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Diet and lifestyle factors as causes of human cancers as exemplified by three model systems Chan, Peter Ka-Lin 1984

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DIET AND LIFESTYLE FACTORS AS CAUSES OF HUMAN CANCERS AS EXEMPLIFIED BY THREE MODEL SYSTEMS By PETER KA-LIN CHAN B.Sc, The Un i v e r s i t y of V i c t o r i a , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1984 ^ P e t e r Ka-Lin Chan, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive 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 of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT There i s epidemiological evidence demonstrating the involvement of dietary and l i f e s t y l e factors i n the development of a large proportion of human cancers. The objectives of t h i s study were to simulate in vitro the genotoxic e f f e c t s of various chemicals introduced i n t o the body through d i e t and l i f e s t y l e habits, and to inves t i g a t e t h e i r r e l a t i o n s h i p to human carcinogenesis. Three model systems were used i n t h i s study: (1) in vitro formation of N-nitrosoproline (NPRO), (2) Chinese sa l t e d f i s h extract, and (3) be t e l nut extracts. Nitrosamines have long been suspected of being r e l a t e d to the development of nasopharyngeal, esophageal, stomach and urinary bladder carcinomas. The formation of NPRO was used to examine the e f f e c t of plant phenolics, major components of the human d i e t , on the n i t r o s a t i o n reactions. The t e s t system consisted of n i t r o s a t i n g (pH 2, 1 hr, 37°C) p r o l i n e with or without the phenolics to be used. Catechin, chlorogenic a c i d , g a l l i c a cid, p y r o g a l l o l and tannic a c i d suppressed the formation of NPRO. Their e f f i c i e n c i e s i n i n h i b i t i n g the n i t r o s a t i o n reaction were comparable to that of ascorbic a c i d . However, there was another family of phenolics with a re s o r c i n o l moiety which enhanced the n i t r o s a t i o n reaction at a c e r t a i n r a t i o of p h e n o l i c s : n i t r i t e : p r o l i n e . This indicated that some phenolics may act as i n h i b i t o r s while others may act as c a t a l y s t s i n the n i t r o s a t i o n reaction. Several teas, which are phenolic-containing beverages, were also studied. A Chinese, Japanese and Ceylanese tea a l l i n h i b i t e d the formation of NPRO at doses which are normally consumed by man. i i Chinese s a l t e d f i s h was used as a model to demonstrate that d i e t may-play a r o l e i n human cancers. Salted f i s h has long been thought to be involved i n human cancers. Employing the Ames Salmonella mutagenicity t e s t system, i t was found that the mutagenic components i n the sa l t e d f i s h e xtract were d i r e c t - a c t i n g and water-soluble. Moreover, there were components present which could be converted to mutagenic n i t r o s o compounds when n i t r i t e was added. This was detected by Salmonella typhimurium ( s t r a i n TA1535). Hence the i n h i b i t o r y e f f e c t of phenolics and phenolic-containing beverages on the formation of mutagenic n i t r o s a t i o n products was studied. The t e s t system consisted of n i t r o s a t i n g (pH 2, 1 hr, 37°C) an aqueous f r a c t i o n of a sa l t e d f i s h (Pak Wik) with or without the i n h i b i t o r s to be tested and estimating the frequency of h i s + revertants per survivors of S. typhimurium ( s t r a i n TA1535) by applying the l i q u i d suspension t e s t . The phenolics and teas were added to the n i t r o s a t i o n mixture. Again, the phenolics tested (catechin, chlorogenic a c i d , g a l l i c a c i d , p y r o g a l l o l and tannic acid) and three tea samples (Chinese, Japanese and Ceylanese), at doses consumed by man, a l l prevented the formation of mutagenic ni t r o s a t e d f i s h products. During the course of a meal the f i s h products, n i t r i t e and n i t r i t e -trapping agents w i l l mix with s a l i v a . Therefore the e f f e c t of s a l i v a on the formation of mutagenic N-nitroso compounds was also studied. S a l i v a exerted an i n h i b i t o r y e f f e c t on the formation of NPRO and mutagenic nit r o s a t e d f i s h extract. The n i t r i t e depletion assay was c a r r i e d out to determine i f the modulators react with n i t r i t e . Phenolics, teas and s a l i v a a l l reacted with the n i t r i t e present, i n d i c a t i n g that they competed with the nit r o s a t a b l e agents f o r the a v a i l a b l e n i t r i t e s . Betel nut was used as an example to demonstrate that l i f e s t y l e f a c t o rs may also be involved i n the development of cancer. Betel nut chewing has been r e l a t e d to the development of o r a l and esophageal cancer. Betel nut water extract and b e t e l tannin (a major component of b e t e l nut) d i d not demonstrate any mutagenic e f f e c t on S. typhimurium t e s t e r s t r a i n s TA98, TA100 and TA102, with or without S9 (a r a t l i v e r microsomal preparation). However, when the Chinese hamster ovary (CHO) c e l l t e s t system was employed, both b e t e l nut water extract and b e t e l tannin exerted chromosome-damaging (clastogenic) e f f e c t s on the CHO c e l l s . In addition, S9 and catalase reduced these clastogenic e f f e c t s . When lime was added to these mixtures, the clastogenic e f f e c t was enhanced. However, S9 and catalase again reduced these clastogenic e f f e c t s . H2°2 W a S therefore suspected to be one of the causative agents since phenolics generate H2°2 u n ^ e r oxidative conditions. Using a c o l o r i m e t r i c assay, i t was found that ^2(~>2 was indeed present and could account, i n part, for the clastogenic e f f e c t of these mixtures. I t has also been reported that b e t e l nut water extract reduced the formation of endogenous n i t r o s a t i o n products. What we put i n t o our mouths i s complex, and a l l kinds of chemical reactions may take place. This study shows that d i e t and l i f e s t y l e f a c t o rs are r e l a t e d to the development of human cancers. However, the extent of these two factors i n the causation of cancers cannot be demonstrated. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS V LIST. OF TABLES v i i i LIST OF FIGURES ix LIST OF ABBREVIATIONS x i ACKNOWLEDGEMENTS x i i INTRODUCTION 1 MATERIALS AND METHODS 8 Chemicals 8 Preparation of Chemical Stock Solutions 8 Nitrosoproline Synthesis 9 Preparation of N i t r o s a t i o n Reaction of Pr o l i n e Mixture 9 Analysis of N-Nitrosoproline 9 Determination of N i t r i t e Depletion 10 Preparation of S9 L i v e r Microsomal Mixture 10 Preparation of Sodium Phosphate Buffer 11 Preparation of Sa l i v a Samples 11 Preparation of Salted F i s h Extract 12 Ni t r o s a t i o n of the Salted F i s h Extract 12 Preparation of Betel Nut Extracts 12 Preparation of Tea Samples 13 Salmonella Mutagenicity Assay 13 Controls f o r Mutagenicity Assay 15 Chromosome Aberration Test •-• 16 Controls f o r CHO C e l l Conditions 17 Assay f o r Hydrogen Peroxide 17 v RESULTS '.' 19 Ef f e c t s of Doses of N i t r i t e , P r o l i n e , Time and pH on Formation of NPRO 19 Ef f e c t s of Five Phenolics, Teas and S a l i v a on NPRO Formation 22 Ef f e c t s of Dihydroxybenzoic Acid With and Without Resorcinol Moiety on N i t r o s a t i o n of Proline 25 E f f e c t s of Dihydroxybenzoic Acid With Resorcinol Moiety at Low Concentration of N i t r i t e 25 Clastogenic A c t i v i t y of Aqueous and N i t r i t e - T r e a t e d Aqueous F i s h Extract 27 Mutagenicity of Aqueous Salted F i s h Extract 27 Mutagenicity of N i t r i t e - T r e a t e d F i s h Extract . . .• 29 E f f e c t of Human S a l i v a on Mutagenicity Resulting from N i t r i t e - T r e a t e d F i s h Extract 34 E f f e c t of Naturally Occurring Phenolics on Mutagenicity of N i t r i t e - T r e a t e d F i s h Extract 36 E f f e c t of Teas on Mutagenicity Resulting from N i t r i t e - T r e a t e d F i s h Extract . 36 Study of the Interaction of Aqueous Salted F i s h Extract with NaNO^ by a Colorimetric Method 38 Interaction of Phenolics, Teas and S a l i v a with NaNO^ by a Colorimetric Method 38 Mutagenicity of Betel Nut Water Extract at pH 7.0 and 10.0 43 Mutagenicity of Betel Tannin at pH 7.0 and 10.0 46 Mutagenicity of Hydrogen Peroxide . . 46 Assay f o r H O^ Generated by Betel Nut Water Extract and Betel Tannin at Two ph Levels 46 Clastogenic E f f e c t of Betel Nut Water Extract at pH 7.0 and 10.0 . . 49 Clastogenic E f f e c t of Betel Tannin and Tannic Acid at pH 7.0 and 10.0 53 v i DISCUSSION 60 Food and Cancer 60 Plant Phenolics and t h e i r Roles i n Human Carcinogenesis 62 Possible Mechanism of I n h i b i t i o n and C a t a l y t i c E f f e c t by Phenolics on N i t r o s a t i o n Reactions 64 Relation of Salted F i s h to Human Carcinogenesis 65 Nit r o s a t i o n Reactions Modulated i n a Complex Mixture 66 Relation of Betel Nut Chewing to Human Carcinogenesis 67 Sali v a and i t s Role i n Carcinogenesis 68 B i o l o g i c a l Protective Mechanism 70 Unresolved Issues 71 REFERENCES 75 APPENDICES 84 I. Extraction Procedure of Salted F i s h 84 I I . N i t r o s a t i o n of Salted F i s h Aqueous Extract 85 I I I . Extraction Procedure of Betel Nut 86 IV. Chemical Structures of Some Modulators 87 v i i L I S T OF TABLES 1. Clastogenic a c t i v i t y of aqueous f i s h extract and n i t r i t e - t r e a t e d f i s h extract 28 2. P o s i t i v e controls f o r d i f f e r e n t t e s t e r s t r a i n s 31 3. . H i s t o r i c a l : ranges for mutagenicity i n experimental runs 32 4. Mutagenic e f f e c t of betel nut water extract at pH 7.00±0.05 44 5. Mutagenic e f f e c t of betel nut water extract at pH 10.00±0.05 ....45 6. Mutagenic e f f e c t of b e t e l tannin at pH 7.00±0.05 47 7. Mutagenic e f f e c t of b e t e l tannin at pH 10.00±0.05 48 8. Amount of H O^ generated by b e t e l nut water.-.extract (108mg/ml f and b e t e l tannin (1.0 mg/ml) 51 9. Clastogenic a c t i v i t y of b e t e l nut water extract at pH 7.00±0.05 .52 10. Clastogenic a c t i v i t y of b e t e l nut water extract with lime at pH 10.00±0. 05 54 11. Clastogenic a c t i v i t y of b e t e l tannin at pH 7.00±0.05 55 12. Clastogenic a c t i v i t y of b e t e l tannin with lime at pH 10.00±0.05 .56 13. Clastogenic a c t i v i t y of tannic a c i d at pH 7.0010.05 57 14. Clastogenic a c t i v i t y of tannic a c i d with lime at pH 10.0010.05 58 15. Proportion of cancer cases a t t r i b u t e d to various factors by d i f f e r e n t authors 61 16. Genotoxic e f f e c t s measured with plant phenolics 63 v i i i LIST OF FIGURES 1. E f f e c t of various concentrations of NaNO^ on the formation of N-Nitrosoproline 20 2. E f f e c t of:various concentrations of p r o l i n e on the formation of N-Nitrosoproline 20 3. E f f e c t of various incubation times on the formation of N-Nitrosoproline 21 4. E f f e c t of various incubation pH l e v e l s oh the formation of N-Nitrosoproline 21 5. Inhib i t o r y e f f e c t of tannic acid, p y r o g a l l o l and g a l l i c a c i d on the formation of N-Nitrosoproline 23 6. In h i b i t o r y e f f e c t of ascorbic a c i d , catechin and chlorogenic a c i d on n i t r o s a t i o n of p r o l i n e 23 7. E f f e c t of three tea samples on the formation of N-Nitrosoproline 24 8. E f f e c t of s a l i v a of f i v e i n d i v i d u a l s on the formation of N-Nitrosoproline 24 9. E f f e c t of r e s o r c i n o l , .2,4-dihydroxybenzoic ac i d , 3,5-dihydroxybenzoic a c i d , p-hydroxybenzoic a c i d and ascorbic a c i d on the formation of N-Nitrosoproline 26 10. E f f e c t of 2,3-dihydroxybenzoic a c i d , 2,5-dihydroxybenzoic a c i d and 2,6-dihydroxybenzoic a c i d on the formation of N-Nitrosoproline 26 11. I n h i b i t o r y e f f e c t of r e s o r c i n o l , 2,4-dihydroxybenzoic a c i d and 3,5-dihydroxybenzoic a c i d on the formation of N-Nitrosoproline at low NaNO^ concentration 26 12a. Mutagenicity of _S. typhimurium (TA98) following exposure to salte d f i s h aqueous extract with and without S9 30 12b. Mutagenicity of _S. typhimurium (TA98) following exposure to n i t r o s a t i o n products of sa l t e d f i s h aqueous extract with and without S9 30 13a, Mutagenic e f f e c t of sa l t e d f i s h aqueous extract on _S. typhimurium (TA100) with and without S9 30 13b. Mutagenic e f f e c t of nitr o s a t e d s a l t e d f i s h aqueous extract on _S. typhimurium (TA100) with and without S9 30 ix 14. Effect of salted fish aqueous extract and nitrite-treated salted fish aqueous extract on reversion frequency of S_. typhimurium (TA1535) using the preincubation test 33 15. Mutagenicity and survival frequencies of S^. typhimurium tester strain TA1535 following exposure to nitrosation products of aqueous fraction of a fish extract 33 16. Effect of whole saliva of five individuals on c e l l survival and mutagenicity of strain TA1535 exposed concurrently to nitrite-treated aqueous salted fish extract 35 17. Inhibitory effect of tannic acid, pyrogallol and g a l l i c acid on mutagenicity on strain TA1535 of the nitrite-treated fish extract 35 18. Inhibitory effect of ascorbic acid, catechin and chlorogenic acid on mutagenicity on strain TA1535. of the nitrite-treated f i s h extract 37 19. Effect of three tea samples on c e l l survival and mutagenicity on strain TA1535 treated with nitrosated fish products in the .' liquid suspension test 37 20. Reaction pathways of coloured compound formation with sodiums n i t r i t e 39 21. Nitrite depletion capacity of aqueous salted fish extract 40 22. Nitrite depletion capacity of g a l l i c acid, pyrogallol and tannic acid 41 23. Nitrite depletion capacity of ascorbic acid, catechin and chlorogenic acid 41 24. Percentage of n i t r i t e remaining in the presence of three tea samples 42 25. Nitrite depletion capacity of saliva at various . :... y. .... concentrations 42 26. Mutagenicity of _S. typhimurium (TA102) following exposure of various concentrations of H2°2 27. Reference curve for the determination of HO concentrations ..50 x LIST OF ABBREVIATIONS B(a)P CHO G6P H 2 ° 2 HEPES + His HPLC i. p . KC1 KH 2P0 4 MEM M Cl„ g 2 MNNG NaAz NaCl NADP Na 2HP0 4 NaNO. NED 2NF NPRO S.D. X Benzo(a)pyrene Chinese hamster ovary Glucose-6-phosphate Hydrogen peroxide N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic a c i d H i s t i d i n e independence High pressure l i q u i d chromatography Intraperitoneal Potassium ch l o r i d e Monobasic potassium phosphate Eagle's minimal e s s e n t i a l medium Magnesium c h l o r i d e N-Methyl-N'-nitro-N-nitrosoguanidine Sodium azide Sodium chl o r i d e Nicotinamide adenine dinucleotide phosphate Dibasic sodium phosphate Sodium n i t r i t e N-l-Naphthylethylenediamine dihydrochloride 2-Nitrofluorene Nitro s o p r o l i n e Standard deviation Mean x i ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my supervisor, Dr H.F. S t i c h , f o r h i s guidance, patience and encouragement throughout the course of t h i s work. I am also indebted to Dr R.H.C. San and Dr M.P. Rosin f o r t h e i r expert advice and encouragement. I would l i k e to thank Dr B. Bohm of the Department of Botany, U.B.C., for the i s o l a t i o n and p u r i f i c a t i o n of some of the phenolic compounds. I would also l i k e to extend my sincere thanks for the help, support and good company which I have received from a l l the s t a f f i n the Environmental Carcinogenesis Unit of the B.C. Cancer Research Centre. Last but not l e a s t , I would l i k e to thank Miss Rosemary Johnson f o r typing t h i s t h e s i s . F i n a n c i a l support was from the National Cancer I n s t i t u t e of Canada through a grant awarded to Dr H.F. S t i c h . x i i INTRODUCTION Environmental factors have been indicated as playing a major r o l e i n human mutagenesis and/or carcinogenesis (World Health Organization, 1964). Epidemiological research has revealed s i g n i f i c a n t v a r i a t i o n s i n cancer incidence both between and within countries (Drasan and Irving, 1973; B l o t et a l . , 1976; Hoover et a l . , 1975). Experimental data also suggest that environmental f a c t o r s could account f o r as much as 90% of current cancer incidence (Wynder and Gori, 1977; D o l l , 1977). Several well-documented examples from occupational sources include coal t a r , v i n y l c h l o r i d e , chromium, n i c k e l and asbestos (Bartsch and Montesano, 1975; Cole and Goldman, 1975; Dean, 1978). Tobacco smoking i s also a well-recognized e t i o l o g i c a l f a c t o r i n cancer of the lung, buccal c a v i t y , pharynx and larynx (World Health Organization, 1975). Other environmental f a c t o r s such as a i r p o l l u t i o n (Pike et a l . , 1975), viruses and other organisms (Heath et a l . , 1975) , chlorinated water (Simmon et a l . , 1977), food additives (Boffey, 1976) , drugs (Fraumeni and M i l l e r , 1972) and h a i r dyes (Kirkland et a l . , 1978) are also r e c e i v i n g a t t e n t i o n as possible causative agents i n human carcinogenesis. Besides the e f f e c t s of dietary d e f i c i e n c i e s or excesses, food has received a d d i t i o n a l attention as a source of cancer r i s k . Epidemiological evidence supports the idea that d i e t a r y and l i f e s t y l e f a c t o rs are involved i n the e t i o l o g y of a large proportion of human cancers (Hirayama, 1981). For example, migrant populations provide valuable evidence of the r o l e of d i e t i n cancer. In general, the cancer experience of migrants changes from t h e i r native population experience to that of the host country. In Japanese 1 migrants to the United States, a s h i f t i s apparent, as early as the f i r s t generation, i n stomach and colon cancer mortality patterns from the rate i n Japan to that prevalent i n the United States. By the t h i r d generation, cancer incidence i s v i r t u a l l y i d e n t i c a l to the host country rate (McMichael, 1978). Numerous genotoxic, and by i m p l i c a t i o n carcinogenic agents enter man through food products (Sugimura and Nagao, 1979; S t i c h et a l . , 1982a). These agents consist of n a t u r a l l y occurring mutagens and clastogens, compounds newly formed during roasting, b r o i l i n g and f r y i n g processes, p e s t i c i d e and herbicide residues, and a host of various food a d d i t i v e s . Moreover, carcinogens can be formed within man from ingested precursors such as n i t r i t e and secondary amines (Spiegelhalder et a l . , 1976; Gruger, 1972). One example used i n t h i s study to show that d i e t may be involved i n human carcinogenesis was the consumption of s a l t e d f i s h (Hirayama, 1966; Ho et a l . , 1978; Yang, 1980). The possible agent causing cancer was suspected to be the nitrosamines present i n the s a l t e d f i s h . The reason for t h i s suspicion was that the s a l t used for preserving the f i s h had a high n i t r i t e concentration (Dr Cai Hai-Ying, personal communication) and was r i c h i n secondary amines (Gruger, 1972). Nitrosamines have been implicated i n the development of nasopharyngeal carcinomas among Cantonese (Ho, 1971; Huang et a l . , 1978), of esophageal carcinomas i n high r i s k areas such as Linxian county (Yang, 1980), of stomach cancer among populations consuming salt-preserved f i s h (Hirayama, 1966), and of urinary bladder cancer (Hawksworth and H i l l , 1974). Carcinogenic nitrosamines are c l a s s i c a l l y produced by the e l e c t r o p h i l i c reaction between n i t r i t e or nitrous a c i d and secondary or t e r t i a r y amines 2 under a c i d conditions s i m i l a r to those i n the human stomach and i n animal experiments (Mirvish, 1978). Ease of n i t r o s a t i o n chemically may be influenced by many factors such as the b a s i c i t y of the amine, pH, substrate concentration and the presence of some inorganic ions, e.g., thiocyanate (Wolff and Wasserman, 1972). I t has been postulated that up to 700 ug of N-nitrosodimethylamine i s formed d a i l y within man (Tannenbaum, 1979). In addition, nitrosamines can be formed i n food systems i f the concentrations of n i t r i t e and n i t r o s a t a b l e amines are high enough and the conditions appropriate (Fan and Tannenbaum, 1973). They have also been detected i n the atmosphere (Fine et a l . , 1977) and i n r i v e r water (Fine et a l . , 1976). N i t r i t e s have been used as food additives to s t a b i l i z e the colour of cured meats (Cho and B r a t z l e r , 1970) , to contribute flavour (Wasserman and T a l l e y , 1972) and to protect against the danger of botulism (Duncan and Foster, 1968). N i t r i t e s can also be produced by the b a c t e r i a l reduction of n i t r a t e s which are widely d i s t r i b u t e d i n vegetables such as spinach, beets, ce l e r y and le t t u c e (Ashton, 1970). Secondary amines have been reported i n f i s h (Gruger, 1972), vegetables ( P h i l l i p s , 1966) and f r u i t j u i c e s (Stewart et a l . , 1960). In some instances, a sin g l e meal may contain as much as 100 mg of secondary amines (Sebranek and Cassens, 1973). Thus the amount of n i t r i t e and secondary amines consumed by humans merits some attent i o n . The second model used i n t h i s study was the chewing of b e t e l nut. For centuries, numerous plant products have been chewed by many d i f f e r e n t popu-l a t i o n groups. Among these products, the betel nut (Areca catechu) has become the best known example because of i t s suspected r o l e i n the etiology of o r a l and esophageal carcinomas (Jussawalla, 1976; Muir and Kirk, 1960; Ramanathan and Lakshimi, 1976) and i t s induction of neoplasms i n experimental 3 animals (Bhide et a l . , 1979; Ranadive et a l . , 1979). In addition, the s a l i v a of b e t e l nut chewers has been shown to contain r e l a t i v e l y large q u a n t i t i e s of phenolics and shows chromosome-damaging a c t i v i t y (Stich and S t i c h , 1982). I t was also of i n t e r e s t to note that the incidence of o r a l cancer i s gr e a t l y increased i n chewers of lime-containing mixtures (H.F. S t i c h , personal communication). However, no s i g n i f i c a n t increase i n o r a l cancer was observed among chewers.of leaves which produce an a c i d i c or neutral pH i n the s a l i v a , such as miang (tea) leaves (Simarak et a l . , 1977). The addi t i o n of lime-containing mixtures would increase the a l k a l i n i t y of the s a l i v a of b e t e l nut chewers, which would cause oxidation of phenolics and give r i s e to H^O^, concomitantly enhancing i t s c l a s t o g e n i c i t y (Hanham et a l . , 1983). One of the major components i n b e t e l nut other than the a l k a l o i d (arecoline), eugenol and quercetin (Stich et a l . , 1981a) i s b e t e l tannin. In t h i s study, the mutagenic and clastogenic e f f e c t of a water extract of the b e t e l nut and b e t e l tannin was examined. In addition, lime was added to these components to simulate the chewing habit of the chewers. This would increase the a l k a l i n i t y of the mixture and hence provide good conditions for the oxidation of the components i n water extract and b e t e l tannin. The new S. typhimurium s t r a i n TA102 was used for the mutagenicity study since t h i s t e s t e r s t r a i n was shown to detect oxidative agents (Levin et a l . , 1982). Hydrogen peroxide assay was c a r r i e d out under these conditions to determine i f B^O£ was a c t u a l l y formed. Catalase was used as a natural i n h i b i t o r and/or i n d i c a t o r f o r H„0„ formation. 4 Considering the large number of procarcinogens or carcinogens which are consumed d a i l y by man, the question as to the type and e f f i c i e n c y of protection mechanisms against these damaging agents must be r a i s e d . Because of the complexity of food products and the multitude of possible chemical i n t e r a c t i o n s , there i s a need f o r model systems to study factors which can suppress or enhance car c i n o g e n i c i t y of various food components. Since chemical carcinogens can induce a v a r i e t y of genetic damages (Stich and Acton, 1979), 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, DNA damage, chromosome aberration and gene conversion (Stich and San, 1979). These t e s t s can also be used f o r 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 , 1979, 1980; Buening et a l . , 1981). In t h i s study, the mutation on S. typhimurium and chromosomal aberration on CHO c e l l s were used to examine the genotoxic and clastogenic e f f e c t of sa l t e d f i s h and be t e l nut. Several possible p r o t e c t i v e modulators were considered i n the n i t r o s a t i o n reaction. During the consumption of a meal, n i t r o s a t a b l e compounds and compounds which may i n h i b i t or enhance the n i t r o s a t i o n reaction are mixed with s a l i v a . Human s a l i v a has been shown to i n h i b i t n i t r o s a t i o n of methylurea (Stich et a l . , 1982b). The question was therefore r a i s e d as to the e f f e c t of s a l i v a on the formation of mutagenic n i t r o s o compounds i n the salt e d f i s h extract. Since the presence of n i t r i t e i s a pr e r e q u i s i t e f or n i t r o s o compound formation, any compound which reacts r e a d i l y with n i t r i t e w i l l , i n e f f e c t , i n h i b i t amine n i t r o s a t i o n . Examples of such known compounds are ascorbic 5 a c i d and a-tocopherol, which can block the n i t r o s a t i o n reaction by reacting with n i t r i t e (Mirvish, 1981; Newmark and Mergens, 1981). In addition, Knowles (1974) showed that n i t r i t e can i n t e r a c t with a wide v a r i e t y of smoke phenols i n bacon during processing and f r y i n g . Several simple and polymeric phenolics have been shown by Mirvish (1981) to compete su c c e s s f u l l y with secondary amines or alkylureas f o r n i t r o s a t i n g species. An understanding of the contribution of phenolics to the t o t a l genotoxic burden of d i e t and l i f e s t y l e f a c t o r s appears to be of s i g n i f i c a n c e because of t h e i r r e l a t i v e l y high concentration i n vegetables, f r u i t s and beverages (Stich and Powrie, 1982) . The phenolic compounds involved i n t h i s study include some common ones which are present i n our d a i l y d i e t such as chlorogenic a c i d , catechin, p y r o g a l l o l , g a l l i c a c i d and tannic a c i d . The conditions which may occur during consumption of a meal c o n s i s t i n g of f i s h , n i t r i t e , phenolics and tea were also simulated. This i s because these phenolic compounds and teas may be j o i n t l y ingested with f i s h products and t h i s may lead to the endogenous formation of carcinogenic nitrosamines or alkylureas (Mirvish et a l . , 1978). There are also studies which i n d i c a t e that some phenolic compounds could act as c a t a l y s t s on the n i t r o s a t i o n reaction (Davies and McWeeny, 1977; Walker et a l . , 1982). Two chemical analyses were therefore used to study the chemistry involved i n the n i t r o s a t i o n reaction i n the presence of phenolics: (1) n i t r o s a t i o n of p r o l i n e in vitro, and (2) the n i t r i t e depletion r e a c t i o n . These two t e s t s provide information on whether the structure of the phenolics had any e f f e c t on the n i t r o s a t i o n reaction and i f the phenolics competed with the a v a i l a b l e n i t r i t e . 6 The removal of carcinogens from man's environment would appear to be the simplest\.and most d i r e c t manner i n which to reduce hazardous exposure. However, t h i s approach i s d i f f i c u l t , perhaps u n r e a l i s t i c , i f a carcinogenic mixture i s part of a regular d i e t (e.g., s a l t e d f i s h i n Chinese populations), provides pleasure (e.g., smoking), or has a r i t u a l i s t i c s i g n i f i c a n c e (e.g., b e t e l nut chewing among some Asian populations). In such cases, chemo-prevention could be applied with the aim of (1) trapping the carcinogens (or promoters) before they can induce c e l l u l a r changes, (2) preventing the mutated c e l l s from d i v i d i n g , or (3) i n h i b i t i n g the formation of colonies of mutated c e l l s from which n e o p l a s t i c a l l y transformed c e l l s could a r i s e . I t i s the inten t i o n of t h i s thesis to explore d i e t and l i f e s t y l e factors which may be involved i n human cancer. In add i t i o n , some possible chemo-preventive agents are studied i n order to prevent the formation of damaging agents. 7 MATERIALS AND METHODS Chemicals Chlorogenic acid, p y r o g a l l o l , catechin, r e s o r c i n o l , sodium n i t r i t e , p r o l i n e , s u l f a n i l i c a c i d , ammonium molybdate, potassium iodide, catalase, a f l a t o x i n B^, sodium azide, benzo(a)pyrene, 2-nitrofluorene, N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C were purchased from Sigma Chemical Co., St Louis, MO. G a l l i c a c i d and tannic acid were obtained from A l d r i c h Chemical Co., Milwaukee, WI. Ascorbic a c i d was supplied by Mallinckrodt Inc., St Louis, MO. N-l-Naphthylethylenediamine dihydrochloride, starch and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic a c i d were purchased from BDH Chemicals Ltd., Vancouver, B.C. Other phenolic compounds, namely, p-hydroxybenzoic a c i d , 2,3-dihydroxybenzoic a c i d , 2,4-dihydroxybenzoic a c i d , 2,5-dihydroxybenzoic a c i d , 2,6-dihydroxybenzoic a c i d , and 3,5-dihydroxy-benzoic a c i d , were prepared by Dr B. Bohm of the Department of Botany, Un i v e r s i t y of B r i t i s h Columbia. The p u r i t y of a l l the phenolic compounds was determined by nuclear magnetic resonance spectroscopy, c a r r i e d out at the Department of Chemistry, Un i v e r s i t y of V i c t o r i a , V i c t o r i a , B.C. L i t t l e or no impurities were observed. A l l other chemicals were reagent grade. Preparation of Chemical Stock Solutions The phenolics and b e t e l tannin were dissolved i n phosphate bu f f e r solution (PBS) for the Salmonella mutagenicity assay or i n "wash" medium f o r c l a s t o -genic a c t i v i t y i n CHO c e l l s . The solutions were warmed i n a hot water-bath' to help dissol v e a l l chemicals. The f i n a l pH was adjusted according to the experimental design. 8 Nitrosoproline Synthesis 10 g of L-proline and 6 g of NaNC^ were dissolved i n 7.5 ml of d o u b l e - d i s t i l l e d water. The mixture was cooled over i c e and 8.0 ml of concentrated HC1 was slowly added while s t i r r i n g f o r a period of 4 hr. A li g h t - y e l l o w s o l i d was obtained which was f i l t e r e d and dr i e d under vacuum. This s o l i d was p u r i f i e d by d i s s o l v i n g i n 10 ml of hot chloroform. The sol u t i o n was f i l t e r e d and l e f t to cool overnight on an ice-bath. This produced l i g h t - y e l l o w c r y s t a l s of pure n i t r o s o p r o l i n e with m.p. of 98 ± 2°C which was very close to the l i t e r a t u r e value ( L i j i n s k y et a l . , 1970). Preparation of N i t r o s a t i o n Reaction of Proline Mixture Reaction mixtures consisted of the following components: L-proline ( f i n a l concentration, 30 mM), various doses of modulators (tea samples, phenolics or s a l i v a ) , and sodium n i t r i t e ( f i n a l concentration, 7 mM). The components were combined i n the order indicated. The whole mixtures were a c i d i f i e d to pH 2.00 ± 0.01 and incubated f o r 1 hr at 37°C. The mixtures were then analyzed f o r the formation of NPRO by high pressure l i q u i d chromatography. Analysis of Nitros o p r o l i n e A 2.0 y l a l i q u o t of the reaction mixture was used to determine NPRO by HPLC equipped with the following: a Beckman 165 v a r i a b l e wavelength detector, a Spectro-Physics SP 8700 solvent d e l i v e r y system, a Whatman P a r t i s i l PXS (5 um p a r t i c l e s i z e , 25 cm i n length, ODS-3) reverse phase column. The solvent was i n i t i a l i z e d at 2% methanol i n d o u b l e - d i s t i l l e d water with a gradient of 1.8% methanol/min. The methanol concentration was rai s e d to 100% at 10 min and then held at 100% for 2 min longer before r e s e t t i n g i t to 2%. 9 The flow rate of the solvent was set at 1.0 ml/min and NPRO was detected at a wavelength of 238 nm. Under these conditions, the peaks of the two isomers of NPRO were well resolved (retention times, 8.8 ± 0.7 min and 9.0 ± 0.7 min). The L-proline, NaNO^ , teas, different phenolic compounds and saliva employed in this study contained no detectable levels of preformed NPRO. In addition, no inter-fering peaks appeared at these retention times. Determination of Nitrite Depletion The n i t r i t e depletion method was modified from the procedure described by Fan and Tannenbaum (1971). The n i t r i t e concentration was determined by the classical Griess reaction with su l f a n i l i c acid and NED under acidic conditions. The colour reagent was prepared by dissolving 0.1 g of NED and 1.0 g of sulfan i l i c acid in 100 ml of 20% hydrochloric acid. The reaction mixture was prepared by adding 1.0 ml of (0.03 M) NaNO^ , 1.0 ml of various concentrations of teas, phenolic compounds, saliva or fish extract and 1.0 ml of glass-distilled water. This reaction mixture was incubated for 1 hr at 37°C, pH 2.00 ± 0.01. A 25 ul aliquot of this reaction mixture was then added to 2.0 ml of colour reagent. The n i t r i t e content was determined from azo dye colour formation at wavelength 540 nm using a Spectronic 21 spectrometer. Preparation of S9 Liver Microsomal Mixture S9 li v e r microsomal mixture was prepared from l i v e r of aroclor 1254-pretreated rats to induce rat liver enzymes for carcinogen activation, as described by Ames et a l . (1975). The aroclor 1254, a polychlorinated biphenyl mixture, was diluted in corn o i l (200 mg/ml) and given to Wistar rats by a 10 s i n g l e i . p . i n j e c t i o n at a dose of 500 mg/kg body weight three days before s a c r i f i c e . The ra t s were given drinking water and Purina Laboratory Chow up to the day before s a c r i f i c e . The ra t s were s a c r i f i c e d by a blow to the head and then decapitated. The l i v e r s were removed from the rats and put i n t o Erlenmeyer f l a s k s ; 0.15 M KC1 at 3 ml/g wet l i v e r was added and homogenized with a Potter-Elvejhem homogenizer. The homogenate was centrifuged f o r 10 min at 9000 x g at 4°C and the supernatant saved. The supernatant was then quickly frozen down i n small portions and stored at -80°C. For every 3 ml of standard S9 reaction mixture there was 0.013 ml of 0.5 M MgCl 2, 0.013 ml of 3.3 M KC1, 0.132 ml of 50 mM G6P, 0.132 ml of 40 mM NADP, 0.293 ml of 20 mM HEPES, and 2.121 ml of l x PBS or "wash" medium. This s o l u t i o n was brought to pH 7.4 and 0.3 ml of l i v e r supernatant added. The s o l u t i o n was prepared fresh before each experiment. Preparation of Sodium Phosphate Buffer The sodium phosphate buffer (PBS) was prepared by d i s s o l v i n g 80 g NaCl, 2.0 g KC1, 11.5 g Na2HPC>4 and 2.0 g KH2PC>4 i n t o i i±ter Q f d o u b l e - d i s t i l l e d water. This mixture was autoclaved at 121°C f o r 30 min. The pH of the PBS was 7.4. Preparation of S a l i v a Samples S a l i v a was obtained from 5 non-smokers (2 males and 3 females) who consumed a regular "Western-type" d i e t . The s a l i v a was c o l l e c t e d between 10 and 11 a.m. which was about 2-3 hr a f t e r breakfast. P r i o r to use, the s a l i v a was adjusted to pH 2.0 and passed through a 20 um pore s i z e M i l l i p o r e f i l t e r (Stich et a l . , 1982b). 11 Preparation of Salted F i s h Extract The salted f i s h , commercially known as "Pak Wik" , was purchased from a l o c a l food store i n Chinatown, Vancouver. The s a l t e d f i s h were extracted by a modification of the procedure of van der Hoeven and van Leeuwen (1980). The s a l t e d f i s h were washed with d i s t i l l e d water to remove the s u p e r f i c i a l s a l t c r y s t a l s p r i o r to extraction. The f i s h samples were minced, homogenized i n a blender, freeze-dried for 48 hr, and extracted three times with hexane (3 ml/g of fish) which was discarded. Residual hexane was removed by a i r -drying. The remaining material was then extracted three times with methanol (3 ml/g of f i s h ) , the methanol evaporated, and the residue reconstituted i n d i s t i l l e d water (1 ml/g of residue). The aqueous f r a c t i o n of the f i s h e xtract was used throughout the e n t i r e study (Appendix I ) . N i t r o s a t i o n of the Salted F i s h Extract The n i t r o s a t i o n mixtures consisted of the following components ( f i n a l concentration i n mg/ml): aqueous f r a c t i o n of the f i s h extract (200-450 mg equivalent of f i s h ) , various doses of modulating agents (phenolics, teas or saliva) and sodium n i t r i t e (0.8 mg or 0.01 M). The components were added i n the order indicated. The whole mixture was a c i d i f i e d to pH 2.00 ± 0.01 and incubated for 1 hr at 37°C. The mixture was then ne u t r a l i z e d to pH 7.00 ± 0.01 and used i n the mutagenicity t e s t (Appendix I I ) . Preparation of Betel Nut Extracts The b e t e l nuts were extracted by a modification of the procedure of Mathew and Govindarajan (1964). The b e t e l nuts were crushed into powder and extracted twice with hexane (1.5 ml/g of b e t e l nut). The residue was a i r -dr i e d to remove the hexane. The remaining material was then extracted twice 12 with water (1.0 ml/g of residue), heated i n a b o i l i n g water-bath, and the mixture f i l t e r e d through Whatman q u a l i t a t i v e 1 (15.0 cm) f i l t e r paper. This was named "water extract". The residue was then extracted three times with n-butanol (1.0 ml/g of residue) and the n-butanol evaporated. The dr i e d butanol extract was taken up i n 200 ml of g l a s s - d i s t i l l e d water; 100 ml of 1.5% caffeine was added to t h i s water mixture to p r e c i p i t a t e the tannin (83.4 mg tannin/nut) (Appendix I I I ) . Preparation of Tea Samples The three tea samples used included a Chinese tea (Pu Erh Beeng brand), a Japanese green tea (Shincha-Ohhashiri brand) and a black Ceylanese tea (Bee brand). The tea samples were prepared by adding b o i l i n g water to the tea leaves (100 mg/ml water). A f t e r 5 min, the samples were centrifuged and the supernatants decanted for the study. Salmonella Mutagenicity Assay Salmonella typhimurium t e s t e r s t r a i n s TA98, TA100, TA102 and TA1535 were obtained from Dr B.N. Ames of the Un i v e r s i t y of C a l i f o r n i a , Berkeley. The b a c t e r i a l s t r a i n s were grown and maintained as described by Ames et a l . (1975). S t r a i n TA98 i s a h i s t i d i n e auxotroph which contains a resistance tran s f e r factor (R factor) and i s s e n s i t i v e to frameshift mutagens. Tester s t r a i n TA100 i s a h i s t i d i n e auxotroph which contains an R factor and 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 . Tester s t r a i n TA153 5 can also be used to detect mutagens causing base-pair s u b s t i t u t i o n s , but i t does not contain the R fac t o r (McCann et a l . , 1975). Tester s t r a i n TA102 contains A-T base p a i r s at the s i t e of mutation i n contrast to the other Salmonella t e s t e r s t r a i n s which detect mutagens damaging G'C base p a i r s . This s t r a i n 13 d i f f e r s from previous t e s t e r s t r a i n s i n that the mutation has been introduced in t o a multicopy plasmid, so that approximately 30 copies of the mutant gene are a v a i l a b l e f or back mutation (Levin et a l . , 1982). Preliminary experiments were conducted using the preincubation modifi-cation (Nagao et a l . , 1977) of the procedure developed by Ames et a l . (1975). Frozen master stock was innoculated into 5 ml of Difco nu t r i e n t broth and grown overnight (14-16 hr) on a Labquake rotary wheel at 37°C i n an incubator. The treatment mixtures were composed of the following which were added i n the order indicated: 0.1 ml of the overnight b a c t e r i a c u l t u r e , 0.5 ml of PBS or the standard S9 l i v e r microsomal mixture, and 0.1 ml of the t e s t samples. The mixtures were then incubated at 37°C i n a water-bath for 20 min; 2.0 ml of molten top agar at 49°C containing 0.455 mM h i s t i d i n e was added to each of the treatment mixtures and poured onto minimal glucose agar plates (Ames et a l . , 1975). These plates were incubated at 37°C f o r 2 days. The colonies were counted on an Artek Model 880 automatic colony counter (Farmingdale, N.Y.). The mutagenic a c t i v i t y was expressed as the number of h i s t i d i n e ( h i s + ) revertants per p l a t e . To t e s t the e f f e c t of increased a l k a l i n i t y of b e t e l nut water extract and b e t e l tannin, 20 y l of lime (10 mg/ml) was added to the b e t e l nut water extract and be t e l tannin to bring the pH to 10.00. These mixtures were incubated for 30 min at 37°C. The pH was adjusted back to 7.00 with HC1 (1 M) before they were applied to the b a c t e r i a . The mutagenicity of the n i t r o s a t i o n reaction products of the aqueous f i s h extract was assayed using a modification (Rosin and S t i c h , 1979) of the method of Ames et a l . (1975). This procedure provides information on the mutagenicity of tested mixtures while at the same time measuring c e l l s u r v i v a l 14 frequencies, a factor of major importance when examining the e f f e c t of modulating agents on induced mutagenicity. Logarithmically growing cultures Q of S. typhimurium (5-8 x 10 cells/ml) were prepared by reinnoculating 0.1 ml of an overnight culture (14-16 hr) in t o 5.0 ml of fresh n u t r i e n t broth. These l a t t e r cultures were grown on a Labquake rotary wheel i n an incubator f o r 4 hr at 37°C. One ml of t h i s culture was placed i n t o centrifuge tubes and the b a c t e r i a p e l l e t e d by c e n t r i f u g a t i o n at 3000 rpm for 5 min. The p e l l e t s were then resuspended i n 0.5 ml of the n i t r o s a t i o n reaction products. The treatment duration was 20 min at 37°C. The b a c t e r i a were p e l l e t e d and washed by resuspension i n PBS and c e n t r i f u g a t i o n . The b a c t e r i a were then re-9 suspended i n 0.5 ml PBS at ca. 10 c e l l s / m l , and aliquots were d i l u t e d with s a l i n e (0.01 ml/10 ml x 0.01 ml/10 ml) and plated (0.3 ml) onto nutrient agar plates for s u r v i v a l studies (Ames et a l . , 1975). Aliquots (0.1 ml) of b a c t e r i a l suspensions were added to low-histidine (0.455 mM) top agar and the agar overlayed on minimal agar plates i n order to estimate the number of h i s + revertants. The plates were scored a f t e r 48 hr incubation at 37°C. Mutagenic a c t i v i t y was ca l c u l a t e d i n terms of the number of h i s + revertants 7 per 10 surviving b a c t e r i a l c e l l s (Rosin and S t i c h , 1979). Controls f o r Mutagenicity Assay The a c t i v i t y of the S9 a c t i v a t i o n mixture was assayed with B(a)P on TA100 at a concentration of 10 ug/plate. The S9 was deemed s a t i s f a c t o r y i f there was an elevated number of revertants per plate from 100 ± 16 to 760 ± 63. B(a)P at the same concentration without S9 present should not elevate the spontaneous frequency of TA100 over that found i n the untreated c o n t r o l . The following chemicals were used to t e s t the a c t i v i t y of the s t r a i n s by showing an elevated number of revertants per p l a t e : NaAz (3 yg/plate) gave 1205 ± 13 15 revertants/plate from 100 ± 16 on TA100; 2NF (5 yg/plate) gave an increase from 26 ± 4 to 887 ± 13 revertants/plate; and mitomycin C (0.5 yg/plate) on TA102 gave an increase from 250 ± 20 to 3108 ± 174 revertants/plate. Chromosome Aberration Test The method employed was that previously described by S t i c h et a l . (1979). CHO c e l l s were grown i n MEM supplemented with 10% f e t a l c a l f serum, a n t i -b i o t i c s (streptomycin s u l f a t e , 29.6 yg/ml; p e n i c i l l i n , 125 yg/ml; kanamycin, 100 yg/ml; and fungizone, 2.5 yg/ml) and sodium bicarbonate (1 mg/ml). For the analysis of chromosome aberrations, approximately 140,000 CHO 2 c e l l s were seeded onto each 22 mm c o v e r s l i p i n 3.5 cm p l a s t i c dishes (Falcon). Experiments were begun when c e l l s were 40-60% confluent. The t i s s u e culture medium was removed from the p e t r i dishes and replaced with 1 ml of reaction mixture. For t e s t s i n v o l v i n g an S9 a c t i v a t i o n mixture or catalase, 0.5 ml of these a l i q u o t s were added to each p e t r i d i s h p r i o r to the ad d i t i o n of 0.5 ml of a double-strength t e s t chemical. Following a 3-hr exposure to the t e s t mixtures, the c o v e r s l i p s were washed twice with "wash" MEM, and 2.0 ml fresh MEM with 10% f e t a l c a l f serum was added to the p e t r i dishes. To t e s t the e f f e c t of lime on b e t e l nut water extract and betel tannin, 20 y l of lime (10 mg/ml) was added to the b e t e l nut water extract and betel tannin to bring the pH to 10.00. These mixtures were incubated f o r 30 min at 37°C and the pH adjusted back to 7.00 with HC1 (1 M) before they were mixed with the CHO c e l l s . Chromosome aberration frequency was estimated by sampling the c e l l s 20 hr a f t e r completion of treatment with chemicals; 4 hr p r i o r to sampling, 0.1 ml of c o l c h i c i n e (0.1% i n "wash" MEM) was added. C e l l s were then treated with a hypotonic s o l u t i o n of 1% sodium c i t r a t e f o r 20 min. The c i t r a t e s o l u t i o n 16 permits the c e l l s to swell. The c e l l s were then f i x e d with Carnoy's (acetic acid/ethanol, 1:3) f o r 20 min. A i r - d r i e d s l i d e s were stained with 2% orcein i n 50% a c e t i c acid/water, dehydrated and mounted i n permount. For each sample, a minimum of 100 metaphase plates were analyzed for chromatid breaks or chromatid exchanges. Exchanges scored included chromatid and chromosome exchanges, mono- and m u l t i - r a d i a l s , and r i n g s . Controls for CHO C e l l Conditions The a c t i v i t y of the S9 a c t i v a t i o n mixture was assayed with the following -2 controls. A stock s o l u t i o n of 2 x 10 M a f l a t o x i n was s e r i a l l y d i l u t e d -4 -5 -5 to concentrations of 2 x 10 M, 2 x 10 M and 1 x 10 M i n "wash" MEM; 0.5 ml of these solutions was then added with 0.5 ml of the S9 mixture or -4 -5 -6 "wash" MEM to a f i n a l concentration of 1 x 10 M, 1 x 10 M and 5 x 10 M a f l a t o x i n B^. The S9 was considered to be s a t i s f a c t o r y i f at l e a s t 80% of the metaphase plates had a minimum of one chromosome break at a concentration -6 of 5 x 10 M a f l a t o x i n B . A f l a t o x i n B^ at the same concentration, but without S9, should not elevate the frequency of chromosome aberrations over that found i n non-treated co n t r o l c u l t u r e s . "Wash" MEM, S9 reaction mixture and catalase were also added to the CHO c e l l s to check f o r any increase i n the spontaneous frequency of chromosome breaks. Assay f o r Hydrogen Peroxide 100 ml colour reagent was prepared fresh each day and contained 0.4 M potassium iodide, 2 x 10 ^ M ammonium molybdate and 3% starch. This mixture was heated and s t i r r e d u n t i l dissolved. Acetic acid was then added to a f i n a l concentration of 0.2 M. Aliquots (1.0 ml) of the sample to be assayed were pipetted i n t o two separate tubes. Catalase (0.1 yg i n 10 yl) was added to one 17 sample, and t h i s sample was allowed to incubate at room temperature for a minimum of 10 min. Aliquots (1.0 ml) of colour reagent were then added to each sample. Absorbance was determined at 575 nm i n a dual beak Perkin Elmer Lambda 3 spectrophotometer, using the (peroxide-free) catalase-treated sample as a blank. The reading was taken at the maximum absorbance. H2^2 l e v e l s were estimated from a standard curve prepared using phosphate-buffered samples containing 0 to 100 uM H^O^. This procedure was a modification of that described by Wang and Nixon (1978). 18 RESULTS In t h i s study, a l l the data shown are the average of two sets of experiments with t r i p l i c a t e analyses for each set ± the standard deviation. E f f e c t s of Doses of N i t r i t e , P r o l i n e , Time and pH on Formation of NPRO Nit r o s a t i o n of p r o l i n e was chosen as a model to study the e f f e c t of modulators on the n i t r o s a t i o n reaction since i t provides a simple system with which to work. Proline also has a chemical structure of an amine. Moreover, p r o l i n e i s water-soluble. This model may also.simulate, at l e a s t to some extent, the s i t u a t i o n of foods coming into contact with n i t r o s a t i n g agents. The four v a r i a b l e s considered were (1) concentration of NaN02, (2) concentration of p r o l i n e , (3) time of the reaction, and (4) the pH of the reaction mixture. The formation of NPRO in vitro was found to be strongly dependent on n i t r i t e concentration. There was only a s l i g h t increase i n the formation of NPRO at up to 2 mM NaNO^. However, when 3 to 6 mM of n i t r i t e was added, 2 to 5 times more NPRO was formed. The formation of NPRO increased exponentially with the dose of n i t r i t e present (Fig. 1). The e f f e c t of the amount of p r o l i n e added on the formation of NPRO was also studied (Fig. 2). When d i f f e r e n t amounts of p r o l i n e were added to 6 mM NaN02 and incubated for 1 hr at 37°C, a l i n e a r dose response was observed up to 100 mM p r o l i n e . Between 100 and 200 mM, the increase began to l e v e l o f f . Thus the formation of NPRO in vitro appeared to be proportional to the dose of p r o l i n e provided up to 100 mM. The increase slowed down between 100 and 200 mM, probably due to the l i m i t e d amount of NaNO a v a i l a b l e . 19 F i g u r e 1. The a b s o r p t i o n peak a r e a s by t h e f o r m a t i o n o f NPRO a t 230nm w i t h v a r y i n g NaN0 2 c o n c e n t r a t i o n s . The a b s o r p t i o n peak a r e a s were i n a b s o r p t i o n u n i t s . The x±S.D. p l o t t e d were from t h r e e e x p e r i m e n t s . The c o n c e n t r a t i o n o f p r o l i n e was lOOmM. The m i x t u r e was i n c u b a t e d f o r l h , a t 37°C and pH 2. F i g u r e 2. The a b s o r p t i o n peak a r e a s by t h e f o r m a t i o n o f NPRO a t 238nm w i t h v a r y i n g p r o l i n e c o n c e n t r a t i o n s . The a b s o r p t i o n peak a r e a s were i n a b s o r p t i o n u n i t s . The x±S.D.- p l o t t e d were from t h r e e e x p e r i m e n t s . The c o n c e n t r a t i o n o f NaNO^ was 6mM. The m i x t u r e was i n c u b a t e d f o r l h , a t 37°C and pH 2. 20 Figure 3. The absorption peak areas by the formation of NPRO at 238nm with varying times. The x±S.D. p l o t t e d were from three experiments. The absorption peak areas were i n absorption u n i t s . The concentrations of NaNO^and p r o l i n e were 6mM and lOOmM res p e c t i v e l y . Figure 4. The absorption peak areas by the formation of NPRO at 238nm with varying pH l e v e l s . The absorption peak areas were i n absorption u n i t s . The concentrations of NaNO^and p r o l i n e were 6mM and lOOmM re s p e c t i v e l y . The mixture was incubated at 37°C, f o r l h . The x±S.D. p l o t t e d were form three experiments. 21 Figure 3 Figure 4 21a The consideration of time and pH on the e f f i c a c y of NPRO formation was studied. A l i n e a r increase was observed from 0 to 30 min and i t reached a plateau a f t e r 2 hr. Again, t h i s may be due to the amount of NaN02 and pr o l i n e a v a i l a b l e f o r the reaction. A f t e r 1 hr, 85% of NPRO had been formed (Fig. 3). The highest response to NPRO formation was observed at pH 3, whereas at pH 4 the formation of NPRO decreased d r a s t i c a l l y (Fig. 4). The a c i d i t y of the reaction mixture therefore seems to play a major r o l e i n the n i t r o s a t i o n reaction. Based on these experimental data, the conditions for the following studies on NPRO formation were chosen at 6 mM NaN02 and 30 mM p r o l i n e . A reaction time of 1 hr at pH 2 and 37°C was chosen to make the experimental conditions comparable to the f i s h extract mutagenicity studies. E f f e c t s of Five Phenolics, Teas and S a l i v a on NPRO Formation The f i v e phenolics, g a l l i c a c i d , tannic a c i d , p y r o g a l l o l , catechin and chlorogenic a c i d , a l l i n h i b i t e d the formation of NPRO. Their i n h i b i t o r y e f f e c t was very s i m i l a r and they were a l l more e f f i c i e n t than ascorbic a c i d (Figs 5 and 6). This observation was i n good agreement with that observed with the i n h i b i t i o n by these phenolics on sa l t e d f i s h extract mutagenicity (Figs 17 and 18). The three tea samples (Chinese, Japanese and Ceylanese) studied also i n h i b i t e d the formation of NPRO. Chinese tea showed the greatest i n h i b i t o r y e f f e c t , whereas the Ceylanese tea was the l e a s t active ( Fig. 7). This may be due to the d i f f e r e n t components i n the tea samples since tea infusions were complex mixtures. S a l i v a at 20% concentration reduced the formation of NPRO by 50%. This showed that s a l i v a as an i n h i b i t o r of n i t r o s a t i o n was e f f e c t i v e i n complex f i s h extract or simple compounds such as pr o l i n e (Fig. 8.). 22 F i g u r e 5 . T h e i n h i b i t o r y e f f e c t o f t h r e e p h e n o l i c s o n n i t r o s a t i o n o f p r o l i n e : t a n n i c a c i d ( • ) ; p y r o g a l l o l ( • ) ; g a l l i c a c i d ( • ) . T h e c o n c e n t r a t i o n s o f n i t r i t e a n d p r o l i n e w e r e 6 m M a n d 3 0 m M r e s p e c t i v e l y . % N P R O c o n t r o l w a s t h e a b s o r b a n c e u n i t s o b t a i n e d i n t h e p r e s e n c e o f t h e p h e n o l i c s c o m p a r e d w i t h t h a t o b t a i n e d i n t h e a b s e n c e o f t h e p h e n o l i c s . P l o t t e d a r e x ± S . D . f r o m t h r e e e x p e r i m e n t s . F i g u r e 6 . T h e i n h i b i t o r y e f f e c t o f t w o p h e n o l i c s a n d a s c o r b i c a c i d o n N P R O f o r m a t i o n : a s c o r b i c a c i d ( A ) ; c a t e c h i n ( • ) ; c h l o r o g e n i c a c i d ( # ) . T h e c o n c e n t r a t i o n s o f n i t r i t e a n d p r o l i n e w e r e 6 m M a n d 3 0 m M r e s p e c t i v e l y . % N P R O c o n t r o l w a s t h e a b s o r p t i o n p e a k a r e a s o b t a i n e d i n t h e p r e s e n c e o f t h e m o d u l a t o r s c o m p a r e d w i t h t h a t o b t a i n e d i n t h e a b s e n c e o f t h e s e m o d u l a t o r s . P l o t t e d a r e x i S . D . f r o m t h r e e e x p e r i m e n t s . 2 3 C h (D 00 *3 H-C CVi Figure 7. The i n h i b i t o r y e f f e c t of three tea samples on NPRO formation at 6mM n i t r i t e and 30mM pr o l i n e : Chinese tea (•) ; Japanese tea (•) ; Ceylon tea ( • ). %NPRO control was the absorption peak areas obtained i n the presence of the teas compared with that observed i n the absence of the teas. P l o t t e d are x±S.D. form three experiments. Figure 8. The i n h i b i t o r y e f f e c t of whole s a l i v a of f i v e i n d i v i d u a l s on the formation of NPRO. The concentrations of n i t r i t e and p r o l i n e were 6mM and 30mM res p e c t i v e l y . %NPRO cont r o l was the absorption peak areas obtained i n the presence of the s a l i v a compared with that observed i n the absence of the s a l i v a . The x±S.D. p l o t t e d were from three experiments. 24 24a E f f e c t s of Dihydroxybenzoic Acid With and Without Resorcinol Moiety on  Nitr o s a t i o n of Proline 2,4- and 3,5-Dihydroxybenzoic a c i d both exhibited an enhancement of NPRO formation within a dose range. 2,4-Dihydroxybenzoic a c i d gave a peak maximum at 1 mM concentration, whereas 3,5-dihydroxybenzoic a c i d reached i t s maximum NPRO formation at 7.6 mM concentration at 30 mM p r o l i n e and 6 mM NaN02. However, 2,4-dihydroxybenzoic acid demonstrated an i n h i b i t o r y - e f f e c t on n i t r o s a t i o n at 10 mM, whereas 3,5-dihydroxybenzoic a c i d did not i n h i b i t n i t r o s a t i o n even at a concentration of 19 mM. Resorcinol showed the greatest enhancement compared to the above, but i t had a narrow range i n which r e s o r c i n o l acted as a c a t a l y s t . A f t e r a concentration of 3 mM, r e s o r c i n o l became an i n h i b i t o r at the dose used for p r o l i n e and NaN02. p-Hydroxybenzoic acid, which has been shown not to i n h i b i t n i t r o s a t i o n product formation (H.F. S t i c h , personal communication), and ascorbic acid, which i n h i b i t s n i t r o s a t i o n , are included f or comparison (Fig. 9). 2,3-, 2,5- and 2,6-Dihydroxybenzoic acids, which do not have r e s o r c i n o l moiety, d i d not show any enhancement of NPRO formation at the dose of p r o l i n e and NaN02 used (Fig. 10). E f f e c t s of Dihydroxybenzoic Acid with Resorcinol Moiety at Low Concentration of  N i t r i t e When the n i t r i t e concentration was reduced from 6 to 0.5 mM, 2,4-, 3,5-dihydroxybenzoic acid and r e s o r c i n o l a l l showed an i n h i b i t i o n of NPRO formation. Resorcinol was the most e f f e c t i v e i n h i b i t o r , whereas 3,5-dihydroxybenzoic acid was the l e a s t e f f e c t i v e i n i n h i b i t i n g the n i t r o s a t i o n of p r o l i n e (Fig. 11). These r e s u l t s were i n good agreement with those obtained as shown i n F i g . 9. These r e s u l t s indicate that there was some optimal r a t i o between NaN02, pr o l i n e and phenolics with r e s o r c i n o l moiety f o r the phenolics to act as c a t a l y s t s or i n h i b i t o r s . 25 F i g u r e 9 . E f f e c t o f r e s o r c i n o l ( • ) ; 2 , 4 - d i h y d r o x y b e n z o i c a c i d ( # ) ; 3 , 5 -d i h y d r o x y b e n z o i c a c i d ( A ) ; p - h y d r o x y b e n z o i c a c i d ( A" ) ; a s c o r b i c a c i d ( ® ) o n N P R O f o r m a t i o n . T h e c o n c e n t r a t i o n s o f n i t r i t e a n d p r o l i n e w e r e 6 m M a n d 3 0 m M r e s p e c t i v e l y . % N E R O . c o n t r o l w a s o b t a i n e d f r o m a b s o r p t i o n p e a k a r e a s i n t h e p r e s e n c e o f t h e m o d u l a t o r s c o m p a r e d t o t h a t i n t h e a b s e n c e o f t h e m o d u l a t o r s . P l o t t e d a r e x ± S . D . f r o m t h r e e e x p e r i m e n t s . F i g u r e 1 0 . E f f e c t o f t h r e e d i h y d r o x y b e n z o i c a c i d s : 2 , 3 - ( • ) ; 2 , 5 - ( A ) ; : . 2 , 6 - ( • ) o n NPRO f o r m a t i o n . T h e . c o n c e n t r a t i o n s o f N a N O ^ a n d p r o l i n e w e r e 6 m M a n d 3 0 m M r e s p e c t i v e l y . I n c l u d e d f o r c o m p a r i s o n ' w e r e p - h y d r o x y b e n z o i c a c i d ( * ) a n d a s c o r b i c a c i d ( ® ) . %NPRO c o n t r o l w a s o b t a i n e d f r o m a b s o r p t i o n p e a k a r e a s i n t h e p r e s e n c e o f t h e s e m o d u l a t o r s c o m p a r e d t o t h a t i n t h e a b s e n c e o f t h e m o d u l a t o r s . P l o t t e d a r e x ± S . D . f r o m t h r e e e x p e r i m e n t s . F i g u r e 1 1 . T h e i n h i b i t o r y e f f e c t o f r e s o r c i n o l ( • ) ; 2 , 4 - d i h y d r o x y b e n z o i c a c i d ( • ) a n d 3 , 5 - d i h y d r o x y b e n z o i c a c i d ( A ) o n N P R O f o r m a t i o n a t 0 . 5 m M n i t r i t e a n d 3 0 m M p r o l i n e . % N P R O c o n t r o l w a s o b t a i n e d f r o m a b s o r p t i o n p e a k a r e a s i n t h e p r e s e n c e o f t h e s e m o d u l a t o r s c o m p a r e d t o t h a t i n t h e a b s e n c e o f t h e m o d u l a t o r s . T h e x ± S . D . s h o w n w e r e o b t a i n e d f r o m t h r e e e x p e r i m e n t s . 2 6 Clastogenic A c t i v i t y of Aqueous and N i t r i t e - T r e a t e d Aqueous F i s h Extract The a b i l i t y of aqueous f i s h extract and n i t r i t e - t r e a t e d aqueous f i s h extract to induce chromosome damage was monitored with a conventional CHO c e l l chromosome aberration assay. The clastogenic a c t i v i t y of the f i s h extract and the n i t r i t e - t r e a t e d f i s h extract i s shown i n Table 1. The aqueous f i s h extract alone d i d not induce chromosomal breakage. However, the clastogenic a c t i v i t y increased by approximately 10 times when exposed to n i t r i t e - t r e a t e d f i s h extract. S9 l i v e r microsomal a c t i v a t i o n d i d not have any e f f e c t on the c l a s t o g e n i c i t y . The aqueous f i s h extract became t o x i c to the CHO c e l l s at a dose of 360 mg equivalent of s a l t e d f i s h per ml mixture ( i . e . , no metaphase observed). Mutagenicity of Aqueous Salted F i s h Extract The a b i l i t y of the aqueous f r a c t i o n of the f i s h extract to induce mutagenicity was examined with the preincubation modification (Nagao et a l . , 1977) of the Ames Salmonella mutagenicity assay (Ames et a l . , 1975). Figs 11a, 12a and 13 show the e f f e c t of adding t h i s aqueous f i s h extract to the b a c t e r i a l t e s t e r s t r a i n s TA98, TA100 and TA1535, r e s p e c t i v e l y . The mutagenic a c t i v i t i e s of the aqueous f i s h extract were expressed as the number of h i s + revertants per p l a t e . The concentrations of the aqueous f i s h extract were expressed i n terms of gram equivalent of s a l t e d f i s h per ml of extract. The r e s u l t s shown here were based on the average number of h i s + revertants per p l a t e ± S.D. of two sets of experiments, each with t r i p l i c a t e p l a t e s . The aqueous f i s h extract was strongly mutagenic to both t e s t e r s t r a i n s TA98 and TA100 (Figs 12a and 13a). No s i g n i f i c a n t enhancement of mutagenicity was observed on s t r a i n TA1535 (Fig. 14). The aqueous f i s h extract became toxic at a dose higher than 0.12 g equivalent of fish/ml extract on both 27 TABLE 1 CLASTOGENIC ACTIVITY OF AQUEOUS FISH EXTRACT AND NITRITE-TREATED FISH EXTRACT Percent Metaphase Plates with Chromatid Breaks and/or Exchanges (±S.D.) F i s h Extract N i t r i t e - T r e a t e d F i s h Extract''" H Equivalent of fish/ml -S9 +S9 -S9 +S9 0.36 T 2 0.18 3±2 1±1 37±5 28±7 0.09 0 0 14±3 9±3 N i t r i t e at 0.8 mg/ml mixture alone = 0%. "'"Nitrite-treated f i s h extract was composed of the indicated concentration of f i s h extract, 0.8 mg/ml mixture of n i t r i t e , incubated f or 1 hr at 37°C, pH 2. 2 T = t o x i c . 28 s t r a i n s TA98 and TA100. However, t h i s was not observed on s t r a i n TA1535. S9 l i v e r microsomal metabolic a c t i v a t i o n did not appear to a f f e c t the mutagenicity of the aqueous f i s h extract. Of the p o s i t i v e controls, the number of h i s + revertants per plate can be seen i n Table 2. These r e s u l t s for the p o s i t i v e controls are i n good agreement with those obtained by the Environmental Carcinogenesis Unit of the B.C. Cancer Research Centre (Table 3). Mutagenicity of N i t r i t e - T r e a t e d F i s h Extract N i t r i t e - t r e a t e d (pH 2, 1 hr, 3 7°C) f i s h extract again showed an increase i n the mutagenic e f f e c t on s t r a i n s TA98 and TA100. However, no s i g n i f i c a n t d i f f e r e n c e i n mutagenicity was observed between n i t r i t e - t r e a t e d and non-n i t r i t e - t r e a t e d aqueous f i s h extract (Figs 12b and 13b). A s i m i l a r trend was obtained i n both cases. In the case of s t r a i n TA1535, a s i g n i f i c a n t increase was observed and no t o x i c e f f e c t was seen when exposed to the doses of n i t r i t e - t r e a t e d f i s h extract used (Fig. 14). Once again, S9 l i v e r microsomal a c t i v a t i o n had no e f f e c t on the mutagenicity of the n i t r i t e - t r e a t e d f i s h on any of the above s t r a i n s . S t r a i n TA1535 was therefore used throughout the study of n i t r o s a t i o n of s a l t e d f i s h extract without S9 l i v e r microsomal a c t i v a t i o n . Since the preincubation t e s t p r o tocol 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. The l i q u i d suspension t e s t (Rosin and S t i c h , 1979), which i s another modification of the method of Ames et a l . (1975), was used to better assess the i n h i b i t o r y e f f e c t of the modulators. The advantages of using t h i s assay are t h r e e f o l d : (1) both reversion frequencies and s u r v i v a l frequencies can be estimated; (2) i t provides information on whether a reduction of h i s + revertants/plate was due to a to x i c e f f e c t on the b a c t e r i a or an i n h i b i t o r y e f f e c t exerted by the modulators; and (3) the exposure of the b a c t e r i a to the chemical i s better c o n t r o l l e d . 29 F i g u r e 1 2 a . M u t a g e n i c i t y o f S . t y p h i m u r i u m ( T A 9 8 ) f o l l o w i n g e x p o s u r e t o s a l t e d f i s h a q u e o u s e x t r a c t w i t h ( • ) a n d w i t h o u t ( • ) S 9 i n t h e p r e - i n c u b a t i o n t e s t . P l o t t e d a r e x + S . D . f r o m t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . F i g u r e 1 2 b . M u t a g e n i c i t y o f S . t y p h i m u r i u m ( T A 9 8 ) f o l l o w i n g e x p o s u r e t o n i t r o s a t i o n p r o d u c t s o f s a l t e d f i s h a q u e o u s e x t r a c t w i t h ( A ) a n d w i t h o u t ( • ) S 9 i n t h e p r e - i n c u b a t i o n t e s t . P l o t t e d a r e x ± S . D . f r o m t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . F i g u r e 1 3 a . M u t a g e n i c e f f e c t o f s a l t e d f i s h a q u e o u s e x t r a c t o n S . t y p h i m u r i u m ( T A 1 0 0 ) w i t h ( A ) a n d w i t h o u t ( • ) S 9 . T h e x ± S . D . s h o w n a r e f r o m t w o p r e - i n c u b a t i o n e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . F i g u r e 1 3 b . M u t a g e n i c e f f e c t o f n i t r o s a t e d s a l t e d f i s h a q u e o u s e x t r a c t o n S . t y p h i m u r i u m ( T A 1 0 0 ) w i t h ( A ) a n d w i t h o u t ( • ) S 9 . T h e x ± S . D . s h o w n a r e f r o m t w o p r e - i n c u b a t i o n e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . 3 0 30a TABLE 2 POSITIVE CONTROLS FOR DIFFERENT TESTER STRAINS yg/plate TA98 TA100 TA1535 -S9 +S9 -S9 +S9 -S9 +S9 B(a)P (10) NT 1 NT 93±8 510±21 NT NT NaAz (3) NT NT 1310±43 NT NT NT 2NF (5) 795±33 NT NT NT NT NT MNNG (20) NT NT NT NT 1310±43 NT Solvent 24±3 23±5 96±3 100±4 10±2 11±3 NT.= not tested. 31 TABLE 3 HISTORICAL RANGES FOR MUTAGENICITY IN EXPERIMENTAL RUNS Revertants per Plate TA98 TA100 TA1535 TA102 Concentration — Treatment (yg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 PBS 1 8-56 11-66 74-197 73-215 10-18 11-19 233-275 B(a)P 1 10 4 NT NT 71-194 201-702 NT NT NT NT MNNG2 20 NT NT NT NT 1310±43 NT NT NT NaAz 1 3 NT NT 735-1606 NT NT NT NT NT 2NF 1 5 238-942 NT NT NT NT NT NT NT 3 Mitomycin C 0.5 NT NT NT NT NT NT 2772 NT From Environmental Carcinogenesis Unit, B.C. Cancer Research Centre. 'From Marquardt et a l . (1977) . From Levin et a l . (1982). NT = not tested. F i g u r e 1 4 . T h e e f f e c t o f s a l t e d f i s h a q u e o u s e x t r a c t a n d n i t r i t e t r e a t e d s a l t e d f i s h e x t r a c t o n r e v e r s i o n f r e q u e n c y o f S . t y p h i m u r i u m ( T A 1 5 3 5 ) u s i n g p r e - i n c u b a t i o n t e s t . S a l t e d f i s h a q u e o u s e x t r a c t w i t h ( A ) a n d w i t h o u t ( • ) S 9 ; n i t r i t e t r e a t e d s a l t e d f i s h a q u e o u s e x t r a c t w i t h ( y ) a n d w i t h o u t ( • ) S 9 a r e p l o t t e d w i t h x ± S . D . b a s e d o n t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . F i g u r e 1 5 . M u t a g e n i c i t y a n d s u r v i v a l f r e q u e n c i e s o f S . t y p h i m u r i u m ( T A 1 5 3 5 ) f o l l o w i n g e x p o s u r e t o n i t r o s a t i o n p r o d u c t s o f a n a q u e o u s f r a c t i o n " o f a s a l t e d f i s h e x t r a c t : f i s h e x t r a c t a l o n e ( # ) ; n i t r i t e t r e a t e d f i s h e x t r a c t ( A ) i n t h e l i q u i d s u s p e n s i o n t e s t . T h e x ± S . D . s h o w n a r e o b t a i n e d f r o m t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . 33 At the doses used f o r n i t r o s a t i o n , the f i s h extract by i t s e l f showed no detectable mutagenic a c t i v i t y (Fig. 15). For t e s t i n g the i n h i b i t o r y or enhancing e f f e c t s of phenolics, s a l i v a and teas, the n i t r o s a t i o n reaction mixture was chosen to consist of 340 mg equivalent of f i s h per ml and 0.8 mg sodium n i t r i t e . These reaction conditions r e s u l t e d i n the production of mutagenic products which induced a reversion frequency of 15.1 ± 0.1 h i s + revertants per 10 7 survivors (n = 8) (182 ± 6 S.D. h i s + revertants/minimal agar plate) and re s u l t e d i n no t o x i c i t y i n the treated b a c t e r i a . The sodium n i t r i t e concentration used also d i d not show any mutagenic e f f e c t . The spontaneous reversion frequency observed f o r t h i s t e s t e r s t r a i n was + 7 1 ± 0.07 S.D. h i s revertants per 10 survivors (n = 8) or 7 ± 0.8 S.D. h i s + revertants/minimal agar p l a t e . E f f e c t of Human S a l i v a on Mutagenicity Resulting from N i t r i t e - T r e a t e d F i s h  Extract Human s a l i v a was chosen since most chemicals ingested through food w i l l i n t e r a c t with s a l i v a . I t i s therefore reasonable to look at the e f f e c t of human s a l i v a on the n i t r i t e - t r e a t e d f i s h extract. The addition of human s a l i v a to the n i t r i t e and f i s h extract mixture at pH 2 reduced the formation of reaction products which induce h i s + revertants i n S. typhimurium. F i g . 16 shows the r e s u l t s obtained with s a l i v a samples from f i v e i n d i v i d u a l s (2 males and 3 females, a l l non-smokers). There was a dose-related reduction of mutagenicity as the concentration of the s a l i v a increased. S a l i v a samples d i l u t e d to as low as 5% (v/v) of the o r i g i n a l s a l i v a concentration s t i l l s i g n i f i c a n t l y reduced the mutagenic a c t i v i t y of the n i t r o s a t i o n products (percent mutagenicity, 77.6 + 8.4, S.D., n = 5). 34 F i g u r e 1 6 . T h e e f f e c t o f w h o l e s a l i v a ( • ) o f f i v e i n d i v i d u a l s o n c e l l s u r v i v a l a n d m u t a g e n i c i t y o f T A 1 5 3 5 e x p o s e d c o n c u r r e n t l y t o n i t r i t e t r e a t e d a q u e o u s s a l t e d f i s h e x t r a c t . T h e c o n c e n t r a t i o n s o f f i s h e x t r a c t a n d n i t r i t e u s e d i n c o m b i n a t i o n w i t h w h o l e s a l i v a w e r e 3 4 0 m g e q u i v a l e n t o f f i s h p e r m l a n d . 0 . 8 m g r e s p e c t i v e l y . P l o t t e d a r e x ± S . D . f r o m t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g f o r e a c h i n d i v i d u a l . % m u t a g e n i c i t y i s t h e r e v e r s i o n f r e q u e n c y o f n i t r o s a t e d f i s h e x t r a c t - t r e a t e d b a c t e r i a i n t h e p r e s e n c e o f s a l i v a c o m p a r e d w i t h t h a t o b s e r v e d i n b a c t e r i a w i t h n i t r o s a t e d f i s h e x t r a c t o n l y . T h e v a l u e s h a v e b e e n c o r r e c t e d f o r s p o n t a n e o u s r e v e r s i o n . F i g u r e 1 7 . T h e i n h i b i t o r y e f f e c t o f t h r e e p h e n o l i c s o n m u t a g e n i c i t y o n s t r a i n T A 1 5 3 5 o f t h e n i t r i t e - t r e a t e d f i s h e x t r a c t : t a n n i c a c i d ( • ) ; p y r o g a l l o l ( • ) ; g a l l i c a c i d ( A ) . T h e c o n c e n t r a t i o n s o f f i s h . e x t r a c t a n d n i t r i t e u s e d w e r e 3 4 0 m g e q u i v a l e n t o f f i s h p e r m l a n d 0 . 8 m g r e s p e c t i v e l y . T h e v a l u e s h a v e b e e n c o r r e c t e d f o r s p o n t a n e o u s r e v e r s i o n . P l o t t e d a r e x ± S . D . f r o m t w o e x p e r i m e n t s w i t h t r i p l i c a t e p l a t i n g e a c h . 3 5 E f f e c t of Naturally Occurring Phenolics on Mutagenicity of N i t r i t e - T r e a t e d  F i s h Extract Five n a t u r a l l y occurring phenolic compounds were assayed f o r modulating a c t i v i t y on the formation of mutagens r e s u l t i n g from the n i t r o -sation of the f i s h extract. These phenolics are known to be present at high concentrations i n teas, vegetables and edible f r u i t s (Stich and Powrie, 1982). Figs 17 and 18 show the i n h i b i t o r y e f f e c t of tannic a c i d , p y r o g a l l o l , g a l l i c a c i d , catechin and chlorogenic a c i d on the mutagenicity of the n i t r o s a t i o n reaction products. A l l these phenolics show s i m i l a r e f f i c i e n c y i n i n h i b i t i n g n i t r o s a t i o n reaction f i s h products-induced h i s + reversion. Concentrations of the phenolics as low as 1 mg/ml already reduced the mutagenicity by approximately 50%. For comparison, the e f f e c t of ascorbic a c i d on t h i s reaction was included i n F i g . 18, since i t i s known that t h i s compound can react with the n i t r i t e . The i n h i b i t o r y e f f e c t s of the f i v e phenolics were comparable to that of ascorbic a c i d . A l l the reactions were performed at concentrations which d i d not a f f e c t the s u r v i v a l of S. typhimurium ((upper curves of Figs 17 and 18) . E f f e c t of Teas on Mutagenicity Resulting from N i t r i t e - T r e a t e d F i s h Extract Since single phenolic compounds e x h i b i t an i n h i b i t o r y e f f e c t on nitrosated f i s h extract-induced h i s + reversion, the question was r a i s e d as to whether complex mixtures of phenolics also behave i n the same manner. Teas are phenolic compound-containing mixtures (Stich and Powrie, 1982) which may be ingested j o i n t l y with f i s h products during a meal. The mutagenic a c t i v i t y of the n i t r i t e - t r e a t e d f i s h extract was markedly i n h i b i t e d i f the n i t r o s a t i o n reaction proceeded i n the presence of tea inf u s i o n s . F i g . 19 shows the i n h i b i t o r y e f f e c t of Chinese, Japanese and 36 Figure 18. The i n h i b i t o r y e f f e c t of ascorbic acid and two phenolics on ;.v± mutagenicity on s t r a i n TA1535 of the n i t r i t e treated f i s h extract: ascorbic acid ( A ) ; catechin ( • ); chlorogenic acid ( • ). The concentration of sal t e d f i s h extract and n i t r i t e used were 340mg equivalent per ml and 0.8mg re s p e c t i v e l y . The values have been corrected for spontaneous reversion. The x+S.D. shown are obtained from two set of experiments with t r i p l i c a t e p l a t i n g each. Figure 19. The e f f e c t of three tea.samples on c e l l s u r v i v a l and mutagenicity of TA1535 treated with ni t r o s a t e d f i s h products i n l i q u i d :. suspension t e s t : Chinese tea ( • ) ; Japanese tea ( • ); Ceylon tea ( A ) . Plotted are x±S.D. from two experiments with t r i p l i c a t e p l a t i n g each. Values have been corrected f o r spontaneous reversion. 37 Ceylanese teas on the mutagenicity of the t e s t system. Once again, the observed decrease i n mutagenic a c t i v i t y of the n i t r o s a t i o n mixtures was not due to a t o x i c e f f e c t on the treated b a c t e r i a (upper curve of F i g . 19). Study of the Interaction of Aqueous Salted F i s h Extract with NaNO^ by a  Colorimetric Method Since n i t r i t e - t r e a t e d f i s h extract showed an enhanced mutagenic a c t i v i t y on b a c t e r i a l t e s t e r s t r a i n TA1535, there was a need to explore whether the f i s h extract reacts with the a v a i l a b l e n i t r i t e . The procedure involved mixing the sodium n i t r i t e , f i s h extract and a colour reagent together i n the presence of hydrochloric a c i d . The a c i d reacted with the sodium n i t r i t e to y i e l d nitrous a c i d (HlsK^) . This nitrous a c i d then formed a diazo compound with s u l f a n i l i c a c i d which i n turn coupled to NED to produce an azo compound with an absorption maximum at 550 nm (Fan and Tannenbaum, 1971). This s e r i e s of reactions i s ou t l i n e d i n F i g . 20. A s i g n i f i c a n t reduction i n the production of the azo compound resu l t e d when s a l t e d f i s h extract was incubated with the n i t r i t e . At the dose used f o r the mutagenicity studies (340 mg equivalent of f i s h / m l ) , 50% of the n i t r i t e content was depleted (Fig. 21). The aqueous s a l t e d f i s h extract did not i n t e r f e r e with the absorption of the coloured azo compound. Interaction of Phenolics, Teas and S a l i v a with NaNO^ by a Colorimetric Method The f i v e phenolics used a l l showed a reduction i n n i t r i t e content as the concentration of the phenolics increased. Chlorogenic a c i d reacted with the n i t r i t e the best among the f i v e phenolics tested. Catechin showed the l e a s t n i t r i t e depletion a b i l i t y . Ascorbic a c i d was included for comparison. A l l f i v e phenolics used c o n s i s t e n t l y showed a better n i t r i t e depletion capacity than ascorbic a c i d (Figs 22 and 23). 38 Figure 20: The reaction pathways of colored compound formation with NaNO (Fan and Tannenbaum, 1971). Figure 21. The n i t r i t e depletion capacity of aqueous sa l t e d f i s h extract. The i n i t i a l concentration of NaNO^ used was 0.01M i n the f i n a l mixture. % n i t r i t e remained was obtained from the absorbance of the colored azo compound formed i n the presence of f i s h extract compared with that of the o r i g i n a l concentration of n i t r i t e . The x±S.D. shown was;obtained from three experiments. 40'. HH 1 H 0 200 300 400 M G EQV OF FISH/ ML Figure 21 40a Figure 22. The n i t r i t e depletion capacity of three phenolics: g a l l i c a c i d ( A ) ; pyrogallol ( • ); tannic acid ( • ) . The i n i t i a l concentration of ni t r i t e used was O.OlmM in the finalmixture. % n i t r i t e remained was obtained from the absorbance of the color generated by the azo compound formed in the presence of the phenolics compared with that of the original concentration of n i t r i t e . The x±S.D. shown was obtained from three experiments. Figure 23. The n i t r i t e depletion capacity of ascorbic acid ( A ) ; catechin (•); and chlorogenic acid ( • ) . The i n i t i a l concentration of NaNO^  used was O.OlmM in the fi n a l mixture. % n i t r i t e remained was the absorbance of the coloured azo compound formed in the presence of the modulators compared with that of the original concentration of n i t r i t e . Plotted are x±S.D. from three experiments. 41 C n CD to to r o p-c n CD 2 M r N I T R I T E R E M A I N E D | i i % N I T R I T E R E M A I N E D Figure 24. The % n i t r i t e remained i n the presence of three tea samples: Chinese tea ( • ); Japanese tea ( •) and Ceylon tea ( A ) . The concentrations of n i t r i t e i n i t i a l l y was O.OlmM i n the f i n a l mixture. % n i t r i t e remained was the absorbance of the coloured azo compound formed i n the presence of the tea samples compared with that of the o r i g i n a l concentration of n i t r i t e without the teas. P l o t t e d are x±S.D. from three experiments. Figure 25. The n i t r i t e depletion capacity of the s a l i v a at various concentrations. The i n i t i a l concentration of NaNO^used was O.OlmM i n the mixture. % n i t r i t e remained was determined from the absorbance of the coloured azo compound formed i n the presence of the s a l i v a compared with that of the o r i g i n a l concentration of n i t r i t e without the s a l i v a . The x±S.D. shown were obtained from three experiments. 42 (D Tea infusions (Chinese, Japanese and Ceylanese), at the doses used fo r mutagenicity studies, a l l reacted with n i t r i t e . A dose response was observed. A l l the teas had s i m i l a r r e a c t i v i t y towards the n i t r i t e a v a i l a b l e . At a dose of 10 mg equivalent of tea per ml, only about 10% of the o r i g i n a l n i t r i t e remained (Fig. 24). Sa l i v a also showed a reduction i n n i t r i t e content as the concentration of s a l i v a increased. I t s e f f i c i e n c y seemed to be lower than that of phenolics and teas. However, i t i s not absolutely comparable since the concentrations were expressed d i f f e r e n t l y (Fig. 25). Under the conditions used f o r mutagenicity studies of salted f i s h extract on S. typhimurium s t r a i n TA1535, a l l these modulators reacted with more n i t r i t e than the sal t e d f i s h extract. Mutagenicity of Betel Nut Water Extract at pH 7.0 and 10.0 The preincubation t e s t with three Salmonella t e s t e r s t r a i n s (TA98, TA100 and TA102) was used f o r t h i s study. S t r a i n TA102 used used to detect i f any oxidative mutagens were generated. At pH 7.00 ± 0.01, no increase i n h i s + revertants was observed i n t e s t e r s t r a i n s TA98 and TA100 with or without S9 l i v e r microsomal mixture. There was a s l i g h t increase i n h i s + revertants on s t r a i n TA102 at 108 mg equivalent of b e t e l nut per plate i n the absence of S9. At t h i s dose, a s l i g h t reduction i n mutagenicity was observed i n the presence of S9. However, no d e f i n i t i v e conclusion could be made based on t h i s r e s u l t (Table 4). At pH 10.00 ± 0.01, a condition which was f e a s i b l e f o r oxidation of the phenolic compounds i n the water extract to generate H 2 ° 2 ' n ° i n c r e a s e i n mutagenicity was observed i n a l l t e s t e r s t r a i n s . No differ e n c e i n reversion frequency was observed between the two pH l e v e l s (Table 5). 43 TABLE 4 MUTAGENIC EFFECT OF BETEL NUT WATER EXTRACT AT pH 7.00 ± 0.05 Average Number of Revertants per Plate ± S.D. TA98 TA100 TA102 Concentration (mg/plate) -S9 +S9 -S9 +S9 -S9 +S9 108.0 29±2 27±2 95±4 92±5 303±28 254±8 86.4 27±2 28±3 94±4 94±3 282±4 26313 64.8 2713 2713 9116 9913 29318 252110 43.2 2812 3311 9415 9212 26114 254111 21.6 2914 2713 10616 9214 251120 243118 10.8 2912 2813 9416 9113 22517 222115 0 2711 2712 8913 9311 237114 23713 44 TABLE 5 MUTAGENIC EFFECT OF BETEL NUT WATER EXTRACT WITH LIME AT pH 10.00 ± 0.05 Average Number of Revertants per Plate ± S.D. Concentration (mg/plate) TA98 TA100 TA102 -S9 +S9 -S9 +S9 -S9 +S9 108.0 24±3 23±1 103±5 113±8 290+6 292+9 86.4 25±3 24±2 102±7 109±2 27817 297111 64.8 21±1 23±3 103±6 115±8 292±8 288115 43.2 23±2 23±3 105±9 98±7 282+9 30515 21.6 23±3 27±3 101±5 98±6 270112 252112 10.8 22±3 25±1 99±1 95±3 243115 240111 0 22±2 27±1 97±7 98±1 235116 234115 Lime alone 20 y l (10 mg/ml) 24±3 26±2 94±3 101±5 249110 23718 45 Mutagenicity of Betel Tannin at pH 7.0 and 10.0 No enhancement i n mutagenicity on a l l the three t e s t e r s t r a i n s , TA98, TA100 and TA102, was observed. S9 l i v e r microsomal a c t i v a t i o n showed no e f f e c t on mutagenicity. No d i f f e r e n c e i n reversion frequency was observed between pH 7.00 ± 0.01 and 10.00 ± 0.01. At a concentration of 5 mg/plate, the b e t e l tannin became tox i c to the b a c t e r i a s t r a i n s (Tables 6 and 7). Mutagenicity of Hydrogen Peroxide B a c t e r i a l t e s t e r s t r a i n TA102, which was shown to respond to by Levin et a l . (1982), was used i n t h i s study. Reagent grade ^2^2 a t a concentration as high as 100 mM showed only a doubling e f f e c t i n the mutagenicity on t h i s t e s t e r s t r a i n . Any concentration lower than 100 mM showed e i t h e r a negative e f f e c t or only a s l i g h t increase i n the number of h i s + revertants per plate (Fig. 26). Assay for H2°2 G e n e r a t e d b v Betel Nut Water Extract and Betel Tannin at Two  pH. Levels'. . Since there was no e f f e c t on mutagenicity on s t r a i n TA102 by the b e t e l nut water extract and b e t e l tannin at pH 7 and 10, a t e s t was c a r r i e d out to determine whether an oxidative product of phenolic compounds, namely, H 2 ° 2 ' W a s a c t u a H y produced. The t e s t was a c o l o r i m e t r i c analysis performed by mixing a colour reagent containing potassium iodide, ammonium molybdate and starch with the b e t e l nut water extract and b e t e l tannin. The molybdate catalyzed the formation of from potassium iodide by which reacted with starch to give a purple coloured.solution. This purple colour had a maximum absorption wavelength at 575 nm. The b e t e l nut water extract and b e t e l tannin d i d not i n t e r f e r e with the absorption at t h i s wavelength. 46 TABLE 6 MUTAGENIC EFFECT OF BETEL TANNIN AT pH 7.00 ± 0.05 Average Number of Revertants per Plate ± S.D. Concentration (mg/plate) TA98 TA100 TA102 -S9 +S9 -S9 +S9 -S9 +S9 10 11+2 12±2 69±7 71±4 90±7 12315 8 10±2 15±3 77±2 73±4 133±8 14416 5 23+3 21±3 79±6 73±8 16119 15617 4 24±1 26±2 92±5 90±6 233113 239115 2 23±3 26±3 98±7 108±4 234110 232114 1 23±3 25±4 97±5 97±10 263112 255112 0 24±3 26±2 102113 98±9 279122 258111 47 TABLE 7 MUTAGENIC EFFECT OF BETEL TANNIN WITH LIME AT pH 10. 00 1 0. 05 Average Number of Revertants per Plate 1 S.D. Concentration (mg/plate) TA98 TA100 TA102 -S9 +S9 -S9 +S9 -S9 +S9 10 T 1 T 93±2 97+2 17319 17113 8 T T 97±2 9216 17616 17715 5 21±3 19±1 10813 10419 25715 261111 4 25±1 22+3 97±4 10416 233111 255116 2 27±1 25±1 116115 110111 231110 277115 1 22±2 24±3 10012 9814 277115 251110 0 23±3 25±3 11317 103110 24614 263110 Lime alone 20 y l (10 mg/ml) 24±5 23±3 9614 9915 234110 25117 T = to x i c 48 At pH 7.00, the be t e l nut water extract produced 21.7 uM H^O^, whereas b e t e l tannin generated 36.3 uM at the doses used (Table 8). However, there was a s i g n i f i c a n t increase i n ^ 2^2 P r°d u c t ;>- o n a t P H 10.00 by the two mixtures. The b e t e l nut water extract and b e t e l tannin produced 51.2 uM and 83.7 uM of H 2 ° 2 ' r e sP e c t :'- v e ]-y• This showed that at higher pH, the oxidation process occurred more r e a d i l y and generated more U^d^. The values obtained were compared to a standard curve by adding a known amount of pure ^2^2 t 0 ^ S colour reagent (Fig. 27). Clastogenic E f f e c t of Betel Nut Water Extract at pH 7.0 and 10.0 Since the Salmonella mutagenicity assay showed a negative response to be t e l nut water extract and b e t e l tannin, and since the ^ 2°2 c o l o r i m e t r i c assay supported the idea that ^2(~>2 ^ia<^ e v°lved, another b i o l o g i c a l t e s t system was required. A study on clastogenic a c t i v i t y i n CHO c e l l s by these mixtures was c a r r i e d out. The frequencies of chromosome aberrations i n CHO c e l l s exposed f o r 3 hr to the mixtures are shown i n Table 9. At a concentration as low as 1 mg equivalent of be t e l nut per ml, there was a s i g n i f i c a n t increase of 15% metaphase plates with at l e a s t one chromatid break or exchange. Exchanges which were scored as p o s i t i v e included chromatid breaks and exchanges, mono-or m u l t i - r a d i a l s . Catalase, which changed ^2°2 tC> w a t e r anc^ oxY9en> w a s used as a possible i n d i c a t o r . At a concentration of 0.1 mg catalase/ml added concurrently to the extract, the t o x i c i t y by the betel nut water extract at 5 mg equivalent/ml was reduced. A 5% S9 microsomal a c t i v a t i o n mixture added concurrently to the be t e l nut water extract showed a reduction i n t o x i c i t y and c l a s t o g e n i c i t y on the CHO c e l l s at 1.0 to 5.0 mg equivalent/ml. 49 Figure 26. Mutagenicity of S. typhimurium (TA102) following exposure of various concentrations of (• ). The x±S.D. shown were obtained from two prer-iiicubation experiments with t r i p l i c a t e p l a t i n g each. Figure 27. Reference curve for the determination of H^O^ concentrations. Known qu a n t i t i e s of commercially- a v a i l a b l e H^O^ were assayed with J ' J . the potassium iodide-starch color reagent and p l o t t e d vs absorbance at 575nm. This assay was l i n e a r between 1 and lOOuM. Plot t e d are x±S.D. obtained from three set of experiments. 'SO 500H 200 H O C m M ] 2 2 Figure 26 (above). Figure 27 ( r i g h t ) . 50 100 H 2 Q 2 C M M ) 50a TABLE 8 AMOUNT OF H 20 2 GENERATED BY BETEL NUT WATER EXTRACT (10.8 mg/ml) AND BETEL TANNIN (1.0 mg/ml) H 20 2 ( U M ) pH = 7.00 ± 0.01 pH = 10.00 ± 0.01 21.7 ± 3.1 51.2 ± 4.8 36.3 ± 4.5 83.7 ± 7.2 Betel nut water extract Betel tannin 51 TABLE 9 CLASTOGENIC ACTIVITY OF BETEL NUT WATER EXTRACT AT pH 7.00 ± 0.05 Betel Nut Water Extract (mg equivalent/ml) Average Percent Metaphase Plates with Chromatid and/or Exchanges (±S.D.). Breaks +Catalase Wash Medium (0.1 mg/ml) +S9 (5%) 5.0 T 1 16±2 8±2 3.0 MI 2 11±1 7±2 2.0 MI 9±2 5+2 1.0 9±2 5±1 4±1 0.5 6±2 3±1 2±1 0.25 4±2 2±1 1±1 0.125 3±1 1+1 1±1 0 0 0 1±1 1 T = toxi c 2 MI = mi t o t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 52 When the pH of the be t e l nut water extract was adjusted to 10 by the addition of lime, the clastogenic a c t i v i t y was increased. Once again, 5% S9 and catalase decreased the frequency of chromosome aberrations. S9 appeared to exert a greater reduction e f f e c t . The increase i n c l a s t o g e n i c i t y at 1.0 mg equivalent/ml by r a i s i n g the pH of the be t e l nut water extract was not due to the addition of lime (Table 10). This increase may be due to more ^2°2 k e i n9" generated. Clastogenic E f f e c t of Betel Tannin and Tannic Acid at pH 7.0 and 10.0 Betel tannin at pH 7 showed a s i g n i f i c a n t increase i n chromosomal breakage at 0.031 mg/ml and became t o x i c a f t e r t h i s dose. Catalase (0.1 mg/ml) reduced t h i s e f f e c t by more than 50%, and i t also reduced the t o x i c i t y applied by the b e t e l tannin. A 5% S9 l i v e r microsomal mixture completely destroyed t h i s clastogenic e f f e c t . In addition, S9 also reduced the toxi c e f f e c t as observed i n the case of the b e t e l nut water extract (Table 11). When lime was added to the be t e l tannin s o l u t i o n to increase the a l k a l i n i t y to pH 10, both the t o x i c and clastogenic a c t i v i t y were r a i s e d compared to that at neutral pH. The frequency of chromosome breakage was increased by approximately 50% at 0.015 mg/ml. Catalase (0.1 mg/ml) and 5% S9 microsomal mixture added to the b e t e l tannin s o l u t i o n reduced both the t o x i c i t y and c l a s t o g e n i c i t y exerted by the be t e l tannin. Again, lime d i d not show any clastogenic a c t i v i t y (Table 12). Tannic a c i d was also analyzed f o r i t s clastogenic e f f e c t f o r comparison. At the same concentration, pure tannic a c i d c o n s i s t e n t l y showed a smaller frequency of chromosomal aberrations than b e t e l tannin (Table 13). When lime was added to tannic a c i d to bring the pH to 10, an enhancement of the c l a s t o -genic e f f e c t was observed (Table 14). Catalase and an S9 mixture reduced both 53 TABLE 10 CLASTOGENIC ACTIVITY OF BETEL NUT WATER EXTRACT WITH LIME AT pH 10.00 ± 0.05 Betel Nut Water Extract (mg equivalent/ml) Average Percent Metaphase Plates with Chromatid and/or Exchanges (±S.D.) Breaks +Catalase Wash Medium (0.1 mg/ml) +S9 (5%) 5.0 1 T T 9±2 3.0 T 2 MI 5±2 2.0 MI 12±2 4±1 1.0 15±1 8±1 2±1 0.5 12±1 6±1 1±1 0.25 9±2 4±1 1+1 0.125 5±1 2±1 1±1 0 1±1 0 0 Lime alone 20 u l (10 mg/ml) 1±1 0 0 T = to x i c MI = mi t o t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 54 TABLE 11 CLASTOGENIC ACTIVITY OF BETEL TANNIN AT pH 7.00 ± 0.05 Average Percent Metaphase Plates with Chromatid and/or Exchanges (±S.D.) Breaks Betel Tannin (mg/ml) Wash Medium +Catalase (0.1 mg/ml) +S9 (5%) 0.50 T 1 T 8±2 0.25 T MI 2 6±2 0.125 MI MI 3±1 0.062 MI 13±3 2±1 0.031 19±3 8±2 1±1 0.015 12±2 5±1 1±1 0.007 7±2 3±1 0 0 1±1 0 0 T = t o x i c i 'MI = m i t o t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 55 TABLE 12 CLASTOGENIC ACTIVITY OF BETEL TANNIN WITH LIME AT pH 10.00 ± 0.05 Average Percent Metaphase Plates with Chromatid Breaks and/or Exchanges (±S.D.) Betel Tannin (mg/ml) Wash Medium +Catalase (0.1 mg/ml) +S9 (5%) 0.50 T 1 T 15±3 0.25 T T 10±2 0.125 T MI 2 7±2 0.062 T 9±3 4±1 0.031 MI 6±2 2±1 0.015 20±2 4±2 1±1 0.007 14±2 2±1 0 0 0 1±1 1±1 Lime alone 20 y l (10 mg/ml) 1±1 0 2±1 T = toxi c > MI = mi t o t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 56 TABLE 13 CLASTOGENIC ACTIVITY OF TANNIC ACID AT pH 7.00 ± 0.05 Average Percent Metaphase Plates with Chromatid Breaks and/or Exchanges (±S.D.) Tannic Acid (mg/ml) Wash Medium +Catalase (0.1 mg/ml) +S9 (5%) 0.25 T 1 MI 2 13±2 0.125 MI 12±2 8±1 0.062 16±1 9±1 5±2 0.031 12±1 5±1 2+1 0.015 7±2 3±1 1±1 T = t o x i c i 'MI = mito t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 57 TABLE 14 CLASTOGENIC ACTIVITY OF TANNIC ACID WITH LIME AT pH 10.00 ± 0 . 0 5 Tannic Acid (mg/ml) Average Percent Metaphase Plates with Chromatid Breaks and/or Exchanges (±S.D.) Wash Medium +Catalase (0.1 mg/ml) +S9 (5%) 0.25 1 T 2 MI 14±2 0.125 MI 12±2 9±2 0.062 25/MI 6±1 4±1 0.031 18±3 4±1 2±1 0.015 10±2 2±1 1±1 T = toxi c • i 'MI = mi t o t i c i n h i b i t i o n : fewer than 40 d i p l o i d metaphases per plate observed. 58 the to x i c and clastogenic e f f e c t at both pH l e v e l s . Again, 5% S9 exhibited a greater e f f e c t than catalase at 0.1 mg/ml on reduction of the to x i c and clastogenic e f f e c t s . 59 DISCUSSION Food and Cancer Epidemiological investigations have implicated several aspects of d i e t and l i f e s t y l e i n the et i o l o g y of cancer (Table 15). Peers et a l . (1976) have shown a l i n e a r r e l a t i o n s h i p between the dietary l e v e l s of the hepato-carcinogenic a f l a t o x i n s and primary l i v e r cancer i n c e r t a i n areas of A f r i c a . Nitrosamines (Issenberg, 1976) and saccharin (Reuber, 1978) are other chemical components which may be important contributors to chemical carcino-genesis. However, an increasing number and d i v e r s i t y of compounds with the capacity to i n h i b i t the occurrence of neoplasia when administered p r i o r to or simultaneously with exposure to cancer-causing agents are being i d e n t i f i e d . For example, butylated hydroxyanisole (BHA), an antioxidant u t i l i z e d to increase s h e l f - l i f e , prevents the induction of experimental tumours i n rodents (Wattenberg, 1975). Other n a t u r a l l y occurring non-nutrient constituents of foods have been implicated i n modifying c a r c i n o g e n i c i t y . Cruciferous vegetables such as Brussel sprouts, cabbage and c a u l i f l o w e r , contain indoles which have been shown to increase the metabolism of carcinogenic p o l y c y c l i c aromatic hydro-carbons (Pantuck et a l . , 1976; Wattenberg and Loub, 1978). I t i s generally believed that most chemical carcinogens are compounds which contain highly reactive e l e c t r o n - d e f i c i e n t regions. In addition, chemical carcinogens can also induce the mixed function oxidases to a c t i v a t e the carcinogens into e l e c t r o p h i l i c species (Wattenberg et a l . , 1976). They can 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, RNA and proteins i n target t i s s u e s ( M i l l e r and M i l l e r , 1977). 60 TABLE 15 PROPORTION OF CANCER CASES ATTRIBUTED TO VARIOUS DIFFERENT FACTORS BY DIFFERENT AUTHORS Percent of a l l Cancer Cases i n : Factor or Class of Factors England, Birmingham Region, Based on Higginson and Muir (1979) United States, Based on Wynder and Gori (1977)* Male Female Male Female Tobacco 30 7 28 8 Tobacco/alcohol 5 3 4 1 Diet - - 40 57 L i f e s t y l e 30 63 - -Occupation 6 2 4 2 Sunlight Ionizing r a d i a t i o n 10 1 10 1 8 8 Iatrogenic 1 1 - -Exogenous hormones - - - 4 Congenital Unknown 2 15 2 11 16 20 *Deduced from histograms; non-environmental factors equated with congenital and unknown. From D o l l and Peto (1981). 61 Plant Phenolics and Their Role i n Human Carcinogenesis Plant phenolics constitute a major portion of the components consumed i n man's d a i l y d i e t . The estimated range i s between 600 mg and several grams per day (Maga, 1978). Their b i o l o g i c a l a c t i v i t y i s therefore of some i n t e r e s t i n determining the r o l e which they may play i n human carcinogenesis. Many deleterious e f f e c t s of plant phenolics have been reported. A composite l i s t of genotoxic events caused by phenolics under a v a r i e t y of t e s t conditions i s shown i n Table 16 (Hanham, 1983). However, c o n f l i c t i n g reports have been published on t h e i r r o l e i n carcinogenesis. Extensive studies on phenolic compounds have been c a r r i e d out f o r t h e i r i n h i b i t o r y e f f e c t s on the t o x i c and carcinogenic actions of a wide v a r i e t y of chemical carcinogens (Wattenberg, 1972, 1979). In p a r t i c u l a r , three n a t u r a l l y occurring phenolic d e r i v a t i v e s of cinnamic a c i d (o-hydroxy-cinnamic a c i d , c a f f e i c a c i d and f e r u l i c acid) were e f f e c t i v e i n suppressing B(a)P-induced neoplasia i n the forestomach of r a t s (Wattenberg et a l . , 1980). S t i c h and Rosin (1984) have also reported that phenolic d e r i v a t i v e s i n h i b i t the mutagenic a c t i v i t i e s of both d i r e c t - a c t i n g carcinogens and pre-carcinogens i n the presence of mixed function oxidases. The report also goes on to demonstrate that they are able to reduce the in vitro formation of mutagenic and carcinogenic n i t r o s o compounds. In man, phenolics were observed to reduce l e v e l s of n i t r o s o p r o l i n e i n urine following administration of n i t r a t e , p r o l i n e and phenolic t e s t substances. The in vitro n i t r o s a t i o n of p r o l i n e i n the presence of phenolics used i n t h i s study, as demonstrated by HPLC a n a l y s i s , also shows a c o r r e l a t i o n with t h i s observation (Figs 5-7). To further complicate the issue of phenolics and t h e i r r o l e in human carcinogenesis, other i n v e s t i g a t o r s have suggested that some phenolic compounds 62 TABLE 16 GENOTOXIC EFFECTS MEASURED WITH PLANT PHENOLICS PHENOLIC GROUP ASSAY ORGANISM REFERENCE Simple Phenols: Phenol Catechol P y r o g a l l o l Resorcinol Benzoic Acids: G a l l i c a c i d Protocatechuic a c i d V a n i l l i c a c i d Cinnamic Acids: C a f f e i c a c i d Chlorogenic a c i d S i s t e r chromatid exchange Point mutation Mi t o t i c crossover S i s t e r chromatid exchange Nephrotoxicity Chromosome aberrations Point mutation Point mutation Micronuclei Chromosome aberrations DNA i n h i b i t i o n Micronuclei Point mutation Chromosome aberrations Chromosome aberrations Chromosome aberrations Gene conversion Chromosome aberrations Gene conversion Chromosome aberrations Human lymphocytes S_. c e r e v i s i a e S^ . c e r e v i s i a e Human lymphocytes Rats CHO c e l l s _S. typhimurium E. c o l i Mice CHO c e l l s Mice Mice S_. typhimurium CHO c e l l s CHO c e l l s CHO c e l l s _S. c e r e v i s i a e CHO c e l l s _S. c e r e v i s i a e  _ CHO c e l l s Morimoto and Wolff (1980) Kunz et a l . (1980) Kunz et a l . (1980) Morimoto and Wolff (1980) Calder et a l . (1975) S t i c h et a l . (1981a) Ben-Gurion (1979) Yamaguchi (1981) B i l i m o r i a (1975) Mitra and Manna (1977) Gocke et a l . (1981) S t i c h et a l . (1981a) S e i l e r (1977) Mitra and Manna (1977) Yamaguchi (1981) S t i c h et a l . (1981a) S t i c h et a l . (1981a) St i c h et a l . (1981a) St i c h and Powrie (1982) St i c h et a l . (1981a) S t i c h et a l . (1981a) S t i c h et a l . (1981b) From Hanham (1983). such as r e s o r c i n o l , p-nitrosophenol, catechin ( P i g n a t e l l i et a l . , 1982) and p h l o r o g l u c i n o l (Walker et a l . , 1982) , showed a c a t a l y t i c e f f e c t rather than an i n h i b i t o r y e f f e c t . P i g n a t e l l i et a l . (1982) also indicated that phenolic compounds with r e s o r c i n o l moiety, which can r e a d i l y form C-nitroso d e r i v a t i v e s by reaction with n i t r i t e , can catalyze the formation of n i t r o s o compounds. This may explain the observed c a t a l y t i c a c t i v i t i e s of 2,4- and 3,5-dihydroxybenzoic a c i d on the formation of n i t r o s o p r o l i n e , molecules which carry r e s o r c i n o l moiety (Fig. 9). Possible Mechanism of I n h i b i t i o n and C a t a l y t i c E f f e c t by Phenolics on  N i t r o s a t i o n Reactions Those phenols which consume n i t r i t e , e i t h e r by the formation of C-nitroso phenols or by n i t r i t e reduction to NO, coupled with oxidation of the phenol to guinones, act as scavengers; they may therefore i n h i b i t n i t r o s a t i o n by reducing the n i t r o s a t i n g agent to n i t r i c oxide. A possible mechanism for the c a t a l y s i s of phenolic compounds on n i t r o -sation has been proposed by Walker et a l . (1982). During an i n i t i a l step, C-nitroso d e r i v a t i v e s may be formed which further react with the n i t r o s a t i n g species to generate a more powerful n i t r o s a t i n g agent (thought to be a nitrosoquinone oxime d e r i v a t i v e ) . This postulated mechanism implies that assay systems containing increasing amounts of a c a t a l y t i c a l l y active phenolic compound lead to increasing concentrations of the C-nitroso intermediate, and consequently to a reduced concentration of the n i t r o s a t i n g species. This mechanism would explain the optimum r a t i o f o r n i t r i t e to phenolic compound, as shown i n F i g . 9. Accordingly, a large excess of phenolic compound would i n h i b i t n i t r o s a t i o n since the n i t r o s a t i n g agent would be completely used up i n forming the c a t a l y s t and there would be l e f t none to continue the c a t a l y t i c reaction (Fig. 11). 64 How are these two phenomena - i n h i b i t o r y e f f e c t and c a t a l y t i c e f f e c t -applied to a p h y s i o l o g i c a l situation? The average concentration of n i t r i t e found i n human s a l i v a i s 0.097 mM (Walters et a l . , 1979) and, as mentioned above, the amount of phenolics consumed by humans i s between 600 mg and several grams per day. Thus, p h y s i o l o g i c a l l y , i t would seem that there would not be enough n i t r i t e to continue the c a t a l y t i c reaction. Hence the c a t a l y t i c e f f e c t of n i t r o s a t i o n by phenolic compounds i s chemically f e a s i b l e . However, p h y s i o l o g i c a l l y , i t i s only probable, but nonetheless an unreasonable proposition. Relation of Salted F i s h to Human Carcinogenesis Salted f i s h has long been thought to be involved i n human cancers (Ho, 1971; Ho et a l . , 1978; Huang et a l . , 1978; Yang, 1980). I t has been shown that the urine excreted from humans eating s a l t e d / d r i e d Hawaiian f i s h contains mutagens which can be detected by HPLC and the Ames Salmonella mutagenicity assay (D. Ichinotsubo and H.F. Mower, personal communication). There are also reports which i n d i c a t e that there are more than one type of n i t r o s o compound i n s a l t e d f i s h . Huang et a l . (1978) reported that mutagenic a c t i v i t i e s were observed i n both TA98 and TA100, and that S9 mix greatly enhanced i t s mutagenic a c t i v i t y . They proposed that these mutagens were a type of nitrosamine. Other reports (Marquardt et a l . , 1977; Yano, 1981) indicate that the mutagens i n s a l t e d f i s h were d i r e c t - a c t i n g n i t r o s o compounds, probably nitrosamides. In t h i s study, the r e s u l t s i n d i c a t e that the mutagenic components i n s a l t e d f i s h extract are water-soluble and d i r e c t - a c t i n g compounds (Figs 12 and 13). In addition, there are components present which can be converted to mutagenic n i t r o s o compounds with the addition of n i t r i t e (Fig. 14; Table 1). 65 However, the actual chemical nature of the mutagenic f r a c t i o n s w i l l have to l be determined by HPLC and/or gas chromatography combined with a mass spectrometer. N i t r o s a t i o n Reactions Modulated i n a Complex Mixture There has always been a concern as to whether or not the above chemical studies on p u r i f i e d compounds can be used to p r e d i c t how n i t r o s a t i o n reactions are modulated i n a complex mixture such as food products. Attempts were therefore made to simulate in vitro those conditions which may a c t u a l l y occur within the en t i r e organism. Such approaches may bridge the gap between epidemiological evidence pointing to a l i n k between the consumption of food products and an elevated r i s k f o r g a s t r i c cancer and lead to an understanding of the chemistry of n i t r o s a t i o n reactions. In t h i s study, we t r i e d to simulate the events which may occur during the consumption of a meal c o n s i s t i n g of s a l t e d f i s h , teas and plant phenolics which are present i n vegetables. An aqueous f r a c t i o n of a Chinese sa l t e d f i s h was nitrosated i n the absence or presence of several n a t u r a l l y occurring plant phenolics, teas and human s a l i v a . Catechin, chlorogenic a c i d , g a l l i c a c i d , p y r o g a l l o l and tannic a c i d , when added to the n i t r o s a t i o n mixtures, exerted a strong i n h i b i t o r y e f f e c t on the formation of d i r e c t - a c t i n g mutagens which were detected by the Ames Salmonella assay. These r e s u l t s obtained on the complex mixture of a f i s h extract are i n good agreement with the observation that these phenolics i n h i b i t NPRO formation, and i n other simple model systems such as the mutagenicity r e s u l t i n g from in vitro n i t r o s a t i o n of methylurea (Stich et a l . , 1982c). A r e l a t i v e l y strong i n h i b i t o r y e f f e c t was also observed with the Chinese, Japanese and Ceylanese teas at concentrations which are a c t u a l l y ingested. 66 These observations provide the basis that i n h i b i t i o n of n i t r o s a t i o n by n a t u r a l l y occurring phenolics and teas on simple p u r i f i e d compounds can also occur i n complex food mixtures in vitro. Hence i t provides an i n d i c a t i o n as to what may occur i n vivo. In order to prove that phenolics do compete with the amines for the n i t r o s a t i n g agents, the n i t r i t e depletion assay was c a r r i e d out. I t was observed that a l l phenolics, teas and s a l i v a depleted the n i t r i t e a v a i l a b l e (Figs 22-25). In a d d i t i o n , s a l t e d f i s h aqueous extract also reacted with n i t r i t e to generate the azo compound (Fig. 21). Thus the i n h i b i t i o n of n i t r o s a t i o n product formation by phenolics, teas and s a l i v a i s due to t h e i r a b i l i t y to compete with s a l t e d f i s h extract and p r o l i n e f or the a v a i l a b l e n i t r i t e . Relation of Betel Nut Chewing to Human Carcinogenesis Betel quid chewing has been a common pr a c t i c e f o r a long time i n Southeast Asia, Central Asia and countries of the Western P a c i f i c . The high incidence of o r a l cancer i n these areas i s reported to be associated with the habit of b e t e l quid chewing (Muir and Kirk, 1960; Jussawalla, 1976). In addition, b e t e l quid induces neoplasms i n experimental animals (Bhide et a l . , 1979; Ranadive et a l . , 1979). There are also reports on the clastogenic a c t i v i t y of the s a l i v a of b e t e l quid chewers (Stich and S t i c h , 1982; S t i c h et a l . , 1983a), i n that i t elevated the frequency of micronucleated c e l l s i n the buccal mucosa and caused chromosome breakage i n CHO c e l l s . Shivapurkar et a l . (1980) reported that the n i t r i t e and nitrosamine l e v e l s i n the s a l i v a of b e t e l nut chewers were also elevated. However, b e t e l nut extract has also been shown to i n h i b i t the endogenous n i t r o s a t i o n i n humans (Stich et a l . , 1983b). Thus b e t e l nut chewing i n regard to human carcinogenesis s t i l l remains an unresolved issue. In t h i s study, b e t e l nut water extract and betel tannin (one of the major phenolic components of b e t e l nut) showed a dose-related increase i n the clastogenic a c t i v i t y on CHO c e l l s (Tables 9 and 11). These results:' c o r r e l a t e with the data on the s a l i v a of b e t e l quid chewers as shown by S t i c h and S t i c h (1982). I t i s of i n t e r e s t to note that the incidence of o r a l cancer i s greatly increased i n chewers of lime-containing mixtures which cause an a l k a l i n e s a l i v a (H.F. S t i c h , personal communication). The s a l i v a of b e t e l nut chewers contains r e l a t i v e l y large q u a n t i t i e s of phenolics. Lime added to the chewing mixture enhanced oxidation which gave r i s e to ^2°2 an<^ ^ r e e r a < 3 i c a l s (Hanham et a l . , 1983). The generation of H 2 ° 2 ' superoxide, hydroxyl and semiquinone r a d i c a l s concomitantly enhanced the c l a s t o g e n i c i t y of the phenolics. The data on the clastogenic e f f e c t of b e t e l nut water extract and b e t e l tannin i n the presence of lime supported t h i s proposition (Tables 10 and 12). An assay f o r H^O^ also showed that at a l k a l i n e pH l e v e l s more H^O^ was generated from b e t e l nut water extract and b e t e l tannin (Table 8). Salmonella mutagenicity t e s t s on t e s t e r s t r a i n s TA98, TA100 and TA102 were not p o s i t i v e . This was probably because these s t r a i n s were not s e n s i t i v e to the oxidative mutagens at the concentration used. The amount of H^O^ required f o r TA102 to be p o s i t i v e i s much higher than the amount generated by both b e t e l nut water extract and b e t e l tannin (Table 8; F i g . 26) . S a l i v a and i t s Role i n Carcinogenesis During the consumption of a meal, chemicals consumed through food and beverages w i l l become mixed with s a l i v a . This mixing can lead to a reduction or enhancement of mutagenic dr carcinogenic a c t i v i t y of the ingested mutagens and carcinogens. S a l i v a has proven to be an a c t i v e i n h i b i t o r of the mutagenicity 68 of several man-made (Nishioka et a l . , 1981) and n a t u r a l l y occurring (Stich and Rosin, 1984) carcinogens. The question was therefore r a i s e d as to the e f f e c t of s a l i v a on the formation of mutagenic compounds. The presence of n i t r i t e i n human s a l i v a i s well-known (Savostianov, 1937). The n i t r i t e appears to be the product of microbial reduction of n i t r a t e , which c i r c u l a t e s through the s a l i v a r y glands (Tannenbaum et a l . , 1974). I t i s the presence of n i t r i t e i n human s a l i v a which causes nitrosamine formation i n human s a l i v a (Tannenbaum et a l . , 1974, 1978; Spiegelhalder et a l . , 1976). In addition, Shivapurkar et a l . (1980) showed that t h i s increased n i t r i t e l e v e l may be r e l a t e d to the development of o r a l cancer i n India. However, i n t h i s study, s a l i v a exerted a r e l a t i v e l y strong suppressive e f f e c t on the appearance of mutagenic n i t r o s a t i o n products when present during n i t r o s a t i o n of the f i s h extract and NPRO formation (Figs 8 and 16). This observation was i n good c o r r e l a t i o n with the study on model n i t r o s a t i o n reactions i n the presence of s a l i v a by S t i c h et a l . (1982b). The complex composition of s a l i v a w i l l make i t d i f f i c u l t to i d e n t i f y those chemicals which can reduce n i t r o s a t i o n by i n t e r a c t i n g with n i t r i t e . Moreover, whole s a l i v a i s not only a so l u t i o n of chemicals, but contains a wide array of b a c t e r i a , e p i t h e l i a l c e l l s and various blood c e l l s which can influence the rate of formation of nitrosamines (Tannenbaum et a l . , 1978). The question must be r a i s e d as to the amounts of mutagens and carcinogens which would saturate the i n h i b i t o r y capacity of s a l i v a . The chewing of betel nut releases numerous chemicals into the s a l i v a . I t appears l i k e l y that saliva-borne mutagens and carcinogens can then a f f e c t the mucosal tissues of the o r a l c a v i t y , pharnyx and, on swallowing, the mucosa of the esophagus. S t i c h and S t i c h (1982) demonstrated the appearance of potent clastogenic, mutagenic and convertogenic agents i n the s a l i v a of volunteers who chewed b e t e l 69 nuts for a few minutes. Despite the intensive s a l i v a t i o n due to chewing of the b e t e l nuts, the s a l i v a of chewers was not able to suppress the a c t i v i t i e s of the released genotoxic agents. A comparable suggestion was made by Tannenbaum et a l . (1981) who suggested that the depletion of the p r o t e c t i v e a n t i n i t r o s a t i o n f a c t ors of the g a s t r i c j u i c e was an e s s e n t i a l condition f o r high cancer r i s k . B i o l o g i c a l Protective Mechanism Organisms have obviously evolved mechanisms to protect themselves against the genotoxic e f f e c t s of oxidation by-products, phenolics and nitrosamines. In f a c t , an early explanation for the d i f f e r e n t oxygen tolerances of aerobes and obligate anaerobes was based on ^2^2 t o ± e r a n c e s • Thus aerobes were thought to contain catalase as a defence mechanism against E^O^, whereas anaerobes lacked t h i s enzyme and were subsequently k i l l e d by ^2°2 w h e n e x P o s e < ^ to oxygen l e v e l s present i n a i r . Catalases and peroxidases catalyze the conversion of ^2^2 t 0 H 2 ° a n c ^ °2" These enzymes catalyze the d i v a l e n t reduction of H2°2 t 0 2 H 2 ° us-'-n9 H2°2 a s the electron donor i n the case of catalases, or a v a r i e t y of reductants i n the case of peroxidases (Fridovich, 1976) . Commercially prepared catalase was observed to eliminate the clastogenic a c t i v i t y i n the solutions of b e t e l tannin and b e t e l nut water extract (Tables 9-12) . Whether catalase had indeed removed a l l ^2^2 w a s ver^^-e^ with the potassium iodide-starch c o l o r i m e t r i c assay. The clastogenic a c t i v i t y of commercial H 2 ° 2 ~ t r e a t e ( ^ s o l u t i o n was also eliminated by catalase (Hanham, 1983). A s i m i l a r degree of a c t i v i t y was observed i n the b e t e l nut water extract and b e t e l tannin. This would permit one to i n f e r that some portion of the c l a s t o -genic a c t i v i t i e s of the b e t e l nut water extract and b e t e l tannin (treated with or without lime) was due to the presence of hydrogen peroxide. 70 There are enzymes which can metabolize or detoxify the mutagens or carcinogens. For example, mixed function oxidases are a c l a s s of enzymes involved i n the biotransformation and oxidative a c t i v a t i o n of chemical carcinogens and other exogenous compounds (Autrup, 1982). These enzymes are l o c a l i z e d p r i m a r i l y i n the microsomal membrane and contain one or more of the various forms of cytochrome P-450 and the associated electron transport enzymes such as NADPH cytochrome P-450 reductase, cytochrome b r and NADH b cytochrome b^ reductase. S9 mix i s one type of these mixed function oxidases which have been shown to enhance the clastogenic a c t i v i t y of several phenolic compounds such as v a n i l l i c a c i d , p-coumaric a c i d and p-hydroxybenzoic acid; S9 mix also shows no e f f e c t on others such as r e s o r c i n o l and p y r o g a l l o l (Stich et a l . , 1981b). However, S9 shows a decrease i n clastogenic a c t i v i t y of other phenolics such as catechol, g a l l i c a c i d (Stich et a l . , 1981b), d r i e d f r u i t s (Stich et a l . , 1981c) and caramel (Stich et a l . , 1981d). In t h i s study, S9 showed no e f f e c t on the mutagenic and clastogenic a c t i v i t i e s of s a l t e d f i s h aqueous extract (Figs 12, 13, 14; Table 1). However, when S9 was added to the b e t e l nut water extract and b e t e l tannin mixture, i t exerted a great reduction i n clastogenic and t o x i c a c t i v i t i e s of the b e t e l nut water extract and b e t e l tannin (Tables 9-12). These observations again point to the f a c t that i t i s very d i f f i c u l t to define the genotoxicity of a complex mixture such as food. Unresolved Issues 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 and l i f e s t y l e f a c t o rs such as smoking. Numerous epidemiological data have supported the idea that smoking i s r e l a t e d to human cancer (Doll and Peto, 1981; World Health Organization, 71 1975). The composition of food, upon entering man, w i l l become continuously changed. The p o s s i b l e i n t e r a c t i o n s which can occur between the various components of food among themselves as well as with c e l l u l a r substances are inumerable. Some of these i n t e r a c t i o n s may contribute to an enhancement, while others lead to the suppression of the net genotoxic a c t i v i t y of the substances involved. With regard to genotoxicity and c a r c i n o g e n i c i t y , phenolics can exert a spectrum of apparently contradictory e f f e c t s . They can be clastogenic (Stich et a l . , 1981b) and mutagenic (Brown, 1980). S i m i l a r l y , tea infusions were found to have a r e l a t i v e l y strong genotoxic e f f e c t (Nagao et a l . , 1979). This genotoxic a c t i v i t y of phenolics and teas could i n d i c a t e p o t e n t i a l carcinogenic properties. On the other hand, phenolics have the capacity to trap n i t r o s a t i n g species (Mirvish et a l . , 1978), which r e s u l t s i n a reduced n i t r o s a t i o n of amines and amides i n vitro ( P i g n a t e l l i et a l . , 1976; Walker et a l . , 1975) and in vivo (Stich et a l . , 1983b). Does epidemiological evidence support such a conclusion? There were high mortality rates due to esophageal carcinomas i n Japanese population groups consuming large q u a n t i t i e s of tea gruel (Hirayama, 1979), among chewers of phenolic-containing b e t e l nuts (Jussawalla, 1976) , and among matte'-drinking i n d i v i d u a l s i n Argentina and B r a z i l (Prudente, 1963). However, the mortality rates for stomach cancer are r e l a t i v e l y low i n groups with a high incidence of esophageal cancers, e.g., tea gruel-consuming Japanese (Segi, 1975) and b e t e l quid-chewing G u j r a t i s (Jussawalla, 1976). There have also been many arguments on whether d i e t and l i f e s t y l e f a c t o rs are the cause of cancer. Epstein and Swartz (1981) argued that cancers are caused by environmental and occupational exposure to cancer-causing agents. They claim that the r o l e of l i f e s t y l e f a c t o rs has been exaggerated by those with 72 an economic or i n t e l l e c t u a l investment i n t h i s theory, l a r g e l y by excluding involuntary exposures to carcinogens and minimizing the r o l e of occupational carcinogens. They supported t h e i r point of view by the r e l a t i v e r i s k s of cancer of the bladder and pancreas which were v a r i o u s l y characterized as r e l a t e d to or caused by smoking (Cole et a l . , 1971; Wynder et a l . , 1973). However, the r e l a t i v e r i s k s f or these cancers were s e v e r a l f o l d l e s s i n smokers than i n non-smokers. In addition, excess bladder cancer rates had been i d e n t i f i e d i n several occupational categories, in c l u d i n g rubber, paint manufacturing and t e x t i l e - d y e i n g workers (Cole and Goldman, 1975). They also claimed that from an epidemiological standpoint, a c o r r e l a t i o n between a high f a t d i e t and breast and colon cancer was weak since there were populations with'.a high f a t intake and l i t t l e bowel cancer (Berg, 1975). To further complicate the issue, Cairns (1981) suggested that the o r i g i n of human cancers i s not from conventional mutagens but that they are more l i k e l y to be the r e s u l t of genetic t r a n s p o s i t i o n s . He claims that the issue i s not simply whether conventional mutagens are capable of causing cancer, but whether they reach our c e l l s i n s u f f i c i e n t quantity to make a major contribution to our present national incidence of cancer. I t i s a question of the balance between the dose of mutagens and the e f f i c i e n c y of our various pathways f o r DNA r e p a i r . Cancer i s a disease of m u l t i f a c t o r i a l e tiology. There i s no doubt that l i f e s t y l e and d i e t a r y factors are involved, but these two factors are c e r t a i n l y not the only cause of cancer. Other factors such as occupational, genetic and environmental f a c t o r s i n r e l a t i o n to cancer cannot be eliminated. Based on epidemiological data that most cancers are caused by l i f e s t y l e , d i e t , occupation and the environment, cancer would thus seem to be preventable. 73 Following the awareness of the health hazards of a d e t e r i o r a t i n g external environment (Higginson and Muir, 1973; Wynder and Gori, 1977) , a further awareness i s currently developing that the part of the man-made environment which we casually place i n t o our mouths and swallow has more far-reaching health consequences than previously believed. 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Gann 12.' 451-454 (1981). 83 APPENDIX I EXTRACTION PROCEDURE OF SALTED FISH Rinsed with d i s t i l l e d water V Freeze-dried Extracted 3x with hexane Centrifuged Salted f i s h Hexane evaporated Residue a i r - d r i e d Extracted 3x with methanol Residue reconstituted i n d i s t i l l e d water Methanol evaporated 84 APPENDIX II NITROSATION OF SALTED FISH AQUEOUS EXTRACT 10 ml aqueous extract 10 ml NaNO 2(3.0xl0~ 2M f i n a l ) pH adjusted to 2 with HC1 Incubated f o r 1 hr at 37°C V pH adjusted to 7 with NaOH Mutagenicity and c l a s t o g e n i c i t y tested 3-5 APPENDIX III EXTRACTION PROCEDURE OF BETEL NUT Betel nut Crushed into powder \1/ Extracted 2x with hexane V Hexane removed by a i r - d r y i n g , residue extracted 2x with d i s t i l l e d water heated i n water-bath V F i l t e r e d through Whatman q u a l i t a t i v e 1 f i l t e r paper (water extract) Residue extracted 3x with n-butanol n-Butanol evaporated V Dissolved i n d i s t i l l e d water, 1.5% ca f f e i n e added to p r e c i p i t a t e tannin 86 APPENDIX IV Resorcinol P y r o g a l l o l G a l l i c a c i d O H O H C O O H Ascorbic acid Benzoic acid 87 

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