ADVANCES I N CANCER RESEARCH VOLUME 1 1
Contributors to This Volume Joseph C. Arcos
Saul Kit
M a r y F. Argus
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ADVANCES I N CANCER RESEARCH VOLUME 1 1
Contributors to This Volume Joseph C. Arcos
Saul Kit
M a r y F. Argus
Sidney S. Mirvish
D. Keast
William Regelson
ADVANCES IN CANCER RESEARCH Edited by
ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital, London, England
SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania
Volume 7 7
ACADEMIC PRESS
NEW YORK AND LONDON
COPYRIGHT
@ 1968, BY ACADEMIC PRESS, INC.
ALL RIQHTS RESERVED. NO PART OF T H I S BOOK MAY BE REPRODUCED I N ANY FORM,
BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM T H E PUBLISHERS.
ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published b v ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W.l
PRINTED I N T H E UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 1 1 Numbers in parentheses refer to thc pagcs on which tlic authors’ contributions begin.
JOSEPHC . ARCOS,Seamen s Memorial Research Laboratory, U . S. Public Health Service Hospital, N e w Orleans, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, N e w Orleans, Louisiana (305)
MARYF. ARGUS,Seamen s Memorial Research Laboratory, U . S. Public Health Service Hospital, N e w Orleans, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, New Orleans, Louisiana (305) D. KEAST,Department of Microbiology, University of Western Australia, Perth, Western Australia (43) SAULKIT, Division of Biochemical Virology, Baylor University College of Medicine, Houston, Texas (73) SIDNEYS. MIRVISH,Department of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel; and the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin ( 1 ) WILLIAMREGELSON, Division of Medical Oncology, Department of Medicine, Medical College of Virginia, Richmond, Virginia (223)
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CONTENTS CONTRlRUTORS To V O L U M E
11
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CONTENTS OF PREVIOVS VOLIJMl<S
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The Carcinogenic Action and Metabolism of Urethan a n d N-Hydroxyurethan SIDNEY
I . Introduction . . I1. TTrethan . . . I11. N-Hydroxyurrthan IV . Urinary Metabolites V . Conclusions . . Refercnces . .
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1 3 26 31 34 35
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Runting Syndromes. Autoimmunity. and Neoplasia
D . KEAST
I . Introduction . . . . . . . . . I1. Homologous Disease and the Clinical Syndromc I11. Situations Which Have Been or Could Be Classified Runting Syndromes . . . . . . . I V . Tlic Important Fcatiirtls of Riinting . . . V . Discussion . . . . . . . . . V I . Comments . . . . . . . . . Rcferenccs . . . . . . . . . . . . . . . . . . Addendum
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Viral-Induced Enzymes and the Problem of Viral Oncogenesis
SAULKIT I . Introduction . . . . . . . . . . . . . I1. Virus-Indiiccd Antigen Syntlicsis . . . . . . . . I11. Viral-Inductd Enzymes of D(.osyl.il~oiiiicIt,ic.Acid M~~t:~l)olisiii . IV Viral-Induced Enzymes That Hydrolyze or Modify . . . . . Deoxyribonucleic and Ribonucleic Acids . V . Viral-Induced Riboniicloic Acid Synt.lirtase (Replicase) . . . \'I . Eff(ds of Virus Infection on Host-Cell Nucleic Acid aiid Protein Syntlirsis . . . . . . . . . . VII . Biorlic.iiiicul Asllihcts of Vir:tl Oiicogcric4s . . . . . .
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CON TEN 'I'S
Vlll
The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology
WILLIAMREOELSON I . Introduction . . . . . . . . . . . I1. Biological Evidence . . . . . . . . . I11. Growth Control . . . . . . . . . . IV . Radiation . . . . . . . . . . . . V . Morphological Alteration . . . . . . . . VI . Cell Membrane . . . . . . . . . . VII . Surface Charge . . . . . . . . . . VIII . Adenosine 5'-Triphosphate and Polyanions . . . . I X . Calcium . . . . . . . . . . . . X . Adhesion . . . . . . . . . . . . X I . Polysaccharides . . . . . . . . . . XI1. Colloidal Effects . . . . . . . . . . XI11. Hydrophilic Gels . . . . . . . . . . XIV . Surface and Enzyme Activity . . . . . . . XV . Enzyme Inhibition and Activation . . . . . . XVI . Respiratory Enzymes . . . . . . . . . XVII . Hyaluronidase and Glycosidases . . . . . . XVIII . Ribonuclease . . . . . . . . . . . X I X . Deoxyribonuclease . . . . . . . . . X X . Polyphosphatm . . . . . . . . . . XXI . Lipase and Esterase Activity . . . . . . . XXII . Clinical Antimitotic Side Effects, and Clinical and Experimental Antitumor Activity . . . . . XXIII . Summation . . . . . . . . . . . References . . . . . . . . . . .
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223 226 234 241 242 244 246 247 251 257 259 265 268 271 275 278 279 280 282 283 284 286 287 287
Molecular Geometry and Carcinogenic Activity of Aromatic Compounds N e w Perspectives
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JOSEPH C . ARCOSAND MARYF. ARGUS I . Introduction . . . . . . . . . . . . . I1. Condensed Polycyclic Aromatic Compounds . . . . . I11. Conjugated Arylamincs and Compounds Generating Arylamines . Arylhydroxylamines . . . . . . . . . . . IV . Covalent Binding to Proteins and Nucleic Acids . . . . Rcferences . . . . . . . . . . . . .
. SUBJECTINDEX .
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305 308 363 433 454
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CUMULATIVE INDEX .
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AUTHOR INDEX
514
CONTENTS OF PREVIOUS VOLUMES Carcinogenesis and Tumor Pathogenesis I . Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogcn Metabolism in Cancer Leonard D . Penninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards calvin T. ~l~~~ and jeanne c. Bateman Genetic Studies in Experimental Cancer L. W . L~~ The Role of Viruscs in the Production of Cancer C . Oberling and M . Guerin Experimental Canccr Chemothcrnpy C . Chester Slock
Volume 1
Electronic Configuration and Carcinogencsis C . A . Coulson Epidermal Carcinogcnesis E . V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis ?'. U. Gardner Properties of the Agent of Rous NO. 1 Sarcoma R. J . C . Harris Applications of Radioisotopes to Studics of Carcinogencsis and Tumor Metabolism Charles Heidelberger The Carcinogenic Aminoazo Dycs James A. Miller and Elizabeth C. Miller The Chemistry of Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Silverstone Plasma Proteins in Cancer Richard J . Winzler AUTHOR INDEX-SUBJECT
AlJTIIOIl INDEX-SUBJECT
INDEX
Volume 3
Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P . Morris Elrctronic Structurc and Carcinogenic Activity and Aromatic Molecules: New Developmcmts A . Pullman and B . Pullman Some Aspects of Cnrcinogcncsis P . Rondoni Pulmonary Tumors in Experimental Animals Michael B . Shimkin
INDEX
Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M . Badger
ix
X
CONTENTS 017 I’REVIOUS VOLUMES
Oxidative Mrlabolism of Nwplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT
INDEX
Volume 4
Advances in Chcmoth-iapy of Cancer in Man Sidney Farber, Rudolj Toch, Edward Manning Sears, and Donald Pinlcel The Use of Myleran and Similar Agents in Chronic I~enltrmias D . A . G. Galton The Employment of Mctliods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian Organism Abiaham Goldin Some Recent Work on Tumor Immunity P. A . Gorcr Inductive Tissue Interaction in Development Cliflord Grobstein 1,ipids in Cancer Frances L . IIaven and W . I$. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Propertics of Angular Brnzacridines A. Lacassagne, N . P. Buu-Hoi, R. Daudel, and F. Zajdela The Hormonal Genesis of Mammary Cancer 0.Miihlbock AUTHOR INDEX-SUBJECT
INDEX
Volume 5
Tumor-Host Relations R. W . Begg Primary Carcinoma of the Liver Charles Bemnan Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N . Campbell
Tlie Ncwcir Concept of Cancer Toxin
War0 Nakahara and Fumiko Fukuoka Chcmically Induced Tumors of Fowls P. R . Peacock Anemia in Cancer Vincent E . Piice aiid Robert E . Greenfield Specific Tumor Antigens L. A. Zilber Clirniistry, Carcinogenicity, and Mctabolism of 2-Fluorenaminc and Related Compounds Elizabeth K . Weisburger and John H . Weiibeiger AUTHOR INDF.X-S~lR~lF~CT INEEX
Volume 6
Blood Enzymes in Cancer and Other Diseases Oscar Bodansky T l i ~Plant Tumor Problem Armin C . Braun and Henry N . Wood Cancer Chcmothrrapy by Perfusion Oscnr Creech, Jr., and E d u m d T . Krementz Viral Etiology of Mouse 1,cukcmia Ludwik Gross Radiation Chimcras P. C . Koller, A . J. S. Dairies, and Sheila M . A . Doak Etiology and Pathogenesis of Mouse Leukemia J. F. A . P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. iM. Timmis Behavior of Liver Enzymes in Hcpatocarcinogenesis George Weber ATJTIIOR INDEX-SIJBJECT
INDEX
Volume 7
dvian Virus Growths and Their Etiologic Agents J . W . Beard
xi
CONTENTS O F PREVIOIIS \'OI,CJMES
Mcchanisins of Hcsistanve to Antic*anc.cr Agents R . W . Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W . M . Court Brown and Ishbel M . Touah Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hails L. Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT
Volume 9 Urinary Enzymcs and Thrir Diagnostic Valuc in Human Cancer Richard Stambaugh and Sidney Weinhouse The Relation of thc Immune Ilraction to Cancer Louis V . Caso Amino Acid Transport in Tumor Cells R. M . Johnstone and P . G. Scholefield Studies on the Development, Biocheniistry, and Biology of Experimental Hepatomas Harold P. Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells I . F . Seitz AUTHOR INDEX-SUBJECT
INDEX
INDEX
Volume 10
The Structure of Tumor Viruses and Its Bcaring on Their Relation to Viruses in General A . P . Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Steele Nncleolar Chromosomes: Structures, Interactions, and Perspectives M . J . Kopac and Gladys M . Mateyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Mctabolites H . F. Kraybill and M . B. Shimkin Experimental Tobacco Carcinogenesis Ernest L. W v n d e r and Dietrich H o f f mann
Carcinogens, Enzyme Induction, and Gene Action H . V . Gelboin I n Vitro Studies on Protcin Synthesis by Malignant Cells A . Clark Grifin The Enzymic Pattern of Neoplastic Tissue W . Eugene K n o x Carcinogenic Nitroso Compounds P . N . Mngee and J. M . Barnes The Snlfhydryl Group and Carcinogenesis J . S. Flarington Thv Treatment of Plasma Ccll Myeloma Damel E . Bergsagel, K . M . Grifith, A . Haut, and W . J . Stuckey, Jr.
AUTHOR INUEX-SUBJECT
AUTHOR INDEX-SUBJECT
Volume 8
INUKX
INDEX
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THE CARCINOGENIC ACTION AND METABOLISM OF URETHAN AND N-HYDROXYURETHAN' Sidney S. Mirvish Deportment of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel; and the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin
I. Introduction . . . . . . . . . . . . . 11. Urethan . . . . . . . . . . . . . . A. Carcinogenic Effects . . . . . . . . . . B. Biological Activity of Compounds Related to Urethan . . C. Relationships with Nuclcic Acid Metabolism and Other Effects of Possible Relevance to Carcinogenesis . . . . . D. Metabolism of Urethan . . . . . . . . . 111. N-Hydroxyurethan . . . . . . . . . . . A. Chemistry . . . . . . . . . . . . . B. Effects Other than Carcinogenesis . . . . . . . C. Carcinogenic Effects . . . . . . . . . . D. Metabolites in Blood and Tissucs . . . . . . . IV. Urinary Metabolites . . . . . . . . . . . A. Urethan . . . . . . . . . . . . . B. N-Hydroxyurethan . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .
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I. Introduction
Urethan (ethyl carbarnate; NHz.COOEt) has long been known t o cxhibit a narcotic action and was used for many years as an anesthetic in man. A related effect is its inhibition of sea urchin egg cleavage, which has also bccn known for many years (Cornman, 1950, 1954). I n 1943, Ncttleship et al., while examining the effects of irradiation under uretlian ancsthesin, discovered that injcctions of urethan rapidly induce lung adenomas in mice. For many years the carcinogenic action of urethan was believed to be confined to the lung. Much attention was meanwhile focused on its cytotoxic :rnd anticancer actions. It was found that urethan produced chromosome damage, especially in the rapidly dividing cells of 'This review was begun while the author was an Eleanor Roosevelt Fellow a t the University of Wisconsin, and finished at the Weizmann Institute of Science with the nid of n grant from the International Agency for Research on Cancer. 1
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SIDNEY S. MIRVISH
thr intehtincs ( I h s t i n , 1947; Boyluncl niitl Kollcr, 1954), induced leukopenia (Skipper ef al., 1949; Moeschliii and Bodiner, 1951) , showed antiIcukeinic activity in rodents (Skippel‘ r t a/., 1949), and antitumor action against the Walkcr carcinoma 256 i n rats (Haddow and Scixton, 1946). Urethan was also tested as an anticancer agcnt in man (reviewed by Haddow, 1963) and has been used in particular to treat multiple myeloma (Loge and Rundles, 1951). Finally, urethan is mutagenic for Drosophila (Vogt, 1948) and Escherichia coli (Bryson, 1949) but not for Neurospora (Rogers, 1955). Some of thcse effects are discussed later in more detail. I n 1953 it was shown that urethan is not uniquely a lung carcinogen, as it initiated skin tumors in mice, as revealed when the skins were subsequently painted with croton oil (Graffi e t al., 1953; Salaman and Roe, 1953). Later developments showed that urethan is a “multipotential” carcinogen (Tannenbaum, 1964) and can induce many types of tumor, notably malignant lymphomas of the thymus, hepatomas, mammary carcinomas, and hemangiomas. These tumors were particularly prominent when repeated doses of urethan were administered, by injection or in the drinking water, and when newborn or very young mice were treated with the carcinogen. Also, most of these tumors were only observed whcn the mice were maintained for a t least 1 year after treatment, whereas lung adenomas mostly develop after 2 to 6 months (malignant lymphomas also show a relatively short latent period of 4 to 12 months). Urethan is structurally one of the simplest carcinogens; it is soluble in both water and lipids, and, in fact, was the first water-soluble carcinogen to be discovered. Urethan readily sublimes even a t room temperature, so that weighing small amounts is not easy. It contains amide and ester groups modified by resonance between each other and is readily hydrolyzed by hot acid and alkali to give NH,, CO,, and ethanol, and also cyanate under alkaline conditions; but it shows few other specific chemical reactions. The chemistry of carbamates was reviewed by Adams and Baron (1965). I n this review the carcinogenic actions of wethan will be discussed separately for each type of tumor, with emphasis on possible mechanisms of action, and then the chemical specificity for some of the varied actions of urethan will be examined. Certain metabolic effects of urethan, and the metabolism of urethan itself, will be discussed in relation t o its carcinogenic action. It was recently proposed that urethan might act after metabolic conversion into N-hydroxyurethan (HONH .COOEt) , and, accordingly, the biological actions of N-hydroxyurethan and its metabolism will be discussed, and reasons will be given for the provi-
URETHAN AND N-HTDROXYURETHAN
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sional rejection of this hypothesis. N-Hydroxyurethan is also of interest as it and related hydroxylamine derivatives show certain biological effects probably depending on interactions with deoxyribonucleic acid (DNA) metabolism. The author is indebted t o reviews by Haddow (1963) on the carcinogenic and other effects of urethan, which may be referred t o for various effects not discussed here, and by Shimkin (1955) on lung tumorigenesis, by Handschumacher and Welch (1960) on nucleic acid metabolism, and by Tannenhaum ( 1964) on the “multipotential” carcinogenic actions of urethan. I I . Urethan
A. CARCINOGENIC EFFECTS 1 . Lung Adenonins
I n the classic investigation I y Nettlesliip ef nl. (1943) it was found tliiit multiplc injections of u r e t h n into C3II mice a t the anesthetic dose of 1 mg./gm. body weight induced a 75% incidence of lung adenomas after 7 months (in most work discussed in this review, a single dose of urethan consisted of 1.0 to 1.5 mg./gm.). Strain A mice were originally bred for spontaneous development of lung adenomas, which occurs in 75% incidence a t an age of 18 months, but multiple injections of urethan induced 100% incidence, with an average of 50 adenomas per mouse appearing after 6 months. Most of these adenomas were 2-3 mm. in diameter a t this time, and situated just beneath the pleura. The spontaneous incidence at the same age was 10%. The early work on lung tumorigenesis by urethan, mostly in A and C3H mice, was summarized by Shimkin (1955). It was shown that there is no sex difference, that urethan is active whether injected or given by mouth, and that the tumor yield is proportional to the number of doses (Henshaw and Meyer, 1944, 1945; Larsen, 1946; for recent dose-response curves, see Kaye and Trainin, 1966; Shimkin e t ul., 1966). Similar tumors had previously been induced by intravenous injections of carcinogenic hydrocarbons (see Shimkin, 1955). Urethan also induces lung adenomas when painted on the skin (Cowen, 1950h; Roe and Salaman, 1954; Berenblum and Haran-Ghera, 1957a) or administered in aerosols (Otto and Plotz, 1966). Anesthetics other than urethan were not carcinogenic (Larsen, 1946), and lysergic acid diethylamide, which prevents urethan anesthesia, did not affect its wrcinogenicity (Berenblum et nl., 195911). Althougli t h v tuiriois arc. o f t c w siiiall nix1 do not inctastasize, so111c~later evolve iiito c:irciiioni:ts (Allen, 195G). Also, tlic :id(wom:is (#:in I)e trans-
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SIDNEY S. MIRVISH
planted and may then develop sarcomatous areas (Klein, 1957). I n large doses urethan also induces a low incidence of lung adenomatoses and lung squamous cell carcinomas (Tannenbaum and Maltoni, 1962; Deringer, 1965). Swiss and BALB/c mice show a fairly high spontaneous incidence of lung tumors and are fairly sensitive to lung tumorigenesis by urethan (Law, 1954; Berenblum et al., 1959a; Trainin et al., 1964), e.g., in adult Swiss mice a dose of 1 mg. urethan/gm. induces about three lung adenomas per mouse after 40 weeks (Berenblum e t al., 1959a). Six mouse strains showed correlations between their susceptibilities t o spontaneous, hydrocarbon-induccd, and urethan-induced lung adenomas, with the order of susceptibility A > Swiss > CBA > DBA (Shapiro and Kirschbaum, 1951). I n an investigation by Cowen (1950a) the order was A > CBA > C57BL (the last strain was originally bred for resistance to spontaneous lung tumorigenesis) . The parallel susceptibilities to spontaneous and urethan-induced lung tumors indicate that these mouse strains differ in some parameter which is not peculiarly related to urethan, e.g., the parameter is unlikely to involve differences in the metabolism of urethan. I n genetic experiments, Cowen (1950a) found that crosses between the resistant C57BL and sensitive A strains showed a susceptibility to urethan lung tumorigenesis rather less than that of A mice, and backcrosses with C57BL mice showed segregation into two groups, as expected from Mendelian genetics. Similar results were obtained by Falconer and Bloom (1962; Bloom, 1964; Bloom and Falconer, 1964), except that the sensitivity of the F, and A mice were found to be almost identical. These results indicate that A mice possess a single dominant gene responsible for most of the strain difference, as was prcviously indicated for spontaneous lung tumorigenesis (Bittner, 1938). A heterogenous mouse strain was selectively bred within nine generations t o give two lines showing large differences in their susceptibility to urethan (Falconer and Bloom, 1964). Urethan is particularly suitable because of its even distribution throughout the body (see later) for use in transplantation experiments. Thus Shapiro and Kirschbaum (1951) implanted mouse lung tissue of the susceptible albino and resistant DBA strains into the ears of F, hybrids, and then injected urethan into the hybrids. They found that 12 of 17 albino grafts developed tumors, but oidy 1 of 17 DRA grafts, showing that susceptibility to urethan is mainly an intrinsic propcrty of the lung tissue and not of the whole animal. Then hlalmgren and Saxen l tissue from strain A mice was (1953) showed that ~ h c nn ~ i * n i n lung transplnntcd subcutaricously into host micc previously treated with
U 1 W ~ ' I I A N AND N-IITDROXYURETHAN
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urethan, adenomas developed in the transplants only when the urethan was injected less than 24 hours beforehand, proving that the active carcinogen must have disappeared after this period. Conversely, when lung tissue was transferred from mice previously treated with urethan to untreated mice, 22% of the transplants developed tumors, confirming that urethan acts directly on the lungs. Transplantation experiments by Rogers (1955)attempting t o demonstrate formation of an active urethan metabolite are discussed later. Histological studies have shown that urethan-induced lung tumors mostly arise from the alveolar epithelium in mice (Mostofi and Larsen, 1951 ; Brachetto-Brian, 1951.; Klarner and Gieseking, 1960; Svoboda, 1962; Driessens e t al., 1963) and rats (Rosin, 1949),unlike human lung cancer which usually arises from the bronchial epithelium. As an example of the observable short-term effects, Brachetto-Brian (1951) showed that in Swiss mice, urethan induced necrotic pycnosis of most of the septa1 alveolar cells within 24 hours, followed after 48 hours by proliferation of these cells, which later showed polymorphism and nuclear atypia. The first adenomas appeared after 24 days. Shimkin and Polissar (1955) described the development in the lungs of urethan-treated A mice of numerous hyperplastic foci after 3 to 5 weeks and of tumors after 3 t o 7 weeks, though it was not clear whether the tumors arose from the hyperplastic foci. I n confirmation of the histological findings, radioautography of the lungs after injection of tritiated thymidine revealed a 70% suppression of mitosis 1 day after urethan treatment, followed by a rise to 3 times the normal value on the fourth to seventh days (Foley e t aE., 1963). I n an important in vitro study, treatment of lung organ cultures with urethan led to a loss of loose connective tissue and a slowing of epithelial budding, and the latter was restored to normal by contact with untreated mesenchyme of lung or submandibular gland (Globerson and Auerbach, 1965). Immunological factors appear to be involved in lung tumorigenesis, as the yield of lung adenomas in Swiss mice after injecting urethan or feeding 7,12-dimethyl-1,2-henzanthracenewas increased by neonatal thymectomy (Trainin e t al., 1967). Contrary to a brief report by Imagawa e t aZ. (1957),Casazza et al. (1965) found that infection with influenza virus a t thc tinie of urethan injection did not enhance lung tumorigenesis, which was consirlercd not surprising since the virus mainly affects the bronchial epithelium, whereas urethan affects the alveolar cpithelium. l'uiiitiiig the mouth with cig:irette tar was stated by Di Paolo an(l Pht~c~lic(19G2) to eiiliance uret1i:iii lung tumorigenesis, but the tar alone w t h tiimoi*igchnic,:Ind a ~yrrc~i~gistic (i.e., inorc than additive) effect was not clcarly tlrmon~trated.
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SIDNEY S. MIRVISH
Several reports have appeared on the effects of varying the oxygen pressure on urethan lung tumorigenesis in strain A mice. Thus tumorigenesis was increased by exposing mice to 70% oxygen for 2 days after injection of the urethan (Di Paolo, 1959), and it was also increased in the progeny of pregnant mothers exposed to 10% or 100% oxygen after injecting the urethan, provided that the urethan was injected less than 24 hours before parturition (Di Paolo, 1962). Also, tumorigenesis by urethan was reported by Mori-Chavez (1962) to be increased a t high altitudes in Peru, but was not affected by exposure of the mice to reduced pressure in decompression chambers (Ellis et al., 1966). Some of these effects could be due to changes in the rate of urethan catabolism (see later). Large doses of X-rays have the effect of suppressing lung tumorigenesis by urethan (Gritsiute, 1961; Duplan et al., 1962; Foley and Cole, 1963). I n the last report, a lethal dose of 880r X-rays was administered 3-24 hours after the injection of urethan into (C57BL x A)F, mice, and this suppressed lung tumorigenesis (expressed as tumors per mouse) to 18% of the value for urethan alone. Syngeneic bone marrow was injected after the irradiation to protect the mice. Some effect was observed when the mice were irradiated a t any time from 8 weeks before to 1 week after the urethan injection, and the irradiation was moderately effective when fractionated into 9 X 100 r, or given as a single dose of 500 to 700 r, without protection by bone marrow (Foley and Cole, 1964, 1966). The effect was prevented by shielding the thorax during the irradiation (Foley and Cole, 1964) and was apparent in only one lung when this lung alone was irradiated (Duplan et al., 1962). Finally, irradiation suppressed the urethan-induced hyperplasia in the lung, as measured by the incorporation of tritiated thymidine (Foley et al., 1963). These results suggest that irradiation inhibits urethan tumorigenesis by a direct action on the lung, probably by prcventing proliferation of the cells sensitive to urethan. Small “subliminal” doses of X-rays followed by urethan produced a synergistic increase in lung tumorigenesis, so that irradiation may have opposite effectsdepending on the dose (Cole and Foley, 1966).
3. S k i n Papillomas I n 1953 it was found that skin papillomas develop on application of urethan to the skin, followed by repeated painting with croton oil (Graffi et al., 1953; Salaman and Roe, 1953; Berenblum and Haran, 1955), so that urethan is here an “initiator” according to the terminology of Berenblum and Shubik, in which the carcinogenic hydrocarbons acted as “initiators” and croton oil as “promoters” of skin tumorigenesis. The same effect was obtained when uretltan was administered by irioutli or Iiy in4jcctioit (1T:tr:tn and Rerenbhittt, 19Fi6; Bet*cnhluniaud €Int~an-Ghcr:t,
URETJ3AN AND N-HYDROXYTJRETI-IAN
7
1957:i; Ilitchie, 1957). LJrrt1i:tn :i])pli(~ilt o tlw ~ l i i i iwas cqiizilly cffectivc i n acetone or Carbowas solution (Roc, n t ~ lSalanim, 1954) : ~ n dis ahsorlwd directly itnd not l)y helug licakctl : i i i t l sw:illowrd, as collars which prevented licking did iiot affect thc turnor yicld (Berenblum and HarariGhera, 1957a). The tumor yield was proportional to the dose of urethan, both when the urethan was applied to the skin, where a total dose of 240 mg. per mouse induced five papillomas per mouse (Roe and Salaman, 1954), and when administered by stomach tube, where the same tumor yield was induced by 64 mg. per mouse (Berenblum and Haran-Ghcra, 1957a). The latter workers found that the mean latent period fell from 16 to 8 weeks as the total oral dose was raised from 1 to 64 mg. per mouse; and when a total oral dose of 64 mg. per mouse was divided into five or twenty subdoses, the tumor yield fell about 50%. The promotion with croton oil may be delayed for 8 weeks with no change in tumor yield (Berenblum and Haran-Ghera, 1957a), but when the promotion was delayed for 24 to 30 weeks the tumor yield fell about 50% (Roe and Salaman, 1954; Pound, 1966). Promotion may also be carried out with the noriionic detergent, Tween 60 (Van Esch et al., 1958). Only a few skin tumors arc induced by urethan acting alone, and even this low incidence may depend on traumatic injury acting as a promoter (Chieco-Bianchi et al., 1964). Urethan is thus virtually a pure initiator for the skin, unlike the hydrocarbon carcinogens which are complete skin carcinogens a t repeated doses. This difference may be associated with the fact that urethan, unlike the hydrocarbons, produces little hyperplasia of the skin (Salaman and Roe, 1953). Thus experiments with tritiated thymidine showed that, in the epidermis, urethan induced a short-term reduction in DNA synthesis, perhaps associated with tumor initiation, whereas 7,12-dimethyl-1,2-benzanthraceneinduced in addition a long-term increase in DNA synthesis, associated with the hyperplasia (Garcia and Leiva, 1966). I n exception to the general rule, painting of tiitthan alone induced epidermoid carcinomas but not papillomas in linirless hr/hr mice, though no effect was found in the hybrid haired Hr/ hr mice (Deringer, 1962). Pound et nl. (Pound and Bell, 1962; Pound and Withers, 1963; Pound, 1966) applied croton oil to the skin of mice, then injected urethan, and finally carried out the usual promotion with croton oil. By this means they obtained a fourfold increase in tumor yield, as compared with controls where the croton oil pretreatment was omitted. Similar results were obtained by preliminary trcatment with acetic acid and other chemicals, or by scarring, all of which lead to inflammation and cellular proliferation. A critical time interv:d of 15 to 18 hours between the croton
8
SIDNEY S. MIRVISH
oil (or acotic acid) and urethan treatments gave the maximum tumor yield, and this corresponds to thc period of maximum DNA replic at'ion in the epidermis, prior to a burst of mitoses about 27 hours after the croton oil treatment. 3. Maliytiurd LympIwiiLus
The most usual type of mouse leukemia induced by urethan is a malignant lymphoma (lymphosarcoma) originating in the thyinus (Pietra e t al., 1961), as with mouse leukemia occurring spontaneously or induced by other agents. The induction of this neoplasm by X-rays was stated by Kaplari (1964) to be mediated by ( 1 ) release of a leukemogenic virus, ( 2 ) injury to the thymus, and (3) injury to the bone marrow, influencing thymic regeneration. Investigations into urethan leukemogenesis have mainly attempted to elucidate similar questions, and, in particular, (I) the interactions between urethan, X-rays, and viruses as leukemogenic agents, ( 2 ) the question whether urcthan acts directly on the thymus or whether other organs, eg., bone marrow, are involved, and (3)the reasons for the greater effect of urcthan in newborn and very young mice. The first demonstration that urethan is a coleukemogenic agent was provided by Kawamoto et al. (1958), who showed that leukemogenesis by X-rays, estrogens, and 3-methylcholanthrene in adult mice was augmented by simultaneous treatment with urethan. Then, in 1961, it was discovered that newborn mice are particularly sensitive to urethan leukemogenesis, as a single injection of urethan into newborn Swiss mice induced a 20-30% incidence of leukemia within 15 to 30 weeks (FioreDonati e t al., 1961, 1962; Pictra e t al., 1961; Della Porta et al., 1963). The leukemia incidence was 80-1000/0, with a latent period of 20 to 30 weeks, when multiple doses of urcthan were administered to mice of the C57BL (Doell and Carnes, 1962), (C57BL X A/J)Fl (Klein, 1962), dd (It0 e t al., 1964, 1965), and (C57BL X C3H)Fl (Vesselinovitch and Mihailovich, 1966) strains, starting a t birth or 1 week later. The incidence was higher in female than male dd mice, though there was no sex difference in the Swiss mice of Della Porta e t al. (1963), or in C57BL mice examined by Della Porta e t al. (1967). I n addition, Liebelt e t al. (1964) reported that urethan injected into newborn female C3Hf mice induced ft 30% yield of reticulum cell sarcomas (Type A) after 1 year. In adult mice, urethan acting alone usually shows little leukemogenic activity (Kawamoto e t al., 1958; Berenblum and Trainin, 1960), but very large doses of urethan in the drinking water induced 10-30% incidences of leukemia in adult Swiss and C3H mice after 30 to 50 weeks (Toth et al., 1 9 6 1 ~ Tanncnhaum ; and Maltoni, 1962; Della Porta e t al., 1963). Following on thc rcport of I s simplex virus was extrtmely sensitive to actinoniyciii D inhibition; at a drug concentration of 0.5 pg./ml., d T kinasc induction was inhibited by 9776 (Table I V ) . At actinoniycin D concentrations of 1 to 2 pg./nil., induction of the enzyme was virtually undctectable. However, much higher concentrations of actinomycin D wcrc TABLE I V ACTINOMYCIN
D
INHIBITlON OF
THYMIDINE ICINASE
INDUCTlON I N
LM(T1I-) CELLSBY VACCINIAAND HERPESSIMPLEX VIRUSES~ Thymidine kiriase activity (prmoles dTMPb formed per pg DNA in 10 minutes a t 38°C.) Herpes simpleuinfected Actinomyciii D cells Concentration (P&h1.) Exp. a 0 0.5 1 2 4 0 8
356 9
Vacciniainfected cells Exp. b
Exp. c
Exp. d
16.1
-
1 0
0
-
12
12
24
4
Noninfected Lhl(TIC-) (.ells exhibited 110 delecta1)le enzyme activity. Experiment a-actinomycin D was added to the cultures 30 minutes prior to herpes simplex iiifec6oii arid d T kiiiase was assayed 5 hours after infection. Experiments b and c-actinomyciii D was added 1 hour prior to vaccinia infection arid dT kiriase was assayed a t 4 hours after infection. Experiment d-actinomycin D was added 1 hour prior to vaccinia infect l o l l niitl d T kinuse was assayed 5 holm after infection. * Thyinidylate.
rcquired to inhibit tlT kinase formation after vaccinia infcction. Altliougli 2.4 pg./nil. actinomycin D suppressed the incorporation of radioactive uridinc into crllular RNA by 95% or more within 1 hour of drug treatment, 4 pg./nil. :ictinomycin D permitted 18-31 '/. of thc normal amounts of dT kinasc to he induced by vaccinia, and cvcn a t nctinomycin concentrations of 8 pg./ml., significant amounts of dT kinase were made. Actinomycin D bincls t o gu:inine groups of DNA. The :mount of actinoniycin D bound by any single D N A is a function of its guanine
102
SAUL KIT
content. It may be recalled that lierpcs simplex 1)NA cout:lins about 37% guanine, whereas vaccinia DNA has a guanine content of only 18%. Thus, the fact that lower actinomycin D concentrations were required to inhibit d T kinase induction by herpes simplex than by vaccinia may be understood in terms of the differences in their DNA guanine content. The experiments imply that the messenger RNA’s functioning in the induction of d T kinase are different in vaccinia and herpes simplexinfected cells (Kit and Dubbs, 1963b; Kit et al., 19G3d). McAuslan (1963b) has investigated the time of synthesis and the comparative stability of the d T kinase messenger RNA. He observed that if the addition of actinomycin D to cowpox virus-infected HeLa cell cultures was delayed until 2 to 4 hours after virus infection (but not earlier), a maximal rate of d T kinase synthesis took place. Therefore, the synthesis of messenger RNA for d T kinase must have been completed within 2 hours after infection. Not only was d T kinase induced in infected cells to which actinomycin D was added 2 to 4 hours after infection, but the synthesis of the enzyme was not shut off a t 5 to G hours as was normally the case. Instead, enzyme synthesis continued for 18 hours, a result indicating that the messenger RNA for d T kinase was very stable. These observations have been confirmed by Jungwirth and Joklik (1965). I n addition, Jungwirth and Joklik (1965) have shown that the messenger RNA for DNA polymerase is synthesized by 2 hours after vaccinia infection and that the DNA polymerase messenger RNA is also functionally active for relatively long periods of time.
3. RNA Polymerase Activity in Cells Infected with DNA-Containing Viruses The way in which DNA-containing viruses initiate either new RNA or protein synthesis is perplexing since, in order for the virus to begin RNA synthesis, an RNA polymerase must be available, and before a new protein can be made, an RNA messenger must be made to code for the new protein. There are four ways in which viral RNA synthesis can be initiated: ( 1 ) by using a pre-existing host-cell RNA polymerase; ( 2 ) by bringing into the cell an RNA polymerase as part of the virus particle; (3) by using a new induced host-cell RNA polymerase; and ( 4 ) by bringing an RNA into the cell with the virus particle that would serve as a messenger for the synthesis of an RNA polymerase. Mechanisms (S) and ( 4 ) require that a new RNA polymerase be formed in infected cells-a process requiring protein synthesis-whereas mechanisms ( 1 ) and ( 2 ) permit the synthesis of RNA without prior protein synthesis. It is known that RNA polymerase activity is not increased in T-even
phage-infected cells; indeed, the activity of this enzyme is reduced after phage infection (Skold and Buchanan, 1964). Moreover, Jungwirth and Joklik (1965) failed to detect a new DNA-dependent RNA polymerase in the cytoplasm of vaccinia-infected HeLa cells. Using a polyauxotrophic mutant of Escherichiu coli deficient in the ability to synthesize thymine, uracil, and histidine, Sekiguchi and Cohen (1964) studied the synthesis of phage-specific RNA after T6r+ infection. Ribonucleic acid was made in the TGr+-infected E . coli cells in the absence of the required amino acid and also in the presence of chloramphenicol. The newly synthesized R N A had the characteristic base ratio and electrophoretic mobility of nornial phage-induced R N A and it was mainly associated with ribosomes. Incubation of the isolated RNAcharged ribosomes with inorganic phosphorus resulted in the selective degradation of the phage-induced RNA. Furthermore, after charging ribosomes with phage-induced RNA in the absence of protein synthesis, it was found t h a t the addition of the essential aniino acid to infected cells produced a stimulated rate of synthesis of two early enzymes, d C M P hydroxymethylase and d T M P synthetase. However, such prior synthesis of RNA did not significantly affect the rate of synthesis nor even the time of appearance of a late enzyme, lysozyme. The formation of T 4 phage-messenger RNA has been demonstrated in cells pretreated with chloromycetin (Nomura e t nl., 1960, 1962; Okamoto et al., 1962) ; therefore, mechanisms ( 3 ) and ( 4 ) can be ruled out for phages T 4 and T6 since the latter mechanisms presuppose protein synthesis prior to phagemessenger RNA synthesis. I n designing experiments on whether protein synthesis is required for the induction of RNA synthesis by vaccinia virus, Munyon and Kit (1966) made use of the following observations. First, Salzinan et nl. (1964) and Becker and Joklik (1964) had shown that, in noninfected cells, RNA, synthesized after short pulses with radioactive precursors, was localized primarily in the nucleus, hut after vaccinia infection, “pulse”-labeled RNA was found in the cytoplasm. This newly synthesized cytoplasmic RNA had thc base composition of vaccinia D N A and formed specific hybrids with vncciiiia DNA. Second, the induction of d T kinase by vwcini:i w t s highly resistant to :~ctinomycin D inhibition (Tahlc 1 1 7 ) . R/Iouw fil)roliIa>t c c ~ l ln~. c i ~f i~r h t 1)rxd r w t c v l with liigli conccntratiotis of actiiioiiiycin 1) to iiiliibit thc. iiicoi,l)ol.iition of ‘13-iiridiiic into hostcell RNA. B y drastically reduciirg the base line of host-cell RNA syntliesis, it was possible to reveal :i \.accinia-clepcndent stimulation of ?ITuridine 1ncorpor:ition into cytoplaemic RNA. Having established that cytoplasmic RNA synthesis was stimulated
104
SAIII, K I T
in vaccinia-ixifectcd cells pretreated with actirioinycin D, it then bccamc possible to test whether protein synthesis was required for this stirnulation. Cells were incubated with both cyclohexiniide and actinomycin D, washed, then infected with vaccinia in the presence of cycloheximide, and further incubated. The presence of cycloheximide before and during infection prevented 3H-leucine incorporation into LM cell proteins or the induction by vaccinia of d T kinase. However, cycloheximide treatment did not inhibit the vaccinia-dependcnt synthesis of cytoplasmic RNA. Similar results were obtained with cells pretreated with puromycin or p-fluorophenylalanine. It would appear that, as in the case of T-even phage-induced RNA synthesis, vaccinia-stimulated cytoplasmic R N A synthesis did not require prior protein synthesis. Thus, the RNA polymerase which catalyzed the formation of the virus-induced RNA was probably made before infection. It was either a preexisting host-cell enzyme or was brought into the cell as part of the infecting virus particle. Joklik (1964a,b) has shown t h a t FPA and puromycin prevent the disassembly of infecting-vaccinia virus so t h a t the virus D N A is not degraded by DNase digestion. Corroborative work by Dales (1965) has been reported showing that infecting-vaccinia particles do not release their D N A into the cytoplasm of the cell in the presence of Streptovitacin A, another inhibitor of protein synthesis. The results of Munyon and Kit (1966) in conjunction with the conclusions of Joklik (1964a,b) and Dales (1965) suggest that vaccinia infection can initiate messenger RNA synthesis without the disassembly of the virus to the point that viral DNA is degradable by DNase and before the DNA is relcascd into the cytoplasm of the cells. 4. Inhibitors of D N A Synthesis itnd Enzyme Induction Although RNA and protein syntheses are required for the iriductioii of early enzymes by phage and animal viruses, D N A synthesis is not needed. Normal levels of d C M P hydroxymcthylase and FH, reductase were made by dFU-treated Bsclieiichia coli cells after T6rC infection, I n addition, a t least one “late” protein, namely, lysozyme was formed (Sckiguchi and Cohen, 1964). T o learn whether DNA synthesis was required for d T kinase induction by wccinia, mouse fibroblast cells were treated with dFU, niitoniyciii C, or l-~-~-:ii~:il~ii~oFUl‘:111OSY]CYtohil~ (:iix-(’) (Kit Pt ( ~ l . ,1963t1, niitl u i 1 ~ ~ u l ~ l i u 4 l1)dw r ~ ~ : ~ t i. oNoilc* n ~ ) of Ilicsc. (Irugs appreciably rcducc~l (IT kinase foimttiou ~llthougli :ill three drugs stlinost coinl)lctcly inhibited D N A biosynthesis. Experiments with vaccinia-infected HeLa cells and with adenovirus
VIRAL-INDUCED E N Z Y M E S A N D VIRAI, ONCOGENESIS
105
FV15-infected inoiiliey kidney cell+ also showed tlint tlFU :ml ara-C, rvbpectively, inhiliited ncitlier d T kinn>e nor DNL1polymerase formatioiih (Jiingwirth :ind Jolilili, 1964; Kit c t ( I / . , 1!167r).
5 . h irz!/me Indiwtion b y U V LicJiit-Ir,atliatcd Virus Particles
It is generally assumed that DNA lesions account for loss of infcctirity of viruses irradiated with UV light. A comparison has been made of the effects of UV radiation on viral infectivity and on enzymc-indiicing c:tpacity (Dirksen et al., 1960; Flaks et aZ., 1959; Keck e t al., 1960; McAuslan, 1963a). \Vhen bacterial cells were infected with phage inactivated by UV light, the DNA content of cells remained constant, whereas in normal infection, it increased rapidly after an initial lag. Early synthesis of RNA was more resistant to damage caused by UV light than was subsequent synthesis of viral DNA, although both processes were progrcssively impaired (Minagawa et al., 1964; Sekiguchi and Colien, 1964). The induction of dCMP deaminase by phage T2 was also more resistant to UV radiation than was phage infectivity. When tlie irradiation reduced infectivity to 0.21 and 0.78% of normal, respectively, enzynieinducing activity was reduced to only 18 and 47% of normal (Kcck et al., 1960). Heavily-irradiated, T-even phage retained partial ability to induce the formation of d T M P and dGMP kinases, dCTPase, d T h l P synthetase, dCMP hydroxymethylase, and FH, reductasc (Dirksen et al., 1960; Flaks et al., 1959; Kozinski and Bessman, 1961 ; Sekiguchi and Cohen, 1964) . Phage of which the DNA had been heavily labeled with 3zPlost their ahility to form plaques a t a rate proportional to the specific activity of the isotope and tlie amount of DNA in the phage. All available cvidencc suggests that the primary lethal action of tlie 32Pdecay is the destruction of genetic material, presumably by scission of the DNA doublc helix. The capacity of cells infected with the 32P-labelcd phage to foriii early proteins was strongly affected by decay of the incorporated isotopc. However, the rate of decay of the initiation of synthesis of dCMP hydroxymethylase, d H M P kinasc, and dCMP clcaniinase was oiily 40 to 50% of that of tlie rate of loss of infective centers (Ebisuzaki, 1962). Also dGMP kinase-inducing activity was more resistant to 32P decay than was loss of infectivity (Kozinski and Bessman, 1961). As first shown by McAuslaii (1963a), the capacity of poxviruses to induce d T kinase was progressively impaired by irradiating virus particles with increasing doses of UV light. Figure 4 illustrates the finding that the ability to induce dT kinase was considerably more resistant to UT’ radiation than was infectivity. Whereas the synthcsis of d T kinase
106
SAUL KIT
0.01 0
30
60
90 120 UV- irradiation (seconds)
150
180
FIG.4. Ultraviolet (UV) light inactivation of vaccinia infcctivity and ability to induce thymidine kinase in LM(TK-) cells (Munyon and Kit, unpublished experiments). A preparation of vaccinia having an initial titer of 2.3 X 10’ PFU/ml., suspended in media containing 0.1% bovine serum albumin, but no calf serum, was irradiated at 4°C. with a 25-watt General Electric germicidal lamp. At 30-second int,ervals, samples were withdrawn and assayed for infcctivit,y and for thymidine kinase-inducing activity (3 hours after infection). (dT = thymicline.)
was induced by vaccinia virus inactivated with UV radiation, syntheses of DNA polymerase and DNase were not. When the proportion of survivors was d T kinase was still induced but even a t a survival of to very little induction of DNA polymerase and DNase occurred (Jungwirth and Joklik, 1965). Vaccinia virus irradiated with UV light is either not “uncoated” a t all or to a very small extent. The results, therefore, suggest that normally “uncoated” viral genomes cause induc-
VIRAL-ISDUCED ENZYMES AND VIRAL ONCOGENESIS
107
tion of the synthesis of DNA polymerase and DNase, whereas only limited ‘(uncoating” is needed for the induction of d T kinase. Although several early enzymes were induced by UV-irradiated bacteriophage, the formation of coat proteins and of lysozyme was almost completely prevented. Lysozynie is normally made a t about 15 minutes after infection with nonirradiated phage; however, a t 69 minutes after infection, the activity of lysozyme in irradiated phage-infected cells was only 10% of the normal value, i n marked contrast t o dCMP hydroxymethylase in the same extract, which was almost twice as high as that in normal infection. Since early enzymes increased during the entire period of incubation of cells infected with irradiated phage, the failure of late proteins to increase in these cells cannot be ascribed to the overall cessation of protein synthesis a t the later period of incubation (Sekiguchi and Cohen, 1964). 6. Extended E n z y m e Synthesis i n Cells Infected with U V -Irradia t ed Virzises
The pattern of enzyme induction ascribed to the phenomenon of “extended enzyme synthesis” is as follows: Early in the infectious cycle, irradiated virus induces enzyme a t the saiiic rate or nearly the same rate as nonirradiated virus. At later times, the amount of enzyme in cells infected with nonirradiated virus levels off or decreases, whereas the enzyme levels in cells inoculated with irradiated virus continue to increase, frequently surpassing the maximum levels obtained in cells inoculated with nonirradiated virus. Extended synthesis of dCMP hydroxyinethylase was observed by Dirksen et al. (1960) when infection of Escherichia coli was produced by W-irradiated T2 phage. Normally, the synthesis of this enzyme was shut off a t about 12 minutes after infection. With phage irradiated with UV light to to survivors, dCMP hydroxymethylase synthesis continued for about 60 minutes. Ultraviolet irradiation had caused a lesion in some system responsible for the cessation of enzyme formation during normal infection. Formation of dCTPase, dTRlP kinase, and dTMP synthetase also continued beyond the normal period whenever infection was produced by irradiated phage. With these three enzymes, however, the initial rate was significantly decreased a t a radiation level that did not affect the initial rate of liydroxymetliylase formation (Dirkscn et al., 1960; Sekiguchi and Cohen, 1964). It seems that tlie factors responsible for the initial formation of these enzymes were more sensitive to W light t t i i t i i tlie oiie fur tlic furniatioii of liyclroxyinethyl:~~~. T h c initial rate of induction of H I 2 rcrluctase activity Ijy UV-irrxli:ttcd phagc was : h o
108
SAUL
Iirr
UV exposure
0.5 min I .5min
Control (no UV exp.)
2 min
2.5 min
Hours after infection
FIG.5 . Extcndrd thymidine kinase synthesis after inoculation of LM(TK-) cell cultures with vaccinia virus irradiated with increasing doscs of ultraviolet light (Munyon and Kit, unpublished experiments). (dUMP = deoxyuridylate.)
slower, and FH, reductase increased to only one-half the normal level a t 30 minutes after infection. Extended synthesis of dT kinase was observcd by McAuslan (1963a) after HeLa cell infection with UV-irradiated poxviruses. At 24 hours after infection, the d T kinase induced by irradiated virus reached values up to 38 times that of noninfected cells. Experiments illustrating extended synthesis of d T kinase in LM(TK-) cells infected with UVirradiated vaccinia are shown in Fig. 5 . After 1 minute of irradiation (1.13% survival of infectivity), the initial rate of d T kinase induction was almost normal. However, the lcvels of d T kinase reached a t 10 hours were o w r twice thobc attaincd by nonirradiatc(1 virus. With more heavily irradiated vaccinia, both the initial rates and the 10-hour levels of d T kinase artivity w r c subnormal. McAuslan ( 1 963:~) t1rir~onstr:~ted t h t (17’ liinase induction by UV-
iiw(liattv1 cowl)os vii,iis c*onltl Iw sliiit off by s.ul)ei.iiifcctioii witli :I i~cllatetl livr virus. The :iiwlal)ility of niritant inoiisc fil)rol)la~tcells [ LRI (TI< ) 1 , deficient in (IT kinaw :trtivity, an[I of :L iriutant vwcinia strain (Vtk ), deficient in enzyme-inducing activity, 1)erriiittcd a confirmation of McAuslan’s findings in :t system where noninfected and Vtk--infected cells exhibited essentially no d T kinase activity (Fig. 6 ) . Since nonirradiated virus prevented the rxtendcd d T kinaw synthcsi:, by irradiated virus whcn cells were simultaneously infectcd with both viruses, the shutoff mechanism of enzyme synthesis must be cytoplasmicnlly mediated. Irradiation damages some property of poxvirus DNA wliicli affects the repression of enzyme induction hoiiie time after infection. Repression dominates over induction by irradiated virus. also occurs in poxThe observation t h a t extended enzyme synthc
‘oool
Vtk+(UV) infection
-,P I
//
;
i I
I I
In
Vlk’
I
400-
I
infection I
3 200 -
a‘/
I0
3
Vtk’ (UV) and Vtk- infection (sirnu;toneous)
6
9
12
Hours after infection
FIG.6. Inhibition of extendcd enzyme synthesis in LM(TK-) cells simultaneously infected with UV-irradiated parental vaccinia (Vtk’) and a nonirradiated mutant vaccinia (Vtk-), deficient in thymidine kinase inducing activity (Munyon and Kit, unpublished experiments). (dUMP = deoxyuridylatc.)
110
SAUL K I T
virus-infected cclls treated zlt 2 to 4 hours after infection with actinonlycin D suggested an experimental approach toward defining the chemistry of the cytoplasmic repression phenomenon (McAuslan, 1963b). AS actinomycin D blocks the shutoff mechanism, the function of shutoff appears t o be mediated either by some species of RNA or by the product of such an RNA. If thc repressor were RNA, its synthesis should take place even though protein synthesis were inhibited by puromycin ; in this case, removal of puroinycin would not lead to the resumption of enzyme synthesis. If the repressor were protein, it would not accumulate in the presence of puroinycin. I n this case, removal of purornycin would permit enzyme synthesis to continue until the repressor was synthesized. T o test these alternatives, HeLa cells were infected with C O W ~ O X virus; 2 hours later, when synthesis of dT kinase iiiessenger RNA was completed, puroinycin was added. Five and one-half hours after infection, the puroiiiycin block was removed, whereupon enzyme synthesis was resumed a t the preinhibition rate. The repression of enzyme synthcsis did not occur until 4 hours after the release of protein synthesis. Thus, the shutoff mechanism could not be established in the presence of puroinycin, indicating that thc repressor was not simply a species of RNA. Whereas addition of actinomycin D a t 2 hours prevented the shutoff, addition a t the end of the 3.5-hour period of puromycin inhibition failed to prcvent repression, which occurred 4 hours after enzyme synthesis WRS resumed. Therefore, the actinomycin-sensitive step for the establishment of the shutoff must have occurred prior to the addition of actinomycin D, i.e., during the period of puromycin inhibition. Thus, the establishment of the shutoff required the syntheses of both RNA and protein. 7. Extended Enz yine Formation in Cell Cultures Treated with Aminopterin or dFU
It is known that viral D N A is not made by cells infected with UVirradiated viruses. I n order t o inhibit D N A synthesis without affecting the D N A molecule itself, aminopterin or dFU have been applied to infected cells. I n the case of poxvirus-infected HeLa cells, aminopterin treatment resulted in a n impairment of the dT kinase shutoff mechanism, and extended synthesis of d T kinase took place. However, if the infected cultures were incubated with both d T and aminopterin, the inhibition of DNA synthesis was reversed and, concomitantly, d T kinase formation was repressed a t the normal time (McAuslan, 1963a). The addition of dFU to vaccinia-infected HeLa cell cultures also resulted in extended syntheses of d T kinase and of D N A polymerase (Jungwirth and Joklik, 1965). When the dFU-containing media was supplemented
VIRAL-INDUCED ENZYMES A N D \‘IRAL ONCO(;ENE‘IS
11 1
with thymidine, the d F U block of D N A synthesis was overcome and the switchoff of enzyme synthesis took place in the normal manner. Fluorodeoxyuridine inhibits the incorporation of l*C-uracil into DNA of T6r’-infected bacterial cells ; however, the inhibition is not complete. There is no net increase in DNA, but a sniall and significant amount of DNA formation does take place. Possibly, this is because small amounts of d T are generated through the breakdown of bacterial DNA. I n dFUtreated bacterial cells, dCMP hydroxynicthylase and FH, reductase activities increase in a manner similar to normal infection and enzyme synthesis is shut off a t the normal time. There is also a considerable increase in lysozyme, in contrast to the behavior of cells infected with irradiated phage in which the increase in lysozyme activity is inhibited (Sekiguchi and Cohen, 1964). Moreover, phage-structural proteins are made in dFU-treated culturcs a t 33 to 50% of the rate of controls (Ebisuzaki, 1963). Oric can imagine that the transcription of the genes coding for lysozyme actually occurs on thc few replicas of the initial infecting DNA which are formed in the presence of dFU. These results lead t o the conclusion that the cessation of synthesis of early enzymes and inception of synthesis of late proteins require D N A synthesis.
F. MUTANTVIRUS STRAINSDEFECTIVE I N ENZYME-INDUCING ACTIVITY 1. Amber Mutants of Bacteriophage T4
Amber mutants of T 4 are conditional lethal mutants capable of growing on some strains of Escherichia coli K12 (i.e., CR63, a permissive host), but not on E . coli B (a nonpermissive host). Benzer and Champe (1962) have described a similar relationship between certain strains of E . coli and some rII mutants of T4. They suggested that these rII mutants contain a codon which is nonsense in the nonperniissive host, whereas the permissivc host is able to supprcss the mutation making the mutated codon LLiiiissen+e” rather than nonsense. The results obtained by Sarabhai et a l . (1964), using T4 mutants containing amber mutations in genes corresponding to the head protein, suggest that this explanation may apply to :tnibcr mutations, since chain termination occurs during synthesis of the head proteins of E . coli B infected with these mutants. Genetic studics by Epstcin et al. (1963) with ambcr mutants have lr11 to the construction of :t genetic niap for T4 coiitainiiig approximatcly scventy genes oi’ which twenty are believed to function in D N A biosynthesis. Some of the aiiiber mutants (i.e., am N82 and am N122) are completely unable t o reproduce in E . coli B and produce practically no DNA (DO mutants; T:il)le V ) . Other mutants (i.e., a m N81, am N116, and am N130) multiply to a limited extent and phage production is
112
SAUL KIT
TABLE V AMBER MUTANTS O F BACTERIOPHAGE T4 Mutant gene No.
Amber mutant
Mutant type"
Enzyme controlled by gene
1 39 56 41 42 43 43 44 46 47 52 30
am B24 am N l l 6 am E56 am N81 am N122 am B22 am NlOl am N82 am N130 am A456 X 5 am H17 am H39X
DO DD DS DS
Deoxyiiucleoside nioiiophosphute kinase dCTPaseh dCMPc hydroxymethylase DNA polymerase DNA polymerase Unable to cause breakdown of host DNA Unable to cause breakdown of host DNA Polynucleotide ligase
DO DO
DO DO DA DA DD DS
0 DO-no DNA synthesis in Escherichia coli B ; DD-delayed DA-arrested DNA synthesis; DS-some DNA synthesis. Deoxycytidine triphosphatase. c Deoxycytidylate.
DNA synthesis;
roughly related t o the level of DNA production in infected cells. With am N81, DNA synthesis is negligible during the first 20 minutes, but later there is a slow but significant rate of synthesis (DS mutant). With am N116 (DD mutant), DNA synthesis is delayed until approximately 20 minutes and then commences a t a rate comparable t o that observed with cells infected with normal T4 phage (Wiberg e t al., 1962). In the case of am N130, DNA synthesis starts a t a normal rate but stops abruptly a t about 15 minutes after infection (DA mutant). A number of amber mutants have been examined for their ability to induce in E. coli B (the nonpermissive host) the formation of enzymes related to the synthesis of DNA (Warner and Barnes, 1966; Warner and Lewis, 1966; Wihcrg et al., 1962). Amber mutants of T 4 defective in genes 1, 42, 43, and 56, respectively, fail to produce deoxyribonucleoside monophosphate kinase, dCMP hydroxymethylase, DNA polymerase, and deoxycytidine triphosphatase (dCTPase) . Studies with the amber mutants support the hypothesis that phagc DNA synthesis is closely related to the regulation of the synthesis of early enzymes. Iiifcction of E. coli B with DO or DD amber mutants (Table V) generally results in extended synthesis of phage-induced early enzymes. Thus, although mutant an] N122 does not induce thc formation of dCMP hydroxyrnethylasc i n E . coli B, dTMP synthetase, dcoxyribonucleoside inonophosphate kinase, DNA polymerase, dCTPase, H M C
VIRAIA-INDUCED ENZYMES AND VIRAL ONCOGENESIS
113
0-glucohyl t ransferase, antl dCRlP deaniiiiase attain activities two- to fi\clfoltl greater than with wild-type phage, and enzyme synthesis continut~sfor almit 60 minutes after infection. With am N82, all of thew cnzyni~santl :dso dCMP hydroxymethylase are synthesized for an cxtended period of time. With amber mutants am N81 arid am N116, the enzymes are all induced a t higher levels than normal ; however, enzyme synthesis is repressed a t about 20 to 30 minutes after infection when DNA synthcsis commences. Amber mutants am 90 and am H17 induce phage DNA synthesis in E . coli B, although only limited phage production takes place. These mutants are dtfectivc in essential steps other than DNA replication. In the case of am H17 and am 90 and also mutant aiii N130, normal enzyme levels arc inducctl and enzyme synthesis is shut off a t the usual time. Although FH, rcductase activity is induced by a t least t n ~ n t yamber mutants, seldom do the FH, reductase levels induced by the mutants exceed that induced by the wild-type phage, and, in contrast to the other enzymes, extended synthesis is not observed (Warner and Lewis, 1966). These observations suggest that the increase in FH, reductase activity occurring in T4-infected cells may not be subject to the same control as other phage-induced early enzymes. The control of the synthesis of other enzymes is lost if DNA synthesis is not initiated on tiiiit, whereas the control of FH, reductase is unaffected. 2. Temperature-Sensitive Mutants
Temperature-~ensitive(ts) mutants are a second type of conditional lethal mutant. Thc ts mutants generally replicate poorly a t about 40°C. hit do grow a t about 30°C. Among the ts mutants of bacteriophage T4 are two, ts L13 and t s G25W, that contain sites of mutation located in the same gene as that of aniber mutant aiii N122 (gene 42). In infections carried out a t 425°C. with mutant t:: L13, no DNA was made and no dCMP hydroxymethylase WRP detectable. Either the enzyme was not formed a t 42.5"C. or it wa6 rapidly and irrcvcrsihly inactivated a t that temperature. At 30"C., however, mutant ts L13 did induce dCMP hydroxymcthylase activity. With wild-type phage, neither DNA synthesis nor dCMP hydroxymethylase production were impaired a t 42.5"C. (Wiberg and Buchanan, 1964). With mutant ts G25W, just as much dCMP hydroxymcthylase activity was detected with high-temperature infections as with low-temperature infections but very little DNA was synthesized a t 425°C. Although dCMP hydroxymethylase was made a t 42.5"C., the enzyme was very unstable and exhibited other abnormal properties. Probably, mutant ts G25W failed to make DNA a t 425°C.
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SAUL KIT
because the dCMP hydroxymethylase induced by this mutant functioned poorly. Temperature-sensitive mutants defective in gene 43 have been tested for their ability to induce T4 DNA polymcrasc (de Waard et al., 1965). Of the ts mutants, t s L97 and ts 1,107 induced the synthesis of H M C ,8-glucosyl transferase a t 37"C., thus indicating that infection proceeded successfully. Deoxyribonucleic acid polymerase was not produced by cells infected a t 37"C., but the enzyme was formed a t 25°C. The enzyme induced a t 25°C. showed a significantly higher in vitro activity a t 37°C. than a t 25°C. This suggests that there was a thermolabile step in the formation of the tertiary structure of the protein so that a functional protein was formed a t 25°C. but not a t 37°C. With a third mutant, ts L91, equivalent levels of enzyme were produced a t 37" and 25°C. However, activity measured a t 25°C. was nearly twice as great as that measured a t 37°C. The ratio of activity a t 37°C. to that a t 25°C. The QiiZ for this DNA polymerase was 0.5. On the other hand, the DNA polymerases purified from Escherichia co2i B cells after either wildtype virus or mutant am N82 infection (gene 44) showed a &,;:' of 2.8. Moreover, the ts L91-induced enzyme was considerably more labile than the wild-type enzyme a t 37°C. A DNA polymerase from a bacteriophage T 5 mutant, ts 53 has also been purified. Infection a t either 30" or 43°C. resulted in the induction of polymerase activity. The activity of the T 5 ts 53-induced DNA polymerase, in contrast to the enzyme induced by the wild-type phage, was less active when assayed a t 43°C. than a t 25°C. The Q!gX of the sixtyfold purified polymerase from ts 53 was 0.04 compared t o a value of 1.4 for the wild-type T5-induced enzyme. 3. Mutant Phage Defective in Thymidylate Synthetase-Inducing Activity Using Escherichia coli strain B3, which requires thymine for growth, Wulff and Metzger (1963) have isolated mutant strains of T4 which require exogenous thymine for growth. Similar T4 mutants (td mutants) have been isolated by Simon and Tessman (1963). The mutants map close to amber mutant am 134, in the tail fiber region, a t a position far removed from the region controlling other functions that are expressed early in phage development. I n E . coli B3 infected with td mutants, DNA synthesis was absolutely dependent upon the presence of thymine in the medium. However, aftcr infection of E . coli B, which contains dTMP synthetase activity, DNA was synthesized a t about two-thirds the rate in cells infected by mutant td8 as those infected with td'. I n dilute cultures of E . coli B,
VIRAL-INDUCED ENZ’I-MES ANI) \‘IRAL ONCOGENESIC;
115
the eventual phage yield and the phage growth rate were two- to threefold lower with the mutant than in cultures infected with the wild-type strain. Addition of thymine not only increased DNA synthesis but also mutant-phage production. The ability to induce d T M P synthetase formation in infected cells probably confers a selective advantage on the wild-type strain (Mathews, 1965). Enzyme studies have shown directly t h a t little or no d T M P synthetase is induced in E . coli infccted with three of the T 4 td mutants and a fourth mutant was “leaky” (synthetase induction 19% of normal). However, inductions of d C M P hydroxymethylase, dCTPase, and FH, rcductase were normal with all of the T 4 td mutants (Shapiro et al., 1965). 4. T 4 Miitants Unable to Induce dCilIP Deaminase Activity
Hall and Tessman (1966) have isolated mutants (cd-) of bacteriophage T 4 that were unable to induce d C M P deaminase activity. Extracts of Escherichia coli B cells infected by these mutants showed less than 1% of the activity of extracts from cd+-infected cells. However, the induction by the cd mutants of d T M P synthctabe and d C M P hydroxymethylase activities were normal, suggesting that the mutation was specific for the deaminase. The lack of activity in extracts from cd mutant-infected cells was not due to an excess of inhibitors of d C M P deaniinasc. This was shown by experiments in which extracts from cd- and cd+-infected cells were mixed; the cd- extract did not inhibit the activity of the cd’ extract. Fleming and Bessman (1965) have found that the d C M P dearninase induced by phage T 6 is subject to feedback control, being activated by d C T P and inhibited by dTTP. The deaminase activity of extracts of T 4 cd+-infected E . coli B was increased sixfold by the addition of d C T P to the reaction mixture, suggesting that the activity of the T4-induced deaminase was also regulated by feedback inhibitors and activators. Nevertheless, extracts from cells infected with cd- mutants showed no significant activity even in the presence of dCTP. The isolation of the cd- mutants demonstrates that a t least one T 4 cistron controls the production of dCMP deaminase activity. However, the cd+ function does not appear to be essential for T 4 growth in E . coli B ; the burst size of the cd- mutants was a t Icast one-half that of the wild-type virus. 5 . M u t a n t Virus Strains Defective in H M C cu-Glucosyl Transferuse-Ind ir ciny A ctiv it 1~ From T 2 : t t i ( I T6 hnctt~riopliagcstocks which l i d I)crn treated with Iiydroxyl:itiiiii(i +oluCion, Rc\.cbl :uuI (w-woikrrs (1965) liave isol;itetl
I IG
SAUL KIT
rllutant virus strains defective in H M C a-glucosyl transferase-inducing activity. Pliage were selected which grew on Shigella dysenteriae Sh (permissive host) but not on Escherichia coli B (restrictive host). The mutants were designated T2gt and T6gt, respectively. Upon infection of Shigella dysenteriu Sh, the mutants failed to initiate the synthesis of HMC a-glucosyl transferase, whereas dCMP hydroxymethylase and dTMP kinase were made in normal amounts. I n its reactions with the restrictive host (Escherichia coli B) , T2gt-1 behaved similarly to host-modified T2 (T"2) (see Section IV,I). Adsorption to E . coli B was normal, but thc killing efficiency per particle was only one-half to onc-third. Small amounts of dCMP hydroxymethylase and dTMP kinase were formed. I n mixed infection on E. coli B the mutant T2gt-1 complemented amber mutants of cistron 42 (dCMP hydroxymethylase defective), but not amber mutants of cistron 44 (function unknown). As with T"2, multiplicity activation occurred a t high multiplicities of infection. The DNA of T2gt-1 was as good a substrate for HMC CY-glucosyltransfcrwsc of T2 as was T"2 DNA, indicating the DNA of tliesc phages contained little or no glucose. 6. Vaccinia and Herpes Simplex Mutants Using dBU as a selective agent, a mutant mouse fibroblast line [strain LM (TK-) 1 with the following properties was isolated: 1. The LM(TK-) cells were highly resistant to growth inhibition by dBU, dIU, or excess dT. 2. They were more resistant to (1FU inhibition than parental LM cells. 3. They lacked dT or dU phosphorylating activities although the cells were capable of phosphorylating deoxycytidine, uridinc, or dTMP, and the cells contained normal levels of FH, rerluctase, dTMP synthetaw, and DNA polymerase activities. 4. They failed to incorporate "-dT into DNA but did incorporate 3H-deoxycytidine into DNA (Dubbs and Kit, 1964a; Kit et al., 1 9 6 3 ~ ) . Following infection of LM(TK-) cells by either normal vaccinia or herpes simplex viruses, d T kinase activity was induced, and the capacity of the cells to incorporate 3H-dT into DNA was restored. By infecting LM(TK-) cells with vaccinia or herpes simplex viruses at low input multiplicities in the presence of dBU, mutant virus strains were isolated deficient in d T kinase-inducing activity (Dubbs and Kit, 19641),r, 1965; Kit and Dubbs, 1963b). The vaccinia (Vtk-) and 1i~rl)eshiinplt~s (Htk ) mutants proved to be relatively stable, since thry could be p least 5 times in either IJn4 01' LM(TK-) cells in tllc :il)spllccl of clT3lJ without gross revcrsion.
Thc wrccinin mutants (Vtk-) not only failcd to induce d T kinase activity in IAI (TIX DNA 01’ X n i c ~ s c ~ ~ liNA, i g ~ ~ rattclhting to tlio drficiciicay of other X-associated functions. The inability of N mutants to syrithesize
exonuclease and endolysin is not due simply to in:tbility to produce h DNA. Mutants in cistrons, N , 0, and P all fail to synthesize X DNA, but the mutants in the 0 and P cistrons do produce both enzymes. It, therefore, appears that N may be a regulatory cistro11, the protein product of which “turns on” functions by allowing the initiation of transcription of early h cistrons. Although defective lysogens carrying mutations in the N cistron of h phage produced little or no A-exonuclease activity upon induction, defective lysogens in cistron TI1 showed a fivefold greater rate of increase in exonuclease activity and 10 to 30 times greater yields of activity than wild-type (Radding, 1964b). When the X exonuclease was highly purified after induction of lysogcn, XTI1, it was found that the hTll-associated exonuclease and the wild-type exonuclease were similar in the following properties: ( 1 ) strong specificity for native DNA; ( 2 ) p H optimum; ( 3 ) divalent cation reyuirement; ( 4 ) K,n value; and ( 5 ) inhibition by sodium chloride. Inhibition and competition experiments with antiserum prepared against partially purified hTll exonuclease demonstrated immunological similarity with the wild-type enzyme. These data indicate that the active site of XTll exonuclease had not been mutationally altered. Presumably, the T11 mutation indirectly enhances the synthesis of X nuclease (Radding, 1966; Radding and Shreffler, 1966).
E. HERPES SIMPLEX-A N D POXVIRUS-INDUCED DEOXYRIBONUCLEASES Pronounced increases in deoxyribonuclease activities occur in cultures infected with poxviruses and with herpes simplex virus (Hanafusa, 1961; Jungwirth and Joklik, 1965; Keir arid Gold, 1963; McAuslan, 1965; Russell et al., 1964). The increases begin a t about 2 hours after infection and closely follow those for virus-induced d T kinase. Further, as in the case of dT kinase, puromycin addition a t the time of infection prevents the increases in DNase activities, and addition of puromycin to infected cultures after thc DNase increases have begun halts further enzyme inductions (.Jungwirth and Joklik, 1965; McAuslan et al., 1965). It appears that a t least three deoxyribonuclease activities are involved in these increases. The three nucleases can be conveniently described by their substrate preference and pH of maximal activity. One DNase (alkaline SS-DNA) was maximally active for heat-denatured DNA at about p H 7.8 to 8.5 (Eron and McAuslan, 1966; Jungwirth and Joklik, 1965; Keir and Gold, 1963). The second nuclease (alkaline DS-DNA) hydrolyzed native D N A a t p H 9.2 and was induced in chick embryo fibroblasts or HeLa cells by cowpox, rabbit pox, and vaccinia viruses
: L i d i l l I-IeLa cell nioiioluycrs I)y Iicrpes siiiil)lox virus. Tlic tliircl iiiicleasc (acid IINase) degracletl thermally denatwed D N A at p H 6 . This latter nucleasc increased five- to tenfold in activity after poxvirus infection of HeLa cell cultures. The alkaline DS-deoxyribonuclease and the acid DNase have been tentatively characterized as exonucleases. NO increase in any DNase, (single- or double-stranded substrate a t alkaline, neutral, or acid pH) was denionstratcd after adenovirus Type 2 infection of KB cells. I n contrast to the puromycin inhibition of the synthesis of poxvirusinduced d T kinase, the puromycin inhibition of the increase in acid DNase was not reversible, suggesting that the mcseenger RNA for acid DNase was comparatively unstable. I n support of this suggestion it was found that R rapid inhibition of the increase in acid 1)Nase followed addition of actinomycin D. Similar results have been obtained with alkaline DS DNase. As previously discussed, UV-irradiated poxviruses induced d T kinase a t the same rate as live virus, but with W-irradiated virus, the normal shutoff of kinase synthesis was impaired. Induction of alkaline D S DNase was found to be more sensitive to UV irradiation than was induction of d T kinase. However, repression of both d T kinase and alkaline DS DNase was abolished by the same UV dose (McAuslan and Kates, 1966). I n contrast to this, when HeLa cells were infected with vaccinia virus irradiatcd with graded doses of UV light (survival lo-* to very little induction of alkaline SS DNase was observed (Jungwirtli and Joklik, 1965). Further evidence that somewhat different control mechanisnis exist for different poxvirus-induced enzymes has been obtained through studies of the effects of inhibitors of DNA synthesis on the regulation of enzyme inductions. I n vaccinia virus-infected HeLa cells, induction of alkaline SS DNase occurred at the same time whether viral DNA was allowed to replicate or not. The enzyme increased rapidly in the presence of dFU or aminopterin, and this increase continued beyond the time when synthesis of the enzyme was switched off under conditions permitting viral-DNA replication. Thus, similar switch-off mechanisms were observed for d T kinase, DNA polymcrase, and alkaline SS DNase in vaccinia-infectetl IleLa cells (Jungwirtli and Joklik, 1965). Similarly, in cowpox- or rabbit pox-infected HeLa cells, dFU addition prior to about 2.5 hours PI prevented the termination of the increase in induced alkaline D S DNase (hl cAuslan and Kates, 1966). In striking contrast to the effect on the regulation of the two alkaline DNases, the addition of dFU to HeLa cells a t the start of infection completely prevented thc normal increase in cowpox virus-induced acid
134
SAUL KIT
DNase activity. Tlic b:t111[7 resultb twre o1)t;tiiicd uhing :iiiiiii~i)t~~riil ill place of dFU. Furthermore, after dFU was removed from cowpox virusinfected cells and dT was added to insure prompt reversal of viral-DNA synthesis, acid DNase activity rapidly increased. This indicates that a mechanism for terminating the increase in acid DNase activity was not established during the delay in initiating DNA synthesis. In a study of pseudorabies virus-infected rabbit kidney cells, Kamiyn et nl. (1964) found that dBU prcvented infectious virus formation although dBU-labeled DNA was produced. Thymidine kinase and the enzymes for incorporating 3H-dCMP into DNA were also induced in the dBU-treated cells. Normally, enzyme synthesis stopped at about 6 hours but the substitution of dBU for d T in the progeny DNA allowed the enzyme increases to continue up to 14 hours after infection. If dBU was added to HeLa cell cultures before 2.5 hours after poxvirus infection, the drug was also effective in preventing thc onset of d T kinase repression (McAuslan and Kates, 1966). Similarly, dBU treatment prevented the termination of alkaline DS DNase formation in poxvirus-infected HeLa cells. I n dBU-treated cultures, an increase of acid DNase activity did take place, but the rate was somewhat lower than i t was in poxvirusinfected cells not treated with dBU. Thus, induction of acid DNasc differed from that of the other DNases in that the control of the increase in its activity was dependent on viral-DNA synthesis.
F.
PHOSPHORYLATION O F
NUCLEICA C I D
BY AN ENZYME: FROM
T4
BACTERIOPHAGE-INFECTED Escherichin coli Bacteriophage T 4 infection of E. coli leads to the appearance of an enzyme, polynucleotide kinase, which catalyzes the transfer of orthophosphate from ATP to the 5’-hydroxyl termini of a wide variety of nucleic acid compounds (Richardson, 1965). The specificity of the enzyme permits the phosphorylation of DNA, RNA, small oligonucleotides, and even nucleoside 3’-monophosphates. Novogrodsky and Hurwitz (1965) have described an enzyme with similar properties from T2 phageinfected bacteria. The T4-induced polynucleotide kinase could he detected 5 minutes after infection and increased to a maximum a t about 20 minutes. The enzyme has been purified and requires divalent cations, 5’-hydroxyl-terminated DNA, and the addition of mercaptoethanol or reduced glutathione for niaxiiuum activity. The Mn++ions can partially replace the Mg++ions. The pH optirnurn for enzyme activity was about 7.4 to 8.0 in tris-fIC1 buffer. The function of polyriucleotide kiiiase is not known. Perhaps the presence of 5’-phosphoryl termini may serve to prevent initiation of hydrolysis by exonucleases. The presence of 3’-phosphoryl end groups
VIRAL-ISDUCED ENZYMES AND L‘IHAL ONCOGENES18
135
in D N A has been shown to prevent exonuclease attack in addition to eliminating their template activity for D N A polymerase. Another possibility is that polynucleotide kinase, together with other enzymes, might lead to the synthesis of a polynucleotide chain bcaring an activated 5’-terminus. This, in turn, could result in the condensation of performed polynucleotide chains.
G. INDUCTION O F I)EOXYRIRONUCLElC ACIDi\/lETHYLASE INFECTED CELLS
BY PHAGE-
Infection of Escherichia coli B with bacteriophages T1, T2, or T 4 results in a pronounced increase in the activity of DNA methyla~e.The largest increase was observed after infection with T 2 phage; T 4 and T1 phages produced smaller increases in t h a t order of decreasing magnitude. After infection with T3, T5, or T 6 phages, a decrease in activity was observed; T 7 and h phages had no effect (Gold e t al., 1964; Hausniann and Gold, 1966). The kinetics of the increase in DNA methylase activity after T 2 phage infection was simihr t o t h a t found with other “early enzymes” induced by T-even phages. The increase was first detected a t 2 minutes after infection and rcaclied inaxinial levels a t about 10 to 15 minutes after infection when phage-DNA synthesis occurred. Mixing extracts from noninfected and from T 2 phage-infected cells resulted in additive activity, suggesting t h a t T 2 phage infection did not inactivate an inhibitor present in excess in normal cells. T h a t de nova protein syntlic.sis was esscntial for the increased DNA methylase activity was shown i n two ways. First, when chloramphenicol was added a t the time of infection, the incrcasc in DNA methylase activity was not observed. Second, a methionine-requiring strain of bacteria was infected in the absence of the necessary amino acid, and enzyme activity followed. I n the presence of mcthionine, D N A methylase activity increased. However, in the absence of methionine, no significant change in enzyme activity took place after infection. When niethionine was added to the culture a t 25 minutes after infection, the rise in D N A methylase activity began without an appreciable lag. The ability of T 2 phage to induce nn increase in D N A methylase activity could be innctiv:ited by preirradiation of the phage with UV light. l3otli the nictliyl:isc~-iiiclucing cap:icity :ind the infectivity of the phage were inactivated oxponentially with single-hit kinetics when bacteria were infected a t a ~nultiplicity of 0.3 particle per bacterium. However, the rate of inactivation of inethylase-inducing activity was about 30% of that of phage-producing activity. With a UV dose of survival, both the rate of increase and the
maximum level of rnetliylasc activity attained were lower than with nonirradiated phage. Even though the increase in activity continued past the normal shutoff time of 10 to 15 minutes, the results were in marked contrast to those found with other early enzymes such as dGMP kinase, where enzyme synthesis continued for relatively long periods of time. Higher doses of UV radiation severely depressed both the rate of increase and the final levels of DNA methylase activity. The UV light experiments tend to discount the possibility that it is a nongenetic factor associated with phage infection that brings about the changes observed. A study of mutant strains of phage T2 has yielded information concerning the regulation of DNA methylase synthesis. All amber mutants tested in permissive hosts had the ability to induce an increase in DNA methylase activity, and the enzyme levels attained were quite similar to those obtained with wild-type phage. In the nonpermissive hobt, however, amber mutants blocked in DNA synthesis induced increased levels of enzyme activity and DNA methylase synthesis continued for a considerable length of time beyond the normal “shutoff” time. Thus, although the regulatory mechanism for shutting off DNA methylase synthesis was relatively resistant to UV light, it was impaired in amber mutants defective in DNA synthesis. The rII mutants of T-even phage are unable to grow normally in Escherichia coli K12 (A) although DNA and protein syntheses proceed until about 10 minutes after infection and then decline abruptly. The total DNA made corresponds to about 10 phage units per cell. The DNA synthesized in E . co2i K12(X) after abortive infection with rII mutants of phage T4 contains hydroxymethylcytosinc, so that the defect associated with the r I I mutation is not a failure to form dCMP hydroxymethylase (Nomura, 1961). When DNA methylase was measured in E . coli K12(A) after infection with TBrII, the increase in x t i v i t y was similar to that obtained with wild-type phage in initial rate, duration, and total amount of enzyme induced (Hausmann arid Gold, 1966). Deoxyribonucleic acid methylase has been highly purified from extracts of phage T2-infected cells. The properties of the induced enzyme differed from those of the host-cell enzyme suggesting that the phage and the host-cell enzymes were distinct proteins. The phage-induced enzyme was more stable than the host enzyme a t all stages of purification. Reagents such as Twccn greatly stimulated the purified phage enzyme but they were without effect on tlie host-cell enzyme. Unlike tlie niethylases of E . coli W or K12 strains, that of E . coZi U catalyzed the transfer of a methyl group only to adenine residues producing 6-methylaminopurine. In strains W and K12, 5-nietliylcytosine was also formed. The methylase purified from phage T2-infected cells, however, catalyzed only
thc form:ition of 6-nid Ii~laniiiioliuriiie,n-lirtlier the hobt w a b the B 01’ the K12 strain of E . c d i . Ah htnted v:irlior, D N A nict I i y l : t ~ (i~i o t uiily w a s not induced in h r toria infcotctl witlr I)li:igc T3 but the wtivity of the pre-existing hostcell enzyme rapidly tlcclinetl. 1l:xtr:tcts of T3-infected cells, when mixed with extracts from either normal cells or T2-infected cells, markedly inhibited the activity of the latter (Gold e t al., 1964). These inhibitions were caused by the induction by T3 phage of an enzyme catalyzing the hydrolysis of S-adeiiosylmethioiiine, the methyl donor rcquired for the DNA methylation reaction (Gefter et nl., 1966) : 8-iidenosylmethionine
---f
thioniethyladenosine
+ homoserine
(11)
This enzyme appeared only after T3 infection. Cell-free extracts from noninfected bactcria or from bactcria infectcd with T1, T5, T7, T-even, or phagea contained no significant liyrlrolytic activity. Enzyme formation required de novo protein synthesis, appeared 2 minutes aftcr infection, reached a maximum a t about 8 niinutcs and then dcclined. The enzyme was isolated from normal T3-infected E . coli and from E . coli infected with UV-irradiated T3. The activity of cell-free extracts induced by UV-irradiated T3 was sevcral times liiglicr than extracts obtained from normal T3-infectctl cclls, and “cxteridcd” enzyme synthesis was observed. The function of the niethylatcd bases in DNA is not unknown. A possible role of inethylation in host-induced modifiration has been suggcsted mid will bc discussed in Section IV,I,5.
H. INDUCTION OF GLUCOSYL TRANSPERASE ACTIVITIESBY T-EVENPHAGE The T-even phage differ from Escherichiu coli in containing H M C in place of cytosine in their DNA’s. I n addition, the DNA’s of the Tcven phages contain glucose linked to the hydroxymethyl group of H M C in characteristic ratios. I n T 2 phage DNA, 25% of the H M C is nonglucosylated, 70% is monoglucosylated, and 5% is diglucosylated. The binding of glucose to the hydroxymethyl group of H M C is by means of an a-glucoside linkage. 111 T 4 phage DNA, all the H M C is in the monoglucosylated form but 70% has the glucose attached in the LYconfiguration and 30% has a /3-linkage. I n T 6 DNA, 25% of the H M C is nonglucosylated, 3% is monoglucosylated with the linkages the same a s in T2, and 72% is diglucosylated. The diglucosyl-HMC is a disaccharide in which the 2 glucose residues are linked t o each other in ;t p linkage and the hydroxymcthyl group of H M C in an N linkage (Kuno and Lehman, 1962; Lichtenstein and Cohen, 1960; Lehman and Pratt, 1960). Enzymes that transfer glucose from UDPG to DNA-containing H M C
138
SAUL KIT
:ire iiitluccd i n Ixicteriii infcctctl with the T-even pl~agosbut not by
phage T5. Enzyme activity is not detectable in lioninfected bacteria (Kornberg e t nl., 1959). The increases in the DNA-glucosylating enzymes (glucosyl transfernscs) commence a t about, 7 minutrs and are normally shut off :it :ibout, 20 niinlitcs aftcr infe(%ion. The fitil11l.c of certain amber inutants of T4 to turn off glucosyl transferase synthesis a t the normal time has been discussed in Section III,F,l, and the isolation of mutant virus strains defective in glucosyI transferase-inducing activity has been described in Section 111,F,5. From the evidence cited, i t is apparent that the induction of glucosyl transferasc activities is a function mediated by T-even phage genes. The glucosyl transferases have been highly purified from T-even phageinfected cells and are conveniently described by their substrate specificities aild the nature of the glucoside linkage formed (Kornberg e t al., 1961). All of the T-even phages induce HMC a-glucosyl transferases. In addition, phages T 4 and T 6 induce H M C p-glucosyl transferascs (Table VII). All three a-glucosyl transferases add glucose to cnzymically TABLE VII EXTENT OF GLUCOSYLATION OF DEOXYRIBONUCLEIC ACID ACCEPTORS BY GLUCOSYL TRANSFERASES~ ~
~
hccept,or DNA:
Trarisferases
Enzymically synthesized HMC-DNA T 2 D N A
T4DNA
T6DNA
(% of total HMCb residues glucosylat,ed) T2 HMC L Y - ~ ~ U C O S Y ~ T4 HMC Oc-glucosyl T6 HMC Cu-glucosyl T4 HMC j3-glUcosyl T6 glucosyl-HMC ~-glllCoSyl
50-58 66-75 50-7 1 70-78 hatconverted single-stranded viral R N A to double strands, and it was this enzyme which was presumably thermolabile. Since the synthesis of single-stranded R N A proceeded normally for a t least 10 minutes after the shift up, it appears that a second enzyme (enzyme 11) was responsible for that synthe,qis. The sccond enzyme could he either the normal cellular RNA polynierase or a second phage-induced enzyme. The fact that, in time after the increase in temperature, the synthesis of single-stranded RNA was also inhihited implies t h a t this synthesis is coupled to the synthesis of double-stranded RNA. Further genetic evidence for the existence of an f2 phage-induced enzyme which converts single strands to double-stranded RNA has been obtained by using host-dependent, amber mutants (Lodish et al., 1964; Lodish and Zinder, 19661)). Amber mutations in the coat-protein cistron o f phage f2 nltcr the n o m : ~ lprocess of control of synthesis of both viral RNA and of RNA synthetnee. These effects depend on the position of the amber mutation and on the suppressor properties of the host bacterium. Phage mutant sus-3 carrics mi nmbcr codon near the N-terminus of the virus-coat protcin cisti~on.Whcn thi.: mutant is i ~ tod infect Su- h.%cri:i, wliic~li lack :L supi)ttssor gtwc’, inifi:illp little sitigle- or ~ l o i i l ~ l t ~ - ~ t i ~ :\,it ~ n:ilt l ~KN.1 tl 01’ \.it :iI-RNA bytitlieta~c:ti iuadr, althoi~glt t l i c i ~ ~ : i t x ~ n t :plitigv 11 liN.1 i h c o i i \ ~ c . t t t ~ till l t o :L ~ ~ ~ L I I ~ ~ f0t.m ~ ~ nl- ~ ~ I ~ A ~ (1
‘The
QP
Spiegelman.
reglicasr einplovrd in tlic study nns n gift from Drs. Haiunn and
162
SAUL K I T
most as efficiently a s is RNA from wild-type phage. The functional lesion of sus-3 infected Su- bacteria appears to be in the synthesis of single-strandcd viral RNA. Infection with sus-3 of bacterial hosts such as Su 11, which are iionpermissive but partially suppress the amber mutation, results in normal synthesis of single-stranded and 14-18 S doublestranded viral RNA. As in f2 wild-type cells, all enzymes needed for this synthesis are made very early in infection. However, late in infection, all available single-stranded RNA (including RNA from superinfecting wild-type phngc) are converted to small (7 S) double-stranded RNA; this transformation is not merely the consequence of lack of viral-coat protein to encapsulate the RNA, but is dependent on protein synthesis late in infection. Furthermore, the amount of virus-induced RNA synthetase in extracts late in infection by sus-3 is 5-10 times that of extracts from wild-type cells. The excess mutant-induced enzyme has properties expected of a phage-induced enzyme (enzyme I) which converts singlestranded viral RNA into a double-stranded form. An amber mutant (sus-11) carrying a mutation near the middle of the virus-coat protein cistron makes, on both hosts (Su I1 and Su-), normal amounts of single-stranded RNA, but excess amounts of RNA synthetase (enzyme I) and 7 5 double-stranded RNA. Thus, when single-stranded RNA is made normally, but sufficient coat protein is not produced, early cessation of enzyme synthesis does not occur. The cell synthesizes large excess amounts of enzyme I. Acting directly or indirectly, virus-coat protein is the repressor of phage-enzyme synthesis. These results were interpreted in terms of a model coupling, genetic translation and transcription. Viral RNA is translated in two distinct states; as free (parental) RNA, which makes RNA synthetase (s), and as a nascent messenger still bound to its double-stranded RNA template, which makes coat and other phage proteins synthesized late in infection. The translation into protein of the N-terminal region of the coat cistron is necessary for continued synthesis of the nascent messenger RNA. When RNA is synthesized under coiiditions when sufficient coat protein is not made, as in mutant-infected cells, single-st,randed progeny RNA molecules appear analogous to parental RNA molecules and synthesize enzyme I. VI. Effects of Virus Infection on Host-Cell Nucleic Acid and Protein Synthesis
A. SHUTDOWN OF BACTERIAL N~ETABOI~ISM I N CEILSINFECTED WITH T-EVEN AND T5 PHAGES The T-even and T5 coliphages, which are known to destroy the nuclear structurc of Escherichiu coli, arc also thosc phagcs for whicli radiatior~
VIRAL-INDUCED ENZTBlES AND V I R A L ONCOGENESIS
163
experiments inclicate that tlicir iiiultiplication is relatively indepenrlent of the integrity of the host cell. Within minutes of infection with Teven phage, the nuclei of E . coli are disrupted into small blocks of chromatin material, which collect a t the periphery of the ccll. After T5 infection, there is a rapid and progressive deformation of the nucleoids :ind they disappear. This involves the chemical degradation of host DNA and, hence, the destruction of the gcnctic infornintioii of the lioht (Kcllenberger, 1961). After T-even phage infection of growing bacteria, the syntheses of bacterial DNA and all classes of bacterial RNA stop (Volkin and Astrachan, 1956). Protein formation continues but the majority of the proteins synthesized appear to be phage proteins or enzymes associated with phage formation (Cohen, 1948; Nomura et al., 1962). The synthesis of bacterial enzymes comes to a sudden and complete halt and remains a t the preinfectiori level. The bacteria can 110 longer be induced or dercpressed to form P-galactosidase, lysine decarboxylase, aspartic transcarbamylase, dihydroorotic decarboxylase, or alkaline phosphatase (Benzer, 1953; Levin and Burton, 1961 ; Pardee and Kunkee, 1952). During T-even phage growth, the major part of the host DNA is degraded to substrates of low molecular weight. With phage T5, the total DNA decreases very rapidly to reach about one-third the initial level 20 minutes after infection. The profound inhibitions of host-cell metabolic processes and breakdown of host-cell structures are in marked contrast to effects produced by other bacteriophage strains. With phages T1, T7, A, and P1, nuclear breakdown, if it occurs a t all, takes place so late that these phages can use home of the genetic information of the bacteria for their own syntheses. The syntheses of bacterial enzymes and other proteins may also continue during the first part of the infectious cycle. With phage f2, all bacterial metabolic processes continue normally until late in infection, and there is no inhibition of cellular DNA or RNA syntheses. Sucrose-gradient fractionation of RNA labeled after infection reveals csseritially the same pattern as docs RNA from noniiifected cells, and enzymes such as P-galactosidase and alkaline phosphatase can be induced in a normal manner (Zinder, 1965).
B. RIBONUCLE~C ACID POLYMERASE ACTIVITYIN T-EVENPHAGEINFECTED CELLS
It appears tliat a Itlwtively heat-stable substance is produced after T 4 phage iiifection that can effectively inhibit purified DNA-dcpentlent RNA polynierase of noninfected cells (Skold and Buchanan, 1964). The production of this inhibitor can account for the decrease in turnover of messenger RNA and the eventual cessation of bacterial protein synthesis. Ribonucleic acid polymerase activity decreases to 15 to 20% of
164
SATJL K I T
I)I*cinf(!ctioi~ ;icLtivity tluriirg tIro f i r h t 10 to 12 niiiiutes alter '1'4 iiifccation. The decline i i i IiNA polymerase activity is preventcd by the addition of chloraniphcnico1 to cells shortly after their infection, suggesting that polymeruse inhibition may be mediated by :L protcin. Sephadex G25 experiments indicate that the inhibitor has a minimum molecular wcight of about 5000. A similar inhibitor of RNA polymerase is apparently elaborated aftcr T2 phage infection (Khesin e t al., 1962). tllr
C. Loss OF POLYADENYLATE POLYMERASE ACTIVITYAFTER PHAGE INFECTION The abrupt inhibition of R N A polymerase activity in cells infected with T 2 phage was not confirmed by Oritz and co-workers (1965). Although a gradual and variable decrease in thc activity of this enzyme was sonictimes found in cells infected for 20 minutes, Ortiz et al. (1965) never observed a significant loss of RNA polymerase activity with bacteria infected for less than 4 minutes. Ribonucleic acid polymerase activity was also examined in cxtracts of bacteria infected with phages T1 and T7 with similar results to those seen with phage T2. I n contrast, a rapid loss of polyadenylate polymerase activity was observed; a decrease of about 50% occurred within 1 minute of T 2 infection and less than 10% of the normal activity remained after 8 minutes. Inhibition of 32P-orthophosphate incorporation into RNA occurred within 2 minutes after phage addition. Thus, the inhibition of polyadenylate polymerase and cessation of RNA synthesis took place within 1 minute of each ot,lier. Moreover, decreased levels of polyadenylate polymerase were also found in all cases in which cessation of net RNA synthesis was a consequence of phage infection, i.e., with Teven and T5 phages. Convcrscly, following infection with f2, T3, or induction of h phage, when RNA synthesis did not decrease, normal polyadenylate polymerase activity was observed. The inhibition of polyadenylate polymerase appeared t o be specific. Neither the ribonucleotide kinases, polynucleotide phosphorylase, nor RNA adenylatc (cytidylate) pyrophosphorylase showed a Significant change after T 2 phage infection. Of the possible causes for the loss of enzyme activity there was evidence only for induction or releasc by bacteriophage infection of a n inhibitor of the polyadenylate polymerasc reaction. The loss did not occur when cells were infected in the presence of chloramphenicol or a s a rcsult of destruction of Escherichia coli D N A in bacteria grown in the presence of mitomycin. Inhibition was not produced in vitro by components of phage T2, i.e., phage T2 DNA, internal protein, spermidine, or putrescine, but inhibition in vitro did occur when extracts of normal and
VIIIAL-ISI)UCED ENZYMES AND VIRAL ONCO(;ENESIS
165
infected cells wcre incubated together. The iiihibitor was prcsent i n the supernatant fluid of infected cells after high-speed centrifugation, whereas, polyadenylate polymerase itself, was sedimented with ribosomes. It must be granted that the role of polyadenylate polymerase in bacterial metabolism is still unclear; nevertheless, the intriguing finding that a specific inhibitor of this enzyme is elaborated soon after infection by the same phages that shut off bacterial RNA synthesis docs not appear to be coincidental and merits further study. D. T4 PHAGE-CONTROLLED BREAKLXIWN OF BACTERIAL
DEOXYRIBONUCLEIC ACID When one measures the total DNA as a function of time after infection by wild-type phage T4, the breakdown of Escherichia coli DNA, which takes place in these cultures, is obscured because of the concomitant synthesis of phage DNA. However, if infection is carried out with a mutant of T4 that is unable t o induce synthesis of any phage DNA, one observes clearly and directly the conversion of a large portion of the bacterial DNA from an acid-insoluble to an acid-soluble form. sl prior), one might suppose that this conversion involves a DNase. However, although the induction of three new DNases in T-even phage-infected cells has been reported, evidence is lacking that any of them has a direct role in host-cell DNA breakdown (Oleson and Koerner, 1964; Short and Koerner, 1965; Stone and Burton, 1962). I n order t o test the hypothesis that bacterial DNA breakdown was controlled by coliphage genes, Wiberg (1966) tested a series of T 4 phagc mutants known to be defective in DNA synthesis. It was found that mutants in genes 46 and 47 were unable to cause host-cell DNA breakdown (Table V). These results imply that genes 46 and 47 control one or more DNases and that these DNases preferentially attack DNAcontaining cytosine rather than hydroxymethycytosine. It is not known, however, whether genes 46 and 47 are structural genes for a DNase or, instead, control an inducer or activator of a DNase. I n addition to host-cell DNA breakdown, other functions occurring after phage infection that have been suspected of involving DNase activity are (1) genetic exlusion of related superinfecting phage and ( 2 ) genetic recombination. These functions were virtually unimpaired in the T 4 mutants defective in genes 46 and 47.
E. INDUCTION OF DEOXYCYTIDINE TRIPHOSPHATASE ACTIVITYBY T-EVENPHAGE Following T-even phage infection, a new enzyme, dCTPase, is induced which specifically catalyzes the hydrolysis of dCTP and deoxycytidine 5’-
166
SAUL KIT
cliphosphilte (clCDl-’) (Iiocriici. ct d., 3959; Iioi*~il)rrg ct d., J 959; Zinimerman and Kornberg, 1961). ( M g + + )dCTPase
dCTP dCDP
( M g t f ) dCTPase
dCMP
+ pyrophosphate
dCMP + orthophosphate
(17)
This enzyme has a dual function in the promotion of the synthesis of phage DNA. First, it degrades dCTP so that i t cannot be utilized l ~ y DNA polymerase to make Escherichia coli DNA and, second, it generates dCMP which is the precursor of dHMP. Extracts of noninfccted cells have 0.1% or less of the dCTPasc activity observed in extracts of T 2 phage-infectcd cells. The enzyme is not induced by coliphagc T5, which contains cytosine rather than hydroxymethylcytosine in its DNA. Studies with UV-irradiated phage and with phage mutants have provided additional evidence that this enzyme is controlled by T-even phage genes. Normally, the enzyme is detected in about 4 minutes and increases to a plateau at about 15 minutes after infection. With UV-irradiated T 4 phage, the initial rate of enzyme synthesis is decreased; however, dCTPase synthesis is not shut off a t 15 minutes and extended enzyme synthesis is observed (Dirksen e t al., 1960). Infection of E . coli B with amber mutants defective in T 4 phage genes 41, 42, and 44 (Table V) also leads to extcnded synthesis of dCTPase (Wiberg e t al., 1962). Gene 56 appears to control the formation of dCTPase. Amber mutants defective in gene 56 fail completely to induce dCTPase activity (Wiberg, 1966).
F. THYMIDYLATE SYNTHETASE AND THYMIDYLATE NUCLEOTIDASE OF PHAGE-INFECTED Bacillus subtilis ACTIVITIES
The B. subtilis phages, SP8, +e, and SP5C, contain hydroxymcthyldeoxyuridylate in place of d T M P in their DNA’s. I n another B. subtilis phage, SP2, deoxyuridylate rcplaced dTMP in the DNA. The metabolism of dTMP is abnormal in bacteria infected with these phages. Phages SPSC, SP8, and SP2 all induce in infected cells increases in the activity of n 5’-nuclcotidase, specific for rlTMP (Aposhian, 1965; K n h n , 1963) : dTMP
dTMP nucleotidase
dT
+ orthophosphate
The appearaiice of this activity is inhibited by chloramphenicol, indicating that de TLOVO protein synthesis is needed for enzyme formation. The enzyme from phage SP5C-infected bacteria has been purified, and i t
rat;llyzch thc lly~lroly of dThIP, but iiot that of t1coxp:itlciiosiiic 5’iiionol)lio~pliatr (dAMP) , dChIP, ant1 cl(:nlP (Aposhi:in, 1965). The increase in dTAlP nucleotidase is not relxtcd to tlic induction of defective phages known to bc present in Iz. subtilis, since treatnictit of noninfected cells with niitoniycin C, an inducer of thew prophages, does not produce c1TMP nucleotidase activity. The dTh1P nucleotidase of phage ye-infected l m t c r i a has not been stutlictl a t this writing. However, it has been shown that in hactcria infcctcd with this phage, the dTlClP synthetase activity of the host cell declines t o very low levels. This rcduction in dTMP synthctase activity 5eciiis t o bc caused hy the presence of an inhibitor in the infected cell extracts (perhaps d T M P nucleotidase) . If extracts from infected and noninfected bacteria are mixed, the synthetase activity of the latter decreased (Roscoe and Tucker, 1964). As a consequence of thc increased d T M P nucleotidase activity and the rapid inhibition of dTMP synthetase activity, formation of thyminecontaining DNA is prevented. Concurrently, the inductions of d C M P deaminasr, d U M P hydroxyrnethylase, and deoxyuridylate aiid hydroxy~~icthylclcoxyuridylate kinnse activities facilitate phage-DNA synthesis.
G.
SIIUT1)UU‘N OF
BIOSTNTHETIC PROCE5SES
IN
VIRUh-INFECTED
ANIMAL CELLS
1. R N A - Containing Animccl Viruses
The 1-eplication of many RNA-containing animal viruses is relatively independent of host-cell functions. Thus, the picornavii~uses,Newcastle disease virus (NDV) , am1 vesicular stoinatitis virus (VSV) grow normally despite the addition to infected cultu,cs of actinoniycin D, a drug which prevents host-cell RNA polymcrasc function. The replication of thwc viruses is aleo uiiitnpaired by drugs t h a t inhibit DN.4 biosynthesis. In ccIl culture,. infected hy mengovirus, poliovirus, ME virus, NDV, niitl VSV, there ia an early inhibition of cellular RNA and protein syntheses followed by niorpliologiral c l i m g ~ awhich culniiiiatc in the rounding and rlisruption of cell:, (I3al~laniane t nl., 19651; Baltimore and Franklin, 19621); Baltimore et al., 19631); Fenwick, 1963; Hausen and Vcnvoerd, 1963 ; Holl:ind, 1962 ; I-Iomma, ancl Graham, 1963; Martin and Work, 1961 ; Vcnvoeid and Hausen, 1963; Wlicelork aiid Tamni, 1961 ; Zimmermnii c’f rrl , 1963). Ti1 iiiciigo\.ii i i h - i i i f c i ( + d I, c ~ l l s ,DN-4 *yritlicsis is r 1 o r i l l : k I for more t h i 3 h u i 3 a t t e r i n f t d i o i i and t h greatly decieabt~.s.Thii5, the rapid : i r i t l c:115ly iiili~l~itioii of Iio-t-cell RNA syiithcsis is not due to a generalized inhibition of all DNA-dependent processes (Baltimore and Franklin,
168
SAIJL K I T
1962b). A progrcssivc decrease of DNA biohynthesis also occurs in NDV-infcctcd HeLa cells. This decrease coincides in time with a rapid increase in virus antigen and infective particles, and synthesis of DNA stops as the amounts of virus materials produced reach maximal levels (Wheelock and Tanim, 1961). Both puromycin and FPA suppress the inhibition of host-cell RNA and protein syntheses when added early in the infectious cycle (Bablanian et al., 1965a,b; Baltimore et al., 1963b; Bolognesi and Wilson, 1966; Huang and Wagner, 1965; Verwoerd and Hausen, 1963; Wagner and Huang, 1966). These rcsults could meal1 that the inhibitions are caused by proteins that are synthesized under the control of the viral RNA. A t the same time that nuclear RNA synthesis is depressed in mengovirus-infected cells, a pronounced inhibition of RNA polymerase is observed. Within 1 hour of virus infection, RNA synthesis falls to less than 10% of normal and RNA polymerase to less than 50% of normal (Balandin and Franklin, 1964). These inhibitions appear to be mediated by an inhibitor made in the cytoplasm of the infected cells. If control nuclei and cytoplasm from infected cells are mixed, RNA polymerase activity of control cells is inhibited by 61%. However, mixing control nuclei with nuclei of infected cells has no effect on the enzyme activity of the control nuclei and mixing control cytoplasm with nuclei from infected cells does not restore the polymerase activity of the nuclei from infected cells. The cytoplasmic inhibitor of RNA polymerase is present in the postmitochondrial fraction; it is inactivated by trypsin but not by RNase. I n a study of poliovirus-infected HeLa cells, Holland (1962) confirmed the finding of Balandin and Franklin (1964) that RNA polymerase activity is rapidly inhibited after virus infection. This inhibition is not due to the breakdown of HeLa DNA nor to permanent alterations preventing DNA priming nor even to firm masking of DNA by protein in some manner to prevent contact with polymerase. Holland (1962), however, was unable to detcct the presence of a protein inhibitor in the cytoplasm of infected HeLa cells. The inhibition of protein synthesis noted prcviously could be an indirect result arising from virus-induced suppression of messenger RNA synthesis. However, this does not appear to be the primary reason for the arrest of host-protcin bynthesis. Protein synthesis is inhibited by poliovirus infec4ioii tnur(~ntpidly thttii Ly :~c.tinornycinD (Holland, 1962)-a result coinpatible with the hypothesis that virus infection interferes directly with protein-synthesizing mechanisms in intact cells. The release of ribosomes from complexes with cellular messenger RNA might well be one reason for the inhibition of protein synthesis. Consistent
VIRAL-INDUCED ENZYMES AND VIRAI, ONCOGENESIS
169
with this concept, it his heeii found that polyril,osomes are degraded after poliovirus infection of HeLa cells (Penman ef nl., 1963; Rich et nl., 1963). T h e 1.att: of polyrihn,iornal clisriiption increabecl linearly with thc t inic of iiifrvtion. R v l ( ~ i , s c of ~ Iio~t-iiiC~~sc~ligc~i~ R N A fro111polyrihosomcs was not mercly a coriscqucnce of co~npctitionfor ribosomes with virus RNA. Polyribosome breakdown and inhibition of host-cell protein synthesis took place even when viral RNA replication was prevented by guanidine (Bablanian et al., 1965a,b; Penman and Summers, 1965). However, polyribosome breakdown was manifested only after a period of protein synthesis following virus adsorption. This disruption thus may be due to a product of the viral genome which is stable for a t least 1 hour in the absence of protein synthesis and seems to be specific for the host-cell messenger RNA (Penman and Summers, 1965; Willems and Penman, 1966). The inhibitory factor probably acts by affecting messenger RNA on the polyribosomes of the host cell so that it is rendered incapable of attaching ribosomes. The irihibition of protein synthesis by poliovirus does not appear to result froin an effect on the cellular ribosomes. The protein-synthesizing system of the cell is capable of functioning with viral messenger later in infection t o produce viral-specific proteins. Moreover, the host-cell ribosomes are not simply inhibited from functioning with host-cell messenger since newly synthesized messenger RNA, produced in cells infected in the absence of actinomycin D, apparently functions with the existing ribosomes (Willems and Penman, 1966).
2. Metabolism of Reovirus-Infected Cells Reovirus differs from most of the RNA-containing animal viruses in that mature reovirus particles contain double-stranded RNA. Moreover, reovirus replication is inhibited by actinomycin D, whereas that of the picornaviruses, NDV, and VSV is not. Reovirus is a comparatively slowly replicating virus. In L cells, thc latent period for reovirus Type 3 was 8 hours, and maximal yields were not reached until about 17 hours (Gomatos and Tanim, 1963a). Virusspecific RNA formation commenced a t about 6 hours and continued through most of t.he infectious cycle. In contrast, formation of poliovirus particles in HeLa cells increased exponentially beginning a t about 3 hours after infection, and maximal titers were obtained by about 6 hours. With NDV, newly made virus antigens and the first infective virus particles appeared a t 2.5 hours, and infective virus reached a peak a t about 5 hours after infection (Wheelock and Tamm, 1961). Capsid antigen of influenza A virus was detected in infected HeLa cells a t 3 to 4 hours and hecamc promincnt a t 6 to 7 hours (Whcclock and Tamm,
170
SATTI, TiIT
1959) . In I, cclls infectecl with ni~ngovirus,formation of infectious RNA began a t about 4 hourh and ivah completed by 7 hours after infcction; at 6.5 I I O U ~ a11cl ~ t . 1 1 d ~ dby 0 110~1’sPI VIIWS I i i w t i i r a t i o i i i’oiii~ii(~ii(~d (llolllnls :uld G r : h t l l l , 1963). The syntliescs of protein : ~ n dL)NA wcrc not iiiliibited during tlic first 8 hours of infection of L cells with reovirus Type 3 (Gomatos and Tamni, 1963a; Kudo and Graliam, 1965). However, late in infection when infectious reovirus increased exponentially, a pronounced inhibition of DNA synthesis and a moderate inhibition of protein synthesis were observed. There was very little inhibition of either ribosomal RNA or nuclear RNA syntheses up to 12 hours after reovirus infection. 3. Metabolism of Cell Cultures Infected with DNA-Contairwig Aninial Viruses
A severe inhihition of 3H-dT incorporation into host-cell DNA ensues early after infection of cell cultures with vaccinia, cowpox, herpes simplex, and pseudorabies viruses (Ben-Porat and Kaplan, 1963; Dubbs and Kit, 1964b; Hanafusa, 1960; Kaplan and Ben-Porat, 1963; Kato et nl., 1964; Kit and Dubbs, 1963c; Kit et nl., 1963a; Roizman and Roane, 1964). Although host DNA synthesis was inhibited, viral DNA was made. I n cultures infected with poxviruses, 3H-dT labeling of nuclear structures decreased while foci of cytoplasniic labeling developed. In either exponentially growing or stationary phase cells infected with herpes viruses, formation of “light,” cellular DNA synthesis was arrested early in the infectious cycle while the formation of “heavy” viral DNA was accelerated. The inhibition of cellular DNA synthesis was not due to the degradation of that DNA (Kaplan and Ben-Porat, 1963; Kit and Dubbs, 1962b). In pseudorabies virus-infected cells i t was also not attributable to greater affinity of the DNA polymerase for viral DNA than for cellular DNA, nor to the successful competition of viral DNA with cellular DNA to act as a template for DNA replication. Thc inhibitory process was arrestcd by the addition of puromycin to pscudorabies virus-infected cells a t 1 to 2 hours after infection suggesting that a protein was responsihle for the inhibition of the syntliesis of cellular DNA (Bcn-Porat and Kaplan, 1963, 1965). Inhibitions of host R N A and protein syntheses were also observed after poxvirus or herpes virus infections. The capacity of infected LM cells to incorporate 3H-uridine into RNA was reduced within 1 hour after vaccinia infection, and by 6 hours declined to 2570 the rate of noninfected cells. The incorporation of 2-I4C-alanine into protein was inliil)itctl 25% at I hour : ~ n r l 56% hy 7 hours after infection (Kit : L I I ~
I h b b s , 1962a). During the first 4 hours of vaccinia infection of HeLa cells, about 30 to 40% of tlic proteins made were viral proteins; after 4 hours, net ccllular protein synthesis diminished considerably and mostly viral proteins were made (Shatkin, 1963). Vaccinia RNA was detected in the cytoplasm of HeLa cells as early as 0.5 hour after infection. By 1.5 to 2.5 hours, about 50% of the newly formed RNA had the properties of vaccinia-messenger RNA and a t 2.5 to 3.5 hours, 80% was viral-specific RNA. New formation of ribosomal RNA did not occur in the cytoplasm of cultures 3-4 hours after infection (Becker and Joklik, 1964; Stllzman et al., 1964). Between 1 to 3 hours after infection of cell cultures with herpes simplex virus, 3H-uridine incorporation into RNA was reduced by half, relative to that of noninfected cells. The rate of synthesis of ribosomal RNA declined to 39% of the control levcl in the period 3.5-6.0 hours after herpes simplex virus infection. This decline paralleled the time course of inhibitions of soluble RNA (4 S) and host-cell messenger RNA. By 90 minutes after infection, a new RNA species exhibiting a sedimentation coefficient of 2 0 s and capable of hybridizing with herpes simplex DNA was observed. Although host-cell RNA synthesis decreased the formation of herpes-specific RNA increased. A 4 5 fraction capable of hybridizing with herpes simplex DNA was also made (Hay e t al., 1966; Roizman et al., 1965). During the first 3 hours after infection, protein synthesis, as measured by the incorporation of radioactive amino acids into protein, decreased to about 70% of the control rate. Between 3 to 6 hours, this incorporation was generally stimulated but 6 to 10 hours after infection, protein synthesis declined to approximately 60% of that of noninfected cells. Adenovirus Type 2 infection inhibited multiplication of KB cells, but overall nucleic acid and protein syntheses continued throughout much of the infectious cycle a t essentially unchanged rates (Green, 1959; Green and Daesch, 1961; Polasa and Green, 1965). Noninfected cells multiplied three- to four-fold during a 48-hour experimental period. In contrast, multiplication of cells in infected cultures was limited to the first 12 hours and was never more than 1.2- to 1.3-fold. Infected cells did not lyse but increased in size. Protein, DNA, and RNA accumulated continuously in infected KB cells starting within 12 hours after infection. At 36 hours, the content per cell of these polymers was double that of noninfected cells. cell cultures includcs soliiblc The RNA fomed in :Idcnovirus-iiifc~t(~(l RNA, rilmsotiiiil HN-4, :itiil RNA4c.oiiipIrtiirntaiy to hoth liust ~ ( viral 1 DNA's (Rose ct nl., 1965). The presence of viriil-conip1einental.y RNA was detected ns e:trly ns 9 Iiours after infection. A t 28 liours, ahout 36%
172
SAUL K I T
of the newly synthesized RNA formed duplexes with adenovirus Type 2 DNA. I n contrast to viral-complementary RNA, KB cell-complementary RNA remained an almost constant fraction of newly synthesized RNA after adenovirus infection. Overall RNA and protein syntheses were not inhibited in adenovirus Type 5-infected HeLa cells. In fact, an increase in the overall rate of RNA synthesis (presumably viral-messenger RNA) was noted a t 8 to 9 hours and was still evident 24 hours after infection. A significant increase in the rate of protein synthesis occurred a t 14 to 17 hours and a markedly accelerated rate continued until approximately 20 hours after infection (Flanagan and Ginsberg, 1964; Wilcox and Ginsberg, 1963). 4. Decrease in Cellular Enzyme Activities after Virus Infection The uridine kinase activity of LM mouse fibroblast cclls varies with the metabolic condition of the cell. The enzyme exhibits highest activity during exponential growth and declines to about one-third the peak level during the stationary phase. Infection of growing LM cell cultures with either vaccinia or herpes simplex viruses resulted in a rapid decline in uridine kinase activity. Within 5 hours after infection, the enzyme activity fell to less than half the value of noninfected cells. However, uridine phosphorylase activity was not significantly changed in the infected cultures (Dubbs and Kit, 1964b,c; Kit et al., 1964). Extracts from vaccinia-infected cells, when added to those from noninfected cells, did not reduce the in vitro activity of the latter extracts. Thus, the decline in uridine kinase activity was probably not caused by free inhibitors in the infected cell extracts. It is more probable that the uridine kinase decrease was due to a greater turnover of the enzyme after virus infection. As discussed previously, the formation of messenger RNA was inhibited in vaccinia- and herpes simplex-infected cells. This inhibition of messenger RNA synthesis would be expected to contribute t o a decline in the activity of enzymes with a high turnover rate. The inhibition of uridine kinase, in turn, would reduce the capacity of infected cells to incorporate "-uridine into RNA. The d T kinase activity of LM cells was enhanced about threefold after infection by vaccinia or herpes simplex viruses, Since formation of new virus-specific enzymes was taking place, changes in the activities of pre-existing host-cell d T kinase were obscured. However, by infecting LM cells with mutant viral strains deficient in d T kinase-inducing activity, it was possible to follow the effects of virus infection on cellular dT kinase activity (Dubbs and Kit, 1964b,c). Within 5 hours after infection of LM cells with mutant vaccinia (Vtk-) or herpes simplex (Htk-) strains, LM cell clT ltinnse activity declined to about half the
TI
1 ~ ~ 1IS , -D u CED EK z Y M ES A N D
v IHAL
ONCOG ICNESIS
173
iioriiial ~ : 1 1 1 1 1 ~This . t1ccre:~sew:ih not duo to frcv inhibitors in tlie cxtracts from infcctctl cells. Thc results suggest that vaccinia and herpes simplex viruses also enhanced the turnovcr of cellular d T kinase in infected LM ccll Cultures. VII. Biochemical Aspects of Viral Oncogenesis
A. REPLICATIOK CYCLE? OF SV40 AND POLYOMA VIRUS
I n previous sections, the general aspects of viral gene functions havc been considered. Against this background we can now discuss the gene functions of tumor-producing viruses. Considerable progress has recently hccn made in studies of polyoma virus and simian papovavirus SV40, and most of our discussion will be concentrated on thesc two viruses. The SV40 and polyomn viruses have dual capacities; they can actively niultiply in certain types of cells, which arc then killcd, or they can transform other cells. Let us firbt consitlcr the events taking place during active replication of these viruses. Virus SV40 replicates in primary cultures of green monkey kidney (GMK) cells and in CV-1 cells-an established line of GMK cells. The duration of the growth cycle is long compared with other types of animal viruses. Also, the growth of SV40 depends t o some extent on the metabolic condition of the host cells. I n confluent monolaycr cultures of GMK or CV-1 cells, the eclipse period lasted for about 20 to 24 hours (Fig. 7 ) . Intracellular infectious virus, then, increased to a maximum a t about 48 to 55 hours; total infectious virus (intracellular and extracellular) increased until about 72 hours after infection. Vacuolation was first detected about 60 hours after infection, and, by 72 hours, virtually all the cells in the culture displayed typical cytoplasmic vacuolation. At an input multiplicity of 5 to 10 PFU (plaque forming units)/ceIl, 3% of the cells were positive for the SV40 T-antigen at 18 hours, and this percentage increased so that a t 48 hours after infection, approximately 80% of the cells were positive. About 80% of the cells were also positive for the SV40-capsid (V)antigen. At SV40 input multiplicities of about 25 PFU/cell or more, 6080% of the cells were capable of forming infectious centers (Carp and Gilden, 1966; Kit et al., 1966e; Mayor et al., 1962). The rcsults were quite diffeient if rapidly growing cells were infected with SV40. Whereas the yield of SV40 was about 100 PFU/cell in stationary-phase cultures, this yield was less than 2 PFIJ/cell in replicating-phase cultures. The SV40 T-antigen was detected in only 1 to 10% of the rcplicating-phase cells and few cells showed cytopathic changes. Cells infected while rapidly growing continued to multiply despite in-
174
SAUL KIT I o8
x
10'
/?
-E a b v) ._ c t
P
.-
E
b
L
g3 10'
I
I 16
1
I 32
I
I 40
I
I 64
I
1 80
Hours postinoculation
Fm. 7. Growth of SV40 in CV-1 cells (established line of Green monkey kidncy cells).
fection and have been subcultured serially for at least fourteen passages (Carp and Gilden, 1966). Polyoma virus replicates actively in mouse kidney or mouse embryo cells. In confluent monolayer cultures of mouse kidney cells, the eclipse period lasted about 21 hours after which virus titers increased until about 60 hours after infection. At an input niultiplicity of about 100 to 200 PFU/cell, 40-600/0 of the cells were productively infected by polyoma virus (Dullmco et al., 1965; Kit e t al., 196Gd).
B. RIBONUCLEIC ACID AND PROTEIN SYNTHESES IN PAPOVAVIRUS-INFECTED CELL CULTURES The overall rate of 'H-uridine incorporation into RNA was not significantly changed during the first 24 hours after acute infection of
175
VIR.iI,-ISUUC'EU ENZYMES A N D I'INAL ONCOGENEhlS
$ 1 c
c
0
loot
-u-LuJ 0
4
8
12
16
20
24
Hours oostinoculotion
FIG.8. Incorporation of 3H-uridine into RNA of noninfected or SV40-infected CV-1 cells. Seven-day-old cultures (5.4 X loG cclls/culture) were inoculated with 180 PFU/cell of SV40 at t h e zero. At the times indicated in the figure, 3H-uridinc was added and the cultures were incubatcd for an additional 30 minutes.
CV-1 cells by SV40 (Fig. 8 ) . Moreover, protein synthesis, as measured by the 3H-leucine incorporation into protein continued a t an unaltered rate (Carp and Gilden, 1966). Similai-ly,there werc no large differences in the rates of incorporation of "-uridine into total cell RNA or of 'Hleucine into total cell protein during the first 30 hours after polyoma virus infection of confluent mouse kidney cell cultures (Dulbecco et al., 1965). These findings are in marked contrast to the early inhibitions of cellular nucleic acid and protein syntheses observed with most other viruses (Section VI 1 and :ittest to the fact that papovavirus infcctions are moderate.
C. TEMPORAL R E L A T I O N ~ HOFI PPROTEIN ~ A K D SUCLEIC ACIDSYNTHESES DURING PAPOVAVIRLS DEVELOPMENT 1. Virus-Specific RNA
Virus-specific RNA can be detected by pulse-labeling infected cells with RNA prwur>orb :tnd studying the calmcity of the labcled RNA to hybridize with virus DNA. During the first 16 hours aftcr polyoma infection of mouse kidney cells, a .~iiiallbut iiiri.cnsing :mount of hybritlizable RNA was detected. From 16 to 32 hours aftvr infection, the
176
SAUL KIT
relative amount of virus-specific RNA was much greater. However, even during the period of maximum synthesis of polyoma virus RNA (2428 hours), not more than 1% of the labeled RNA could hybridize to polyoma DNA. These results support the conclusion that polyoma infection does not block the synthesis of cellular RNA. I n fact, a comparison of the capacity of RNA from noninfected and from infected cells t o hybridize with cellular DNA suggests that celluIar RNA synthesis may be stimulated by polyoma virus infection (Benjamin, 1966). 2. Virus-Specific Proteins Drug-inhibitor and immunologica1 studies have shown that synthesis of early proteins essential for polyoma virus DNA formation starts a t about 9 hours after infection and that synthesis of virus-capsid proteins commences several hours later. Infectious DNA formation was prevented if puromycin was added to infected cultures a t any time during the first 9 hours after infection. Polyoma virus production was completely blocked by drug addition from 0 to 14 hours after infection. When applied after 14 hours, virus formation was only partly blocked. If puromycin was present for 16 hours and then removed, a normal yield of polyoma virus was obtained at 48 hours (Gershon and Sachs, 1964). Exposure of polyoma virus-infected cells to FPA from 4 t o 15 hours after infection delayed virus maturation by 19 hours, but a t 106 hours, virus yields from FPA-treated and infected control cultures were equal (Munyon e t al., 1964). During lytic infection of mouse embryo cells, T-antigen appeared about 12 hours after polyoma virus infection, reached a peak a t 24 to 48 hours, and then decreased (Habel et al., 1966). Twenty-four hours after infection, over 50% of the cells showed nuclear fluorescence similar to that described for SV40 (Takemoto et al., 1966). As measured by immunofluorescence, polyoma capsid-protein formation was first detected about 14 hours after infection. B y 16 t o 18 hours, most of the cells were producing virus protein (Sheinin, 1964; Sheinin and Quinn, 1965). The kinetics of SV40 tumor (T)-antigen and of viral-capsid antigen formation in CV-1 cells are shown in Fig. 9. An increase in T-antigen was detected a t 10 hours and attained a maximum a t about 30 hours. Formation of viral-rapsid antigen began several hours after that of Tantigen (Kit ef nl., 19GGg). Synthesis of SV40 T-antigen in primary cultures of GMK C C ~ was S abolished by cycloheximide addition froni 0 to 10 hours after infection (Gilden and Carp, 1966). However, inhibitors of DNA synthrsis (i.e., fluorouracil, dFU, ara-C, or dIU) neither reduced nor delayed SV40 T-antigen production (Butel and Rapp, 1965; Gilden and Carp, 1966;
177
VIRAL-INDUCED ENZYMES AND VIRAIJ ONCO(iENES1S
Cril(lcn et (11.. 19G.5; Rlclnick : i i d R:ipp, 1965; R:ipp et al., 1965a). Viral DNA ~ynthc+is also 1va3 not, required for polyoma virus T-antigen protluctioii siuw CF (coinl)lmc~nt3 fixing) titers rrlaclied those of infected control cclls whrn viral rq)lic.:ttioii wis in1iil)itccl by tiFU or ara-C (Habel et ul., 1966). I n contrast to the lack of inhibition of T-antigen formation, the inhibitors of D N A synthesis greatly reduced viral capsid-antigen production (Melnick and Rapp, 1965; Muriyon et al., 1964; Rapp et al., 1965a; Sheinin, 1964). Addition of ara-C a t 8 to 12 hours after infection T - anttqen ( C V - I cells) 256[
-1
80
Hours postinoculation S V 4 0
FIG.9. Kinetics of T-antigen and viral-capsid antigen formations in CV-1 and in mouse kidney cell cultures inoculated with SV40. (CF = complement fixing.)
inhibited viral capsid-antigen formation, but addition of the inhibitor 16 hours after infection or later failed to do so. These results suggest t h a t viral capsid-antigen formation may be dependent on D N A replication. Inhibition of RNA synthesis by actinomycin D treatment prevented either SV40 T-antigen or viral capsid-antigen production.
3. Metabolic Antagomsts and Viral D N A Synthesis From the following experiments, it appears t h a t synthesis of polyonia virus D N A began a t approximately 14 to 1.5 hours PI, and that of SV40 DNA a t about 18 hours PI. When dBU, dIU, or mitomycin C were added to mouse embryo cultures earlier than 14 hours after polyoma virus infection, virus formation was cornpletely blocked. The addition of these drugs a t progressively later intervals of time resulted in the formation of correspondingly greater yields of infectious virus. No inliibition of infectious virus formation wiis obscrved if dIU was added
178
SAUL KIT
a t a time later ttiun 40 hours after infection (Gershon and Saclis, 1964; Rlunyon c f nl., 1964). Tllt~(.ff(\cthof (IFTI and ;ira-C, rcqwctivcly, on SV40 replication have been htucliul hy Uutel and Rnpp (1965) and by Gilden and Carp (1966). No infectious SV40 was detected when dFU or ara-C were added as late as 18 to 22 hours after infection. Addition of the inhibitors a t later times permitted increasing amounts of infectious SV40 formation. The inhibitory effects of DNA antagonists on virus production were reversible (Butel and Rapp, 1965; Gilden and Carp, 1966; Sheinin, 1964). The presence of dFU in cultures of mouse embryo cells infected with polyoma virus for up to 17.5 hours had no effect on the subsequent time course of virus formation when the inhibitor was removed. Also removal of ara-C after 40 hours of treatment of infected GMK cells permitted normal yields of infectious SV40 24 hours later.
4. Stimulation of $H-dT Incorporation into D N A of Znfected Cultures Radioautographic and radioisotope-incorporation studies have provided additional data concerning the timing of DNA synthesis in papovavirus-infected cell cultures. Studies by several laboratories have shown that when confluent monolayer cultures were infected with these viruses the overall ratc of DNA biosynthesis was stimulated (Dulbecco et al., 19fj5; Kit et al., 1966d,e; Minowada, 1964; Minowada and Moore, 1963; Molteni et al., 1966; Weil e t al., 1965; Winocour et al., 1965). Figure 10b illustrates pulse-labeling experiments on the incorporation of 3H-dT into DNA of SV40-infected GMK cell cultures. Similar experiments have been performed with 3H-deoxyadenosine as DNA precursor with csseiitially the same rcsults. A stimulation of DNA biosynthesis was first detected about 16 hours after SV40 infection. A t 30 to 32 hours, the rate of 3H-dT incorporation into DNA of infected cultures was over 3 times that of noninfected cultures. This high rate of DNA biosynthesis continued until about 50 hours after infection. Radioautographic experiments on the same cell populations are shown in Fig. 10a. Initially, less than 10% of the nuclei of noninfected GMK cells were labeled after a 2-hour ‘H-dT pulse. This value increased to about 20% a t about 16 hours after i‘rnock-infection” and then declined. I n SV40-infected cultures, the percentage of cells with 3H-labeled nuclei increased sharply at about 16 hours and exceeded that of noninfected cultures. By 30 to 32 hours after infection, 7 0 4 0 % of the cells exhibited nuclear labeling. The radioautographic experiments demonstrate that many cells not synthesizing DNA initiated DNA synthesis a t about 16 hours after SV40 infection. Colorimetric assays of the total DNA synthesized in infected cultures
VIRAL-INDUCED ENZYMES A N D VIRAL ONCOGENESIS
179
I00
a
SV40 - infected
ao 70 60 -
.-m
50 -
c
40 -
0
8.
30 20 10 I
0 800-
I
I
I
I
b.
700
600
% 500
,E
400
300
'""I I00
u
0
Noninfected
-
L
10
l
~
20 30 Hours postinoculation
-
40
50
FIG. 10. Fh3dioautogral)hic (a) and biochemical (b) studies on the uptake of 'H-thymidinc ('H-dT) by SV40-infected GMK cells. Replicate 9-day-old cultures (14.7 X lo6 cells/culture) were inoculated with approximately 10 PFU/cell of SV40. A t the indicated times, 'H-tlT was nddcd and the cultures wcre furthcr incubated for 2 hours at 37" C.
confirmed that DNA synthesis was enhanced. The total D N A of SV40iiifccted cultures exceeded that of noninfected cultures a t about 30 hours, and a t 48 hours after infection was 43-9076 greatcr than that of noninfected cultures (Kit e t al., 1966e). The 3H-dT incorporation into DNA of confluent monolqcr cultures of inouse kidney cells was significantly increased a t 16 t,o 24 hours after polyoma virus infection; a t 30 Iioiirs, incorpoixtion into 1)NA was 5 times grcltt,ei*i i i infrctctl t l i a i i i i i noninfcxrttvl rult ~ i i ' t + (Kit, rl u L . ) 1966~1). Polyoliia virus infection :tlso incre:wctl ty sovei*alfold the ii~coi~~~orat.iona of J"'-oi.thopliosl,Ii:ltc, "C-Toi.iii:itc~, : i i i ( l ' i ~ ~ I ~ ~ : - L - i i i c t l l i ointo i i i l ~ DNA c
180
SAUL KIT
of mouse kidney cultures. The total DNA syr~thcsized by infected cultures a t 36 hours was 21-63oJo grcater than that of noninfected cultures (Dulbecco et al., 1965; Winocour et al., 1965). The nuclei of about 5% of noninfected mouse kidney cells were labeled after a 2-hour 3H-dT pulse. However, a t 24 to 26 hours after polyoma virus infection, this number increased to 34% and, by 48 t o 50 hours, 65% of the nuclei were labeled (Kit et al., 1966d). 5. Viral-Induced Synthesis of Host D N A
I n confluent monolayer cultures productively infected with papovaviruses, some of the newly synthesized DNA must be viral DNA. HOWever, the total amount of DNA synthesized appears to be far in excess of that needed for infectious virus formation alone, and this suggests that induction of cellular DNA synthesis had also taken place. Experiments from several laboratories strongly support this inference. The evidence is as follows: 1. The rate of DNA biosynthesis a t 16 to 30 hours after infection of stationary-phase, mouse kidney cell cultures with polyoma virus was 10 times greater than that of mock-infected cultures. Chromatography on methylatcd albumin columns revealed that about two-thirds of the newly synthesized DNA was cellular DNA. Therefore, ccllular DNA synthesis in infected cultures exceeded that in rioninfected cultures by a factor of over 6 (Dulbecco et al., 1965). 2. Polyoma DNA contains 0.9 residue of 5-methylcytosine per 1000 DNA nucleotides, whereas mouse kidney DNA has 9-10 residues per lo00 nucleotides of DNA (Winocour et al., 1965). The methyl group of 14CH,-methionine is a precursor of 5-methylrytosine and of thymine, guanine, and adenine. During the period of stimulated DNA synthesis, the pattern of incorporation of radioactivity from 14CH,-methionine into the DNA bases of polyoma virus-infected cultures was similar to that of noninfected cultures but differed from that of purified, polyoma virus DNA. Thus, a substantial portion of the newly made DNA must have been cellular DNA. 3. Noninfected mouse kidncy monolayers incorporate little 14C-dBU into DNA. Following CsCl density gradient centrifugation of this DNA, two bands are observed-a large band corresponding to normal mouse DNA and a small, labeled band corresponding to “hybrid” DNA (onc strand containing 14C-dBU and the other only dT). The uptake of I4CdBU into DNA is markedly stimulated a t 24 to 50 hours after polyoina. virus infection of mouse kidney monolayers. Aiialysis of the DNA of thc infected cultures revealed that most of tllc DNA h : ~ lshiftetl fi-onl tlle positioll of normnl tlcnsity to :i position coriwponding to hybrid DNA.
VIRAL-1NDUCED ENZYMES AND VIRAL ONCOGENESIS
181
Moreover, the hybrid DNA was radioactive proving conclusively that cellular DNA synthesis had been induced by polyoma virus infection (Weil et al., 1965). I n a study of crowded cultures of GMK cells, Hatanaka and Dulbecco (1966) have shown that cellular DNA synthesis is also induced by SV40. The induction of cellular DNA synthesis was initiated about 24 hours after infection and reached a maximum at 36 hours. At about 48 hours, the rate of cellular DNA synthesis decreased rapidly whereas SV40 DNA synthesis increased. I n the experiments so far described, crowded monoIayer cultures were crnployed and about half of the cclls were productively infected. Sheinin and Quinn (1965) have investigated DN-4 synthesis in rapidly growing cultures of mouse embryo cells infccted with approximately 5000 PFU/ cell of polyoma virus. Under the conditions of their experiments, practically all the cells werc productively infected. In contrast to the results obthined with crowded monolayer cultures, Sheinin and Quinn (1965) found that in the rapidly growing cultures, synthesis of host-cell DNA was inhibited by polyoma virus. During the eclipse period, practically all of the DNA made was cellular DNA. Thereafter, cellular DNA synthesis decreased rapidly to very low levels. Polyoma virus DNA synthesis began a t the end of the eclipse period and increased progressively until all the DNA synthesized in the infected cultures was viral DNA (Sheinin, 1966b). I n view of the results of Sheinin (196613) and Sheinin and Quinn (1965), the question arises as t o whether induction of celluIar DNA synthesis occurred only in cells that were not productively infected, i.e., cither in abortively infected cells or, as an indirect phenomenon, in noninfccted cclls. In order to clarify this question, Vogt and co-workers (1966) carried out a combined radioautographic and irnmunofluorescence study. Polyoma-infected confluent monolayer cultures of mouse kidney cells were pulse-labeled with 3H-dT at a time when a high proportion of the DNA synthesized was cellular. The cells were fixed after an appropriate time of incubation to allow for the synthesis of the viral-capsid protein. At least 90% of thc cells synthesizing DNA a t the time of the pulse also made viral-capsid antigen a t the time of fixation. These rcsults showed that the induction of cellular DNA synthesis took place in productively infected cells. The following interpretation of the preceding experiments may be suggested. Iiicluction of cellular DNA synthesis by papovaviruses depends 011 tlic physiologicd conditioii of the cclls. When few cells are in the ‘W-period of t h r initotic cycle, DNA syntliesis in:iy l)c initiiited in ~ n a n y of tlic rcmaining cclls of tlic cultiirc. T-Towever,if the cultures are actively
182
SAUL KIT
growing so that a large proportion of the cells already are synthesizing cellular DNA a t a rapid rate, cellular DNA will not be stimulated. Indeed, late in the infection as viral-DNA synthesis increases and infectious virus particles are formed, cellular DNA synthesis will be progressively inhibited.
D. INDUCTION OF ENZYMES FUNCTIONING IN THE TERMINAL PATHWAY OF THYMIDINE METABOLISM 1. Kinetics of Enzyme Formation
Since DNA synthesis was grossly stimulated in cells infected with papovaviruses, it seemed likely that some of the enzymes of DNA metabolism would also be increased. Seven enzymes catalyze reactions in the terminal pathway of d T metabolism leading to DNA synthesis. This pathway and the pertinent enzymes are schematically depicted in Fig. 11. Six of these enzymes (i.e., dCMP deaminase, d T kinasc, d T M P
4,
(Dihydrofolate reductase)
(dCMP Deaminase)
1,
AT P, Mg++
TPNH
(TMP synthetase)
(TDP kinase)
ATP Mg*
(dTMP kinase)
... ........( Thymidine . . . . .... ATP, kin& Mgt+
synthetasc, dihydrofolate (FH,) reductase, dTMP kinase, and DNA polymerase) increased in cell cultures infected with papovaviruses (Kit e t al., 1965, 1966d,e). The seventh enzyme, d T D P kinase, is present in great excess, and it seems unlikely that this enzyme would be induced by virus infection. Deoxyribonucleic acid polymerasr mid tlT kitlahe activities we^^ tn:irkrtlly stfiiiiul:itorl i n c w r i f l u n i t tiionolayc~r cultuws of citlier nio~i>e
183
\'I RA L- 1N DT 1C ED EX Z Y M ES A N D 1'1R A L ON CO( iEN ESIS
liitliicy oi* iiioiiw t.iiibryo w l l h iiifrcleil wit11 polyoilia v i r w and in either (iAIK or ( X - 1 cells infwtfvl n i t h PV40 (Diilhwco clt (7/., 196.5; F r e a r w i c f ( I / . , 1W5; Iiit r / ( I / . , 1965, 19(if%I,o,g;Sliviniii, 19(i(k). Tho 1iiiicBtic.s of tlic rneyiiic IIICTC:LMX i l l SV40-iiitc~tctlCV-1 cells :ire sliown i n Fig. 12. The d T kiiiase increascs began a t about 12 to 16 hours after SV40 infection, and at 28 to 48 hours tho onzyine activity 1ws 4-15 times greatcr
l2
t
Infected kinose-
/'
\ \
/
0 I
/
/
\
\
\ \
I
\
I
I
\
Infected polymerase
I
\ \
I
I I
I
-a0
10
20
30
40
50
60
70
80
Hours postinoculation SV40
FIQ.12. Kinetics of thymidine kinasc and DNA polymerase formation in confluent monolayer cultures of CV-1 cells inoculated with SV40 at an input multiplicity of 140 PFlJ/cell. Thyinidinr kinase nct,ivity : micromicromoles deoxyuridylatc fornied per microgram protein in 10 niinritcs at 38°C. Dcoxyribonucleic acid polymerase activity : micromicromoles 'H-deoxythyniidinc triphosyhatr incorporated into DNL4per microgram protein in 30 minutcs at 38°C.
than that of noninfected cells. The increase in DNA polymerase activity was less pronounced than t ha t of d T kinase but paralleled that of the latter enzyme. I n polyoma-infected mouse kidney monolayers, the kinetics of d T kinsse and DNA polymerase inductions were about the same as that in SV40-infected monkey kidney cell cultures (Dulbecco e t al., 1965; Hartwell et al., 1965; Hatanaka and Dulbecco, 1966). Two- t o threefold increases in d T M P synthetasc :tncl FH, reductase activit,ics were found following SV40 infection of CV-1 cells or polyoina
SAUL KIT 60
-
1
l2
50 -
40 -
w
3 s
--
30-
0
0
s
2
R 20G
P
10-
5\, Noninfected ( dT kinase) \ a . V-
/'
-
A /
'4 0
I
I
I
10
20
30
'Q-,
I
40 Hours postinoculation of SV40
c
--7
I
50
0
Fro. 13. Kinetics of dihydrofolate (FH,) rednctase and thymidine kinasc induction following infection of 7-day-old CV-1 cells (4.7 X 10' cclls/culture) with 314 PFU/cell of SV40. Dihydrofolate reductase activity : micromicromoles FH, reduced per microgram protein in 10 minutes at 23°C. Thymidine kinase activity: micromicromoles deoxyuridylate formed per microgram protein in 10 minutes at 38°C. (TdR = thymidine.)
virus infection of mouse kidney cells (Frearson e t al., 1965, 1966; Kit e t al., 1966e). Figure 13 illustrates experiments with SV40-infected CV-1
cells in which dihydrofolate (FH,) reductase and d T kinase activities were measured on the same cell extracts. The percentage increase of FH, reductase activity also was not as great as that of d T kinase but occurred over the same time interval. The dCMP deaminase activity of mammalian cell culturcs is very unstable, but the enzyme can be stabilized during extraction and activated during assay by dCTP. As mentioned previously, d T M P kinasc can be stabilized and activated by its substrate, dTMP. Even when extracts were prepared with buffer containing dCTP or dTMP, the activities of dCMP deaminase and d T M P kinase were very low in confluent mouse kidney cell monolayers. The activities of both enzymes were increased severalfold after polyoma virus infection. The time a t which 50% of the increase was achieved was almost the same as for d T kinasc (Dulbccco et al., 1965; Hartwcll e t al., 1965; Kit et al., 1966d).
I n contrast, tlic, :wt ivity of t1CMP dr:uninabe was uiiusually high in GMK or CV-1 cells (about 80 to 100 times greater than in mouse kidney cells). Moderately high levels of d T M P kiiiasc are also found in monkey kidney cells. Thebe two enzymes did not increasc in activity in GMK cells after SV40 infection. Scvrral additional enzymes have h e n stutlictl in papovavirus-infected cell cultures. Uridine kinase, d T M P phosphatase, dAMP kinase, and dCMP kinase did not change appreciably in activity after papovavirus infections (Dulbecco et al., 1965; Kit et al., 1966d,e). Experiments with puromycin and cycloheximide have shown that the papovavirus-induced cnzyrne increases did not occur in the absence of protein synthesis. Addition of puromycin during the first 12 hours after polyoma virus infection prevented the increases in d T kinase, dCMP deaminase, and DNA polymerase in murine cell cultures. Cycloheximide inhibited the stimulation of d T kiiiase and DNA polymerase in SV40infected monkey kidney cell cultures. If the inhibitors of protein synthesis were added to cultures after the enzyme increases had begun, further enzyme induction was curtailed. Moreover, removal of the drugs permitted a renewal of enzyme synthesis after a lag period (Hartwell et al., 1965; Kit et al., 1966d,e; Sheinin, 1966:~). Experiments in which mixtwes of enzymes from both infected and noninfected cells were assayed tlcmonstrated that the increased enzyme activities in infected cells were not attributable to activators in the infected cell extracts. Similarly, the low activities in extracts from noninfected cells were not due to an excess of free inhibitors in the extracts from noninfected cells. 2. Effect of Actinomycin Il
OIL
SV4O-Iduced Enzyme Synthesis
I n order to learn whether RNA synthesis was required for the induction of d T kinase by SV40, CV-1 cells were treated with 1 to 5 pg./ml. actinomycin D a t 2 hours after SV40 infection. Actinomycin D completely inhibited the SV40-induced incrcnw nornially observed a t 26 hours after infection (Kit et nl., 1965, 19GGf). If actinomycin D addition was delayed until 10 to 14 hours after infection, a partial induction of d T kinase took place. If the actinoniycin D was added at 17 to 21 hours after infection, almost normal levels of d T kinase were induced. These results siiggcst that most of thc nicssengvr RNA rrquirctl for (IT kinase formation was niacle by I7 hours aitrr SV40 infcctiun. similarly, nctinoniycin D (1&2.5 pg./'ml.) iitldetl at 2 hours after SV40 iiifection of CV-1 cells completely inliihitctl the increase in FH, rccluctase activity noimally observed 41 hourh after infection. If actiaomycin I)was added a t 12 hours, the SV40-infected cells showed a signifi-
1%
SbtJL KIT
cant increase in FH, reductasc activity a t 41 hours, but still 1 ~ : than s in infected but nontreated cells. Addition of actinomycin D a t 19 hours had little or no effect on the induction of FH, reductase (Frearson et al., 1966).
3. Effect of dBU on Papovavirus-Induced dT Kinases Halogen-containing analogs of d T are known to inhibit cell growth and t o prevent the development of infectious SV40 and other DNAcontaining animal virubes. However, both T-antigen and virus-capsid antigen are synthesized in infected cell cultures treated with the d T analogs. The addition of dBU to SV40-infected cell cultures did not appreciahly affect the ratc of dT kinase induction (Kit et al., 1966e). I n infected cultures not treated with dBU, d T kinase activity was elevated for about 48 hours and then declined sharply a t the time that cytopathology became pronounced. This decline was delayed in SV4O-infected cultures treated with dBU. At 96 hours after infection of dBU-treated cultures, the d T kinase activity was still sixfold greater than the activity of noninfected cultures. Treatment with dBU also did not inhibit the induction of d T kinase by polyoma virus (Kit et al., 1966d) nor the induction of FH, reductase by either SV40 or polyoma virus (Frearson et al., 1966). 4. E f f e c t of Mitomycin C on SV4O-Induced dT Kinase Formation Mitomycin C is a potent inhibitor of cell growth and DNA synthesis. Treatment of bacterial or mammalian cell cultures with this drug leads to an induction of DNase activity and the breakdown of DNA. At concentrations which inhibited DNA synthesis, mitomycin C addition to cultures a t 2 hours did not block the increase in d T kinase observed at 30 hours after SV40 infection. I n some experiments, GMK cell cultures were pretreated with mitomycin C for 16 hours prior to SV40 infection. The infected cultures were then further incubated in the absence of mitomycin C. Despite the pretreatment of GMK cell cultures with mitomycin C, d T kinase activity was 24 times higher in SV40-infected than in control cultures a t 30 hours after infection. Moreover, in cultures in which mitomycin C was present both in the preinfection and postinfection periods, the dT kinase activity of infected cultures was about 10 times greater than that of noninfected cultures. It is probable that prolonged treatment with mitomycin C not only inhibited DNA synthesis hut caused damagc, t o the host-cell DNA. The failure of mitoniyciii C to prevent the increase in d T kinase therefore suggests that nornial undamaged GMK cell DNA w i s not recluirccl for SV40-induced d T kinase synthesis.
5 . Effect of Ara-C on Papovavirus-Induced Enzyme Synthesis l-P-D-Arabinofuranosylcytosine is a potent inhibitor of cell growth, DNA synthesis, and the replication of DNA-containing animal viruses. Tlie mcchilnisin of action of ara-C is different froin that of either initomycin C or dBU. It is thought that ara-C curtails d C T P synthesis by preventing the reduction of ribonucleotides to deoxyribonucleotides. The addition of ara-C (10 pg./inl.) a t 2 hours after SV40 infection of CV-I cells or polyoiiia virus infection of iiiouse kidney cells had no inhibitory effect on the inductions of d T kinase or FH, reductase by these viruses (Frearson et al., 1966; Kit et al., 1966d,e). This concenh t i o n of ara-C did suppress "H-(IT iticorporation into the DNA of the cells. Ara-C treatment of CV-1 cells :ilso fniled to inhibit the induction by SV40 of DNA polymerase nctivity. It may be concluded froin the experiments with dBU, mitomycin C, anti ara-C that DXA synthesis, cell growth, and infectious-virus forinatioii were not required for the inductions of d T kinase, FH2reductase, and DNA polymerase by the papovaviruses.
6. Effect of Ultraviolet Light on the Infectivity and dT Kinuse-Inducing Activity of SV4O Ultraviolet irradiation of SV40 inactivates both infectivity and Tantigen-inducing activity. Loss of T-antigen-inducing activity occurs a t a slower rate than loss of infectivity (Carp and Gilden, 1965). Ultraviolet radiation also reduced the clT kinase-inducing capacity of SV40 (Carp et al., 1966). The loss of virus infectivity occurred 2-6 times faster than enzynie-inducing capacity. It is probable that virus DNA is the primary target of UV radiation. Since increasing doses of UV radiation progressively inactivated the d T kinase-inducing capacity of SV40, it appears that d T kinase synthesis in infected cells is controlled by the virus DNA. However, the UV irradiation experiments do not elucidate the mechanism by which the SV40 nucleic acid controls the forination of d T kinase. This could be due either to effects that act directly on the coding properties of the virus genome or to effects on the DNA of tlic virus which then arc translatrd to t,he control mcclianisnis of tlic cell. 7. Stiniulation by Ara-C and Illitomycin C of the dT Kinase Activity of Noninfected Monkey Kidney Cell Cultures I n connection with the problem of the mechanism of viral-induced enzyme synthesis, it would be useful t o have a method for inducing enzymes in noninfected cell cultures. Comparisons could then be made
188
SAUL KIT
of the enzyme induced in noninfected cells with those induced by virus infection. During study of the effects of drug treatment on the iiiduction of d T kinase in papovavirus-infected cells, a simple procedure was discovered for increasing the d T kinase activity of noninfected cells hy a factor of 3 to 10. It was found that the addition of ara-C (10 to 20 pg./ ml.) to noninfected GMK or CV-1 cells causecl pronounced increases in the activity of d T kinase starting a t about 12 hours after drug treatment (Fig. 14). The ara-C-induced stimulation of dT kinase activity occurred
O6
I
I
I
I
10
20
30
40
Hours after Ara-C addition
FIG.14. Kinctics of ara-C-induced stimulation of dT kinase nciivity of 9-dny-old G M K cell cultures. Arn-C concentration : 10 pg./nil.
in HeLa cell cultures as well a s monkey kidney cell cultures but did not occur after ara-C treatment of LM and LM(TK-) mouse fibroblast cells, primary mouse kidney cells, or HeLa (BU-100) cells (Kit e t al., 1966a,b). I n noninfected CV-1 cell cultures treated with ara-C, a n 84% increase of FH? reductase and two- to threefold increases of dCMP deaminase and DNA polymcrase activities were observed. However, dTMP kinase activity was not significantly changed. The ara-C-induced incrcascs could be reversed by deoxycytidine (dC) . It was also found that an appreciable increase in d T kinase activity occurred after DNA synthesis was inhihited by mitomycin C or amethopterin. The amethopterin inhibition of DNA synthesis could he reversed
189
\ l I t A I ~ - I N L ) U C E D ENZYMES AND \‘1RAL ONCOGENEHIS
by d T ; after d T reversal of the inhibition, the activity of d T kinase rapidly declined. It is attractive to suppose that cell cultures were pseudosynchronized by drug treatment. Cells not in the G, phase of the mitotic cycle entered this phase but were prevented by the inhibitors from entering the “S” phase of DNA synthesis. An accumulation of enzymes related to DNA biosynthesis would appear to be charactcristic of late G1-phase cells. TABLE IX AND ARA-C OR BROMODEOXYURIDINE TREATMENT EFFECTSOF SV40 INFECTION ON THE THYMIDINE KINASEACTIVITY OF GMK CELLS dT kinase activity (ppmoles dUMPn formed per fig. protein in 10 min. at 38°C.) (:onr?mt,m.- - .- -.... I
tion Inhibitor
(pg./ml.)
None Ara-C dBU
None 20 25
a
48 hr. PI 28 hr. P I Nonirifected SV40-infected Noriinfected SV40-infected 1.3 9.9 1.8
6.4 14.7 7.8
0.8 4.8 0.9
5.9 10.9 9.2
Deoxyuridylate.
Table IX shows the dT kinase activity of GMK cclls infected with SV40 and also treated with ara-C or dBU for 28 and 48 hours. Either ara-C treatment or SV40 infection increased the d T kinase of GMK cells, but dBU treatment did not. The effects of ara-C and SV40 infections on d T kinase induction were additivc. Additive effects of ara-C and SV40 infections on the induction of DNA polymerase were also observed. 8. Properties of Partially Purified dT Kinnse
Using a relatively simple procedure consisting of ammonium sulfate fractionation and negative phosphate gel adsorption, d T kinase has been purified approximately twenty- to fortyfold compared with crude-cell cxtracts (Kit et al., 1966d,e). To learn whether SV40 infection produced a d T kinase with altered properties, the I(, of the SV40-induced eiizyme was determined and a comparison was m : d ~with the I (Table YII) . C'aixinogcnicity i> lo5t outright by iuetliyl s u l ~
332
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE V I I CAR(:INOGENICITY OF TRICYCLOQUINAZOLINE DERIVATIVES TOWARD THE SKIN OF MICE"
+
Papilloma epithelioma incidence Compound
( %)
Tricycloquinasoline (TCQ) 1-Methyl-TCQ 2-Methyl-TCQ 3-Methyl-TCQ 4-Methyl-TCQ 3,8-Dimethyl-TCQ 3,8,13-Trimethyl-TCQ 2-Methoxy-TCQ 3-Methoxy-TCQ 3-Ethyl-TCQ 3-tert-Butyl-TCQ 2-Fluoro-TCQ 3-Fluoro-TCQ 4-Fluoro-TCQ 3,8-Difluoro-TCQ 2-Chloro-TCQ 2-Bromo-TCQ 3-Bromo-TCQ 2,3-Benzo-TCQ 3,CBenzo-TCQ
81 55 9 59 44 9 17 12 12 55 54 79 76 31 40 58 74 68 2 0
Epithelioma Mean latent Iball index incidence period (epitheliomas (%) (days) only) 75 45 0 48 44 0 9 6 0 24 20 23 65 11 16 53 68 0 0
186 3 16 46 1 296 282 244 277 32 1 464c 53w 544 454c 375 3 10 55P 360" 33OC 274 487" 425c
Compiled from Baldwin et al. (1962a,c, 1963b, 1964a, 1965a,b). The 2-hydroxy derivative is also inactive (cited in Baldwin et al., 1962a, 196513). Total length of experiment (days). Estimated transitory values based on available data: totul tumor incidence and/or totul duration of experiment.
stitution in the 2-position, but the isomeric 1-, 3-, arid 4-methyl derivatives are still appreciably active. The 3-position is surprisingly insensitive to the nature or size of the substituent; appreciable ability to induce malignant epitheliomas is preserved in 3-methyl-, 3-fluoro-, 3-bromo-, 3-ethyl-, and even 3-tert-butyltricycloquinazoline.Annelation of another benzene ring in the 2,3- or 3,4-positions brings about total loss of activity. All these observations suggested to Baldwin and his associates that stereocheniical factors play a preponderant if not exclusive role and that the critical interaction of tricycloquinazoline in the cell requires a highly specific orientation or fit of the molecular frame with respect to or on the receptor site(s) (Baldwin and Partridge, 1964; Baldwin e t al., 1965a). They thought to derive support for this view from the fact that substitution in the. 2-pnsition by n fluoriiic atom, wliich is only slightly larger than
hydrogen (atomic radius 1.35 A, compared to 1.1 A for hydrogen), lowers the epithelioma incidence to less than one-third. Thus, it was held that steric orientation during cellular interaction is critically controlled by the 2- and equivalent positions. However, an important piece of evidence furnished recently by Partridge and Vipond (1966) showed that deactivation by the 2-substituents in the hitherto tested derivatives should be ascribed to electronic rather than to steric effects. Thus, it appears t h a t deactivation is due to the +M effect of the substituent. This follows from the observation that 2-trifluoromethyltricycloquinazoline gives a tumor incidence of 60%. Methyl groups are well known to promote mesomeric shift by hypereonjugation, whereas a trifluoromethyl group gives a n overall -1 effect owing to the electronegativity of the fluorine atoms. With 2-substituents which have an overall -1 effect, activity is retained, whereas a substituent having a +I or +M effect abolishes activity. This also provides a rationale for the substantial activity of 2-bromotricycloquinazoline over that of 2-fluorotricycloquinazoline (Table V I I j which cannot be explained stereochemically.
D. METABOLISM 1. Carcinogenic Activity and Rate of Elimination from the Tissues
The relationship between the rate of elimination from the target tissue and carcinogenic potency has continued to attract interest. A study (Domsky et al., 1963) of the rate of disappearance contributed in drawing attention to the high susceptibility of newborn mice t o carcinogenic chemicals (Kelly and O’Gara, 1961 ; Pietra et al., 1961; Roe et al., 1961), in particular to 9,10-dimcthyl-1,2-benzanthracene, which has been found to induce a high incidence of lymphomas and other tumors in the newborn a t dosages t h a t induce only few such tumors in the adult. I n Fig. 4 are shown the results of Domsky et al. (1963), indicating t h a t the free 9,10-dimethyl-l,2-benzanthracene,solvent-extractable from the homogenized whole carcass, decreases much faster in the adult than in newborn mice. Thus, the greater susceptibility of the newborn to tumor induction is due t o the longer persistence of the compound in the newborn than in adult mice. This is consistent with the earlier finding of Cramer et al. ( 1960a) that young aiiirrials mctabolize hydrocarbons less actively than adults. Correlation between slow rate of elimination from the tissues and a high degree of carcinogenic potency was also the conclusion of Unseren and Fieser (1962) who found t h a t most, if not all the potent 3,4,9,10dibenzopyrene remains unmetabolized in mice a t the site of subcutaneous injection, No aromatic metabolites were detected either in the feces or
334
.JOSEPH C. ARCOS AND M A R Y F. ARGUS
2
4
6
8 10 Days
12
14
FIG.4 . Rate of disappearance of 9,10-dimethyl-1,2-benzanthracene (DMBA) from adult ( X ) anti newborn ( A ) whole Swiss mice. The logarithm of the percentage of injccted hydrocarbon rerovered from thc carcass is ploi t ~ dagainst thc time aftcr injrction. (From Domsky et al., 1963.)
the emerging tumors despite the fact that the hydrocarbon may be readily oxidized chcmically to 5,g-quinone and 5,8-diacetoxy derivatives. This substantiates the earlier findings of Lacassagnc e t al. (e.g., 1961a) on the long persistence of this and other large hydrocarbons in the tissues. I n contrast, Goodall e t nl. (1963) observcd no correlation betwcen tissue localization and rarcinogcnic potency in fcrriale Spraguc-Dawlcy rats when 14C-labeled 20-methylcholanthrcne, which is known to producc mammary tumors in females of this strain (e.g., Huggins e t al., 1959a), was administered orally ; neither labeled carcinogen nor a labcletl metabolite was localized in the mammary tissue. The relation between rate of elimination and the resistance of different tissues to topical carcinogenic action was investigated by Pozdnyakov (1963a,b). This author found that using 9,10-dimethyl-1,2-benzanthracene by direct intramuscular route in roosters there is a higher tumor incidence in the thoracic muscle from which resorption is slow than in the femoral muscle from which resorption is much faster. It seems, therefore, that the higher mctaholic activity of the latter muscle contributes to increased resistance to local carcinogenic effect. The rate of excretion of tricycloquinazolinc arid its persistence in thc target tissue has been briefly investigated by Baldwin e t al. (196313, 1964a). Intravenously injected l-lC-tricyclocluinazoline concentrates very rapidly in the abdominal tract and carcass ; following intraperitoneal administration, over 50% of the radioactivity is excreted in 48 hours through the feces. Twenty-four hours following skin painting, as much
hlOLECIlL.4R (;EOhIETRT AND CARCINO(;ENIC’ ACTIVITY
335
:is 80y0 of thc radioactivity is rccoverable froiii the skin. However, this low rate of ahsorption hliould not ncccssarily be regarded as a limiting factor in carcinogenic activity. One should recall in this regard, the ~ ~ r s u lof t s Bock and Bui*nliaiii (1961) who found no relation hetween skin pcwc’tration ui~lci. stnndnrrl cxpcriincntal conditions and carcinogenic activity in ti ect of twclvc polycyclic hydrocarbons. 2. A‘onbo wit1 M e tci bol ites u . Metabolism of 1,2-Benzantlzracene uiid I t s Relation to Bond Order. The early finding that polycyclic hydrocarbons containing a 1,2-benzantliracene nucleus undergo metabolic hydroxylation predominantly in the 4’-position suggested that the 3’,4’-bond represents a molecular “region” of metabolic perhydroxylatiori p a r excellence; hence the term M region designating this bond (Pullman and Pullman, 1955a). However, in recent years, Boyland and his associates (e.g., Boyland and Sims, 1964; reviewed by Boyland, 1964a,b) carried out exhaustive investigations on the nietabolisni of 1,2-benzantliracene and plienanthrene, and showed t h a t metabolic reactions involve other bonds of tlic iiiolccule as well. What is highly significant is that tlie relative ninouiits of total inetaholites involving thc different bonds vary with tlic chemical reactivity of the latter. A measure of this reactivity is the bond order which, in turn, is an expression of electron presence a t an aromatic double bond (Table VIII) . These TABLE VIII Bond Orders and Relative Amounts of Metabolites of 1,2-Benzanthracene Produced by Rodentsa 2’
10
5
4
Bond order
Relative amount of metabolites
6,7
0.593
-
2‘,3‘
0.628
-
1‘,2’
0.695
-I-
3’,4‘
0.700
++
7,8
0.731
+
Bond
5,6
0.132
3,4
0.783 a From Boyland (1964a).
tit
i
+++ t
336
JOSEPH C. ARCOS AND MARY F. AROUb
and similar data for phenanthrene seem to concur with the notion t h a t a specific molecular region of metabolic perhydroxylation may not be a tenable one. Despite the fact that all metabolites of polycyclic hydrocarbons have proved so far to be less active than the parent compounds or inactive, Boyland (1950, 1964a) hypothesized that epoxides might be proximate carcinogens of the hydrocarbons. Epoxidation may take placc preferentially a t the K-region which has a high bond order. I n an attempt to substantiate this hypothesis, E. C. Miller and Miller (1967) found t h a t 3,4epoxy-3,4-dihydro-10-methy1-1,2-benzanthracene1 the K-region epoxide of the potent carcinogenic hydrocarbon, lO-rnethyl-1,2-benzanthracene,has only low carcinogenic activity when tested subcutaneously in the rat or on thc skin of the mouse. Boyland and Sims (1967), testing subcutaneously in C57 mice, confirmed t h a t the cpoxide is significantly less active than the parent hydrocarbon. I n the same test system, the K-region epoxides of other hydrocarbons, chrysene, 1,2-benzanthracene1 1,2,5,6dibenzanthraceiie (Boyland and Sims, 1967), and of 20-methylcholanthrene (Sims, 1967b), were consistently less active than the parent hydrocarbons. E. C. Miller and Miller (1967) found the K-region epoxide of l12-benzanthracene t o be devoid of tumor-initiating activity in surface application. The K-region dihydrogenated derivative of 20-methylcholanthrene was also inactive (Sims, 1967b). b. Metabolism of J,.&Renzopyrene. With the exception of thc socalled F, metabolite, the identity of the 3,4-benzopyrene biliary metabolites remains unresolved. Falk et al. (1962) reported the isolation of a total of twenty-seven biliary metabolites in the rat. These have been tentatively identified as being various sulfo and glucuroconjugates of the following hydroxy derivatives of 3,4-benzopyrene: 5-, 8-, 5,8-, 5,lO-, 8,9-dihydroxy-8,9-dihydro-, 6,7-dihydroxy-6,7-dihydro-,and 1,5-dihydroxy-l15-dihydro-. Awaiting detailed confirmation of this very complete work, it should bc already noted t h a t Pihar and SpAleny (1956a,b) isolated 8-hydroxy-, 10-hydroxy-, and a small quantity of 5-hydroxy-3,4benzopyrene from the feces of rats rcceiving 3,4-benzopyrene and that rat liver homogenates were found to metabolize the hydrocarbon to the 8- and 10-hydroxy and 5,8-diliydroxy derivatives (Conney et al., 1957). Nevertheless, a t present there is far from general agreement concerning the identity of Weigert and Mottram’s XI, X,, and F, biliary metabolites (reviewed by Boyland and Wcigert, 1947) a s illustrated in Table IX. Moreover, Sinis (1967s) working with liver hornogcnatcs clainrctl that F, corresponds to 8-hytfroxyl)enzo[a ] pyrenc (also known as 3’-hytlroxy-3,4benzopyrene) wliich is actually a n artifact resulting from the deconiposition of 7,8-dihydro-7,8-diliydroxybenzo [ a ]pyrene present probably in its
IDEXTITY O F
Metabolite
BILl.4RY B P X
AND
TABLE IX BPF ~IETABOLITES O F 3,PBENZOPYRENE
Weigert and Mottram (1943, 1946)
Bereiddurn and Schoerital (1946, 1955)
IN
IVTRAVENOUS MICE FOLLOWING
Harper (1958, 1959b)
.4DMINISTR4TlOrrl
Falk ef al. (1962)
8,9-Dihydroxy-8,9-dihydro- 10-Hydrnxy-3,4-benzopyrene, 3,4-benzopyrene, conconjugated with unknown jugated a t the 8-hydroxyl group with unknown group
Sulfoconjugates of l-hydroxy- and 8-hydroxy3,4-benzopyrene
S-Hydroxy-3,4-benzopyrene, conjugated mit,h an unknown group
8,9-Dihydmxy-S,S-dihydro- X-Hydroxy-3,4-beiizopyrene, 3,4-benzopyrene, both hydroxyls conjugated with unknown groups
conjugated with unknown group
(;lucuronides of l-hydroxyand 8-hydroxy-3,4benzopyrene
8-Hydrosy-3,4-t,eiixopyreiie glucositlnronic acid
8-Hydroxy-3,Pbenzopyrene. conjugated with unknown group
10-Hgdroxy-3 4-benzopyrene
1-Hydroxy-3,Pbenzopyrene
Sulfoconjugates of 8-hydroxyand 5,8-dihydroxy-3,4benzopyrene
8-Hydroxy-3,Pbenzopyreiie
8-Hydroxy-3,Pbenzopyrene
8-Hydroxy-3,4-benzopyrene
8-Hydroxy-3,4-benzopyrene
338
JOSEPH C. AHCOS AN0 MARY 17. ARGUS
sulfo-conjuga ted form. Sims found no c~itleiiccfor the presence of 3,4benzopyrenc metabolites hydrosylated in the K-region. Ingenious cxpcriinents by Kotin et n l . ( 1962) gave furthcr support to tlic lwlivf that tlit. 1iydroctirl)ons t h c ~ n w l ~ratlicr ~ ~ h than sonic inetabolitcs are the cruci:il carcinogeiiic. stimuli. In thcse experiments, where mice received simultaneously subcutaneous injections of 3,4-benzopyrene and the hepatotoxic solvent, carbon tetrachloride, a strong enhancement of the tumorigenic response was brought about by impairment of thc detoxicating ability of the liver. Since the XI, X,, and F, metabolites are noncarcinogenic and F, is a wcak carcinogen compared t o the parent compound, the enhancement can be reasonably ascribed to slower detoxication of the hydrocarbon by the carbon tctrachloride-injured liver and, hence, longer persistence a t the injection site. These and subsequent studies with 3 , 4 - b e n ~ o p y r e n e - ~ ~have, C in fact, shown a considerable slowdown of the elimination of total radioactivity through the bile in the carbon tetrachloride-injure(1 animals (Falk, 1963). However, not only is the rate of elimination drastically altered in these animals, but also the hepatic damage results in qualitative and quantitative alterations of the metabolic profile; in particular, there is blockagc of conjugation of the dihydrodiol alcoholic hydroxyl groups. These effects are not uniquc to carbon tetrachloridc, and identical or similar alterations are produced by other hepatotoxic agents such as tannic acid, thioacetamide, and bromobenzene. c. Side-Chain Oxidation of Methyl-Substituted Hydrocarbons. Up to 1959, all the identified nontissue-bound metabolites of polycyclic hydrocarbons were exclusively various typcs of aromatic arid hydroaromatic ring-hydroxy compounds in conjugated or unconjugated form. Testing of the metabolites showcd invariably that they are less activc than the parent compounds or inactive. Identical conclusions have now been reached by Siins (196713) who tested various ring-hydroxy and keto derivatives of 20-mcthylcholanthrene. Harper (1959a) was the first to describe a metabolite corresponding to the oxidation of a substituent carbon chain. H e found t h a t in micc, 20-methylcholanthrenc is metabolized, in addition t o phenolic derivatives, to an acidic product which he tentativcly identified as cholanthrene-20carboxylic acid 011 thc strength of spectral and chemical evidence. The typical biliary metabolite of 9,10-dimethyl-l,2-benzanthracene is the 4'-hydroxy derivative (reviewed by Boyland and Weigert, 1947). However, rat liver honiogenates oxidize the side chains and convert this hydrocarbon into a mixture of 9-hydroxymethyl-10-methyl and 10-hydroxymethyl-9-methyl derivatives. The 4'-, 3-, 9-, and IO-methyl-1,2henzanthrarcncs arc also converted into the corresponding hydroxyniethyl
MOLECULAR GEORIETRY AND CARCIKOGESIC ACTIVlTY
339
derivatives. To a minor extent, ring hydroxylation occurs with all tliese Iiydrocarbons to yield phenols and diliydrodihydroxy compounds. As with 3,4-benzopyrene, no hydroxylation (Boyland et al., 1964c, 1965) was detected a t the K-region. Interestingly, using liomogcnates from animals pretreated with 20-niethylcholanthrene or phenobarbital, both of which induce microsomal enzyme synthesis, the yield of ring hydroxylated products was increased a t the expense of the hydroxymethyl derivatives (Sims, 1966). Boyland and Sims (1967), testing by subcutaneous route in C57 black mice, found that hot11 9-hydroxymethyl-10-methyl- and 10liydroxymethyl-9-methyl-1,2-l~enzanthracene are much less carcinogenic than the parent 9,10-dimethyl compound. Thus, these metabolites may not be regarded as proximate carcinogcns. d . Metabolisnx of Tricycloquinazoltne. Studied exclusively by Baldwin and his colleagues (Baldwin et al., 1963b, 1964a), biliary metabolism yields the 1- and 3-hytlroxy derivatives; the 2- and 4-hydroxy derivatives are definitely absent. On the other hand, in witro metabolism with r a t or iiiousc liver homogcnates appears to yield the 3-hydroxy derivative only. No acid-labilc dihydrodiol-typc compounds were detected. A fraction, h i t apparently not all, of the two hydroxy metabolites formed arc conjugated with glucuroiiic acid ; no bulfoconjugatcs were detected. In interesting contrast to biliary metabolism, metabolism in the mouse skin (which is priniarily the target tissue) hydroxylates tricycloquinazoline in all four positions. However, the significance of these metabolites is questionable as they account for not more than 2% of the tricycloquinazoline-14C applied to the tissue. About 5% of the compound is transformed into nontricycloquinazoline-like but unidentified metabolites. At least 90% of the tricycloquinazoline remains unchanged 6 hours after skin painting.
E.
P R E S E N T %FATI’S OF’ THE
K-REGIONHYPOTHESIS
The multitude and variety of carcinogens discovered in recent years brought about an apparent decrease of interest in the K-region hypothesis. A strong defense of the orthodox point of view, against competing physicochemical liypotlieses, has been voiced by A. Pullman ( 1964). Howevcr, L: possil)ly inore fruitful, eclectic attempt of generalization has b c v n made (in lhiidel and Daudcl, 1966). An early detected inconsistency of the L ‘ e l e ~ t r ~ ntheory i ~ ’ ’ was t h a t sullstitution of 1,2-benzanthraccne in position 10 by either electron-C-N, attracting or clectroii-rlonating subxtituents (e.g., -0CH -G‘HO, or, 011 the other l i : m l , -CH, or -CLH5) inv:triably converted the parent liytlroc~url)oii,v-liicli h l i o ~ r doiily 1)ortlerlinc cai~inogenicity tow:ircl the siil)cutuncour tishue of stock niicc1, into a inow 1)otent agcnt $,
340
JOSEPH C. ARCOS AND MARY F. ARGUS
(e.g., reviewed by Badger, 1954). Yet, in the framework of the theory, electron-attracting substituents should rather decrease carcinogenicity since they withdraw the electron charge from and, hence, decrease the reactivity of the K-region. Rcccnt work shows better accordance with the theory in the 7,8-benxacridine series. Buu-Hoi et al. (1966) reported that substitution of the highly active IO-methyl-7,8-benzacridine with a trifluoromethyl group (which has a powerful -1 effect) in position 2 (LXX) brings about total loss of activity. Substitution of 7,8-benzacridine with a carboxamide or cyano group in position 10 leads to the
CHs
CH,
inactive
weakly active
(LXXIV)
(LXXV)
highly active
(=I)
highly active
weakly active
(LXXVII)
(IZXVIII)
slightly active or inactive compounds (LXXI) and (LXXII). On the other hand, substitution of the inactive 3-methyl-7,8-benzacridine with a formyl group in position 10 yields the highly active 3-methyl-10formyl-7,8-benzacridine (LXXIII) . Since the isosteric 3,10-dimethyl-7,8benzarridinc is alw ii potent c'wrciriogen (Ltwissagne ct ( i l . , 1956) , the
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
341
carcinogenicity of ( L X X I I I ) is in accordance with the observation in the anthanthrene and dibenzopyrcne scries (Section II,A,l) t h a t the effects of a methyl and a formyl group are cquivalcnt. Using methyl-l,2-benzaiithracenes fluoro-substituted a t the K-region, other investigations attempted to providc more specific evidence that covalent interaction via the K-region is a requirement for carcinogenicity. The central idea of these studies was derived iE. C. Miller and Miller, 1960) from the earlier findings that a number of fluoro-substituted derivatives of 4-diniethylaniinoazohenzene a i d 2-acctylarninofluorene are highly carcinogenic. This was interpreted to mean (e.g., ,J. A. Miller et nl., 1953) that the ring position(s) substituted by fluorine is not involved in the process of carcinogenesis but rather, because of the strength of the C-F bond, they are protected from metabolic inactivation. Conversely, loss or decrease of carcinogenicity owing to fluoro substitution was taken as a n indication t h a t the respective positions required for the cellular interactions leading to tumorigencsis are blocked by the fluoro substituent (s). Guided by this concept, E. C. itfiller and Miller (1960), J. A. Miller and Miller (1963), and Bergmann e t al. (1963) tested several methylbenzanthraceiies and niethylbcnzacridines, fluoro-substituted in the K-region, in order to gain insight into the metabolic importance of the 3and 4-positions for tumorigenesis. The results obtained with fluoro derivatives of the highly active lO-methyl-l,2-benzanthracene would seem to indicate that a free 3-position is critical for tumor induction, whereas a free 4-position is not. Both compounds, 3-fluoro-l0-methyl-l,2-benzanthracene (LXXIV) (E. C. Miller and Miller, 1960) and 3-fluoro-9,lOdiniethyl-1,2-benzanthracene (LXXV) (Bergmann e t al., 1963) are inactive or very slightly active, whereas 4-fluoro-l0-methyl-l,2-benzanthracene (LXXVII) is about as carcinogenic as the nonfluorinated parent hydrocarbon. However, no clear-cut conclusion can be made in view of the unexpected activity of two 3-fluoro-substituted polycyclics : 3-fluoro9-methyl-l,2-benzanthracene (LXXVI) is clearly a highly potent carcinogen (Bergmann et al., 1963), and 6-fluoro-2,10-dimethyl-7,8-benzacridine (also called 3-fluoro-7,l O-diniethyl-l,2-benzacridine) (LXXVIII) has well detectable although low carcinogenic activity (Bergmann et al., 1963). Although i t is true that considerably more detail is available in thc full reports of E. C. Miller and Miller (1960) and J. A. Miller and Miller (1963) than in the note of Bergmann, Blum, and Haddow (1963), there is no reason to disregard the important results in the latter. Further complexity is introduced into this picture by the fact t h a t 3,9-dimethyl1,2-benzaiitliraceiie (a stcrir analog of I X X V I ) is an inactive compound (HartwcJll,1951, 1). 151).
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JOSEPH C. AHCOS AND MARY F. ARGUS
The totality of these data suggest, then, t h a t the inactivity or activity of these methylbenzanthraceiies is not related to the effectiveness of the fluorine atom to block covalent bond formation a t the K-region, but t h a t the activity level depends on the relative position of the hyperconjugating methyl group(s) and the fluoro substituent; the latter may have a net -1 or fM effect depending on its position on the resonant frame. These, in turn, indicate that activity or inactivity depends on the overall x-cloud distribution in the polycyclic frame, modified and shaped by the resultant electronic effect of the substituents. T h a t covalent binding a t the K-region is not involved in the activity or inactivity of these compounds is also borne out from the carcinogenic potency of 4,9- and 4,10-dimethyl-l,2-benzanthracene (Hartwell, 1951, p. 151), since in these compounds the supposedly critical 3-position, although not substituted, is sterically hindered by the neighboring methyl group. Another argument which cautions against a simple interpretation of the results with the fluorinated hydrocarbons is t h a t the strength of the C-F bond is not invariable, but is likely t o be influenced by the electron distribution of the whole moleculc. The metabolic removal of fluorine from p-fluoroaniline has been shown many years ago by Hughes and Saunders (1954). Very recently, Westrop and Topham (1966a,b) reported the enzymatic defluorination of 4’-fluoroaiiiinoazobenzenes. Unfortunately, investigations are lacking on the metabolic removal of fluorine suhstituen ts from polycyclic hydrocarbons. Chalvet and Mason (1961) predicted t h a t in lO-methyl-1,2-benzanthracene, fluorine substitution in 1’,2’,3’,4,6,8, or 9 will lend to carcinogenic coinpounds, whereas in 3 and possibly in 5 or 8 to inactive substances. The studies of the Millers did, however, support the idea that blocking the sites of metabolic inactivation by fluorosubstitution potentiates carcinogenicity. This has been illustrated with 4’-fluoro-l,2-benzanthracene which has considerable carcinogenicity toward the subcutaneous tissue of the rat ( J . A. Miller and Miller, 1963). Here, the substituent occupies the typical position of metabolic ring hydroxylation. The high carcinogenicity of 4’-fluoro-1,2-benzanthracene stands in striking contrast to the virtual inactivity of 4’-methyl-1,2-benzanthracene (Dunning and Curtis, 1960 ; Imassagne e t al., 1962). Studies of the metabolic fate of methyl substituents in the beiiz ring of 1,2-beiizaiithr~cene,as a possible clue for the inactivity of these derivatives, are needed. One of the instances often cited to point out the paramount importance of the K-region is the carcinogenicity of 1,2,5,6- and 1,2,7,8-dibenzanthracene, each having two K-regions, versus the inactivity of 1,2,3,4tlil~crizniitlirncc~ie which lias no K-rcgioii (ci.g., Pullman nnd Pullni:in,
1955:r,l);Hci~lell~erger et nl., 1962).Tile high level of binding of the latter to skin proteins was ascribed to the presence of a reactive L-region ( Heitlelhcrger, 1959 ; Heidclherger and Rloldenhauer, 1956). Considerable doulh h:is I)cc~n tlirown on t h t ~vahlity of t l i i h rc~isoningnow by the important finding of Uuu-Hoi (1964)that it suffices to introduce a methyl group in position 5 to evoke carcinogenicity in 1,2,3,4-dibenzanthracene [see ( X X I ) in Section II,A,l]. Evidence for tlie importance of the clelocalization of the x-electrons in polycyclic carcinogens has been provided hy Buu-Hoi et al. (1963). These workers obscrved tlie total absence of carcinogenic activity in a series of coiijugatcd aromatic polyacctylenes despite the pronounced number of x-electrons in these compounds. The great unsaturation of the conjugated polyacetylenic chain, that is, tlie much greater localization of the x-electrons than in the condensed aromatics, and the long, linear molecular shape are the likely c a u m of inactivity. The much greater localization of the x-electrons is shown by the fact t h a t none of the polyacetyleiies can act as an electron-donor to form colored r-complexes, a behavior typical of the condensed aromatic polycyclics. Thus, it would appear that neither extreme localization of the x-electrons (as in an ethylene or acetylene bond) nor great clelocalization is favorable for carcinogenic activity (cf. Badger, 1954). Evidence which has been accumulating in the last 12 years (see also Arcos and Arcos, 1962) points inescapably to the fact t h a t interaction through a ,neso-plienanthreiiic region is not the exclusive way by which polycyclic carcinogens initiate neoplastic changes. I n many highly active polycyclics, the K-region (9) may bc important zone (s) of interaction. I n other aromatic polyriuclear compounds, different reactive zones produce cellular alterations leading to the same biological result. Buu-Hoi (1950) already envisioned that van der Waals forces may play an important role in kcy cellular interactions. Arcos and Arcos (1956) proposed that covaleiit bond formation, hydrogen arid chargc-transfer bonding, dipole interactions, resoii:ince, arid dispersion forces act siinultancously and that the critical interaction ( s ) between the carcinogenic molecule and the cellular receptor sitc(s) is duc to tlie totality of these forces. A chemical agent is a “potelit” carcinogen when the cliffcrent optimal conditions prevail siinultancously a t the b i t r of action. The ahsence of one of the factors responsible for the intermolecular forces, for example, lack of a K-region, may limit carcinogenirity but not necessarily eliniinate activity altogether, since various types of stable molecular coinplexes can he inaintained hy secondary forces alone. Similar views ivew adopted recently 1)y .J. A. RIiller and Rfiller (1963).
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From the mass of sotnetimes contradictory experimental evidence and maze of interpretations, there emerges a newer, more simple picture of the carcinogenic hydrocarbons so aptly described by Buu-Hoi ( 1964) : The conjugated frame offers certain sites and areas of high r-electron densities, hence a greater covalent and noncovalent reactivity, through which the interaction with cell components is facilitated; in many instances, there is a meso-phenanthrenic K-zone, whose involvement in the metabolic degradation of the carcinogen has been experimentally established but whose existence is essential neither for protein-binding nor for carcinogenicity. Where meso-anthracenic L-zones are present (case of naphthacene hydrocarbons), their reactivity does not necessarily preclude carcinogenicity. Replacement of =CH- groups by tervalent nitrogen heteroatoms may have a positive or a negative effect on the carcinogenicity, depending on the number and the position of the nitrogen atoms and on the nature of the molecule ; when several nitrogen heteroato.ms are present, the prime importance of their position in relation to one another suggests that they act as centers for binding with cell components, in place of, or in conjunction with, other zones of biochemical interaction. Where there are substituents, these may be either electron-donating or electron-accepting groups (except acid functions), and their contribution to the carcinogenicity may be positive or negative, depending on the type of molecule. In the case of alkyl substituents, lengthening of the chain has an adverse effect on activity, owing to increase in the encumbranw area of the carcinogen, the degree of loss of activity depending on the site of substitution. The introduction of substituents with acid hydroxyl groups (carboxyl, sulfonic acid, and phenolic functions) invariably results in a sharp deerease or total loss of carcinogenicity-an effect which must be due to a departure from the “normal” molecular orientation of the carcinogen within cellular lipid structures, produced by the strong hydrophilic radical. These, then, are the basic physico-chemical characteristics which are to be borne in mind when formulating or assessing general theories on the mode of interaction of polycyclic aromatic hydrocarbons and their heterocyclic analogs with cell components. The electronic theory of carcinogens which has proved of such great value in the past, as a guide in the search for nctive compounds through the .maze of organic chemistry, can probably continue to play that role if through adequate refinements and/or modifications it, can integrate the new experimenhl data.
F. NONCOVALENT INTERACTIONS OF POLYCYCLIC AROMATICS 1. Solubilization by Surfactants and Proteins Demisch and Wright (1963) investigated the nature of solubilization of polycyclic hydrocarbons by deoxycholate in a study of the partition coefficients of twenty-eight polynuclear aromatic hydrocarbons between aqueous monoethanolammonium deoxycholate and hexane. Their data are
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consistent with the much earlier results of Fieser and Newrnan (1935) who observed that some hydrocarbons, but not others, yield well-defined crystalline addition compounds with deoxycholic acid. The complexes conform to a coordination principle inasmuch as they contain two, three, or four molecules of deoxycholic acid per molecule of hydrocarbon. Solubilization and complexing depend on a favorable molecular shape rather than molecular size proper. It is likely t h a t these complexes represent inclusion compounds since deoxycholic acid even alone tends to form helical aggregates in solution (McCrea and Angerer, 1960). Relevant to the interaction of polycyclic aromatics with cellular lipid constituents is the report of Snart (1967) who studied the surface behavior of mixed monolayers composed of a carcinogenic or noncarcinogenic hydrocarbon and cholesterol or lecithin. This study extends the early investigations of Clowes, Davis, and Krahl (Clowes e t al., 1939; Davis e t al., 1940). Hydrophobic bonding is probably the sole interaction between polycyclic hydrocarbons and proteins and lipoproteins in body fluids during transport. This is suggested by the study of Sahyun (1966a,b) on the solubilization of aromatic hydrocarbons and other nonionic aromatic compounds to bovine serum albumin. Following Sahyun’s solubility model, when a planar nonionic aromatic compound is dissolved in water alone, both surfaces are exposed to the solvent. On the other hand, when the compound is bound in a plane-parallel fashion to planar surfaces of the protein, only one surface is exposed. Hence, the energy of the hydrogen-bonded water cage in contact with the aromatic compound is twice as great in the former case as in the latter. Since the solubilities give a measure of the energy of thc dissolved state, Sahyun could confirm this model by determining the solubilities in the presence and absence of bovine serum albumin. Related to the problem of transport is the brief study of Anghileri (1967a,b) on the effect of different hydrocarbons on the binding of tritiated 3,4-benzopyrene to plasma proteins. 2. Solubilization by Purines
Probably more closely related to the critical cellular interaction of polycyclic hydrocarbons is their noncovalent combination with and solubilization by purines. Boyland and Green (1962a) have extended the investigations initiated by Weil-Malherbe (1946a,b) and Booth and Boyland (1953). The solvent power of purines shows a i l approximate parallelism with the number of N-inetliyl groups. The solvent effect is shared by the ~iucleosideu,adenosine arid guanosine. The l o w r solvent power of these, relative to the component purines, is likely to be a reflection of the fact t h a t the binding ability of the nucleosides is due t o
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JOSEPH C. ARCOS AND MARY F. ARGUS
the purine moiety only. The pyrimidines, tliyniidine, vytitline, and uracil, have very little solvent powcr compared to the purines (Table X ) . Hydrogen bonding appears unimportant in the solubilization phenomenon since urea and lithium nitrate, both rcgarded as strong hydrogen bond breakers, h a v ~no cffect on the soluhilization of 3,4-hcnzopyrenc by caffeine (Boyland and Green, 1962a). Purines arc also cffirieiit quenchcrs of the typical fluorescence of polycyclic 1iydroc:trbons (Weil-Malherbe, 1946b; Boyland and Green, 1962a). Structural changes in purines which bring about loss of solvent power also bring about loss of quenching effect. The efficiency of quenching incrcascs linearly with purine concentration, but does not increase with the temperature, indicating t h a t collisional deactivation is not involved. The quenching action is not due to solubilization per se since deoxycliolate ion, a powerful solubilizer by hydrophobic bonding, is not a fluorescence quenchcr. Two hypotheses have becn proposed to account for the nature of the TABLE X RELATIVE SOLVENT POWER OF PURINES AND PYRIM IDINES TOWARD 3,4-BENZOPYRENE" Compound Caffeine
Caff eine-Ng-methiodide Caffeine-Ng-methochloride R-€Iydroxy-9-methyl-8,9-dihydrocaffeine 3,7-Dimetliyl-4,5-dihydroxy-2,6,8-trioxypurine 1,3,7-Trimet hyl-4,5-dimethoxy-2,6,8-trioxypurine Tetramethyluric acid 6-Dimet~hyl'aminopuriiie G'uanine Guanosine Adenine Adenosine Hypoxari thine Thymidine Orotic acid Cytidine Uracil Tryptophan
Molecular ratio
2 ,430 24,'LO0 16,050 87 ,400 00
m
468 9,620 18,500 23,130 43,600 50 ,400 92 ,000 110,000 209,000 272,000 1 ,255,000 ;W,4OO
Solvent power ( %)
100 10 0 15 1 2 8 0 0 520 25 2
13 1 10 5 56 48 26 2.2 12 09 0 2 8 0
u Compiled from Weil-Malherbe (1946s)and Boyland arid Green (19CLtt). The niolecular ratio represents the number of moles of purine required to dissolve one mole of hydrocarbon. The solvent power is calculated relative to that of caffeine. In order to obtain romparal)le values for the solvent power, the molecular ratios froin Boyland a ~ d Oreeii's paper were adjusted t hrorigh it coiiversioii factor obtained 1)y dividing the catfeirie molecular ratios from the two reports.
Pu1lni:tn :in(I PuIIiii:in (19.58, 1960) were tlir first to 1)i*opow,on t h v hihis of calculations of the relative electron-donor :tnd acceptor :t Mities of purines and pyrimidines, that charge transfer is involved. In charge transfer, polycyclic hydrocarbons usually play tlie role of electron-donor. However, I,ovelock e t nl. (1962) conclucled from an estimation of the electron affinities that aromatic polycyclics can also act as clcctroii-acceptorh. From the X-ray crystallographic data of DcSantis e t al. (1961) and Leela and Mason (1957), and the investigations of Ts’o and his co-workers (Ts’o e t nZ., 1963; Akinrimisi and Tb’o, 1964) on tlie absor1)tion and fluorescence spectra, it appears that the purine-1iydrocarl)on complexes consid of columnlike aggregates in which purine and hytlrocarbon molecules are alternately stacked. This is consistent with the geometry of aromatic charge-transfer complexes. Furthermore, any structural modification of the purine partner which impairs the planarity of the molceulc and, thus, brings about a decrease of plane-parallel adlineation to the hydrocarbon, produces decrease or loss of solubilizing power. This is exemplified by purine derivatives in which the tervalcnt nitrogen in position 9 is transformed into a quaternary ammonium salt (caffeine nicthiotlidc or methochloride) or by purines in which the double bond bctuwn the 8,9- and/or 4,5positions is lost (Table S ) ;probably for the same reason, the highly carcinogeiiic but nonplanar 9,10-dimethyl-1,2-benzanthracene is only slightly solubilized by aqueous caffeine. On the other hand, Boyland and Green (1962a) pointed out that the purine-hydrocarbon complexes do not show a new almorption band in the visible, which is so characteristic of the charge-transfer complexes of hyilrocarbons with chloranil, trinitrobenzene, iotlinc, etc. The only spectral change observed is a hathochromic shift in the ultraviolet absorption spectrum of the hydrocarbon. Hence, they suggested that solubilization depends on polarization forces or van der W:tals interactions. However, it should be noted that the same structural modifications which bring about decrease of charge transfer also bring about decrease in electrostatic attraction forces which are highly sensitive to tlie increase of intermolecular distance. Furthermore, increasing the molecular size and introducing coplanar mrthyl substituents, which generally enhance electron-donor ability, also increase tlie polarizability. Support for Boylitnd’b thesis was providcd by Pullman et al. (1965) who calculated the bonding energies by dipole-induced dipole attraction and by London dispersion force between 3,4-benzopyrene and various purines and pyrimidines. They found a fair parallelism between tlie order of London force bonding energies and purine solvent power. It is important to note, however, t h a t neither the correlation between London ~~iii~i~ic-liydi~oc.;ll.l,oll inter:ictions.
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C. .211(’OS AND M A R Y
18.
ARGTJS
force and solvent powcr, nor the correlittion between the solvent power and the energy of the highest-filled or lowest-empty molecular orbital (Pullman and Pullman, 1958, 1960) is satisfactory enough to account alone for the total intermolecular attraction. It is more likely that these forces are only components of thc overall binding force which is, in addition, also influenced by the actual geometric orientation of the partners and the physicochemical variables (dielectric constant, ionic strength, viscosity, etc.) of the medium. It is known that the stability of charge-transfer complexing is, in many instances, reinforced by simultaneously operating dipole attraction and London forces (“polarization bonding”). It is perhaps significant in this regard that in the calculations of Pullman et al. (1965) the correlation is improved if the sum of the two bonding energies is considered.
3. Interaction with Nucleic Acids Boyland and his associates resumed their investigations on the solubilization of polycyclic hydrocarbons by DNA (Boyland and Green, 1962b). Solubilization of pyrene and 3,4-benzopyrene by DNA, just as solubilization by purines, is accompanied by bathochromic shifts of the spectra and by fluorescence quenching, and the changes are larger than are observed with the single purines, caffeine, and tetramethyluric acid. Destruction of the double-helix structure of D N A by heat denaturation brings about great loss of the solvent power. Similarly, in the presence of ethylene glycol or formamide, which also abolish the double-helix structure, DNA does not induce the bathochromic spectral shift. Ribonucleic acid, which unlike DNA has a low helix content, has also very little solubilizing power. These results were confirmed by Liquori et al. (1962) with the exception, however, t h a t in their experiments, solutions of denatured DNA had a greater solvent power than solutions of native DNA. T h a t noncovalent binding does take place between the polycyclic hydrocarbons and nucleic acid was also confirmed using a different technique. Robert (1963), taking up the earlier investigations of Brigando (1956), demonstrated interaction between 20-methylcholanthrene and DNA in monomolecular film. Solubilization by DNA can be accounted for by two binding mcchanisms: (a) “internal” binding, i.e., insertion between base-pairs; (6) “external” binding to DNA perpendicular t o the planes of the purine bases and interaction with them a t points not involved in the maintenance of the double helix (Fig. 5 ) . Boyland and Green (196213) showed on a molecular model of DNA that planar polycyclic hydrocarbon molecules, such as 3,4-benzopyrene and 1,2,5,6-dibenzanthracene,can be accommodated between the base-pairs by slight untwisting, “straightening” of
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
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the sugar-phosphate backbone and intcrcalation of the hydrocarbon molecule into the spacc thus arisen. This stcric accommodation would then he stabilized by polarization bonding betwccn the hydrocarbons arid the purines and by a hydrophobic effect involving the entire doublehelix strand. The ability of hydrocarbons to complex with DNA is dependent on the pH (Ball et al., 1965) and is sensitive to the presence of inorganic ions and small polar molecules. This is consistent with the polyelectrolyte nature of DNA and the intercalation model of the complexing. The
I I
I
A
I
I
B
Fro. 5. Schematic representation of possible spatial orientations of polynuclear aromatic molecules in complexing with DNA. I n ( A ) is seen slight untwisting of the helical backbone and intcrcalation of thc aromatic compound into the spacc aristw, parallel to thc basc-pairs; in (B) is shown external binding to DNA without disturbance of the double helix, the planes of the aromatic molecules lying more or less parallel to the helix axis.
solubilization experiments of Boyland and Green were usually carried out in 0.001 M NaCl to protect DNA against spontaneous denaturation. Increasing the salt concentration reduces the solubility of the hydrocarbons in DNA solutions. Since the solubility of hydrocarbons in water is not affected by such concentrations of salt, an explanation based on simple solubility effect appears to be excluded (Boyland and Green, 196213). Sodium ions, as well as K+, Ca++,Mg++,aliphatic amines, urea, and dimethyl sulfoxide all lower the solubility of hydrocarbons in DNA solutions and can also, upon addition, release the bound hydrocarbon from the soluble complex (Boyland e t nl., 1964~1).The ion effect may be interpreted in terms of the intercalation hypothesis. Increasetl ion conccntr;ition t c ~ n ( l xto clccrc:~sciq)uIsion 1)c~twcwithe pliosp11:ttc~groups in
350
,JOSEPH C. ARCOS AND MARY 1“. ARGUS
the sugar-phosphate backbone and would also increase hydrophobic bonding between the bases by rendering all nonpolar substances less soluble. The overall effect amounts to an increase of the stability of the helix (Boyland and Green, 1962b; Boyland e t al., 1964d). The solubility and spectral studies of Kodama e t al. (1966) indicate, indeed, t h a t the ions bring about a more compact packing of the bases resulting probably in a shortening of the pitch of the helix. Hence, the macromolecule becomes less accessible to intercalation. Boyland and Green (1964a) presented evidence that the ion effect accounts for the conflicting observation of Liquori e t al. (1962) on the greater solubility of 3,4-benzopyrene in denatured rather than nativc DNA. Other evidence for the combination of polycyclic hydrocarbons and D N A was provided by interesting results on the in vitro effect of hydrocarbons on the T, of DNA. Boyland and Green found t h a t anthracene, pyrene, 3,4-benzopyrene, 1,2-benzanthracene, and 9,10-dimethyl-1,2-benzanthraccne appreciably increase the T , of calf thymus D N A (Boyland and Grcen, 1963; reviewed by Boyland, 1964a). Thc increase of the T , could reflect contribution to polarization bonding or, more likely, enhancement of hydrophobic bonding. The latter effect may consist in shielding sites of the D N A molecule-at which fluctuations in the rigid hydrogen-bonded structure occur most readily-against the surrounding water structure. These regions are thereby stabilized causing a n increase of the temperature necessary for “unzipping” the double helix (Boyland and Green, 1 9 6 2 ~ ) . However, Daniieriberg and Sonncnbichler (1965) were unable to bring about a significant increase of the T , of D N A with the above and other polycyclic hydrocarbons, and also with aromatic amines. The very drastic methods used 1)y these authors (consecutive chloroform and cyclohexane extraction) to remove the “unbound” hydrocarbon are likely to be responsible for the negative rcsults. This is actually indicated by their own tabulated values of the spectrophotoinetrically determined levels of compounds in the solution following extraction ; the nonpolar hydrocarbons are extensively removed while the more polar 3-aminophennnthrene (the only aromatic amine studied in this regard) remains in solution a t levels 50-100-fold higher. Finally, from the DNA-proflavinc and DNA-acridine orange complexes, extraction removes even less of the non-DNA component; this is consistent with the highly polar character of these dyes. With these solutions, in which a high level of non-DNA component remained, an appreciable increase of the T, was observed. It is of interest, in view of the possible gene mediation of thalidomide embryopathy, t h a t N-phthalylglutarylimide produced an increasc of thc T,,, comparnhlc t o the above ncridine Iiiutagens.
MOLECI'1,AR
tiEOMETRY AND CARCINOCIESIC ACTIVITY
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I n Boyland and Green's ( 1962c, 1963) experiments, botli the carcinogenic and the noncnrciiiogrnic hydrocartions gave the same effect in ? ) { f r ophenomenon IJe involved raising t,hr T,. Howeiw, should this in the niwhanisni of hiological :ivtioii, qii:intit:atir.cI diffcwiicoh inay hc of i of the T , was 10°C. critical importance. I n fact, tlic i i i : ~ x i i ~ i u i iiiicrc:w with 9,10-dimethyl-l,2-benzanthracene (one of the most rapidly acting skin carcinogens known), but thc iiicrease was only 6°C. with the less active 3,4-benzopyrene. Also, whereas this latter increase of 6°C. was brought about with as little as 0.3 phf 3,4-l)enzopyrene, when using the inactive pyrene 1.4 piM was needed to bring about the same increase of the T,,&. These quantitative differences bring to iiiiiid and may be related to the investigations of Nagata and his co-workers on the quantzty of charge transfer-as distinct from the stabilization energy due to complex formation (i.e., thc charge-transfcr force) --betuwn a hydrocarbon carcinogen and purine-pyrimidine base-pairs. Nagata e t al. (1963b) found that although there is only a small difference in the stabilization energy calculated for pyrelie and for the potent carcinogen, 3,4,9,10-dibenzopyrene, the quantity of charge transfer is far larger for the carcinogen than for the inactive pyrene. It is interesting, moreover, that with either hydrocarbon, both the quantity of charge transfer and the stabilization energy is greater with the guanine-cytosine than with the adeninethymine base-pair. Results consistent with the latter finding were obtained when these two charge-transfer parameters were calculated for the heterocyclic carcinogens, tricycloquinazoline (LVII) and its oxygencontaining structural analog ( L I X ) (Nagata e t al.,196613). Since aniong all the constituents of the nucleic acids, guanine is the best electron donor, this base has probably a dominant role in charge-transfer complexing with nucleic acids. Experimental cvidencc supporting this is found in the preponderant solvent power of guanine aniong the bases in nuclcic acids (Table X ) . These impressive inrestigations on the bolubilization of polycyclic aromatics by DNA came under strong attack by Giovanella et al. (1964). I n a refutation of the work of the 13oylancl aiitl Liquori groups, Giovanella et nl. reported that when a O.OSC/, solution of DNA is ground with 3,4benzopyrene or 1,2,5,6-dibeiizantlirace1ie, the suspension of the hydrocarbon (nonsedimentable a t low speed) can be completely sedimcnted under their experimental conditions by high-speed centrifugation, or filtered off by uniform 0.45-p pore-size Millipore filters. They concluded that the hydrocarbons are not truly solubilized by D N A hut form finedisperse aqueous suspensions stabilized by the macromolecule, and they reported t h a t this stabilization effect can actually be duplicated with
352
JOSEPH C . ARCOS A N D MARY F. ARGUS
the m e of soap (approx. O . o l y 0 ) instead of DNA. In tlie light of their findings, Giovanella et al. related the lowering of solubility in or release of hydrocarbons froni DNA solutioiis by ion6 and p01m molecules to thc wcll-knowii fact th:it d t s prccipit:itr colloids. O i i the other hand, the concentration of 3,4-benzopyrenc in saturatc>tlcaffeine solutions could not be diminished by high-speed centrifugation for a prolonged period of time; thus, they regarded these as true solutions. I n an examination of the criticism of Giovanella et al. (1964), Boyland and Green (1964b) presented evidence that accounts for the conflicting observations. Pyrenc and 3,4-benzopyrene are, indeed, removed from aqueous caffeine or aqueous DNA solutions if the high-speed centrifugation is carried out in plastic tubes (polypropylene, polypropyleneethylene copolymer, cellulosc acetate). No loss of hydrocarbons occurs, however, upon centrifugation in quartz or glass tubes. Boyland and Green also confirmed the spectrophotonietrically demonstrable loss of hydrocarbons when filtering the solutions (in aqueous caffeine or DNA) through a pad of several Millipore filters. There is consequently no difference in the actual results obtained, but rather in their interpretation. Boyland and his associates attributed the losses of hydrocarbons to adsorption by the filter material or to the formation of solid solution with the polymer material of the centrifuge tubes. I n retrospect, the controversy is even more surprising in view of evidence from the earlier work of Steele and Szent-Gyorgyi (1957) that 3,4-benzopyrene complexes with and is solubilized by caffeine as well as DNA. These authors noted in accordance with the observations of Brock et al. (1938) and Weil-Malherbe (1946b) that the hydrocarbon has a yellow fluorescence either in solid form or in fine-disperse colloidal solution. For example, by adding a drop of acetone solution of 3,4-benzopyrene to water, the resulting colloidal solution emits a greenish-yellow fluorescence under ultraviolet light. Howevcr, the color of fluorescence turns dramatically to blue if the solution is shaken with solid caffeine or with DNA, indicating quite evidently that, in the latter case, a-electron interaction with the nucleic acid took place. 3,4-Benzopyrene is also fairly potent in quenching the phosphorescence emission of frozen aqueous solution of DNA from which i t can reasonably be interpreted that the collective energy conduction through the bases is cut by interposition (i.e., intercalation) of a “dud,” the hydrocarbon molecule (Steele and Szent-Gyorgyi, 1957). The interaction of polycyclic hydrocarbons with DNA brings to mind the well-investigated interaction of nucleic acids and polynucleotides with various planar acridine dyes, acriflavine, proflavine, and acridine orange (e.g., Heilweil and Van Winkle, 1955; Bradley and Felsenfeld, 1959; Steiner and Beers, 1959; Lerman, 1964). The complex of DNA with proflavine was studied by Luzzati et al. (1961)
ILIOLECULAR (XOMETBY A N D CARCINOGENIC ACTIVITY
353
by X-ray crystallography aiid found to have a planar, “sandwichlike,” inclusion compound structure in which proflavine molecules are intercalated between the base-pairs of DNA. The recent work of Ball et al. (1965) provided further support for the solubilization of hydrocarbons by DNA. Heating the DNA-3,4bcnzopyrene complex over a temperature rnnge, which produces the separation of control DNA into single polynucleotide strands, brings about a loss of absorption at 395 nip, the wavelength of iiiaximuni absorption of the complex. The dissociation of the complex into its component parts (over this temperature range) is also indicated by the increase of relative viscosity. Essentially identical conclusions were reached subsequently by Isenberg et al. (1967) from studies of the complexing of hydrocarbons with native and denatured DNA, RNA, aiid single-stranded poly A. Particularly interesting is their finding that the degree of complexing appears to depend on the molecular dimensions of the hydrocarbon. Definitive confirmation of Boyland’s interpretation was provided by the elegant experiments of Nagata et 01. (1966a,b). The Japancse workers applied the method of flow dichroism to dctcrmine the spatial orientation of the polycyclic molecules with respect to the lengthwise axis of DNA. The basis of this important but little-known technique, as applied by Nagata and his co-workers, is as follows. In planar aromatic molecules, such as 3,4-benzopyrene1 polarized light can induce electronic transitions only if the plane of polarization (i.c., the electric vector) is parallel to the molccular plane; in this case light energy is absorbed. No electronic transition and, hence, no light absorption occurs when the plane of polarization is perpendicular to the molecular plane (Fig. 6 ) . Figure 7 gives a schematic representation of the flow dichroism spectrophotometer arI
poloriled light plane of polorimtion
y
0 ABSORPTION
polarimllan
FIG.6. .1 representation of electronic: tr:uiiitions induced by polarized light in 3,4-benzopyrene.
354
JOSEPH C. AIlCOS A N D MARY F. ARGUS L iahl source
mi
mofo mulliplier
FIG.7. Schematic representation of the apparatus of Wada and Kozawa (1964) for the measurement of flow dichroism. C1 and C, are coaxial cylinders made of optical glass. The clearance between them is 0.5 mm. and the total light path in the solution is 1 mm. The inner cylinder is rotated a t or above 1000 rpm. P is the calcite polarieer which can be positioned so that the plane of polarization is either parallel to or perpendicular with the flow line.
rangement of Wada and Kozawa (1964) used by the Nagata group in their studies, and Fig. 8 illustrates the principle of determination of differential dichroism. Owing to the flow of the solution containing the DNA-hydrocarbon complex, in the space between the stationary optical glass cylinder C, and the rotating glass cylinder C,, the double-helix strands of the complex are aligned parallel to the flow line. Therefore, the position of the molecular planes of the bound hydrocarbon molecules, with respect to the plane of polarization, will be different depending on whether they are intercalatcd between the bases (Fig. 8A) or adsorbed to the surface of the double helix (Fig. 8B). Hence, the difference of the light absorption values in the two positions of the polarizer will directly indicate the geonietry of the complex. The differential dichroism, AE, is obtained by subtracting the molar extinction coefficient toward the light polarized perpendicular to the flow line, e,, from the niolar extinction coefficient toward the light polarized parallel to the flow line, ell, that is Ae =
€11
- e,
Since € 1 1 and el are both positive, Ae can be either positive or negative depending on whether € 1 1 or el is greater. If the niolecular planes of the chromophores are oriented perpendicular to the flow line, then e, > t l I and Ae is negative, whereas Ae is positive if the niolecular orientation i s parallel to the [!ow line Iwxaiisc thcii € 1 1 > el. Thus, froin the sign of A€, the orientation of the plaiir of electronic transitions (i.c., the spatial orientation of the nioleculcs) can be tleterinined (k’ig. 8). Nagata d 01. (lSCiGa,l)) showed that Ae is negative for phenanthrene, pyrene, 3,4-benzopyreiie, tric’yclocluiiiazolirle (LYII), ant1 the tricycloquinazoline analog (LIX), whereas A€ has a positive sign for 20-methyl-
cliolantlirene, ( ~ o I ' o ~ I ~iiaplithacwc, wc~, niill pciit:tcc~no. Thus, tlic formcr compounds are oricnted 1)arallcl to tlic p1anc.s of the bases so that interaction hy intercalation is unequivocally established. T h e latter compounds, on the other hand, are oriented perpendicular to the planes of the bases :tnd bound to the external surface of the DNA strands. The cliffcreiitial dichroibtn spectra arc excniplified and compared to the respective absoiptiou spectra in Fig. 9. Distinct dichroism was not observed for 9,10-diniethyl-1,2-benzanthracenc and 1,2,5,6-dibenzanthracene so that their mode of orientation could not be dcteriiiincd. However, the changes in soluhility in the presence of various concentrations of NaCl are similar to the oiics ohserved with thobe hydrocarbons that complex by intercalation. I n addition, the AE spectra provide unainbiguous evidence for the noncovalent interaction with DNA and for the true solubilization of polycyclic aromatics. I n fact, the bathochromic shift produced by thc complexing iii the hydrocarbon absorption spectrum (Boyland and Green, 1962b; 1,iCjuori et al., 1962) also occurs in the dichroism spectrum, and the peaks of the two types of spectra coincide quite accurately (e.g., Fig. 9). Nonspecific soaplike adsorption to D N A molecules or colloid stabilization by DNA, as suggestcd by Giovanclla et al. (1964), can certainly be excluded bccaube 110 tlichroism can occur in such cases (cf. Nagata e t al., 1966a). From the parallel orientation of the hydrocarbons to the bases, as observccl for 3,4-benzopyrene, overlapping of the r-orbitals is a virtual certainty so that charge transfer and polarization bonding are almost to be expectcd. The phenomena of hathochromic shift and fluorescence quenching are entirely consistent with this view. For intercalation into DNA (also to some extent for adlineation to the external surface of the doublc helix) as well as for complexing with purine-pyrimidine base-pairs, conditions of steric fit and geometric similarity must he met. The requirement of geometric similarity between a hydrocarbon moleculc and a single purine or pyrimidine, or a haw-pair, is implicit in the calculations of charge-transfer parameters, since a-type overlap of atomic orbitals is assumed (Nagata e t al., 196311, 1966a,b). I n order to fit into the narrow but flexible and stretchable space between the base-pairs, the moleculc must have within limits an adequate shape. Haddow (1957) has poiiitcd out that scveral carcinogcnic hydrocarbons are of a sizc and shapc >imilar to purine-pyrimidinc pairs; for cxainple, the potent carcinogen, 3,4,8,9-dibcnzopyrei~~~, occupies an area similar to that of an adenine-thyminc pair (Fig. 10A). I n some instaiices, intercalation into and interaction with DNA docs not appear to involve the entire polycyclic moleculr. This is the case with tricycloquinazolinc-a coinpound complexing by intercalation-which is similar to the g u a n i n e
w cn Q,
A. Hydrocarbon intercalated parallel to the bases. therefore A € IS negative >
EL El,
I I
clearance 015 mm.
polorizer
-
Monochromator electric vectors
DN4 dwble helix ___)
flow line
r Photomultiplier
I
plane of polorization
polorizer
Monochrornotor
-@
+El=
osciIli+ory plane porollel to flow line
-
flow line
done of phorizotion
Photomultiplier
0
B Hydrocarbon bound externally and oriented perpendicular to the bases.
< E,I
therefore Ac is positive
I Photomultiplier
,~-./’
hydrocobon h n d to surface of DNA
flow line
’
cross s?ction of M A hbte helix
plane of polarization
I ~
-
Photomultiplier
Monochromotor
oscil latory plane parallel to flow Ime
flow line
plane of polarization
FIG.5. Principle of the determination of differential dichroism of polycyclic hydrocarbons complexed with DNA. In (A) is shown why the negative sign of h e indicates intercalation parallel to the bases; in (B) the basis of a positive value, indicative of external coniplexing parallel to the lengthwise axis of DNA, is depicted.
358
JOSEPH C. ARCOS AND MARY F. ARGUS
370 380 390 400 m p UI
a
380 390 400 410 420rnp
-0.1-
FIG.9. Comparison of absorption spectra to flow dichroism spectra for 3,4benzopyrene and 20-methylcholanthrene complcxcd with DNA. (A) The negative sign of Ae for 3,4-benzopyrene indicates that there is intercalation between the base pairs; (B) 20-methylcholanthrene has a A€ spectrum in the positive region indicating that the hydrocarbon is bound parallel to the lengthwise axis of the double helix. (From Nagata et al., 1966a.) Dotted curves, absorption spectra; solid curves, Aclc spectra.
cytosine pair (Fig. 10B), as pointed out by Baldwin e t al. (1963a), Of special interest in this regard is the recent finding of Isenberg e t al. (1967) mentioned above that molecular size is an important parameter determining the extent of solubilization by DNA. The concatenation of
3,4,8,9-Dibenzopyrene
Tricycloquinazoline
A
B
FIG.10. Geometric similarity of polynuclear aromatic carcinogens with purinepyrimidine base-pairs in DNA. (A) 3,4,8,%Dibenzopyrene and adenine-thymine. (After Haddow, as modified by Boyland, 1964a.) (B) Tricycloquinazoline and cytosine-guanine.
these findings and concepts now provides a beginning for understanding, a t the molecular level, the requirement of an optimum molecular size range for carcinogenic activity. 4. Significance of Hydrocarbon-DNA Interaction for Mutagenesis
and Carcinogenesis: Some Conceptual Advances Watson and Crick (1953) have postulated years ago that spontaneous mutations may occur as the result of amine -+ imine-type tautomeric shift in one of the purine or pyrimidine bases of DNA and that this results in the miscoupling of bases. Subsequently, Lawley and Brookes (1962; reyiewed by Brookes and Lawley, 1964a) and Nagata et al. (1963a) suggested that the ionization of guanine due to alkylation of the base may also lead to anomalous pairing. This would explain the well-known mutagenic effect of alkylating agents. A somewhat different molecular mechanism should account for the mutagenicity of planar aromatic molecules that complex with DNA by intercalation. Brenner et al. (1961) put forth the idea that the mutagenic activity of aminoacridines-which were shown to intercalate between the bases of DNA-may be due to the increase of the distance between certain neighboring base-pairs and, thus, to the creation of a gap in the orderly periodicity of DNA structure. This could conceivably lead to the deletion of a base from or to the inclusion of a randomly selected additional base into the replica DNA, in the region of the gap, during cell division. Hence, the replica would become a mutant DNA species. Intercalation of hydrocarbons and other polycyclic carcinogens could, similarly, bring about alteration of the base sequence (Boyland and Greeii, 1962b). It is likely, moreovcr, that the polarization bonding between the alternately stacked hydrocarbon molecules and bases promotes plane-parallel molecular adlineation and, hence, keto + enol-type, lactam-lactim tautomerism in the latter (Arcos and Arcos, 1962). The observation that there is an approximate parallelism between the mutagenicity and basicity of the aminoacridines (Orgel and Brenner, 1961) and between their mutagenicity and ability to form charge-transfer complexes (Brenner et nl., 1961 ; Pullman, 1962), suggests that this mechanism may be a participant in the niutagenicity, in addition to the purely steric iriterfcrcnce suggeated by Brenncr et nl. Credence to the idea of lactam + 1:ictiin rc:i1.1.nrlgc.lncltit is lent l y thc rarly experiments of Henclricks ( 1941) who hliowcd th:tt (Iiiriiig t l w :icl~otptionof gunnitw by tlie 1nriit~ll:ir t t i o l t ~ u l ~&vc, ~r iiiontiiioi~illoititt~,t l i c ~ w ih :L shift of the tautoirirr eyuililxiuin toward the more plmar enolic f o i ~ i iwhich provides greater contact for molecular adlinentioil. For example, following the Watson-Crick model, guanine and thymine niust be in the keto form for
360
JOSEPH C. A R C 0 6 AND MARY F. ARGUS
providing the required sitcs for hydrogcn bonding. Thus, displacement of the tautomer equilibrium would bring about a change in the hydrogenbonded partners. Hence, guanine (enol) would couple with thymine (instead of cytosine), and thymine (enol) would couple with guanine (instead of adenine) during the formation of the first replica DNA strand (Fig. 11). At the second replication (occurring in the absence of the carcinogen) the bases in the first replica single strand, thymine and guanine, would couple with adenine and cytosine, respectively. Therefore, a t the site of intercalation, the bases in the parent single strand, guanine and thymine, are now permanently replaced by adenine and cytosine, respectively, so that the DNA alteration has become self -perpetuating. Thymine
Hd
Guanine (enol)
\
Thymine (enol)
HO
Guanine
I
\
FIG.11. Miscoupling of the cnol forms of guanine and thymine.
Calculations by the Pullmans (B. Pullman and Pullman, 1962; B. Pullman, 1964) of the resonance energies of the lactam and lactim tautomeric forms provide other support for the participation of this phenomenon in the mechanism of mutations. I n fact, the lactam + lactim rearrangement is paralleled by an increase of resonance energy, and this gain represents the “driving force” toward the increase of the proportion of the lactim form. On the other hand, in the amine + imine-type tautomerism proposed by Watson and Crick (1953), the shift is accompanied by n clccrcnhe of thc rcmianw rnergy ; thcrcforc, tlic snialler this rlectwsc, the grcittcr will IN, tllc proportioil of the iiiiiiie form (Table X I ) . The prediction that cytosine would h~ the greatest tendency to exist in a tautorneric forin (lactiin or imino) beems to be substantiattxl by iiuclear magnetic resonance studies (Kokko et al., 1961 ; Gatlin and Davis, 1962).
361
MOLECIJ1,AR GEOMETRY ZND C' \RC'TKOC:ESIC ACTIVITY
ItESONAXCE
Gurtiiiiie
Cytosine Iiracil
Thymine C; mini rie Adenine
Cytosiiie
TABLE XI ENERGIES OF THE TAUTOhIERIC F O R M S ~
1,acI an1 3 84 2 28 I %2 2 05 Aniine 3 x4 3 89 2 28
irn 4.16 2.69 2.14 2.27 Imine
1,itc.l
3.68 :; . A2
2.15
0.32 0.41 0.22 0.22 -0. 16 -0.27 -0.13
l h m B. Pullniaii and Pullman (196'2). = Variation of resonance energies arcompitnyiiig the transformation from the ~ I a l ~tloe the less stable form. 'I
* AH
Karreman (1962) calculated that charge-transfer coinplexing of 4-nitroquinoline-N-oxide with adenine actually promotes the amine + imine tautomerism by increasing the reboiiance energy of the iiiiino form above that of the aniine. Evidence for the cornplexing of 4-nitroq~inolinc-~Voxide with DNA by intercalation was provided by the flow dicliroisrn studies of Nagata et al. (1966a). Of cowbe, such ti specific mechanistic picture based on amine + imiiie or lactam -+ luctim tautomerism can not directly explain alterations in base-pairing brought about by externally complexed planar aromatics. It should be borne in mind, however, t h a t except for hydrophobic bonding, all types of noncovalent interactions can bring about a redistribution of 7-clectrous. Hence, given an optiinum geometry and charge distribution, :in externally bound polycyclic aromntic molecule may weakeii hydrogen bonding and the stability of the helix a t certain loci. The prohleni of the causal relationship betwcen mutagenicity and cnrcinogenicity retilains unresolved since tlie time of the rcvirws by Boy1:uid (1964) :intl Burclctte (1955). Altliougli :Lgreat nuniher of rcports :~1)poiiredin recent yc:irs on the niutagcwicity of various nit1 osiiiiiinc,~ (rcvicwetl by hlagcc an({ Bariies, 1967) nu(l other :ilkylating cnrcinogenh (e.g., Frccse, 1963; A1ex:indcr :in([ Glnngcs, 1965), the present reviewers feel that these do not constitute unambiguous support for the causal rclationdiip between the two phenomena. In fact, these agents are chemically highly reactive molecular species and, thus, their mutagenicity is likely to be due to randoin alkylations (cf. Trams e t al., 1961). Therefore, their mutngcnicity rnny not be construed as evidence for the relatedness of the two p1ienonien:i. 4 t any rate, the niutagrnirity of these ciircinogens of
362
JOSEPI-I C. ARCOS A N D MARS F. ARGUS
high chemical reactivity certainly does iiot seem to be relevant to the rarcinogenicity of thc rather unreactive polyryclic aromatics, which gave ambiguous and often irreprociucihle rewlts as mutagens in a nuniher of cxperimcnts (rrvicwc(1 hy Boyl;iii(l, 1954 ; B u ~ ~ l r t t 1955). c, TIE spccultltions of Clayson (1962) throw frcbli liglit on this question a l i c l possibly reconcile the opposing views: If cancer is thc result of a mutation at a specific locus or loci in the genetic system, the mere demonstration of the mutagenicity or otherwise of a chemical is of little relevance to thc induction of cancer. I t is necessary to show that carcinogens induce mutations a t the correct loci. That is to say, tissue, species and strain specificity should be partially explicable in terms of the ease of induction of the required mutations.
G. EVIDENCE FOR HYDROCARBON FREERADICALS Szent-GyGrgyi e t al. (1960) claimed that a correlation exists between the carcinogenic activity of a series of polycyclic hydrocarbons, aromatic amines and azo compounds, and the ability of these to form chargetransfer complexes with iodine. They have measured the magnitude of the electron spin resonance (ESR) signals of these complexes and concluded that carcinogenic compounds give strong signals whereas noncarcinogens give wcak or no signals. I n an extension of this study, Damerau and Lassmann (1963) examined the iodine complexes of a greater variety of azo compounds by ESR spectrometry and could not distinguish between carcinogens and noncarcinogens. Similarly, Jones e t al. (1966) used this technique to study the cornplexing between purine and pyrimidine bases, on one hand, and carcinogenic hydrocarbons and aromatic amines, on the other, and failed to detect any ESR signal. However, Wilk et al. (1966) concluded from a new dimerization and tetramerization reaction of polynuclear hydrocarbons that the reaction must pass through an intermediate radical cation species. Nagata et al. (1966d) used ESR spectrometry in a study of the interaction of polycyclic hydrocarbons with animal tissues. They observed a strong signal-indicating the presence of free radicals-in rat liver homogenates following in vitro treatment with 3,4-benzopyrene1 but only very weak signals in control tissue samples and in tissue samples treated with the noncarcinogen, pyrene (Fig. 12). On the other hand, no differencc in ESR signals was detectable in rat skin tissue 2 days following the final painting of a 5-day treatment period with the same two hydrocarbons. It is, therefore, possible that the radicals formed during the in vitro treatment of liver homogenates, and detectable immediately after treatment, are short-lived and are destroyed by metabolism, as seems to be indicated by the results of the in vivo experiments. Thus, there is now a beginning
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
363
5 0 gauss H
C
FIQ.12. Free radicals detccted by ESR spectrometry in rat liver homogcnates. (A) control; (B) following treatment with pyrene; (C) following treatment with 3,4-benzopyrene. The arrow points in the direction of increase of the magnetic field strength H. (From Nagata et al., 1966d.)
of evidence that carcinogenic hydrocarbons may give rise to reactive radical species in the tissues. Ill. Conjugated Arylamines and Compounds Generating Arylamines. Arylhydroxylamines
A. ARYLAMINESAND ARYLNITROCOMPOUNDS 1. Monocyclic Compounds
It is increasingly accepted that aniline itself is not carcinogenic, a t least in man (Scott, 1962). However, the question of the activity or inactivity of its ring-methylated derivatives is not yet definitely settled. There is some suggestive evidence that o-toluidine might be carcinogenic in man (Vigliani and Barsotti, 1962). Deichmann (1967), however, was unable to induce tumors in dogs by administering the three toluidines for 6 years. Carcinogenicity, although still of very low order, is more readily detectable in the ring di- and trimethylanilines. In addition to 3,4-dimethylacetanilide which produces mammary tumors in a scattering of animals (E. C. Miller et al., 1956), 2,4-dimethylacetanilide (Klein and Weisburger, 1966) anti 2,4,6-t,rimethylacetarlilide (Morris and Wagner, 1964; Klein :tnd Weisl)iii*gcr, 1966) were shown to induce malignant or near malignant 1iep:ttic lesions in occnsioiinl nnimals (rats or mice). Consistent
364
JOSEPH C. AHCOR AND MARY
11’.
AllGVS
with thc carcinogenic activity is tlic observcd hepatotoxic effect whcn large amounts of 2,4-xylidine were fed to rats (Lindstrom et al., 1963). Nonetheless, because of the a t most borderline tumorigenic activity of all these monocyclic compounds, it may not be excluded that activity is due to trace amounts of potent impurities. I n view of the industrial importance of the ring methylanilines, further assaying with highly purified compounds, in large test groups and for longer periods of time is urgently needed. It may be of interest to remember, accepting conditionally these amines as truly carcinogenic, that the methyl substitutions raise the conjugating power of the ring so that the aromatic moieties represent intermediates betwecn an unsubstitutcd phenyl and a naphthyl group. Searle (1966b) has shown that another group of monocyclic compounds which may be transformed metabolically to amines and hydroxylamines are active both as tumor initiators and as complete carcinogens. Treatment of the mouse skin with pentachloronitrobenzene or with any of the thrce isomeric tetrachloronitrobenzcnes produces multiple papillomas during subsequent promotion with croton oil. On epithelial application, these compounds did not induce tumors without promotion. However, subsequent work (Searle, 1966a) showed that they act as complete carcinogens when assayed in mice by subcutaneous route. Metabolism of these compounds, with the exception of the 2,3,4,5-tetrachloro compound, results in the formation of mercapturic acids by replacement of the -NOz group to the extent of 36 to 37%. The 2,3,4,5-tetrachloro compound was the most effective, both as a tumor initiator and as a coinplete carcinogen. Furthermore, N-ethylmaleimide, a sulfhydryl reactor par excellence, was found inactive as initiator in these tests. For these reasons, Searle (1966b) discounted the irnportancc of SH- reactivity in the biological activity of the compounds. A nonbenxenoid group of monocyclic aromatic compounds which manifests carcinogcnic activity is the 5-nitrofurans. A variety of 2-sub-
(LXXJX)
stituted 5-nitrofurans (LXXIX) have been known for soinc tirric to he effective antibacterial agents (Eaton Laboratories, 1958) and have found widcsprcd c1inir:il al)plications for trcating infcctioris of the urinary tract and gynecological bacterial disorders. It is assumed that metabolism transforms these nitrofurans to amines and hydroxylamines. Price et al. (1966) and Stein e t 02. (1966) found that several 5-nitrofuran derivntivec arc potent carcinogens having an ubiquitous tissuc spectrum upon atl-
MOLECTTLAR GEOMETRY A K D CARCINOGENIC ACTIVITY
365
iiiiiiist,r;ition to Slmiguc-D:iivley i x t h :it the lcvels of 0.1 to 0.3% i i i Llic tliet. Tumors of the following Iiistologir:~l typcs and tissue localizations were found: maminary :idcnornas arid ;i~Ie~ioc~rci~ioiias, multiplc papillomas, squainous cell carcinomas and adenocarcinomas of the forestomach, adenocarcinomas of the small and large intestine, renal adenomas, and adenocarcinomas. Moreover, a high incidence of fibroaderioinns and occasional ear duct tumors were observed. 2. Diphenyl- and Triphen ylinethane Derivatives
There is a scarcity of systematic structure-activity studies on amines of the diplienylmethane and triphenylmethane series. Case and Pearson (1954) concluded from statistical data that bladder cancer is an orcupatiorial hazard among workers employcd in the manufarture of auraminr
(LXXXI) R = H (LXXXII) R = CH,
(LXXX). Subsequent testing in rats showed that this dye is a fairly potent carcinogen producing exclusively hepatomas when administered in the diet (Williams and Bonser, 1962; Walpole, 1963), and liver and intestinal tumors and local sarcomas when administered by subcutaneous
366
JOSEPH C. ARCOS AND MARY F. ARGUS
injection (Walpolc, 1963). N o tumors were lwoduccd in Walpole’s experiments by repeated injection of dimethylaniline, the starting material in the manufacture of auramine, or of the intermediate tetramethyldiaminodiphenylmethane (LXXXII) . Dogs appear to tolerate high dietary levels of auramine in chronic experiments without adverse effects. However, recent results of Munn (1967) suggest that the complete absence of carcinogenicity of (LXXXII) should be taken with reservation. I n fact, 4,4’-diaminodiphenylmethane (LXXXI) was slightly carcinogenic in Munn’s experiments. With the latter compound, when a total dose of 600 mg./100 gm. body weight was administered by gastric intubation to 24 male Wistar rats in a n 18-month period, and the animals then kept in observation, liver tumors were found in 2 rats. Two subcutaneous fibromas, one pituitary tumor, and one tumor of the small intestine were detected in other animals. Also, Michler’s ketone (tetraniethyl-4,4’diaminobenzophenone) which is thc last step in a synthesis of auramine produces papillomas of the stomach and neoplastic changes in the liver (in Hueper and Conway, 1964). Carcinogenicity of (LXXXI) is preserved or even somewhat augmented by methyl substitution of the central carbon atom. Deichmann (1967) fed 2,2-bis (4-aminopheny1)propane (LXXXIII) a t a total dose level of 178 gni. to 3 female beagle dogs for 6 years and found, upon autopsy, multiple bladder tumors in one dog. Considerably greater potentiation of carcinogenic activity is brought about if methyl substitution is in the two rings instead of the central carbon atom. Munn (1967) administered 3,3’-dimethyl-4,4’-diaminodiphenylmethane (LXXXIV) by gastric intubation, a t a dose level of 1020 mg./100 gm. body weight over a period of 10 months to 24 malc Wistar rats. I n a total observational period of 487 days, among 23 survivors, 20 rats had liver tumors (18 malignant and 2 benign) ; moreover, subcutaneous fibromas were found in 11 animals. Munn suggests a possible connection between the potent carcinogenicity of this compound and the epidemiological finding of Case and Pearson (1954) of cancers that have occurred in workmen employed in the manufacture of Magenta dye. Compound (LXXXIV) is, in fact, the first intermediate, known as the “ditolyl base,” of Magenta manufacture. The closely related 3,3’-dichloro4,4’-diaminodiphenylmethane, increasingly used in industry must also be regarded with suspicion. This compound has not yet been tested for carcinogenic activity. However, Mastromattco (1965) reported hematuria in 2 workmen who had acute exposure to this substance. The simple aminotritane, 4-dimethylaminotriphenylmethane(LXXXV) was tested by Druckrey and Schmiihl (1955). No tumors were obtained by administering a total dose of 7.3 gm. (LXXXV) orally during an
MULECU1,AR GEOMETRY AND CARCINOGENIC; ACTIVITY
367
observational period of 800 days. However, by subcutaneous injection of a total of 360 mg. of the compound, local sarcomas were produced in 5 of 9 surviving rats in 28 months. Comparison of the test data obtained in various conditions suggests that carcinogenic activity probably increases with the number of amino groups on the tritanc nucleus. Druckrey et al. (1956) obtained a 7/12 sarcoma incidence in 11 months following subcutaneous injection to rats of a total of 650 mg. Parafuchsinc (LXXXVI) hydrochloride. Kaump et al. ( 1965) fed (LXXSVI) as the panioatc [ 4,4’-iiietliyleiiebis (3-hydroxy2-naphthoic acid) ] salt to Sprague-Dawley rats and obtained in females, but not in males, a marked incrc:isc in the incidence (and tlecrease of the induction times) of tumors of the skin and of the sebaceous and mammary glands; tumors were also induced in the small intestine, subcutaneous tissue, and auditory canal gland. With the hexamethyl derivative (LXXXVII) , Kinosita (1940) induced, by oral administration, gastric papillomas and slight adcnoinatous proliferation of the liver in the same species. Some investigations have been carried out in the past on the carcinogenicity of complex substituted aminotriphenylmethane derivatives used as biologic stains and industrial dyes. A review of these studies is given in the “Discussion” of the report by Kaump e t al. (1965).
3. Derivatives of Naphthalene and Anthracene Since 1961, evidence continues to accumulate that nonmetabolized 2-naphthylamine is inactive or is at most a very weak carcinogen, and must undergo metabolic conversion t o the proximate carcinogen (s) prior to tumor induction. Intraperitoneal injection of 50 mg./kg. of 2-naphthylamine twice weekly for 3 months to random-bred rats produced abdominal sarcomas in only 2 out of 14 rats surviving for more than 600 days (Boyland et al., 1963a). Nor could the carcinogenicity of the arninc be shown by taking advantage of the special sensitivity of newborn animals to carcinogenic stimuli (Section II,D,l). Roe et al. (1963) and Walters et al. (1967) found no significant increase, relative to the control, in the incidence of lung and other tumors in newborn BALB/c mice injected subcutaneously with 50 or 100 pg. of 2-naphthylamine. These results stand, however, in some contradiction with previous results of Bonser et al. (1956a; rf. Bonser and Ckiyson, 1961) who observed the emergence of n high incidence of hcpntoinas in mice injected sutxutaneously with freshly prep;Lred solutioiis of thv :iniinc. Tested by t l i t i bladdcr iniplaiitatioii tc~cliiiiquc.,it was ~ l i o w ni)reviously that 2-naphthylaniirre is inactive or a t most marginally active (Bonser et ul., 1956b), and, riiore recently, also I-risphtliylamiiie ( u s hydrochloritle) was found inactive by the same route (Bonser ct al., 1963). Tlic
368
JOSEPH C. A R W S AND MART F. ARGUS
inactivity of Z - n ~ ~ ~ h t h y l a n i (as i r ~ etlic acctamide) in testing by bladder implantation in mice was confirrncd by Bryan et al. (1964~).Nonetheless, the inactivity of 2-naphthylamine toward the bladder remains an intriguing problem because ( a ) the bladder mucosa of various species appears to possess a high level of N-hydroxylating activity (e.g., Uehleke, 1966b, 1967), and ( b ) 2-naplithylhydroxylamine appears to be a proximate carcinogen on the grounds that, a t least by intraperitoneal route in rats, the N-hydroxy compound is much more active than the parent amine (Boyland et al., 1963a). However, the superior carcinogenic activity of the N-hydroxy compound in rats could not be unequivocally confirmed by testing via subcutaneous injection in newborn mice. I n the experiments of Roe et al. (1963), a t 50-pg. dose level, both 2-naphthylamine and 2-naphthylhydroxylamine were inactive or a t most marginally active. I n subsequent experiments (Walters et al., 1967), using doses of 100 pg., 2-naphthylhydroxylamine but not the parent amine increased the incidence and multiplicity of lung tumors above those of the control. However, the significance of these results remains open to question since neither local sarcomas (compare to Boyland et al., 1963a) nor hepatomas (compare to Bonser et al., 1956a) were obtained. Brill and Radomski (1965b, 1967) and Belman et al. (1967) investigated the finding of Bonser et al. (1956a) that aged solutions of 2-naphthylamine in oil produce a high incidence of local sarcomas upon subcutaneous injection to mice. Brill and Radomski found that the development of the red coloration of these solutions can be prevented by storing the solutions in the dark, or in vacuo, or under nitrogen atmosphere. This indicates that a photochemical oxidation takes place. Three major products of this oxidation have been identified as 2-amino-l,4-naphtho-
(LXXXVIII)
(LxxxrX)
~uiiione-2-naphtliyliiiiiiie(IXXXVIII), its hydrolysis product, 2-amino1,4-naph thoqui~ionc,and 1,2,5,6-dil~~~1izophcnal,inc (LXXXIX). l-Aniii1o-1,4-1ia~~l1lhoyuinone was fourid noiicarcinogeiiic fur rats (Belman et al., 1966). However, testing by early workers indicated that the closely related compounds, benzoquinoi~e and 1,4-naplithoquinone, are fairly potent carcinogens toward the skin of mice (in Hartwell, 1951).
In view of the mtrtivity of quinoneiiiiinc~toward proteins (Irving a d Gutniaiin, 1959; Belman and Troll, 1962), Brill and Radomski (1967) :ttkrit>iitcrl tho iiirwawl local rarc+iogenirity of the aged soliltions to thp ( ~ ~ i i i i ~ ~ i i ( ~ i (i 1 i i,iS i iS~S V I I I J . Howo~~c.i*, dt~hl)it(~ : i n o:trly rq)ort, ( i n H:tttwell, 1951) 011 tlic in:trtii~ityof I ,2,5,li-clil~cnz01~~1~1i~~zi11~ (LSXXIX) as a carcinogen in cpitlielial application, it should be recalled here that, according to a more recent report, testing by bladder irnplantation into rats (Rudali e t al., 1955) indicates this eonipound to be quite a potent carcinogen. The testing now in progress of the carcinogenicity of the quinoneimine (LXXXVIII) and of 1,2,5,6-dibenxophenazine, in oral application in beaglc dogs, by Brill and Radomski (1967) has not yielded tumors up to 14 to 16 months. Up to recently, the dog was the only experimental species in which malignant bladder tumors were known to arise by oral administration of 2-naphthylamine (Hueper e t al., 1938; Bonser, 1943). Saffiotti e t al. (1967) have found now that 2-naplithylamine is highly potent to induce bladder tumors iii hamsters when fed a t the dietary level of 1%; the induction time was 45-49 weeks. Already a t 0.1% dietary level, proliferative changes in the bladder epithelium can be seen after about 100 days, but no cancers develop. Also, Coiizelrnari e t al. (1967) reported the induction of carcinomas in the urinary bladder of rhesus monkeys by daily oral administration of 200 mg./kg. of 2-naphthylamine. It was already known that in rats, and in rabbits even up to 5 years, administration of the amine produces only papillomatous changes in the bladder (Bonser e t al., 1952). The problematic status of tlic requirement of N-hydroxylation for thc carcinogenicity of 2-naphthylaniine was brought to focus by the finding of Shenoy e t al. (1964) that 3-niethyl-2-naphthylamine (XC) is a potent
mNHa /
/
CH,
(
Oc1
p
Cl
(XC)
(XCI)
(XCII)
carcinogcii wliicli induccs upoii subcutancow iiijcction to mice, suhcutaneous sarcomas with a high incidence and short latent period; no tumors were obtained in these experiments in the control mice injected with 2-naphthylamine. The carcinogenicity of the methyl derivative was confirmed by J. H. Weisburger et al. (1967a) who administered it by stomach tube to male and female rats. Tested by this route, the coinpound produced skin and ear duct tumors, and a high incidence of tumors of the gttstrointestinal tract in males ; rnnmmary gland tumors
370
JOSEPH C. ARCOS AND MARY F. ARGUS
arose in almost all females. In another experiment (Griswold et (~1.)1966) in which this compound (in a single dose) was tested by the same route and found to be carcinogmic toward t0w mammary gland, R kidney carcinoma was found in one rat. Thus, 3-mcthyl-2-naphthylarnine, proposed originally as a substitute for the parent amine, is a. potent and ubiquitously acting carcinogen. The related 3-nitro-2-naphthylamine (XCI) was less active toward the mammary gland in the experiments of J. H. Weisburger et al. (1967a) but somewhat more active to induce tumors of the gastrointestinal tract. No tumors of the skin or ear duct were observed. 1,2-Dichloro-3-nitronaphthalene (XCII) , which expectedly undergoes reduction to the corresponding amine in vivo, produced only mammary tumors with a notably lower incidence. 2-Nitronaphthalene, the nonchlorinated compound corresponding to (XCII), is inactive just as 2-naphtliylamine, when tested by bladder implantation (Bryan et al., 1 9 6 4 ~ ) . Whatever the final outcome of the investigations on 2-naphthylaminej available evidence seems t o suggest that its higher benzolog, 2-anthramine, does not require metabolic activation for carcinogenicity. It has been known for some time that 2-anthramine is carcinogenic when tested in bladder implantation (Bonser et al., 1958). 2-Anthramine is an unusual aromatic amine carcinogen. It is highly active by epithelial application toward the skin of rats, but not of mice. An interesting feature of 2-anthramine carcinogenesis is that croton oil has a retarding effect on the emergence of skin tumors in the rat (reviewed by Arcos and Arcos, 1962). I n several respects, however, the biological action of 2-anthramine resembles that of typical carcinogenic hydrocarbons. Administered orally to female rats as a single dose, i t produces malignant mammary tumors in 6 months in a sizable proportion of the animals (Griswold et al., 1966). Dobson and Griffin (1962) and Dobson (1963a) studied the histogenesis of tumors and the alterations of the pilosebaceous apparatus (Dobson, 1963b) during anthramine-induced skin tuinorigenesis. Zackheim et al. (1959) and Zackheim (1964) made comparative histopathological investigations of the effects of 2-anthramine, 20-methylcholanthrene, and 9,10-dimethyl-1,2-benzanthraceneon the skin of mice and rats of various strains. The conclusions of Dobson and Zackheim agree that mainly basal cell epitheliomas are produced by anthramine and methylcholanthrene, while with dimethylbenzanthracene, squamous cell carcinomas are the predominant type of tumor. Inspite of the commercial availability of 2-anthramine, the biochemical changes and alterations of electronmicroscopic morphology brought about by this interesting carcinogen appear to be totally unexplored.
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
371
In the reviewers’ opinion, this compound which structurally is a typical aromatic amine, yet gives biological responses resembling those of polycyclic hydrocarbons, could possibly hold interesting clues for the mechanism of carcinogenesis. I n the experiments of Griswold et al. (1966), 2-chryseneamine was found to be noncarcinogenic when administered as a single dose (at the maximum tolerable level) by gastric intubation to Sprague-Dawley rats which were observed for 6 months. 4. Derivatives of Biphenyl and Fluorene I n the last 6 years, investigations on the structure-activity relationships of 4-aminobiphenyl and benzidine have been carried out mainly by E. C. Miller et al. (1962) and by Pliss (1963, 1964). The totality of the data available now (also see review by Arcos and Arcos, 1962) gives a fairly complete picture of the structural requirements for carcinogenicity (Table XII) and of the effect of ring substituents in influencing tissue target specificity in the rat (Table XIII). It must be recognized t h a t t i e s p i t e the extensiveness of the accumulated data-no consistent picture can be drawn about the role of the ring substituents, except for those in the 4- and 4’-positions. Although tumor distribution in the target tissues varies, carcinogenicity is maintained if the functional group in 4-aminobiphenyl is replaced by amine- or hydroxylamine-generating groups, such as acetylamino, dimethylamino, nitro, or acethydroxamic acid. 4-Fluorobiphenyl is a noncarcinogenic compound. Surprisingly the ability to produce tumors of the small intestine is not lost by replacing the amine by a methoxy group (Arcos and Simon, 1962). The effect of 4’ substituents on the carcinogenicity of 4-aminobiphenyl brings to mind the analogous situation with 2-acetylaminofluorene and 4-dimethylaminoazobenzene. I n 4’-fluoro-4-aminobiphenyl, carcinogenicity toward the mammary tissue and acoustic sebaceous gland is maintained, but in addition, this compound also produces a high yield of tumors in the liver and the kidney. However, activity is absent if the 4’-substituent is a chlorine or bromine atom, and 4’-methyl-4-aminohiphenyl possebses probably only borderlinc carcinogenic activity (Table X I I , footnote a ) . In this respect also, the pattern resembles that seen with 4-dimethylaminoazobenzene. The 4’-bromo derivative of the azo dye is inactive, and the 4‘-chloro and 4’-methyl derivatives are w r y weak carcinogens. However, similarity apparently ends here since, whereas both benzidine and certain of its derivatives and itlho 4,.l’-diiiitrobi~~t1~~11yl ( T A n l i : i i n et ul., 1964) ure ciircinogenic, thc 4’-nitro, 4’-acety1:iiiiino, :tiit1 4’-tlimetliyla1uiiio deriixtives of 4-tlinictliyl-
372
JOSEPH C. ARCOS AND MARY B’.
ARGUS
TABLE XI1 Synoptic Tabulation of the Structural Requirements for Carcinogenicity of 4-Aminobiphenyl and Benzidine Derivatives
t F o r 4-aminobiphenyl Very active if Active if
Very active if 3-methyl 3,2‘-dimethyI 3,3’-dimethyl 3 -fluoro 3’-fluoro
Very active if ,OC .CH, -N ‘OH Active if -NH * OC * CH, --N(CH,), -NOz -OCHs
Active if Inactive ifasb
-B r
Active if
-S-
Inactive if -NH-
3- chloro 3 - methoxy 3, 2’, 5‘-trimethyl 3, 2’, 4’, 6’-tetramethyl
Inactive if -F
Weakly active if 3-hydroxy Inactive if 2- methyl Z’-methyl 2’ -fluoro 3-amino
R I -CHTransition to diphenylmethane and triphenylmethane amines
For benzidine Very active if 2-methyl 3,3’-dihydroxy 3,3’-dichloro Weakly active if 3,3‘-dimethyl 3,3’-dimethoxy Inactive if 2,2‘-dimethyl 3,3’-bis-oxyacetic acid
a
-CH=CHTransition to aminostilbenes -N=N-
t -
Transition to amino azo dyes
‘E. C. Miller ef a/. (1962)found the4‘-methylderivative inactivewhen fedto rats a t t h e level of 1.62 mmoles for 8 months. However, Walpole and Williams (l958)found this compound hepatocarcinogenic in the s a m e species when it was administered by subcutaneous injection and given at a comparatively very high total dose of 10.1 gm/kg body weight. bWhile 4’-hydroxy-4-aminobiphenyl does not appear to have been tested, it is generally assumed to be an inactive compound.
‘E. C. Miller et a/. (1956)found the2-methylderivative inactive by dietary administration. However, according to Walpole and Williams (1958) this compound is a hepatic carcinogen when tested by subcutaneous route.
aiiiiiio:izobenzenc arc inactivc by oral routc (.J. A. Miller et al., 1957; Arcoh and Simon, 1962). Data are lacking on the testing of the 4’-amino derivatives of 4-dimethylaminonzobe~izene by other routes, in other species, and a t higher dose levels. The rationale appears to be less clear concerning the influence of other ring substituents on carcinogenic activity. In 1952, it was proposed on the grounds of correlations between ultraviolet spectral shifts and carcinogenic activities that 2- (or 6-) or 2’- (or 6’)-methyl groups depress the carcinogenic activity of 4-aminobiphenyl by distorting the coplanarity of (and, thus, lowering the resonance between) the two benzenic nuclei (J. A. Miller et al., 1952; Sandin et al., 1952) ; the generally greater biological activity of 2-aminofluorene derivatives has been attributed to the -CH2bridge which helps maintaining a coplanar arrangement (E. C. Miller et al., 1949). In 1962, this explanation appeared still acceptable, and the high potcncy of 3,2’-dimethyl-4-aminobiphenyl and 2-methylbenzidine versus tlic inactivity of 2-methyl-4-aminobiphenyl could be explained by the contribution of the hyperconjugating 3-methyl group (in 3,Y-dimethyl-4-aminobiphenyl) and of the second amino group (in 2-methylbenzidine) to the 4-4’ resonance which counteracts the crowding effect of the 2-methyl substituent (Arcos and Arcos, 1962). Support for this view was actually provided by the finding of Walpole and Williams (1958) that 3-methyl-4-aminobipheny1, tested by subcutaneous route in rats, is a more potent carcinogen on the basis of the total dose used than either the parent compound or the 3,P-dimethyl derivative. Subsequently, E. C. Miller et al. (1962) tested 3-methyl-4-aminobiphenyl (as the N acetyl) by oral administration and concluded that it is only moderately active. However, analysis of their results clearly indicates that, although this compound has a somewhat different target tissue spectrum (Table X I I I ) than 4-acetylaminobiphenyl (tested simultaneously a t the same dose level), in the target tissues actually affected the 3-methyl derivative produced a much higher incidence of tumors in a notably shorter period of time. Thus, on the strength of the results, obtained either by parenteral or I)y oral routc, 3-nicthyl-4-nminobiphcnyl should be regarded as inarkcdly morc potent than tlic parent compound. However, different consitlcrntions introduce added complexity in this attractive interpretation of the sul)+tituent effects, as mediated through influence on intcrnuclcar conjugation. In fact, in the tcsting of Walpole :LII(I 1Villi:iiiis (1958) ( ~ I T I Itlir Iiiglrly rrowtlr(l 3,~,4’,C,’-tct,i.:in~rthyl-4: I I I I ~ I ~ ~ ~ ~ ~ J\\’ah ~ I I i~~ui~krclly ~ ~ I I ~ I arti\rr. Chi llle other hand, ill tlir oral testing experiment hy E. C AIiller rt ul. (1956) , 2’-fluor0-4-aminobiphenyl, which is only sliglitly iiiore cro\vtlctl than the pnreiit amine (compnre the vitn der Wnals r:itliuh of 1 . 1 A of Irydrogcn to tlic radius of 1.35 A of
TABLE XI11 EFFECTOF RING SUBSTITUENTS ON THE TISSUE TARGET SPECIFICITY OF ~AMINOBIPHENYL AND BENZIDINE DERIVATIVES IN THE RAT’ Substituent (s) PAminobiphenyl* None None 2-Methyl 2-Methyl 3-Methyl 3-Methyl 3-Fluoro 3-ChlOrO 3-Hydroxy 3-Methoxy 3-Amino 2’-Methyl 2’-Fluoro 3’-Fluoro 3,2‘-Dimethyl 3,3’-Dimethyl 3,2’,5’-Trimethyl 3,2’,4‘6’-Tetramethyl
Routec
s. c. Oral
s. c. Oral s. c. Oral Oral
s. c.
Intestine
Liver
Bladder
Mammary Ear duct
Salivary gland
Other sites
Uterus; kidney(?) Thymus & lung(?) -
-
Oral
s. c. Oral Oral Oral Oral
s. c. s. c. s. c. s. c.
-
Uterus(?) -
Bemidine* None None 2-Methyl 2,2’-Dimethyl 3,3’-Dimethyl 3,3’-Dimethyl 3,3’-Dihydroxy 3,3’-Dihydroxy 3,3’-Dimethoxy 3,3’-Dichloro 3,3’-bis-Oxyacetic acid 3,3’-Disulfonic acid
s. c. Oral Oral Oral
s. c. Oral
s. c. Oral
s. c. s. c. s. c. s. c.
+ + + + + + + +
Skin & S. C. sarr. Skin & lung(?) -
-
Uterus(?j Stomach
-
-
Skin Stomach Ovary Skin 8: S. C. sarc. -
-
-
-
a Compiled from Spitz et al. (1950);Walpole and Williams (19%);Xalpole et al. (1952, 1955); Baker (19.53);E. C. Miller 1962); Pliss (1959, 1963, 1964) ; Laham et al. (1964) ; Bremner and Tange (1966). * Nitro, acetylamino, and dimethylamino groups have been regarded as equivalent to an amino group. S. C. = subcutaneous.
f f (I(.
(1356,
376
,JOSEPH C. ARCOS AND MARY F. ARCXTS
fluorine) , was devoid of activity. Contrastiiig with tlic above fin4tig of Walpole and Willianis, the similarly crowded 2,2’-dirnethylhenzidine (as N,N’-cliacet,yl) is inactive hy oral route (E. C. hIiller ct a!., 1956). It, alioiiltl I w n o t c ~ It h t while iii Iho Iwiizifliiicsrriva tlic 3,3’-dihytlrosy derivativc is one of tlic most wtive compounds, in the 4-aininobiphenyl series, hydroxy substitutioii in the 3-position brings about considerable loss of activity. Now, it is well known that among the aromatic carcinogens, in general, ring hydroxylation causes a partial or total loss of activity, and that “shielding” of the free hydroxyl group by methylation brings about a considerable regain of activity, sometimes beyond the activity level of the parent compound itself. Allowance made to the evident possibility that differences in experimental conditions may completely obscure the relative order of activities, the data available suggest that 3,3‘-dimethoxy benzidine is much less potent than the 3,3”-dihydroxy derivative; there is, moreover, a change in target specificity when passing from the dihydroxy to the dimethoxy compound. Change in target specificity is also seen between 3-hydroxy- and 3-methoxy-4-aminobiphenyl, although, here again, the difference in the routes of administration could well be responsible (Table XIII) . The overall picture becomes even less consistent if the structural requirements for carcinogenicity in the xenylamine and benzidine series are related to the structural features of the two compounds tested recently by Munn (1967) (see Section III,A,2). These compounds, 4,4’-diaminodiphenylmethane (LXXXI) and 3,3’-dimethyI-4,4’-diaminodiphenylmethane (LXXXIV) are analogous t o benzidine and 3,3‘-dimethylbenzidine, respectively, with the essential difference, however, that in the diphenylmethane derivatives, conjugation between the two benzenic nuclei is blocked by the sp9 hybridized -CH2group, Moreover, in these diphenylmethane derivatives, the two benzenic nuclei are not colinear as in the biphenyl derivatives, but their lengthwise axes form an angle approxicarbon. Yet, despite the commating the valence angle of the -CH2partmentation of conjugation into two quasi-independent halves and the great difference in molecular shape, methyl substitution in orfho to the amino groups-just as in the 4-aminobiphenyl series, but not in the henzidine series-has a powerful potentiating effect on carcinogenic activity (cf. Walpole e t al., 1952). The totality of evidence summarized above illustrates the scarcity of consistent correlations between carcinogenic activity and ring substitutions in the xenylamine and benzidine series. For a more comprehensive view of the rapidly evolving panorama of the structure-activity relationships, activating metabolism, leading to the formation of N-arylhydroxylamines (Section II1,D), must be considered.
MOLECDLAR GEOMETRY A N D CAI{CINO(:I~:NIC A C ' I ' I V I l Y
377
In an invebtigation of the dosing schedules for bladder tumor induction, Deichmanri (1967) found 4-aminobiphenyl t o be considerably more carcinogenic to the dog than 2-naphthylarnine. Bladder tumors were produced with the former agent in female beagles after a total oral dose of 5.4 to 7.3 gni. per dog over a pcriod of 31 to 37 months; similar doses of 2-naphthylamine failed to induce tumors over thc same period of time. Benzidine, administered to hamsters a t 0.1% dietary level for the entire life-span, induces a high incidence of livcr tumors; no bladder tumors were found. 3,3'-Dimethyl- and 3,3'-dichlorobenzidine are inactive under identical conditions (Saffiotti et ai., 1967). Histopathological studies on aromatic amine-induced tumorigenesis have been carried out with benzidine by Pliss (1964) and with N,N'diacetylbenzidine by Bremner and Tange (1966). Laham et aZ. (1964) reported on the tumor distribution in 4,4J-dinitrobiphenyl-inducedcarcinogenesis. Their preliminary results suggest that 2,2'-dinitrobiphenyl is a weak carcinogen on the basis of the average cumulative intake recluired to produce a significant percentage of malignant tumors above the control level. Spjut and Spratt (1965) and Cleveland et al. (1967) investigated the genesis of intestinal neoplasia by 3,2'-dimethyl-4-aminobiphenyl in defunctionalized intestinal tracts (in cecal pouch and colon segment, respectively) and concluded that direct surface contact of the compound or its metabolite through the fecal stream with the intestinal mucosa is necessary for tumor induction a t intestinal sites. Spjut and Spratt (1965) and Navarette-Rejna and Spjut (1966) injected the amine directly into the isolated intestinal segment and concluded that the amine was active as such since no ncoplastic lesions were observed a t distant sites. It is not impossible, however, that, just as the bladder mucosa (Uehleke, 1966b, 1967), the intestinal iniicosa may possess N-hydroxylating activity. Progress has been made in the study of the structure-activity relationships of derivatives of 2-aminofluorene (XCIII) and related compounds. These are discussed in the following paragraphs.
(XCIII)
n. Amino Subatituents unil Amiire-Generutiny Groups. It appears now to be definitely establislicd that for high level of carcinogenic activity, there must be a t least one aniine or amine-generating group in para
378
JOSEPH C. ARCOS AND MARY F. ARGUS
position with respect to the biphenyl linkage. Replacement of the 2-amino group by CH3*S- brings about total loss of activity; substituting by CH, CO S-, however, the resulting acetylthiofluorene retains trace activity toward the small intestine of Sprague-Dawley rats (E. C. Miller et al., 1962). Also, acetylthiofluorene is inactive in mice by oral administration (Argus and Ray, 1956) ; in Wistar male rats, however, by the same route, this compound was found to induce syuarnous papillomas of the stomach in 9 out of 29 animals (Argus and Ray, unpublished). It was already established that, just as with 4-aminobiphcnyl, the 2-amino group of 2-aminofluorene may be replaced by a nitro group with only partial loss of activity. E. C. Miller et al. (1962) have now shown that also 2,7-dinitrofluorenc is a carcinogen toward the mammary tissue of the rat, having a potency cqual to that of 2-acetylaminofluorene; liowever, this dinitro compound is inactive toward the liver and the ear duct gland and has only trace activity toward the intestinal tract. Activity toward the mammary tissue is reduced to a very low level but not totally abolished by positioning one of the nitro groups ortho to the biphenyl linkage, as in 2,5-dinitrofluorene. The marginal activity of the 2,5-dinitro derivative is the compounded result of ( 1 ) the unfavorable position of the second amine-generating group and ( 2 ) the fact that the nitro groups must be reduced in the animal’s body. This is borne out by the finding (E. C. Miller et al., 1962) that the corresponding 2,5-fluorenylenebisacetamide is a notably active but still a less potent carcinogen than 2-acetylaminofluorene. The noncarcinogenicity of 2-dimethylamino-3-nitrofluorene exactly parallels the situation in the 4-aminobiphenyl series, since both 3-amino-4-acetylamino- and 3-amino-4-dimethylaminobiphenylare virtually inactive (E. C. Miller et al., 1956, 1962). The prediction of Argus and R a y (1959) that the carcinogenic activity of various N-acyl-2-aminofluorenes parallels the ease of hydrolysis of the N-substituent [which was partially verified by Morris and his associates (reviewed by E. K. Weisburger and Weisburger, 1958)j is borne out by the moderate carcinogenicity of 2-formylaminofluorene (E. C. Miller e t al., 1962). b. Ring Substituents. Until 1962, 7-fluoro-2-acetylaminofluorene was the only fluoro-substituted derivative tested. It was found to be much more active in the rat for the induction of liver tumors, but with an activity comparable to the parent amide toward other target tissues. E. C. Millcr et nl. (1962) have ussaycd all the rcniaining isomcrir :iromatic ring-monofluoro tlerivativcs, that is the 1-, 3-, 4-, 5-, 6-, and 8-inonofluoro-2-acetylamiiiofluorenes. With the cxception of the 4-fluoro derivative, whicli is inactive towaid the liver, cnch compound is activc to a level comparable to the parent amide in the four typical target tissues:
ALOLECULAR GEOMFTKY A N D CAKCXNOGENIC A C T I V I T Y
379
liver, inaniniary gland, ear duct, and small intestine. The fact that all aromatic ring-fluoro derivatives of 2-acetylaminofluorene have high levels of carcinogenicities has been construed to mean that covalent bond formation between cell components and the substituted positions is not a prerequisite for carcinogenesis by this amide. The potency of the ring-fluoro derivatives stands in striking contrast with the virtual inactivity of the ring-hydroxylated compounds. It may be recalled that of these, the 5- and 7-hydroxy derivatives are major and the 1- and 8-hydroxy derivatives are minor metabolites. It has been known for sonic time that 1-, 3-, and 7-hydroxy-2-acetylaminofluorene are inactive or a t most slightly active in the rat by oral or intraperitoneal administration, and Boiiser (cited in Clayson, 1962) found l-hydroxy-2miinofluorene inactive also by bladder implantation in iiiice (using crushed paraffin as vehicle). Very recently, Gutinann et al. (1967) found the 1- and 3-hydroxy derivatives inactivc also in the N-benzoylatcd form, administered intraperitoneally. In an attempt to test the validity of the ortho-hydroxylation hypothesis, Irving et al. (1963) have assayed in mice the hydrochlorides of the 1-, 3-, 5 - , and 7-hydroxy derivatives of 2-aniinofluorene by bladder iniplantation, using paraffin as vehicle. None of these compounds proved to be carcinogenic and the only tissue changes noted were squamous metaplasia and epithelial liyperplasia. A possible objection which may be raised against the very careful experimental work of Irving et al. is the unquestioning acceptance of paraffin as a vehicle in an effort to reproduce the exact experimental conditions of Boiiser et nl. (1956b). Since the detection of carcinogenic activity by the bladder implantation technique is highly dependent on the rate of elution from the pellet (in Arcos et al., 1968), the total absence of carcinogenic activity with all the aminofluorenols may have been due to the too slow rate of elution (compare also Bryan et al., 1964a,b). This is, in fact, suggested by the elution experiments with 2-amino1-fluorenol-”C. It is stressed t h a t this should not be construed as a defense of the ortho-hydroxylation hypothesis on the part of the reviewers. The inactivity of the aminofluorenols is due to the hydrophily of the phenolic hydrosyl groups. “Shielding” of these groups by methyl substitution brings about partial or total regain of activity. 7-Methoxy-2-acetyl:miinofluorenc is a highly carcinogenic compound (Morris et al., 1960), whereas l-nietlioxy-2-acctylaminofluorene is moderately active, and 3methoxy-2-acetylaminofluorene inactive (Gutinann et al., 1968). c. Other Aspects. Hackmarin (1956) was the first to show that 2amino-3-niethoxydiphenylene oxide is highly specific toward the urinary bladder of Wistar rats without the requirement of indologenic substances
380
JOSEPH C. ARCOS A N D MARY F. A R G U S
in thr diet. Tliat s1)ccificity tow:ircI Llic I ) l a t l t l ( ~ r is t l u c to the -01)rirlgc is further supportrd now hy thc fintliiig C1i:it tlio p r r c n t ) coIlil)ouli(l, 2aininodipliciiylciic oxide, is :i bladder cart*iiiogcii I)y oral at1iiiiiiistr:itioii to C57 x IF mice (Bonser et al., 1965). In addition to the individuals bearing bladder tumors, all mice developed malignant hepatomas. It was known for a number of years that 2-aminofluorene produces only rarely bladder tumors in rats maintained on a normal high pioteiii diet, and the addition to the diet of indole, indole acetic acid, or tryptophan is necessary to direct target specificity toward the bladder. Recent reports on this subject are due to McDoniild et al. (1962) arid Dunning (1967). In the rabbit, on the other hand, it had already been known that 2-acetylaminofluorene produces tumors of the urinary tract (Bonser and Green, 1950). Their findings have now been confirmed by Irving et al. (196713). Tumors of the liver and mammary gland have not been observed, which stands in marked contrast to the systemic carcinogenicity of the amide in susceptible rodents. Instances of production of tumors in the skin by dietary administration of carcinogens are rare. How and Snell (1967) have now shown that a high incidence of tumors of sebaceous gland origin and a number of epidermal tumors develop in ACI/N female rats following the feeding of 2,7-fluorenylenebisacetamide. Second to the well-studied instance of the guinea pig, macaque monkeys are now found to be completely refractory to the carcinogenic action of 2-acetylaminofluorene and 2,7-fluorenylenebisacetamide tested by oral administration (for periods varying from 10 to 43 months) and by repeated subcutaneous injections (as the cupric chelate of 2-fluorenylhydroxylamine). With a few exceptions, the animals were newborn when the treatment was initiated (Dyer et al., 1966). The failure of these two fluoreneamides to show any carcinogenic effect in monkeys is possibly related to their metabolic behavior. Thus, 98% of the 2,7-fluorenylenebisacetamide given orally to rhesus monkeys is recoverable unmetabolized from the feces within 1 week. Orally or intraperitoneally administered 2-acetylaminofluorene is excreted within 72 hours (97% in the urine and 3% in the feces); 87% of these metabolites consist of the 7-hydroxy derivative (partially conjugated), the rest is a mixture of unchanged amide, free amine, and a small amount of the N-hydroxy metabolite (Dyer et al., 1966).
5. Tryptophan Metabolites Because of the increasingly recognized causal relationship of tryptophan metabolites to spontaneous bladder cancer in humans, there is continued interest in the testing of these metabolites for carcinogenic
activity, i n the seiircli for new nictabolites, and in asccrtaining the increased excrction of carcinogenic tryptophan metabolites in patients with bladder tumors. A careful study of the coiiditions of testing for carcinogenic activity by the bladder implantation technique has been carried out by Bryan et al. (1964b). These workers studied the rate of rlution of tryptophan metabolites and other aromatic nitrogen-containing compounds from cholestcrol pellets implanted into mouse bladders, in order to determine thc probable extent ant1 duration of exposure of the bladder mucosa to potential carcinogcns. The importance and necessity of such elution studies, previous to actual tcsting, have been first pointed out by Allen et al. (1957) and recently reemphasized by Arcos et al. (1968). Price arid his co-workers liavc reported a comparative testing study using paraffin (Bryan et ul., 19644a) and cholesterol (Bryan et nl., 1964c) as “inert” pellet material. They concluded that cholesterol (compressed into pellets in a pellet preqs), but not paritffin, is a satisfactory vehicle for testing urinary tryptophan ~nctabolitcsfor carcinogenic activity. Using cholesterol, five metabolites were found active to various degrees: xanthurenic acid and its 8-methyl ether, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and 8-hytlroxyquinaldic acid. The 8-methyl ether of xanthurenic acid was the most active of all the compounds tested in these experiments and confirms the carcinogenicity of this compound to the bladder first noted hy Allen et al. (1957). Xanthurenic acid was found earlier to he inactive (cited in Boyland, 1958). 8-Hydroxyquinaldic acid had not been previously tested. Using cholestero1 as vehicle, these tryptophan metabolites were as active as the reputedly potent carcinogens, 2-naphthylhydroxylamine and N-acetyl-2-fluorenylhydroxylamine. Three out of the five active tryptophan metabolites are quinoline derivatives and are closely related to 8-hydroxyquinoline which has repeatedly been found (Allen et ul., 1957; Hueper, 1965) to possess a t least weak carcinogenicity. Thus, Bryan et al. (1964~)were led to explore the possible activity of other dihydroxyquinoline derivatives. The 2,8-, 4,8-, and 2,6-quinolinediols are inactive by the same statistical standards. A synoptic tabulation of the structural requirements for carcinogenicity of quinoline compounds is given in Table XIV. The work of Bryan e t al. contributes a needed confirmation of thc carcinogenicity of 3-hydroxyanthranilic acid to the mouse bladder. I n fact, the carcinogenic activity of 3-hydroxyanthranilic acid toward this tissue (tested in clwlrsterol) , w1)urtetl origiiially by Allen et ul. (1957), could not be confirmed b y Clayson et nl. (1958). Inasmuch as doubts hare heen expressed (e.g., ,J. .4. IIiller et nl., 1961; Irving r t al., 1963) ahoiit thc re1i:tI)ility :mti/or validity of tlic hlnd~lcrimplantation tech-
w
00
t3
TABLE XIV Synoptic Tabulation of the Structural Requirements f o r Carcinogenicity of Quinoline Compounds in Rats and Micen
4
1
2
Positions of substituents 3 4 5 6
0
?n
7
8
-OH
-0 -0
-OH
Activity
+ -
-OH
-OH -OH
-OH
-
-COOH
-OH
-OH
+ +?
-COOH
-OH
-OCH,
r '
-OH
-0
Bladder implantationb Bladder implantationb B1adder implant at ionb Bladder implantationb
Urinary bladder
Bladder implantationb
Urinary bladder"
Bladder implantationb
Urinary bladder
Skin, S. C.b Skinb
-NO, -CH3
-0
-c1
-0
-Br -NO,
-
Skinb
+ ?
s. C.b s. c . b s. C . b
z d P
E
8
Skin, S. C.b
-NO,
-0
Bladder implantationb; intrarectal o r intraUrinary bladder? UterusC vaginal instillationC
Bladder implantationb
-OH
-COOH
-0
Target tissue
s. CP
-OH
. -OH
Route
Skin & subcutaneous t i s s u e e
-0
+
-NO,
Skin, S. C b , c ; oral
Skin, s u b c u t a n e o u s tissue
(& all local sites), lung & uterus.6.c M a m m a r y & lymphoid t i s s u e -0
NO,
-0
- NO,
-0
-NO,
-0
-NO,
-0
-NO,
-0
-NO,
-0
--NHZ
-0
NH.OH
-0
-NH.OH
T h e 2-, 5 - , 6 - , 7 - , and 8methyl, 2-ethyl, 5,6-, and 7 - c h l o r o , - 6 , 7 - d i c h l o r o & 6-carboxy der i v a t i v e s are a c t i v e to various d e g r e e s ~
NO,
-NO,
Skin, S. C . b
-
s. c.6 s. c.6 s. C.b
-
-NO2 -NO,
-t
-
-NO2
-
-NO,
-
.4ctive sites
at local & d i s t a n t
0
m 0
S. C.*
s. c.6 s. c.6 s.C.b*C
Papillomas. s a r c o m a s L leukemia.* S a r c o m a s C
Skin
“Compiled from Allen 1 1 ~vcrtiI n.or1wi.h (cb.g.,T,uaznti :icricliiies with 1)N.i Iias l ~ h iii\xL-tig:itc.tl t l ( ~ l . ,19G1 ; ldeiiii:tiiJ1964) , :iiuI tlic c * u i i w i h i i h of evi(leiict. i. i i i favor of Scctiuii 1 I,E',4). E'ollowiiig tlic discovery 11i e i 1i te r c x 1:t tio I i I i 1 wt Iit+ is ( by hlunn (1967) of the carcinogenicity of Acricline Oixnge, thc :tbove findings and concepts lead directly to the idea that the testing for car%
392
JOSEPH C. ARCOS AND MARY F. ARGUS
cinogenic activity of a series of aminoacridiiies of graded mutagenic potencies might prove fruitful. B. AMINOAzo DYES Investigations in recent years on the effect of ring substituents on the carcinogenic activity of alkylamino azo dyes have been carried out mainly by Brown and his co-workers (Brown et al., 1961 ; E. V. Brown, 1963; Napier, 1964; Brown and Hamdan, 1966) and by Arcos and Simon (1962). An excellent review on the methods of study and problems of amino azo dye carcinogenesis has been given by Terayama (1967). 1. Auxocarcinogenic Effect of 4 -Substituents
Poteiitiation of the carcinogenic activity of 4-dimethylaniinoazobenzene (C) by certain substituents in the 3’- (or equivalent) and 4’-positions has been known for some time. In particular, 4’-ethyl substitution brings about a reinarkable enhancement of the hepatocarcinogenic activity of 4-monomethylaminoazobenzene (MAB) and 4-dimethylaminoazobenzene (DAB) in the rat (Sugiura et al., 1954; J. A. Miller et al., 1957) . The 4’-isopropyl-, 4’-n-propyl-, 4’-n-butyl-, and 4’-te&butylDAB are all active in this decreasing order (Brown and Hamdan, 1961). On the other hand, 4’-methyl-DAB is a very weak hepatic carcinogen (J. A. Miller and Miller, 1953), and this could be due to oxidation to the inactive 4’-carboxy derivative or to replacement by a hydroxyl group. Besides an ethyl (or higher alkyl) group in the 4’-position, other groups can enhance the activity of the parent dye. Thus, 4’-fluoro substitution brings about a doubling (.J. A . Miller and Miller, 1953; J. A. Miller et al., 1953), and replacement of the “prime” ring by a 4-pyridylN-oxide brings ahout a three- to fivefold increase (Brown et al., 1954a; Brown and Hamdan, 1966) of activity relative to the parent compound (C). The coordinatively bonded oxygen atom in the N-oxide analogs studied by Brown and his associatcs corresponds sterically to a substituent in the respective positions.
(XCIX)R = H (C) R = CH, (CI)R = C,H,
True potentiatioii of activity, a t least with the alkyl and N-oxide groups, sippears to be specific to thc 4’-position since both P’-ethyl-MAB (Sugiura et al., 1954) and pyridine-1-oxide-2-azo-p-dimethylaniline
MOT,ICCTJLAR GEOMETRY AND C A R C I S O C E N I C ACTIVITY
393
(PO2) (Brown c t a l , 1954a) are inactive, No potentiation is tee11 either i n tlie correspondiiig 2’-fliioro-ei~hstitiited coinpound, since 2’-fliioro-DAB Irws a potency e q i i ~ lto or nt niost slightly Irigher than tlic paicwt dye (,J. A. Miller et al., 1953). The 3’-position is much less sensitive to the nature of the substituents than the 4’-position. I n particular, while the 4’-methyl, 4’-methoxy, 4’cliloro, and 4’-nitro derivatives of DAB are inactive or a t most weakly active, the same substituents in the 3’-position givc rise to medium active or potent hepatic carcinogens. There is an inverse situation with the pyridine-N-oxide analogs: pyridine-1-oxide-3-azo-p-dimethylaniline( P 0 3 ) is only about :is active as DAB, while pyridine-1-oxide-4-azo-p-dimethylaniline ( P 0 4 ) is 3-5 times as potent (Table XVI). On tlie other hand, fluoro substitution, irrespective of whether in the 3’- or 4’-position, brings about doubling of the activity of the parent dye (J. A. Millcr et al., 1953). Similarly, Burkhard et al. (1962) reported that 3’- and 4’-methylthioDAB are about equally active hepatic carcinogens in the rat. Unfortunately, a DAB control group was not included in the testing, so t h a t relative potencies cannot be calculated. However, judging from the tumor incidences (16/19 in 16 weeks for the 3’-isomer and 13/16 in 20 weeks for the 4’-isomer) , their activity levels are betwecn those of the parent compound and the 3’-methyl derivative. The 2’-methylthio derivative mts found inactive when fed to 21 rats for 23 weeks under identical conditions. I n an attempt to gain insight into tlie mechanism of potentiation by 4’- and 3’-substituents, Arcos and Simon (1962) carried out a comparative study of the effect of 3’-methyl, 4’-ethyl, and 4’-fluoro substitution on hepatocarcinogenic activity in Sprague-Dawley rats. The inactive azo compounds, 2-niethyl-DL4B, 4-diethylaminoazobenzene (CI), 4-aminoazobenzene (XCIX) , and 4-hydroxyazobenzene, were substituted with these groups in the respective positions. All derivatives of 4-aminoazobenzene and 4-hydroxyazobenzene were inactive. However, 4’-ethyl substitution was found to confer appreciable cnrcinogcnic activity to tlic inwtive 2-iiiethyl-DAB and 4-dietliylnniiiioazo~eiizeiic (CI) . 4’-Ethyl2-metliyl-DAB and 4’-ethyl-4-diethylaniino:~zohenzene have relative activities of 12 and 5, respectively. 4’-Fhoro substitution is much less effective in bringing about carcinogenic activity, as 4‘-fluoro-2-methylDAB is only weakly active (relative activity 1-2) and 4’-fluoro-4diethylaminoazobenzene is inactive. A methyl group in the 3’-position is the least effective of all three substituents in bringing about carcinogenic activity, as both 2,3’-dimethyl-DAB and 3’-methyl-4-diethylaminoazohenzene are not carcinogenic. Recently, Brown and HRmdan (1966) have provided the iiitcresting
394
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE XVI Carcinogenicity Toward the Rat Liver of "Prime" Ring Analogs of 4-DimethylaminoazobenzeneTested by O r a l Administrationa
Active compoundsC PO 3 P4 2'-Me-P4 PO4 2'- Me-PO 4 3'-Me-P04 2, P-diMe-PO4 3,2'-diMe-P04 a', 6'-diMe-PO4 N, N-Methylethyl-PO q d N,N-Diethyl-P04d 1- Naphthyl Z-NWhthyl 44 QO 4 Q5 QO 5 66 W 6 4 - Isoquinolyl 5- Isoquinolyl 5- Isoquinolyl-N- oxide 5- Isoquinolyl-N- oxide 7-Isoquinolyl
Lowest dietary level
0.06 0.06 0. 06 0.03 0.01 0.06 0.03 0. 03 0.01 0.03 0.03 0.075 0.075 0.03 0.03 0.01 0.01 0.01 0.01 0.03 0.03 0. 03 0. 01 0.03.
(56)
Activityb -6 50 15 -13 > 18 > 50 > 18 12 Moderate Potent 15 22 150 200 200 100 15 40 > 50
-
zoo -6
Inactive compoundsC P2 PO 2 P3 4' - Me-P 2 4'-Me-P02 6'-Me-P 2 6'-Me-P02 N,N-Dipropyl-PO 4 2-Thiazolyl 4-Xenyl QO 2 Q3 QO 3 Q7 go7 Q8 QO 8 2- Anthryl 1- Anthraquinonyl 2- Anthr aquinonyl 6-Quinoxalinyl 2-Dibenzofuryl 3- Dibenzothienyl 2- Benzothiazolvl
a Compiled from Brown el a/. (1954a,b.1961); E. V. Brown (1963); Brown and Hamdan (1966); Lacassagne e! a/. (1952); Mulay and Firminger (1952); Mulay and Congdon (1953); Napier (1964).
b The values for the quinoline and isoquinoline dyes were taken from the reports of Brown
er a/. (1961) and E.V. Brown (1963). Other values were approximated from other published data of Brown and co-worker8 where 4-dimethylaminoazobenzene controls w e r e available. C The xenyl, quinoline, isoquinoline, quinoxaline, dibenzofuran, dibenzothiophen, and benzothiazol dyes were fed for 6 months. All other compounds were fed for 10 to 12 months or for the time necessary to reach 100% tumor incidence,
These compounds correspond to 4-methylethylaminophenyl-, 4-diethylaminophenyl-, and 4-dipropylaminophenyl-azo-4-pyridine-N-oxide,respectively.
information that also an N-oxide group in 4' confers carcinogenicity upon the inactive 4-diethylaminoazobenzene (CI) . Pyridine-l-oxide-4-azo-pdiethylaniline (N,N-diethyl-P04) is a hepatic carcinogen in the rat with potency roughly comparable to that of 3'-methyl-DAB (relative activity 12). Thus, the totality of the data availahle shows that the relative effec-
R1OLEC;ULAR C;EOMWIIY AND CAIK‘INOGENIC ACTIVITY
395
tivcncss of tlic four substitucirts to confer activity upon iiiactivc carcinophile structurcs is
4 ’ e N - 0 > 4’-CzHS > 4’-F > 3’-CHy The relative ineffectiveness of a 3’-methyl group is also indicated by the fact t h a t such substitution of the parent compound (C) raises activity only twofold. In Table XVI, the N-oxides PO4 and 2’-Me-P04 (the steric analog of 3’-methyl-DAB) show roughly the same ratio of activities. The potentiating effect of a 4’-ethyl group seems to be related t o the conditions t h a t ( a ) it is in thc 4’-position and ( b ) it is linked dircctly to the “prime” ring. In fact, whereas 4’-methoxy-DAB is a weak t o moderately active carciiiogcn, the 4’-cthoxy derivative is inactive (Arcos and Simon, 1962). Thc graded nuxocarcinogenic effect of tlic four substituents strongly suggests that also noncovalent interactions arc involved, in a nonspecific fashion, in binding the dye molccule to critical ccllular site (s) . Speculatively, EN + 0, -C,H,, and -F groups can interact by means of coordination bonding, 1iydi.ophobic bonding, and hydrogen bonding, respectively. The bond cnergy of these interactions (consider the short length of the cthyl group) decreases in this order and parallels the observed carcinogenicities. T h a t a 3’-methyl group is the least active as a n auxocarcinogen may be due to the possibility that, here, potcntiation results froin the positive inductive effect of the group, and this effect increases the electron charge a t tlic 4’-carbon atom. This, then, strengthens the electrostatic interaction bctween the 4’-position and tlic cellular site(s) ( M . Arcos and Arcos, 1958). Neverthcless, such an electrostatic or fractional valence interaction is of lower energy than the above considered bonding types. Hence, a 3’-CH, group is the ]cast effective auxocarcinogen. The inactivity of 4”ethoxy-DAB has been interpreted (Arcos and Arcos, 1962) as due to the loss of hydrophobic bonding ability because of the hydropliilic character of the oxygen atom. I n addition to neutralizing hydrophobic bonding, the electroriegative oxygen atom may act as an “elcctron sink” reducing electrostatic interactions with the 4’-carhon atom. Support for the latter vicw is provided by thc notably higher potency of 4’-rnethylthio-DAB (Burkliard e t ul., 1962), which contains thc less clectronegative sulfur atom, than of 4’-methoxyDAB (*J. A. Miller e t al., 1957; Arcos and Simon, 1962), which contains the more clectroncgative oxygen atom. I n the light of their results, Arcos and Simon (1962) have questioned the current iiiterprctation of the phenomenon t h a t fluoro substitution in (wtaiir Imsitionh increases the activity uf vnrioub carcinogtmic cum1 ) o i i n i l h . AccotS(1iiig to this i~itcrpi~ct:~tion ( c , . ~ . , J . A. Rliller ef u l . , 1953;
396
JOSEPH C. A R C 0 6 AND MARY F. ARGUS
E. C. Miller et nl., 1962; E. C. Miller and Miller, 1960; J. A. Miller and Miller, 1963), carcinogenicity is increased because fluoro substitution diminishes the extent of metabolic ring hydroxylation by virtue of the strength of the C-F bond. While this working hypothesis has led to a number of valuable data, i t appears to be difficult to reconcile with the inactivity of 4‘-fluoro-4-diethylaminoazobe~1zene and the weak activity of 4’-fluoro-2-methyl-DAB. We have scen above t h a t substitution by 4’-ethyl or 4’-N-oxide groupings, instead of 4’-flUorO, leads to potent compounds. Yet, according t o Westrop and Topham (196Ga), a 4’-ethyl substituent protects DAB against 4’-hydroxylation lcss than a 4’-methyl substituent, although thc 4’-cthyl-substituted compound is considerably more activc than the 4’-methyl-substituted compound. Clearly, then, the problcin of potentiation of carcinogenicity is a phenomenon more complex than merely protection of ring positions against metabolic hydroxylation. The work of Westrop and Topham is discussed in more detail in Section III,C,3. At any rate, whatever the ultimate significancc of Westrop and Topham’s work is, their results do not appc:ir to support a correlation between carcinogenic activity and protective cffect of 4’-suhstitucnts (including fluoro) against 4’-hydroxylation.
2. The Requirement of N-Alkyl Groups The reports of Arcos and Simon (1962) and Brown and I-Iamdan (1966) prompt a reformulation of the conclusion of the Millers that a t least one N-methyl group is required for hepatocarcinogenic activity of amino azo dyes (reviewed by J. A. Miller and Miller, 1953; see also Kinosita, 1937). This requirement must now be broadened to include N-alkyl groups other than methyl, in view of the carcinogenic activity of 4‘-ethyl-4-diethylaminoazobenzene and N,N-diethyl-P04. Although i t is true t h a t these compounds are somewhat less active than the corresponding N-methyl dycs, the differences of activities arc insignificant in view of the considerable widening in recent ycars of the activity spcctrum of hepatocarcinogenic azo dyes (Table XVI). Regarding the possible importance of a two-step enzymatic activation mechanism (N-hydroxylation and N-hydroxy esterification) for hepatocarcinogenicity of amino azo dycs, Poirier et nl. (1967) assume t h a t an N-alkyl group may be necessary for fitting the dye on the cnzymc(s) involved. This suggestion appears to be in contradiction, however, with the results of the carcinogenicity tests of N-hydroxy dyes. I n fact, while N-hydroxy-2-ttcctylaii~iiiofluo1~e1ic is a potcnt agent to inducc sarcomas in rats by subcutaneous injection, 4-hydroxylaminoazobenzene, its N acetyl derivative, and the O-acc>tyl ester of the latter are inactive when n i ~ lin long-tcrni fcc~ling(Sato tested in tlic s:me spccies pni~ciitc~r:illy
hIOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
397
et al., 1966; Poirier et al., 1967). Yet, N-acetoxy-4-aminoazobenzene, a t least, represents an esterified N-hydroxy compound similar to the end product (s) of the postulated two-step in vivo activation, and, therefore, an alkyl group should logically not be required for activity. At any rate, tliis picture is further complicated by the recent important discovery that amino azo dyes are potent carcinogens also toward extrahepatic tissues of tlie rat (e.g., Fare, 1966). In particular, 4-;21iiinO-, 4-nionomethyl:mino-, and 4-diniethylaminoazobenzene and their 3-niethoxy derivatives are potent carcinogens to the skin of the rat by topical application. Any “all-or-none” requirement for an N-alkyl group is absent for activity toward this target tissue, since all six dyes give 100% tumor incidence; Iiowcvcr, it remains that tlie N-methyl dyes have shorter induction times than the corresponding free amines (Fare, 1966). 3. Heterocgrlic and Lnrye-Sizc Priine Ring Aiinlogs of ~ - D i n z e t h y Z a ~ ~ i i n o a x ~ b e n(zD e ~AiB e)
Tlie extremely high lcvels of carcinogenic activity encountered in certain nieriibers of this class (Table S V I ) represent a n important developnient. Tlie suggestion of J. H. Il’eisburger and Weisburger (1963) should he reiterated here, that, because of the considerable levels of activities, “application of these compounds to studies on the possible existence of a threbhold in cheinical carcinogenesis might prove worthwhile.” Moreover, the investigations of Brow11 and his associates may stimulate new studics on the structure-activity relationships of tlie amino azo dyes and ttic inechaiiim of auxocarcinogen effect. T h e carcinogenicity of the 1- and 2-naphthyl and 4-xenyl analogs has h e m rcvicwed previously (Arcos and Arcos, 1962). Although the 2-anthryl :tnalog has been fed to Sprague-Dawley rats for 1 year a t 0.06% level in :Lcontrolled riboflavin semisyntlietic diet (Napier, 1964), the length of time may have been insufficient and the strain of test animals may have h e n rcnistant t o this agent. In this regard, a similar instance should be recalled in which J. A. Miller and Baumann (1945) found the l-naphthyl analog inactive in Holtzmnnn (Sprague-Dawley) rats before its carcinogenicity in Oshorne-Mendel rats was shown by Mulay and Firminger (1952). It is also possible t h a t differcnt routes of administration may have to be used for tcsting. Nevcrthelcss, conditionally accepting Napier’s testing as :itlcquatc, it is not surpribing that other tricyclic LLprinie’lring analogs, 1- ~ t n d2-:inthraqui1ioiiyl, dibenzofuryl, and dibenzothienyl, are inactive (E. V. Brown, 1963; Napier, 1964). Molecular size may not be, however, a limiting factor hcrc since Dittmar (1942) found 4-nitrobenzeneazo-3,4-benzopyrene quite potent to induce heinangiosarcomas in mice by subcutaneous injection.
398
.IOSEI’H C. ARCOS A K U RIAltT
17.
ARGUS
,Judging from thc f:wt t1i:it N:Lpicr t1escril)ctl no mammary tumors wit11 the 2-tnthryl aii:iIog, it, should IIC :issunid thitt the compound does not undergo significant metabolism involving reductive clcavage of the azo linkage. I n fact, one moiety of such cleavage would be 2-anthramine which has been shown by Griswold e t al. (1966) to yield an 8/18 incidence of malignant mammary tumors in rats, in as little as 6 months, following an oral administratioii of the maximum-tolerated single dose. Another possibility is that the insolubility of this high-molecular-weight dye may have limited its adsorption from the intcstinal tract. Testing thc 2-anthryl analog by subcut,aneous route might prove worthwhile since Mulay and O’Gara (1957) found thc 1-naphthyl analog also active by this route. The pyridine-N-oxide analogs have been discussed in some detail in relation to tlie auxocarcinogenic effect of 4’ substituents. Table XVI shows that the potent carcinogenicity of PO4 is further enhanced by a 2‘-methyl group. (Note that, because of the numbering of the pyridine ring system, 2’-Me-P04 is actually the structural analog of 3”methylDAB.) On the other hand, introduction of the methyl group into the 3’-position (3’-Me-P04) reduces the activity of P04. Here, again, the pattern follows that of the parent compound series, since 3’-Me-P04 is the analog of a’-methyl-DAB, which has a relative activity of only 2 to 3. Potentiation by tlie N-oxide grouping in the 4‘-position (‘overrides” the deactivating effect of unfavorable methyl substitutions ; thus, 2,2’- and 2’,6’-diMe-P04 are potent carcinogcns. Except for the presence of the 4’-N-oxide grouping, these compounds are identical to the inactive dyes, 2,3’- and 3’,5’-dimcthyl-DAB (Arcos and Sinion, 1962; J. A. Miller and Miller, 1953). The inactivity of both N,iV-dipropyl-P04 (Table XVI) and of N,N-dipropyl-DAB (J. A. Miller and Miller, 1953) gives a definite indication that the stereochemical limitations compatible with carcinogenicity are much more stringent for the amino substituent than for a substituent occupying thc 4’-position. The most potent hepatic carcinogcns known to date can be found among the quinoline and isoquinoline analogs and their respective N oxides. It should be noted in passing that Cosgrove e t al. (1965) reported the carcinogenicity of the styryl analog of Q4, 4- (p-dimethylaminostyryl) quinoline, which produces a high incidence of hepatomas in mice following single intravenous injection. Whilc the rationale for the high activity of the azo derivatives remains to be cluciclated, it is clcar that, in the quinoline series, linking of the 4-dimethylaminophcnylazo group to the 2-, 3-, 7-, or 8-position does not give rise to carcinogenic compounds. Speculatively, this is possibly due to ( a ) tlie rcquircment of a certain distance between the dimethylamino group and the region of highest electron density in the heterocyclic nucleus and ( b ) the fact that because of the electronegativity of the nitrogen or N-oxide grouping,
MOLECULAR GEOMETRY A N D CARCINOGENIC ACTIVITY
399
conjugation tlirough the heterocyclic nucleus is oriented in a specific fashion. Morcover, obscurc steric eflects arc probably additional determinants of carcinogenic activity. To the reviewers, it seems that investigations with these most unusual compounds have, in truth, barely begun. Correlated biochemical and theoretical studies with them could open new levels of understanding of the relationship between the electronic propcrties and carcinogenic activity of aromatic compounds. 4. Activity of Azo D y e s to ExtTahepatic Tissues For about three decades, the notion pervaded the literature that the amino azo dyes related to 4-dimethylaminoazobenzene (DAB) and oaiiiinoazotolucne have an exclusive spccificity toward the liver. J. A. Miller and hhller (1961) reported that ingestion by the rat of 3-methoxy-4aminoazobenzenc or its N-monomethyl and N,N-dimethyl derivatives a t the level of 2.67 mmoles/kg. diet iiiduccs high incidences of tumors in extrahepatic tissues, in particular, squamous cell carcinomas of the ear duct. However, these dyes manifested only trace activity toward the liver. 3-Methoxy-4-aminoazobcnzene also iiiduccd low incidences of tumors in the small intestine and the mammary gland. Moreover, the dyes induced skin tumors in occasional animals. No tumors were noted in rats which received the corresponding 2-nicthoxy or 2-hydroxy dyes in the diet. Essential features of the Millers’ results were confirmed by Fare and Howell (1964) ; however, these authors found a notably higher incidence of skin tumors with 3-mctlioxy-4-aminoazobenzene. Simultaneous administration of cupric oxyacetatc protected against liver tumor induction by the methoxy dyes, just as it was noted earlier in DAB-induced hepatic tumorigenesis (Howell, 1958) ; the copper salt did not affect, however, the incidence of ear duct and skin tumors. Subsequently, Fare and Orr (1965) showed that 3-methoxy-DAB is a potent agent to induce tumors on the skin of rats. Painting twice wcekly with 1 ml. of a 0.2% solution of the dye in acetone on the shaved intcrscapular region yielded 100% tumor incidence with an average induction time of 46 weeks for the appearance of the first tumor. All rats developed multiple skin tumors, and a number of them also developed ear duct tumors. The sampling of skin tumors was identified mainly as squanious carcinomas, keratoacanthomas, basal cell carcinomas, and trichoepithelionias. Just as in the 1964 report, feeding of cupric osyacctatc to the rats did not protect against skin tumorigenesis, ~ l i i c l110 col~pc’i’;ic.cuinul:ttion was noted in the skin. No bound or free dye mas detected ill thi. li\.cbi*b of thv rats. Previous to this study, only two (unswces>ful) attenipts to iiicluce skin tumors in rats by epithelial a1)pIie:itioii of tliffcrctit :izo compounds appear to have beeii rccordcd in the literature (Ray et nl., 1952; Mulay and Congdon, 1953). Prompted I Jliis ~ results, Fare (1966) undertook a systematic investi-
400
JOSEPH C. AHCOS A N D MARY F. A R G I J S
gation of the carcinogenicity of specially purified amino azo dyes toward the rat skin. As we have briefly mentioned in the discussion of the requirement for N-alkyl groups, the methoxy grouping in the 3-position is not necessary for carcinogenic activity since 4-aminoazobenzene, 4-monomethylarninoazobenzene (MAB) , and DAB are all active and produce multiplo skin tumors of histologic types observed with other skin carcinogens. With or without the 3-methoxy substitucnt, the N-monomethyl dyes proved consistently to bc the most potent. Since all compounds eventually produced tumors in 100% of the animals, the relative activities of the dyes toward the rat skin are defined on the basis of the average induction times and have the following order: 3-methoxy-MAB > MAB > 3-methoxy-DAB > DAB > 3-methoxy4-aminoazobenzene > 4-aminoazobenzene Tumors of thc ear duct arose only with the methoxy dyes, and whereas the 3-methoxy group is not necessary for epithelial activity as i t was believed in early studies, its introduction into the molecule does increase the potency. I n contradistinction to the high susceptibility of the r a t skin to the epithelial carcinogenic action of azo dyes, mice were found to be totally resistant to skin tumorigenesis by 3-methoxy-DAB. Another newly found, extrahepatic tissue target of amino azo dyes is the bladder. Saffiotti et al. (1967) reported that feeding of o-aminoazotoluene to hamsters a t 0.1% dietary level for 49 weeks produces a high incidence of liver tumors as well as a high incidence of bladder tumors. Trypan Blue has been known to produce histiocytic tumors in the liver and other reticuloendothelial organs in rats by prolonged weekly subcutaneous administration. Driessens et al. (1962) and D. V. Brown (1963) described the induction by this dye of subcutaneous tumors a t the injection sites. The dye was injected every 2 weeks (1 ml. of a 1% solution) until subcutaneous tumor formation began; the tumor incidence was 50-60% with a latent period of 7 t o 18 months. Histologically, the tumors were described as being probably histiocytosarcomas.
C. DETOXICATING METABOLISM Except for ortho hydroxylation, which possibly plays some role in the carcinogenic activity of 2-naphthylamine, the metabolic ring hydroxylation of aromatic amines-just as the ring hydroxylation of polycyclic 1iydrocai.bons-invnria~)ly brings about decrease or loss of carcinogenicity. The clcawge of tlic YZO double bond and the removal of N-alkyl groups from amino azo dyes also cause loss of activity. Such metabolic routes of aromatic amines and azo dyes, since they a11 bring about decrease of carcinogenicity, represent pathways of true detoxication. On
MOLECI’LAR (;EOl.II.:TRT AKD C A R C I S O G E S I C ACTIVITY
401
the other hand, -hytlroxylatiou anti sulwqiient v4cmficwtion of the N-hy droxy metaholi t es I’PP I I It 111 111 c rt’n *(+ of c :i r r i 11 ogciiir a c t i vi t y . The latter pathways ni:ty I)c dehigii:itcJ(l:t* rcc.//zw/rrr!/ / u c / a I d r s m . Unfortunately, itii uneyuivocnl clenionstr:itioii that a irietabolite is a proximate carciiiogeii, i.e., that the metabolic path leading to it is a truly essential step in the carcinogenic effect (s) of the parent compound, presents some inherent difficulties. Several metabolites along the pathway (s) originating a t the pareiit compound may be carcinogenic. However, the higher potency of :t metabolite, and even its local carcinogenicity versus the local inactivity of the parent conipouiid-although it furnishes strong circumstantial evidence that the particular metabolite is a proximate carcinogen-does not logically rule out that the parent compound, prior to metabolism, is also carcinogenic. Thc circumstance that a parent compound is active toward certain target tissue (s) when systemically administered, but inactive locally, can certainly mean that the target tissue (s) represent site (s) of metabolic activation. However, activity in specific target tissues, following systemic administration, can also be the result of the particular physicochemical properties of the parent coinpound together with the permeability aiid circulatory characteristics of the tissues affected. That both niechanisms may be responsible for determining the target specificity of carcinogens cannot be excluded a t the present time. In the following, some salient results of investigations on detoxicating metabolism are summarily reviewed. Because the role of ortho hydroxylation for the carcinogciiicity of 2-naphthylamine is still unresolved, this topic is discussed in the section (II1,D) 011 “Activating Metabolism.” It should be recalled in passing, that the carcinogenic benzidine derivative, 3,3’-dihydroxybenzidiiie, is not a riictabolite of benzitline. A\
1. %Acetylanainofluorene, 4-Acetylnnzinobipheny1, and 4-Acetylaininostilbene
A considcrablc amouiit of work has lwei1 carried out in the last 10 years 011 the metabolism of 2-acetylarninofluorene in a variety of species, including man, arid these investigations arc summarized in Table XVII. The 7-position is a major site of rnctabolic ring hydroxylation in all species studied. In the dog, monkey, and iiiaii, the 7-hydroxy metabolite is actually the only urinary ring hydroxy compound detected to date. There appears to be no evidence for hydroxylation in the 9-position in any species. The hydroxy metabolites arc excreted both free aiid conjugated with glucuronic and/or sulfuric acid. Hydroxylation of 2-acetylaminofluorerie in vitro by hepatic microsomes from normal and 20-incthylcholanthrcne-treated mice, rats, ham-
TABLE XVII METABOLIC HYDROXYLATION OF 2-.h2ETYLAMINOFLUORENE IN DIFFERENT SPECIES Species
Rat
Mouse
Hamster Guinea pig Steppe lemming Rabbit Dog Cat Monkey Man Rainbow trout
Positions of ring hydroxylation"
.Y-Hydroxylation
Reference
G, S
E. K. Weisburger and Weisburger (1958); J. H. Weisburger et al. (1958, 1959); J. A. Miller t t nl.
+ +
7, 5, 3, 1 (trace) 7, 5, 3, 1 7, 5, 8, 3 (trace)
G,S Gi S
G, S
+(low)
+ + + +Oow) +
7 7, 5 7 7 7, 5
G G S, G G 0, S G
~
(1960) J. A. Miller et al. (1960) J. H. Weisburger et al. (1964a) J. H. Weisburger et al. (1958); J. A. Miller et al. (1960); Kiese and Wiedemann (1966); \*on Jagow et al. (1966) J. H. Weisburger et al. (1965) Irving (1962, 1963) Poirier et al. (1963); Dyer et al. (1965) J. H. Weisburger et al. (196413) Enomoto et al. (1962); Dyer et al. (1966) J. H. Weisburger et al. (1964~) Lotlikar et al. (1967b)
?
?
7, 5 7, 3, 5
~~
Conjugat.ion6
+
5, 7, 1, 3, 6, 8
~~
(URINARY METABOLITES)
~
~
Bold-faced numbers represent positions of hydroxylation in major ring-hydroxy metabolites. b Glucuro and sulfo conjugation are represented by G and S, respectively. Bold-faced letter indicates the major conjugatiou. a
P
>
8 >
3
sters, mbbits, aiitl guinw pigs has been stutlicd by Lotlikar et al. (19674. There were notable differences in these enzyme activities in normal animals of the species studied. The lowest level of in vitro 7-hydroxylation was found in the rat, the most susceptible among these species, and the highest level in the guinea pig, which is refractory t o the carcinogenic action of the amide. Only rat and mouse liver microsomes showed large increases of ring-hydroxylation activity following trcatment with 20methylcholant,hrene. It is consistent with the preference to the 7-position as a hydroxylation site in 2-acetylaminofluorcne that fluoro substitution in this position notably iitcreases carcinogcnic activity. However, a s alrcady noted in Sections II,E and III,B,l, fluoro substitution does not give complete protection against hytlroxylation. WwtroI) and Topliam (1965) reported preliminary evidence that 7-fluoro-2-acetylaminofluorene undergoes defluorohydroxylation in v i m . Urine from rats and guinea pigs, to which this compound was administcrcd, contained the glucuronide of 7-hydroxy2-acctylaniinofluorcne, although in a much smaller amount than t h a t which was prorluccd from the unsubstitutecl amidc. T h a t similar rationale may account for the potentiation of carcinogenic activity of 4-acetylaminobiphenyl by 4'-fluoro substitution is indicated by the finding of Booth and B o y h i d (1964) that rabbit liver microsomcs ring-hydroxylate this amide in the 3- and 4'-positions besides metabolic conversion to the N-hydroxy form. Similarly, Baldwin et ul. ( 1 9 6 3 ~ )found 4'-hydroxy-4-acetylaminostilbene inactive by oral administration t o rats under conditions in which t,he nonhydrosylated amide is highly carcinogenic. Metabolism studies by Ancicrsen et a l . (1964), Baldwin and Smith (1965), and Baldwin and Romerii (1965) have shown bubsequently t h a t the 4'-hydroxy and (probably to a lesser cxtent) 3-hydroxy derivatives, free and conjugated, are actual urinary metabolites of 4-aminostilbenc (as N-acetyl or N,Ndimethyl), in the rat. Surpi-isingly, in tumor inhibitory studies (which property roughly parallels carcinogenic activity in this series), 4'-fhoro4-acetylaminostilbene had little activity a t the dose used, but the N-hydroxy derivativc-following expectations-was much more active than the parent arnidc (Anderscri et al., 1964).
2. l-Pherz ylnzo-2-naphthol Exhaustive studies 011 thc mc~tnhoIismof l-plic1iylazo-2-n:iphthol have kwen c:ii.rietl out by Daniel (1956, 1962) :i11(1 by Chilcla aiitl Clayson (1966). The IJI'CWntly knowit 1)atliways of this c o ~ i ~ p o uarc ~ ~ dsumm:rriactl ill Tal~lcM ' I I I . I t ~ l ~ o u llw d recalled that 1-~~lic~iyl:~zo-2-~i:~plitho1 is prob:il)ly it wrak c:ircinogcn, but W:LS usc(l c:irlicr for coloring
404
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE XVIII The Known Pathways of Metabolism of l-Phenylazo-2-Naplithola
0-Gluc
Q I
NHCOCIL,
t pH2
\
SL ,hate of p-am iophenc
/
Probably sulfoconjugate of 4', 6'-dihydroxy-1phenylazo- 2-naphthol
Modified, after Child8 and Clayson (l9fX)
margarine. Certain derivatives are still in use as food colors. Kirby and Peacock (1949) induced hepatomas with this compound by injection into random-bred mice. However, Clayson et al. (1965) found it inactive by oral administration t o :uiolhcr litlo of riuidoin-l~iwl niicc ant1 to CBA
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
405
mice. Bonser e t al. (1956b) and Clayson and Bonser (1965) found it active by bladder implantation.
3. Ring Hydroxylation of 4-Dimethylanzinoazobenzene Derivatives The mechanism by which amino azo dyes are diverted toward noncarcinogenic pathways has been investigated from different approaches. The work of Westrop and Topham on the removal of 4’-substituents from 4-dimethylaminoazobenzene (DAB) derivatives has been succinctly mentioned in earlier sections, with particular reference to the fluoro substituent. Aromatic ring-linked fluorine atoms have generally been regarded as metabolically inert despite the carlicr work of Hughes and Saunclers (1954), and Kaufman (1961) 011 the removnl of the fluorine atom from p-fluoroaniline and 4-fluorophenylalanine, respectively. I n a first study, Westrop and Tophain (1966b) identified the nonbound mctabolitcs ether-extractable from the livers of rats, intragastrically dosed with 4’-fluoro-nA~. The compounds separatctl hy thinlayer chroinatography w ( w tlie 4’-fluoro and 4’-hydroxy derivativcs of 4-aminoazobciizcuc.ie~ and of ~ ~ 7 - ~ ~ i o ~ i o r nanrl c t l ~N,N-diinethyl-4-aniinoylazobenzcne; 4’-fluoro4-aii1i1ioazot~~1izciic was also present, N-acctylatcd. I n their second study (using tlie same cxpcrimental procedure) , Westrop and Topham (19GGn) mwbiired the extent of 4’-hydroxylation of DAB and various ring-methyl and 4’-substitutcd derivatives. Taking thr 4’-substituted dyes as a separate group, they have rioted in a total of eight compounds that the amount of 4’-hydroxylated metabolites increases in the same order as the carcinogenic activities. No such correlation was observed with dyes having the 4’-position free. On the grounds of the data obtained with the 4’-suhstitutccl dyes, n’estrop and Topham concluded that the 4’-hydroxylation is thc result of the p-p intramolecular rearrangement of metabolically formed, respcctivc N-hydroxy compounds. T h at is, bince S-hydrosy tlcrivativcs (a5 tlic 0-esters) arc considered as likcly candidates to bc the active fornib of amino azo dyes (E. C. Miller and Miller, 196G), the greater amounts of N-hydroxy derivatives formed from the more carciriogenic tlyes bring about, in turn, highcr lcvcls of the 4’-hydroxy metabolites. Although, in any event, the claimcd corrcltition does not support tlie idea t h a t ring suhstitucnts (fluorine or other) incrcasc carcinogcnicity by blocking hyclroxylation, s:oniu aspects of Westrop and Topham’s hypothesis may be qucstioncd. First, the N-hgtlroxy O-(Jsters, assurncd ultimate carcinogc~nic metabolites of nmino nzo dyes, show high reactivity toward proteins and nuclcic acidh ( Poirier e t nl., 1967). Second, Funakoshi a n r l Ter:iy:tnia ( 1965) conclutlerl, 011 tlic 1-m.is of q ~ c t r n evitlencc, l tli:tt 110 plrenolic liytlixxyl gioul) i b present i n tlic (lye moiety of tlic
406
JOSEPH C. ARCOK AND MARY F. ARGUS
“polar dye” extracted from liver tissue. These two findings appear to lessen the significance of the metabolic 4‘-hydroxylation for the mechanism of carcinogenesis and lead t o two alternatives: ( a ) that carcinogenesis and 4’-hydroxylation follow divergent pathways, both originating at and requiring the prior formation of an N-hydroxy metabolite and/or its 0-ester ; ( b ) that the observed partial correlation between carcinogenic activity and amount of 4’-hydroxy metabolites may be entirely fortuitous. That the latter alternative may be valid is strongly suggested TABLE XIX 4’-HYUROXYLATION AND HEPATOCARCINOCiENlClTY I N THE R.ATOF AMINO Azo DYEP
Compoundh 4‘-Nitro-DAB 4‘-Trifluoromethyl-DAB 4-Ethov-DAB 2-Methyl-D AB 4’-Me t hyl-D AB 3-Methyl-MAB 4’-Chloro-DAB a’-Methyl-DAB 4’-Methoxy-DAB DAB 4’-Ethyl-DAB 4’-Fluoro-DAB 3‘-Meth yl-DAB
Relative activity 0 0 0 0 (trace) 1 1 1-2 2-3 3 6 10 10-12 10-12
4’-Hydroxylated metabolites (mpmoleslgm. liver) 0 0 6.6 24.3 6.6 18.7 5.0 20.4 10.0 47.7 21.6 16.2 18.4
a From Westrop and Topham (1966a). The compounds listed in Tables 2 and 3 of these authors have been combined and arranged in order of increasing carcinogenic activities * DAB designates 4-dimethylamiiioarobenzene; MAB designates 4-monomethylaminoazobenzene. 4’-Ethoxy-DAB is an inact,ive compound (Arcos and Simon, 1962).
by the finding that the relationship holds only for 4’-substituted compounds of which only a small number were studied. Actually, if all of thc dyes in Westrop and Topham’s report are arranged in order of increasing carcinogenic activities (Table XIX) , the correlation vanishes (correlation coeff. = 0.44). Biliary metabolites of DAB, retaining the cliromophore group, were examined by Ishidate e t al. (1963) in the rat. 4’-Hydroxy derivatives of DAB, of 4-nionoiriethyla1iiiiioazobeiiaene, and of 4-an1ii1onzobeiizenc were detectetl I)oth as glucuro t i t i d :is sulfo coiijugutes.
MOIXXXJLAR GEOMETRY AND CARC~IKOGENIC ACTIVITY
407
4. Reductive Clenvnge of the A z o Double Bond Rlecke and Schmiihl (19.57) were thr first, to iiivestigatp the possible existence of it rorrelatioii in azo dyrs hetwcw cwcinogrnic activity and the ability to unclcrgo recluctivc, c1e:ivagcI. A similar study was carried out recently by Matsunioto arid Terayama (1965a). These authors measured the rates of azo double-bond reduction using rat liver homogenates fortified with a NADPH,-generating system, in nitrogen atmosphere. As with the yeast suspensions in Mecke and Schmahl’s experiments, no correlation was found between azo reduction rate and carcinogenic activity with liver homogenates.
5 . Methylation and Demethylation of Amino Azo Dyes The problem of the interconversion of primary, secondary, and tertiary amino azo dyes requires further study. In fact, although Matsumoto and Terayama (1965b) appeared to have confirmed the earlier view (J. A. Miller and Miller, 1953) of the rapid metabolic interconvertibility of 4dimethylaminoazobenzene and 4-monomethylaminoazobenzene, the Miller’s group concluded in the same year that-contrary to their previous hypothesis-the metabolic product of 4-monomethylarninoazobenzene, hitherto regarded as the N,N-dimethyl dye, is actually the 3-methylmercapto derivative (Scribner et al., 1965; E. C. Miller and Miller, 1966). Primary amino azo dyes, such as o-arninoazotolucne and the 4’-fluoro and 3’-methyl derivatives of 4-aminonzobenzene, are not methylatecl (Matsumot0 and Terayama, 1965b). Although unlikely to be of any significance for the mechanism of carcinogenic action, it is of interest to note that l’C-labeled N-methyl carbon of 4-dimethylaminoazobenzene is incorporated into purines in nucleic acids (Berenbom, 1962; Terayama and Yang, 1964). This is probably a normal metabolic route for the one carhon fragment liberated by oxidative N-demethylation. 6. Some Special Metabolic Pathways of o-Aminoaxotoluene
In their above-mentioned study on the interconvertibility of azo dyes having tertiary, secondary, and primary amino groups, Matsumoto and Terayama (1965b) noted that primary amino azo dyes, such as o-aminoazotoluene, 4-aniinoazobenzene1 and the 3’-methyl and 4‘-fluoro derivatives of the latter, gave several unknown metabolic products which seemed to be of a complex nature. Subsequent work (Matsunioto and Terayama, 1 9 6 5 ~ )allowed the identification of a high molecular weight metabolite of o-aminoazotoluene (CII) which corresponds to a product of oxidative dimerization of the dye. The tentative pathways leading
408
TABLE XX Tentative Pathways of Metabolic Reductive Dimerization of o-Aminoazotoluenea
I
JOSEPH C. ARCOS A N D MARY F. ARGUS
I
a From Matsumoto and Terayama (1965~).
from o-:iiriirioazotolueii~ to 4,4’-bis (0-tolylazo) -2,2’-dimethylazobenzene ( C I I I ) are given in Table XX. The identity of the metabolically formed ( C I I I ) was ascertained by comparing it to a synthetic sample obtained by oxidative dinierization of the parent dye ( C I I ) with M n 0 2 . There is no experiment:il evidence as yet for the formation of the postulated Nhyclroxy and nitroso interrnediatcs ; however, inference from results with other dyes strongly supports this possibility. The metabolic fate of the ring methyl groups of o-aminoazotolucnc was investigated by S:iniejima et nl. (19437). Interestingly, the 2J-niethyl group alone undergoes oxidation, first to hydroxymethyl and, subsequclntly, to cnrl)oxyl. Tlierr is no evitlencc for the oxidation of the 3-methyl group. 4’-Hydi~oxylation is ;ilsu a major metabolic route for o-amino:tzotolucne. Thebe inetabolitcs appcar in the bile as N-glucuroi d e s and 4’-O-~ulfo conjugates. N-Glucuronidation appears to occur predominantly prior to oxidation of the 2’-hydroxymethyl group. This was inferred from the fact that rats, admiiiistercd the 2’-hydroxymethyl dye, excrete it, largely unaltered :IS the N-glucuronide, and only a notably smaller amount of 2’-carbosy-N-glucuronide is formed. 7. Possible Significance of Amino A m Dye Amine Oxides
The possibility that the amine-oxide form of 4-diinethylaminoazobenzene may be mi intermediate in the oxidative demethylation of the dye has been prol’osed by Terayama. Using, a t first, the amine oxide of the azoxy form of the dye, Terayama and Hanaki (1959) observed a great loss of carcinogenicity and tissue binding. On the other hand, in subsequent work, the ainine oxide of the azo form proved to be significantly more potent than the parent amine dyc in inducing hepatic tumors by oral administration (Terayama antl Orii, 1963) as well as in binding to tissue constituents (Terayama, 1963a,h; Terayama and Orii, 1963). 4-Dimethylamino:izot,enzene-N-oxide (D.4B-N-oxide) is notahly reactivc and decomposes rapidly in the presence of ii,oii-porphyrin coiiipountls to yield mainly DAB antl 4-iiionomethyla1iiiii~~:tzob~rizeiie (MAB) and 3OH-DAB, and in lesser amounts, 3-OH-R9AB, 4-aminoazobenzene, antl its 3-hydroxy derivative (Terayama, 1963n,b). Tcrayaiiia i1967) proposed st scheme (Table X X I ) to account for the products of decomposition. There is evidence for the metaholic formation of niiiine oxides (Baker :mI Chaykin, 1960, 1962; Zieglcr and Pettit, 1964; Tielileke and Stahn, 1966) , and the cnt:tljtic~( iiuu-KAl)PH,-i tquiring) cleiiirthylation of dimethylaniline-h--oxitle hy liver inicrosonics has been shon-n (Pettit and i ~ Zieglei., 1963). The iiiiiine ositle of DAB ib clealkylate(l ( i l l tlic ~ i I ) w uf NADPH?) by isolated rat liver rnicrosomcs niorc rapidly t1i:in tlinictliyl-
~
e
TABLE XXI
0
Hypothetical Sequence for the Decomposition of DAB-mine-N-oxide by Iron Porphyrin Compounds'*b Q/ - \ N = N o f / ;
DAB-N-oxide
\ 0
- .....Fe-Porphyrin
"activation"
I
deoxygenation
? ,OH
a activated oxygen
From Terayama (1967) DAB = 4-dimethylaminoazobenzene; MAB = 4-monomethylaminoazobenzene; AB = 4-aminoazobenzene.
OH
RIOLECULAH (IEOMETRY ANI) CAHCINO(iEN1C ACTIVITY
41 1
: ~ i i i l i i i ~ - ~ - o ~:Lid i i l ( ,t,licw , i h rii1)id I)iiiding to protc4ns rliiring (lw~lkyl:ttion (Uehleko id Slalin, 1966). ,4 comprrlic~nsivetreatisc 011 thc clicmistry, physical chemistry, and biological properties of aromatic arnine N-oxides has been written by Ochiai (1967). The N-oxide of 4-dimethylaminoazobenzene could also represent the intermediate of a pathway leading to an unusual type of active metabolite. Furst (1963) hypothesized that an arigular arrangement of DAB is necessary to fit and complex with hnsc-pairs in DNA. Actually, the possibility of such angular molecular arrangenicnt of the dye-corresponding ( c ) cinnoline (C1V)-has been aftcr ring closure to 2-di1iietliyl:i1~iiiiol~e1izo
(CIV)
considered earlier by the Millers and their associates. In view of testing the cyclized compound for carcinogenic activity, DAB was treated in an AlC1,-NaCI-KCl-NaF eutectic using a procedure from an I. G. Farbenindustrie patent for the synthesis of benzo (c) cinnoline and its amino derivatives (Arcos et al., 1956). The resulting compound-what was then believed to be 2-dimethylaminobenzo (c) cinnoline on the strength of its elementary analysis, the preparatory procedure, and certain physicochemical considerations-was tested by J. A. Miller e t al. (1957) for carcinogenic activity in rats and found to be inactive. However, subsequent work by M. Arcos (1958) on the spectra of benzo(c) cinnoline derivatives led to a questioning of the structure of the assumed cyclized derivative. She has demonstrated that the AlCl, dehydrogenation procedure yields benzo (c) cinnoline derivatives only with RZO compounds having no electron-donor group in para to the azo linkage. If such an electron-donor group is present, as in DAB, the reaction takes a different orientation to yield biphenyl derivatives. Thus, the presumed cyclized derivative of the dye was shown to be actually 4,4'-bis (dimethylaminophenyl) -bisazobiphenyl (CV) . (CHAN-@=N-N=
N-@(CH), (CV)
Recently, Lewis and Reiss (1967) synthesized 2-dimethylaminobenzo(c) cillnoline (CIV) by photochemical cyclization of DAB-N-oxide; the structure of the compound was ascertained by its identity with the con-
412
.JOSEPH C. 4RCOS AND XIART F. ARGTJS
(Lensation product of 2-clilorol~enzo( c )cinnoline and dimethylamine. These authors have also confirmcd the identity of 4,4’-bis (dimethylaminophenyl) -bisazobiphenyl (CV) , This, then, reopens the problem of the carcinogenic activity of 2-dimethylaminobenzo (c) cinnoline and suggests the interesting possibility that an in vivo-formed, azo dye N-oxide could undergo metabolic cyclization to a heterocyclic aromatic amine.
D. ACTIVATING METABOLISM : PRESENT STATUSO F THE Ortho-HYDROXYLATION HYPOTHESIS ; THE CARCINOGENICITY OF N-ARYLHYDROXYLAMINES 1. Metabolites of W-Naphthylamine and Their Carcinogenic Activity
A considersblc amount of ingenious work has bcen carried out to unravel the fascinating jigsaw puzzle which is the carcinogenically significant pathways of naphthylamine metabolism. The validity of the ortho-hydroxylation hypothesis, i.e., that aromatic amines are carcinogenic by virtue of their metabolic oxidation to o-aminophenols (Clayson, 1953), appears now to be limited to 2-naphthylamine. The demonstration since 1953 that synthetic ortho-hydroxylation of 4-aminobiphenyl, benzidine (to 3-monohydroxy), 2-aminofluorene1 and 4-dimethylaminoazobenzene brings about a total or almost total loss of activity, rules out that this metabolic route is a participant in their mechanism of carcinogenic action. Even regarding 2-naphthylamine-especially since the discovery of the metabolic N-hydroxylation of aromatic amines and subsequent studies along those lines-the role of ortho-hydroxylation in carcinogencsis has bcen seriously questioned. Certain aspccts of this problem have already becn touched upon in Section 1111A,3.Metabolic pathways leading to probable proximate carcinogens of 2-naphthylamine are given in Table XXII. Despite the uncertain results of carcinogenicity testing with 2-naphthylhydroxylamine (CVI) in newborn mice by subcutaneous administration (Roe et al., 1963; Walters et al., 1967), repeated intraperitoneal injection into random-bred rats produced a much higher abdominal sarcoma incidence than the parent amine (9/15 versus 2/14) (Boyland et al., 1963a). I n bladder implantation, both 2-amino-l-naphthol and 2-naphthylhydroxylamine showed a highly significant tumor incidence relative to the controls, the latter compound giving a somewhat higher incidence (Bonser et al., 1963). The high level of carcinogenic activity of 2-naphthylhydroxylamine toward the mouse bladder epithelium was confirmed by Bryan et al. ( 1 9 6 4 ~ )Regarding . the above report of Bonser et al., it must be pointed out that 2-naphthylhydroxylamine certainly did not induce “a higher incidence of bladder tumours than any other compound tested” as stated by Boyland et al. (1963a) about that investiga-
41 3
I\IOLECUL.ZR GEOMETRY A N D CARCINOCXKIC A C T I V I T Y TABLE XXII Metabolic Pathways Leading to Probable Proximate Carcinogens of 2-Naphthylamine
_
_
_
_
'
~
Compounds in parentheses represent hypothetical metabolites analogous to those found with other aromatic amines and azo dyes. Solid lines represent established routes of metabolism, and the broken lines are hypothetical pathwaye.
tion. This is a n important point to strehs, since in that study of Uonser et aZ., by far thc highcst tumor incidence among 2-naphthylaminc mctabolites was actually observed with bis(2-amino-l-naphthy1)sodium phosphate, which compound has probably a special significance for 2-naphthylamiiie carcinogenesis. The urinary presence of 2-naphthylhydroxylami~~e has been actually clctected in the dog (Boyland et al., 1960, 1964a; Troll and Nelson, 1961) and in man and the rabbit (Troll et al., 1965; Troll and Belni:tii, 1967). However, 2-n:iphthylamine is also N-hydroxylated in the cat in vivn aiitl mny he detected 111 the 1)lood (Uchlckc, 1963) ; the definite absence of the AT-hyclrosy t1eriv:itivc i l l t h c ui*inc of t,liis species clocs not appcitr to havc heen reported. X mctaholic. pntliwny csi>ts whicli osiclizes 2-1iaplithylli~d~oxyl~mine to 2-nit1~0~011:1~~Iitl1:~le1~~ :111(1, c.onwrwIy, nnother whicli i*educcs the hy-
414
JOSEPH C. AHCOS AND MARY F. ARGUS
clroxylamine to thc parent amine. Boyland c t al. (1964a) detected the preseiicc of 2-nitrosonaphthalene (CVII) in the urine of dogs dosed with 2-naphthylamine ; 2-nitrosonaphthalene does not appear to have been tested for carcinogenicity. Lotlikar et al. (1965) reported that total rat liver homogenates (from weanlings) reduce N-hydroxy-2-acetylaminonaphthalene to the respective acetamide. Recent results of Uehleke (1966a,b, 1967) indicate that N-hydroxylation is not unique to liver tissue but is actively carried out by the bladder mucosa of various animal species. I n view of this observation it would seem surprising that 2-naphthylamine is a t most slightly active in bladder implantation, although the enzymatic modality is present to convert it in situ to 2-naphthylhydroxylamine. Other aspects of the relationship between N-liydroxylation and carcinogenicity of the two naphthylamines complicate this picture further. As late as 1963 it was still assumed that 1-naphthylamine-which has, in comparison with the 2-isomer, very low activity by oral administration in dogs-is not metabolized to l-naphthylhydroxylamine (Clayson and Ashton, 1963). However, the following year l-naphthylhydroxylamine was detected as a metabolite in occasional animals among dogs dosed with l-naphthylamine (Boyland et al., 196411). l-Naphthylhydroxylamine is almost as active in bladder implantation in the mouse as 2-naphthylhydroxylamine (Boyland e t al., 1962a). Surprisingly, by intraperitoneal administration to rats, the former compound appears to be considerably more potent than the latter, to produce abdominal fibrosarcomas (Belman e t al., 1966; Troll and Belman, 1967) ; in fact, although these authors used exactly the same dosing schedule and route of administration as Boyland et al. (1963a), they found after 10 months a tumor incidence of only 1/15 with 2-naphthylhydroxylamine against 11/14 with l-naphthylhydroxylamine. The considerable discrepancy in the findings of the Boyland and Belman-Troll teams on the activity level of 2-naphthylhydroxylamine may be due to the difference in strains (random-bred albino by the former and Wistar by the latter) or the nature of the oil vehicles used. Nevertheless, this discrepancy could raise some doubts about the significance of topical carcinogenicity results as a basis for considering a metabolite a true proximate carcinogen. Paralleling the p a r e n t e d testing results of Belman e t al., Perez and Radomski (1965) found 1naphthylhydroxylamine to be more mutagenic than the 2-isomer. I n connection with the doubtful carcinogenicity of intraperitoneally administered 2-naphthylhydroxylamine, i t should also be noted that Boyland et al. (1964b) failed t o obtain tumors in guinea pigs which received 24 closes (20 nig./kg. body weight) of 2-naphthylhydrosyl~ininc ant1 survived for as long as 26 riiontlis ; howcvcr, c11:uiges descril)cd as
MOLECI’1,AR GEOMETRY A N D CAHCINO(>ENIC ACTlVlTY
41 5
“pnthologir:tI” WCI‘C ol)scrved i n the 1ii.cl.s a n t 1 lii(1iicys. The inactivity of thih compound w:i\ ii\criI)cd to its lwing r:zpicIIy rrducerl to 2-iiaphthylaiiiiiio 111 this +1)0rit’s. (hi(, rhoiilrl a1.o i ~ c a l lI i ( w that the N-hydroxy dcrivtitive of 2 - : ~ ~ c ~ t v l : ~ 1 ~ i i n r ~ fi,l 1raiwiiogtwtti ~ 1 ~ t ~ 1 i ~ ~ i n t l i i h s])ccics (which is refractory to tlic 1):irc‘iit :miinc~) citlrcr by oral or intrapcritoneal administration (E. C. hliller et al., 1964b), and this is regarded as an evidence supporting the view t h a t the N-hydroxy derivative is a proximate carcinogen of the pnrent amine. Regarding 2-amino-l-naphthol (CVIII)-the long assumetl proximate carcinogen of 2-naplitliylaii~ine-its prescncc in the urine of different species, susceptible and nonsusccptiblc, lias been known for some time. The claim t h a t the susceptibility of different species to bladder tumor induction by 2-naphthylamine depends on the proportion of the dose metabolized to 2-amino-l-naphthol slid excreted in the urine (Bonser et al., 1951) has been criticizcd by Arcos and Arcos (1962). However, Conzelman et al. (1963) have shown subsequently that, maintained on the same dosing schedule, dogs are murh inore susceptible to bladder carcinogenesis than monkeys; dogs excrete about 70% of the ingested amine as ortho-hydroxy derivative, whereas monkeys excrete only about 19% in t h a t form. Although earlier authors found 2-amino-l-naphthol active toward the mouse bladder, in recent testing experiments its carcinogenicity could not be consistently demonstrated (Bonser et nl., 1963; coinpare Bryan et al., 1 9 6 4 ~ ) .Moreover, in subcutaneous administration the activity of 2amino-l-naphthol is definitely low (Bonser et a l , 1952) so as to be hardly compatible with the expected activity level of a proximate carcinogen. In line with the demise of this compound from its status of proximate carcinogen, Dewhurst ( 1963) found that young rodents, which arc notoriously more suscepti1)le to various carcinogenic stimuli than adults, convert a smaller perccntagc of a dose of 2-naphthylainine to 2-amino-l-naphthol conjugates th:m do adults. Troll et 01. (1959, 196311) isolated from urine of both dog and man bis(2-amino-l-naphthyl) phosphate (CIX) and a second unidentified phosphate ester as nietaholites of 2-1ia~~htliylai~iinc. Their findings were confirmed fly Boyland et al. (1961) working with dogs only, and thc latter authors also provided a definitive proof of structure for the mctabolite (CIX) by comparing it with n synthetic sample. In R recent investigation (R:idomski et al., 1967) the presence of a siniilar diester could not be detected in the urine of (logs dosed with l-naphthylamine. Bis (2-amino-l-iiaphtliyl) phosphate has the distinction in having been regarded, up to recently, as the ultimnte carcinogenic metabolite of 2-naphthylamine (c.g., Clayson, 1964). There is justification for this
416
JOSEPH C . ARCOS AND MARY F. ARGUS
view (cf. Ratloiiibki ct nl., 1967) since, a t least in bladder implantation, metabolite (CIX) is btrongly carcinogenic, and actually appreciably more bo than 2-naplitliyIIiydroxyl~miii~ ( E3onsc.r ct nl., 1963) . Recently, l'roll nnd Iklinn~i( 1 (367) took u p the bO-ucturc dclrtniination of the :Ibovc-mciitioiictl unitlentifietl pliohpliate ester metabolite. This metabolite has been shown to be bis (2-hydroxylamino-1-naphthyl) phosphate (CX). The two N-hydroxy hydrogen atoms are probably hydrogen bonded to a phosphate oxygen; this explains the ether-solubility of the metabolite, which property may be essential for cell penetration. This compound, if its structure is confirmed in other laboratories and its carcinogenicity demonstrated by different methods of testing, may well prove to be the true ultimate carcinogenic metabolite of 2-naphthylamine. It combines, interestingly, ortho- and N-hydroxylation in one and the same structure, both features regarded a t diffcrent times a s essential for the carcinogenic activity of the parent aminc. 2. N-Hydroxylation of 4-Nitroquinoline-N-Oxide Investigations showing the carcinogenic activity of 4-hydroxylaminoquinoline-N-oxide, the tissue targets thereof, and the inactivity of 4aminoquinoline-N-oxide have been reviewed in Section III,A,6. Both microorganisms and mammalian tissues possess pathways t o reduce the parent 4-nitroquinoline-N-oxide to the hydroxylamino and amino derivatives. 4-Nitroquinoline-AT-oxide is rcduced by microorganisms in the following manner (Oliabayashi, 1962; Okabayashi and Yoshimoto, 1962) :
+
b
0
J
d-d
NH*OH
4
0
The mutagenicity of 4-nitroquinoline-hT-oxide appears t o depend on the relative rates of these pathways. For example, the compound is not mutagenic in Escherichin coli in which the entire process progresses
ra1)idly to 4-an1inoc~uinoline. On the other h m d , tlie conipoun~l is mutagenic toward Aspergillus nigw in which reduction from -NH . O H to NH, is slow; this results in an :mumulation of the former which is the mutagenic form. An interesting survey of the possible role of -SH groups which react with 4-nitroyuinoline-N-oxide in biological systems in relation to mutagenesis and carcinogeiiesis is given in the L ‘ D i s c ~ s ~ i o of n’’ the paper by Hutner et al. (1967). Invcstigations on mammalian tissue enzymes catalyzing thc convcrsion of 4-nitroquinoline-AT-oxideto the hydroxylaniino and amino f o r m have been initiated independently by two Japanese teams (Hashimoto et al., 1964; Sugimura e t al., 1965). Considerable extension t o these initial studies was given by the Sugimura group (Hoshino et al., 1966; Sugimura et al., 1966a,b). The reduction of 4-nitroquinoline-N-oxide t o 4-hydroxylaminoquinoline-N-oxide and t h e reduction of the latter to 4-aminoquinoline-N-oxide are catalyzed by two different enzymes. The enzyme reducing -NO, t o --NH.OH has been identified as the DT diaphorase in the r a t liver supernatant (Sugimura et al., 1965, 1966a,b). Typically, reduction requires NADH, or NADPH,, and it is inhibited by dicoumarol and stimulated by albumin and Tween 80. Normal liver and slowly growing hepatomas contain relatively large amounts of this enzyme, while its level is close to nil in fast growing hepatomas. The enzyme that produces 4-aminoqui~ioline-N-oxideis almost equally distributed in the mitochondria1 and microsonial fractions; NADH,- or NADPH,-generating system can serve as hydrogen donor. Although p chloromercuribenzoate inhibits tlie overall reaction, reduction does not seem to depend on the functioning of the main electron transport chain since 2,2’-bipyridyl, amytal, antimycin A, and cyanide do not inhibit thc reduction (Sugimura et al., 1965). The in vivo conversion in rats of subcutaneously injected 4-nitroquinoline-N-oxide to the --NH.OH and -NH, forms and t o 4-hydroxyquinoline-N-oxide has been shown by Hoshino et al. (1966) and Sugimura c f al. (1966b).
3. N-Hydroay Derivatives of 2-Acetylaminofiuorene, 4-Acetylaminobiphenyl, and 2-Acetylaminophenanthrene Investigations on arylhydroxylamincs aiitl their carcinogenicity began with the important diycovcry of N-ltgclt,osyl:itiori, n new nictnl)olic reus substrate action o l ) ~ . r v c t al t firht i n llir rat witlt 2-~irctylnniiiiofluoi,~,ii~ (Cr;imer e t al., 1960h). -1conqweliensiw r e ~ ~ i rof w tlie hiologjcal oxidation ant1 iecluction of nromatic amiiio and nitro derivatives was contributed by Uehleke (1965) . Varioub natural iV-hydroxy derivative+-
418
JOSEPH C. ARCOS A N D MARY F. ARGUS
hydroxamic acids-play a rolc in iron metabolism and possibly in othcr metabolic processes in microorganisms (revicwed by Neilands, 1967) ; on the grounds of the structural similarity of some of thcse compounds with purine N-oxides, quinoline-type carcinogens, and N-hydroxy urethan, they probably represent a new field of investigation for unsuspected carcinogens which may be present in the normal environment. I n the first extensive report of their finding, J. A. Miller et al. (1960) showed that the product of N-hydroxylation of 2-acetylaminofluorenc, N - (2-fluorenyl) acetohydroxamic acid, is a major metabolite of the amide in the rat. It is excreted in the urine as a conjugate in amounts which increase considerably with the time of administration. This was interpreted by them as probably being related to the progressive liver damage caused by this carcinogen. Since that time, a variety of susceptible species was found to excrete the N-hydroxy metabolite, free and conjugated, following administration of 2-acetylaminofluorene (Table XVII, Section III,C,l). The N-hydroxy metabolite is absent or a t a low level in the urine of species which are resistant or refractory. Thus, in their initial study, J. A. Miller et al. (1960) found no N-hydroxy metabolite (following 2-acetylaminofluorene administration) in the urine of guinea pigs, a species which is notoriously refractory to the carcinogenic action of this amide and to arylamine-induced cancer, in general. Similarly, in the steppe lemming (cited in J. H. Weisburger et al., 1964c), monkey, and rainbow trout, in which 2-acetylaminofluorene is inactive or weakly active, the N-hydroxy metabolite (free or conjugated) is absent or low in the urine. Also, man metabolizes, in uiuo, the amide to the N-hydroxy form. There is no record of human malignancy due to accidental exposure to the amide. The amide is also N-hydroxylated in vitro by isolated human liver microsomes (Enomoto and Sato, 1967). In view of their finding that microsomes from human liver with jaundice or with fatty changes concomitant with acromegaly do not N-hydroxylate, the increase of N-hydroxy metabolite excretion in rats may have been due t o adaptive enzyme synthesis rather than liver damage as interpreted earlier by J. A. Miller e t al. (1960). Contrary to the Millers' findings, Kiese and his co-workers reported the excretion of the iV-hydroxy metabolite in the urine of guinea pigs dosed with 2-acctylnminofluo1,cne (Kicsc nnd Wicdcinann, 1966 ; Kiese f t nl., 1966; voii Jagow f t ul., 1966). Coiisistcwt with their in vi7)o fincling, Kiese e l a l . (1966) idso olxwvecl i/t 7 1 i t ~ oi\'-liy~lrorryIation of tllc. frcc aniiiie by guinea pig liver microso~ncs.Yet, 1,otlikar et (11. (1967~)coulcl find no N-hydroxylation with liver iiiicrosoiiies from intact guinea pigs previously treated with 20-methylcholanthrene; in the rat, hamster,
moube, and rwhhit , gix~ntincwltseb i n A‘-hytlroxylalioii were not~tvlfollowing treatinelit with the hydrocarbon. Hence, further investigations will be necessary to clarify this question. N-Hydroxylation considerably enhances the carcinogenic potency of and the variety of tissue targets affected by 2-acetylaminofluorene. N(2-Fluorenyl) acetohydroxamic acid is more active than the parent arnide in producing tumors of the liver, ear duct, and small intestine by ingestion or by multiple intraperitoneal injection into adult rats of both sexes. When administered by intraperitoneal injection, the hydroxarnic acid also produced a variety of sarcomas in the peritoneal cavity. Administered orally, about 60% of the animals also developed benign tumors, and another 30% of them developed malignant tumors of the forestomach. By injection into weaiiling female rats, N- (2-fluorenyl) acetohydroxarnic acid was much more :ictive than 2-acetylaminofluorene in inducing mamniary tumors (E. C. Illiller et al., 1961). I n parallel experiments, the parent arnide was inactive toward the forestoninch and the connective tissue a t the sites of injection. The hydroxamic acid is also a uhiquitous carcinogen in other species. E. C. Miller et a l . (1964b) have studied the comparative carcinogenicity of the N-hydroxy metabolite in mice, hamsters, and guinea pigs by oral and pareiiteral aclministration. Just as in rats, the compound produces tumors a t sites of tissue contact; on oral administration, it induces tumors of the forestomach in mice and hamsters, and tumors of the small intestine in guinea pigs; by injection, the metabolite induces abdominal sarcomas in all three species. The parent nmide is inactive toward these tissues in mice and hamsters and toward all tissue targets in the guinea pig. On the othcr hand, toward the liver, mammary gland, and urinary bladder in the mouse and toward the liver in the hamster, the hydroxamic acid and the parent amitle have about equal carcinogenic activities. The generally higher activity of the metabolite toward local and systemic tissue targets is, however, surprisingly contrasted by the higher tumor-initiatory activity toward the mouse skin of orally administered 2-acetylaminofluorene upon croton oil promotion. Nevertheless, these findings provide strong evidence (with the reservations pointed out above in connection with the work of Kiese and his associates) that N-(2fluorenyl) acetoliytlroxamic :icid is a major proximate carcinogenic metabolite of 2-acetylaminofluorenc, since “the apparent inability of the guinea pig to N-hydroxylate 2-acetylaminofluorene parallels the failure of 2-acetylaminofluor~~ne to produce tumors in this species.” I n the rabbit, however, N-hydroxy-2-acetylamiiiofluorene is not more active than the parent nmide upon oral administration, and both induce tumors in the urinary tract only (Irving et al., 1967b). On the other hand,
420
JOSEPH C. ARCOS AND M A R Y F. ARGUS
the liyciroxamic wid is inuch more carcinogenic in this species thau the parent alnide when injected intraperitoncally (or intramuscularly in the form of its cupric chelate), and a high incidence of peritoneal sarcomas results, confirming its topical carcinogenicity observed in other species by the Millers' group. Peritoneal sarcomas cannot be produced by intraperitoneal injection of the amide. The potent local carcinogenic action of N- (2-fluorenyl) acetohydroxamic acid was also demonstrated in other ways. Goodall and Gasteyer (1966) obtained a 100% incidence of benign and malignant skin tumors in the rat following skin painting with this agent (as a 2% acetone solution) for 37 weeks; the first skin tumor arose a t 21 weeks. Several rats also developed distant primary tumors arising in the ear duct, mammary gland, ant1 lungs. These authors also found that a single subcutaneous
FIG.13. A possible modality of binding of N-hyclroxy-2-acetylaniinofluorrn~~ iricttil chclates to proteins and nucleic acids. (Froin Poiricr e t id., 1965.)
injection of 5 mg. of the N-hydroxy compound sufficed to induce subcutaneous sarcomas in 7 out of 9 rats in 44 weeks. I n the tumor-induction studies reviewed above, multiple injections were given. Toward the bladder epithelium in mice, however, N-hydroxy-2-fluorenylacetamide is somewhat less carcinogenic than free or AT-acetylated 2-naphthylhydroxylamine (Bryan et al., 1 9 6 4 ~ ) . Paralleling the findings with other types of locally acting carcinogens, the topical carcinogenic action of N- (2-fluorenyl) acetohydroxamic acid is roughly proportional to the length of retention a t the site. This was the conclusion of Poirier e t al. (1965) who studied the carcinogenic activities of various metal cliclates of the N-hydroxy compound in relation to their carcinogenic activities. The greater carcinogenic activities of these chelates a t the subcutaneous injection site are generally associated with a longer persistence, so that the increase of carcinogenicity due to chelation with the heavy metals appears a t first sight, solely as a matter of solubility decrease. However, Poirier e t al. also considered (cf. Furst,
MOLICCIJLAR GEORIETRY AND CARCISOGENIC ACTIVITY
421
1963) tliat tht inctal iiiay :wt a h ~1 coorc1in:iting atoni to facilitatc h i i i c l ing to proteins a i d nucleic acids and, thus, interfere with normal cell metabolism (Fig. 13). Hence, the carcinogenic activity is enhanced. Taking advantage of the prolonged retention time of N - (2-fluorenyl) acetohydroxamic acid in the chelated form, Stanton ( 1967) iiiducrtl primary bone and lung tumors in rats by local depositioii of the cupric chelate. The alterations of electron-microscopic ultrastructure of the rat liver following N-hydroxy-2-acetylarninofluorene administration have been studied by Hartniann (1965). His findings are in general agreement with reports on the effects of other carcinogens on hepatic ultrastructure. However, the disorganization of the parallel arrays of the rough enrloplnsrnic reticulum begins somewhat earlier tliaii with tlic other carciriogens so f:ir studied electroiirriicroscopically, and this ih consistent with tlic generttlly high level of carcinogenicity of this agent. N-Hydroxy-7-f~uoro-2-acetylaminofluorer~eis a urinary mctabolitc of 7-fluoro-2-acetylaminofluoi ene in the rat. The N-hydroxy derivative is considerably more active than the parent 7-fluorinated amide. It is probably the most active of all fluorene carcinogens tested to date. Administered a t the 0.01% dietary level for 10 to 15 weeks, i t produces high incidences of malignant tumors of the forestomach, small intestine, liver, and of the mammary gInn(1 ( i n females). It is also notably active toward the ear duct and urinary bladder (E. C. Miller et nl., 19662~). Thc cause of inactivity of 7-hyrlroxy-2-acetylaniinofluorene appears to be that this compouncl docs not uiiclergo inctabolic &\\’-hydroxyld t’ 1011. This niay be infcrred from thc recent finclings of Gutiiiann et al. (1967) that this compound may be converted by synthetic AT-hydroxylation t o the highly carcinogenic N - (7-hydroxy-2-fluorenyl) acetohydroxamic acid. Since 7-fluoro substitution decreabes hydvoxylation in this position (Westrop and Tophnni, 1965), one reason for the high carcinogenicity of 7-fluoro-2-acctylaminofluorene should be that a greater proportion of the total dose is N-hydroxylated than in the case of 2-acetylaminofluorenc. Howevcr, that an additional factor is involved here is readily cliscerned since synthetically obtained iV- (7-fluoro-2-fluorenyl) acetohydroxamic acid is more carcinogenic than either N - (Pfluorenyl) acetohydroxamic acid or iY-(7-hydroxy-2-fluorcnyl) acetoliydi.oxamic acid. Possible reasons for this will be discussed in the following scction in connection with Scrihner’s theoretical investigations. Again, inferring from the investigations of Gutmann et ul. (1967), 2-aminofluorenes, iV-suhstitutcd with bulky groups, have low activity or are inactive because the substituents sterically hinder N-hydroxylation. Under conditions in which 2-benzoylaminofluorene is a very weak car-
422
.JOSEPH C. ARCOS A N D M A R Y F. ARGUS
cinogen, Gutmauu ct ( I I . fouird the synthetically i\7-lylroxylalecl derivative, N - (2-fluorcnyl) benzohydroxamic acid, to be a highly potent agent; the tumor incidences were 8 and loo%, respectively. This is probably the true rationale for the observation that the ease of hydrolysis of various N-acyl-2-aniinofluorenes roughly parallels carcinogenic activity (Section III,A,4), since the rate of hydrolysis also depends on steric factors. In the same investigation, Gutmann e t al. made some highly interesting observations on metabolic peculiarities of the benzohydroxamic acid which contribute to its unexpectedly high carcinogenicity. Thus, whereas TABLE XXIII Metabolism of 2-Hydroxylaminofluorene in the Rat a
I
I
isomerization
dehydroxylation
t
t
1
\OH
acetylation
A
&&c*'"
h
HO&{o*c%
ydroxylation
H
OH "From J. H.Weisburger eta!, (l966a)
2-fluorenylbenzamicle is comparatively resistnnt to metabolic debenzoylation, the benzoliydroxamic acid is rapidly debcnzoylated to yield 2-hydroxy lamiriofluoreiic. Furthcrniorc, unlike N- (2-fluorenyl) acetohydroxainic acid (Lotlikar et nl., 1965), N - (2-fluorcnyl) bcnzohydroxamic mid is not reduced to thc coi,respontling ncylarylamine. Several reports appcarcd on the metabolic fate (Table XXIII) of N - (2-fluorenyl) acetoliydroxitmic acid in the rat (e.g., E. K. Weisburger et al., 1964; Grantham et al., 1965; Lotlikar et al., 1965; .J. H. Weisburger et al., 1966s). Moreover, Irving (1964) p.csented evidence that
2-iiitrosofluoreiie is formed from N-liydroxy-2-:icetyIxiiiiiioflnore1ic by rabbit liver microsoiiies. 2-Nitrosofluorene is also a highly potcnt, locitlly acting carcinogcn and likely a proximatc carcinogenic metabolite of 2acetylaminofluorene (E. C. Aliller e t al., 1964a). The most receiit investigations from the Millers' laboratory suggest that 2-acetylaminofluorene may undergo a sccond metabolic activation step following N-hydroxylation. Work was begun already in 1964 (E. C. Miller et al., 1964a) in a mmli for further proximate carcinogens. This culminated in the finding that the acetyl (CXI), pliosphatc ( C X I I ) , and
sulfate esters of A'- (2-fluorcnyl)acetohydroxamic acid are all more reactive than the nonesterified N-hydroxy compound toward amino acids and iiucleosicles in v i t r o (Lotlikar e t nl., 1966, 1967a; Kriek e t al., 1967; DeBaun e t al., 1967). The phosphate arid sulfate esters are more reactive than the acetoxy derivative, and they may represent the actual in wivo ester form. The in vivo reactive metabolites may also include carboxylic acid esters and 0-glucuronides. The one 0-rstci. tested for carcinogenic activity so far, N-acetoxy-2-acetyl:~minofluor~~1ie ((2x1) , is a stronger subcut:incous carcinogen than thc nonesterified A'-liytlroxy compound in :iccoid:tnce with the higher chemical reactivity in the in vitm systems (Lotlikar et nl., 1967a). i\'-Iiytlroxy nietaholiteo of otlicr corijugatctl arylaniincs have bcert dcrnoiist ra tecl to he proximate carcinogeiis. Thc respective N-hytfroxy metabolites are present in substantial amounts, mostly as glucuronides, in the urine of rats and dogs fed 4-acetylxminobiphenyl (,J. A. Miller e t al., (E. C. 1961 ') :ml in the urine of rats fed 2-~crtyl:~mi1iopheii~iiitlirr1ie Millcr et nl.. 1966a). Fefcr et al. (1967) have detectrtl the presence of 4-nitrosobiphenyl in the ivine of dogs rlohed with 4-ainiiiobiphenyl. The metabolites, A T - (4-xenyl) acetohydroxamic acid and N - (2-phenanthryl) :icctoliydi~oxnmicacid arc iiiorc potent carcinogcns t h i n the parent amitlcs. I3;v httbeiit:Ltio(>il* or ititi.:il)et*itotic:~ltxoiltr, tllcy pro(lrtc~loc:il si1rcoIii:lP : i t i t 1 :i Iiigli i i i ( * i i l t ~ i i c tof ~ iii:itiiiii:iry tuii~or..:1.1 .I. llillct. P / ( i l . , 19til ; 14:. C. 3Iillt.r tit ( { I . , I !%(j:t) . ?'lit. coiivc.i.tit)ility of iV-l~yd~~uxy-2-at~ctyla~~~i~to~,lit~n;~ritlii~eiie t o tlir 1):iwnt :iiriicle by r a t liver lioiiiogenatcs has Iwen s1ion.n (1,otlikar et nl., 1965).
424
JOSEPH C. ARCOS AND MARY B. ARGUS
4. N-Hydroxy Derivatives of Amino Azo Dycs and 4-Acetylaminostilbene Amino azo dyes and aminostilbenes follow the general pattern of other arylamines in that they are N-hydroxylated in mammalian organisms. Already in 1964, preliminary results were available illustrating that derivatives of 4-aminoazobenzene are N-hydroxylated in the r a t in vivo (J. A. Miller et u,L,1964; for the full report of this investigation, see Sato et al., 1966). Rats which parenterally received 4-aminoazobenzene or its N-acetyl, N-methyl, or N,N-dimethyl derivatives excrete in tlic urine appreciable quantities of N-hydroxy-N-acetyl-4-aminoazobenzenc, mainly as glucuronide, besides 4?- and 3-hydroxy derivatives of 4-acetylaminoazobenzene (also in conjugated form). Extension of these experiments to mice and hamsters indicates that these species follow the same metabolic pattern. Injection of 3’-methyl-4-monomethylaminoazobenzene into rats resulted in the excretion of two metabolites which were tentatively identified a s N-hydroxy- and 3-hydroxy-3’-methyl-4-acetylaminoazobenzene. Surprisingly, unlike the N-hydroxy and N-acetoxy derivatives of 2-acetylaminofluorene, N-hydroxy- and N-acetoxy-4-acetylaminoazobenzene (CXIII) and 4-hydroxylaminoazobenzene are inactive in rats either in long-term feeding, by repeated intraperitoneal injections, or by repeated subcutaneous injections as the cupric chelate of the acetohydroxamic acid form (Sato et al., 1966).
Q-N=N+”s
\
0-C-C,H,
II 0
highly active (CXIV)
The unexpectc(1 inactivity of these derivatives was the first indication that N-hydrouylation and even conversion to an N-acyloxy ester may be a necessary, but not sufficient condition for carcinogenicity and that the structure of and conjugation in thc aromatic moiety largely determines
MOLECIJ1,AR GEOMETRY AND CAIK!Ih’OCENIC ACTIVITY
425
carcinogenic acti\.ity. A’-Hydroxylation is not restricted to carcinogenic aromatic amiites (Uchleke, 1965), and, therefore, i t cannot be regarded as a metabolic stel) leacling inc\it:ihly t o carcinogenic metabolites. For cxaniplr, plirnylliycl~o~yI:i~~iinc~, l)li(~iiyl(~f Iiylliycli,osyl:iiiiitie ( 14. C. 1LIillcr et al., 1966a), aiicl N-plietiylbenzohytlroxwtiiio :kcid (Gutiiiann e t d., 1967) were totally inactive under conditions in wlticli N-hydroxy-2-acetylaminofluorene was highly carcinogenic. Yamamoto et al. (1967) tested the simplest N-hydroxyamine, hydroxylamine and, also, hydroxyurea and methoxyamine in long-term oral administration. None of these compounds appeared to have any carcinogenic effect in C3H/HeN strain mice, and rather had a lowering effect on the spontaneous tumor incidence of the strain. Hydroxylamine is a mutagen which is considered to be highly specific in acting on cytosine (Kihlman, 1966) ; methoxyamine (O-methylhydroxylamine) is reputed to be a more potent mutagen than hydroxylamine (Turbin et al., 1964). A subsequent study by Poirier et al. (1967) showed that the inactivity of the above N-hydroxy and N-acyloxy derivatives of 4-aminoazobenzene must be ascribed to the lack of an N-alkyl group. As attempts to synthesize N-hydroxy-N-niethyl-4-aminoazobenzene were unsuccessful, the 0benzoyl derivative, N-benzoyloxy-N-methyl-4-aminoazobenzene (CXIV) , was prepared. Paralleling the findings with N-acetoxy-2-acetylaminofluorene, the benzoyl ester of the N-hydroxy dye (CXIV) was sarcomatogenic locally in rats. One hundred percent tumor incidence was obtained in 9 t o 12 months by intramuscular injection of twenty-four 3.9-mg. doses. Parallel control experiments with 4-monomethylamino- and 4-dimethylaminoazobenzene, 4-benzoyl-4-monomethylaminoazobenzene,N-hydroxy-4-aniinonzobenzene, 4-dimethylaminoazobenzene-N-oxide,and benzoylperoxide yielded no tumors, while positive control groups injected with N-hydroxy2-acetylaminofluorene reached 50-6570 tumor incidence in 12 months. The carcinogenic activity of N-hydroxy-4-acetylaminostilbene in the rat has been reported by Smith and Baldwin (1962) and Baldwin et al. 41963c,d), and coiifirmctl by Aiitlerseii et aL. (1963, 1964). The only organ in which tumors were observed (following oral administration) by Bnldwin and his associates, is the ear duct gland. In these experiments, the activity of the N-hydroxy derivative appeared not greater than that of 4-acetylaminostilbene, but higher than the activity of 4-diniethylaminobtilbene. On the other hand, in the Millers’ group, Andersen et aL. found the N-hydroxy derivative to be a definitively stronger carcinogen than either 4-amino- or 4-acetylaminostilbene toward the mammary gland, forestomach, subcutaneous tissue, and small intestine in the rat. In agreement with Baldwin’s findings, the stilbene derivatives were about rqually carcinogeiiic toward the etkr tliict glands. ni iickwy et al. (1955)
426
JOSEPH C. ARCOS AND MART F. ARGUS
previously reported the carcinogenicity of 4-nitroetilhene toward the forest,omach of khe rat, and Andersen et al. (1964) suggested in this connection that forcstomnrh tissue may h a w the capacity to rcrliic~the nitro to L: liytlroxyluniiiio grou]). Andersen et al. (1964) liave tilao syntliesized the N-acetoxy and iV-acetoxy-7-fluoro derivatives of 4-acetylaminostilbene.The former but not the latter compound was somewhat more active than the N-hydroxy in inhibiting the growth of the Walker 256 tumor, which may be regarded as an indication of the relative carcinogenicities of these compounds. Smith and Baldwin (1962) were the first to report the detection of N-hydroxy-4-acetylaminostilbene in the urine of rats fed 4-acetylaminoor 4-dimethylaminostillene. The metabolic results (for detailed accounts, see Andersen et ul., 1964; Baldwin and Smith, 1965) show that 4-aminostilbene (free or N-acetylated) follows the general metabolic pattern of fully conjugated arylamines. ortho-Ilydroxylation, N-acetylation, and N-hydroxylation, as well as reduction of the N-hydroxy group occur. Following parenteral administration of N-hydroxy-4-acetylaminostilbene, Andersen et 01. detected an increase in the excretion of 3-hydroxy4-acetylaminostilbene, which they regarded as supporting the thesis that the N-hydroxy compound is an in vivo precursor of the ortho-hydroxy metabolite. On the other hand, Baldwin and Smith observed that the 4'-hydroxy derivative is the only major ring-hydroxy metabolite following oral administration of N-hydroxy-4-acetylaminostilbene. The latter finding appears to lend circumstantial support to the hypothesis of Westrop and Topham (1966a) that the 4'-hydroxy metabolites result from a rearrangement of the N-hydroxy forms. The data available a t present on carcinogenic N-hydroxy arylamines indicate that for conferring carcinogenicity upon a hydroxylamine or a hydroxamic acid by attachment of an aryl moiety alone, this moiety 1967). must have a t least a certain minimum size (cf. Gutmann et d., Hydroxylamine, phenylhydroxylamine, and the N-benzoyl derivative of the latter are not carcinogenic, although the mutagenicity of hydroxylaminc is well known. Carcinogenicity arises with an N-linked 1- or 2nxplithyl moiety, and activity is maintained and even augmented by replacing the naphthyl by a 4-xenyl, 2-fluorenyl, 2-phenanthryl, or 4-stilbenyl group. These are conjugated systems, and, therefore, the resonance in the aromatic skeleton must strongly influence the bond strength of the 0-ester linkage. Hence, the reactivity of the hydroxamic acid group (and the carcinogenic activity of these compounds) depends on the force of conjugation. The more the electrons are withdrawn toward the aromatic skeleton, the greater is the reactivity of the hydroxamic acid ester grouping toward nucleophilic reagents. Evidently, electron withdrawal, i.e., bond activation, is lower with a plienyl than with the higher
:cry1 groups. This is tlic basis for thc inactivity of ~ ~ l i c n y l l i y ~ ~ ~ o x y l : ~ i i i i i ~ ~ and N-phenylbenzohydroxaniic acid. A similar inbtance has been obser~etl with respect to the effect of the aryl moiety on the rate of hydrolybis of N-aryl iiitrogen mustards (reviewed by ROSS,1953). This was excellently demonstrated by Scribner (1967; also Lotlikar e t al., 1967a) who calculated tlic rcsonance activation energicb for certain aryl moieties, labilizing the ester bond. I n parallel with these thcoretic:tl studies, the corresponding AT-arylacetohydroxamic ebters were tested in the Millers' laboratory for sarcoinatogcnic activity in the rat undcr standardized conditions. A good correlation was found hetween tlic calculated aryl resonance activation energies and the sarcoma incidences of the acetoxy esters, which ranged in the following order: 2-fluorenyl > 4-xenyl > 4-stilbenyl > 2-phenanthryl. In this theoretical framework, the very high carcinogenic activity of N - (7-fluoro-2-fluorenyl) acetohydroxamic acid (E. C. Miller et ul., 1966a) is duo to the electronegativity of the fluorine atom, increasing tlirrcby clectron withdrawal toward tlic aromatic nucleus. The samc molccu1:ir mechanism may account for potentiation of carcinogenic activity, ill general, by fluoro substitution a t various points on an amine-linked aromatic skeleton. The unexpected finding that with an azobeneene grouping, the presence of an N-alkyl group is required for carcinogenicity of the hydroxamic acid form should now be considered. It may be speculatively advanced that because the resonance activation provided by azobeneene is too low, a n N-alkyl group is necessary to increase conjugation by the hyperconjugation increment beyond a certain thrcshold value. The influence of steric factors, which may limit the reactions involved in the metabolic activation steps, may account for the absence of carcinogenicity with N-alkyl groups longer than ethyl. The N-substituent and the azobenzene frame both may contribute in linking the molecule during the activation process t o enzyme site(s) (cf. Burkhard et al., 1962). This may account for the fact that, for an increase of chain length as little as passing t o N-ethyl groups (which are sterically less favorable than N-methyl groups), an auxocarcinogenic suhstitucnt is required in the 4'-position for reinforcing noncovalent interactions with the activatioii site (s) , if carcinogenic activity is to be maintained (Section III,B,l) .
E. FREERADICALS IN ARYLAMINE CARCINOGENESIS. INTERACTIONS WHICH APPEARTO B R NONCOVALENT WI'I'TI PROTEINS AND NIlcLIClC ACIDS I , Evidence for and the Possible Role of Soine Arylumine Free Radicals
The ESR spectronietric study of Damerau and Lassninnn (1963) on iodine coriiplexcs of boiiic aniiiio :mo dyes : ~ n dnonJ)asic, largcr molecular
428
JOSISPH C. AllCOS AND MARY F. A R G U S
sizc azo compounds lias becn bricfly mentioned in Scction II,G. Althougll the number of compounds cxamiiied was small and, perhaps, not fortunately selected for such a study, it clearly appears that there is no correlation between carcinogenicity and either spin concentration or bandwidth in their system. Nagata et al. (1966c,e) more recently observed the formation of large amounts of free radicals in solutions of 4-dimethylaminoazobenzene and of 1-amino-2-naphthol and 2-amino-lnaphthol. They proposed that these radical forms may play a role in the interaction of these compounds with DNA. Perhaps the most interesting findings by this approach have been obtained with 4-hydroxylaminoquinoline-N-oxide. At first Nagata e t al. (1966~)showed that 4-hydroxylaminoquinoline-N-oxide gives rise to a large amount of free radicals in the solid state, or in solution in water or certain organic solvents. The ESR signals are strong a t p H 11 to 12, decrease to about a third of that intensity a t p H 7 t o 8, and are absent a t pH 3. The frce radicals are produced by an oxidative process. This was convincingly demonstrated by the facts that ( a ) the ESR signal was absent when 4-hydroxylaminoquinoline-N-oxidewas dissolved in dioxane and rigorously degassed, but as soon as the solution was exposed to air, it appeared instantly, and ( b ) the signal of the free radical was quenched immediately by addition of the reducing agents, bensaldehyde and ascorbic acid, or of catalase. The temperature dependence of radical TABLE XXIV Participation of 4- Hydroxylaminoquinoline-N-Oxide Free Radical in the Oxidation-Reduction Process of 4-Nitroquinoline-N-Oxide Derivatives= N=O
I
0
0
1
0
.N/OH I
b a From Nagata et d.( 1 9 6 6 ~ ) .
1 0
MO1,ECIJLAR GEOMETRY A N D CARCINOGENIC ACTIVI'L'Y
429
foriuation hliows, iirterehtingly, :t +Ii:irl) r i s ~to :i ni:txi~iiuni :It, 30"(:. followed by it grar111:tl ciec.rcase. Howcvcr, tlic aniount of radicals present is still close to thc maxinium up to about 40°C. This free radical formed by oxidation and destroyed by reducing agents is a participant in the oxido-reduction processes of 4-nitro- and 4-hydroxylaminoquinoline-N-oxide, for which Nagata et al. proposed the scheme shown in Table XXIV. Actually, the unpaired electron is delocalized and distributed throughout the whole molecule. That this radical form may play an unusual role in the carcinogenic activity of 4-hydroxylaminoquinoline-N-oxideis suggested by an interesting report by Hozumi e t al. (1967). These authors have shown that glutathione and cysteine are oxidized in the presence of air by 4-hy(lroxylaminoquinoline-N-oxide in vitro a t pH 7 and 37°C. The pH clependence of the oxidation of glutathione by this compound parallel:, the intensity of ESR signals in the experimcnt of Nagata and his associates. There is no detectable chemical combination between the yuinoline compound and the sulfhydryl agents or any chemical alteration of the former. The role of 4-hydroxylaniinoqi~inoline-N-oxideappears to be purely catalytic. The overall reaction is dcacribeci by Hozunii et al. as
H*O
wherc RSH, RSSR, HAQO, and HAQO. are the reduced and oxidized forms of glutathione (or cysteine) and 4-hydroxylaminoquinoline-A'oxide, respectively. It is an interesting possibility that the greater carcinogenic potency of this compound in comparison with the 4-nitroquinoline-N-oxide may be related to the difference in their modes of reaction with tissue sulfhydryls, since the former, unlike the latter carcinogen, is not deactivated by chemical combination with sulfhydryl agents. An investigation with two arylamine carcinogens-which in some respects parallels that of Nagata et al. (1966d) on the presence of free radicals in benzopyrene-treated liver homogenates-was reported by Vithayathil et al. (1965). These workers observed the general appearance of a special type of ESR signal (g = 2.035 signal) in the livers (slices) of rats fed 2-acetylaminofluorene, 4-dimetliylaminoazobenzene, or thioacetamide. The plot of the intensity of this signal (relative to the normal g = 2.005 signal in the same liver samples) against the time of carcinogen administration is shown in Fig. 14. It is of interest to observe that the times of occurrence of the maxima follow the same order as the carcino-
430
JOSEPH C. ARCOS A N D MARY F. ARGUS
20
40 Days on dlel
60
80
FIG.14. Amplitude ratio of g=2.035 over g=2.005 ESR signals in rat liver slices as a funvtion of the time of administration (at 0.06% dietary level) of 2-acetyl4-dimethylaminoazobenzene (O), and thioacetamide ( V 1. aminofluorene (01, (From Vithayathil et al., 1965.)
genicities of these agents toward the liver. Administration of various drugs with no known carcinogenic activity does not bring about the appearance of the g = 2.035 signal. Both the g = 2.005 “normal)’ signal and the g = 2.035 “prccunccr” signal arc absent i n tissue from tumors induced by the azo dye. The generation of the free radicals detccted, suggested to Vithayathil et al. that the primary effect of thcse agents may be on cellular electron transport. In view of the very stnall tissue samples needed, an exploration of the possible diagnostic value of this system was planned. 2. Apparently Noncovalent Interactions of A9,ornatic Amines with D N A
The demonstration by Belman and Troll (1967) that 2-naphthylhydroxylamine brings about a lowering of the T,,, of DNA only a t pH 5, but not a t pH 7, suggests that the N-hydroxy compound reacts via the liydrogen-ion-catalyzed mcchanism described by Heller et nl. (1951), and which is considered to account for the covaleiit interactions of arylIiydroxyla~nines,in general. On the other hand, tlie T,,-lowering reaction of l-amino-2-naphthol (Troll et al., 19G3a) and of 2-amino-l-naphthol (Troll et al., 1963a; Beltnan and Troll, 1967) with DNA (salmon sperm, calf thymus, or Escherichiu coli) does not require specific p H conditions. Testing the T,-lowering effect of aminonaphthol on synthetic polynucleotides suggests that guanine is the base that reacts in DNA. Reducing agents, such as hydrosulfite, inhibit the T,-lowering effect, and this
effect is paralleled hy a strong binding of the amitlonaphthol to DNA. Aniinonaphthol in the presence of hydrosulfite neither binds to nor affects the T , of DNA. Although it is highly probable from the foregoing evidence that 2-amino-l-naphthol is oxidized to ortho-quinoneimine before undergoing interaction with DNA, the evidence advanced by Belman and Troll-that insep:irability during CsCl density gradient centrifugation demonstrates the covalent nature of binding-is not entirely convincing. I n view of the findings of Nagata et nl. (1966c,e), another possibility, which is equally consistent with inhibition by liydrosulfite is t h a t interaction with DNA involves the formation of aminonaphthol free radicals. It seems, howcver, that further work will be necessary on the entire problem of the aminonaphthol-DNA interaction as a consequence of the report of King and Kriek (1965) of their inability to observe the reduction of the T , under the conditions used by Troll e t al. and Belman and Troll. Although the exact nature of the miinonaphthol-DNA interaction remains t o be elucidated, the breakage of some hydrogen bonds in the native helical structure becomes manifest not only in the lowering of the T, (if confirmable), but also in the greater reactivity of the DNA amino groups toward formaldehyde. However, a much more sensitive system for detecting structural changes in D N A is the change in RNApriming ability (Belman et al., 1964). Interaction of 2-amino-l-naphthol with calf thymus D N A resulted in total loss of RNA-priming ability, whereas there is only about 50% loss following reaction with N-hydroxy2-acetylaminofluorene. Structural changes induced in D N A by aminonaphthol are also shown by the decrease of the rate of its hydrolysis by niicrococcal nuclease. The kinetic data indic:ite that aminonaphtholtreated D N A is more tightly bound to nucleabc (Belman and Troll, 1967).
3. I n Vitro Coinplcxing of ~-Nitroqi~ir~olinc-h -Ozide with 1)NA The quantum nicchanical calculations of Karreman (1962) on the alterations of the charge distribution of adenine by 4-nitroquinolineN-oxide have already been discussed in relation to hydrocarbon-DNA interaction, mutagenesis, and carcinogenesis (Section II,F,4) . Nagata et 01. (1966a) were the first to show that, in vitro, 4-nitroquinoline-Noside complexes with DWA by intercalation, and concluded in agreement with Karrenian’s calculations that either adenine or guanine is the principal paitnor of intcmctiotr witliii~tlie Iielis. C:iffeincb coinpetitively inhibits the intertictioil betweell 4-nitroyuiuoliiie-~~~-oxide a i d DNA. A distinct parallel was found i n their aystcm between the carcinogenicity and extent of complcxlng to DNA with $everal 4-1iitroc~uinolin~-~-oxide derivative?. These experimental findings are in good agreement with the earlier theoretical prediction of Nagata e t al. (1963b) that in the 4-nitro-
432
JOSEPH C. ARCOS AND MARY F. ARGUS
quinoline-N-oxidc-DNA complex, the quantity of charge transfer rather than the strength of the charge-transfer bonding is the determinant factor. According to Paul et al. (1967), guanine is the interacting partner in the in vitro interaction of 4-nitroquinoline-N-oxide with DNA. I n their studies, the complex proved to be quite stable, withstanding extensive dialysis without loss of complexed 4-nitroquinoline-N-oxide. There is, however, no evidence so far that covalent binding is involved. This is noteworthy regarding the previously discussed view of Belman and Troll on the nature of binding between 2-amino-1-naphthol and DNA. Both Nagata et al. (1966a) and Paul et al. (1967) have noted the similarity between the 4-nitroquinoline-N-oxide-DNA and actinomycin D-DNA complexes. The thin-layer chromatographic study of this interaction by Malkin and Zahalsky (1966) supports the intercalation mechanism, since neither heat-denatured DNA nor soluble RNA complex. There is slight interaction with synthetic, low-molecular-weight polynucleotides and histone. The great stability of the 4-nitroquinoline-N-oxide-DNA complex (as compared to the weak hydrocarbon-DNA complexes, Section II,F,3) is also indicated in Malkin and Zahalsky’s experiments by the resistance to ionic strength increase and by the lack of competitive replacement of the nitroquinoline compound by proflavine. 4. Noncovalent Interactions of Azo Dyes with Nucleic Acids and Proteins There is some indication for weak noncovalent interactions between 4-dimethylaminoazobenzene (DAB) and RNA (Marmasse, 1964) or DNA (Nagata et al., 1966e) in in vitro systems. Evidence is much more solid, however, for similar interactions between carcinogenic amino azo dyes and proteins. Szafarz and Galy-Fajou (1966) carried out a spectral study of DAB complexed in vitro with various proteins. Complexing with histones resulted in the greatest changes in the 410/452 mp absorbancy ratio in the limited series of proteins examined. Watters and Canter0 (1967) reported a careful and interesting study, using optical rotation and viscometry, on the interaction of bovine serum albumin and eighteen amino azo dyes of graded carcinogenic activities. There is a reasonably good parallelism between the structural features required for carcinogenicity nnd those for inrrease of optical rotation. Thus, optical rotation is little nffectcd by 4-nmino:~xobenzen~and its 3’-methyl Iioniolog, is nollal)ly more so by tlie N-iiiethyl aiitl N,ATdiinethyl derivatives, arid by far the highest optical rotation increase is produced hy 3’-fluoro-DAR. The results tend to suggest that such secondary valence forces (independently from covalent binding) may play a role in azo dye carcinogenesis by changing the helix content and structural rigidity of cellular proteins, thereby influencing their functional
MOLECTJLAR GEOMETRY AND CARCINOGENIC ACTIVITY
433
contributions to cell metabolism. The finding of Whitcutt e t al. (1960) that in the soluble proteins of liver from rats, which received a single oral dose of 3’-methyl-DAR1 there i s a11 immediate qualitative change of ekctrophoretir behavior in a gruul) of m l i i l ~ l vprotcin5 which do not covalently bind the dye, tends to suppor1 the view that structural clia~rgesin proteins may be brought about by direct noncovaleiit interactions. Although :izo dye interaction did not significantly affect the reduced viscosity of ovalbumin in Watters and Cantero’s system (suggesting that there was little or no change in tertiary structure), it is not known whether the changes in protein helicity are accompanied by sulfhydryl4isulfide changes such as observed by Argus e t al. (196610) with ovalbumin in the presence of water-soluble carcinogens. IV. Covalent Binding to Proteins and Nucleic Acids
A. POLYCYCLIC HYDROCARBONS AND TRICYCLOQUINAZOLINE Abell and Heidelberger (1962) reported that protein-bound hydrocarbons in mouse skin are predominantly bound t o a slightly basic fraction of soluble proteins, electrophoretically similar to the h, proteins of rat liver supernatant. I n a series of twelve hydrocarbons of graded carcinogenic activities, a good quantitative correlation was found t o exist between the extent of binding and carcinogenic activity. I n hydrocarbon-induced epithelial carcinomas and subcutaneous sarcomas, a considerable reduction of this h-like protein fraction was observed in analogy with the reduction of h-protein level in liver tumors induced by amino azo dyes and 2-acetylaminofluorene. Daudel et al. (1962) carried out a low-temperature fluorescence spectroscopic study of tissue-bound metabolites of 3,4-benzopyrene following application to mouse skin. The bound metabolites were liberated by a modification of the hydrazinolynis technique used earlier for obtaining bound metabolites of 1,2,5,6-dibenzzanthracene.Comparison of the fluorescence spectra of the metabolites with those of chrysene and 1,2benzanthracene appears to indicate that hincling OCCUI*S through both K-regions:
434
JOSEPI3 C. ARCOS A N D MARY F. ARGUS
Anthanthrene is a typical exception to the K-region hypothesis of hydrocarbon-induced carcinogenesis in that this compound, while it possesses an electronically favorable K-region and no L-region, is not carcinogenic. Daudel et al. (1960) have shown t h a t j u s t as the noncarcinogenic 1,2,3,4-dibenzanthracene-anthanthrene is bound to mouse skin proteins. Actually, the amount of tissue-bound anthanthrene was found by them to be greater than the amounts of bound 9,10-dimethyl-1,2,5,6dibenzanthracene or l0-methyl-7,8-benzacridine, both of which are potent carcinogens. Howell (1958) has shown that administration of cupric oxyacetate gives a good degree of protection against 4-dimethylaminoazobenzeneinduced hepatic tumorigenesis in the rat. Subsequently, Fare (19644 was successful in demonstrating that this protection against tumorigenesis parallels the considerable delaying by the cupric oxyacetate t o attain the maximum amount of bound dye in the liver. A similar study carried out and cupric oxyby Fare (196413) with 9,10-dimethyl-l,2-benzanthracene acetate could not demonstrate an analogous situation in mouse skin tumorigenesis. On the contrary, this fluorimetric study shows that, whereas the addition of 0.15% cupric oxyacetate to the acetone solution of the hydrocarbon does lower the binding to skin proteins, i t also accelerates the rate of appearance of the tumors. Despite the considerable body of evidence that polycyclic hydrocarbons interact with DNA as such and do not require metabolic activation for carcinogenicity, Brookes and Lawley (3964b) reported a remarkable correlation between covalent binding to DNA and the activity of six polycyclic aromatic hydrocarbons (Fig. 15). Covalent binding is not specific t o DNA, and fixation to RNA has also been observed. The DNA-bound hydrocarbon persists for a longer time than the proteinhound hydrocarbon. Goshman and Heidelberger (1966) confirmed these results and provided important additional evidence that the nature of the DNA hydrocarbon combination is, in fact, covalent binding. Among others, Goshman and Heidelberger have ascertained that the binding is not affected by treating the mice and isolating the DNA in the dark. This is a significant point toward determining the metabolic origin of this binding, in view of the observation of Ts’o and Lu (1964) that irradiation of noncovalent DNA-3,4-benzopyrene complex a t the absorption band of the hydrocarbon (above 340 mp) yields a covalently linked photoproduct. Unlike with the hydrocarbons, there is no firm evidence that covalent binding to cell constituents occurs with tricycloquinazoline. An extensive study by Baldwin et nl. (1962b) failed to detect any firm binding to nucleic acid or protein fractions in the mouse skin painted with tricyclo-
MOLECULAR GEORCETRY A N 0 C.AHCINOGICNIC AC'I'IVI'I'Y
435
quinazoline. Suhoequent work has revealed some protein-bouiicl tricycloquinazoline (Baldwin e t d.,1964b) which is, apparently, covalently hound (Baldwin e t aZ., 1965b) ; however, because of the extremely low lcvel of bound material (1 molecule of tricycloquinazoline per 4.3 X lo4 molecules of soluble protein of mol. wt. lo"), the significance of this binding for carcinogenesis is questionable. Attempts to demonstrate in vivo binding to skin nucleic acids have so far given negative results.
Iball's index
FIG. 15. Number of micromoles hydrocarbon bound per mole DNA phosphorus, divided by the dose of hydrocarbon given (in micromoles), a t the maximum cvtrnt of binding. The symbols represent the following hydrocarbons : A naphthalcnr ; A 1,2,3,4-dibenzanthracene ; 0 1,2,5,6-dibenzanthracene; @ 3,4-benzopyrene ; 20-methylcholanthrenc ; 0 Q,lO-dimethyl-1,2-benzanthracene. (From Brookes and Lawley, 196413.)
B. 4-NITROQUINOLINE-N-OXIDE A demonstration that this compound actually becomes covalently bound to cell components in its many tissue targets appears to be lacking despite much in vitro evidence of its high chemical reactivity in both the -NO, and -NH.OH forms (Sections III,A,6 and III,E,l) and the observation by Hayashi (1959) that there is a decrease of the intraepithelial -SH content following a single application of 4-nitroquinolineN-oxide. There is also qualitative in vitro evidence that, in addition to simple sulfhydryl compounds, 4-nitroquinoline-N-oxide interacts with the -SH groups of proteins (Searle and Woodhoube, 1963). Nevertheless, this interaction may well represent a detoxication mechanism rather than a facet of its carcinogenic action, since the rat liver, which is not a tissue target of 4-nitroyuinoline-N-oxide (Table XV) , contains an enzyme that catalyzes its conjugation with sulfhydryl compounds such a~ glutathione (Al-Kassab ct (11.. 1963).
436
JOSEI'II C. AI3COS A N D MARY I?, ARGUS
Just as arylhydroxylamines, in general, condense with sulfhydryl compounds in vitro to give S-aminoaryl derivatives (Boyland et aZ., 1962b, 1963b), 4-hydroxylaminoquinoline-N-oxidecould react with tissue sulfhydryls, and this may well prove to be involved in the mechanism of action. Parallel studies of the sulfhydryl levels in target tissues using the -NOz and the --NH.OH forms would be of importance. It may not be excluded, however, that the catalytic effect of 4-hydroxylaminoquinolineN-oxide free radicals in oxidizing -SH groups (Hozumi et al., 1967) plays a key role, in which case a correlation may not exist between tissue binding and carcinogenic activity of various ring-substituted derivatives.
C. ARYLAMINES AND AMINOAzo DYES 1. 2-Naphthylamine Although the early experiments of Henson et al. (1954) indicated that the bladder epithelium and the red blood cells are the only tissues capable of retaining 2-naphthylamine-14C in the rat and rabbit after iqtraperitoneal injection, Roberts and Warwick (1966b) found that tritiated 2-naphthylamine binds t o liver, kidney, and spleen of the rat. The extent of binding to different cell components in all three tissues ranked in the following order: cytoplasmic proteins > nuclear proteins >> ribosomal RNA. No binding to DNA is indicated by their results. On the other hand, in the urinary bladder of the dog, which is a typical tissue target of 2-naphthylamine, no bound metabolite was detected following feeding this agent (Brill and Radomski, 1965a). Although it may not be excluded that the fluorescence method employed in the latter study is not sensitive enough to detect low levels of metabolites or that metabolites may lose their fluorescence by tissue binding, in appearance this observation agrees with the earlier conclusion of Scott and Boyd (1953) that the carcinogenic action of 2-naphthylamine is related to prolonged physical contact rather than tissue retention. 2. dcetylaminofluorene I n arialogy with their earlier observations on amino azo dyes, Sorof et al. (1960, 1965) found that 2-acetylaminofluorene is localized in the fast h, proteins separated by electrophoresis. The results of Barry and Gutmann (1966) essentially confirm the finding of Sorof et al. despite differences in experimeiital coiiditions. Protein-bound derivatives are not detectable in hepatic tumors induced by this carcinogen, following administration of 2-acetylaminofl~orene-'~C(Sorof et al., 1965), and this is in ugreemeiit with their early finding that in these tumors there is a large decrease of the level of h, protein (Sorof et al., 1958). Although the
MULIK!ULBR GEOMETRY AND CAHCINOGENIC ACTIVITY
43 7
level of total h proteins, as a class, is considerably higher in the “minimal deviation” hepatomas than in 2-acetylaminofluorcne-induced liver tumors, tlie former hepatomas contain little or no h,-fluorenyl proteins following administration of 2-acetylaminofluorerie or its N-hydroxy derivative (Sorof et al., 1966). Following intravenous injection, 2-acetylaminofluorene becomes rapidly bound in an unextractable form to red blood cells (J. H. Weisburger et al., 1966b), but it is tightly as well as loosely bound to plasma proteins and this may represent the modality of its circulatory transport (Bahl and Gutmann, 1964; J. H. Weisburger et al., 1966b). The protein binding to liver proteins of the inactive metabolite, l-hydro~y-2-acetylaminofluorene-~~C, has been shown to occur in vitro (Nagasawa and Osteraas, 1964) and in vivo (Irving and Williard, 1964). Whereas in vitro much more bound radioactivity was observed in this study with the metabolite than with 2-a~etylaminofluorene-’~C,in the in vivo study in most tissues, radioactivity was much higher following administration of the parent amide than following administration of equivalent doses of the metabolite. Even the hydrocarbon corresponding to the aryl moiety of the amide, fluorene, binds to Iiver proteins to a notable extent when administered to rats a t high doses (Grantham, 1963), but with low doses the extent of binding is less than with the amide or the N-hydroxy derivative (Marroquin and Farber, 1965). The essentiality of protein binding for carcinogenesis is illustrated by the observations that chloramphenicol which inhibits liver carcinogenesis by 2-acetylaminofluorene (Puron and Firminger, 1965; Oster and Firminger, 1966), also inhibits the binding of the carcinogen to liver proteins (J. H. Weisburger et al., 1967b). I n view of the often-considered possibility that the antibiotic exerts its effect through the ribosomes, which arc attached to the endoplasmic reticulum membrane, it is of interest that 2-acetylaminofluorene-binding proteins are present in the microsomes (Kitagawa et al., 1966) and the level of these proteins is considerably decreased in the amide-induced hepatoma (Tanigaki et al., 1967). Binding of 2-acetylaminofluorene to RNA was reported by Marroquin and Farber (1962, 1965) and confirmed by Williard and Irving (1964), E. C. Miller et al. (1964a), and Irving et al. (1967a). E. C. Miller et al. (1964a) have a150 shown that the level of binding to RNA of 2-nitrosofluorene, 2-fluorenylhydroxylamine, and N - (2-fluorenyl) acetohydroxamic acid is 2-4 times greater than tlie kvel of binding of tlic parent aiuicle. Binding of 2-acetylaminofl~oreiie-’~Cto rat liver RNA is wveral times higher than to liver RNA of guinea pig$, hamsters, atlid cotton rats (Marroquin and E’iirl~er, 1965) . Nuclcur riboson1:~1RNA and cytoplasmic (soluble) RNA are labeled to :m equal extent and their specific activities
438
JOSEPH C. ARCOS AND MANY I?. ARGUS
are about 3 times higher than that of ribosomal RNA; the pattern of labeling suggests binding to pre-formed RNA rather than incorporation during synthesis (Henshaw and Hiatt, 1963). Investigations by Irving et al. (1967a) have shown that unlike the above carcinogenic fluorene compounds, injection of the noncarcinogenic metabolite l-hydroxy-2-acetylaminofluorene-14C does not result in any binding of radioactivity to rat liver RNA. In a single experiment, 2acetylaminofl~orene-'~Cwas found to bind to liver RNA of the rabbit, and in repeated experiments N - (2-fluorenyl) acetohydroxamic acid-14C was noted to bind to liver RNA of the but not 2-acetylaininofl~oreiie-~~C guinea pig. Neither 2-acetylaminofluorene nor its N-hydroxy derivative are hepatocarcinogenic to these species; however, in all cases the extent of binding was notably low, only about 30% of that found in the rat liver. Experiments with the acetohydroxamic acid labeled with 14Cin positions 9 or 1' indicate that no deacetylation occurs prior to binding to RNA. Although Henshaw and Hiatt (1963) could not find clearly demonstrable labeling of rat liver DNA following intraperitoneal injection of radioactive 2-acetylaminofluorene, significant specific radioactivity in this DNA could be demonstrated by Williard and Irving (1964) following administration of 14C-labeled 2-acetylaminofluorene or its N-hydroxy derivative. Binding of 2-a~etylaminofluorene-~~C to rat liver DNA was fully confirmed by Sporn and Dingman (1966) ; there is no binding of 14C-20-methylcholanthrene to DNA of this organ which is generally not a target of the carcinogenic action of the hydrocarbon. This stands in interesting contrast with the investigations of Brookes and Lawley (196413) (see Section IV,A) showing that 20-methyl~holanthrene-~~C binds appreciably to DNA of the mouse skin which is a highly sensitive tissue target for the hydrocarbon. Binding to RNA and DNA may not be the exlusive mechanism by which 2-acetylaminofluorene metabolites alter cellular information transfer in the target tissues. This is suggested by the finding of Barry e t al. (1967) that after a single intraperitoneal injection of 2-acetylaminofluorene-14C to rats the carcinogen becomes extensively bound to histones in the liver.
3. Amino Azo Dyes Following the well-known classic demonstration in 1947 by the Millers of the binding of amino azo dyes to liver proteins (e.g., reviewed by E. C. Miller and Miller, 1952), the cytoplasmic h-protein components of this combination have been extensively investigated by Sorof and his associates (e.g., Sorof e t al., 1963). Freed and Sorof (1966) have provided evidence that the h, proteins function in normal cells as met,abolic regulators.
MOLECULAR GEOMETRY A N D C A R C I S O G E S I C ACTIVITY
439
Isolated h, protein fraction drongly inhibited the growth of L-strain mouse fibroblasts in suspension tissue culture, and the inhibition of cell multiplication is reversed by rcnioval of the h, proteins. The inhibitory fraction centered a t the slow h, proteins has been recently identified as arginase (Sorof e t al., 1967). Protein-bound dye has also been found in the livers of rats which received the N-oxide of 4-tli1nethylaminoazobellaene (DAB) orally (Terayama and Orii, 1963). Protein-bound amino azo dyes are present in both cell targets in rat liver tissue-the parcnchynial and the bile duct cells (DeLamirande, 1964). The amiiio acid composition of the peptide segment to which the dye is bound was studied following 3’-methyLDAB adininistration and alkaline hydrolysis of the total liver homogenate by Prodi (1963). A simi1:irly oriented and very careful investigation has been carried out by Ketterer et 01. (1967) (following DAB administration) on electrophoretically separated, dye-binding protein preparations submitted to Pronase digestion. Bakay and Sorof (1964) have investigated a small dye-bound, salinephosphate extrartable, soluble, nuclear protein fraction and found that it exhibits electrophoretic properties similar to the cytoplasmic h proteins ; also these nuclear proteins are markedly reduced in dye-induced liver tumors. Dijkstra and Griggs (1967) studied the binding in the rat liver of 3’-methyl- and 2-methyl-DAB to the acid-insoluble nuclear proteins of the chromatin and extrachromatin fractions. The amount of bound 3’-methyl-DAB was significantly higher than the amount of bound inactive 2-methyl isomer, and this differential binding was specific to the chromatin fraction. That dye binding to nuclear proteins may be related to alteration of repression and derepression of gene function is more specifically suggested by the finding of Rees and Varcoe (1967) that, in vivo, histones in the rat liver bind administered, tritiated DAB. The distribution of protein-bound dye in subcellular fractions of thr rat liver, following oral administration of 3’-methyI-DAB, was studied ill inore recent year$ by Y:imnda e t al. (1963). They have reported that a consistently high concentration of protein-bound dye is present in the ribosome fraction (separated by deoxycholate treatment) throughout the whole period of dye administration. On the other hand, J. C. Arcos and Arcos (1958) found previously that practically all the bound form of this dye localized in the deoxycliolate-soluble membrane fraction of the microsomes, and the amount of membrane-bound dye had a sharp maximum a t 2 weeks. This is the same pattern as was noted for whole homogenates (reviewed by E. C. Miller and Miller, 1952). T h a t dye binding in the microsomes is preferentially to the lipoprotein membrane fraction is consistent with the mnrkcd tlcpression of activities of meml,rane-localized,
440
JOSEPH C. ARCOR AND MARY F. ARGUS
microsoinal drug-iiirt:tt~olizirlg riizymcs hy varioiis inicro~oiiic-l,iiitljng amino azo dyes, irrespectii-e of thcir carciuogenic activities (Baldwin and Barker, 1965). Data OII t l i t b inc,ol.l”)i’:itioii iiilo RNA of 1 -co:tr.l)oii f r a y i i i w t h , origitiatiiig from :~niiiio: ~ Z O dyc N-iiietliyl groiil)s, arc 1)id);ibly iiot grmume to the meclianisni of carcinogenic action. Roberts and Warwick (196613) used DAB tritiatcd in the “prime” ring to study the time course of binding to protein, RNA, and D N A in different tissues of rats and guinea pigs. A high level of radioactivity was noted in the livcr RNA from albino rats; the label was also detectablc in guinca pig liver RNA although the level a t the maximum time of binding was a t most one-sixth of t h a t in rat liver RNA. Binding to DNA was comparatively very low in these experiments. Actinoniycin D, which depresses the incorporation of orotic acid into ribosomal RNA does not inhibit the binding of tritiated as with 2-acetylDAB t o RNA (Roberts and Warwick, 1 9 6 6 ~ )Hence, . aniinofluorene-14C (Henshaw and Hiatt, 1963), binding of DAB is to pre-formed RNA. Persistent binding of amino azo dycs to r a t liver DNA was reported simultaneously by Warwick and Roherts (1967) using DAB (tritiated in the “prime” ring) and 1)y Dingmsn and Sporn (1967) using DAB (I4C-labeled in the ‘Lprime”ring or tritiatcd in the amine ring) and the 2- and 3’-methyl derivatives (ring tritiated) . Dingnian and Sporn reported that binding of thc highly active 3’-methyl derivative is 6 times greater than binding with the comparatively inactive 2-methyl isomer, and 9 times greater than binding with the noncarcinogenic, radioactive, 3’-trifluoromethyl derivative. High dietary riboflavin protects against binding. Thus, the bound metabolitc probably possesses a n intact azo linkage. The pattern of DNA labeling with the different radioactive dyes suggests that covalent binding of D N A to both rings may occur. Burkhard et al. (1962) reached an analogous tentative conclusion regarding the binding of DAB derivatives to liver protcins.
4. Oxidation of o-Anainophenols to o-Quinoneimines As a Possible Activation for Binding. Mechanisms of ( ovalent Binding of hT-llyd?.ol?/ Arylamines and Their Esters to Cellular iYucleophiles Nagasawa and Gutmaiin (1959) and Nagasawa et al. (1959) reported that o-aminophenol, 2-amino-1 -fluorenol, and 3-hydroxy-4-aminobiphenyl are oxidized by cytochronie c and cytochrome oxidase to indophenols and isophenoxazones. These oxidative dimerizations pass through highly reactive intermediates, thc corresponding o-quinoneimines (Gutmann and Nagasawa, 1960). Addition of bovine serum albumin and 2-amino-1 -fluoreno1 to the cytochrome c plus cytochrome oxidase system leads to cxtensive protein
hlOLECULAR GEOMETRY A N D CAHCINOCENlC ACTIVITY
44 1
tinding of the transitory oxidation intcrmecliatc, 2-i1iiino-l,2-fluorenoquinone (Nagasawa and Gutmann, 1959). Synthetic 2-iniino-l ,a-fluorenoquinone readily combines with serum albumin nonenzymatically in an in vitro system (Gutmann and Nagasawa, 1960). These findings appeared to provide experimental support for the view (Gutmann e t al., 1956) that quinoneimides and -imines derived from the phenolic intermediates of %aminofluorenc, in particular l-hydroxy-2-aminofluorene, may play a role in the protciii 1)incling and carcinogenicity of 2-acctylaniinofluorenc. 1-Hydroxy-2-acctylaminofluorene appears to be gencrated metabolically from 2-acetylaminofluorene via N - (2-fluorenyl) acctohydroxamic acid (.J. A. Miller et al., 1960), and in in vitro experiments, 1-hydroxy-2-acetylaminofluorene binds to liver proteins much more extensively than 2acetylaminofluorene (Nagasawa and Osteraas, 1964). However, an in vivo study (Irving and Williard, 1964), which is more germane to the actual process of carcinogenesis, indicated more protein binding in most tissues following intraperitoneal administration of 2-a~etylaminofluorene-'~C than following injection of equimolar amounts of the labeled 1-hydroxy metabolite. Furthermore, both o-hydroxy metabolites of 2-acetylaminofluorene are virtually inactive as carcinogcns (Section 111,A,4). For thcse reasons it appears unlikely that o-c~uinoneimincsplay a role in the carcinogenicity of 2-acetylaminofluorene. It is possible, however, that o-quinont~imincintermediates do play a role in carcinogenesis by other arylaminrs. Thc interactions of these interrnediatcs, which are assumed to result in covalent binding to cellular nucleophiles a t it position metn to the amino group (J. A. Miller e t al., 1960), may hc exemplified as follows:
B
@ \
/
S-R
& IOP
R-S:H
\
/
0
I t is still a reasonable assumption a t prescnt that 2-ai~iiiio-l-1i:~plltlio] and/or its bis-phosphate ester is one of the proximate carcinogens of
442
JOSEPH C!. ARCOS A N D M A R Y F. ARGUS
.
2-naphthylamine (Section III,D,l) Morcover, there is evidence that tryptophan metabolites bearing an o-hydroxy group are causative agents in spontaneous bladder cancer (Section III,A,5). The interaction of these with their target tissues may involve the above mechanism following metabolic activation by oxidation to the respective o-quinoneimines. Another reaction in which proximate carcinogens behave as arylating agents is the condensation of arylhydroxylamines with sulfhydryl compounds to give aminoaryl mercapturic acids (Boyland et aZ., 1962b), as exemplified with 2-naphthylhydroxylamine and N-acetylcysteine: OH
NH
a
S--C,H,O,N I
I
HS--C,H&”,
NH
c
Similarly, reaction of phenylhydroxylamine with N-acetylcysteine gives the corresponding o- and p-aminophenyl mercapturic acids. The same mercapturic acids have been detected in the urine of animals treated with the parent amines (Boyland et al., 1963b), and it is likely that these mercapturic acids arise in vivo subsequent to the formation of N-hydroxy metabolites. Boyland et al. (196313) have proposed that N-linkage of the sulfhydryl compound is a possible intermediate in mercapturic acid formation. I n fact, they found that 2-naphthylhydroxylamine, and arylhydroxylamines, in general, react readily with sulfhydryl compounds in neutral solution a t room temperature (cited in Boyland, 1963). On the other hand, the direct formation of a ring-linked nucleophile would require catalysis by hydroge? ions in order to generate, by a Bamberger-type rearrangement, the clectrophilic o-quinolimide ion (Heller et al., 1951). The rearrangement from N-linked to ring-linked acetylcystcine appears to parallel a tnetabolic pattern in which N-hydroxyarylamines are the metabolic precursors of the o-hydroxyarylamines (J. A. Miller et al., 1960). In view of the discovery by the Millers and their associates that the N-acyloxy derivatives of arylamines are more carcinogenic and also more reactive than the N-hydroxy derivatives toward proteins and nucleic acids, it is not clear whether activation of the latter by in vivo esterification is a mandatory step preceding binding to cell structures and carcinogenesis. In fact, arylatioii of cell components by N-hydroxyarylamines could occur in vivo, without previous esterification, in lowpH-gradient regions following the h3’drogen-ion-catalyze~ Bambergertype rearrangement of :irylhydroxylamines described by Heller e t nl. (1951). This is exemplified in Table XXV. The importance of low pH
MOLECULAR G E O M E l R Y AXD CARCIXOGEK I C AC'I'LVI'TY
443
TABLE X X V Hydrogen-Ion-Catalyzed Reaction of 2-Fluorenylhydroxylaine with a Cellular Nucleophile
t
\*
,S'R
for arylation is dramatically illustrated by esl)erinients 011 the in vitro interaction of arylhydroxylamines with DNA. Kriek ( 1965) reported that 2-fluorenylhydroxylamine interacts with DNA and alters its spectrum a t pH 5, but not a t p H 7. Similarly, Belnian and Troll (1967) found that 2- and especially 1-naphthylhydroxylaniine lower the T, and modify the spectrum of DNA following interaction a t pH 5, but not a t neutrality. Considerable effort has been devoted recently to the study of the structure of protein- and nucleic acid-bound forms of acyloxy arylamines. Not only are these esters more carcinogenic and chemically more reactive than the parent N-hydroxy compounds, but what is of particular importance is that, unlike the latter, they rcact with cell components a t neutrality. Lotlikar et ul. (1966, 1967:i), E. C. Miller e t nl. (1966b), and Poirier e t ul. (1967) have shown tli:\t N-acetoxy-2-acc~tyl:i1;iinofluorenc and N-benzoyloxy-4-monomethyl:~1tii11o:tzo~~e1iz~1ic react readily in vitro a t pH 7 with proteins, RNA, and DNA to form niaci~omolecular bound dye. Under similar experimental conditions five nuclcophilic components of proteins and nucleic acids (tyrosine, tryptophan, cysteine, methionine, and guanosine) react with the above two N-acyloxy compounds to form polar, bound carcinogens. No reaction occurs a t pH 7 with sixteen other
444
JOSEPH C. ARCOS AND MARY 11’. ARG‘CTS
ainino acids, nor with thyinicline, cyticlitie, ancl uricliiic. With N-:hcctoay2-acetylaminofluorene (Lotlikar et al., 1967a), but not with N-benzoyloxy-4-monomethylaininoazobenzetie (Poirier e t al., 1967), adenosine gave about 4% as much reaction as guanosine. In accordance with the lower Carcinogenic potency of N-acetoxy derivatives of 4-acetylaminobiphenyl, 4-acetylaminostilbene, and 2-acetylaminophenanthrene, these derivatives are less reactive than the more carcinogenic N-acetoxy-2acetylaminofluorene toward methionine and guanosine in vitro (Lotlikar e t al., 1967a). The reaction of N-acetoxy-2-acetylaminofluorene with guanine in DNA and RNA in vitro a t pH 7 causes a marked increase in absorption from 280 to 320 mp (E. C. Miller e t al., 1966b). These authors confirmed the observation of Kriek (1965) that N-hydroxy-2-acetyla1ninofluorene does not react a t neutrality. Unlike the carcinogenic alkylating agents, which are known to alkylate the 7-nitrogen atom of guanine in nucleic acids in vivo or in vitro (e.g., reviewed by E. C. Miller and Miller, 1966; and by J. A. Miller and Miller, 1966), N-acetoxy-2-acetylaminofl~1orene arylates guanine (as guanosine) in vitro in the 8-position. The site of arylation in guanine (in RNA and DNA), in vitro and in vivo, is invariably the 8-position (De Baun e t al., 1967; Troll and Rinde, 1967). Kriek (1965) assumed that, in the reaction of 2-fluorenylhydroxylamine with guanine derivatives a t pH 5, substitution occurred a t the same position. Expectedly, reaction with N-acetoxy-2-acetylaminofluorene brings about drastic alteration in the T,,, and the functionality of DNA. Troll and Rinde (1967) found that DNA treated for as little as 1 minute loses as much as 50% of its RNA polymerase priming activity, and there is complete loss after 1 hour, as well as lowering of the T,. In elegant experiments, Lotlikar e t al. (1966) and Poirier et al. (1967) in the Millers’ group demonstrated that both N-acetoxy-2-acetylaminofluorene and N-benzoyloxy-4-monomethylaminoazobenzenereact with methionine in an essentially identical way. The detailed mechanism proposed by Poirier et al. (1967) to account for the reaction of the azo dye with methionine is given in Table XXVI. The reaction products of both carcinogens with methionine have been characterized as the 3-methylmercapto derivatives. These same derivatives have also been detected as alkaline degradation products of liver proteins from rats which were fed 2-acetylaminofluorene or 4-monomethylaminoazobenzene (MAB) (Scribner et al., 1965; De Baun et al., 1967). 3-Methylmercapto-MAB has also the distinction of being the artifactual constituent isolated by the alkaline digestion procedure from the liver of rats fed MAB; this mercapto derivative was incorrectly assumed earlier to be 4-dimethyl-
MOLE,ClJL.\R (;EOMETHY A N D CAltCINOGENIC ACTIVITY
TABLE XXVI Possible Mechanisms for the Reaction of N-Benzoyloxy-N- methyl-4-aminoazobenzene (N-Benzoyloxy-MAB) with Methionine a
3-Methylmercapto-MAB' a From Poirier el al. (1967).
b MAR
7
4-monomethylaminoazobenzene .
Homoserine lactone
445
446
JOSEI’H C. A R C 0 8 AND M A N Y 14’. ARGUS
aminoazobenzerie (DAB) and, thus, led to the belief that MAB unclergoes niethylation to DAB in vivo (Section 111,C15). There is significant evidence, a t least for the amino azo dyes derived from DAB that much of the dye is attached to the protein by means of a methionine moiety. This is consistent with the following observations: ( a ) the facile release of the dye from the protein upon treatment with alkali a t room temperature, but not by ethanol, lauryl sulfate, phenol, 01‘ hot trichloroacetic acid; ( b ) treatment of the liver homogenate in 957% ethanol a t 60°C. greatly diminishes the amount of 3-methylmercaptoMAB that can be obtained by subsequent alkaline digestion from the livers of rats admjnistered DAB or MAB; this is presumably due to demethylation of the sulfonium derivative hy the hot ethanol trcatment to an alkali-stable thioether (Scribner et al., 1965) ; (c) the livers of rats administered MAB together with m e t h i ~ n i n e - ~but ~ s , not with cystine-”S, yielded 35S-labeled 3-methylmercapto-MAB (Scribner e t al., unpublished, cited in E. C. Miller and Miller, 1966). Funakoslii and Terayama (1965) investigated thc reaction between 3-hydroxy-4-amino~tzobenzcne or 3-hydroxy-MAB and amino acids or ulkylamines, on the grounds that the o-quinoneimine metabolically generated via the o-hydroxy derivative might be responsible for protein binding. I n the same report, a spectral study of the natural polar dye suggests that the dye moiety does not possess a phenolic hydroxyl group, a t least in the free state. The difficulties encountered in investigating the presence of a phenolic group are discussed by Terayama (1967). In the in vitro reaction of the above two azo dyes with lysinc, histidine, and tyrosine, the a-amino and carboxyl groups of the amino acids do not seen1 to be involved, and the 3-hydroxy group in the dye moiety is preserved (Funakoshi and Terayama, 1965). Thus, in view of the observation with thc natural polar dye, such reactions are unlikely to occur in vivo. In the experiments of Terayama and his associates with the natural polar dye, enzymatic and alkaline hydrolysis yielded four fractions of polar dyes (Terayama and Takeuchi, 1962; Higashinakagawa et al., 1966). Subsequently, Higashinakagawa et al. (1966) and Terayama (1967) detectcd 10 conthe presence of sulfur in their polar dye fractions. Their results alL firm the conclusions of Scribner et al. (cited in E. C. Miller and h/lillcr, 1966) in that methionine-’W, but not cystinc-’Y3, is overwhelmingly incorporated into the polar dye. However, they assigned a different position of binding of the methionyl group to thc dye. I n fact, they found that the polar dye in three out of the four fractions contains a aecondary amino group since it could be methylated by dimethylsulfatc. Furtherinow, comparison of the spertm of the polar dye and of 3 - l n r t h y l - D ~ R
suggested the a l w n c e of a substituent in the 3-position in the polar dye. The latter point ~ ' R Sthen more firmly ascertained by oxidative degradation (liy I,'.roxytrifliioroaretic acid) and siilisequent, reduction (by SnCl,) of llic polnr (lye, which yiclcled p-pheiiylc~ncdiamiiie.Other experiments with MAB and DAB bearing 14C-methyl groups showed t h a t the N methyl carbon of the former dye is completely retained in the polar dye, whereas half of tlie N-iiicthyl carbon of the latter dye is lost during protein binding. Hence, since the polar dye has a secondary amino group in the 4-position and the amine ring is unsubstituted in the 2-, 3-, 5 - , and 6-positions, tlie following formula was assigned to thc polar dye:
Other possible structures for the polar dye were proposed by Terayama, Matsumoto, and Higashinakagawa (cited in Terayama, 1967). The problem of the conflicting assignments of the position of substitution in the polar dye remains unresolved, a t present. However, Lin et al. (1967) concluded in an ingenious study that. the polar dye contains the methyl group of the administered MAB in an intact form. These investigators have prepared labeled MAB using a mixture of CH,I-3H and CH,I-14C. They found that the dyc administcred to the rats and the polar dye isolated from the liver had very similar ?H/I4C ratios, indicating that no hydrogen in the methyl group was replaced by a substituent group. I n tlie discussion of their data, Lin e t nl. implied that the p-phenyleriediamirie obtained by Higashinakagawa e t nl. may have resulted from the removal of the methionine side chain by the reducing agent. The reactivity of the N-hydroxymethyl drrivativcs formed a s probable intcrrnediates in the oxidative tlemethy1:ition of tlie dyes continued to attract interest. It 1ias been known for many years t h a t hydroxymethyl groups combine with rcactive C H and N H groups in proteins by a Mannich base type of binding (E. C. Miller and Miller, 1952). Roberts and Warwick (1963) sliowcd more recently that 4-aminoazobenzene in the presence of formaldehyde binds covalently also to RNA and D N A in vitro. With cytosine derivatives, 4-aminoazobenzene (with two molecules of formaldehyde) undergoes Mannich-type condensation a t two points (with the pyrimidine amino group and the ring N, atom) so as to form a triazine ring structure, stable in a wide pH range around neutrality (Robcrts and TV:irwirk, 1966a).
448
JOSEPH C. ARCOS AND MARY F. ARGUS
N O T E A D D E D I N PROOF: ( 1 ) 1’0 Section II,AJ. The full report on the carcinogenic activity of 5-methyl-l,2,3,4-dibenzanthracene (XXI), also known as 10-methyldibenz[a,c]anthracene has appeared [A. Lacassagne, N . P. Buu-Hoi, and F. Zajdela., Eirrop. ,I. Cancer 4, 123-127 (1968)l. Irpon suhrutaneous injection into tiiicr, this cornpoitiid proditc:cs local sarwnii\s w i t h a m t w i I:il,c$nt pmiocl of 250 days; the noiiniuthylatcd parenl compound is itmtivc i i n tlor i(Ii~iit,ii*:ilc*oiitliiions. A. Lacassagne, F. Zajdela, N. P. Buu-Hoi, 0. Chalvct, and G. €1. Daub [Intern. J. Cancer 3, 238-243 (1968)l have investigated the carcinogenicity of fourteen mono-, di-, and trimethylated 3,4-benzopyrenes. Many of these homologs are distinctly more active than the nonsubstituted parent compound. There is an approximate correlation between carcinogenic activity and the calculated electronic charge of the respective K-regions. I t is predicted that introduction of more than three methyl groups will lead to loss of activity. D. Lavit-Lamy and N. P. Buu-Hoi [Bull. SOC. Chim. France, pp. 2613-2619 (19S6)l have shown that the compound believed to be 1,2,3,4-dibenzopyrene (XV) is, in fact, 3,4,6,7-dibenzofluoranthene,also known as dibenso [a,elfluoranthr~nc.The true 1,2,3,4-dibenzopyrene, also known as dibenso[a,llpyrene, which was synthesized by an unequivocal route, has now been shown to be a potent sarcomatogcnic agent (Iball sarcoma index: 82) by subcutaneous injection in XVII nc/ZE mice [A. Lacassagne, N. P. Buu-Hoi, F. Zajdela, and F. A. Vingiello, Naturwksew9mjten 55, 43 (1968)l. That the problem of the endogenous transformation of naturally occurring steroids to carcinogens cannot yet be ruled out is suggested by the discovery of the potent carcinogenicity of ll,l2-dimethylcyclopentano[alplienanthrene and the 11,12methoxy compound [cited in F. Homburger, Science 161, 190 (1968)l. T. Arata, S. Tanaka, and C. M. Southam [J. Natl. Cancer Znst. 40, 623-627 (1968)1 have shown that the halogenated nucleosides, iododeoxyuridine and fluorodeoxyuridine, and also cliloramphenicol produce a statistically significant doubling of 20-methylcholanthrene-induced skin papilloma incidence in mice. This contrasts with an earlier study [H. V. Gelboin and M. Klein, Science 14& 1321-1322 (1964)l showing that another agent interfering with DNA, artinomycin D, inhibits skin tumorigenesis by 9,10-dimethyl-1,2-benzanthracene. (8) To Sections II,A,2 and II,B. An exhaustive review on chemical carcinogenesis by hydrocarbons and other agents using newborn mice and rats has been given by B. Toth [Cancer Res. 28, 727-738 (1968)l; F. J. C. Roe, R. L. Carter and W. H. Percival [Brit. J. Cancer 21, 815-820 (1967)l reported on carcinogenesis in newborn rabbits induced by 9,10-dimethyl-1,2-benzanthracene.A good survey on “Chemical and Environmental Carcinogcnesis in Man” has been made by D. B. Clayson [Europ. J. Cancer 3, 405-415 (1967)l. (3) To Section ZZ,DJ. The absorption, distribution and cxcretion of 20-methyladminischolantlirene, Q,lO-dirnrthyl-1,2-bcnsunthraccnc and 1,2,5,6-dibcnzantItr~~cne tered by stomach tube t,o rats has been studied by P. M. Daniel, 0. E. Pratt, and M. M. L. Prichard [Nulure 215, 1142-1146 (1967)l. Much of the absorbed carcinogen is taken up and retained for a long period of time by the body fat, but there is only little carcinogen in the brain in which tissue thc lipids are mainly polar. The extensive storage of carcinogen in fat adjacent to the mnmmary gland could explain the systemic specificity of these agents toward this organ. (4) To Section II,D$. P. Sims [Bfochem. J. 105, 591-598 (1967)l and P. Sims and P. L. Grover [Nature 216, 77-78 (1967)l continued to investigate the metabolism of 9,10-dimethyl-1,2-benzanthraccne by rat liver homogenates and the conditions of
MOLECI'L.iR
(iEOMETRY A N D CARCINOGENIC ACTIVlTY
449
animal age and dict influencing this metabolism. D. N. Wheatley and M. S. Inglis [Brit.J . Cancer 82, 122-127 (1908)l found that, in contrast t o the potent mammary t,umor inducing properties of 9,10-dimethyl-1,2-benzanthracene,the 9- or 10-hydroxymethyl derivatives induced tumors in only an occasional animal, and the 9,lOhishydroxymethyl derivative was inactive when given by stomach tube t o SpragueDawlcy rats. This concurs with the previous results of E. Boyland and P. Sims [Intern. J . Cancer 2, 500-504 (1967)l working with C57 black mice and subcutaneous administration, on the relative inactivity of these hydroxy metabolites. W. Levin and A. H. Conney [Cancer Res. 27, 1931-1938 (196711, P. H. Jellinck and B. Goudy [Biochem. Phnimacol. 16, 131-141 (196711 and P. Sims and P. L. Grover [Brit. Empire Cancer Campaign 45, 18-19 (196711 investigated the effect of pretreatment of the animals with polycyclic hydrocarbons on the subsequent It appears that although pretreatmetabolism of 9,10-dimethyl-1,2-benzanthracene. ment, in general, increases metabolism, it alten the pattern of metabolism from sidecalrain to ring hydroxylation. Since the hydroxymethyl metabolites are active in inducing adrenal necrosis, it is likely that this shifting of metabolic pattern is responsible for the protective effect of hydrocarbons and other compounds against adrenal necrosis by 9,lO-diinethyl-l,2-benzanthracene. A study of the structure-activity relationships of flavones, flavonones and chalcones t o induce increased 3,4-benzopyrene-hydroxylaseactivity in the liver and lung of the rat has been carried out by L. W. Wattenberg, M. A. Page, and J . 1,. Leong [Cancer Res. Za, 934-937 (196S)I. One of the very rare instances in which polpcyclic hydrocarbon administration brings about inhibition rather than induction of a microsomal mixed function oxidase is the basis of the finding of C. HochLigeti, M. F. Argus, and J. C. Arcas [ J . Null. Cancer Inst. 40, 535-549 (1968)l that simultanrous administration of 2O-methylcho1,znthrcne inhibits hepatic tumorigenrsis by dimethylnitrosamine. Consistcnt, with this is thr sithsrqurnt ohsrrvation by N. Venkatesan, J. C. Arcos and M. F. Argus [Life Sciences 7, 1111-1119 (1968)l that 20-methylcholanthrene is an inhibitor of the dfmethylation and incrrases the LD,, of dimethylnitrosamine. Also other aromatic polycyclics bring ahout this inhibition which appears to depend on the molecular size of the hydro(-arbon. The inhibition is possibly gene-mediated since 20-methylcholanthrene in vilro, unchanged or after metabolism, does not inhibit the demethylating activity of microsomrs. Study of the biliary metabolism of intraperitoneally injected tricycloquinazolinc by R. W. Baldwin, J. A. Nilrolic, H. C. Palmrr, and M. W. Partridge [Bioehem. Pharmacol. 17, 1349-1363 (1068)I showed the prrsence of the 1- and 3-hydrosy metabolites in the urine and feces; the major metabolites, however, were unidentified polar compounds. Mt~tabolim of Y"tricgc1oquinazolinc in the mouse skin yields all four monohgdrosy dcrivativcs tR. W. Baldwin, M. Moore, J. A . Nikolic, and M. W. Partridge, Biochpna. P h a i m a c d . 17, 1365-1375 (1968)l. A small amount of radioactivity was prescnt in the skin as strongly bound conjugates to protein. This radioactivity was liberatrd only by drastic hydrolytic conditions. (6) To Section II,P. R. Franke and M. Biichncr [A& Biol. Med. German. 19, 1047-1051 (1967)I csrrird out an invrstip:ition on t.he soluhilimtion of pgrcnc and 3.4-I~i~iizopvrenr1 y 1lliin:ui srruni :illwinin, niitl c : i n i t ~ to c.onvliisions rswnti:~llg itlrntic.:il l o tliose of Fal~giin ( r i ~ v i i ~ \ v v t li n TI.F,1) regarding tllc i l ~ ~ ~ t ~ l l a nofi s i ~ ~ s o l i ~ ~ d i z a t i oI i I~ proteins. ~ D. I> 2-met,liyl-Q5, 3-methyl-Q5 >> 3’DAB 6’-mcthyl-Q5. methyLQ5 (11) T o Section III,D,l. E. Brill and J. Radomski [Life Sciences 6, 2293-2297 (1967)1 confirmed that 1-naphthylhydroxylamine is a urinary metabolite of l-naphthylamine in the dog. H. Uehleke, F. Geipert, F. Schnitger, E. Brill, J. L. Radomski, end W. B. Deichmann [Abstract, Naunyn Schmiedebergs Arch. Pharmak. E x p . Puthol. 260, 213 (1968) 1 have shown that in the same species the N-hydroxylation of 2-naphthylamine is enhanced by phenobarbital pretreatment. The full report on the comparative carcinogenicities and mutagenicities of 1- and 2-naphthylhydroxylamine has appeared [S. Belrnan, W. Troll, G. Teebor, and F. Mukai, Carlcer Res. 28, 535-542 (1968)I. (12) To Section III,D,3. New results strengthen the conclusion of H. R. Gutmann, 8. B. Galitski, and W. A. Foley [Cancer Res. 27, 1443-1455 (196711 that synthrtic. N-hydroxylation transforms inactive or weakly active N-substituted derivatives of 2-acetylaminofluorene to highly active compounds. For cxamlilr!, syntlirtic N-llytlroxylation of the inactive 2-benzenesulfonamidofluorene transforms it int,o 11 highly active agent producing 100% mammary tumor incidence in frmalc rats; similarly, N-hydroxylation transforms the inactive 3-acetylaminofluorrnc to the highly active 3-acetohydroxamic acid. The general validity of this principle is also shown by the considerable enhancement of activity by N-hydroxylation of 4-benzenesulfonamidobiphenyl (H. R. Gutmann, personal communication). Linking of an amino, hydroxylamino, or acetohydroxamic acid group to a sufficiently large hydrocarbon-type grouping is, however, not per se a sufficient condition for carcinogenic activity. This is exemplified by the finding of H. Dannenberg, I. Bachmann, and C. Thomas [Z. Krebsforsch. 71, 74-80 (l96S)l and E. Hecker, M. Traut, and M. Hopp [Z. Krebsforsch. 71, 81-88 (1968)l that the introduction of a 3-amino, 3-acetylamino, 2-acetylamino, or 3-acetohydroxamic acid grouping onto the steroid skeletons, A’,*~G(’a)-oe~tratriene and A‘~s~G‘‘0’-oestratrienol-(17~), dors not evoke carcinogenic activity. Thus, in addition to a sufficiently large size (cf. Gutmann, Galitski, and Foley, Zoc. cit.), the hydrocarbon moiety must be planar and aromatic in accordance with Scribner’s theoretical framework (discussed in Srction III,D,4). E. Hecker, M. Traut, and M. Hopp [Z. Krebsforsch. 71, 81-88 (1968)l confirmed the pot.ent carcinogenic act,ivity of 2-nitrosofluorene. Fed by stomach tube to rats for 42 weeks it yielded, a t an activity level equal to that of 2-acetylaminofluorene, ear duct tumors in all animals and liver tumors in males. The nitroso compound also produced squamous epithelial carcinomas of the forestomsch. The results of C. C. Irving, R. Wiseman, and J. T. Hill [Cancer Res. 27, 23092317 (196711 suggest that it is the formation of 0-glucuronide which is the major metabolic reaction of 2-fluorenylacetohydroxamic acid in the rat liver in vivo, rather than reduction or deacetylation. Subsequently, E. C. Miller, P. D. Lotlikar, J. A. Miller, and B. W. Butler [Mol. Pharmacol. 4, 147-154 (196813 demonstrated that the 0-glucuronide is, in fact, reactive at neutral p H in vitro toward methionine, tryptophan, and guanosine. However, the reactions are considerably slower than those with esters of N-hydroxy-2-acetylaminofluorene, such as the N-acrtoxy derivative. I n accordancc with the lesser reactivity of the 0-glucuronide, the conjugate was found to be a notably weaker carcinogrn than the unconjugated arctohydroxamic acid (see “Note added in proof” in the report of E. C. Miller et ul., Zoc. cit.). Thus, whethcr metabolically formed 0-glucuronide is involved in the carcinogenic activity of 2-acetylaminofluorene and its N-hydroxy derivative still remains questionable.
>
>
MOI.ECUI,:\R
GEOMETRY A N D CARCINOGENIC ACTIVITY
453
'l'Ii~* frill rcqwrt, froin Ualtlwin's group 011 coniparat,ivc carcinogenicity in thc rut of 4-acet,amidost,iIhene and its N-hydrosy derivative has appeared [Brit. 3. 1 1 i t b
Cancer 22, 133-144 (196S)l. The N-hydroxy derivative is highly active by three routes of administration and produces invariably ear duct tumors in the majority of animals; local tumors were seldom seen in the subcutaneously injected animals. The parent amide anti the N-hydroxy derivative were equally active orally ; howcvw, by subcrrtnnrous and intrnperitoncxl injection t,he N-hydroxy derivative was more potmt. (13) To Sectwn 111,E,1. M. Hozumi [Bwchem. Phurmacol. 17, 769-777 (196S)I showed that 4-hydroxylaminoquinoline-N-oxideis a powerful inhibitor of sulfhydrylrequiring enzymes, such as catalase, alcohol dehydrogenase, and urease, by virtue of its catalytic property to oxidize -SH groups. Significantly, the inhibition is rwersed by the addition of glutathione. ( 1 4 ) To Section IV,A. In the full report [R. W. Baldwin, M . Moore, and M. W. Partridge, Intern. J . Cancer 3, 244-253 (1968)l of the rccent work of Baldwin's group on the interaction of "C-tricycloquinazoline with mouse skin proteins, i t wu shown that; this cbarcinogcm bocomes covalently bound to soluble and particwlnt c skin proteins, although the binding levels are lower than those obscrved with carcinogenic and noncarcinogenic hydrocarbons. Furthermore, the level of binding reaches a saturation level at the applied carcinogen dose of 0.03 fiM. Thc bound radioactivity can only be liberated by drastic hydrolytic treatment and it is not identihble as unchanged tricycloquinaeoline. The soluble proteins with which tricycloquinazoline is associated in the skin have the electrophoretic mobility of albumin and axe not comparable to h-like proteins. (16) To Section IV,B. Ma. Tada, Mi. Tada, and T. Takahashi [Biochem. Biophys. Res. Commwn. 29, 46-77 (1967)l investigated the complex formation in vivo between 4-hydroxylaminoquinoline-N-oxide,on one hand, and DNA and RNA, on the other hand, during exposure of ascitcs tumor cells to the action of thc carcinogen. The isolated DNA complexes show strongly decreased RNA-priming ability, and there is s reciprocal relationship between the amount of cornplexcd carcinogen and RNA-priming ability. Surprisingly, heating to 100°C brings about a release of the carcinogen in a probably metabolized form and the template activity of the DNA increases to that of heat-denatured control DNA; .this would suggest that the binding to the bases is not of covalent nature. However, when the DNAcarcinogen complex was submitted to enzymatic degradation and the hydrolysatilc: cliromatographed, about one-half of the carcinogen was eluted together with thc nucleotides; these results would indicate that covalent binding between the carcinogen and the nucleotides has taken place. (10) T o Section IV,C. M. Sluyser [Biochirn. Biophys. Acta 154, 606609 (19f38)J studicd the in vitro interaction of 4-dimethylaminoazobeneene and 3,4-benzopyrene with various histones. The association a.ppears to be rather nonspecific and is duc! to noncovalent bonding to large hydrophobic rcgions of the histones. Administration of 2-acetylaminofluorene to rats, however, results in covalent binding of the carcinogen to lysine-rich and arginine-rich histones [E. J. Barry, C . A. Ovechka, and H. R. Gutmann, J . Biol. Chem. 243, 51-60 (1968)l. B. Bakay [Biochem. Phurmucol. 17, 689-698 (1968)l produced rcsults indicating that in the liver nuclei of rats f d 3'-methyl-4-dimethylaminoazobenzene some of the protein-dye conjugates are aseociated with, or are an integral part of, nuclear ribosomes, Regarding the role of the liver h proteins a9 growth regulators, M. F. Argus, J. A. Walder, J. A. Fabian, and J. C. Amos [Brit. J . Cancer 22,330-341 (1968)l have shown that during liver regeneration following partial hepatectomy there is an
454
JOSEPH C. ARCOS A N D MARY F. ARGUS
iiivcrst, rt~l:ttiondii~ibetween total h protvin levcl on one Iland, and mitotic intlcs ant1 solrhlc cytolilasmic sidfhydryl level, on the ot lirr. ITonPvc~r,in 20-metliylcholanthrene-induced liver growth the total h protein lrvel remains uuchanged despite the considerable increase in the mitotic index and the sulfhydryl level. There is now substantial evidence indicating that o-quinoneimines are not instrumental in the mechanism of carcinogenesis by 2-acetylaminofluorene [reviewed in C. M. King and B. Phillips, Sczence 59, 1351-1353 (1968)l. These investigators have shown that a protein fraction of the 105,000 X g supernatant of rat liver catalyzes the reaction of 2-fluorenylaeetohydroxan~ic acid wit11 sRNA, and also with DNA and protein. The cofactor requirements and labeling studies suggest that sulfate and phosphate esters of the acetohydroxamic acid may be the reactive ultimate metabolites. J. K. Lin, J. A. Miller, and E. C. Miller [Biochemislry 7, 188s1895 (196811 cstablished that the major polar dye derived from the liver proteins of rats fed 4-monomethylaminoazobenzene is 3-(lioinocystein-S-yl)-N-mrtliyl-4-aminoazobenzcne. The protein bound sulfonium dye (see Table XSVI), 3-(methion-S-yl)-Nmethyl-4-sminoazobcnzcne appears to be the lilrcly precursor of bolli this polar dye and 3-methylmerc:tpto-4-monomethylaminoazobenzene.
REFERENCES Abell, C. W., and Heidelberger, C. (1962). Cancer Res. 22, 931-946. Akinrimisi, E. O., and Ts’o, P. 0. P. (1964). Biochemislry 3, 619-626. Alexander, M. L., and Glanges, E. (1965). Proc. Natl. Acad. Sci. U . S. 53, 282-288. Alifano, A., Papa, S., Tancredi, F., Elicio, M. A., and Quagliariello, E. (1964). Brit. J . Cancer 18, 386-389. Al-Kassab, S., Boyland, E., and Williams, K. (1963). Biocliem. J. 87, 4-9. Allen, M. J., Boyland, E., Dukes, C. E., Homing, E. S., and Watson, J. G. (1957). Brit. J . Cancer 11, 212-228. Andersen, R. A., Enomoto, M., Miller, J. A., and Miller, E. C. (1963). Proc. Am. Assoc. Cancer Res. 4, 2. Andersen, R. A., Enomoto, M., Miller, E. C., and Miller, J. A. (1964). Cancer Res. 24, 128-143. Andervont, H. B., and Shimkin, M. B. (1940-41). J. Null. Cancer Znst. 1, 225239. Anghileri, L. J. (1967a). Naturwissenschaften 54, 249-250. Anghileri, L. J. (1967b). Ezperientiu 23, 661-662. Arcos, J. C. (1961). Bull. Tulane Univ. Med. Fac. 20, 133-150. Arcos, J. C., and Arcos, M. (1955). Naturwissenschajten 42, 608. Arcos, J. C., and Arcos, M. (1956). Bull. SOC.Chim. Belges. 65, 5-16. Arcos, J. C., and Arcos, M. (1958). Bwchim. Biophys. Acta 28, 9-20. Arcos, J. C., and Arcos, M. (1962). Progr. Drug Res. 4, 407-B1.*
* The
following errors in this review should be noted: p. 419, first line: ref. (749) should be deleted and placed on p. 425, end of 2nd paragraph. p. 427, Table 6: references 56, 58, 59, 60 should read 84, 83, 270, 821, respectively. p. 439, second line: ref. (838) should read (383). p. 462, 29th line: “resting” should read “testing” p. 404, 30th line : triphenyl-stilbene should read triphenylethylene ; 1,1,3-tris-(4methoxyphenyl)-3-chl0r0-stilbene should read 1,1,Z-tris(4-methoxyphenyl)-2chloroethylene. p. 510, 5th line: sentence should read “Selye recorded the induction with croton
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1).
p. p. p. p.
oil, :inotlier agent known origin:illy as tumor promotor, of sarcomas in grannIoiii:Ltoiis ~ O I I ( * ~ I I i~~Hr i - : i t c din 1 I I V rat (748) ,” 510, 3rd paragrapli : 2nd sentriirt~should rc(m1 “Plienol WMS Tuuii(l 1 0 intiiicil ptqJilloirias 111 suriacci application on the I I I O I I S I ~skin (713, 717) ,” 510, line 40: after “to!vald thc 1110usz;(~ skill’’ insrrt “and Y ~ I k J ~ ’ 1 l t i l l l C lissuc.” ~ll~ 511, Section 2.223: second ref. should reaa! (796). 514, 4th line: a t end of first sentence insert ref. (557). 560, 13th line: “hydrolytic” should read “metabolic.”
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46 1
(.~raiilliaiii, 1’. H., Weisburger, E. Iolisin and, 20-22 (*:itabdizingciizvrne for, 26 cornpounds rrlated to, biological a(#tlvlty. 15-20 l l ~ J l l ~ ~ ~ ~ r l ' l uIS o~f'nl~, t l l ' t ( ' l 1 1 1 1 1 1 : ~ ~ 1 ~ J 1Or, 1 2" N-liytlro\yiirelliaii froni, 26-27 N-substitiit ed, rarcinogenic action, 1617 metabolism of, 22-26 rate of elimination, 24-26 urinary metabolites of, 31-34 UV light, bacteriophage genes controlling sensitivity to, 148-149 UV radiation, effect on viral induction of enzymes, 105-110
V Vaccinia virus DNA of, biosynthesis of, 96-97 enzyme induction by, 123 in mutants, 116-119 replication cycle, 95 UV effects on infectivity, 106 Viral-induccd enzymes distinctive properties of, 119-125 of DNA-containing animal viruscs, 94100 of DNA metabolism, 89-125 host-controlled modification and, 140148 DNA methylation, 147-148 hcat sensitivity, 146-147 of phage A, 143-144 of T-cvm phage, 14CL143 hydrolysis or modification of DNA or RNA by, 126-151 induction system rharartcristics, 100111 induction by UV-irradintcd virus pnrticles, 105-110 inhibitors of, 104-105 mutant virus strains and, 111-119 of thymidine metabolism, 182-193 Irinetics, 182-185 viral onrogenesis and, 73-221 Viral oncogenesis, biochrmical asprcts of, 173-207 genetic factors, 204-207
Virus (cs) animal, see Animal viruscs -induced antigen syntliesis, 84-89 “early protein” synthesis, 87-89 viral-capsid proteins, 8 4 8 7 -infected cells, “early protein” syntliesis in, 87-89 nucleic acids of, iiioleculsr weights of, 83 polyanion inhibition of, 225-226 runting and, 45, 54-55 -transformed cells, continued viral genome in, 201-203
l,ririior-protliiciii~, sco Tunlor-protlucing viruses
W Wasting tliscuse, runting syndroinc and, 50-51
X Xenylaniincs, carcinogenic activity, 376377
Y Yabs monkey poxvirus, as tumor-producing virus, 74, 78
CUMULATIVE INDEX VOLUMES 1-1 1 A Akylating agcnts, cytotoxic, chemistry of, 1, 397 Amino acid transport, in tumor cells, 9, 143 Aminoazo dyes, carcinogenic, 1, 339 Anemia in cancer, 5, 199 Animals, experimental, pulmonary tumors in, 3, 223 Anticancer agents, mechanisms of resistance to, 7, 129 Aromatic compounds, molecular geometry and carcinogenic activity of, 11, 305 Aromatic molecules, electronic structure and carcinogenic activity of, 3, 117 Autoimmunity, 11, 43 Avian virus growths, their etiologic agents, 7, 1
B Benzacridines, angular, and carcinogenic activity, relation between, 4, 315 enzymcs~ in and othrr diseases, 6, 1 Bone marrow, normal and lrirlteinic, hiochemistry of, 9, 303
C
cross resistancc and collateral scnsitivity in, 7, 235 experimental, 2, 425 in man, 4, 1 Cancer production, role of viruses in, 2, 353 Cancer toxin, newer concept of, 5, 157 Carcinogenesis, application of radioisotopes to studies of, 1, 273 aspects of, 3, 171 and tumor pathogenesis, 2, 129 electronic configuration and, 1, 1 epidermal, 1, 57 ethionine, 7, 383 related to contaminated foods, 8, 191 sulfhydryl group and, 10, 247 tobacco, experimental, 8, 249 Carcinogenic activity, and angular benaacridines, 4, 315 of aromatic compounds, 11, 305 of aromatic molecules, 3, 117 Carcinogenic aminoaao dyes, 1, 339 Carcinogenic nitroso compounds, 10, 163 Carcinogcnicity of 2-flnorenaminr and rclntcd compounds, 5, 331 Carcinogens enzyme induction, and genc action, 10, 1 with macromolecules, reactions of, 2, 1 Carcinoma, primary, of the liver, 5, 55 Cell physiology, activity of polyanions in, 11, 223 Chrmical constitution, and carrinogenic activity, 2, 73 Chirkm tumor virnws, and pnssmgrr
Cancer energy and nitrogen mctabolism in, 2, 229 experimental, genetic studies in, 2, 28 1 human, urinary enzymcs in, 9, 1 ionizing radiations and, 2, 177 lipids in, 4, 237 niitrition in relation lo, 1 , 151 viriiws, 7, 51.5 1 ) l r l h i n i t prolc,ins i n , 1, 503 icalatiori of iinniiiiic. r c w t i o i r l o , 9. E 47 Electronic strnctnrc, of niomntic molrCanccr chemothernpy, cules, 3, 117 by perfusion, 6, 111 Energy metabolism in cancer, 2, 220 514
I ~ n z ~ . n iiiiiIu(~t,iun, c gi'iic~:I(,( i o i i , varciiiogl'lls ~ l l i t l , 10, 1 l~~nzymicpat,lrriis I ) ( ncol)laalic t,issuc, 10, 117 Etliioninc carcinogonrsis, 7, 3S3 Etiology of lung cancer, 3, 1 ol nioiiw loukcmia, 6, 291
F 2-l;luoren:iniine, chemistry, carcinogenicity, and nic~t;ibolisrii of, 5, 331 Folic acid, antagonists of, 6, 369 Fowls, clieniically induccd l.uniors of, 5, 179 Fungal iiietat)olit,c~s,foods contaminakd by, :ind carcinogc>nr&, 8, 191
G Gene action, carcinogr~na,enzyme induction and, 10, 1 Growth liroccwcs, 1)rotrin synthcsis referc~nce to, 5, 97 H
Hepatocarcinogoncsis, behavior of livcr enzymes in, 6, 403 Hrpatomns, expc~rimc~ntiil,development of, 9, 227 bioclic.mistry of, 9, 227 biology of, 9, 227 Hormonal aspects, of i~xperimi~nt:il tumorigencsis, 1, 173 Hormonal gcnesis, of mammary cancchr, 4, 371 N-Hydroxyurethan, carcinogenic action, and metaholisni of, 11, 1
I , t i i ki:III i:t cliroiiit,, iisc ol i ~ i y l ~ ~ r iin, i n 4, 73 cliroiiic Iiiycloid, cytogenic studies in,
7, 351 Lilids in cancer, 4, 237 Liver, primary carcinoma of, 5, 55 Liver enzymes, beliavior, in liepatocarcinogcncsis, 6, 403 Lung cancer, etiology of, 3, 1 Lung cancer pathogenvsis, atmos1)heric fuctors in, 7, 475
M P\/lacrornoleculcs, reactions of carcinogens with, 2, 1 Malignant cclls, in U ~ N J studies on protein synthesis by, 10, 83 Mammalian organism, normal and tumor-bcaring, inhibition analysis in, 4, 113 Mainmary cancer, hormonal genesis of, 4, 371 Mamrn:iry tumors in micc, milk agent in, 1, 103 M[yt.abolism, of 2-fluorenamine and relatcd compounds, 5, 331 Motabolites, purine and pyrimidine, antngonists of, 6, 360 Milk agent, in mammary tumors, 1, 103 Molecular geometry, of aromatic compounds, 11, 305 Mouse leukemia, etiology and pathogrnc,sis of, 6, 201 viral etiology of, 6, 140 Myleran, use of, in chronic I(,rikemias, 4, 73
I
N
Immune rcaction, relation of, t,o cancer, 9, 47 Inhibition analysis in normal niainmalinn organism, 4, 113 in tu,mor bearing I 1i:iiii i i i ali:in orgnnism, 4, 113 Ionizing radiations, ant1 cxnccr, 2, 177
Neoplasia, 11, 43 Neoplastic cells, nurlcar protc,ins of, 8, 41 Neol jlastic tissue (s) rnzyinic pattern of, LO, 117 oxidative metabolism of, 3, 269 Nitrogen metabolism in r a n w r , 2, 229 Nitrogen muqtards, cliniral use of, 2, 255 Nitroso compounds, carcinogenic, 10, 163 Nuclear proteins, of neoplastic cells, 8, 41
1 Leucocytes, normal and Icultrmic, biochemistry of, 9, 303
516
CUMULATIVE INDEX VOLUMES
Nuclcolrtr cliromosomcs, structiircs, in1.wa.ctions, and pi’rslwotivcs, 8, 121 Nutrit,ion, relation to wnner, 1, 451
0 Oxidative metabolism, of neoplastic tissues, 3, 269
P Patliogencsis, of nioiise Icukcniin, 6, 291 Perfusion, cmccr chemolhcritpy by, 6, 111 Plant tumor problem, 6, 81 Plasma cell myclorna, treatmcnt of, 10, 311
Plasma proteins in cancer, 1, 503 Polyanions, in intercellular cnvironinmt and cell physiology, 11, 223 Protein synthesis, in vitro studies by malignant cells, 10, 83
1-1 1
T Tlii oiiiboc~i,i*s,iioriiial auil Icukcinic, biuc4ieinistry of, 9, 303 Thyroid gland tumors, development and mctabolism, 3, 51 Tissue, inductive interaction in development, 4, 187 Tobacco carcinogenesis, experimental, 8, 249
Tumor antigens, specific, 5, 291 Tumor cells, amino acid transport in, 9, 143
Tumor-host relations, 5, 1 Tumorigenesis, experimental, hormonal aspects of, 1, 173 Tumor immunity, recent work on, 4, 149
Tumor metabolism, npplirntion of radioisotopes to studics of, 1, 273 Tumor pathogenesis, carrinogenesis and, 2, 129
with reference to growth processes, 5, 97 Pulmonary tumors, in experimental animals, 3, 223 Purine and pyrimidine metabolitcs, antagonists of, 6, 369
Tumor viruses of chickens and mammals, 7, 515 structure of, and relation to general viruses, 8, 1 Tumors, fowl, chemically induced, 5, 179 Tumors, frozen, survival and preservation of, 2, 197
R
U Urethan, carcinogenic action and metaholism of, 11, 1 Urinary rnaymcs, and human cancer, 9,
Radiation chimeras, 6, 181 Radioisotopes, application to cnrcinogenesis and tumor metabolism, 1, 273
Rous no. 1 sarcoma agent, properties of, 1, 233 Runting syndromes, 11, 43
S Sulfhydryl group, and carcinogencsis, 10, 247
1
V Viral-induced cnzymcs, and viral oncogenesis, 11, 73 Viral etiology, of mouse leukemia, 6, 149 Viruses, tumor, of chickens and mammals, 7, 515