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Inhibitory effect of Browning reaction products and phenolic compounds on carcinogen-induced mutagenesis Chan, Robin Isaac Man-Hang 1983

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INHIBITORY EFFECT OF BROWNING REACTION PRODUCTS AND PHENOLIC COMPOUNDS ON CARCINOGEN-INDUCED MUTAGENESIS b y ROBIN ISAAC MAN-HANG CHAN B . S c , The U n i v e r s i t y of B r i t i s h Columbia, - 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of M e d i c a l Genetics We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1983 tr © Robin Isaac Man-hang Chan, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department Of Medical Genetics The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 25, 1983 DE-6 (3/81) ABSTRACT Chemical carcinogens are highly reactive e l e c t r o p h i l i c substances capable of i n t e r a c t i n g with n u c l e o p h i l i c s i t e s i n -discr i m i n a t e l y . The use of nuc l e o p h i l i c trapping agents that react with e l e c t r o p h i l e s could thus provide possible protection for c r i t i c a l c e l l u l a r targets against the action of chemical carcinogens. It was on the basis of t h i s general concept that substances i n the present study were examined for possible i n h i b i t o r y a c t i v i t i e s against carcinogen-induced mutagenesis. Non-enzymatic browning reactions occur i n v i r u t u a l l y a l l heated food s t u f f s . Phenolic compounds are also widely d i s -t r i b u t e d i n plants and are consequently present i n many foods. Antimutagenic a c t i v i t y of two non-enzymatic browning r e a c t i o n products (caramelized sucrose and lysine-fructose M a i l l a r d reaction products) and several phenolic compounds were deter-mined i n the present study. At non-toxic concentrations, the two browning reaction products and three phenolic compounds ( g a l l i c a c i d , c a f f e i c a c i d and chlorogenic acid) s i g n i f i c a n t l y suppressed the mutagenicity of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) i n Salmonella typhimum-um s t r a i n TA 1535 and Sacchavomyces cerevis-iae s t r a i n XV185-14C. Interaction between phenolic compounds and MNNG was also studied i n a c e l l -free system. The amount of MNNG, detected by a co l o r i m e t r i c method, following i t s incubation with phenolic compounds de-creased s u b s t a n t i a l l y compared to that i n the untreated MNNG controls. The re s u l t s are consistent with the assumption that phenolic compounds scavenged reactive e l e c t r o p h i l i c MNNG de-gradation products thereby preventing t h e i r action on c r i t i c a l c e l l u l a r targets. The two browning r e a c t i o n products and seven phenolic compounds (tannic acid, g a l l i c a c i d , c a f f e i c a c i d , chlorogenic acid, s a l i c y l i c acid, p-hydroxybenzoic acid and dopamine) also reduced the mutagenicity of the precarcinogen, a f l a t o x i n (AFB^), when assayed on Salmonella typh-imurium s t r a i n TA 98 i n the presence of a rat l i v e r microsomal a c t i v a t i o n system. The e f f e c t of phenolic compounds on the a c t i v a t i o n of AFB^ by rat l i v e r microsomes was also studied by high-pressure l i q u i d chromatography (HPLC). The r e s u l t s from the HPLC analysis suggested that one mechanism whereby the phenolic, compounds suppressed the mutagenic a c t i v i t y of AFB^ i s by i n t e r f e r i n g with i t s metabolic a c t i v a t i o n . However, the p o s s i b i l i t y re-mains that part of the antimutagenic a c t i v i t y observed may be due to int e r a c t i o n s between the phenolic compounds and the reactive metabolite(s) of AFB^. The present study does not permit an assessment of the r e l a t i v e contribution of the two mechanisms of antimutagenic action. TABLE OF CONTENTS 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 L i s t of Abbreviations x Acknowledgements x i 1. INTRODUCTION 1 2. MATERIALS AND METHODS 6 2.1 Chemicals 6 2.2 Preparation of phosphate buffered saline 7 2.3 Preparation of S9 l i v e r microsomal mixture 7 2.4 Salmonella mutagenicity assay 8 2.5 Yeast mutagenicity test 11 2.6 Colorimetric determination of MNNG 13 2.7 Analysis of AFB^ a c t i v a t i o n by high-pressure l i q u i d chromatography 14 3. RESULTS 16 3.1 I n h i b i t i o n of MNNG-induced mutagenesis by browning reaction products 16 V 3.2 I n h i b i t i o n of MNNG-induced mutagenesis by phenolic compounds 19 3.3 I n h i b i t i o n of MNNG-induced mutagenesis i n the yeast mutagenicity t e s t . 23 3.4 I n h i b i t i o n of AFB^-induced mutagenesis by browning rea c t i o n products 26 3.5 I n h i b i t i o n of AFB^-induced mutagenesis by phenolic compounds 29 3.6 E f f e c t of pre- and post-treatment with i n h i b i t o r s on carcinogen-induced mutagenesis 32 3.7 Study of the i n t e r a c t i o n between phenolic compounds and MNNG i n a c e l l - f r e e system 37 3.8 Study of the i n h i b i t o r y e f f e c t of phenolic compounds on AFB^ metabolism using high-pressure l i q u i d chromatography 38 3.9 Interaction between phenolic compounds and preformed AFB^ metabolites 53 4. DISCUSSION 58 4.1 Concurrent administration of mutagen and i n h i b i t o r required for antimutagenic a c t i v i t y 58 4.2 Phenolic compounds i n h i b i t MNNG mutagenesis by a scavenging r e a c t i o n 59 4.3 Phenolic compounds i n h i b i t AFB^-induced mutagenesis by suppression of i t s metabolism 61 4.4 The ro l e of browning reaction products and phenolic compounds i n the prevention of human cancer 69 References 74 v i i LIST OF TABLES I. E f f e c t of pre- and post-treatment with caramelized sucrose on MNNG-induced mutagenesis 33 I I . E f f e c t of pre- and post-treatment with chlorogenic acid on MNNG-induced mutagenesis .34 I I I . E f f e c t of concurrent- and post-treatment with caramelized sucrose on AFB^-induced mutagenesis 35 IV. E f f e c t of concurrent- and post-treatment with chlorogenic acid on AFB^-induced mutagenesis 36 V. Inh i b i t o r y e f f e c t of phenolic compounds on the production of color compounds from MNNG 41 VI. The occurrance of phenolics i n food products and beverages 73 v i i i LIST OF FIGURES 1. E f f e c t of browning rea c t i o n products on reversion frequency of MNNG-treated b a c t e r i a i n the preincubation test 18 2. Ef f e c t of browning reaction products on c e l l s u r v i v a l and reversion frequency of MNNG-treated b a c t e r i a i n the suspension test 18 3. E f f e c t of phenolic compounds on reversion frequency of MNNG-treated b a c t e r i a i n the suspension test 22 4. E f f e c t of chlorogenic acid on c e l l s u r v i v a l and reversion frequency of MNNG-treated b a c t e r i a i n the suspension test 22 5. E f f e c t of c a f f e i c acid on c e l l s u r v i v a l and reversion f r e -quency of MNNG-treated b a c t e r i a i n the suspension t e s t . . .22 6. E f f e c t of browning reaction products on c e l l s u r v i v a l and mutagenic a c t i v i t y of MNNG-treated yeast cultures 28 7. E f f e c t of phenolic compounds on the mutagenic a c t i v i t y of MNNG-treated yeast cultures 28 8. Reaction pathways of MNNG 31 9. E f f e c t of browning rea c t i o n products on reversion frequency of AFB^-treated b a c t e r i a i n the preincubation test 35 10. E f f e c t of browning r e a c t i o n products on c e l l s u r v i v a l and reversion frequency of AFB^-treated b a c t e r i a i n the suspension test 35 i x 10. E f f e c t of phenolic compounds on reversion frequency of AFB^-treated b a c t e r i a i n the suspension test 31 11. E f f e c t of tannic acid on c e l l s u r v i v a l and reversion f r e -quency of AFB^-treated b a c t e r i a i n the suspension test...31 12. E f f e c t of chlorogenic acid on c e l l s u r v i v a l and reversion frequency of AFB -treated b a c t e r i a i n the suspension test 31 13. Reaction pathways of MNNG 40 14. Chromatograms of AFB^ incubated with S9 for various time periods 46 15. A c t i v a t i o n of AFB^ by S9 with d i f f e r e n t rat l i v e r microsome concentrations 46 16. E f f e c t of phenolic compounds on the production of an AFB^ metabolite t e n t a t i v e l y assigned to be AFB^-dihydrodiol...49 17. Comparsion of the e f f e c t of phenolic compounds on the production of AFM and the metabolite t e n t a t i v e l y assigned to be AFB^-dihydrodiol 49 18. Chromatograms of AFB^ incubated with S9 i n the presence of chlorogenic acid 51 19. E f f e c t of g a l l i c a c i d on the production of AFB^ metabolites from d i f f e r e n t concentrations of AFB^ 51 20. E f f e c t of c a f f e i c acid on the production of AFB^ meta-b o l i t e s under d i f f e r e n t incubation conditions 57 21. Known metabolic fate of AFB, i n animal hosts 63 X LIST OF ABBREVIATIONS AFB X — A f l a t o x i n B^ AFB^ d i h y d r o d i o l — 2,3-dihydro-2,3-dihydroxy-aflatoxin B^ AFM1 — A f l a t o x i n M^  AFQ X — A f l a t o x i n DMSO — Dimethyl sulfoxide G6P — Glucose-6-phosphate his — H i s t i d i n e independence HPLC — High-pressure l i q u i d chromatography i.p. — Intraperitoneal KC1 — Potassium chloride MgCl 2 — Magnesium chl o r i d e MNNG — N-methyl-N'-nitro-N-nitrosoguanidine NaCl — Sodium chloride NADP — Nicotinamide adenine dinucleotide phosphate PBS — Phosphate buffered saline x i ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to his supervisor, Dr. R.H.C. San, for a l l h i s guidance, encouragement and patience throughout the course of t h i s work. He i s also indebted to Dr. H.F. Stich for the use of h i s laboratory f a c i l i t i e s and Dr. M.P. Rosin for her expert advice and encouragement. Thanks are also due to Dr. D.P. Dunn for his assistance i n the HPLC portion of the study, to Dr. W.D. Powrie and Dr. CH. Wu (Department of Food Science, UBC) for the prepar-ation of browning reaction products and to Dr. D.P.H. Hsieh (Department of Environmental Toxicology, University of C a l i f o r n i a at Davis) for the a f l a t o x i n M^  and standards. F i n a n c i a l assistance from the National Research Council of Canada and National Cancer I n s t i t u t e of Canada (grants to Dr. Stich) i s g r a t e f u l l y acknowledged. He wishes also to thank Miss S. Tong and his s i s t e r , Miss L. Chan, for c o r r e c t i o n of the typed manuscript. F i n a l l y , he would l i k e to extend his thanks to the members of the Environ-mental Carcinogenesis Unit for t h e i r help and good company. 1 1. INTRODUCTION It i s now generally believed that most i f not a l l chemical carcinogens are compounds that contain highly reactive e l e c t r o n -d e f i c i e n t regions. If a p a r t i c u l a r chemical carcinogen i s not highly e l e c t r o p h i l i c pev se, then i t can be in e v i t a b l y activated into e l e c t r o p h i l i c species by mixed-function oxidases or other enzyme systems. Because of t h e i r high r e a c t i v i t y , they bind covalently and non-enzymatically to the abundant n u c l e o p h i l i c or e l e c t r o n - r i c h s i t e s present i n DNA's, RNA's and proteins i n target tissues ( M i l l e r , 1970; M i l l e r and M i l l e r , 1977). Although a l l chemical carcinogens adequately studied have shown to be able to bind to macromolecules such as nucl e i c acids and proteins covalently ( M i l l e r , 1978), the role of macromolecular binding i n carcinogenesis i s not c l e a r . Nevertheless, because of th e i r high r e a c t i v i t y , chemical carcinogens attack n u c l e o p h i l i c s i t e s i n d i s c r i m i n a t e l y . Therefore, the use of n u c l e o p h i l i c trap-ping agents that can react with the e l e c t r o p h i l i c carcinogens can provide possible protection for the c r i t i c a l c e l l u l a r targets against the action of chemical carcinogens. It was on the basis of t h i s general concept that substances i n the present study were examined as possible i n h i b i t o r s of chemical carcinogens. 2 Because chemical carcinogens can also induce a v a r i e t y of genetic damages, numerous short-term in vitro t e s t systems have been developed to detect chemical carcinogens as genotoxic substances. Some of the genetic damages employed for t h i s purpose include mutation, bacteriophage induction, DNA damage, chromosome damage as w e l l as gene conversion ( S t i c h and San, 1979). These tests are also useful i n uncovering factors with enhancing or i n h i b i t i n g e f f e c t s on genotoxic compounds (Rosin and S t i c h , 1978a,b, 1979, 1980; Rosin, 1981; Buening et al., 1981). One important area of current research i s i n the a p p l i c a t i o n of these t e s t s to the i d e n t i f i c a t i o n of genotoxic substances as well as modifying factors i n foods. This study was focused on the i n h i b i t o r y e f f e c t of non-enzymatic browning re a c t i o n products and plant phenolic compounds on chemical carcinogen-induced mutagenesis. Browning reactions are complex reactions that occur during the processing or storage of food. These reactions r e s u l t i n the production of brown pigments, which contribute to the color of the food, and v o l a t i l e products, which contribute to the f l a v o r and aroma of the food. The antimutagenic e f f e c t of products from two model browning reactions were examined. Caramelized sucrose was used as an example of the caramelization reaction which occurs when sugars are heated above t h e i r melting points i n the absence of 3 amino acids or proteins. The M a i l l a r d reaction represents another t y p i c a l browning reaction, which occurs when reducing sugars are heated i n the presence of amines, amino acids or proteins. Heated lysine-fructose s o l u t i o n was used as an example of the M a i l l a r d reaction. Because of the abundant nuc l e o p h i l i c s i t e s present i n these browning reaction products (Shallenberger and B i r c h , 1975), they may o f f e r p rotection against e l e c t r o p h i l i c substances capable of producing genotoxic e f f e c t s . Plant phenolic compounds constituted the second group of chemicals studied i n t h i s research project for possible a n t i -mutagenic a c t i v i t y . The phenolic compounds examined were simple substituted benzoic acids and cinnamic acids. Phenolic compounds have been extensively studied for t h e i r i n h i b i t o r y e f f e c t s on the toxic and carcinogenic actions of a wide v a r i e t y of chemical carcinogens (Wattenberg, 1972, 1979; Wattenberg et al., 1976). In p a r t i c u l a r , three n a t u r a l l y occurring phenolic derivatives of cinnamic acid (o-hydroxycinnamic a c i d , c a f f e i c acid and f e r u l i c acid) were e f f e c t i v e i n suppressing benzo(a)-pyrene-induced neoplasia i n the forestomach of rats (Watten-berg et al., 1980). Since these phenolic compounds have a n t i -oxidant properties, they are n u c l e o p h i l i c compounds that can int e r a c t with e l e c t r o p h i l i c carcinogens and prevent t h e i r 4 binding to c e l l u l a r n u c l e o p h i l i c macromolecules. The p o s s i b i l i t y that phenolic compounds i n h i b i t the action of chemical carcinogens through t h e i r antioxidant properties has been suggested although a l t e r n a t i v e mechanisms of i n h i b i t i o n cannot be ruled out (Wattenberg et al., 1976; Wattenberg, 1979). The present study examined the i n h i b i t o r y e f f e c t s of browning reaction products and plant phenolic compounds on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and a f l a t o x i n (AFB^) i n the Salmonella mutagenicity assay. MNNG was selected as a representative of the a l k y l a t i n g N-nitroso compounds, many of which are mutagens and carcinogens. Some of these N-nitroso compounds may be endogenously produced by the e l e c t r o p h i l i c r e a c t i o n between n i t r i t e and secondary amines under the a c i d i c condition of the stomach. AFB^, one of the most potent carcinogens known, was used as an example of a carcinogen which requires metabolic a c t i v a t i o n i n order to e l i c i t i t s genotoxic a c t i v i t y . It i s also a food-borne mycotoxin that may be an environmentally important human carcinogen i n some parts of the world (Shank et al., 1972; Peers and L i n s e l l , 1973; van Rensbury et al., 1975). Non-enzymatic browning reactions occur i n v i r t u a l l y a l l heated food s t u f f s . Plant phenolic compounds are also widely 5 d i s t r i b u t e d and are consequently present i n many foods. Both of these two groups of substances are probably consumed by the vast majority of the human population. Therefore, any e s t i -mation of the genotoxic hazard i n the human diet would be incomplete i f the e f f e c t s of these compounds are not considered. 6 2. MATERIALS AND METHODS 2.1 Chemicals The two model browning reaction products were prepared by the Department of Food Science, University of B r i t i s h Columbia. The lysine - f r u c t o s e model M a i l l a r d r e a c t i o n products were prepared by d i s s o l v i n g 0.01 mole of L-lysine and 0.01 mole of D-fructose i n 10 ml of d i s t i l l e d water and adjusting the pH to 10.0. The re a c t i o n s o l u t i o n was brought up to 12.5 ml so that the f i n a l concentration of the reactants was 0.8 M. The re a c t i o n tubes were capped and autoclaved at 121°C for 1 hr. The pH of the sample was then ad-justed to pH 7.0. Caramelized sucrose was prepared by heating a 38% sol u t i o n of D-sucrose i n evaporating dishes to a temperature of 180°C for 1.5 hr i n an a i r - c i r c u l a t i n g oven (Powrie et al., 1981; Stich et al., 1981a; Chan et al. , 1982). The following phenolic compounds: tannic acid, c a f f e i c a c i d , chlorogenic a c i d , s a l i c y l i c a c i d , p-hydroxybenzoic acid and dopamine were purchased from Sigma Chemical Co. (St. Louis, MO). G a l l i c acid was supplied by A l d r i c h Chemical Co. (Milwaukee, WI). Other biochemicals were obtained from Sigma Chemical Co. except nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P), which were obtained from Calbiochem-Behring Corp. 7 (La J o l l a , CA) , and methionine-freed leucine, which was obtained from General Biochemicals Co. (Chagrin F a l l s , OH). Dimethyl sulfoxide (DMSO) was purchased from Burdick & Jackson Laboratory (Muskegon, Ml). The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was obtained from A l d r i c h Chemical Co. while the precarcinogen a f l a t o x i n B^ (AFB^) was purchased from Sigma Chemical Co. 2.2 Preparation of phosphate buffered saline (PBS) One l i t e r of PBS contains 80 g sodium c h l o r i d e , 2.0 g potassium c h l o r i d e , 11.5 g dibasic sodium phosphate and 2.0 g monobasic potassium phosphate dissolved into double d i s t i l l e d water. Phosphate buffered s a l i n e has a pH of 7.4. 2.3 Preparation of S9 l i v e r microsomal mixture Standard S9 l i v e r microsomal mixture was prepared with l i v e r from Aroclor 1254 pre-treated rats as described by Ames et al. (1975). The rats used i n the present study were one-month-old male Wister rats of about 150 g each. Aroclor 1254, a polychlorinated biphenyl mixture, d i l u t e d i n corn o i l (200 mg/ml), was given to each rat at a dosage of 500 mg/kg body weight by a single i . p . 8 i n j e c t i o n 5 days before s a c r i f i c e . The rat s were given water and Purina Laboratory Chow ad libitum u n t i l 1 day before s a c r i f i c e . The rats were s a c r i f i c e d by a blow to the head and then decapit-ated. The l i v e r s were removed from the r a t s , added to 0.15M KC1 (approx. 1 ml/g wet l i v e r ) and homogenized. The homogenate was then centrifuged for 10 min at 9000 g and the supernatant was c o l l e c t e d and stored at -80°C i n small portions. The l i v e r supernatant was thawed when required before experiments. Each ml of the standard S9 reac t i o n mixture consists of 0.1 ml l i v e r supernatant, 0.02 ml 0.4 M-MgCl 2, 0.02 ml 1.65 M-KC1, 4.0 mmol NADP and 10.0 mmol G6P dissolved i n sodium phosphate buffer (pH 7.4). The so l u t i o n was fr e s h l y prepared before each experiment. 2.4 Salmonella mutagenicity assay Salmonella typhimurium tester s t r a i n s TA 1535 and TA 98 were obtained from Dr. B.N. Ames, Un i v e r s i t y of C a l i f o r n i a at Berkely. They were grown and maintained as described by Ames et al. (1975). TA 1535, a h i s t i d i n e auxotroph which reverts to h i s t i d i n e independence by base-pair s u b s t i t u t i o n , was used as an indicato r organism for the mutagenic a c t i v i t y of the carcinogen MNNG. The 9 mutagenic action of the precarcinogen AFB^ was assayed by TA 98 i n the presence of S9 l i v e r microsomal preparation. Tester s t r a i n TA 98 i s a h i s t i d i n e auxotroph sen s i t i v e to frame-shift mutagens (Ames et al. , 1975; McCann et al. 1975). Preliminary experiments were conducted using the preincuba-t i o n modification (Nagao et al. , 1977) of the procedure developed by Ames et al. ( 1975). Frozen master stock was inoculated i n Difco nutrient broth and grown overnight (16-18 hr) on a rotary wheel at 37°C i n an incubator. Treatment mixtures consisted of the follow-ing were added i n the indicated order: 0.1 ml of the overnight b a c t e r i a l suspension, 0.4 ml of eith e r PBS or the standard S9 l i v e r microsomal mixture, 0.1 ml of the test sample, and 0.1 ml of either MNNG dissolved d i r e c t l y into PBS or AFB^ dissolved i n DMSO. The treatment mixtures were incubated at 37°C i n a water bath for 20 min. Two ml of molten top agar (47°C) containing minimal amounts of h i s t i d i n e (0.455 mM) was then added to each of the treatment mixtures and o v e r l a i d on minimal glucose agar plates (Ames et al., 1975). Following incubation at 37°C for 2 days, the colonies were counted on an Artek Model 880 automatic colony counter (Farmingdale, NY). The mutagenic a c t i v i t y was expressed as the number of h i s + revertants per pla t e . Subsequent studies were done using the suspension procedure 10 (Rosin and St i c h , 1978a,b), which i s another modification of the basic method of Ames et at. (1975). This procedure permits an estimation of the s u r v i v a l as well as the incidence of reverse mutations of the b a c t e r i a . Logarithmically-growing cultures g (5x10 cells/ml) was prepared by reinoculating 0.1 ml of an over-night culture (16-18 hr) into 5.0 ml of fresh nutrient broth. These l a t t e r cultures were grown on a rotary wheel i n an incubator at 37°C for 4 hr. One ml aliquots of t h i s culture were placed i n centrifuge tubes and the b a c t e r i a were p e l l e t e d by ce n t r i f u g a t i o n (3000 rpm for 5 min). The p e l l e t s were resuspended i n 0.5 ml of treatment media and incubated for 20 min at 37°C i n a water bath. The treatment media consisted of e i t h e r equal volumes of test chemicals and MNNG or equal volumes of test chemicals, AFB^ and S9 preparation. MNNG i s dissolved d i r e c t l y into PBS while AFB^ i s dissolved i n 15% DMSO i n PBS ( f i n a l DMSO cone. = 5%). After the incubation period, the chemicals were removed by ce n t r i f u g a t i o n (3000 rpm for 5 min) and the b a c t e r i a were washed once by resus-pension i n 0.5 ml PBS and cen t r i f u g a t i o n . The b a c t e r i a were then 9 resuspended i n 0.5 ml PBS at ca. 10 c e l l s / m l . Reversion of the ba c t e r i a to h i s t i d i n e prototrophy were assayed by adding 0.1 ml aliquots of each sample to 2.0 ml molten top agar (0.455 mM h i s -t i d i n e ) and ov e r l a i d on minimal glucose agar plate s> (Ames et at. , 1975). Aliquots of the treated b a c t e r i a l culture were also d i l u t e d with 0.9% sodium chloride s o l u t i o n (0.01 ml/10 ml x 11 0.01 ml/10 ml) and plated (0.3 ml) onto nutrient agar plates (Ames et at., 1975) to determine c e l l s u r v i v a l frequencies. The nutrient agar plates were incubated f o r 1 day while the minimal agar plates were incubated f or 2 days p r i o r to scoring on the Artek automatic colony counter. The mutagenic a c t i v i t i e s were calculated i n terms of the number of h i s + revertants per 10^ surviving b a c t e r i a l c e l l s (Rosin and S t i c h , 1978a,b). Some experiments involved 2 consecutive incubation periods of 20 min each at 37°C. The b a c t e r i a were centrifuged and washed before they were resuspended with the second treatment mixture. These experiments were conducted i n order to determine the e f f e c t of t r e a t i n g the b a c t e r i a with test chemicals before or af t e r t h e i r exposure to MNNG or AFB^ and S9 on the mutagenic a c t i v i t y of the carcinogens. 2.5 Yeast mutagenicity test The Sacchavomyces cevewisiae s t r a i n XV185-14C was obtained from Dr. R.C. von Bo r s t e l of U n i v e r s i t y of Alberta, Edmonton, Alberta. XV185-14C i s a haploid s t r a i n with the genotype a, ade2-l, arg4-17, l y s l - 1 , trp5-48, h i s l - 7 , hom3-10. It requires six amino acids and one base f or growth as the homoserine mutation blocks the synthetic pathway of both threonine and methionine. 12 Mutations at the h i s l - 7 locus were used i n assaying the mutagenic a c t i v i t y of MNNG. Hisl-7 i s a missense mutation that can be reverted either by true back mutations or by second s i t e base substitutions (Shahin and von B o r s t e l , 1978). Ce l l s from a stock culture were inoculated into l i q u i d YEPD medium (2.0% Difco bactopeptone, 1.0% Difco yeast extract and 2.0% glucose) and incubated at 28°C i n a gyrotory water bath u n t i l they reach late logarithmically-growing stage (ca. 1-5x10^ c e l l s / m l ) . The c e l l s were harvested by ce n t r i f u g a t i o n (3000 rpm g for 5 min) and the c e l l concentration was adjusted to 10 cel l s / m l by resuspending i n D7 medium (6.7 g/1 Difco yeast nitrogen base without amino acids, 60 mg/1 each of adenine, tryptophan and isoleucine and 2.0% glucose) (Zimmermann, 1975). Treatment mixtures consisted of 0.5 ml yeast suspension, 0.25 ml test chemical and 0.25 ml MNNG, added i n the indicated order, were incubated i n a 28°C gyrotory water bath for 20 min. A l l chemicals were dissolved d i r e c t l y into t r i s buffer (pH 7.4). After the incubation period, the chemicals were removed by cen t r i f u g a t i o n and the yeast c e l l s were washed by resuspension i n t r i s buffer and ce n t r i f u g a t i o n . The yeast c e l l s were then resuspended i n 1.0 ml t r i s buffer and d i l u t e d to 1x10^ c e l l s / m l by adding 4.0 ml of s t e r i l e d o u b l e - d i s t i l l e d water. Mutagenic 13 a c t i v i t y was estimated by p l a t i n g 0.5 ml aliquots on h i s t i d i n e d e f i c i e n t agar plates (2.0% agar, 0.67% Difco bacto-yeast nitrogen base without amino ac i d , 2.0% glucose and a l l required supplements except h i s t i d i n e ) while s u r v i v a l frequencies were determined by p l a t i n g 0.5 ml aliquots of further d i l u t e d samples (0.1 ml/10 ml x 0.05 ml/10 ml) onto YEPD plates (15% agar, 2.0% Difco bacteropeptone, 1.0% Difco yeast extract and 2.0% glucose) (Zimmermann, 1975). A l l plates were incubated at 28°C for 3 days before scoring on an Artek automatic colony counter. The mutagenic a c t i v i t i e s were ca l c u l a t e d i n terms of the number of his revertants per 10 survivors. 2.6 Colorimetric determination of MNNG Samples containing MNNG and a phenolic compound (1 ml t o t a l volume) were placed i n 10 ml c a l i b r a t e d vessels and incubated at 37°C for 20 min i n a water bath. At the end of t h i s time, the samples were treated according to the c o l o r i m e t r i c method of F o r i s t ( F o r i s t , 1964; Preussmann and Schaper-Druckrey, 1972) to determine the amount of MNNG present i n the samples. Color reagent was prepared by mixing equal volumes of N-(l-naphthyl)-ethylenediamine (0.1% w/v) i n aqueous acetic acid (30% v/v) and s u l f a n i l i c acid (0.5% w/v) i n aqueous acetic acid (30% v/v). The color reagent was f r e s h l y prepared before use and was not 14 stored because i t i s l i g h t s e n s i t i v e ( F o r i s t , 1964; Preussmann and Schaper-Druckrey, 1972). Five ml color reagent and 1 ml 6M hydrochloric acid were added to each sample. The samples were then capped, vortexed and incubated i n a 60°C gyrotory water bath for 45 min. After cooling the samples to room temperature, the volume of each sample was made up to 10 ml. The absorbance of the samples was measured at 550 nm using a Perkin-Elmer Lambda 3 UV/VIS spectrophotometer (Oak Brook, IL) and an 1-cm cuvette. 2.7 Analysis of AFB^ a c t i v a t i o n by high-pressure l i q u i d  chromatography (HPLC) Treatment mixtures c o n s i s t i n g of 0.5 ml test chemical, 0.5 ml AFB^ and 0.5 ml standard S9 preparation were incubated at 37°C for 20 min i n a gyrotory water bath. The test chemicals were dissolved i n PBS and the pH adjusted to 6.5. AFB^ was dissolved i n 15% DMSO i n PBS (pH 6.5, f i n a l DMSO cone. = 5%). The pH of the S9 preparation was also adjusted to 6.5 (Lin et al. , 1978). After the incubation period, the samples were processed as described by L i n et al. (1978). The reaction was stopped by adding 1 ml of the treatment mixture to 2.0 ml ice cold ethanol and 0.075 ml 2M NaCl i n a centrifuge tube, vortexed and l e t stand on ice for 30 min. At the end of t h i s 15 time, the samples were centrifuged at 3000 rpm for 5 min and the supernatants were removed for analysis by HPLC without further treatment. HPLC was c a r r i e d out using a Vydac 201 TP, lOum p a r t i c l e reverse phase column with 3.2 mm x 250 mm inside dimensions (Spruce Hesperia, CA) and a Spectro-Physics SP 8700 solvent d e l i v e r y system (Santa C l a r a , CA). The solvent was i n i t i a l i z e d at 0% methanol i n d o u b l e - d i s t i l l e d water with a gradient of 12.5% methanol/min r a i s i n g the methanol concentration to 50% at 4.0 min and then held at 50% u n t i l 6.0 min before r e s e t t i n g to 0% methanol. The flow rate was 1.0 ml/min. The samples were monitored by a Varian Fluorichrom fluorescence detector (Palo A l t o , CA) using an e x c i t a t i o n wavelength of 360 nm and a 400 nm emission cut-of f f i l t e r . The data were analysed by a Perkin-Elmer Sigma 10 data s t a t i o n . A l l HPLC data p l o t t e d are the means of the detector response (integrated peak area) for three i n j e c t i o n s from a single sample i n each experiment. The AFM^ and AFQ^ standards used for the HPLC analysis were obtained from DR. D.P.H. Hsieh of the University of C a l i f o r n i a at Davis. 16 3. RESULTS 3.1 I n h i b i t i o n of MNNG-induced mutagenesis by browning re a c t i o n products The e f f e c t of the products from two model browning react-ions on the mutagenicity of MNNG, a d i r e c t - a c t i n g mutagen and carcinogen, i n Salmonella typhimurium was studied using the preincubation modification (Nagao et al. , 1977) of Ames' method (Ames et al., 1975). Caramelized sucrose and a heated l y s i n e -fructose mixture were used as model browning reaction products. Figure 1 shows the e f f e c t of adding these products to the b a c t e r i a l t e s t e r s t r a i n TA 1535 during t h e i r exposure to MNNG. The mutagenic a c t i v i t i e s of MNNG were expressed as h i s + revert-ants per p l a t e . Since the active products i n the M a i l l a r d reaction s o l u t i o n have not been i d e n t i f i e d , the concentrations of lysi n e - f r u c t o s e browning re a c t i o n products were expressed i n terms of the i n i t i a l l y s i n e concentration i n the s o l u t i o n p r i o r to the st a r t of the browning re a c t i o n (Powrie et al., 1981). The r e s u l t s indicated that the presence of caramelized sucrose or lysi n e - f r u c t o s e browning re a c t i o n products caused a decrease i n the number of MNNG induced h i s + revertants. Although browning reaction products alone at these same concentrations were not mutagenic, the p o s s i b i l i t y remains that the reduction Figure 1. E f f e c t of browning rea c t i o n products on reversion frequency of MNNG-treated b a c t e r i a i n the pre-incubation t e s t . Concentrations of MNNG used i n combination with caramelized sucrose (figure la) were: 9 . 7xlO~ 6M ( A ) , 6.8xlO~ 6M ( # ) , 5.8xlO~ 6M ( • ) , 4.9x10 M (ifr) and 3.9x10 M ( • ) . Concentrations of MNNG used i n conjunction with lysin e - f r u c t o s e M a i l l a r d reaction products (figure lb) were 7.8x10 ^ M —6 ( I ) and 3.9x10 M ( # ) . Control samples treated with browning rea c t i o n products alone i n the absence of MNNG were also shown (O). Plotted are x+S.D. from one experiment with t r i p l i c a t e p l a t i n g . Figure 2. E f f e c t of browning rea c t i o n products on c e l l s u r v i v a l and reversion frequency of MNNG-treated b a c t e r i a i n the suspension t e s t . Concentrations of MNNG used i n combination with caramelized sucrose (figure 2a) were 6xlO _ 5M (•) and 3xl0~ 5M (A ) . Concentrations of MNNG used i n conjunction with l y s i n e - f r u c t o s e M a i l l a r d r e a c t i o n products (figure lb) were 9x10 M (•) and 7x10 M^ (A) . Control samples treated with browning r e a c t i o n products alone i n the absence of MNNG were also shown ( 0 ) « Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . 18 i n h i s revertants might have been due to a syngerism between the toxic e f f e c t s of the browning re a c t i o n products and MNNG. Since the preincubation test protocol does not provide s u r v i v a l data, there was a need to use another modified version of the Salmonella mutagenicity assay. To better assess the i n h i b i t i o n e f f e c t of browning reaction products, subsequent follow up studies were c a r r i e d out using the l i q u i d suspension test ('Rosin and S t i c h , 1978a,b), which i s another modification of Ames' basic method (Ames et al. , 1975). The advantage of using t h i s assay i s twofold: (1) both reversion frequencies and s u r v i v a l frequencies can be estimated and (2) the exposure of the b a c t e r i a to the chemical can be better c o n t r o l l e d . Using t h i s assay, the i n h i b i t o r y e f f e c t of the browning react-ion products on MNNG mutagenicity was confirmed (figure 2). The reverse mutation frequencies were reduced i n the presence of increasing concentrations of browning reaction products. This reduction was observed over a range of dose combinations for the browning re a c t i o n products and MNNG which did not show a decrease i n the s u r v i v a l of the b a c t e r i a . 3.2 I n h i b i t i o n of MNNG-induced mutagenesis by phenolic compounds Three n a t u r a l l y occurring plant phenolic compounds were 20 tested using the Salmonella suspension assay. Chlorogenic ac i d , c a f f e i c acid and g a l l i c a c i d a l l exhibited antimutagenic a c t i v i t y . Figure 3 shows the r e l a t i v e effectiveness of the three phenolic compounds i n i n h i b i t i n g MNNG-induced h i s + reversion. C a f f e i c a c i d was the most potent i n h i b i t o r followed by g a l l i c acid while chlorogenic acid exhibited a r e l a t i v e l y weak i n h i b i t o r y e f f e c t . The mutagenic a c t i v i t y of 3x10 "*M MNNG was reduced by more than 90% with the i n c l u s i o n of 2.78xl0" 2M (5.0 mg/ml) c a f f e i c acid or 5.32xlO _ 2M (10.0 mg/ml) g a l l i c a c i d i n the incubation mixture. Chlorogenic a c i d at 1.13x10 "^M (40 mg/ml) decreased the mutagenic a c t i v i t y of MNNG by only 80%. The same r e l a t i v e capacity of these three phenolic compounds to i n h i b i t MNNG-induced mutagenesis was also observed at a higher concentration of MNNG (6x10 **M) . At th i s higher concentration of MNNG, the weaker i n h i b i t o r chlorogenic acid did not suppress the mutagenic action of MNNG. The phenolic compounds themselves did not induce h i s + reversion above the background l e v e l of spontaneous mutations ( i . e . <1 h i s + revertant / 10^ surviving c e l l s ) . No toxic e f f e c t of these phenolic compounds on the ba c t e r i a was observed i n these experiments. The i n h i b i t o r y e f f e c t of chlorogenic acid on MNNG mutagenicity was reproducible i n d i f f e r e n t experiments (figure 4). Figure 3. E f f e c t of phenolic compounds on the reversion frequency of MNNG-treated b a c t e r i a i n the suspension t e s t . Two concentrations of MNNG were used: 6x 10 °M ( D , A , 0 ) and 3x10 "*M ( I , A , The phenolic compounds used were g a l l i c acid (L~H,H), c a f f e i c acid ( A , A ) and chlorogenic acid ( 0 » G ) » Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Reversion frequencies of phenolic compound controls were <1 revertant / 10^ survivors. Figure 4. E f f e c t of chlorogenic acid on c e l l s u r v i v a l and reversion frequency of MNNG-treated b a c t e r i a i n the suspension t e s t . Concentration of MNNG used was 3x10 M ( • ) . Controls containing chlorogenic acid alone were also shown (O)-Plotted are x±S.D. from three experiments, each with t r i p l i c a t e p l a t i n g . Figure 5. Ef f e c t of c a f f e i c acid on c e l l s u r v i v a l and reversion frequency of MNNG-treated b a c t e r i a i n the suspension t e s t . Concentrations of MNNG used were 9x10 "*M (V ) , 6xlO~ 5M (•) and 3xlO~ 5M (A ) . Control samples without MNNG were also shown (O)* % mutagenic a c t i v i t y i s the reversion frequency of MNNG-treated b a c t e r i a i n the presence of c a f f e i c acid compared with that observed i n b a c t e r i a treated with MNNG only. Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Values have been corrected for spontaneous reversion. 22 23 The antimutagenic e f f e c t of c a f f e i c acid on three d i f f e r e n t concentrations of MNNG was studied (figure 5). MNNG alone at - 5 - 5 6x10 M and 9x10 M concentrations were s l i g h t l y t o x i c r e s u l t i n g i n 80% and 74% su r v i v a l r e s p e c t i v e l y as compared to that of the untreated c o n t r o l . The addition of c a f f e i c acid to the b a c t e r i a during t h e i r exposure to MNNG i n h i b i t e d the toxic e f f e c t as well as the mutagenic a c t i v i t y of MNNG. The degree of i n h i b i t i o n depended on the concentrations of both the i n h i b i t o r and the carcinogen. As the concentration of MNNG increased, a greater concentration of c a f f e i c acid was required to exert the same l e v e l of i n h i b i t i o n of mutagenic a c t i v i t y . This requirement for greater concentrations of i n h i b i t o r to suppress the higher doses of MNNG may (1) indicate a stoichiometric r e l a t i o n s h i p between i n h i b i t o r and carcinogen and (2) explain the ineffectiveness of chlorogenic acid i n i n h i b i t i n g 6x10 "*M MNNG (figure 3). 3.3 I n h i b i t i o n of MNNG-induced mutagenesis i n the yeast  mutagenicity test The antimutagenic. e f f e c t of the two model browning reaction products, caramelized sucrose and lysine- f r u c t o s e M a i l l a r d r e a c t i o n products, was also demonstrable i n the yeast mutagen-i c i t y t e s t (figure 6). The three n a t u r a l l y occurring plant phenolic compounds also showed i n h i b i t o r y e f f e c t on MNNG-induced Figure 6. Ef f e c t of browning r e a c t i o n products on c e l l s u r v i v a l and mutagenic a c t i v i t y of MNNG-treated yeast cultures. The MNNG concentration used was 1x10 ^M (,%). Values plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . The values have been corrected for spontaneous reversion. The c e l l s u r v i v a l of samples treated with browning re a c t i o n products alone were also shown (O)-Figure 7. Ef f e c t of phenolic compounds on the mutagenic a c t i v i t y of MNNG-treated yeast cultures. The MNNG concentration -4 used was 1x10 M. The phenolic compounds tested were g a l l i c acid (Q) , c a f f e i c acid (A) and chlorogenic acid CO)- Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Values have been corrected for spontaneous reversion. Phenolic Compounds Cone, mM 26 mutagenesis i n t h i s test system (figure 7). 3.4 I n h i b i t i o n of AFB^-induced mutagenesis by browning  reaction products The i n h i b i t o r y e f f e c t of caramelized sucrose and l y s i n e -fructose M a i l l a r d reaction products on the mutagenic a c t i v i t y of AFB^, a precarcinogen.'requiring metabolic a c t i v a t i o n was examined using the preincubation modification (Nagao et al., 1977) of the Salmonella mutagenicity test (Ames et al., 1975). In these experiments, the b a c t e r i a were exposed to the browning reaction products, AFB^, as well as a r a t l i v e r microsomal preparation, S9, to provide the a c t i v a t i o n system for AFB^. Figure 8 shows the reduction i n the number of h i s + revertants per plate when browning reaction products were present during the time the b a c t e r i a were exposed to AFB^ and S9. Subsequent studies using the suspension assay confirmed that the reduction was due to the i n h i b i t i o n of AFB^-induced mutagenesis rather than due to a t o x i c e f f e c t (figure 9). The browning reaction products alone or the AFB^ i n the absence of S9 had no mutagenic a c t i v i t y . Figure 8. E f f e c t of browning rea c t i o n products on reversion frequency of AFB^-treated b a c t e r i a i n the pre-incubation t e s t . Concentrations of AFB^ used i n combination with caramelized sucrose (figure 8a) were 2.5xlO~ 6M (•) and 1.25xlO~6M ( # ) . Concentrations of AFB^ used i n conjunction with l y s i n e - f r u c t o s e M a i l l a r d r e a c t i o n products (figure 8b) were 4x10 M ( I ) and 2x10 ( 0 ) . Control samples treated with browning rea c t i o n products alone i n the absence of AFB^ were also shown (O) • Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Figure 9. E f f e c t of browning r e a c t i o n products on c e l l s u r v i v a l and reversion frequency of AFB^-treated b a c t e r i a i n the suspension t e s t . Concentrations of AFB^ used i n combination with caramelized sucrose (figure 9a) were 3xl0 _ 5M (•) and lxlO~ 5M ( A ) . Concentrations of AFB^ used i n conjunction with l y s i n e - f r u c t o s e M a i l l a r d reaction products (figure 9b) were 2x10 M^ (O) and 1x10 "*M ( A ) . Control samples treated with browning reac t i o n products alone i n the absence of AFB^ were also shown (O). Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . 28 CaramelizedSucroseConc.,mg/ml Lysine-fructoseConc, mM Figure 8, above. Figure 9, below. Caramelized Sucrose Cone., mg/ml Lysine-fructose Cone, mM 29 3.5 I n h i b i t i o n of AFB^-induced mutagenesis by phenolic compounds The i n h i b i t o r y e f f e c t of a more extensive series of phenolic compounds on AFB^-induced mutagenesis was studied using the Salmonella suspension t e s t . Tannic a c i d , g a l l i c acid, chlorogenic a c i d , c a f f e i c a c i d , dopamine, p-hydroxybenzoic acid and s a l i c y l i c acid were a l l found to have i n h i b i t o r y a c t i v i t y towards AFB^-induced mutagenesis (figures 10, 11). S a l i c y l i c acid and p-hydroxybenzoic acid were less potent i n h i b i t o r s than the other phenolic compounds tested. The concentrations of the phenolic compounds used had no e f f e c t on the s u r v i v a l of the bacte r i a . AFB^ at 3x10 "*M was toxi c decreasing the s u r v i v a l of the Salmonella to 70% of that i n the untreated c o n t r o l (data not shown). However, the addition of phenolic compounds i n a l l cases reduced the toxic e f f e c t of AFB^ as well as i t s mutagenic a c t i v i t y . The data on tannic acid are presented separately because i t i s a polymeric macromolecule and i t s concentrations cannot be expressed i n molar concentrations. This p a r t i c u l a r compound i s a highly e f f e c t i v e i n h i b i t o r as only a concentration of less than 0.4 mg/ml was needed to suppress the mutagenic action of AFB^ completely (figure 11). Figure 12 shows the i n h i b i t o r y e f f e c t of chlorogenic acid on two d i f f e r e n t concentrations of AFB.. AFB. alone was toxic Figure 10. E f f e c t of phenolic compounds on reversion frequency of AFB^-treated b a c t e r i a i n the suspension t e s t . AFB^ concentration used was 3x10 M^. Phenolic compounds tested were g a l l i c acid ( • ) , c a f f e i c acid ( | ) , chlorogenic acid (A) , s a l i c y l i c acid CO)» p-hydroxy-benzoic acid (Q) and dopamine ( A ) . Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Reversion frequencies of phenolic compound controls were <5 revertants / 10^ survivors. Figure 11. E f f e c t of tannic acid on c e l l s u r v i v a l and reversion frequency of AFB^-treated b a c t e r i a i n the suspension test (LZ1). AFB^ concentration used was 3x10 M^. Control samples containing tannic acid i n the absence of AFB^ were also shown (O) • Plotted are x±S.D. from one experiment with t r i p l i c a t e p l a t i n g . Figure 12. E f f e c t of chlorogenic acid on c e l l s u r v i v a l and reversion frequency of AFB^-treated b a c t e r i a i n the suspension t e s t . Concentrations of AFB^ used were 6xl 0 _ 5 M (A) and 3xlO~ 5M (V ) . Plotted are x±S.D. from three experiments each with t r i p l i c a t e p l a t i n g . Control samples containing chlorogenic acid i n the absence of AFB 1 were also shown (O) • 32 with 67% and 53% s u r v i v a l at 3xlO~ 5M and 6xlO~ 5M AFB^ respect-i v e l y . The addition of chlorogenic acid reduced the toxic as well as the mutagenic e f f e c t of AFB^. Chlorogenic acid at 6 mg/ml completely eliminated the toxic e f f e c t of 6x10 AFB^ while only 1 mg/ml of the i n h i b i t o r was needed to restore c e l l s u r v i v a l to 97% of that i n the untreated buffer c o n t r o l . The i n h i b i t i o n of AFB^-induced mutagenesis by chlorogenic acid was reproducible i n d i f f e r e n t experiments (figure 12).. The i n h i b i t i o n of AFB^-induced mutagenesis by the chemicals being investigated was not studied using the yeast mutagenicity test due to d i f f i c u l t i e s encountered i n inducing mutation i n th i s yeast s t r a i n by AFB^. 3.6 E f f e c t of pre- and post-treatment with i n h i b i t o r s on  carcinogen-induced mutagenesis The e f f e c t of pre-treatment and of post-treatment with caramelized sucrose or chlorogenic acid on the mutagenicity of MNNG was studied using the Salmonella suspension t e s t . The re s u l t s are presented i n Tables I and I I . The addition of caramelized sucrose or chlorogenic acid to ba c t e r i a p r i o r to or afte r t h e i r exposure to MNNG had no e f f e c t on either the muta-genic response or the s u r v i v a l of the b a c t e r i a compared to Table I. EFFECT OF PRE- AND POST-TREATMENT WITH CARAMELIZED SUCROSE ON MNNG-INDUCED MUTAGENESIS3 Revertants per b 7 c 1st treatment 2nd treatment % Survival 10 survivors PBS c.s. f PBS MNNG MNNG C. S. + MNNG MNNG MNNG C.S. + MNNG PBS C.S. PBS 92±3 6 99±4 93±4 89±5 93±5 99±4 20 7 ±4 192±15 29±l g 227±15 171±5 22±2 g Samples of Salmonella were exposed to one of s i x treatment combinations. See Section 2.4 for incubation procedure. C e l l s u r v i v a l compared to that observed i n b a c t e r i a samples treated with PBS only. Mutagenic a c t i v i t y of co n t r o l sample was <1 revertants / 10 survivors. WlNG concentration used was 5x10 "*M. x±S.D. (Three r e p l i c a t e plates from one experiment). Caramelized sucrose concentration used was 50 mg/ml. 'S i g n i f i c a n t l y d i f f e r e n t from MNNG controls (p<0.0005). 34 Table I I . EFFECT OF PRE- AND POST-TREATMENT WITH CHLOROGENIC ACID ON MNNG-INDUCED MUTAGENESIS3 Revertants per 1st treatment 2nd treatment % Survival' 3 , n7 . c 10 survivors PBS MNNGd 97±4 e 183±21 Ch.A.f MNNG 73±7 175112 PBS Ch.A. + MNNG 85±9 76 ±6 8 MNNG PBS 97±14 187±22 MNNG Ch.A. 94±7 188+10 Ch.A. + MNNG PBS 66±3 61±5 8 Samples of SalmonetZa were exposed to one of six treatment combinations. See Section 2.4 for incubation procedure. ^ C e l l s u r v i v a l compared to that observed i n b a c t e r i a samples treated with PBS only. Mutagenic a c t i v i t y of cont r o l sample was <1 revertants / 10 survivors. M^NNG concentration used was 3x10 "*M. ex±S.D. (Three r e p l i c a t e plates from one experiment). ^Chlorogenic acid concentration used was 20 mg/ml. g S i g n i f i c a n t l y d i f f e r e n t from MNNG controls (p<0.0005). 35 Table I I I . EFFECT OF CONCURRENT- AND POST-TREATMENT WITH CARAMELIZED SUCROSE ON AFB,-INDUCED MUTAGENESIS3 Revertants per b 7 c 1st treatment 2nd treatment % Survival 10 survivors A F B ^ PBS 61±6 6 90112 AFB X C.S.f 4811 8816 C S . + AFB 1 PBS 6915 22+l g Samples of Salmonella were exposed to one of s i x treatment combinations. See Section 2.4 for incubation procedure. 'Cell s u r v i v a l compared to that observed i n b a c t e r i a samples treated with PBS only. Mutagenic a c t i v i t y of control samples was <3 revertants / 10 survivors. ^AFB^ concentration used was 3x10 "*M. xiS.D. (Three r e p l i c a t e plates from one experiment). Caramelized surcose concentration used was 70 mg/ml. 'S i g n i f i c a n t l y d i f f e r e n t from AFB 1 controls (p<0.05). Table IV. EFFECT OF CONCURRENT- AND POST-TREATMENT WITH CHLOROGENIC ACID ON AFB,-INDUCED MUTAGENESIS3 1st treatment 2nd treatment % Survival Revertants per 10 survivors PBS 69±12 79±3 e 49±8 33+2 AFB 1 76 ±8 77±9 44+5 37±6 Ch.A. + AFB^ PBS 97±6 111±18 7±1 9 ±2 g Samples of Salmonella were exposed to one of s i x treatment combinations. See Section 2.4 for incubation procedure. 'Cell s u r v i v a l compared to that observed i n b a c t e r i a samples treated with PBS only. Mutagenic a c t i v i t y of co n t r o l sample was <3 revertants / 10 survivors. 'AFB^ concentration used was 3x10 "*M. x±S.D. (Three r e p l i c a t e plates from one experiment. Data from two separate experiments are reported). Chlorogenic acid concentration used was 9 mg/ml. 'S i g n i f i c a n t l y d i f f e r e n t from AFB controls (p<0.05). 37 th e i r exposure to MNNG alone. I n h i b i t i o n of MNNG mutagenesis was observed only when the bac t e r i a were exposed concurrently to MNNG and one of the i n h i b i t o r s . Similar experiments were also performed i n order to study the suppression of AFB^-induced mutagenesis by these i n h i b i t o r s . However, b a c t e r i a pre-treated with PBS and then subsequently treated with AFB^ re s u l t e d i n very low c e l l s u r v i v a l . Consequently, comparison of the e f f e c t s between pre-treatment and concurrent-treatment of the i n h i b i t o r s on AFB^-induced mutagenesis could not be made. It was observed, however, that post-treatment of the ba c t e r i a with either i n h i b i t o r after t h e i r exposures to AFB^ had no e f f e c t on the mutagenic a c t i v i t y of AFB^ (Tables I I I and IV). 3.7 Study of the i n t e r a c t i o n between phenolic compounds and MNNG i n a c e l l - f r e e system The e f f e c t of three phenolic compounds, chlorogenic acid, c a f f e i c acid and g a l l i c acid, on MNNG was studied by using a col o r i m e t r i c method. The procedure involved mixing MNNG and a color reagent together i n the presence of 6M hydrochloric a c i d . The acid cleaves the N-nitroso group from MNNG to y i e l d nitrous acid (HNO^). The nitrous acid then forms a diazo compound with s u l f a n i l i c a c i d which i n turn couples to N-(l-naphthyl)-38 ethylenediamine dihydrochloride to produce an azo compound with an absorption maximum at 550 nm ( F o r i s t , 1964). This series of reac t i o n i s diagrammed i n figure 13. S i g n i f i c a n t reduction i n the production of the azo compound resulted when MNNG was incubated with one of the phenolic compounds (Table V). In c o n t r o l experiments where sodium n i t r i t e (NaNO^) was substituted for MNNG, the phenolic compounds did not reduce the formation of the color compounds. 3.8 Study of the i n h i b i t o r y e f f e c t of phenolic compounds on AFB metabolism using high-pressure l i q u i d chromatography The i n h i b i t i o n of AFB^ metabolism was studied using high-pressure l i q u i d chromatography (HPLC). AFB^ and the a c t i v a t i o n system, S9, were incubated with phenolic compounds for 20 min at 37°C. At the end of t h i s period, the a c t i v a t i o n of AFB^ was terminated by p r e c i p i t a t i n g out the enzymes with a NaCl-ethanol mixture. The processing of the test materials was e s s e n t i a l l y i d e n t i c a l to that of L i n et at. (1978) except that the samples were not f i l t e r e d before i n j e c t i n g into the HPLC column. The eluates were monitored at wavelengths greater than 400 nm by a fluorescence detector. Using t h i s system, fluorescent compounds were eluted from the column i n the order of t h e i r decreasing p o l a r i t y . In other words, the more polar a compound was, the Figure 13. Reaction pathways of MNNG. Pathway A. Reactions involved i n the co l o r i m e t r i c determination of MNNG (from F o r i s t , 1964). Pathway B. Proposed decomposition pathway for MNNG at p h y s i o l o g i c a l pH's (from Neale, 1976). 40 SO3H NMCHjCHjNHj^HCI 6M HCI f 0 06 H H j N-(1-naprm>yl)Bthyl«oocUamine sulfanitic acid \ S 0 3 H NHO, ^ * f J — ^ Color Compound . _ Umax-550 nm) NjCI hk II / C H , Pathway A , N - C - N ' 0 2 N / X N = 0 MNNG CH,-N=N-OH—HCH 3 NJ HCH Methyldiazonium Carbonium ion ion Pathway B Figure 13. 41 Table V. INHIBITORY EFFECT OF PHENOLIC COMPOUNDS ON THE PRODUCTION OF COLOR COMPOUND FROM MNNG Absorbance at 550 nm Phenolic compounds MNNG NaN02 a —4 b e c (mg/ml) none 1x10 M net net Chlorogenic acid 60 0.005 0.079±0.002 0.074 1.944 40 0.003 0.099±0.001 0.096 20 0.004 0.118±0.003 0.114 10 0.008 0.131±0.002 0.123 0 0 0.143±0.006 0.143 1.951 G a l l i c acid 20 0.023 0.066±0.004 0.043 1.932 15 0.027 0.073±0.001 0.046 10 0.021 0.08710.003 0.066 5 0.016 0.102±0.002 0.086 0 0 0.130±0.002 0.130 1.951 (Con't next page) 42 Table V. (Con't) Absorbance at 550 nm Phenolic compounds MNNG NaN02 (mg/ml) a none l x l 0 ~ 4 M b ,_c net n e t C C a f f e i c acid 20 0.065 0.112±0.007 0.047 1.945 15 0.045 0.113±0.007 0.068 10 0.035 0.11510.001 0.080 5 0.031 0.12310.003 0.092 0 0 0.12710.003 0.127 1.951 Absorbance of phenolic compounds alone. bxiS.D. (n=3). c -4 Difference i n absorbance between 1x10 M MNNG or NaM^ and phenolic compound c o n t r o l s . 43 sooner i t would be eluted out of the column. Figures 14a-d show the chromatograms of mixtures of AFB^ and S9 incubated for various time periods. The f i r s t peak eluted from the column appears to be r e l a t e d to polar fluorescent substances associated with the S9 preparation. Incubation of S9 alone resulted i n the appearance of t h i s same peak (figure 14e). The l a s t peak eluted from the column with a retention time of 7.76 min was the AFB^ parental compound. In j e c t i o n of AFB^ alone into the column resulted i n the e l u t i o n of a single peak at t h i s same re t e n t i o n time (figures 14f,g). The other peaks i n the chromatograms (figures 14a-d) increased i n si z e as the incubation time increased i n d i c a t i n g that they were peaks representing fluorescent AFB^ metabolites. The major meta-b o l i t e peak with a retention time of 6.79 min had been i d e n t i -f i e d as AFM^ (4-hydroxy-AFB^) by v i r t u e of i t s having an i d e n t i c a l retention time, as that of an AFM^ standard (figure 14h). The other peaks remained to be i d e n t i f i e d . The peak with the retention time of 6.28 min i s most l i k e l y 2 ,3-dihydro-2 ,3-dihydroxy-AFBj^ (AFB -dihydrodiol) . AFB^-dihydrodiol was reported to be the major AFB^ metabolite produced by rat l i v e r microsomal preparation ( L i n et at. , 1978) . The other possible i d e n t i t y for t h i s major peak i s AFQ an AFB1 metabolite possessing a hydroxyl group on the carbon 44 atom 3 to the carbonyl of the cyclopentenone r i n g (Neal and Colle y , 1978). But according to Masri et al. (1974), AFQ^ only represents a very small f r a c t i o n of the AFB^ metabolites produced by rat l i v e r preparation. The fluorescence detector used i n the present study also f a i l e d to detect an AFQ^ standard. The f a i l u r e to detect AFQ^ i s most l i k e l y due to the f a c t that AFQ^ has fluorescent properties d i f f e r e n t from those of AFB^, AFM^ and AFBj-dihydrodiol (Neal and C o l l e y , 1978) . In spite of the appearance of metabolite peaks, the incubation of AFB^ with S9 did not r e s u l t i n any substantial decrease i n the l e v e l of the AFB^ parental peak (figures 14a-d). This observation suggested that only a small f r a c t i o n of AFB^ was a c t u a l l y metabolized by the standard S9 preparation. By increasing the concentration of l i v e r homogenates i n the S9 preparation, a greater proportion of AFB^ was metabolized (figure 15). Incubation of AFB^ alone without S9 did not r e s u l t i n the appearance of any metabolite peaks or i n any s i g n i f i c a n t changes i n the fluorescent l e v e l of the AFB^ parental peak (figures 14f,g). When AFB^ was incubated with S9 i n the presence of phenolic compounds, a l l AFB^ metabolite peaks became reduced i n size but there were no changes i n any of t h e i r retention times. The Figure 14. Chromatograms of AFB^ incubated with S9 for various time periods (figures 14a-d). Figure 14e, S9 c o n t r o l incubated for 20 min. Figures 14f,g, AFB^ c o n t r o l s , not incubated and incubated for 20 min, r e s p e c t i v e l y . Figure 14h, AFM^ standard. AFB^ concentration used was 3x10 ~*M. Figure 15. A c t i v a t i o n of AFB^ by S9 with d i f f e r e n t rat l i v e r microsome concentrations. E f f e c t s without S9 (a), with standard (lx) S9 (b), with 2x S9 (c) and with 4x S9 (d) are shown. Plotted are detector responses for AFB^ peak (O—O) , AFM^ peak (#-—-#) and the u n i d e n t i f i e d major metabolite peak t e n t a t i v e l y assigned as AFB^-dihydrodiol ( • * • - • ) . AFB^ concentration used was 3x10 M^. 46 47 amount of reduction increased as the concentrations of the phenolic compounds increased ( f i g u r e 16). S a l i c y l i c acid and p-hydroxybenzoic acid, the two phenolic compounds that were less potent i n h i b i t o r s of AFB^-induced mutagenesis i n the Salmonella suspension test (figure 10), were also less e f f e c t i v e than the other phenolic compounds i n suppressing the production of AFB^ metabolites. Figure 17 shows that s i m i l i a r l e v e l s of i n h i b i t i o n were observed f or both major AFB^ meta-b o l i t e peaks by the phenolic compounds tested. The chromatograms of AFB^ incubated with S9 and chlorogenic acid are shown i n figures 18a-d. S i m i l i a r r e s u l t s were obtained with a l l phenolic compounds tested. There were no new peaks appearing when the phenolic compounds and AFB^ were incubated together with S9 suggesting that covalently bound fluorescent complexes between the phenolic compounds and AFB^ metabolites were not formed. Chromatograms of incubation mixtures contain-ing chlorogenic acid exhibited a broad fluorescent peak with approximately the same re t e n t i o n time as that of the S9 peak. The l e v e l or retention time of t h i s peak was not affected by the addition of e i t h e r S9 or AFB^ (figures 18e-h), but the peak increased i n l e v e l as the concentration of chlorogenic acid increased (figures 18a-d). The other phenolic compounds, whether incubated with S9 or not, did not r e s u l t i n any Figure 16. E f f e c t of phenolic compounds on the production of an AFB^ metabolite t e n t a t i v e l y assigned to be AFB^-dihydrodiol. AFB^ concentration used was 3x10 ~*M. Phenolic compounds tested were g a l l i c acid ( 0 ) , c a f f e i c a c i d ( B ) , chlorogenic acid ( A ) , s a l i c y l i c a c i d ( 0 ) » p-hydroxybenzoic acid ( D ) and dopamine ( A ) . Plotted are detector responses of the tentative AFB^-dihydrodiol peak i n the presence of phenolic compounds compared with that observed i n the absence of phenolic compounds. Figure 17. Comparison of the e f f e c t of phenolic compounds on the production of AFM^ and the u n i d e n t i f i e d metabolite t e n t a t i v e l y assigned to be AFB^-dihydrodiol. AFM^, ( # — # ) . AFB - d i h y d r o d i o l , ( # - - # ) . Phenolic Compound Cone, mg / ml Figure 18. Chromatograms of AFB^ incubated with S9 i n the presence of chlorogenic acid (figures 18a-d). Figure 18e, chlorogenic acid c o n t r o l . Figure 18f, chlorogenic acid incubated with S9. Figure 18g, chlorogenic acid incubated with AFB^ . Figure 18h, AFB^ c o n t r o l . Figure 19. E f f e c t of g a l l i c acid on the production of AFB^ meta-b o l i t e s from d i f f e r e n t concentrations of AFB^. Three concentrations of AFB^ were used: 9x10 (a), 6x10 (b) and 3x10 ~*M (c). P l o t t e d are detector responses for AFB^ peak (O—O) , AFM^ peak (#--•#) tentative AFB^-dihydrodiol peak (•-•-•) . and the 51 5 2 fluorescent peaks except s a l i c y l i c acid which exhibited a fluorescent peak with s i m i l a r properties as the chlorogenic acid peak. Incubating AFB^ with phenolic compounds i n the absence of S9 (figures 18g,h) also did not r e s u l t i n the appearance of new peaks or changes i n the fluorescent properties (peak area, peak height, r e t e n t i o n time) of the AFB^ parental peak. These r e s u l t s suggest that AFB^ and phenolic compounds do not react covalently to produce fluorescent complexes. The preceding HPLC experiments were performed using an AFB^ concentration of 3x10 which was the concentration used i n the Salmonella experiments. As shown e a r l i e r (figure 14d) , t h i s con-centration of AFB^ incubated with the standard S9 preparation did not r e s u l t i n s i g n i f i c a n t change i n the parental AFB^ peak s i z e . It was therefore only possible to show the i n h i b i t i o n of AFB^ meta-b o l i t e s formation which i n turn can be taken as an i n d i r e c t i n d i -cation of an i n h i b i t i o n of AFB^ metabolism. In order to show a d i r e c t i n h i b i t i o n of AFB^ metabolism by phenolic compounds, higher concentrations of AFB^ were used to study the i n h i b i t o r y e f f e c t of g a l l i c acid (figure 19). The r e s u l t s indicated that not only were the AFB^ metabolites production suppressed, but less AFB^ was metabolized as well when g a l l i c acid was present during the 53 incubation of AFB^ with S9. 3.9 Int e r a c t i o n between phenolic compounds and preformed AFB. metabolites To provide further support for the conclusion that phenolic compounds i n h i b i t AFB^ mutagenesis by suppressing the metabolism of AFB^, an attempt was made to separate the mutagenically active AFB^ metabolites from the rea c t i o n mixtures. I f phenolic compounds were able to i n h i b i t h i s + reversion induced by t h i s metabolite, then scavenging of t h i s metabolite by the phenolic compounds would at least be p a r t l y responsible f o r the observed i n h i b i t i o n . I f i n h i b i t i o n of mutagenesis induced by t h i s metabolite cannot be demonstrated, then the suppression of AFB^ metabolism would be the only l i k e l y mechanism of i n h i b i t i o n . The experiment was c a r r i e d out by an incubation procedure d i f f e r e n t from the normal method. AFB^ was f i r s t incubated with S9 at 37°C for 20 min i n the absence of b a c t e r i a to form meta-b o l i t e s . This incubation mixture was then heat-treated i n a water bath at 80°C for 2 min to in a c t i v a t e the enzymes present. The heated AFB^-S9 mixture was then cooled to room temperature with ice and incubated with the b a c t e r i a f o r a further 20 min i n the absence or presence of phenolic compounds. However, t h i s heat-treated mixture did not e l i c i t any mutagenic a c t i v i t y i n the 54 Salmonella assay. Consequently, the i n h i b i t o r y e f f e c t of the phenolic compounds could not be studied using t h i s procedure. In order to gain some insight i n the i n t e r a c t i o n between phenolic compounds and preformed AFB^ metabolites, the reaction mixture was also analyzed by HPLC. Figure 20a represents the i n h i b i t i o n of AFB^ metabolism i n the presence of c a f f e i c acid. Figure 20b shows that the l e v e l of AFM^ present i n the heat-treated mixture was not decreased by increasing concentrations of c a f f e i c a c i d . Furthermore, the l e v e l of AFM^ present i n Figure 20b i s comparable to that present at zero c a f f e i c acid concentration i n Figure 20a. Thus, i t seems that the AFM^ produced during the f i r s t 20 minutes of incubating AFB^ with S9 was not affected by the heat-treatment and subsequent incubation with c a f f e i c acid. The l e v e l of the t e n t a t i v e l y assigned AFB^-dihydrodiol peak was diminished s u b s t a n t i a l l y by the heat-treatment. Further incubation of the heat-treated mixture with c a f f e i c acid did not have any e f f e c t on t h i s peak (figure 20b). This lack of reduction i n the AFB^ metabolite peaks suggests that c a f f e i c a c i d , and may be other phenolic compounds as w e l l , did not i n h i b i t AFB^-induced mutagenesis by covalently binding to AFM1 or A F B ^ d i h y d r o d i o l . In another experiment, AFB and S9 were f i r s t incubated 55 together to form AFB^ metabolites and then, without heat-t r e a t i n g the mixture, followed by the addition of c a f f e i c acid (figure 20c). The two AFB^ metabolites decreased i n l e v e l as expected when the concentration of c a f f e i c acid was increased. However, the two metabolites were decreased only to l e v e l s comparable to those at zero c a f f e i c acid concentration i n Figure 20a. This suggests that the metabolites formed during the f i r s t 20 minutes of incubation were not affected by the subsequent incubation with c a f f e i c acid. The decrease that was seen i n Figure 20c can thus be a t t r i b u t e d s o l e l y to the i n h i b i t i o n of AFB1 metabolism during the second 20 minute incubation period. Figure 2 0 . E f f e c t of c a f f e i c acid on the production of AFB^ metabolites under d i f f e r e n t incubation conditions. Plotted are detector responses for AFB^ peak (Qi Q), AFM^ peak ^ ) and the t e n t a t i v e l y assigned AFB^-dihydrodiol peak 57 Figure 2 0 . 58 4. DISCUSSION 4.1 Concurrent administration of mutagen and i n h i b i t o r  required f o r antimutagenic a c t i v i t y The present study demonstrated that caramelized sucrose, ly s i n e - f r u c t o s e model M a i l l a r d browning re a c t i o n products and a number of n a t u r a l l y occurring phenolic compounds have a s i g n i f i c a n t e f f e c t i n reducing the mutagenic a c t i v i t i e s of two potent carcinogens, MNNG and AFB^. However, the e f f e c t was observed only when the i n h i b i t o r and the carcinogen were administered concurrently. The necessity of concurrent treatment for antimutagenic action was also reported f o r the i n h i b i t i o n of N-acetoxy-2-acetyl-aminofluorene-induced mutagenesis by cysteine (Rosin and S t i c h , 1978a). The lack of antimutagenic e f f e c t from the pretreatment of bacte r i a with i n h i b i t o r p r i o r to exposing them to the mutagen MNNG suggests that the i n h i b i t i o n was not caused by an i n t e r a c t i o n between the i n h i b i t o r and the b a c t e r i a l c e l l wall thereby preventing the passage of MNNG into the c e l l s . These i n h i b i t o r s also did not seem to have any i n h i b i t o r y e f f e c t on MNNG present on the surface of or inside the b a c t e r i a since the addition of chlorogenic acid to b a c t e r i a following t h e i r exposure to mutagens 59 did not r e s u l t i n any i n h i b i t i o n of mutagenic a c t i v i t y . Furthermore, i f an enhancement of the repair of carcinogen-induced DNA damage leads to a decrease i n the mutagenic e f f e c t , then the lack of an i n h i b i t o r y response from post-treatment with i n h i b i t o r s suggests that chlorogenic acid and caramelized sucrose did not exert t h e i r antimutagenic a c t i v i t y v i a t h i s mechanism. 4.2 Phenolic compounds i n h i b i t MNNG mutagenesis by a scavenging r e a c t i o n For the i n h i b i t i o n of MNNG-induced mutagenesis, the simplest explanation that can account for the necessity of concurrent treatment i s that the i n h i b i t o r s act on MNNG by a scavenging ac t i o n . MNNG i s chemically unstable at ph y s i o l o g i c a l pH and decomposes spontaneously to a reac t i v e a l k y l a t i n g d e r i v a t i v e believed to be a carbonium ion (Montesano and Bartsch, 1976; Neale, 1976). The proposed pathway for the degradation of MNNG i s shown i n Figure 13. The evolution of the carbonium ion i s supported by the transfer of int a c t deuterated methyl groups to DNA by the treatment of N-trideuterio-methyl l a b e l l e d MNNG to DNA in vitro (Haerlin et al., 1970) or to Escherichia coli DNA in vivo (Lingens et al., 1971). Browning reaction products and phenolic compounds can prevent MNNG from exerting i t s mutagenic a c t i v i t y on the c r i t i c a l target of the 60 c e l l s by ei t h e r i n t e r a c t i n g with MNNG d i r e c t l y or with some of i t s degradation products including the carbonium ion. This mechanism of i n h i b i t i o n i s supported by the r e s u l t s obtained using a c o l o r i m e t r i c determination of MNNG. In t h i s c e l l - f r e e system where only MNNG and one of the phenolic compounds were incubated together, the amount of MNNG detectable by the c o l o r i m e t r i c method was s u b s t a n t i a l l y reduced compared to that i n the untreated MNNG con t r o l s . With reference to the scheme for the c o l o r i m e t r i c determination of MNNG (figure 13), phenolic compounds can reduce the amount of color compounds formed by i n t e r a c t i n g d i r e c t l y with MNNG. However, phenolic compounds, being n u c l e o p h i l i c , are more l i k e l y to in t e r a c t with one of the reactive e l e c t r o p h i l i c decomposition products of MNNG. This i n t e r a c t i o n should reduce the amount of carbonium ions available to cause mutagenesis. At the same time, t h i s scavenging r e a c t i o n should accelerate the degradation of MNNG, thus decreasing the amount of MNNG available f o r the formation of the color compound. Phenolic compounds do not suppress the production of color compounds by i n t e r f e r i n g with the c o l o r i m e t r i c determination procedure. For instance, the phenolic compounds did not reduce the production of color compounds when sodium n i t r i t e was 61 substituted f o r MNNG. 4.3 Phenolic compounds i n h i b i t AFB^-induced mutagenesis by suppression of i t s metabolism The i n h i b i t i o n of AFB^-induced mutagenesis i s a more complex s i t u a t i o n than the one presented f or MNNG. Since AFB^ i s a precarcinogen and premutagen, i t requires metabolic a c t i v a t i o n before i t s mutagenic e f f e c t can be demonstrated (Garner et al., 1972; McCann et al. , 1975; Wong and Hsieh, 1976; Campbell and Hayes, 1976; Ong, 1975). Consequently, a reduction i n i t s mutagenic a c t i v i t y on b a c t e r i a can be due to an i n h i b i t i o n of the a c t i v a t i o n process or .to the scavenging of the activated AFB^ metabolite analogous to that proposed for the i n h i b i t i o n of MNNG-induced mutagenesis. AFB^ i s mainly metabolized by the microsomal mixed-function oxidase system (Campbell and Hayes, 1976). The known metabolic pathways of AFB^ i n animals are shown i n Figure 21'. The ultimate reactive species of AFB^ i s cu r r e n t l y believed to be AFB^-2,3-oxide. Although t h i s metabolite has never been i s o l a t e d , i t s presence can be i n f e r r e d by several l i n e s of evidence. Studies have shown that the 2,3-double bond i s required for a c t i v i t y because AFB i s more active i n i t s gure 21. Known metabolic fate of AFB^ i n animal hosts (from Neal and Colley, 1976). 63 Aflatoxin Di dihydrodiol Figure 2 1 . 64 carcinogenic, mutagenic and macromolecular binding a c t i v i t i e s than AFB^ which does not have a double bond at the 2,3-p o s i t i o n (Butler et al., 1969; Wogan et al. , 1971; Wong and Hsieh, 1976; Swenson et al. , 1977). S i m i l a r l y a l l other metabolites without a 2,3-double bond are p r a c t i c a l l y devoided of mutagenic a c t i v i t y (Wong and Hsieh, 1976). Other l i n e s of evidence i n support of the AFB^-2,3-oxide as the ultimate reactive metabolite include (1) the formation of covalent adducts of AFB^ and nucleic acids in vivo (Swenson et al. , 1974 , 1977) and in vitro i n the presence of l i v e r microsomal preparation (Swenson et al., 1973; L i n et al., 1977); (2) the i s o l a t i o n of AFB^-2,3-dihydrodiol from nu c l e i c acid adducts formed in vivo or in vitro by mild acid hydrolysis (Swenson et al., 1973, 1974) and (3) the i s o l a t i o n of an AFB^-guanine adduct from nucleic acid by mild acid hydrolysis and then protected from contact with weak a l k a l i ( L i n et al. , 1977). AFB^-2,3-oxide was thus proposed as the ultimate nucleic acid-attacking species that resulted i n the AFB^-guanine adduct. The i n a b i l i t y to i s o l a t e the reactive metabolite of AFB^ also pointed to the high r e a c t i v i t y of AFB^-2,3-oxide (Garner et al., 1972; Garner, 1973; Gorst-Allman et al., 1977). A l l known metabolites of AFB^ are l e s s mutagenic and carcinogenic than the AFB^ parental compound. A f l a t o x i c o l , the most potent genotoxic metabolite of AFB , has only 22.8% of the mutagenicity 65 demonstrable with AFB^ i n Salmonella and only h a l f the tumori-genic a c t i v i t y of AFB^ i n rainbow trout. AFM^, the next most potent metabolite of AFB^, ex h i b i t only 3.2% of the mutagenicity and one t h i r d of the tumorigenicity compared to AFB^ i n Salmonella and rainbow trout r e s p e c t i v e l y (Wong and Hsieh, 1976). A l l known AFB^ metabolites, l i k e AFB^ i t s e l f , require metabolic a c t i v a t i o n before t h e i r mutagenic e f f e c t can be detected i n the Salmonella mutagenicity test (McCann et at., 1975; Wong and Hsieh, 1976). I t i s not known whether these metabolites are converted by enzymes back to AFB^ and then further activated or whether they are activated to reactive species v i a d i f f e r e n t routes from that of AFB^. Because these metabolites have weaker mutagenic and carcinogenic a c t i v i t i e s than AFB^, i t i s not clear whether the conversion of AFB^ to these metabolites are also a c t i v a t i o n processes. Because AFB^ requires metabolic a c t i v a t i o n , i n h i b i t o r y actions on i t s mutagenicity can be caused e i t h e r by i n h i b i t i o n of i t s metabolism or by trapping of the AFB^-2,3-oxide to prevent i t from acting on c r i t i c a l c e l l u l a r targets. In order to d i s t i n g u i s h between these two p o s s i b i l i t i e s , a study using HPLC was conducted. HPLC analysis of the incubation mixture of AFB^ and S9 could be used to monitor the metabolism of AFB^ and the metabolites formed (Neal and Colley , 1978). Because of the' 66 high r e a c t i v i t y of AFB^-2,3-oxide, i t i s expected to become hydrolyzed to form AFB^-dihydrodiol spontaneously. Thus the amount of AFB^-dihydrodiol detected should r e f l e c t the net e f f e c t of the epoxidative a c t i v i t y of the enzyme system and the amount of the AFB -2,3-oxide trapped by any nucleophiles present (Lin et al., 1978; Neal and Colley, 1978). The present study indicates that AFB^ metabolism i s i n h i b i t e d by phenolic compounds. From the HPLC studies, the addition of phenolic compounds reduced the si z e of a l l major and minor metabolite peaks of AFB^. In some cases, when the concentration of the phenolic compounds was increased to a c e r t a i n l e v e l , a l l metabolite peaks were completely suppressed. At the same time, the AFB^ parental peak increased as the concentration of phenolic compounds was increased. This indicates that less AFB^ was metabolized when phenolic compounds were present so that more AFB^ was retained i n the incubation mixture. Incubation of AFB^ with phenolic compounds, i n the absence of S9, did not a l t e r the fluorescent l e v e l or the retention time of the AFB^ peak. I f AFB^ and phenolic compounds could i n t e r a c t covalently to form a complex, i t was not detectable by the fluorescent method. Garner et al. (1972) have previously reported that AFB^ does not bind to nucleic acid covalently i n the absence of metabolic a c t i v a t i o n . Only non-covalent i n t e r -67 actions between non-activated AFB^ and nu c l e i c acid have ever been described ( C l i f f o r d and Rees, 1967; Sporn et at., 1966). A l l these observations point to the fact that AFB^, by i t s e l f , i s not a r e a c t i v e e l e c t r o p h i l e , and consequently, i t cannot be expected to form a covalent adduct with n u c l e o p h i l i c phenolic compounds. Like AFB^, a l l known AFB^ metabolites require metabolic a c t i v a t i o n to become mutagenic (Campbell and Hayes, 1976; Wong and Hsieh, 1976). Their requirements for a c t i v a t i o n imply that they also do not contain re a c t i v e e l e c t r o p h i l i c centers and must be metabolized to become e l e c t r o p h i l i c ( M i l l e r , 1971; M i l l e r and M i l l e r , 1977). Therefore, without reactive s i t e s , these metabolites should not be expected to i n t e r a c t covalently with n u c l e o p h i l i c phenolic compounds. In an experiment where a mixture of AFB^ and S9 were f i r s t incubated together to form AFB^ metabolites and then the mixture was heated to i n a c t i v a t e the microsomal enzymes, further incubation of t h i s mixture with the addition of c a f f e i c acid did not reduce any of the meta-b o l i t e s preformed during the f i r s t incubation. The HPLC r e s u l t s from the present study did not detect any covalent interactions between phenolic compounds and the reactive e l e c t r o p h i l i c AFB -2,3-oxide. The HPLC study detected AFB -68 2,3-oxide as i t s breakdown product, AFB^-dihydrodiol (Lin et at., 1978). I f covalent binding between phenolic compounds and AFB^-2,3-oxide did occur, only the AFB^-dihydrodiol peak should show a reduction i n si z e with the p o s s i b i l i t y for the appearance of a new peak representing the bound complex. When AFB^, S9 and phenolic compounds were incubated together, a l l AFB^ metabolite peaks were reduced i n size without the appearance of any new peaks. The p o s s i b i l i t y of course remains that the bound complex i s non-fluorescent. In another experiment, where AFB^ and S9 were f i r s t incubated together to form AFB^ metabolites and then incubated i n the presence of c a f f e i c acid, there was no further metabolism of AFB^. The peaks representing the preformed metabolites of AFB^ were also not reduced. Thus, phenolic compounds had no detectable e f f e c t i n reducing any of the AFB^ metabolites observed i n t h i s study. These r e s u l t s therefore suggest that phenolic compounds do not reduce the mutagenic a c t i v i t y of AFB^ by i n t e r a c t i n g with the non-reactive metabolites of AFB^. This study, however, has demonstrated that one mechanism of t h e i r i n h i b i t i o n of AFB^-induced mutagenesis i s through an interference with the metabolism of AFB^. The p o s s i b i l i t y remains that the phenolic compounds may i n t e r a c t with the reactive AFB^-2,3-oxide, but t h i s a l t e r n a t i v e mechanism of antimutagenic a c t i v i t y cannot be established i n the present study. 69 Phenolic compounds also modulate the a c t i v i t y of a number of other enzymes. Some phenolic compounds i n h i b i t mitochondrial r e s p i r a t i o n (Cheng and P a r d i n i , 1978, 1979). Phenolic compounds also i n h i b i t r a t l i v e r mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase a c t i v i t i e s (Shama Bhat and Ramasarma, 1979). Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), both phenolic anitoxidants, a l t e r the pattern of metabolism of the carcinogen benzo(a)pyrene (Wattenberg, 1979). I t i s therefore not s u r p r i s i n g that the phenolic compounds studied have i n h i b i t o r y action on AFB^ a c t i v a t i o n enzymes. 4.4 The r o l e of browning reaction products and phenolic compounds i n the prevention of human cancer The present study demonstrated that products from two model non-enzymatic browning reactions and some plant phenolic compounds have a s i g n i f i c a n t e f f e c t i n reducing the mutagenic a c t i v i t y of two potent carcinogens. However, browning reaction products at c e r t a i n concentrations also exhibited genotoxic a c t i v i t i e s by themselves. Both caramelized sugars and products from M a i l l a r d r e a c t i o n model systems have clastogenic a c t i v i t y i n Chinese hamster ovary c e l l s ( S t i c h et al. , 1981a; Powrie et at., 1981). Products from M a i l l a r d reaction model systems were 70 also mutagenic to Salmonella TA 100 and convertogenic i n Saccharomyaes cerevis-iae s t r a i n D5 (Powrie et al., 1981). Phen-o l i c compounds are genotoxic agents as w e l l . Chlorogenic acid and c a f f e i c a c i d , for example, caused gene conversion i n S. oevevisiae s t r a i n D7 and chromosome aberration i n Chinese ham-ster ovary c e l l s . Both of these compounds, however, lacked a c t i v i t y i n the Salmonella mutagenicity test ( S t i c h et al., 1981b). Several phenolic acids and cinnamic acids were also examined for clastogenic a c t i v i t y i n Chinese hamster ovary c e l l s . Among the tested compounds relevant for the present study, c a f f e i c acid and g a l l i c acid were c l a s t o g e n i c a l l y active while p-hydroxybenzoic acid and s a l i c y l i c acid were inactive (Stich et al., 1981c) . Since the two groups of chemicals studied have both genotoxic and antigenotoxic e f f e c t s , i t i s d i f f i c u l t to assess the i m p l i c a t i o n of the r e s u l t s . For the browning reaction products, which contain numerous compounds (S t i c h et al., 1981d; Powrie et al., 1981), the mutagenic and antimutagenic a c t i v i t i e s might not have come from the same group of chemicals. In general, at subthreshold dose l e v e l s , where genotoxicity was not evidenced (such as the concentrations employed i n the present study), these chemicals exhibited protective e f f e c t s against other more potent mutagens and carcinogens. But at 71 higher concentrations, the e f f e c t of the genotoxic components became more prevalent. Some genotoxic components i d e n t i f i e d so far i n the browning r e a c t i o n products include pyrazines ( S t i c h et at., 1980), furans ( S t i c h et al., 1981e) and some 1,2-dicarbonyl compounds (Bjeldanes and Chew, 1979). The antimutagenic components of browning re a c t i o n products, however, remain to be i d e n t i f i e d . But nevertheless, the r o l e of other protective mechanisms against genotoxic agents should not be overlooked. T r a n s i t i o n metals, such as F e l l l and C u l l , and several enzyme systems, including rat l i v e r S9 preparation, have i n h i b i t o r y e f f e c t s on the genotoxic a c t i v i t y of browning rea c t i o n products as well as phenolic compounds ( S t i c h et al., 1981b,c,f; Powrie et al., 1981; Rosin et al., 1982). The question has been r a i s e d many times as to whether one can define at a l l the genotoxicity of a complex mixture such as food. The composition of food, upon entering man, w i l l become continuously changed. The possible in t e r a c t i o n s that can occur between the various components of food among themselves as well as with c e l l u l a r substances are innumerable. Some of these i n t e r a c t i o n s may contribute to an enhancement while others to a suppression of the net genotoxic a c t i v i t y of the substances involved. In order to assess the p o t e n t i a l health hazard posed by a p a r t i c u l a r food component, i t i s therefore important to 72 further these -in vitro studies i n the int a c t animal. In view of the fact that many compounds have i n h i b i t o r y e f f e c t s on the a c t i v i t i e s of genotoxic substances, the question arises as to the ro l e of these compounds i n reducing the impact of environmental carcinogens on man and in the prevention of human cancers. The consumption of green-yellow vegetables has been associated with a lower r i s k for cancers of the lung as well as other s i t e s , such as the prostate and stomach, i n both smokers and non-smokers (Hirayama, 1979). The dose lev e l s of phenolic compounds used i n the present study (0.6-40 mg/ml) are comparable to the concentrations of these compounds found n a t u r a l l y i n green-yellow vegetables and some beverages (Table VI). This suggests a possible r o l e for phenolic compounds i n lowering the r i s k of some cancers among i n d i v i d u a l s with a high intake of green-yellow vegetables. Other supportive studies for the p o s i t i v e e f f e c t of green-yellow vegetables i n reducing cancer r i s k s include works by Wattenberg et al. (1980) on antineoplastic properties of plant phenolic compounds and by Buening et al. (1981) on antimutagenic plant flavonoids. 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