., 603 Bittner, D., 125 Blackett, P. R., 516 Blake, D. R., 555,569 Blakely, W. F., 3, 4(8), 5-6, 6(8), 9(32), 11(28) Blamey, R. W., 590 Blanchard, J. M., 231 Blanchard, J. S., 612 Blanchflower, W. J., 299 Blank, V., 161 Blasig, I. E., 422,423(34), 425(34) Blattner, F. R., 176, 216(8)
646
AUTHOR INDEX
Blavet, N., 475 Blazovics, A., 422,425(26) Bligh, E. G., 609 B1fcher, D., 100, 101(26) Block, G., 269 Blouin, F. A., 415,416(25) Blum, H., 185 Bockstette, M., 135 Boehme, P., 620, 628(6), 629(6) Boisnard, M., 438 Boiteux, S., 3, 10, 16, 16(14), 82, 117, 123, 125, 129-130 Bokn~ov~i, B., 578 Boli, R., 523 B61ker, M., 220 Bolle, A., 218 Bolli, R., 515, 523(23) Bolme, P., 487 Bolognesias, C., 96 Bolton, D. C., 254, 257 Bolton, P. H., 113 Boman, G., 491 Bomford, A., 543,544(13) Bonfanti, R., 296 Bongiorno, A., 401 Bonhomme, B., 462 Bonkowsky, H. L., 438 Bonner, W. M., 50 Bonomi, F., 457 Bonomo, R. P., 421 Boobis, A. R., 491 Boozer, C. E., 312,403 Borek, C., 252 Borel, J. P., 421 Borgstr6m, L., 487 Borish, E. T., 619 Borner, C., 591 Borrello, S., 601 Bors, W., 384, 420, 422, 424(24), 425, 425(24, 25), 426(25, 65), 427(25, 65), 428,428(24, 25), 429(65), 437 Borst, P., 603 Bothe, E., 426 Botnen, J., 278 Bottazzi, B., 602 B6ttcher, C.J.F., 616 Bourel, D., 438 Bourel, M., 438 Bourgeois, C., 295 Bourgeois, C. F., 299
Bourre, F., 45, 80, 119 Bourrier, M. J., 416 Boveris, A., 66, 437 Bowry, V. W., 354, 372 Box, H. B., 24, 36, 38(19) Boyer, V., 79 Bradford, M. M., 441 Bradley, K., 136 Brand, J. G., 616 Brankow, D. W., 237 Braquet, P., 421,423,462, 475 Brasch, R. C., 580 Brasseur, N., 81,635 Brattsand, R., 479 Braughler, J. M., 376, 402, 548, 550-553 Braun, A. G., 34 Braun, A. M., 384 Braunstein, S. N., 125 Bray, T. M., 525 Breimer, L. H., 34, 40, 235 Bremner, I., 546 Brent, R., 221 Brewer, G. J., 545-546, 547(18) Bridge, R. J., 493 Bridgeman, M.M.E., 488 Bridges, R. J., 493 Brigelius, R., 370 Briggs, M. R., 165 Brin, M., 327 Brissot, P., 437-438,441,443 Britigan, B. E., 561,566(31) Brittenham, G. M., 438 Briviba, K., 384, 574, 578(13) Brodskii, A. V., 421 Brodsky, M. H., 375 Brooks, G. A., 368.371(13) Brotherton, J. E., 435 Brown, D. M., 40, 44(27) Brown, E. D., 397 Brown, M. S., 606 Brown, R. A., 446 Brownlie, I. T., 581 Brownstein, B. H., 202 Brubacher, G., 295 Bruchelt, G., 337 Bruchhausen, F. V., 411 Bruener, B. A., 253, 254(11), 255(11) Bruice, T. W., 77 Brun, M., 416 Bruning, P. F., 592
AUTHOR INDEX Brunner, K. T., 135 Bruno, A., 415,416(19, 21), 418(19), 419(19) Bruno, G., 415,416(32) Bryan, C. L., 551 Bryant, P. E., 100, 101(26) Buchko, G. W., 79, 81-82, 83(25), 85(25), 87, 87(25) Buckpitt, A. R., 258 Budowski, P., 424 Buege, J. A., 566, 569(39), 570(39) Buettner, G. R., 70-71, 71(32), 375 Bujard, H., 166, 167(14) Bukley, G. B., 619 Bunnell, R. H., 295 Buran, L., 578 Burgunder, J. M., 488 Burk, R. F., 316 Burkitt, M. J., 66-67, 70, 70(16), 71, 71(16, 36), 72(38), 73, 73(16), 74(16), 75(16), 76, 76(16), 77(35) Burlakova, E. B., 345 Burlakova, Y. B., 356 Burr, J. A., 312-313,313(14), 315(12) Burtiss, J. L., 295 Burton, G. W., 269, 280, 286(1), 303-304, 310, 310(2), 316, 322,331,355-356, 362, 362(8), 372, 381(3), 403, 406, 506, 533, 543-544, 601,633 Burton, K., 35, 37(17), 40(17), 104 Busch, S. J., 505,506(4) Bush, K. M., 273,475 Butler, J., 68, 69(27), 489, 557, 571(18) Butler, L. G., 429,432,434-435,435(12, 15) Butta, A., 591 Bylund-Fellenius, A. C., 582, 586 C Cabrini, L., 344 Cadenas, E., 66, 79, 468, 476, 479(1), 480(1) Cadet, J., 3, 17, 55, 79-81, 81(12), 82, 83(23, 25, 29), 85(25), 87, 87(25) Cahill, D. S., 17 Caldarera, M., 373 Caldwell, K. A., 477-478, 47804) Caleffi, M., 590 Camerini-Otero, R. D., 125 Cameron, L., 590 Campbell, C. R., 162
647
Campbell, E., 496 Campbell, W. B., 514 Campion, J. P., 438 Canada, A. T., 424 Canney, A. H., 411 Cannon, M., 591-592, 592(32, 33), 594(33, 37, 39), 595(32, 37, 39), 596(33, 37, 39), 597(33, 39), 598(47, 48), 599(48), 600(47), 601(33, 37, 39, 47, 48) Cannone, P., 385 Cantilena, L. R., 393 Cantin, A. M., 256 Cantineau, R., 477 Cantley, J. C., Jr., 410 Cantoni, O., 77 Cao, W., 524 Carbone, P. P., 590 Carew, T. E., 505, 513-514 Carey, F., 446, 449(15), 450(15), 453,453(15) Carey, J., 153, 160(8), 165, 171(11) Carini, M., 415, 416(22) Carlin, G., 557, 582, 586 Carlsson, D. J., 384 Carney, J. M., 515, 523-524, 524(1), 5250), 526(1) Carri6re, I., 475 Carrillo, M.J.H., 321-322, 322(12), 323(14), 325(14), 326(14) Carter, D. E., 542 Carvalho, A. P., 591 Carven, P. A., 557 Catania, J., 82 Cathart, R., 331 Cathcart, K.N.S., 494 Cathcart, M. K., 513-514 Catignani, G. L., 394, 397 Cattabeni, F., 77 Catteau, J. P., 421,423(13), 425(13) Cavalieri, L. F., 4 Cavatorta, P., 601 Ceasrone, C. F., 96 Celeste, M., 591 Celotti, L., 100 Cerimele, B. J., 505 Cerottini, J.-C., 135 Cerutti, P., 77, 191, 192(22) Cerutti, P. A., 88, 187, 191(19), 203(19), 235 Cesarone, C. F., 489-490, 490(27) Ceva, P., 415, 416(29) Chait, A., 506, 514
648
AUTHOR INDEX
Chambon, P., 602 Chamulitrat, W., 70 Chan, B. C., 433 Chan, G. L., 35, 37(14), 103 Chan, H. C., 580 Chanal, S., 591 Chance, B., 66, 344 Chander, C. L., 590 Chandler, L. A., 396 Chapelat, M., 462 Chaplin, D. D., 202 Chase, R. L., 548, 550,553 Chatopadhyaya, R., 51 Chatterjee, A., 381 Chaudary, M. A., 590 Cheek, J. M., 258 Cheeseman, K. H., 601,616 Chen, C.-H., 166, 167(13) Chen, G., 525 Chen, Q., 16 Chen, S.-L., 68 Chen, W.-J., 505 Chenard, M. P., 602 Cheng, K. C., 17 Cheng, M. S., 523,524(1), 525(1), 526(1) Cherian, M. G., 547 Chevion, M., 524, 544 Chi, E. M., 505(8), 506, 509(8), 512(8) Chiba, S., 630 Chida, H., 322 Chiesa, G., 515 Ching, T.R.A.M.I.P., 423 Chipault, J. R., 300, 356 Chirico, S., 254 Chisolm, G. M., 513-514 Chiu, R., 170 Chiu, S., 3 Chiu, S.-m., 78 Choi, D. W., 552 Choisy, H., 511 Chomczynski, P., 193,227 Chong, S., 491 Choo, Y. M., 528 Chow, C. K., 621 Christ, B., 416, 417(40) Christensen, A., 557, 569(22), 570(22) Christiaens, L., 477 Christian, M. F., 214 Christman, M. F., 175(3), 176, 181(3) Chuang, S. E., 176, 216(8)
Chung, M.-H., 24, 80 Church, D. F., 252, 506 Churley, M., 237 Ciaccio, M., 401 Cilento, C., 79 Cillard, J., 421,437,441 Cillard, P., 421,437,441 Claeys, M., 416 Claiborne, A., 227 Clark, G. R., 68 Clarke, R., 601 Clarkson, B., 592 Claycamp, H. G., 19, 20(13) Clayton, D. A., 42 Cleland, L. G., 557 Cleland, W. W., 612 Clemetson, C.A.B., 421 Climent, I., 254 Clostre, F., 462,475 Clostre, P., 462 Clough, R. L., 314 Coassin, M., 425,478,479(20), 480(20) Cochrane, C. G., 77 Cogrel, P., 437 Cohen, G., 542, 564, 566(34) Cohen, L. A., 314 Cohen, M., 77 Cohen, M. S., 561,566(31) Cohen, N., 309 Cohen, R. M., 283 Cohen, S. S., 5 Colditz, G. A., 269 Collins, A. R., 77, 78(52) Collins, M., 601 Combs, G. F., 327 Conn, P. F., 385, 387(14) Connor, J. A., 236 Constantinescu, A., 363(34), 366 Cook, H. W., 481 Coon, M. J., 411 Cooney, R. V., 236-237, 238(17), 239(8), 241(9), 243(9) Cooper, M. J., 284 Coquerelle, T., 100 Corsaro, C., 421 Corthout, J., 416 Cosgrove, T. P., 273 Costa, R., 487 Costa de Oliveira, R., 121 Cotelle, N., 421,423(13), 425(13)
AUTHOR INDEX Cotgreave, I. A., 477-478,478(13), 479,482, 484, 485(5), 486, 486(5), 487, 487(7), 488-489, 489(8) Cotter, R. J., 57 Cotton, F. A., 69 Courison, C., 591 Coussio, J. F., 437 Coutant, J. E., 512 Cox, A. C., 327 Cox, D., 446 Craft, D. A., 615 Craft, N. E., 278, 397 Crain, P. F., 6-7, 7(30), 24 Cramp, W. A., 66, 78(4, 5), 79(4-6) Crane, F. L., 375 Crapo, J. D., 584 Crastes de Paulet, A., 423 Crawford, D., 191, 192(22) Crawford, D. R., 175, 181, 187, 191(19), 203(19) Crawford, J. M., 494 Crepaldi, G., 511 Crockett, R., 573,574(5), 575(5), 576(5) Cross, C. E., 252-254, 254(11), 255(11, 26), 256, 475, 619 Crystal, R. G., 256 Csallany, A. S., 439 Culcasi, M., 475 Cundy, K. C., 67, 117 Cunningham, R. P., 33, 37, 38(22), 40, 40(5), 41(28), 42(28), 102, 104(1), 105(1), 110(1), 123, 125 Curran, T., 163-164, 165(1,2, 8, 9), 166(2, 7, 9), 167, 167(2, 8), 168(2, 16), 170, 174(7, 9), 224 Cursted, T., 603 Cutler, R. G., 24, 31(27) Czapski, G., 77
D Dagan, A., 606 D'Agostini, F., 489, 490(27) Daikh, Y., 181 Dallner, G., 344 Dammann, A. L., 615 Damon, M., 423 D'Andrea, A. D., 34
649
Dani, C., 231 Daniel, V., 135-136, 139(6), 140(11), 492 Daniels, D. L., 176, 216(8) Danks, D. M., 542, 547(7) d'Arbigny, P., 475 Darley-Usmar, V. M., 423 Darnell, J. E., Sr., 199 Das, A. K., 604 Das, B., 252 Das, D. K., 410 Das, M., 411 Das, N. P., 401,421,437 Davies, D. S., 491 Davies, J.M.S., 175, 175(7), 176 Davies, K.J.A., 175, 175(4-7), 176, 254 Davies, M. J., 284, 423 Davis, K. A., 270 Davis, L. G., 226 Davis, P. A., 254, 255(26), 612, 614(46) Davis, W. B., 256 Davison, P. F., 98 Dawson, J., 488 Day, B. W., 631 Day, J. S., 553 DeB Butter, J., 337 De Bernardi di Valserra, M., 487 Debey, H. J., 145, 147(26) DeBoer, C. J., 147 Deby, C., 462 De Carro, L., 487 Decarroz, C., 55, 82, 83(29) Decuyper-Debergh, D., 121 Deeble, D. J., 3 Deelstra, H., 299 De Felippis, M. R., 573 DeFeudis, F. V., 462, 463(1) De Flora, S., 489-490, 490(27) Degan, P., 17, 24, 24(9), 26, 27(9), 32(9), 80 DeGraff, W., 588 DeGraff, W. G., 582, 587(17), 589(17) de la Harpe, J., 147 DeLange, R. J., 282 De Leenheer, A. P., 393 Della Loggia, A., 446 del Negro, P., 446 Delorenzo, O., 416 Demediuk, P., 619 Demet, D. L., 590 Demets, D. L., 590 DeMore, W. B., 262
650
AUTHOR INDEX
Demple, B., 123, 151, 175, 181,214(2), 217, 224 Denda, A., 24 Denny, R. W., 384 Dereu, N., 477, 480-481 Derguini, F., 394 Derian, C. K., 514 de Rooij, B. M., 478 DeRubertis, F. R., 557 Desa, F. M., 590 Desai, I. D., 295 Deschuytere, A., 299 Desvergne, B., 438 Dethmers, J. K., 493 Deugnier, Y., 438 Devary, Y., 224 Devasagayam, T.P.A., 45, 59, 80, 385, 386(18), 387(18, 24), 388,456, 575 de Vries, H.M.J., 631 Dewar, J. A., 590 Dhariwal, K. R., 270, 337 Dianzani, M. U., 616 Diatchkovskaya, R. F., 582 di Bilio, A. J., 421 Dibner, M. D., 226 Dick, R. D., 545-546, 547(18) Dickens, B. F., 620, 621(10), 626(10) DiCorleto, P. E., 514 Dietz, J. M., 397 Dignam, D., 159 Di Iorio, E. E., 469 Dikomey, E., 89, 93, 100, 101(5, 16, 28) Di Mascio, P., 45, 79-80, 116, 118(2, 3), 119, 120(3), 121, 129, 384-385, 385(12), 386(18), 387(12, 18), 575 Di Monte, D., 628 Dinarello, C. A., 141 Diplock, A. T., 295 Dirksen, M.-L., 6, 9(32) Distel, R. J., 170 Dixon, A. K., 547 Dizdaroglu, M., 3-4, 4(8), 5-6, 6(8, 21, 25), 7, 7(24), 8(24, 37), 9, 9(6, 26, 27, 31, 32), 10, 11(6, 25, 28), 13(21, 24), 14(21), 15(6, 21, 45), 16, 16(14), 17, 67, 70(15), 73(15), 75(15), 76(15), 82, 86, 117, 131 Djuric, Z., 5, 13(22) Djursater, R., 557 Doba, T., 322, 356 Dobberstein, B., 166, 167(14)
Dobie, J., 590 Doebeli, H., 167 Doelman, C.J.A., 489 Doetsch, P. W., 33-35, 37, 37(14), 38, 38(22), 40, 40(4, 5), 41(28), 42(8, 28), 102-104, 104(1, 3), 105(1), 110, ll0(l, 3), 112(15), ll3 Doghieri, P., 416 Doleiden, F. H., 384 Doly, M., 475 Donner, L. W., 337 Dorc, J. C., 591 Dorian, R. L., 497 Dormandy, T. L., 280(3), 281 Doroshow, J. H., 3 Dorozhko, A. I., 421 Dostalova, L., 296 Dougherty, M. H., 416 Douglas, K. T., 67, 73(14), 75(14) Doura, F., 4 Douste-Blazy, L., 423,606 Downs, D., 515 Dowsett, M., 591 Doyle, M. P., 469 Draper, H. H., 316 Dratz, E. A., 569 Dreano, Y., 592 Dresel, H. A., 601 Dri, P., 446 Drieu, K., 462-463,476 Drings, P., 135, 139(6) Driscoll, J. S., 638 Driskel, W. J., 397 Dr6ge, W., 135-136, 136(9), 137, 139, 139(6, 9, 16, 17), 140, 140(5, 11), 141(17), 142, 142(5, 8, 17), 143(17, 22), 144, 144(4, 5, 8, 16, 23), 145(7,24), 146(7, 8), 148, 148(9), 149, 149(7, 24), 152, 156(5), 157(5), 158(5), 492 Droy-Lefaix, M. T., 462, 474 Dubbelman, T.M.A.R., 69 Dubose, C. M., 523 Duchon, A., 524 Duck, M. V., 474 Duda, C. T., 328 Duhault, J., 423 Duigou-Osterndorf, R., 592 Duker, N. J., 131 Dulin, D., 311,313(7) Dull, B. J., 557
AUTHOR INDEX Duncan, L. A., 550 Dungworth, D. L., 257, 258(3) Dupuis, M., 462 Durand, R. E., 100 Durckheimer, W., 314 Duroux, E., 421 Dusting, G. J., 491 Dutton, H. J., 415 Duvall, T. R., 254, 257,475 Dyer, W. J., 609 Dypbukt, J. M., 76 Dzido, T. Z., 415,417(24) l)urba, A., 578, 579(31)
E Eakins, N. N., 437 Eaton, J. W., 568, 569(45) Ebashi, I., 341 Ebert, B., 422, 423(34), 425(34) Ebert, M. H., 108 Eck, H.-P., 135-136, 136(9), 137, 139(6, 9, 16, 17), 140, 140(5, 11), 141(17), 142, 142(5, 8, 17), 143(17), 144(5, 8, 16, 23), 146(8), 148(9), 492 Eckfeldt, J. H., 327 Edbauer-Nechamen, C. A., 175 Eddy, L. J., 578 Edelstein, S., 400 Edlund, P. O., 275 Edvardsson, K. A., 89, 94(9), 96(9), 100(9), 102 Edwards, J. C., 66, 79(6) Edwards, K. J., 591 Egan, D., 446 Eichhom, G. L., 68 Eisenberg, W. C., 257, 258(4) Eklow, L., 628 Eklund, A., 487, 488(12) Ela, S. W., 492 Elinder, L. S., 512 Elkins, D., 538 Ellis, I. O., 590 Ellison, E. G., 300 Ellman, A. L., 495 Ellman, G. L., 273,458, 502 EI-Saadani, M., 513 Elstner, E. F., 420 Elwell, J. H., 244
651
Emery, S., 601 Emslie, E. A., 200, 201(33) Endo, H., 282 Eneff, K. L., 3, 21, 59, 80 Engel, J., 250, 251(7) Engel, R. R., 284 Engemann, R., 462 Engers, H. D., 135 Englert, G., 390, 393, 394(14) Engman, L., 477-478, 478(13) Engmann, L., 477 Ennen, J., 137, 492 Epe, B., 79, 122-123, 125-126, 129-130 Epp, O., 478 Eppenberger, U., 592 Epps, D. E., 283,345,551,554 Epstein, S., 590 Erben-Russ, M., 422, 425(25), 426(25), 427(25), 428, 428(25) Erdman, J. W., 389, 397 Erickson, L. C., 128, 129(7) Erixon, K., 67, 78(18), 89, 94(14), 96(14), 100(14) Erjefalt, I., 253,256(15) Ernster, L., 344 Eskins, K., 415 Esko, J. D., 609 Espenson, J. H., 426 Essigmann, J. M., 17 Estabrook, R. W., 577 Esterbauer, H., 513,616 Estrela, J. M., 367 Etienne, A., 462 Evans, P. J., 411,424, 446, 453(11, 12), 543, 544(13) Evces, S., 615 Evers, M., 480 Ewertz, M., 24, 31(28) Ewig, R.A.G., 128, 129(7)
F Fabbro, D., 591 Facino, R. M., 415, 416(22) Fahey, R. B., 497 Fahey, R. C., 367 Fahrenholtz, S. R., 384 Fairweather, D. S., 82 Falck, J. R., 514, 606
652
AUTHOR INDEX
Fanger, M. W., 135 Fanidi, A., 591 Faraggi, M., 573 Fargnoli, J., 200, 204,205(34, 38), 208(38) Fariss, M. W., 367, 368(8), 369(8), 371(8) Farjanel, J., 438 Farmilo, A., 385 Farr, A. L., 516, 594, 598(50) Farr, S. B., 151, 156(1), 181, 217, 217(12), 224 Farrell, P. M., 514 F~itkenheuer, G., 135, 139(6) Faure, M., 423 Fausel, M., 476, 479(2), 482(2) Favier, A., 17, 80 Fayard, J. M., 591 Fedelo~ov,q, V., 574 Feelisch, M., 472 Feldman, R. S., 601 Feidstein, M., 262 Feletti, F., 487 Fellin, R., 511 Fells, G. A., 256 Fentiman, I. S., 590 Fernandes, A. C., 552 Ferrand, B., 438 Ferrfindiz, M. L., 446, 453,453(14), 454(14, 21) Ferrari, M. B., 601 Ferrari, R., 373 Ferraro, G. E., 437 Ferraro, P., 100 Ferrero, J. A., 371 Feskens, E.J.M., 454 Fewtrell, C.M.S., 411 Fey, W., 590 Feyzi, J., 590 Fibach, E., 606 Fiers, W., 244 Filipe, P. M., 552 Filipski, R. F., 327 Finkelstein, E., 582 Fischer, C., 557 Fischer, H., 477,481,482(33, 34) Fischer, V., 638 Fitchett, M., 73 Flamm, D. L., 264 Flanders, K. C., 591 Fleming, W. E., 553 Flemstrom, G., 527
Flenley, D. C., 488 Floyd, R. A., 3, 17, 21, 59-60, 62(6), 79-80, 87, 515, 523-524, 524(1), 525(1), 526(1) Fogelman, I., 590 Foglia, T. A., 604 Folch, J., 599 F61des-Papp, Z., 422 F61diak, G., 422, 425(26) Fong, K. L., 316 Fong, L. G., 513 Foote, C. S., 79, 314, 384, 605, 606(29), 607(29) Ford, D. A., 620 Ford, G. D., 458 Ford, P. C., 331 Forder, R. A., 446, 449(15), 450(15), 453, 453(15) Fornace, A. J., Jr., 128, 200, 204, 205(34, 38), 208(38) Forsmark, P., 344 Fort, P., 231 Forte, T., 254, 317, 363,373,467 Foster, D. O., 303, 310(2) Fraga, C. G., 24, 437 Franceschini, G., 515 Francis, F. J., 415 Franklin, R. M., 125 Franza, B. R., Jr., 164 Franzke, J., 93, 100, 101(16, 28) Frayer, W., 492(5), 493,494(5) Freedman, A. M., 620 Freeman, B. A., 256, 273,475 Frei, B., 253-254, 269-270, 273(10), 344, 348(7), 372 Frei, J. V., 547 Freisleben, H.-J., 371-372, 381(6), 401, 409(11), 460, 467, 533,534(23) Freistleben, H.-J., 358 Frelinger, J. A., 194 Frenkel, K., 3, 58 Fridovich, I., 3, 338, 357, 469, 536, 558, 566(25), 584, 621,625,625(14) Friedberg, E. C., 34, 37, 123 Friedman, C. A., 128, 129(7) Friedman, L. R., 78 Friedman, P. A., 411 Friedman, T., 40, 44(27) Filmer, A. A., 605, 606(30), 607(30) Fritsch, E. F., 48, 50(4), 117, 121(8), 170, 171(21), 193, 197(25), 202(25), 203(25),
AUTHOR INDEX 207(25), 213(25), 214(25), 226, 227(15), 228(15), 229(15), 230(15),232(15) Froncisz, W., 580, 588 Fuchs, J., 310, 317, 318(6), 580 Fuciarelli, A. F., 5, 9(26), 11(28) Fujiki, Y., 603-604 Fujimori, E., 384 Fujimoto, M., 52 Fujioka, S., 446 Fujisawa, H., 425 Fujiwara, Y., 601 Fukunaga, Y., 275 Fukuzawa, K., 322, 366 Funahashi, T., 514 Fung, H. L., 491 Furr, B.J.A., 600 Furukawa, M., 411,453 Furukawa, T., 371 Furuya, Y., 601
G Gabard, B., 484 Gabauer, I., 578 Gabe, E. J., 356 Gabor, M., 411 Gabriel, H., 479, 480(22), 481,482(33) Gabrielsson, J., 487 Gagne, E., 167, 168(16) Gaitonde, M. K., 148 Gajewski, E., 3, 5, 9(26, 27), 11(28), 16(14), 67, 70(15), 73(15), 75(15), 76(15), 82,117 Galeotti, T., 601 Gallas, H., 142, 144(23) GaUatin, W. M., 206 Galmozzi, M. R., 487 Galos, R., 189 Ganesan, A. K., 34, 127 Ganguly, T., 131 Gantchev, T., 638 Gantchev, T. G., 631,635 Gardana, C., 415,416(21, 22) Gard~s-Albert, M., 462 Garen, A., 52 Garland, D., 254 Garland, L. G., 423 Garner, M. M., 165, 171(10) Garry, P. J., 331 Gasc6, E., 371
653
Gasc6, M. A., 446 Gasparotto, A., 511 Gates, F. T., 123 Gatt, S., 604, 606, 619(23, 24) Gavarron, F. F., 616 Gawthorne, J. M., 543 Gaydou, E., 421,423(13), 425(13) Gedik, C. M., 77, 78(52) Geher, K., 516, 523(31) Geierstanger, B. H., 68, 70(23) Gentz, R., 164, 165(2), 166, 166(2), 167(2, 13, 14), 168(2) George, A. M., 66, 78(4, 5), 79(4-6) Gerber, G., 422 Gerber, L. E., 388 Gerdin, B., 557 Gergel, D., 574-575, 577(18) Gerlt, J. A., 113 Gerrard, J. M., 327 Gerrity, R. G., 513 Gesteland, R. F., 218 Getoff, N., 3 Ghizzi, A., 487 Giacomoni, P., 17, 80 Giannella, E., 424 Giannoni, P., 490 Gibson, D. D., 316 Gilbert, B. C., 67, 73 Gilbert, H. F., 367 Gilbert, H. S., 328 Gilbert, J., 591 Gilbert, W., 35, 38(18), 103 Gimeno, C. J., 24, 25(25), 26(25), 67, 117 Giovanniello, T. J., 53 Giovannucci, E., 269 Giulivi, C., 254 Glajch, J. L., 417 Glaze, W. H., 256 Glazer, A. N., 273,282,406, 460, 531 Glende, E. A., 404 Glinz, E., 302-303,307(3), 390, 394(14) Glockner, J. F., 580 Glogowski, J., 472 Gluzman, Y., 116 Gmiinder, H., 135-137, 139(16, 17), 140(5, 11), 141(17), 142, 142(5, 17), 143(17, 22), 144(5, 16, 23), 149, 492 Gober, K. H., 416, 417(40) Goddard, P. M., 591 Godfrey, L., 592
654
AUTHOR INDEX
Godinger, D., 586, 589(27) Goeddel, D. V., 244, 250, 251(8, 9) Gogolewski, M., 320 Gohil, K., 274, 368, 371(13) Goiffon, J. P., 416 Gold, E., 590 Goldman, D., 108 Goldman, P., 557 Goldman, R., 318 Goldstein, J. L., 606 Goldstein, M. S., 58 Goldstein, S., 77 Goli, M. B., 398 Gomperts, B. D., 411 Gonzalez, F. J., 588 Goodenough, D. A., 236 Goodwin, T. W., 388 Gooneratne, S. R., 543 Gopinathan, V., 284 Gordon, L. K., 42 Gorecki, M., 250, 251(6) Gorman, A. A., 384 Goscin, S. A., 621,625(14) Gossett, J., 37, 38(22), 102, 104(1), 105(1),
110(1) Gotchev, G. P., 635 Goth, S., 357, 358(22), 359(22) Goto, A., 425 Gotoh, N., 402 Gottlieb, R. A., 224 Goulding, A., 590 Goutier, R., 462 Gouyette, A., 123 Govil, G., 528 Gown, A. M., 513 Graca, M., 591 Graf, E., 477, 481,568 Graf, P., 476, 479(1), 480(1), 482 Grafstr6m, R., 487 Graham, D. A., 581,583(12) Grajewski, E., 17 Gramatica, P., 384 Graminski, G. F., 505 Granger, D. N., 256, 555, 557, 568, 569(44), 570(49), 571 GranstrOm, E., 449 Grant, R. D., 422 Gray, G. M., 614, 615(47) Grayer, R. J., 429 Graziani, Y., 411
Green, J., 355 Green, L. C., 472 Greenberg, J., 181 Greenberger, J. S., 181 Greenley, T. L., 423 Greenstein, J. P., 496 Gregolin, C., 317 Greiff, L., 253,256(15) Greim, H., 546 Griesenbach, U., 89, 90(12), 91(12), 94(12), 96(12), 100(12) Griffith, O. W., 492(8), 493,494(8), 496 Grill, H. J., 592 Griller, D., 422 Grimek-Ewig, R. A., 89, 90(8), 94(8) Grimm, S., 163 Grisham, M. B., 256, 555-558, 560, 561(29), 562(29), 563(29), 564(29), 565, 566(24, 29, 36), 567(36), 568, 568(36), 569(41, 44), 570(41, 49), 571 Groilman, A. P., 80 Grosch, W., 424 Gross, H. J., 185 Gross, J., 388,389(2), 390(2) Gross, R. W., 603, 620 Grossman, S., 424 Grunicke, H., 602 Gryglewski, R. J., 411,421,422(16), 42306) Guan, D. M., 439 Guardiola, B., 423 Guarnieri, C., 373 Gudej, J., 415, 417(24) Guguen, C., 438 Guguen-Guillouzo, C., 438 Guillaume, M., 477 Guillon, J. M., 462 Guillot, F. L., 553 Guillouzo, A., 438 Gulz, P.-G., 415, 416(23) Gunning, P., 250, 251(7) Gunter, E. W., 397 Guo, H., 236, 241(14) Gupta, R. C., 18 Gupta, S. P., 9 Gustafsson, B., 253, 256(15) Gutteridge, J.M.C., 3, 16, 66, 67(1), 80, 253, 256, 256(13), 269, 280(3), 281,372, 506, 528, 542-543, 544(12), 547(12), 561, 561(33), 563(33), 566(32, 33), 594,596, 601(52)
AUTHOR INDEX Gy6rgy, I., 422, 425(26) Gyorgy, P., 327 Gysel, D., 296
H Haagsman, H. P., 252 Haas, S. M., 536 Haenen, G.R.M.M., 362,455,478, 489 Haeuptle, M. T., 166, 167(14) Haga, S., 533 Hagaman, K. A., 505 Hagan, M. P., 3, 4(8), 6(8) Hagar, W. L., 257 Hagerman, A. E., 429, 431-432, 435, 435(12), 436, 436(20) Hagmaier, V., 337 Hahn, S. M., 582, 589(22) Haidle, C. W., 127 Haim, N., 631,638(6) Hajra, A. K., 603-604, 612, 614(46) Hakusui, H., 482 Halbrook, J. H., 175, 214(2) Halevy, O., 401 Hall, E. D., 548, 551-553 Hall, K., 591 Hallaway, P. E., 568, 569(45) Hallberg, A., 477, 479, 488 Halliwell, B., 3, 16-17, 66-67, 67(1), 70(15), 73(15), 75(15), 76, 76(15), 252-254, 254(11), 255(11, 26), 256, 256(13), 269, 273, 372, 411, 424, 446, 453(11-13), 459, 475, 489, 506, 528, 543, 544(13), 557, 561, 566(32), 571(18), 591-592, 592(32, 33), 594, 594(33, 37, 39), 595(32, 37, 39), 596, 596(33, 37, 39), 597(33, 39), 598(48), 599(48), 600(51), 601(33, 35, 37, 39, 40, 48), 619, 621 Hamada, A., 582 Hamano, M., 421,423(22) Hamberg, S., 141 Hamernyik, P., 335, 337(7) Hamilos, D. L., 135 Hamilton, C., 590 Hamilton, C. E., 403 Hamilton, C. S., 312 Hamilton, K. K., 33, 38, 40(4), 102-104, 104(3), ll0(3), 112(15), 113 Haming, W. J., 262
655
Hammes, G. G., 410 Hammond, G. S., 312, 403 Han, D., 321,361,363(26, 34), 366, 366(26), 383 t Han, F., 554 Han, J., 492(8), 493,494(8) Han, L.-P.B., 504 Hanahan, D., 120 Handelman, G. J., 381,398,569 Handleman, G. E., 524 Hankovszky, O. H., 580 Hanna, P. M., 69-70, 70(30) Hans, P., 462 Hansch, C., 538 Hansen, H.-J., 384 Hanusch, M., 394, 398(27) Hara, K., 491 Hara, S., 335, 416 Harada, T., 256 Haraikawa, K., 422, 425(40) Harborne, J. B., 420, 446 Harel, S., 568 H~iring, M., 130 Hariton, C., 429 Harm, W., 35, 36(15), 104 Harman, D., 524, 619 Harper, D. A., 5, 13(22) Harris, G., 66, 78(4), 79(4, 6) Harris, J., 17 Harris, L., 620, 621(9) Harrison, A. G., 57 Hart, B. A., 423 Hart, D. A., 135 Hart, L. E., 66, 78(4), 79(4, 6) Hartley, D. M., 552 Hartman, J. R., 250, 251(6) Hartmann, M., 136, 140(11), 492 Hartzell, W. O., 270 Haseltine, W. A., 34-35, 37(14), 42, 42(8), 103 Hashimoto, H., 393 Haslam, E., 431 Hatayana, K., 411 Hatch, G. E., 256 Hatefi, Y., 275 Haugan, J. A., 390 Haupt, R., 256 Havsteen, B., 446 Hawkes, W. C., 270, 271(13) Hawley, C. J., 557
656
AUTHOR INDEX
Hay, R. T., 153 Hazen, S. L., 620 Hearse, D. J., 523 Heath, R. L., 560,566(27) Heeg, J. F., 511 Hegler, J., 122, 125-126, 130 Heidelberger, C., 237 Heidemann, H. T., 462 Heijman, M.G.J., 426 Heilmaier, H. E., 546 Heimler, D., 415 Heinecke, J. W., 506, 514 Heinkel, K., 557 Heinola, K., 527 Heintz, N. H., 191 Heinz-Erian, P., 296 Heitzman, A.J.P., 426 Helbock, H. J., 24 H616ne, C., 79 Helland, D. E., 34, 40, 41(28), 42(8, 28), 79 Heller, W., 422, 424(24), 425, 425(24), 426(65), 427(65), 428, 428(24), 429(65), 437 Hemingway, R. W., 430, 433(3) Hemler, M. E., 481 Hems, R., 367, 370(7) Henderson, B. E., 590 Hengartner, U., 390, 394(14) Henglein, A., 426, 427(70) H6nichart, J. P., 421,423(13), 425(13) Henle, E. S., 51 Henner, W. D., 40, 41(28), 42(28) Hennig, B., 621 Hennighausen, L., 172 Henriksen, T., 514 Henry, P. D., 630 Hepp, M., 463 Hernanz, A., 331,335 Herrlich, P., 170 Herscovitz, H., 604 Hersey, A., 423 Hertog, M.G.L., 454 Herzenberg, L. A., 146, 492 Hess, D., 296 Heuvel, H.V.D., 416 Hewitt, S., 569 Heymans, H.S.A., 603 Hicks, K. B., 337 Hideg, K., 580 Hiermann, A., 415
Higashida, M., 410 Higgs, E. A., 475 Hildebrand-Zanki, S. U., 638 Hillis, W. E., 432 Hilton, J., 100 Hinzmann, J. S., 554 Hiraishi, H., 256 Hiramatsu, M., 401,409(9) Hiramitsu, T., 404 Hirono, I., 422,425(40) Hiser, M. F., 511 Hixson, C. S., 460 Ho, W.-T., 385 Hochstein, P., 331,578 Hochuli, E., 167 Hodnick, W. F., 424 Hoefler, G., 604 Hoefler, S., 604 Hoekstra, J. W., 469 Hoey, B. M., 489, 557, 571(18) Hofer, E., 199 Hoffman, M. Z., 284, 288(15) Hogeboom, G. H., 535 Hogsett, W. E., 21 Holbrook, N. J., 200, 204, 205(34, 38), 208(38) Holden, S. A., 494 Holdiness, M. R., 487 Holec, V., 578 Hollander, M. C., 200, 205(34) Hollman, P.C.H., 454 Hollunger, B. G., 344 Holm, B. A., 256 Holwitt, E., 3, 4(8), 6, 6(8), 9(32) Honkanen, E., 527 Hoogland, H., 619 Hope-Ross, P., 619 Hopwood, L. E., 580, 588 Hor¢tkov~i, L., 572, 574, 576, 578, 578(13), 579, 579(20) Horie, T., 453 Horiguchi, K., 24 Hornbrook, K. R., 316 Hornig, O., 337 Hornsey, S., 66, 78(4), 79(4) Horobin, J. M., 590 Horowitz, J. D., 491 Horrocks, L. A., 603,619 HorswiU, E. C., 312 Hosomi, M., 393
AUTHOR INDEX Hossain, M. Z., 236, 241(14) Hough, K., 491 Houghton, J., 590 Hoult, J.R.S., 411, 424, 443, 446, 449(15), 450(15), 452-453, 453(11-13), 557 Howeland, S. K., 328 Howell, J. McC., 543 Howes, T. W., 397 Hrstich, L. N., 433 Hsie, A. W., 582 Hu, M. L., 254, 475 Hua, X., 415,416(26) Huang, M. T., 411 Huang, R.-R.C., 477 Hubbard, R. C., 256 Huber, C. O., 581 Huber, L. A., 601 Huber, R., 478 Hubert, N., 443 Hudson, B. S., 375,460 Huff, D. L., 397 Hughes, L., 303,310(2), 533 Hughes, R. E., 332, 334(5) Huguet, A. I., 421 Hui, B., 581 Hull, W. E., 148 Humplov~, J., 576 Hunziker, F., 296 Huque, T., 616 Husain, S. R., 421 Hutchinson, F., 33, 35(2) Hutchison, M., 335, 337(7) Hutin, P., 602 Huynh, H., 591 Hyde, J. S., 580 Hyland, K., 530 Hyslop, P. A., 77 Hyuga, T., 547
I Ibrahim, C., 555 Ibrahimi, I., 166, 167(14) Ida, Y., 275 Ide, H., 34 Igarashi, O., 304 lijima, F., 630 Iizuka, K., 321, 356 Ikeno, H., 322
657
Ikenoya, S., 274 Ilan, Y., 426, 427(70) Imagawa, M., 170 Imai, K., 484 Imbra, R. J., 170 Imlay, J. A., 53 Inada, T., 321 Ingold, K. U., 269, 280, 286(1), 303-304, 310, 310(2), 312,316, 322,354-355,362, 362(8), 372, 374, 381(3), 403, 406, 422, 506, 533, 543-544, 581,601,633 Inoue, M., 338, 341-343 Inselmann, G., 462 Ionnone, A., 580 Ippendorf, H., 387(24), 388 Irvin, B., 269, 304 Ishi, K., 505,506(6) Ishibashi, E., 415,416(31) Ishii, T., 137, 139(15), 148 Ishikawa, H., 338 Ishizu, K., 366 Isler, O., 295, 388, 390(1), 392(1) Israel, A., 161 Itaka, Y., 312 Ito, E., 506, 565 Ito, Y., 415,416(26) Itoh, S., 351 Ivanov, S. A., 360 Ivanov, St. A., 320 Ivanov, V. I., 69 Ivey, K. J., 256 Iwata, T., 335 Iwatsuli, K., 630 Iyengar, V., 546 Izzotti, A., 489, 490(27)
J Jackson, I., 590 Jackson, J. H., 77 Jackson, R. L., 505, 505(7, 8), 506, 506(4), 507, 509(8), 512(7, 8) Jacob, R. A., 24 Jacobs, A., 438 Jacobs, L. W., 491 Jacobsen, E. J., 550, 552 Jacobson, F. S., 175(3), 176, 181(3) Jacolot, F., 592 Jaffe, I. J., 542
658
AUTHOR INDEX
Jaggy, H., 463 Jain, A., 492(5-7, 9), 493,494(5-7, 9) Janero, D. R., 626 Janeway, C. A., Jr., 141 Janssen, Y.M.W., 191 Janzen, E. G., 525 Jarman, M., 591 Jayaraman, J., 372 Jeanteur, P., 231 Jedstedt, G., 527 Jefferson, M. M., 571 Jego, P., 443 Jenkinson, S. G., 551 Jensen, C. D., 397 Jensen, G. L., 492-493,493(3) Jensen, N. H., 390 Jergelovfi, M., 578 Jeroudi, M. O., 515, 523, 523(23) Jevcak, J. J., 89, 90(10), 91(10), 94(10), 96(10) Jewet, S. L., 578 Jiang, J. L., 546 Jin, H. L., 394 Jin, R., 51 Jocelyn, P. C., 147 Johanson, K. J., 100 Johansson, M., 487 Johnson, A., 181 Johnson, A. W., 123 Johnson, D., 126 Johnson, F. F., 411 Johnson, G., 475 Johnson, M. C., 322 Johnson, P. F., 164 Johnsrud, L., 34 Jolly, R. A., 551 Jonat, C., 170 Jones, A., 478 Jones, A. L., 590 Jones, C. L., 603 Jones, D. A., 253,254(11), 255(11) Jones, T. J., 488 Jongkind, J. F., 627 Jordan, V. C., 590, 600 Joseph, J., 522 Joshi, V. C., 372 Jovanovi~, S. V., 573,574(5), 575(5), 576(5) Joyce, A., 316, 355 Julius, M. H., 146 Jurfinek, I., 576, 578-579, 579(20) Jfirgens, G., 513
K Kaakkola, S., 527 Kadiiska, M. B., 70 Kadle, R., 236, 241(14) Kadomatsu, K., 236 Kadonaga, J. T., 165, 172 Kagan, V., 321-322, 357-358, 358(22), 359(22), 363, 366(33), 382(25), 383,638 Kagan, V. E., 311, 316-318, 343-345, 346(5), 351, 353(10), 354, 361, 363, 363(26), 366(26), 371-373, 373(9), 381, 381(6), 382(9), 401,404,456, 460,467, 527-528, 533,534(23), 537(4), 577, 631, 638 Kagawa, T. F., 68, 70(23) K~gedahl, B., 484, 487 Kageyama, H., 181, 191(16) KAhlberg, M., 484 Kahmann, R., 220 Kahn, A. U., 619 Kaiser, S., 384, 385(12), 387(12), 575 Kakata, A., 220 Kakemi, M., 491 Kfillay, Z., 574 Kaitchev, M. G., 635 Kalyanaraman, B., 522, 632, 633(10), 638(10) Kamb, A., 244 Kamiya, Y., 317,356, 372,374, 382(10), 402, 404(12), 506, 533 Kan, M., 280(4), 281 Kaneda, H., 402, 480, 481(29) Kanematsu, S., 459 Kanner, J., 568 Kanofsky, J., 555 Kanofsky, J. R., 256, 619 Kanter, P. M., 89, 90(11), 91(11), 94(11), 96(11), 100(11) Kaplan, L. A., 389 Kappock, T.J.I., 237 Karimi-Booshehri, F., 87 Karin, M., 170, 224 Karlstr6m, O., 95 Kartha, V.N.R., 401 Kasai, H., 17, 24-25, 59, 61(1), 63(1), 64(1), 65(1), 80 Kasai, K., 80 Kasai, N., 547 Kashiwazaki, S., 282 Kass, G.E.N., 76
AUTHOR INDEX Kasurinen, J., 606 Katan, M. B., 454 Katayama, K., 274 Katcher, H. L., 40 Kato, A., 320, 321(4), 356 Kato, K., 311,313,313(6), 314, 314(6), 406 Kato-Jippo, T., 393 Kauf, H., 253,254(11), 255(11) Kaur, H., 254 Kawai, C., 505,506(6) Kawakami, A., 356, 372, 382(10), 402, 404(12) Kawamura, I., 592 Kawanishi, S., 70 Kaye, J., 141 Kaysen, K. L., 311-312, 312(8), 313(8, 14), 314(8), 315(11) Keana, J. F., 580 Kedes, L., 250, 251(7) Keenan, B. C., 82 Kehrer, J. P., 320(15), 328 Keilin, D., 469 Keller, H. E., 296 Kelley, D. G., 426 Kellogg, E. W., 625 Kennedy, T. A., 312, 315(11) Kennett, F. F., 620, 621(9) Keown, W. A., 162 Kern, D. H., 638 Kerppola, T. K., 164 Keshavarzian, A., 555 Keskinova, E., 3 Kessner, A., 620, 621(9) Keyse, S. M., 200, 201(33), 224-225 Kezdy, F., 554 Kezdy, F. J., 283, 345 Khachik, F., 396, 398 Khan, H., 66, 78(5), 79(5) Khan, S., 318,456 Khoo, J. C., 513 Khorana, H. G., 52 Khrapova, N. G., 345, 351(12), 356 Khwaja, S., 343,461 Kiesow, L. A., 53 Kikuchi, G., 225 Kikuchi, K., 181, 191(16) Kikuchi, S., 315,351 Kim, E. H., 175(4), 176 Kim, E. K., 422 Kim, M., 344, 348(7) Kim, P.M.H., 38, 112(15), 113
659
Kim, Y. K., 175 Kimmel, A. R., 197 Kimura, M., 422 Kindahl, H., 449 King, M. M., 294 Kingsbury, R., 219 Kingston, R. E., 221 Kinscherf, R., 139 Kirkkola, A.-L., 281,282(5) Kirkland, J. J., 417 Kishi, T., 275 Kishino, B., 514 Kissinger, C. M., 202 Kita, T., 505, 506(6) Kitagawa, S., 425 Kitanova, S. A., 344, 346(5), 528 Kittler, L., 79 Kitzler, J. W., 17, 24(9), 27(9), 32(9), 80 Kiyosawa, H., 24 Klapper, M. H., 573 Klebanoff, S. J., 556 Klein, P. D., 58 Kling, O. R., 516 Klotz, U., 557 Kluin, K. J., 545,547(18) Kogoma, T., 181,217, 217(12) Kohn, K. W., 89, 90(8), 94(8), 128, 129(7) Kohno, M., 422, 425(40) Kohno, N., 601 Kohsaka, M., 592 Kolachana, P., 17, 24(9), 27(9), 32(9), 80 Kolonel, L. N., 235 Komarov, P. G., 582 Komiya, T., 446 Komiyama, K., 320-321,322(12), 356, 360 Komura, S., 591 Komuro, E., 322, 348, 356, 383, 402, 506, 565 Kondo, N., 592 Konishi, Y., 24 Kono, Y., 378 Konovalova, N. P., 582 Koop, D. R., 411 Koppenol, W. H., 51,422 Korbut, R., 411 Korkolainen, T., 527 Kormann, A. W., 302-303,306, 310(4) Korn, T. S., 17, 24(9), 27(9), 32(9), 80 Kornitzer, D., 219 Korstanje, L. J., 553 Kosai, K., 4
660
AUTHOR INDEX
Kosower, E. M., 367, 497 Kosower, N. S., 367, 497 Koster, A. S., 367, 368(10) Koster, J. F., 561,566(30) Kostyuk, V. A., 421 Kourai, H., 453 Kourilsky, P., 161 Kow, Y. W., 34 Kowaluk, E., 491 Koyama, K., 338, 343 Koyama, Y., 390, 391(16), 392(16), 393, 393(16) Koynova, G. M., 344, 346(5), 528 Kozlov, Yu.P., 404 Kraemer, K. H., 35, 67, 73(13), 75(13), 121 Kramer, J. H., 620, 621(8, 10), 626(10), 629(7) Krasnovskii, A. A., Jr., 386, 387(22) Krebs, H. A., 367, 370(7) Krezowski, A. M., 327 Krinsky, N. I., 235, 372, 381,384, 393, 398 Krishna, M. C., 580-582, 583(12), 587(17), 588, 589(17) Krishnamurthy, S., 401 Kr/anov~i, L., 576 Krolikowska, M., 421,424(7) Kromhout, D., 454 Krstulovic, A. M., 397 Krueger, V.P.M., 8, 13(39) Ku, G., 507 Kubo, K., 34 Kucherlapati, R. S., 162 Kuchino, Y., 24 Kuchtina, Y. E., 356 Kuhn, D. C., 327 Ktihnau, J., 411,420 Kuhnlein, U., 53 Kuksis, A., 516, 523(31) Kulesz, M. M., 24 Kulesz-Martin, M. F., 36, 38(19) Kumar, N. B., 590 Kumazawa, T., 324, 326(24) Kume, N., 505, 506(6) Kung, W., 592 Kuninaka, A., 52 Kunitomo, R., 342 Kunitz, M., 52 Kunze, D., 256 Kuo, W. H., 515 Kurihara, K., 324, 326(22-24)
Kuroki, T., 181, 191, 191(16) Kurth, R., 137,492 Kurt-Jones, E. A., 141 Kusterer, K., 528 Kutnink, M. A., 270, 271(13) Kutty, R. K., 225 Kuypers, F. A., 345,373,382 Kvaltinov~l, Z., 579 Kyogoku, K., 411
L Labadie, R. P., 423 Labataille, P., 82 Labb6, R. F., 270, 335, 337(7) Labeque, R., 605 Lachman, L. B., 141 Ladenstein, R., 478 Laemmli, U. K., 66, 78(3), 108, 180, 242 Lafleur, M.V.M., 95 Lagarde, M., 591 Lahti, R. A., 552-553 Lai, C. S., 580, 588 Lai, E. K., 515, 523,523(23) Lakshman, M. R., 388 Lalonde, M., 303, 307(3) Lambert, C., 477 Lambert, H., 66, 79(6) Lamola, A. A., 384 Lamon, S., 511 LaMont, J. T., 256 Land, E. J., 68, 69(27) Landi, L., 344 LandoR, P. A., 257 Lands, W.E.M., 481 Landschulz, W. H., 164 Landum, R. W., 523,524(1), 525(1), 526(1) Lang, C. A., 368 Lang, J. K., 269, 274, 304 Langford, S. D., 253,256(9) Langholz, E., 557, 569(22), 570(22) Langlois, R., 81, 83(23), 635 Lanks, K., 147 Lanzer, M., 166, 167(14) Lardy, H., 126 Larson, R. A., 446 Larsson, K., 487,488(12) Larsson, S., 491 Lashley, D. W., 331
AUTHOR INDEX Laskawy, G., 424 Last, J. A., 254, 257-258, 264(12) Lau, J. M., 389 Lau, L. F., 224 Laughton, C. A., 591 Laughton, M. J., 411,424, 446, 453(11, 12), 591,592(32), 595(32) Laugier, C., 591 Laura, R., 411 Lauterberg, B., 488 Lautier, D., 225 Laval, J., 3, 10, 16, 16(14), 80, 82, 117, 123 Lawrence, R. A., 316, 551 Lazarow, P. B., 603-604 Leavitt, R., 252 Lebovitz, R. M., 159 Le Cam, A., 438 Lederer, F., 123 Le Doan, T., 79 Le Doucen, C., 423 Lee, C. R., 397 Lee, C. Y., 424 Lee, C.S.L., 601 Lee, D. M., 513,515 Lee, K., 33, 102-103, 104(1, 3), 105(1), llO(1, 3) Lee, M. S., 250,251(8) Lee, S.-H., 384 Lee, W., 172, 270, 335, 337(7) Lees, M., 599 Lefebvre, S. P., 591 Lefer, A. M., 475 Lehmann, J., 327 Leibowitz, M. E., 322 Lennard, M. S., 592, 594(39), 595(39), 596(39), 597(39), 601(39) Lenz, A. G., 254 Lenz, J., 33, 40(3), 104 Leo, A., 538 Leonard, W. J., 152 LeRosen, A. L., 390 Lescoat, G., 437-438, 441,443 Lesellier, E., 397 Lesser, R., 477,481(6) Levade, T., 606 Levanon, A., 250, 251(6) Leventhal, H., 590 Levin, J. D., 123,224 Levine, A., 189 Levine, M., 270, 337
661
Levine, R. L., 254 Levine, Y. K., 426 Levinthal, C., 52 Levy, E., 497, 498(29), 499(29) Levy, E. J., 492, 499-500, 501(1), 502, 502(1), 503, 503(1, 3), 504(3, 5) Lewis, C. D., 66, 78(3) Lewis, D. F., 514 Lewis, M., 331 Lewis, P. A., 484 Leyck, S., 481 Li, C., 258 Li, P., 415,416(26) Li, Y., 547 Liaaen-Jensen, S., 390 Liang, P., 207 Lichter, P., 241 Liebler, D. C., 311-312, 312(8), 313, 313(8, 14), 314(8), 315(11, 12) Liehn, H. D., 600 Lien, E. A., 591-592 Lightner, D. A., 273 Lim, B. P., 403 Lim, J.-S., 137, 139(17), 141(17), 142(17), 143(17) Limacher, P., 602 Lin, Y., 494 Lindahl, T., 34, 40, 95, 123 Lindan, C. P., 34 Lind6n, I.-B., 527 Lindhal, R., 615 Lindsay, D. A., 303,310(2) Linn, S., 51, 53, 123 Linseman, K. L., 551-552 Lion, Y., 462 Lippmann, M., 252 Lissi, E. A., 423 List, T., 555 Liu, F., 77 Liu, G. J., 515,524 Liu, G. T., 421 Liu, T. K., 590 Liuzzi, M., 54 Livrea, M. A., 401 Lloyd, R. S., 127 Lobos, E., 557 Locke, J. T., 533 Locke, S., 269, 280, 286(1) Locke, S. J., 269,423,506, 543 Lodola, E., 487
662
AUTHOR INDEX
Loeb, L. A., 17 Loewe, H., 422, 423(34), 425(34) Loewenstein, W. R., 236 Loft, S., 24, 31(28) L6hr, J. P., 481,482(34) Loman, H., 95 Lonchampt, M., 423 Longo, A., 487 Lonning, P. E., 591 Lopes, F., 591 Lopes, M.C.F., 591 L6pez-Torres, M., 332, 333(4), 334(4), 337(4) Lopresti, R. J., 309 LoSardo, J. E., 104 Loser, R., 600 Lou, Z. C., 416 Louie, S., 254, 475 Love, R. R., 590 Lowry, C. V., 175-176 Lowry, O. H., 516, 594, 598(50) Loyer, P., 438 Lualdi, P., 487 Lubon, H., 172 Luchette, C. A., 494 Luczynski, A., 320 Luethy, J. D., 200, 205(34) Luk, D., 164, 165(1, 2), 166(2), 167, 167(2), 168(2, 16) Lukovi~, L., 576, 579, 579(20) Lundberg, W. O., 617 Luo, Y., 51 Luongo, D. A., 5, 13(22) Lusby, W. R., 396, 398 Luscher, P., 225 Luts, A., 253,256(15) Lutzke, B. S., 551 Lyall, V., 458 Lyman, G. H., 590 M Macdermott, R. P., 557 MacDonald, H. R., 135 Machlin, L., 327 Machlin, L. J., 270, 310, 355 Ma~i~kov~t, 578, 579(31) Macintyre, J., 590 Mackenzie, C. G., 145, 147(26)
Mackenzie, J. B., 145, 147(26), 327 Maclennan, K., 591 MacNee, W., 488 MacNeil, J. M., 423,506, 533 Maddix, S. P., 601 Maddock, J., 484 Madere, R., 270, 271(12) Madzak, C., 116, 118(3), 120(3) Maeda, H., 515 Maes, D., 422 Maguire, J. J., 317, 351,354, 363, 381 Mahadevan, V., 617 Mahoney, E. M., 514 Maidt, L., 80 Maier, K., 557 Maines, M. D., 225 Maiorino, M., 317, 476, 478, 479(20), 480(4, 2O) Mak, I. T., 620-621,621(8, 10), 624(2), 625, 625(3), 626(10), 628(6), 629(6, 7) Makheja, A. N., 514 Makino, K., 220 Malek, T. R., 141 Malin, M. J., 431 Malva, J. O., 591 Manabe, M., 371 Manera, E., 415, 416(29) Manez, S., 421 Maniatis, T., 48, 50(4), 117, 121(8), 170, 171(21), 193, 197(25), 202(25), 203(25), 207(25), 213(25), 214(25), 226, 227(15), 228(15), 229(15), 230(15), 232(15) Manitto, P., 384 Miinnel, D., 152, 156(5), 157(5), 158(5) Mannisto, P., 527 Manohar, M., 191 Manoharan, M. A., 113 Manso, C. F., 552 Manwaring, J. D., 439 MaD, S.J.T., 505, 505(7, 8), 506, 509(8), 512(7, 8) Marai, L., 516, 523(31) Marchaj, A., 426 Marcinkiewicz, S., 355 Marcocci, L., 462, 526 Margison, G. P., 82 Margolis, S. A., 5, 9(27) Markland, S. S., 558,566(26) Marko, V., 574 Markwell, M.A.K., 536
AUTHOR INDEX Manner, W. N., 604 Marnett, L. J., 406, 605 Marsden, M., 488 Marsh, A. C., 396 Marsh, J. P., 191 Martelli, E. A., 415,416(32) Martensson, J., 492, 492(5-9), 493, 493(4), 494(4-9) Martin, A. M., 102 Martin, H.-D., 387(24), 388 Martin, J. S., 435 Martin, M. M., 435 Martino, V. S., 437 Marry, L., 231 Martz, B. L., 505 Marx, J. J., 588 Marzo, A., 415,416(32) Masarykovfi, M., 577 Masher, H., 484 Mashiko, S., 425 Maskos, Z., 422 Mason, J., 546, 547(20) Mason, K. E., 327 Mason, R. P., 66, 69-70, 70(30), 71, 71(32), 72(38), 424, 638 Masotti, L., 601 Massaro, E. J., 316 Matalon, S., 256 Mathews-Roth, M. M., 384 Matsubara, S., 236 Matsui, T., 311,313, 313(6), 314, 314(6) Matsumoto, S., 312-313 Matsuo, M., 312-313 Matsuqawa, Y., 514 Matsuzaki, Y., 415, 416(31) Matthews, J. R., 153 Mauer, S. I., 327 Maurer, R., 303,393 Manri, P., 415, 416(19, 22), 418(19), 41909) Mauri, P. L., 415, 416(21) Mautone, G., 487 Maxam, A. M., 35, 38(18), 103 Maxwell, S.R.J., 284 Mazess, R. B., 590 Mazumder, A., 77 Mazumder, S. C., 113 Mazur, P., 394 McCague, R., 591 McCall, J. M., 548, 550-551 McCann, J., 59
663
McCay, P. B., 294, 316, 362, 515, 523, 523(23), 525 McCloskey, J. A., 8-9, 13(39) McConathy, W. J., 516 McCord, J., 474, 541 McCord, J. M., 338, 584, 619 McDonagh, A. F., 273,406 McDonald, C. C., 590 McGowen, E. L., 331 McGrath, R. A., 89, 94(7) McHale, J. B., 355 Mclntosh, D. D., 505 McKenna, R., 283,345 McKenna, R. L., 554 McKinna, A., 590-591 McKinney, P. E., 487 McKnight, S. L., 164, 219 McLafferty, F. W., 9 McLean, L. R., 505 McManus, T. T., 252 McMillan, R. M., 454 McMiUin, D. R., 77 McMurray, C. H., 299 McNally, A. K., 513-514 McQuaid, A., 546-547, 547(20) Meadows, K. A., 77 Means, E. D., 552, 619 Medler, E. M., 491 Meduna, V., 393 Mehlhorn, R., 581 Mehlhorn, R. J., 322, 363,580, 588 Mehlman, M. A., 252 Meier, B., 152, 156(5), 157(5), 158(5) Meinkoth, J. L., 197 Meister, A., 367, 492, 492(5-9), 493,493(14), 494, 494(1, 4-9, 16), 496(2, 21), 497, 497(1, 2, 21), 498(21, 29), 499, 499(29), 500, 501(1), 502, 502(1), 503,503(1, 3), 504, 5O4(3, 5) Melander, B., 491 Melhorn, R. J., 317, 318(6), 351 Mello-Fihlo, A. C., 77 Mellors, A., 344, 362 Menck, C.F.M., 3, 45, 80, 115-116, 118(2, 3), 119, 120(3), 121 M6ndez, J., 446 Meneghini, R., 77 Meng, T., 416 Menzel, D. B., 252 Menzen, H., 456
664
AUTHOR INDEX
Merril, C. R., 108 Mertens, T., 135, 139(6) Messing, J., 40, 42(26) Metsii-Ketelfi, T., 281,282(5), 527 Meunier, M. T., 421 Meyer, K., 390, 394(14) Meyer, M., 154, 155(10), 157(10), 162(10), 163(10) Meyskens, F. L., 235 Miao, G., 164, 165(9), 166(9), 174(9) Michel, C., 384, 420, 422, 424(24), 425, 425(24), 426(65), 427(65), 428, 428(24), 429(65), 437 Michel, F., 423 Michielis, C., 373 Middleton, E., 446 Mihm, S., 135, 136(9), 137, 139, 139(9), 142, 142(8), 144(8, 23), 146(8), 148(9), 149, 492 Miki, M., 542-545, 547(17) Miles, A. M., 555, 558 Milhorak, A. T., 327 Mill, T., 311,313(7) Millart, H., 511 Miller, H., 47 Miller, K. W., 278 Miller, N. J., 279, 284 Miller-Eberhand, V., 411 Millest, A. J., 454 Milne, D. B., 278 Milne, L., 67, 70, 70(16), 71(16), 73(16), 74(16), 75(16), 76(16), 77(35) Milner, A. D., 284 Milosavljevic, E. B., 424 Min, D. B., 384 Minakami, S., 338 Minchenkova, L. E., 69 Mino, M., 321,544-545, 547(17) Miquel, J. F., 591 Mirabelli, F., 73 Mi~k, V., 574-575,577(18) Misra, H. P., 621 Mitchell, J. B., 581-582, 583(12), 586, 587(17), 588, 589(17, 22, 27) Mitchell, P., 172, 344 Miura, Y., 582 Miyake, N., 406 Miyamoto, T., 24 Miyata, A., 393
Miyauchi, Y., 342 Mizel, S. B., 141 Mizota, T., 592 Mizumoto, Y., 24 Mizuno, G. R., 300 Mizuta, E., 446 M6ckel, H., 426 Mokady, S., 400 Mold6us, P., 482, 484, 485(5), 486, 486(5), 487, 487(7), 488, 489(8), 490 Mold6us, P. M., 479 Mold6us, P. W., 489 Molnar, I., 416, 417(40) Monboisse, J. C., 421 Moncada, S., 475 Montesano, R., 26 Monti, D., 384 Monyer, H., 552 Moon, R. C., 235 Moore, D. D., 221 Moore, P. D., 118 Moore, P. K., 557 Moorhouse, C. P., 594 Mora, A., 421 Moran, E., 416, 446 Morand, O., 606 Morand, O. H., 603-604, 605(27), 606, 606(21, 27), 608(27), 609(27), 610(27), 611(27), 616(27), 618(21, 27), 619 Morehouse, L. A., 66, 67(2) Morel, D. W., 514 Morel, I., 437,441,443 Morgan, D. L., 257 Morgan, R. W., 175(3), 176, 181(3) Morgenstern, R., 477-478,478(13) Morgunov, A. A., 582 Morimoto, H., 351 Morino, Y., 341 Moroi, M., 491 Moroney, M. A., 411,424,443,446,449(15), 450, 450(15), 452(17), 453,453(12, 15) Morris, C., 569 Morris, S., 581 Morse, P. D. II, 580 Morton, M. R., 217 Moser, A., 604 Moser, A. B., 604 Moser, H. W., 603-604 Mosley, S. T., 606
AUTHOR INDEX Mossman, B. T., 191 Motchnik, P., 475 Motchnik, P. A., 24, 253, 254(11), 255(11), 269 Motoyama, T., 545,547(17) Mouret, J.-F., 17, 80, 82, 83(29), 87 Moustacchi, E., 79 Mower, R., 327 Muckel, C., 370 Mudd, J. B., 252 Mueller, M., 166, 167(14) Mueller, P. K., 262 Mukai, K., 315,351,366 Mukohato, H., 410 Miiller, A., 476, 479,479(1), 480(1, 22), 481, 482(33) Miiller, E., 123, 125 Muller, J. M., 553 Miiller, L., 456 Mfiller-Hiicker, J., 603 Mulloy, D., 475 Murahashi, S., 4 Muramatsu, T., 236 Murasecco-Suardi, P., 384 Murata, K., 24 Murphy, M. E., 328, 329(15), 384, 455 Murphy, R. F., 601 Murray, R.D.H., 446 Murray, T. K., 396 Murthy, C. P., 573 Musgrove, E. A., 601 Mustafa, M. G., 252 Mutoh, H., 256 M0tzel, P., 129 Myher, J. J., 516, 523(31)
N Nabel, G., 153, 162(9) Nackerdien, Z., 5, 6(25), 11(25) Nagano, Y., 505, 506(6) Naganuma, A., 493 Nagao, A., 403 Nagayama, K., 393 N/~her, H., 135, 139(6) Nakabayaski, S., 410 Nakabeppu, Y., 123 Nakae, D., 24
665
Nakagawa, S., 321 Nakahara, T., 321,322(12) Nakamura, K., 282 Nakamura, M., 335 Nakamura, T., 321,357 Nakane, S., 411 Nakanishi, K., 394 Nakano, M., 321,357 Narayanaswami, V., 479 Nartey, N. O., 547 Nash, G. S., 557 Nathan, C. F., 147 National Regional Council, 269 Naumov, V. V., 345, 351(12) Nauta, H., 426 Nebert, D. W., 200, 205(34) Neff, G. L., 553 Negre-Salvayre, A., 429 Neidle, S., 591 Neijt, J. P., 588 Nelis, H.J.C.F., 393 Nelson, D. P., 53 Nelson, J. H., 424 Nemec, J., 631-632,633(10), 638(6, 10) Netscher, T., 303,307(3) Neukom, C., 309 Newcombe, P. A., 590 Newcombe, R. A., 590 Newman, C. M., 491 Newton, G. L., 367,497 Ng, S. Y., 250, 251(7) Nguyen, T. D., 424 Nicholls, P., 469 Nicholson, B. J., 236, 241(14) Nicholson, R. I., 590 Nick, S., 135, 144(4) Nicotera, P., 67, 70(16), 71(16), 73(16), 74(16), 75(16), 76, 76(16) Nicotra, C., 401 Nielson, A. B., 390 Nielson, O. H., 557,569(22), 570(22) Nierenberg, D. W., 393 Nigro, R. G., 116, 118(2), 121 Niki, E., 311-312, 317, 322, 348, 356, 372, 374,382(10), 383,402,404(12), 423,460, 467, 480, 481(29), 506, 532-533, 534(19), 565,633 Nikula, K. J., 257-258, 258(3), 264(12) Nilsson, E., 488
666
AUTHORINDEX
Nilsson, U. A., 582 Nilsson, U. L., 586 Nishimura, M., 315 Nishimura, S., 17, 24-25, 59, 61(1), 63(1), 64(1), 65(I), 80 Nishiyama, J., 300 Nisonoff, A., 135 Nissinen, E., 527 Nitschmann, W. H., 580 Noack, E. A., 472 Nogala-Kalucka, M., 320 Noguchi, N., 402, 480, 481(29) Nomoto, T., 425 Nomura, H., 482 Nomura, T., 324, 326(22-24) Nordenbrand, K., 344 Norling, B., 344 Nose, K., 181, 191, 191(16) Nottenburg, C., 206 Novel, N., 462 Novikov, K. N., 404 Nungesser, E., 604 Nyberg, B., 95, 123
O Obendorf, M.S.W., 45, 59, 80 Oberley, L. W., 244 Oberlin, B., 296 Oberreither, S., 513 Oberritter, H., 337 Ocaktan, A., 443 Ocaktan, A. Z., 441 Ochial, M., 491 O'Connor, T. R., 123 Odin, F., 17, 80, 87 O'Donnell-Tormey, J., 147 O'Farrell, P. H., 181 Offermanns, H., 542 "Official Methods of Analysis" (Association of Official Analytical Chemists), 295 Ogasawara, T., 280(4), 281 Ogden, R. C., 49 Ogino, T., 341 Oguni, I., 425 Ohara, J., 141 Ohata, K., 453 Ohnishi, S., 416 Ohno, M., 491
Ojasoo, T., 591 Okamoto, T., 275 Okamura, M., 337 Okayasu, T., 547 O'Kennedy, R., 416, 446 Okenquist, S. A., 33, 40(3), 104 Oki, T., 321,357 Okoli6~iny, J., 578 Okuda, H., 446 Okudaira, H., 24 Olcot, H. S., 581 Oleinick, N. L., 3, 78 Olinski, R., 5, 6(25), 11(25) Olive, P. L., 100 Oliver, C. N., 254, 515, 523-524, 524(I), 525(1), 526(1) Oliveros, E., 384 Ol'khovskaya, I. P., 356 Ollis, W. D., 414 Olson, J. A., 235, 388 Olsson, B., 487 Olsson, L. I., 582 Olszewski, J., 514 Olton, D. S., 524 Omaye, S. T., 270, 271(13) Ondreji6v~i, O., 576, 578-579, 579(20, 31) OndriaL K., 574-575, 577(18) O'Neil, C. A., 394 O'Neill, C. A., 254 Ongun, A., 252 Ooshima, A., 505, 506(6) Oosting, R. S., 252 Ootsuyama, A., 24 Op den Kamp, J.A.F., 345, 373, 382 Oppenheim, A. B., 219 Orbell, J. D., 68 Oren, R., 250, 251(6) Orrenius, S., 67, 70(16), 71(16), 73, 73(16), 74(16), 75(16), 76, 76(16), 488, 628 Orunesu, M., 490 Osborne, D. J., 452 Osebold, J. W., 254, 257 Osheroff, N., 631 Oshino, N., 66 Oster, K. A., 619 Oszmianski, J., 424 Ota, S., 256 Otsuka, H., 446 Otter, R., 476, 479(2), 482(2) Ouchi, S., 366
AUTHOR INDEX Ouellet, R., 81, 83(23) Overvad, K., 24, 31(28) Owen, G. M., 331 Owens, K., 603,609(5), 611(5), 620, 621(9) Owyang, C., 546 Oyama, T., 415,416(31)
P Pace, D. M., 257 Pacht, E. R., 256 Pacifici, R. E., 175(5-7), 176 Packer, L., 156, 254, 255(26), 274, 310-311, 316-318, 318(6), 321-322, 343-344, 346(5), 351, 353(10), 354, 357-358, 358(22), 359(22), 361,363, 363(26, 34), 366, 366(26, 33), 368, 371,371(13), 372373, 373(9), 379(2), 381, 381(6), 382(9, 25), 383, 401, 409(9, 11), 454-456, 458(5), 459(8), 460-462,467, 526-528, 533,534(23), 537(4), 577, 580, 638 Padrini, R., 511 Padulo, G. A., 422 Paganga, G., 592, 601(40) Pagani, S., 457 Pageaux, J. F., 591 Pagonis, C., 423 Pallardo, F. V., 367 Pallard6, F. V., 371 Palma, L. A., 273 Palmer, R.M.J., 475 Palombini, G., 601 Palozza, P., 372 Paltauf, F., 612,614(43) Pan, Y.-C.E., 164, 165(9), 166(9), 174(9) Panalaks, T., 396 Pang, H., 9 Panter, S. S., 568, 569(45) Papathanasiou, M., 200, 205(34) Paramova, L. I., 386, 387(22) Parathasarathy, S., 513 Pardee, A. B., 207 Pardini, R. S., 424 Park, C. H., 438 Park, D., 403 Park, E. M., 17, 24, 24(9), 27(9), 32(9), 80 Park, J. W., 24, 67, 117 Parker, R. A., 505(8), 506, 509(8), 512(8)
667
Parker, R. S., 389 Parnham, M. J., 477, 481 Parr, I. B., 591 Parthasarathy, S., 513-515, 522 Paschke, E., 604 Pascoe, G. A., 328 Pascual, C., 425 Pasdeloup, N., 437,441,443 Pasquali, P., 344 Patel, B. S., 515,523,523(23) Patel, L., 164, 166(7), 174(7), 224 Paterson, M. C., 54 Patterson, B., 269 Pattison, T. S., 397 Paul, D. L., 236 Paul, W. E., 141 Pauling, L., 390 Paulsen, O., 487 Pavlotsky, N., 423 Payli, M., 421,443,446, 453(13) Pechard, M. R., 397 Pelle, E., 422 Penhoet, E. E., 53 Perdrix, L., 423 P6rez-Campo, R., 332, 333(4), 334(4), 337(4) Periasamy, M., 191 Perin, L., 602 Perruchot, T., 423 Perry-O'Keefe, H., 202 Persson, C.G.A., 253,256(15) Pesek, C. A., 392 Pessara, U., 137,492 Peters, M., 557 Peters, T., Jr., 53,482 Petry, T. W., 551 Petzoldt, D., 135-136, 139(6), 140(11) Pfander, H., 388 Pfanstiei, J., 557 Pflaum, M., 129-130 Phillips, F., 617 Phillips, M. W., 615 Phillips, T. M., 620, 621(10), 626(10) Photo, P., 527 Pickett, C. B., 217 Pidoux, M., 226 Piechaczyk, M., 231 Pierson, H. F., 397 Pierson, T. S., 554 Piestri, S., 475 Pieters, L.A.C., 416
668
AUTHORINDEX
Pietta, P., 415, 416(19, 21, 22, 29), 418(19), 419(19) Piette, J., 79, 121 Pihan, G., 528 Pincemail, J., 462 Pinedo, H. M., 631 Pinson, A., 582, 589(22) Pipkorn, U., 253,256(15) Pippurl, A., 527 Piretti, M. V., 416 Piris, J., 557 Plenevaux, A., 477 Plopper, C. G., 257-258, 258(3) Podhajer, O. L., 602 Pohl, C., 135, 139(6) Polgar, A., 390, 393 Poli, G., 616 Poll, T., 601 Pollack, S. E., 52 Pollak, M. N., 591 Pollow, K., 592 Polverelli, M., 6, 17, 80, 87 Pomerantz, S. H., 632 Pomeroy, J. J., 438 Pons, M., 591 Ponte, P., 250, 251(7) Poor, C. L., 389, 397 Popov, A., 320 Porter, L. J., 430, 433 Postlethwait, E. M., 253,256(9) Potapovitch, A. I., 421 Pottmeyer-Gerber, C., 135, 144(4) Pou, S., 77, 561,566(31) Poulsen, H. E., 24, 31(28) Poulsen, L. L., 482 Povirk, L. F., 129 Powell, R., 318, 456 Powles, T. J., 590 Powrie, F., 504 Powrie, R., 492, 493(2), 496(2), 497(2) Pradhan, D. S., 456 Preece, P. E., 590 Pregenzer, J. F., 376,402, 550-551 Price, M. L., 434-435, 435(15) Price, M. P., 432 Price, S., 80 Pries, C., 616 Prosser, E., 446 Proudfoot, K., 601 Proudrnan, K. E., 454
Priitz, W., 66, 67(7), 68(7), 69(7, 27), 73(7), 74(7) Priitz, W. A., 68, 73 Pryor, W. A., 252-253,506, 619 Przybyszewski, J., 24, 36, 38(19) Ptzoldt, D., 492 Puhl, H., 513 Pung, A., 237 Puppo, A., 421,422(20) Purl, R. N., 492, 493(1-3), 494(1), 496(2), 497(1, 2), 504 Pystynen, J., 527
Q Qimin, L., 416 Quackenbush, F. W., 396 Quayle, J. R., 612 Quigley, G. J., 68 Quilliam, M. A., 7 Quinlan, G. J., 594 Quinn, M. T., 513 Quinn, P. J., 322,344-345,346(5), 528,591, 601(35)
R Rabani, J., 426,427(70) Rabenstein, D. L., 498 Rabinowitz, J. L., 616 Race, M., 373 Radany, E. H., 34 Radford, I. R., 89 Radi, R., 273,475 Radom, J., 606 Raetz, C.R.H., 604, 605(27), 606(21, 27), 608(27), 609, 609(16, 27), 610(27), 611(27), 616(27), 618(21, 27), 619 Rahmsdorf, H. J., 170 Raisys, V. A., 335, 337(7) Raju, P. A., 492 Ramakrlshnan, N., 3 Ramasarma, T., 343 Ramos, C. L., 561,566(31) Rampton, D. S., 555 Ranalder, U. B., 302-303 Ranasarma, T., 372 Randall, R. J., 516, 594, 598(50)
AUTHOR INDEX Randoux, A., 421 Rangaswamy, S., 604 Ranney, H. M., 497 Ransom, J. A., 113 Rao, G.H.R., 327 Rao, N. U., 581 Rao, R.D.N., 638 Rapkin, L., 33, 40(3) Rasmussen, R. E., 257,258(5) Rasokat, H., 135, 139(6) Rattner, A., 438 Ratty, A. K., 421,437 Rauscher, F. J. III, 164, 165(2), 166(2, 7), 167(2), 168(2), 170, 174(7), 224 Rava, A., 415 Ravanat, J.-L., 79, 81-82, 83(23-25), 85(25), 87(25), 88(24) Raynaud, J. P., 591 Razandi, M., 256 Razell, W, E., 52 Reames, S. A., 393 Rechtin, A. E., 505(7), 506, 512(7) Recknagel, R. O., 404, 438 Reddy, J.M.C., 316 Reddy, P., 5, 9(27) Redegeld, F.A.M., 367, 368(10) Reed, C. J., 67, 73(14), 75(14) Reed, D., 328 Reed, D. J., 367, 368(8), 369(8), 371(8) Reif, D. W., 475 Remacle, J., 373 Renson, M., 477 Retel, J., 631 Rettenmaier, R., 294 Rettmer, R. L., 270 Revzin, A., 165, 171(10) Reznick, A. Z., 254, 255(26), 461 Reznikoff, C. A., 237 Riazzudin, S., 123 Ribeiro, D. T., 45, 80, 116, 118(3), 119, 120(3), 121 Rice, D. A., 299 Rice-Evans, C., 279,284, 592, 601(40) Richard, M.-J., 17, 80 Richardson, C. C., 42, 44(30) Riche, C., 592 Richelmi, P., 73 Richie, J. P., 368 Richter, C., 24 Rickard, R. C., 60, 62(6), 80
669
Riddell, R. H., 555 Rieber, P., 152, 153(3), 155(3), 157(3), 162(3), 217, 224 Riesz, P., 582 Rigo, M., 317 Riis, P., 557, 569(22), 570(22) Riley, D., 590 Riley, W. T., 35, 37(17), 40(17), 104 Rimm, E. B., 269 Rink, H., 89, 90(12), 91(12), 94(12), 96(12), 99, 100(12) Rio, M. C., 602 Rios, J. L., 421 Rippstein, A., 306 Riss, G., 302-303,306, 310(4) Rissel, M. Y., 438 Rizzardo, E., 422 Rizzo, W. B., 604, 615 Robak, J., 411,421,422(16), 423(16), 424(7) Robberson, D. L., 127 Robbins, C. T., 435,436(20) Roberfroid, M., 490 Roberts, R., 523 Robertson, J.F.R., 590 Robins, A., 590 Robinson, C., 452, 555 Rochette, L., 462 Rockholm, D. C., 435 Rodgers, M.A.J., 384-385 Roeder, R. G., 159, 167, 168(16) Roederer, M., 492 Roelofsen, B., 345, 373, 382 Rogers, M., 236, 241(14) Rogers, S. G., 123 Rojas, C., 332, 333(4), 334(4), 337(4) Roman, J., 631,638(6) Romanovsky, J. C., 262 Romay, C., 425 R6mer, A., 481,482(33, 34) Roosen, O., 4 Rose, C. S., 327 Rose, J. R., 511 Rosebrough, N. J., 516, 594, 598(50) Rosen, C. A., 166, 167(13) Rosen, G. M., 561,566(31) Rosen, H., 506, 514 Rosenfeld, M. E., 514 Rosenkrantz, H., 327 Rosolowsky, M., 514 Ross, D., 628
670
AUTHORINDEX
Ross, R., 513 Ross, R. K., 590 Ross, S., 189 Roth, E. E., Jr., 328 Roth, S., 135, 140(5), 142, 142(5), 144, 144(5, 23), 145(7, 24), 146(7), 149, 149(7, 24) Rotheneder, M., 513 Rouge6, M., 480 Rouseff, R. L., 416 Roved, A., 476, 478,479(20), 480(4, 20) Rowlands, M. G., 591 Roy, P. K., 411 Rfidiger, H., 130 Rumyantseva, G. V., 66 Rush, J. D., 51,422 Rushin, W. G., 397 Rushmore, T. H., 217 Rusnack, M. G., 331 Russett, M. D., 398 Russo, A., 581-582, 583(12), 586, 587(17), 588, 589(17, 22, 27) Rustow, B., 256 Rusznyak, S., 429 Rydberg, B., 89, 94(15), 96(15), 99(15), 100, 102 Ryle, A. P., 488 Ryter, S. W., 175(5), 176
S
Saad, Z., 590 Sabovljev, S. A., 66, 78(4), 79(4, 6) Sabrouty, S. E., 231 Sacchi, N., 193, 227 Sacks, N.P.M., 591 Sadrzadeh, S.M.H., 568, 569(45) Sfiez, J. C., 236 Safadi, A., 527, 537(4) Safayhi, H., 476, 479(2), 482(2) Saffitz, J. E., 620 Safsten, B., 527 Sage, E., 79 Sagfipanti, J.-L., 35, 67, 73(13), 75(13) Saiki, K., 390, 391(16), 392(16), 393(16) Salnsbury, J.R.C., 590 Saint-Jean, R., 385 St. John, T., 194, 206 Salto, M., 374 Saito, R., 557
Saito, T., 356, 372, 382(10), 402, 404(12) Saitoh, Y., 601 Sakiyama, S., 181, 191(16) Sakurai, H., 425 Salata, K., 557 Salditt, M., 125 Salim-Hanna, M., 423 Salin, M. L., 474 Salmenper~i, L., 296 Salmon, S. L., 175 Salser, W., 218 Saltzman, B. E., 262 Salvati, A. M., 569 Salvayre, R., 423,429, 606 Sambrook, J., 48, 50(4), 117, 121(8), 170, 171(21), 193, 197(25), 202(25), 203(25), 207(25), 213(25), 214(25), 226, 227(15), 228(15), 229(15), 230(15), 232(15) Sambucetti, L. C., 170 Samokyszyn, V. M., 406 Samuni, A., 580-582, 583(12), 586, 587(17), 588, 589(17, 22, 27) Samuni, U., 582, 587(17), 589(17) Sanadi, D. R., 455 Sander, L. C., 397 Sandu, I. S., 565, 566(36), 567(36), 568(36) Sandy, M. S., 479, 489 Santi, L., 96 Santos, M. J., 604 Saotome, H., 411 Saran, M., 384, 420, 422, 424(24), 425, 425(24, 25), 426(25, 65), 427(25, 65), 428, 428(24, 25), 429(65), 437 Sarasin, A., 45, 80, 115-116, 118(2, 3), 119, 120(3) Sarett, H. P., 491 S~irkioja, T., 514 Sarycheva, I. K., 356 Sasajima, M., 411 Sasaki, H., 415,416(31) Sastre, J., 367, 371 Satake, Y., 311,313(6), 314(6) Satoni, D. K., 512 Satonin, D. K., 511 Saucy, G., 309 Saul, R. L., 619 Saunders, R. D., 619 Saziki, R., 411 Scaiano, J. C., 422 Scala, J., 397
AUTHOR INDEX Scalia, M., 421 Scandura, O., 270 Scarabelli, L., 490 Scarpa, M. A., 317 ~asnfir, V., 574, 578 Schaap, A. P., 605 Schacher, A., 167 Schalch, W., 385, 387(14) Schalkwijk, C., 345,373 Schalkwijk, C. G., 604 Schapira, D. V., 590 Schaus, E. E., 270, 271(13) Schemer, E., 601 Scheinberg, I. H., 546 Scherberich, P., 542 Scherch, H. M., 552 Schiff, L. J., 257,258(4) Schillaci, M., 269, 304 Schilsky, M., 546 Schloemann, S. R., 557 Schmid, R., 303,307(3) Schmidt, H., 135, 144(4) Schmidt, K., 337 Schmitz, H. H., 389, 397 Schneider, G., 422 Schneider, J. A., 136 Schneider, J. E., 3, 21, 59, 80 Scholich, H., 455 Scholz, R. W., 316 Sch6neich, C., 479 Schrakamp, G., 603-604 Schram, K. H., 7, 9 Schramel, P., 546 Schrappe, M., 135, 139(6) Schraufstatter, I., 77 Schreck, R., 151-152, 153(3), 154, 154(4), 155(3, 10), 156(4, 5), 157(3, 5, 10), 158(5), 160, 161(15), 162(3, 10), 163(10), 217,224 Schreurs, W.H.P., 335, 337(9) Schrijver, J., 335, 337(9) Schroeder, W. A., 390 Schubert, M. P., 144 Schuchmann, H.-P., 67 Schuchmann, M. N., 3 Schiiep, W., 294, 296 Schuler, R. H., 428 Schulte-Frohlinde, D., 426 Schultz, E., 527 Schultz, W. A., 80
671
Schulz, H., 415,416(28) Schulz, W. A., 45, 59 Schuppe, I., 488 Schutgens, R.B.H., 603-604 Schwartz, H. S., 89, 90(11), 91(ll), 94(11), 96(11), 100(11) Schwartz, S. J., 394, 396-397 Schwarz, W., 388-389, 394, 398(10, 27) Schwenke, D. C., 505, 514 Schwiers, E., 331 Scita, G., 317-318, 358, 363, 372-373, 381(6), 401,460, 533,534(23) Scott, C. G., 309 Scott, T. W., 603 Scurlock, R., 480 Searls, R. L., 455 SeaweU, P. C., 34 Seawell, P. S., 127 Sechi, M., 344 Sedghi, S., 555 Sedlfik, J., 578, 579(31) Sedman, S. A., 108 Seegmiller, J. S., 136 Sefranka, J. A., 505 Seher, A., 320 Seher, V. A., 360 Seibel, K., 600 Seidman, J. G., 221 Seidman, M. M., 121 Seifart, K., 199 Sekaki, A. H., 462 Sekiguchi, M., 123 Sekiya, K., 446 Sellers, R. M., 284, 288(15) Sen, J. N., 312,403 Sen, R., 172 Sentjurc, M., 580 Serbinova, E., 321, 357, 358(22), 359(22), 363,366(33), 382(25), 383,456, 461,577 Serbinova, E. A., 317-318, 343-344, 346(5), 351, 353(10), 354, 361, 363, 363(26), 366(26), 372-373, 373(9), 381, 382(6), 528, 638 Sergent, O., 437, 441 Serra, D., 490 Sestili, P., 77 Setchell, B. P., 603 Sethy, V. H., 553 Sevanian, A., 175(4), 176, 553 Shaltiel, S., 254
672
AUTHOR INDEX
Sharma, M., 603 Sharma, O. P., 411 Sharp, P. A., 172 Sharp, R. J., 280(3), 281 Shedlofsky, S., 438 Sheffner, A. L., 491 Shen, B., 398 Shenk, T., 189 Shevach, E. M., 141 Shibahara, S., 225 Shibanuma, M., 181, 191, 191(16) Shibutani, S., 80 Shieh, J. J., 284, 288(15) Shigenaga, M. K., 16-17, 24, 24(9), 25(25), 26(25), 27(9), 32(9), 67, 80, 117 Shimamura, T., 393 Shimasaki, H., 402, 506 Shimizu, K., 402 Shimomura, K., 592 Shinagawa, H., 220 Shing Ho, P., 68, 70(23) Shinitzky, M., 324, 356 Shoemaker, R. H., 638 Shridi, F., 421,424(7) Shull, S., 191 Shvedova, A., 318,456 Shvedova, A. A., 404 Sichel, G., 421 Sicot, N., 423 Siemsen, F., 553 Sies, H., 3, 45, 59, 79-80, 116, 118(2, 3), 120(3), 121, 129, 338, 367, 368(9), 370, 384-385, 385(12), 386(18), 387(12, 18, 24), 388-389, 394, 398(10, 27), 455456, 468, 476, 479, 479(1), 480, 480(1, 22), 481,481(3), 482, 482(34), 572-574, 574(5), 575,575(5), 576(5), 577,578(13), 581 Sigal, E., 514 Sigman, D. S., 77 Silbert, L. S., 428 Sima, P., 256 Simic, M. G., 24, 31(27), 422, 573, 620 Simmon, V., 59 Simmonds, N. J., 555 Simmons, R. D., 475 Simon, I., 592 Simon, M., 438 Simon, T. C., 514 Simoni, R. D., 375,460
Simons, E. R., 423 Simpson, E., 146 Simpson, K. L., 388 Sinclair, P. R., 438 Singh, H., 172 Singh, S., 515 Singhal, R. K., 493,494(16) Sinha, B., 631,638(6) Sinha, B. K., 631-632, 632(7), 633(10), 638(10) Sinkina, Y. B., 356 Sirtori, C. R., 515 Sive, H. L., 206 Sj6din, K,, 488 Skipper, P. L., 472 Skipsky, V. P., 603 Sklan, D., 401 Sklar, L. A., 375,460 Slater, T. F., 437, 535, 601,616 Slevin, M. L., 631 Slezfik, J., 578 Sloane-Stanley, G. H., 599 Smedegard, G., 557 Smith, B. F., 256 Smith, C., 592, 594(37, 39), 595(37, 39), 596(37, 39), 597(39), 601(37, 39) Smith, D. L., 9 Smith, I., 591 Smith, J. C., 397-398 Smith, M. T., 479, 489 Smith, S. L., 552 Smith, W. E., 328 Smith, W. P., 422 Snodderly, D. M., 398 Snyder, F., 603, 604(2) Snyder, L. R., 415,417 Soares, J. H., 278 Soczewinski, E., 415,417(24) Solar, S., 3 Solheim, E., 592 Solomon, D. H., 422 ~olt6s, L., 574 Somerharju, P., 606 Sorenson, J.R.J., 542 Sosnovsky, G., 581 Sowell, A. L., 397 Sparrow, C. P., 514-515 Spector, A., 477 Speder, A., 490 Speek, A. J., 335, 337(9)
AUTHOR INDEX Speranza, G., 384 Spiegelman, B. M., 170 Spikes, J. D., 607, 633 Spiller, G. A., 397 Spinnewyn, B., 475 Spinnewyn, S., 462 Sporn, M. B., 591 Spray, D. C., 236 Staab, H. J., 600 Staal, F. J., 492 Stadtman, E. R., 254, 515,524 Stafford, H. A., 429 Stahl, W., 388-389, 394, 398(10, 27), 482 Staicup, A. M., 394 Stampfer, M. J., 269 Stanley, W. C., 368, 371(13) Starke-Reed, P. E., 515, 523-524, 524(1), 525(1), 526(1) Stag, A., 574-575, 577(18) Steenken, S., 3, 45, 59, 80, 83, 572-573, 574(5), 575(5), 576(5) ~tefek, M., 573-574, 576-578 Steighner, R., 129 Stein, B., 170 Stein, E. A., 389 Steinberg, D., 505, 513-515 Steiner, M., 327 Steinherz, R., 492(6), 493,494(6) Stenson, W. F., 557 Stern, G., 579 Sternlieb, I., 546 Stettmaier, K., 422, 428 Stevens, P. A., 256 Stevens, T.R.J., 555 Stewart, H. J., 590 Stivala, L., 73 Stocker, R., 254, 269-270, 273,273(10), 344, 354, 372,406 Stockert, R. J., 546 Stocks, J., 280(3), 281 Stoewe, R., 66, 67(7), 68(7), 69(7), 73(7), 74(7) ~tolc, S., 572, 573(1), 578(3), 579 Stole, E., 492(9), 493,494(9) Stoll, I., 602 Storz, G., 151, 156(1), 187, 214, 216(18), 217-218, 220, 224 Sttisser, R., 422 Stoyanovsky, D., 638 Stoyanovsky, D. A., 343,631
673
Stoytchev, T. S., 344, 346(5), 528 Strait, L. A., 504 Strauss, B. S., 118 Streitwieser, D., 59 Stremmenos, C., 601 Strickland, T., 506 Striegl, G., 513 Strong, L., 189 Struhl, K., 37, 40(21), 42(21), 110,221 Strumeyer, D. H., 431 Stueber, D., 166, 167(14) Stump, D. D., 328 Styk, J., 578 Subar, A., 269 Subbiah, M.T.R., 283 Subramanian, M., 456 Sugimoto, T., 256 Sugioka, K., 321,357 Sugita, Y., 137, 139(15) Suko, M., 24 Sumida, S., 317, 318(6), 322 Summer, K. H., 546 Sun, F., 553 Sun, W., 258, 264(12) Sundler, F., 253,256(15) Sundquist, A. R., 384-385,386(18), 387(18), 388-389, 394, 398(10, 27), 573, 574(5), 575,575(5), 576(5) Suprunchuk, T., 384 Surawicz, T. S., 590 Sussman, M. S., 619 Sutherland, M. W., 421 Sutherland, R. L., 601 Sutton, C. L., 77 Suzuki, N., 425 Suzuki, Y., 458 Suzuki, Y. I., 156 Suzuki, Y. J., 254, 255(26), 454-456, 458(5), 459(8), 461,526-528, 537(4) Svensson, C., 253, 256(15) Swaak, A.J.G., 561,566(30) Swain, T., 432 Swallow, A. J., 350 Swanson, C., 456 Swanson, C. E., 588 Swansson, C., 318 Swartz, H. M., 580-581 Sweeney, J. P., 396 Sweet, F., 257 Sweet, W. E., 257
674
AUTHOR INDEX
Swies, J., 411 Syenson, W. F., 557 Szabo, M. E., 475 Szabo, S., 528 Szent-Gy6rgyi, A., 429 Sz6csovfi, H., 574
T Tabor, S., 37, 40(21), 42, 42(21), 43(29), 110 Tachizawa, H., 511 Taller, J. M., 603-604 Tainsky, M., 189 Takada, M., 274 Takahama, U., 420 Takahashi, M., 322, 378, 383,506, 565 Takahashi, T., 547 Takamatsu, K., 366 Takamoto, M., 446 Takatsuki, K., 343 Takeda, S., 415, 416(31) Takegoshi, T., 482 Takei, H., 422 Takeichi, N., 547 Takekoshi, Y., 547 Takenaka, Y., 544 Takeshida, M., 80 Takii, T., 390, 391(16), 392(16), 393(16) Tamaka, A., 356 Tamura, K., 24 Tamura, S., 588 Tanabe, T., 545, 547(17) Tanaka, A., 320, 321(4) Tanaka, I., 411 Taniguchi, N., 545, 547(17) Tanimura, R., 317 Tankanow, R., 545, 547(18) Tarmenbaum, S. R., 472 Tano, K., 24 Tanooka, H., 17, 24 Tao, K., 220 Taoukis, P. S., 392 Tappel, A. L., 344, 355, 362, 477-478, 478(14), 560, 566(27) Tapper, D. P., 42 Tarboletti, G., 602
Tarkington, B. K., 254, 257-258, 264(12), 475 Tartaglia, L. A., 151, 156(1), 187, 216(18), 220, 224, 244, 250, 251(8, 9) Tatsuta, T., 321,356 Taub, I. A., 284, 288(15) Tauber, A. 1., 423 Tavendale, R., 590 Tavill, A. S., 438 Taylor, B. M., 553 Taylor, G. W., 491 Taylor, H. A., 505 Taylor, K. B., 454 Tchapla, A., 397 Tchou, J., 80 Tedeschi, R. E., 505 Teebor, G. W., 58 Teff, D., 219 Tegelaers, W.H.H., 603 Teicher, B. A., 494 Tentori, L., 569 T6oule, R., 6, 33 Terano, A., 256 Terao, J., 372, 403 Terao, K., 506, 565 Terekhova, S. F., 345 Terlinden, R., 481,482(33, 34) Tesoriere, L., 401 Tezuka, T., 422 Thiange, G., 477 Thirion, A., 462 Thomas, C., 66, 67(2) Thomas, C. E., 316, 506-507 Thomas, E. L., 571 Thomas, P. S., 196 Thompson, D. H., 604 Thompson, D.F.T., 401 Thompson, J. A., 577 Thompson, K. E., 544 Thor, H., 488 Thornally, P. K., 502 Thornes, R. D., 416, 446 Thorpe, G.H.G., 284 Tithe', 572, 573(1) Tiegs, G., 476, 479(2), 482(2) Tietze, F., 627 Tillotson, J. A., 331 Ting, S. V., 416 Tingey, D. T., 21
AUTHOR INDEX
Titani, K., 338 Tjian, R., 165 Tjonneland, A., 24, 31(28) Tochimaru, H., 547 Toda, S., 478 Togashi, Y., 547 Tokfirov~t, J., 578, 579(31) Tokumura, A., 322 Tolbert, N. E., 536 Toledano, M. B., 152, 218 ToUes, R. L., 325 Tomasi, A., 580 Tomomura, M., 236 Toney, J. H., 40, 41(28), 42(28) Torel, J., 421,437 Torresi, J., 491 Tosaki, A., 474, 523 Towsend, L. B., 9 Toyo6ka, T., 484 Trakshel, G. M., 225 Treutter, D., 415, 416(27) Tromvoukis, Y., 225 Trozzolo, A. M., 384 Truelove, S. C., 557 Trull, F. R., 273 Trumble, M. J., 316 Truscott, T. G., 385, 387(14) Trush, M., 631 Tsai, C. L., 590 Tsao, P. S., 475 Tsuchiya, J., 317, 356, 402 Tsuchiya, M., 357-358, 358(22), 359(22), 371-372, 381(6), 401, 454-456, 458(5), 459(8), 460-461, 467, 526-528, 533, 534(23), 537(4), 577 Tsuchiya, N., 321,356 Tsuiji, T., 24 Tsukada, T., 513 Tsukamoto, T., 604 Tsukatani, H., 322 Tsukayama, M., 453 Tsukida, K., 390, 391(16), 392(16), 393(16) Tsuneyoshi, H., 491 Tubaro, A., 446 Tuck, L. D., 504 Tunek, A., 488 Tunstallpedoe, H., 590 Tur~iny, P., 579 Turi-Nagy, L., 578
675
Tyler, K., 257, 258(4) Tyrrell, R. M., 224-226 Tyurin, V. A., 344, 346(5), 528
U Uchiyama, T., 150 Udardy, A., 199 Ueda, T., 304 Ueland, P. M., 592 Ueno, I., 422, 425(40) Ueno, Y., 311,313, 313(6), 314, 314(6), 406 Ulick, S., 327 Umezawa, I., 321,356 Underwood, M., 493 Urano, S., 351,506, 565 Uraz, V., 576, 579, 579(20) Ursini, F., 317,425,476,478,479(20), 480(4, 20) U.S. Code of Federal Regulations, 262 Usui, N., 631,632(7) Utsumi, H., 582 Utzinger, G. E., 504
V V~iclavincowi, V., 296 Vahoouny, G. V., 620, 621(9) Vairetti, M., 73 Vale, M.G.P., 591 Vale, P., 591 Valente, A. J., 515 Valenza, M., 401 Valerio, G., 511 van Asbeck, A. B., 588 van Fassen, F. E., 588 Van, N. F., 580 van Bennekom, W. P., 367, 368(10) Vance, J. E., 603 van den Berg, H. W., 601 van den Berg, J.J.M., 345, 373, 382 Van den Bosch, H., 603-604 Vander Jagt, D., 504 Van der Veen, J., 581 van der Vliet, A., 254 van der Zee, J., 69 Van de Vorst, A., 121
676
AUTHOR INDEX
van Dijk, H., 423 Vane, J. R., 411 Van Eijk, H. G., 561,566(30) Van Ginkel, G., 553 Van Golde, L.M.G., 252 Van Greevenbroek, M.M.J., 252 van Hees, P.A.M., 557 Van Kessel, J., 423, 506, 533 van Kuijk, F.J.G.M., 381 Van Langenhove, A., 557 van Lier, J. E., 55, 81-82, 83(23, 29), 631, 633, 635 Van Maanen, J.M.S., 631 Van Scoyoc, S., 434, 435(15) Van Sickle, W. A., 505(7), 506, 512(7) Van Someren, R.N.M., 555 van Steveninck, J., 69 van Tongeren, J.H.M., 557 van't Veld, A. A., 553 van Zandvoort, M.A.M., 553 Varfolomeev, V. N., 582 Varriale, A., 488 Vatassery, G. T., 327-328 Vecchi, M., 303,307(3), 309(1), 390, 393 Ventresca, G. P., 487 Verheof, J., 252 Verkerk, A., 627 Verly, W. G., 111, 113, 113(12) Vermeulen, N.P.E., 478 Vernon-Roberts, B., 557 Vesioli, O., 373 Vetter, W., 303,307(3) Videla, L. A., 423 Vieira, J., 40, 42(26) Vigny, P., 80, 81(12) Viguie, C., 368, 371(13) Vile, G. F., 401 Villar, A., 446 Villarejio, M. R., 167 Vifia, J., 367, 370(7), 371 Virelizier, J.-L., 153 Vistisen, K., 24, 31(28) Vitale, E., 511 Vliegenthart, J.F.G., 423 Vlietinck, A. J., 416 Voest, E. E., 588 Vogt, T., 415,416(23) Voisin, E., 530 Volkmer, C., 557, 566(24), 568, 569(41), 570(41)
Volkova, L. M., 582 von Bruchhausen, F., 421 von Ritter, C., 256, 570(49), 571 yon Sonntag, C., 3, 67 yon Voigtlander, P. F., 552 Voulalas, P. J., 164 Vuilleumier, J. P., 296
W Waggoner, A. S., 325 Wagner, D. A., 472 Wagner, G. R., 420 Wagner, J. R., 55, 81-82, 83(29) Wagner, R. J., 635 Wahl, G., 189 Wahl, G. M., 197 W~hlander, L., 491 Wakabayashi, T., 321 Wakasugi, N., 153 Wakefield, L. M., 591 Wakui, Y., 415,416(31) Wallace, D. M., 48, 193 Wallace, S. S., 33-34, 40, 40(3) Walldins, G., 512 Wallet, J. C., 421,423(13), 425(13) Walshe, J. M., 542, 547 Waither, J. U., 603 Walther, W., 302-303,307(3) Wanders, R.J.A., 603-604 Wang, A.H.-J., 68, 70(23) Wang, B. E., 421 Wang, C. H., 325 Wang, F., 164, 165(9), 166(9), 174(9) Wang, Y., 337 Wang, Y. M., 328,421 Ware, K., 565, 566(36), 567(36), 568(36) Warthesen, J. J., 392 Washko, P. W., 270, 337 Wasil, M., 557, 571(18) Wassail, S. R., 528 Wassen, J. B., 491 Watanabe, H., 137, 139(14), 321, 356, 478 Watanabe, N., 341 Watanabe-Kohno, S., 411,453 Watson, J. J., 17, 60, 62(6), 80 Watson, J. T., 8, 11, 11(38), 14(46) Watson, W. A., 487 Watterson, J. J., 434
AUTHOR INDEX Wayner, D.D.M., 269, 280, 286(1), 543 Weast, R. C., 374 Webb, A., 303-304, 310(2) Webb, A. C., 601 Weber, G., 309 Weber, R., 477 Wedner, H. J., 135 Wegher, B. J., 5, 11(28) Weglicki, W. B., 620-621, 621(8-10), 624(2), 625, 625(3), 626(10), 628(6), 629(6, 7) Wehr, C. M., 17, 24(9), 27(9), 32(9), 80 Weibezahn, K. F., 100 Weigert, W. M., 542 Weil, T. J., 581 Weimann, B. J., 294 Weiner, L. M., 66 Weinfeld, M., 54, 87 Weinges, K., 463 Weiser, H., 294, 303, 309(1), 310(4) Weiss, B., 123 Weiss, J. H., 202 Weiss, R., 477,481(6) Weiss, R. H., 577 Weiss, S. J., 619 Welankiwar, S., 393 Welch, R. W., 337 Wellman, R. B., 389 Wellner, V. P., 492, 493(3) Wells, J. V., 135 Wendel, A., 476, 478, 479(2), 482(2), 560, 566(28) Wenzel, D. G., 257 Werner, T., 387(24), 388 West, M., 17 West, M. S., 3, 21, 59, 80 Westmore, J. B., 7 Wever, R., 619 Wheeler, T., 590 Whitburn, K. D., 284, 288(15) Whitcutt, J. M., 257 White, E., 8, 13(39) White, G. P., 438 White, J. G., 327 Whitehead, T. P., 284 Whitehouse, M. W., 557 Whitfield, M. K., 256 Whittam, J. H., 397 Wiebe, D. A., 590 Wieland, S., 514
677
Wiese, A. G., 175(4, 7), 176, 181, 186(14) Wilbrandt, R., 390 Wild, C. P., 26 Wild, D., 126 Wiles, D. M., 384 Wilkins, L. R., 237 Wilkinson, F., 385 Wilkinson, G., 69 Willerson, J. T., 514 Wilier, W. C., 269 Williams, C. W., 552 Williams, E. C., 590 Williams, J., 189 Williams, R., 89, 94(7) Williams, V. M., 430 Willis, A. L., 327 Willson, R. L., 426 Wilpart, M., 490 Wilson, A., 590 Wilson, D. W., 257-258, 258(3), 264(12) Wilson, J. H., 119 Wilson, R. B., 565, 566(35) Wilson, S. R., 477 Wilson, T., 384 Winitz, M., 496 Winkelmann, J., 481 Winkelsberg, D., 582, 589(22) Winship, D., 555 Winter, R., 428 Winterbourn, C. C., 401 Winterle, J., 311,313(7) Wise, R. M., 620, 621(9) Wise, S. A., 278 Wiseman, H., 590-592, 592(32, 33), 594(33, 37, 39), 595(32, 37, 39), 596(33, 37, 39), 597(33, 39), 598(36, 47, 48), 599(48), 600(47), 601(33, 35-37, 39, 40, 47, 48), 602(36) Wishnok, J. S., 472 Wistort, P. M., 123 Witt, E., 318 Witting, L. A., 355,362(3) Witztum, J. L., 513 Wolbis, M., 421,424(7) Wolf, C., 602 Wolfgang, G.H.I., 551 Wollmer, P., 253,256(15) Wolohuis, J., 95 Wong, G.H.W., 244, 250, 251(8) Wong, P. K., 17, 21, 59-60, 62(6)
678
AUTHOR INDEX
Wong, P. T., 80 Wood, M. L., 17 Wood, R.A.B., 590 Woodward, A. J., 484 Wosikowski, K., 592 Wratten, M. L., 553 Wu, J. F., 523,524(1), 525(1), 526(1) Wu, R., 257-258, 264(12) Wurm, G., 411,421 Wyatt, G. R., 5 X Xanthoudakis, S., 163-164, 166(9), 167(8), 174(9) Xue, L., 3 Xue, L.-y., 78
165(8, 9),
Y Yagi, K., 508, 591 Yalowich, J., 638 Yalowich, J. C., 631 Yamada, T., 556-557,566(24) Yamada, T. I., 568,569(41), 570(41) Yamaguchi, M., 335 Yamaizumi, Z., 80 Yamamoto, A., 514 Yamamoto, F., 25 Yamamoto, H., 422 Yamamoto, K., 70 Yamamoto, S., 453 Yamamoto, S.H.T., 411 Yamamoto, T., 422 Yamamoto, Y., 254, 273,348, 356, 374-375, 402, 406, 480, 481(29), 506, 533 Yamamura, T., 514 Yamaoka, M., 320-321, 321(4, 5), 322, 322(12), 323(14), 325(14), 326(14), 356, 360 Yamashita, K., 123 Yamauchi, R., 311,313,313(6), 314, 314(6), 406 Yamazaki, S., 478 Yanagisawa, E., 415, 416(31) Yang, C. S., 278 Yaniseh-Perron, C., 40, 42(26) Yanishlieva-Maslarova, N., 320
Yasuda, H., 544-545, 547(17) Yasuda, K., 478 Yates, M. T., 505, 505(7, 8), 506, 509(8), 512(7, 8) Yatvin, M. B., 66, 78(5), 79(5, 6) Yavin, E., 604, 619(23, 24) Yee, B. G., 314 Yeo, H. C., 24, 79 Yin, Y., 189 Yin Foo, D.D.Y., 187, 191(19), 203(19) Yl~i-Herttuala, S., 514 Yodoi, J., 150 Yodoi, Y., 153 Yohannes, P., 102 Yokode, H., 505, 506(6) Yokota, S., 604 Yokoyama, C., 453 Yokoyama, S., 514 Yonei, S., 220 Yonkers, P. A., 548, 552-553 Yoshida, H., 505,506(6) Yoshida, T., 225 Yoshida, Y., 402, 480, 481(29) Yoshiji, H., 24 Yoshikawa, Y., 374 Yoshimoto, T., 453 Yoshimoto, Y. T., 411 Yoshino, H., 52 Yoshino, S., 569 Young, A. B., 545,547(18) Youngman, R. J., 88, 420 Yuki, H., 4 Yumibe, N. P., 577 Yuzbasiyan-Gurkin, V., 545, 547(18) Yuzuriha, T., 274 Z Zabel, U., 160, 161(15) Zabin, I., 167 Zaklika, K. A., 605 Zannoni, C., 601 Zarins, Z. M., 415, 416(25) Zaslavsky, Y. A., 345 Zbinden, I., 191, 192(22) Zblewski, R., 99 Zechmeister, L., 389-390, 390(12), 392(12), 393 Zee, Y. C., 257
AUTHOR INDEX Zelnik, V., 573 Zeng, L., 416 Zhang, L.-X., 235-236, 239(8), 241(9), 243(9) Zhang, R. Y., 416 Zhang, T. M., 421 Zhang, T. Y., 415,416(26) Zhdanov, R. I., 582 Zhee, Y. C., 254 Ziegler, D. M., 136, 482 Ziegler, R., 601
679
Ziegler-Heitbrock, L., 155 Zilli, C., 446 Zimmer, L. T., 542 Zitomer, R. S., 176 Zivkovic, Z., 580 Zoeller, R. A., 604, 605(27), 606(21, 27), 608(27), 609(16, 27), 610(27), 611(27), 616(27), 618(21, 27) Zucker, M. B., 328 Zucker, P. A., 477 Zwelling, L. A., 631
680
SUBJECT INDEX
Subject Index
A Absorption spectra antioxidant assay, 282, 284-290 /3-carotene cis isomers, 390-392 Ginkgo biloba extract hydroxyl radical effects, 466-467 superoxide radical effects, 464 Acetaminophen toxicity, N-acetylcysteine therapy, 490 N-Acetyl-5-aminosalicylic acid, antioxidant effects on hemoglobin-catalyzed lipid peroxidation, 569-570 on hydroxyl radical formation, 562-565 on myeloperoxidase, 571 on peroxyl radical-mediated lipid peroxidation, 566-567 N-Acetylcysteine anticarcinogenic properties, 489-490 antimutagenic properties, 501-502 antioxidant capacity, 291-293 antioxidant properties, 488-489 assay in biological systems, 483-487 derivatizing reagents, 484 metabolism, 488 pharmacokinetics in human, 487 substitution for cysteine, 144-145 therapeutic applications, 482-483,490492 N-Acetylglutathione diethyl ester, preparation, 503-504 N-Acetylglutathione monoethyl(glycyl) ester, preparation, 503 Activator protein-1 DNA binding activity, regulation, 163174 oligonucleotide probe, preparation, 170171 N-Acylglutathione derivatives, biological applications, 505 preparation, 503
Adipose tissue, a-tocopherol extraction, 305-306 Adult respiratory distress syndrome, Nacetylcysteine therapy, 491 Albumin assay, 291-293 bovine serum, precipitation of tannin, 436 Aldehydes, fatty, see Fatty aldehydes Alkaline elution assay damage profiles of DNA from mammalian cells, 128-129 DNA strand breaks, 88-91, 94-95 Alkaline phosphatase bacterial, post-Fenton reaction digestion of DNA, 54 hydrolysis of DNA, 6-7, 21 8a-Alkyldioxytocopherones, formation by oxidation of t~- tocopherol by azo initiators, 313-314 by peroxyl radicals, 311-312 Amino acids, crosslinked to DNA bases, GC-MS, 9-11 Amino acid sequencing, N-terminal, oxidant stress-induced proteins, 186-187 2-Amino-3-mercapto-3-methylbutanoic acid, see Penicillamine Aminosalicylates, antioxidant properties, 555-572 4-Aminosalicylic acid, effect on myeloperoxidase, 571 5-Aminosalicylic acid, antioxidant effects, 557 on hemoglobin-catalyzed lipid peroxidation, 569-570 hydrogen peroxide decomposition, 559560 on hydroxyl radical formation, 562-565 hypochlorous acid scavenging, 571-572 on myeloperoxidase, 571 on peroxyl radical-mediated lipid peroxidation, 566-567 superoxide radical scavenging, 558-559
SUBJECT INDEX
21-Aminosteroids, antioxidant action, 548555 Ampholytes, 183 Anthocyanidin, in assay of condensed tannins, 433-434 Antibodies, incubation, 188 Antioxidants assay, general principles, 402-403 lipid-soluble, assay, 274-279 plasma assay, 269-279 loss after ozone exposure, 254-255 total, in plasma and body fluids, assay, 279-293 analytical imprecision, 290-291 automated assay, 289 clinical applications, 293 manual assay, 290 treatment of cells, 156-157 water-soluble, assay, 270-273 Apurinic/Apyrimidinic lyase, 111-115 Arachidonic acid, in assay of eicosanoid metabolism, 450-452 ARDS, see Adult respiratory distress syndrome Ascorbate antioxidant capacity, 291-293 interaction with ~-tocopherol, 380 interaction with vitamin E phenoxyl radical, 319-320 reduction of 8a-substituted tocopherones to a- tocopherol, 315-316 Ascorbic acid assay, 270-272 serum, HPLC assay, 335-337 tissue, HPLC assay, 332-334 Ascorbyl radicals, in vivo assay, 338-343 Astroglial cells, lazaroid antioxidant effects, 552 Atherosclerosis, oxidized LDL role, 513514 Azide radicals, in generation of peroxyl radicals, 428 2,2'-Azinobis(3-ethylbenzothiazoline 6sulfonate) cation, in assay of antioxidants, 284-290 2,2'-Azobis(2-amidinopropane) dihydrochloride peroxyl radical generation, 282, 565566 VP-16 phenoxyl radical generation, 633
681
2,2'-Azobis(2-amidopropane) hydrochloride, peroxyl radical generation, 281 2,2'-Azobis(2,4-dimethylvaleronitrile) cis-parinaric acid fluorescence induced by antioxidant effects, 377-380 in assay of radical scavenging, 35736O time course and spectra, 376-377 peroxyl radical generation, 282 rate studies, 374, 375-376 c~-tocopherol oxidation, 313-314 VP-16 phenoxyl radical generation, 633
B Bacteria, see also Escherichia coil; Salmonella typhimurium
colonies lysis, 203 replicas, 202-203 DNA damage profiles, 126 Bacteriophage PM2, DNA damage profiles, 129 Benzaldehydes, substituted, in assay of condensed tannins, 434-435 Bilirubin, assay, 273,291-293 Binding assay, ethidium-based, copperdependent DNA oxidation, 73-75 Biotinylation, mRNA, 206 Bisbenzamide, fluorescence enhancement by cell DNA, 96-97 Bis(3,5-di-tert-butyl-4-hydroxyphenylthio)propane, see Probucol Bisphenol, serum, assay, 512-513 Blood, see also Plasma; Serum /3-carotene and lycopene geometrical isomers, separation, 397-398 collection, 328-329 oxidized glutathione, HPLC assay, 367371 Brain gerbil, spin trap antioxidant activity, 523-526 rat, a-tocopherol extraction, 305-306 Bronchitis, N-acetylcysteine therapy, 490491 C Calcium channels, drugs blocking, antioxidant activity, 620-630
682
SUBJECT INDEX
Capillary elution, for RNA transfer to filter, 197 Carbon monoxide, production from DOPA, in assay of antioxidants, 284 Carcinogenicity, inhibition by N-acetylcysteine, 489-490 B-Carotene antioxidant activity in membranes, 371383 assay, 274-279 cis isomers, absorption spectra, 390-. 392 effect on fluorescence decay of cisparinaric acid, 377-380 geometrical isomers, separation, 388400 in blood, 397-398 in fruits and vegetables, 396-397 in model systems, 393-394 in tissue, 400 interaction with a-tocopherol, 380 isomerization, induction, 392 Carotenoids delivery to target cells, 237-238 geometrical isomers, 390 quenching of singlet oxygen, 384-388 assay, 386 rate constants, 386-388 regulation of gap junctional communication and connexin expression, 235244 Cell cultures aortic endothelial cells, 626 CHO-9 cells, 89-90 fibroblasts C3H/10T1/2, 237 V79, 589 HeLa cells, 154 Jurkat cells, 154 ozone exposure, systems for, 257-265 preparation for DNA isolation, 18 rat hepatocytes, 438-439 Cell extracts, nuclear and whole, preparation, 158-160 Chemiluminescent assay antioxidants, 281-282, 284 connexin 43,243 peroxyl radical scavenging by Ginkgo biloba extract, 467-468 Chloroform, extraction of RNA, 218, 227228
CHO-9 cells cell culture, 89-90 X irradiation, 90 CHO-K1 cells [14C]ethanolamine-labeled, plasmenylethanolamine breakdown, 610-612 [14C]hexadecanol-labeled fatty aldehyde formation, 614-616 radioactive formic acid formation, 612-614 [32p]Pi-labeled, plasmalogen breakdown in, 608-610 Cholesterol and tamoxifen and estrogens, comparative antioxidant effects, 590-602 unesterified, in LDL, enzymatic oxidation, 517-518 effect of c~-phenyl N-tert-butylnitrone, 520-521 Cholesterol coefficients, 597-598 Cholesteryl esters, in LDL enzymatic oxidation, 517-518 effect of a-phenyl N-tert-butylnitrone, 520-521 fatty acid composition, 518 Chromanols, incorporation in membranes, 363 Chromanoxyl radicals electron spin resonance, 364-365 generation, 361-366 reduction, 365-366 Chromatography, see also specific techniques neutral lipid separation from phospholipids, 617 OxyR protein, 220-221 singlet oxygen DNA damage products, 79-88 yeast redoxyendonuclease on DEAE-cellulose, 105-106 on Mono S, 106-108 on phosphocellulose, 106 on Superose 6-Superose 12, 108-110 Cloning complementation technique, 213-214 in differential display techniques, 210211 in subtractive hybridization, 207-209 Colitis, ulcerative, 555-558 Colorimetric assay, lipid peroxidation in sarcolemmal membranes, 622-626
683
SUBJECT INDEX Complementation, cloning of genes identified by mutagenesis, 213-214 Connexin 43 analysis and quantitation, 244 detection, 242-243 electrophoresis, 242 expression, regulation by carotenoids, 235-244 membrane, isolation, 241-242 Western blot analysis, 241-244 Coomassie blue, in 2D electrophoresis, 185 Copper catalysis of ghost membrane oxidation, 544-545 Cu(I) binding to DNA, 69 preparation from Cu(lI), 69 stabilization of DNA, 68-69 Cu(II), destabilization of DNA, 68 detoxification in penicillamine therapy of Wilson's disease, 546-547 - D N A adducts hydroxyl radical formation dependent on, ESR detection, 70-73 overview, 66-67 physical properties, 68-69 DNA oxidation dependent on, assay, 73-75 endogenous, catalysis of DNA oxidation in intact cells, 75-78 hydroxyl radical formation dependent on, detection, 69-70 penicillamine in presence of, prooxidant effect, 543-545 role in DNA oxidation, probes of, 7778 COS-7 cells, introduction of IO2-damaged shuttle vectors, 119-120 Coumarins effects on cylooxygenase and lipoxygenase, 443-454 structures, 445 Culture media, bacterial, 8-oxoguanine assay, 30-31 Cyclooxygenase, eicosanoid generation, effects of flavonoids and coumarins, 443-454 Cysteine assay, lactate and pyrnvate effects, 147149 deficiency, effect on T cells, 142-144
extracellular, effect on intracellular glutathione levels, 140-145 intracellular, assay, 135-137 membrane transport systems, 137-139 requirement during T cell activation, 141-142 requirement of T lineage cells, lactate and pyruvate effects, 147-149 Cystine intracellular, assay, 135-137 membrane transport systems, 137-139 substitution for cysteine, 144-145 Cytochrome c, acylated derivative conjugated to poly(styrene-co-maleic acid) properties, 342-343 synthesis, 339-342 in in oioo assay of superoxide and vitamin C radicals, 338-343
D Decolorization assay, antioxidants, 284285 Dehydroascorbate serum, HPLC, 335-337 tissue, HPLC, 332-334 Denaturation DNA fluorometric analysis, 91-93, 95-99 kinetics, 97-98 RNA, 196 Deoxycytidine, HPLC, 22 Deoxycytidine monophosphate, products after Fe2+/H202 exposure, radiochromatogram, 56 Deoxyguanosine HPLC, 22 isotope effects, 58 products after Fe2+/H2Oz exposure, radiochromatogram, 55 2'-Deoxyguanosine, photosensitized reactions, 82-83 Deoxyribonuclease I footprinting, in mapping of OxyR binding, 222-223 hydrolysis of DNA, 6-7 post-Fenton reaction digestion of DNA, 53-54 Derivatization DNA, 7
684
SUBJECT INDEX
fatty aldehydes and plasmalogens with 2,4-dinitrophenylhydrazine, 616618 a-tocopherol to a-tocopherol methyl ether, 306-307 Dialysis, Fos and Jun renaturation during, 167 1,2-Dichloroethane, radical cations, scavenging by ebselen, 479 Diethylenetriaminepentaacetic acid, complex with Fe 2. hydroxyl radical generation, 563 preparation, 564 Differential display techniques, DNA, 200201,207-211 4,8-Dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine, 4R* and 4S* diastereomers formation by 2'-deoxyguanosine photooxidation, 83 HPLC, 81, 83-85 mass spectrometry, 85-88 preparation, 81 and related base component, chromatography and mass spectrometry, 8688 Dihydrolipoic acid antioxidant properties, 458-460 detection, 458 generation from a-lipoic acid, 456-458 by lipoamide dehydrogenase, 456-457 by sodium borohydride, 457-458 homologs, structure, 455 peroxyl radical scavenging, fluorescence assay, 460 reaction with superoxide radicals, 458459 structure-antioxidant activity relationships, 454-461 7,8-Dihydro-8-oxo-2'-deoxyguanosine, 17, 80, 83 in biological fluids, analysis, 24-31 radiolabeled standards, synthesis, 25 sample preparation and isolation, 2631 in DNA, steady-state levels, 18-24 excretion by cells, 29-30 in plasma, recovery, 30 production in tissue culture, 28-29 in urine, recovery, 26-27
3-(3,4-Dihydroxy-5-nitrobenzylidene)-2,4pentanedione, s e e Nitecapone 3,4-Dihydroxyphenylalanine, CO production, 284 Diltiazem effect on loss of endothelial cell glutathione and viability, 627-629 inhibition of lipid peroxidation, 622-626 5,5-Dimethyl-l-pyrroline N-oxide, in detection of hydroxyl radical formation, 70, 71 2,4-Dinitrophenylhydrazine, fatty aldehydes derivatized with, HPLC, 616618 Dioleoylphosphatidylcholine, effect on cisparinaric acid fluorescence, 376 Diphenoquinone, serum, assay, 512-513 1,6-Diphenyl-1,3,5-hexatriene, fluorescence polarization, vitamin E-induced changes, 324-326 3,3'-Di-propylthiocarbocyanine, fluorescence, vitamin E-induced quenching, 324-326 Disodium 3,3'-(1,4-naphthylidene) dipropionate, singlet oxygen generation, 116 Dissolution, ~-carotene and lycopene, isomerization induced by, 392 1,4-Dithioerythritol, effect on serum ascorbic acid levels, 337 DNA acidic hydrolysis, 5-6 bacterial, damage profiles, 126 bacteriophage PM2, damage profiles, 129 bases crosslinked to amino acids, GC-MS, 9-11 free, GC-MS, 8 cell, enhancement of bisbenzarnide fluorescence, 96-97 cell-free, damage profiles, 125 cleavage products analysis, 38-39 redoxyendonuclease-generated, posttreatment, 43-44 complementary, labeling, 202 -copper adducts hydroxyl radical formation dependent on, ESR detection, 70-73
SUBJECT INDEX
overview, 66-67 physical properties, 68-69 damage profiles, GC/MS/SIM acquisition, 131 derivatization, 7 differential display, 200-201,207-211 differential hybridization, 200-204 7,8-dihydro-8-oxo-2'-deoxyguanosine in, steady-state levels, 18-24 enzymatic hydrolysis, 6-7, 20-21 ethanol precipitation, 20 Fenton reaction-mediated damage, detection, 51-58 Fos binding, dependence on Cys redox state, 163-174 isolation, 18-21 Jun binding, dependence on Cys redox state, 163-174 L1210 leukemia cell, damage profiles, 129-130 mammalian cell, damage profiles, 128129 mitochondrial, damage profiles, 125-126 mobility shift, 221 nuclear, oxidative damage biomarkers in, HPLC assays, 16-33 oxidation copper-dependent, assay, 73-75 in intact cells, catalysis by endogenous copper, 75-78 role of copper and iron, probes of, 7778 oxidative damage biomarkers, HPLC assays, 16-33 endonuclease fingerprinting, 122-131 eukaryotic enzymes recognizing, detection and characterization, 33-44 GC-MS analysis, 3-15 by reactive oxygen species, assay, 4850 by singlet oxygen assay, shuttle vector for, 115-122 electrophoretic assay, 117-118 product analysis, 79-88 OxyR binding DNase I footprinting assay, 222-223 gel retardation assay, 221 phenol extraction, 19-20 plasmid, s e e Plasmids
685
post-Fenton reaction digestion to nucleosides, 53-54 preparation for HPLC, 18-21 strand breaks alkaline elution assay, 90-91 dose-effect curves, 93 fluorescence assay, 91-93, 95-99 induction, 90, 99-100 rejoining, 90 rejoining curves, 93-94 relaxation assay, 127-128 repair, 100-102 strand cleavage, mode of, determination, 42-44 substrates incubation with eukaryotic enzymes, 37 osmium tetroxide-damaged, preparation, 40, 42 ultraviolet-damaged, preparation, 37 subtractive hybridization, 200, 204-207 supercoiled, endonuclease-sensitive modifications, relaxation assay, 127-128 thymine glycol-containing, redoxyendonuclease digestion, 40-41, 42-43 ultraviolet radiation effects, 36-37 unwinding fluorometric analysis, 91-93, 95-99 kinetics, 97-98 DNA N-glycosylase, 34 DNase I, s e e Deoxyribonuclease I DOPA, s e e 3,4-Dihydroxyphenylalanine Dot blotting, heine oxygenase 1 transcription assay, 225-226 DTPA, s e e Diethylenetriaminepentaacetic acid Dye microinjection assay, gap junctional communication, 238-239
E Ebselen biological effects, 481 disposition, 481-482 glutathione peroxidase-like activity detection, 479 reaction scheme for, 477-479 metabolism, 481-482
686
SUBJECT INDEX
protective effect against lipid peroxidation, 480-481 radical scavenging, 479-480 singlet oxygen quenching, 480 synthesis, 477 Eicosanoids, generation by leukocytes inhibition by coumarins, 445 by flavonoids, 444 radiochromatographic assay, 450-452 radioimmunoassay, 449-450 stimulation, 449 Electroblotting, oxidant stress-induced proteins, 188 Electron spin resonance chromanoxyl radicals, 364-365 copper-dependent hydroxyl radical formation, 69-70 copper-DNA adduct-dependent hydroxyl radical formation, 70-73 nitroxides, 583-584 vitamin E phenoxyl radicals, 316-320, 351-353 VP-16 phenoxyl radicals detection, 632-637 reduction reactions in aqueous solution, 638-640 in cell and nuclear homogenates, 641-642 Electrophoretic mobility shift assay, 153 N F - K B activity, 160-162 Elution alkaline, see Alkaline elution assay capillary, for RNA transfer to filter, 197 End-labeling analysis, plasmid DNA strand breaks, 50 Endonucleases fingerprinting of oxidative DNA damage, 122-131 preparations, 124-125 supercoiled DNA modifications sensitive to, assay, 127-128 Endothelial cells aortic culture, 626 loss of glutathione and viability, effects of calcium channel blockers, 627-629 oxidative incubation, 626-627
brain microvessel, membrane physicochemical properties, effects of lazaroids, 553 Epoxy-8a-hydroperoxytocopherones, formation, 312 Epoxytocopherones, preparation, 313-314 Epoxy-a-tocopherylquinones, formation, 312 EPR, see Electron spin resonance Erythrocytes antioxidant effects of lazaroids, 552 ghosts chromanoxyl radical generation, 363 membranes, copper ion-catalyzed oxidation, 544-545, 547 glutathione diethyl ester transport into, 500-501 isolation, 328-329 saponification, 329 tocopherols and tocopherolquinone, assay, 327-331 Escherichia coil
culture media, 8-oxoguanine assay, 3031 expression and purification of recombinant Fos and Jun, 166-169 oxyR-cont r ol l e d regulon, 217 OxyR purification, 220-221 in screening of mutation-containing plasmids, 120-121 shuttle vector rescue, 120 ESR, see Electron spin resonance Estradiol-17/3, membrane antioxidant activity comparison with cholesterol, 596-598 in liposomal and microsomal systems, 594-596 Estrogen receptors, binding affinity of tamoxifen and tamoxifen metabolites, 600 Estrogens, membrane antioxidant activities, 590-602 Ethanol, precipitation of DNA, 20 Ethanolamine, ~4C-labeled, labeling of plasmenylethanolamine, 610-611 Ethidium bromide, in assay of copperdependent DNA oxidation, 73-75 N-Ethylmaleimide, quenching of reduced glutathione, 368-369
SUBJECT INDEX Etoposide, phenoxyl radicals ESR detection, 632-637 generation by azo initiators of peroxyl radicals, 633 by peroxidase, 632-633 photosensitized generation, 633-635 by tyrosinase, 632-633 interactions with reductants, ESR and HPLC studies in aqueous solution, 638-640 in cell and nuclear homogenates, 640642 Exonuclease, hydrolysis of DNA, 6-7 Extraction DNA with phenol, 19-20 flavonoids, 413-415 primary transcripts, 199-200 RNA, 193-196 with acid phenol and chloroform, 218 with guanidinium thiocyanate, t93-194 guanidinium thiocyanate-phenolchloroform method, 227-228 poly(A) + mRNA preparation, 194-196
F Fast protein liquid chromatography, yeast redoxyendonuclease on Mono S, 106-108 on Superose 6-Superose 12, 108-110 Fatty aldehydes 2,4-dinitrophenylhydrazine-derivatized, HPLC, 616-618 formation in [U-~4C]hexadecanol-labeled cells, 614-616 Fenton reaction DNA damage mediated by, 51-58 hydroxyl radical generation, 422 Ferric nitrilotriacetate, 438,440 Ferricytochrome c, reduction by nitroxides, 584-585 Ferrylmyoglobin radicals, generation of 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) radical cation, 284-285 Fibroblasts Chinese hamster lung, V79 culture, 589
687
intracellular nitroxide localization, 588-589 human dermal 7,8-dihydro-8-oxo-2'-deoxyguanosine, 8-oxoguanine, and 8oxoguanosine levels in vitro, 28-29 glutathione diethyl ester transport into, 500-501 hydrogen peroxide treatment, 227 UVA irradiation, 226-227 murine, C3H/10T1/2, culture, 237 Flavonoids antioxidant mechanisms, 421 chemistry, 412-413 effects on cylooxygenase and lipoxygenase, 443-454 extraction, 413-415 55Fe mobilization from hepatocytes, 443 functions in mammalian cells, 411 from Ginkgo biloba leaves, HPLC, 417418 HPLC, 413-420 oligomers, assay, 429-437 reactions with oxygen radicals, 420-429 structures, 412-413,445 tannin-related, assay, 429-437 Fluorescence bisbenzamide, enhancement by cell DNA, 96-97 cis-parinaric acid in assay of antioxidants, 283 azo initiator-induced decay antioxidant effects, 377-380 in assay of radical scavenging, 358360 time course and spectra, 376-377 effect of dioleoylphosphatidylcholine, 376 in liposomes, measurement, 374-375 quenching and polarization measurement, 323-324 vitamin E partitioning effects, 325-326 Fluorescence assay antioxidants, 282-284 peroxyl radical scavenging by dihydrolipoic acid, 460 by nitecapone and OR-1246
688
SUBJECT INDEX
in membranes, 533-535 in solution, 531-533 Fluorometric analysis, DNA unwinding, 91-93, 95-99 Footprinting, DNase I, in mapping of OxyR binding, 222-223 Formate dehydrogenase, in assay of formic acid formation from plasmalogens, 613-614 Formic acid, radioactive, formation in [l-J4C]hexadecanol-labeled cells, 612614 N-Formylglutathione, preparation, 504 N-Formylglutathione monoethyl(glycyl) ester, preparation, 504 Freedox, see U74006F Free radical reductase, 316-317 G Gap junctions communication dye microinjection assay, 238-239 regulation by carotenoids, 235-244 plaques, immunofluorescent detection, 239-241 Gas chromatography, capillary c~-tocopherol methyl ether stereoisomers, 307-309 all-rac-a-tocopherol stereoisomers, 302310 Gas chromatography-mass spectrometry acquisition of DNA damage profiles, 131 chemical determination of oxidative DNA damage, 3-15 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine and related base component, 86-88 DNA base-amino acid crosslinks, 9-11 free bases, 8 instrumentation, 7-8 nucleosides, 8-9 reference compounds, 4-5 with selected ion monitoring, low annlyre concentration measurements, 11-15 c~-tocopherol stereoisomers, 310 Gel electrophoresis, see also Isoelectric focusing assay of DNA damage by 1Oz, 117-118 connexins, 242
heme oxygenase 1 mRNA, 225, 229-230 monitoring of RNA quality, 246-247 Northern blot, mRNA, 196-199 oxidant stress-induced proteins, 179-181 oxidative DNA damage assay, 48-50 two-dimensional, oxidant stress-induced proteins, 181-187 Gel retardation assay Fos and Jun redox state, 171-174 OxyR binding to DNA, 221 Genes complementation cloning, 213-214 expression during oxidative stress, assessment comprehensive survey strategies, 177 DNA techniques, 200-211 in eukaryotes and prokaryotes, 177179 genetic strategies, 211-216 overview, 175-177 protein techniques, 179-191 RNA techniques, 191-200 supF, 119
mutations, 120-121 Ginkgo biloba extract
antioxidant action, 462-475 composition, 463 effect on xanthine oxidase, 469 flavonoids from, HPLC, 413-418 hydroxyl radical scavenging, 465-467 nitric oxide scavenging, 469-473 biological consequences, 475 nitrite-based detection, 472-473 oxyhemoglobin-based analysis, 469471 peroxyl radical scavenging, 467-468 superoxide radical scavenging, 464-465 without terpenes hydroxyl radical scavenging, 465-467 superoxide radical scavenging, 464465 Glutamate, extracellular, effect on lymphocyte function, 139-140 Glutamine syntbetase, effect of a-phenyl N-tert-butylnitrone, 526 Glutathione assay, 291-293 delivery by glutathione diethyl ester, 499-500 by glutathione monoesters, 492-493
SUBJECT INDEX depletion, effect on T cells, 142-144 intracellular levels assay, 135-137 effect of extracellular cysteine, 140145 oxidized, in blood, HPLC assay, 367371 reduced, quenching, 368-369 substitution for cysteine, 145 Glutathione diester, conversion to monoester in murine plasma, 500 Glutathione diethyl ester preparation, 501 transport into human cells, 500-501 Glutathione monoesters formation from diester in murine plasma, 500 free, preparation, 496 as glutathione delivery agents, 492-493 high-performance liquid chromatography, 497-498 metal impurities, 498-499 oxidation in vitro and in vivo, 499 preparation and use, 492-499 recrystallization, 496 thin-layer chromatography, 497 Glutathione monoethyl ester hemihydrosulfate, preparation, 495-496 Glutathione monoethyl ester hydrochloride, preparation, 494-495 Glutathione peroxidase, mimicry by ebselen, 476-482 N-Glycosylase, 33-34 activity of yeast redoxyendonuclease, 110-111 Gradient gels, in 2D electrophoresis, 184185 Guanidinium thiocyanate, extraction of RNA, 193-194, 227-228 H Heine oxygenase 1 agents inducing, 224-225 mRNA, accumulation assay selection, 225-226 Northern analysis, 229-235 oxidative stress-induced increase, 233-235 transient enhancement, 224-235
689
Hemoglobin, lipid peroxidation catalyzed by, effects of aminosalicylates, 568570 Hepatocytes, see also Liver iron-loaded, 55Fe mobilization, 443 rat, isolation and culture, 438-439 Hexadecanoi, 14C-labeled, labeling of plasmalogens, 612-614 Hexane, a-tocopherol and a-tocotrienol radical scavenging in, 357-359 High-performance liquid chromatography N-acetylcysteine, 485-487 bilirubin, 273 /3-carotene geometrical isomers, 393-400 in blood, 397-398 in fruits and vegetables, 396-397 in tissue, 400 dehydroascorbate from serum, 335-337 from tissues, 332-334 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine 4R* and 4S* diastereomers, 81, 83-85 DNA hydrolysates, 21-24 fatty aldehyde moieties after 2,4-dinitrophenylhydrazine derivatization, 616-618 flavonoids, 413-420, 416 glutathione monoesters, 497-498 8-hydroxyguanine nucleosides, 63-65 immunoaffinity-purified biological fluids, 31 lipid hydroperoxides, 375 lipid-soluble antioxidants, 276-279 lycopene geometrical isomers, 393-400 in blood, 397-398 in fruits and vegetables, 396-397 in tissue, 400 malondialdehyde, 439, 441 nucleosides produced by post-Fenton reaction digestion, 54-58 oxidative DNA damage biomarkers, 1633 oxidized glutathione in blood, 367-371 probucol and metabolites in serum, 511513 tocopherol, 329-330 ct-tocopherol methyl ether stereoisomers, 307 tocopherolquinone, 329-330
690
SUBJECT INDEX
all-rac-c~-tocopherol stereoisomers, 302310 uric acid from serum, 335-337 from tissues, 332-334 vitamin C from serum, 335-337 from tissues, 332-334 vitamin E homologs, 294-302 fluorescence detection, 299 reproducibility, 300 reversed-phase experiments, 298 straight-phase experiments, 298 VP-16 phenoxyl radical reduction in aqueous solution, 638-640 in cell and nuclear homogenates, 642 water-soluble antioxidants, 271-272 Horseradish peroxidase, radical generation, 284 Human immunodeficiency virus, type 1 long terminal repeat, in assay of NF-KB, 162 replication, inhibition by N-acetylcysteine, 492 Hybridization differential, 200-204 in Northern analysis of heme oxygenase 1 mRNA, 230-232 for Northern blot electrophoresis, 197199 in one-day Northern blotting, 248-249 subtractive, 200, 204-207 in transcriptional run-on assay, 200 Hydrogen peroxide decomposition by 5-aminosalicylic acid, 559-560 DNA damage induced by, detection, 5158 reaction with hepatic cytosol, associated copper release and membrane oxidation, 546-547 role in lymphocyte activation, 145-147 treatment of cells, 155-156 treatment of fibroblasts, 227 Hydrolysis acidic, DNA, 5-6 enzymatic, DNA, 6-7, 20-21 8a-Hydroperoxytocopherone, formation, 312
8-Hydroxy-2'-deoxyadenosine HPLC with elecrochemical detection, 63-65 synthesis by methylene blue and light, 62, 65 ultraviolet spectra, 62 8-Hydroxyguanosine HPLC with elecrochemical detection, 63-65 photochemical synthesis, 59-65 by methylene blue and light, 60-61, 65 Udenfriend system, 61-62, 65 ultraviolet spectra, 62 Hydroxyl radicals copper-dependent formation, detection, 69-70 copper-DNA adduct-dependent formation, ESR detection, 70-73 DNA-bound copper-catalyzed formation, 66-67 generation copper-binding sites as catalytic centers for, 544-545 by dihydrofumarate oxidation in presence of Fe-ADP, 621-622,625626 Fenton system, 422-423 by neutrophils, 560-561 site-specific methods, 561-562 inhibition by aminosalicylates, 562-565 interaction with N-acetylcysteine, 489 reaction with aliphatic structures, 426 scavenging by Ginkgo biloba extract, 465-467 by stobadine, 575-576 site-specific reactivity, 561 8a-Hydroxytocopherone, formation, 312 Hypochlorous acid interaction with N-acetylcysteine, 489 scavenging by 5-aminosalicylic acid, 571-572
Immunoaffinity columns, 26-28 Immunofluorescent microscopy, gap junctional plaques, 239-241 Immunoprecipitation, oxidant stressinduced proteins, 188-191
SUBJECT INDEX Iron chelated to DTPA hydroxyl radical generation, 563 preparation, 564 chelation, role in flavonoid antioxidant action, 437-443 DNA damage induced by, detection, 5158 55Fe, mobilization from hepatocytes, 443 interactions with nitroxides, 586-588 reaction with 5-aminosalicylic acid, 562563 role in DNA oxidation, probes of, 77-78 Ischemia, stobadine effects, 578-579 Isoelectric focusing, see also Gel electrophoresis gel used in, application to SDS gel, 184 oxidant stress-induced proteins, 181-184 Isomerization,/3-carotene and lycopene, induction, 392 Isotope effects, in HPLC of deoxyguanosine, 58
K Kinetics competition, in generation of oxygen radicals, 424-425 dihydrolipoic acid reaction with superoxide radicals, 458-459 DNA unwinding, 97-98 peroxyl radical scavenging by retinoids, 408-410 all-trans-retinol reaction with linoleic acid-derived peroxyl radicals, 404408
L Lactate, effect on cysteine requirement of T lineage cells and on cysteine assay, 147-149 Lazaroids, antioxidant action, 548-555 LDL, see Lipoproteins, low-density Leaf, Ginkgo biloba, flavonoids from extraction, 413-415 HPLC, 417-418
691
Leukocytes eicosanoid generation inhibition by coumarins, 445 by flavonoids, 444 radiochromatographic assay, 450452 radioimmunoassay, 449-450 stimulation, 449 mixed, preparation from rat peritoneal cavity, 447-448 polymorphonuclear, suspensions, preparation, 448-449 Light, induction of fl-carotene and lycopene isomerization, 392 Linoleic acid, peroxyl radicals derived from generation, 402-403 reaction with all-trans-retinol, 404-408 Linoleic acid methyl ester, oxidation, 402404 assays, 403-404 inhibition by all-trans-retinol, 404-405 by vitamin analogs, 408-410 Lipid hydroperoxides formed by cis-parinaric acid peroxidation, assay, 375 production during fluorescence decay of cis-parinaric acid, 376-377 Lipid peroxidation ebselen effects, 480-481 hemoglobin-catalyzed, aminosalicylate effects, 568-570 in hepatic microsomes, effects of ubiquinol and vitamin E, 345-348 iron-induced, analysis, 439-442 peroxyl radical-induced aminosalicylate effects, 565-566 nitecapone and OR-1246 effects, 535536 in sarcolemmal membranes, assay, 622626 Lipids neutral, separation from phospholipids, 617 peroxidation in hepatic microsomes, effects of ubiquinol and vitamin E, 345-348
692
SUBJECT INDEX
yeast membrane, antioxidant ability after tamoxifen treatment, 598-599 Lipoamide dehydrogenase, dihydrolipoic acid formation with, 456-457 a-Lipoic acid characteristics, 454-455 dihydrolipoic acid generation from, 456458 NMR spectra, 458 solution, preparation, 456 Lipoperoxyl radicals, scavenging by vitamin A and analogs, 401-410 Lipoproteins, low-density cholesterol molecular species, enzymatic oxidation, 517-518 effect of a-phenyl N-tert-butylnitrone, 520-521 chromanoxyl radical generation in, 363 isolation, 515 oxidized, role in atherosclerosis, 513514 probucol antioxidant activity in, assay, 508-509 triglyceride molecular species, enzymatic oxidation, 518-520 effect of a-phenyl N-tert-butylnitrone, 520-521 vitamin E phenoxyl radical in, 319-320 Liposomes 17/3-estradioi antioxidant effects, 594596 cis-parinaric acid-incorporated fluorescence, measurement, 374-375 model of antioxidant activities in membranes, 371-383 stobadine antioxidative effects, 577 tamoxifen antioxidant effects, 594-596 ct-tocopherol and a-tocotrienol radical scavenging in, 359-360 and water, partitioning of nitecapone and OR-1246, 538-539 Lipoxygenase eicosanoid generation, effects of flavonoids and coumarins, 443-454 oxygen radical generation, 423-424 and phospholipase A2, oxidative effects on LDL molecular species, 517521 Liquid chromatography /~-carotene geometrical isomers, 393
carotenoid isomers in fruits and vegetables, 396 lycopene geometrical isomers, 393 Liver, see also Hepatocytes cytosol, reaction with hydrogen peroxide, associated copper release and membrane oxidation, 546-547 homogenates, preparation, 297 tx-tocopherol extraction, 305-306 vitamin E homologs, HPLC, 294-302 Lucifer Yellow CH, 238-239 Luminogenic assay, nitroxides, 585 Lycopene assay, 274-279 geometrical isomers, separation, 388-400 in blood, 397-398 in fruits and vegetables, 396-397 in model systems, 393-394 in tissue, 400 isomerization, induction, 392 Lymphocytes, T, see T cells Lysophosphatidylethanolamine, formation in [2-14C]ethanolamine-labeled cells, 610-612
M Malondialdehyde, HPLC assay, 439, 441 Mass spectrometry, see also Gas chromatography-mass spectrometry 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine, 85-88 singlet oxygen DNA damage products, 79-88 Membranes /3-carotene antioxidant activity, 371-383 chromanol incorporation, 363 connexin 43, isolation, 241-242 cysteine and cystine transport systems, 137-139 dihydrolipoic acid scavenging of peroxyl radicals, 460 erythrocyte ghost, copper ion-catalyzed oxidation, 544-545 estrogen antioxidant effects, 590-602 comparison with cholesterol, 596-598 in liposomal and microsomal systems, 594-596 fluidity, effects of tamoxifen and estrogens, 600-602
SUBJECT INDEX lazaroid antioxidant effects, 550-552 peroxyl radical scavenging by nitecapone and OR-1246, 533-535 physicochemical properties, effects of lazaroids, 553-554 sarcolemmal lipid peroxidation, assay, 622-626 preparation, 621 tamoxifen antioxidant effects, 590-602 comparison with cholesterol, 596-598 in liposomal and microsomal systems, 594-596 ~-tocopherol antioxidant activity, 360361, 371-383 c~-tocotrienol antioxidant activity, 360361 tocotrienol restriction within, 320-327 ubiquinol antioxidant activity, 371-383 vitamin E distribution to, associated fluorescence properties, 325-326 and water, partitioning of nitecapone and OR-1246, 538-539 yeast, lipid fraction, antioxidant ability after tamoxifen treatment, 598-599 2-Mercaptoethanol, substitution for cysteine, 145 Metallothionein, role in penicillamine therapy of Wilson's disease, 546-547 Metals impurities in glutathione monoester preparations, 498-499 interactions with nitroxides, 585-588 Methylene blue in 8-hydroxy-2'-deoxyadenosine synthesis, 62, 65 in 8-hydroxyguanosine synthesis, 60-61, 65 Methyl linoleate, s e e Linoleic acid methyl ester Metmyoglobin in assay of antioxidants, 285-290 purification, 287-288 Microsomes chromanoxyl radical generation, 363 17/3-estradiol antioxidant effects, 594596 hepatic, lipid peroxidation, effects of ubiquinol and vitamin E, 345-348 stobadine antioxidative effects, 577-578 tamoxifen antioxidant effects, 594-596
693
yeast, lipid fraction, antioxidant ability after tamoxifen treatment, 598-599 Mitochondria chromanoxyl radical generation, 363 DNA, damage profiles, 125-126 stobadine antioxidative effects, 578 Monobromobimane, 485-487 Monocytes, treatment with hydrogen peroxide, 155 Mononuclear cells, glutathione diethyl ester transport into, 500-501 Mutagenicity, inhibition by N-acetylcysteine, 489-490 Mutations oxidative damage resistance detection in higher eukaryotes, 215216 hypersensitivity-inducing, 211-214 resistant phenotype-inducing, 214-215 pleiotropic assessment, 213 causes, 215 s u p F gene analysis, 121 screening, 120-121 Myeloperoxidase, effects of aminosalicylates, 571-572 Myoglobin, in assay of antioxidants, 285289
N NADPH oxidase, oxygen radical generation, 423 3,3'-(1,4-Naphthylidene) dipropionate endoperoxide, 385-386 NDGA, s e e Nordihydroguaiaretic acid Neocuproine, probe of copper and iron roles in DNA oxidation, 77-78 Neurons, antioxidant effects of lazaroids, 552 Neutrophils human, suspensions, preparation, 448449 hydroxyl radical generation, 560-561 Nicardipine effect on loss of endothelial cell glutathione and viability, 627-629 inhibition of lipid peroxidation, 622-626
694
SUBJECT I N D E X
pharmacologically active and inactive enantiomers, comparison, 629-630 Nifedipine effect on loss of endothelial cell glutathione and viability, 627-629 inhibition of lipid peroxidation, 622-626 Nitecapone antioxidant activity, effect of structural modification, 526-541 effect on peroxyl radical-induced lipid peroxidation, 535-536 effect on xanthine oxidase, 536-538 gastroprotective effects, 527-528 partition between aqueous and membrane phases, 538-539 peroxyl radical scavenging in membranes, 533-535 in solution, 531-533 physicochemical properties, 528-529 superoxide radical scavenging, 530-531 Nitrate tolerance, N-acetylcysteine therapy, 491 Nitric oxide, scavenging by Ginkgo biloba extract, 469-473 biological consequences, 475 nitrite-based detection, 472-473 oxyhemoglobin-based analysis, 469-471 Nitrite, in assay of nitric oxide scavenging by Ginkgo biloba extract, 472-473 Nitroxides antioxidant properties, 580-589 electron paramagnetic resonance, 583584 interaction with metals, 585-588 intracellular localization, 588-589 luminogenic assay, 585 midpoint redox potentials, 581 reaction with semiquinone radicals, 588 reduction of ferricytochrome c, 584585 superoxide dismutase mimetic activity, 582-583 Nordihydroguaiaretic acid inhibition of Mn superoxide dismutase mRNA induction, 250-251 reduction of 8a-substituted tocopherones to c~-tocopherol, 315-316 Northern blotting heme oxygenase 1 mRNA, 225-226, 229-235
capillary blot procedure, 230 quantification, 232-235 mRNA, 196-199 one-day, for mRNA detection, 244-252 reverse, 203-204 Nuclear magnetic resonance dihydrolipoic acid, 458 a-lipoic acid, 458 Nuclease P1 hydrolysis of DNA, 20-21 post-Fenton reaction digestion of DNA, 54 Nuclei homogenates, VP-16 phenoxyl radicaltyrosinase interaction in, 640-642 isolation, 199 Nucleic acids, precipitation, 193 Nucleosides GC-MS, 8-9 mixtures, HPLC, 54-58 production by post-Fenton reaction digestion of DNA, 53-54 Nucleotides, 32P-labeled, labeling of overlapping oligonucleotides, 249-250
O Oligo(dT)-cellulose, preparation, 247 Oligonucleotides AP-I probe, preparation, 170-171 overlapping, labeling, 249-250 OR-1246 antioxidant activity, 526-541 effect on peroxyl radical-induced lipid peroxidation, 535-536 effect on xanthine oxidase, 536-538 partition between aqueous and membrane phases, 538-539 peroxyl radical scavenging in membranes, 533-535 in solution, 531-533 physicochemical properties, 528-529 superoxide radical scavenging, 530-531 Osmium tetroxide, DNA substrates damaged by, preparation, 40, 42 Ovarian cells, glutathione diethyl ester transport into, 500-501 Oxidation, see also Photooxidation cerebral proteins, effect of a-phenyl Ntert-butylnitrone, 526
SUBJECT INDEX DNA assay, 73-75 in intact cells, catalysis by endogenous copper, 75-78 role of copper and iron, probes of, 7778 ghost membranes, catalysis by copper ions, 544-545,547 glutathione monoesters in vitro and in vivo, 499 low-density lipoprotein molecular species, 517-520 effect of c~-phenyl N-tert-butylnitrone, 520-521 cis-parinaric acid, effects of ubiquinol and vitamin E, 345 phospholipids, inhibition by tocotrienols and tocopherols, 321 photosensitized cultured cells, 606-608 plasmalogens, 605-606 a-tocopherol by peroxyl radicals, 310313 Oxidative stress damage and repair pathways, 176, 178 eukaryotic cells, heme oxygenase 1 as marker, 224-235 gene expression during, assessment comprehensive survey strategies, 177 DNA techniques, 200-211 in eukaryotes and prokaryotes, 177179 genetic strategies, 211-216 overview, 175-177 protein techniques, 179-191 RNA techniques, 191-200 8-Oxo-2'-deoxyguanosine, HPLC assays, 16-33 8-Oxoguanine in bacterial media, assay, 30-31 in biological fluids, analysis, 24-31 radiolabeled standards, 25 sample preparation and isolation, 2631 excretion by cells, 29-30 HPLC assays, 16-33 production in tissue culture, 28-29 8-Oxoguanine endonuclease, detection, 38-39
695
8-Oxoguanosine in biological fluids, analysis, 24-31 radiolabeled standards, 25 sample preparation and isolation, 2631 excretion by cells, 29-30 production in tissue culture, 28-29 2-Oxo-4-thiazolidine carboxylate, substitution for cysteine, 145 Oxycarotenoids, quenching of singlet oxygen, 386-388 Oxygen activated, treatment of plasmids, 48 reactive species enzymes metabolizing, overexpression, 157-158 plasmid strand breaks induced by, localization, 45-51 singlet DNA damage products formed by, analysis, 79-88 generation, 79, 116, 385 induced DNA damage and mutagenicity, assay, shuttle vector for, 115122 quenching by carotenoids, 384-388 by ebselen, 480 by stobadine, 575 reactivity to plasmalogens, 603-620 treatment of cells, 156 uptake, in assay of antioxidants, 281 Oxygen radicals generation, 422-425 reactions with flavonoids, 420-429 Oxyhemoglobin, in assay of nitric oxide scavenging by Ginkgo biloba extract, 469-471 Ozone biomolecular damage, evaluation in vitro, 252-256 experimental design, 253-255 extrapolation to in vivo situation, 256 contact with respiratory tract lining fluids, 252-253 effects on plasma, 254-255 exposure of cultured cells and tissues, 257-265 large system, 261-262
696
SUBJECT I N D E X
small system, 258-260 uniformity, 262-265 monitoring, 262
P
cis-Parinaric acid fluorescence in assay of antioxidants, 283 azo initiator-induced decay antioxidant effects, 377-380 in assay of radical scavenging, 358360 time course and spectra, 376-377 effect of dioleoylphosphatidylcholine, 376 in liposomes, measurement, 374-375 oxidation, effects of ubiquinol and vitamin E, 345 peroxidation, lipid hydroperoxides formed from, assay, 375 PDTC, inhibition of NF-KB activation, 157 Penicillamine antioxidant effects, 542-547 prooxidant effect in presence of copper ions, 543-545 therapy of Wilson's disease, 542 copper detoxification role, 546-547 2,2,5,7,8-Pentamethylchroman-6-ol, 313 Peritoneal cavity, leukocyte preparation, 447-448 Peritonitis, induction, 447-448 Peroxidase, generation of VP-16 phenoxyl radicals, 632-633 Peroxidation, cis-parinaric acid, lipid hydroperoxides formed from, assay, 375 Peroxisomes, plasmalogen biosynthesis, 603-604 Peroxyl radicals generation, 423,427-428 in aqueous phase, 281-282 by 2,2'-azobis(2-amidinopropane) dihydrochloride, 565-566 by 2,2'-azobis(2,4-dimethylvaleronitrile), 374, 375-376 in lipid phase, 282 interactions with a-tocopherol and c~tocotrienol
in hexane, 357-359 in liposomes, 359-360 linoleic acid-derived, reaction with alltrans-retinol, 404-408 lipid peroxidation aminosalicylate effects, 565-566 nitecapone and OR-1246 effects, 535536 oxidation of a-tocopherol, 310-313 reactions with flavonoids, rate constants, 426-428 reactivity with ubiquinol and vitamin E, 344-345 scavenging by dihydrolipoic acid, fluorescence assay, 460 by ebselen, 479-480 by Ginkgo biloba extract, 467-468 by nitecapone and OR-1246 in membranes, 533-535 in solution, 531-533 by retinoids, kinetics, 408-410 pH, effect on HPLC of ascorbic and uric acids, 333 Phagemids, subtractive hybridization based on, 204-205 Pharmacokinetics, N-acetylcysteine in human, 487 Phase partitioning, nitecapone and OR1246, 538-539 l, 10-Phenanthrolene, probe of copper and iron roles in DNA oxidation, 77-78 Phenol DNA extraction, 19-20 RNA extraction, 218, 227-228 Phenolics general assays, 432-433 protein precipitable, assay, 435-436 Phenoxyl radicals, etoposide ESR detection, 632-637 generation by azo initiators, 633 by peroxidase, 632-633 photosensitized generation, 633-635 by tyrosinase, 632-633 interactions with reductants, ESR and HPLC studies in aqueous solution, 638-640 in cell and nuclear homogenates, 640642
SUBJECT INDEX 2-Phenyl- 1,2-benzisoselenazol-3(2H)-one, see Ebselen ~-Phenyl N - t e r t - b u t y l n i t r o n e antioxidant activity in brain, 523-526 antioxidant for low-density lipoproteins, 513-523 effect on oxidation of LDL molecular species, 520-521 preparation, 516 o-Phenylenediamine, radical generation, 282 Phosphodiesterase, post-Fenton reaction digestion of DNA, 54 Phospholipase A2, and lipoxygenase, oxidative effects on LDL molecular species, 517-521 Phospholipids oxidation, inhibition by tocotrienols and tocopherols, 321 separation from neutral lipids, 617 Photochemistry, 8-hydroxyguanine nucleoside synthesis, 59-65 Photoemission assay, singlet oxygen quenching by carotenoids, 386 Photolysis, oxygen radical generation, 422 Photooxidation, 2'-deoxyguanosine to 4,8dihydro- 4-hydroxy-8-oxo-2'-deoxyguanosine, 83 Phycoerythrin assay, antioxidants, 282283 Plasma, see also Blood; Serum 7,8-dihydro-8-oxo-2'-deoxyguanosine recovery, 30 glutathione diester conversion to monoester, 500 lipid-soluble antioxidants, 274-279 model for respiratory tract lining fluids, 253 ozone exposure, 254-255 ct-tocopherol extraction, 305-306 all-rac-c~-tocopherol stereoisomers, separation, 302-310 total antiooxidant status, 279-293 vitamin E homologs, HPLC, 294-302 water-soluble antioxidants, assay, 270273 Plasmalogens antioxidant function, 604-605 breakdown in [32p]Pi-labeled cells, 608610
697
decomposition by photosensitized oxidation, 605-606 derivatization with 2,4-dinitrophenylhydrazine, 616-618 fatty aldehyde formation in [U-~4C]hexadecanol-labeled cells, 614-616 formic acid formation in [l-t4C]hexade canol-labeled cells, 612-614 peroxisomal biosynthesis, 603-604 reactivity to singlet oxygen and radicals, 603-620 Plasmenylethanolamine, breakdown in [2~4C]ethanolamine- labeled cells, 610612 Plasmids, see also Shuttle vectors oxidative damage, assay, 48-50 pDS56, in expression of recombinant Fos and Jun, 166-169 redoxyendonuclease substrates, generation, 103-104 strand breaks, localization, 45-51 treatment with activated oxygen, 48 Platelets isolation, 328-329 saponification, 329 tocopherols and tocopherolquinone, assay, 327-331 Polydeoxyadenosine, products after Fe2÷/ H202 exposure, UV absorbance profile, 57 Polyenes, synthetic, quenching of singlet oxygen, 387-388 Polymerase chain reaction in differential display techniques, 209210 subtractive hybridization based on, 204205 Poly(styrene-co-maleic acid), conjugates of cytochrome c and superoxide dismutase, in in vivo assay of superoxide and vitamin C radicals, 338-343 Precipitation, see also Immunoprecipitation DNA with ethanol, 20 nucleic acids, 193 yeast redoxyendonuclease, 105-106 Primer extension assay, OxyR regulon, 219-220 Probucol antioxidant activity, assay, 506-510
698
SUBJECT INDEX
in low-density tipoproteins, 508-509 in whole serum, 509-510 metabolic pathway, 506 and metabolites, assay in serum, 511513 Proteins cerebral, oxidation, effect of a-phenyl N-tert-butylnitrone, 526 digestion, 18-19 Fos recombinant bacterial expression and purification, 166-169 properties, 164 redox-dependent DNA binding activity, 163-174 redox state, assay, 171-174 reduction by cellular proteins, 165-166 renaturation during dialysis, 167 Jun recombinant bacterial expression and purification, 166-169 properties, 164 redox-dependent DNA binding activity, 163-174 redox state, assay, 171-174 reduction by cellular proteins, 165-166 renaturation during dialysis, 167 oxidant stress-induced amino N-terminal microsequencing, 186-187 gel electrophoresis on polyacrylamide, 179-181 two-dimensional techniques, 181187 identification by pulse labeling, 185186 immunoprecipitation, 188-191 Western blotting, 187-188 OxyR activation of transcription, in vitro assay, 223 binding of DNA DNase I footprinting, 222-223 gel retardation assay, 221 purification, 220-221 thiol groups, assay, 273-274 Prussian blue assay, phenolics, 432-433
12-(1'-Pyrene)dodecanoic acid, in photosensitized oxidation of cultured cells, 606-608 Pyrimidine dimer endonuclease, yeast, detection, 38-39 Pyruvate, effect on cysteine requirement of T lineage cells and on cysteine assay, 147-149
R Radial arm maze test, gerbils, effect of aphenyl N-tert-butylnitrone, 525-526 Radial diffusion assay, tannin, 436-437 Radiochromatographic assay, eicosanoid metabolism, 450-452 Radiofluorography, 189-191 Radioimmunoassay, eicosanoid release from leukocytes, 449-450 Radiolabeling cDNA probes, 202 cell extracts for immunoprecipitation studies, 189 overlapping oligonucleotides, 249-250 phospholipids with [32p]Pi, 608 plasmalogens with [1-~4C]hexadecanol, 612-614 plasmenylethanolamine with [2J4C] ethanolamine, 610-611 pulse, oxidant stress-induced proteins, 185-186 Radiolysis, 422 pulse, oxygen radical generation, 424425 Recrystallization, glutathione monoesters, 496 Redox factor-l, reduction of Fos and Jun, 165-166 Redoxyendonucleases comparison, 40-42 digestion of thymine glycol-containing DNA, 40-41, 42-43 DNA cleavage products generated by, posttreatment, 43-44 substrates, generation, 103-104 yeast apurinic/apyrimidinic lysase activity, 111-115 assay, 103-105
SUBJECT INDEX detection, 38-39 N-glycosylase activity, 110-111 properties, 110 purification, 105-110 substrate specificity, 110-115 Relaxation assay DNA strand breaks, 127-128 endonuclease-sensitive modifications in supercoiled DNA, 127-128 Renaturation, Fos and Jun during dialysis, 167 Reperfusion, stobadine effects, 578-579 Respiratory tract, ozone exposure epithelial cells or explants, in vitro systems, 257-265 exposure uniformity, 262-265 large system, 261-262 small system, 258-260 lining fluids, 252-253 extrapolation of in vitro results to in vioo situation, 256 plasma as model, 253 Retinoids, peroxyl radical scavenging, 408-410 Retinol, see Vitamin A Ribonuclease, inactivation during RNA extraction, 193 RNA denaturation, 196 denatured, transfer to filter, 197 digestion, 18-19 extraction, 193-196 guanidinium thiocyanate-phenolchloroform method, 227-228 hybridization, 197-199, 200 isolation, 218 messenger biotinylation, 206 detection by one-day Northern blotting, 244-252 heme oxygenase 1 Northern analysis, 229-235 oxidative stress-induced increase, 233-235 transient enhancement, 224-235 Mn-superoxide dismutase, induction by TNF, 244-252 Northern blot electrophoresis, 196199
699
poly(A) ÷, preparation, 194-196 oxidant-modulated, basic study techniques, 191-193 poly(A) ÷, isolation, 247-248 primer extension assay, 219-220 quality, electrophoretic monitoring, 246247 total cellular, isolation, 227-228 cytoplasmic, isolation, 245-246 preparation, 193-194 transcriptional run-on assay, 199-200 Runoff transcription assay, heme oxygenase 1,225 S Saccharomyces cerevisiae
crude cell extracts, preparation, 105 proteins recognizing oxidative DNA damage, identification and characterization, 36-39 tamoxifen-treated, membrane lipid fraction antioxidant ability, 598-599 Salmonella typhimurium, oxyR-controlled
regulon, 217 Saponification, red cells and platelets, 329 Semiquinone radicals, reaction with nitroxides, 588 Serum, see also Blood; Plasma bisphenol, assay, 512-513 dehydroascorbate, assay, 335-337 diphenoquinone, assay, 512-513 preparation for HPLC of vitamin E homologs, 297 probucol antioxidant activity, 509-510 assay, 511-513 spiroquinone, assay, 512-513 uric acid, assay, 335-337 vitamin C, assay, 335-337 Shuttle vectors, for assay of ~O2-induced DNA damage and mutagenicity, 115122 rescue into Eseherichia coli, 120 transfection of mammalian cells, 119120 treatment with ~O2, 116-119 Silver staining, 185
700
SUBJECT INDEX
Sodium borohydride, dihydrolipoic acid synthesis with, 457-458 Sodium dodecyl sulfate gel, application of isoelectric focusing gel, 184 Spectrophotometric assay dihydrolipoic acid, 458 heme oxygenase 1 transcription, 225 hydrogen peroxide decomposition by 5aminosalicylic acid, 560 nitecapone and OR-1246 effects on peroxyl radical-induced lipid peroxidation, 535-536 scavenging of superoxide radical, 530531 nitric oxide scavenging by Ginkgo biloba extract, 469-473 ozone, 262-263 phenolics, 432-433 protein thiols, 273-274 superoxide scavenging by aminosalicylates, 558-559 xanthine oxidase, 469, 536-538 Spin traps, antioxidant activity in brain, 523-526 Spiroquinone, serum, assay, 512-513 Spleen exonuclease, hydrolysis of DNA, 6-7 Stobadine antioxidative effects, 572-580 in liposomes, 577 in microsomes, 577-578 in mitochondria, 578 assay, 574 chemical properties, 573 effects in ischemia and reperfusion, 578579 hydroxyl radical scavenging, 575-576 quenching of singlet oxygen, 575 radical formation, 574-575 superoxide radical scavenging, 576-577 synthesis, 572-573 Sulfapyridine, antioxidant effects on hemoglobin-catalyzed lipid peroxidation, 569-570 on hydroxyl radical formation, 562-565 on myeloperoxidase, 571 on peroxyl radical-mediated lipid peroxidation, 566-567 Sulfasalazine, antioxidant effects, 556557
on hemoglobin-catalyzed lipid peroxidation, 569-570 on hydroxyl radical formation, 562-565 on peroxyl radical-mediated lipid peroxidation, 566-567 Supercritical fluid chromatography, r-carotene geometrical isomers, 397 Superoxide dismutase conjugated to poly(styrene-co-maleic acid), in in vivo assay of superoxide and vitamin C radicals, 338-343 mimetic activity of nitroxides, 582-583 MnmRNA, induction by TNF, 244-252 overexpression, effect on NF-rB activation, 158 Superoxide radicals generation, 422 by dihydrofumarate oxidation in presence of Fe-ADP, 621-622, 625626 enzymatic, 424 reactions with N-acetylcysteine, 489 with dihydrolipoic acid, 458-459 with ct-tocopherol and c~-tocotrienol, 357 role in lymphocyte activation, 145-147 scavenging by aminosalicylates, 558-559 by Ginkgo biloba extract, 464-465 by nitecapone and OR-1246, 530-531 by stobadine, 576-577 treatment of cells, 156 in oioo assay, 338-343
T Tamoxifen membrane antioxidant activity, 590-602 comparison with cholesterol, 596-598 in liposomal and microsomal systems, 594-596 metabolites, 592 yeast treated with, membrane lipid fraction from, antioxidant ability, 598-599 Tannins, condensed assay, 429-437
SUBJECT INDEX biological sources, 431 extraction, 431 functional group assays, 433-435 protein precipitation-based assays, 435437 purification, 431-432 structure, 429-431 T cells activation cysteine requirement, 141-142, 147149 role of reactive oxygen intermediates, 145-147 function extracellular glutamate effects, 139140 intracellular cysteine effects, 140-145 glutathione diethyl ester transport into, 500-501 Molt-4 cysteine, cystine, and methionine levels, 137 glutathione levels, 136-137 subsets, effects of cysteine deficiency and glutathione depletion, 142-144 Tetrahydrofuran, in delivery of carotenoids to target cells, 237-238 Thin-layer chromatography fatty acids formed from plasmalogens, 615-616 glutathione monoesters, 497 two-dimensional plasmalogen breakdown products, 609-610 plasmenylethanolamine breakdown products, 611-612 Thiobarbituric acid-reactive substances assay aminosalicylate antioxidant properties, 561-563,566,569 antioxidants, 283 17fl-estradiol membrane antioxidant action, 594-596 lipid peroxidation nitecapone and OR-1246 effects, 535536 in sarcolemmal membranes, 622-626 probucol antioxidant activity in low-density lipoproteins, 508-509 in whole serum, 509-510
701
tamoxifen membrane antioxidant action, 594-596 Thiols, protein, assay, 273-274 THP-1 cells, cysteine, cystine, methionine, and glutathione levels, 137 Thymidine, HPLC, 22 Thymine glycol, DNA containing, redoxyendonuclease digestion, 40-41, 42-43 Tirilazad mesylate, s e e U74006F Tissues /3-carotene and lycopene geometrical isomers, separation, 400 dehydroascorbate, uric acid, and vitamin C, assay, 332-334 ozone exposure, systems for, 257-265 all-rac-a-tocopherol stereoisomers, separation, 302-310 TNF, s e e Tumor necrosis factor c~-Tocopherol, s e e Vitamin E 3,-Tocopherol, 274-279 a-Tocopherol methyl ether formation from ct-tocopherol, 306-307 stereoisomers capillary gas chromatography, 307-309 chiral phase HPLC, 307 Tocopherolquinone, platelet and red blood cell, assay, 327-331 Tocopherols antioxidative activity in heterogeneous system, 321-323 in homogeneous system, 320-321 HPLC, 294-302 inhibition of phospholipid oxidation, 321 platelet, assay, 327-331 red blood cell assay, 327-331 lability, 331 Tocopherones, 8a-substituted, reduction to a-tocopherol, 315-316 Tocopheroxyl radicals generation, 310 reactions, 310-311 a-Tocotrienol antioxidant properties in membranes, 360-361 overview, 354-357 interaction with superoxide radicals, 357 radical scavenging activity in hexane, 357-359 in liposomes, 359-360
702
SUBJECT INDEX
recycling efficiency, 361-366 Tocotrienols antioxidative activity in heterogeneous system, 320-327 in homogeneous system, 320-321 HPLC, 294-302 inhibition of phospholipid oxidation, 321 Transactivation assay, NF-KB activity, 162 Transcription, activation by OxyR, in vitro assay, 223 Transcriptional run-on assay, oxidantmodulated RNA, 199-200 Transcription factors NF-KB activation, effect of Mn-superoxide dismutase overexpression, 158 electrophoretic mobility shift assay, 160-162 regulation in vitro and in vivo, 152153 transactivation assays, 162 oxyR, 151-152 soxRS, 151-152 Transfection, mammalian cells with I02damaged shuttle vectors, 119-120 TRAP assay, antioxidants, 280-282 Triglycerides, LDL-associated, molecular species enzymatic oxidation, 518-520 fatty acid composition, 519-520 Tumor cells breast carcinoma, NF-KB activation, effect of Mn- superoxide dismutase overexpression, 158 HeLa culture conditions, 154 hydrogen peroxide treatment, 155 leukemia K-562, homogenates, VP-16 phenoxyl radical-tyrosinase interactions in, 640-642 L1210, DNA damage profiles, 129-130 U937 cysteine, cystine, and methionine levels, 137 glutathione levels, 136-137 pituitary, antioxidant effects of lazaroids, 553 T lymphoma Jurkat culture conditions, 154
hydrogen peroxide treatment, 155 L5178Y, cysteine, cystine, methionine, and glutathione levels, 137 Tumor necrosis factor, induction of Mnsuperoxide dismutase mRNA, 244-. 252 Tumor necrosis factor receptors, type 1, activation, Mn-superoxide dismutase mRNA induction via, 244-252 Tyrosinase generation of VP-16 phenoxyl radicals, 632-633 interactions with VP-16 phenoxyl radicals in cell and nuclear homogenates, 640-642
U U-74006F, inhibition of lipid peroxidation, 549-554 in membrane systems, 550-552 physicochemical effects on membranes, 553-554 in whole cells, 552-553 U-74500A, inhibition of lipid peroxidation, 549-554 in membrane systems, 550-552 physicochemical effects on membranes, 553 -554 in whole cells, 552-553 Ubiquinol antioxidant activity assay, 343-354 mechanisms, 344 in membranes, 371-383 assay, 274-279 effect on fluorescence decay of cisparinaric acid, 377-380 effect on lipid peroxidation in hepatic microsomes, 345-348 in vivo studies, 346-348 effect on cis-parinaric acid oxidation, 345 interaction with a-tocopherol, 380 prevention of vitamin E oxidation, 348351 reactivity toward peroxyl radicals, 344345 reduction of vitamin E phenoxyl radical, 351-354
SUBJECT INDEX Ubiquinones antioxidant function assay, 343-354 mechanisms, 344 assay, 274-279 Udenfriend system, 8-hydroxyguanosine synthesis, 61-62, 65 Ulcerative colitis, 555-558 Ultraviolet ozone analyzer, 262 Ultraviolet radiation DNA substrates damaged by, preparation, 37 effects on DNA, 36-37 generation of vitamin E phenoxyl radical, 318-319 UVA, human fibroblast exposure, 226227 Ultraviolet spectra, 8-hydroxyguanine nucleosides, 62 Urate, assay, 291-293 Uric acid assay, 270-272 serum, HPLC, 335-337 tissue, HPLC, 332-334 Urine 7,8-dihydro-8-oxo-2'-deoxyguanosine isolation, 26-27 HPLC with electrochemical detection, 31 8-oxoguanine isolation, 26-27 8-oxoguanosine isolation, 26-27
V Vanillin, in assay of condensed tannins, 434-435 Verapamil effect on loss of endothelial cell glutathione and viability, 627-629 inhibition of lipid peroxidation, 622-626 Vitamin A and analogs, lipoperoxyl radical scavenging in homogeneous solution, 401-410 reaction with linoleic acid-derived peroxyl radicals, 404-408 Vitamin C, s e e Ascorbic acid Vitamin E, s e e a l s o Tocopherols; Tocotrienols
703
antioxidant properties in membranes, 360-361,371-383 overview, 354-357 assay, 274-279, 291-293 derivatization to a-tocopherol methyl ether, 306-307 effect on fluorescence decay of cisparinaric acid, 377-380 effect on lipid peroxidation in hepatic microsomes, 345-348 in v i v o studies, 346-348 effect on cis-parinaric acid oxidation, 345 extraction from plasma and tissue, 305306 formation by reduction of 8a-substituted tocopherones, 315-316 homologs HPLC, 294-302 overview, 294-296 structures, 295 interaction with antioxidants, 380 interaction with superoxide radicals, 357 occurrence in nature, 294 oxidation by peroxyl radicals, 310-313 phenoxyl radicals ESR studies, 316-320 interaction with reductants, 319-320 reduction by ubiquinol, 351-354 UV-induced generation, 318-319 radical scavenging activity comparison to ubiquinol, 344-348 in hexane, 357-359 in liposomes, 359-360 reactivity toward peroxyl radicals, 344345 recycling efficiency, 361-366 regeneration, assessment, 316-320 stereoisomers, separation, 302-310 ubiquinol-dependent regeneration, 348354 VP-16, s e e Etoposide
W Water, and membrane phases, partitioning of nitecapone and OR-1246, 538-539 Western blotting connexin proteins, 241-244 oxidant stress-induced proteins, 187-188
704
SUBJECT INDEX
Wilson's disease, penicillamine therapy, 542-547 X Xanthine oxidase effect of Ginkgo biloba extract, 469 effects of nitecapone and OR-1246, 536538
oxygen radical generation, 423 Xanthophylls, quenching of singlet oxygen, 386-388 X irradiation, CHO-9 cells, 90
Y Yeast, see Saccharomyces cerevisiae