Genetic Response to Metals

Genetic Response toMetals Proceedings of the First International Symposium Metals and Genetics held May 24-27, 1994,...

Author: Sarkar B.

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Genetic Response toMetals

Proceedings of the First International Symposium

Metals and Genetics held May 24-27, 1994, in Toronto, Ontario, Canada, under the auspices of the Hospital for Sick Children, Toronto, Canada, and the International Association of Environmental Analytical Chemistry, Basel, Switzerland

Cover illustration adapted k m

R e m 3 in Genetics ZO, 1994; used with permission.

Genetic HesDonse I

edited by

Bibudhendra Sarkar The Hospital for Sick Children Toronto, Ontario, Canada

Marcel Dekker, Inc. New York.Basel.Hong

Kong

Library of Congress Cataloging-in-Publication Data

Genetic Response to metalsl edited by Bibudhendra Sarkar cm. p. Includes bibliographical references and index. ISBN 0-8247-9615-2 (hbk.) 1 . DNA-ligand interactions--Congresses. 2. Metals-Carcinogenicity--Congresses. E. Mutagenesis--Congresses. 4. Zinc fmgerproteins--Congresses. 5. GeneticToxicology--Congresses. I.Sarkar,Bibudhendra. II. InternationalSymposiumon"Metalsand Genetics"(1st : 1994 : Toronto,Ont.) QP624.7.G46 1995 615.9'253--d~20 95-7010 CIP

The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special SaledProfessional Marketing at the address below. This book is printedon acid-free paper. Copyright 0 1995 by MARCEL DE-,

INC. All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York

10016

Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

Preface The past few years have seen scientific discoveries greatly contribute to our understanding of the relationships between metals and DNA. The fields which have contributed to this area range from genetics to biochemistry and chemistry. This book provides a forum for investigators from these diverse fieldsto reflect on the broad impactof the direct and indirect interactions of metals and DNA. This volume contains29 chapters contributed by scientists who are known for their special expertise and outstanding contributions to the current state of knowledge in the field. Although chapters have been chosen from various subject areas,the general theme of the book is on metals and genetics. The book begins by discussing the latest advances in our knowledgeof the molecular mechanisms of metal-induced mutagenicityandcarcinogenicity.Thisdiscussionisfundamental to our understanding of how cancer may be caused by various metals as a result of occupationalandenvironmentalexposure.Further,recentinvestigations have demonstrated that various metal compounds are able to cleaveDNA.Therearechapters thatdealwith the designofsuch agents, their mechanisms of action, as well as possible applications for site-specific DNA cleavage. There are also exciting developments inthe area of DNA-binding proteins having zinc-finger motif. Aspectsof these proteins’ DNA recognition, effect of metal replacement, and their ture and function are also presented. In the area of genetics and biochemistry of metal-related diseases the identification of genes for Menkes andWilsondiseaseshasbeenamajorbreakthroughinrecenttimes. There are chapters written by the authors who discovered these genes. Advances in the understanding of the pathophysiology as well as advances in new therapies for Menkes and Wilson diseases are presented as well. Finally, there are chapters devoted to major advances in the area of gene regulation involving metals. Certainly,it is difficult to comprehensivelycoverallareas of metals and genetics. An attempt has been made, nonetheless, to highlight recent important advances. This book will be of infinite value to researchersinthefieldsofbiochemistry,environmentalchemistry, inorganic chemistry, genetics, molecular biology, physiology, pharmacology, toxicology, and medicine. iii

iv

Preface

ACKNOWLEDGMENTS This volume contains the proceedings of the First International Symposium on Metals and Genetics, held May24-27, 1994, at the Hospital for Sick Children, Toronto, Ontario, Canada. We are indebted to Laura Faiczak, who assisted in the planning and operation of the Symposium, andto the following for financial support: the Hospital for Sick Children Foundation, the Research Institute of the Hospital for Sick Children,the Ministry of Environment and Energy, the Province of Ontario, the Samuel Lunenfeld Charitable Foundation, the municipalityofmetropolitanToronto, the NationalInstituteofEnvironmental Health Sciences (National Institutes of Health), the Nickel Producers Environmental Research Association (NipERA), the Nickel Development Institute(NiDI), the National Cancer Institute of Canada, Sandoz Canada, Inc.,Allelix Biopharmaceuticals, Falconbridge Limited, DiaMed Lab Supplies, Life Technologies, Inniskillin Wines, Hillebrand Estates,HenryofPelhamEstates,MarynissenEstates,CaveSpring Cellars, and Brick Brewery.

Bibudhendra Sarkar

Contents iii

ir Molecular Mechanismof Metal-Induced Mutagenicity and Carcinogenicity 1. Regulation of Nuclear Calcium and Zinc Interference by Toxic Metal

Ions

Stefan Hechtenbergand Detmar Beyersmann 2.

Effects of Antioxidants. Zinc, and Chelators on Free Radical Status of Children Living in the Chernobyl Area Ljudmila G. Korkina and Igor B. Afmas’ev

3. The Roleof Ascorbate in Metabolism and Genotoxicityof

Chromium(VI) Karen E. Wetterhahn, DianeM. Steam, Manoj Misra, Paloma H. Giangrande, Laura S. PhiGer, Laura J. Kennedy. and Kevin D. Courtney

1

21

37

4. Inactivation of Critical Cancer-Related Genes by Nickel-Induced

DNA Hypermethylation and Increased Chromatin Condensation: A New Model for Epigenetic Carcinogenesis Max Costa 5. Oxidative Mechanisms of Nickel@) and Cobalt@) Genotoxicity Kazimierz S. Kaspnak 6. The Antimutagenic Effects of Metallothionein May Involve Free

Radical Scavening E. I. Goncharova and T,G. Rossman

53 69

87

7. Protection from Metal-Induced DNA Damage by Metallothionein

in an in Vino System Lu a i , Jim Koropatnick, and M. George Cherian

101

8. DNA Strand Breakage and Lipid Peroxidation as Possible

Mechanisms of Selenium Toxicity

J. Kitahara, Y. Seko, N. Imura, H. Utsumi, and A. Hamada

v

121

vi

Contents

9. Role of Metal in Oxidative DNA Damage by Non-mutagenic

Carcinogen Shosuke Kawanishi, Shinji Oikawa. and Sumiko Inoue

131

Metal-DNA, DNA Cleavage, and Zinc-Finger Proteins

10. Sequence-Selective Cleavage of DNA by Cationic Metalloporphyrins

153

Genevi2ve Pratviel, Pascal Bigey, Jean Bernadou. and Bernard Meunier

11. LanthanidemI) Complexes as Synthetic Nucleases: Hydroxyalkyl Group Participation in Catalysis Janet R. Morrow, K. 0. Aileen Chin, and Kelly Awes

173

12. Initiation of DNA Strand Cleavage by Iron Bleomycin: Key Role of DNA in Determining the Pathway of Reaction David H. Petering, Patricia Fulmer, Wenbao Li, QunkaiMao,

185

and William E. Antholine

13. Nickel Compiexes in Modification of Nucleic Acids

201

Steven E. Rokita. Ping Zheng, Ning Tang, Chien-Chung Cheng, Ren-Hwa Yeh. James G. Muller, and Cynthia J. Burrows

14. New Methods for Determining the Structure of DNA and DNAProtein Complexes Based on the Chemistry of I r o n 0 EDTA

217

Thomas D. Tullius

15. DNA Recognition by Steroid Hormone Receptor Zinc Fingers: Effects of Metal Replacement and Protein-Protein Dimerization Interface Bibudhendra Sarkar

16. HIV-1Tat Protein Forms a Zinc-Finger-like Structure Jean-Pierre Laussac, Honord Mazarguil. Dani2le P r o d ,

237 255

Monique Erard, and Manh-Thong Cung

Metal-Related Genetic Diseases:Menkes and Wilson Diseases

17. Menkes Disease: From Patients to Gene

S. Packman. C. Vulpe, B. Levinson. S. Das, S. Whimey, and J. Gitschier

275

Contents

vii

18. Variability in Clinical Expression of an X-Linked Copper

Disturbance, Menkes Disease Nina Horn, Tonne Tonnesen,and Zeynep Tiimer 19. Development of Copper-Histidine Treatmentfor Menkes Disease Bibudhendra Sarkar

285 305

20. Copper-Histidine Therapy in Menkes Disease: Clinical, Biochemical, and Molecular Aspects Stephen G. Kaler

317

21. Biochemical and Clinical Benefits of Copper-Histidine Therapy in Menkes Disease J. Kreuder. A. Otten. A. Borkhardt, F. Lampert, K. Baerlocher, H. J. BGhles. and A . D6rries

323

22. The Wilson Disease Gene: A Copper-Binding ATPase Homologous to the Menkes Disease Gene Diane W.Cox and Gordon R. Thomas

343

23. Zinc Therapy: An Advance in the Treatment of Wilson’s Disease Tjaard U. Hoogenraad

361

Metals and Gene Regulation 24. Transcriptional Regulation and Functionof Yeast Metallothioneh Genes Zhiwu Zhu, Mark S. Szczypka. and Dennis J. Thiele

379

25. Transcriptional Regulationof the Metallothionein Gene: MetalRegulatory 397 Responsive Zinc Factor Element and Shinji Koizumiand Fuminori Otsuka

26. Gene Disruption of the Transcription FactorMTF-l Leads to Loss of Metal Regulation of Mouse Metallothionein Genes 1 41 Freddy Radtke, Rainer Heuchel, Oleg Georgia? Gerlinde Stark, Michel Aguet, and Walter S c m e r 27. Characterization and Purification of MEP-l, a Nuclear Protein Which Binds to the Metal Regulatory Elements of Genes Encoding Metallothioneins Simon Labbi, Lucie Larouche, Jacinth Pr&ost, Paolo Remondelli, and CarlSkguin

425

viii

Liver

Contents

28. CopperAccumulation, and MetallotbioneinStability and Developmental Regulation, in the Toxic MiZk Mouse Jim Koropatnick, Greg Stephenson, and M. George cherian

443

29. Metallothionein Synthesis Is Selectively Enhanced by Copper in the of LEC Rats Kazuo T. Suzuki

467

I&

485

Contributors Igor B.Afanas’ev 117820, Russia

VitaminResearchInstitute,Nauchnypr.14A,Moscow

Michel Aguet Institut f i r Molekularbiologie I, UniversitiitZurich,CH-8093 Zurich, Switzerland William E. Antholie Medical College of Wisconsin, Milwaukee, W1 53226 Kelly Awes ChemistryDepartment,AchesonHall,StateUniversity York,Buffalo,NY14214

of New

K. Baerlocher Children’sHospital,St.Gallen,Switzerland Jean Bernadou Labomtoire de Chimie de Coordination, Centre Nationalde la Recherche Scientifique,.205, routede Narbome, 31077 Toulouse cedex,France

Detrnar Beyersmann DepartmentofBiologyandChemistry,Universityof Bremen NW2, D-W-2800Bremen 33, Germany

Pascal Bigey Laboratoire de Chmie deCoordination,CentreNationaldela Recherche Scientifique, 205, route de Narbome, 31077 Toulouse cedex, France Department of Pediatrics, Johann Wolfgang Goethe University, DdOOO Frankfurt, Germany

H. J. Bohles

A. Borkhardt DepartmentofPediatrics,Children’sHospital, University, Feulgenstrasse 12, D-35385 Giessen, Germany

Justus Liebig

Cynthia J. Burrows Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794

Lu Cai

DepartmentofPathology,UniversityofWesternOntario,London, Ontario N6A 5C1, Canada Chien-Chung Cbeng Department of Chemistry, State Universityof New York at Stony Brook, Stony Brook, NY11794

M. GeorgeCherian

Department of Oncology,LondonRegionalCancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada ix

X

Contributors

K. 0 . Aileen Chin Chemistry Department, Acheson Hall, State University of NewYork,Buffalo,NY14214 Max Costa Nelson Instituteof Environmental Medicine,N W Medical Center,

LongMeadowRoad,Tuxedo,NY10967 Kevin D. Courtney NH 03766

DartmouthCollege,6128BurckeLaboratory,Hanover,

Diane W. Cox Department of Genetics, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada Manh-Thong Cung Laboratoire de Chimie Physique Macromolhlaire, C Nancy, France

N S ,

S. Das

Department of MedicineandtheHowardHughesMedicalInstitute, University of California, San Francisco, CA 94143

A. Diirries Department of Pediatrics, University Clinics, Wilrzburg, Gennany CNRS,

MoniqueErard InstitutdeBiologieCellulaireetdeGhnetique, route de Narbonne, 31077 Toulouse cedex, France Patricia Fulmer Department of Chemistry, University Milwaukee,Milwaukee, WI 53201

205,

of Wisconsin-

Oleg Georgiev Institut fir Molekularbiologie 11, Universitiit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland Paloma H. Giangrande Hanover, NH 03766

Dartmouth College, 6128 Burcke Laboratory,

J. Gitschier DepartmentofMedicineandtheHowardHughesMedical Institute, University of California, San Francisco, CA 94143

E. I. Goncharova Nelson Institute of Environmental Medicine, NYU Medical Center,LongMeadowRoad,Tuxedo,NY10967 A. Hamada Japan

SchoolofPharmaceuticalSciences,ShowaUniversity,

Tokyo,

StefanHechtenberg Department of BiologyandChemistry,University Bremen NW2, D-W-2800 Bremen 33, Germany Rainer Heuchel Institut fiir Molekularbiologie 11, Universitiit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland

of

xi

Contributors

Tjaard U. Hoogenraad Department of Neurology, University Hospital, 4584 CX Utrecht, The Netherlands Nina Horn Denmark

The John F. KennedyInstitute, G1. Lmdevej7,Glastrup2600,

N. h u r a School of PharmaceuticalSciences,Kitasat0university,5-9-1, Shirokane, Minato-h, Tokyo 108, Japan SumikoInoue Department of PublicHealth,Faculty University, Kyoto 606, Japan

of Medicine,Kyoto

Stephen G. Kaler Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 8C429, 9OOO Rockville Pike, Bethesda, MD 20892 Kazimien S. Kaspnak Department of HumanHealth,FrederickCancer Research & Development, Building 538, Room 205E, Frederick, MD 217021201 Shosuke Kawanishi Department of Public Health, Facultyof Medicine, Kyoto University, Kyoto 606, Japan Laura J. Kennedy Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766 J. Kitahara School of PharmaceuticalSciences,KitasatoUniversity,5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan Shinji Koizumi Department of Experimental Toxicology, National Institute of Industrial Health, 6-21-1 Nagao, Tama-Ju, Kawasaki 214, Japan Ljudmila G. Korkina Lab, Cell Biophysics and Biochemistry, Russian Institute of Pediatric Hematology, Leninskii pr. 117, Moscow 117513, Russia

Jim Koropatnick Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L.6, Canada J. Kreuder Department of Pediatrics,Children’sHospital, University, Feulgenstrasse 12, D-35385 Giessen, Germany

Justus Liebig

Simon Labbe CentredeRechercheenCandrologiedel’Universit6Laval, l’H6tebDieu de Qu6bec. 11 C6te du Palais, Quebec GlR 2J6, Canada Department of Pediatrics,Children’sHospital,JustusLiebig University, Feulgenstrasse 12, D-35385 Giessen, Germany

F. Lampert

Contributors

LucieLarouche Centre de Recherche en Canc6rologie de l’Universit6 Laval, l’H6tel-Dieu de Qu6bec, 11 CBte du Palais, Quebec GlR W6, Canada Jean-Pierre Laussac Laboratoire de Chimie de Coordination, C N R S , 205,route de Narbonne, 31077 Toulouse &ex, France

B. Levinson Department of MedicineandtheHowardHughesMedical Institute, University of California, San Francisco, CA 94143 WenbaoLi Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee. WI 53201 Qunkai Mao Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201 Honor6 Mazarguil Institut de Pharmacologie et de Biologie Structurale, CNRS,

205,route de Narbonne, 31077 Toulouse cedex, France Bernard Meunier Laboratoire de Chimie de Coordination, Centre National de laRechercheScientifique, 205, routedeNarbonne, 31077 Toulousecedex, France ManojMisra 03766

DartmouthCollege, 6128 BurckeLaboratory,Hanover,

NH

Janet R Morrow ChemistryDepartment,AchesonHall,StateUniversity NewYork,Buffalo, NY 14214

of

James G. Muller Department of Chemistry, State University of New York at 11794 StonyBrook,StonyBrook,NY

ShinjiOikawa Department of PublicHealth,Faculty University, Kyoto 606,Japan

of Medicine,Kyoto

A. W e n

Department of Pediatrics, Children’s Hospital, Justus Liebig University, Feulgenstrasse 12. D-35385 Giessen, Germany FuminOri

Otsuka

TeikyoUniversity,Kanagawa,Japan

S. Packman

Department of Pediatrics,DivisionofGenetics,University California, Box 0748,San Francisco, CA 94143

of

David H. Petering Department of Chemistry, University Wisconsin-Milwaukee,Milwaukee, WI 53201

of

xiii

Contributors

Laura S. Phieffer Dartmouth College, 6128 Burcke Laboratory, Hanover, 03766 GeneviBvePratviel Laboratoirede ChmiedeCoordination,CentreNational de la Recherche Scientifique, 205. route de Narbonne, 31077 Toulouse cedex, France

Jacinthe Pr6vost Centre de Recherche en Candrologie de l’Universit6 Laval, 1’HBtel-Dieu de Quebec, 11 CBte du Palais, Quebec G1R 276, Canada Dani&le Prom6Institut de Phannacologie et de Biologie Structurale, CNRS, 205, route de Narbonne, 3 1077Toulouse cedex, France Freddy Radtke Institut fir Molekularbiologie II, Universittit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland Paolo Remondelli Centre de Recherche en Candrologie de l’Universit6 Laval, 1’HBtel-Dieu de Q u C b e c , 11 CBte du Palais, Quebec GlR 276, Canada

Steven E. Rokita Department of Chemistry, State University of New York at StonyBrook,StonyBrook,NY11794

T. G. Rossman

NelsonInstitute of EnvironmentalMedicine, N W Medical Center,LongMeadowRoad,Tuxedo,NY10967

Bibudhendra Sarkar Biochemistry Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario MSG 1x8, Canada Walter Schaffner Universitslt Zurich, Institut fiir Molekularbiologie Winterthurestrasse 190, CH-8057 Ziirich, Switzerland

11,

Carl S*in CentredeRechercheenCandrologiedeI’UniversitCLaval, 1’HBtel-Dim de Qu6bec, 1 1 CBte du Palais, Quebec GlR 256, Canada

Y. Seko

School of PharmaceuticalSciences, Shirokane, Minato-ku, Tokyo 108, Japan

Kitasato University,5-9-1,

Gerlinde Stark Institut fir MolekularbiologieI, Universitslt Zurich, CH-8093 Zurich, Switzerland Diane M. Stearns Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766 Greg Stephenson Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4M. Canada

xiv

Contributors

KazuoT. suzuki FacultyofPharmaceuticalSciences,ChibaUniversity, Yayoi, Inage, Chiba 263, Japan Mark S. Szczypka DepartmentofBiologicalChemistry,TheUniversityof Michigan Medical School, M5416 Medical Science 1, 1301 Catherine Road, Ann Arbor, MI 48109-0606

Ning Tang Department of Chemistry, State University of New York at Stony Brook,StonyBrook, NY 11794 Dennis J. ThieleDepartmentofBiologicalChemistry,TheUniversityof Michigan Medical School,M5416 Medical Science1, 1301 Catherine Road,h Arbor, MI 48109-0606 Gordon R. Thomas Department of Genetics, The Hospital for Sick Children, S55 University Ave., Toronto, Ontario MSG 1x8,Canada TenneTannesenThe 2600,Denmark

John F. KennedyInstitute,G1.Landevej

7, Glastrup

Thomas D. Tullius Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 Zeynep Tiimer The John F. Kennedy Institute, GI.Landevej 7, Glastmp2600, Denmark H. Utsumi Japan

SchoolofPharmaceuticalSciences,ShowaUniversity,Tokyo,

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143

C. Vulpe

Karen E.Wetterhahn Dartmouth College, 6128Burcke Laboratory, Hanover, NH 03766 S. Whitney Departments of Pediatrics and Medicine, University of California,

San Francisco, CA 94143 Ren-HwaYehDepartmentofChemistry,StateUniversity StonyBrook,StonyBrook, NY 11794

of NewYork at

Ping Zheng Department of Chemistry, State University of New York at Stony 11794 Brook,StonyBrook,NY

Zhiwu Zhu Department of Biological Chemistry, The University of Michigan Medical School, M5416 Medical Science 1, 1301 Catherine Road, Ann Arbor, MI 48109-0606

1 Regulation of Nuclear Calcium and Zinc Interference by Toxic Metal Ions Stefan HechtenbergandDetmarBeyersmann Department of Biology and Chemistry, University of Bremen NW2, D-W-2800 Bremen 33, Germany

L

INTRODUCTION

The genetic toxicologyof metals is very complex, since toxic metal ions of bind to multiple cytoplasmic and nuclear target molecules. Low levels heavy metals interfer with crucial nuclear functions as DNA replication, DNA repair, and gene expression [l]. A pertinent mechanismof action of toxic metals may be the interference with nuclear uptake, homeostasis and function of essential metal ions. Calcium and zinc ions are believed of celldifferentiation,proliferationand toparticipateinthecontrol apoptosis. Calciumis involved directly or via calcium binding proteins in likecalmodulin in nearly all majorsignaltransductionchains multicellular organisms [2]. These include the transferof messages from of gene extracellularsignalsintothecellnucleusandthecontrol function. Table 1 lists some relevant findings about the role of Ca2+ or Ca2+/calmodulin in the regulation of gene expression. A comprehensive has review of the functionsof calcium and calmodulin in the cell nucleus been published by Carafolis Laboratory [3]. In at least one instance, the direct interaction of Ca2+/calmodulin with a subclass of transcription factors has been demonstrated, recently [4]. 1

2

Hechtenberg and Beyersmann

Table 1. Control of gene expressionby Ca2+ or Ca2+/calmodulin

1 Calcium specifically prolaction-gene expression

rsuda et al. 1986 [l91 Morgan k Curran 1986 [20] Sheng et al. 1990 [21] Sch6nthaI et al. 1991 [22]

Sheng et al. 1991 [23]

Can et al. 1991 [24]

Kapiloff et al. 1991 [25]

(h2+, PKC and CAMP regulate c-fos expression ~ a 2 flux + contro1s c-fos expression

Ca2+/calmodulin controls c-fos expression Mobilization ofCa2+ by inhibition of Ca2+ ATPase causesc-fos and c-jun expression Ca2+/calmodulin dependentkinases activate transcription factor CREB (CAMP responsive) Both Ca2+ and PKC regulate expression ofthe gene for the thyrotropin B-subunit

Ca2+/calmodulindependentprotein kinase activated the rat prolactin gene independently of PKC

Bartlett et al. 1991 [26]

Ca2+ ionophore A 23187 induces expression of the gadd153 gene (growth arrest and DNA damage

Liu et al. 1992 [27]

inducible) Transcription factors ATF-l and CREB mediate Ca2+ and CAMP-induced transcription

Corneliussen et al. 1994 [4]

Ca2+/calmodulin *%its directly DNA binding of helix-loophelix transciption factor domains -

Regulation of Nuclear Calcium and Zinc

3

Interference

Whereas the regulatory functions Ch2+ of are wellestablished, little hard data are available about the role of Zn2+ in cellular controls. Certainly, key zinc is a structural constituent of hundreds of proteins comprising enzymes involved in cellular controls and transcription factors, namely as well as protein kinase C, RNA polymerase and reverse transcriptase the zinc-finger transcription factors. The intracellular free Zn2+ concentration is probably regulated by metallothionein and other zincis binding proteins, and the expression of the metallothionein gene itself controlled by Zn2+ via regulatory zinc binding proteins[5].However, the evidence for the involvement of Zn2+ in reproduction andgrowth control is relatively indirect. Dietary zinc is rapidly taken up by cell nuclei [6], zinc deprivation inhibits specifically cell growth and differentiation [7] whereas zinc restoration seems to trigger DNA synthesis and mitosis [S]. The intracellular homeostasis of essential metal ions is a sensitive target of toxic metal ions. Especially the cellularfree Ca2+ is highly vulnerable by heavymetals [9,10].Lowconcentrationsof cadmium ionsinduce inositol1,4,5-trisphosphateand Ca2+ mobilization in fibroblasts [l l], submicromolarlevelsofHg2+ionsamplifyreceptor-mediatedCa2+ signals in PC12 cells [l21 and micromolar concentrations of lead ions cause a sustained increase in the basal free Ca2+ level but diminish the hormone-stimulatedrise in free Ca2+ inbonecells [13 . It may be presumed that the nuclear calcium homeostasis and the dependent nuclearactivitiesalso are susceptibletointerference by toxicmetals. Cadmium activates nuclear protein kinase C [14],it inhibits the Ca2+ activated DNA fragmentation [ 151 and it stimulates the transcription of theprotooncogenesc-jun,c-myc [l61 and c-fos [17]. It still has to be elucidated whether the latter effects are caused by interference withCa2+ dependent gene expression or by direct interactions of toxic metal with proteins controlling transcription. Last but not least, the genotoxicity of radiation and chemical mutagens may be enhanced if toxic metal ions DNA. interfer with the function of Ca2+ in the repair of damaged

CaJ+

IL

CONTROL OF FREE Ca2+ CONCENTRATIONS IN CELL NUCLEI

To accomplish its control of nuclear activities, the intranuclear free Ca2+ concentrationmust be finelyadjusted in timeandspace.Table2 free Ca2+ summarizes reports whichdemonstratethatthenuclear concentration is not simply following changes in cytoplasmic free ~ a 2 + but is undergoingspecificmodulationwhencellsarestimulated by

Table 2. Reports on the nuclear control of free Ca2+ c

S'mooth muscle cells Williams et al. 1987 [28] Neylon et al. 1990 [29] Himpens et al. 1992 [30] lveurons and neuralcells Lipscombe et al. 1988 [3 l]

-

Nuclear Ca2+ is shielded from cytoplasmic stimulation Nuclear Ca2+ is lower in resting cells, but exceeds cytoplasmic Ca2+ upon stimulation No difference between cytoplasmic and nuclear ~ a 2 +in resting cells

Hernandez-Cruz et al. 1990 [32] P r z y w a r a et al. 1991 [33] Nuclear Ca2+ signal is larger than cytoplasmic Birch et al. 1992 [34] Nuclear Ca2+ signal is larger than cytoplasmic during neurite regeneration M-Mohanna et al. 1994:[35] The neuroblastoma cell nucleusis insulated from large cytosolic ~a2+ changes, but follows small changes rapidly G r M h f d o r - and hormone-stimulatedc e k Tucker & Fay 1990 [36] Waybill et al. 1991[37] Yelamarty et al. 1990 [38]

PDGF-stimulated fibroblasts exhibit similar Ca2+ transients in nucleus and cytoplasma EGF-stimulated hepatocytes show higher Ca2+ peaks in cytoplasma than in nucleus Eryhropoetin-inducedincrease in nuclear Ca2+in erythroblasts

Ca2+ uutake and releasefiver in cell nuclei Nicotera et al. 1989 [39], Hechtenbergsi Beyersmann 1993 [40] ATp-stimulated uptake of Ca2+ into isolated nuclei Nicotera et al. 1990 [41] Ip3-induced releaseof Ca2+ from rat liver nuclei Malviya et al. 1990 [42] 1p3 mediates ~ a 2 +release from rat liver nuclei 4

Regulation of Nuclear Calcium

and Zinc Interference

5

variousextracellularfactors. This conclusion has beenreachedfor stimulatedsmoothmusclecells,triggeredneuralcellsorregenerating neurons and hormone or growth factor stimulated cells. With isolated liver cell nuclei it has been demonstrated that ATP elicits the uptake of Ca2+ whereasinositol1,4,5-trisphosphateinducesthereleaseofCa2+ from nuclei (Table 2). All these findings suggest that the nucleus has additional mechanismsto controlits free Ca2+ levels.

EL

FURTHER CHARACTERIZATION OF THE CALCIUM UPTAKE AND RELEASE FROM LIVER NUCLEI

Bovine liver nuclei were loaded with the fluorescent metal chelator Fura2 in its acetoxymethyl ester form. The measurement of intranuclearfree Ca2+ was feasible since (i) the Fura-2 ester was taken up and (ii) it was hydrolyzed to the free dye by intranuclear esterase(s). The fluorescence was calibratedtogivethe intensityoftheCa2+-Fura-2complex Ca2+ intranuclearfree Ca2+ concentration.Figure1Ashowsthatthe uptake into liver nuclei is stimulated by ATP, whichis in agreement with nM extranuclear Ca2+, an results of other laboratories [39]. With 200 intranuclear Ca2+ concentration of 500 n M was reached.ATP also stimulated an increaseintotalnuclear C@+. I ~ M ATP causedthe intranuclear Ca2+ levelto rise to 150 nmoVmg DNA, i.e.,twicethe amount taken upin the absence of ATP. Also shown in Figure 1A is the Ca2+ rapidlossofaccumulatednuclearfreeCa2+afteradditionof ionophoreA23187.Subsequentapplication of thedetergentNP40 brought the nuclearCa2+ level down to the equilibrium concentration. TheATP-stimulated Ca2+ uptakeintolivernuclei was prevented by thapsigargin, an antagonistofthe Ca2+ pumpsof theendoplasmic with further reticulum (Figure 1B). Figure 2 summarizes results obtained an inhibitors.Vanadate, a typicalATPaseinhibitorandtributyltin, antagonist of membrane ion transport,also inhibited the uptake of Ca2+ into liver nuclei. At variance verapamil, a blocker of plasma membrane Ca2+ channels, was not effective as inhibitor of nuclear Ca2+ uptake. All these results are consistent with the assumption that the nuclear ~a2+ pump is related to that of the endoplasmic reticulum. In concordance, Lanini et al. did not find any difference in molecular weight, antibody Ca2+ ATPases of the binding and phosphoenzyme formation between the nucleus and the endoplasmic reticulum from rat[43]. liver

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800

Time (S)

Figure lk ATP-stimulated increaseof intranuclear free Ca2+. B. Inhibition of ATP-stimulated Ca2+ accumulation by thapsigargin.

Isolated bovine liver nuclei were treated with the following additions at thetimes indicated:200nM free Ca2+, 1 m M ATP, 250 nM thapsigargin (TG), 2 p M A 23187, and 0,05% NP40.

Regulation of Nuclear Calcium and Zinc Interference

7

I

Figure 2. Efffeets of inhibitors on ATP-stimulated intranuclear free Ca2+ uptake.

Isolated bovine liver nuclei were treated with 200 IIM C@+, 1mMATP and the following concentration of inhibitors5 min: for 100 pMvanadate, 250 n M thapsigargin, 5 p M tributyltin chloride, 100 p M verapamil. Regarding the release of Ca2+ from liver nuclei, two laboratories have 1,4,5-trisphosphate stimulates Ca2+ demonstrated that inositol mobilization [41,42]. Since the same second messenger also causes Ca2+ release from endoplasmic reticulum stores, it may be concluded that the Ca2+ mobilization mechanisms of both nuclear and endoplasmic This conclusion is further reticulum compartments are similar. substantiated by our finding thatalso GTP triggers a part~alrelease from liver nuclei (Figure 3A). This effect is dependent on GTP concentrations and it can be further enhanced in the ph siological range(50 - 500 m, if the Ca& reuptake is inhibited by thapsigargin (Figure 3B). The effect of GTP on Ca2+ release from nucleiis again similar tothe effect of GTP on Ca2+ mobilizationfromendoplasmicreticulumvesicles[44,45]. However, the mobilization of Ca2+ from the nucleus seems tobe subject of the to additional control, since the inositol l,4,5-trisphosphate receptor nuclear membraneis activated by proteinkinase C [46].

700

IA

GTP

3 300 V

3

2 200

L

E

U

100 0 0

100

200

300 500 400

600

Time (S)

B "

0

0

100

200

300 400 600 500 GTp(IrM)

Figure 3A. ATP-stimulated uptake and GTP-induced release of intranuclear Ca2+. B. Dependence of Ca2+ release from nuclei on the concentration of GTP.

Isolated bovine liver nuclei were treated with 200 n~ free and 1 mM ATP. At the times indicatedGTP was added at the NP40 was added concentrations given. Subsequently the detergent to afinal concentration of O,O5%. In the experimentshown in Figure 3B, 250 nM thapsigargin was added prior tothe administration of GTP. C $ +

8


Interference

9

NUCLEAR UPTAKE OF ZINC, CADMIUM AND LEAD IONS

N.

The same fluorescent dye, Fura-2, which is used to estimate free W + concentrations, can be used for the determination of free Zn2+; CM2+ and &+[47]. Figure 4 shows the spectral titration curves with Fura-2 in

800

f

a

B

E;

"1 D 1.6

l .o

0.5

a

. m

. p

. o

. y

. o

. ~

r

y

)

.

y

.

Y

)

. . . . - 3 2 0 3 1 o m m m

Excitation wavelength (nm) Excitation wavelength (nm). Figure 4. Excitation spectraof Fura-2 in the presenceof various concentrations of free metal ions. A. Spectrawith Ca2+ C. Spectra with Cd2+ B. Spectrawith Zn2+ D. Spectrawith Pb2+

The fluorescence emissionwas recorded at 5 10 nm. Free Ca2+ and Zn2+ concentrations are given in n M , free CM2+ and P@+ concentrations inPMin increasing order from bottom to top curve.

10


comparison. The Zn2+-Fura-2complex is excitated at a slightly longer complexwithanevenmore wavelength than therespective pronounced increase in fluorescence intensity. Similarly, Cd2+ and form fluorescent complexes with Fura-2. From the titration curves, the stoichiometry and the dissociation constants were calculated. the stoichiometry was 1:l in all cases, and the dissociation constants for the Ca2+, Zn2+, Cd2+, and P@+ complexes were 2.0.10-7 M, 1.5.10-9 M, 2.7*10-13M, and 2.1.10-12 M, respectively. The allinities of the metal ions forFura-2 are several ordersof magnitude greater that that of(h2+. To avoidfurther the interference of Ca2+ withtheZn2+-sensitive Ca2+ bywashingwith fluorescence,livernucleiweredepletedof chelatorcontaining bufferprior to the experiments with zinc. G & +

F @ +

Figure SA shows that the uptake of Zn2+ into liver nuclei was time- and of a membraneconcentrationdependent as with Ca2+. Addition impermeant metal chelator (DTPA) stopped the further uptake of Zn2+ butdidnotsigmiicantlychangetheZn2+dependentfluorescence. In a membrane-permanentchelator contrast,thesubsequentadditionof ("PEN) caused rapid a decrease in the Zn2+-Fura-2complex free Zn2+ fluorescence.Figure 5B demonstratesthattheintranuclear concentration is limited compared with thefree Zn2+ concentrationof the medium. this conclusion does not apply to the total nuclear zinc level whichincreased three- to fivefold over theextranucleartotalzinc 5A concentration, probably due to tight binding to nuclear protein. Figure further shows that the increase in nuclearfree Zn2+ is not stimulated by ATP. Furthermore, the intranuclear free Zn2+ level is not changed by thapsigargin andby the addition ofGTP to the medium(data not shown). These findings clearly demonstrate that the mechanism of Zn2+ uptake from that of uptake. into liver nuclei differs C @ +

Theuptake of Cd2+ and intoisolatedlivernuclei also can be measured by the Fura-2 fluorescence technique as described for Zn2+, still tighter than Zn2+. since the heavy metal ions are binding to the dye The nuclear uptake ofCd2+ and P$+ is shown in Figure 6 as a function AS withZn2+, 'the ofextranuclear free metalionconcentrations. intranuclear. free Cd2+ and &+concentrations stay well below the medium concentrations. Furthermore, as with Zn2+, no stimulation of Cd2+ or &+ uptake into nucleiby ATP was observed (datanot shown). In conclusion, thereis no activetransport system available to pumpCd2+ or &+ into cell nucleus, and the intranuclearfree ion concentrations of these heavy metals stay below those offered in the medium. p b z +

11


:i 2

0 100 200

0

300 400 500

600

700

Time (S)

-9

-0

-7

-6

Extranuclear fm Zn2+ (log M)

figure 5. Uptake of zn2+ into liver nuclei A. ~acreasein nuclearfree zn2+ concentration. Isolated liver nuclei were incubated with 10 nM (lower trace), 100 nM (middle trace), and316 nM free Zn2+ (upper trace).1 mM ATP, 200 p M DTPA, and 200 @ TPEN l were each added at the times and to the samples indicated in the figure. B. Dependence of Zn2+ uptake on the extranuclear free Zn2+ concentration.

Isolated bovine liver nuclei were incubated5 for min with various concentrationsof free Zn2+. (From ref.40 with permission)

12

Hechtenberg and Beyersmann 0.20

0.15

0.10

0.05

I

-12

I

I

-11

-10

1

I

I

I

-8

-7

I

-9

I

I

Extranuclear freeCd2+ (log M) 8 -

7

6

"

B

--

5

"

4

"

3

"

2

"

1

"

0-? -12

I

I

I

-1 1

-1 0

-9

I

II

-8

-7

Extranuclear freePb2+ (log M)

Figure 6. Uptake of Cd2+ and Pt?+ into livernuclei. Isolated bovine liver nuclei were incubated for 5 min with various concentrations of free Cd2+(A) and P@+ (B).


V.

Interference

13

INHIBITION OF NUCLEAR Ca2+ TRANSPORT BY Cd2+ AND Pb2+ IONS

Since the heavy metal ions interfer with fluorimetric assay of intranuclear special experimentalrecautionshadtobeused.Isolatednuclei P$+ solutions, washed with chelator were preincubated with Cdg or Ca2+ and ATP thereafter. With containing buffer, and supplemented with thenuclear Ca2+ accumulationwasdiminished bothCd2+and (Figures 7 and 8). From the concentration dependence (Figures 7B and 8B) it is estimated that halfmaximal inhibition fo Ca2+ uptake is obtained at 8 n M free Cd2+ and 14 nM free P@+, respectively. These results are Ca2+ pumpsof the consistentwith a closerelationshipbetweenthe nuclearenvelopeandthatoftheendo-/sarcoplasmicreticulum.Ina Ca2+ uptakeinto previous s t u d y , wealsoobservedaninhibitionof sarcoplasmic reticulum vesicles by cadmium and lead and estimated the halfmaximal inhibitory concentrations to be 450 n M free Cd2+ and 60 n M free P@+, respectively [48]. Ca2+,

p b 2 +

M.

CONCLUSIONS AND PERSPECTIVES

Thenuclearlevelsoffree Ca2+ and free Zn2+donotfollowthe cytoplasmicconcentrationsoftheseionspassivelybutarecontrolled differentially. the uptake ofCa2+ into nuclei is an active, ATP-stimulated process,and Ca2+ mobilizationfromnuclei is stimulated by inositol 174,5-trisphosphate and GTP. These controls allow a distinct regulation of intranuclear Ca2+ and Ca2+'calmodulin dependent nuclear functions. The uptake of Ca2+ into nuclei is inhibited by nanomolar concentrations of F@+, and probably by Hg2+, too. This mechanism of heavy free Cd2+ and metal toxicity extends thelist of known interferences of toxic metals with signal transduction steps such as signal reception, mobilization of second messengers and gene expression. Whereas the regulatory role of nuclear free Ca2+ is well established, a corresponding function of free in cell nucleiis still speculative. our data show that the level of free nuclear Zn2+ is limited, suggesting a barrier function of the nuclear membrane. Up to now, no mechanism for the stimulation of Zn2+ mobilizationin nuclei has been detected. On the otherhand,there are distinctexamplesofZn2+andCd2+mediated [49] controls of gene expression, i.e., via the activation of metallothionein and heat shock protein [50] promotors. The observed induction of the early control genes c-fos, c-myc and c-jun by Cd2+ may be mediated by &+

14


100

0

200

300

500

400

Time (S)

-12

-11

-10

-9

-8

-7

-6

Extranuclear free Cd2+ (log M)

Figure 7. Effect of Cd2+ on nuclearCa2+ uptake.

A. Time dependenceof inhibition by Cd2+. Nuclei were preincubated for10 min with l O n M free Cd2+, washed, and supplemented with200 nM free Ca2+ and 1 m M ATP at the times indicated. B. Dependence of the inhibition on the extranuclearfree Cd2+ concentration duringthe preincubation period.


15

600

Control

500 400

300 200 100

100

0

200

300

500

400

Time (S) I L"

IB

T

100

0

I

I

I

I

I

I

-11

-10

-9

-8

-7

-6

Extmnnclerr f m P@+ (log M)

Figure'8. Effect of P$+ on nuclear uptakeof Ca2+. k Time dependenceof inhibition by Pp+ B. Dependence of inhibition on extranuclear free Pp+ concentration during the preincubation period.

Experimental conditions werethe same as described in the legend to Figure 7 .

inte~erence of the metal ion wi homeos~sis. Thus, cellular balance r e n ~ a ~ oton p r o l ~ e r a ~ oinn a nongenoto interac~on.

A. PreDaration of nuclei Cell nuclei were prepared from bovine liver by homogenization in 50 mM Tris-HC1, 120 mM KC1, 4 mM MgC12, 0.2 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, 0.25 M sucrose adjusted to pH 7.5, and dfierential centrifugation as decribed elsewhere [40]. The nuclear fraction was virtually free of contamination by plasma membranes, microsomes and mitochondria, as checked by marker enzyme activities (5'-nucleotidase, alkaline phosphatase I, glucose-6-phosphatase7 succinate-IN" reductase). Purified nuclei were suspended in standard incubation medium containing 25 mM Hepes, 125 mM KCl, 2 mM K;?HPO4, 4 mM MgC12, 0.5 mM EGTA, 0.5 mM EDTA, 2 mM NTA, adjusted to pH 7.0.

C. Loading of nuclei with fura-2 and fluorescence measurements Isolated nuclei were preloaded with 7.5 jiM fbra-2 acetoxymethyl ester for 45 min at 4°C. The nuclear suspension was then washed twice in standard incubation medium. Fluorescence measurements were performed at 25°C in a dual-wavelength fluorescence spectrometer (Perkin-Elmer LS 50). Fluorescence intensity was monitored at an emission wavelength of 510 nm, by using a pair of excitation wavelengths at 340 nm and 380 nm. Free ion concentrations were calculated from ratio data as described by Grynkiewicz et al. [53], by using the Perkin-Elmer intracellular biochemistry application sohare. The apparent dissociation constants for the fura-2-complexes were determined as described by Kwan and Putney [54].

18


15.

RD. Lohmann and D. Beyersmann, Biochem. Biophys. Res. 190: 1097-1103 (1993). CO-.

16.

P. Jin and N.R. Ringertz, J. Biol. Chem. 265: 14061-14064 (1990).

17.

N. Tang andD.Enger, Toxicology81: 155-164 (1993).

18.

B. A. White,L.RL. Bauerle and F.C. Bancroft, J. Biol. Chem. 256: 5942-5945 (1981).

19.

T. Tmda, Y. Hamamori, Y.Fukumoto and Y. Takai, FEBS208: 39-42 (1986).

20.

J.I. Morgan andT. Curran, Nature 322: 552-555 (1986).

21.

M. Sheng, G. McFadden andM.E. Greenberg, Neuron4: 571-582 (1990)

22.

A. Schenthal, J. Sugarman, J. Heller-Brown, M.R Hanley andJ.R Feramisco, Proc. Natl. A d . Sci USA 88: 7096-7100 (1991).

23.

M. Sheng, M.A. Thompson andM.E. Greenberg, Science252: 1427-1430 (1991).

24.

E. C a r r , R. J. Galloway, A.H. Heid, L.L. Kaseem, G. Dhillon, H.G. Fein andRC. Smallridge, Biochemistry 30: 3721-3728 (1991).

25.

M.S. Kapiloff, J.M.Mathis, C.A. Nelson, C.R Lin and M.G. Rosenfeld, Proc. Natl. Acad. Sci. USA88: 3710-3714 (1991).

26.

J.D. Bartlett, J.D. Luethy, S.G. Carlson,S. J. Sollott and N. J. Holbrook, J.Biol. Chem. 267: 20465-20470 (1992).

27.

F. Liu, M.A. Thompson,S. Wagner, M.E. Greenberg and M.R Green, J. Biol. Chem.268: 6714-6720 (1992).

28.

D.A. Williams, P.L. Becker andF.S. Fay, Science 235: 1644-1648 (1987).

29.

C.B. Neylon, J. Hayland, W.T. Masonand RF. Irvine, Am. J. Physiol. 263: C95-C105 (1990).


Interference

30.

B. Himpens, H. desmedt, G.Droogmans andR.Casteels, Am.J. Physiol. 263: C95-ClO5 (1992).

3 1.

D.Lipscombe, D.V. Madison, M. Poenie, H. Reuter,RY. Tsien

19

and R.W. Tsien, Proc. Natl. Acad. Sci. USA85: 2398-2404 (1988) 32.

A. Hernandez-C-, F. Sala and

P.R Adams, Science 247 858-

862 (1990).

33.

D.A. Przywara, S.V. Bhave, A. Bhave, T.D. Wakade andA.R Wakade, FASEB J.5: 217-222 (1991).

34.

B.D. Birch, D.L. Eng andJ.D. Kocsis, Roc. Natl. Acad. S c i . USA 89: 7978-7982 (1992).

35.

F.A. Al-Mohanna, K.W.T. Caddy andS.R Bolsover, Nature367 745-750 (1994).

36.

RW. Tucker and F.S. Fay,Eur. J. Cell Biol. 51: 120-127 (1990).

37.

M.M. Waybill, RV. Yelamarty, Y. Zhang, RC. Scaduto, Jr., K.F. LaNoue, C.-J. Hsu, B.C. Smith, D.L. Tillotson, F.T.S. Yu and J.,Y. Cheung, Am. 3.Physiol. 261: E49-57 (1991).

38.

RV. Yelamarty, B.A. Miller,RC. Scaduto, Jr., F.T.S. Yu,D.L. Tillotson and J.Y. Cheung, J. Clin. Invest. 85: 1799-1809 (1990).

39.

P. Nicotera, D. J. McConkey, D.P. Jones andS. Orrenius, Proc. Natl. Acad. Sci. USA 86: 453-457 (1989).

40.

S. Hechtenberg andD. Beyersmann, Biochem. J.289: 757-760 (1993).

41.

P. Nicotera, S. Orrenius, T. Nilsson and P.-O. Berggren,Proc. Natl. Acad. Sci. USA 87: 68584862 (1990).

42.

A.N. Malviya, P. Rogue andG. Vincendon, Proc. Natl. Acad. Sci. USA 87: 9270-9274 (1990).

43.

L. Lanini,0.Bachs andE. Carafoli, J. Biol. Chem.267: 1154811552 (1992).

20


44.

V. Heme and H.-D. Stiling, FEBS 202: 267-273 (1986).

45.

D.L. Gill, T. Ueda, S.-H. Chueh and M.W. Noel, Nature 320: 461464 (1986).

46.

N. Matter, M.-F.R&, S. Feyermuth, P. Rogueand A.N. Malviya, J. Biol. Chem. 268: 732-736 (1993).

47.

P.M. Hinkle, E.D. ShanshalaII and E.J. Nelson, J. Biol. Chem. 267 25553-25559 (1992).

48.

S. Hechtenberg and D. Beyersmann, Enzyme45: 109-115 (1991).

49.

D. Hamer, Annu. Rev. Biochem. 55: 913-951 (1986).

50.

M. Fischbach, E. Sabbioniand P. Bromley, Cell. Biol. Toxicol. 9:: 177-188 (1993).

51.

A. Fabiato, Meth. Enzymol.157 378-417 (1988).

52.

RM. Smith and A.E. Martell, Critical stabilityconstants. Vol. 1-6, Plenum, New York.

53.

G. Grynluewicz, M. Poenie and RY. Tsien, J: Biol. Chem. 260: 3440-3450 (1985).

54.

C.-Y. Kwan and J.W. Putney, Jr., J. Biol. Chem. 265: 678-684 (1990).

2 Effects of Antioxidants, Zinc, and Chelators on Free Radical Status of ChildrenLivingintheChernobyl Area Ljudmila G . Korkina Lab, Cell Biophysics and Biochemistry, Russian Institute of Pediatric Hematology, Leninskii pr. 117, Moscow 117513, Russia

Igor B. Afanas’ev 117820, Russia

VitaminResearchInstitute,Nauchnypr.

14A, Moscow

L INTRODUCTION Is the exposure of a human organism to prolonged low level irradiation really dangerous? This question now is not a theoretical one but one of utmost.importance for the people (the adults and especially children) living in the areas contaminated with radioisotopes after the accidents at atomicpowerstations and atomicreactors(ChemobylinUkraine, Chelyabinsk in Russia, etc) and exposed to low-levelbutcontinuous irradiation..Untilnowtheattempts to answer it wereunsuccessful possibly due totwo major remons: (1) Many classical hematologicaland biochemical testsarenotspecificallydesignedfordetection of such effects; (2) To make statistically significant conclusions, it is necessary to study a sufficiently large human populationexposed long enough to lowlevel irradiationto detect any real changes. Itis nowwellestablishedthat damaging effects of irradiationare mediated by the active o&gen species formed as a result of the direct interaction of y- and p-rays with the molecules of oxygen, water, and organiccompounds [ 1,2] . Moreover,incorporatedradioisotopesmay influencetheendogenoussystemsproducingactive oxygen species, 21

22

and

Korkina

Afanas'ev

for example, blood leukocytes, which is thought to be a main endogenous sourceofthesespecies [3]. Thesespecies(theinorganicradicals superoxide ion and hydroxyl radical, oxygen-centered organic radicals and hydrogen peroxide) are ableto interact practically with all important biologicalsubstances(lipids,proteins,nucleicacids,polysaccharides etc.) changing their properties and causing irreversible damage. As long as radiation has always been the part of environment, human beings have adapted to some extent to the potential risk of irradiation. Therearepowerfulantioxidantsystems in humanorganismswhich protectthecellsandnon-cellularmaterialagainstfreeradicals.They consist of enzymes such. as superoxidedismutases (SOD), catalase, glulathioneperoxidase,andglutathionereductaseandvariousnonenzymatic compounds (reduced glutathione, iron-binding proteins, antioxidant vitamins,uric acid, and so on). It is believed that disbalance between the rates of fkee d i c a l production and their inactivation by antioxidant defense systems may lead to pathologiessuch as tumors, degenerative, age-associated, environment-induced diseases and inflammatory and allergic states [4]. Most of these pathologies may be initiated by high-level or chronic low-level irradiationof an organism[5]. Therefore, it is veryimportant to detectthe fust subtlesignsof prmxidant/antioxidant disbalance before the development of irradiationinduced disorders. The main aim of present work is to study comparatively large groups of children from irradiated areas in orderto evaluate the very first features of thedeleteriouseffectsofaccidentalirradiation, to obtainnew knowledgeofpossiblemechanismofthedevelopment of disorders associated with low-level irradiation, andto develop non-toxic long-term administration capable of preventing irradiation-induced disorders. We investigated the effects of irradiation on the children who had been living for 6 years now in the regions contaminated with radioisotopes aftertheChemobylaccident.Contaminationlevelsintheseregions ranged from 15 to 40 Gykm2. Several major parametem of the organism's free radicalstatus including oxygen radical generation in the wholebloodandbyisolatedleukocytes,theactivitiesofantioxidant enzymes, and the changes in glutathione metabolism have been determined. The frequency of spontaneous chromosome breakage in the blood lymphocytes as well as the amount of &-staining material in the lymphocyte nucleolus wasalso determined.

Free Radical Status of Chernobyl Children

23

IL SUBJECTSAND METHODS k Children 156 Chernobyl .children of both sexes (62 males and 94 females) were practically healthy without any serious disorders in the main biochemical and hematologicalparameters.Thechildren'agerangedfrom 8 to 14 years (an average agewas of 11.4-2.1 years). For comparison, 21 healthy childrenofthesameagerange f i o i the Moscow region were tested after their informed consents. After preliminary testing, 70 Chernobyl children with the enhanced level of spontaneous luminol-amplified CL in isolated leukocytes were selected for clinical trial and randomized into 5 groups: Group A:16 children were given 400 lipoic mg acid day. a Group B: 14 children were given 400 mg lipoic + acid 200 mg vitamin E a day. Group C: 14 children were given 200 mg vitaminE a day. Group D: 14 children were given 100 mg zinc aspartate a day. Group E: 12 children without any treatment (control group). h the beginning and after completing the alinical trial, pediatrician examinationandallstandardhematologicalandbiochemicalanalyses essential for the evaluation of general living functions have been carried out. It was shown that there were no any side effects, allergic reactions, and other adverse events induced by the drug administration.

B. Chemicals Vitamin E was t?om Henkel Co. (Germany), lipoic acid (Tioctacid)was fi-om ASTA Medica (Germany), zinc aspartate was from Dr. F.Kohler ChemieCO(Germany).Luminol,lucigenin, phorbol-12-myristate-l3acetate (F'MA), reduced (GSI-I) and oxidized glutathione (GSSG), glutathione reductase, latex particles of 1 pm diameter,andcolchicine werepurchased from SigmaChemical Co., USA. Bovineerythrocyte superoxidedismutase(CuZnSOD, EC 1.15.1 .l) and thymol free bovine liver catalase @C I .l 1.1.6) were from Serva, Germany. Dextran sulfate and sodium metrizoate were from Phamacia, Sweden.

24

Korkina and Afanas'ev

Phitogemagglutinin (PGA) was h m Difco-P, USA All commercialreagentsandsolventswere of the highest purity.

other

C. Leukocyte and erythrocyte preparation.

5.0 m1 venousbloodwas drawn .beforebreakfast at 7.30 a.m.by disposable syringes with20 U/ml of heparin as an anticoagulant. 1.5 m1 blood sample was layered on the equal volume dextranof metrizoate mixture ( 5 2 v/v of 6.2% dextran and 38% metrizoate) and sedimented at 25% for 30 min. The cell-rich Supernatant was centrifuged at 15% for 10 min, and cell pellets were washed twice with cold Hanks' balancedsaltsolution (HBSS). Finally,thecellswere resuspended in medium 199 endstoredat 4% duringexamination. Cell viability was assessed by exclusion of 0.1% trypan blue; usually, it exceeded 95%. Cell differential count was confimed microscopicallybyGiemza , & S a &l suspensionconsisted of PMNs (50-60%), monocytes (4-10%) andlymphocytes (3045%). The contaminationwithplateletsanderythrocytes was negligibleanddid notaffectchemiluminescenceparameters. Erythrocytes wereobtained after the blood sedimentation on dextran-metrizoate gradlent. and washed twice with a cold 0.1M potassium phosphate buffer (pH 7.2). Washed cells were resuspended in the same buffer and their amountwas ajusted to 2% of hematocrit.

D. Chemiluminescence measurement of oxygen radical production by blood celis.

Luminol-amplifiedchemiluminescence(CL) was measuredon a LKB luminometer(mod. 1251,Sweden)equippedwith a computer to control experimental conditions, the data registration and estimation. 20 pL whole blood diluted 10 times with HBSS or 20 pL leukocyte swpension ( 2 ~ 1 0cells/ml) ~ wereadded to 0.85 m1 HBSS containing 50 p M luminol and incubated in the lml polysterene cuvette of the CL unit at 37O C andcontinuousmixingfor 5 min.Stimuli (PM 100 ng/mlor0.1%latexsuspension)weredispensedintothe CL cuvette automatically, and the light emission was recorded each 10 sec. Maximal intensity ofspontaneousandactivated CL was registered,andresults were expressed in mV/ 106 PMNs.

Radical Free

25

Status of Chernobyl Children

E. Determination of glutathione metqbolism in erythrocytes. The contents of reduced (GSH) and oxidized (GSSG) glutathione were determined bythe Beutler method[6].

F. The measurement of CuZnSODandMnSODactivitiesin erythrocytes and leukocytes.

Superoxide dismutase (SOD) activity was determined by the adrenaline method [7], in which the rate of superoxide production WBS measured by lucigenin-amplified CL [8]. Heparinized venous blood (0.2 ml) was hemolyzed with ice-cold water. Lysate was added to the equal volume of ethanol-chloroform mixture (1:1 v/v) and centrifuged at 1 500 x g for 30 min. Protein content in supernatantwas determined by the Lowry method [g]. The total SOD activity was calculated fiom calibration curve using commercial SOD as a standard and expressed as U/mg protein. For the determination of MnSOD activity, CuZnSOD WBS inhibited bythe additionof NaCN (4 to the supernatant. After that, the CL measurement of WSOD activity was performed as described above. CuZnSOD activity was estimated as a differencebetween the total and &SOD activities.

m

G. Measurement of spontaneous chromosome aberrations in lymphocytes

The whole blood was cultured accordingto the standard procedures [lo] with culture medium containing 75% Minimal Essential Medium, 15% fetal calf serum, and 10% whole blood. Lymphocytes were stimulated to divide by the addition of PHA at 0 h o€ culture. Blood was cultured at 37% in the 25 mL culture flasks for 56 h. 2.5 h before cell fixation, colchicine was added to a final concentration of 0.25 mg/ml. Cell fixation and the cytogenetic analysis were carried out by standard methods [l l]. 200-300 cells were analyzed for each subject.

H. Determination of &-staining material

in the lymphocyte

nucleolus.

Blood smears were prepared,fmed with methanol,-andstained with silver nitrate accordmgto method described previously[121. The stained smears

26

Afanas'ev

Korkina and

were examined microscopically and the results were expressed as average number of granules for300-350 nuclei analyzed.

L Statistical evaluation Statistical analysis was performed using Student's t-test assumed 5% as a level of significant difference.

EL RESULTS All Chernobyl children exhibited the strong featuresofinternal irradiation: the individual doses of incorporated radioisotoS being equal to 3 m 4 0 nCi and the excretion of radioactive isotope 7Cs with urine was w b i the rangeof 12-180 B&. Cytogenetic analysis showed that in comparison with normal cells the circulating lymphocytes from Chernobyl childrenhadasignificantlyhigherlevel of chromosomeaberrations especially slngle and double chromosome breakages comparing with the control children (Table 1). We found a greater number of Ag-staining granules in the nucleolus of Chernobyl children' lymphocytes (82% of children had from 1 to 5 granules per a nucleolus, 15% - from 6 to 10 granules, and 3% - more than 10 granules per a nucleolus) comparing with normalgroup (97% ofchildrenhadfrom1 to 5 granules pera nucleolus and3% - from 6 to 10 granules).

r

TABLE

1 3romosome aberrations in the blood lymphocytes. Number of Number of Subject . aberrant cells metaphases scored (percentage) 526 18,568 Chernobyl children (2.83%) (n = 98) p 0.001 Control 65 5,604 children (1.16%) (n = 22)

"

Frequency of aberrations ".

0.030 p 0.001 0.013

Radical Free

27


To evaluate the oxygen radical productionbybloodleukocytes, the luminol-amplified CL ,a sensitive and specific methodof measuring therelease of reactiveoxygenspecies @OS) bystimulated and nonstimulated phagocytic cells [131 was applied. We measured spontaneous CL and the CL producedby theleukocytesstimulated with PMA (thesolubleactivatoractingthroughprotein kinase C receptor) and latex particles (an activator of phagocytosis). As is seen from Table 2, bothnonstimulatedandstimulatedleukocytesisolated from the blood of Chernobyl children produced a significantly greater amount of ROS than those of Moscow children.

TABLE 2

CuZnSOD activity

133&21

(24)

61k5

MnSOD activity (Wmg protein)

3139

(24)

15G

(Uhg protein)

*) Standard deviation

(22)

28

and

Korkina

Afanas'ev

Thus, the initial spontaneous CL in isolated leukocytes nearly doubled that in noma1 children Thesametendency m observed for ROS production in the whole blood: the intensities of spontaneous and latexstimulated CL were equal to 10 and 210 mV/lO3 cells, respectively, for irradiated children andto 3 and 70 mV/lO3 cells for donors.We found a good correlation between spontaneous CL intensities and the number of Ag-staining granules in Chernobylchildren'lymphocyteswiththe correlationcoeffkient being equal to 0.83 and 0.80 forisolated lymphocytes and the whole blood, respectively. There was no significant difference in the erythrocyte GSH content between Chernobyl and Moscow children, however, the GSSG concentration and,Correspondingly, the GSSG/GSH ratio was 1.S times higher for Chemobyl children (Table 2). It was also found that both CuZnSOD andMnSOD activities were two times higherin the blood of Chemobyl children (Table 2) than in the blood of Moscow children Surprismgly, SODS activitiesdid not correlate with the intensities of spontaneous or activated CL as well as with the content of Ag-staining material in the nucleolus. Administration of the combinationof lipoic acid + vitamin E (Group C), zincaspartate (Group D), and a-lipoic acid .(Group A) led to significant changes in spontaneous CL of isolated leukocytes: its values diminished by4.67, 3.42, and 1.59 times, respectively (Fig. 1). It is seen that the levels of spontaneous CL achieved normal values in groups C and D that was confirmed statistically. In contrast, neither the combination of vitamin E + a-lipoic acidnor its componentstaken separately affected significantly the levelsof P M - and latex-stimulated CL @8ta are not shown). At the s m e time, Zn aspartate adminiStr3tiOn resulted in a decrease in PMA- and latex-stimulated CL by 40% and 25%, respectively. Thecombination of vitamin E and a-lipoicacid as well as Zn aspartate turned out to be very effective inhibitorsof PMA-activated CL in the wholeblood(Fig. 2). The same antioxidants significantly improvedglutathionemetabolism by decreasingthe GSSG level and, correspondingly, lowering theGSSG/GSH ratio down to its normal value (Data are notshown).Neitherlipoicacidnovitamin E takenalone affected the GSSG content.


Group Healthy Group Group Group B C D donors A

29

Group E

Fig. 1. Spontaneous chemiluminescencein the isolated blood leukocytes before ( 0 ) and after ( H ) the clinical trial. Group A:children were given 400 mg a-lipoic acid a day for 28 days; Group B: 400 mg a-lipoic acid + 200 mg vitamin E; Group C: 200 mg vitamin E, G~oupD. 100mg zinc aspartate a day, Group E: control.

TV.DISCUSSION Our results show thatcontinuouslow-levelirradiationdoesaffect the Gee radical status of children living in the areas contaminated with radioisotopes. There are three different important parameters characterizing the intensity of the organism's oxidative stress, which were affected by low-level irradiation. 1. Spontaneousluminol-amplified CL of nonstimulated("dormant") leukocytesin the absence of anystimulusand CL of theleukocytes stimulated with either a soluble receptor-associated agonist (Pm)or a

30

Korkina and Afanas'ev

3004

v

Healthy Group Group Group Group Group

donors

A

B

C

D

E

Fig. 2. PMA-activated chemiluminescence in the whole blood after the clinical trial.

particular activator acting through an unreceptor pathway (latex particles) were enhanced by 2.2,5.0, and 1.8 times, respectively. The same tendency was observed for the whole blood. 2. The GSSG level andthe GSSG/GSH ratio in erythrocytes increased by 1.5times. 3. There was a significant (approx.200%) increase in the CuZnSOD and

MnSOD activities. It is known that under physiological conditions the level of oxygen radicals released' by "dormant" i.e. nonstimulated leukocytes normally is very low, but it sharply increases during a respiratory burst triggered by endogenous and exogenous stimuli such as microorganisms, tumor cells, immunecomplexes,complementcomponents,lymphocytes,etc.[ 1l]. Certain pathological condltions may drastically enhance oxygen radcal production by blood leukocytes through the so-called "priming"effect, which can be induced by inflammatory agents, allergenes, y-interferon,

Radical Free


31

tumor necrosis factor,growth factors, and some other biologically active compounds [123. In this study we have found for the fmt time that continuous low-level irradiation couldbe the in vivo 'priming" agent enhancing the production of active oxygen species by circulating blood leukocytes (and perhaps tissue phagocytes and bone marrow cells). It is a potentially dangerous symptom because "primed" cells can continue the inflammatory process which in turn can be the cause of the enhanced frequency of mutations, tumor initiation and promotion [13]. Thus, a significantly higher than normal level of chromosome breakage, which was found for Chernobyl children (Tablel), may be partly dueto the oxidative stress mediated by c i r c u l w leukocytes (Table2). The occurence of a pennanent source of oxygenradicaloverproductionwithmutageniccapacityapparently significantly enhances therisk of tumor development. It should be noted that occasional CL measurements, which were perfomed by us 3 years ago, did not show a large increase in the oxygen radical production by leukocytes of Chernobyl children. Therefore, the possibility of developing "free radical" pathologies obviously becomes higher with the prolongation of living in the contaminated areas. An increaseintheGSSGlevel(Table 2) is directevidence of the beginning of antioxidant depletionin the blood of Chernobyl children.It is known that free radicals fmtly attack the low-molecular antioxidants such as glutathione reduced, ascorbic acid (vitamin C), and a-tocopherol (vitamin E). A is virtually impossible to detect the effect of low-level irradiation on ascorbic acid and a-tocopherol because the amount of these vitamins in the organism dependson the qualityof the food consumed. It isalsoimpossible to detectsmallchanges in GSHconcentration; however, there was a significant increase in the GSSG concentration and the GSSG/GSH ratio. It is known that GSSG is a toxic compound andits formation is specificfortheconditions of oxidativestress [l 31. Therefore, the GSSG/GSH ratio is apparently one of the most important. an parameterscharacterizingtheprooxidantlantioxidantbalancein organism,and itsincreaseadditionallyindicatestheprevalence of oxidative processesin an irradiated organism. An increase in SODS activities (Table 2) in the blood of Chernobyl children maybeinterpreted as theresponseof an organism to the radiation-inducedoxygenradicaloverproduction.Indeed,it has been shown [ 14,151 that human and animal organisms as well as

32

and

Korkina

Afanas'ev

microorganismsexposedtoradiationareable to enhanceantioxidant defense systems via the induction of antioxidantenzymes,whichare thought to beregulatedbythelevel of ROS. It was shown by us previously [161 and in present work that Chemobyl children exhibited ROS overproduction.Actually,theexpressionof growing number of genesencoded SODS, DNA repair' enzymes,proto-oncoproteins,heat stress proteins, and others is now known to be modulated by oxidative stress [17]. This gene overexpression leadsto enhanced FWAdependent proteinsynthesis[l8]. It has beenreportedpreviously[l9]thatthe variability of the amount of nucleolar Ag-staining material reveals the degree of nucleolar, mainly transcriptional activity. Therefore, it seems to be of importance that there is a strong correlation between intensities of. oxygenradicalproductionandtheamountof &-staining materials, because this fact may reflect the process of adaptationto oxidative stress, which is talung place in Chernobyl population. Our results apparently suggest the benefits of longterm administration of nontoxic compounds topreventthedevelopmentof"freeradical"pathologies in subjects (children and adults) living in the areas contaminated with radioactive materials.Thesepreventingandtherapeuticagentsarelikely to be classicalantioxidants(such as vitamin E), zinc-containing drugs, and transition metal chelators.To the bestof our knowledge, there areat least four major parthways of suppressing free radical-initiated processes and reducingoxidative stress: a) the scavenging of freeradicals ; b)the stimulation of the antioxidantenzyme activities; c)the suppression of the activities of enzymescatalyzingtheoxygenradicalformation;d)the chelation and inactivation of active transition metalions, which catalyze the productionof hydroxyl and hydroxyl-like radicals. There is a Jarge body of evidence that shows lipoic acid exhibits both antioxidant a d chelatingactivities[20,211,and that vitamin E is a powerfid natural lipid-soluble antioxidant[22]. It was also sugested that non-transitionmetalsarecapableofindirectlyaffectingfreeradical processes.Atpresent,aspecialattention is drawn to theantioxidant activity of zinc compounds. Thus it wasfound that zinc inhibited the formation of free radicals in the in w'tro systems containing iron ions and cysteine, decreased the level of peroxidation products formed in erythrocyte membranes exposed to x a n h e oxidase, suppressed damagng free radical processes in cultured hepatocytes and superoxide production by human neutrophils, and protected E.coZi bacteria against


33

copper-mediated paraquat-induced damage [23-271. Animal studies confirm the antioxidant capacity of zinc in vivo [28]. We found that short-term admjnistration of the combination of lipoic acid with vitamin E and zinc aspartate substantiallydecreased the level of spontaneous oxygen radical production by isolated blood leukocytes.At the same time, the combination of vitamin E and lipoic acid failed to inhibit PMA- orlatex-activatedoxygenradicalgenerationbyisolated cells. h contrast, zinc aspartate inhibited both spontaneous and activated oxygen radical production (Fig. 1 and 2). However, both the combination of lipoicacid with vitamin E andzincaspartatewereveryeffective inhibitors of spontaneous andactivated oxygen radical productionin the whole blood. Theabovefmdmgs could be explainedbythe direct inhibition of leukocyte NADPH-oxidase by zinc cations (our unpublished d ata)but this is not the case for vitamin E or a-lipoic acid. Apparently, vitamin E and a-lipoic acid acted as effectivescavengemof free radicals, while zinc aspartate is believed to be a regulator for cellular oxygen radicalproducing systems [27]. Thus our results show that vitamin E and lipoic acid are effective inhibitors of oxygen radical production by "primed" leukocytes but practicallydo not influenceNADPH oxidase activity and leukocyte activation. At the same time, vitaminE + lipoic acid and zinc aspartate significantly enhance the oxygen radical-scavenging activity of bloodplasma,that is apossiblecause of theinhibitionoflatexstimulated CL in the whole bloodof Chemobyl children.

ACKNOWLEDGMENTS Dr. F. Kohler Chemie GmbH company provided financialsupport for the clinical trial. We appreciate very much the advice of Elena Samochatova

in themedicalpartoftheclinicalstudy.TatianaSnigireva, Galina Ibragimova, and Irina Deeva provided valuable technical assistance in the laboratory work. REFERENCES 1. C.L.Greenstock, in Free Radicals, Aging, and Degenerative Diseuses, Alan R. Liss, Inc., 1986, pp.509-526.

34Afanas'ev

and

Korkina

2. J.F.Ward, A h . Radiat. BioL 5: 181, (1975). 3. B.M.Babior, Blood 64: 959 (1984). 4. B.Halliwel1and J.M.C.Gatterige, Free Radicals in Biology and Medicine. W o r d University Press, 1984. 5. C.L.Greenstock, Advances in Radiation Biology 11: 269 (1982). 6. E.Beutler, Red Cell Metabolism,Grune & Stratton, N.Y.1975, pp. 6971 and 112-1 17 7. H.Misra andL Fridovich, J. BioL Chem. 247: 3170 (1972). 8. H. Gyllenhanunar, J. ImmunoL Methods 97: 209 (1987). 9.O.H.Low1-y~ N.J.Rosebrough,AL.Fm, and R.J.hdal1, LBioLChem 193s 265 (1951). 10. RC-Allen and L.D.Loose, Biochern Biophys Res Commun 69: 245 (1976). 11. I.B.Afanas'ev, Superoxiide Ion: Chemistry artd Biological Implicatiom CRC Press, Bocs Raton, 1991; v. 2: pp. 87-134. 12. T.HFinkel, MJ.Rabst, RSuzulu, L.AGuthrie, J.RForehand, W.APhillips, and RB.Johnston, JBiolChem 262: 12589 (1987). 13. C.W.White and J.E.Repine, in Superoxide and Superoxide (G.Rotilio, Ed.), Dismutase in Chemistry,BiologyandMedicine Elsevier Sci. Publ., Amsterdam, 1986:pp. 524-527. 14. IFridovich,HorizBiochemBiophys., 1: 1 (1974). 15.T.Nakata, K.Sumki, J.Fujii, M.khikawa, H.Tatsumi,T.Sugiyama, T-Nishida,TShimizu, M.Yakushiji, and N.Taniguchi, Carcinogenesis 13: 1941 (1992). 16. L.G.Korkma, LB.Afanas'ev, and AT.Diplock, BiochemSoc.Trans 21: 314s (1993). 17. HSies, Amer: . l Mea! 91(3C): 31S(1991). 18. G.Storz,L-ATartaglia,and 'B.N.Ames, Science 248: 89 (1990). 19. D.Hemandez-Verdun,J. Cell S&. 9 9 : 465 (1991). 20. Y .J . S d , MTsuchiya, and L.Packer, Free RadRes. Comms. 15: 255 (1991). 21. L.Mullerand H.Menzel, Biochim Biopkys. Acta 1052: 386 (1990). 22. G.W.Burton, AJoyce, and K.U.Ingold, ArchBiochemBiophys. 221: 281 (1983). 23. C.Coudray, S.Rachidi, and AFavier, Biol Trace Element Res. 38: 273 (1993). 24. AW.Girotti,J.P.Thomas, and J.E.Jordan, Free Rad BioL Med 1: 395 (1 985).

35


25. P.H.Bemrik, P.C.Branneh and S.S.Hurles,J.Clin

Lab.ImrnunuL 21:

71 (1986).

26. M.-J.Richard,P.Guiraud, U-T.Lessia, J.-C.Beani, and AFavier, Bioi!

Trace Element Res. 37: 187 (1993). 27. M.Chvapi1, L.Stankova, C.Zukoski LLAClinMed 89: 135 (1977).

W, and C.Zukiski III,

28. D.E.Coppen, D.E.Richar&on, and R.J. Cousins, Proc. Soc. Exper.

Bioi! Med. 189: 100 (1988).

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3

The Role of Ascorbate in Metabolism and Genotoxicity of Chromium(VI) Karen E. Wetterhahn,DianeM. Stearns, Manoj Misra,Paloma H. Giangrande, Laura S. Phieffer, Laura J. Kennedy, and Kevin D. Courtney Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766

I.

INTRODUCTION

Epidemiological studies have shown that chromium(VI) [Cr(VI)] compoundscause cancer, chromeulcersand allergiccontactdermatitisinpopulationsexposedto chromiumintheworkplace [1,2]. In addition to occupational exposure, significant environmental exposure to carcingenic chromium has been shown to occur because of landfill and waste sites contaminated with Cr(VI) [3,4]. For example, over 130 different sites in HudsonCounty,NewJerseyhavebeenfoundto contaminatedwithwastefrom Cr(V1) manufacturing plants [3]. Topical solutions of ascorbate have been used to prevent skin problems, i.e., ulcers and contact dermatitis, in workers exposed to Cr(VI) [2,5]. However, there is a considerable lack of information regarding the role of 37

38

Wetterhahn et al.

ascorbate (vitamin C) in Cr(VI)-induced genotoxicity both in vitro and in vivo. Intracellular reduction of the carcinogen Cr(VI) is widely considered to be a prerequisite for Cr(VI)-induced DNA damage [6]. Ascorbate has been shown to be the major reductant of Cr(VI) in rat liver, kidney and lung tissues [A. In vitro EPR studies indicate that reaction of Cr(VI) with ascorbate produces Cr(V), Cr(IV), ascorbate radical and othercarbon-centered radicals species [8]. Spectroscopic studiesindicatethatthefinal chromium(II1) product complexes with ascorbate and oxidized forms of ascorbate that are produced in the reaction [g]. Although Cr(VI) itself is unreactive toward DNA, chromium binding to DNA and DNA strand breaks are observed when DNA is reacted with Cr(VI) in the presence of ascorbate [lo]. The level of DNA damage induced by Cr(VI) in the presence of ascorbate appears to correlate with the levels of Cr(V) and radicals detected by EPR. In contrast to the glutathionemediated reaction of Cr(VI) with DNA which results in glutathione-chromium-DNA adducts [ll], it appears that the ascorbate-mediated reaction does not result in crosslinking of ascorbate to DNA by chromium. Ascorbate is required as a vitamin in the diet for the Osteogenic Disorder Shionogi (ODS) rat, making it an ideal in vivo animal model in which to study the effect of ascorbate on Cr(VI)-induced genotoxicity. Ascorbate was required for binding of chromium to DNA in liver of ODS rats treated with Cr(VI). Little hepatic Cr-DNA binding occurred upon Cr(VI) treatment of ascorbate-deficient ODS rats fed 5 ppm ascorbate in the diet. High Cr-DNA binding was observed in liver of ODS rats fed200 ppm ascorbate in the diet, a level which supports normal growth, andlower but significant Cr-DNA binding was observed at800 pprn dietary ascorbate. The hepatic Cr-uptake and glutathione

Ascorbate in Metabolism and Genotoxicity

of Chromium(V1)

39

levels were similar at 5, 200, and 800 ppm ascorbic acid in the diet. Depletion of glutathione caused a decrease in hepatic Cr-DNA binding in the ODs rats fed 200 and 800 ppm dietaryascorbate,suggesting thatglutathione is involved in Cr-DNA binding in the presence of normal ranges of ascorbate concentrations. These results indicate that ascorbate plays a critical role in activation of Cr(VI) to produce Cr-DNA binding in vivo. 11.

SPECTROSCOPICCHARACTERIZATION OF SPECIES FORMED IN THE REACTIONOF CHROMIUM(VI) WITH ASCORBATE

Thegeneralaim of the invitro studieshasbeento determine the types of species that are produced during the reaction between Cr(VI) and ascorbate, and to evaluate the factors which may influence the reactivity of these species. Varying the buffer or the ascorbate concentration relative to Cr(VI) was found to produce intermediates in differentrelativeamounts.Oncethesespecieswere identified and their relative stabilities established, their reactivity with DNA could be studied in vitro. A.

Reactive intermediates observed by EPR spectroscopy

Thereaction of Cr(V1) (1.8 mM, from K2Cr207) with (0, 0.5, 1.0, 1.5, 3.0, 5.0, and 10 sodiumascorbate stoichiometric equivalents) was studied by EPR spectroscopy in twodifferent buffers, N-[2-hydroxyethyl]piperazine-N"[2-ethanesulfonic acid] (HEPES, 0.10 M) and tris(hydroxymethy1)aminomethane hydrochloride (Tris. HC1, 0.050 M) at pH 7.0, at room temperature. Unstable free radicals were detected by reaction with 5,5-dimethyl-lpyrroline-l-oxide, (DMPO, 0.10 M). Chromium(V)and free radical EPR signals were quantitated as described [8]. Representative spectra havebeen published [8,10].

40

Wetterhahn et al.

A Cr(V) EPR signal was observed in HEPES buffer at g = 1.980 at the 0.5:l and 1:l reaction ratios of ascorbate to Cr(VI). Chromium(V) levels corresponded to 0.3 k 0.1 per cent of chromiumasCr(V)forthesereactionratios, No Cr(V) was respectively at a 1.3 minreactiontime. detected above the 1:l ratio in HEPES, or at any reaction ratio in Tris-HC1buffer.

Chromium(IV) levels were estimated by reaction of Cr(VI) (1.8 mM) with ascorbate (0 - 18 mM) in the presence of 3.6 mM manganese(I1) (from MnC12.4H20) through quantitation of the decrease of themanganese(I1) EPR signal after a 1.3 min reaction [8]. Concentrations ofCr(IV) were found toincrease with increasing ascorbate to Cr(VI) reaction ratio and ranged from 15 k 5 to 37 k 8 per cent of chromium as Cr(IV) for the 0.5:l to 10:l reaction ratios in both buffers. Carbon-based radicals were observed as DMPO-radical adducts in both HEPES and Tris-HC1 buffers [8]. These radicalswerepresumedtobeproducts of the Cr(V1)induced fragmentation of ascorbate. Carbon-based radicals were observed only at low reaction ratios (0.5:l and 1:l) of ascorbate to Cr(VI). Unlike the Cr(V) intermediate, levels of carbon-based radicals were lower in HEPES than in Tris-HC1buffer by approximately 3-fold, corresponding to 0.06 per cent starting ascorbate for the 1:l reaction ratio in HEPES buffer and 0.2 per cent starting ascorbate for the 1:l reaction in TrissHCI buffer [g]. The ascorbate radical anion was observed in both buffers during reaction of Cr(VI) with ascorbate [g], and was not trappedby DMPO. Levels of ascorbateradical anion increased with increasing reactant ascorbate concentration

Ascorbate in Metabolism and Genotoxicity of Chromium(VI1

41

for the 1.3 min reaction and ranged from 0.5 to 1 pM for the 0.5:l to 10:l ratios in HEPES and Tris-HC1buffer. B.

Finalchromium(II1)product observed by Whisible spectroscopy

Chromium(II1)levels weredeterminedbyUV/visible spectroscopy for the reaction of 1.8 mM Cr(VI) with 0 - 18 mM ascorbate in HEPES and Tris-HC1buffers (pH 7.0) at 1.3 min reaction and 30 minreactiontimes atroom temperature [9]. For all reaction ratios the spectra over time showed a decrease in the Cr(VI) charge transfer band at 370 nm and an increase in chromium(II1) visible bands at -400 nmand 570-580 nm. Levels of chromium(II1) increasedwithincreasingascorbateto Cr(V1) reaction ratios. Concentrations of chromium(II1) for the 1.3 min reaction ranged from 37 to 92 per cent of total chromium for the 0.59 to 1O:l ratios in HEPES, and 37 to 87 per cent in Tris-HC1. The finalconcentrations of chromium(II1) afterreactionshadgonetocompletion at 30 min correspondedto 43 to 100 percentchromiumas chromium(II1)forthe 0.5:l to 1O:l reactionratios, respectively, in bothbuffers. The final km,, for chromium(III) varied between buffers and reaction ratios. For 30 min reactions in HEPES buffer the 0.5:l reaction showed a visible absorbance at 568 nm spectrum with an whilethe 1 O : l reactiongaveafinal absorbance at 584 nm.Reactions in TrisaHCl buffer showed a final kmax of 558 nm and 580 nm for 0.5:l and 1O:l reactions, respectively. These differences likely reflect of thefinalchromium(II1) a differentcoordination productdependingonthe relativeascorbatereaction concentration.Inthepresence of excess ascorbatethe chromium(II1)wouldpresumablybecoordinatedby ascorbate, whereas in reactions with excess Cr(VI) the only

42

Wetterhahn et al.

potential ligands for chromium(II1) would be dehydroascorbate or, more accurately, its ring open form diketogularic acid [12]. Spectra of reactions of 10-fold ascorbate or dehydroascorbate withchromium(III) support this hypothesis. Differences between HEPES and Tris-HC1 bufferssuggestinteraction of theTrisbufferin chromium(II1) coordination. C.

Summary of intermediates and products produced from reaction of chromium(V1) with ascorbate

To summarize, the reaction of Cr(VI) with ascorbate at pH 7.0 produced Cr(V), Cr(IV), chromium(III), carbon-based radicals, andascorbateradical anion. The relative concentrations of these intermediates could be altered by changing the reaction ratio and/or buffer. Chromium(V) and carbon-basedradicalswereonlyobserved at low reaction ratios in HEPES buffer, and carbon-based radicals butnoCr(V)wereobservedin Tris.HC1 buffer. Chromium(IV),chromium(II1)andascorbateradical predominatedathigh reactionratios inbothbuffers. These differences allowed for evaluation of the reactivity of these intermediates toward DNA. 111.

DNA DAMAGE RESULTING FROM THE REACTION OF DNA WITH CHROMIUM(VI) AND ASCORBATE

Unliketheothergenotoxicmetalsknowntomediate indirect oxidative damage of DNA[l31 Cr(VI)-induced DNA damage also includes direct chromium binding to DNA, as well as single-strand breaks, DNA interstrand cross-links and DNA-protein cross-links [14]. The role of theabovedescribedintermediatesintheformation of chromium-DNAadductsandsingle-standbreakswas evaluated in calf thymus and pBR322 plasmid DNA.

Ascorbate in Metabolism and Genotoxicity of Chromium(V1)

A.

43

Direct chromium binding to calf thymus DNA

Binding of chromium to calf thymus DNA was determined for DNA (1.8 mM DNA-P) incubated with Cr(VI) (1.8 mM, from K2Cr207) and ascorbate (0 - 18 mM) in HEPES and Tris-HC1 buffers (pH 7.0) after a 30 min incubationat 37°C. Non-covalently boundchromium was separated from DNA by NENsorb chromatography following published procedures [1l]. Concentrations of chromium and DNA in reaction samples were determined by atomic absorption spectroscopy and the diaminobenzoicacid (DABA) fluorometricassay [15], respectively, from which a chromium to DNA ratio was calculated (Cr/DNA-P). Levels of chromiumboundto DNA werehighestfor reactions of 1:l ascorbate to Cr(VI) in HEPES buffer and correspondedto 29.3 & 6.1 x 10-3 Cr/DNA-P. The 1:l reactioninTris-HC1bufferresulted in 8-fold lower binding, or 3.7 f 1.1x 10-3 Cr/DNA-P [lo]. Preincubation of Cr(VI) with ascorbatefor 30 min at 37°C priorto incubationwith DNA resultedindecreasedlevels of chromium bound to DNA, as did reaction of calf thymus DNAwithsolutions of chromium(II1) andvarying ascorbate that had been preincubated for 30 min at 37°C. Based on the above spectroscopic studies, these results are consistent with the interpretation that Cr(V) is the form of chromium reacting with calf thymus DNA. B.

No formation of ascorbate-chromium-DNAadducts

The reduction ofCr(V1) by GSH in the presence of calf thymus DNA was shown to result in a GSH-Cr-DNA of adduct [ll]. In order to evaluate the analogous role ascorbate in chromium-DNA adduct formation the DNA bindingexperimentsdescribedabovewerecarried out

44

Wetterhahn et al.

with 14C(l)-labelled ascorbic acid. Calf thymus DNA (1.8 mM) was incubated with Cr(VI) (1.8 mM) and varying Wascorbic acid (0 - 9.0 mM) in either 0.10 M HEPES or 0.050 M Tris.HC1 buffers at pH 7.0 for 30 min at 37°C. Amounts of chromium and DNA-P were determined as described above, and levels of W-ascorbate were determined by scintillationcounting. The presence of Cr(V1) didnot result in ascorbate binding to DNA above control levels. C.

Single-strand breaks in pBR322 plasmid DNA

Single-strand breaks in pBR322. DNA were measured by gelelectrophoresisafterreaction of plasmid (0.36 mM DNA-P, 82 nMplasmid)with Cr(V1)(1.8mM, from K2Cr207) and ascorbate (0 - 18 mM) in HEPES and TrisoHC1 buffers (pH 7.0). Plasmidwasincubated with reaction solutions for 30 min at 37°C. Relaxation of supercoiled plasmid was visualized by ethidium bromide staining and quantitated by scannirig densitometry. Amounts of relaxedplasmidwerehighest for the 1:l reaction ratios and were 2-fold higher for reactions in Tris-HC1comparedto HEPES buffer. Based onthe spectroscopic studies presented above, this observation suggested that carbon-based radicals rather than Cr(V) were responsible for the plasmid relaxation. This hypothesis was tested by carrying out reactions at the 1:l ascorbate to Cr(VI) ratio in the presence of 0.10 M DMPO whichwasshownby EPR spectroscopytoreactwith carbon-based radicals but not Cr(V). Levels of plasmid nicking were found to decrease in both buffers in the presence of DMPO. Fromtheseexperimentsitwas concludedthatplasmidrelaxationwaspredominantly caused by the carbon-based radicals produced during the reduction of Cr(VI) by ascorbate.


IV.

45

IMPLICATIONSFOR IN W O GENOTOXICITY

The results of the in vitro spectroscopic and DNA damage experiments with Cr(VI) and ascorbate are summarized in Table I.

Effect of the ascorbate concentrationon reaction intermediates andDNA damage produced during the in vitro reduction of chromium(V1) by ascorbate.

TABLE I.

Asc : Cr(VI) Reaction Ratio

1O:l

1:l

Chromium(V) Chromium(1V) Chromium(II1) Carbon-Based Radicals Ascorbate Radical

none

++

++ + + ++ +

Cr-DNA Adducts Plasmid Relaxation

none none

max max

++ ++

none

Chromium(III) was found toreact with DNA quite slowly when it was fully coordinated by ascorbate or oxidized products compared to "uncoordinated" hexaaquo chromium(II1). Fullycoordinatedchromium(II1)isthe likely form that will be produced by the in vivo reduction of Cr(VI); therefore, our results are consistent with the hypothesis that the more reactive intermediates Cr(V) and carbon-basedradicalsareresponsible for the Cr-DNA adducts and plasmid relaxation in vitro, and that these types of intermediates will likely be the genotoxic agents in vivo. Theformationand/ordecay of the reactive intermediateswasstrongly affected bythereactant ascorbate concentration. The hypothesis that the in vivo

et

46

Wetterhahn

al.

Cr(VI)-induced DNA damage would also be affected by intracellular ascorbate concentration was tested in a rat model. V.

EFFECTS OF ASCORBATE ONCHROMIUM(V1)-INDUCED DAMAGE IN ODSRATS IN W O

A.

Animal model

Most in vivo experimentalanimals,includingnormal species of rats, biosynthesize L-ascorbic acid. Humans cannot synthesize L-ascorbic acid and thus require it as a vitamininthe diet. In order tomimicthe range of vitaminCfoundinhumans,ananimalrequiring Lascorbic acid as a vitaminis needed. The ODS rat is a mutantWistarratwhich lacks Lgulonolactone oxidase (GLO, EC1.1.3.8), the enzyme which catalyzes the last step in the biosynthesis of ascorbic acid [16,17]. Studies by Horio et al. [l81 have shown that the ODs rat grows normally and shows nosigns of vitamin C deficiency when maintained on a diet containing -300 ppm ascorbic acid. TheODS rat was used to study the effects of ascorbate on Cr(VI)-induced genotoxicity in vivo. B.

Diets andtreatments

The ODS rats were initially purchased from Clea Japan, Inc., Japan and bred at Veterans Administration Medical and RegionalResearchServiceCenter,Vermont.The ascorbic acid-free (basal) and supplemented diets were purchased from Zeigler Bros.,Inc., Gardners, PA, USA. Six-eight week old male ODS rats were usedin the present study. The rats were kept at 24°C with a 12-h light cycle (from 0800-2000 h) and dark. All ratswerehousedin duplicate and were provided feed and water ad libitum.


47

Before the experiment, rats were divided into six groups each consistingof 6-8 rats and two of each group were fed abasaldietsupplementedwith 5, 200, and 800 pprn ascorbate for 14-days. On the 14th day, groups of each diet were injected intraperitoneally (i.p.) with 10 mg of sodium dichromate/kg body weight for 4 h with or without 5 h pretreatment of 4 mmol of L-buthionine-S,R-sulfoximine (L-BSO)/kg body i.p. Before diets were fed to rats, diets supplemented with ascorbic acid were tested for actual ascorbic acid content by the HPLC-ECD method described below. C.

Dietary ascorbate and hepatic ascorbate levels

Ascorbatelevelsweremeasuredbyhighperformance liquidchromatographywithelectrochemical detection (HPLC-ECD) using a modified method of Berger et al. [19]. The hepatic ascorbate level was about 10-fold higher in rats fed 200 pprn ascorbate and about 16-fold higher in rats fed 800 pprn ascorbate than ratsfed 5 ppm ascorbate in diet for 14 days (0.05 f 0.007, 0.51 k 0.072, and 0.93 k 0.1 pm01 ascorbate/g tissue in rats fed with 5, 200, and 800 ppm ascorbate in diet for 14-days, respectively). BSO treatment hadno effect onhepaticascorbate levels although glutathione (GSH) levels were decreased 60-65%, suggesting that there is no direct relationship between GSH and ascorbate levels in ODS rats. Cr(VI)-treatment in BSO-pretreated rats fed 800 pprn ascorbate increased the ascorbate level as compared to saline or Cr(VI)-treated rats suggesting asynergistic effect of Cr and GSH-depletion on ascorbate levels. However, no effect of Cr(VI) on ascorbate 5, 200, and 800 ppm levels wasobservedinratsfed ascorbate and in BSO-treated rats fed with 5 and 200 ppm ascorbate in the diet.

48

D.

Wetterhahn et al.

Ascorbate levels and Cr-uptake

Pre-weighed liver was digested with nitric acid and H 2 0 2 essentiallyasdescribed by Veillon and Patterson [20]. Chromiumwasdeterminedbyatomicabsorption spectroscopy on a Perkin-Elmer 503 atomic absorption spectrophotometer with an HGA-2100 graphite furnace. Different levels of hepatic ascorbate did not affect Cruptake levels. In rats fed 5, 200, and 800 pprn ascorbate, the levels of hepatic Cr-uptake were0.54-0.75 pm01 of Cr/g tissue in Cr(V1)-treated rats. Also, BSO-pretreatment did not affect the levels of Cr taken up in livers of Cr(V1)treated rats. This result suggests that hepatic Cr-uptake is not dependent on ascorbate or glutathione levels. E.

,

Ascorbate and hepatic Cr-DNA binding

Liver nuclei were isolated [21] and DNA was isolated and analyzed using the fluorescent dye Hoechst 33258 method [22]. Chromiumwasdetermined by atomicabsorption spectroscopy as described above. The level of Cr-DNA binding in liver was about 9-fold higher in Cr(VI)-treated rats fed 200 pprn ascorbate than rats fed 5 pprn ascorbate, but Cr-DNA binding was about 2.5-fold lower in Cr(V1)treated rats fed 800 ppm ascorbate than rats fed 200 ppm ascorbate. BSO-pretreatment decreased Cr-DNA binding in rats fed 200 and 800 pprn ascorbate in diet. A significant 2.8-fold decrease in hepatic Cr-DNA binding was observed 200 pprn inCr(V1)-treatedBSO-pretreatedratsfed ascorbate. These results indicate concentrationa dependent role of ascorbate in Cr-DNA binding in liver and also suggest that glutathione is involved in Cr-DNA binding in the presence of normal ranges of ascorbate concentrations.

Ascorbate in

G.

Metabolism and Genotoxicity of Chromium(VI1

49

Summaryof in vivo results

This is thefirstworkinanintactanimalsystem demonstrating the effect of dietary ascorbate on Cr-DNA binding in vivo. The results of the in vivo experiments with Cr(VI) in ODs rats are summarized in Table II.

II. Effect of different levels of ascorbate in diet on liver ascorbate, hepatic Cr-uptake and Cr-DNA binding in ODS rats after 4 h treatment with sodium dichromate.

TABLE

Ascorbate in Ascorbate diet (ppm)

5 200 800

+ ++

+++

Cr-DNA binding Cr-uptake -BSO -BSO +BSO +BSO

+ l +

+ +

+ +

+

+++ ++

I none

++ +

The availability of the ODS rats, which like humans, do not biosynthesize ascorbate, allowed us to evaluate the effect of dietary ascorbate levels on Cr-DNA binding in liver. Despite similar Cr-uptake at all levels of ascorbate tested (5, 200, and 800 ppm), Cr-DNA bindingwas significantly different. We found that dietary supplementation of ascorbate in amounts that supported normalgrowth of theanimals (200 ppm)weremost potent in inducing Cr-DNA binding inliver,whereas (5 ppm) little Cr-DNA binding was observed at lower dietary ascorbate, and higher (800 ppm) ascorbate levels caused less Cr-DNA binding than was induced by 200 ppm.Our resultsalsosuggestarole for glutathione, another endogenous antioxidant and reductant of Cr(VI), in influencing Cr-DNA binding. Thus for the first time, we have identified a function of ascorbate in facilitating

50

Wetterhahn et al.

Cr-DNA bindinginanintactanimal. These results indicate that ascorbate plays a critical role in activation of Cr(VI) to produce Cr DNA binding in vivo. ACKNOWLEDGEMENTS

This work was supported by PHS Grant No. CA34869, National Cancer Institute, DHHS (KEW). DMS was supported bya postdoctoral fellowship from the Norris Cotton Cancer Center, DartmouthHitchcock Medical Center, and an NRSA Fellowship (CA59292) from the National Cancer Institute, DHHS.PHG wassupported bya HowardHughesUndergraduate Biological Science Research Internship. LSP was supported by a Dartmouth College Presidential ScholarResearchAssistantship and a Waterhouse Research Grant. LJK and KDC were supported by the NSF Research Experiences for Undergraduates Program (NSF CHE-9100493).

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9.

S. Langard, Am. J. Ind. Med. 17189-215 (1990). N.B. Pederson, Top. Environ. Health 5:249-274 (1982). T. Burke, J. Fagliano, M. Goldoft, R. Hazen, R. Iglewicz, and T. McKee, Environ. HealthPerspect. 92:131-137 (1991). C.D. Palmer, and P.R. Wittbrodt, Environ. Health Pwspecf. 92:2540 (1991). D. Burrows in Chromium:Metabolism and Toxicity, CRC Boca Raton, Fla, 1983, pp. 137-163. P. H. Connett, and K. E. Wetterhahn, Structure and Bonding 5493124 (1983). (a) Y. Suzuki, and K. Fukuda, Arch. Toxicol. 64169-176 (1990). (b) A. M. Standeven, and K. E. Wetterhahn, Carcinogenesis 12:1733-1737 (1991). (c) A. M. Standeven, and K. E. Wetterhahn, Carcinogenesis 13:1319-1324 (1992). D.M. Steams, and K. E. Wetterhahn, C h . Res. Toxicol. 7219230 (1994). D. M. S t e m , L. J. Kennedy, K. D. Courtney, P. H. Giangrande, L. S. Phieffer, and K. E. Wetterhahn, Biochemistry, submitted for

publication.

Ascorbate in Metabolism and Genotoxicity of Chromium(VI1

51

10. D. M. Steams, K. D. Courtney, P. H. Giangrande, L. S. Phieffer, and K. E. Wetterhahn, Environ. Health Perspect. 202(Suppl 3):2125 (1994). 11. K. M. Borges, and K. E. Wetterhahn, Carcinogenesis 10:2165-2168 (1989). 12. B. M. Tolbert, and J. B. Ward, (1982) in Ascorbic Acid: Chemistry,

Metabolism and Uses (P. A. Seib, and B. M. Tolbert, Eds.), 200, Washington, DC., 1982, pp. Advances in Chemistry Series 116-118. 13. K. S. Kasprzak, Chem. Res. Toxicol. 4:604-615(1991). Z16914. (a) S. De Flora, and K. E. Wetterhahn, LifeChem.Reports 244 (1989). (b) S. De Nora, M. Bagnasco, D. Serra,and P. Zanacchi, Mutat. Res. 23899-172(1990). 15 J. M. Kissane, and E. Robins, J. Biol. Chem. 233984-188 (1958). 16. (a) S. Makino, and K. Katagiri, Exp. Anim. (inJapanese) 29:374375 (1980). (b) Y. Mizushima, T. Harauchi, T. Yoshizaki, and S. Makino, Experientia, 40: 359-361 (1984). 17. N. Nishikimi, T. Koshizaka, H. Mochizuki, H. Iwata, S. Makino, Y. Hayashi, T. Ozawa, and K. Yagi, Biochem. Int., 16:615-621 (1988). 18. F. Horio, K. Ozaki, A. Yoshida, S. Makino, and Y. Hayashi, J. Nutr. 115~1630-1640(1985). 19. J.Berger, D. Shepard, F. Morrow, and A. Taylor, J. Nutr. 119:734740 (1989). 20. C. Veillon and K.Y. Patterson in Environmental carcinogens

.

selected methods of analysis. Vol. 8--Some metals: As, Be, Cd, Cr, Ni, Pb, Se, Zn. (ONeill, I.K., Schuller, P., and Fishbein, L., Eds.) M C ,1986, pp 433-439. 21. H.S. Lilja, E. Hyde, D.S. Longnecker, and J.D. Yager, Jr. Cancer Res. 373925-3931 (1977). 22. C.Labarca and K. Paigen, Anal. Biochem. 102:344-352 (1980).


Inactivation of Critical Cancer-Related Genes by Nickel-InducedDNAHypermethylation and Increased Chromatin Condensation: A NewModelfor EpigeneticCarcinogenesis Max Costa Nelson Instituteof Environmental Medicine, N W Medical Center, Long MeadowRoad, Tuxedo, NY 10967

I.

INTRODUCTION

For a numberof years ithas been known that nickel carcinogenesisadds a unique component and is synergistic to the carcinogenesis process of other genotoxic carcinogens such as W, benzopyrene, and x-rays (1). The most carcinogenicnickelcompounds

are generally water insoluble andare

phagocytized by target cells (2). Phagocytosis permits large quantities of nickel to enter cells allowing rapid dissolution of Ni2+ by the acid pH of the phagocytic vacuoles (3). Solublenickel salts are generally not highly carcinogenic

in

experimental animals because the uptakeof soluble Ni*+is very poor, however, in tissue culture situations where extracellular exposure can be maintained, soluble nickelsalts are active (2). Crystalline NiS induces a number of genotoxic effects in cells including the production of strand breaks,DNA-protein crosshks, and generationof oxygen radicals that produce oxidized DNA bases (2). However, the majorityof these effects occurin heterochromatic DNA which

is not genetically active andthus has little mutagenic consequence (2).This may

explain why nickel compounds that are potently carcinogenic are generally not mutagenic in most systems (2). 53

54

Costa In the present report, we describe a new model forthe mechanism by

whichnickelmayproduce

its mostprominent

genetic effects during

that carcinogenesis. This model is based upon our previously reported findings

nickel inducesDNA hypermethylation and therebyturns off a senescence gene during the courseof nickel-induced transformation of Chinese hamster embryo cells (4). 11.

PHAGOCYTOSISAND DELIVERY OF NICKELCOMPOUNDS INTO THE CELL Using two model compounds, carcinogenic crystalline nickel sulfide and

non-carcinogenicamorphousnickel

sulfide that havebeen

studied by

Sunderman and colleagues for carcinogenic activity in experimental animals(5), we investigated the molecular mechanisms responsible for the differences in the carcinogenicity of these compounds(2). In cultured hamster cells, we found that the crystalline nickel sulfide particles w.ere actively phagocytized to a much greater extent than the amorphous nickel sulfide particles (3). Phagocytosis depended upon the surface charge, and we found that the crystalline nickel sulfide particles were negatively charged whereas amorphous nickel sulfide of particles were positively charged(6,7). When we changed the surface charge amorphous nickel sulfide particles by treating them with lithium aluminum hydride, we activated their phagocytosis and their cell transforming activity to levels similar to thatof crystalline nickel sulfide(8). This is shown in Tables 1 and 2. Water soluble nickel saltsare not potentially carcinogenic because they do not yieldhigh intracellular levelsof ionic nickel(2). Thus, the abilityof nickel is of obvious critical importance to its carcinogenic action (3). It to enter the cells

is puzzling though with nickel oxide that thelow temperature calcined black

form is more potentthan the high temperature green form, but it was difficult to

Gene Inactivation by Ni-Induced DNA Hypermethylation

55

Table 1 Phagocytosis of solvent washed andLiAlH4 reduced crystalline and amorphous NiS particles in CH0 Cells

Treatment compound Amorphous NiS Untreated Pyridine washed LiAlH4 reduced in pyridine

9.9 f 2.05 27.7 f 0.90

CrystallineaNiS Untreated Pyridine washed LiAlH4 reduced

29.2 f 252 33.3 f 9.8 53.1 f4.59

4.8 f 0.16

CH0 cells grownin monolayer culture were exposedin 60 mm diameter tissue

culture dishes to 10 pg/ml (1.78 pg/an3 surface growth area) of the particle preparations for 24 hr. Following treatment, cells were fixed, stained, and the percentage ofcells containing intracellular particles was detennined by light microscopy. The particle size of the preparations ranged from1p m to 3.6 p, and within this range there was little effect of particle size on phagocytosis. Each number shownin the tableis the meanof 4 plates where at least 500 cells were examined in each plate. No. of cells with phagocytized NiS particles/total no. of cells examined, meanf S.D.for 4 plates. Uptake differs from untreated particles. Pd.05 Student t-test

Reproduced with permission fromRef. 9.

study phagocytosis of the oxide particles. Perhaps there is some component involving membrane interactions that contributes to the carcinogenic process as well. This phenomenon has not been well investigated, however, there is evidence that signal transduction can substantially affect gene expression and one cannot rule out the possibility that these particles may perturb membrane function related to signal transduction. Following phagocytosis of insoluble particles, Ni2+ dissolves from the particle and high intracellular levels of nickel are generatedas modeled in Figure 1.

56

Costa

Table 2 Enhancement of cellular transformation following lithium hydride reductionof Crystalline and amorphous nickelsulfide particles Treatment compound

Transformed coloNes/total pg/d growth area

AmorphousNiS particles 1.0 p, (0.39) untreated 2/510 1.27

pyridine 2.66 pm,washed 1.27

17 5

10

6

1.27 2.07 pmwashed ,pyridine

0.13 0.63

(0.49)

10/2054

8

(0.41)

8/1949

9

1

0.13

5

5 10

0.63

8

1/636 (0.15)b 1/614 (0.16)b 3/248 (1.21)b

3

5/1088 (0.46)b

L i A l Q reduced 1 2.26 pm,reduced in pyridine 5 1.27 10

Crystalline aNiS particles 237 p, untreated

No.ofplatessurvivingcolonies(%.)a

0.13 3 (2.42)c 0.620/826

5

6 5

(1.47) 0.63 17/115!5

6

10 10/1784 1 6 5

10

6/183 (3.27)c

(2.67)

0.13 0.63 li7

5

4

27/1026 (2.63)c W268 (4.47)b

0.13 0.63 1.27

3 9 7

4/264 (151)d 24/972 (2.47)c 40/534 (7.49)C

(0.56)d

"

U A l Q reduced

1 3.76 pm,reduced in pyridine 5

10

The cell transformation assay was conducted as d e s c n i . ?he particle sizeof each preparation is shown with the treatment compound. aNo. of transformd colonies/total no. of surviving colonies bNot statistically significant from exposure to corresponding untreated particles CDiffers significantly from corresponding untreated particles: P 4 0 0 1x2test dcorresponding concentration not available for statistical analysis Reproduced withpermission from Ref. 9.


57

Figure 1 111.

INTERACTION OF NICKEL WITH HETEROCHROMATIN

A promhent site for genetic damage induced by nickel compounds in

mammalian cellsis within heterochromatic chromosomal regions. This has been shownforbothChinesehamster

and mousecellswhichhave

different

organizations of their heterochromatin(10). In fact, the modeof delivery of Ni2+ into the cellis very important in inducing heterochromatin damage.If Ni2+ is delivered throughliposomesorby

particle phagocytosis, it damages

heterochromatin (11). If Ni2+ is presented to a cell as a soluble salt, it cannot enter the cell very well, andit also undergoes ligand exchange reactions such that most of the nickel ionsare not available for reactingwith nuclear proteins (11).

Figure 2 shows the damage that nickel selectively induces in

heterochromatin in Chinese hamster cells. This damage is illustrated by what appears to be decondensation of the heterochromatin, however, a mitotic chromosome is maximally condensed and this apparent decondensationis not relevant in a non-mitotic cell. In non-mitotic cells Ni*+ inducesan increase in

58

Costa .

...

d

Fig. 2 Chromosome damage inducedin Chinese hamster ovary cellsby NiC12 and NiS. Cells were treated for 16 h with 1 mM NiC12 and mitotic cells were collected following colcemid exposure. Figure 2A shows a cell where the heterochromatic long arm of the X chromosome was not properly condensed during mitosis. Figure 2B is a photograph of a Chinese hamster ovary cell X chromosome after the cells had been treated with crystalline NiS particles. Cells were treatedwith crystalline NiS particlesranging in concentration from10 to 20 pg/ml for 24 to 48 hr. The figure shows an increasing degreeof fragmentation of the long arm of the X chromosome with higher dosages and longer exposure times (a-i). Note the absence of fragmentation of the euchromatic short arm. from Refs. 11and 17. Reproduced with permission

Gene Inactivation Ni-Induced by

DNA Hypermethylation

59

A

i

=-

A

Fig. 3A C-banding of parental BALB/c 3T3 (1)and B200 cell (2) chromosomes. Arrows, regions of heterochromatic DNA.

Fig. 3B Localization of mouse satellite sequencesof BALB/c 3T3 (1) and B200 cells (2). Chromosomes of BALB/c 3T3 cells and B200 cells were hybridizedin situ with atritium-labeledmousesatellite DNA probelabeled by nicktranslation. The hybridized sequences were visualized by autoradiography and by Giemsastaining. Reproduced with permission from Ref. 13.

60

Costa

chromatin condensationas the resultof substituting for divalent metal ions, such as magnesium

(12).

The acquisition of nickel resistance in mousecells is

associated with fusionsof heterochromatin as shown in Figure3 (13). It should be noted that DNA-protein crosslinks were found to be induced selectively in heterochromatin in cells treated with nickel compounds (2) and that nickel bound selectively to magnesium insoluble fractions of heterochromatin (14,15). There is substantial evidence that nickel interacts with heterochromatin, and the selective interactionsof nickel with heterochromatin may explain why nicke! compounds are not generally mutagenic.

W.

INTERACTIONS OF NICKEL WITH MAGNESIUM BINDING

SITES IN CELLS

It has been known for some time that excess magnesium can antagoniz nickel-induced carcinogenesisin uivo (16). We have previously reported that magnesium can antagonize nickel-induced damage to heterochromatin (17) and that nickel-induced cell transformation can be inhibited by elevating the extracellular levelsof magnesium (17). Nickel and magnesium have very similar is thought to interact with magnesium binding in sites the atomic radii, and Ni2+

cell (17). This is particularly interesting with regard to heterochromatin iswhich maintained in an increased condensation state by several factors of which magnesium is thought tobe of great importance. Table3 shows the antagonism of nickel-inducedcelltransformation magnesium, and Table

4

by increasingtheconcentration

of

illustrates the antagonism of nickel-induced

heterochromatic damage by increasing the magnesium concentrations in the cell (17). Clearly, magnesium can inhibit nickel-induced heterochromatic damage

and its carcinogenesis. Therefore, there mustbe some relationship between the carcinogenesis. heterochromatic damage induced by nickelitsand


61

Table 3 The effect ofM S 1 2 on NiC12 or K2CrOq-induced SHE cell transformationa

Treatment MgC12 Incidence morphological of survival SHE cell (mM) transformation

in SHE cells

(transformants/survivingcolonies)

0/680

0.8

0

5oow NE12

0.2 0.8 5 20

0/235 (0) (0) 0/130 (0)

94 100 71 59

0.2

11/901(1.2) 1/431(0.2) 1/565(0.2) 0/755 (0)

24 29 24 29

ND 21/149(1.4) 9/1039 (0.9) 1/1667 (O.l)b

ND 24 18 24

5/984(0.5) 10/1240 (0.8) 10/1028 (1.0)

18 24 18

5 20 0.2lOOOpM NE12

0.8

5 20 10W 0.8K2CrO4

("huntreated) of

0.2 20

om1

(0)

%HE cells were incubated for 2 h with Mg2+a "-free E M supplemented with 10% FBS and varying concentrations of MgC12. NiCl2 or K2CI04 were then addedanfor

additional 24 h. Following treatment,cultures were allowed to incubate for 14 days. Cultures were fixed, stained, and morphological transformation was determined. bp4.005 compared with 0.8 mM Mg&, Xi-ltest. Reproduced with permission from Ref. 17. How did nickel produce suchhigh a incidence of 6-TG resistance in G12 cells? There wasno structural mutations detected ingptthe gene, however,in 27 out of 31 "mutants" induced by nickel, there was hypermethylation in the coding and flanking regions of this gene (24). T h i s DNA methylation was associated

with the acquisition of decreased DNase1 sensitivity and decreased MSPl degradation of the gene (24). Additionally, thegpt gene was readily reactivated by treatment of cells with azacytidine which induces hypomethylation of DNA (24). It is clear that nickel can induce hypermethylation of genes that inactivates theirexpression.

This is alsoassociated

with increases in chromatin

is no longer expressed. condensation such that the gene

62

63

64 V.

Costa

NICKEL-INDUCEDOMDATIVEDNADAMAGE There is no question that nickel compounds can produce oxygen radicals

both in vi& and in vim. However, the way they produce oxygen radicals is very iron,cobalt different than the way most metals with active Fenton chemistry(e.g.,

and copper) produce oxygen radicals. Oxygen radicalsare very important in metal carcinogenesis processes (18). We have shown that nickel increases oxygen radical levels in intact cells (19). Kasprzak and others have shown that oxygen radicals can be produced by nickel in vitro (20). The interesting point about nickel-induced oxygen radical formationis that Ni2+ binds to certain peptide of Ni2+ from1.09 v to about0.79 ligands andthis lowers the oxidation potential v such that Ni2+ can now be oxidized by hydrogen peroxide to generate Fenton chemistry (21). Nickel may thus produce oxygen radicals at selected binding sites. However, the end result is that mostof the oxidative damage produced by nickel

OCCUTS

in heterochromatin which is genetically inactive DNA and,

therefore, nickel does not produce mutations in most cells. This may explain why the oxygenradicalsinducedbyNi2+

are of little direct mutagenic

consequence.

VI.

NICKELINDUCED DNA METHYLATION A number of years ago, in studies attempting to understand the

mechanism by which nickel induces carcinogenesis through its interaction with heterochromatin, we discovered

that male Chinese hamster embryo cells

transformed by nickel compounds exhibited the of losssenescence gene activity associated with the X chromosome (4,22). The

X chromosome contains the

longest contiguous regionof heterochromatin in the Chinese hamster genome and, therefore, nickel was targeted this to chromosome preferentially over other


0

0.1

65

l .2

l .8

NiO Black (pglcrn')

Fig. 4 Comparative mutagenesisof NiS (A) and NiO black (B) in three cell lines -G12 (m), G10 (A) and V79 ( 0 ) . Results are meanof 2 independent experiments for each nickel compound. Bars are SD.

chromosomes.Theinactivatedsenescencegenewasnotfoundwithin heterochromatin of the X chromosome but rather was believed to be on the short arm of the X chromosome. Thus, this senescence gene was not deleted from within heterochromatin but was inactivated by DNA methylation as evidenced (4,22). The mechanism by which nickel from our experiments with azacytidine

could stimulate DNA methylation was puzzling. However, in studies using a transgeniccell

line (G12) wheretheendogenous

hpgrt gene had been

permanently inactivated and a bacterialgpt gene under the controldf an SV 40 promoter was inserted in the heterochromatic region of chromosome 1 (G12), nickel produced a very high incidence of 6-thioguanine (6-TG) (23). In contrast, placing the gene in another cell line, G10, on chromosome 6 far away from heterochromatin rendered cells unresponsive to nickel(23). Figure 4 illustrates the responsesof G12, G10 and wild typeV79 cells to carcinogenic crystalline NiS or NiO(23).

66 W.

Costa

MODEL FOR HOW NICKEL EXERTS ITS

EFFECTS ON

TRANSCRIPTION OF GENES NEAR HETEROCHROMATIN Figure 5 is a model for how nickel inactivates the transcription of genes. Nickel selectively binds to heterochromatin producing caged oxygen radicals at that site. It binds to histone H-1 substituting for magnesium and increases chromatin condensation.These

interactions cause the extension of

This can be modeled by aspool heterochromatin into neighboring euchromatin.

(heterochromatin) with thread (euchromatin) where the spool will pull in more thread. T h i s process is activated by Ni*+ binding to heterochromatin. These effects would possibly be transient, except that

DNA methylase enzymes

recognize that the new thread of DNA on the spool is not as heavily methylated

is, therefore, a as is the restof the DNAon the spool. The newly condensed DNA

Other CriticalGenes that

DNA Methyl

Cytosine Methylation

Figure 5


67

substrate for theDNA methylase enzyme. DNA methylation causes the pulled in thread

to remain on the spool for subsequent generations since methylation

patterns are inherited. Thus by interacting with heterochromatin and inspooling moreDNA,nickelcaneffectivelyinactivateexpressedgenes,

such as the

senescence gene and other critical genes by hypermethylation leading to a genetically inactive state for subsequent generations.

ACKNOWLEDGMENTS I would liketo thank Dr. Catherine Klein for discussions and editorial assistance and Jane Galvin for secretarial help. This work was supported by NIEHS grant numbers ES 00260, ES 04895, ES 04715, ES 05512 and CA 16087 from the National Cancer Institute.

REFERENCES 1.

N.T.Christie, D.M. Tummolo, C.B. Klein,and T.G. Rossman, The role of Ni(I1) in mutation, in Nickel and Human Health: Current Perspectives (E. Nieboer, Ed.),JohnWiley, 1990.

2.

M. Costa, Annu. Rev. Phamcol. Toxicol. 31:321-337 (1991).

3.

M. Costa and H.H. Mollenhauer, Science 209515-517 (1980).

4.

C.B. Klein, K. Conway, X.W. Wang, RK. Bhamra, X. Lin, M.D. Cohen, L. Annab, J.C.Barrett, and M. Costa,Science 251:796-799 (1991).

5.

F.W. Sunderman, Jr., Carcinogeniaty of nickel compoundsin the animals, in Nickel in the Human Environment, IARC Monogr. Ser. (F.W. Sunderman, Jr., Ed.), IARCSci. F'ubl., Lyon, France,1984,53127-142.

6.

M.P. Abbacchio, J.D. Heck, R.M. Caprioli,

and M. Costa, Chemosphere

10897-908 (1981). 7.

M.P. Abbacchio, J.D. Heck,and M. Costa,Carcinogenesis 3175-180 (1982).

68

Costa

8.

M. Costa and J.D.Heck, Cancer Res. 435652-5656

9.

J.D. Heckand M. Costa,Cancer Left. 1519-26

10.

P. Sen, K. Conway, andM.Costa, Cancer Res. 472142-2147

11.

P. Senand M.Costa, Toxicof.Appf. Phurmucof. 29:606-613

12.

N. Borochov, J. Ausio, and H. Eisenberg, Nucfeic Acih Res. l230893096

(1983).

(1982). (1987). (1986).

(1984). 13.

X.W. Wang,R J. Imbra, and M. Costa, CancerRes.486850-6854

14.

S.R Patiemo andM.Costa, Chem.-Biof.Interucf.55:75-91

15.

S.R. Patiemo, M. Sugiyama, J.B. Basilion, and M. Costa, Cancer Res. 455785-5794

16.

(1985).

(1985).

K.S. Kaspnak, M.P. Waalkes, and L.A. Pokier, Toxicol. Appl. Phurmucol. 82336-343

17.

(1988).

(1986).

KXonway, X-W. Wang,L.S.Xu, and M. Costa, Carcinogenesis81115-1121 (1987).

18.

C.B. Klein, K. Frenkel, and M. Costa, C h . Res. Toxicof.4592-604

19.

X. Huang, K. Frenkel, C.B. Klein, and M. Costa, Toxicof.Appf. Phurmucof. 12029-36

(1991).

(1993).

20.

K.S. Kasprzak, Chem. Res. Toxicof.4604615 (1991).

21.

D.W. Magerum and S.L. Anliker, Nickel(II1) chemistry and propertiesof thepeptide complexesof Ni(II) and Ni(III), in The Bioinorgunic Chemistryof Nickef (J.R. Lancaster, Ed.), VCH Publishers, New York, 1988,pp. 29-551.

22.

X.W. Wang, X. Lin, C.B. Klein, RK. Bhamra, Y.W., Lee, and M. Costa, Carcinogenesis 13555-561

(1992).

23.

B. Kargadn, C.B. Klein, and M.Costa, Mutut. Res. 300:63-72

24.

Y.W. Lee, C.B. Klein, B. Kargacin, K. Salnikow, N.T. Christie, and M. Costa, Mol. Ceff.Bwf. (submitted, 1994).

(1993).

5 Oxidative Mechanisms of Nickel(II) and Cobalt(II) Genotoxicity Kazimien S. Kaspnak Department of Human Health, Frederick Cancer Research & Development,Building 538, Room 205E,Frederick,MD217021201

I. INTRODUCTION There is experimental evidence that following in vitro and in vivo exposures, transition metals bind to cell nuclei[l-51. Therefore, current hypotheses suggest that such binding may play a direct [6,7l. role in the mechanisms of metal-induced carcinogenesis Infact,thebinding is oftenaccompaniedbygenedamaging effects,suchasconformationalchanges in DNA andnuclear proteins, strand breakage and depurination ofDNA the molecule, DNA base cross-linking chromatin of components, and modification.Thoseeffectsmaylead to mutationsdueto erroneous repair or replication of the damaged DNA template. Metal binding to enzymes which control DNA replication and 69

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Kasprzak

repair may further contribute to increased error frequency. And, competitionbetweentoxicandessentialmetalsinnuclear regulatory proteins, e.g., proteins containing "zinc fingers" [2,4], may cause abnormal gene expression. More details onthe above are presented in reviews by Sunderman [5-71 andCosta [8]. But, the essentialquestionremains:Isdirecttoxic metal binding (DNA-metal"adduct"formation)aloneresponsibleforthe observed genotoxic effects? It is likely that depurination, conformational alterations, and certain types of cross-links in chromatin are a direct result of "metaladduct"formation.However, the DNAbasedamage found in vitro [S] and in vivo [lo-131 cannot be explained in this way since the damaged bases are not metal adducts but oxidation products. A common explanation may be that the DNA damaging action of chromatin-bound metal is, at least in part, redox catalyticin nature. This assumptionis consistent with both the experimental thechemistryof the metalsinquestionand evidence in cell-free and in vivo systems, reviewed recently by several authors [14-181.

II. OXIDATIVEDNADAMAGE Carcinogenicmetals,suchasnickelandcobalt,aretransition elements and thus have rich coordination and redox chemistries. These factors, among others, allow carcinogenic transition metals to activate oxygen species. The most important reactions include 'Abbreviations and Formulae: 02,ambient oxygen;H202,hydrogen peroxide; O;', superoxide anion radical;'OH,hydroxylradical;A,adenine;G, guanine; C, cytosine; 5-me-C; 5-methylcytosine; T, thymine; His, L-histidine; NTA, nitrilotriacetate; GSH, glutathione; 8-oxodG, 8-oxo-2'deoxyguanosine; 8-oxo-dGTP, 8-oxo-2"deoxyguanosine triphosphate; intraperitoneal; iv, intravenous; GC-MS/SIM, gas chromatography-mass spectrometry with selected ion monitoring. For abbreviations of thedamaged DNA bases see Figure 1.

Oxidative Mechanisms of Nickel(ll1andCobalt(ll1 Genotoxicity

71

0; conversion to superoxide (0;')and/or H202in autoxidation H202conversiontohydroxylradicals ('OH) reactions,and through Fenton/Haber-Weiss chemistry [14,151. Formation of otherpotentoxidantssuchasmetal-oxoandmetal-peroxo [15,19]. Infact,increased complexeshasalsobeenobserved levels of oxygen activation products, which may damage chromatin, have been found in cells cultured in the presence of metals [20-241. Besides catalyzing the generation of reactive oxygen species, transitionmetalsmayassistintheattackofthosespecieson chromatinbyinhibitingcellularantioxidantandDNArepair systems [25-291and by directing oxidative attack to specific sites on DNA [30]. It is not surprising, therefore, that both in vitro and in vivo exposures of cells to carcinogenic transition metals result in production of modified DNA bases, strand breaks, and avarietyofcross-linksthat are alsocharacteristicforDNA damage caused by 'OH and other oxidants generated by ionizing radiation [14,15,311. Let us look at some examples from our own work. A. Nickel

Experiments were conducted under conditions already proven to initiaterenalcarcinogenesis in rats [10,32]. PregnantFischer rats were injected ip with Ni(II) acetate at the end of gestation [ll]. Chromatin was isolated from kidneys and livers of both 1 or 2 daysafterthelastinjectionand mothersandfetuses l), analyzedfor 11 oxidativelydamagedDNAbases(Figure usingthegaschromatography-massspectrometry/selectedion monitoring(GC-MS/SIM)techniquedevelopedbyDizdaroglu [33]. The results are presented in Figure 2. As can be seen in thisfigure,DNA in thekidney(atargetorganfornickel carcinogenesis) contained significantly elevated levels of promutagenic 8-oxo-Gua and Thy glycol. In contrast, DNA in liver (a non-target organ) did not contain increased levels of any establishedpromutagenicDNAbaseproducts.Thisfinding

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Kasprzak

supports our former suggestions of a possible role of 8-oxo-dG (DNA lesion the same as 8-oxo-Gua) in Ni(II)-induced carcinogenesis [10,34,35]. To confirm in animals our in vitro findings that L-histidine (His) enhances Nio-mediated oxidation of 2’-deoxyguanosine [35,36], male Fischer rats were injectedwith a single iv dose of the Ni(His), complex [l31 or, for a comparison,with equivalent doses of Ni(II) acetate, sodium acetate, or His. In addition to measuring the extent of DNA base damage, as above, the effect of Ni(His), on DNA-protein cross-linking was evaluated the using alkaline elution technique [13]. Asin the in vitro experiments, chelation with His was found to enhance Ni(II)-induced oxidative

Figure 1. Structures of active oxygen-generatedproductsof DNA bases, as identified by the use of the GC-MS/SIM technique according to Dizdaroglu 1331.

OxidativeMechanisms of Nickel(ll1andCobalt(ll1Genotoxicity

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Figure 2. Levels of damaged DNA basesin kidneys and liversof pregnant rats and their fetuses following systemic exposure to Ni@) [ll]. The rats were injected with single ip dosesof 45 pm01 N i o acetatekg body wt each on days 16 and 18 of gestation and killedon day 19 of gestation. Control rats received equivalent doses of sodium acetate. A l l levels shown as > 100% wereincreasedsignificantly (P < 0.05 or better). See Figure 1 for abbreviations.

DNA base damage in the rat kidney (Figure 3). The extent of DNA-protein cross-linking was increased as well (Figure 4). At the dose levels applied, treatments other than with Ni(His), did not produce any significant increase in the base damage above control. To verify in vivo our in vitro findings that lipid peroxidesmay play a role in metal-mediated genotoxic effects [37,38], N i o -

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1

1 Pyrimidinederived bases

Purinederived bases

Figure 3. Levels of damaged DNA basesinkidneys of rats following systeniic exposure to the Ni(His), complex [13]. The rats were injected with a single iv dose of 20 pm01 Ni(His)Jkgbody wt and killed 18 hr later. Control rats received an equivalent doseof sodium acetate. All levels shown as > 100% were increased significantly (P -C 0.05 or better). See Figure 1 for abbreviations.

induced DNA base oxidation was correlated with Nio-induced lipid peroxidation in the kidneys of mice [39,40] and rats [41]. In those studies, BALB/c, C57BL, B6C3F1, and C3H male mice and male Fischer rats received single ip injections of Ni(II) acetate. The levels of lipid peroxides and 8-oxo-dG in kidneys were determined 3 - 48 hr post-injection in mice (Figure 5 ) and 1 - 72 hr in rats (Figure 6), using the HPLC technique with electrochemical detection [lo]. A significant increase in 8-oxodG level was observed only in BALB/c mice, i.e., animals which were also very susceptible to Ni(II)-induced lipid peroxidation. In mice, both effects were temporary, indicating efficient repair of the damage. In rats,however, a transient increase in lipid

Oxidative Mechanisms of Nickel(ll1andCobalt(ll1

75

Genotoxicity

wan o.* Nws,

0..

0.10 0

6

S

12

0

3

6

9

12

Figure 4. Alkaline elution curves for renal DNA of rats killed 18 hr after a single iv injection of compounds specified in the legend. Closed symbols, samplesirradiatedwith 400 rads of y-radiationpriortotheelution. Open symbols, nonirradiated samples. A, without proteinase K digestion. B, after protein& K digestion [13].

peroxidation was followed by a persistent elevation of the8-oxodG level. In bothmiceand rats, the maximum levels of lipid peroxidation preceded those of 8-oxo-dG by at least12 hr. This finding casts some doubts on the possibility that lipid peroxides participate directly in the inducticn of DNA base damage in vivo. The observed species- and strain-related differences in response to Ni(I1)-mediated DNA base damage may, however, be a good indication of corresponding differences in susceptibility to renal carcinogenesis.

B. Cobalt Spectra of damaged DNA bases, similar to those described for nickel, have also been established in rats for cobalt 1121. Male and female Fischer rats were injected ip with different dosesof CO(@ acetate and killed 2 or 10dayslater.Renal,

76

Kaspnak BALWC

C57BL

BbC3F1

C3H

T

.

24 48

mln (h0

Figure 5. Lipid peroxide GPO) and 8-oxodG levels in kidneys

of mice

injected with a single ip dose of 170 pm01 of N i O acetatekg body wt and killed 3 48 hr later.Thecontrolmicereceived 340 pm01 of sodium acetatekg body wt. Asterisks indicate statistically significant differencesvs. the control levels, with P < 0.05 or better.

-

hepatic, and pulmonary chromatin from those rats was analyzed by the GC-MS/SIM techniqueaccordingtoDizdaroglu [33]. Some of the results are shown in Figure 7 [12]. No significant differences in the response were found between male and female rats. The damagedepended on CO@) doseandtargettissue, withkidneybeingthemostaffectedorgan. The renaland hepatic, but not pulmonary, DNA base damage tended increase to with time, implying possible inhibition CO@) by of DNA repair.

Oxidative Mechanisms of Nickel(ll) andCobalt(l1) Genotoxicity

77

the baseproductsfound are promutagenic the resultssuggestapossibilityofinitiationof carcinogenesis by cobalt in the investigated tissues. A bioassay to test this prediction is under way in our laboratory. The carcinogenic potential of cobalt is lower than that of nickel [43] which may be reflectedin relatively lower levels of damaged DNA bases observed in tissues of cobalt-treated rats. However, in vitro cobalt appears to be a much more potent catalyst DNA of base oxidationthan nickel [g]. Those differences are most likely due to substantial differencesin bioavailability of both metals at target sites.

Sincesomeof [14,15,42],

d

-

1

0

3

24

72

0

3

24

72

Time (hr) Figure 6. Lipidperoxide &PO) and 8-oxodG levelsinkidneys of F344/NCr rats injected with a single ip dose of 105 pmol of N i o acetatekg body wt and killed 3 - 72 hr later. The control rats received 200 pm01 of sodium acetatekg body wt. Asterisks indicate statistically differences vs. the control levels with P < 0.05 or better.

significant

l

Pyrimidine-derived bases

1

-

Punne-denved bases

Figure 7. Levels of damaged DNA bases in kidneys, livers, and lungs of rats following systemic exposure to C o o [12]. The rats were injected with a single ip dose of 100 pm01 C o o acetatekg body wt and killed 10 days later. Control rats received equivalent doses of sodium acetate. *, the control level in this case was below the detection limit. All levels shown as > 100 % were increased significantly (P < 0.05 or better). See Figure 1 for abbreviations. 78

Oxidative Mechanisms of Nickel(ll1and CobaltW Genotoxicity

79

III. DISCUSSION The significance of the above effects to carcinogenesis relieson evidence that at least some types of oxidative DNA damage are mutagenic. As shown above, the variety of DNA base damage in vitro and in vivo has been established for carcinogenic Ni(II) and C o o [g-121, without, however, verification of the resulting the otherhand,mutational spectra havebeen mutations.On determined for other metals, such as Fe(II), C u o , and Cu(II), with reversion and forward mutation assays using single stranded DNA templates exposed to the metal salts under air [44-461, but the underlying types ofDNAlesionswerenotresolved.The mutationswerepredominantlysinglebasesubstitutionswhich clusteredatdistinctgenepositionscharacteristicallyforeach metal. The most frequent mutations produced by Fe@) were G + C transversions followed by C + T transitions and G -* T Cue, the mutationswere transversions[45].WithCu(1)and predominantly C + T transitions followed by G +T transversions [46]. The G -* T transversions are characteristic for base mispairing by 8-oxo-dG (8-oxo-Gua) lesion [47,48]. A significant increase in 8-oxo-dG production has been found in renal DNA of male of carcinogenic Wistarratsgivenasingleparenteraldose in ratstreated with noncarcinogenic Fe(III)NTA, but not Na(I)NTA orFe(III)chloride[49].Interestingly,ratrenal mesenchymaltumorsinducedwithanothercarcinogen,nickel K-ras oncogene subsulfide,havebeenshowntocontaina activatedatcodon12exclusivelyby the G -* Ttransversion mutation [50]. Most recently, the 8-oxo-dG lesion hasalso been associated with altered methylation of adjacent cytosines, which, in turn, may affect proper gene expression [51] and lead to cell transformation and cancer. The origins of the remaining types ofpointmutations, mentioned above, are not clear. The mutations may be produced by various DNA lesions, including the damaged bases shown in Figure 1. For example, the C + T transitions are likely to be

80

Kasprzak

caused by mispairing on 5-OH-Cyt lesion [52], or through metalin enhancedoxidativedeaminationofCand5-me-Cresidues DNA [53]. Deamination of other bases may, perhaps, contribute Thy glycoland tothosemutationsaswell.Mutagenicityof 5-0HMe-Ura, arising in DNA of Ni(I1)-, or CO@)-treated rats, respectively [ll,121, is still debatable [15]. ReidandLoeb [54] andTkeshelashvili et al. 1551 have established that tandem double CC + TT mutations, known to occurvia W damagetoDNA, can alsobeproducedby treatmentsgeneratingactiveoxygenspecies,e.g.,by NiO, F e O , Cu(I),and C u O plus O2 and/or H202. The authors speculate that the mutations occur because of base mispairing at sites of cytosine dimers (intrastrand cross-linking), known to be produced by 'OH. The significance and specificity of cross-links, e.g., the DNAproteincross-linksobservedinrenalchromatinofNi(Hisbtreated rats [13], to certain types of mutation and carcinogenesis remain to be defined. It is believed that persistent cross-links may impair functions of the nuclear matrix, such as replication and transcription, and thus introduce genetic and/or epigenetic alterations into the affected cells [56]. Other metal-induced effects, such as DNA depurination and strand scissions,arepromutagenicevents [45,57], but they cannot be ascribed solelyto the catalytic modeof metal action on needs DNA.Theircontributiontometal-relatedmutagenesis further elucidation. Besidesmediatinggeneticdamagedirectlyatchromatin binding sites, metals may also affect genetic material remotely through promotion of lipid peroxidation, inhibition of cellular oxygenhandlingsystems,and/orinhibitionofDNArepair [14,15]. Lipid peroxidation products were found to mediatethe formationof8-oxo-dG [58] andstrandbreaks [59] inDNA. Lipid peroxidation by N i O in mice was found to be high in certainstrains(e.g.,BALB/c)that are lowinGSHandGSH B6C3F1 or C3H) peroxidasecomparedtootherstrains(e.g., [29,39,40]. Catalase and GSH peroxidase, which protect cells

Oxidative Mechanisms of Nickel(l1)andCobalt(l1) Genotoxicity

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against metabolic peroxides, are inhibitedby N i O 1271. Carcinogenicmetalcations,including N i O and C o o , also have the ability to bind to cellular antioxidants such as ascorbate, cysteine, histidine, GSH, and others and to modify their reactions with oxygen species to produce free radicals [14,15]. Oxidative DNA damage can be prevented and/or repaired in living cells by various mechanisms [60-651. The cellular protection against 8-oxo-dG mutagenicity includes several enzymesactingagainst the 8-oxo-dGlesion.Dataonthose enzymes have been recently reviewed by Grollman and Moriya 1631. It is noteworthy that the activity of at least two enzymes the engaged in the repair depends on essential metals: that of 8-oxo-dGTP triphosphatase (MutT protein), which removes this damaged nucleotide fromthe nucleotide pool, depends on Mg(II) [66], while that of formamidopyrimidine DNA glycosylase (Fpg protein),whichremoves8-oxo-dGandsomeotherdamaged purinesfromDNA [67l, depends on Z n o (Fpg isa."zincfinger"protein [68]). Therefore, we maysuspectthatboth to inhibitionbyotherdivalent enzymesshouldbesensitive cations. In conclusion, among the various pathogenic effects produced by nickel, cobalt, and other transition metals, the mediation of oxidative damage appears to have a primary role in metal-induced carcinogenesis. REFERENCES 1. 2. 3.

S.E. Bryan, Heavy metals in the cell's nucleus, in Metul Zons in Genetic Information Trunsfer (G.L. Eichhornand L.G Marzilli, Eds.), Elsevier, New York, 1981, pp. 87-101. J.M. Berg, Science 232: 485-487(1986). A. Leonard,Chromosomedamageinindividuals exposed to heavy metals, in Metul Zons in Bwibgicul Systems Sigel, Ed.), Vol. 20, Marcel Dekker, Inc., New York, 1986, pp. 229-258.

(H.

82 4.

Kasprzak

F.W. Sunderman, Jr. and A.M. Barber, Ann.Clin. Lab. Sci. 18: 267-288 (1988).

5. F.W. Sunderman, Jr., Ann. Clin. Lab. Sci. 14: 93-122 (1984). 6.

F.W.Sunderman, Jr., Carcinogenicity and mutagenicity of some for Research on metals andtheir compounds, in International Agency Cancer Scientific Publications,Vol. 71, IARC, Lyon, 1986, pp. 17-

7.

F.W. Sunderman, Jr., Scand. J. Work Environ.Health 15: 1-12

8.

(1989). M. Costa, Annu. Rev. Phamacol. Toxicol. 31: 321-327 (1991).

43.

9.

10.

11. 12.

13. 14. 15. 16.

Z. Nackerdien, K.S. Kaspnak, G. Rao, B. Halliwell, and M. Dizdaroglu, Cancer Res. 51: 5837-5842 (1991). K.S. Kasprzak, B.A. Diwan, N. Konishi, M. Misra, and J.M. Rice, Carcinogenesis 11: 647-652 (1990). K.S. Kasprzak, B.A. Diwan, J.M. Rice, M. Misra, C.W. Riggs, R. Olinski, andM. Dudaroglu, Chem Res. Toxicol.5: 809-815 (1992). K.S. Kaspnak, T.H.Zastawny, S.L. North, C.W. Riggs, B.A. Diwan, J.M. Rice, and M. Dudaroglu, G e m Res. Toxicol. 7: 329335 (1994). M. Misra, R. Olinski, M. Dizdaroglu, and K.S. Kaspnak, Chm Res. Toxicol. 6: 33-37 (1993). K.S. Kasprzak, Chm Res. Toxicol. 4: 604-615 (1991). K.S. Kaspnak, Cancer Invest. (In press). C.B. Klein, K. Frenkel, and M. Costa, Chem Res. Toxicol. 4: 592604 (1991).

17. E.T. Snow, Pharmac. mer. 53: 31-65 (1992). 18. A.M. Standeven, and K.E. Wetterhahn, Chem Res. Toxicol. 4: 616625 (1991). Wink, R.W. Nims,and P.C. Ford, Environ. 19. D.A.Wink,C.B. Health Perspect. (In press) 20. F.W. Sunderman, Jr., Acta Phamacol. Toxicol. 59: Suppl. W, 248-255 (1986).

21.

J. Sole, J. Huguet, L. Arola, and A. Romeu, Bull. Environ. Contam

Toxicol. 44:686-691 (1990). G.R. Buettner, Arch. Bwchm Bwphys. 300: 535-543 (1993). T.A. Dix and J. Aikens, Chm Res. Toxicol. 6: 2-18 (1993). X. Huang, K. Frenkel, C.B. Klein, and M. Costa, Toxicol. Appl. Pharmacol. 120: 29-36 (1993). 25. A. Hartwig and D. Beyersmann, Biol, nace E l m Res. 21: 359-365

22. 23. 24.

(1989).

Oxidative Mechanisms of Nickel(l1) andCobalt(l1) Genotoxicity

83

A. Hartwig, R.D. Snyder, R. Schleppepll, and D. Beyersmann, Mutat. Res. 248: 177-185 (1991). 27 R.E. Rodriguez and K.S. Kasprzak, Effects of nickel on catalase and the glutathione peroxidase-reductase system in vitro, in Nickel and Human Health: Current Perspectives (E.Nieboer and J.O. Nriagu, Eds.), J. Wiley h Sons, New York, 1992, pp. 375-385. 28. R.E. Rodriguez, M. Misra, and K.S. Kasprzak, Toxicology 63: 4526.

52 (1990). 29. R.E. Rodriguez, M. Misra, S.L. North, and K.S. Kasprzak, Toxicol. Lett. 57: 269-281 (1991). 30. S. Inoue and K. Yamamoto,. Carcinogenesis 12: 2231-2235 (1989). 31. M. Dizdaroglu, Methods Enzymol. 186: 530-544 (1990). Kasprzak, and J.M. Rice, Carcinogenesis 13: 32. B.A.Diwan,K.S. 1351-1357 (1992). 33. 34.

M. Dudaroglu, Free Radic. Biol. Med. 10: 225-242, (1991). K.S.Kasprzakand L. Hernandez, Cancer Res. 49: 5964-5968

35.

K.S. Kasprzak, S.L. North, and L.Hernandez, Chem Biol. Interact. 84: 11-19 (1992). A.K. Datta, M. Misra, S.L. North, and K.S. Kasprzak, Carcinogenesis 13: 283-287 (1992). X. Shi, N.S. Dalal, and K.S. Kasprzak, Arch. Bwchem Bwphys. 299: 154-162 (1992). X. Shi, N.S. Dalal, and K.S. Kasprzak, J. Inorg. Biochem. 50: 211225 (1993). M. Misra, R.E. Rodriguez, S.L. North, and K.S. Kasprzak, Toxicol. Lett. 58: 121-133 (1991). K.S. Kasprzak, M. Misra, R.E. Rodriguez, and S.L. North, ToxicoZogist 11: 233 (1991). M. Misra, R.E. Rodriguez, and K.S. Kasprzak, Toxicology 6 4 : 1-17

(1989).

36. 37. 38. 39.

40. 41.

(1990).

L. Shirname-More, T.G. Rossman, W. Troll, G.W. Teebor, and K. Frenkel, Mutat. Res. 178: 177-186 (1987). 43. A. Leonard and R. Lauwerys, Mutat. Res. 239: 17-27 (1990). 44. L.A. Loeb, T. J. McBride, T.M. Reid, and K.C. Cheng, Mutagenic spectra of oxygen free radicals,in New HorizonsinMolecular Toxicology (G.S. F’robst, M.J. Vodicnik, and M.A. Dorato, Eds.), FASEB h b l . , Beth=&,, 1991, pp. 35-39. 45. T.J. McBride, B.D. Preston, and L.A. Loeb, Biochemistry 30: 207-

42.

213 (1991).

84

Kaspnak

46.

L.K. Tkeshelashvili, T.J. McBride, K. Spence, and L.A. Loeb, J. Biol. Chem 266: 6401-6406 (1991). K.C. Cheng, D.S. Cahill, H. Kasai, S. Nishimura, and L.A. Loeb, J. BWl. Chem 267: 166-172(1992). M.L. Wood, A. Esteve, M.L. Morningstar, G.M. Kuziemko, and J.M. Essigmam, Nucleic Acids Res. 20: 6023-6032 (1992). T. Umemura, K. Sai, A. Takagi, R. Hasegawa, and Y. Kurokawa, Carcinogenesis 11: 345-347 (1990). K.G. Higinbotham, J.M. Rice, B.A. Diwan, K.S. Kasprzak, C.D. Reed, and A. Perantoni, Cancer Res. 52: 4747-4751 (1992). S.A.Weitzman,P.W.'Turk,D.HowardMilkowski,and K. Kozlowski, Proc. Natl. Acad. Sci. USA 91: 1261-1264 (1994). D.1 Feig, L.C. Sowers, and L.A. Loeb, Proc. Am. Assoc. Cancer Res. 35: 588 (1994). K.S.Kasprzak,S.L.North,andL.K.Keefer, Proc.Am. Assoc. Cancer Res. 32: 108 (1991). T.M. Reid and LA. Loeb, Proc. Natl. Acad. Sci. USA 90: 3904-

47. 48. 49.

50. 51. 52. 53. 54.

3907 (1993).

55. 56. 57. 58.

L.K.Tkeshelashvili,T.M.Reid,T.J.McBride,andL.A.Loeb, Cancer Res. 53: 4172-4174 (1993). N.L.Oleinick, S. Chiu,N.Ramakrishnan,andL.Xue, Brit. J. Cancer 55, SUPPI.VIII: 135-140 (1987). T.M. Runger and K.H. Kraemer, EMBO. J. 8: 1419-1425 (1989). J.W.ParkandR.A.Floyd, Free Radic. Bwl. Med. 12: 245-250 (1992).

Hartwig, H. Klyszcz-Nasko, R. Schleppegrell, and D. 59. A. Beyersmann, Carcinogenesis 14: 107-112 (1993). 60. T.Bessho, R. Roy, K. Yamamoto, H. Kasai, H. S. Nishimura, K. Tano,and S. Mitra, Proc. Natl. Acad. Sci. USA 90: 8901-8904 (1993). 61.

M.H. Chung, H.K. Kim, E. Ohtsuka, H. Kasai, F. Yamamoto, and S. Nishimura, BiochemBiophys. Res. Commun. 178: 1472-1478 (1991).

62. 63.

B. Demple, and C.F. Amabile-Cuevas, Cell 67: 837-839 (1991). A.P.GrollmanandM.Moriya, Trends in Genetics 9: 246-249

64.

J.C.Klein,M.J.Bleeker,C.P. Saris, H.C.P.F.Roelen,H.F. Brugghe, H. van den Elst, G.A. van der Marel, J.H. van horn, J.G. Westra, E. Kriek, and A.J.M.Berns, Nucl. Acid Res. 20: 4437-

(1993).

4443 (1992).

OxidativeMechanisms of Nickel(l1)andCobalt(l1)Genotoxicity 65.

85

F. Yamamoto, H. Kasai,T. Bessho, M.H. Chung, H. Inoue, E.

Ohtsuka, T. Hori, and S. Nishimura, Jpn. J. Cbncer Res. 83: 351357 (1992). 66. J.Y. MO,H. Maki, and M. Sekiguchi, Proc. Natl. Acad. Sci. USA 89: 11021-11025 (1992). 67. S. Boiteux, E. Gajewski, J. Laval, and M.Dizdaroglu, Biochemistry 31: 106-110 (1992). 68. T.R. O’Connor, R.J. Graves, G. de Murcia, B. Castaing, and J. Laval, J. Biol. Chm 268: 9063-9070 (1993).


6 The Antimutagenic Effects of MetallothioneinMayInvolveFree Radical Scavenging E. I. Goncharova and T. G. Rossman

NelsonInstitute of Environmental Medicine, NYU Medical Center, Long Meadow Road, Twtedo, NY 10967

I. INTRODUCTION Metallothionein (MT) is a metal-binding sulfhydryl rich protein in which cysteine residues makeup one-third of the amino acid residues. MT's occur in vertebrates, invertebrates, plants, eukaryotic microorganismsandsome prokaryotes [l]. It has been well-documented that cadmium, zinc and copper salts, among others, induceMT expression [2]. Since its recognition ithas been suggested that MT plays a crucial role in detoxification of heavy metals, in the storage of metal ions and in the regulation of cellular Zn(I1)andCu(I1) metabolism [l]. MT has also been reported to play a protective rolein oxidative stress by scavenging free radicals [3,4]. V79 cells) The transgenic cell line G12 (derived from Chinese Hamster contains a single copy of the E. coli gpt gene as a target for mutagenesis [S]. The gpt target is sensitive to both genetic and epigenetic events [6] and is superior to the endogenoushprf gene for detecting mutagenesis byW, X-rays and agents causing oxidative damage [71. G12 cells have a very low level of endogenous MT. expression. A number of G12 derived MT.-over-producinga ll lines have been isolated after transfection with a vector containing the mouse MT-I gene under control of its own promoter region [81. We recently reported that M T - I transfectants have lower spontaneous mutation frequencies compared with the G12 parental cell line, and that the spontaneous mutation frequencyis by the inverselyrelatedtothelevel of MT expression[8].Mutagenesis alkylating agents N-methyl-N'-nitro-nitrosoguanidineandethylmethane 07

88

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Goncharova

sulfonate were not altered by MT expression. In this study, we examine the effects of MT on mutagenesis by the chemotherapeuticdrug amsacrine and the synthetic vitamin K analogue menadione. We demonstrate that MT can block "A-induced oxidative stress. In addition, we reporta protective role forMT in the survival of cells exposed to arsenite. Each of these agents is reported to produce free radicals by variousmechanisms.Ourresultssupportthe hypothesis that a protective role of MT against mutagenicity and toxicity of a number of cytotoxic agents is basedon its ability to act as a free radical scavenger.

It. MATERIALS AND METHODS A. CellCulture: The cell lines used were line G12, a gpt transfectant of Chinese hamster V79 cells [5], and MTl-2 and MT1-2A, MT transfectants of G12 cells[8]. Cells are grown in Hams mediumF-12,supplementedwith 5% fetal bovine serum (Gibco, Grand Island, NY) and penicillin-streptomycin (Gibco, Grand Island, N Y ) at 37O C in an atmosphere of 5% C02 and 95%

air.

B. Toxicity assay:

For clonal toxicityassay, cells wereseeded at a density of 300 cells/60 mm dish in normal medium. Test agents were added to the cells following attachment, and remained on the cells for the time chosen. The cultures were incubated in C02 for five to seven days, during which time clones developed to macroscopic size. Clones were fixed with methanol, stained with Giemsa (Gibco, Grand Island, NY) and counted. Agents used: mAMSA was a gift from Dr. C. Klein, NYU, menadionewaspurchasefromSigma(St.Louis, MA), andsodium (Fair Lawn, NJ). arsenite was purchased from Fisher Scientific C. Mutagenesis assay: The mutagenesis assayused has been previouslydescribed [5].

D. Gene Amplification: Gene amplification assays were performed according to the method Ottoof er a2 [91. The selective agent PALA(N-(phosphonoacety1)-L-aspartate)was added to the medium at sufficient concentration to give 10 x LD50. &D50 is the dose required to reduce clonal survival by 50%). The medium was changed weekly. After 4 weeks, PALA-resistant clones were fixed with methanol, stained with

89

Effects of Metallothionein and Free Radical Scavenging

Giemsa and counted. PALA was kindly provided Institute.

by the National Cancer

E. Fluorescent measurementof intracellular oxidants producedby TPA Cells were treated with 500 ngml TPA (phorbol 12-myristate 13-acetate) 50 PM (Sigma, St. Louis, MO) for 6 hoursandthenincubatedwith dichlorofluorescin diacetate (DCF-dAC)(Kodak,Rochester,NY)foran additional 30 min. Fluorescence was measuredas previously described forCH0 cells [lo]. Briefly, cells were washed twice with ice cold PBS, scraped from the late, and resuspended at lo6 cells/ml for fluorescencemeasurement. fluorescence was analyzed using a Fluorescent Spectrophotometer (Perkin Elmer, Norwalk, CT), using 502 nm excitation and 522nm emission.

f

3

m. RESULTS AND DISCUSSION A.

Protection by MT against mutagenesis and killing

' ' chemotherapeutic agent amsacrine (mAMSA). '

by the

Resistance of tumors and cellsin vitro to some anticancer agentswas found to correlate with elevated levelsof M T. Tumor cell lines selected for resistance to cis-platinum over-expressed MT and were cross-resistant to the alkylating agents chlorambucil and melphalan. Induction of MT by Cd(II) in these cells also conferred resistance to the same agents [ll]. Mouse C127 cells transfected with the MT IIa gene and showing high levels of its expression were resistant to melphalan and chlorambucil but showed no increased resistanceto bleomycin, doxorubicin, 5-fluorouracil or vincristine [l l]. DNA topoisomerases are unique enzymes thatcan break and rejoin the phosphodiester backbone ofDNA and thus change the topology of DNA. Topoisomerase I1 is localized at the baseof chromatin loops [12], where there aresequencescontainingconsensustopoisomerase I1 binding sites [13]. Topoisomerase I1may be involved in DNA replication, transcription, and recombination. DNA topoisomerase 11is a target of antineoplastic drug therapy [ 141. Several topoisomerase inhibitors including mAMSA, adriamycin, [ 151. ellipticines, and epipodophylotoxins are potent antineoplastic agents Type 11 DNA topoisomerases promote the passage of one double An intermediate step in topoisomerase I1 strand ofDNAthroughanother. reactions involves cleavage of DNA and covalent linkage of the enzyme to a DNA phosphate group. The binding is reversible and is normally followed by a subsequent rejoiningof the DNA ends after the strand passage [16]. mAMSA is a 9 anilinoacridine derivative developed as an antileukemicagent[17]. Originally it was thought that the DNA cleavage produced by mAMSA was

90

Gonchatova and Rossman

caused by free radicals. However, a number of lines of evidence now point toa connection between the cytotoxic actionof mAMSA and its ability to inhibit topoisomerase I1 [13,14,18]. Normally, topoisomeraseII catalyzes the breakage and rejoining of DNA strands. In the presence of mAMSA, both strands of DNA are broken and covalently attached to topoisomerase I1 [ 191. This is called stabilization of a “cleavable complex” between DNA and topoisomerase 11. Upon replication of the complex, DNA strand breaks occur [16]. Broken DNA ends are potentially recombinogenic, and may result in deletions, amplifications and translocations. The mutagenicity of mAMSA was first demonstrated inS. typhimirium, where frameshift mutations were induced [20]. mAMSAwas found to have weak but significant mutagenic activity at the hprt, but not at the Na/K ATPase locus in Chinese hamster V79 cells [21,22], and at the thymidine kinase locus of mouse lymphoma cells [23]. Cytogenetic analysis of these mutants showed that mAMSA is a potent clastogen [24], an effect also seen at the S-l (cell surface antigen) locus in a human-hamster hybrid cell line, where 92% of all mutants induced by mAMSA had megabase pair deletions [25]. The authors suggest that mqMSA-induced deletions may involve a series ofmany topoisomerase 11-DNA loop complexes, because it is unlikely that such large deletions are mediatedby the loss ofa single replicon. The toxicity and mutagenicity of mAMSA were compared in G12 cells and in MT1-2A cells, a G12 derivative which produces high levels of MT. MTl-2A cells are more resistant to the cytotoxicity of mAMSA compared with the parental G12 cells. mAMSA causes a dose-dependent increase in 6TG resistantvariantsinG12cells.However,thismutageniceffect is almost abolished in MT1-2A cells (Figure 1). To c o n f m that the MT content affects the mutagenic response to mAMSA, the mutagenicityof mAMSA was examined in MT transfectants with different levelsof MT expression (Figure 2). The cell line MTl-2, which has a lower MT content than MT1-2A, exhibited a higher mutant fraction after MT contentwasestimated by sensitivity to mAMSAadministration.The of mutant accumulation. Parental G12 cells witha cadmium chloride at the time low basal level of MT expression had the highest mutant fraction induced by mAMSA. The mechanismsby which MT reduce the toxicityand mutagenicity of mAMSA are not clear. One might speculate that its protective effect could related to its ability to scavenge free radicals. Besides acting as a topoisomerase inhibitor, mAMSA is also reported to generatefree radicals upon oxidation [26] A second possibility is that MT may act in some way to stabilize the cleavable complex, thereby preventing DNA strand breaks.

91


+c12 mwivsl "T1-2A Mwival +G12 mutagenesis -p"T1-2A mutagenesis

120

A

100

2

-> 0

5

n

80

F

i

(D

z

0

3

S

L

60

S

h

W

'E

-

3 -.

0

40

Y

0

c

U

e

r

E

20

I r 0

mAMSA (nglml)

Figure 1 Effects of

MT

expressionontoxicityandmutagenicity

of m A M S A

92

Goncharova and Rossman

MTl-2

-: e

0 2

MTl-2A

c

Cd" LDSo:

Figure 2

38pM

50pM

73pM

Mutagenesisinduced by mAMSA in cells expressing different levels

B. protection by MT against menadione(MD) toxicity and mutagenicity. Menadione (2-methyl-1.4 napthoquinone)is asynthetic analogueof vitamin K1. MD is capable of redox cycling and subsequently generating toxic oxygen species [27]. The cytotoxicity of MD probably results from hydroxyl radicals, since single strand DNA breaks are induced in MD-treated cells [28]. MD is also reported to cause depletion of glutathione pools [29] and oxidation of sulfhydryl groups in cytoskeletal proteins [30]. All of these effects point toan involvement of hydroxyl radical production byMD. MD has also been shown to induce MT synthesis [3l]. At low concentrations,MD reduces mutagenesis in G12 cells almost to the level seen in MTl-2A (Figure 3). MT1-2A cells are more resistant to the cytotoxic action of MD compared withG12 cells, especially at higher concentrations. MD is mutagenic to G12 cells at a concentration which allows approximately 35% survival. At the same concentration, MTl-2A cells show


Figure 3 Effects

of

M" expressionontoxicityandmutagenicity

93

of

menadion

little mutagenesis. Thus, MT protects cells fromthe cytotoxicity and Since the major mechanism of MD toxicity is related to mutagenicity of the production of reactiveoxygen species such as hydroxylradical, the protective roleof MT against MD-induced cytotoxicityand mutagenicity might be mediated by its free-radical scavenging ability. Low concentrations of MD are antimutagenic perhaps because MD can act as a free radical scavenger as well as a free radical generator.

MD.

C. MT Protects Against the Cytotoxicity of Arsenite Although the evidenceof a role for M" in metal resistance comes mainlyfrom studies on Cd(II), C u m , and Zn@) [32], a protective role for MT was also

1

W

0

Arsenite (pM)

metallot~ionein e x p r e ~ i o non a r ~ n i t ecytoto~icit

age in r ~ e via n f~ o ~ a of~ the o d~ i m e t h y ~ ~ n i c ies [36,37]. The i n d ~ ~ of ~on

binds arsenite in! vitro, ~senite

were grown in the 0 dose, which was th eoulsy sin^ clones resis ifica~onin GI2 and MTl-2A cells

450

4500

330.6

450

500

320.

96

Rossman

and

Goncharova

by which M" decrease spontaneous mutagenesis has no bearing on this other aspect of genetic stability.

E. Metallothionein Blocks Spontaneous and TPA-induced OxidativeStress There is much evidence that TPA acts as a tumor promoter at least in part by causing the formation of reactiveoxygen species [45, 463. Levels of intracellular oxidants were measured G12 in and MT1-2A cells grown in normal medium and after treatment for 6 hours with TPA (Figure 5). Basal levels of oxidants are lower in MTl-2A cells compared withG12 cells. Treatment with TPA leads toa two fold increase of the level of oxidants in G12 cells butonly a small increase in MT1-2A cells. Even after treatment with TPA, the level of oxidants in MT1-2A cells was lower than the basal level in G12 cells. These

6-

-s . 1

.

a

L

-

4-

e -

!!-

g- 3:$

E (D

8

9

(D

E

-

.

: 2.

u.. v . 0

.

1.

0-

GG 1 21+2

MT1-2A

TPA

Figure 5

MTl-2A+ TPA

Metallothioneinprotectsagainstspontaneous andTPA-inducedoxidativestress

97


mults provide further evidence for the protective roleMT ofagainst reactive oxygen species.

IV.Conclusion The agents used in this study (mAMSA, menadione, arsenite and "PA) are all one feature in common. They am all different in structure and function, but have able to produce reactive oxygen species which may as serve a key point for their MT toxicity. The data presented in this paper clearly demonstrate that high expression correlated withincreasedresistanceto the mutagenic and/or cytotoxic action of these agents. We hypothesize that a major mechanism for the increased resistance to these agents is the ability of MT to act as a free radical scavenger. The radical scavenging capability ofMT in tissues and cells was recently reviewed [47]. Data presented here provide additional support for this important physiological role h4T. of ACKNOWLEDGMENTS This workwas supported by NCI grants CA57352 and CA61319 and is part of NYU Institute of Environmental Medicine Centerprograms supported by grant CA13343 from the National Cancer Institute and grant ES00260 from the National Institute of Environmental Health Sciences. We thank Ms.Eleanor in document preparation. Cordisco for her expert help REFERENCES

1. D.H. Hamer, Ann. Rev. Biochem. 52913-951 (1986). 2.

M. Karin, Cell 41:9-10 (1985).

3. P.T. Thomalley, B.A. Teicher, M.P. Hacker, D.M. Hamer, and

K.V&,

Biochem. Biophys.Acta 82236-66 (1985).

4.

A. Bakka, A.B.S. Johnsen, I. Endersen, andU.E. Rugstad, Expenentia 28381-383 (1982).

5. C. Klein, andT. Rossman, Environ. Mol. Mutagen. 161-12 (1990). 6. C.B. Klein, Environ. Mol. Mutagen. 23(Suppl. 23):31 (1994). 7. C.B. Klein, L. Su, T.G. Rossman, and E.T. Snow,Mutat. Res. 304:217-228 (1994).

98 8.

Rossman

and

Goncharova

E.I. Goncharova and T.G. Rossman, CancerRes., (1994) (In Press).

9. E.Otto, S. McCord, and T.D. Tlsty,J. Biol. Chem. 264:3390-3396 (1989). 10. X.Huang, K. Frenkel, C.B. Klein, and M. Costa, Toxicol.Appl. Phurmucol. 12029-36 (1993). 11. S.L. Kelley, A. Basu, B.A. Teicher, MP. Hacker, D.M. Hamer, and J.S. Lazo,Science 241:1813-1815 (1988). 12. W.G. Nelson, andD.S. Coffey, NCIMonographs 423-29 (1987). 13. W.C. Earnshaw, andM.M.S. Heck,J. Cell Biol. 100:1716-1725 (1985). 14. L.A. Zwelling, E. Estey, M. Bakic, Monographs 4:79-82 (1987).

L. Silberman, and D. Chan, NCI

15. L.F. Ziu, Ann. Rev. Biochem.58:351-375 (1989). 16. T.-S. Hsieh, in DNA Topology and its Biological Effects. Cold Spring Habor Laboratory Press, Cold SpringHarbor, W, 1990, pp. 243-262. 17. B.F. Cain, andG.J. Atwell, Eur. J. Cancer 10537-549 (1974). 18. L.A. Zwelling, MJ. Mitchell, P. Satipanway-cha,J. Mayer, E. Altshuler, M. Hinds, and B. Bugully, Cancer Res. 52:209-217 (1992).

19. T.C. Rowe, C.L. Chen, and Y.H.Hsiang, CancerRes. 46:2021-2026 (1986). 20. L.R. Ferguson, and W.A. Denny, J . Med. Chem. 22:251-255 (1979). 21. W.R. Wilson, N.M. Harris, and LR.Ferguson, Cancer Res. 44:4420-4431 (1984).

22. M.M.

Moore, D. Clive, J.C. Hozier, B.E.Howard,A.G. Turner, and J. Sawyer, Mutat. Res. 151:161-174 (1985).

Batson, N.T.

23. D. DeMarini, C.L. Doerr, M.K., Meyer, K.H. Brock, J. Hozier, and M.M. Moore, Mutagenesis5349-355 (1987). 24.

L.L. Deaven, M.S. Oh,and R.A. Tobey, J . Natl. Cancer Inst. 6 0 11551161 (1978).

99


25. NL. Shibuya, A.M. Meno, D.B. Vannais, P.A. Craven, and C.A. Waldren, Cancer Res. 541092-1097 (1994). 26. J.L. Jurlina, A. Lindsay, J.E. Packer, B.C. Baguley, and W.A. Denny, Jr.,

Med. Chem.30473-480 (1987). 27. H. Thor, M.T. Smith, P. Hartzell, G. Belommo, S.A. Jewell,and S. Orrenius, J . Biol. Chem. 25212419-12425 (1982). 28. E.O. Ngo, T.-P. Sun, J.-Y. Chang, C.-C., Wang, K.-H.

Chi, A.-L. Cheng, and L.M. Nutter, Biochem. Pharmacol.42:1%1-1968 (1991).

29. D. DiMonte, D. Ross, G.Bellomo,

L. Eklow, and S. Orrenius, Arch.

Biochem. Biophys. 232334-342 (1984).

30. F. Mirabelli, A. Salis, M. Perotti, F. Taddei, G. Bellomo, and S. Orrerius, Biochem. Pharmacol.37:3423-3427 (1988). 31. K . 4 . Min, Y. Terano, S. Onosaka,and Phatmacol. 11374-79 (1992).

K. Tanaka, Toxicol. APPl.

32. L.D.H. Petering, and B.A. Fowler, Environ. HeallthPerspect. 65:217-224 (1986). 33. J. Liu, W.C. Kershaw, and C.D. Klaassen, Toxicol. Appl. Pharmacal. 10227-34 (1991). 34.

J. Liu, W.C. Kershaw,and D. Klaassen, J. Toxicol.Environ. Health.35:5162 (1992).

35. T.G. Rossman, in Handbook of Experimental Pharmacology, Toxicology of Metals -Biochemical Aspects (R.A. Goyerand M.G. Cherian, Eds.) Springer-Verlag, New York, 1994. 36. K. Yamanaka, M. Hoshino, M. Okamoto, R. Sawamura, A. Hasegawa, and S . Okada, Biochem. Biophys.Res. Commun.16858-64 (1990). 37. K. Yamanaka, A. Hasegawa, R. Sawamura, and S. Okada, Toxicol. Appl. Pharmacal. 108205-213 (1991). 38. Keyse, S.M., and Tyrrell, R.M. Proc. Natl. Acad. Sci USA 85:99-103 (1989).

100

Goncharova and Rossman

39. A. Albores, J. Koropatnick, M.G. Cherian, and A.J. Zelazowski, Biol. Interactions 85:127-140 (1992).

Chem.-

40. T. Maitani, N. Saito, M. Abe, S. Uchiyama, and Y. Saito, Toxicol.Le#. 3963-70 (1987). 41. M. Kreppel, J.W. Bauman, J. Liu, J.M. MC-, Appl. Toxicol. 20:184-189 (1993).

and C.D. Klassen, Fund.

42. 2. Wang, Y. Shore, and T.G.Rossman, A cadmium-sensitive Chinese hamster cell line with low constitutive level of metallothionein gene expression. WC.First International Symposium onMetals and Genetics, Toronto, 1994, p. 2 6 . 43. A. Albores, J. Koropatnick, M.G. Cherian and A. Zelazowksi, Chem.Bio1. Interactions85:127-140 (1992).

44. G.R. Stark, M. Debatisse, E. Giulletto, and G. Wahl, Cell 52901-908 (1989). 45. Wei, H.and K.Frenkel, Carcinogenesis 14(6):1195-1201 (1993).

46. R.A. Floyd, FASEB J. 4~2587-2597 (1990). 47. M. Sato, and I. Bremner, Free Rad. Biol. Medicine 14:325-337(1993).

7 Protection from Metal-Induced DNA DamagebyMetallothioneininan in Vitro System Lu Cai Department of Pathology,University of WesternOntario,London, Ontario N6A 5C1, Canada Jim Koropatnick and M.George Cherian Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada

I. INTRODUCTION Chemicalalterationsin DNA resulting From theactionofoxygen free radicals formed during oxidant stress incells can result in mutagenic lesions. Although free radicals are formed during normal cellular metabolism, are most detoxified by physiological antioxidant systems [ 1-41. Most of the toxicity of in vivo is thought to arise from transition metal oxygen and hydrogen peroxide ion-catalyzed production of highly reactive hydroxyl radicals (.OH) by the Fenton reaction[2,5-81. For example, iron (a participant in the Fenton reaction) can promote the formationof reactive radicals that initiate oxidative damage to nucleic acids, proteins, and lipids [6-141. Cu(II) ions can also participate in 101

Cai et al.

102

formation of *OHby the Fenton reaction, and appear to be potentially more reactive in mediating oxygen radical-induced cytotoxicity and genotoxicity than Fe ions 115-191. may also enhance the effects of other oxidizing agents, including ionizing [20,21] and UV [22] radiation. Therefore, elucidating the mechanism of iron-or copper-induced oxidative damage to biological molecules is important in understanding the initiation and progress of several diseases, including cancer. Oxidant stress and DNA damage havebeen suggested to be critical in aging, mutagenesis, and carcinogenesis [23-251. Itis clear that severalmetal ions can bind with DNA or chromatin in vivo, or oxidative stress could liberate them from intracellular storage sites with subsequent binding to DNA [8-121. DNA could, in fact, be damaged in the presence of reagents capable of generating reactive oxygen radicals: hydrogen peroxide in the presence of iron [4,13-151 or copper [lS-191 is an example of such a situation. Although oxidizing radicals are produced at the cellular level,organisms can counter their deleterious effects through enzyme systems (catalase, superoxide dismutases and glutathione peroxidase) and antioxidants (vitamin C,vitamin E and thiolcontaining molecules) that inhibit cell damage, and can utilize DNA repair enzymes to correct DNA damage [25-271. Metallothioneins (MTs) are metal-binding proteins with a high (approximately 30 %) cysteine content: it has been suggested that they play a role in both essential metal homeostasis and resistance to heavy metal toxicity [28]. They may also play an antioxidant role [29-321. Similar to glutathione,MT maybindandinactivateavarietyof radicals, including hydroxyl and organic radicals induced by metals [33,34], radicals induced by ionizing radiation [35,36], and radicals induced by other chemical reactions in vivo [37]. Direct addition ofMT can inhibit hydroxyl radical-induced DNA damage in an aqueous in vim reaction system using Fe-EDTA [38]. On the other hand, Cd/Zn-MT has been shown to induce damage to supercoiled plasmid DNA through the generation of radicals [39]. The effect of MT on the ability of radicals to damageDNA in intactcellsis,therefore, an openquestion. However,thecellularcompartmentalization of MTwithinnucleiduring development [40,41] and in certain human tumours [42], and the importance of metals in chromatin structure and gene regulation [43-46] suggest that an

Cum

Protection from DNA

Damage by Metallothionein

103

intimate association of MT and DNA is of biological significance. We explored the possibility thatMT may protect DNA from radical-mediated damage using a free radical-generating system in which 'DNA was cleaved in vitro in the presenceofactivatedoxygen. These freeradicalsweregeneratedfrom hydrogen peroxidein the presence of ascorbic acid by a Fenton reaction, which is critically dependent on metals.

II. COMPARISON

OF DNADAMAGEINDUCED COPPER COMPOUNDS

BY R O N AND

When calf thymus DNA (66 pglml) was incubated with hydrogen peroxide (2 mM H,OJ and ascorbic acid (2 mM) in the presence of iron (50 PM)or copper (50 pM) salts at 30" C for 30 min in Chelex-treated 20 mM phosphate buffer @H 7.0), several degrees ofDNA damage were observed (Fig. 1). The EDTA to a concentration of10 mM reaction was terminated by the addition of and the intact DNA content was measured by fluorescence in the presence of l mM ethidiumbromide(EB)(excitationat 510 nm andemission at 590 nm)[47]. Enhanced fluorescence following interaction of EB and DNA is a measure ofthe integrity ofDNA a solution containing all reagents exceptKO, was assumed to have 100%fluorescence and0% DNA damage. DNA damage was measured as a loss in fluorescence[48]. In control experimentsEDTA was added before the addition ofcopper or iron and ascorbate to prevent the action of metal ions. Zero fluorescence wasassessed in a solution identical to control except that DNA was absent. 50 p M Cu reduced EB/DNA fluorescencebymorethan 95% while equimolarconcentrationsofironresultedinonly 30% damage. Thus, the ability of copper to damageDNA was much higher than that of iron under our experimental conditions (Fig. 1). A. Iron

Fifty p M Ferric ammonium sulfate reNH,(SO,)J induced only a 30% loss of fluorescence (Table1) compared to a95%! loss induced by equimolar Cum

104

Cai et al.

(Fig. 1) in the presence of 2 mh4 H202. Maximum damage was 67%.and was achievable only by increasing theH202 concentration to 6 mh4 and iron to 100

x 100 -

6

F 75E

a

50-

4

n 250(v

* * 0 0 c/)

3

0

v, 6,

LL

(v

0 6, LL

n

v

m n m

0 v, W

0

Z

W

a

G

0 a, LL

v I Z Q) LL

figure I Cu(I1) induces DNA damage in the presence of H202 and ascorbate more effectively than Fe(II) and Fe(I1I). Addition of 50 pM Cu(II), Fe(1I) or Fe(III) in the presence of 2 mM H202 and 2 mM Na-ascorbate caused loss of fluorescence of the ethidium bromide @B)-DNA complex. DNA damage was assessed as the loss of fluorescence. 100% damage results in complete loss of fluorescence.

Protection from DNA

105

Damage by Metallothionein

Table 1 DNA damage induced by FeNH,(SO,), in the presence and 6.0 mM H,O, and 2 mM ascorbic acid.

0 0.1 1.o 5.0 10 20 50 100 200

0

0

3.4 f 1.5 14.6 f 5.4 16.7 f 5.2 13.7 f 2.3 15.6 f 2.2 15.3 f 1.2 21.6 f 0.8 27.5 f 2.4

10.1 15.6 22.6 24.5 28.4 29.1 29.9 42.4

f 2.3 f 2.7 f 5.9 f 0.7 f 5.6 f 0.9 f 0.3 f 0.9

of 0.8, 2.0

0 6.9 f 0.1 11.7 f 3.1 26.2 f 6.6 30.5 f 0.9 28.0 f 1.7 41.4 f 6.8 67.5 f 1.5 65.4 f 2.0

also mediated a significant increase in oxygen radical-induced

DNA

damage that was dependent upon both hydrogen peroxide (Table2) and metal concentration (Table3). Relatively high levelsof F e 0 (125 pM) or hydrogen peroxide (4 mM) were required to induce a 50% loss in fluorescence.

B. Copper

Cum

was amoreeffectivemediator than of DNA damagecausedby hydrogen peroxide. It caused a 50% loss in fluorescence at less than 5 pM Cum in the presence of only 2 mM hydrogen peroxide (Table 4). Maximum loss of fluorescence in 2 mM hydrogen peroxide occurred at 10 pM copper chloride; in 0.2 mM hydrogen peroxide maximumDNA damage was induced

per chloride. Thus, Cu(I1) and Fe(I1) had gen peroxide to generate free radicals, w1 effective of the two.

t effwts on

0,on F ~ I I ) - ~ DNA d u ~damage"

1

DNA damage (95)

0

0

0.2 1.0 2.0

6.6 f 0.1

.o

A

12.4 j, 0.5 33.1 j, 0.5 49.52 f 0.02

ascorbic acid and 50 pM Fe(II).

ffwt of Fe(I1) on DNA damage in the presence of 2

F

DNA damage (%)

0 0.5 1.25

0

16.5 f 3.2 15.8 f 2.3 17 f 1 31 j , 4 47 I: 2

2

mci

Effect of Cu(I1) concentration on DNA damage in the 0.2 or 2.0 DIM H202

NA damage ((270)

2.0

0 0.1 1 .o 5.0 10.0 20.0 40.0 60.0 100.0 200.0

0

3.0 f 0.1 6.1 f 0.1 9.2 f 0.5 23.6 f 1.6 1.8 f 0.1 59 f 1 70 f 3 63.4 f 0.6 68 f 2

0 2

0 17 f 1 31 f 13 56 f 5 90 f 1 93.6 f 0.7 not done 98.2 f 0.7 98 f 2 not done

108

Cal et ai. 100.

L

i

*

95 -

90 -

*+ 85

~

0

300

900 600

1200

1500

Zn-MT (ug/ml>

100

50

I

0.0

0.5

1.0

I

1.5

2.0

H202

figure 2

Zn-MT protectsDNAfromdamageinducedby

&(IQ inthe

presence o f H202 andNa-ascorbate.(A)DNAdamageinducedby 50 p M Cu(I1) in the presence of 2 mM H202 and 2 mM Na-ascorbate'and increasing amounts of Zn-MT; (B) DNA damage induced by50 pM &Ci2 in the presence of increasing amounts of H202, (0) in the absence of Zn-MT, (A) with 4.12 pg Zn-MTlml; (0) with 41.2 pg Zn-MTlml.

109

Protection from DNA Damage by Metallothionein

III.

DNA DAMAGE INDUCED BY CU(Il)-MEDIAmD OXYGEN RADICAL IS INHIBITED BY METALLOTEXONEIN

Zinc-bound MT (Zn-MT) inhibited the ability ofCum to mediate radicalinduced DNA damage in the presence of hydrogen peroxide(2 mM) and Naascorbate (2 mM). The data are presented in Figure 2 (A,B). The inhibitory effect of Zn-MT in DNA damage was dosedependent in .the presence of hydrogen peroxide and ascorbate (Fig. 2A).As little as 40 pg MT/ml exerted a significant protective effect(Fig 2B). At a concentration of 500 pg/ml, ZnMT effectively protected 80% or more of DNA at risk for oxygen radicalinduceddamageatcopperconcentrations as high as 200 pM (Fig. 3). Compared to the marked protective effect of 100 p g / d MT. other proteins such as calf thymus histone (100 pglml) did not protect Cu(II)-induced DNA damage, and bovine albumin (100 pg/ml) exerted little protection (Fig. 4).

x

6

m a E

l

o

t

o

-

75

x

M1

a U

< 7

(3

0

@we

20

40 60 80 100120140160

1

180 200

3 Zn-MT (500 pg/rnl) protects DNA fromdamageinducedby increasing concentrations ofCu(I1) in the presence of 0.2mM H202 and 2 mM Na-ascorbate.

110

Cai et al.

100

Cu(I1) alone

+ Cu(I1)

+ MT (100 clg/ml)

7 5 +- Cu(I1) + His (100 clg/ml)

+ Cu(I1)

+

AL (100 clg/ml)

50

25

0l

I

0

10

20

figure 4 Calf thymus DNA damage induced by Cu(I1) is markedly inhibited by Zn-MT. Bovine albumin exerted little protection, and calf thymus histone did not protect. All reactions were camed out in the presence of 2 LUMH202 and 2 mh4 Na-ascorbate. DNA damage was assessed by loss of fluorescence loss of the DNA-EB complex. AL: albumin; His: histone.

111


W . FORMATION OF CU(I) FROM CU(n) AND ITS INHIBITION BY METALLOTHIONEIN

Cum

generated from Cu(I1)may be the active ion mediating free radical from Cu(II) in thepresenceof production[16,17]. Generation of hydrogen peroxide and ascorbate was determined using bathocuproinedisulfonic u O and acid (BCS)and the method of Li and Trush [ 161. Concentrations of C BCS (pH7.0) were varied to achieve a final BCS concentration of 0.3' mM in a total volume of 2 ml at 25" C The concentration of stable BCS-Cu(I) complex formed wasmeasured by absorption at 480 I"The amount ofmetal available for Cu(I) formation (and not hydrogen peroxide concentration) was

Cum

.

rate-limiting, with Cu(1) formation reaching a plateau at approximately 75 pM CuCl, (data not shown). The addition of Zn-MT (500 pg/ml) to a copper chloridehydrogen peroxidelascorbate system inhibited production of active from Cu(I1) (Fig. 5).

Cum

z

.75

,,P

C

0

aJ

d-

v

C 0

CUCl* (MM) Figure 5 Cu(1) production from Cu(I1) in the absence of MT (0) or presence of 500 p g / d h - M T (A). 2 mM H202+ 2 mM Na-ascorbate were included in reaction mixtures. Cu(1) formation was assessed by absorption at 480 m.

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V. DISCUSSION Superoxide radicals (O;-) are formed in all aerobic cells. Most damaging effects of systems capable of generating qhave been attributed to the metaldependent formation of more reactive species, such as hydroperoxyl radicals (Hop),singlet Oz, and hydroxyl radicals(-0H)[2,5-8]. Highly reactive *OH radicals can be produced by a reaction between Fe@) and H,O,: the process is termed the Fenton reaction and proceeds as shown in (1): Fe@)

+ H202+ Fe@) + -OH + OW

(1)

Many metal ions and their complexes in lower oxidation states (Cum, C@), and C oo,for example) can participate in this reaction to produce radicals capable of inducing oxidative DNA damage [5]. The present study confirms the previous reports on both Fe and Cu induced DNA damage in the presence of H202and ascorbate (Fig. 1). The hydroxyl radicals could also be generated from 0,- and H202in a Haber-Weiss reaction as shown in (2) and (3):

Fe@)

+ H,O,

+

Fe@)

+ *OH + OH-

(3)

Reactions (l), (2) and (3) are interrelated in that the Fenton reaction is the second step of the Haber-Weiss reaction. In reaction (2), 4"[which reduces Fe@) to Fe@)] can be replaced by alternate reducing agents- glutathione or ascorbic acid, for example [49-521. We reportthe differences in Fe andCu induced DNA damage in vitro in the presence of HzOz and ascorbic acid. The phenomenon displays two different features: (1) C u O induces much more extensive DNA damage compared to iron, confirming data from previous studies[48,53-551, and (2) DNA damage induced by Cum is markedly dependent on metal and H202 concentration. DNA darnage in thepresenceof iron, on theotherhand, was minor at equimolar metal and hydrogen peroxide levels, and became significant only at (6 mh4) and iron (125 PM). high concentrations of H202


113

Muiras et al. [l41 demonstratedthatthedose-responsecurveforFe@)At afixed inducedDNAdamageinthepresenceofHzOziscomplex. concentration of Fe(I1) the maximum extent of DNA damage occurred at low HzOz concentrations (10-30 PM). Higher hydrogen peroxide concentrations markedly suppressed DNA damage: it was suggested that high levels of HzOz scavengedhydroxylradicalsandinhibitedthedamagingeffect[14,57,58]. Furthermore, their results also indicated that the dose-response curve Fe@)- for induced DNA damagein the presence of HzOz critically depended on the nature of the reaction buffer, ionic strength, temperature and pH [14]. However, the lack of suppressionof Cu(II)-induced DNA damage in the presence of ascorbic acid and high HzOz concentrations had previously been reported [48]: at the highest levels of peroxide applied mM), (10 almost complete DNA degradation occurred within 8 min. DNA degradation induced by Hz02 in the presence of catalyzing iron was about50 times slower than with copper (DNA damage was much less extensivethanthatobservedwithcopperunderthesame experimental conditions). They suggested that the phenomenon was due to differenca in the binding of Cum and Fe@) to DNA: scavengers of .OH radicalsprotectedagainstirondependent,butnotcopperdependent,DNA damage. It is well-establishedthatFeions[4,13-151 or Cu ions[15-19], can participate in the formation of *OH from Oz and HzOz, both in vitro and in vivo: the pattern of radical-mediated base modification in the presence of these metals is similar to that produced by ionizing radiation. However, the formation has been disputed. of .OH in reactions involvingCu(II) ions and HzOz [56,57] It has been reported that nitrilotriacetic acid inhibited DNA damage in systems containing Cu(II), butincreasedthereactivity of Fe [13]. In addition,the extensiveDNAdamageproduced by Cu(II)/HzOz/ascorbic acid is not significantlyinhibited by superoxidedismutase or by the .OH scavenger mannitol.Theinabilityofhydroxylradicalscavengerstoprotectagainst damage in several systems has been the basis of arguments that those radicals are not responsible forDNA damage [57,58]. Subsequent to those arguments, it was suggested that the production of modified DNA in systems containing ions and Hz02and/or 0,- or ascorbic acid is mediated by .OH [i.e., Cu(II) ions bound to DNA could react with HzOzand ascorbic acid to generate

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hydroxyl radicals, which then immediately attack and damage DNA bases]. Thus, in terms of its ability to promote damage to DNA, Cu(II) is an extremely active metal, much more than Fe(III) and FNII).Cu(I1) is also a very effective in promoting oxidation of some lipids [7]. The high activity of Cum in increasing hydroxyldependent damagetoDNAand tootherbiomolecules suggests that the intranuclear availability of Cum ions in vivo is likely to be carefblly controlled. The protection of DNA from Cu-induced oxidative damage may be important in limiting mutagenesis and consequent carcinogenesis. There is increasing evidence that MT can act as a free radical scavenger. Rat liver Cu-MT, for example, enhanced dismutation of superoxide radicals (W) in vifro [59]. MT has also been shown to decrease the toxic effects of both hydroxyl and superoxide radicals produced by the xanthinexanthine oxidase and whole animal reaction in virro [31]. In cultured cell [29,30,36] [35,37,60,61] experiments, a role for MT in scavenging free radicals generated from a varietyof chemical and radiation-induced sourceshas been implicated. This suggests that MT is ofimportance in cellular defense mechanisms directed against free radicals. In addition to its ability to protect, there is controversy with regard to the ability of MT, under some circumstances, to potentiate cellular damage. Cu,ZnMT actually stimulated microsomal lipid peroxidation initiated by xanthinexanthine oxidase in virro [62]. DNA damage could, potentially, be caused by unidentifiedradicalspeciesformedbyZn/Cd-MT[39]. Reports existthat overexpression of foreign MT genes in transfectedCH0 cells did not increase cellular resistance to subsequent oxidative challenges [63-66]. Here, however, it isclear thatMT did not increase Fe-or Cu-induced DNA damage,butrathermarkedlyreducedit. This supportsthereportthat FeinducedDNAdamagecould be inhibitedby MT [38],andextendsthe suggestion thatMT could directly protect calf thymus DNA from Cu-inducing damage in vitro. At the level of whole cells, induction ofMT expression may protect against subsequentfree radical-induced damage: zinc-induced Chinese hamster V79 cells exhibit both increased MT content (without increased GSH levels).and reduced susceptibility to HzOz-inducing DNA damage, while cells transfectedwithantisense MT-1RNAexpressionvectors aresensitiveto oxidant stress [60].

Protection from

DNA Damage by Metallothionein

115

The mechanismbywhichMTprotectsDNAfromfreeradical-induced in vitro ionizingradiation damagemay be bybindingthosefreeradicals: studies have shown that Zn,Cd-MT can effectively protect against radiationinduced damage to the tagadphosphate DNA backbone [67]. However, the mechanism by which MT inactivates free radicals is still unclear. Although MT does not normally bindFe in vivo, it appears to preventfree radical formation [68]. It shouldbe mentioned that anFe-MT complex can in the Fenton reaction be formed in vitro underanaerobicconditions [as]. WereportthatDNA damage caused byCu(I) generated fromCu(II) was inhibited by addition of MT (Fig. 5). These results suggest sequestration of Cu(II) by MT and thereby inhibition of the availabilityCu of m for reduction toCuo.The higher affinity of MT for copper than for zinc suggests that Zn-MT found under uninduced cellular conditionsin vivo would be available for such sequestration. In addition to a role for MT in preventing formation of damaging radical species by interaction with metals, it is also possible that MT could bind with DNA to reduce the possibility ofDNAlCuO interactions (considered tobe a key step in Cu-induced DNA damage). The sitespecific DNA damage induced in the presence of Cuhydrogen peroxide suggests that copper binds to specific DNA sites and forms singlet oxygenor copper-peroxide radicals [53]. In our studies, DNA in the presence of Zn-MT formed a precipitate in the presence of 50 pM C u m and 2 mM YO,: thatprecipitatecontainedbothDNA ( a s d by ethidium bromide fluorescence and agarose gel electrophoresis) and MT(assessedbyWesternblotanalysis). It is possiblethat Zn-MT complexed with DNA can sequester metals and thus reduce copper-induced damage to DNA. Recent studies, however, show that copper-MT can increase lipid peroxidationin vitru and can act as a pmxidant [70]. Thus the specific metals bound to MT may influence the properties of MT in oxidant stress. In summary, we report that mammalian Zn-MT has the capacity to inhibit DNA damage induced by free radicals generated from reactive oxygen donors in the presence of copper. It may do so by direct interaction with copper to prevent its participation in redox reactions. The localization of MT in cell nuclei under certain circumstances (for example, during embryonic and postnatal mammalian development, and in certain human tumours) may suggest a role for MT in protecting against DNA damage induced by free radicals.

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W. ACKNOWLEDGMENTS This research wassupportedby Research Council of Canada.

grants to MGCand

JK bytheMedical

REFERENCES l. I. Fridovich, Arch. Biochem. Biophys. 247, 1-1 1 (1986). 2. K. Brawnand I. Fridovich, Arch. Biochem.Biophys. 206, 414-419 (1981). 3. D.A.RowleyandB.Halliwell,Biochim.Biophys.Acta 761, 86-93 (1983). 4. 0.1. Aruoma, B. Halliwelland M. Dizdaroglu, J. Biol.Chem. 264: 13024-13028 (1989). 5. S. Goldstein, D. Meyerstein and G. Czapski. Free Radic. Biol. Med. 15, 435-445 (1993). 6. S.D. Aust, C.F. Chignell, T.M. Bray, B. Kalyanaraman and R.P. Mason, Toxicol. Appl. Pharmacal. 120, 168-178 (1993). 7. M.K.Eberhardt,C.SantosandM.A. Soto, BBAGEN 1157, 102-106 (1993). 8. S.D. Aust, L.A. Morehowse, C.E. Thomas, J. Free Radic. Biol. Med. 1: 3-25 (1985). 9. D.W. Feif, Free Radic. Biol. Med. 12,417-427 (1992). 10. R.F. Castilho, A.R. Meinicke, A.M. Almeida, M. Hermes-Lima and A.E. Vercesi, Arch. Biochem. Biophys., 308, 158-163 (1994). 11. E. Kukielka and A.I. Cederbaum, Arch. Biochem. Biophys., 308,70-77 (1994). 12. E.S.Driomina, V.S. Sharovand Y.A. Vladimirov,FreeRadic.Biol. Med., 15, 239-247 (1993). 13. 0.1. Aruoma, B. Halliwell, E. Gjewskiand M. Dizdaroglu, J. Biol. Chem., 264, 20509-20512 (1989). 14. M.L. Muiras, P.U. Giacomoni and P. Tachon, Mutation Res. 295,47-54 (1993).


117

15. M. Dizdaroglu, G . Rao,B. Halliwell and E. Gajewski, Arch. Biochem. Biophys. 285, 317-324 (1991). 16. Y.B. Li and M.A. Trush, Arch. Biochem. Biophys. 300, 346-355 (1993). 17. Y.B. Li and M.A. Trush, Carcinogenesis, 14, 1303-1311 (1993). 18. 0.1. Aruoma, B. Halliwell, E. Gajewski and M. Dizdarogl, Biochem. J. 173, 601-604 (1991). 19. L. Miline,P.Nicotera, S. Orrenius andM.J. Burkitt, Arch. Biochem. Biophys. 304, 102-109 (1993). 20. A. Samuni, M. Chevion andG. Czpaski, Radiat. Res. 99,562-572 (1984). 21. M.A. George, S.A. Sabovljev, L.E. Hart, W.A. Cramp, G. Harris andS. H O ~ S ~Brit. Y , J. Cancer, 55 (Suppl. VIII), 141-144 (1987). 22. R.E.Lloyd,R.A. Larson, T.L. AdairandR.W.Tuveson,Photochem. Photobiol. 57, 1011-1017 (1993). 23. R.M. Rose, Evolutionary Biology of Aging, OxfordUniversity Press, 1991. 24. D.I. Feig, M.M. Reid, L.A. Loeb, Cancer Res., 54,11890~-1898~(1994). 25. M.G. Simic, Cancer Res., 54, 1918s-1923s (1994). 26. T. Miura, S. Muraoka andT. Ogiso, Pharmacology &Toxicology, 74,8994 (1994). 27. A. Meister, Cancer Res., 54, 1969s-1975s (1994). 28. D.H. Hamer, Annu. Rev. Biochem., 55, 913-951 (1986). 29. D.M. Templeton and M.G. Cherian, Meth. Enzymol. 205, 11-24 (1991). 30. N. Imura, M. Satoh and A. Naganuma, inMetalbthionein in Biobgy and Medicine (C.D. Klaaassen, andK.T. Suzuki, W), CRCpress,Boca . Raton,BostonandLondon, 1991, pp.375-382. 31. P.J. Thornalley and M.Vasak, Biochim. Biophys. Acta827,364(1985). 32. M. Sato and I. Bremner, Free Radic. Biol. Med. 14, 325-337 (1993). 33. H.M. Chan, R. Tabarrok, Y. TamuraandM.G. Cherian, Chem.Biol. Interactions 84, 113-124 (1992). 34. M.G. Cherian and M. Nordberg, Toxicol. 28, 1-15 (1983). 35. A. Bakka, A.S. Johnsea,L. Endresen and H.E. Rugstad, Experientia, 38,

381-383

(1982).

36. M. Satoh, A.Naganumaand N. Imura, Cancer Chemother. Pharmacol., 21, 176-178 (1988).

118

Cai et al.

37. J.P. Thomas, G.L.Bachowski andA.W. Girotti, Biochem. Biophys. Acta, 884, 448-461 (1986). 38. J. Abel and N. deRuiter, Toxicol. Lett., 47. 1991-1996 (1989). 39. T. Muller, R. Schuckelt andL. Jaenicke, Arch. Toxicol. 65,20-26 (1991). 40. M.Panemangalore,D.Banerjee, S. OnosakaandM.G.Cherian,Dev. Biol., 97: 95-102 (1983). 41. N.O.Nartey, D. BanerjeeandM.G.Cherian,Pathology 19:233-238 (1987). 42. N.O. Nartey, M.G. Cherian and D. Banerjee, Am. J. Pathol., 129:177182 (1987). 43. C.E. Castro, Annu. Rev. Nutr., 7: 407421 (1987). 44. J.K. Chesters, Nutr.Rev., 50: 64-71 (1992). 45. T.V. O’Halloran, Science 261: 715-725 (1993). 46. C.O. Pabo and R.T. Sauer, AM. Rev. Biochem., 61: 1053-1059 (1992). 47. G.R. Buettner, J. Biochem. Biophys. Methods 16, 27-40 (1988). 48. R. Stoewe and W.A. Prutz, Free Radic. Biol. Med. 3, 97-105 (1987). 49. B. Halliwell and J.M.C. Gutteridge, Arch. Biochem. Biophys. 246, 501514 (1986). 50. D. Jamieson, Free Radical Biol. Med., 7: 87-108 (1989). 51. I. Karuzina and A. Archakov, Free Radical Biol. Med., 16, 73-97 (1994). 52. C.J.Reedand K.T. Douglas,Biochem.Biophys.Res.Commun. 162, 1111-1117 (1989). 53. K. Yamamoto, S. Inoue and S. Kawanishi, Carcinogenesis. 14, 1397-1401 (1993). 54. P. Techon, 1989, Free Radical Res. Commmun, 7, 1-10 (1989). 55. J.A. M a y and S. LW, Science 240, 1302-1309 (1988). 56. J.A. M a y , S.M. Chin and S. Linn, Science, 240, 640-642 (1988); 57. G.R.A. Johnson, N.B. Nazhat and R.A. Saadalla-Nazhat, J. Chem. Soc. Chem. Commun., 404408 (1988). 58. H.C. Sutton andC.C.Winterbourn,FreeRadicalBiol.Med. 6, 53-60 (1989). 59. M. Shiraishi, K. Utsumi, S. Morimoto, I. Joja, S. Iida, Y. Takeda and K. Ano, Physical. Chem. Phys.. 14, 533-537 (1982). 60. L.S. Chubatsu and R. Meneghini, Biochem. J. 291, 193-198 (1993).


119

61. T.P. Coogan, R.M. Bare and M.P. Waalkes, Toxicol. Appl. Pharmawl. 113, 227-233 (1992). 62. J.R. Arthur, I. Bremner, P.C. Morrice and C.F. Mills, Free Radic. Res. C O ~, 4,. 15-20 (1987). 63. B. Kaina, H. Lohrer, M.KarinandP.Herrlich,Proc.NatlAcad.Sci. USA., 87, 2710-2714 (1990). 64. H. Lohrer and T. Robson, Carcinogenesis 10, 2279-2284 (1989). 65. M. Miura and T. Sasaki, Radiat. Res., 123,171-175(1990). 66. J. Koropatnick and J. Pearson, Mol. Pharmacol., 44, 44-50(1993). 67. C.L. Greenstock, C.P. Jinot, R.P. WhitehouseandM.D.Sargent, Free Radic. Res. &mm. 2, 233-239 (1987). 68. A.C. Mello-Filhoand R. Meneghini, Biochim. Biophys. Acta, 847, 82-89 (1985). 69. W.A. Prutz, J. ButlerandE.J.Land,Int. J. Radiat.Biol., 58,215-234 (1990). 70. G.F.Stephenson,H.M.ChanandM.G.Cherian,Toxicol.Applied Phannacol., 125, 90-96 (1994).


DNA Strand Breakage and Lipid Peroxidation as Possible Mechanisms of Selenium Toxicity J. Kitahara, Y. Seko, and N. ImuraSchool of PharmaceuticalSciences, Kitasato University, 5-9-1, Shirokane, Minato-h, Tokyo 108, Japan H. Utsumi and A. HamadaSchool University, Tokyo, Japan

of PharmaceuticalSciences,Showa

I. INTRODUCTION Selenium isknown as an essential trace element constitutingthe active site of glutathione peroxidase which handles the active oxygen suchas hydrogen peroxide and organic hydroperoxides to protectanimalsfromoxidativestresses.Ontheotherhand, selenium has long been recognized as a toxic element which causes alkaline disease andblind stagger disease in farm animals and human poisoning in some areas of high selenium content in the soil such as Enshi district in China. Further, it was reported recently that a health food supplement having unusually high selenium content caused human intoxication [l]. Although the molecular basis of its desirable functions has recently been well documented, the mechanism of selenium toxicity has not clearly beenelucidated yet. Cytotoxicity of selenite was reported to be enhanced by glutathione (GSH) [2-4]. Further, the addition of selenite to rat hepatocyte cultures caused lipid peroxidation [5]. A few papers 121

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have suggested superoxide anion (m-)generation by the reaction of selenitewithsulfhydrylcompounds in vitro [6-81.These results seem to indicate a possible involvement of active oxygen species in selenite toxicity. However, we recently found that the experimental procedures used for proving superoxide anion formation with cytochrome c or acetyl-cytochrome c were inadequate and the results could not be an evidence for active oxygen formation in the reaction of selenite with sulfhydryl compounds [9]. These facts prompted us to re-examine a possibility o f active oxygen generation in the reaction of selenite with sulfhydryl compounds in vitro and to study its rolein selenite toxicity exertedin vitro and in vivo.

11.

DNA STRANDBREAKAGEANDLIPID PEROXIDATION IN ORGANS OF MICE ADMINISTEREDSELENITE

ICR mice (female, 6-weeks) were intraperitoneally administered 60 pmol/kg of sodium selenite dissolvedin saline. DNA strand break was examined athr2 after the selenite administration by the method of Sina etal. [lo]. Thiobarbituric acid-reactive substances (TBA-RS) were determined fluorometrically according to the method of Ohkawa et al. [1l] at 24 hr after the administration using 5% homogenate of various organs of mice. Dose dependent increases inTBA-RS and DNA single strand break were observed in various organsof mice treated with selenite, demonstrating that in vivo. selenite exerted oxidative stress

III. CYTOTOXICITY OF SELENITE Rat hepatocytes (2x106 cells/ml) obtained by the method of Hogberg and Kristoferson [l21 were incubated with or without selenite and/orthe other chemicals at37°C in a rotating tube under 95% 0 2 and 5% C02 atmosphere.Analiquot o f thecell suspensionwasremovedandusedformeasuringoxygen consumption rate. Another aliquot was centrifuged at10,000 x g for 15 sec. The supernatant was used for detenninig TBA-RS and extracellular lactate dehydrogenase(LDH) activity. Cellular GSH was determined fluorometrically by the method of McNeil et al.

DNA Breakage,LipidPeroxidation,andSeleniumToxicity

E

F

h

E

F

123

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[13]. LDH activity was assayed bythe method of Wroblewskiet al. [14]. Oxygen content in rat hepatocyte suspension was decreased (Fig. la) by the exposure of the cells to seleniteas well as GSH level (Fig. lb) and followed by the increasesin TBA-RS (Fig. IC) and LDH (Fig. Id) released from the cells. These changesin the indicators for celllesionweresignificantlyreduced by desferrioxamine-manganese(DFMn), an SOD mimic, suggesting a roleof active oxygen speciesin the cytotoxic action of selenite ~51.

IV. DNA STRAND BREAKAGE BY SELENITE AND GSH IN VITRO pUCl18 plasmid DNA (2 pg/ml) was incubated with 50 pM sodium selenite and2 mM GSH in phosphate buffer (pH 7.4) at 37°Cfor 4 min. The DNAwasextractedandanalyzedby agarose-gel electrophoresis [16]. DNA single strand breakage wasoccurredbythereactionwithseleniteandGSH.The presence of oxygen was shown tobe essential for the breakage. The breakage was not inhibited by the addition of Cu, Zn-SOD or catalase. The SOD appeared to enhance the DNA cleavage to some extent. Desferrioxamine and DETAPAC, iron chelators, failed in depressing the DNA cleavage. While mannitol and DMSO, hydroxyl radical scavengers, efficiently inhibited the DNA strand break. These results indicate a role of hydroxyl radical in the DNA strand break caused by selenite in the presence of GSH.

V. HYDROXYLATION OF SALICYLATE BY SELENITE AND GSH Sodium salicylate (2 mM) was subjectedto the reaction with50 pM selenite and2 mM GSHat 37°C for20 min in the presenceor absence of various scavengers or an iron chelatorin phosphate buffer (pH 7.4). The reaction was terminated by the addition of N-ethylmaleimide (final 9.1 mM) and TCA (final 5.2%). The mixture was centrifuged at 12,000 x g for 10 min and 2,5dihydroxybenzoic acid in the supernatant was determined by HPLCusingShodexODSpakcolumn(4.6x150mm).

DNA Breakage,LipidPeroxidation,andSeleniumToxicity

125

Ammonium acetate (0.2%, pH 5.5)/methanol(95:5) was used as the elution buffer (0.6mvmin). Fluorescence (excitation 3at 2Onm and emission at 460 nm) was monitored. It was confirmed that 2,5-dihydroxybenzoicacid,amajoroxidationproduct of salicylate, was formed by selenite and GSH. The amount of2,5dihydroxybenzoic acid formed from salicylate was decreased by the addition of Mn-SOD (25 units/ml) to50% of the control, but of the enzyme. not completely inhibited even with excess amount Heat-denatured Mn-SOD did not affect the hydroxylation. Cu, Zn-SOD (50 units/ml),ontheotherhand,enhancedthe hydroxylation by40%. Ironchelatorcoulddepressthe hydroxylation only partly. However, when xanthinexanthineoxidase system was usedin place of selenite and GSH, both Mn-SOD and Cu, Zn-SOD diminished the hydroxylation almostcompletelyanddesferalalsoinhibitedthereaction markedly.

VI. DEOXYRIBOSE DECOMPOSITION BY SELENITE AND GSH Deoxyribose (0.98mM)was incubated at 37°C for 20 min with 50 pM sodium selenite and 2 mM GSH in phosphate buffer @H 7.4) in the presence or absenceof scavengers or an iron chelator. The reaction was terminated by the addition of Nethylmaleimide (final 9.1 mM). The reaction mixture(0.55 ml)was added with 0.5 ml of 2.8% TCA and 0.5 m1 of 1% TBA and incubated at 100°C for 10min before determination of TBA-RS as decomposed products from deoxyribose[17]. The inhibition profile of the formation of TBA-RS from deoxyribose by the scavengers or desferrioxamine was similar to that obtainedin the hydroxylation of salicylate.

VII. ELECTRON SPIN RESONANCE (ESR) SPECTRUM OF THE MIXTURE OF SELENITE AND GSH The ESR spectrum of the mixture of selenite and GSH was measured by Electron Spin Resonance Spectrometer (JEOL, JESRElX)using 5,5-dimethyl-l-pyrrolineN-oxide @ W O ) as a spin

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trapping agent. The signals corresponding to those of hydroxyl radicaladductofDMPO(DMPO-OH)wereobtained.The addition of ethanol to the mixture gave the signals obviously corresponding to these of a-ethylhydroxy radical adduct of DMPO. The intensity of theESR signals of DMPO-OHwas not affected by Cu,Zn-SODandcatalaseandwasreducedto approximately half of the control by mannitol and DMSO, which are hydroxyl radical scavengers.

VIII. CONCLUSION As shown in Table 1, Cu,Zn-SODappears to substantially stimulate these reactions induced by hydroxyl radical. Mn-SOD partly inhibits the reactions, but not completely. Desfenioxamine, anironchelator,alsopartlydepressthedeoxyribose

Table 1 Summary :Effects of radical scavengers and iron chelator onH00 formation by selenite and GSH

.

j

n

DNA Breakage, Lipid Peroxidation, and Selenium Toxicity

................ )....................... ...:...... ... ! ..v e

.. .... ... ..

.......... U

m

.m Y

Q

127

128

Kitahara et al.

decompositionandhydroxylationofsalicylate.Mnnitoland DMSO which are known as hydroxyl radical scavengers almost completely inhibitthese reactions. The presence of oxygen seems to be essential. Further, it was confirmed in separate experiments that hydrogen selenide caused the same reactions in the presence of oxygen as those induced by selenite and GSH. In addition to the above mentioned data obtained by selenite, selenocystine, a selenoamino acid, also induced DNA strand break and lipid peroxidationinorgans ofmice. In in vitro reaction of selenocystine with GSH also caused DNA strand break and deoxyribosedecomposition.However,thesereactionswere effectively depressed by Cu, Zn-SOD and iron chelator. These results may suggest that superoxide radicalis a major species of active oxygen initially generated in the reaction of selenocystine with GSH. Finally we would like to suggest that selenide formed by the reduction of selenite with GSH or through the metabolism of as a final molecular selenoamino acid generates hydroxyl radical species of active oxygen by reducing oxygen molecule. A part of hydroxylradical is formedfromsuperoxideradicalthrough hydrogen peroxide and Fenton-tyte reaction in the presence of iron.However,majorpartofthehydroxylradicalmaybe generated through another unknown processin the presence of selenide as shown in Fig. 2. REFERENCES 1. R. Jensen, W.Closson and R. Rothenberg, Bobid. Morta1,Weekly Rep. 33,157-158 (1984) 2. J.H. Ray and L.C.. Altenburg, Mutat. Res. 54:343-354 (1978) Cancer Res. 3. G. Batist, A.G. Katki, R.W. Klecker Jr. and C.E. Myers, 465482-5485 (1986) 4. R.D. Snyder, Cancer Lett. 3473-81 (1987) 5. N.H. Stacey and C.D. Klaassen, J . Toxicol. Environ. Health 7:139-147 (1981) 6. P.Garberg, A. S a , M. Warholm and J. H6gberg, Biochem. Phamcol. 37:3401-3406 (1988) 7. G. F. Kramer and B.N.Ames, Mutat. Res. 201,169-180 (1988) 8. Y. Seko, Y. Saito, J. Kitahara and N. Imura, Active oxygen generation by the reactionof selenite with reduced glutathione in vitro in Selenium in Biology and Medicine (Wendel A, Ed) , Springer-Verlag, Berlin Heidelberg New York, 1989, pp. 70-73. 9. Y. Seko,J. Kitahara, H. Utsumi, A. Hamada and N. Imura,Reexamination of the proposed mechanismof active oxygen generationby the reaction of selenite with reduced glutathione (GSH), Fifth

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International SymposiumonSelenium in Biology and Medicine, Vandrbilt University School of Medicine, Nashville, 1992, p.107 10. F. Sina, C.L. Bean, G.R.Dysart, V.I. Taylor and M.O. Bradley, Mutat. Res. 113,357-391 (1983) 11. H. Ohkawa, N.Ohisi and K. Yagi,Anaf. Biochem. 95,351-358 (1979) 12. J. Htigberg andA. Kristoferson, Eur. J . Biochem. 74,77-82 (1977) 13. T.L. McNeil and L.V. Beck ,Anal. Biochem. 22,431-441 (1968) 14. F. Wr6blewski and J.S. LaDue, Proc. Soc. Exp. Biof. Med. 90, 210213 (1955) 15. J. Kitahara,Y. Seko andN.Imura, Arch. Toxicol. 67,497-501 (1983) 16. S. Toyokuni and J.-L. Sagripanti, J. Inorg. Biochem.47,241-248 (1992) 17. J.M.C. Guttenridge, FEBS Lett. 128,343-346 (1981)


Role of Metal in Oxidative DNA Damage by Non-mutagenic Carcinogen Shosuke Kawanishi, Shinji Oikawa, and Sumiko InoueDepartmentof Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Free radicals and other active oxygen species are constantly formed in the human body. Many of themserveuseful physiological h c t i o n s , but they can be toxic when generated in excess and this toxicity is often aggravated by the presence of ions of transition metals [l].DNA damage by active oxygen species has drawn much interest in relation to carcinogenesis. Active oxygen species may be involved in initiation, promotion and conversion of multistage carcinogenesis. The active oxygen species in metal-induced DNA damage play important roles in the metal carcinogenesis and the unknown carcinogenic mechanism of some organic carcinogens. Metal ionsreact with superoxide anion radicals (02-)and hydrogen peroxide m02)to produce highly reactive species such as hydroxyl freeradicals (*OH)and metal-oxygen complexes in biological systems (Figure 1).

Figure 1. A mechanism of metal-mediated oxidative DNA damage. 131

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Kawanishl et al.

The Fenton reaction of Fe(I1) with KZ02 is the well-known mechanism for the generation of *OHand/or the ferry1 ion 121. For the first time, Kawanishi et al. reported that carcinogenic Cr(V1) reacts with H- to produce *OHand singlet oxygen (102) which of oxygen free cause DNA damage,andemphasizedtherole radicals in metal carcinogenesis 133. Other carcinogenic metal compounds such as Fe(II1) nitrilotriacetate [41, Ni(I1) [5, 61 and Co(I1) 171produce various types of active oxygen species from H202. Cu(I1) plus H202caused damage to isolated DNA. The main active species causing the DNA damage are more likely copper-oxygen complexes with similar reactivity to Q 2 and/or *OH,rather than *OHitself [SI. These active oxygen species were suggested to give different kinds of site specific DNA damage (l'hble 1). Since H202 reaches the nucleuswhen it survives in significant concentrations, and can be produced even in nucleus [g, 103, these DNA damages mayoccur in cells. Dizdaroglualso observed oxidative DNA modifications in chromatin of cultured mammalian cells treated with H202 and in chromatin of organs of animals treated with carcinogenic metal salts C113. Table 1. Active oxygen species formation and site specificities of DNA damage induced by metal compounds in the presence of hydrogen peroxide M e a cardnoaenldtv

&(VI) ++

Ni(ll)

++

Fe(lll)-NTA

+

Co(l1)

Cu(ll)

Wl)

+

7

7

G>T-C>A

T-G20>A

.OH @T&A -R

PI

[5,61

0-T-C-A [41

m

PI

T of S-GTC

I

++; sufticient evidence of carcinogenicity in humans and animal experiments + ;evidence o f carcinogenicity in animal experiments On the other hand, Cu(I1) and Mn(I1) have ability of mediating both Hzoz formationand oxidative DNA damage by certain carcinogenswhichhave no or weak mutagenicity. Benzene, ophenylphenol (OPP) and caffeic acid, pentachlorophenol (PCP), tryptophan metabolites have not been proved to be mutagenic i n bacterial test systems, either. Our previous work showed that in

Metal and DNA Damage by Non-mutagenic Carcinogen

133

the presence of Cu(II), those compounds or their metabolites caused damage to isolated DNA through H202 formation (Figures 1 and 2). It is of interest that Ames-test negative "non-mutagenic" carcinogens or theirmetaboliteshave beenshown t o cause oxidative DNA damage in the presence of transition metal ion. I n this chapter, we reviewed the role of metal in carcinogenesis of non-mutagenic carcinogens such as benzene [12],OPP [13],caffeic acid [14],PCP [l51and tryptophanmetabolites. benzene metabolite

tryptophan acid caffeic metabolites OH

OH

OH

tm

I I

3-hydroxyanthranllic acid

CH

1,2,4-benzenetrlol

COOH

OPP metabolite

PCP metabolite

I

OH

2,s-dlhydroxyblphenyl

tm 3-hydroxykynureine

ai tetrachlorohydroquinone

Figure 2. Chemical structures of non-mutagenic carcinogens and theirmetabolites; they are derivatives of dihydroxybenzene or hydroxyaminobenzene. 11. Oxidative DNA Damage

& B e n z e n eMetaboIit.4~

Benzene, widely used in the chemical industry, hasbeen shown to cause serious hematological disorders and carcinogenic effects on humansandanimals. Extensive epidemiological evidence has beenpublished on the highincidence of leukemias inmen occupationally exposed to benzene [16, 171. The administration of

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Kawanishi et al.

benzene to animalsproducedleukemia [181, lymphomaand carcinomas of the Zymbal gland, mammary gland and liver C16, 191. Previous studies showed that benzene induced sister chromatid exchanges in mouse bone marrow [16,20,211. However, benzene has not been shown to be mutagenic in bacterial test systems.

OH I

0-

OH hydroqumone

f

benzene

0"

l,2,4-benzenetriol

catechol

Figure 3. Benzene and itsmetabolite. We previously investigated reactivitiesof benzene metabolites (phenol, hydroquinone, catechol, 1,2,4-benzenetriol)(Figure 3)with DNA by a DNA sequencing technique using =P 5I-end-labeledDNA fragments C123. Among benzenemetabolites, 1,2,4.-benzenetriol caused strong DNA damage, and hydroquinone caused slight DNA damage.Benzene,phenol,catechol and resorcinol showed no effect. lkace amounts of Cu(I1) were shown to be necessary for the induction of DNA damage by 1,2,4-benzenetriol. The cleavage sites induced by 1,2,4-benzenetriol were determined by utilizing the Maxam-Gilbert procedure C221. The result showed that cleavages at the positions of guanine and adjacent thymine weremore frequent thanthose of other bases. The cleavagewithout piperidine treatmentindicatedthebreakage of deoxyribose-phosphate backbone.Theincrease of oligonucleotideformationwith piperidine treatment suggests that the base alteration(s1 and/or liberation(s) are induced by 1,2,4-benzenetriol plusCu(I1) and subsequently the cleavages at those bases occurred. We examined the effects of superoxidedismutase (SOD), catalase, and *OH


135

scavengers on 1,2,4-benzenetriol-inducedDNA damage. SOD and catalase almost completely inhibited DNA damage, suggesting the involvement of 02- and H202. Methional significantly inhibited DNA damage, whereas sodium formate did not inhibit it. Since buffers and reagents areknown to be invariably contaminated with trace amounts of metal ions [23], and the addition of Cu(I1) is reported to accelerate drug-induced DNA damage [24], the effects of chelating agents and metal ions on 1,2,4-benzenetriol-induced DNA damage were examined. 1,2,4-Benzenetriol-inducedDNA damage was inhibited by the addition of a Cu(1)-specific chelating agent,bathocuproine,andwasaccelerated by theaddition of Cu(I1). On the other hand, the addition of Fe(II1) did not accelerate the DNA damage induced by 1,2,4-benzenetriol. ESR studies using spin traps demonstrated that addition of Fe(II1) increased *OHproduction during the autoxidation of 1,2,4benzenetriol,whereastheaddition of Cu(I1)did not. Several papers suggested that DNA damage was caused by H202 through Fenton reaction in vivo [2, 251. Therefore, there still remains the possibility that Fe(I1) participates in =OH production from H202 and in DNA damage. Recently, measurements of 8-hydroxy-2deoxyguanosine (8-OH-dG) have been shown to be useful to clarify the participationof oxygen radical in DNA damage. We measured benzene 8-OH-dG content in calf thymus DNA treatedwith metabolites in the presence of Cu(I1) or Fe(II1) by using a n electrochemical detector (ECD) coupled to a HPLC (HPLC-ECD). Formation of 8-OH-dGby 1,2,4-benzenetriol in the presence of Cu(I1) or Fe(II1) increased with the increasing concentration of 1,2,4-benzenetriol. The 1,2,4-benzenetriol plusFe(II1)-induced 8 OH-dG formationwassignificantlyinhibited by typical *OH scavenger,ethanol,whereasethanol did notinhibitthe 1,2,4benzenetriol plus Cu(I1)-induced 8-OH-dG formation (Figure 4). Therefore, it is considered that thespecies causing DNA damage i n the case of Cu(I1) are active oxygen species other than *OH. The inhibitory effect of catalase on DNA damage indicated that H202 plays an important role in producing active oxygen species causing DNA damage. Inthe presence of Cu(I1) or Fe(III), 1,2,4benzenetriol caused DNA damage more efficiently than H202. It can be speculated that 1,2,4-benzenetriolenhances DNA damage by H202 in the presence of metal ions, probably by promoting the conversion of Cu(I1) to Cu(1) or Fe(II1) to Fe(I1) and/ or inducing the change of DNA conformation.

Kawanishi et al.

cu

r

Fe

ij.

P'

I 0 o

b

,

0 Figure 4. The 8-OH-dG formation in DNA induced by 1,2,4-benzenetriolin the presence of Cu (11) or Fe (111) and theeffects of scavengers. Calf thymus DNA (140pM per base) was incubatedwith 50 pM 1,2,4-benzenetriol in the presence of10 p M metal in buffer(pH 7.9 ) at 37 "Cfor 10 min. The treated DNA was analyzed by a HPLCECD.

Our idea that the metal-mediated DNA damage through H202 is relevant for the expression of the carcinogenicity of benzene has been supported by Kolachana et al.'s observations regarding with the inductionof oxidative DNA damage by benzene metabolites in HL60 cells in uitmand in thebone marrow of mice in vivo[26].

m. OxidativeDNA Damagebyehenylphenol o-Phenylphenol (OPP) and its sodium salt have beenused a s fimgicides for citrus fruits [273. It has been reported that long-term administration of OPP and its sodium salt a t high dose induces carcinoma of the urinarybladder in rats [28], although OPP and its sodium salt have not been proved to be mutagenic in bacterial test systems [29]. Morimoto et al. reported that DNA damage was induced in urinary bladder epithelium of male rats treated with


137

OPP metabolite, 2-phenyl-1,4-benzoquinone (PBQ) [30]. However, the mechanismsof DNA damage remain to be clarified. Reactivities of OPP and its metabolites (2,5-dihydroxybiphenyl (Di-OH-BP), PBQ) with DNA wereinvestigated by a DNA sequencing technique, and the reaction mechanism was studied by UV-visible and ESR spectroscopies [131. In the presence of Cu(II), PBQ CU(I1)

NADH

- - - + + + + + -- -+ ++ -- +- -+ ++ ++

incubation time 20 20 20 20 20 20 5

20 (min)

Figure 5. Autoradiogram of =P-labeled DNA fragments incubated with OPP metabolite in the presence of NADH. .. . The reaction mixture contained the=P 5' end-labeled 337basepairfragment (PstI 234!j-AuaI* 2681)obtained from human c-Ha-ras-l protooncogene, 1@M per base of sonicated calf thymus DNA, 60 W PBB, 250 W NADH and 20 p.MCuCh in 200 p1 of 10 mM phosphate buffer at pH 7.9 containing 5 @l DTPA. After theincubation at 37."C, themixture was heated with lM piperidine at 90"Cfor 20min. and theDNA fragments were electrophoresed on an 8 % polyacrylamide/8 M urea gel and the autoradiogram was obtainedby exposing X-ray film ta the gel.

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Di-OH-BP caused DNA damage even without piperidine treatment. Catalase, methionine and methional inhibited the DNA damage completely, whereas -OH scavenger (mannitol, sodium formate, ethanol, tert-butyl alcohol) and SOD did not. Di-OH-BP plus Cu(I1) induced piperidine-labile site frequently a t thymine and guanine residues. The addition of Fe(II1) did not induce DNA damage with Di-OH-BP. ESR-spin trapping experiments showed that the addition of Fe(II1) produced *OH during the autoxidation of Di-OH-BP, whereas the addition of Cu(I1) hardly did so. The results suggest that DNA damage by Di-OH-BP plus Cu(I1) is due to active species other than *OH. We also studied DNA damage by PBQ plus NADH. PBQ alone did not induce DNA damage in thepresence of Cu(111, but addition of NADH induced the DNA cleavage even in theabsence of NADHFMN oxidoreductase (Figure 5). These results demonstrated that semiquinone radical produced by the reduction of PBQ by NADH reacts with0 2 to produce 02-and subsequently H202, which may be activated by transition metalsto cause DNA damage.

DbIA damage

H24

4

Figure 6. A possible mechanism of DNA damage induced by OPP metabolites.


139

UV-visible spectroscopicstudies showed that the autoxidationof Di-OH-BP was accelerated by Cu(I1) and the additionof catalase did not inhibit the accelerating effect of Cu(I1). ESR studies showed thattheinitialrate of semiquinoneradicalproductionwas increased by the addition of Cu(I1) and the increaseby Cu(I1) was not affected by catalase. On the other hand, DNA damage by DiOH-BP plus Cu(I1) was inhibited by catalase. SOD facilitated the autoxidation of Di-OH-BP and the production of semiquinone radical, whereas SOD itself did not induce DNA damage with DiOH-BP. These results suggest that neither semiquinone radical 2 'is the reactant to DNA.On the basis of these data, we nor 0 proposed a possible mechanism of DNA damage induced by OPP metabolites as shown in Figure 6.

IV. Oxidatim DNA Damage by Caffeic Acid Caffeic acid is a phenolic compound widely distributed in plants [31]. Caffeic acid was reported to induce forestomach squamous cell carcinoma of rats 1321. However,caffeic acid has not been shown to be mutagenic in bacterial testsystems [33,341. Previous studiesdemonstratedthat caffeicacidinducedchromosomal aberrations in Chinese hamster ovary cells L351 and DNA double strand breaks in cultured rat fetal lung cells and HeLa cells [36]. However, the mechanism of DNA damage induced by caffeic acid remains to be clarified. Our previous experiments with the isolated DNA showed that caffeic acid caused extensive DNA damage in the presence of Cu(I1) but not in thepresence of Mn(I1) [14]. The inhibitory effects of bathocuproine and catalase on the DNA damage suggest that Cu(1) and H202 have important roles in the production of active species causing DNA damage. Caffeic acid plus Cu(I1) caused cleavage frequently a t thymine residues especially of the 5'-GTC-3' sequence and 5'-CTG-3' sequence. The site specificity cannot be explained by *OH,since it is generally considered that *OHcauses DNA cleavage at every nucleotide with little marked site specificity [37]. Moreover, typical *OH scavengers (mannitol, sodium formate, ethanol, tert-butyl alcohol) did not inhibit caffeic acid plus Cu(I1)induced DNA damage,whereasmethionalandmethionine completely inhibited it. Therefore, it can be speculated that Cu(1)

140

Kawanishi et al.

and H z 0 2 produce a complex such as Cu(I)OOH, other than fiee hydroxyl radical, and that the complex participates in the DNA damage. In recent years, pulsed field gel electrophoresis (PFGE) has emerged as a powerful tool for the study of high-molecular weight DNA. PFGE is generally used for detection of cellular DNA doublestrand breaks. CafFeic acid was shown to produce strand breaksi n DNA of the cells treatedwith Mn(I1). We havedesigned a n experimental protocol which allows detection of DNA single-strand breaks andalkali-labile sites by PFGE. With this procedure, caffeic acid was shown to produce single-strand breaks and alkali-labile sites in DNA of the cells treated with Mn(I1). The enhancing effects of 3-aminotriazol(acatalaseinhibitor)andbuthionine sulphoximine (a GSH synthesis inhibitor) and the inhibitory effect of catalaseon caffeicacidplus Mn(I1)-induced DNA damage indicate the participation of H202. Theinhibitory effect of ophenanthroline indicates that endogenous transition metals such as copper and iron participate theoxidative DNA damage. Thus, it

I

.OH, metal-oxygen complex

t

DNA damage Figure 7. A possible mechanism of DNA damageinduced by caffeic acid in the cell treated with Mn (11).


141

is considered that through Mn(I1)-catalyzed autoxidation caffeic acid produces H202, which is activated by endogenous transition metals to cause damage to cellular DNA. As for the isolated DNA, The Cu(I1)-mediated DNA damage was enhanced by preincubation of caffeic acid with Mn(I1). The rate of H202 formation by Mn(I1) was greater than that by Cu(I1). Theseresultssupportthe mechanism that caffeic acid causes cellular DNA damage by the Mn(I1)-catalyzed formation of H202 and metal-activation of H202 (Figure 7).

V. Osidative~DNA Damam byPentacMomphen01 Pentachlorophenol (PCP) is a wide-spectrum biocide. PCP has let to a substantial environmental contamination and accumulated i n normal human population in addition to PCP-exposed workers 1381. PCP has shown to be carcinogenic for mice [391, although it does not seem to be mutagenic in bacterial test systems. A significant increase in chromosome-type abberrations has been observedin the lymphocytes of PCP-exposed workers 1383. M&onnel et al. showed that PCP is carcinogenic for mice 1391. However, PCP has not been shown to be mutagenic in bacterial test systems, whereas weak mutagenicity has been reported in other systems [38]. Studies on the metabolism of PCP in vivoand in vitrorevealed that tetrachlorohydroquinone (TCHQ) is a major metabolite of PCP in mice and rats' [401. Oxidation of TCHQ to tetrachloro-p-benzoquinone(TCBQ) can occur enzymatically [41]. Regarding PCP metabolite-induced DNA damage, two possible mechanisms have been proposed. Witte et d . reported that TCHQ covalently boundto calf thymus DNA [42]. Covalent binding of PCP to protein and DNA was observed in vitro by incubation with a metabolic activation system [41]. On the other hand, TCHQ can produce reactive oxygen species which may cause DNA damage [43]. Part of the strandbreak formation by TCHQ in human cells is supposed to be due to the action of *OH[44]. In this study, we examined DNA damage by TCHQ in thepresence of metal ions. We also analyzed the8-OHdG formation in calf thymus DNA by using a HPLC-ECD, and investigated the reaction mechanism by UVvisible and ESR spectroscopies.

142

Kawanishi et al.

NADH

N A D +

M OH tetrachiorohydroquinone

c'

0 2

oi tetrachiorobenzoquinone

+metal

Figure 8. A possible mechanism of DNA damage induced by PCP metabolites.

TCHQ caused DNA damage in thepresence of Cu(I1) but not i n thepresence of either Mn(I1) or Fe(II1).TCHQ plusCu(I1) induced piperidine-labile sites frequentlya t thymine residuea and guanineresidues.Themostpreferredsites were thethymine residues of the 5'-GTC-3' sequence. TCHQ increased 8-OH-dG i n calf thymus DNA inthe presence of Cu(I1). Typical *OH scavengers showed no inhibitory effectsonTCHQ plus Cu(I1)induced DNA damage. Bathocuproine and catalase inhibited the DNA damage, suggesting that Cu(1) and H202 have important roles in the production of active species causing DNA damage. We also studied DNA damage by TCBQ plus NADH. TCBQ alone did not induce DNA damage in the presence of Cu(II), but additionof NADH induced the DNA cleavage even in the absence of NADHFMN oxidoreductase. UV-visible and ESR spectroscopies revealed that TCHQ was rapidly autoxidized into semiquinone even in the absence of metal ions, indicating thatsemiquinone radical itself is not the main active species inducing DNA damage. These results suggest that semiquinone radical produced by the autoxidation of TCHQ and/or the reduction of TCBQ by NADH reacts withdioxygen


143

to form 02- and subsequentlyH202,which is activated by transition metals to cause DNA damage (Figure 8).

Boyland and Watson reported that 3-hydroxyanthranilic acid (3HAA) and 3-hydroxykynureine (3-HKyn). tryptophan metabolites ( Figure g), were carcinogenic in the bladders of mice 145,461. Implantation of cholesterol pellets containing 3-HAA or 3-HKyn into bladders in mice induced a significantly greater number of bladder cancers than cholesterol pellets alone 1471. 3-HAA and 3HKyn weredemonstrated t o inducechromatidbreakageand chromatid translocations in mammalian cells 1481. 3-HAA showed promotional effect on X-ray-initiated transformation of BALB/3T3 cells [49]. However, the mechanism of DNA damage induced by 3HAA and 3-HKyn remains to be clarified.

Figure 9. nyptophan metabolites.

In order to clarify the mechanism of the DNA damage, we examined the induction of DNA strand breaks in human cultured cells treated with 3-HAA and 3-HKyn in thepresence of metal ions. Pulsed field gel electrophoresis showed that in the presence of Mn(II), 3-HAA and 3-HKyn induced DNA strandbreaks i n cultured human cells. Enhancing effect of catalase inhibitor and inhibitoryeffect of o-phenanthroline on thestrand breakage indicated the involvement of H 2 0 2 and endogenous transition metal ion. As for the isolatedDNA,we examined DNA damage by 3-HAA and 3-HKyn in the presence of metal ions. 3-HAA and 3-HKyn induced piperidine-labile sites frequentlyat thymine and guanine residues in thepresence of Cu(I1). The inhibitory effects of catalase and bathocuproine on Cu(I1)-mediated DNA damage suggest that

144

Kawanishi et al.

Hzoz and Cu(D produce adive species causing DNA damage. The Cu(II>mediated DNA damage was enhanced by preincubation of 3-

HAA with Mn(I1). UV-visible spectroscopy showed that Mn(I1) and Cu(I1) enhanced the autoxidationof 3-HAA in differentway.

60-

" H A A e 3 - H K y n

0

25

50

75

100

125

Concentration of tryptophan metabolites

Figure 10. The 8-OH-dG formation in DNA incubated with tryptophan metabolites in the presence of Cu(I1). Calf thymus DNA(140 pMper base)was incubated with3-HAA or concentrations inthe presence of 2opM CuC12 in 4 0 0 ~ o1 f 4 mM phosphate buffer at pH 7.9 containing 5 pM DTPA at 37"c for 60 min. Treated DNA was subjected l.~ enzyme digestion and analyzed by a HPW-ECD. 3-HKyn ofthe indicated

By using a HPLC-ECD, we measured 8-OH-dG content in calf thymus DNA treated with 3-HAA or 3-HKyn in the presence of metal ions (Figure 10). The 8-OHdG formation increased with the concentration of 3-HAA in the presence of Cu(I1). On the other hand, concentration dependence was not clearly observed with 3HKyn. At a low concentration, 3-HKyn caused Cu(I1)-dependent formation of 8-OH-dG more efficiently than .3 " 3-HAA plus Cu(I1) induced 8-OH-dG more efficiently than H 2 0 2 plus Cu(I1). Neither H202 alone, 3-HM alone, 3-HKyn alone, nor Cu(I1) alone increased 8-OH-dG content.These results suggest that in the presence of Mn(I1) or Cu(II), these tryptophanmetabolites produce

Metal and

DNA Damage by Non-mutagenic Carcinogen

145

Hzoz, which is activated by transition metalion to cause damage to DNA both in the case of isolated DNA and culturedcells.

VII.

Oxidative DNA Damage byNon-mutagenicCarcinogen

Certain carcinogensmay induce both H202formation andoxidative DNA damage in the presence of endogenous metal ions [12-14,50541. The benzene metabolite 1121, the OPP metabolite [131, caffeic acid [14], the PCP metabolite and tryptophan metabolites caused oxidative damage to isolated DNA through 02- formation, although quinone type metabolites required NADH. Mn(I1) has ability of mediating oxidativedamage of cellular and isolated DNA by certain carcinogens [14,541. Figure U. shows a possible mechanism for DNA damage due to Cu(I1)- or Mn(I1)-mediated formation of Hz02 which reacts with endogenous metal ions such as Fe(I1) and Cu(1) bound to or close to the DNA to produce active oxygen species. Similarly, the active oxygen species were shown to participate in

I caffeicacid I metabolite +benzene

YO2 +peroxisome(fatty acid B-oxidation)

clofibrate phthalate esters

*OH, metal-oxygencomplex

t

DNA damage Figure 11. A possible mechanism of active oxygen formation and DNA damage by certain non-mutagenic carcinogens.

146

Kawanishi et al.

Cu(n)-dependent DNA damage by hydrazine, hydroxylamine and their derivatives, which are no or weakly mutagenic carcinogens [50-553.

Certain peroxisome proliferators have carcinogenicity and may cause DNA damage through Hz02 formation. A major role of the peroxisomes is the breakdown of long-chain fatty acids. A wide range compounds, including clofibrate, di(2-ethylhexyl)phthalate, trichloroethylene and 2,4-dichlophenoxyacetic acid increase in the number of hepatic peroxisomes. Thus, the activity of peroxisome system (including acyl CoA oxidase) for the P-oxidation of fatty acids to produce Hz02 often increases more than that of catalase. Manyperoxisomeproliferators havebeenshown to induce hepatocellular tumours when administered at high dose levels to rats and mice for long periods [561. These peroxisome proliferators have not been proved to be mutagenic in bacterial systems. Several mechanismshavebeen proposed to explain the induction of tumours. One is based on increased production of active .oxygen species due to imbalanced production of peroxisomal enzymes; it has been proposed that these active oxygen species cause indirect oxidative DNA damage with subsequent tumour formation [57]. Endogeneous transition metal ions should participate in these oxidative DNA damage.

1973

1976

1987-

90%

60%

Mutagen Carcinogen

I

Concordance 40-60%

Figure 12. Historical change in concordance between mutagenicity and carcinogenicity. Short-term tests for genotoxic chemicals were developed to assess the potentialgenetic hazard of chemicals to humans. Ames


147

et al. developed the Salmonella mutagenicity test and reported that 90 % of the carcinogens tested were mutagens and 90 % of the

noncarcinogens were nonmutagens [58].However, concordance between carcinogenicity and mutagenicity decreased from 90 % to 60 96 according to subsequent reportst59-611 (Figure 12). There is a need for a short-term test to detect those carcinogen that missed by the Ames test. On the basisof our data and reported data, we classified the non-mutagenic carcinogens into genotoxic type and non-genotoxic type as shown in Table 2. Hormone and Table 2. Classification of non-mutagenic carcinogen. nickel sulfides,cobalt oxide, Metal iron nitrilotriacetate Genotoxic Benzene (oxidative DNA derivatives damage) Peroxisome proliferators

benzene, OPP, caffeic acid, PCP,

tryptophan metabolites,

+ diethylstilbestrol, hydroquinone clofibrate, phthalate esters, 2,4dichlophenoxyaceticacid, WV-14.643

"

Nongenotoxic

Hormone

estradiol-l7/3, estriol, estron, ethinylestradiol, mestranol

Polychlorinated

polychlorinated biphenyl,

aromatic hydrocarbon

2,3,7,8-tetrachlorodibenzo-pdioxin

"

Unclassified

dioxin, chloroform, arsenic, thioacetamide

halogenated aromatic hydrocarbons are truly non-mutagenic or non-genotoxic carcinogens.Halogenatedaromatichydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxincan recognize specific intracellular proteins and appearto mimic the action of hormones and growth factors and perturb signal transduction pathways, resultingincarcinogenesis C621. On theotherhand, nonmutagenic carcinogens such as metals, benzene derivatives and peroxisome proliferators .can participate inactive oxygen formation and oxidative DNA damage. Shibutani et al. reported that the formation of 8-OHdG, one of the oxidative DNA products, caused misreplication of DNA that might leadto mutation or cancer [63]. Thus, it seems reasonable to conclude that the metal-mediated oxidative DNA damage through H 2 0 2 formation is relevant for the

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expression of the carcinogenicity of certainnon-mutagenic carcinogens.

1. B. Halliwell and J. M.C. Gutteridge, Methods Enzymol186 185 (1990). 2. J.A. Imlay, S. M. Chin, and S. Linn, Science 240: 64o-(iQ2 (1988). 3. S. Kawanishi, S. Inoue, and S. Sano, J. Biol. Chem. 261: 595% 5958 (1986). 4. S. Inoue and S. Kawanishi, Cancer Res. 47: 6522 6527 (1987). 5. S. Kawanishi, S. Inoue, and K Yamamoto, Carcinogenesis 1 0 2231-2235 (1989). 6. S. Inoue and S. Kawanishi, Biochem. Biophys. Res. Commun. 159 445451 (1989). 7. K. Yamamoto, S. Inoue, A. Yamazaki, T. Yoshinaga, and S. Kawanishi, Chem. Res. Ibxicol. 2: 234-239 (1989). 8. K Yamamoto and S. Kawanishi, J. Biol. Chem. 264: 15435 15440 (1989). 9. A. V. Peskin andL. Shlyahova, FEBS Lett 194: 317-321 (1986). 10. S. Puntarulo, and A.I. Cederbaum, Biochim Biophys Acta 1116 17-23 (1992). 11. M.Dizdaroglu, Mutation Res. 275 331342 (1992). 12. S. Kawanishi, S. Inoue, and M.Kawanishi, Cancer Res. 42164-168 (1989). 13. S. Inoue, K. Yamamoto, and S. Kawanishi, Chem. Res. Zbxicol. 3 (2): 144-149 (1990) 14. S. Inoue, K. Ito, K. Yamamoto, and S. Kawanishi, Carcinogenesis l3 (9): 1497-1502 (1992) 15. S. Naito, Y.Ono, I. Somiya, S. Inoue, K. Ito, K. Yamamoto, and S. Kawanishi, Mutation Res. (in press). 16. IARC Working Group. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 2 9 , pp.93148.Lyon, France, 1982. 17. M. Aksoy, S. Erdem, and G. DinCol, Acta. Haemat. 55: 65-72 (1976). 18. E.l? Cronkite, J. E. Bullis, T. Inoue, and R. T.Drew, Ibxicol. Appl. Pharmacol. 75: 358-361 (1984).


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19. C. Maltmi, B. Conti, and G.Cotti, Am. J. Ind. Med. 4 589630 (1983). 20. R. R. Tice, D. L. Costa, and R.T.Drew, Proc. Natl. Acad. Sci. USA 74: 2148-2152 (1980). 21. K Morimoto and S.Wolff, Cancer Res. 4 0 ll89-1193 (1980). 22. A. M. Maxam and W. Gilbert, Methods Enzymol. 6 5 499560 (1980). 23. J. Diguiseppi and I. Fridovich, Arch. Biochem. Biophys. 205 323-329 (1980). 2 4 . A. Wong, C-H. Huang, S.T.Crooke, Biochemistry 23 2946-2952 (1983). 2 5 . L.Nassi-Calb, A. C. Mello-Filho, and R. Meneghini, Carcinogenesis 109055- 1057 (1989). 26. l? Kolachana, V.V.Subrahmanyam, KB. Meyer, and L. Zhang, Cancer Res. 5 3 : 1023-1026 (1993). 27. IARC Working Group on the Evaluationof the Carcinogenic Risk of Chemicals to Man. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, IARC, Lyon, vol. 30, pp. 329344,1983. 28. T.Fujii, H. Mikuriya, N. Kamiya, and K. Hiraga, Food Chem. lbxicol.2 4 : 207-211 (1987). 29. R. H. Reitz, T. R. Fox, J.F. Quast, E. A. Hermann, and P.G. Watanabe, Chem.-Biol. Interactions 4 3 99119 (1983). 30. K Morimoto, M. Fukuoka, R. Hasegawa, A. Tanaka, A. ‘hkahashi, and Y.Hayashi, Jpn. J. Cancer Res. (Gann) 78 1027-1030 (1987). 31. H. F. Stich, Mutation Res. 259 307-324 (1991). 32. M. Hirose, S. Fukushima, T. Shirai, R. Hasegawa, T. &to, H. Tanaka, E. Asakawa, and N. Ito, Jpn. J. Cancer. Res. 81: 207212 (1990). 33. H. F. Stich, M. P. Rosin, C. H. Wu, and W. D. Powrie, Mutation Res. 90: 201-212 (1981). 3 4 . R.H. C. San and R. H. C. Chan, Mutation Res. 177:229239 (1987). 35. A. F. Hanham, B. l? Dunn, and H.F.Stich, Mutation Res. 116: 333-339 (1983). 36. K Yamada, S.Shirahata, H. Murakami, K Nishiyama, K. Shinohara, and H. Omura, Agric. Biol. Chem. 4 9 1423-1428 (1985). 37. D. W. Celander and T.R. Cech, Biochemistry B1355-1361 (1990).

150

Kawanishi et al.

38. J. P. Seiler, Mutation Res. 257: 27-47 (1991). 39. E. E. MaConnell, J.E. Huff, M. Hejtmancik, A.C. Peters, and R. Persing, Fundam. Appl. l'bxicol. 17: 519532 (1991). 40. U.G. Ahlborg, K. Larsson, and T.Thunberg, Arch.Tbxicol.40: 4553(1978). 41. B. Van Ommen, A. Adang, F. Muller, and P.J. Van Blanderen, Chem.-Biol. Interaction 60:1-11(1986). 42. I. Witte, U. Juhl, and W. Butte, Mutation Res. 145 71-75 (1985). K. Jasson and V. Jansson, Mutation Res. 260: 83-87 (1991).

43.

44. C.-€? Carstens, J. K. Blum, and I. Witte, Chem.-Biol. Interactions 7 4 305-314 (1990). E.Boyland and G.Watson, Nature 177: 837-838 (1956). 46. M. J. Allen, E. Boyland, C. E. Dukes, E. S. Horning, and J. G.Watson, Brit. J. Cancer 11:212228 (1957). 47. G. T.Bryan, R. R. Brown, and J. M. Price, Cancer Res. 24: 5% 602 (1964). 48. L.E.Kuznezova, Nature 222 484-485(1969). 49. M.Umeda, K. Tanaka, and T.Ono, Carcinogenesis 1 0 16651668 (1989). M). K. Yamamoto and S. Kawanishi, Chem. Res. l'bxicol. 5: 44@ 446 (1992). 51. K. Yamamoto and S. Kawanishi, Biochem. Pharmacol. 41: 905 45.

914 (1991). 52. K Yamamoto and S. Kawanishi, J.Biol. Chem. 266: 15091515 (1991). 53. S. Kawanish, and K. Yamamoto, Biochemistry 30: 30693075, (1991). 54. K. Ito, K. Yamamoto, and S. Kawanishi, Biochemistry 31: 11606-11613 (1992). 55. K. Yamamoto, S. Inoue, and S. Kawanishi, Carcinogenesis 1 4 1397-1401 (1993). 56. M. S. Rao and J. K. Reddy, Environ. Health. Perspect. 9 3 205 209 (1991). 57. l? Bentley, I. Calder, C. Elcombe, P. Grasso, D. Stringer, andH. J. Wiegand, Food. Chem. l'bxicol. 31(11): 857-907 (1993). 58. J. McCann, E. Choi, E. Yamasaki and B. N.Ames, Proc. Natl. Acad. Sci. USA 7 2 5135-5139 (1975). 59. E. Zeiger, Cancer Res. 47: 1287-1296 (1987).

60. R. W. Tennant, B.H.

Margolin, M. D. Shelby, E. Zeiger,J. K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz,


151

B. Anderson and R. Minor, Science 236 933-941 (1987). 61. R.W.Tennant and E.Zeiger, Envimn. Health Perspect. 100 37315 (1993). 62. S. Green, l k n d s in Pharmacological Science 13 251-255 (1992). 63. S. Shibutani, M. Takeshita, and A.€?Grollman, Nature 349 431434,(1991).


10 Sequence-Selective Cleavage of DNA by Cationic Metalloporphyrins Genevihe Pratviel, Pascal Bigey, Jean Bernadou, and Bernard Meunier LaboratoiredeChimiedeCoordination,CentreNationaldelaRecherche Scientifique, 205, route de Narbonne, 31077 Toulouse cedex, France

I. INTRODUCTION Transition metal complexes endowed with redox properties and DNA affinity have been developed as “chemical nucleases”. Reagents capableof efficient DNA cleavage would have potential application in medicineas antitumoral or antiviral agents and in molecular biology as footprinting reagents or artificial restriction enzymes. Well known examples of such compounds are FeEDTA [1,2], Cu(oP), [3], metalloporphyrins [4] and COor Ru 153

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complexes [5]. Their mechanismof DNA cleavage is an oxidative degradation of DNA that can be classified in two types: (i) generation of superoxide anion from molecular oxygen or/and formation of hydroxyl radicals from hydrogen peroxide (Fenton reagent) catalyzedby metal ion salts and (ii) direct oxidationof DNA by high-valent transition metal-oxo complexes. Unlike natural enzymes, theydo not hydrolyze phosphodiester bonds of DNA,but can cleave nucleic acids by oxidation of deoxyribose units. The non-specificityof the diffusible hydroxyl radical attack and the usual destruction of sugar ring by chemical nucleases is a driving force to develop the design of hydrolytic reagents for producing DNA fragments that canbe religated and cloned[6-91. Up to now, these compounds are far less efficient than natural enzymes or oxidative chemical nucleases. We will describe here the oxidativeDNA cleavage by a highvalent metal-oxo species of Mn cationic porphyrin. Oxidative damage to sugar in this case (mprecisely hydroxylationof the S’-carbon of an intrastrand sugar) can be reverted by a mild reduction,theequivalent of a ‘‘pseudo-hydrofysis”of a phosphodiester linkage.This reagent combinesthe high reactivity of a metal-oxo species with no destruction of DNA sugarphosphate backbone. For the development of a chemical nuclease, an efficient reactivity is necessary but another important point is the sequence specificity of DNA cleavage. It can be improved by tethering a DNA recognition moiety to the active metal complex and should lead to a family of synthetic cleaving reagents with tailored specificity [lo-141. We will also present some results specific on DNA cleavage by cationic metalloporphyrins covalently linked to oligonucleotides.

Sequence-Selective Cleavage

by Cationic Metalloporphyrins

155

XI. Mn-TMPyP:OXIDATIVEDNACLEAVAGE Synthetic metalloporphyrins are able to mimic heme-enzymesmediated oxygenation and oxidation reactions 141. Beside this mode ofmtivity,iron or manganese porphyrins with peripheral positivecharges, e.g. Fe- or Mn-mso-tetra(4-N-methylpyridiniumy1)porphyrins(Fe- or Mn-TMPyP, Figure 1; for crystallographic data onMn-TMPyP see [15]), exhibit a strong interaction with DNA that brings into the vicinity of the targeta powerful oxidizing species. The active oxidative speciesin the case of DNA cleavage is probably the same as that involved in catalytic oxygenation and oxidation reactions described for metalloporphyrins in general, namely, a high-valent metal-oxo porphyrin complex able to hydroxylate a C-H bond or to epoxidize an olefin [4]. This active speciesis generated in the c

Figure 1 Structure of Mn-TMPyP(eachaxialposition occupied bya water molecule).

is

et

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al.

p s e n c e of oxygen atom donor compounds like iodosylbenzene [1Q ,hydmgen peroxide[171, potassium monopersulfate[17- 191 or magnesium monoperphthalate [20]. Another way to generate an oxo-metalloporphyrin species is to use molecular oxygen associated with an electron source, as does cytochromeP450 in vivo [16, 21,221. Among all these different methods, the most efficient for performing oxidative cleavage of DNA is the oxidation of cationic Mn-porphyrins by KHS05 [17, 20, 231. Data on DNA cleavage by Mnm-TMPyP/KHS05 suggest that diffusible radical species are not involvedin the reaction, because the breaks arc well-defined, and also because HzOz is at least three orders of magnitude less efficientin generating the active species leading to cleavage [17]. A catalytic activatiodreaction cycle analogousto cytochrome P-450is possible : TMPyp-Mnm+ KHSOS

? TMPyP-Mnm + ROH

+

TMPyP- mv=0 + RH

e

TMPyP- "v-OH

3.

+R.

The high-valent m P " n v = O species responsiblefor DNA cleavage is too reactive to be characterized. Turnovers of the catalyst can be observed only whenprotected fkom self-oxidation (leading to the chmmophm bleaching) by strong interaction with its target. For example when the DNA/Mn-porphyrin ratio is high (75 pM bp/5 nM), up to 5 SSBs per Mn-TMPyP molecule were observed 1173. Mn-TMqrP binds in the minor groove of AT-rich regions of DNA [23-331. The Mn-porphyrin frameworkis devoid of any Hbonding donor/acceptor capacity and intercalation is precluded by the presence of an axial ligand on manganese [H]. Thus the

Sequence-SelectiveCleavage by Cationic Metalloporphyrlns

157

ability of Mn-TMPyP to select AT-rich regions of DNA is apparently electmstatic/stericin origin. It has been proposed that this cationic metalloporphyrinis attracted by the high negative potential at the surface of the minor groove [34] of AT-rich sequences. A close contactwith the minor grooveis necessary to improve the binding interaction and the cleaving efficiency of the metalloporphyrin, as deduced fromstudies on variations in total charge andin charge distribution at the macrocycle periphery [2527,35361. At high ionic strength, the reagentis unable to bind and/or cleave DNA[17,371. Considering the size of MnTMPyP, it could span over5 to 6 base-pairs in the minor groove of B-form DNA, butthe preferred cleaving site consistsof three consecutive AT base-pairs creating a suitable “box” for highly selective DNA cleavage [la, 19, 28, 29, 381. At this site MnTMPyP is strictly mediating C5’ oxidation on nucleosides on both 3’-sides of the “AT box”[39]: one single-strandbreak (SSB) on each strand leads to double-strand cleavage with a 4 base-pair shift to the 3’-end of the opposite DNA strand (Figure 2). Double-strand cleavage is possible on the same binding site because the metal-oxo entity can be generated on either side of the symmetric porphyrin ring.So far, thereis no definite proof that such a cleavage pattern is the result of two SSBs by the same metalloporphyrin activated twice inside the minor groove, or whether it is due to two different activated metalloparphyrins. On large DNA substrates, SSBs prevail [l71 and on oligonucleotides substrates (with one or two “AT boxes”) DSBs are easily obtained 1391. Besides cleavage at (AV3 cleaving sites, some secondary reaction sequences are also noted. They consist of one base-pair changein the (AV3 site (one GC base-pair outof three, no matter what the position of the GC bp)

1 58

Pratviel et al.

[25]. This secondary reactivity is particularly noticeable when drastic cleaving reactionsare performed r39-401.The reactivity at these secondary sites is one order of magnitude less than for (AV3sites.

I

Figure 2 Interaction of high-valent metal-oxo TMPyP-Mnv=O in the minor groove -of (An3sequences. Four basepair 3’ stagger of oxidative attack(4).

The mechanism of DNA cleavage is shown in Figure 3. The activated cationic metalloporphyrin initiates the reaction by CS’ hydroxylation of the deoxyribose giving the 5’-OH derivative a. Spontaneous cleavagefollows, with formation of a 3’-phosphate

Sequence-Selective Cleavage by Cationic Metalloporphyrins

a

b

C

159

d

Figure 3 Mechanism of DNA cleavage.

end and a 5”aldehyde ending derivativeb (direct break of the DNA backbone). Further transformation (first p-elimination) produces a second break on DNA the backbone with release of a S-phosphate end andthe a,p-unsaturated aldehyde compoundc. Finally a second p-elimination gives rise to freebase and furfural d as sugar degradation product. Sometimes, depending on the extent of cleavage and on the sequence at the 3’-side of (An3 site, a second oxidative reactionof activated Mn-TMPyPon the same site leadsto 5’-COOH ending fragments[40-41]. This selectiveC S hydroxylation can take place on both 3’ sides of the (An3 double-strand cleavage site of the Mn-TMPyP/ KHS05 system. This leadsto direct strandbreaks with a4 basepair 3”stagger of the cleaved residues, which conf‘iis that oxidative attackof the activated metalloporphyrin occurs from the minor groove of DNA (Figure 2). The two DNA hgments are bearing 3”protruding single-stranded termini overlapping on three base-pairs that are reminiscent of restriction enzymes cleavage sites. Both strand nicks are identical and consist of 3’phosphate termini facing a 5’-aldehyde residue. By treatment

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with NaBH4, this terminus canbe readily convertedto a 5’-OH end. Thus the oxidative cleavage followed by a reduction stepis equivalentto the hydrolysis of the phosphodiester bond: hydroxylation step+ reduction step= “pseudo-hydrolysis”.

reduction

{NIB&

S’TGCGG~TT~ 5a%ACGACI’G3’ ” A C G C C ~ ~~AMCTGCMACS

Figure 4 Pseudo-hydrolysis of a DNA phosphodiester linkage. In order to develop such chemical toolsfor gene engineering,it wastempting to check .ifthese chemically cleaved DNA fragments couldbe ligated to construct a covalently linked new strand of DNA. For that purpose, natural ligases could not be used because fragments to b e , joined ..were not carrying 5’phosphate and 3”OH ends, the usual termini, but the situation was the opposite. Fortunately, chemical ligation methods described in the literature seemed especially appropriate since the 5’-OH/3’-phosphate configuration is the most favorable for alcohol nucleophilic attack ontoan activated phosphate [42-441. We chose to use the “BrCN method‘’ described by Shabarova et al. 1421on short double-strandedDNA sequences containing one (An3site.


by Cationic Metalloporphyrin8

161

R ' I check thata chemically cleaved and religated DNA shows no chemicalbasemodificationsandpresents a re-formed of the cut,we tested whetherthe phosphodiester bond at the site religated duplex could be a substrate for a restriction enzyme. We used the 35-mer duplex that is bearing a restriction site forBgl I enzyme [41]. A s shown on Figure 5, Mn-TMPyPIKHSOd NaBH4 and BgZ I are cleaving the same phosphodiester bonds but leave the phosphate at the site of the cut at the 3' or the 5'end, respectively. The 35-mer duplex was chemically cleaved (route 1, Figure 5), and religated (route2, Figure 5). It was then tested asa substrate forBgl I (route 3, Figure 5). The enzymatic hydrolysis was complete. Furthermore, alkaline phosphatase removed the S-phosphate group from the Bgl I generated fragment, The S-OH 1 6 " single-stranded frasment generated by Bgl I + alkaline phosphatase had the same electrophoretic migration as the S-OH 16-mer generated by the Mn-TIWyP/ KHSO#NaBH4 system.In a control experimentBgl I digestion of control duplex showed the same cleavage pattern. One problem remaining for the development of the cationic manganese porphyrinas a chemical DNA restriction toolis that the (AV3 cleaver affinity sequence is too often encountered on random double-stranded DNA.Oneway to improvethe sequence selectivity of this chemical nuclease would be to covalently link such cleaver with a sequence-recognition vector like an oligonucleotide.

m. CATIONIC

PORPHYRIN-OLIGONUCLEOTIDES

The manganeseporphyrinconjugateMn-trisMPyP-5'" I T m G G G G G T was synthesized [13,45] (see Figure

162 Pratviel et al.

z

d 0L

\o

m

82

0

ru


r,

X

Ti

u, ru 0

U

0

Y 0

ru

3

C

4 X

0

Y

Q

a

Q)

L

em kl

.I

163

164

Pratvlel et al.

6 for structure). The tether has 17 bonds and the 16-mer vectoris complementary to the 29-mer target sequence 5'AGCTAKCCCCAAAAGAAAAGTAGAX(X. In vitro assays of the nuclease activityof the vectorized manganese porphyrin were performed by usingKHS05,to generate activated manganese-oxo porphyrin complexes (such metal-oxo species can be generated inside of a cell by molecular oxygen and an electron source a c d n g to a mechanism similar to thatof P450 enzymes and activated bleamycin).In the experimental conditions each DNA cleavage reaction(Figure 7) contained 7 n M of the 5'labeled 29-mer, from0.1 n M to 1 pMof Mn or Fe-trisMPyP-16mer, and 400 p M in nucleotides of double-stranded salmon testes DNA (2000 equivalents with respectto the 29-mer)in a solution of 125 mM NaCl and 50 mM TrisHC1buffer (pH8). Annealing of the free metallo-trisMPyP-16-mer with the 29-mer was obtained by heating at 90"C for 3 min and followedby a cooling to 37 "C within 4 h and stored overnightat 4 "C. All assays (total volume = 16 pL) were performed at 4 'C. For reactions without conjugate, the free 16-mer was hybridized with the 29-mer and then pre-incubated with 100 nM and 1 p M of Mn-"yP, the parent DNA cleaver without vector, for 15 min at 4 "C before addition of KHS05. DNA cleavage reactions were initiatedby addition of 1 mM KHSO, for Mn-or 1 mM ascorbate for Feporphyrins and maintained at 4 "C for 1 h. Reactions were then quenched by'40 mM HEPES (pH 8) and heated at 90 "C for 30 min (all concentrations listed are final concentrations). After cooling at 4 "C samples were dilutedwith 1 p.L of yeast tRNA (10 mg/ml) and 100 pL of 0.3 M sodium acetate (pH 5.2) and precipitated with300 pL of ethanol and finallyMsed with 75% ethanol and lyophylized. Fragments of DNA were analyzed by

165

Sequence-SelectiveCleavage by Cationic Metalloporphyrins

metal number of bonds G CTAG

45

“n 17 6-10

24 11-15

1 30 16-20

,

Fe

17 2 1-25

C T

.“#l

* G21

TA20

TA C G 16 TA 15 TA TA TA GC G C io GC

GC GC GC TA T C A

GI Figure 7 Analysis of the cleavageof a 5’ labeled 29-mer singlestranded DNA by oligo”n or Fe-porphyrin conjugates(* is the location of the metalloporphyrin reagent).

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20% polyacrylamide gel electrophoresis under denaturing conditions (7 M urea). The manganese porphyrin conjugate Mn-trisMPyP-l 6-meris able to cleave the single-stranded 29-mer target at a very low concentration, 100 nM,with only 14 equivalents of the vectorized DNA cleaver with respectto thetarget.Such is obtained in the presenceof a large remarkable nuclease activity excess of a random double-stranded DNA of salmon testes.From previous studies, free Mn-TMPyP was expected to only cleave the duplexf m e d by the 29-mer and the free 16mer vector at the vicinal nucleotides on the3' side of each AT base-pair triplet, 4 and G21, A20, A15 andG16. This is effectively the case (lanes 5 ) for the non-vectorized manganese porphyrin, but at higher concentration (1 PM). Almost no cleavage was detected at 100 nM concentration (lane 4). The presence of an intense smear (lanes 9 and 10) near the junction of single and double-stranded region of the duplex suggests that the mechanism of the DNA is not restricted cleavage by the vectorized manganese porphyrin to thespecific 5' mechanismobserved forthefree metalloporphyrin Mn-TMPyP. We suspected that the porphyrin was not free enough tooptimise its interaction within the minor groove of double-strandedDNA due to a too short linker.. The length of the tether was then increased from 17 to 30 bonds in order to improve the cleavage efficiency of the metalloporphyrin and to mediate non dispersed lesions. In lanes 14 and 15, the cleavage pattern was cleaner and located on the same bases T22, G21, A20 ofthe junction between single and doubleregions. In lanes 19 and 20 smears were observed in the single-stranded part of the 29-mer and they span over more bases as the lengthof the A20 cleavage sites tether rised30 bonds butthe classical G21 and


167

persist. As the lengh of the tether increased the specificity of cleavage of thereagentresembledthat of thefree metalloporphyrin (Mn-TMPyP): atG21 and A20 sites. If the arm is too long, the rea&ive moiety was reaching new remote sites. Interestingly, theFe derivative could be activated with molecular oxygen in the presence of a reductant, confirming the possibility of in vivo activationof these metalloporphyrin compounds (lanes 21 to 25). The cleavage sites were unexpectedly located on the single-stranded region and were remarkably restricted to two sites G21 and "22. Withoutquestion,the attachement of thecleaver motif tris(methylpyridiniumyl)porphyrinato-manganese(III) to an oligonucleotide vectorallowed selective recognition and cleavage of the complementary target, even in the presence of alarge excess of random DNA. Site selectivity of cleavage within the selected sequence is more challenging in order to obtain artificialDNA restriction tools. Among allpossible DNA cleaversto be attached to oligonucleotides to prepare active antisense oligonucleotides, the motif tris(methylpyridiniumy1)porphyrinatomang) (MntrisMPyP) has some significant advantages:(i) (Mn-TMPyP) is an efficient DNA cleaver; (ii) manganese is not removed from n& (iii) these cationic manganese synthetic porphyrins in vivo a porphyrins exhibit a non-negligible anti-HIV activity [4q. Inhibition of the expression of the Hnr transactivation factorTAT or other viral proteins likeREV or ENV has been obtainedwith antisense oligonucleotides[47-48]. These data provide support for further investigations with nuclease-resistant metalloporphyrin conjugates to target RNA or DNA via triple-helix approach at cellular level [49].

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ACKNOWLEDGMENTS. Thefinancial support of the CNRS, the ‘Association pour la Recherche contre le Cancer’ (ARC, Villejuif), the ‘Agence Nationale de Recherches sur le Sida’ ( A N R S , Paris), the ‘Region Midi-PyrMes’ and Genset (Paris) is gratefully acknowledged.

REFERENCES

1. T. D. Tullius, Annu. Rev. Biophys. Chem. 18, 213-237 (1989). 2. P.B. Dervan, Science 232,464-471 (1986) 3. D. S. Sigman, T. W. Bruice, A. MazumderandC.L. Sutton, Acc. Chem. Res.26,98-l04 (1993). 4. B. Meunier, Chem. Rev.92,1411-1456 (1992). 5. a) A. M. Pyle, J. K,Barton, Prog. Inorg. Chem. 38,413 475 (1990); b) A. Sitlani, E. C.Long,A.M. Pyle, J. K. Barton, J . Am. Chem. Soc.114,2303-2312 (1992). 6. B. K Takasaki and J. Chin, J. Am. Chem. Soc. 116,11211122 (1994). 7. M.T. Schnaith, R. S. Hanson and R. S. Que, Proc. Natl. Acad. Sci. USA 91,569-573 (1994). 8. H.Schneider, J. R a m 0 and R. Hettich, Angew. Chem. Int. Ed. Engl. 32, 1716-1719 (1993). 9. Chin, J. Acc. Chem. Res. 24, 145-152 (1991). 10. S. A. Strobel and P.B. Dervan, Science 249,73-75(1990). 11, C.B. Chen, M. B. Gorin and D. S. Sigman, Proc. Natl. Acad. Sci. USA 90, 4206-4210 (1993). 12. J. C. Franqois, T. Saison-Behmoaras, M. Chassignol, N.T.



169

Thuong and C.HClbne, J. Biol. Chem. 264, 5891-5898 (1989). 13. M. PitiC, C. Casas, C. J. Lacey, G. h t v i e l , J. Bernadou 32,557-559 and B. Meunier, Angew.Chem.Int.Ed. (1993). 14. E. I. Frolova, 0. S. Fedorova and D. G. Knorre, Biochimie 75,512 (1993). 15. S. Prince, F. Ktirber, P.R. Cooke, J. R. Lindsay Smith, M.A. Mazid, Acta Cryst.C49, 1158-1160 (1993). 16. B. Ward, A. Skmobogaty, J. C. Dabrowiak, Biochemistry 25,6875-6883 (1986). 17. J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Biochemistry 28,7268-7275 (1989). 18. E. Fouquet, G. Pratviel, J. Bernadou, B. Meunier, J . Chem. Soc., Chem. Commun. 1169-1171 (1987). 19. B. Ward, R. Rehfuss, J. C. Dabrowiak, J. Biomol. Struct. & Dynami~s4,685-695 (1987). 20. G. Pratviel, J. Bernadou, M. Ricci, B. Meunier, Biochem. Biophys. Res. Commun. I60,1212-1218 (1989). 21. T. J. McMurry, J. T. Groves in Cytochrome P-450: S t r w w e , Mechanism and Biochemistry, (Ed.:P. Ortiz de Montellano), Plenum Press, New-York, 1986, pp. 1-28. 22. R. J. Fiel, T. A. Beerman, E. H. Mark, N. Datta-Gupta, Biochem. Biophys. Res. Commun.107, 1067-1074 (1982). 23. R. W. Bymes, R. J. Fiel, N. Datta-Gupta, Chem.-Biol. Interac. 67,225-241 (1988). 24. M. Rodriguez, T. Kodadek. M. Torres, A. J. Bard, Bioconjugate Chem. I , 123-131 (1990). 25. S. D. Bromley, B. W. Ward, J. C. Dabrowiak, Nucleic Acids Res. I#, 9133-9148 (1986).

170

Pratviel et al.

26. B. Ward, A. Skorobogaty, J. C. Dabrowiak, Biochemistry 25,7827-7833 (1986). 27. G. Raner, B. Ward, J. C. Dabrowiak, J. Coord Chem 19, 17-23 (1988). 28. G. Raner, J. Goodisman, J. C. Dabrowiak, in Metal-DNA Chemistry (Ed.:T.D. Tullius), ACS SymposiumSeries 402, 1989, pp. 74-89. 29. R. F. Pasternack, E. J. Gibbs, in Metal-DNA Chemistry (Ed.:T D. Tullius), ACS Symposium Series 402, 1989, pp. 59-73. 30. R. F. Pasternack, E. J. Gibbs, J.J. Villafranca, Biochemistry 22,5409-5417 (1983). 3 1. J. A. Strickland, L. G. Marzilli, K.M. Gay, W. D. Wilson, ibid 27, 8870-8878 (1988). 32. R.J. Fiel, J. Biomol. Struct. & Dynamics 6, 1259-1273 (1989). 33. L. G. Marzdli, New J. Chem 14,409-420 (1990). 34. a) X. Hui, N. Gresh, B. Pullman, Nucleic Acids Res. 18, 1109-1114 (1990); b) P.K. Weiner, R. Langeridge, J. M. Blaney, R. Schaefer, P. A. Kollman, Proc. Nutl. A c d Sci. USA 79,3754-3758(1982). 35. L. G. Marzilli, G. Pethti, M. Lin, M. S. Kim, D.W. Dixon, J. A m Chem Soc. 114,7575-7577 (1992). 36. a) M. A. Sari, J. P. Battioni, D. Dupd, D. Mansuy, J.B. Le Pecq, Biochemistry 29,4205-4215 (1990); b) U. Sehlstedt, S. K. Kim, P. Carter, J. Goodisman, J. F. Vollano,B. Norden, J. C. Dabrowiak, ibid 33,417-426 (1994). Atta, J. Bernadou, B. Meunier, S. M. Hecht, 37. R.B.Van Biochemistry 29,4783-4789 (1990). 38. J. C. Dabrowiak, B. Ward, J. Goodisman, Biochemistry



171

28,3314-3322 (1989).

39. M. PitiC, G. Pratviel, J. Bernadou, B. Meunier, Proc. Natl. Acad Sci. USA 89,3967-3971(1992). 40. M.PitiC, G. Pratviel, J. Bernadou,B.Meunier in The

Activation of DioxygenandHomogeneousCatalytic Oxidation (Eds.:D.H. R. Barton, A. E. Martell, D. T. Sawyer), Plenum, New-York, 1993, pp. 333-346. 41. G. Pratviel, V. Duarte, J. Bernadou, B. Meunier, J. Am. Chem. Soc. 115,7939-7943 (1993). 42. N. I. Sokolova, D. T. Ashirbekova, N. G. Dolinnaya and Z.A. Shabarova, FEBSLetters 232,153-155(1988). 43. K. J. Luebke and P.B. Dervan, J. Am. Chem. Soc. 113, 7447-7448 (1991).

44. N. Dolinnaya, N. I. Sokolova, D. T. Ashribekova and 2.A. Shabarova, Nucl. Acids Res. 19,3067-3072 (1991). 45. C. Casas, C. J. Lacey and B. Meunier, Bioconjugate Chem. 46.

4,366-371 (1993).

L.Ding, J. Balzarini, D. Schols, B. Meunier, E. De Clercq,

Biochem Phurmucol. 44, 1675-1679 (1992). 47. E ' Shimada. H. Fugii, B. Maier, S. Hayashi, H. Mitsuya, S. Broder, A.W. Nienhuis, Antiviral Chem Chemotherapy , 2, 133 (1991). 48. a) G. Sczakiel, M. Pawlita, J. Wrol. 65,468 (1991); b) S. T. Cload, A. Shepartz, J. Am.Chem.Soc. 116, 437-442 (1994); c) C.HClbne, J.J. ToulmC, Biochem Biophys. E. Uhlmann, A. Peyman, Acta 1049,99-125(1990); Chem. Rev. 90,543-584 (1990). 49. a) H. Han, P. B. Dervan, Proc. Natl. Acad. Sci. 90,38063810 (1993); b) C. H6li?ne, N. T. Thuong, Angew. Chem. Int. E d Engl. 32,666-690 (1993).


l1 Lanthanidemu Complexes as SyntheticNucleases:Hydroxyalkyl Group Participation in Catalysis Janet R Morrow, K. 0.Aileen Chin, and Kelly Aures Chemistry Department, Acheson Hall, State University of New York, Buffalo, NY 14214

I. INTRODUCTION It has been known for many years that metal ions efficiently promote substitutionreactions o f phosphateestersandphosphoricanhydrides. Thereiscurrentlyarenewedinterest in thedesign of metalion goal of producing complexcatalysts for thesereactionswiththe catalysts for the specific cleavage of RNA (l), DNA (2) or the cap structure of RNA (3). Here we present new lanthanide@) complexes of macrocycles the 1,4,7,10-tetrakis(2-hydmxyethyl)-1,4,7,10tetraazacyclododecane (THED) and lS, 4S, 7S, lOS-tetrakis(2hydroxypropyl)-tetraazacyclododecane (S-THP) thatmay be useful as synthetic nucleases. The mechanism o f transesterification of RNA and phosphateestersbytheselanthanide(1II)complexes is discussed. In addition, the lanthanide(1II) complexes of THED promote an unusual substitution reaction o f a phosphate diester whereby a bound alkoxide group of themacrocycleactsasanucleophiletowardthephosphate diester to form a covalent adduct. This type o f reaction is reminiscent 173

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of the covalent adducts formed in the hydrolysis of phosphate esters by metalloenzymes such as alkaline phosphatase.

r H0

H0

L~I(THED)~+

L~(s-THP)~+

Ln = La, Eu

Ln = La, Eu, Lu

Figure 1 Lanthanide(II1)HydroxyalkylMacrocyclicComplexes.

II.

LANTHANIDE@I) HYDROXYALKYL MACROCYCLIC COMPLEXES

Although lanthanide ion salts promote rapid RNA cleavage, o f common ligands such as the lanthanide(II1) complexes as cleavingagents (4). Ligands polyaminocarboxylatesareinactive must be designed such that a positive overall charge is maintained on the macrocycliccomplex. In addition,theligandmustbindstrongly all availableexchangelabile to thelanthanideionwithoutblocking coordination sites. We have prepared lanthanide(III) macrocyclic complexes that catalyzeRNA cleavage by transesterification (5, 6). An importantclassofligands for the lanthanide(II1)ionscontains the 1,4,7,1O-tetraazacyclododecanemacrocycle with four additional pendent donor groups that are neutral (6-9).

LanthanideWl) Complexes as Synthetic Nucleases

175

The Eu(n1)and La(III) complexes of THED and the La(III), Eu(III) and Lu(III) complexes of S-THP have been prepared (Figure 1) as their trifluoromethylsulfonate salts (7, 8).Solution'H, 13C and ? L a N M R studies and solid-state structural data support octadentate coordination of the THEDand S-THP ligands to thelanthanideions.Withthe exception of the La(THED)(CF,SO,), complex, all of the lanthanide(II1) hydroxyalkyl macrocyclic complexes are extremely inert to lanthanide ion release in water at 37 "C,at pH 6.0 or pH 7.4. In the presence of excess Cu2+ as a trapping agent, half-lives for the dissociation of the are 0.87 and 11 days, THED complexes of La(II1) and Eu(III) respectively, and the half-livesfor dissociation of the S-THP complexes of La(III), Eu(II1) and Lu(III) are 73, 100 and 53 days respectively. The ionic radii of the trivalent lanthanide ions decrease by approximately 15% upon traversing the series from La(III) to Lu(III). This contraction has two importanteffects from the standpointof catalysis. First, the coordination number will be greater for the lighter lanthanide ions than for the heavier lanthanide ions. If the THED and S-THPmacrocyclesremainoctadentate,thenonewouldpredictthat Eu(I1I)andLu(II1) therewill be fewersites for catalysisforthe complexes than for the La(III) complexes. Solid state studies bear this out. The La(III) complex of an octadentate macrocyclic ligand that is structurally similar to THED has a ten coordinate La(II1) cation that contains two sites for smallmoleculebinding(6).Thesolidstate structure of Eu(S-THP)(H,O)(CF,SO,), featuresa nine-coordinate Eu(II1) cationwithasinglecoordinationsite for bindingwater(8)and is 2. Fluorescencelife-timestudiesof the Eu(II1) showninFigure complexes of S-THP or THED in aqueous solution indicate that 1.3 f 0.5 water molecules are bound to the Eu(1II) cation in these complexes (10). Second, the decrease in ionic radius across the lanthanide series has the effect of increasing the Lewis acidity of the later lanthanide ions. One would anticipate that this would increase the potency of the lanthanideion as atransesterification or hydrolysiscatalyst.The anticipated Lewis acidity order is observed with the 1anthanideaI) STHPcomplexes(9). AS determinedbypotentiometrictitrations,pK, values for a lanthanide-bound water or lanthanide-bound hydroxyalkyl groupdecreaseacrosstheseries:La(S-THP)(CF,SO,),,8.40(kO.05); Eu(S-THP)(CF,SOJ,, 7.80 (kO.1) and Lu(S-THP)(CF,SO~)~, 6.40 (fo.1) and9.30 (kO.1).

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Figure 2

IIL

The structure of the [Eu(TtP)(H,O)I3+ cation. Reproduced with permission from ref. 8.

RNA CLEAVAGE

The S-THP complexes of L a m ) and Eu(III) and the Eu(III) complex of THED promote cleavage of the oligomers of adenylic acid A&,, at37 "C,pH7.60 (11). Productsinclude2',3'-cyclicadenosine monophosphate, consistent with cleavage by transesterification of RNA. Pseudo-first-orderrateconstants for thecleavage of &-A,, in the presence of 2.00 x 104 M complex are 7.1 (fo.8) x l@'S", 4.2 (B.5) x 10'' S" and1.9 (fo.3) x lo4 S" forLa(S-THP)(CF3S0d3, Eu(STHP)(cF3so3)3and Eu(THED)(CF,SO,), respectively.Incomparison, a hexadentate Schiff-base macrocyclic complex of Eu(III) has a pseudo-

177

Lanthanide(lll1 Complexes Synthetic as Nucleases

first-order rate constant of 4.2 x lo4 S" under similar conditions (5). Cleavage of A,-A,, by Eu(THED)(CF3S03),is first order in complex for a complex concentration ranging from 1.00 to 2.00 x lo4 M, and 0.95 (M.15) M"s" isobtained. In asecondorderrateconstantof contrast, the Lu(II1) S-THP complex does not promote substantialRNA cleavage under similar conditions.

7.0

Figure 3

7.5

8.0

8.5

9.0

The pHdependence of thesecondorderrateconstant for the transesterification of 1 by La(S-THP)(CF3SOJ, at 37°C.

The pHdependence(1 1) of thepseudo-first-orderrateconstant for cleavage of adenylic acid oligomers by the Eu(III) THED complex and

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for transesterificationofthemodel RNA compound, 1 U = 4nitrophenylphosphate ester of propylene glycol), suggests that a lanthanide(II1)-bound hydroxide or a lanthanide(II1)-bound alkoxide is the activecatalyticform ofthecomplex.ThesigmoidalpH-rate profiles are fit to equation (1) below with K, the apparent kinetic K, second-order the and constant. rate Values of k, for transesterification of 1 byEu(THED)(CF,SO,),, La(S-TW)(CF3S03),, and Eu(”IED)(CF,SO,), at 37°C ‘are respectively: 7.2 x lo2,6.3 x 10, M1s” with K, values of 1.0 x lo’, 7.9 x lo9and 10’and1.4x 4.0 x lo4. K, and k, values for the cleavage of A,,-A,, oligomers by EuWD)(CF,SOJ, at 37°C are 7.9 x lo-’ and 1.1 M”s”, respectively. An example of the pH dependence of the rate constants for transesterification of 1 by the La(1II) complex of S-THP is shown in Figure 3.

The most striking aspect of the kinetic data for RNA cleavage by the various complexes is that the most Lewis acidic complex as indicated bypK,values(Lu(S-THP)(CF,SOJ,) is theleasteffectivepromoter. In addition, the La(1II) S-THP complex is a better promoter than the Eu(II1) analog although pK, values indicate that the reverse should be true. In fact, the order that is followed for the S-THP lanthanide(II1) complexes correlates to that of the basicity of the bound hydroxide or alkoxide group which presumably participatesin general base catalyzed RNA cleavage.Alternately,anotherfactorwhichwouldproduce the observedtrend is thedecrease in coordinationnumber for trivalent lanthanide ions on traversing the lanthanide series from lanthanum(II1) to lutetium(I1I).Withanencapsulating ligandsuch as S-THP or THED,there are relativelyfewsitesopen for bindingandcatalysis. As discussed in section 11, solid state and solution studies indicate that S-THP and THED have at least one the Eu(II1) complexes of coordinationsite for smallmoleculebindingwhereas the La(I1I) two sites.Therearenosolid-state complexesarelikelytohave structural studies of analogousLu(II1) macrocyclic complexes. Solution N M R data of similar macrocyclic complexes indicate that the= is one small molecule binding site for complexes of the heavier lanthanide ions such as Lu(II1) (12). l is J catalytic in Transesterification of RNA and the phosphate ester ( lanthanide(III) complex. Several turnovers are observed in the transesterificationof a dinucleotide by a Eu(III) hexadentate Schiff-base

179

Lanthanide(lll1 Complexes Synthetic as Nucleases

complex. The Eu(III) complex THED of appears to be a tiansesterificationcatalyst as well. In thepresence of atwenty-fold catalytic turnovers are observed for the excess o f 1, several transesterification of 1 by Eu(THED)(CF3S03), at 3TC, pH 7.40. The sole phosphorus product of the reaction of 1 with the Eu(III) THED catalyst even after several days at 37°C as observed by use of 31PNMR is the cyclic phosphate ester (Figure 4).

-

4 OH

0

I

-o-p=o I

+

4-N02PhU

+ I"+

4-NO2Ph 0

1 Figure 4

W.

Productsfromthetransesterificationofthephosphate ester 0.

SUBSTITUTION REACTIONS'OF PHOSPHATE ESTERS

There are manyexamplesofmetalionpromotedhydrolysisof phosphate diesters (13-15) and phosphoric anhydrides (16-21). For the are thought to very efficient metal complex catalysts, these reactions proceed by metal ion binding to the phosphate ester followed by attack of ametal-boundhydroxide or metal-boundwatermoleculeatthe (22) phosphoruscenter.Elegantstudies bySargesonandcoworkers demonstrated this important pathway. An ''0 label in a water molecule Co(II1) complex was incorporated into the product bound to a phosphate. Therearerelativelyfewstudies ofphosphate ester substitution by metal-boundnucleophilesother than hydroxide or water.Examples include phosphate ester transesterification by a Co(II1)-amido complex

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(23), and more recently phosphate ester substitution reactions promoted An interestingearly bylanthanide(III)peroxidecomplexes(24-25). example is the nucleophilic attack of a Zn(II)-carbaldoxime anion on aphosphorylimidazole (26). Thelanthanide(III)complexesdiscussed hereprovide an additionalexample.Productanalysisandkinetic studies indicate that a bound hydroxyalkyl group acts as a nucleophile toward a phosphate diester. Treatment ofbis(4-nitropheny1)phosphate (BNPP) with eitherthe L a m ) or Eu(III) complex of THED at 37 "C, pH 7.40 results in therapid production of 4-nitrophenolate. Pseudo-first-order rate constants for the production of 4-nitrophenolate (0.1M NaCl and 0.01 M Hepes buffer) are 2.7 (fo.2) x W S-' and1.0 (fo.05) x 10' S" in the presenceof 1.00 x lo-, M La(I1I) or Eu(III) THED complexes, respectively. Rate constants for Eu(THED)(CF,SO,), are slightly higher in the absence of NaCl (1.9 f 0.2 x lo4 S"). The production of 4-nitrophenolate is first order in Eu(THED)(CF,SOJ,. ThepH-rateprofile is sigmoidaland computer-assisted fitting of the data to equation (1) gives an apparent pK, of theactivecatalyst(7.38)that is close to that measuredby potentiometric titration (7.50) and a k, of 0.19 M's". In contrast, the rate constant for production of nitrophenolate from treatment of BNPP (1.8 f 0.2 x l o 6 S") withEu(S-THP)(CF3S03), is fifty-fold less than This isin contrastto the five-fold that for Eu(THED)(CF,SOJ,. difference in rate constants for RNA cleavage by the two complexes.

In contrast to the catalytic nature of phosphate ester transesterification by Eu(THED)(CF,SO,),, the production of 4-nitrophenolate from BNPP in the presence of the Eu(III) complex is stoichiometric in nature. An equivalent of 4-nitrophenolateis produced under conditions where there is atwenty-fold excess o f BNPP to Eu(III) THED complex. Product o f this inhibitionbynitrophenylphosphate(NPP) is notthecause behavior.Additionofanequivalentof NPP didnotinfluencethe initial rate of 4-nitrophenolate production. The reaction products were examined by use of 31PNMR. A solution of 0.01 M La(THED)(CF,SOJ, or Eu(THED>(CF,SOJ, was added to a 0.01 M solution of BNPP and the pH was adjusted to 7.6. A new NMR resonance was observed in the Eu(THED)(CF,SOJ, reaction (-5.2 ppmversus Hp0.J andtwonewresonanceswereobserved for the La(THED)(CF,SOJ, reaction (-4.7 ppmand -5.0 ppm). These ,'P resonances are assigned to a covalent adduct of the macrocycle with 4nitrophenylphosphate. 'H N M R data of the La(I1I) complex adduct is

Lanthanide(ll1) Complexes Synthetic Nucleases as

181

supportive of this assignment. The two 31PN M R resonances observed with the La(III) complex are attributed to an adduct with the La@) ion bound or to an adduct of the free macrocycle. This is consistent with the faster dissociation rate of the La(III) complex of THED. The adducts are readily isolated as solids from treatment o f solutions of the of potassium Ln(THED)(CF,SO,),complexeswithoneequivalent hydroxide and BNPP in ethanol. Elemental analysis of the products are consistent with covalent adducts of the formula [Ln(THED-H+)(P03(4NO,C,H,O))][CF,SO,],where thephosphateester is bound tothe hydroxyethyl group of the THED ligand as shown in Figure 5. HPLC analysisand 31P studiesindicatethat NPP is produced in small amounts. Studies are underway to determine whether NPP is produced by the hydrolysis of the covalent adduct.

OR I

0 Figure 5

Production of a covalent adduct from attack boundalkoxidegroupoftheTHEDligand phosphatediester. R is 4-nitrophenyl.

of a

on a

preliminary studies as monitored by N M R suggestthatsimilar substitutionreactionspromotedbyLn(THED)(CF,SO,),occur for phosphate esters with good leaving groups. Cyclic phosphate estexs do not react measurably with the Eu(III) complex of THED after 24 hours at 37°C. In contrast phosphoric anhydrides are rapidly hydrolyzed by both theLa(III)andEu(I1I)complexesofTHED.Thenucleotide product ofATP treatment with La(THED)(CF,SOJ, is exclusively ADP.

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Other 31PN M R resonances indicate that inorganic phosphate is produced as well as a new species with a chemical shift of 0.58 ppm. This resonance is tentativelyassignedtoacovalentadductofthe macrocycliccomplexwithphosphatebasedon its appearancewhen La(THED)(CF,SO,), is treated with other phosphorylating agents such as NPP. In addition, over time the 31PNMR resonance for inorganic phosphate increases at the expense of the resonance for the covalent adduct. The proposed pathway for production of 4-nitrophenol from BNPP and Ln(THED)(CF,SOJ, is shown in Figure 5. This pathway is congruent with the fact that theEu(THED)(CF,SO,), complex has a single site for binding small molecules (10) and with the stoichiometric nature of the reaction. In addition, thelesserreactivity of the Eu(II1) S-THP complex toward BNPPis consistent with the rate determining formation of an adductbynucleophilicattackofadeprotonatedhydroxyalkyl group.Nucleophilicattackonthephosphatebythemorebulky be anticipatedtoproceed deprotonatedhydroxypropylgroupwould more slowly than attack by a deprotonated hydroxyethyl group. It is ofinterestthatmetalloenzymessuch as alkalinephosphatase catalyze the hydrolysis of phosphate monoesters through the formation of a similar covalent adduct (27-29). Hydrolysis is thought to proceed in two steps. First, a serine group attacks the phosphate ester to form a covalent adduct and it is thought that one of the two Zn(I1) ions may facilitate this step by lowering the pK, of the serine group. This step is analogous to the pathwayobservedherewiththelanthanide(1II) complexesof THED. Finally, the adjacentzinc(II)-boundhydroxide hydrolyzes the covalent adduct to complete the hydrolysis reaction.

In summary,hydroxyalkylmacrocycles are good ligands for the trivalentlanthanides.Thependenthydroxyalkylgroupsparticipate in general base catalyzed transesterification of RNA and phosphate esters and participate as nucleophiles in stoichiometric substitution reactions of phosphate esters.

ACKNOWLEDGMENTS

the NationalInstitutesofHealth (GM46539)andby the donorsof the PetroleumResearchFund, administered by the American Chemical Society.

This work was supported by

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as Synthetic Nucleases

183

REFERENCES 1.

2. 3.

4. 5. 6. 7.

8. 9. 10. 11. 12.

13. 14.

15. 16. 17.

18.

For a review see: J. R. Momw, Artificial Ribonucleases, in Advances in Inorganic Biochemistry (G.L. Eichhorn and L. G. Mamilli, Eds.), prentice Hall, EnglewoodCliffs, N.J., 1994, vol. 9, ch. 2. L. A. Basile, A. L. Raphael, and J. K. Barton, J. Am. Chem.

SOC. 109: 7550-7551(1987). B. F. Baker, J. Am. Chem. Soc. 115: 3378-3379(1993). J. R. Morrow and V. M. Shelton, New Journal of Chemistry 18: 371-375(1994). J.R. Morrow, L. A. Buttrey, V. M. Shelton, K. A. Berback, J. Am. Chem. Soc., 114: 1903-1905 (1992). S. A. Amin, J. R. Morrow, C. H. Lake, M. R. Churchill, Angew. Chem. Int. Ed. Engl., 33: 773-775 (1994). J. R. Morrow,and K. 0. A. Chin, Inorg.Chem. 32: 3357-3361(1993). K. 0. A. Chin, J.R. Morrow, C. H. Lake, M. R. Churchill, M. R., Inorg. Chem. 33: 656-664 (1994). J. R. Morrow, S. A. Amn i , C. H. Lake, M. R. Churchill, Inorg. Chem. 32: 4566-4572 (1993). J. R. Morrow, S. A. Amin, D. A. Voss, Jr, C. H. Lake, M. R. Churchill and W. Dew. H o m k s , Jr., in

preparation,

K. 0.A.

Chin,and J. R. Morrow, submitted. Bryden, C. N. Reilley and J. F. Desreux, Anal. Chem. 53: 1418-1425(1981). See ref. 1 for references through 1992. J. H. Kim and J. Chin, J . Am. Chem. Soc. 114: 97929795(1992). J. N. Burstyn and K. A. Deal, Inorg. Chem. 32: 35853586 (1993). H. Sigel and P. E. Amsler, J . Am. Chem. Soc. 98: 73907400(1976). H. Sigel, F. Hofstetter, R. B. Martin, R. M. Milburn, V. Scheller-Krattiger and K. H. Scheller, J. Am. Chem. Soc. 106: 7935-7946(1984). R.M. Milburn, M. Gautam-Basak, R. Tribolet and H. Sigel, J. Am. Chem. Soc. IO7 3315-3321 (1985). C.C.

184

19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29.

Morrow et al.

J.Visscherand A. W.Schwartz, NucleicAcids Res. 20(21): 5749-5752(1992). R. N. Bose, R. D. CorneliusandR. E. Viola, Inorg. Chem. 24: 3989-3996 (1985). G.P. Haight, Jr., Coord. Chem.Rev. 79:293-319 (1987). D. R. Jones, L. F. Lindoy and A. M. Sargeson, J . Am. Chem. Soc. 105: 7327-7336 (1983). J. MacB Harrowfield, D. R. Jones, L. F. Lindoy and A. M. Sargeson, J. Am. Chem. Soc.102: 7733-7741 (1980). B. K. TakasakiandJ.Chin, J. Am.Chem.Soc. 115: 9337-9338(1993). B. K. Takasakiand J. Chin, J. Am. Chem. Soc. 116: 1121-1 122 (1994). G. L. LloydandB. S. Cooperman, J. Am. Chem. Soc. 93: 4883-4893(1971). E. E. Kim and H. W. Wyckoff, J. Mol. Biol. 218: 449464(1991). J. E. Butler-Ransohoff, S. E. Rokita, D. A. Kendall, J. A. Banzon, K. S. Carano, E. T. Kaiserand A. R Matlin, J. Org. Chem. 5 7 142-145(1992). P. Gettins and J. E. Coleman, J. Biol. Chem 258: 408416(1983).

12 Initiation of DNA Strand Cleavage by Iron Bleomycin: Key Role of DNA in DeterminingthePathway of Reaction David H. Petering,Patricia F’ulmer, Wenbao Li, andQunkaiMao Departmentof Chemistry, Universityof Wisconsin-Milwaukee, Milwaukee, WI 53201 William E. Antholine Medical College of Wisconsin, Milwaukee, WI 53226

I.

INTRODUCTION

Metallodrugs are rare agents in the modem apothecary. Next to the multitude of organic c~mpw~lds, coapuatively few metal complexes, metal bmding ligands, and other structures that interact with intracellular metals are used to treat human disease. For example, among theapproximately 30 drugs c o d y employed in cancer chemotherapy, onlythree or four require metals, cisdiamminedichlom Ptand its cyclobutanedicarboxylatederivative, bleomycin, md possiblyadriamycin [l-31. Amongthese,only the second generation platinum complexresulted from deliberate inorganicpharmacological research. Considering the remarkable development of inorganic chemistry over the past several decades and the rmccess of the drugs mentioned above,it is surprising that relatively little attention has been devoted to metal complexes and related compounds as sources of drugs. 185

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Petering et al.

An importaut site of reaction of chemdhenpevtic agents is DNA. Among the types of damageresultingfrom such d o n s are oxidative alterations in St~cture initiated bythe attack of .ctivoted forms of oxygea m the bases or backbone of the polymer. Metal complexes that include redox active metal ions are particularly adeptat catalyzing the reductionof dioxygen in d l s to species such as hydroxyl radical, which readily attack organic molecules in their vicinitysuch as DNA [4]. Bleomycin is the model for a number of inorganicnucleaseswhichhave been designed during the past decade [5]. Shown in Figure 1, its structureand that hypothesizedfor Fe bleomycin display the essentialfeatures of all of thesecomp0undS"thepresence of a metal coordination site where dioxygen activation occurs, a domain that has affinity for DNA, and a linker between them. In this type of structure, a dox-active metal ion becomes associated with the polymer and so generates an activated species of oxygen in the vicinity of the target DNA. Reaction of Iron Bleomycin with Dioxygen

A.

Bleomycin is isolated froma streptomycete as a partial copper complex [6]. Considering its strong biological activity in mammalian systemsand even greater potency against microrganisms such as Euglena gracilis, it probably tames as an antibiotic in nature which has been designed to cause lethal DNA damage [7]. As described below, Blmacts against tumor cells as an iron not a copper complex. In an elegantset of papers by Peisach, Horwitz, and coworkers [8-10], it has been shown that FeBlm can be converted to an activated form, probably HO2-Fe(III)Blrn, in three general ways(Figure 2). Fe(II)Blm c811 seme bothas site of activation and reductant, Fe(III)Blm can m t with 02 and two electrons from another source, or Fe(IIQBlm and H2% can react directly to form the activated intermediate. Once formed in the presence of DNA, H02-Fe(III)Blm reacts with theC4'H of deoxyribose to initiate strand scission or base release in a reaction equivalent to hydrogen abstraction by an hydroxyl radical [l 1,121. Inits absence, H02-Fe(III)Blm~dergoesan ill-defined self-inactivation mction which renders the drug much less capable of causing further DNA damage when reacted again with reductant, 0,.and DNA [13,14]. The reaction of HO2-Fe(IIr)Blm with DNA Ic+ds either to outright single strand scission as a 3carboo base propad from deoxyribose is formed W h base releaseleaving alkaline labilesites (Figure 3) [15]. The first PJhWay requiresdioxygen and another electron. As writtea, starting with H02'

'Abbreviations:bathophenanthroline disulfonate, BPS;bleomycin,Blm; major congeners of Blm, Blm A2 andB.& redox-inactivated bleomycin, RIBlm.

Initiation of DNA Strand Cleavage by Iron Bleomycin METALBINDING DOMAIN BINDING DOMAIN DNA I

CO-TERMINAL AMlNE

Figu~? 1 Structures of bleomycins. (a) Metal-free ligand and (b) metallobleomycin.

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188 ~

al.

SELF INACTIVATION

Figun 2 Routes of formation and reaction of activated FeBlm. Fe(III)Blm, the reaction produces one hydroxyl did,which is not deteded by spin trapping: H02-Fe(IQBlm

+ DNA + O2 + e' ->F.(m)Blm + cleavage products

+ 'OH

(1)

The other pathway can occur under anaerobicconditiws and effectively d u c e s the peroxide ligand to two water m o l e c u l e s U 8 base is released from DNA leaving a modified alkaline labile sugar: H02-Fe(III)Blm

+

+ DNA -> -k

Fe@I)Blm free base modified DNA 2H20

+

(2)

This paper examinesthe m e c h a n s im ofcellular DNA damage caused by bleomycin, focusing attentionon the chemistrywhich occurs when metallodrug and DNA interact to initiate DNA damage and considering bow the drug may cause double strand cleavage.

B.

Cellular DNA Strand Cleavage by Bleomycirrs

Both single and double straad damage is ddected in cdls. It hs beat shown that the formation of double strrnd damage follows the same Blm concentration dependace as inhibition of cell proliferatioa [16,17]. Furthermore, damage is prognssive over tims unless hlted by the dditim of 1, IO-phenmthroline [18]. Once inhibited, angoing DNA np.ir is observed in

which a significant fraction of double but not single s t z d piof DNA remains fragmented. Thus,8 strong relrtionshipbetwaen double strand scimion and cytotoxicityhas beenestrblished. Importantly, under conditionsleuiing the

0

I

0

a

.

1 I = 0-a-o

189

l90

Petering et al.

cellular DNA damage, the concentration of drug in the nucleus is less than 1 molecule per 16base pairs [16]. Chemicalstudiesdescribedabovedemonstratethat under suitable conditions FeBlmcan cleave the DNA backbone in the pl.lesence of O2 [8-lo]. Other reports indicatethat a'+, Blm,and 0, also produceDNAstrand scission [19]. Blm, FeBlm, and CuBlm CIUI also inhibit cell proliferation and cause DNA damage [16,20]. However, in HL-60 and Euglena gracilis cells which were made iron deficient by reducing iron available to them in their growthmedia,onlyFeBlmretained full DNA strandscissionactivity[2]. Similarly, in preparations of nuclei, Blm was unable to causemore than 1% of the DNA double strand breakage resulting from exposure to Fe(I1I)Blm [21]. Thus, it is evident in cells that Blm requires iron for its activities and that the comparable activity ofBlm, CuBlm and FeBlm is due to the facile conversion of the first two forms into FeBlm.

II.

REACTIONS OF METALLOBLEOMYCINS WITH DNA

The DNA damaging action of Blm is striking because it occurs when the base pair to drug ratio in the nucleus is on the orderof 16:1 and because it results in both single and double strand damage at all concentrations of drug as discussed below.To determine which of the pathways of activation of FeBlm might take place under physiological conditions and how double strand scission might occur despite the presence of only onesite of activationof 0, in the drug, studies of the reactionsof DNA-bound CO- and FeBlm havebeen undertaken.

A.

Redox Reactions of Co(IQBlm with 0,: Influence of DNA and Structure of Produds

The following set of reactions characterizethe oxidation of Co(II)Blm by 0, [22]: Co(II)Blm O2 e O2400Blm Ft BlmCo(II)~~-Co(lI)Blm + 02 202-Co(lI)Blm BlmCo(II)-02-Co(II)Blm + H+ "> H02-Co(III)Blm co(III)Blm (Form I) (Form II)

+

+

(3) (4) (5)

A dioxygenated species of Fe(II)Blm and H02-Fe(III)Blm are thought to form during the corresponding reaction of F@)Blm withO2 so that reactions 3-5 appear to be an excellent model for the reaction of Fe(I1)Blm with OT A similar set of reactionsoccurs when Co(I1)Blmreacts with O2 in the presence of DNA [23]. Nevertheless, there are significant differences. First, the rate of conversion of 02-CoBlmto Form Iand 11 is dramatically dependent

Initiation of DNA Strand Cleavage

by Iron Bleomycin

191

upon the base pair to drug ratio. Given a binding site si= of 2-3 bum pairs, OSKC this Wio 6-8: 1 reactions4 and 5 almost leaving st.ble %Co(II)BlmDNA. Apparently, once the ratio of base pairs to CoBlm molecules iacrersesbeyond the size of the bindingsite of 2-3base pairs and adjwxntdrug molecules can not make direct contact, reaction 4 can not take place without raorgaaizrtian'of drug molecule to establish conditionsfor dimerization. The dioxygmted adductassociatedwithoriented DNA f i h was examined by ESR spectroscopy to determine whether the presence of DNA ccmstrriwd the orientation of the paramagnetic center [24]. It was found that the dioxygen species was fixed in a plane nearly perpendicular to the double helical axis (Figure h). This finding provides strong evidence that the metal coordination site 1s well as the DNA binding domain interacts with DNA. Combined with the i n d i m evidence that Blm binds in the minor groove of DNA, these dah arc .Is0 consistent with a model in which the oxygen+xygen bond of O+cQI)Blm a n be approximately colinear with the C4"H bond on either strlad that is the initialsite of attack in the cleavage reaction (Figure 4b). NMR structures of Form I and I1 have been completed,revealing conformntid complexity not reported for the structures of ZnBlm andOCFe(II)Blm [B-271. As seen in Figure 5, the bithimle is folded back upon the pyrimidine in Form I to constitute a compact structure with a central pocket containing the peroxy ligand to co(m). Form I binds strongly to DNA and provides a model for how both themetal and DNA domains mayjointly interact with DNA.

I

0

I

O;Jgt;.....&0:j

2'

3'

a

4' H

I'

......... f 1'

0

4' t0 i )

0-

/

\o

b

Figure 4 Structure of O,-CoBlm bound to DNA. (a)Orientation of the oxygensxygen bond with respect to the DNA helix axis. (b) Hypothetical orientation of dioxygen in the minor groove of DNA. The dot represents the double helix axis.

I

Bithiazole Peroxide ligand ("L"in cleft) COW) (Hexagon in cleft)

+I

Charged

I Pyrimidine Figure 5 Energy minimized structure of H02..Co(III)Blm NMR analysis.

4 determined by

B. Redox Reaction of Fe@)Blm with 0,: Influence of DNA The oxidation of Fe(II)Blm by 0, occurs rapidly in the absence of DNA. As with CoBlm, the rate of d o n slows in the presence of DNA. Recent studies illustrated in Figure 6 support a pathway of reaction in which 0, rapidly bmds to Fe(I1)BlmDNA (step 1, reaction 6) [28]. This is followed by a slower releaseof dioxygea into solution that occurs with kineticsthat are seoond order in 02-FeOBlmDNA (step 11, reaction 7 and 8).

+

F@)BlmDNA 02 it OyFe(II)BlmDNA (6) 202-Fe(lI)BlmDNA P [DNABlmF@)-O2-Fe(II)BlmDNA] +0 2 (7) [DNABlmFe(II)-02-Fe0BlmDNAI+H+ -> H02-FQ)BlmDNA F m B l m D N A (8)

+

Although a dimer intermediate may form in reaction 7, that is uncertain. The important finding is that the secondader rate constant for step II decreases dramaticallyfrom 1180 M"s" to 160 as theDNA base pair to drug ratio increases from 511 to 8/1. Thus, as in the oxidatim of Co(lI)Blm bound to DNA,theseparation of FeBlmmolecules-alongtheDNAdoublehelix effectively inhibitsthe biomolecular reaction described in reactions 7 and 8. As the base pair to FeBlm ratio increases to 20 to 1, large amounts of 02-

Initiation of DNA Strand Cleavage by Iron Bleomycin

193

100

90

z

P l-

a LL 2

5cn

80

0"

ml

60 0

IO

20 30

TIME (MINUTES)

F'igure 6 Reaction of 0, with Fe(I1)BlmDNA. (IUptake ) of O2 a h Fe2+ is added to BlmDNA. Ratio of base pairs to F e o B l m is 1 0 1. (II) Release of 0, from 02-Fe(II)BlmDNAduring redox reaction. (m) Release of O2 from 02-Fe(II)BlmDNA after reaction of the in the d u c t with bathophenanthroline disulfonate.

Fe(II)BImDNA can be detected by addition of the F e 0 chelating ageat, htbophenanthroline disulfonate to the reaction mixture, which liberates s t o i c h i d c amounts of Fe@) and 0, (step III of Figure 6 ) from bound dioxygearted drug. These results show clearly thatat large base pair to drug ratiossuch m are found in cells treated with Blm, 02-FeBlm willbe stabilized on DNA and will require the input of electrons from sources other than Fe(II)Blmto cany out the activation and DNA cleavage reactions (Figure 2). Examination of the properties of dioxygenated metallobleomycinshas revded that it is difficult to remove 0, from O 2 ~ B l m D N A and 0,Fe(II)BlmDNA.

Petering et al.

194

The rate constant for dissociationof 0, From the latter is 0.003 S-', which is remarkably small in comparison with that for the loss of 0, from the a chain of hemoglobin,28 s-l[28]. This fmding suggeststhat the irondioxygen adduct l i e that of 02€o(II)BlmDNA exists in a constrained site that restricts the rate of dissociation of 0,. Anothermodel for 02-Fe(II)BlmDNA is ON-Fe(II)BlmDNA. A comparison of the interaction of nitric oxide with Fe(II)Blmin the absence or presence of DNA by electron spin resonance spectroscopy has shown that the ESR spectrum of theNO adductis altered when the drugis bound to DNA [29]. As with O 2 ~ ) B l m D N Ait, appears that ON-Fe coodination site 6 ~ l s e sthe presence of the polymer, consistent with a folded conformationof the molecule and perhaps the association of metal both and DNA binding domains with DNA.

m.

REDOX-INACTIVATED IRON BLEOMYCIN

Figure 2 indicates that once the drug is rcciv.tsd to HO2-Fe(IIl)Blm, it is competent to initiate DNA damage. A l t d v e l y , in the abseace of DNA it undergoes a suicide-like reaction thatmodifies its own structure such that it is W longer effective in DNA strand scission chemistry. h l i e r work hd demonstrated that this material, called redox-inrctivated bleomycin (RIBlm), was fully capableof activating 0, to H~-Fe(III)RIBlmin the pnsenceof Fe2+ 1151. However,therewasevidence that tbe bithiamle chromophore in the molecule had been modified [30]. Returning to the structure of H02-Co(III)Blm 4 as a model for the corresponding iron species,theperoxideligand is positioned in close proximity to the bithiazola moietyd, thus, might be ex ted to react with this portion of the molecule. In addition, unpublished ll&lm Nh4R experiments show that the '13Cd resol~ancesfor the Blm 4 aud 4 complexes differby several ppm (Figure7a). This result shows that the metal center senses the positively charged tails ofthe two, othewise identical molecules, as might be expected in flexiblefolded conbmations for such structures. Indeed, in an ongoing NMR analysis ofRIBlm 4,it has bees found that the sole differences in the modified structure are the destruction of the native bithiamle group and the loss of one of the methyl groups from the dimethyl sulfonium group in the tail of the molecule (Figure 7b). All other rotons in the molecule are accounted for sccording to detailed 2dimeasional NMR analysis of m l m A2 From these combined results, it appears that RIBlm 4 fails to cleave the DNA backbone because neither of the DNA binding elementsof Blm 4remains intact. As a consequence, RIBlm A2does not strongly associate with DNA and like &methylBlm A2, which also lacks a methyl group in the dimethyl sulfonium moiety, has little DNA cleavage activity [15].

L

.

Initiation of DNA Strand Cleavage by Iron Bleomycin

-3

1 3

t c

l$

195

1

196

Petering et al.

N.

MECHANISM OF DOUBLE STRAND SCISSION BASIC CONSIDERATIONS

Cellular studies point to a relationship between double straad DNA cleavage and cytotoxicity [16,17]). While past studies have ducidatcd many aspects of the alternative pathwaysof DNA damage, single strand scissiaa and base release (Figure 3), much less is known about double strand cleavage. Nevertheless,thefollowingpropertieswill need to be inoorporrted into a mechanism for this process. First, it has been shown that after reaction of Fe(n1)Blm and ascorbate with supercoiled plasmid DNA (Figure 8), double strand cleavageis observed (band III) dong with single strand brealrage (band II) at every concentration of FeBlm. In contrast, d o n of the plasmid with Fe2+ first convertsthesupercoiled structure (band I) to the single strand scission product, relaxed circularDNA, and then further cleaves it to double strand linear mol~ules,probably as a result of producing enough proximate singlestrandbreaks on opposite strands to get double strand breakage. Therefore, as in cells single and double strand p d u c t s are produced at all effective concentrations of FeBlm, suggesting that the two types of damage are mechanistically related. The kinetics of formation of single and double strand bmaks am also similar (Figure 9). As such, they are not consistentwith independent, sequential formation of singleandthendoublestrandbreaks. I n s t e a d , they support reaction to c a w single or double strand breaks by single FeBlm molecules at particularsites. Indeed, because the base pair to dmg ratio is large, one hypothesizes that dissociation of FeBlmfrom the site of doublestrand cleavage does not occur prior to complete double strand brenkage; othewise random rebinding of the drug would prevent further reaction at the original site. n d e d DNA Finally, double strand damage to produce largely blunt e breaks or ones offset by one nucleotide on the two strands occurs at the same sites where base releasetakes place [3 1.321. Pmbbly, single strand damage is also confined to these same sites (321. The presenoeof these site-spccific alternative reaction productssuggests that reaction of activatedFeBlm is kinetically partitioned among three pathways. A d i n g to a racent modd. H%-Fe(III)Blm, reacting at a site, initidly auses either bre Felersa or the introduction of a single strand break at a sitaspscific G-pyrimidine [32]. If the former, no further &ion occurs on the other straad. If tbe latter, no reaction may occur, another break might be formed on the second otrpnd leading to double strand cleavage,or thc second stranddso may be damaged by base release. A constraint on the mechanism foreach pathway is that little free radical production is observed in these DNA damage reactions [33]. To 8ccoullt for resction on both strands, the same model wggeskd a collcerted mechanism for reactivation of FeBlm following the initiationof single strand breakageat the primary site of attack that involves reactionof Fe(III)Blm

Figure 8 DNA strand scission of pBR322plasmidby species of F$+. Reaction of 15 FM plasmid base pairs, 25°C in phosphatebuffer, pH 7.4, with specified concentrations of (left) Fe2+ added as Fe(NHd2(S0d2 and (right) Fe(II1)Blm plus 100 mM ascorbate. Reaction times: left, 15 min and right, 30 min.

W

2

5 W

U

10

30 40 TIME, MINUTES

20

50

60

Figure 9 Kinetics of single and double strand scission of pBR322 plasmid by Fe(In)Blm andascorbate.Form I, nativeplasmid;form XI, r e l a x e d circular DNA with single strand breaks; form 111, linear DNA with double strand breaks. 15 p M base pairs was reacted with 0.1 CM Ft(l1I)Blm in phosphate (pH 7.4) buffer at FWN)$.

25".

Reactions were stopped at each time point with 0.2 mM

Petering et al.

198

with the hydroperoxide intermediatein the strand scission pathway in analogy to its reaction with hydrogen peroxide (Figures 2 and 3). In this model, the hydroperoxide provides the hydroxyl radical equivalent to abstract the C4"H from a deoxyribose on the second strand. The problem with this hypothesis is that in the process the first strand is left without the necessary hydroperoxide intermediate to proceed to cleavage products. Two other possiblemechanisms are consistentwith much of the information about double strand scission. First, a um~rtedreaction might occur as follows to initiate cleavage at C4"H and C4"-H on both strands:

+ C4'-H-> (oOH)Fe(III)BlmDNA + C4'. + H20(10) (oOH)Fe(nI)BlmDNA + C4"-H->Fe(III)BlmDNA + C4"' + Hz0 (11)

HOZ-Fe(III)BlmDNA

In this mechanism, the drug%ound peroxide homolytidy provides two hydroxyl radicalsto abstract thetwo hydrogen atom. Like the base-release path (reaction 2), which also converts the peroxide ligand to two water molecules, reactions 10 and 11 do not produce residual hydroxyl radicals. h the dbet mechanism*~OH)F~(III)B~~DNA must lmd~rgoa secund cycle of activation to HO2-Fe(II1)BlmDNA, which thm initiatcs cleavage011 the other strand. Two hydroxyl radicals arc produced sequmtially in this mechanism which m not deteded, making it a less attractive alternative to reactions 10 and 11 [M].However, the first mechanism does not provide an obvious to achieve a mixture of siugle and double strand cleavage, whereas in the second mechanism a mixture of these damaged products could result if reactivation occufi at a rate comparable to the rate of dissociation of Fe(I1I)Blmfrom the single stranddamaged site. Underthatcondition, the dissociation event would effectively limit DNA damage at the site to single strand cleavage. V.

CONCLUSIONS

The bindmg interactions within andbetween various forms of Fe- or CoBlm and DNA play critical roles in determining the chemical reactivity of these species with respect to DNA. The particular stoichiometryof base pairs to Fe@)- or Co(1I)Blm alone specifies whether adduct formation or oxidationreduction in the presence of0, will occur. Adduct formation has consequences for structure and reaction. Binding of 0, to CoBlmDNA is accompanied by rigorous orientation of02 with reqxct to the DNA structure. Thennodynamic andkineticstabilizationofdioxygen species result.Dioxygenbinding also decreasestheligandsubstitutionreactivity of Fe(1I)BlmDNA [28]. Taken together thw results support the hypothesis that both metal and DNA binding

Initiation of DNA Strand Cleavage

by Iron Bleomycin

199

domains interact intimately with the DNA structure. The perturbgtim of the ESR signal of ON-Fe(I1)Blm by DNA also supports this hypothesis. Indeed, the findingthat H02€oBlm 4 existsin a foldedstructurenowprovides a structural model in which bothmetal and DNA binding domainsof the drug may jointly interact with the minor groove. It is expected that the mechanism of initiation of single and double strand damage will depend on these binding interactions between drug and DNA which are now emerging.

ACKNOWGEMENTS The authors thank the United States National Institutes of Health National Cancer Institute, and theAmerican Cancer Society fortheir support of thisresearch through grants NIH-CA-22184and American Cancer Society -DHP 3 1C.

REFERENCES l. 2. 3.

4. 5. 6.

7.

8. 9. 10.

11.

K. Rsdtke, R. Bymes, F. brnitzo,W. E. Antholine, and D. H. Petering, J. Inorg. Chem. 43: 456 (1991). K. Radtke, F. A. brnitzo,R. Bymes, W. E. Antholine, and D. H. u b d i lo n (1994). Petering, Bidem. J., acceptd for p D. H. Petering, R. W. Bymes, .ad W. E. Antholine. Transitim metpl requirements for the nction of antibiotics, in Handbook on Metal-Ligand Interactions in Biological Fluidr, Vol. 2 (G. Berthon. Ed.), Marcel Mer,in p m , 1994. R. W. Bymes, M. M o b , W. E. Antholine, R. X. Xu, d D. H. Petering, Biochemistry 29: 7046-7053 (1990). D. S. Sigman, Biochemistry 29: 9097-9105 (1990). H. Umezawa, Y. Suhara, T.Takita, .ad K. Maeda, J. Antibioz., Sa.A 19: 210-215 (1966). S. Lyman. P. Taylor. F. brnitzo,A. Weir, D. Stoat, W. E. Antholine, and D.H. Petering, Biofhcn Phannaml. 3& 42734282 (1989). E.A. Sausville, J. Pcisach, awl S. B. Horwitz, Bi17: 2740-2745 (1978). E. A. Sausville, R. W. Stein, J. Pcisach, .ad S. B. Honrvitz, Biochemistry 17: 2746-2754 (1978). R. M. Burger, J. Peisach, .ad S. B. Horwitz, J. Biol. Chan. 256 11636-1 1 6 4 4 (1981). J. C. Wu, J. Kozarich, and J. Stubbe. J. Biol. Chem. 257: 3372-3375 (1982).

200 12. 13. 14. 15. 16. 17. 18. 19.

U). 21.

22. 23. 24. 25. 26.

27.

28.

29.

30. 31. 32. 33. 34.

Petering et ai.

J. C. Wu, J. Kozarich, and J. Stubbe, J. Biol. chem 258: 4694-4697 (1983). R. M. Burger, J. Peisach, and S. B. Honrvitz, J. Biol. chem.257: 3372-3375 (1982). J. Templin, L. Berry, S. Lyman, R. W. Byrnes, W. E. Antholiue, and D. H. Petering, B i o h Phunna~url.43: 615-623 (1992). J. Stubbe, and J. Kozarich, c h m r . Rw. 87: 1107-1136 (1987). R. W. Bymes, J. Templin, D. Sem, S. Lyman, and D. H. Petering, Cancer Res. 5& 5275-5286 (1990). R. W. Bymes and D. H. Petering, Rud. Res. 237: 162-170 (1994). R. W. Byrnes and D. H. Petering, Bioahem. Pharmaml. 42: 1241-1248 (1991). 0. M.Ehrenfeld, J. B.-Shipley, D. C. Heimbrodr, H. Sugiyanm, E. C. Long, J. H. van Boom, G. A. van der Mad, N. J. oppenheimer, and S. M. Hecht, Bioahanisrry 26: 931-942 (1987). E. A. Rao, L. A. Suyan, W. E. Antholine, and D. H. Petaing, J. Med. ChRm. 23: 1310-1318(1980). R. W. Byrnes .ad D. H. Petering, Bh&m l%urmumL, in press (19w. R. X. Xu, W. E. Antholine, and D. H. petering, J. BM. chcm 267: 944-949 (1992). R. X. Xu, W. E. Antholine, d D. H. Petering, X BM. chcm 267: 950-955 (1992). M. Chikira, W. E. Antholine, urd D. H. Pehing, J. Bwl. than, 264: 21478-21480 (1989). R. X. Xu, D.Nettesheim, J. D. Otvos, and D. H. Petering, BioCJlcmhtry 33: 907-916 (1994). M. A. J. Merman, C. A. G. Haasaod, U. K. Pandit, & C . W. Hilbers, Eur. J. Bioahem. 273 211-225 (1988). M. A. J. Akkeman, W. J. F. Neijman, S. S. Wijmenga. C. W. Hilbers, and W. Vermel, J. Am. chsn.Soc. 112: 7462-7474 (1990). P. Fulmer and D. H. Petering, Biochemistry 3 3 5319-5327 (1994). W. E. Antholine and D. H. Petering, BioQhan, Biophys. Rcr. C o m m ~ n .91: 528-533 (1979). M. Nakamura and J. Peisach, J. Antibiot. 42: 638-647 (1988). L. F. Povirk, Y.-H. Han, and R. J. Steighaer, Biodtemistly 28: 5808-5814 (1989). R. J. Steighner and L. F. Povirk, Proc. Natl. A d Sci. USA 87: 8350-8354 (1990). L. 0. Rodriguez and S. M. Hecht, Biochem. Biophys. Res. Cbmmun. 104. 1470-1478 (1982).

D. H. Petering, R. W. Bymes, and W. E. Antholine, them.-Biol. I~U~TUCL 73: 133-182 (1990).

13 NickelComplexesinModification NucleicAcids

of

Steven E. Rokita, Ping Zheng, Ning Tang, Chien-Chung Cheng, Ren-Hwa Yeh, James G. Muller, and Cynthia J. Burrows Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794

1. lNTRODUCTlON Issues of nickel metaholism, toxicity, and carcinogenicity inevitably relate back to the umrdination and oxidation chemistry of this metal. The ligand environment strictly controls the efficiencyand diversity of nickel-dependent reaction. For example, certain nickel(I1)-peptide complexes undergo spontaneous oxidation in the presence of the dioxygen [l], whereas free nickel salts such as Ni(l1) chloride are quite inert to oxidation. The laboratories of Burrows and Rokita have combined their respective expertise in nickel-based catalysis and conformation-dependent modification of DNA to explorethe intrinsic reactivity of nucleicacids with biomimetic and macrocycliccomplexes of nickel. A series of square planar complexes were found to promote efficient and selective modification of guanine residues that were highly accessible to solvent. Theorigin of this specificity likely derives from direct interaction hetween guanine N7and nickel and concomitant delivery o f an associated peracid. Most recently, additional nickel complexes have been developed to promote nucleohase arylationand strand scission alternatively. 201

202

Roklta et al.

11. SELECTIVITY OF NICKEL-PROMOTED OXIDATION The initial goal of our collaboration wasto define the fundamental specificity and reaction determinants of macrocyclic nickel complexes with nucleic acids. NiCR (shown below) was one of the first species examined since it appeared to exhibit many properties known to promote effective catalysis also of olefin epoxidation 12,3]. Most subsequentinvestigationshave continued to focus on this example.The initial targets of modification were synthetic oligodeoxynucleotidesthat provide well defined systems for rapid and unequivocal characterization of nucleic acid specificity [4,5].

A. Modification is Specific for GuanineResidues Allguanineresidues (G) of single-strandedoligodeoxynucleotidesare subject to modification in the presence of NiCR and a water soluble oxidant such as KHSO, (Figure l). Typically, equimolar concentrationsOtM) of NiCR and a DNA strand are incubated for minutes in the presence of excess peracid. These conditions induce an alkaline lability that leads to strand scission at the effected guanines as a result of subsequent treatment with hot piperidine (lane 1, Figure 1B) [ 6 ] . The final pattern of fragmentation is equivalent to that generated by theMaxam-Gilbertsequencing reaction for G (alkylation by dimethylsulfate, lane 2, Figure 1B). In contrast, no spontaneous strand scission is evident directly after the metalmediated oxidation reaction (lane 3, Figure 1B). DNA modification based on nickel required the participationof both the peracid and nickel complcx, and accordingly no strand scission was observed after incubating DNA with either the metal or oxidant alone and assaying with piperidine. In addition, nickel chloride could not substitute forNiCRnor could an oxidant such as H,O, substitute for the peracid. Further mechanisticcharactcrization is discussed Collowing thesections below on the conformational specificity of guanine oxidation.

B. Only Solvent Accessible Guanines are Oxidized in the Presence of NiCR Synthetic oligodeoxynucleotides were further used to examine the relationship between DNA secondary structure and modification by NiCR. Guanine residues remained the only target of oxidation throughout our investigations. Most interestingly, the extent of reaction was very dependent on

z" v

m

3

Nickel Complexes

d

U I

In

in Modification Nucleic ofAcids

C

e

u u

h

0

m

W

n

E3

rrl

W

C

CI)

U

v)

.E

2

m

203

204

Rokita et al.

the local environmentsurroundingguanine. Modification wasseverely limited or not detectable for G residues that were paired to their complementary base, C, within the interior of a standard Watson-Crick duplex. In contrast, most non-canonical arrangements of G were readily oxidized by NiCR [7]. While all of the G residues in single-stranded A were susceptible to modification by NiCR, none reacted under equivalent conditions in the presence of the complementary strand A' (Figure 2). The only G-C pairs that exhibited an apparent reactivity were those at a helical terminus, and eventhen modification could have been due to base pair fraying. For example, the 3' terminal G in (he radiolabeled strand B was not protected from reaction in the presence o f the fully complementary strand B'. Other guanine residues accessibleto NiCR werc either mispaired (C) or unpaired as in an extrahelical bulge (A+[A'-C]) or loop (D and E) (Figure 2).

5 ' -

3'

3-T

lG-0 m

+ L

I

A 1

H T-A

-b&---k

B B '

L

4-4 T-3

A

A-T

m;"$c

D

\-I. 3-T

7-T l-A c c

Figure 2 Conformationspecificmodification deoxynucleotides using NiCR.

-

J ~P+G-T+T-@\

4J-A"LLly E t f of

["PI-labeled oligo-

Nickel Complexes in Modification of Nucleic Acids

205

Thisconformationalspecificity is not generally mimicked by other metal or non-metal reagents. The G residues in the loop formed by D were clearly distinguished by NiCR but not by other metal complexes based on iron, copper or manganese [S]. The most commonly applied reagent for G modification, dimethylsulfate, exhibits little or no sensitivity to secondary structure 191. The unique specificity of NiCR then likely derives from the distinctcoordination and redox chemistry of the metal center. Taillander's laboratory had previously demonstrated that Ni(I1) salts selectively coordinate to G N7 [lo], and this might also provide a basis for nucleic acid recognition by NiCR. Our attention next focused on modification of yeast tRNAph"in order to establish a precise correlationbetween reactivity and solvent accessibility of a single functional domain within the guanine nucleotides. This target was quite valuable sinceits surface properties have heen extensively characterized by experimental and theoretical techniques[ 11-13]. NiCR readily modified 12 out ol the 23 residues of guanine and i t s natural derivatives when the RNA was partially denatured by the absence ofMg" [ 14). In the native conformation, only 4 residues were reactive (Figure3) and these residues corresponded exactlyto the guanines with the greatest accessibility and surfacepotential at theirN7 positions 113). Thisrelationship has greatly enhanced the value of NiCR as a probe for nucleic acid conformation. Now, guanine modification induced by this complex can be used to indicate the structural environment of G N7 specifically. 4

G-18

G-l9 G -20

Figure 3 Selective reaction oftRNA*' in thepresence of NiCR (adapted from ref. 15). G-34

64

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Rokita et al.

C. NiCR has Utility as a Probe for Nucleic Acid Folding The structure of the self-splicing ribozymeof Temzlrymena was next examined with NiCR to test the more general applicability of this reagent as a probe for nucleic acid folding. The polynucleotide target in this case contained a total of 409 nucleotides and formed a compact and stahle conformation in solution. Only 11 G residues in the native structure were able to react with NiCR. Most importantly, those results could be anticipated from the exposure of G N7 suggested by a model of the ribozyme structure [16]. Although NiCR is large enough to express a high degree of conformational specificity, it is still small enough to react with a G residue within the ribozyme active site. This residue had not previously been shown to be accessible by the much larger probe, RNase TI, which may he prohibited from entering the active site. The length of the ribozyme represented a maximum for direct analysis of a 5'-I3'P] labeled polynucleotide. An alternative method for characterizing reaction of long sequences relies on a primer extension assay. In these cases, sites of modification are detected by their ahility to terminate chain elongation as catalyzed by reverse transcriptase. This second procedure wasrecently used to reinvestigatethe reaction of the Tefrahymerra ribozyme with NiCR (Figure 4) [ 171, and it essentially confirmed the earlier results. Hydroxyl radical protection studies of the Cech laboratory 1181 have also been summarized in Figure 4 to illustrate the complementary nature of this modification technique. Hydroxyl radical reaction measures the solvent cxposure of the ribose backbone which is clearly independent of the exposure of G N7. Interpreting the results generated by a single modifying reagent can be extremely difficult in the absence of supporting data. Chemical modification reflects the most reactive and not necessarily the most abundant conformation. Both RNA targets described above folded into a unique conformation, and therefore thepattern of reactive sitescould be used to diagnose secondary and tertiary structure directly. For sequences that equilibrate between two or more structures, individual species are not easily distinguished in the ensemble of d a h . Our laboratories attemptedto characterize a model RNA pseudoknot with NiCR but were unable to prevent rapid interconversion between alternative conformations (Figure5). NiCR failed to detect the dominant pseudoknot even after reducing the reaction time to one min and temperature to 4 'C. Analysis of this system was originally attempted because the laboratory of Tinoco had previously characterized

Nickel Complexes in Modification of Nucleic Acids 163

?

207

169 U

d

Q

C A A

S'

226 G A G G G4

-

288

Figure 4 Reaction of the fefrdynenaintron (L-21Sca RNA) with NiCR and hydroxyl radical. Sequences that wereprotected from hydroxyl radical are indicated by shading [ 181. Residues that were modified by NiCR and detected by primer extension are indicated by arrows. A solid arrowhead represents reaction in the presence and absence of Mg"; an open circle, in the ahsence of Mg" only; and an open arrowhead, in the presence of Me" only.

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pseudoknot J

A CU G U c u C-G A-U G C G-C G U-A AUUUC-G 3

3' hairpin

5' hairpin

*

NiCR inducedcleavage not determined

Figwe 5 (A)Conlhrrnationalequilibrationand

(B) reaction of a model

pscudoknot. the structural equilibrium of this RNA quite successfully hy other chemical and physical methods [19, 201.

Nickel Complexes in

Modification of Nucleic Acids

209

Ill. LIGAND CONTROL OF NICKEL ACTIVITY A. Selective Oxidation of Guanine is Not a General Characteristic of Most Metal Complexes A variety of metal complexes related to NiCR were surveyed during the early phase of this project to highlight the major requirementsfor selective modification of G residues. Only positively charged, square planar Ni(I1) complexes represented by NiCR and Ni(cyclam) were active[ h ] . Although the nickel complex of glycylglycylhistidine is square planar and known to promote oxidative reactions [21,22], i t s net negative charge appeared to prevent interaction with the polyanionic DNA in this application. Octahedral species such as Ni(cyclen) and Ni(tren) provided no vacant coordination sites and were unreactive. Square planar copper derivatives, CuCR and Cu(cyclam), with a minimal tendency to gain two axial ligands were also unable to effect modification of DNA in the presence o f 21 peracid.

B. Ligand Donor Strength Regulates the Activity of the Macrocyclic Nickel Complexes The initial studies used to compare coordination geometry and reactivity couldnot easily distinguish hetween the relative contributions of ligand field strength and Ni(III/II)potential. This required synthesis and examination of complexes that expressed independent variation of electrochemical potential and field strength. Fifteen compounds derived fromNiCR, Ni(cylam) and Ni(Me,cyclam) were chosen, and their reactivity was tested with the hairpin-forming oligodeoxynucleotide D 1231. Selectivity for the unpaired guanines remained constant throughout the comparative investigation. Onlytheextent of modification varied. The order of increasing reactivity, 1 c 2 c 3 c 4 c 5, correlated closely to an increase in ligand donorstrength(Figure 6). In contrast, the trend in E,, values(ranging from 0.78 V for 4 to 1.25 V for 1) did not coincide with the nickel reactivity. This suggest.. that the high in-plane ligand field provided by a tetraazamacrocycle is repsonsihle for the appropriate ligand exchangerates necessary for reaction.

210 v)

b

v)

b

0

m

0

U)

cu

v)

v)

cu

(U

NOIlVaIXO VNa %

0

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Rokita et al.

Nickel Complexes

in Modification Nucleic of Acids

21 1

C. Nucleic Acid Recognition and Oxidation May Occur Through Direct Association of the Nickel Complex, Oxidant, and Guanine Thecorrelation between macrocyclic ligand and metal-promoted DNA oxidation suggested that the optimum geometry of the initial Ni(I1)complexis four-coordinate, square planar. Although this would preclude direct interaction between the Ni(I1) species and DNA, it would provide axial coordination sites that were thermodynamically and kinetically accessible once the metal center was oxidized from Ni(I1) to Ni(II1). Accordingly, a macrocyclic complex of Ni(Il1) was expected to bind DNA more strongly then its Ni(l1) derivative. This was confirmed using an electrochemical technique first developed by the laboratory of Bard [24]. For Ni(cyclam), the ratio of binding constants, KNi[111)/KNi(1,), was approximately 30:l (Figure 7) 1231. Preliminary analysis further suggested that this ratio increased to greaterthan 200: 1 when calf thymus DNA substituted for GMP. In contrast,AMP and CMP didnot greatly affecttheelectrochemistry of Ni(cyclam). Spectroscopic investigations arenow in progress to define the nickel-DNA interactions more fully.

l

with 50mM GMP

E(V0LT)

Figure 7 The effect of GMP on the electrochemistry of Ni(cyclam).

Rokita et al.

212

A variety of mechanisms can be proposed for selective oxidation of guanine through intermediate formation of Ni(lI1) complexes. Of the nucleohases, guanine is most susceptible to electron abstraction. Consequently, a pathway involving electron transfer to regenerate theNi(I1) complex and form the radical cation of guanine was consideredhut later found unsatisfactory.Theoxidantdependence of G modificationsupports an alternative process that requires a two electron oxidant and may involve direct oxygen atom transfer. One electron oxidants including K&O, and NazIrCI, were unable to replace KHSO, for modification of DNA. Furthermore, Ni(I1I)cyclam did not induce guanine oxidation alone or in the presence of these one electron oxidants [S]. Theactiveperacids, KHSO, and magnesium monoperoxyphthalate (MMPP),additionally provided theopportunity for coordination to the Ni(II1) intermediate. A simplc peracid such as peracetic acid functioned poorly in this application in accord with its lack of ready ligation to the nickel complex 161. A ternary complex of Ni(IIl), guanine N7 and oxidant might then assemble during reaction to achieve the target specificity and oxidant dependence described here (Figure S). The cis orientation of the oxidant and G is shown in order to indicate that direct oxygen transfer could be possible.

2+ A -

A

S.. ..

.iL'w..,&

&&ne i n d u c e d strand scission at guanine

n

S"0H

__*

residueswith tigWy accessible N7 positions

Figure 8 Proposed mechanism for selective modification of guanine by NiCR.

D. Oxidation Products are under Investigation A common product of deoxyguanosine oxidation, %oxodeoxyguanosine, might also be generated during the nickel-dependent modification of DNA. This guanine derivative could account for the alkaline lahility at sites of reaction [ E ] but , it.. formation has not yet been confirmed. Control studies revealed that 8-oxodeoxyguanosine was degraded by KHSO, (or "PP) alone more rapidly than dG was oxidized by the combined presence of NiCRand KHSO, (MMPP). If the S-oxo derivativedoes indeed form

213

Nickel Complexes in Modification of Nucleic Acids

during the DNA reaction, then it too would likely he oxidized further. The search for possible secondary derivatives is ongoing. The ultimate strand fragmentation induced by piperidine treatment yielded both 3' and 5' terminal phosphate groups(Figure 9). Theseproducts are consistent with a variety of processes that include hase oxidation and deglycosylation.

A

B

5' A

3' T A

T ( A ' T

T

C

A G"

A T

-

i

2'" -=

A T

C

T A

:\ C

T

A T 32PA

c

C

T A

0 G

yr*

*

/

-phosplloglycolate

@@ma

A C T A

32pT

Figure 9 Electrophoretic analysis identified the formation of phosphate termini in (A)5'-13'P1 and (B) 3'-[32P] labeled oligodeoxynucleotides.

E. Alternative Ligands Promote Distinct Reactions of DNA Macrocyclic ligands play a dominant role in the activation and control of nickel-dependent reactions. As descrihed ahove, CR, cyclam and many of their derivatives stahilizea characteristic coordination geometry and oxida-

TMAPES

Rokita et al.

214

tion chemistry of nickel that allows for selective oxidation of guanine. Active nickel complexes formed by an alternative ligand might then be expected to induce nucleic acid modification that is consistent with the distinct ligand chemistry. In addition to the macrocyles described above, salen derivatives also form square planar complexes with Ni(I1) and stimulate the metal redox chemistry. However, unlike CR, salen has the added ability toform ligand-derived radicals. Since Ni(salen)is not water soluble, our laboratories synthesized a cationic salen derivative (NiTMAPES) [26]. Consistent with the unique chemistry of salen, NiTMAPES induced arylation rather than oxidation of DNA in the presence of KHSO,. This modification was indicated by the formation of products with apparent molecular weights greater than the target oligodeoxynucleotides 1261. Only a small fraction of the adducts induced strand scission after piperidine treatment,and consequently, a primer extension assay was uscd to identify the complete set of modification sites. Reaction targets were once again limited to solvent accessible guanine residues of DNA and RNA [17,26].

H

NiCR

A final ligand system used tocontrolnucleic

Ni(N,HF)

acid reactivity in this report was originally developedby the laboratory of Kimura [27] for nickel activation of molecular oxygen. Under these conditions, electron transfer between Ni(1I) and oxygen was facilitated by metal coordinationto a pentadentate macrocycle. A related set of ligands have now been prepared and their complexes tested in a preliminary manner with plasmid DNA. The nickel complex Ni(N,HF) was found to promote spontaneous strand scission in the presence o f 0, without need of a subsequent alkaline treatment 1281. This process apieared independent of hydroxyl radical since standard trapping agents such as mannitol (50 mM) and ethanol (1.7 M) did not inhibit the scission reaction. The sequence and conformation de-

Nickel Complexes

in Modification Nucleic of Acids

215

pendence of modification is now under investigation and not expected to mimic the selectivity of NiCR. As currently designed, Ni(N,HF) should have little affinity for DNA. The overall charge of this complex is neutral, and the nickel would become coordinatively saturated when bound with oxygen. Logical derivatives of this and other ligands are now being synthesized for selective recognition and reaction in vitrQ and in vivo.

ACKNOWLEDGMENTS We greatly appreciate the dedication, enthusiasm and support of our coworkers and collahcmtors. In addition, we thank Professor Woodson for the ferral1ytnerta intron RNAand ProfessorTinoco for thepseudoknot RNA. Research support was provided by the American Cancer Society, National Institutes of Health, National Science Foundation and the Stony Brook Center for Biotechnology sponsoredby the New York State Science and Technology Foundation.

REFERENCES Bossu, E. B. Paniago, D. W. Margerum, S. T. Kirksey, and J. L. Kurlz, Inorg. Cltern. 17: 1034-1042 (1 978). 2. J. F. Kinneary, J. S. Alhert, and C. J. Burrows, J. Am. Cltern. Soc. 110: 6124-6129 (1 988). 3. H.Yoon, T. R. Wagler, K. J. O’Connor, and C. J. Burrows, J. Am. Cltern. SOC.112: 4569-4570 (1990). 4. S. E. Rokita and L. Romero-Fredes, Nucleic Acids Res. 20: 3069-3072 ( 1 992). 5. U. Hiinsler, and S. E. Rokita, J. Am. Cltern. Soc. 11.5: 85544557 (1993). 6. X. Chen, S. E. Rokita, and C. J. Burrows, J . Am. Clrem. Soc. 113: 58x45886 (1 991). 7. X. Chen,C. J. Burrows, and S. E. Rokita, J . Am. Clrem. Soc. 114: 322325 (l 992). 8. J. G. Muller, X. Chen, A. C. Dadiz, S. E. Rokita, and C. J. Burrows, Pure R.Appl. Cltern. 6.5: 545-550 (1993). 9. P. E. Nielsen, J. Mol. Recop?. 3:1-25 (1990). 10. J. A. Taboury, P. Boutayre, J. Liquier, and E. Taillaadier, Nucleic Acids Res. 12: 4247-4257 ( 1 984). 11. S. H.Kim, F. L. Suddath, G.J. Quigley, A. McPherson, J. L. Sussman, A. H. J. Wang, N. C. Seenlatl, and A. Rich, Science 18s: 435-440 ( 1 974). 12. J. D.Rohertus, J. E. Ladner, J. T. Finch, D. Rhodes, R. S. Brown, B. F. 1. F.P.

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C. Clark, and A. Klug, Nature 2.50: 546-551 (1974). 13. R. hvery, and A. Pullman, Bioplrysicnl Clrem. 19: 171-181 (1984). 14. X. Chen, S. A. WOodSo11,C. J. Burrows, and S. E. Rokita, Biochemistry 32: 76 10-76 16 ( 1993). 15. S. R. Holbrook, J. L. Susslllan, R. W. Warrant, and S. H. Kim, J . Mol. Biol. 123: 63 1-660 (1 978). 16. F. Michel, and E. Westhof, J.Mol. Biol. 216: 585-610 (1990). 17. S . A. Woodson, J. G. Muller,C. J. Burrows, and S. E. Rokita, Nr.lcleic Acids Res. 21: 5524-5525 (1993). 18. J. A. htha111, a11d T. R. Cech S C ~ ~ I 24.5: ~ C L 276-282 . (1989). 19. J. D.Puglisi, J. R. Wyatt, and 1. Tinoco, J. Mol. Biol. 214: 437-453 (1990). 20. J. R. Wyatt, J. D. Puglisi, and 1. Tinoco, J. Mol. Biol. 214: 455-470 (1990). 21. T. Sakurai, and A. Nakahara, Inorg. Chim. Actn 34: L243-244 (1979). 22. D.P.Mack, and P. B. Dervan, Bioclremisrry 31: 9399-9405 (1992). 23. J. G. Muller, X. Chcn, A. C. Dadiz, S. E. Rokita, and C. J. Burrows, J. Am. Clrem. Soc. I I4: 6407-641 1 (1992). 24. M. T. Cqrter, M. Rodriguez, and A.J. Bard,J. Am. Clrem. Soc. I l l : 8901891 1 ( 1 989). 25. M. Kouchakdjiaa, V. Bodepudi, S. Shibutani, M. Eisenberg, F. Johnson, R. P. Grollman, and D.G. Patel, Biochemistry 30: 1403-1412 (1991). 26. J. G. Muller, S. J . Paikoff, S. E. Rokita, and C. J. Burrows, J . Inorg. Bioclrem. 54: 199-206 ( 1994). 27. E. Kimura, R. Machida, and M. Kodama, J . Am. Clrem. Soc. 106: 54975498 (1984). 28. C. C. Cheng, S. E. Rokita, and C. J. Burrows,Angew. Clrem. Int. Ed. Ertgl. 32: 277-278 ( I 993).

14 NewMethodsforDeterminingthe Structure of DNA and DNA-Protein Complexes Based on the Chemistry of Iron(1I) EDTA Thomas D. Tullius Department of Chemistry, The Johns Hopkins University, 3400 North CharlesStreet,Baltimore, MD 21218

I.

DNA-PROTEIN COMPLEXES

DNA must be associated with protein in order to function in a biological system. The study of DNA structure inits own right has provide many surprises and new insightsrecently, with the structural characterization of left-handed Z-DNA [l],DNA triplexes [2], four-stranded (telomeric) DNA [3, 41, and the Holliday junction recombination intermediate [5]. But while DNA structural polymorphismis now a well-precedented pheof these unusual structures, let alone nomenon, the biological exploitation simple B-form DNA, invariably requires the agency of DNA-binding proteins. Even RNA, which has been conclusively demonstrated to possess its own [6],most often performs its the capacity for catalytic activity on biological functions in association with protein. So, to understand how nucleic acids participatein the life of the cell it is necessary tostudy their interactions with proteins. By the late 1970's only a single eukaryotic transcription factor had been isolated (the zinc finger protein Transcription Factor mA, about 217

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Tullius

which morewill be said later this in chapter), and only a small handful of prokaryotic DNA binding proteins had been crystallized.But in parallel with the characterization during the last decade of the unusual DNA structures mentionedabove, therehas been aflood of DNA binding proteins discovered. Beyond the cloning and isolation of both eukaryoticand prokaryotic DNA binding proteins, several have been co-crystallized with their DNA binding sites,and so high-resolution three-dimensional [A.N M R has also becomean important structures are available for many technique for structure determination [S] of both DNA binding proteins and in some cases complexesof protein with DNA 191. With this large amount of new structuralinformation has come the recognition of several distinct families of DNA-binding proteins, each using a particular protein structural motif (the zinc finger, the helix-turn-helix, and so on) to bind to DNA [lo]. While it might be thought thatwith the recent successof the crystalstructure deterlographers and N M R spectroscopists, other methods for mination have become less important, this is not the case. Perhaps the most important reason this for is that complexesof protein and DNA that are often highlycomare competentto functional in a biological system plicated, with several proteins bound to a long segment'of DNA. Molecular biologists have been able to reconstitute functional transcription, replication, and recombination systems using purified components, but if not impossible these very large macromolecular assemblies are difficult subjects of study for crystallographyor NMR.

A. Chemical Probe Methods This need to study large assembliesof DNA and protein has resulted in the development of a strategy called "chemical probing" [U, 12,131. A key advantageof this experimental approachis that very small quantities of protein and DNA are required, so that the functional assemblages produced by in vitro reconstitution may easily be studied. Chemical probe DNA sequencexperiments makeuse of the high resolution afforded by ing gel electrophoresisto monitor the reactivityof each DNA nucleotide toward a chemical reagent. A variety of small-molecule DNA cleavage reagents is now available, each having particular recognition properties for the structural features of a DNA-protein complex 1121. Some, like potassiumpermanganate,reactmorereadilywithsingle-stranded regions of DNA. Others, like dimethyl sulfate, alkylate particular positions in the DNA bases, and can be used to demonstrate whether these positions are exposed or hidden in a DNA-protein complex. While the atomic resolution of crystallography or NMR is obviously not available

New Methods of Determining Structure

of DNA

219

from a “map” of the chemical reactivity of the DNA in a DNA-protein complex, the ability to study complicated systems as easily as simple this ones, andthe relative easeof chemical probe experiments, have made experimental strategy widely used.

B. Hydroxyl Radical Footprinting In my laboratory we have been engaged in developing a particular chaical reagent, the hydroxyl radical, for use in structure determination of DNA-proteincomplexesinsolution. We producethehighlyreactive hydroxyl radical by a convenient inorganic reaction, the Fenton reaction [14]:

[Fe(EDTA)]’-

+ H202 + [Fe(EDTA)]l- + OH- + .OH

(1)

We use theEDTA complex ofi r o n 0 as a sourceof electrons to reduce hydrogen peroxide, because the negative charge of this metal complex reducesitsaffinitytowardthepolyanionic DNAmolecule[15].The chemical probe in our system, then, is the neutral hydroxyl radical and not the metal complex. This is unlike the many other DNA chemical probe reagents that are based on inorganic complexes [l61 in which the properties of the metal complex itself (its shape or reactivity, for example) are exploited. In our method we make useof the ability of the hydroxyl radical to cleave the DNA backbone by initial hydrogen atom abstraction from a deoxyribose [lq.The result of this homolytic reaction is a deoxyribosebased radical, which by subsequent chemistry decomposes, leaving a single-nucleoside ”gap”in the DNA strand. Because the hydroxyl radicalso reactive it is quite non-selective in its reactions with the DNA backbone, so that each nucleotide in a normalDNA molecule is cleaved to nearly the same extent. However, if a protein is bound to a specific site in the DNA the deoxyribosesthat are covered by the protein become inaccessible to the hydroxyl radical and no cleavage is seen at the protein binding site [18]. Becausethe hydroxyl radicalis so small, the “footprints”it produces of bound protein are the highest-resolution available. We have used this chemistry to make images of a wide variety of DNA-protein complexes. Hydroxyl radical footprinting is now a widely-used method. Since there is an extensive literature on this technique, covering both applications and experimental methodology[19], I will not discuss hydroxyl radical footprinting further. A newer method that makes use of hydroxyl is aimed at radical chemistry, the Missing Nucleoside Experiment, which

220

Tullius

Obtaininginformationontheenergetically-importantcontactsmade between a protein and its DNA binding site,is the subject ofthe next section.

C. The Missing Nucleoside Experiment The footprint shows where a protein physically covers DNAa molecule. as a direct measure of the The hydroxyl radical footprint can be regarded solvent accessible surface area of the DNA in a protein-DNA complex [19].While the footprint gives a picture of the overall shapeof the complex, the question of how the protein recognizes this particular DNA sequence among all others is not addressed. This question gets at the - what are the problem of molecular recognitionin a DNA-protein complex energetically-importantcontactsmadebyaproteinwith DNA? To answer this question we developed a new experimental strategy, the ”Missing Nucleoside Experiment”[20]. The method is based on the experimental approach called ”chemical interference” [U].It has a more direct antecedent in the missing contact method developed by Brunelle and Schleif[21].In previously-developed interference methods [22]the effect on protein bindingof alkylation of a is monitored. If, for example,the N-7 posinucleotide base or phosphate tion of a particular guanineis methylated, and the proteinis then unable to bind to that modified DNA molecule, it is concluded that the guanine makes an important contact with the protein, likely a hydrogen bond with theN-7 nitrogen. Several studies have found that phosphates that are implicated in proare invariably found tein binding by ethylation interference experiments [B].Recently Verdine to be sites of protein contact in co-crystal structures and coworkers [24]have developed a seriesof modified nucleotides that can be incorporated by automated synthesis into DNA oligomers. This collection of syntheticmodifiednucleotidessigrufrcantlyextendsthe kinds of protein-DNA contacts that may be studied by interference methods. One difficulty with previously developed interference methods is that different chemistry must be used for each of the fourDNA basesin order to detennine which nucleotidesin a DNA molecule make contactwith a in one protein. We sought to develop a method that would identify, experiment, all of the nucleotidesin a DNA molecule that make energetically-importantcontactswithaDNA-bindingprotein.TheMissing Nucleoside Experiment relies on the ability of the hydroxyl radical to generate a family of DNA molecules eachhaving a single-nucleoside gap in one of its strands. Itis the absence of a chemical p u p in theDNA (the


of DNA

221

missing nucleoside)that we monitor for its effect on protein binding. In contrast, in conventional interference experiments it is the addition of a chemical group by alkylation, and thus the added steric bulk or elimination of a hydrogen bond donor or acceptor, that gives information on con tacts of protein withDNA. A diagram of the Missing Nucleoside Experiment is shown in Figure 1. Because the hydroxyl radical is so nonselective in its reactivity with duplex DNA, a uniform population of gapped DNA molecules can be produced by useof the Fenton reaction on a sampleof DNA containing Protein is then added the binding site for the protein of interest (Step0). If a nucleoside thatis importo the mixtureof gapped DNA’s (Step 0). is missing in a particularDNA, the proteinwill be unable tant to binding to bind (left).If, however, the missing nucleosidein a DNA molecule is outside the protein binding site (or, in fact, it is in the binding site but is still able to bind to the does not make an important contact), the protein gapped DNA (right). DNA’s The gapped DNA molecules boundto protein, and the gapped are unable to bind, encode the information concerning which nucleosides necessary for formation of the protein-DNA complex. To recover this information the mixtureof protein-bound and unbound gappedDNA is ). separated by electrophoresis on a native polyacrylamide gel (Step 0 The bands containing boundand unbound DNA are excised from the gel and the DNA is denatured and electrophoresed in separate lanes of a Missing nucleosidesthat interfered with DNA sequencing gel (Step protein binding show up as prominent bands in the lane containing unbound DNA (right). A complementarypattern,inwhichmissing is seen in the lane bands correspond to the important nucleoside contacts, containing boundDNA (left). We first applied the Missing Nucleoside Experiment to the complex of ORl operator site [20].This the bacteriophage lambda repressor with the protein had been studied extensively in many other laboratories. In particular, the X-rayco-crystal structure had been determined [ E ] ,and exhaustivemutagenesisexperiments had establishedtheenergetic importance for protein binding of each base pair in the operator sequence

m).

[261.

The results of the Missing Nucleoside Experiment on lambda repressor are shownin Figue 2 along with a diagramof the contacts made by repressor with DNA from the co-crystal structure [25]. There is a great deal of information in the Missing Nucleoside pattern, which for lack of space cannot be discussed in detail here. To summarize the results, we found that essentially every signal the in Missing Nucleoside Experiment could be explained by a hydrogen bonding or hydrophobic contact that

222

Tullius

* -

l+

4

LIlr Unbound

G9 Sequencing Gel

Native Gel

-

I

Important Contacts

Figure 1 Diagram of the Missing Nucleoside Experiment. was found inthe co-crystal structure. Conversely, nucleosides for which Missing Nucleoside signals were not observed were not assigned in the X-ray structure as contacts. A very interesting feature of the correspondence betweenthe crystal structureand the Missing Nucleoside Experiment is the stmad-specific nature of the contacts between protein and that contacts DNA. That is, for base pairs 1-3the crystal structure shows are made by proteinside chains with the bases on thebottom strand (as drmm in Figure2). For base pairs 4-8 the contacts switch to top thestrand. 'I"& switching of contacts from bottom to top strandis also clearly seen in the Missing Nucleoside pattern. Furthermore, our resultswereinexcellentaccordwithpublished mutagenesis experiments [26]. The correspondence with mutagenesis is

223

New Methods of Determining Structure of DNA

Missing Nucleoside

11111111

TTTACCTCTGGCGGTGAZA IIII I I I

1' 2' 3 ' 4 ' 5' 6' l' 8'

8 l 6 5 4 3 2 l

0,l

X-Ray Crystallography GlyM

---

PhWh* " . . ) Hydrophobic

ATACCACTGGCGGTGATAT 1'2'3' 4 * 5 ' 6 ' 7 ' d

g

8 l 6 5 4 3 1 l

fI

TATGGTGACCGCCACTATA lle54

"\\

011144)

Gin33

OLl

Figure 2 Comparison of the MissingNucleoside pattern and the contacts derived by X-ray crystallography for the lambda repressorDNA complex. Missing nucleoside data are from Hayes & Tullius [20]. The length of a vertical bar is proportional to the effectof that missing nucleoside on repressor affinity. Horizontal bars mark nucleosides that can be lost without effect on repressor binding. Crystallographicallyderived contacts are from the report of the CO-crystal structure [25]. For clarity, crystallographic contacts for only one half-siteare shown.

224

Tullius

worth considering in more detail. Mutagenesis by necessity involves changing both halves of a base pair simultaneously. One particularly interesting aspectof the Missing Nucleoside Experimentis that, in some cases, one half of a base pair gives a strong signal, suggesting thatit is essential to binding, while the other base in the base pair can be remove without effect on complex formation. An example of this phenomenon can be seenat base pair 2in Figure 2, in which the adenine gives a strong signal, but the base-paired thymidine does not. The Missing Nucleoside Experiment, therefore, in contrast to mutagenesis, permitseach base of a base pair to be studied individuallyitsfor contacts with protein.

D. Structure of the TFIIIA-DNA Complex Having established the experimental validity of the Missing Nucleoside Experiment we went on touse it to determine the structureof the comIIIA with the 55RNA plex of the zinc finger protein Transcription Factor gene, a DNA-protein complex for which no X-ray structure has yet been solved. Transcription Factor LUA is the prototypical zinc finger protein [27l. TFIIIA binds to a 50-bp long segment of DNA internal tothe 5s ribosomal RNAgeneof Xenopus, and regulates transcription of this gene. Since the fist suggestion, based on the internal sequence homology of TFIIIA, that small domains of the protein might fold around a zinc ion and form nine ”fingers”, the zinc finger protein structural motif has been found tooccur hundreds of times in the genomesof higher organisms. Berg’s prediction of the three-dimensional structureof the zinc finger [28] was subsequently confirmed by solutionof the structures of single N M R methods [29,30]. Since natuzinc fingers using multi-dimensional ral zinc finger proteins invariably contain multiple tandem repeatstheof zinc finger sequence motif, it was of interest to determine how arrays of zinc fingers bind DNA. to Pavletich& Pabo [31] solvedthe first co-crystal structure of a zinc finger protein, Zif268, which contains three zinc finrn of the DNA gers. The protein was found to wrap around almosttuone helix, following the major groove. Each finger interacts with three base pairs of DNA and the three fingers are related by almost exact threefold screw symmetry. How a protein like TFIIIA, with its nine zinc fingers, might interact with DNA was still an open question. Would it resemble Zif268 taken three times, wrapping for nearlythree complete turns around the major groove of its DNA binding site?Or is there another modeof zinc fingeran X-ray structure? Our earlier DNA interactionnotyetfoundin and a series of delehydroxyl radical footprinting experiments TFIIIA on Zif3 model was tion mutantsof the protein[32]suggested that the simple


225

unlikely to be correct, because the footprints were more complicated than that simple model might predict. We recently proposed a detailed three-dimensional model for TFIthe ITA-DNA complex based on the results of Missing Nucleoside analysis [33]. This work represents arguably the most complicated protein-DNA complex for which only solution chemical probe methods were usedin proposing the structure.It will be interesting to see how close we have of the TFIlIA-DNA comcome in our prediction when a crystal structure plex is solved. .The results of the Missing Nucleoside experiment on the TFIJ”5S DNA complex are shown in Figure 3. It is immediately apparent that there are three separate regions of the DNA that make energeticallyimportantcontactswiththeprotein.Thesethreeregionscorrespond almost exactly tothree “boxes” determined by mutagenesis experiments to be necessary for regulation of transcription by TFIIIA. Each of these regions of interaction covers slightly more than one helical turn of DNA. One might imagine a model for the complex that involves three of subset of DNA. the nine zinc fingers of TFIIIA binding to these segments

Figure 3Missing Nucleosidepattern for the complex of TFIIIA with the Internal ControlRegion of the 5s ribosomal RNA gene of Xenopus.The length of a vertical baris made to be proportional tothe effect of that missing nucleoside onTFIIIA affinity. Taken from Hayes & Tullius [33].

226

Tullius

Our final, detailed, model for the structure was based on a consideration of the symmetryof the Missing Nucleoside pattern in Figure 3. The two envelopes of contacts at the two ends of the DNA binding site have pair DNA - that is, the peaks in the near mirror symmetry across a base of set of contacts on the two strands occu at the same base pair.We conthis sort of symmecluded that a protein-DNA complex that would give try in its contacts would resemble theZif complex, which consists of an of DNA and object (the three fingers of Zif) that follows the major groove acts almost like tahird DNA strand. The envelope of contacts in the tenter of the site have a different strand symmetry. The peaks in the contact pattern are offset from one strand to the otherby 5 base pairs in the 5' direction. We observed an identical offset in the lambda repressorMissing Nucleoside pattern (see Figure 2). Lambda repressor binds to DNA through an alpha helix that is inserted in the major groove. Lambda on one strandof the DNA at the repressor makes contacts with the bases end of the site, and then switches strands to make contacts farther within the site.

IE

C 87

I

77

I

A m

I

57

I

COOH

Figure 4 Model of the TFIIIA-DNA complex, based on the Missing

Nucleoside Experiment. Fingers 1-3and 7-9 were modeled based on the [31]. Thesefingers are foreshortenedto indicate that theygo into and out of the planeof the paper.A, IE, C:the approximate positionsof the three "boxes" defined by mutagenesis as importantfortranscriptionalactivation. C, coding;NC,noncoding strands. Taken from Hayes& T a u s [33].

Zif268 co-crystalstructure

Our model for the TFIIIA-DNA complexis shown in Figure 4. Fingers 1-3and 7-9 were placed on the DNA in the same relative orientationas the three fingers of Zif268, and are shownas wrapping aroundthe major groove. Fingers 4-6 are depicted as making a much different interaction with the DNA, extending head-to-tail along one side of the DNA. This set

of three fingers resembles lambda repressor, which also binds to one side


of DNA

227

of DNA parallel to the helix axis. Finger 5 is the only oneof the three that contacts the major groove. Fingers 4 and 6 cross the minor groove and serve mainly tolink finger 5 with thetwo sets of three fingersat the ends 4 6 make withDNA is of the complex. Whilethe interaction that fingers unprecedentedforzincfingers,footprintingand Missing Nucleoside experiments are wholly consistent with the model. In addition, after we had submitted o m paper two other groups [34,35] used different experiTFImental results to propose similar (but less detailed) models for the IIA-DNA complex, givingus confidence that our analysis is correct.

11. "UNUSUAL" DNA STRUCTURES

In the last decade much evidence has accumulated that the structure of DNA is important to its ability to function in a biological system. Perhap the most ubiquitous example of an "unusual" DNA structure that is essential to biologyis curved (or bent)DNA. It has of course long been known that DNA must bend to associate with the histone octamer to form the nucleosome, the first level of compaction of DNA in the eukaryof DNA bending otic nucleus. Itis only recently that the wide occurrence associated with protein binding has been appreciated[36]. Indeed, it is now standard practice to measure the degree of bending of DNA that is caused by any new DNA-binding protein that is discovered. The assay used for this measurement, the additional retardation in gel mobility caused by the protein being bound at the center compared to being bound at the ends of the DNA fragment [37l, gives information on the overall shape of the DNA molecule when it is bound by protein. Less information is available from this experiment on the local structure of DNA that is associated with bending, curving, or kinking, whether protein-induced or the result of the nucleotide sequence of the DNA itself. Another unusual DNA structure that is inarguably associated with biological functionis the fourstranded Holliday junction, a key intermehasbeenmade diatein DNA recombination.Whilemuchprogress recently in defining the shapeof the Holliday junction and the topology of its strands [5], no atomic resolution structural data are yet available for a DNA junction. Several other non-duplex DNA structures have been the subject of study, but are less clearly implicated in biological processes. The lefthanded &form of DNA, while known for more than a decade, has yet to DNA, and the related be proven essential to a biological process. Triplex H-form structure, is of wide experimental interest [38], but aside from possible use as a drug for inactivatingDNA inside a cell, the biological

228

Tullius

function of triple-stranded DNA remains enigmatic. Similarly although the q u a r t e t structures adoptedby the guanine-richends of the chromo[3,4], their relevance some (the telomere) have been studied extensively is not yet completely understood. to chromosome structure and dynamics What is beginning to emerge from the workof many laboratories is that nature makes use of the structural repertoireof DNA, but in ways that have yet to be defined in many systems and for many of the possible "unusual" structures of DNA. Indeed, perhaps the most unusual DNA structure is the one we besthow, the uniform, symmetrical, straightR form duplexDNA structure that came from the original X-ray fiber diffraction experiments on DNA a half-century ago. A challenge beforeus is to detect the occurrence of unusual DNA structures during biological function, to define in detail the nature of these structures, and to manipulate these structures to affect the biological process.

A. Chemical and Enzymatic Probe Methods forDNA Structure Determinationin Solution To address these structural questions, chemical and enzymatic probe experiments have been found to be useful to work out the structuresof nucleic acids in solution [13]. The major advantage of these methods is that they may be applied to DNA molecules that are much larger than could be studied by crystallography or M.As a consequence, structural features of DNA as it is involved in a biological process are now accessible to determinationand study. Some of the reagents that have been developed recently as probes of [39], osmium tetroxide DNA structure include potassium permanganate [40], theuranylion [41], bis(o-phenanthrolinecopperfl)[Q], 5-phenylphenanthrolinecopper(1) 143, 441, and derivatives of tris(o-phenan[16]. throline) complexesof rhodium, ruthenium and cobalt h general, each of these reagents is sensitive to a different aspectof DNA structure. For example, potassium permanganate or osmium tetroxide are thought toreact preferentially with unstacked DNA bases, which might occur at sites ofDNA kinking [39]. The phenanthroline-based reagents developed by Barton are designed to interact shape-selectively with DNA, and thus to detect the presence of a stretch of the A-form or the left-handed Z-form in a DNA molecule [16]. 5-phenylphenanthrolinecopper(1) seems to react withDNA that is "distorted" as the result of protein binding[M]. These reagentsare most often used by allowing the DNA molecule of interest to react with the probe compound, and then observing of the site


229

reactivity (andthus the segmentof DNA with theunusual structure) by inducing DNA strand cleavage at the placeof reaction. The key to analysis of the experiment is the ability to separate DNA molecules differing in length by a single nucleotide,via electrophoresis of the reaction products on a denaturing polyacrylamide gel. By this means information on the occurrence of the unusual structure can be obtained rapidly for a very small quantity (a few femtomoles) of DNA, at single-nucleotide resolution. Furthermore, quantitative analysisof the gel allows accurate determination of the fractionof DNA moleculeshaving the structure. My laboratory has been engaged in developing the hydroxyl radical as a chemical probe of DNA structure. We have adopted an experimental strategy that is in some ways opposite to those discussed above. Since we generate the hydroxyl radical by the Fenton reaction of a negativelycharged transition metal complex, [Fe(EDTA)]*-, with hydrogen peroxide (see Equationl),the metal complex has a negative charge, andit thereif any affinity for the polyanionic DNA molecule [15]. fore will have little Rather than the metal complex, then, the probe of structure inour system is the hydroxyl radical, perhaps the smallest imaginable chemical probe. The hydroxyl radical reacts to nearly the same extent with each nucleotide in a normalB-form DNA duplex.If the DNA is in an unusualconformation,however,thepatternisdifferent. We usethesecleavage of unusual DNA molecules. patterns to make “images”

B. The Structure of Tn&, Tracts Despite much effortin many laboratories, the detailed naturein solution of the unusual structure (called B-DNA)that is adopted by shortruns of this structure leads toDNA bending, adenines (adenine tracts), and how have yet to be definitively determined. While isolated, phased A-tracts to [37,45,46], the dependence are wellknown to impart curvature DNA is also of importance. of the structureof an A-tract onits sequence context This is not a trivial point, since an important benefit of understanding of natural DNA DNA bending wouldbe the ability to predict the shapes sequences (suchas promoters or originsof replication) for which curvature is suspected to be necessary for optimal biological function.If the sequence context of an A-tract affectsits structure, and therefore the degree of curvature, then simply considering the occurrence and phase of only of a naturalDNA. the A-tractsis not enough to predict the shape An experimentbyHagerman [47]graphicallydemonstrated this point. He discovered that the sequence d(GAAAA’I??TC), ran anomalouslyslowlyon an electrophoresisgel,thehallmark of bent DNA. Remarkably, the simple sequence isomer d(GTTTTAAAAC), ran with

Tullius

normal mobility., leading to theconclusionthat this sequencewas straight. These results were explained[48] by assuming that in the bent A4T4sequence small local bends added in phase, giving theDNA molecule an overall curvature, while these same bends out were of phase with the helical repeat in theT4A4 sequence, making this molecule appear to be straight. The key assumption in this analysis was thatthe structure of the A-tracts in both sequence isomers was the same. We decided to test this assumption by performing hydroxyl radical cleavage experiments on these two sequences [49]. We previously had shown [50] that the adenine tracts in a highly natural bent DNA sequence fromatrypanosomeparasitehadavery unusual and characteristic of cleavhydroxyl radical cleavage pattern. We had found that the extent to 3’ along a age by the hydroxyl radical decreased monotonically5’from Ashort A-tract, and then increased in the mixed-sequence DNA between tracts. The thymine-rich strand showed a similar cleavage pattern, but shifted 2 or 3 nucleotides in the 3’ direction. Our conclusion from these observations was that the structure of the A-tract is not uniform, but B form at the 5’ end of an A-tract into changes gradually from the normal at the 3’ end of a short the unusual B’ form, whichis most fully expressed run of adenines. Becauseof the shift in the phase of the cleavage pattern between the two strands, we also concluded thatkey a characteristicof the unusual structure of an adenine tractis a narrow minor groove,that becomes progressively more narrow from 5’3’toalong the A-tract.This prediction was borneout in subsequentX-ray structures of A-tract-conin whichbothanarrowminor tainingoligonucleotides [51,52,531, groove, and its progressive narrowing from 5’ to 3,were seen. Subsequently, other experiments [X, 551 also have noted the same 5’ to 3’ “polarity” of the structure of adenine tracts that we first observed by hydroxyl radical cleavage. Our experiments with the “Hagerman sequences” [49] showed that 4T4, the bent sequence,had an unusual hydroxyl radical cleavage pattern that was very similar to that of the bent trypanosome sequence, while the straight sequence T 4 4 had a normal cleavage pattern. We concluded that the reason why theT4A4 sequence wasnot bent was thatits structure was close to normal B-form, and not that its small local bends were out of phase. We thus provided evidence for a different explanation funof Hagerman’s data, namely that the two sequence isomers adopted damentally different structures. We ascribed this difference in structure to the presence of a 5’-T-A-3’ step in the straight sequence. Others had asserted that T-A steps would interfere with the abilityof base pairs to propellertwist [56]. Aremarkablecharacteristic of theA-tractsthat emerged from the X-ray structures [51,52] was the extreme of prodegree


23 1

peller twist in the A-T base pairs of the A-tract. It had previously been noted that high propeller twist was associated with narrowing of the minor groove [57. We reasoned that the presence of a T-A step might cause the minor groove to remain wide because of its damping effect on propeller twist, thus leading to the inability of the T4A4 sequence to adopt the unusual structure of A-tracts in bent DNA. Haran and Crothers [58] later showed by gel mobility experiments if the valueof that some sequences of the form TnAn were indeed curved, n were large enough. The sequence ( T ~ A T N for ~ ) ~example, , wasfound to be quite curved. Haranandcrothers explained their data in termsof the model that all A-tracts have the same structure, and that overall curvature depends on the phasing of small local bends.We, however, decided to investigate the hydroxyl radical cleavage patterns of the bent and straight TnAn sequences studied by Haran and Crothers, to detennine whether the local structu~s of all indeed were the same [59]. The cleavage patterns of the T7A7N7 sequence, and for reference the highly bent A& sequence, are shown in Figure 5. We found that the hydroxyl radical cleavage patternof the bent T7A7N7 sequence has the characteristic featuresof the cleavage pattern of other bentDNA's, that is, cleavage decreases monotonically along A-tracts, and increases in the mixed sequence DNA separating the A-tracts. However, we also found that the T-A step in theT7A7N7 sequence, and one or two base pairsto either side,reach a local maximum in cleavage.This observation is consistent with our previous reasoningT-A thatsteps interfere with propeller twisting, and therefore sequences with T-A steps lack the ability to form the narrow minor groove thatis associated with curvature. The observation that the cleavage pattern of T7A7N7is not uniform (in contrast to the T4A4 sequence [49]) shows that the T-A step influences the structure only locally (i.e., over4-6base pairs),and not globally. We used theseand other data to propose a new model for the structure of TnA,, sequences. The model asserts that in aTnA,, sequence, the T-A step and 2 to 3 base pairs to either side are in the normal B-form conformation. However, if the T tract or the A tract are longer than3 or 4 base from the T-A step. pairs, theunusual B structure still can form away base pairsof mixed We tested this model by substituting either 4 6or sequence DNA for the TzAz or T3A3 core sequence of T7A7N7 Wefound that these new DNA molecules had hydroxyl radical cleavage patterns very similar to the patternof T7A7N7, showing that the mixed sequence DNA and the T2A2 or T3A3 sequences adopt the same (presumably R form) structure. Moreover, the new sequences had gel mobilities nearly identical to thatof the ( T T A ~ Nsequence, ~)~ demonstrating that substitution of mixed sequence DNA forA/T DNA had no effect on the curva-

232

Tullius '

"C

5 Hydroxyl radical cleavage patterns of cloned oligonucleotidescontaining four repeats of A5N5 (left), and two repeats of T7A7N7(right). The A$J5 sequence is known to impart substantial curvatureto DNA. The smoothly varying hydroxyl radical cleavage pattern of this synthetic sequence is nearly identical to the pattern of anatural,highlycurvedsequencefromtrypanosome parasites. T7A7N7 has a similar modulated cleavage pattern, and it also is curved[58]. The T-A steps in the T7A7N7 pattern thatare discussed in the text are indicated by arrows.

Figure

ture of T7A7Np

We thus showed [59] that the hydroxyl radical cleavage pattern provides sufficiently detailed structural information DNA on that structural principles can be inferred from the data, andDNA that molecules can be structure but a differentsequence compared to designed that have the same another DNA.

C. Concluding remarks I have describedtwo examples of how metal-based chemistry can be used as a high-resolution structural tool study to DNA and DNA-protein complexes. A key characteristicof the chemistry used,the Fenton reactionof iron@) EDTA with hydrogen peroxide to generate the hydroxyl is radical, the simple and inexpensive natureof the reagents required.This feature its wide use by molecular biologists as of the method has contributed to are interested in nucleic acid structure. well as chemists who The principle behind the experiment, thatan exceedingly non-specific reaction can provide detailed structural information, is different from the This princiidea behind most other metal-based chemical probe methods. is now subple might profitably be used in other systems; indeed, there stantial experimental activity in the use of iron@) EDTA-based hydroxyl radical chemistry for the structural analysis of proteins. A further goal of our work is to use hydroxyl radical chemistry to study the structure of DNA and DNA-protein complexes inside living


of DNA

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cells. My group has already made substantial recent progress toward this end.

REFERENCES 1. A. H. -J. Wang, G. J. Quigley, F. J. Kolpak, J. L. Crawford, J. H. van Boom, G. van der Marel, and A. Rich, Nature 282 680-686 (1979). 2. H. E.Moser and P.B. Dervan, Science 238 645-650 (1987). 3. W. I. Sundquist andA. Klug, Nature 342: 825-829 (1989). 4. J. R. Williamson, M. K. Raghuraman, and T. R. Cech, Cell 59: 871-880 (1989).

5. 6. 7. 8.

D. M. J. Lilley, Current Opinion Cell Biol.2: 464-467 (1990). T. R Cech, Ann. Rev. Biochem 59 543-568 (1990). C. 0.Pabo and R. M. Sauer, Ann. Rev. Biochem 62: 1053-1095 (1992). G. M. Clore andA. Gronenborn, Annu. Rev. Biophys. Biophys. Chem 20

29-63 (1991). 9. G. Otting, Y. Q. Qian, M.Billeter,M.Muller,M. Molter, W. J. Gehring, andK. Wuthrich, EMBO I. 9: 3085-3092 (1990). 10. S. C. Harrison, Nature 353: 715-719 (1991). 11. T. D. Tullius, Ann. Rev. Biophys. Biophys. Chem.2 8 213-237 (1989). 12. P.E. Nielsen, J. Mol. Recognition 3: 1-25 (1990). 13. T. D. Tullius, Current Opinion Struct.Biol. 2: 428-434 (1991). 14. C. Walling, Acc. C h . Res. 8 125-131 (1975).

15. T.G. Wensel, C. F. Meares, V. Vlachy, and J. B. Matthew, P m . Natl.

Acad Sci. (USA) 83: 3267-3271 (1986). 16. A. M. Pyle and J. K.Barton, Progress Inorg. C h . 38 477-516 (1990). 17. R.P.Hertzberg andP. B. Dervan, Biochemistry 23: 3934-3945 (1984). 18. T.D. Tullius and B. A. Dombroski, Proc. Nufl. Acud.Sci. (USA) 83: 5469-5473 (1986). 19. W. J.Dixon, J. J. Hayes, J. R, Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius, Meth. Enzymol.208 380413 (1991). 20. J.J. Hayes andT. D. Tullius, Biochemistry 28 9521-9527 (1989). 21. A. Brunelle and R F. W e i f , Proc. Nufl.Acad. Sci. USA 84: 6673-6676 (1987). 22. U. Siebenlist and W. Gilbert, P m . Nufl. A d . Sci. USA 77: 122-126 (1980). 23. F.D. Bushman,J. E. Anderson, S. C. Harrison, and M. Ptashne, Nuhrre 326: 651-653 (1985). 24. K. C. Hayashibara and G.L.Verdine, Biochemistry 32: 11265-11273 (1992). 25. S. R.Jordan andC.0.Pabo, Science 242: 893-899 (1988).

234

Tullius

26. A. Sarai and Y. Takeda, Proc. Nutl. Acud. Sc.i (USA) 86 6513-6517 (1989). 27. J. Miller, A. D. McLachlan, and A. Klug, EMBO J. 4 1609-1614 (1985). 28. J. M. Berg, P ~ o c N . d . Acud. Sci. (USA) 85 99-102 (1988). 29. G. Parraga, S. J. Horvath, A. Eisen, W. E. Taylor, L. Hood,E. T. Young, and R.E. Klevit, Science 242: 1489-1492 (1988). 30. M. S. Lee,G.P. Gippert, K. V. Soman, D. A. Case, and P. E. Wright, Science 245 635-637 (1989). 31. N. P.Pavletich andC. 0.Pabo, Science 252: 809-817(1991). 32. K.E. Vrana, M. E. A. Churchill, T. D. Tullius, and D. D. Brown, Mol. Ce2f. Biol. 8 1684-1696 (1988). 33. J. J. Hayes andT. D. Tullius, J. Mol. Biol. 227: 407- 417 (1992). 3 4 . K. R Clemens, X. Liao, V. Wolf, P. E. Wright, and J. M. Gottesfeld, Pmc. Natl. Sci.(USA) 89: 10822-10826 (1992). 35. L. Fairall andD.Rhodes, Nucleic Acids Res. 20: 4727-4731 (1992). 36. A. A.Travers, Current OpinionStruct. Biol. 2: 114-122(1991). 37..H-M Wu and D. M. Crothers, Nature 308 509-513 (1984). 38. H. Htun and J. E. Dahlberg, Science 243: 1571-1576 (1989). 39. J. A. Bomwiec, L. Zhang, S. Sasse-Dwight, and J. D. Gralla, I. Mol. B i d . 196: 101-111 (1987). 40. D. M. J. Lilley and E. Palecek, EMBO J. 3: 1187-1192 (1984). 41. P. E. Nielsen, C. Jeppesen, and 0.Buchardt, FEBS Lett. 235 122-124 (1988). 42. C. Yoon, M. D. Kuwabara, R Law, R Wall, and D. S. Sigman, J. Biol. Chem 263: 8458-8463 (1988). 43. T. Thederahn, A. Spassky, M. Kuwabara, and D. S. Sigman, Biochem. Biophys. Res. Comm. 168 756-762(1990). 44. B. Frantz andT. V. OHalloran, Biochemistry 29 47474751 (1990). 45. J. C. Marini, S. D. Levene, D. M. Crothers, and P. T. Englund, Pmc. Natl. Acud. Sci. (USA) 79 7664-7668 (1982). 46. P.J. Hagerman, Biochemistry 24 7033-7037 (1985). 47. P. J. Hagerman, Nature 321: 449450 (1986). 48. L.E. Ulanovsky andE. N. Tiifonov,Nature 326: 720-722 (1987). 49. A; M. B~khoffand T. D.Tullius, Nature 331: 455457(1988). 50. A. M. Burkhoff and T. D. Tullius, Cell 48 935943 (1987). 51. H.C. M. Nelson, J. T. Finch, B. F. Luisi, and A. Klug, Nature 330 221226(1987). 52. M. Coll, C. A. Frederick, A. H. -J. Wang, and A. Rich, Pmc. Natl. Acad Sci. (USA) 84 8385-8389(1987). 53. A. D. DiGabriele, M. R. Sanderson, andT. A. Steitz, Pmc. Natl. Acad Sci. (USA) 86: 1816-1820 (1989). 5 4 . J. G. Nadeau andD. M. Crothers, Proc. Nutl. Auzd. Sci.(USA) 86 2622-


of DNA

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2626 (1989). 55. V. Lyadtichev, NucZeic Acids Res. 2 9 4491-4496 (1991). 56. C. R Cayadine, J. Mol. Bioi.262: 343-352 (1982). 57. A. V. Fratini, M. L. Kopka, H. R Drew, and R. E. Dickerson, J. Bioi. Chem 257: 14686-14707 (1982). 58. T.E. Haran and D. M.Crothers, Biochemistry 28 2763-2767 (1989). 59. M.A. Price and T.D. T a m , Biochemistry 32: 127-136 (1993).


15 DNA Recognition by Steroid HormoneReceptorZincFingers: Effects of Metal Replacement and Protein-ProteinDimerization Interface Bibudhendra Sarkar BiochemistryResearch, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada

I . INTRODUCTION The steroid hormone receptor superfamily is a group of cytoplasmic receptors which act as transcriptional enhancer proteins. These receptors bind specificallyto short DNA sequences and control the transcription of a number of genes (1). Sequence comparisons of regions of varying degrees of conservation revealed that a number are shared by almost all the receptors (Figure 1). The A/B domain, which is the most variable and differs considerably in size from one receptor to another is known to contain promoter- and cell-specific trans-activation function. The C domain isthe most highly conserved of the regions and encodes the DNA binding domain. This region is connected to the regionof next highest conservation, the E region, by the hingeor D region. The E region constitutes the 237

a

230

DNA Recognition by SteroidHormoneReceptorZincFingers

239

hormone binding domain. It also contains transactivating and dimerization functions. The DNA binding domainC of these receptors are highly related and similarly stabilized by two zinc atoms each coordinated to 4 cysteine residues. The two fingers present in the DNA binding domain are not equivalent. The first finger (P box) is responsible for sequence specific DNA recognition while the second finger (D box) is involved in protein-protein cooperative interactionin the dimerization process. The hexameric sequence of the consensus response elements, their directionality and spacing dictate their specificity for receptor binding. Although zinc is generally presumedbetothe endogenous metal ion within the zinc fingers, few studies have actually demonstrated in some this to be the case. In vitro studies have demonstrated that are capable of functioning in zinc instances, metals other than zinc fingers (2-8). Consequently the studies of interaction of metals other than zinc with zinc finger domains have become focus a major in our laboratory. Protein-protein interactions betweenDNA binding domains of these proteins in the dimerization interface is responsible for mediating the recognition of base pair spacing and orientation of response element halfsites (Figure 2). The recognition of specific half site sequence is mediated by direct protein-DNA contacts of individual domains. Two estrogen receptor (ER) DNA-binding domain (DBD) polypeptides bind cooperatively as a homodimerto the half site of 2 estrogen response elements (EREs) consisting of inverted repeatsof the consensus sequence AGGTCA. On the other hand retinoic acid receptor (RAR) and retinoid-X receptor (RXR) DNA-binding polypeptides bind cooperativelyas a heterodimer to two retinoic acid response elements (RAREs) consisting of direct repeats of the AGGTCA consensus sequence. Dimerization interfaces required for homodimerization of steroid receptors bound to symmetric hormone response etements (HREs) have been identified within both the ligand - and DNA-binding domains (911). The DBD interfaces in steroid hormone receptors, through head to head interactions, mediate recognition of half-site orientation and spacing as cooperative binding of DBD polypeptidesto DNA occurs only with proper half-site positioning(12,13).Onthe contrary, the asymmetric nature of direct repeat RAREs suggests that non-reciprocal headto tail DBD dimerization interfaces in and RXR are likely to be required for efficient interactions between each heterodimeric partners. Thus RAR and RXR should bind n preferentially to either the up- or down- stream half site core imotif order to achieve specific and cooperative dimer interaction.

U

G C

C3U

ou

f 240

t3u t3u 4E.c

c!Ju t3u

DNA Recognition by SteroidHormoneReceptorZinc

11.

Fingers

241

EFFECT OF METAL REPLACEMENT

A purified, bacterially expressed polypeptide encompassing the ER DBD was used in all our studies (2). When apopolypeptide was dialyzed against buffer without metal no specific binding could be detected as measured by a mobility shift assay with a consensus ERE hexamer-containing oligonucleotide. However, specific DNA binding was restored by dialysis against buffer containing zinc, as cadmium or cobalt but not with buffer containing copper or nickel showninFigure 3, althoughthesemetalsdoappeartointeract Cd

CO

Cu

W

I

.

Ni

Zn

-bound

c free

Figure 3 Mobility shift assay of cadmium-, cobalt-, copper-, nickel and zincreconstituted polypeptidesas indicated (2).

directly with the polypeptide (2,4). Dissociation constantsfor DNA binding domain polypeptide and zinc, cadmium and cobalt reconstituted apopolypeptide with the ERE consensus hexanucleotide-containingoligonucleotide were determined from mobility shift results using the double-reciprocal binding plot. The native DNA-binding domain polypeptide and zinc- and cadmiumreconstituted polypeptide all had very similar affinity for the ERE hexamer whereas cobalt-reconstituted polypeptide had a decreased affinity compared to the native polypeptide (Table 1).

Table 1 Dissociation constants of polypeptide bindingto ERE hexamer sequence as determined by double reciprocal plot analysis of mobility shift assay results(2) Polypeptide form

Kd nM

Native Zinc Cadmium Cobalt

48

66 48 720

242

Sarkar

Methylation interference experiments showed that native, zinc-, cadmium-, and cobalt- reconstituted forms of the polypeptide interact with theAGGTCA (read TGACCT on the other strand) half site in a qualitatively similar manner (Figure 4). The reduced intensity of the guanine band within the TGACCT half-site indicates that in each case, the polypeptideis interacting specifically with the half-site sequence.

T Q+ 4

I

C

C T

G C

Figure 4

A

Methylationinterferenceassays of native,zinc-, cadmium- and cobalt-reconstituted polypeptides with ERE hexamer containing oligonucleotide. In eachcase, bound (B) and free 0DNA lanes are indicated and the ERE hexameric sequenceis denoted inbold. The specific guanine residue in the ERE hexamer which is "required" for specific binding is indicated by arrow(2).

The ability of zinc, cadmium and cobalt to reconstitute theDNAbinding propertiesof the native polypeptide are consistent with the expected structural contributionof these metals. They are knownto coordinate with tetrahedral geometry and are all capable of binding to cysteine sulfhydryls, which are the zinc ligands. The inability of aporeceptor DNA-bindingto be restoredby copper or nickel is also not unexpected. Square planar geometries are more common for nickel. Nickel-binding may result in a distorted finger from the in a conformationally normally tetrahedral metal site resulting


243

changed polypeptide incapableof specifically interacting with the DNA. Copper on the other hand in the form of copper(1) hasa high affinity for sulfhydryl ligands but has less stringent geometric requirements than zinc. Thus copper may not determine the proper folding of the polypeptide for DNA-binding. These results suggest that misincorporation of copper, for example, would lead to a transcriptionally inactive receptor affecting transcriptional regulation. In fact, if copper binding causes wrong folding, the receptor may be rapidly degraded within the cell. In certain conditions intracellular concentration of copper may become significant. This could occur in Wilson and Menkes diseases in which certain cells and tissue retain elevated levels of copper. Furthermore, in a zinc-deficient state, copper may be able to compete effectively with the decreased cellular concentration of zinc. It is possible that some symptoms of zinc deficiency may directly attributable to misfolding of steroid receptors(14). It has now been shown that iron can replace zinc in the zinc finger motif of ER and still retain the DNA-binding activity (15). Furthermore it has been demonstrated that iron can generate free radicals, specifically hydroxyl radicals, while coordinated to the four cysteines of zinc finger. Hydroxyl radical is known to damage DNA, which may lead to mutagenesis and/or carcinogenesis.

111. PROTEIN-PROTEIN COOPERATIVE INTERACTION WITH INVERTED REPEAT RESPONSEELEMENTS A. Homodimerization of ER It was possible to resolve both the monomeric and dimeric bound forms of the ERDBD upon binding to an actual ERE and to quantitate the extentof cooperativity (3). A model forERE binding is presented in Figure5. According to this model ERDBD bindsto "K'. either of the two hexameric half sites with identical affinity

244

Sarkar

sk A

l

l .t+

rk

B

Figure 5 /“el for estrogen receptor DNA-binding domain interaction with theERE. Free polypeptide (hatched box) binds to either half site with an association constant “ K ’ . The complex formed is the with either siteA or site B occupied monomeric complex, while the complex formed with both sitesA and B occupiedis the dimeric complex (3).

Binding of the second polypeptide occurs with affinity an equal to oK,where K is the polypeptide association constant for a half site and o is the cooperativity parameter. Thus, the binding equation canbe derived from the equilibria:


245

where P=free protein,fAfg=free DNA, bAfg=protein bound to site A only, fAbB=protein boundto site B only,bAbg= protein bound to both sites A and B. Cooperativity values were determined by mathematical best fit of data over a rangeof o values using a computer program developed by us. A cooperativity parameter of >l indicates positive of cooperativity, a valueof 1 indicates no cooperativity and a value .-> W W

Cd-induced

l*-

1

0

g .-U

II-

E

10-

v)

In 0 -

-c

7i 9-

tw[tx; = 29;.

6

1

f 1.8 h

t,Iwtl = 16.4 f 4.1 h

0

20

40

60

80

100

120

Time after [35S]cysteine treatment(hours) Figure 8 Degmaktion of hepatic merallothwnein in toxic milk and wild-type mice. Toxic milk (0) and wild-type (0)mice were treated with cadmium (1 mgkg 6 hoursprior to radiolabeling)("Cd-induced"), or iso-osmoticsaline ("Controln).Specific activity of MT separated by SDS-PAGE was determined between 24 and 120 hours as described in the text.

462

Koropatnick et al.

rather than increased stability for Cu-MT (the predominant species in toxic milk mice) this seems unlikely. Altered MT gene structure, or decreased activity of proteinase@),remaincandidatemechanismstoaccount for altered MT degradation, and are actively being explored. remains The underlying metabolic defect@) leading to reduced proteolysis undefined. IncreasedMT half-life intoxic milk mice does not imply that altered metallothionein regulationis the specific defect leading to pathological changes in mutant mice. Changes in degradation of other proteins,or in regulation of could also contribute to the toxic milk phenotype: other proteins or &As, (as altered regulationof copper transport through a specific transporter protein described for Wilson disease) mustbe considered in this regard. The similarities between human Wilson disease and the consequences of the toxic milk mutation in mice (excessive hepatic copper, low caeruloplasmin, [la], high hepatic reduced rate of incowration of copper into caeruloplasmin MT [13,45],and toxic changes in livers in both toxic milk mice and patients withWilsondisease [57,58] make t h w mice apossiblemodeltostudy perturbedcoppermetabolisminthisdisease. It is not yetclearwhether human disease is caused by MT increased MT and copper accumulation in the induction by excess copper, MT expression induced by toxic stress effects [29,59] co-incidentwith or causedbyexcesscopper, or altered MT metabolism. It is important to note that Cu-MT enhances lipid peroxidation in the presence of pro-oxidantsmore effectively than equimolar inorganic copper [60]: therefore, the accumulation of copper-associated MT in toxic milk mice and Wilson disease may enhance rather inhibit the toxic effects of the metal. We suggest that the increased MT protein stability and altered developmental MT protein and mRNA accumulation in toxic milk mice may be a model to help in understanding human disorders (Wilson disease, for example) involving altered copper metabolism.

V. ACKNOWLEDGMENTS This researchwassupportedby grants to MGC and JK by the Medical Research Council of Canada, and grants from the London Regional Cancer Centre Research Fund and the Victoria Hospital Research Development Fund

Effects of Copper Accumulation in the Toxic Milk Mouse

463

to J.K. We are indebted to Dr. Harold Rauch for providing breeding pairsof toxic milkmice. We thankSusan Vesely andJane Pearson-Sharpefor excellent technical assistance.

REFERENCES 1. R.A. Goyer, ToxicEffects of Metals, in Casarett andDoull's Toxicology (M.O. Amdur,J.Doull,andC.D. Klaassen, Eds.), PergamonPress, 1991, pp. 623-680. 2. B. Sarkar, J.-P. Laussac, and S. Lau, Transport forms of copper in human

serum, in Biological Aspects of Metals and Metal-Related Diseases (B. Sarkar, Ed.), Raven Press, New York, 1983, pp. 23-40. 3. G.J. Brewer and V. Yuzbasiyan-Gurkan, Medicine 71, 139-164 (1992). of CopperTransport,in MetabolicBasis of 4. D.M.Danks,Disorders Znherited Disease (A.L. Beaudet,W.S.Sly,andD.Valle, Eds.), McGraw-Hill, New York, 1989, pp. 1411-1431. 5. C. Vulpe, B. Levinson, S. Whitney, S. Packman and J. Gitschier, Nature Genet. 3, 7-13 (1993). 6. J.F.B. Mercer et al., Nature Genet. 3, 20-25 (1993). 7. J. Chelly et al., Nature Genet. 3, 14-19 (1993). 8. P.C. Bull et al., Nature Genet. 5, 327-337 (1993). 9. P.J. Anderson and H. Popper, Am. J. Pathol. 36, 483 (1960) 10. F. Schaffner et al., Am. J. Pathol. 41, 315 (1962) 11. S. Goldfischer, et al., Am. J. Pathol. 99, 715 (1980). 12. S. Goldfischer and I. Sternlieb, Am. J. Pathol. 53, 883 (1968). 13. N.O. Nartey, J.V. Frei, andM.G.Cherian,Lab.Invest. 57,397-401 (1987). 14. J. Chung, N.O. Nartey, and M.G. Cherian, Arch. Environ. Health 41, 319-23 (1986). 15. N. Sugawara et al., Experientia 47, 1060-63 (1991). 16. D.M. Danks, J. Med. Genet. 23:99 (1986). 17. N. Shiraishi et al., Toxicol. Appl. Pharmacol. 110, 89-86 (1991). 18. G.F. Johnson et al., Hepatology 1, 243 (1981). 19. H. Rauch, J. Heredity 74, 141 (1983).

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20. H. Rauch et al., Hepatic copper and superoxide dismutase activity in toxic

milk mutant mice,in Superoxide and Superoxide Dismutase in Chemistry, 1986, p. Biology and Medicine (G. Rotilio, Ed.), Elsevier, New York, 304. 21. J.F.B. Mercer et al., J. Nutr. 121, 894-899 (1991). 22. L. Biempica et al., Lab. Invest. 59, 500-508 (1988). 23. I. Sternlieb, Gastroenterology 78,1615(1980). 2 4 . L.-C. Su et al., Am. J. Physiol243, G231 (1982). 25. D.C. Twedt, I. Sternlieb, and S.R. Gilbertson, J. Am. Vet. Med. Assoc. 175, 269 (1979). 26. W. Sun, L.W. Oberley and Y. Li, Clin. Chem. 34,497-500 (1988). 27. A.M.ScheuhammerandM.G.Cherian,Toxicol.Appl.Pharmacol. 82, 417422 (1986). 28. J. Koropatnick and M.G. Cherian, Biochem. J. 296-449 (1993). 29. D.H. Hamer, Annu. Rev. Biochem. 55, 913-951 (1985). 30. J.H.R. Klgi and B.L. Vallee, J. Biol. Chem. 235, 3460-64 (1960). 31. J.H.R. KSgi and A. Schlffer, Biochemistry 27, 8509-15 (1988). 32. J. Matsubara, Y. Tajima, and M. Darasawa, Radiat. Res. 111, 267-275 (1987). 33. A.J. Fornace,H.Schalch,andI.Alama,Mol.Cell.Biol. 8, 4716-20 (1988). 34. J. Koropatnick, M.E.I. Leibbrandt, and M.G. Cherian, Radiat. Res. 119, 356-365 (1989). 35. A.J. Ouellette, Dev. Biol. 92, 240-246 (1982). 36. G.K. Andrews, E.D. Adamson, and L. Gedamu, Dev. Biol. 257,294-303 (1984). 37. J. Koropatnick and J.D. Duerksen, Dev. Biol. 122, 1-10 (1987). 38. N.O. Nartey,D.Banerjee,andM.G.Cherian,Pathology 19,233-238 (1987). 39. N.O.Nartey,M.G.Cherian,andD.Banerjee, Am. J.Pathology 129, 177-182 (1987). 40. J. Koropatnick and J. Pearson, Somat. Cell Molec. Genet. 16,529-537 (1990). 41. K.E. Mayo and R.D. Palmiter, J. Biol. Chem. 256, 2621-25 (1981).

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42. S.L. Kelley et al., Science 241, 1813-15 (1988). 43. B. Kaina et al., P m . Natl. Acad. Sei. USA 87, 2710-14 (1990). 44. H. Lohrer et al., Carcinogenesis 11, 1937-41 (1990). 45. C.D. Bingle, S.K. Srai, and 0. Epstein, J. Hepatol. 15, 94-101 (1992). 46. M.E.I. Leibbrandt et al., Biol. Trace Element Res. 30, 245-256 (1991). 47. F. Tietze, Anal. Biochem. 27, 502-522 (1969). 48. K. Cain and D.E. Holt, Chem.-Biol. Int. 28, 91-106, 1979. 49. S.K. Kremski et al. Biochem. J. 255,483-491 (1988). 50. J.C.Waterlow,P.J.Garlick,andA.J.Millward, Protein turnover in mammalian tissuesand the whole animal, Amsterdam:NorthHolland (1978). 51. D.E. Laurin, D.M. Barnes, and K.C. Klasing, P m . Soc. Exp. Biol. Med. 194, 157-164 (1990). 52. S.L. Feldman, M.L. Failla and R.J. Cousins, Biochim. Biophys. Acta 4, 638-646 (1978). 53. M. Karin and H.R. Herschmann, J. Cell. Physiol. 103, 35-40 (1980). 54. S.L. Feldman and R.J. Cousins, Biochem. J. 160, 583-588 (1976). 55. I. Bremuer et al., Biochem J. 174, 883-895 (1978). 56. K.-S. Min, Toxicol. Appl. Pharmacol. 113, 299-305 (1992). 57. I.H. Scheinberg and I. Sternlieb, Majorproblems in internal medicine, vol. 23; Wilson’sdisease, W.B. Saunders, Philadelphia, pp. 1-27 (1984). 58. I. Sternlieb, in Progress in liver diseases,vol.

(H.

4 Popperand F. Schaffner, Eds.), Grune and Stratton, New York, p. 511 (1972). 59. M. Karin, Cell 41, 9-10 (1985). 60. G.F.Stephenson,H.M.Chan,andM.G.Cherian,Toxicol.Appl. Pharmacol. 125, 90-96 (1994).


29 MetallothioneinSynthesis Is Selectively Enhanced by Copper in the Liver of LEC Rats Kazuo T. Suzuki Faculty of Pharmaceutical Sciences, ChibaUniversity, Yayoi, Inage, Chiba 263, Japan

I.

INTRODUCTION

Long Evans rats with cinnamon-like coat color (LEC) were established from Long Evans agouti (LEA)rats as an inbred strain showing hereditary hepatitis and hepatic carcinoma (1). This strain develops acutehepatitis at the age of a b u t 4 months. Those rats that survive the acute hepatitis will suffer from chronic hepatitis and develophepatoma after about 1 year (1). Thecausesand mechanisms leading to chronic hepatitis and/or hepatic carcinoma have been of great concern and LEC rats are recognized to be a valuable experimental model animal to study the whole process of spontaneous hepatic carcinoma.

A.

Accumulation of copperin the liver of LEC rats

The liver of LEC rats was found to accumulate much greater amounts of copper (Cu) than the original type strain (2). The Cu accumulating in the LECrat liver is mostly bound to metallothionein (MT) (2-7). Although the mechanisms how the Cu accumulating in the liver in a form bound to MT causes acute hepatitis and hepatic carcinoma are notknownyet,themetalisthought tocause spontaneous hepatitis and hepatoma. In fact, it was demonstrated 467

468

Suzuki

that LEC rats can be effectively cured from otherwiselethal signs by removing Cu selectively from theliver with tetmthiomolybdate (TTM) treatment (8). As a result, the LEC rat appears to be an interesting experimental model to study not onlyspontaneous hepatic carcinoma but also the toxicity and regulation mechanism of Cu (1- 10). B

LEC rats as an experimentalmodelanimal Wilsondisease

for

Wilson disease is recognized as an inherent disease of abnormal Cu accumulation in the liver (11-14). Patients of Wilson disease are cured of the hepatitis by treatment with chelating agents ( 12- 14). Likewise, Cu in the LEC rat liver was decreased by the administration of chelating agents, andthis treatment also inhibited the development of hepatitis (8),suggesting that the Cu accumulating in the liveris the cause for the toxicity both in Wilson disease and in LECrats. These to observationstogetherwiththeirpathogenicsimilaritylead recognize LEC rats as an experimental model animal for Wilson disease (2) 11. DISORDERED BIOLOGICAL REGULATION FOR COPPER METABOLISM Cu is an essential trace metal and is regulated homeostatically by fundamental metabolicprocesses to avoid any toxic effect by its excessive presence. The liver isa key organ in the regulationof Cu metabolism (11-14). Cu ion is preferentially transported in the blood as a complex of albumin (16), and the metal incorporated in excess into the liverinduces MT and binds to this protein. Cu in the liver is secreted to blood in ceruloplasmin binding form (17-19). However, the metabolic process for Cu homeostasis in the liveris still unclear. Disordered Cu metabolism in hereditay diseases certainly gives a clue to understand the normal functioning mechanism of Cu. LEC rats may demonstrate toxicity due to some disorder@) in the metabolic processes of Cu (2-10). A . Defective expression of the transporter gene efflux of copper in Menkes and Wilson diseases

for

Recently the cause of abnormal Cu regulation for Menkes disease has been suggested to be a lack in normal functioning CudependentATPase (20-22), whichmayindicatethatCuis

Metallothionein Synthesis Selectively Enhanced

by Copper

469

accumulated in the cell owingto the lack in the efflux mechanism of Cu and thatMT is induced to sequester the excessiveCu in thecell. Although thetwo genetic diseases appear to be quite differentin their symptoms, the cause of the other genetically disodered disease, Wilson disease is alsosuggested to be a lack in expressionof normal functioning Cu-dependent ATPase (23-25). Although this transporter for efflux of Cu has only been suggested from the gene sequences similar to that encoding bacterial Cu-dependent ATPase (26), the lack in normal functioning efflux system for Cu certainly results in abnormal accumulation of Cu inthe cell. Accumulation of Cu is toxic to the cell and thecell induces the synsthesis of MT to sequester free Cu ion in a form bound to MT. Therefore, theaccumulation of Cu in a form bound to MT in the liver of Wilson diseasepatients is thought to be the result of abnormal Cu accumulation. Cu does not accumulate in the liver of Menkes disease patients andthesymptomisthat of Cu deficiency.However, cultured Menkes cells accumulate Cu by exposure to Cu (27, suggesting the defective expression of the exporter gene for efflux of Cu at the absorption site which results in prevention of the deliveryof Cu to the liver (28). B Imbalancebetweenthesynthesis metallothionein

and degradation of

An abnormal accumulation of Cu in a form of M" can be caused by a different mechanism from that suggested by a lack in normal functioning transporter for efflux of Cu, Cu-dependent ATPase.Namely, anabnormalaccumulation of Cu-binding MT owing to accelerated induction or restricted degradation of MT may explain an abnormal accumulation of Cu in the liver becauseof the lack in anactive secretion of MT from the cell. The abnormal Cu accumulation ina form bound to MT in the liver of toxicmilk mice is suggested to be caused by an imbalance of MT metabolism by restricted degradation of MT (29). In the liverof this strain, MT is synthesized normally, but degraded at a slower rate than the control, which results in the accumulationof Cu bound t o

MT.

C. Accumulation of copper metallothionein

in a form other than

470

Suzuki

Indianchildhoodcirrhosis is known to be causedby an abnormal accumulationof Cu in the liver. However, the inducibility of MT synthesis by Cu and other heavy metals is lower than the normal level and the Cuaccumulating in the cultured fibroblast is not (30). presentin a formbound to MT inthisgeneticdisease Therefore, Cu seems to accumulate bya mechanism different from those mentioned above, suggesting the third mechanism that may not involve MT in itsaccumulation. 111.ACCUMULATION LEC RATS

OF COPPERIN THE LIVER O F

2.0

300

C' 250

Y

1.5

.I

.-.

-g 200 3 3 .CI

W

1.o

150

gS

9

Y

0

CO

E

c,

E

Y

5

Q E

U

100 0.5

8

50 0.0

0 2 4

6 8 l0 12 14 16 18

20

Age (weeks)

Figure 1. Changes in concentrations and content of Cu in the liver of LEC rats with age. Livers were homogenized in 4 volumes of extraction buffer and thehomogenates were centrifuged at 105,000g for 60 min. Cu levels in the liversof female LEC rats at 2,4,6,9, 12, 15, 16, and 16-18weeks of age were expressed by concentration (pg/g liver, open square) and content (mgliver, closed square) (From Ref. 31).

Metallothionein Synthesis Selectively Enhanced by Copper

471

Since it was suggested that hereditary hepatitis and hepatic carcinoma are caused by abnormal accumulation of Cu in the liverof LEC rats, attention has been focused on LEC rats from the two standpoints; One isthe mechanism leading to acutehepatitis and the whole process causing spontaneous hepatic carcinoma, and the other is the mechanism of disordered metabolism of Cu in relation to that in Wilson disease. Our approach from the latter standpoint is to be described in thepresent communication (8-10,28,31,33).

A. Accumulation of copperintheliver age

of LEC rats with

Although the Cu accumulating in the liver of LEC rats was reported to be present in a form bound to MT (2-3, those were as to of Cu accumulation and certainaspectsofthewholeprocess information about theinteraction with other metals suchas zinc (Zn) and iron (Fe) wasalso not sufficient. Therefore, we tried to find, at first, the whole processof Cu accumulation in the liver togetherwith thoseinthe bloodplasma,kidneys,andurine.Changes in concentrations of Zn and Fe in the liver, kidneys, and blood plasma were also traced to find the interaction of Cu with those metals (3 1). Figure 1 shows the whole process of Cu accumulation in the live of LECrats (31). Concentration of Cu in the liver is alreadyas high as approximately 200 pg/g liver at 2 weeks of age before weaning. Although Cu appears to decrease during and after weaning when the Cu level is expressed by concentration (pglg liver), the metal is shown to increase steadily in the liver when it isexpressed by content (mg/whole liver) (31). The Cu accumulating in the liver was distributed mostlyin thecytosol fraction and the cytosolicmetal was present mostly in the form bound to MT and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (9,31). The Cu level decreased suddenly after 16 weeks of age in accordance with the onset of jaundice. The Zn level started to decrease one week earlier than the Cu level did (data notshown). An abrupt increase of the Fe level in the liver was observed with the onset of jaundice (datanot shown) (31). B.

Changes in copper distribution in the plasma and kidneys of LEC rats following acute hepatitis

472

Suzuki

Following the accumulationof Cu up to the limit in theliver, concentration of the metal in theblood plasma increased dramatically and the metal boundto ceruloplasmin (Cp) increased, suggestng that holoceruloplasmin (holo-Cp) was excreted from the liver instead of apoceruloplasmin (apo-Cp) (10,3 1,32). This result implies further that Cu is supplied differentially to the two Cu-enzymes (Cp and SOD) synthesized in the liver. In addition to the increased distibution of Cu to Cp, the Cu increased in the blood plasma was shown to be present in the forms bound to MT and albumin toward the onset of jaundice (31). MT in the blood plasma is known to be filtered from the glomerulus and reabsorbed by the proximal tubules. In accordance with the increase of MT-bound Cu in plasma, concentrations of Cu in the kidneysand urine increased rapidly (10,31), indicating that Cu overflew from the liver to plasma in the form of MT when the metal accumulated to the limit. The details of the age-related changes in concentrations of Cu, Zn, and Fe are reported elsewhere (31) with intensive observationat the onset of jaundice(9, 10). IV. ENHANCED SYNTHESIS OF METALLOTHIONEIN IN THE LIVER OF LEC RTAS (28,33)

W e aimed to clarify how the LECrat liver accumulates Cuin a form bound to M" at a high concentration. For this purpose, the induced synthesis of MT by Cu was compared in vivo and in vitro between LEC andLEA rats (the original strain of LEC rats). A . Enhanced synthesis of metallothioneinin LEC rats (33)

the liver of

Among possible mechanisms for the accumulation ofCu in a form bound to MT in the liverof LEC rats, we tried to compare the As inducibility of MT by Cu from the following consideration; discussed above in 11. Disordered biological regulation for copper metabolism, accumulationof Cu in a form bound to MT is explained either by the result or by the cause. MT synthesis is tobe induced to sequester free Cu accumulating in the liver owing to imbalance between influx and efflux in the former case, while the protein accumulates in the liver owingto imbalance between synthesis and degradation of MT, resulting in an accumulation of Cu in the latter case.


A

a .-

473

250

3 200 3

c 150 0

1-

c1

m

L: 100

.5

8

8

B

50

h

L

al

.-> S I

3

lo

t

1 0 1 2 3 4 5 6 7 8 9 Cu admistration (days)

Figure 2. Changes in concentrations of Cu and Zn in the whole livers and supernatants of Cu-loaded LEA and LEC rats. LEA rats were injected subcutaneously withCuC12 daily at a doseof 3 mglkg body weight for 2 , 4 , 6 and 9 days. LEC rats were killed without any treatment. Livers were homogenized in 3 volumes of extraction buffer and the homogenates were centrifuged at 170,000 g for 1 hr. Concentrations of Cu (A) and Zn (B) were determined by AAS. The closed circle and triangle indicate the concentrations in the liver and supernatant of LEA rats, respectively, while the open circle and triangle indicate thosein the liver and the supernatant in 5-week-old LEC rats, respectively. The concentration in the supernatant (pglg liver) was calculated by multiplying the value inthe supernatant by 4 (pglml). Data are expressed as means S.D. of 3 samples (From Ref. 33).

*

474

Suzuki

We observed that Cu accumulating in the liverin a form bound to MT can be removed selectively by treatment with a chelating agent, TTM (8). This result suggests that Cu bound to MT is not secreted from the liver, while Cu not bound to MT can be secreted from the liver, indicating that accumulation of Cu ina form bound to MT may be the cause in the liver of LEC rats. Then, we tried to find whether the accumulation of MT is due to enhanced synthesis or restricted degradation (33). For this purpose, the induced synthesis of MT by Cu and the distribution profiles of Cu and zincZn in the liverof LEA rats after injections of Cu wereexamined, and they were compared with those of LEC rats. 1. Accumulation of copper in the liver of LEA rats with injections of copper and comparison with those in LEC rats (33)

Concentrations of Cu in the LEA rat liverand the supernatant gradually increased with consecutive injections of CuC12 as shown in Fig. 2A. The formerconcentration was similarto that in fiveweekold control LEC rats after 4 injections (about 120 pg/g). Although the Cu present in the LEC rat was mainly recovered in the liver supernatant (Fig. 2A), only aboutone half of Cu in the LEArat liver was recovered in the supernatant (about 60 pg/g, Fig. 2A). The Cu concentration continued to increase in thewhole liver with injections reaching more than 200 pglg after 9 injections. However, only 40 % of Cu in the liver wasrecovered in the liver supernatant (about 90 pglg), which was still lower than that in the LEC rat liver supernatant (about 120&g). These results indicate that Cu was mainly retained in soluble forms in the LEC rat liver, but more than a half of Cu accumulating in theLEA rat liver was not recovered in the supernatant and remained in the non-cytosolic fraction. The concentrations of Zn in the whole liver and the supernatant of LEA rats increased slightly after Cu injections for 2 days, but it was less than one half of Zn concentration in LEC rats and was not elevated any moreby additional injections (Fig. 2B).

2. Disribution profiles of copper accumulatting in the liver of LEA rats by injections of copper (33) Chemical forms of the Cu accumulating in the LEA and LECrat livers were determined together with those of Zn by gel filtration chromatography (by HPLC-ICP onan SW column) as shown in Fig.


475

LEA rat

1

"

10

15

20

l . . . . I .

10

15

...

.... 25

I . . . . l

20

LEC rat

LEA rat

MT-2,

I

.... I ....I

10

15

.

20

.

I . . . . I . . . . l . . . . ,

10

15

20

Retention time (min)

Figure 3. Changes in distribution profiles of Cu and Zn in the liver supernatants of Cu-loaded LEA and LEC rats. The distribution profiles of Cu (A) andZn (B) in the liver supernatants that correspond to those in Fig. 2 were determined by the HPLC-ICP method on an SW column. The vertical bar indicates the detection level for each element at0.05 pglml (From Ref.33).

476

Suruki

3. In the profile of control LEA rats, superoxide dismutase (Cu,ZnSOD) was eluted as distinct Cu and Zn-bindingpeaks at a retention time of 15.1 min (Figs. 3A and 3B). Injections of Cu into LEA rats induced the synthesisof MT in the liver, and Cu was sequestered by the protein. Two isoforms of MT (MT1 and MT2) were eluted at retention times of 19.4 and 18.1 min, respectively, MT2 being the major isoform in the Cu-loaded LEA rat liver. The amount of MT was gradually increased by 4 consecutive injections of Cu, but was not elevated by anyadditional injections. Excessive Cu not bound to MT was distributed to unidentified proteins which were eluted as high molecular weight proteins between the void volume of the column and MT peaks. The amountof SOD stayed constant during consecutive loadings of Cu to LEA rats. Cu in the liversupernatant of LEC rats was mainly bound to MT. Unlike in LEA rats, MT1 was the major isoform in the LECrat liver. The elution profiles of Zn changed slightly with loadingof Cu to LEArats (Fig. 3B). The small Zn peakin the MT fraction observed in the control rat liver was gradually decreased by the loading with Cu. In LEC rats, Zn was mainly recovered in the MT fraction, and MT1 was also the major component containing Zn. 3. Expression of MT and MT mRNA inthe LEA rat liver by injections of copper (33)

The amount of MT induced in the liver was estimated by measuring the concentration of Cu bound to MT. The amountof Cu bound to MT was gradually increased with injections of Cu up to 4 times, but did not increase afteradditional injections (at maximum, about 22 pglg of liver), approximately 40 % of Cu in the liver supernatant being bound to MT (data not shown). Hepatic MT mRNA was analyzed by Northern blotting. The MT mRNA content in the LEA rat liver was elevated by 2 injections of Cu, but did not increase beyond this level if additional Cu was injected. The MT mRNA content in the LEA rat liver was much lower than in the LEC rat liver (Fig. 4) (33). Lactate dehydrogenase (LDH) activity was used as an indicator for the cytotoxicity caused by Cu administration. LDH activity was not changed by Cu administration, indicating that Cu did not damage the rat liverunder the conditions used in the present experiment. The results raised a question why the Cu accumulating in the liver of LEA


by Copper

477

l 2 3 4 5 6 7

Figure 4. Amounts of MTmRNA in the livers of Cu-loaded LEA and LEC rats. LEA rats were injected subcutaneously with CuC12 daily at a dose of 3 mglkg body weight. Total RNA was extracted from the liversof LEA rats 1 day after thelast injection at 2,4 and 6 days,andalsofromtheliver of LECratswithouttreatment. Amounts of MT mRNA were determined by Northern blot analysis using a mouse MT1 cDNA probe. Lanes 1 and 2; LEA rats without treatment: Lanes 3,4, and 5; LEA rats with 2, 4, and 6 injections, respectively: Lanes 6 and 7 ; LEC rats without treatment (From Ref. 33).

rat liver inthe forms not bound to MT is not toxic as judged bythe LDH activity (33).

The in vivo studydescribed above in this section suggests that MT synthesis is induced only to a limited extent in the liverof LEA rats even if Cu is taken up andaccumulates to the same level or more than that in the LEC rat liver without any signs of liver damage. In other words, MT synthesis in the liver of LEC rats is enhanced and Cu accumulating in the liver ispreferentially sequestered in a form bound to MT.

B.

Selective enhancement of metallothionein mRNA expression by copper in primary cultured liver parenchymal cells of LEC rats (28) The in vivo study described in the preceding section indicates that Cu was taken up dose-dependently by the liver of LEA rats, however, only a limited amount of Cu was present bound to MT (33). These results suggest that the high MT level is the cause for the

470

Suzuki

0

5

10

15

20

25

30

Cu treatment @M)

Figure 5. Uptake of CUby primary cultured liver parenchymal cells prepared from LEAandTTM-treatedLECrats. Liver Parenchymal cells prepared from LEA rats (open circles) and TT” treated LEC rats (closed circles) were exposed to 0.125, 0.25, S, 10 and 25 PM CUfor 6 hr, and concentrationsof Cu in the cells(pg/mg Protein) were plotted againstCu concentration in the medium (From Ref. 28).

abnormal Cu accumulation in the liver of LEC rats. The low MT level in LEArats was reflected in the low MT mRNA level, while the high MT level in LEC rats correlated with the high MT mRNA level. These results suggest thatCu is accumulated in the liver of LEC rats because of enhanced MT synthesis. To clarify the mechanism functioningin LEC rats, we further compared the inducibility of MT synthesis between the mutant LEC of Cu and MT rats and the control LEA rats by determining the levels

mRNA in liver parenchymal cells prepared from LEC and LEA rats. Levels of MT and MT mRNA in the liver parenchymal cellsof LEC rats were reduced to the same levels as those of LEA rats before preparing the cells by removing Cu selectively with repeated injections of tetrathiomolybdate (TTM) into LEC rats(8). Then, the MT mRNA level was compared in the two cells by exposure to inducers of MT: cadmium (Cd), Cu, dexamethazone (Dex), and Zn (2%. 1. Uptake of copperandexpression of metallothionein mRNAin primary cultured liver parenchymal cells prepared from LEA and ITM-treated LEC rats

The amounts of Cu and MT mRNA were comparable in both up liver parenchymal cells before exposure to Cu. Cu was taken dose-dependently by both cells and the amount was comparable between LEA and LEC rat cells, although the mutant cells seemed to take up moreCu by exposure to a dose higher than 10 p M (Fig. 5). MT mRNA was expressed dose-dependently by exposure to Cu in both parenchymal cells (Fig. 6). However, spots on the gel by Northern blotting shown in Fig. 6A were more dense in the cells prepared from TTM-treated LEC rats than those prepared from LEA rats, and this relation was clearer in Fig. 6B after analysis with an image analyzer. Fig. 6B indicates that MT mRNAwas expressed than in LEA cells by exposure to Cu, both in more efficiently in LEC a dose-dependent manner up to 10 pM, beyond which there was a leveling off. Expression of &actin mRNA was constant by exposure to cu ( 2 8 ) .

2.

Selectiveexpression of metallothwnein mRNA by copper

Expression of MT mRNA by exposure to typical MT inducers including Cu was examined in both parenchymal cells to determine whether the response to Cu is specific to the genetic disorder. MT mRNA expression by the three inducers was comparable in both parenchymal cells as shown in Figs. 7A and 7B, indicating that response to MT inducers other thanCu is not affected by mutationin LEC rats, but is specifically enhanced by Cu(28).

480

Suzuki

A

I

LEA

1 2 3 4 5 6 '

B

I

LEC

7 8 91Ollld

8000 n .c) .L

C

1

L

ie

4

4000

Figure 6. Amounts of M" mRNA expressed by exposure to Cu in primary cultured liver parenchymal cells prepared from LEA and of MTmRNA inboth liver TTM-treated LECrats.Amounts parenchymal cells exposed to Cu for 6 hr were analyzed by Northern blotting for the cellsused in Fig. 5. LEA (lanes 1 - 6) and LEC cells (lanes 7 - 12) were exposedto CuC12 at concentrations of 0 (lanes 1, 7), 1.25 (lanes 2, €9,2.5 (lanes 3, g), 5 (lanes 4, lo), 10 (lanes 5, 1l), and 25 pM (lanes 6, 12). The amounts were expressed by spot density on a gel (A)and by arbitrary units after processing with an image analyzer(B)(From Ref. 28).


A

LEA ' 1 2 3

4 " 5

48 1

by Copper

LEC 6 7

8 '

B

Control

Zn Cd Treatment

Dex

Figure 7. Amounts of MT mRNA expressed by exposure to Zn, Cd and dexamethazonein primary cultured hepatocytespreped from LEA and TTM-treated LECrats. Amounts of MT mRNA in the liver parenchymal cells exposed to Zn (25 PM), Cd (2 PM) and dexamethazone (Dex, 10 PM) for 6 hr were analyzed by Northern blotting. The amounts were expressed by spot density on a gel (A) and by arbitrary units after processing with an image analyzer (B). Control (lanes 1 and 9, Zn (lanes 2 and 6), Cd (lanes 3 and 7 ) , and Dex (lanes4 and 8) (From Ref. 28).

482

Suzuki

CONCLUSION

V.

Cu in the liver of LEC rats is already much higher than in the liver of control LEA rats before weaning. The liver continues to take up Cu from foods andwater and accumulates constantly with age ina form bound to MT up tothe limit of approximately250 pglg liver. Thus, the Culevel was correlated with MT level and, then, the MT level was correlated with MT mRNA level in the liverof LEC rats. Further, MT synthesis in the liver of LEC rtas was shown to be enhanced selectively by exposure to Cu. These results suggest that the accumulation of Cu in the liver of LEC rats is caused by the enhanced synthesis of M T and that theenhanced synthesis of IvlT is the cause of the accumulation of Cu in the liver of LEC rats. The conclusion mentioned above indicates that the mechanism for abnormal accumulation of Cu in the liverof LEC rats is different from thatof the defectiveexpression of the exporterfor effluxof Cu. If the mechanism in LEC rats is the same as that explained by the defective expression of the gene of Cu-dependent ATPase, then a reasonable explanation for the enhanced expression of MT mRNA by Cu in LEC rats must exist. One possible such explanation may be that the transcription factor for the MT gene was present in the nucleus even after the corresponding MT mRNA disappeared by TI" treatment. However, this transcription factor yet to be characterized (28).

REFERENCES 1.

2. 3.

4.

5.

N. Takeichi, H.Kobayashi, M. C. Yoshida, M. Sasaki, K. Dempo, and M. Mori, Acta. Pathol. Jpn. 38: 1369-1375 (1988) Y. Li, Y. Togashi, S. Sato, T. Emoto, J-H. Kang, N. Takeichi, H. Kobayashi, Y. Kojima, Y. Une, and J. Uchino, J. C h . Invest. 87: 1858-1861 (1991). Y. Li, Y. Togashi, S. Sato, T. Emoto, J-H.Kang, N. Takeichi, H. Kobayashi, Y. Kojima, Y. Une, and J.Uchino, Jpn. J. Cancer Res. 8 2 490-492 (1991). N. Sugawara, C. Sugawara, M. Sato, M. Katakura, H. Takahashi,and M. Mori, Res. Commun. Chem.Pathol. Pharmacd. 72: 353-362 (1991). N. Sugawara, C. Sugawara, M. Sato, M. Katakura, H. Takahashi, and M. Mori, Experientia 47: 1060-1063 (1991).


6.

483

H.Sakurai, A. Fukudome, R. Tawa, M. Kito, S. Takeshima, M. Kimura, N. Otaki, K. Nakajima, T. Hagino, K. Kawano, S. Hirai, and S. Suzuki, Biochem. Biophys. Res. Commun. 184: 1393-1397 (1992).

7.

H. Sakurai, H. Kamada, A. Fukudome, M. Kito, S. Takeshima, M. Kimura, N. Otaki, K. Nakajima, K. Kawano, and T. Hagino, Biochem. Biophys. Res. Commun. 185: 548552 (1992).

K. T. Suzuki, K. Yamamoto, S. Kanno, Y. Aoki, and N. Takeichi, Toxicology 83: 149-158 (1993). 9. K. T. Suzuki, S. Kanno, S. Misawa, and Y. Sumi, Res. Commun. Chem. Pathol. Pharmucol. 82: 217-224 (1993). 10. K. T. Suzuki, S. Kanno, S. Misawa, and Y. Sumi, Res. Commun. Chern. Pathol. Pharmacol. 82: 225-232 (1993). 11. I. H.Scheinberg, and I. Sternlieb, in Truce Elemenb in Human Health and Disease. (A. S. Prasad, Ed.), Academic Press, New York, 1976, pp. 415-438. 12. I. Bremner, Nutritional and physiological significance of metallothionein in Metallothionein JZ. (J. H. R. Kagi, and Y. Kojima, Eds.), BirkhauserVerlag,Basel, 1982, pp. 81-107. 13. D.M. Dank, in Metabolic Basis of Inherited Disease. I . (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), McGraw-Hill Information Service Company, New York, 1989, pp. 1411-1431. 14. I. Sternlieb, Hepatology 1 2 1234-1239 (1990). 15. I. H.Scheinberg, M. E. Jaffe, and I. Sternlieb, N. Engl. J. Med. 317: 209-213(1987). 16. K. T. Suzuki, A. Karasawa, and K. Yamanaka, Arch.Biochem. Biophys. 273: 572-577(1989). 17. R. J. Cousins, Physiol. Rev. 6 5 238-309 (1985). 18. I. Bremner, J. Nutr. 117: 19-29 (1987). 19. T.Yamada, T. Agui, Y. Suzuki, M. Sato, And K.Matsumoto, J. Biol. Chern. 268: 8965-8971 ( 1993). 20. C. Vulpe, B. Levinson, S. Whitney, S. Packman, and J. Gitschier, Nature Genet. 3: 7-13 (1993). 21. J. Chelly, Z.Tiimer, T. Tonnesen, A. Petterson, Y. IshikawaBrush, N. Tommerup, N. Horn, and A. P. Monaco, Nature Genet. 3: 14-19 (1993). 22. J. F. B. Mercer, J. Livingston, B. Hall, J. A. Paynter, C.Begy, S. Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, 8.

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Suzuki

D. Siemieniak, and T. W. Glover, Nature Genet. 3:20-25 (1993). 23. P. C. Bull, G. R. Thomas, J.M. Rommens, J. R. Forbes, and D. W. Cox, Nature Genet. 5: 327-337 (1993). 24. K. Petrukhin, S. G. Fischer, M. Pirastu, R. E. Tanzi, I.

Chernov, M. Devoto, L. M. Brzustowicz, E. Cayanis, E. V i d e , J.J.Russo, D. Matseoane, B. Boukhgalter, W. Wasco, A. L. Figus, J. Loudianos, A. Cao, I. Sternlieb, 0. Evgrafov, E. Parano, L. Pavone, D. Warburton, J. Ott, G. K. Penchaszadeh, I. H. Scheinberg, and T. C. Gilliam, Nature Genet. 5: 338-343 (1993).

25. R. E. Tanzi, K. Petrukhin, I. Chernov, J. L. Pellequer, W.

Wasco, B. Ross, D.M. Romano, E. Parano, L. Pavone, L. M. Brzustowicz, M. Devoto, J. Peppercorn, A. I. Bush, 1. Sternlieb, M. Pirastu, J.F. Gusella, 0. Evgrafov, G. K. Penchaszadeh, B. Honing, I. S. Edelman, M. B. Soares, 1. H. Scheinberg, and T.C. Gilliam, Nature Genet. 5: 344-350 (1993). 26. F. Portillo, and R. Serrano, EMBO J., 7, 1793-1798 (1988). 27. D. M. Danks, J. Camakaris, S. Herd, J.R. Mann, and M.

Phillips, Copper Transport inMenkes' Disease, in Biological Aspects of Metals and Metal-related Diseases. (B. Sarkar, Ed.), Raven Press, New York, 1983, pp. 133-146. 28. S. Kanno, J. S. Suzuki, Y. Aoki, and K.T. Suzuki, Res. Commun. Chem. Patlwl. Pharmacol. in press (1994). 29. J. Koropatnick, and M. G. Cherian, Biochem. J. 296 443-

449 (1994). 30. S. H.Hahn, M. L. Brantly, C. Oliver, M. Adamson, S. G. Kaler, and W. A. Gahl, Pediatr. Res., 35: 197-204 (1994). 3 1. K. T.Suzuki, S. Kanno, S. Misawa, and Y.Aoki, Toxicology, in press (1994). 32. M. Sato, N. Hachiya, Y. Yamaguchi, J.Kubota, Y. Saito, Y.

Fujioka, H. Shimatake, Y. Takizawa, T. Aoki, Life Sei., 53: 141 1-1416 (1993). 33. S. Kanno, Y. Aoki, J. S. Suzuki, N. Takeichi, S. Misawa, and K. T. Suzuki, J. Inorg. Biochem., in press (1994).

Index Antimutagenic effects, 87 Antioxidant, 21,22 Arsenite, cytotoxicity of, 93 Ascorbate, 37 ATPases, 5,305,356,357,366, 444,468 Bleomycin: cobalt, 190 copper, 190 iron, 185 Blotchy mouse, 445 Brindled mouse, 445 British Anti-Lewisite(BAL),368 Cadmium, 3 , 9 C-fos, 3, 13 c-jun, 3, 13 c-myc, 3, 13 Calf thymus DNA, chromium binding to, 43 Calmodulin, 2,13 Catalase, 22,102,134 Catecholamines. 329,332,339 Ceruloplasmin, 276,309,319,332, 364,446 Chelation therapy, 368 Chernobyl children, 21 Chromatin, 102,132 oxidative DNA modifications, 132 Chromium(VI), 37,39 485

Chromosome aberrations,25,26 Cobalt, 69 copper: copper deficiency, 305 copper exchange, 307 copper transport, 324 excretion, 333,365 toxicosis, 277 Copper histidine: bioavailability,312 biochemical response to, 330 formulation for treatment, 308 in human serum, 306 Menkm disease treatment, 305, 317,323 stability constant, 306 structure, 306 transport, 324 uptake, 323 Cross-linking, 69,70 CytochromeC oxidase, 275,312, 318,324,380 Cytochrome P-450,156 Cytotoxicity, 102, 121 D-penicillamine,329,340 DNA cleavage: by cationic metalloporphyrins, 153 by i r o n 0 EDTA, 217 by iron finger,243

486

DNA damage: by benxne metabolites, 133 by caffeic acid, 139 by copper, 103 by H202,135 by iron.103 metal-induced, 101 nickel-induced,64 by non-mutagenic carcinogen, 145 by o-phenylphenol, 136 oxidative, 131 by pentachlorophenol, 141 by tryptophan metabolites, 143 DNA depurination,80 DNA hypermethylation,53 64 DNA methylation, nickel-induced, DNA-protein complex, 217 DNA-protein crosslinks, 53 DNA strand breaks,90 Dopamine-p-hydroxylase,275,324, 339,340,312 Ehlers-Danlos type V, 297 Endoplasmic reticulum,57,277 Esterase D, 345 Estrogen receptor, 239 Fenton reaction, 101,112,132,154, 219,229 Footprinting, 427 Free radical,21,22, 101,275 Free radical scavenging, 87 Gene amplification, 88 Gene expression, 2, 13,278

Index

Gene regulation, 102 Genetic toxicology: Hg2+, 3 lead, 3,9 Genotoxic carcinogens,53 Genotoxic effects,73 Genotoxicity: chromium0, 37 cobalt@), 69 nickel(@, 69 oxygen radical,102 Glutathione peroxidase,22,102 Glutathione reductase, 22 Hepatitis, 363 Heterochromatic DNA,53.57 HIV-1, 167,255 Holliday junction, 227 Hydroxyl radical, 22,71, 101, 113, 154, 186,219,224,229,243 Indian childhood cirrhosis, 275 Iron@) EDTA, 217 Irradiation, 22 Kaiser-Fleischer ring,345,368 Lanthanideo, as synthetic nuclease, 173 LEC rats, 355,445,467 Leukocyte, 24 Linkage analysis, 277 Lipid peroxidation, 121 Lipid peroxides,73 Lipoic acid, 23

487

Index

Lou Gehrig’s disease, 380 Lysyl oxidase, 276,312,318 Macrocyclic complexes, 174,201 Mannitol, 308,318 Maxam-Gilbert sequencing, 202 Menadione toxicity, 92 Menkes disease, 275,285,305,307, 323,325 380,444,468 Menkes disease gene, 275 Menkes disease therapy, 305 Metal regulatory elements, 367,398, 425 Metallothionein, 3, 13,87, 101,276, 364,379,425,443,454,467, 469,472 Metallothionein gene,397,411 Metallothionein mRNA, 479 Missing nucleoside experiment, 220 Mottled mouse, 293 MRE, 426 =binding factors: MEP-1,425 MTF, 406,411 W ,406 ZRF,401 MREbindmg proteins,4.40 Mutagenesis, 89,102,101,279 Ni(cyclam), 211 Nickel, 53,69,201 Nickel-promoted oxidation, 202 Nuclear: control of calcium, 4 Wet, 3 uptake of metals, 9

Occipital horn syndrome,288,292 Oxidative damage,l02 Oxidative stress, 96 Oxygen radicals, 53 Phagocytosis, 53 Positional cloning, 277 Protein-protein dimerization, 237 Protein+rotein interaction, 256 P-type ATPase, 280,285,305,356, 366,444 Retinoblastoma, 345 Retinoic acid receptor, 239 Retinoid X receptor, 239 Reverse transcriptase,3 RNA cleavage, 176,201 RNA polymerase,3 Sarcoplasmic reticulum, 13 Selenite, 124, 125 Southwestern analysis, 432 Spl, 417,433 Splicing mutations, 319 Steroid hormone receptor, 237 Strand break, 38,44,53,69,80, 121, 157, 185, 196 Superoxide, 22,71, 122 Superoxide dismutase, 22, 102, 134, 276,380,446 Superoxide radicals, 112 Tat protein, 255 =-DNA complex, 224 Toxic milk mouse, 443

488

Index

Transactivation response element

PAW,256 Transcription factorIIIA, 217 Transcriptional regulation: ACEl,385,407 ACE2.385 AMTl, 386 CUPl, 384 MEP-l, 425 MTF, 406.41 1 ZAP,406 ZRF,401,407 Translocation, 276,277,278 Triplex DNA, 227 Tyrosinase, 276, 312,318

UV cross-linking analysis,435

Vitamin C, 102 Vitamin E, 23,102 Wilson disease, 275,281,343,345, 354,361,365,380,444,468 Wilson disease gene,345 Wilson disease therapy,361

X chromosome, 277 X-linked copper disturbances, 286 Yeast artificial chromosomes,278, 347

Zform of DNA, 227 Zif268,224 Zinc finger, 224,237,243,255,366, 407,412 metal replacement,237,267 Zinc therapy, 361,368

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