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Inhibition of DNA repair by sodium ascorbate in vitro and in vivo Koropatnick, Donald James 1981

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INHIBITION OF DNA REPAIR BY SODIUM ASCORBATE IN VITRO AND IN VIVO by DONALD JAMES KOROPATNICK B . S c , The Un i v e r s i t y of B r i t i s h Columbia, 1974 M.Sc, The Un i v e r s i t y of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Genetics We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1981 Donald James Koropatnick, 1981 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of Graduate Studies (Genetics) The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Ptprf&lflM ABSTRACT Several short-term assays are In use to assess the carcinogenic hazard of chemicals. While the a b i l i t y to induce i n i t i a t i n g events that may lead to carcinogenesis i s measured, compounds and conditions that might modify the a b i l i t y of chemicals to cause those i n i t i a t i n g events are not assessed by such t e s t s . In addition, compounds that a f f e c t the a b i l i t y of c e l l s to react i n a normal fashion to the damaging action of carcinogens are not detected by these methods. Sh i f t s i n a l k a l i n e sucrose gradient p r o f i l e s of centrifuged DNA (as an i n d i c a t i o n of DNA fragmentation) and formation of a r y l and a l k y l DNA adducts (as an i n d i c a t i o n of DNA modification) have been used as short-term assays for carcinogenic and mutagenic p o t e n t i a l . Repair of DNA damage has been measured by r e s t o r a t i o n of near-control sedimentation p r o f i l e s of DNA and the loss of a r y l and a l k y l adducts over time a f t e r damage or modification of DNA by carcinogens and mutagens. In t h i s study, the a b i l i t y of sodium ascorbate to modify the DNA fragmenting and adductf.forming action of the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (BP) was investigated. In addition, the a b i l i t y of c e l l s In vivo and In v i t r o to repair DNA i n the presence of sodium ascorbate was assessed by the two methods described above. I t was found that sodium ascorbate i n h i b i t e d r e p a i r i_n vivo and i n v i t r o . In addition, sodium ascorbate was found to fragment DNA ija vivo and i n v i t r o i n the presence of copper, and to i n h i b i t the action of carcinogens _in vivo and i n v i t r o by n u c l e o p h i l i c - i i i -scavenging of e l e c t r o p h i l i c carcinogens. Sodium ascorbate was also found to i n h i b i t the binding of BP to DNA jLn vivo and In v i t r o . On the other hand, other reducing agents had other e f f e c t s . Propyl g a l l a t e (a sulphydryl reducing compound) i n h i b i t e d binding of BP to DNA i n v i t r o , but enhanced binding of BP to DNA ixi vivo. The sulphydryl reducing agent glutathione enhanced binding of BP to DNA i n vivo and i n v i t r o . A l k a l i n e sucrose gradient analysis of DNA damage and recovery from that damage, and BP adduct formation i n DNA and disappearance over time, appear to be s u i t a b l e methods for assessment of the modifying properties of compounds and conditions on the i n i t i a t i n g events that may lead to mutation or carcinogenesis. - i v -TABLE OF CONTENTS PAGE Abstract i i Table of Contents i v L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements x i Introduction 1 Chemical Carcinogenesis 1 Interaction of Carcinogens with Informational C e l l u l a r Macromolecules In Vivo 3 Metabolic A c t i v a t i o n to Reactive E l e c t r o p h i l e s 3 Sites of E l e c t r o p h i l i c Attack In Vivo 5 Carcinogenic Process 6 DNA Repair 9 DNA Repair I n h i b i t o r s 11 Measurement of DNA Damage and Repair 13 1) A l k a l i n e sucrose gradient sedimentation 13 2) Ex c i s i o n of a r y l and a l k y l adducts from DNA .. 14 Factors A f f e c t i n g Chemical Carcinogenesis 15 1) Factors a f f e c t i n g the i n i t i a t i n g action of carcinogens 16 2) I n h i b i t i o n of non-enzymatic formation of ultimate carcinogens 20 3) I n h i b i t i o n of action of ultimate carcinogens 20 Sodium Ascorbate 21 Types of Carcinogens 22 1) Primary carcinogens 23 -v-PAGE 2) Precarcinogens 23 3) Cocarcinogens 24 S p e c i f i c Carcinogens 25 1) Nitrogen compounds 25 2) Dialkylnitrosamines 25 3) P o l y c y c l i c aromatic hydrocarbons (PAH) 28 Outline of the Problem 31 Materials and Methods 33 Chemicals 33 Radionuclides 33 Chemical Carcinogens 33 Reducing Agents 33 Metal Salt Solutions 34 Ni t r o s a t i o n of Methylguanidine 34 Experimental Animals . 34 C e l l Cultures 35 Preparation of S9 A c t i v a t i o n Mixture 37 Administration of Chemicals 37 Dimethylnitrosamine 37 Ni t r o s a t i o n products of methylguanidine 38 MNNG 38 Sodium ascorbate 39 Benzo(a)pyrene 39 Al k a l i n e sucrose gradient analysis of DNA damage and repair 40 Al k a l i n e sucrose gradients 41 - v i -PAGE BP Adduct Measurement of DNA 42 DNA i s o l a t i o n 42 DNA concentration determination 43 Measurement of r a d i o a c t i v i t y 41 Results 45 I n h i b i t i o n of DNA re p a i r by sodium ascorbate 45 1) A l k a l i n e sucrose gradient analysis of DNA damage and re p a i r 45 a) In v i t r o 45 b) - -In vivo ••• 56 2) BP-DNA adduct analysis of DNA repair 63 a) T_n v i t r o 63 b) In vivo 75 Other e f f e c t s of sodium ascorbate 84 E f f e c t of sodium ascorbate and other agents on binding of BP to DNA 107 Discussion 125 Summary 136 Perspectives 137 L i t e r a t u r e Cited 140 - v i i -LIST OF TABLES TABLE PAGE 1 I n h i b i t i o n of Carcinogenesis by Induction of Microsomal Enzyme A c t i v i t y 17 2 Carcinogenicity of Some Chemicals 30 3 Recovery of Ra d i o a c t i v i t y from A l k a l i n e Sucrose Gradients 55 - v i i i -LIST OF FIGURES FIGURE PAGE 1 Proposed i n t e r a c t i o n of c e l l d i f f e r e n t i a t i o n and neoplasia 8 2 DNA re p a i r pathways 10 3 A c t i v a t i o n of nitr o s o compounds 26 4 A c t i v a t i o n of dimethylnitrosamine 27 5 Metabolism of benzo(a)pyrene 29 6 Sedimentation p r o f i l e of cultured human f i b r o b l a s t s 47 7 Sedimentation p r o f i l e s of cultured human f i b r o b l a s t s : repair over 30 hr 49 8 Sedimentation p r o f i l e s of cultured human f i b r o b l a s t s : i n h i b i t i o n of repair by sodium ascorbate 51 9 Sedimentation p r o f i l e s of cultured human f i b r o b l a s t s : resumption of repair a f t e r removal of sodium ascorbate 54 10 Sedimentation p r o f i l e of cultured human f i b r o b l a s t s : lack of fragmentation by sodium ascorbate alone 58 11 Sedimentation p r o f i l e s of mouse g a s t r i c c e l l s : r e pair over 30 hr 60 12 Sedimentation p r o f i l e of mouse g a s t r i c c e l l s : i n h i b i t i o n of repair by sodium ascorbate 62 13 Sedimentation p r o f i l e s of mouse g a s t r i c c e l l s : A) lack of fragmentation by sodium ascorbate alone, and B) resumption of repair a f t e r cessation of sodium ascorbate treatment 65 14 A v a i l a b i l i t y of BP i n s o l u t i o n 67 15 E f f e c t of BP concentration on BP adduct formation i n CHO c e l l s 69 16 Repair course of BP adducts i n CHO c e l l s 72 17 I n h i b i t i o n of BP adduct excision i n CHO c e l l s by sodium ascorbate - 74 - i x -FIGURE PAGE 18 BP adduct e x c i s i o n i n CHO c e l l s : lack of i n h i b i t i o n by cysteine 77 19 E f f e c t of BP concentration on BP adduct formation i n mouse g a s t r i c c e l l s i n vivo 79 20 Repair course of BP adducts i n mouse g a s t r i c c e l l s i n vivo 81 21 I n h i b i t i o n of BP adduct excision i n mouse g a s t r i c c e l l s treated i n vivo with sodium ascorbate 83 22 DNA fragmentation by sodium ascorbate and copper i n v i t r o 86 23 DNA fragmentation by sodium ascorbate and copper i n vivo 88 24 Sedimentation p r o f i l e of cultured human f i b r o b l a s t s treated with DMN 90 25 I n h i b i t i o n of DNA fragmentation by dimethylnitrosamine i n the presence.of sodium ascorbate 92 26 I n h i b i t i o n of non-enzymatic a c t i v a t i o n of methylguanidine by reaction with ni t r o u s acid i n the presence of ascorbate 95 27 I n h i b i t i o n of DNA fragmentation by MNNG i n v i t r o by incubation with sodium ascorbate 97 28 I n h i b i t i o n of DNA fragmentation by MNNG i n vivo by incubation with sodium ascorbate 100 29 Enhancement of DNA fragmentation by MNNG i n v i t r o by co-administration with sodium ascorbate 102 30 Enhancement of DNA fragmentation by MNNG i n vivo by co-administration with sodium ascorbate 104 31 Lack of fragmentation of DNA by sodium ascorbate alone i n v i t r o and i n vivo 106 32 I n h i b i t i o n of BP adduct formation i n DNA i n the presence of sodium ascorbate i n v i t r o ... 109 -x-FIGURE PAGE 33 E f f e c t of sodium ascorbate on BP adduct formation i n mouse g a s t r i c c e l l s treated i n vivo 112 34 I n h i b i t i o n of BP adduct formation i n DNA i n the presence of propyl g a l l a t e i n v i t r o 114 35 Enhancement of BP adduct formation i n DNA of mouse g a s t r i c mucosal c e l l s treated with propyl g a l l a t e i n vivo 116 36 Enhancement of BP adduct formation i n DNA i n the presence of glutathione i n v i t r o 118 37 Enhancement of BP adduct formation in. DNA of mouse g a s t r i c mucosal c e l l s treated with glutathione i n vivo 120 38 E f f e c t of harman on BP adduct formation i n DNA of c e l l s treated i n v i t r o 122 39 E f f e c t of norharman on BP adduct formation i n DNA of c e l l s treated i n v i t r o 124 - x i -ACKNOWLEDGEMENTS I thank my research supervisor, Dr. Hans S t i c h , for h i s i n t e r e s t and enthusiasm i n the research that led to t h i s t h e s i s . His example and aid w i l l always be appreciated. Thanks also to fellow students and researchers Anne, Bruce, Urs, Bob, Lan, Miriam and Sing, a l l of whom provided happy arm-waving discussions and p r a c t i c a l help. I thank Bruce Dunn and Anne Hanham, who helped with the DNA adduct experiments. Special thanks go to Jane, who not only l i v e d with the w r i t e r , but made the supreme s a c r i f i c e of helping with the typing. The help of the National Cancer I n s t i t u t e of Canada and the National Science and Engineering Research Council, i m t h e form of grants to Dr. H.F. S t i c h , i s g r a t e f u l l y acknowledged. -1-INTRODUCTION CHEMICAL CARCINOGENESIS Although chemical carcinogenesis was f i r s t discovered i n humans over 200 years ago ( M i l l e r , 1978) there are, today, only about 20 compounds or mixtures that are known to increase the r i s k of cancer i n several organ s i t e s i n various subpopulations defined by t h e i r exposure i n i n d u s t r i a l , medical or s o c i e t a l s i t u a t i o n s . However, these are not the causes of the major burden of cancer (aside from the induction of primary bronchogenic carcinoma caused by ci g a r e t t e smoke)(Doll, 1977). While the carcinogens responsible f o r the greater part of human cancers are unknown, much i n d i r e c t evidence indicates that environmental factors (very probably chemicals f o r the most part) are involved i n the genesis of these neoplasms ( D o l l , 1977). A large number of chemicals can cause cancer i n various tissues of experimental animals, and humans are exposed to some of these carcinogens (generally at much lower concentration and sporadic administration). Generally, the concept i s that a large number of cancers i n the general population may be p o t e n t i a l l y preventable by the i d e n t i f i c a t i o n and elimination of these carcinogens. In recent years the number of chemicals recorded i n the l i t e r a t u r e has been a measure of the vast increase i n chemical knowledge (Maugh, 1978) , and the great majority of the approximately 4 m i l l i o n known chemical compounds are synthetic laboratory products ( M i l l e r and M i l l e r , 1979) . A c e r t a i n number are useful enough to be used to the extent they have appeared i n the human environment, and approximately 60,000 are estimated to be i n "common use" i n the U.S. (Maugh, 1978). The exact number may be disputed, but i t i s clear that at le a s t several thousand -2-compounds are brought into contact with the human population i n small amounts d a i l y . A major source of contact of humans with n a t u r a l l y -occurring chemicals i s i n t h e i r d a i l y intake of natural foods, which may contain several thousand low molecular weight, non-nutritive organic compounds among which are carcinogens and mutagens ( M i l l e r and M i l l e r , 1979). Among these low molecular weight synthetic and n a t u r a l l y -occurring compounds e x i s t i n g i n food, a i r , water, c l o t h i n g , comsetics, etc., there e x i s t s a wide v a r i e t y of structures which can produce a large range of e f f e c t s i n l i v i n g systems. A l l of the compounds are toxic at s u f f i c i e n t dose, and toxic and pharmacologic information e x i s t s i n large quantity on these compounds. However, due to the complexity, tedium, and expense of studies necessary to produce the information, knowledge of the mechanism of action i s severely l i m i t e d . Most information consists s o l e l y of gross data on the amount of chemical required to produce gross e f f e c t s . While the majority of toxic and pharmacological e f f e c t s are due to non-covalent (and, therefore, r e v e r s i b l e ) i n t e r a c t i o n s with c e l l u l a r molecules (Goldstein, et^ a l . , 1974), recent years have shown that the toxic e f f e c t of chemical carcinogens, many mutagens, some allergens, and a few drugs are due to covalent i n t e r a c t i o n s of these compounds and t h e i r metabolites ±n vivo with c r i t i c a l c e l l u l a r molecules. The great majority of chemical carcinogens are small organic compounds (of molecular weight les s than 500) and are generally l i p i d - s o l u b l e and not very water soluble, although exceptions e x i s t . There are some inorganic carcinogens, including metals (beryllium, cobalt, cadmium, chromium, and n i c k e l compounds) as well as cis-platinum(II)diamine d i c h l o r i d e (Leopold, et a l . , 1970). -3-INTERACTION OF CARCINOGENS WITH INFORMATIONAL CELLULAR MACROMOLECULES IN VIVO The transformation of normal c e l l s to tumour c e l l s appears to need, at l e a s t , a h e r i t a b l e , quasi-permanent a l t e r a t i o n i n phenotype that involves control of mitosis. This may be due to 1) h e r i t a b l e changes i n DNA, or 2) quasi-permanent changes i n genome t r a n s c r i p t i o n (analogous to the d i f f e r e n t i a l expressions of genomes of normal somatic c e l l s ) . In either case informational molecules are involved, and chemical carcinogens must in t e r a c t d i r e c t l y or i n d i r e c t l y with one or more informational macromolecules (DNA, RNA or protein) that have some e f f e c t on c e l l m i tosis. Direct evidence has been provided for a l i n k between DNA a l t e r a t i o n s and carcinogen-e s i s by UV l i g h t (Hart, eit al_., 1977). However, for chemical carcinogens, the c r i t i c a l m o dification i n any one of the macromolecules has not been demonstrated as the mechanism of action of any chemical or v i r a l carcinogen. On the other hand, a l l adequately studied chemical carcinogens form covalently-bound d e r i v a t i v e s with c e l l u l a r macromolecules i n vivo (with the exception of adriamycin, which binds t i g h t l y to DNA i n vivo non-covalently (Marquardt, et a l . , 1977) and generates free r a d i c a l s , but does not bind other macromolecules; and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which i s an inducer of mixed-function oxidases i n l i v e r (Poland and Kende, 1977) and so may potentiate the action of other carcinogens. Generally speaking, some binding i n t e r a c t i o n occurs between carcinogens and target macromolecules i n c e l l s to i n i t i a t e carcinogenesis ( M i l l e r and M i l l e r , 1979). METABOLIC ACTIVATION TO REACTIVE ELECTROPHILES The majority of chemical carcinogens require a c t i v a t i o n - metabolism to rea c t i v e forms - to react covalently with n u c l e i c acids and proteins i n vivo. -4-The covalent i n t e r a c t i o n i s by means of non-enzymatic n u c l e o p h i l i c s u b s t i t u t i o n . The carcinogens are termed precarcinogens, and must be converted to t h e i r f i n a l r e a c t i v e form - ultimate carcinogens - i n a conversion that i s usually catalyzed by enzymes, and c e r t a i n intermediate metabolites (proximate carcinogens) may be generated i n the process. The ultimate carcinogenic form of most, i f not a l l , chemical carcinogens i s a strong e l e c t r o p h i l i c reactant (Poland and Kende, 1977; M i l l e r , 1970) which i s able to acquire electrons from n u c l e o p h i l i c atoms i n c e l l u l a r components ( e s p e c i a l l y the informational macromolecules such as nucleic acids and pr o t e i n s ) . This i s i n contrast to the fac t that the precarcinogenic forms of substances able to induce cancers may have very l i t t l e i n common i n the way of s t r u c t u r a l features. The common factor i s the strong e l e c t r o p h i l i c i t y of the ultimate reactant to which they are converted. Some carcinogenic a l k y l a t i n g and ac y l a t i n g agents do not require metabolic a c t i v a t i o n , since they are strong e l e c t r o p h i l i c reactants as such, and require only d i s s o l u t i o n i n water to produce carcinogenic species. Some chemicals do not give r i s e to ultimate e l e c t r o p h i l i c d e r i v a t i v e s , but appear to be able to cause development of tumours i n experimental animals nonetheless. These compounds may not be i n i t i a t o r s of tumours i n a s t r i c t sense, but modifiers of secondary responses to i n i t i a t i o n events (promotion, immune response) that permit the development of tumours from previously i n i t i a t e d c e l l s . In a d d i t i o n to the common ultimate e l e c t r o p h i l i c i t y of the diverse pantheon of chemical carcinogens, i t has been shown that whenever the reactive form of the carcinogen can be brought into contact with DNA i n mutagenicity test systems, they show mutagenic a c t i v i t y . When metabolic a c t i v a t i o n i s taken into consideration, approximately 90-95% of chemical carcinogens e x h i b i t mutagenicity i n a v a r i e t y of test systems (Hollaender, -5-1978; McCann, et a l . , 1975; McCann and Ames, 1976; Purchase, et a l . , 1978; Sugimura, et a l . , 1976). Most non-carcinogenic analogs and metabolites have l i t t l e or no mutagenicity, and thus there i s a strongly formal p o s i t i v e r e l a t i o n s h i p between chemical carcinogenicity and mutagenicity. SITES OF ELECTROPHILIC ATTACK IN VIVO One goal of cancer research i s to determine what the macromolecular target of chemical carcinogens i s with respect to the c e l l . Because of the strong r e l a t i o n s h i p between carcinogenicity and mutagenicity, the i n i t i a t i o n step i s generally regarded as involving a mutation or mutations i n DNA, and these mutations may be prevented, or they may be " f i x e d " by various DNA •repair mechanisms. Because of the r a p i d i t y of the i n i t i a t i o n step and the persistence of the i n i t i a t e d state, the r o l e of DNA i s e s p e c i a l l y a t t r a c t i v e . However, chemical carcinogens bind to RNA and protein i n vivo as w e l l , so that epigenetic mechanisms of i n i t i a t i o n can be invoked (i.e_. , stable, h e r i t a b l e states of aberrant c e l l behaviour without precursor le s i o n s i n DNA (Mintz, 1978)). The scenario i s that a carcinogen could bind to a c r i t i c a l s i t e i n DNA, and that RNA and protein-bound carcinogen d e r i v a t i v e s could then induce promotion (Berenblum, 1974; Scribner and Boutwell, 1972). While there i s no experimental evidence to ind i c a t e that t h i s i s true, phorbol myristate acetate (a potent promoter) does not bind to c e l l u l a r macromolecules ±n vivo (Weinstein, e_t a l . , 1979). There i s very l i t t l e data to explain the p l e i o t r o p i c responsed to t h i s agent i n mouse skin or c e l l cultures, and the mechanisms of promotion. Therefore, both genetic and epigenetic mechanisms (with the possible p a r t i c i p a t i o n of v i r a l information) must be considered i n studies on i n i t i a t i o n and promotion i n chemical carcinogenesis. -6-In the case of n u c l e i c acids, many n u c l e o p h i l i c s i t e s are open to attack by carcinogens i n vivo. The most n u c l e o p h i l i c base i s guanine, and 2 6 may be e l e c t r o p h i l i c a l l y substituted at the N-3, N-7, N , 0 and C-8 atoms. Adenine, although le s s p r e f e r e n t i a l a s i t e , i s attacked at N - l , N-3 and 4 2 N-7, and cytosine at N-3 and 0 . Thymine may be substituted at 0 and 4 0 . V i r t u a l l y any s i t e i s open to attack, and even the n u c l e i c acid back-bone may be substituted at phosphate oxygen atoms. In each case, a covalent bond i s formed between the e l e c t r o p h i l i c carbon or nitrogen r a d i c a l present i n the ultimate carcinogen and the n u c l e o p h i l i c s i t e i n the n u c l e i c a c i d . In proteins, the sulphur atoms of methionine and cysteine, the r i n g nitrogen of h i s t i d i n e , and tyrosine (at two positions) may be substituted by carcinogenic e l e c t r o p h i l e s (reviewed by M i l l e r and M i l l e r , 1979). CARCINOGENIC PROCESS The induction of malignant tumours i s a m u l t i f a c t o r i a l process that has a multi-step evolution. There i s a progression of complex i n d i v i d u a l reactions and processes that are thought to lead to the f i n a l overt cancer i n man and animals. Each of these reactions and conditions may be subject to c o n t r o l by a number of possible modifying f a c t o r s . 1) A chemical carcinogen introduced into an i n vivo system may be activated to an ultimate carcinogen, e i t h e r n o n - s p e c i f i c a l l y or by s p e c i f i c enzyme systems. This reaction may be modified by biochemical d e t o x i f i c a t i o n and elimination reactions. 2) The ultimate carcinogen may react with targets i n the c e l l . Although RNA and protein targets have not been ruled out, i t i s postulated that the relevant target i s DNA. This i n t e r a c t i o n i s subject to stereochemical conditions and competitive i n h i b i t i o n that are not yet -7-well-def ined. The a l t e r e d relevant macromolecule (e.j*., DNA) may then be repaired and restored by repair enzyme systems whose s u s c e p t i b i l i t y to error may play a s i g n i f i c a n t part i n a l t e r i n g the c e l l ' s conversion to the malignant state. 3) The a l t e r e d carcinogen receptor i s duplicated so that i t becomes subsequently immune to the operation of repair systems. 4) C e l l s containing the abnormal receptor w i l l d ivide to form extremely malignant tumours, or benign tumours, depending on the i n i t i a l c e l l u l a r targets of the carcinogenic stimulus. If primordial stem c e l l s are the target of carcinogenic s t i m u l i , then r e l a t i v e l y malignant tumours (i..e_., metastasizing tumours, r e l a t i v e l y independent of control by surrounding tissue) w i l l r e s u l t . If r e l a t i v e l y w e l l - d i f f e r e n t i a t e d c e l l s undergoing only the f i n a l stages of maturation are subjected to the carcinogenic stimulus, then r e l a t i v e l y benign tumours (1.e_., non-metastasizing, slow-d i v i d i n g tumours r e l a t i v e l y dependent on the influence of surrounding tissues) w i l l l i k e l y r e s u l t . These benign tumours would contain no c e l l s l e s s d i f f e r e n t i a t e d than t h e i r normal counterpart t i s s u e (1. e_., that t i s s u e targeted by the carcinogenic stimulus). Tumours of intermediate l e v e l s of d i f f e r e n t i a t i o n would be the expected r e s u l t i f c e l l s of intermediate d i f f e r e n t i a t i o n were the targets of oncogenic stimulus (Pierce, 1974; Pierce, et a l . , 1978)(Fig. 1). Benign tumours would tend to appear e a r l i e r than malignant tumours, since, being more d i f f e r e n t i a t e d than stem c e l l s , they would not be so "f o r e i g n " to the t i s s u e i n which they appear, and a threshold number would not need to accumulate to produce a tumour (Grobstein and Z w i l l i n g , 1953; Pierce, et a l . , 1978). Undifferentiated malignant stem c e l l s would have a prolonged l a t e n t period, but would be l a r g e l y r e f r a c t o r y to environmental stimulus when a c r i t i c a l number had been produced. -8-Figure 1 D i f f e r e n t i a t i o n and Neoplasia mitotically active —^postmitotic differentiated malignant cells-CO CO 0 c "0 CD o C U o 0 I c 0 CD O C \j a u stem cell benign cells Q ^ r ^ I A 1 , ^ grade 3 or 4 *x L^~pL^ malignancy i ' CO CO 0 c 0 CD O C U L-o u Q grade 2 malignancy i i -0-3 CD 3 C -H O co r C T Q ) benign tumour tissue renewal o normal senescent differentiated cell - from Pierce, et a l . , 1978 - 9 -DNA-chemical i n t e r a c t i o n i s , as shown here, f a r removed from the f i n a l observation of gross tumours. The model system developed to explain l i v e r carcinogenesis proposed that the " i n i t i a t i o n " process (or early, rapid events occurring i n hours or days) leads to the appearance of new c e l l populations. This model system has been re f e r r e d to as "neoplastic c e l l u l a r evolution" (Farber, et a l , , 1974). A s i n g l e dose of the potent l i v e r carcinogen diethylnitrosamine (DEN) administered during l i v e r c e l l d i v i s i o n following p a r t i a l hepatectomy w i l l not induce c e l l u l a r a l t e r a t i o n s leading to progressive c e l l populations, and hence to l i v e r cancer, u n t i l several weeks following i n i t i a l c e l l damage (Solt and Farber, 1977; Ogawa, et a l . , 1979; F i a l a , et a l . , 1972; Laishes, et a l . , 1978). Since a l i n k must be found between the i n i t i a t e d damage to the c e l l and f i n a l appearance of the tumour, DNA has been proposed as the relevant c e l l u l a r target of carcinogens. In general, the somatic mutation theory of carcinogenesis proposes that chemical carcinogens modify c e l l u l a r DNA i n such a way as to produce a non-lethal, h e r i t a b l e DNA change expressed ultimately as a tumour when such c e l l s are viewed as a group (Foulds, 1969; Weinstein, at a l . , 1979). DNA REPAIR When chemical carcinogens i n t e r a c t with DNA there may be a d i r e c t chemical depurination, either spontaneous or enzyme-mediated, that leads to d i s r u p t i o n of the sugar-phosphate backbone and thus single-strand breaks. This may occur a f t e r treatment with a l k y l a t i n g agents that a l t e r covalent and hydrophobic binding c h a r a c t e r i s t i c s of bases (e.£., nitrogen mustard). For the most part, purine bases are a l k y l a t e d , although pyrimidines may be a l k y l a t e d to a l e s s e r extent (Lawley, 1976). However, t h i s "spontaneous" hydrolysis of DNA strands i s not the - 1 0 -FIGURE 2 DNA duplex containing distortions caused by chemicals \ A. Excision repair: Incision (damage-specific endonuclease B. Post-replication repair: Replication and gap formation Repair replication (DNA polymerase) Recombination (Nuclease(s) + ?) Excision and rejoining (5'-<-3' exonuclease + polynucleotide ligase) Repair r e p l i c a t i o n and rejoining (DNA polymerase + ligase) Replication Replication -v^ w^^— + -11-only method for strand breakage. Enzyme-mediated removal of alk y l a t e d bases from DNA has also been proposed (Ikegami, et a l . , 1970). This c e l l -mediated excision of damaged DNA segments i s thought to be part of a process of DNA r e p a i r - an attempt by the c e l l to maintain true DNA copies for immediate use and transmission to daughter c e l l s . In most eukaryote c e l l s there are three p r i n c i p a l mechanisms for the repair of DNA damage: photo-r e a c t i v a t i o n , e x c i s i o n repair and p o s t - r e p l i c a t i o n repair (Fig. 2). Photo-r e a c t i v a t i o n r e p a i r requires a s i n g l e enzyme to bind and p h o t o c a t a l y t i c a l l y cleave thymine dimers. P o s t - r e p l i c a t i o n repair includes a l l those processes whereby errors i n DNA that p e r s i s t a f t e r nuclear r e p l i c a t i o n has taken place and damaged areas i n DNA strands have been bypassed by the r e p l i c a t i o n enzymes i n such a way as to leave gaps i n daughter strands. The process i s l a r g e l y t h e o r e t i c a l i n mammalian c e l l s . Damage may be bypassed and " l e f t behind"by means of DNA recombination (Hanawalt, 1975). Ex c i s i o n repair i s an enzyme-mediated process by which the c e l l repairs damage to DNA by removing the a f f e c t e d portion and replacing i t with the correct structure before undergoing d i v i s i o n . The covalent or non-covalent binding of chemical to DNA may lead to l o c a l d i s t o r t i o n that can serve as a s i t e for endonuclease attack or as a p o s i t i o n for "spontaneous" h y d r o l y t i c f i s s i o n of the DNA chain (Paul, et a l . , 1971). However, where coincident single-strand breaks take place, a f a i t h f u l reconstruction of the o r i g i n a l structure i s u n l i k e l y , due to the absence of an i n t a c t complementary strand acting as a template for poly-merase a c t i v i t y . DNA REPAIR INHIBITORS The process of i n h i b i t i o n of DNA repair i n mammalian c e l l s i s s t i l l -12-rather poorly understood. According to Cleaver (1974) c a f f e i n e i s an out-standing i n h i b i t o r of e x c i s i o n r e p a i r i n ba c t e r i a , along with chloroquine, quinacrine, chloramphenicol and a c r i f l a v i n e . In the b a c t e r i a l case, t h i s i s due to i n h i b i t i o n of repair r e p l i c a t i o n at concentrations that do not a f f e c t normal semi-conservative r e p a i r . The precise method by which t h i s i s done i s unknown, although binding to single-stranded DNA as well as i n h i b i t i o n of DNA polymerases, endonucleases and exonucleases has been implicated, and some cell-mediated binding of the i n h i b i t o r to DNA may be the necessary p r e r e q u i s i t e (reviewed by Kihlman, 1977). In t e r e s t i n g l y , c a f f e i n e i s not included among those agents that are able to i n h i b i t DNA repair r e p l i c a t i o n or e x c i s i o n of UV dimers i n mammalian c e l l s (although i t s e f f e c t on the e x c i s i o n of other types of chemical le s i o n s i n not known). Compounds observed to i n h i b i t r e pair r e p l i c a t i o n i n mammalian c e l l s w i l l also i n h i b i t semi-conservative DNA r e p l i c a t i o n . These compounds are a c r i f l a v i n e , c r y s t a l v i o l e t , actinomycin D, chloroquine and iodoacetate, and they appear to act by some mechanism that includes covalent or non-covalent binding to DNA (Kihlman, 1977). However, c a f f e i n e has been shown to i n h i b i t the g a p - f i l l i n g associated with p o s t - r e p l i c a t i o n r e p a i r i n rodent c e l l s at concentrations that have l i t t l e e f f e c t on semi-conservative DNA r e p l i c a t i o n (Cleaver and Thomas, 1969; Fujiwara, 1975; N i l s s o n and Lehmann, 1975). In human c e l l s , c a f f e i n e has l i t t l e e f f e c t on normal human f i b r o b l a s t s , but strongly i n h i b i t s gap-f i l l i n g (Buhl and Regan, 1975; Lehmann, et a l . , 1975) and the a b i l i t y to synthesize DNA segments of normal s i z e at long times a f t e r i r r a d i a t i o n (Buhl and Regan, 1974) i n cultured xeroderma pigmentosum skin f i b r o b l a s t s . - 1 3 -MEASUREMENT OF DNA DAMAGE AND REPAIR The only true test f o r carcinogenicity of chemicals i s the induction of tumours i n whole animals. Because of the high cost i n time and e f f o r t involved i n assays of t h i s type, short-term i n vivo and i n v i t r o bioassays have been developed to assess carcinogenic r i s k by measuring the a b i l i t y of compounds to induce primary events i n c e l l s where those primary events can be linked to the causal, i n i t i a t i n g events of carcinogenesis. DNA damage and i t s subsequent repair have been implicated as j u s t such events ( M i l l e r and M i l l e r , 1969; Kihlman, 1977; Weinstein, et a l . , 1979; Foulds, 1969). 1) A l k a l i n e sucrose gradient sedimentation: The f i n a l DNA l i g a t i o n and chromosomal reorganization steps of DNA repair, as well as the o r i g i n a l carcinogen or enzyme-mediated cleavage step, can be monitored by examining the single-strand molecular weight d i s t r i b u t i o n of DNA i n a l k a l i n e sucrose gradient zone sedimentation p r o f i l e s . O r i g i n a l l y adapted to detect DNA fragmentation i n cultured c e l l s by i o n i z i n g r a d i a t i o n (Lett, et a l . , 1967; McGrath and Williams, 1966) i t has been modified to assay chemical-induced fragmentation i n cultured c e l l s (Laishes and St i c h , 1973; St i c h and Laishes, 1973; Andoh and Ide, 1972; Coyle and Strauss, 1970) and mammalian c e l l s i n vivo (Cox, et a l . , 1973; Laishes, et a l . , 1975; Koropatnick and S t i c h , 1976; Abanobi, et a l , , 1977). This procedure w i l l r e g i s t e r breaks i n the a l k a l i - l a b i l e linkages i n the backbone of the DNA structure, although DNA i s s t a b i l i z e d i n t h i s respect due to a lack of 2'-hydroxyl groups on the ribose. We have recently developed a system f o r jLn vivo determination of DNA damage and repair by -14-chemical agents using mouse g a s t r i c e p i t h e l i a l c e l l s as a test system (Koropatnick and Stich , 1976; S t i c h and Koropatnick, 1977). In t h i s method the terms "single-strand break" and "fragmentation of DNA" are used oper-a t i o n a l l y and r e f e r to DNA that sediments slowly i n comparison with control DNA sedimented under i d e n t i c a l conditions (Abanobi, et a l . , 1977). Repair of DNA i s measured by a s h i f t i n sedimentation p r o f i l e peaks, from regions i n d i c a t i n g DNA fragmentation to those approximating p r o f i l e peaks derived from untreated co n t r o l DNA, i n the period following chemical administration. This r e p a i r involves, not only the r e j o i n i n g of si n g l e DNA strands, but also repair-type processes that involve considerably larger DNA segments than those concerned with single-strand r e j o i n i n g , possibly reorganization of chromatin (Elkind and Kamper, 1970). Therefore, substances that may be shown to i n h i b i t r e p a i r (as monitored on gradient p r o f i l e s ) may i n h i b i t complex chromatin reorganizational steps as well as simple l i g a t i o n . 2) E x c i s i o n of a r y l and a l k y l adducts from DNA: A r y l a t i o n or a l k y l a t i o n of macromolecular targets i n c e l l s occurs with almost a l l carcinogenic species (see above). Both a l k y l a t i n g and a r y l a t i n g agents w i l l modify DNA to produce modified bases, which may then be excised by enzymatic or non-enzymatic processes within the c e l l . Benzo(a)pyrene adducts are excised from DNA i n vivo i n the case of primary hamster embryo c e l l s (Ivanovic, jst a l . , 1978), human lung c e l l cultures (Feldman, et a l . , 1978) and mouse embryo f i b r o b l a s t s (Brown, et a l , , 1979). In addition, methylated s i t e s i n mammalian DNA produced by a v a r i e t y of chemical agents administered i n vivo have been shown to be excised over a period of 24 to 72 hours, although a s i g n i f i c a n t amount (10-30% of -15-o r i g i n a l methylated s i t e s ) appears to be r e s i s t a n t to excision (Pegg, 1978; Montesano, e^ t a l . , 1979; Swann and Mace, 1980; Kleihues and Margison, 1974, 1976; Goth and Rajewsky, 1974; N i c o l l , et a l . , 1977; Thorgiersson, et a l . , 1980). In general, cultured c e l l s are treated with r a d i o a c t i v e l y - l a b e l l e d a r y l or a l k y l a t i n g agents by incubation i n medium for from 12 to 24 hours (Ivanovic, et a l . , 1978) and mammals are injected subcutaneously or i n t r a p e r i t o n e a l l y and allowed to remain untreated for 12 to 18 hours (Thorgeirsson, e_t a l . , 1980). DNA is. i s o l a t e d from c e l l s or tissues of i n t e r e s t at up to 72 hours following the procedures described, and the amount of radioactive l a b e l covalently bound to the DNA i s used to estimate the l e v e l of a r y l or a l k y l a t i o n of DNA. Since 0-6 a r y l and a l k y l guanines may be more important than other adducts i n mutagenic and carcinogenic e f f e c t s (Loveless, 1969; Gerchman and Ludlum, 1973) a r e f i n e -ment of t h i s procedure has been to hydrolyse DNA by acid or enzymes, and separate purine bases on Sephadex G-10 columns to determine the extent of 0-6 methylation s p e c i f i c a l l y (Kleihues and Magee, 1973). FACTORS AFFECTING CHEMICAL CARCINOGENESIS In view of the multi-step nature of mammalian induction of cancer, modification of the a b i l i t y of chemical carcinogens to cause tumours may take place at two d i f f e r e n t l e v e l s . F i r s t , environmental factors may a f f e c t the primary " i n i t i a t i o n " steps i n carcinogenesis that take place during the period immediately following exposure to carcinogens (up to several days) and, second, they may a f f e c t the behaviour of c e l l s that already carry the c r i t i c a l change that places them on the path toward becoming tumour c e l l s . The factors f a l l i n g into the f i r s t group include those that a f f e c t the -16-a c t i v a t i o n of precarcinogens, the i n t e r a c t i o n of ultimate carcinogens with macromolecular targets i n c e l l s , and those that a l t e r the c e l l u l a r response to i n t e r a c t i o n of carcinogens (e.£. , c e l l d i v i s i o n , DNA r e p a i r ) . The factors that are included i n the second group are those that modulate programs of gene expression to induce c l o n a l growth of i n i t i a t e d c e l l s (Weinstein, et^ a l . , 1979) or i n h i b i t t h e i r growth (Higginson, 1979) and a host of i l l - d e f i n e d f a c t o r s , including s o c i o l o g i c a l c h a r a c t e r i s t i c s (e.j*., "age at f i r s t intercourse" or "age at f i r s t marriage" with respect to the incidence of adult mammary tumours) that have a complex e f f e c t on tumour induction (reviewed by Higginson, 1979). 1) Factors a f f e c t i n g the i n i t i a t i n g a ction of carcinogens A number of studies have demonstrated that i t i s possible to protect against the carcinogenic e f f e c t s of chemical carcinogens by inducing increased mixed function oxygenase a c t i v i t y . P o l y c y c l i c hydrocarbons (either carcinogenic or non-carcinogenic) have been shown to i n h i b i t the occurrence of hepatic cancer r e s u l t i n g from the feeding of 3'-methyl-4-dimethylaminobenzene (Richardson, .et a l . , 1952; M i l l e r , et a l . , 1958). Also, polyaromatic hydrocarbon (PAH) inducers of mixed function oxygenases have been shown to markedly reduce the incidence of tumours of the l i v e r , mammary gland, ear duct, and small i n t e s t i n e i n rats fed 2-acetylamino-fluorene or 7-fluoro-2-acetylaminofluorene ( M i l l e r , et a l . , 1958). Recently, i t has been shown that i t i s possible to protect against the carcinogenic e f f e c t s of a number of other carcinogens, including urethane, dimethylbenz(a) anthracene (DMBA), a f l a t o x i n , bracken fern, and benzo(a)pyrene (Table 1). In each case, the modifying compounds used are potent inducers of mixed function oxygenase a c t i v i t y . H us-er M (t> Inhibition of carcinogenesis by Induction of microsomal enzyme a c t i v i t y Carcinogen Inducer Species Organ References 3'-methyl-4-dimethyl-aminoazobenzene Polycyclic hydrocarbons a-benzene hydrochloride rat l i v e r M i l l e r , e_t a l . , 1958 Richardson, et a l . , 1972 Urethane g-naphthoflavone mouse . lung Yamamoto, e_t a l . , 1971 7,12-dimethylbenz-(a)anthracene Polycyclic hydrocarbons rat breast Huggins, et a l . , 1964 Aflatoxin Phenobarbltol rat breast McLean and Marshall, 1971 • Bracken fern carcinogen Phenothiazine rat small intestine bladder Pamukcu, et a l . , 1971 Benzo(a)pyrene g-naphthoflavone mouse lung, skin Wattenberg and Leong, 1970 - 1 8 -The paradoxic nature of t h i s induction e f f e c t r e s u l t i n g i n reduced carcinogenesis requires some explanation. I t can be argued that i f a compound i s activated by an enzyme system to a carcinogenic form, then enhancement of that system would r e s u l t i n greater induction of cancers. This would be the case i n s i t u a t i o n s involving an e f f e c t i n which there i s a s ubstantial "threshold l e v e l " for the carcinogenic agent, below which tumours would not be i n i t i a t e d , i r r e s p e c t i v e of length of exposure. However, there appears, i n the case of chemical carcinogens, to be either no threshold or a very low threshold (Dipaolo, et a l . , 1971). Therefore, i t might be expected that slow a c t i v a t i o n would r e s u l t i n greater carcinogenic e f f e c t than rapid a c t i v a t i o n . I t could be that the carcinogenic species must be applied at a c r i t i c a l time or times i n the c e l l c ycle. Also, there i s l e s s l i k e l i h o o d of l o s s of activated species (due to carcinogen i n t e r a c t i o n with non-specific c e l l u l a r targets) which might be expected to occur when an excess amount of carcinogenic species i s produced over that most e f f e c t i v e for the number of c r i t i c a l binding s i t e s (Wattenberg, 1974). In addition, the u l t i m a t e l y r e a c t i v e carcinogenic species are only transient compounds i n a pathway employed by the c e l l to detoxify those compounds. Thus, inducers of the mixed function oxygenases induce d e t o x i f i c a t i o n (usually by r i n g hydroxylation) as well as a c t i v a t i o n (usually by nitrogen hydroxylation) as i s the case for aromatic amines ( M i l l e r and M i l l e r , 1969). In addition to enhancement of mixed function oxygenase a c t i v i t y , c e r t a i n conditions may decrease enzyme a c t i v i t y . Administration of some chemicals depresses enzyme action (diethyldithiocarbamate to reduce metabolism of dimethylnitrosamine (Abanobi, e_t a l . , 1977) and coumarin or a-angelicalactone to reduce metabolism of benzo(a)pyrene and benzo(a)pyrene-induced neoplasia of forestomach (Wattenberg, et a l . , 1979). Also, n u t r i t i o n a l states may also decrease enzyme a c t i v i t y --19-starvation of Sprague-Dawley rats for 24 hours r e s u l t s i n almost t o t a l loss of mixed function oxygenase a c t i v i t y i n small i n t e s t i n e (Wattenberg, 1971) as i s the case for rats fed a f a t - f r e e diet (Wattenberg, 1974). P u r i f i e d d i e t s also resulted i n loss of a c t i v a t i o n a b i l i t y i n small i n t e s t i n e (Wattenberg, 1974). Thus, most, i f not a l l , of the mixed function oxygenase a c t i v i t y of the small i n t e s t i n e i s caused by an exogenous inducer or inducers. The a b i l i t y to enhance the production of ultimate carcinogens or to stimulate t h e i r deactivation i s not n e c e s s a r i l y confined to separate modifying compounds. The p y r o l y s i s products of tryptophan -norharman (9H-pyrido-(3,4-b)-indole), harman, and 3-methyl indole (skatole) -are capable of i n h i b i t i n g the whole metabolic pathway of BP, including both the conversion of BP to ultimate carcinogenic form, and production of water soluble metabolites from the ultimate carcinogenic species (Fujino, est a l . , 1978; Matsumoto, e_t a l . , 1977). On the other hand, i n i n v i t r o systems employing an a r t i f i c i a l a c t i v a t i o n mixture with S-9 microsomes derived from rat l i v e r , high concentrations of harman or norharman r e s u l t i n a decrease i n mutagenic p o t e n t i a l , while low con-centrations produce an increase i n mutagenic p o t e n t i a l (Fujino, et a l . , 1978). This i s due to a d i f f e r e n t i a l i n h i b i t i o n of a c t i v a t i n g or deactivating enzyme systems, since harman and norharman are more ac t i v e i n depressing the deactivating a b i l i t y of enzymes that convert hydrophobic metabolites to h y d r o p h i l i c ones (R^)(Matsumoto, et a l . , 1977) than i n depressing the a c t i v a t i n g a b i l i t y of enzymes that convert the parent compound to the oxygenated intermediate (R-?)• L ° w concentrations of i n h i b i t o r may therefore exert p a r t i a l i n h i b i t i o n of R^ and complete i n h i b i t i o n of R^, r e s u l t i n g i n accumulation of carcinogenic BP* intermediates: BP conjugates -20-2) I n h i b i t i o n of non-enzymatic formation of ultimate carcinogens: The best known example of a compound preventing the non-enzymatic production of carcinogenic species i s the case of n i t r o s a t i o n of n i t r o -satable compounds i n acid conditions. Methylguanidine may be reacted with n i t r i t e at low pH to produce mutagenic and carcinogenic compounds, including methylnitrosourea and N-methyl-N'-nitro- N-nitrosoguanidine (Lo and S t i c h , 1978). Ascorbic acid i s able to prevent n i t r o s a t i o n by p r e f e r e n t i a l l y reducing the nitrous a c i d (Mirvish, et a l . , 1972; Synnot, et a l . , 1975) i n a reaction that i s well understood (Dahn, e_t a l . , 1960). Other reducing agents, including cysteine, cysteamine, and propyl g a l l a t e are also able to i n h i b i t the formation of these n i t r o s a t i o n products, which have been implicated as being b i o l o g i c a l l y a v a i l a b l e by reaction of n i t r i t e with various compounds i n the a c i d conditions of the stomach (Lo and S t i c h , 1978). 3) I n h i b i t i o n of action of ultimate carcinogens: Once the r e a c t i v e form of a carcinogenic species has been created, agents that a l t e r the a c t i v a t i o n of precarcinogens by enzymatic means w i l l have l i t t l e e f f e c t on carcinogenic p o t e n t i a l . I f the reactive species i s s u f f i c i e n t l y e l e c t r o p h i l i c , weak nucleophiles (such as water and protein) may exert a p r o t e c t i v e e f f e c t by acting as n o n - c r i t i c a l "targets" ( M i l l e r and M i l l e r , 1979). However, carcinogens of l e s s e r e l e c t r o p o t e n t i a l may be i n h i b i t e d i n t h e i r a c t i o n by "trapping" of the r e a c t i v e e l e c t r o p h i l i c species through administration of agents that a carcinogen reduces p r e f e r -e n t i a l l y over c r i t i c a l c e l l u l a r macromolecules (Bartsch, et a l . , 1972 1973; -21-M i l l e r and M i l l e r , 1969, 1974, .1976; M i l l e r , 1970; Scribner and Naimy, 1973) . Reducing agents such as butylated hydroxyanisole and butylated hydroxytoluene (Grantham, et a l . , 1973), ascorbic acid (Guttenplan, 1977; Pipkin, et a l . , 1969; Schlegel, et a l . , 1969), selenium (Jacobs, et a l . , 1977), cysteamine (Marguardt, et a l . , 1974) and d i s u l f i r a m (Schmall and Kruger, 1972) have also been shown to i n h i b i t carcinogenesis i n animal test systems, or mutagenesis i n b a c t e r i a l asseys, i f they are applied concurrently with carcinogens. Chemical carcinogens of every type capable of producing r e a c t i v e e l e c t r o p h i l e s are susceptible (Rosin and S t i c h , 1978). Sodium Ascorbate While synthetic chemicals and those found i n exotic foods a t t r a c t most att e n t i o n when carcinogenic and mutagenic hazards of environmental agents are assessed, chemicals which are an i n t e g r a l part of c e l l u l a r metabolism or are of v i t a l n u t r i t i o n a l value are often passed over. Ascorbic a c i d (vitamin C) i s consumed i n large doses by humans (Lewin, 1976; Pauling, 1970). I t has been widely used, and i s generally regarded as safe, as a food a d d i t i v e to prevent formation of carcinogenic n i t r o s a t i o n products, and to prevent "browning" by a i r oxidation ( Cardesa, et a l . , 1974; F i d d l e r , et a l . , 1978; Lo, et a l . , 1978; Mirvish, et a l . , 1975, 1976). Sodium ascorbate has a capacity for i n a c t i v a t i n g b a c t e r i o -phage (Murata, 1975) and mammalian vi r u s e s (Jungeblut, 1935,1939), possibly by the l i b e r a t i o n of peroxide from oxidation of ascorbate since catalase provides protection (Schwerdt and Schwerdt, 1975; Wong, et a l . , 1974), or by the a c t i o n of monodehydroascorbate r a d i c a l produced during the oxidation process ( B i e l s k i , est: ' a l . , 1975). Sodium ascorbate has been shown to i n h i b i t the formation of ultimate carcinogens by acting as a - 2 2 -preferred s i t e of oxidation i n the oxidation-reduction reaction that reduces peroxide by the a c t i o n of peroxidase and an electron donor (Floyd, et a l . , 1976) as well as acting as a "trapping" agent by p r e f e r e n t i a l reduction of ultimate e l e c t r o p h i l e s (see above). In addition, sodium ascorbate has been shown to have antimutagenic properties (Guttenplan, 1978; Rosin and S t i c h , 1979; Marquardt, et a l . , 1977) and anti-cancer properties (Cameron and Campbell j 1974; Pipkin, e_t 'al;-,. 1969; Rainerl-and'Weisburget, 1975) . However, ascorbic a c i d has been reported to exert e f f e c t s other than the p r o t e c t i v e ones outlined above. Sodium ascorbate has been shown to increase the frequency of s i s t e r chromatid exchanges (a s e n s i t i v e i n d i c a t o r of DNA damage) (Speit, et a l . , 1980). I t has been shown to fragment DNA and induce chromosome aberrations i n cultured c e l l s ( S t i c h e_t a l . , 1976) as well as mutations i n S. Typhimurium (Stich, et a l . , 1978). Copper(II) as well as Mn(ll) and F e ( l l ) and (III) enhance the chromosome-damaging e f f e c t of ascorbate by catalyzing the autoxidation of ascorbate to form H2O2, since catalase reduces or abolishes the e f f e c t s (St i c h , et a l . , 1979). Ascorbate w i l l also i n h i b i t m itosis (Stich, et a l . , 1979). Sodium ascorbate has also been shown to convey an increased s e n s i t i -v i t y to c e l l s to treatment with carcinogens and mutagens, causing c e l l death and decreased incorporation of thymidine by unscheduled DNA synthesis at lower carcinogen and mutation concentrations than those > normally required (St i c h , et a l . , 1978, 1979). Sodium ascorbate w i l l also enhance the mutagenicity of N—hydroxy-2-acetylaminofluorene (rhorgeirsson, et a l . , 1980).' TYPES OF CARCINOGENS Chemical • carcinogens may be divided into three classes: : primary or ultimate carcinogens, secondary or precarcinogens, and cocarcinogens (promoting agents and f a c t o r s ) . 1) Primary carcinogens: These chemicals are b i o l o g i c a l l y a c t i v e carcinogens without recourse to metabolic a c t i v a t i o n . They may i n t e r a c t d i r e c t l y with tissues and a l l components to y i e l d modified macromolecules that may produce preneoplastic c e l l s . Primary carcinogens include various types of a l k y l a t i n g agents, such as nitrogen and sulphur mustards, sulphonic esters and sulfones, ethylene imines and imides, strained or a, g-unsaturated lactones, epoxides, peroxides and c h l o r o a l k y l ethers ( M i l l e r , 1978) . They are i n t h e i r f i n a l r e a c t i v e form as administered, and take part i n S^2 (su b s t i t u t i o n , n u c l e o p h i l i c , bimolecular) reactions with c r i t i c a l macromolecules i n vivo (Price, et a l . , 1969; Ross, 1962; Shapiro, 1969). They are not generally strong carcinogens - due, presumably, to the interference of n o n - c r i t i c a l nucleophiles (water, proteins) i n vivo which makes i t necessary to admini-ster large doses at l o c a l t i s s u e s i t e s to induce tumours ( M i l l e r , 1978). Much of the e l e c t r o p h i l e may be disposed of before entry into target c e l l s . Most of the inorganic carcinogens (e.g., metals) are e l e c t r o p h i l e s i n t h e i r i o n i c forms (Forst and Haro, 1969). 2) Precarcinogens: Most carcinogenic chemicals, synthetic or natural, f a l l i nto t h i s c l a s s , so structures d i f f e r widely. They are usually, chemically and b i o l o g i -c a l l y i n e r t with respect to target macromolecules, and require spontaneous -24-or host-mediated and c o n t r o l l e d a c t i v a t i o n reactions to convert them to t h e i r ultimate r e a c t i v e species. Where they are spontaneously converted to primary carcinogens by hydrolysis, they may be a c t i v e for a broad range of organs and species, due to the s i m p l i c i t y and u n i v e r s a l i t y of t h e i r a c t i v a t i o n . However, when s p e c i f i c host-controlled biochemical a c t i v a t i o n i s required, there can be great d i v e r s i t y i n a c t i v i t y from organ to organ, i n d i v i d u a l to i n d i v i d u a l or species to species. A c t i v a t i o n depends on s p e c i f i c enzyme systems (Kasper, 1974; Mukhtar, et a l . , 1979; Grover, et a l . 1974). This i s a possible reason why a chemical which may be carcinogenic or mutagenic i n one system may be non—carcinogenic or mutagenic i n an organism or system where the required a c t i v a t i n g system i s absent. 3) Cocarcinogens: These are agents that cannot be used to induce tumours, but w i l l potentiate the tumour-inducing a c t i o n of ultimate or precarcinogens. Complex mixtures such as tobacco smoke are thought to contain large amounts of cocarcinogens, but small r e l a t i v e amounts or precarcinogens (Wynder and Hoffman, 1967; Van Duurnen, et a l . , 1973; S a f f i o t t i , 1969). Croton o i l i s the best known cocarcinogen (and extract of croton r e s i n ) . I t promotes skin tumour formation a f t e r the a p p l i c a t i o n of a carcinogenic p o l y c y c l i c aromatic hydrocarbon, such as 2-methylcholanthrene (Boutwell, 1974; Sivak and Van Duurnen, 1971). -25-SPECIFIC CARCINOGENS 1) Nitrogen compounds: Two compounds that are p o t e n t i a l a l k y l a t i n g agents are methylnitro-sourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Both are potent carcinogens (Table 2). As a c l a s s , the alkylnitrosamides are impressive because of the wide v a r i e t y of tissues i n which they;' induce tumours, and by the s u s c e p t i b i l i t y to low doses of most of the species i n which they have been tested (Lowry and Richardson, 1976; Magee, et a l . , 1976) . This i s presumably due to the f a c t that both require only reaction with c e r t a i n ubiquitous nucleophiles (water, t h i o l s , amino groups) to be converted into e l e c t r o p h i l e s that can s u c c e s s f u l l y a l k y l a t e c r i t i c a l c e l l u l a r nucleophiles ( F i g . 3). Both of these compounds are found among the n i t r o s a t i o n products of methylguanidine which may be produced by reaction i n the a c i d conditions found i n human stomach (Hedler and Marquardt, 1968; Heath, 1962; Montesarro and Magee, 1970) or at neutral pH by alimentary bacteria (Badger, 1962). Because of the ubiquitous nature of these n i t r o s a t a b l e compounds, they have been implicated i n the production of human carcinomas (Kotin and Falk, 1963; L r j i n s k y and Ross, 1967). 2) Dialkylnitrosamines: The N-nitrosod-ialkylamine's'comprise .a' large c l a s s of compounds, some of which are r o u t i n e l y detectable i n the environment (Hedler and Marquardt, 1968). Dimethylnitrosamine (DMN) i s the simplest of these compounds. They are le s s v e r s a t i l e than the nitrosoamides because enzymic N-demethylation i s the f i r s t step i n production of the ultimate carcinogenic -26-FIGURE 3 O CH3-N-C-NH2 NO methyl nitrosourea NH CH3-N-C-NHN02 NO N-methyl-N-nitro-N-nitrosoguanidine R-SH CH3-NH-NO CH3-N2 "OH alkylation of target macromolecules -27-RGURLi CH- Chk> \ / DMN N NO enzymic oxidation hydrolysis CH 3N^ J N-OH CH3N2 1 CHJ alkylation of target macromolecules ^ 1--28-species, and these compounds are, therefore, precarcinogens (Heath, 1962) . The enzymatically produced metabolites of DMN are d i f f i c u l t <bo prepare s y n t h e t i c a l l y , and are too s h o r t - l i v e d to store for an a l y s i s and t e s t i n g . The exact nature of the a l k y l a t i n g species i s unknown, but a mechanism of a c t i o n has been hypothesized ( F i g . 4) (Heath, 1962). DMN i s a potent - '> l i v e r carcinogen i n the r a t (Table 2) and i s metabolized i n human l i v e r s l i c e s at close to the same rate as i n r a t l i v e r s l i c e s (Montesano and Magee, 1970). 3) P o l y c y c l i c aromatic hydrocarbons (PAH) PAH are formed during the incomplete combustion of organic matter (e.g., f o s s i l f uels) and man i s therefore exposed to them i n a v a r i e t y of ways (Badger, 1962). PAH have been detected i n a i r , water, s o i l , vegetation and food (Kotin and Falk, 1963; L y j i n s k i and Ross, 1967; Shabad, et a l . , 1971;. anon., 1973). Benzo(a)pyrene (BP) i s an example that has been shown to be a potent carcinogen (Table 2). BP was once thought to be carcinogenic as such, since i t was commonly able to induce tumours at the s i t e of a p p l i c a t i o n . However, t h i s observation might better be explained by the low aqueous s o l u b i l i t y of the compound which makes a sin g l e a p p l i c a -t i o n into a chronic exposure. The metabolism of BP, as with other PAH, i s ca r r i e d out by "mixed function oxygenases", enzymes that are NADPH-dependent and catalyze the incorporation of molecular oxygen into the substrate molecules (Holtzman, et a l . , 1967) and which c o n t a i n m u l t i p l e ' forms of the cytochrome P-450 (Fig.5). The oxygenases are also termed a r y l hydrocarbon hydroxylase (AHH) and serve to induce the formation of epoxides at the 4,5:7,8: and 9,10 p o s i t i o n s . Although the 4,5-epoxide i s the most stable, the 9,10-epoxide i s considered to be the precursor most -29-FIHIRE 5 Benzo(a)pyrene u u diol- epoxides water soluble BP-O-SG conjugates -30-TABLF.2 Carcinogenicity of Some Chemicals Carcinogen Animal Sites of tumour formation MNNG rat mouse S.G. hamster rabbit dog glandular stomach, forestomach, intestine, subcutanepus ( s i t e of injec t i o n intestine, forestomach, skin ( s i t e of injection) glandular stomach, intestine lung stomach, intestine MNU rat, mouse central and peripheral nervous system, intestine, kidney, forestomach, glandular stomach, skin and annexes, jaw, bladder, uterus, vagina, lung, l i v e r , pharynx, esophagus, trachea, bronchi, oral cavity, pancreas, ear ducts DMN rat mouse S.G. hamster rabbit l i v e r , kidney, nasal cavities l i v e r , lung, kidney l i v e r , nasal cavities l i v e r B(a)P mouse rats hamsters rabbits newts cervix, hematological tumours, skin, respiratory system, mammary tumours, l i v e r , lung, forestomach (when fed B(a)P) subcutaneous, mammary, lung, ovaries subcutaneous, stomach, trachea, skin skin subcutaneous •from "Survey of Compounds Which Have Been Tested For Carcinogenicity DHEW Publication No. (NIH) 73-35, Public Health Service Publication No. 149, U.S. Dept. of Health, Education and Welfare, U.S.A. (1969) important for ca r c i n o g e n i c i t y and mutagenicity (Baird, et^ a l . , 1975). The reactions to form these epoxides go on i n the endoplasmic reticulum, although cytochrome P-450's and epoxide hydratases also occur at s i m i l a r s p e c i f i c a c t i v i t i e s i n the nuclear membrane (Kasper, 1974; Mukhtar, et al.,'. 1979) . Although t h i s membrane i s much smaller than the endoplasmic reticulum (and so absolute amounts of enzyme are small) the proximity of the membrane to DNA might make the metabolism of PAH that goes on there disproportionately important. Epoxide hydratases do not require NADPH or other.cofactors (Oesch, et a l . , 1971) and have the capacity to convert BP-epoxides to dihydrodiol-s (KapitUlnik, et - a l . , 1977). Glutathione-S-transf erase (GSHT) , a soluble f r a c t i o n enzyme, converts epoxides to water soluble conjugates (Nemata and Gelboin, 1975) although non-enzymatic conjugation of glutathione has been demonstrated (Mukhtar and Bresnick, 1976). Conjugases have also been described which may incorporate BP—epoxides into water soluble conjugates (Nemata and Takayama, 1977). OUTLINE OF THE PROBLEM A wide v a r i e t y of tests have been d i v i s e d to assay the a b i l i t y of environmental compounds to induce cancer. These tests are both i n vivo and i n v i t r o , as well as short-term (to measure pre-cancerous lesions) and long-term (to measure the induction of resultant tumours). Both pure compounds and complex mixtures have been and are to be tested. While these tests are useful i n i n v e s t i g a t i n g chemicals that may cause cancer independently, they are not generally aimed at detecting those chemicals or conditions which may modify the induction of cancer. At present, only those agents important i n promoting the production -32-of tumours a f t e r the i n i t i a t i n g events i n carcinogenesis are complete are being widely investigated. The aim of t h i s thesis i s to demonstrate that modification of two proposed i n i t i a t i o n steps i n carcinogenesis (DNA damage by fragmentation or carcinogen-adduct formation, and DNA repair) may be brought about by the addition of chemical reducing agents (sodium ascorbate, propyl g a l l a t e , glutathione). The a b i l i t y of c e r t a i n chemicals or conditions to modify DNA damage and repair w i l l go undetected i n short and long-term carcinogenesis assays as they are generally applied. It becomes necessary f o r these tests to address themselves to such compounds and conditions by se t t i n g standard carcinogenesis conditions (i..e. , MNNG fragmentation of DNA, BP-adduct formation i n DNA) and t e s t i n g compounds for t h e i r a b i l i t y to modify them. In t h i s way, a better c o r r e l a t i o n between cancer incidence and environmental cause may ultimately be achieved. - 3 3 -MATERIALS AND METHODS CHEMICALS Sucrose, EDTA (ethylene diamlnetetraacetic a c i d , sodium chl o r i d e , sodium hydroxide and other common reagents and solvents were obtained from the Fisher Chemical Co., Vancouver, B.C. RADIONUCLIDES 3 Thymidine-methyl- H (1 mCi/ml, s p e c i f i c a c t i v i t y : 20 Ci/mmole) was 3 obtained from the New England Nuclear Corporation, Dorval, P.Q. (G- H) benzo(a)pyrene (5 mCi/ml, s p e c i f i c a c t i v i t y : 37 Ci/mmole) was obtained from Amersham Corp., Ltd., O a k v i l l e , Ontario. CHEMICAL CARCINOGENS Dimethylnitrosamine (DMN) was purchased from K and K Laboratories, Plainview, N.Y. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was purchased from the A l d r i c h Chemical Comapny, Milwaukee, Wisconsin. REDUCING AGENTS L-ascorbic acid (sodium s a l t ) , glutathione (reduced) and propyl-g a l l a t e were obtained from the Sigma Chemical Company, St. Louis, MD, and were stored i n a desiccator at 4° C to minimize a i r oxidation. _34-METAL SALT SOLUTIONS A s o l u t i o n of 0.1 M cupric sulphate was prepared i n d i s t i l l e d water, as was glycine at a concentration of 0.5 M. Stock solutions of glycine complexed copper were made by mixing appropriate amounts of glycine stock with metal stock, followed by d i l u t i o n with 2.5% minimal e s s e n t i a l medium (MEM plus 2.5% f e t a l c a l f serum). The molar r a t i o of copper to glycine was 1:10. NITROSATION OF METHYLGUANIDINE The n i t r o s a t i o n of 1-methyl-guanidine sulphate (Endo and Takahashi, 1974) with modifications made by Lo and S t i c h (1978) was followed. A 1 ml reaction mixture (0.2 M 1-methyl-guanidine sulphate (Sigma Chemical Co.), 0.6 M NaN0£ and 0.6 M sodium ascorbate) was incubated for one hour at 37° C. pH was adjusted to 7.4 with 2 N sodium carbonate and the volume brought to 2 ml by addition of d i s t i l l e d water. S e r i a l d i l u t i o n with MEM without f e t a l c a l f serum was used to lower the concentration -4 of the o r i g i n a l methylguanidine to 5 X 10 M. A l t e r n a t i v e l y , the above procedure was c a r r i e d out with the omission of sodium ascorbate i n the reaction mixture. EXPERIMENTAL ANIMALS Outbred, 4 month old, male Swiss mice were obtained from the Animal Unit, Faculty of Medicine, Un i v e r s i t y of B r i t i s h Columbia ( o r i g i n : Connaught Laboratories, Willowdale, Ontario). They were maintained during experimentation on a d i e t of Standard Purina Lab Chow and water ad l i b i t u m , -35-and were subjected to a 12 hr l i g h t cycle. Food, but not water, was removed from cages at 5:00 p.m. on the day before carcinogen administration. In the case of density gradient sedimentation analysis of DNA fragmentation, mice were injected i n t r a p e r i t o n e a l l y with 5 X 10 ^ C i 3 (0.05 ml) of ( H)TdR i n d i s t i l l e d water at 72, 24 and 16 hrs p r i o r to use, to l a b e l mucosal c e l l s of the stomach (Koropatnick and S t i c h , 1976). CELL CULTURES Cultured human f i b r o b l a s t s were grown from skin punch biopsies taken from the forearm of a 22 year old Caucasian female. The skin was teased into minute fragments with syringe needles and the pieces sandwiched between glass c o v e r s l i p s and incubated i n MEM (minimal e s s e n t i a l medium) with 12-20% f e t a l c a l f serum for 2 to 3 weeks at 37° C i n a CO2 incubator. Growth medium was changed every t h i r d day. When f i b r o b l a s t s began to migrate from the ti s s u e fragments the c o v e r s l i p s were opened and gross t i s s u e fragments removed, leaving a p a r t i a l monolayer of f i b r o -b l a s t s on the c o v e r s l i p s . These were incubated as above u n t i l the f i b r o b l a s t s became a complete monolayer, at which point the c e l l s were subcultured by standard techniques. Cultures were maintained i n plateau phase at 37° C i n a CO2 incubator i n p l a s t i c P e t r i dishes. Transfer passages 3 to 6 were used i n a l l experiments. The cultures were ro u t i n e l y maintained i n Eagle's MEM, supplemented with 15% f e t a l c a l f serum and a n t i b i o t i c s (200 units p e n i c i l l i n / m l , 4 ug streptomycin/ml). A l i n e of Chinese hamster c e l l s (CHO)(kindly supplied by the laboratory of Dr. L. Skarsgard ( P a l c i c and Skarsgard, 1978) were grown i n -36-MEM (Grand Island B i o l o g i c a l Co.) supplemented with 15% f e t a l c a l f serum, a n t i b i o t i c s (streptomicin s u l f a t e (29.6 ^ ig/ml), p e n i c i l l i n G (204 ^ig/ml) , kanamycin (100 yug/ml) , and fungizone (2.5 pg/ml)) and 7.5% sodium bicarbonate (10 ml/800 ml medium). The stock cultures were maintained i n 250 ml p l a s t i c culture f l a s k s (Falcon p l a s t i c s ) and kept i n MEM with 15% f e t a l c a l f serum at 37° C i n a water-saturated CO2 incubator. For experiments where damage to DNA, but not repair of that damage, was to be determined i n human f i b r o b l a s t s or CHO c e l l s , approximately 1.6 X 10~* c e l l s were seeded into 55 mm p l a s t i c dishes (Falcon p l a s t i c s ) and kept i n MEM with 15% f e t a l c a l f serum at 37° C for 2 to 3 days. Experiments were begun when c e l l s were 70-80% confluent. Where DNA repair was to be determined i n human or CHO c e l l s , f i b r o b l a s t s were grown i n the p l a s t i c dishes i n 15% MEM u n t i l about 50% confluent. In order to i n h i b i t c e l l d i v i s i o n a f t e r reaching 70-80% confluence, the c e l l s were then transferred to arginine-d e f i c i e n t medium (ADM) supplemented with 2.5% f e t a l c a l f serum and incubated f o r another 3 to 4 days, at which point they were 70-80% confluent and ready f o r use. Since these c e l l s were not undergoing scheduled DNA synthesis, t h i s process was not confused with repair synthesis of DNA. In the case of density gradient sedimentation analysis of DNA fragmentation, DNA of cultured c e l l s was p r e l a b e l l e d by incubation 3 with 5 ml of 10% MEM supplemented with H-TdR ( O ^ ^ C i / m l ) for 24 hrs immediately p r i o r to use. -37-PREPARATION OF S9 ACTIVATION MIXTURE Adult male Swiss mice were k i l l e d by decapitation and bleeding. The l i v e r s were quickly removed and minced with i c e - c o l d Dulbecco's phosphate-buffered s a l i n e (PBS) with 0.25 M sucrose, homogenized by a S o r v a l l t e f l o n p e stle t o r s i o n homogenizer at 1000 rpm and centrifuged at 9000 X £ for 10 min at 4° C (Garner and Hanson, 1971). The r e s u l t i n g postmitochondrial supernatant f r a c t i o n was frozen i n l i q u i d nitrogen and stored i n a Revco freezer at -71° C. The a c t i v a t i o n mixture consisted of 4 umoles NADPH, 25 nmoles MgCl 2, 20 p i o l e s glucose-6-phosphate, 0.1 ml NaOH (0.2 N) and 0.4 ml of postmitochondrial l i v e r supernatant f r a c t i o n (which contains approximately 240 mg of organ wet-weight (Laishes and St i c h , 1973)). Ingredients were mixed and 0.25 ml of S9 a c t i v a t i o n mixture added to c e l l cultures within 5 min. ADMINISTRATION OF CHEMICALS Dimethylnitrosamine: To each plate of cultured human f i b r o b l a s t s was added 1) 4.5 ml of MEM without f e t a l c a l f serum, 2) 0.25 ml of DMN dissolved i n MEM without f e t a l c a l f serum (0.1 M ) ( s u f f i c i e n t to produce a f i n a l _3 DMN concentration of 5 X 10 M on the p l a t e ) , and 3) 0.5 ml of S9 a c t i v a t i o n mixture. In the case of sodium ascorbate i n h i b i t i o n of n i t r o s a t i o n product formation, s u f f i c i e n t sodium ascorbate (dissolved i n MEM without f e t a l -2 c a l f serum) was added to produce a concentration of 1 X 10 M immediately p r i o r to addi t i o n of S9 a c t i v a t i o n mixture. -38-C e l l s were exposed at 37° C for 1 hr and washed three times with PBS s o l u t i o n (8.0 g NaCl, 0.2 g KC1, 1.15 g Na 2HP0 4 and 0.2 g KH 2P0 4 i n 1000 ml of d i s t i l l e d water: pH 7.45) and used f o r a l k a l i n e sucrose gradient a n a l y s i s . N i t r o s a t i o n products of methylguanidine: S e r i a l d i l u t i o n of the n i t r o s a t i o n reaction mixture with MEM without f e t a l c a l f serum was made to reduce the s t a r t i n g concentration -4 of methylguanidine to 5 X 10 M. C e l l s were treated for 1 hr and prepared for a l k a l i n e sucrose gradient (ASG) analysis i n the manner described above. MNNG: 1) In v i t r o : MNNG was dissolved i n 1 ml of dimethyl sulphoxide (DMSO) and d i l u t e d to the appropriate concentration by addition of 99 ml of MEM with 2.5% f e t a l c a l f serum. C e l l s were treated for 0.5 hr at 37° C i n a C0 2 incubator followed by preparation for ASG analysis i n the manner described above. 2) In vivo: MNNG was force-fed to experimental animals under ether anaesthesia by esophageal intubation i n a t o t a l volume of 0.5 ml. The 0.5 ml contained 0.1 ml DMSO; 0.4 ml d i s t i l l e d water; MNNG. After 4 hr the animals were k i l l e d by c e r v i c a l d i s l o c a t i o n and bleeding and t i s s u e samples taken. -39-Sodium ascorbate: Sodium ascorbate was dissolved at twice the desired concentration i n MEM without f e t a l c a l f serum. Glycine/CuSO^'SI^O solutions were made as described above at twice the desired concentration. The two solutions were mixed i n equal volumes and added to c e l l s within 10 sec. In cases where sodium ascorbate or CuSO^ -SH^O/glycine were administered alone, the concentrated solutions were mixed with MEM (without f e t a l c a l f serum) alone and added to c e l l s . C e l l s were incubated i n v i t r o with mixtures f o r 0.5 min at 37° C. C e l l s were washed with PBS and used i n ASG analysis of DNA damage. Mice under l i g h t ether anaesthesia were force-fed 0.5 ml of mixtures of the desired concentration. A f t e r 4 hr the mice were k i l l e d and tissues excised f o r ASG analysis of DNA damage. Benzo(a)pyrene: 3 1) In v i t r o : S u f f i c i e n t H-BP i n toluene was placed i n a 1 l i t r e glass b o t t l e to produce 4.16 X 10 ^ M BP when d i l u t e d to stock volume. 3 2 ml of DMSO was added to the H-BP and a i r blown over the surface of the f l u i d with an a i r pump and Pasteur pipet f o r 15 min under the fume hood to remove v o l a t i l e toluene. 498 ml of MEM with 5% f e t a l c a l f serum was added to the mixture, shaken, and allowed to s i t 10 min before use. The mixture was used within 1 hr. Sodium ascorbate, propyl g a l l a t e or glutathione was made up i n MEM without f e t a l c a l f serum and used within 10 min of mixing. -40-The pH of glutathione mixtures was adjusted to 7.0 with concentrated NaOH. A l l concentrations were 2.083 times greater than that required i n the f i n a l mixture, since d i l u t i o n took place i n c e l l culture dishes. 2.4 ml of reducing agent, 2.4 ml of BP mixture and 0.2 ml of S9 a c t i v a t i o n mixture were added to the c e l l s , i n that order. C e l l s were incubated at 37° C i n a CO2 incubator for 2 hr. The c e l l s were then washed 2 times with 5 ml of i c e - c o l d PBS and the DNA is o l a t e d f o r BP adduct analysis. 3 2) In vivo: H-BP i n toluene was added to DMSO i n the r a t i o of 1 u^g BP to 0.1 ml DMSO. Toluene was v o l a t i l i z e d i n the same way as described above. Enough MEM with 2.5% f e t a l c a l f serum was added 3 —6 to produce a f i n a l H-BP concentration of 1 X 10 g/ml ( s u f f i c i e n t to del i v e r 100 ng to each mouse). Mice which had been deprived of food, but not water, at 5:00 p.m. the night before were force-fed 0.5 ml of the BP mixture (500 ng) under l i g h t ether anaesthesia between 10:00 a.m. and 1:00 p.m. the following day. Mice were k i l l e d and stomachs excised at varying times following t h i s prodedure, the DNA extracted, and BP adduct analysis c a r r i e d out. A l k a l i n e sucrose gradient analysis of DNA damage and rep a i r : 1) In vivo: The method of Cox, et a l . (1973) was followed i n a l l of the e s s e n t i a l s . The animal was k i l l e d by c e r v i c a l d i s l o c a t i o n and bleeding. The stomach was excised proximal to the p y l o r i c sphincter and d i s t a l to the esophagus, opened along i t s anterior aspect and rinsed twice i n i c e - c o l d PBS. Gastric surface mucosa was removed by scraping with a cold glass microscope s l i d e . The scrapings were mixed with -41-3.0 ml of i c e - c o l d PBS and spun at 1000 rpm i n a Dynac c l i n i c a l centrifuge for 3 min to sediment c e l l s . The c e l l s were mixed with 3.0 ml PBS, spun down again, and resuspended i n 0.5 ml of i c e - c o l d PBS. A 25 u l i t r e a l i q u o t was layered on the gradient. 2) In v i t r o : Chemically treated c e l l s were rinsed 3 times with i c e - c o l d PBS. 0.5 ml of cold PBS was added and the c e l l s were scrubbed away from the dish with a rubber policeman. The c e l l suspension (0.5 ml) was placed i n a 3 ml centrifuge tube and spun at 2600 rpm i n a c l i n i c a l centrifuge f or 5 min. The c e l l - f r e e supernatant was removed and discarded. 100 u l i t r e s of cold PBS was added and the c e l l s were kept on an ice-bed i n preparation for layering on gradients. A l k a l i n e sucrose gradients: Gradients were prepared an hour before use, according to the method of Cox, et a l . , (1973). Into n i t r o c e l l u l o s e centrifuge tubes (Beckman Instrument Co., Vancouver, B.C.) was l a i d , i n succession: 1 ml of 2.3 M sucrose; 5-20% a l k a l i n e sucrose gradient (0.9 M NaCl, 0.3 M NaOH); 0.3 ml l y s i n g s o l u t i o n (0.3 M NaCl, 0.03 M EDTA, 0.1 M t r i s - H C l , 0.5% sodium dodecyl sulphate (SDS); 1 X 10 5-5 X 10^ c e l l s or i n t a c t c e l l n u c l e i i n a volume not exceeding 50 j j l i t r e s ; 0.3 ml l y s i n g s o l u t i o n ; iso-octane to within 0.5 cm of the top of the tube. Gradients were placed i n the buckets of a Beckman SW 40 u l t r a c e n t r i f u g e rotor and spun at 77,561 X j> at an average radius of 11.10 cm (25,000 rpm) for 30 min at 20° C with the brake o f f i n a Beckman L2B u l t r a c e n t r i f u g e . F i f t e e n sequential f r a c t i o n s were taken from the bottoms of the pierced tubes, p r e c i p i t a t e d with 8-10% t r i c h l o r o a c e t i c a c i d and c o l l e c t e d on n i t r o c e l l u l o s e membrane f i l t e r s . -42-Acid soluble r a d i o a c t i v i t y was removed by washing the f i l t e r s with 8-10% t r i c h l o r o a c e t i c a c i d and ethanol. Acid insoluble r a d i o a c t i v i t y was counted by immersing the dried f i l t e r s i n toluene s c i n t i l l a t i o n f l u i d and counting for 10 min per v i a l on the Searle Delta 300 l i q u i d s c i n t i l l a t i o n counter. BP adduct measurement i n DNA: DNA i s o l a t i o n prodedure was a modification of that used by Diamond, et a l . (1967). 1) Tissues: Mouse stomach was excised as described for ASG a n a l y s i s , rinsed by holding with forceps and plunging i n two washes of i c e - c o l d PBS, and the whole stomach placed i n a 25 ml Pyrex glass test tube with 5 ml of 1% sodium dodecyl sulphate/25 mM EDTA. The stomach was dispersed using a Polytron t i s s u e homogenizer at s e t t i n g 5, number 2 head, for 20 sec. The t i s s u e s o l u t i o n was transferred to a 15 ml teflon-capped glass centrifuge tube. 2) C e l l s : CHO c e l l s (approximately 6 X 10 c e l l s per plate) were treated with activated BP for 2 hr as described above and were rinsed 2 times with 5 ml a l i q u o t s of PBS. 2.5 ml of 1% SDS/25 mM EDTA was added to each plate and the c e l l s scraped o f f with a rubber policeman. C e l l s from two plates were transferred to a 15 ml teflon-capped glass centrifuge tube. DNA i s o l a t i o n : 1) Solutions were extracted 4 times with 5 ml t r i s - e q u i l i b r a t e d phenol (90% phenol, e q u i l i b r a t e d with equal volumes of 0.5 M t r i s --43-HC1 (adjusted to pH 8.0 with concentrated HC1)). 2) Solutions were then extracted 2 times with 5 ml e t h y l ether. 3) 1 ml of beef pancreatic RNAase (200 yug/ml, Sigma Chemical Corp.) was added and solutions incubated 1 hr at 37° C. 4) 1 ml of 0.02% pronase (Sigma Chemical Corp.) was added and solutions incubated overnight at 37° C. 5) Solutions were extracted 4 times with 5 ml of chloroform/ isoamyl alcohol (24:1 v/v). 6) Solutions were extracted three times with 5 ml ether, and the l a s t ether traces were b o i l e d off by placing the open tubes i n warm tap water (approximately 60° C) for 15 min. 7) 6 ml of 2% sodium acetate i n 99% ethanol was added to p r e c i p i t a t e DNA, and the tubes l e f t at -4° C overnight. 8) DNA was p r e c i p i t a t e d by spinning i n a Dynac c l i n i c a l centrifuge at s e t t i n g 100 for 20 min (approximately 2000 rpm). 9) Supernatant was discarded and the DNA p e l l e t dried and resuspended i n 4 ml of 0.03 M sodium acetate. DNA concentration determination: P u r i f i e d c a l f thymus DNA (Sigma Chemical Corp.) was dissolved i n a 0.03 M sodium acetate s o l u t i o n and s e r i a l l y d i l u t e d to produce a curve of DNA concentration versus absorbance at 260 nm. I t was found that: A260 „„ A . , .-1. = DNA concentration tug*ml ) 0.024 A l l samples were measured i n 1 ml quartz cuvettes i n a Bausch and Lomb -44-Spectronic 21 UV spectrophotometer. DNA solutions were d i l u t e d , i f necessary, to ensure that absorbance readings f e l l between 0.1 and 0.8 absorbance units (the range i n which the graph was most l i n e a r ) . The absorbance of each sample at 260 and 280 nm was taken and any samples with A„,_/A 0 0_ f a l l i n g below 2.00 were discarded. 1.0 ml Z b U ZoU of each sample was used for DNA concentration determination. Measurement of r a d i o a c t i v i t y : 2.5 ml of DNA s o l u t i o n was placed i n a 25 ml p l a s t i c s c i n t i l l a t i o n v i a l and 15 ml of PCS water-miscible s c i n t i l l a t i o n f l u i d (Amersham Corp.) was added and mixed by shaking. The tubes were kept i n the dark at room temperature for 24 hr to minimize . chemiluminescence. 3 Samples were counted for H r a d i o a c t i v i t y on the Searle Delta 300 l i q u i d s c i n t i l l a t i o n counter for a s u f f i c i e n t period to allow no more than 1% error i n count-reading. Disintegrations per minute were calculated f o r each sample using channels-ratio quench cor r e c t i o n . Using the s p e c i f i c a c t i v i t y of the BP, the BP concentration i n the DNA s o l u t i o n was calculated, and the r e s u l t expressed as "ng of benzo(a)pyrene per g of DNA". -45-RESULTS I n h i b i t i o n of DNA repair by sodium ascorbate 1) A l k a l i n e sucrose gradient analysis of DNA damage and rep a i r : a) In v i t r o : DNA from cultured human f i b r o b l a s t s , when released by c e l l l y s i s on a l k a l i n e sucrose gradients, has been shown to sediment to the saturated sucrose cushion (Fig. 6) (Stich, et a l . , 1979,a,b). When cultured human f i b r o b l a s t s were treated with MNNG (1 X 10 M) for 30 min, and sampled immediately (1. e_., placed on i c e within 5 min and then into l y s i n g s o l u t i o n within 20 min) a s h i f t i n the peak of DNA concentration from the regions occupied by fast-sedimenting DNA to slow-sedimenting DNA was seen, between f r a c t i o n s 10 and 15 (Fig. 7A). However, c e l l s treated i d e n t i c a l l y , but switched to MEM with 5% f e t a l c a l f serum a f t e r treatment with MNNG and then allowed to incubate for 30 hr at 37° showed DNA sedimenting at close to control regions (Fig. 7B). This s h i f t i n DNA p r o f i l e s from damage to repair regions was taken to indica t e repair. On the other hand, c e l l s treated with MNNG for 0.5 hr (Fig. 8A) _3 but which have a so l u t i o n of sodium ascorbate (1 X 10 M) i n the 5% MEM bathing medium i n which they repaired during the following 30 hr showed decreased a b i l i t y to approach co n t r o l DNA p r o f i l e s a f t e r 30 hr (Fig. 8B). When c e l l s were treated with MNNG as above, but were allowed to _3 re p a i r i n 5% MEM supplemented with sodium ascorbate (1 X 10 M) which was renewed every 4 hr up to 20 hr a f t e r MNNG treatment -46-Figure 6 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s . -47-0 5 10 15 <- SEDIMENTATION -48-Figure 7A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 for 0.5 hr. Figure 7B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 for 0.5 hr followed by incubation i n 5% MEM f o r 30 hr at 37° C. -49--50-Figure 8A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 ~* for 0.5 hr. Figure 8B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 for 0.5 hr followed by incubation i n 5% MEM supplemented with sodium ascorbate (1 X 10~ 3 M) for 30 hr at 37° C. -51-0 5 10 15 -*—SEDIMENTATION -52-(5 medium changes i n a l l ) , then r e s t o r a t i o n of control DNA p r o f i l e s was even more i n h i b i t e d (Fig. 9A). This indicated that continued incubation of sodium ascorbate may i n h i b i t i t s r e p a i r - i n h i b i t i n g properties, either by autoxidation to other products, or by c e l l u l a r i n a c t i v a t i o n of the r e p a i r - i n h i b i t i n g species. Because apparent lack of repair on a l k a l i n e sucrose gradients might be due to c e l l death, i t was necessary to demonstrate that sodium ascorbate had not i n f l i c t e d s u f f i c i e n t i n j u r y on the f i b r o b l a s t c e l l s to prevent them from being able to recover. Therefore, c e l l s were treated with MNNG, then with 5 doses of sodium ascorbate over a 20 hr period, and then allowed to rep a i r i n a medium containing 5% MEM up to 72 hr following MNNG treatment (Fig. 9B). DNA from these c e l l s once again sedimented i n a manner that approached that of cont r o l DNA. It might be argued that sodium ascorbate contributes to c e l l k i l l i n g such that c e r t a i n c e l l s die while others are able to go on to rep a i r DNA to restore i t s fast-sedimenting a b i l i t y . 3 A measure of t h i s was the amount of radioactive H l a b e l recoverable from the gradients. C e l l s that have undergone repair should have 3 decreased recovery of H l a b e l (a measure of the amount of DNA) i f some c e l l s had been unable to recover and had been lysed. Table 3 shows r e s u l t s obtained from the experiment i l l u s t r a t e d i n F i g . 9. The r e s u l t s of three separate experiments are shown. Of the 5 r e s u l t s , two came from the f i r s t experiment, two from the second, and one sample from the t h i r d experiment. In t h i s case, c e l l s that had undergone repair a f t e r MNNG and sodium ascorbate treatment s t i l l retained 70-93% of the 3 H l a b e l recoverable from control c e l l s untreated with MNNG or sodium ascorbate. I f higher concentrations of sodium ascorbate were used, -53-Figure 9A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 M) for 0.5 hr followed by incubation i n 5% MEM supplemented with sodium ascorbate (1 X 10 M) for 30 hr at 37° C. The MEM with sodium ascorbate was replaced with freshly-mixed sodium ascorbate so l u t i o n every 4 hr for the f i r s t 20 hr aft e r MNNG treatment. Figure 9B 3 A l k a l i n e sucrose gradient sedimentation p r o d i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 M) for 0.5 hr followed by incubation i n 5% MEM supplemented with sodium ascorbate (1 X 10~ 3 M) for 30 hr at 37° C. The MEM with sodium ascorbate was replaced with freshly-mixed sodium ascorbate s o l u t i o n every 4 hr for the f i r s t 20 hr af t e r MNNG treatment. At 3 hr post-MNNG treatment, the sodium ascorbate s o l u t i o n was removed and replaced with 5% MEM without sodium ascorbate, incubated f o r a further 72 hr at 37° C, and sampled. -54-O 5 10 15 <—SEDIMENTATION -55-TABLE 3 Recovery of radioactivity from alkaline sucrose gradients Source of DNA c.p.m. (mean of 5 samples + standard error) % of radioa c t i v i t y i n control c e l l s recoverable from gradients 3 H-treated control c e l l s (200,000 c e l l s ) 1392 + 320 100% MNNG treatment followed by incubation with 5 sodium ascorbate treatments over 20 hours 1580 + 107 114% MNNG treatment followed by sodium ascorbate treatment followed by repair in MEM without sodium ascorbate up to 72 hours following MNNG treatment 1220 + 66 88% -56-3 the r e c o v e r a b i l i t y of H counts tended to decrease, although the shape of the a l k a l i n e sucrose gradient p r o f i l e s was not found to change. Therefore, sodium ascorbate concentrations for treatment of c e l l s were chosen such that they were as high as possible without 3 decreasing H r e c o v e r a b i l i t y to any great extent. MNNG concentrations 3 chosen for use were those great enough to cause the mass of H l a b e l to accumulate between f r a c t i o n s 9 and 13, but no greater. In t h i s way, the lowest possible MNNG concentrations (those j u s t causing enough damage to observe repair) and the highest possible sodium ascorbate concentrations (those that did not contribute to enhanced c e l l k i l l i n g ) were used. Sodium ascorbate treatment alone did not fragment DNA (Fig. 10). b) In vivo: DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg/kg body weight) waas shown to be damaged 4 hr a f t e r f orce-feeding (Fig. 11A). However, repair occurred that returns DNA to near con t r o l sedimentation p r o f i l e s by 30 hr following force-feeding (Fig. 11B). When, on the other hand, MNNG was force-fed to mice i n the same concentration (Fig. 12A) followed by force-feeding sodium ascorbate (20 mg/kg body weight at 15 min, 4 hr, 8 hr, 12 hr, 16 hr, 20 hr and 24 hr following MNNG treatment) no evidence of repair was apparent by 30 hr and sedimentation of DNA did not approach near-control p r o f i l e s , but remained slow-sedimenting (Fig. 12B). Sodium ascorbate treatment alone did not fragment DNA (Fig.10, . In the same manner as cultured human f i b r o b l a s t s ±n v i t r o , g a s t r i c mucosal c e l l s JLn vivo retained the a b i l i t y to repair DNA (as measured by t h e i r a b i l i t y to restore fast-sedimenting properties to DNA) -57-Figure 10 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from c u l t u r e d human f i b r o b l a s t s exposed to sodium ascorbate _3 (1 X 10 M) f o r 20 hr. The sodium ascorbate s o l u t i o n was replaced w i t h f r e s h l y mixed s o l u t i o n every 4 hr f o r the f i r s t 20 hr a f t e r the beginning of treatment. -58-CO l _ 1 z ZD O u 20 I ro _J < TOT 10 L L O o o 0 5 10 15 SEDIMENTATION - 5 9 -Figure 11A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr and sampled at 4 hr. Figure 11B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr and sampled at 30 hr. -60-20 00 8 1 0 u IE ro _j 0 , 0 - 0 ' L L O 20 10 / "\ / o \ \ O-r 0 / 0 - 0 0 5 10 15 *— SEDIMENTATION -61-Figure 12A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr and sampled at 4 hr. Figure 12B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr followed by force-feeding of sodium ascorbate (20 mg per kg body weight) at 15 min, 4 hr, 8 hr, 12 hr, 16 hr, 20 hr and 24 hr, and sampled at 30 hr. -62-0 5 10 15 *— SEDIMENTATION -63-since f a s t sedimentation was restored when mice that had been force-fed MNNG, 7 doses of sodium ascorbate (as i n F i g . 12) and were allowed to repair without further sodium ascorbate treatment up to 72 hr a f t e r MNNG administration (Fig. 13B). 2) BP-DNA adduct analysis of DNA re p a i r : a) In v i t r o : i ) One of the problems associated with the administration of p o l y c y c l i c aromatic hydrocarbons to cultured c e l l s or mammalian systems i s the hydrophobicity associated with them. BP tends to leave water solutions by attachment to the glass or p l a s t i c container, or by separation of the non-polar solvent carrying the BP from the water s o l u t i o n . In order to solve t h i s problem, 3 H-BP i n toluene was dissolved i n DMSO, the toluene blown o f f by a stream of a i r , and the r e s u l t i n g water-miscible DMSO/BP so l u t i o n dissolved i n MEM supplemented with 5% f e t a l c a l f serum. BP i n t h i s s o l u t i o n (presumably bound to the protein component of f e t a l c a l f serum) remains a v a i l a b l e i n so l u t i o n f or at lea s t 70 min following s o l u t i o n , while BP treated i d e n t i c a l l y , but dissolved i n MEM without FCS, rap i d l y becomes unavailable i n the water s o l u t i o n (Fig. 14). BP i n solu t i o n was measured by s c i n t i l l a t i o n counting of an aliquo t of so l u t i o n . i i ) BP binding to DNA was determined for 2 hr incubations of concentrations ranging from 0.1 X 10 ^  M to 3 X 10 ^  M produced by d i l u t i o n of a si n g l e parent BP stock with MEM with 5% f e t a l c a l f serum (Fig. 15). Since the 2 X 10 ^  M concentration f a l l s i n a region of the curve with constant slope, i t was deemed an appropriate concentration to use rou t i n e l y f or binding and repair studies. -64-Figure 13A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed sodium ascorbate (20 mg per kg body weight) at 15 min, 4 hr, 8 hr, 12 hr, 16 hr, and 20 hr and sampled at 24 hr. Figure 13B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr followed by force-feeding of sodium ascorbate (20 mg per kg body weight) force-fed at 15 min, 4 hr, 8 hr, 12 hr, 16 hr, 20 hr and 24 hr and sampled at 72 hr. - 6 6 -Figure 14 3 H-BP a v a i l a b l e i n s o l u t i o n at various times a f t e r mixing with MEM without f e t a l c a l f serum (• ) or with MEM with 3 5% f e t a l c a l f serum ( O ) . H-BP i n a 0.5 ml aliqu o t was measured by s c i n t i l l a t i o n counting i n water-miscible s c i n t i l l a t i o n f l u i d at the times indicated. A sin g l e sample i s pl o t t e d for each time. -67-"D T 3 O CQ 100 "D <D c_ <D > O u <D L . Q_ CD 0 10 30 50 70 t ime after mixing (minutes) -68-Figure 15 3 Binding of H-BP to DNA of cultured CHO c e l l s exposed 3 to various concentrations of H-BP i n combination with an S9 a c t i v a t i o n system for 2 hr and sampled immediately. The mean of 10 samples i s plotted for each concentration ± standard error. -69-B P concentration (M x 100 -70-i i i ) To follow the disappearance of covalently bound 3 H-BP from DNA, CHO c e l l s that had been i n h i b i t e d i n the G.^  stage of c e l l d i v i s i o n by incubation i n a r g i n i n e - d e f i c i e n t medium(ADM)(Stich and San, 1970) were treated with BP ( i n 5% ADM), rinsed twice with ADM without f e t a l c a l f serum, and then allowed to repair i n 5% ADM without BP. CHO c e l l s rather than human f i b r o b l a s t s were employed because greater rates of c e l l d i v i s i o n allowed larger numbers of c e l l s to be used. BP binding to DNA was determined at various times a f t e r reaction, up to 72 hr (Fig. 16). There was a s l i g h t r i s e i n t o t a l BP adducts between 3 0 and 1.5 hr a f t e r H-BP reaction, which may be due to binding of BP that had not been completely removed from the system (Ivanovic, et a l . , 1978). Thereafter there i s a rapid decline i n adducts up to 12 hr, 3 at which time about 30% of the i n i t i a l bound H l a b e l remained. Up to 72 hr, the rate of decrease i n bound BP was much l e s s and about 70% 3 of the bound H l a b e l at 24 hr remained at 72 hr. iv) When CHO c e l l s were treated i d e n t i c a l l y to those described _3 above, but were subjected to sodium ascorbate (1-5 X 10 M) dissolved 3 i n the post-BP incubation medium, loss of bound H l a b e l was s i g n i f i c a n t l y -3 i n h i b i t e d (Fig. 17). At 5 X 10 M, the rapid decrease i n bound BP 3 was l o s t altogether. DNA-bound H l a b e l remained r e l a t i v e l y constant up -3 to 24 hr, at which time i t began to drop o f f to co n t r o l l e v e l s . 1 X 10 M 3 sodium ascorbate induced l e s s rapid i n i t i a l loss of bound H l a b e l . 3 By 24 hr the bound H l a b e l reached the same l e v e l as control samples taken at the same time. It appears that sodium ascorbate i n h i b i t s the early, rapid e x c i s i o n of bound BP products to DNA. However, the r e l a t i v e l y rapid autoxidation of sodium ascorbate to dehydroascorbate i n so l u t i o n might explain the return to normal excision l e v e l s by 24 to 48 hr. -71-Figure 16 3 Binding of H-BP to the DNA of non-dividing cultured CHO 3 -7 c e l l s exposed to H-BP (2 X 10 M) i n combination with an S9 a c t i v a t i o n system for 2 hr followed by r i n s i n g with PBS and addition of 5% ADM. Samples were taken at up to 3 72 hr a f t e r exposure to H-BP. Three i d e n t i c a l runs are plotted f or each time. -72-120 h < z a E o o) 8 0 j_ cu Q. Q_ m £ o O) o c o c 4 0 0 " o 8° - o -o o o o 0 8 o o o 8 o o o o o • o o 1 8 o o o -0 24 48 72 time after BP administration (hours) -73-Figure 17 3 Binding of H-BP to the DNA of non-dividing cultured CHO 3 -7 c e l l s exposed to H-BP (2 X 10 M) i n combination with an S9 a c t i v a t i o n system for 2 hr followed by r i n s i n g with PBS and addition of 5% MEM supplemented with sodium -3 -3 ascorbate, 5 X 10 M ( O ) or 1 X 10 M (-*-), or 5% MEM without sodium ascorbate ( • ) . The mean of 5 samples i s p l o t t e d f o r each time, ± standard error. -74-time after BP administration (hours) -75-v) Although sodium ascorbate could be used to i n h i b i t r e p a i r , the sulphydryl reducing agent cysteine did not apparently i n h i b i t repair of BP adducts (Fig. 18). b) In vivo: i ) BP binding to mouse g a s t r i c mucosal c e l l DNA was determined 3 for various amounts of H-BP force-fed to mice, and the r e s u l t s plotted as the mean of 8-10 t r i a l s ± standard error (Fig. 19). The curve i s r e l a t i v e l y l i n e a r from 100 ng per mouse to 750 ng per mouse, with 3 a steeper r i s e up to 1000 ng per mouse. In order to use a H-BP concentration for further experiments that f e l l into the region of the graph where slope v a r i e d the l e a s t , a concentration of 500 ng per mouse was chosen as a standard for further experiments. 3 i i ) In order to determine the time course of H-BP binding 3 to mouse g a s t r i c c e l l DNA a f t e r force-feeding 500 ng of H-BP, stomach samples were taken 1.5 to 72 hr a f t e r administration and the amount of BP bound to DNA calculated (Fig. 20). BP binding increased up to 12 hr, followed by a r e l a t i v e l y rapid decrease to approximately 50% of the 12 hr binding by 48 hr. The 48 hr binding was not s i g n i f i c a n t l y reduced by 72 hr. i i i ) In order to determine whether sodium ascorbate had an 3 e f f e c t on the l o s s of H-BP l a b e l jm vivo, mice were force-fed 500 ng 3 of H-BP at 0 hr followed by 100 mg of sodium ascorbate per g body weight (i n a t o t a l volume of 0.5 ml of MEM) at 15 min, 4, 8, 12, 24 and 36 hr 3 (Fig. 21). H l a b e l remained constant from 12 to 36 hr while DNA from 3 control mice l o s t approximately 50% of H l a b e l . Following cessation of 3 force-feeding of sodium ascorbate, H l a b e l f e l l r a p i d l y from 40 hr to 72 hr, by which time i t had reached the same l e v e l as that observed i n c o n t r o l mice sampled 72 hr a f t e r BP force-feeding. -76-Figure 18 3 Binding of H-BP to the DNA of non-dividing cultured CHO 3 -7 c e l l s exposed to H-BP (2 X 10 M) i n combination with an S9 a c t i v a t i o n system for 2 hr followed by r i n s i n g with PBS and addition of 5% MEM supplemented with _3 cysteine (5 X 10 M)(o) or 5% MEM without cysteine ( • ) . The mean of 5 samples i s plotted for each time, ± standard error. -77-0 12 24 36 4 8 time after BP administration (hours) -78-Figure 19 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice force-fed BP of various concentrations at 0 hr and sampled at 18 hr. The mean of values from 10 mice i s p lotted f o r each concentration, ± standard error -79-VN 20 Q E o c _ 15 O ) <D CL Q_ 10 CQ am 5 t _ o c D C 0 0 200 4 0 0 6 0 0 8 0 0 1000 BP administered (nanograms) -80-Figure 20 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice force-fed 3 H-BP (500 ng per mouse) at 0 hr and sampled at various times thereafter. The mean of values from 10 mice i s plo t t e d for each time, ± standard error. - 8 1 -_ J I I L . 0 24 48 72 time after BP administration (hours) -82-Figure 21 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice 3 force-fed H-BP (500 ng per mouse) at 0 hr followed by force-feeding sodium ascorbate (100 mg per kg mouse) at 15 min, 4, 8, 12, 24 and 36 hr ( O ) or by no further treatment ( • ) . The mean of values from 10 mice i s plot t e d f o r each time, ± standard error. -83-time after BP administration (hours) -84-Other e f f e c t s of sodium ascorbate While sodium ascorbate was able to i n h i b i t the repair of damage i n f l i c t e d on DNA by MNNG and BP, i t was able to produce other clastogenic or protective e f f e c t s both ±n v i t r o and i n vivo. 1) Sodium ascorbate fragmented DNA of cultured human f i b r o -b l a sts i n the presence of copper (Fig. 22B), while CuSO^'5^0 alone exerted no detectable e f f e c t (Fig. 22A). In a s i m i l a r way, sodium ascorbate (0.5 ml of 0.15 M solution) or CuSO^•SH^O/glycine complex force-fed to mice did not, i n i s o l a t i o n from each other, fragment g a s t r i c mucosal c e l l DNA (Fig. 23A). However, when i d e n t i c a l amounts of sodium ascorbate and copper/glycine complex were force-fed together, DNA fragmentation i n g a s t r i c c e l l s was apparent, by 4 hr (Fig. 23B). These damaged c e l l s were able to repair DNA so that i t sedimented at near-control l e v e l s by 48 hr post-treatment (Fig. 23B). 2) Sodium ascorbate has been used as a "trapping" agent to scavenge rea c t i v e e l e c t r o p h i l e s (see introduction). This was demonstrated by the a b i l i t y of sodium ascorbate to i n h i b i t the action of activated DMN. Cultured human f i b r o b l a s t s exposed to DMN alone (Fig. 24) showed no DNA fragmentation, while addition of S9 a c t i v a t i o n system induced considerable breakage (Fig. 25A). 3 Only 12% of recoverable H counts were found i n f r a c t i o n s 1-5 (the region into which 80-90% of undamaged DNA normally f a l l s ) . When sodium ascorbate _2 (1 X 10 M) was added to c e l l s immediately p r i o r to addition of the S9 a c t i v a t i o n system, l e s s fragmentation of DNA was observed. Approximately 3 40% of recoverable H counts were found i n f r a c t i o n s 1-5 (Fig. 25B). The decrease i n fragmentation was probably not due to i n h i b i t i o n of the -85-Figure 22A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to CuSO,'5H„0 -5 4 2 (1.8 X 10 M) for 0.5 hr. Figure 22B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to sodium ascorbate (1.8 X 10~3M) i n the presence of CuSO4'5H20 (1.8 X 10~ 5 M) for 0.5 hr. -86-5 10 15 ^-SEDIMENTATION -87-Figure 23A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed CuSO '5H-0 -5 -4 (5 X 10 M) plus glycine (5 X 10 M)(O), or 0.5 ml of sodium ascorbate (0.15 M)(#) at 0 hr and sampled at 4 hr. Figure 23B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed CuSO *5H90 -5 -5 (5 X 10 M) plus glycine (5 X 10 M) plus sodium ascorbate (0.15 M) i n a t o t a l volume of 0.5 ml at 0 hr and sampled at 4 hr (O) or 48 hr ( • ) . -89-Figure 24 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to dimethylnitrosamine _3 (5 X 10 M) without S9 a c t i v a t i o n system for 0.5 hr. -90-LO h -Z ZD O u 25-20-oo _J 15-;< 10-i L L 5-O 0-o 5 10 15 SEDIMENTATION -91-Figure 25A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to dimethylnitrosamine _3 (5 X 10 M) i n the presence of an S9 a c t i v a t i o n system for 0.5 hr. \ Figure 25B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to dimethylnitrosamine _3 (5 X 10 M) i n the presence of an S9 a c t i v a t i o n system _2 and sodium ascorbate (1 X 10 M) for 0.5 hr. -92-i 1 1 r 25-20 -_1 I I | 0 5 10 15 < SEDIMENTATION -93-a c t i v a t i o n system, since the precarcinogen sterigmatocystin was not -2 i n h i b i t e d i n i t s a b i l i t y to induce DNA repair i n the presence of 1 X 10 M sodium ascorbate and S9 a c t i v a t i o n system (Lo and Sti c h , 1978). S t e r i g -matocystin requires epoxidation to form carcinogenic species, i n a manner analogous to BP (Wogan, et a l . , 1979). Therefore, i n h i b i t i o n of activated DMN i s implicated (although some c e l l u l a r i n t e r a c t i o n of sodium ascorbate cannot be ruled out i n t h i s case. S9 a c t i v a t i o n system alone exerts no fragmenting e f f e c t on cultured human f i b r o b l a s t DNA (Laishes, 1974). 3.) While sodium ascorbate may i n h i b i t the action of activated carcinogens, i t may also i n h i b i t the non-enzymatic a c t i v a t i o n of those carcinogens. When cultured human f i b r o b l a s t s were incubated i n the presence of the n i t r o s a t i o n products of methylguanidine f o r 1 hr, DNA -3 fragmentation resulted (Fig. 26A). However, when 1.5 X 10 M sodium ascorbate was added to the reaction vessel where nitrous acid was used to n i t r o s a t e the methylguanidine, the r e s u l t i n g n i t r o s a t i o n products were unable to exert as great a fragmentation e f f e c t (Fig. 26B). The implication i s that the formation of n i t r o s a t i o n products was i n h i b i t e d by the presence of sodium ascorbate. 4) As demonstrated above, sodium ascorbate may i n h i b i t the action of a c t i v a t i o n of precarcinogens. In addition, d i r e c t - a c t i n g , ultimate carcinogens may be affected as we l l . When equal volumes of MEM containing sodium ascorbate and MNNG were added to c e l l cultures, i n -3 that order, such that the f i n a l concentrations are 1 X 10 and 1 X 10 M, respectively, considerable fragmentation of DNA resulted (Fig. 21k). However, when the sodium ascorbate and MNNG were incubated for 30 min at 37° C i n a closed vessel p r i o r to addition to c e l l s , much l e s s fragmentation occurred (Fig. 27B). The same s i t u a t i o n e x i s t s ^n vivo. When MNNG (20 mg per kg body weight) and sodium -94-Figure 26A 3 Alk a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to the n i t r o s a t i o n products of methylguanidine ( s t a r t i n g concentration of -4 methylguanidine was 5 X 10 M) for 1 hr. Figure 26B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to the n i t r o s a t i o n products of methylguanidine ( s t a r t i n g concentration of -4 methylguanidine was 5 X 10 M) where n i t r o s a t i o n had taken _3 place i n the presence of sodium ascorbate (1.5 X 10 M). Exposure was for 1 hr. -95-4 0 3 0 to 2 0 i — z: O 10 u I n 0 _J £ 401 O 3 0 o-o-o.0_0/0-o'°''0Vo 2 0 10 0 \ / o o 0 5 10 15 < — SEDIMENTATION -96-Figure 27A 3 Alk a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 M) _3 i n the presence of sodium ascorbate (1 X 10 M) for 0.5 hr. Figure 27B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 "* M) _3 i n the presence of sodium ascorbate (1 X 10 M) that was incubated at 37° C for 0.5 hr p r i o r to addition to c e l l s . Exposure was for 0.5 hr. -98-ascorbate (100 mg per kg body weight) were mixed and immediately (within 30 sec) force-fed to mice i n a volume of 0.5 ml, damage to DNA re s u l t s (Fig. 28A). On the other hand, incubation of the mixture f o r 0.5 hr at 37° C before force-feeding resulted i n decreased DNA fragmentation of mouse g a s t r i c mucosal c e l l s (Fig. 28B). The concentrations used here (100 mg per kg body weight) were higher than those used to i n h i b i t DNA repair (cf. F i g . 10,11)(40 mg per kg body weight). The low ascorbate concentrations were used previously to minimize long-term t o x i c i t y to c e l l s being tested f o r t h e i r a b i l i t y to repair damage over 72 hr, while the high concentrations (Fig. 27) were used to maximize the observed i n h i b i t i o n of damage over short-term 4 hr treatments. 5) Although incubation of sodium ascorbate with some carcinogens (e_.j*. , DMN) decreased t h e i r DNA-f ragmen t i n g a b i l i t y , sodium ascorbate may also enhance the DNA-fragmenting a b i l i t y of others. MNNG (1 X 10 M) , when administered to cultured human f i b r o b l a s t s , fragmented DNA (Fig. 29A). When equal volumes of MEM containing sodium ascorbate and MNNG were added to c e l l cultures, i n that order, such that the f i n a l -3 -5 concentrations were 1 X 10 M and 1 X 10 M, resp e c t i v e l y , considerably more fragmentation of DNA resulted ( Fig. 29B). In vivo, when MNNG (40 mg per kg body weight) was force-fed to mice, fragmentation resulted (Fig. 30A). When MNNG (40 mg per kg body weight) was mixed with sodium ascorbate (100 mg per kg body weight) and immediately (within 10 sec) force-fed, increased fragmentation of g a s t r i c mucosal c e l l s resulted ( Fig. 30B). The increased damage to DNA was not due to the independent action of sodium ascorbate, since sodium ascorbate alone did not fragment DNA i n vivo (Fig. 31A) or i n v i t r o (Fig. 31B). -99-Figure 28A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed a mixture of MNNG (20 mg per kg body weight) and sodium ascorbate (100 mg per kg body weight) immediately a f t e r mixing. Samples were taken 4 hr a f t e r force-feeding. Figure 28B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed a mixture of MNNG (20 mg per kg body weight) and sodium ascorbate (100 mg per kg body weight) a f t e r a 0.5 hr incubation of the mixture at 37°. Samples were taken 4 hr a f t e r force-feeding. -100-0 5 10 15 < S E D I M E N T A T I O N -101-Figure 29A 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 M) for 0.5 hr. Figure 29B 3 Al k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to MNNG (1 X 10 M) _3 i n the presence of sodium ascorbate (1 X 10 M) for 0.5 hr. -102-0 5 10 15 ^ — SEDIMENTATION - 1 0 3 -Figure 30A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) at 0 hr and sampled at 4 hr. Figure 30B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed MNNG (40 mg per kg body weight) i n the presence of sodium ascorbate (100 mg per kg body weight) at 0 hr and sampled at 4 hr. -105-Figure 31A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from g a s t r i c mucosal c e l l s of mice force-fed sodium ascorbate (100 mg per kg body weight) at 0 hr and sampled at 4 hr. Figure 3IB 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e of H-DNA from cultured human f i b r o b l a s t s exposed to sodium ascorbate (5 X 10" 3 M) for 0.5 hr. -106-20 h ?. CO § 10 O (J CO I 0 ~ 0 - 0 0 O 20 10 ' o / \ \ \ o-o'°-o. 0 0 5 10 15 <— SEDIMENTATION -107-6) I t might be argued that the decrease i n fragmenting a b i l i t y of MNNG observed a f t e r incubation with sodium ascorbate might have been due to loss of sodium ascorbate from the system by oxidation, binding to pro t e i n i n so l u t i o n , etc., during the incubation period. By t h i s argument, sodium ascorbate would have only the a b i l i t y to enhance fragmentation i n t h i s system, and the decrease i n fragmentation seen a f t e r incubation would be due to loss of enhancement only. However, when sodium ascorbate was incubated for 0.5 hr with MNNG and force-fed to mice (Fig. 28B) fragmentation of DNA was le s s than that observed when MNNG alone was force-fed to mice, even when the MNNG force-fed alone was at a dose 25% higher than that force-fed with sodium ascorbate (Fig. 30A). Thus, the decrease i n DNA-damaging a b i l i t y of MNNG a f t e r incubation with sodium ascorbate was l i k e l y due to the action of acorbate or dehydroascorbate on MNNG, rather than merely the loss of sodium ascorbate from the system. E f f e c t of sodium ascorbate and other agents on binding of BP to DNA: 1) Since sodium ascorbate may i n h i b i t damage to DNA by scavenging e l e c t r o p h i l i c carcinogenic and mutagenic species, the a b i l i t y of sodium ascorbate to prevent binding of BP to DNA i n v i t r o and i n vivo was investigated. When sodium ascorbate was incubated with BP and S9 ac t i v a t i o n mixture over CHO c e l l s , increasing concentrations decreased 3 the amount of H-BP l a b e l associated with p u r i f i e d DNA (Fig. 32). When BP (500 ng per mouse) was mixed with sodium ascorbate of various 3 concentrations and force-fed to mice, H-BP l a b e l associated with DNA decreased with increasing sodium ascorbate concentration. However, a low concentration of sodium ascorbate (15 mM) produced an increased -108-Figure 32 3 Binding of H-BP to DNA from cultured CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and various concentrations of sodium ascorbate i n the presence of an S9 a c t i v a t i o n system for 2 hr. Plates were rinsed with PBS and DNA i s o l a t e d immediately. The mean of 5 samples i s plotted for each concentration, ± standard error. -109-^ 5 0 0 < Q E o c_ D) £_ <D Q. Q_ DQ C/) E o c_ D) O c o c 3 0 0 r 100 0 5 0 100 150 Sodium ascorbate (mM) -110-l e v e l of bound BP (Fig. 33) i n two separate experiments. 2) Other reducing agents besides sodium ascorbate may a f f e c t BP binding to DNA. When the reducing agent propyl g a l l a t e was incubated with BP and S9 a c t i v a t i o n system over CHO c e l l s , reduced binding of BP resulted (Fig. 34) i n a manner s i m i l a r to that observed with sodium ascorbate. However, when propyl g a l l a t e was mixed with BP and force-fed to mice, binding to g a s t r i c c e l l DNA increased with increasing propyl g a l l a t e concentration (Fig. 35). Due to the l i m i t e d s o l u b i l i t y of propyl g a l l a t e , only about h a l f the molar concentration of sodium ascorbate used to exert e f f e c t i n the case of propyl g a l l a t e . Sulfhydryl as well as hydroxyl reducing agents may exert an e f f e c t on binding of BP to DNA. The s u l f h y d r y l reducing agent glutathione, when incubated with BP and S9 a c t i v a t i o n system over CHO c e l l s , increased the binding of BP to DNA by over 3 f o l d (Fig. 36). When glutathione was mixed with BP and force-fed to mice, the same increase i n binding of BP to DNA occurred (Fig. 37). In t h i s case, the extreme a c i d i t y of glutathione solutions allowed molar concentrations of only about one-sixth that of sodium ascorbate to be used. These solutions were neutralized by the addition of concentrated 10 M NaOH. 3) Since a l t e r a t i o n i n binding of BP by reducing agents may be due to d i f f e r e n t i a l i n h i b i t i o n of a c t i v a t i n g and deactivating enzyme systems, the e f f e c t of two compounds known to exert t h i s e f f e c t - harman and norharman - was investigated i n t h i s system. Incubation of harman with BP and S9 a c t i v a t i o n system over CHO c e l l s exerted no e f f e c t on BP binding over the concentration range studied (Fig. 38). The same lack of e f f e c t was observed with norharman (Fig. 39). -111-Figure 33 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice 3 force-fed H-BP (500 ng per mouse) i n the presence of various concentrations of sodium ascorbate. A t o t a l volume of 0.5 ml was administered to each mouse. Samples were taken at 12 hr a f t e r force-feeding. The mean of values from 10 mice i s plotted f o r each concentration, ± standard error. The concentrations indicated are those of the force-fed solutions, not stomach contents a f t e r force-feeding. See discussion f o r further consideration. -112-12 [ Sodium ascorbate (mM) -113-Figure 34 3 Binding of H-BP to DNA from cultured CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and various concentrations of propyl g a l l a t e i n the presence of an S9 a c t i v a t i o n system for 2 hr. Plates were rinsed with PBS and DNA i s o l a t e d immediately. The mean of 10 samples i s plotted for each concentration, ± standard error. - 1 1 4 -5 0 0 < z Q E o c_ U) t_ Q. CL CD to E o t_ D) O o c 3 0 0 100 h Propyl gallate (mM) -115-Figure 35 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice 3 force-fed H-BP (500 ng per mouse) i n the presence of various concentrations of propyl g a l l a t e . A t o t a l volume of 0.5 ml was administered to each mouse. Samples were taken 12 hr a f t e r force-feeding. The mean of values from 10 mice i s plotted f o r each concentration, ± standard error. The concentrations indicated are those of force-fed solutions, not stomach contents a f t e r force-feeding. See discussion f o r further consideration. -116-U) o c o c 2 -• -• >-0 1.4 14.1 Propyl gallate (mM) -117-Figure 36 3 Binding of H-BP to DNA from cultured CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and various concentrations of reduced glutathione i n the presence of an S9 a c t i v a t i o n system for 2 hr. Plates were rinsed with PBS and DNA i s o l a t e d immediately. Results from duplicate plates at each concentration are plotted. -118-5 0 0 r 3 0 0 100h Glutathione (mM) -119-Figure 37 3 Binding of H-BP to DNA from g a s t r i c c e l l s of mice 3 force-fed H-BP (500 ng per mouse) i n the presence of various concentrations of reduced glutathione. A t o t a l volume of 0.5 ml was administered to each mouse. Samples were taken at 12 hr a f t e r force-feeding. Results from duplicate samples at each concentration are p l o t t e d The concentrations indicated are those of the force-fed solutions, not stomach contents a f t e r force-feeding. See discussion f o r further consideration. - 1 2 0 -< 20 r Q E o i_ CD <L> Q. Q_ CD cn E o t_ cn O c o c Glutathione (mM) -121-Figure 38 3 Binding of H-BP to DNA from cultured CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and various concentrations of harman i n the presence of an S9 a c t i v a t i o n system f or 2 hr. Plates were rinsed and DNA i s o l a t e d immediately. Results from t r i p l i c a t e samples f or each concentration are plotted. - 1 2 2 -< z Q E o CD c_ <D Q. CL CD 10 E o L. CD o C O c 3 0 0 2 0 0 100 h 0 100 HARMAN (uM) 200 -123-Figure 39 3 Binding of H-BP to DNA from cultured CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and various concentrations of norharman i n the presence of an S9 a c t i v a t i o n system for 2 hr. Plates were rinsed and DNA i s o l a t e d immediately. Results from duplicate samples f o r each concentration are plotted. -124-o 100 200 NORHARMAN (uM) -125-DISCUSSION Studies aimed at i d e n t i f i c a t i o n of chemical carcinogens have h i s t o r i c a l l y focussed on two aspects of the problem. F i r s t , the i n v e s t i g a t i o n of substances that are chemically constructed for t h e i r a b i l i t y to induce cancer, and which are v i r t u a l l y absent from the environment, have been useful because of the information they have provided on the process of cancer i n i t i a t i o n (e.j*., 7,12-dimethyl benz(a)anthracene and 4-ni t r o q u i n o l i n e - l - o x i d e ) . Secondly, those r e l a t i v e l y exotic chemicals made by man for some i n d u s t r i a l , medical or domestic purpose have proved a f r u i t f u l source of carcinogenic substances. Their r e a c t i v i t y (the source of t h e i r usefulness i n the above situa t i o n s ) makes them good candidates for the p o t e n t i a l e l e c t r o -p h i l i c reactants that have been implicated as ultimate carcinogens and mutagens. Only recently have the great mass of ubiquitous, n a t u r a l l y -occurring substances come under the eye of i n v e s t i g a t o r s . R e a l i z a t i o n of the ultimate e l e c t r o p h i l i c nature of carcinogens has made i t possible to "screen" the previously intimidating number of compounds so that p a r t i c u l a r l y promising candidates are recognizable by t h e i r p o t e n t i a l carcinogenic nature (capable of being r e a l i z e d by cell-mediated or n o n - c e l l u l a r chemical modification) rather than by t h e i r i n i t i a l , unmetabolized r e a c t i v i t y . In order to recognize a l l possible carcinogens i n the environment, a v a r i e t y of screening tests have been proposed to i d e n t i f y them. These range from actual induction of tumours i n rodents and other mammals to the detection of short-term c e l l u l a r and ti s s u e changes that have been causally or e m p i r i c a l l y associated with induction of cancer. This second group includes short-term tests of three types: 1) those that assay - 1 2 6 -for changes induced by chemicals i n i s o l a t e d DNA or chromatin ( e . , unwinding of bacteriophage DNA cleaved by chemicals (Kuhnlein, 1980), 2) those that assay for chemical or morphological changes i n cultured mammalian or b a c t e r i a l c e l l s (induction of DNA rep a i r , chromosome aber-rat i o n s , DNA a l t e r a t i o n s , germ c e l l anomalies, DNA-carcinogen adducts, etc.(reviewed by S t i c h and San, 1980)), and 3) those that assay for chemical or morphological changes i n mammalian c e l l s i n vivo. These include many of the tests a v a i l a b l e f o r cultured c e l l s , as well as those possible only i n systems containing organs capable of ex h i b i t i n g develop-ment of preneoplastic tissue (Solt and Farber, 1977) or s p e c i f i c locus mutations (Maier and Zbinden, 1980). This array of tests i s a useful one f o r detection of chemicals which may induce cancer, but i t cannot be used to detect chemicals which may modify the induction of cancer by chemical or other agents. It would be highly desirable' ;to -have' an--assay -system that^could* be' used for the detection of such modifying chemicals, for use both ^n v i t r o and jin vivo, and one of the aims of t h i s work was to evaluate the usefulness of the a l k a l i n e sucrose gradient and BP-DNA adduct tests for DNA damage and repair as suitable candidates for such an a p p l i c a t i o n . As an a p p l i c a t i o n of these t e s t s , both were used to assay the a b i l i t y of sodium ascorbate to modify the capacity of both cultured c e l l s and mammalian e p i t h e l i a l c e l l s i n s i t u to repair damage to DNA caused by d i r e c t - a c t i n g carcinogen (MNNG) or a precarcinogen (BP) applied i n the presence of an appropriate a c t i v a t i n g system. It was found that sodium ascorbate, supplied to c e l l s a f t e r the a p p l i c a t i o n of MNNG to fragment DNA, i n h i b i t e d t h e i r a b i l i t y to restore the capacity of DNA to sediment quickly i n a l k a l i n e sucrose gradients. This r e s t o r a t i o n normally takes place by 30 hours a f t e r -127-carcinogen treatment (Koropatnick, 1978). I n h i b i t i o n of repair took place for the period that unoxidized sodium ascorbate was present, and repair capacity was restored, both to cultured c e l l s i n v i t r o and g a s t r i c c e l l s i n vivo, when sodium ascorbate treatments were stopped. Sodium ascorbate treatment alone had no DNA-fragmenting e f f e c t on DNA. In addition, sodium ascorbate supplied to c e l l s a f t e r the appoication of BP to form covalent DNA adducts i n h i b i t e d t h e i r a b i l i t y to excise those adducts. It was found that, i n the absence of sodium ascorbate, cultured CHO c e l l s r a p i d l y l o s t appro-3 ximately 60% of bound H-BP during the f i r s t 12 hours a f t e r BP 3 treatment, and thereafter l o s t only very small amounts of H-BP l a b e l up to 72 hours post-treatment. This i s s i m i l a r to r e s u l t s abtained 3 when rats were injected with H-BP and the extent of s p e c i f i c a r y l s u b s t i t u t i o n at the 0-6 s i t e of guanine i n l i v e r was determined (P. Kleihues, personal communication). The presence of sodium ascorbate i n the medium present during the repair period strongly i n h i b i t e d the e x c i s i o n during the f i r s t 12 hours, but, at low concentrations -3 3 of sodium ascorbate (1 X 10 M), H-BP binding returned to near-control l e v e l s by 24 hours a f t e r BP treatment. At high concentration _3 (5 X 10 M), c o n t r o l l e v e l s were not reached u n t i l 36-48 hours post-treatment. The same s i t u a t i o n was observed i n vivo i n mouse g a s t r i c c e l l s . Bound BP reached a peak i n these c e l l s 12 hours a f t e r force-feeding 3H-BP. 3H-binding f e l l to 50% of t h i s high l e v e l i n the f i r s t 36 hours a f t e r peak binding, and then did not s i g n i f i c a n t l y change up to 48 hours a f t e r peak binding. In the presence of sodium ascorbate, 3 force-fed a f t e r the peak of H-binding was reached, v i r t u a l l y no 3 increase i n bound H-BP l e v e l s occurred. Within four hours a f t e r - 1 2 8 -3 the end of sodium ascorbate treatments, bound H-BP l e v e l s f e l l r a p i d l y , and normal binding l e v e l s were reached between 12 and 36 hours a f t e r cessation of sodium ascorbate treatment. One of the questions raised by the observed i n h i b i t i o n of r e s t o r a t i o n of normal DNA i s : at which stage of r e s t o r a t i o n of DNA does sodium ascorbate exert i t s effect? Since sodium ascorbate i n h i b i t e d the excision of BP adducts and r e s t o r a t i o n of fast sedimentation of DNA, i t appears that the i n h i b i t o r y e f f e c t i s exerted at a very early point i n r e p a i r , at or before the excision stage of damaged regions of DNA (assuming a common repair mechanism for BP and MNNG-induced DNA r e p a i r ) . Since MNNG may a l k y l a t e DNA to produce apurinic s i t e s that are converted to the single-strand breaks that are observed on a l k a l i n e sucrose gradients, such excision of repairable regions of DNA i s a necessary pr e r e q u i s i t e i n the repair of MNNG-induced damage, as well as repair of BP-adducts. In addition, r e s t o r a t i o n of fast sedimentation to DNA on a l k a l i n e sucrose gradients i s dependent upon l i g a t i o n of s i n g l e -strand breaks, as well as excision of damaged regions. The i n h i b i t i o n of repair of DNA observed i n the presence of sodium ascorbate might there-fore be due to i n h i b i t i o n of DNA l i g a t i o n or i n h i b i t i o n of any of the processes leading up to i t . Thus, sodium ascorbate may i n h i b i t r e pair at one or more steps. A second question thay may be asked i s : does the sodium ascorbate alone i n h i b i t e x cision of BP adducts and r e s t o r a t i o n of fast sedimentation of DNA, or are the oxidative or other metabolic products of sodium ascorbate responsible for t h i s effect? Sodium ascorbate i s oxidized i n vivo to dehydroascorbate by a r e v e r s i b l e process, and dehydroascorbate i s i r r e v e r s i b l y oxidized to diketogulonate and oxalate. The i n i t i a l oxidation step may be catalyzed by sodium ascorbate i t s e l f , -129-e s p e c i a l l y i f metal ions are added ( H a l l i w e l l and Foyer, 1976; Michelson, 1973). Thus, because of the i r r e v e r s i b l e nature of l a t e r oxidation steps, sodium ascorbate concentration i n a s o l u t i o n w i l l quickly decline i n favour of increasing concentration of i t s oxidation products. If these oxidation products were responsible for repair i n h i b i t i o n , then older sodium ascorbate solutions would be more e f f i c i e n t r e pair i n h i b i t o r s than fresh solutions. However, when a single s o l u t i o n of sodium ascorbate was added to cultured human f i b r o b l a s t s recovering, over a period of 30 hours, from damage i n f l i c t e d by MNNG, repair was l e s s i n h i b i t e d than when fresh solutions of sodium ascorbate were added every 4 hours up to 20 hours. Thus, either sodium ascorbate, or some substance produced by i t i n the i n i t i a l oxidation step, are necessary for i n h i b i t i o n of DNA r e p a i r . Third, the question of relevance of the concentrations used to retard DNA i n these experiments must be raised. The concentrations of sodium ascorbate used to i n h i b i t repair i n cultured c e l l s ranged _3 from 1 X 10 M to i n h i b i t r e s t o r a t i o n of fast sedimentation to -3 DNA from cultured human f i b r o b l a s t s to 5 X 10 M to i n h i b i t excision -3 of BP adducts from cultured CHO c e l l s (although 1 X 10 M sodium ascorbate could be used to i n h i b i t excision of BP adducts i n the f i r s t 12 hours a f t e r administration of BP). C a l c u l a t i o n of the e f f e c t i v e concentration of sodium ascorbate i n experiments involving force-feeding, however, are more d i f f i c u l t , although empirical comparisons to human consumption are easier. There are two ways to consider the e f f e c t i v e sodium ascorbate concentration on g a s t r i c mucosal c e l l s i n mice force-fed the sodium ascorbate: 1) by assuming the sodium ascorbate i s d i l u t e d equally throughout the whole body volume, and 2) by assuming that sodium ascorbate i s d i l u t e d only to the -130-volume of the stomach. Because complete dissemination of chemicals i n the body requires a lengthy time period, the f i r s t method i s better suited to observation of long-term e f f e c t s of administered chemicals. Since r e s t o r a t i o n of fast-sedimentation of DNA from cultured human f i b r o -b l a s t s on a l k a l i n e sucrose gradients occurs between 12 and 18 hours a f t e r MNNG administration (Koropatnick, 1978) and p r e r e q u i s i t e metabolic steps (e.j>., excision of DNA adducts) occur at t h e i r greatest rate i n the f i r s t 24 hours a f t e r carcinogen administration, long-term incubation i s not s u i t a b l e here. Also, increase or decrease of concentration of chemical by excretion, d e t o x i f i c a t i o n , or chemical i n s t a b i l i t y , are not taken into account by the whole-body d i l u t i o n system. The second method - c a l c u l a t i n g e f f e c t i v e concentration by stomach volume - seems more appropriate here. The test c e l l s of the g a s t r i c mucosa l i n e the stomach, and so are subjected to bathing chemicals before they are absorbed into the blood stream. Concentrations of sodium ascorbate d i f f e r here from those administered to cultured c e l l s . Because of food present i n the stomach, non-specific binding or oxidation of ascorbic a c i d may decrease the e f f e c t i v e concentration. V a r i a t i o n i n mouse stomach volume (estimated to be 0.002 to 0.005 l i t r e s ) may also a f f e c t concentration. Also, mice do not require sodium ascorbate as a dietary supplement, and produce t h e i r own supply by biosynthesis from glucuronic acid. Unlike humans, they also possess an ascorbic a c i d reductase enzyme that has the a b i l i t y to metabolize ascorbic acid and remove i t from the system (Brown and Reynolds, 1963). If t h i s enzyme i s a v a i l a b l e i n s u f f i c i e n t l e v e l s i n g a s t r i c mucosal c e l l s , rapid meta-bolism could reduce the length of time over which the sodium ascorbate i s e f f e c t i v e . F i n a l l y , movement of stomach contents into the small i n t e s t i n e w i l l also reduce the e f f e c t i v e time of action of sodium ascorbate. -131-With t h i s i n mind, the concentration of sodium ascorbate to which g a s t r i c mucosal c e l l s were exposed, based s o l e l y on the volume of -2 -2 mouse stomach, was 1.4 X 10 M to 3.5 X 10 M i n the case of a l k a l i n e -2 -2 sucrose gradient analysis of DNA repair and 3.5 X 10 M to 8.75 X 10 M i n the case of BP-DNA adduct analysis of DNA r e p a i r . For reasons outlined above, these may be considered the upper l i m i t s of ascorbate concentrations a c t u a l l y applied to the c e l l s . In humans, stomach volume va r i e s roughly between 0.5 and 2.0 l i t r e s . In order to achieve sodium ascorbate concentrations i n the range e f f e c t i v e i n reducing repair i n mice, doses of approximately 1-35 grams per treatment, administered p e r i o d i c a l l y over the period of repair of mucosal c e l l DNA, would have to be given. This i s a high l e v e l i n comparison with that required for normal health (30-40 mg per day)(Pett, 1955). However, some recommended l e v e l s of sodium ascorbate administration for the prevention of colds (Pauling, 1976; Stone, 1972) and treatment of cancers (Cameron, e_t .al. , 1975; Cameron and Pauling, 1978) are within t h i s range. While sodium ascorbate was able to i n h i b i t r e pair as measured by a l k a l i n e sucrose gradients and excision of BP adducts to DNA i n vivo and i n v i t r o , removal of sodium ascorbate (in the case of a l k a l i n e sucrose gradient analysis of repair i n cultured human f i b r o b l a s t s ) and non-renewal of sodium ascorbate treatments ( i n the case of ASG analysis of r e p a i r i n mouse g a s t r i c mucosal c e l l s , and BP adduct analysis of repair i n both cultured CHO c e l l s and mouse g a s t r i c mucosal c e l l s ) resulted i n the eventual return of the DNA of the c e l l s i n question to a state of repair that was close to that observed i n c e l l s untreated with sodium ascorbate. This i s an i n d i c a t i o n that a) the i n h i b i t o r y e f f e c t i s r e v e r s i b l e , and b) that sodium ascorbate i s r a p i d l y decomposed i n the - 1 3 2 -system to remove i t s e f f e c t , or the c e l l becomes a c c l i m a t i z e d to the pre s e n c e of the a s c o r b a t e . S i n c e mice f o r c e - f e d a s c o r b a t e r e t u r n e d to normal l e v e l s o f e x c i s i o n o f BP adducts from DNA more q u i c k l y than c u l t u r e d c e l l s a f t e r c e s s a t i o n o f sodium a s c o r b a t e t r e a t m e n t s , and f r e s h t r e a t m e n t s of sodium a s c o r b a t e i n c r e a s e d the l e n g t h o f time over which r e p a i r i n h i b i t i o n took p l a c e i n c u l t u r e d human f i b r o b l a s t s t r e a t e d w i t h MNNG, i t seems l i k e l y t h a t r a p i d d e c o m p o s i t i o n o f sodium a s c o r b a t e and i t s removal from the system i s r e s p o n s i b l e f o r i t s e v e n t u a l l o s s o f e f f e c t , r a t h e r than a d a p t a t i o n o f c e l l u l a r r e p a i r systems t o the pr e s e n c e of a s c o r b a t e . While the o b s e r v a t i o n s made i n bot h systems appear to f a v o u r the s u g g e s t i o n t h a t sodium a s c o r b a t e i n h i b i t s r e p a i r , o t h e r e x p l a n a t i o n s can be invok e d to e x p l a i n the obs e r v e d e f f e c t s . One s u g g e s t i o n i s t h a t sodium a s c o r b a t e may p o t e n t i a t e damage caused by c a r c i n o g e n s such t h a t , when c a r c i n o g e n i s removed from c e l l systems and sodium a s c o r b a t e added, the o b s e r v e d r e t e n t i o n o f damage i s not due to l a c k o f r e p a i r , but r a t h e r to p r o m o t i o n o f damage by r e s i d u a l c a r c i n o g e n l e f t b e h i n d by i n c o m p l e t e removal and r i n s i n g . I t was obs e r v e d t h a t sodium a s c o r b a t e may enhance the f r a g m e n t a t i o n o f DNA i n c u l t u r e d human f i b r o b l a s t s i n v i t r o and mouse g a s t r i c mucosal c e l l s jin v i v o , caused by MNNG. Sodium a s c o r b a t e i n the pre s e n c e of g l y c i n e - c o m p l e x e d copper c o u l d be used t o fragment t h e DNA o f c u l t u r e d human f i b r o b l a s t s jin v i t r o and mouse g a s t r i c mucosal c e l l s j i n v i v o . Because o f the p o s s i b i l i t y t h a t enhancement o f DNA damage was the cause r a t h e r than the e f f e c t o f the observed r e t e n t i o n o f damage, the e f f e c t of sodium a s c o r b a t e on i n i t i a l damage to DNA was measured. When sodium a s c o r b a t e and BP were a d m i n i s t e r e d to mice by f o r c e -f e e d i n g , an i n i t i a l i n c r e a s e i n b i n d i n g of BP was obs e r v e d a t low sodium a s c o r b a t e c o n c e n t r a t i o n s , f o l l o w e d by a de c r e a s e i n BP b i n d i n g w i t h -133-increasing sodium ascorbate concentration. This small but s t a t i s t i c a l l y s i g n i f i c a n t and reproducible increase i n binding was observed at close to the same concentration (100 mg sodium ascorbate per kg body weight) that was used to cause the maintenance of high l e v e l s of BP adducts and slow-sedimenting DNA c h a r a c t e r i s t i c s (40 mg per kg body weight) of mouse g a s t r i c mucosal c e l l s treated i n vivo. However, sodium ascorbate admini-stered to cultured c e l l s i n combination with BP did not induce any increase i n BP binding to DNA at ascorbate concentrations as high or higher than those used to i n h i b i t r e pair of DNA fragmentation or BP adducts i n cultured CHO c e l l s or human f i b r o b l a s t s . Thus, even when BP i s administered with sodium ascorbate i n concentrations much higher than those l i k e l y to be l e f t behind by incomplete r i n s i n g , only a very small amount of increased binding i s observed, and then only jLn vivo. This small increase does not appear to be enough to account for the retention of high l e v e l s of BP adducts i n DNA observed i n the presence of sodium ascorbate i n the bathing medium of r e p a i r i n g c e l l s . Therefore, i t seems l i k e l y that the retention of MNNG and BP-induced DNA modification seen i n the presence of sodium ascorbate i s due to an e f f e c t on repair of DNA rather than on the i n i t i a l DNA-damaging events. While sodium ascorbate could be used to i n h i b i t the repair of DNA fragmentation and BP adducts, the "scavenging" a b i l i t y of sodium ascorbate may also be used to i n h i b i t the action of several carcinogens. The DNA fragmenting property of S9-activated DMN was i n h i b i t e d by the presence of sodium ascorbate, presumably by i n h i b i t i o n of action of the carcinogen rather than by i n h i b i t i o n of the a c t i v a t i o n system (Lo and St i c h , 1978). Sodium ascorbate i n h i b i t e d the non-enzymatic formation of n i t r o s a t i o n products of methylguanidine, as shown by the decreased DNA-fragmenting -134-a b i l i t y of methylguanidine reacted i n the presence of ascorbate. Sodium ascorbate, when incubated with MNNG for 30 minutes p r i o r to t r e a t -ment i f cultured c e l l s or mouse g a s t r i c mucosal c e l l s , i n h i b i t e d the DNA-fragmenting action of the MNNG. This was i n contrast to the enhancement of MNNG fragmentation seen when mixed MNNG and sodium ascorbate were applied i n v i t r o or i n vivo without the 30 minute incubation. This enhancement was due, presumably, to i n h i b i t i o n of repair of DNA. This, the reducing p o t e n t i a l of sodium ascorbate could be employed to "scavenge" e l e c t r o p h i l e s and prevent t h e i r action. However, sodium ascorbate could be used to fragment DNA when co-administered with copper, both i n v i t r o and i n vivo. This fragmentation was repairable, since mouse g a s t r i c mucosal c e l l s treated i n vivo were able to restore near-control sedimentation properties to t h e i r DNA by 48 hours a f t e r treatment. This fragmenting a b i l i t y was due, presumably, to DNA damage caused by hydrogen peroxide produced by autoxidation of sodium ascorbate i n the presence of t r a n s i t i o n metals (Stich, et a l . , 1979). Since the b i o l o g i c a l reducing agent sodium ascorbate had an ef f e c t on scavenging reactive e l e c t r o p h i l e s , an attempt was made to measure the scavenging a b i l i t y of two other reducing agents -propyl g a l l a t e and glutathione. Propyl g a l l a t e , a food additive used to prevent oxidation (as i s sodium ascorbate), was found to i n h i b i t the binding of activated BP i n cultured CHO c e l l s , as expected from data gathered using sodium ascorbate. However, when propyl g a l l a t e was force-fed to mice i n the presence of S9 a c t i v a t i o n system, an increase i n bound BP i n r e l a t i o n to propyl g a l l a t e concentration was found. In addition, reduced glutathione was found to increase binding of BP, both i n v i t r o and i n vivo. Because of the l i m i t e d s o l u b i l i t y of propyl g a l l a t e , only half the molar concentration of sodium ascorbate -135-required to a l t e r BP-adduct formation was used. In addition, only r e l a t i v e l y low concentrations of glutathione (approximately one-sixth of the molar concentration of sodium ascorbate required for e f f e c t ) were used. In view of t h i s d i s p a r i t y , the difference i n e f f e c t might be due to concentration rather than q u a l i t a t i v e e f f e c t s . In f a c t , the i n i t i a l increase i n BP-binding to DNA i n vivo observed i n the presence of sodium ascorbate occurred at close to the same molar concentration of propyl g a l l a t e that caused a two-fold increase i n binding i n vivo. This may i n d i c a t e that, at higher propyl g a l l a t e and glutathione concentra-tions in vivo, the same i n h i b i t i o n of BP-binding to DNA as that observed i n the presence of sodium ascorbate might be seen. Because of the d i f f e r e n t i a l e f f e c t of propyl g a l l a t e ( i n h i b i t i n g BP binding jin v i t r o but enhancing BP binding jin v i v o ) , the tryptophan p y r o l y s i s products harman and norharman (already shown to either enhance or i n h i b i t BP mutagenicity i n b a c t e r i a , depending on concentration) were administered to CHO c e l l s i n the presence of BP to observe whether such an e f f e c t was observable i n t h i s system. No s i g n i f i c a n t i n h i b i t i o n or enhancement of BP binding was observed over the range of concentrations where i n h i b i t i o n or enhancement of BP mutagenicity i n b a c t e r i a was seen (Fujino, et a l . , 1978). Therefore, i t seems u n l i k e l y that such a d i f f e r e n t i a l e f f e c t , due to differences i n degree of i n h i b i t i o n of a c t i v a t i n g and i n a c t i v a t i n g enzyme systems associated with BP, i s at work here. While the reducing properties of propyl g a l l a t e (as the d r i v i n g force behing a "scavenging" e f f e c t on r e a c t i v e e l e c t r o p h i l e s ) may be invoked to explain decreased BP binding, i t i s i n t r i g u i n g to speculate that the increase i n bound BP observed may be due to i n h i b i t i o n of repair mechanisms. However, i t must be kept i n mind that d i f f e r e n t i a l enzyme i n h i b i t i o n may s t i l l be a factor - 1 3 6 -i n v i v o , as d i f f i c u l t i e s i n d e t e r m i n a t i o n of p r o p y l g a l l a t e c o n c e n t r a t i o n and time of exposure i n the stomach, as o u t l i n e d above, e x i s t . A l s o , the enzymes r e s p o n s i b l e f o r BP m e t a b olism i n g a s t r i c mucosal c e l l s may d i f f e r from the l i v e r enzymes used i n the S9 a c t i v a t i o n system. On the o t h e r hand, g l u t a t h i o n e w i l l i n c r e a s e b i n d i n g of BP i n v i t r o w i t h c o n t r o l l e d g l u t a t h i o n e exposure and S9 a c t i v a t i o n , as w e l l as i n v i v o . Thus, i n h i b i t i o n o f r e p a i r may w e l l be a r e a s o n a b l e mechanism to e x p l a i n i n c r e a s e d b i n d i n g of BP i n the p r e s e n c e of g l u t a t h i o n e . Summary In g e n e r a l , sodium a s c o r b a t e was shown to i n h i b i t r e p a i r of l e s i o n s i n t r o d u c e d i n t o DNA by MNNG or BP. I n h i b i t i o n of b o t h the e a r l y l e s i o n e x c i s i o n s t e p s and l a t e DNA l i g a t i o n and c h r o m a t i n r e o r g a n i z a t i o n s t e p s was o b served i n c u l t u r e d CHO c e l l s o r c u l t u r e d human f i b r o b l a s t s , r e s p e c t i v e l y , as w e l l as i n g a s t r i c c e l l s o f mice t r e a t e d w i t h c a r c i n o g e n and sodium a s c o r b a t e i n v i v o . W h ile sodium a s c o r b a t e was demonstrated to i n h i b i t DNA damage by " s c a v e n g i n g " r e a c t i v e e l e c t r o p h i l e s , and c o u l d a l s o fragment DNA i n v i v o and jin v i t r o when c o - a d m i n i s t e r e d w i t h copper, t h e s e e f f e c t s do not appear to be r e s p o n s i b l e f o r the o b served r e t a r d a t i o n o f e x c i s i o n o f BP a dducts from DNA o r the r e s t o r a t i o n of f a s t - s e d i m e n t i n g a b i l i t y o f DNA on a l k a l i n e s u c r o s e g r a d i e n t s . In a d d i t i o n , the r e d u c i n g agents p r o p y l g a l l a t e and sodium a s c o r b a t e were shown to i n h i b i t BP b i n d i n g i n v i t r o , but p r o p y l g a l l a t e enhanced B P - b i n d i n g ±n v i v o . The r e d u c i n g agent g l u t a t h i o n e a l s o enhanced BP b i n d i n g , b o t h i n v i v o and jm v i t r o . I t i s p o s s i b l e t h a t i n h i b i t i o n o f DNA r e p a i r may be r e s p o n s i b l e f o r the o b served -137-increase i n DNA l e s i o n s . Perspectives While the reducing agent sodium ascorbate has been implicated i n reducing the extent of DNA r e p a i r of damage caused by chemical carcinogens, i t remains to be seen what relevance t h i s observation has to the e f f e c t of vitamin C l e v e l s i n human health. The best l i n k of reduced repair capacity and induction of cancers comes from data gleaned from studies of human patients. Sufferers of xeroderma  pigmentosum, Louis-Bar syndrome, Fanconi's anaemia, f a m i l i a l r e c t a l polyposis, and Cockayne's syndrome (German, 1977; Paterson, 1977; German, 1978) a l l show p r e d i l e c t i o n for cancer i n as s o c i a t i o n with reduced excision repair capacity. However, there are some i n d i v i d u a l s s u f f e r i n g from these diseases that exhibit normal repair capacity. Therefore, the p o s s i b i l i t y e x i s t s that enhanced s u s c e p t i b i l i t y to cancer depends upon the q u a l i t y of repair of damage, and not only on reduced quantity of r e p a i r . I t seems reasonable to suppose that a decrease i n DNA repair may lead to increased c e l l death rather than i n i t i a t i o n of tumours (Kihlman, 1977). Sodium ascorbate has been shown to potentiate the t o x i c i t y of nitro-aromatic compounds (Koch, et a l . , 1979) and ascorbic acid has been shown to i n h i b i t the expression of cultured mouse c e l l f o c i transformed by 3-methyl cholanthrene (Benedict, et a l . , 1980). Sodium ascorbate has also been shown to promote m i t o t i c i n h i b i t i o n i n c e l l s treated with carcinogenic chemicals (Stich, et a l . , 1979). In the l i g h t of t h i s , i n h i b i t i o n of the extent of DNA repair a f t e r carcinogen treatment may prevent tumour i n i t i a t i o n i n mammals by promoting the death of affected - 1 3 8 -c e l l s . I t i s desirable that jiLn vivo experiments be done i n which sodium ascorbate i s applied to animal test subjects a f t e r carcinogen administration to i n h i b i t the short term repair that occurs i n hours or days. The e f f e c t on the production of tumours or preneoplastic ti s s u e would then be observed. In t h i s way, the e l e c t r o p h i l e -scavenging e f f e c t and anti-cancer c e l l properties implicated for vitamin C may be excluded, and only the e f f e c t of i n h i b i t i o n of DNA repair events observed. Also, the e f f e c t of sodium ascorbate on repair of UV-induced lesions i n DNA would be desirable. This would exclude the e f f e c t of electrophile-scavenging by sodium ascorbate, as well as avoid the d i f f i c u l t i e s associated with the presence of r e s i d u a l carcinogen a f t e r treatment of c e l l s . In the case of propyl g a l l a t e and glutathione, the ultimate e f f e c t of increased DNA damage by carcinogens i n t h e i r presence remains unknown. It has been observed that, while sodium ascorbate enhances c y t o t o x i c i t y of nitro-aromatic compounds, sulphydryl reducing agents such as glutathione, cysteine, cysteamine and mercaptoethanol i n h i b i t c y t o t o x i c i t y of these compounds (Koch, e_t al_. , 1979). While the mechanism of these phenomena remains unknown, further i n v e s t i g a t i o n i s c e r t a i n l y warranted. Experiments using combinations of glutathione and sodium ascorbate i n v i t r o , to determine whether there i s a chemical competition between the e f f e c t s , would be a f r u i t f u l avenue of i n v e s t i g a t i o n . As general methods for observing the e f f e c t of compounds that may modify the short-term e f f e c t s of carcinogenic compounds, BP adduct and ASG sedimentation analysis of DNA appear to be w e l l - s u i t e d . The most widely used short-term assay for DNA repair employs 3 unscheduled incorporation of H-TdR (Stich and San, 1980). This -139-assay cannot be used to d i f f e r e n t i a t e between decreased damage and 3 decreased r e p a i r , so that i n h i b i t i o n of incorporation of H-TdR observed i n the presence of some modifying compound could be due to c i t h e r e f f e c t . Also, repair jin vivo i s t e c h n i c a l l y d i f f i c u l t to observe using t h i s method. The BP adduct assay may be used i n v i t r o or jin vivo. 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