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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 U n i v e r s i t y o f 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  in THE FACULTY OF GRADUATE  STUDIES  Department o f Zoology  We a c c e p t 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 K a - L i n Chan, 1984  In p r e s e n t i n g  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  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f 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  department o r by h i s or her  granted by  the head o f  representatives.  my  It is  understood t h a t 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  permission.  Department o f The U n i v e r s i t y o f B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6  (3/81)  Columbia  written  ABSTRACT  There i s e p i d e m i o l o g i c a l e v i d e n c e d e m o n s t r a t i n g t h e involvement o f dietary  and l i f e s t y l e  human c a n c e r s . the  factors  The o b j e c t i v e s  o f t h i s study were t o s i m u l a t e in  vitro  g e n o t o x i c e f f e c t s o f v a r i o u s c h e m i c a l s i n t r o d u c e d i n t o t h e body through  d i e t and l i f e s t y l e h a b i t s , carcinogenesis.  and t o i n v e s t i g a t e  t h e i r r e l a t i o n s h i p t o human  Three model systems were used i n t h i s study:  formation o f N-nitrosoproline and  i n t h e development o f a l a r g e p r o p o r t i o n o f  (NPRO), (2) Chinese s a l t e d f i s h  (1) in extract,  (3) b e t e l n u t e x t r a c t s . N i t r o s a m i n e s have l o n g been s u s p e c t e d o f b e i n g r e l a t e d  to the  development o f nasopharyngeal, esophageal, stomach and u r i n a r y carcinomas.  bladder  The f o r m a t i o n o f NPRO was used t o examine t h e e f f e c t o f p l a n t  p h e n o l i c s , major components o f t h e human d i e t , on t h e n i t r o s a t i o n The  vitro  t e s t system c o n s i s t e d  of nitrosating  o r w i t h o u t t h e p h e n o l i c s t o be used. acid, pyrogallol  and t a n n i c  (pH 2, 1 h r , 37°C) p r o l i n e  Catechin, chlorogenic acid,  a c i d suppressed t h e f o r m a t i o n o f NPRO.  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 of ascorbic  acid.  This indicated  a c t as i n h i b i t o r s w h i l e o t h e r s may a c t as c a t a l y s t s  studied.  gallic Their  o f phenolics with a  r e s o r c i n o l moiety which enhanced t h e n i t r o s a t i o n r e a c t i o n  reaction.  with  were comparable t o t h a t  However, t h e r e was another f a m i l y  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 .  reactions.  that  at a certain some p h e n o l i c s may  i n the n i t r o s a t i o n  S e v e r a l t e a s , which a r e p h e n o l i c - c o n t a i n i n g beverages, were a l s o A C h i n e s e , Japanese and Ceylanese t e a a l l i n h i b i t e d t h e f o r m a t i o n  of NPRO a t doses which a r e n o r m a l l y consumed by man.  ii  Chinese  s a l t e d f i s h was used as a model t o demonstrate t h a t d i e t may-  p l a y a r o l e i n human c a n c e r s . i n v o l v e d i n human c a n c e r s .  S a l t e d f i s h has l o n g been thought  Employing the Ames S a l m o n e l l a  t o be  mutagenicity  t e s t system, i t was found t h a t the mutagenic components i n t h e s a l t e d e x t r a c t were d i r e c t - a c t i n g and w a t e r - s o l u b l e .  fish  Moreover, t h e r e were  components p r e s e n t which c o u l d be c o n v e r t e d t o mutagenic n i t r o s o compounds when n i t r i t e was added. ( s t r a i n TA1535).  T h i s was d e t e c t e d by Salmonella  typhimurium  Hence t h e i n h i b i t o r y e f f e c t o f p h e n o l i c s and p h e n o l i c -  c o n t a i n i n g beverages on t h e f o r m a t i o n o f mutagenic n i t r o s a t i o n was s t u d i e d .  The t e s t system c o n s i s t e d o f n i t r o s a t i n g  an aqueous f r a c t i o n o f a s a l t e d f i s h  suspension mixture.  test.  (pH 2, 1 h r , 37°C)  (Pak Wik) w i t h o r w i t h o u t t h e  i n h i b i t o r s t o be t e s t e d and e s t i m a t i n g t h e frequency per s u r v i v o r s o f S. typhimurium  products  of h i s  +  revertants  ( s t r a i n TA1535) by a p p l y i n g the l i q u i d  The p h e n o l i c s and t e a s were added t o t h e n i t r o s a t i o n  Again, the phenolics t e s t e d (catechin, chlorogenic a c i d ,  gallic  a c i d , p y r o g a l l o l and t a n n i c a c i d ) and t h r e e t e a samples  (Chinese,  and C e y l a n e s e ) , a t doses consumed by man, a l l p r e v e n t e d  t h e f o r m a t i o n of  mutagenic n i t r o s a t e d f i s h  Japanese  products.  D u r i n g t h e course o f a meal t h e f i s h p r o d u c t s , n i t r i t e and n i t r i t e t r a p p i n g agents w i l l mix w i t h s a l i v a .  T h e r e f o r e t h e e f f e c t o f s a l i v a on  the f o r m a t i o n o f mutagenic N - n i t r o s o compounds was a l s o s t u d i e d .  Saliva  e x e r t e d an i n h i b i t o r y e f f e c t on the f o r m a t i o n o f NPRO and mutagenic nitrosated f i s h extract. The  n i t r i t e d e p l e t i o n assay was c a r r i e d o u t t o determine i f the  modulators r e a c t w i t h n i t r i t e .  P h e n o l i c s , t e a s and s a l i v a a l l r e a c t e d w i t h  the n i t r i t e p r e s e n t , i n d i c a t i n g t h a t they competed w i t h t h e n i t r o s a t a b l e agents f o r t h e a v a i l a b l e  nitrites.  B e t e l nut was may  a l s o be  used as an example to demonstrate t h a t l i f e s t y l e  i n v o l v e d i n the development of c a n c e r .  been r e l a t e d t o the development o f o r a l and water e x t r a c t and b e t e l t a n n i n demonstrate any TA100 and  mutagenic e f f e c t on S.  TA102, w i t h o r w i t h o u t S9  (CHO)  ( c l a s t o g e n i c ) e f f e c t s on the CHO  m i x t u r e s , the c l a s t o g e n i c e f f e c t was  nut  was  b e t e l tannin exerted cells.  H  d i d not  preparation).  c e l l t e s t system  When lime was  2°2  agents s i n c e p h e n o l i c s  chromosome-  In a d d i t i o n , S9  enhanced.  reduced these c l a s t o g e n i c e f f e c t s . of the c a u s a t i v e  Betel  t e s t e r s t r a i n s TA98,  (a r a t l i v e r microsomal  c a t a l a s e reduced these c l a s t o g e n i c e f f e c t s .  be one  has  esophageal c a n c e r .  typhimurium  employed, b o t h b e t e l nut water e x t r a c t and  again  chewing  (a major component of b e t e l nut)  However, when the C h i n e s e hamster ovary  damaging  B e t e l nut  a  S  and  added t o these  However, S9 and W  factors  catalase  t h e r e f o r e suspected to  generate 2 ° 2 H  u  n  ^  e  r  oxidative conditions.  U s i n g a c o l o r i m e t r i c assay, i t was  was  c o u l d account, i n p a r t , f o r the c l a s t o g e n i c e f f e c t  indeed p r e s e n t  and  found t h a t ^2 ~ 2 (  >  of these m i x t u r e s . I t has formation  a l s o been r e p o r t e d  t h a t b e t e l nut water e x t r a c t reduced  of endogenous n i t r o s a t i o n p r o d u c t s .  i s complex, and  a l l k i n d s of c h e m i c a l r e a c t i o n s may  shows t h a t d i e t and human c a n c e r s .  What we  lifestyle  i n t o our mouths  take p l a c e .  This  study  f a c t o r s a r e r e l a t e d t o the development of  However, the e x t e n t  of c a n c e r s cannot be  put  the  of these two  demonstrated.  iv  f a c t o r s i n the  causation  TABLE OF CONTENTS  ABSTRACT  i i  TABLE OF CONTENTS  V  LIST. OF TABLES  viii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  x i i  INTRODUCTION  1  MATERIALS AND METHODS  8  Chemicals  8  P r e p a r a t i o n o f Chemical Stock S o l u t i o n s  8  N i t r o s o p r o l i n e Synthesis  9  Preparation of N i t r o s a t i o n Reaction  of P r o l i n e Mixture  9  Analysis of N-Nitrosoproline  9  Determination  10  of N i t r i t e Depletion  P r e p a r a t i o n o f S9 L i v e r Microsomal M i x t u r e  10  P r e p a r a t i o n o f Sodium Phosphate B u f f e r  11  P r e p a r a t i o n o f S a l i v a Samples  11  Preparation of Salted F i s h Extract  12  N i t r o s a t i o n o f the S a l t e d F i s h E x t r a c t  12  P r e p a r a t i o n o f B e t e l Nut E x t r a c t s  12  P r e p a r a t i o n o f Tea Samples  13  Salmonella  13  Mutagenicity  Assay  Controls f o r Mutagenicity  Assay  15  Chromosome A b e r r a t i o n T e s t  •-•  16  C o n t r o l s f o r CHO C e l l C o n d i t i o n s  17  Assay f o r Hydrogen P e r o x i d e  17  v  RESULTS  '.'  19  E f f e c t s o f Doses o f N i t r i t e , P r o l i n e , Time and pH on Formation o f NPRO  19  Effects of Five Phenolics, NPRO F o r m a t i o n  22  Teas and S a l i v a on  E f f e c t s o f D i h y d r o x y b e n z o i c A c i d With and Without R e s o r c i n o l Moiety on N i t r o s a t i o n o f P r o l i n e  25  E f f e c t s o f D i h y d r o x y b e n z o i c A c i d With R e s o r c i n o l Moiety a t Low C o n c e n t r a t i o n o f N i t r i t e  25  C l a s t o g e n i c A c t i v i t y o f Aqueous and N i t r i t e - T r e a t e d Aqueous F i s h E x t r a c t  27  M u t a g e n i c i t y o f Aqueous S a l t e d F i s h E x t r a c t  27  Mutagenicity of N i t r i t e - T r e a t e d F i s h Extract  . . .•  E f f e c t o f Human S a l i v a on M u t a g e n i c i t y R e s u l t i n g Nitrite-Treated Fish Extract E f f e c t of Naturally Occurring Phenolics of 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 E f f e c t o f Teas on M u t a g e n i c i t y R e s u l t i n g Fish Extract .  29  from 34  on M u t a g e n i c i t y 36 from N i t r i t e - T r e a t e d 36  Study o f t h e I n t e r a c t i o n o f Aqueous S a l t e d F i s h w i t h NaNO^ by a C o l o r i m e t r i c Method  Extract 38  I n t e r a c t i o n o f P h e n o l i c s , Teas and S a l i v a w i t h NaNO^ by a C o l o r i m e t r i c Method  38  M u t a g e n i c i t y o f B e t e l Nut Water E x t r a c t a t pH 7.0 and 10.0  43  M u t a g e n i c i t y o f B e t e l T a n n i n a t pH 7.0 and 10.0  46  M u t a g e n i c i t y o f Hydrogen P e r o x i d e  . .  46  Assay f o r H O^ Generated by B e t e l Nut Water E x t r a c t and B e t e l Tannin a t Two ph L e v e l s C l a s t o g e n i c E f f e c t o f B e t e l Nut Water E x t r a c t a t pH 7.0 and 10.0 . . C l a s t o g e n i c E f f e c t o f B e t e l T a n n i n and Tannic a t pH 7.0 and 10.0  vi  46  49  Acid 53  DISCUSSION  60  Food and Cancer  60  P l a n t P h e n o l i c s and t h e i r R o l e s i n Human C a r c i n o g e n e s i s  62  P o s s i b l e Mechanism o f 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 P h e n o l i c s on N i t r o s a t i o n R e a c t i o n s  64  R e l a t i o n o f S a l t e d F i s h t o Human C a r c i n o g e n e s i s  65  N i t r o s a t i o n R e a c t i o n s Modulated i n a Complex M i x t u r e  66  R e l a t i o n o f B e t e l Nut Chewing t o Human C a r c i n o g e n e s i s  67  S a l i v a and i t s Role i n C a r c i n o g e n e s i s  68  B i o l o g i c a l P r o t e c t i v e Mechanism  70  Unresolved  71  Issues  REFERENCES  75  APPENDICES  84  I. II. III. IV.  E x t r a c t i o n Procedure  of Salted Fish  84  N i t r o s a t i o n o f S a l t e d F i s h Aqueous E x t r a c t  85  E x t r a c t i o n Procedure  86  Chemical  o f B e t e l Nut  S t r u c t u r e s o f Some Modulators  vii  87  L I S T OF  1.  TABLES  C l a s t o g e n i c a c t i v i t y of aqueous f i s h e x t r a c t  and  nitrite-treated f i s h extract 2.  28  Positive controls for different tester strains  3. . H i s t o r i c a l : ranges f o r m u t a g e n i c i t y  31  i n experimental  runs  32  4.  Mutagenic e f f e c t of b e t e l nut water e x t r a c t a t pH  7.00±0.05  5.  Mutagenic e f f e c t o f b e t e l nut water e x t r a c t a t pH  10.00±0.05 ....45  6.  Mutagenic e f f e c t o f b e t e l t a n n i n a t pH  7.00±0.05  47  7.  Mutagenic e f f e c t o f b e t e l t a n n i n a t pH  10.00±0.05  48  8.  Amount of H O^ (108mg/ml f  g e n e r a t e d by b e t e l nut  and b e t e l t a n n i n  water.-.extract  (1.0 mg/ml)  51  9.  C l a s t o g e n i c a c t i v i t y o f b e t e l nut water e x t r a c t a t pH  10.  C l a s t o g e n i c a c t i v i t y of b e t e l nut water e x t r a c t w i t h a t pH  7.00±0.05  .52  lime  10.00±0. 05  54  11. C l a s t o g e n i c a c t i v i t y o f b e t e l t a n n i n a t pH  7.00±0.05  12. C l a s t o g e n i c a c t i v i t y o f b e t e l t a n n i n w i t h lime a t pH 13.  44  C l a s t o g e n i c a c t i v i t y o f t a n n i c a c i d a t pH  55 10.00±0.05  7.0010.05  C l a s t o g e n i c a c t i v i t y of t a n n i c a c i d with lime a t pH 10.0010.05 15. P r o p o r t i o n o f cancer cases a t t r i b u t e d t o v a r i o u s f a c t o r s by d i f f e r e n t authors 16. Genotoxic e f f e c t s measured w i t h p l a n t p h e n o l i c s  .56 57  14.  viii  58 61 63  LIST OF FIGURES  1.  2.  3.  4.  5.  6.  7.  8.  9.  10.  E f f e c t of various concentrations of N-Nitrosoproline  o f NaNO^ on t h e f o r m a t i o n  E f f e c t of:various concentrations formation o f N-Nitrosoproline  of proline  20 on t h e 20  E f f e c t o f v a r i o u s i n c u b a t i o n times on t h e f o r m a t i o n o f N-Nitrosoproline  21  E f f e c t o f v a r i o u s i n c u b a t i o n pH l e v e l s oh t h e f o r m a t i o n of N - N i t r o s o p r o l i n e  21  Inhibitory e f f e c t o f tannic acid, pyrogallol on t h e f o r m a t i o n o f N - N i t r o s o p r o l i n e  and g a l l i c  acid 23  I n h i b i t o r y e f f e c t o f a s c o r b i c a c i d , c a t e c h i n and c h l o r o g e n i c a c i d on n i t r o s a t i o n o f p r o l i n e  23  E f f e c t o f t h r e e t e a samples on t h e f o r m a t i o n o f N-Nitrosoproline  24  E f f e c t o f s a l i v a o f f i v e i n d i v i d u a l s on t h e f o r m a t i o n o f N-Nitrosoproline  24  E f f e c t o f r e s o r c i n o l , .2,4-dihydroxybenzoic a c i d , 3,5d i h y d r o x y b e n z o i c a c i d , p-hydroxybenzoic a c i d and a s c o r b i c a c i d on t h e f o r m a t i o n o f N - N i t r o s o p r o l i n e  26  E f f e c t o f 2,3-dihydroxybenzoic a c i d , 2,5-dihydroxybenzoic and 2,6-dihydroxybenzoic a c i d on t h e f o r m a t i o n o f N-Nitrosoproline  acid 26  11.  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-dihydroxybenzoic a c i d and 3,5-dihydroxybenzoic a c i d on t h e f o r m a t i o n o f N - N i t r o s o p r o l i n e a t low NaNO^ c o n c e n t r a t i o n 26  12a.  M u t a g e n i c i t y o f _S. typhimurium (TA98) f o l l o w i n g exposure t o s a l t e d f i s h aqueous e x t r a c t w i t h and w i t h o u t S9  30  M u t a g e n i c i t y o f _S. typhimurium (TA98) f o l l o w i n g exposure 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 aqueous e x t r a c t w i t h and w i t h o u t S9  30  12b.  13a,  Mutagenic e f f e c t o f s a l t e d f i s h aqueous e x t r a c t on _S. typhimurium (TA100) w i t h and w i t h o u t S9 30  13b.  Mutagenic 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 aqueous e x t r a c t on _S. typhimurium (TA100) w i t h and w i t h o u t S9  ix  30  14.  E f f e c t of salted f i s h aqueous extract and n i t r i t e - t r e a t e d salted f i s h aqueous extract on reversion frequency of S_. typhimurium (TA1535) using the preincubation test  33  15.  Mutagenicity and survival frequencies of S^. typhimurium tester s t r a i n TA1535 following exposure to n i t r o s a t i o n products of aqueous f r a c t i o n of a f i s h extract 33  16.  E f f e c t of whole s a l i v a of f i v e individuals on c e l l survival and mutagenicity of s t r a i n TA1535 exposed concurrently to n i t r i t e - t r e a t e d aqueous salted f i s h extract  35  17.  Inhibitory e f f e c t of tannic acid, pyrogallol and g a l l i c acid on mutagenicity on s t r a i n TA1535 of the n i t r i t e - t r e a t e d f i s h extract 35  18.  Inhibitory e f f e c t of ascorbic acid, catechin and chlorogenic acid on mutagenicity on s t r a i n TA1535. of the n i t r i t e - t r e a t e d f i s h extract 37  19.  E f f e c t of three tea samples on c e l l survival and mutagenicity on s t r a i n TA1535 treated with nitrosated f i s h products i n the .' l i q u i d suspension t e s t 37  20.  Reaction pathways of coloured compound formation with sodiums nitrite  39  21.  N i t r i t e depletion  22.  N i t r i t e depletion capacity tannic acid N i t r i t e depletion capacity chlorogenic acid  23. 24. 25. 26.  capacity  of aqueous salted f i s h extract of g a l l i c acid, pyrogallol  and  of ascorbic  and  40  41 acid, catechin  41  Percentage of n i t r i t e remaining i n the presence of three tea samples N i t r i t e depletion concentrations  capacity  of s a l i v a at various .  :... y.  42 .... 42  Mutagenicity of _S. typhimurium (TA102) following exposure of various concentrations of 2 ° 2 H  27.  Reference curve for the determination of HO  x  concentrations  ..50  LIST OF ABBREVIATIONS  B(a)P  Benzo(a)pyrene  CHO  Chinese hamster ovary  G6P  Glucose-6-phosphate  H  Hydrogen p e r o x i d e  2°2  HEPES  N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic  +  Histidine  His  High p r e s s u r e l i q u i d  HPLC  Potassium  KC1 2  chromatography  Intraperitoneal  i.p. KH P0  independence  chloride  Monobasic p o t a s s i u m  4  phosphate  MEM  E a g l e ' s minimal  M Cl„ g 2  Magnesium c h l o r i d e  MNNG  N-Methyl-N'-nitro-N-nitrosoguanidine  NaAz  Sodium a z i d e  NaCl  Sodium c h l o r i d e  NADP  N i c o t i n a m i d e adenine d i n u c l e o t i d e  Na HP0 2  4  Dibasic  e s s e n t i a l medium  phosphate  sodium phosphate  NaNO.  Sodium  nitrite  NED  N-l-Naphthylethylenediamine  2NF  2-Nitrofluorene  NPRO  Nitrosoproline  S.D.  Standard  X  Mean  deviation  xi  dihydrochloride  acid  ACKNOWLEDGEMENTS  I wish t o express my s i n c e r e g r a t i t u d e t o my s u p e r v i s o r , Dr H.F. S t i c h , f o r h i s guidance, p a t i e n c e and encouragement throughout t h e course o f t h i s work. I am a l s o i n d e b t e d t o Dr R.H.C. San and Dr M.P. R o s i n f o r t h e i r a d v i c e and encouragement.  expert  I would l i k e t o thank Dr B. Bohm o f t h e  Department o f Botany, U.B.C., f o r t h e 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 t h e p h e n o l i c compounds. I would a l s o l i k e t o extend my s i n c e r e thanks f o r t h e h e l p , and  support  good company which I have r e c e i v e d from a l l t h e s t a f f i n t h e  E n v i r o n m e n t a l C a r c i n o g e n e s i s U n i t o f t h e B.C. Cancer Research C e n t r e . Last but not l e a s t , typing t h i s  I would l i k e t o thank M i s s Rosemary  Johnson f o r  thesis.  Financial  s u p p o r t was from t h e N a t i o n a l Cancer I n s t i t u t e  through a g r a n t awarded t o Dr H.F. S t i c h .  xii  o f Canada  INTRODUCTION  E n v i r o n m e n t a l f a c t o r s have been i n d i c a t e d as p l a y i n g a major r o l e i n human mutagenesis and/or c a r c i n o g e n e s i s  (World H e a l t h O r g a n i z a t i o n ,  1964).  E p i d e m i o l o g i c a l r e s e a r c h has r e v e a l e d s i g n i f i c a n t v a r i a t i o n s i n cancer i n c i d e n c e b o t h between and w i t h i n c o u n t r i e s B l o t e t a l . , 1976;  Hoover e t a l . , 1975).  (Drasan and I r v i n g , 1973; Experimental  data a l s o suggest  t h a t e n v i r o n m e n t a l f a c t o r s c o u l d account f o r as much as 90% o f c u r r e n t incidence  (Wynder and G o r i , 1977;  examples from o c c u p a t i o n a l sources  D o l l , 1977).  cancer  S e v e r a l well-documented  include coal tar, v i n y l chloride,  chromium, n i c k e l and a s b e s t o s  ( B a r t s c h and Montesano, 1975;  Goldman, 1975;  Tobacco smoking i s a l s o a w e l l - r e c o g n i z e d  etiological  Dean, 1978).  f a c t o r i n cancer  (World H e a l t h O r g a n i z a t i o n , pollution  o f t h e l u n g , b u c c a l c a v i t y , pharynx and l a r y n x 1975).  Other e n v i r o n m e n t a l f a c t o r s such as a i r  (Pike e t a l . , 1975), v i r u s e s and o t h e r organisms (Heath e t a l . ,  1975) , c h l o r i n a t e d water 1976) , drugs 1978)  C o l e and  (Simmon e t a l . , 1977), food a d d i t i v e s  (Fraumeni and M i l l e r ,  1972) and h a i r dyes  (Boffey,  (Kirkland et a l . ,  a r e a l s o r e c e i v i n g a t t e n t i o n as p o s s i b l e c a u s a t i v e agents i n human  carcinogenesis. Besides  t h e e f f e c t s o f d i e t a r y d e f i c i e n c i e s o r excesses,  r e c e i v e d a d d i t i o n a l a t t e n t i o n as a source evidence  supports  o f cancer  risk.  t h e i d e a t h a t d i e t a r y and l i f e s t y l e  the e t i o l o g y o f a l a r g e p r o p o r t i o n o f human cancers  I n g e n e r a l , t h e cancer  native p o p u l a t i o n experience  experience  factors are involved i n For  of the r o l e of d i e t  o f m i g r a n t s changes from t h e i r  t o t h a t o f the host country.  1  Epidemiological  (Hirayama, 1981).  example, m i g r a n t p o p u l a t i o n s p r o v i d e v a l u a b l e evidence i n cancer.  food has  I n Japanese  m i g r a n t s t o the U n i t e d S t a t e s , a s h i f t i s apparent, as e a r l y as the g e n e r a t i o n , i n stomach and  c o l o n cancer m o r t a l i t y p a t t e r n s from the r a t e i n  Japan t o t h a t p r e v a l e n t i n the U n i t e d S t a t e s . cancer  first  incidence i s v i r t u a l l y  By the t h i r d  generation,  i d e n t i c a l t o the h o s t c o u n t r y  rate  (McMichael,  1978). Numerous g e n o t o x i c , through food products  and by i m p l i c a t i o n c a r c i n o g e n i c agents e n t e r  (Sugimura and Nagao, 1979;  S t i c h e t a l . , 1982a).  These agents c o n s i s t o f n a t u r a l l y o c c u r r i n g mutagens and newly formed d u r i n g r o a s t i n g , b r o i l i n g and h e r b i c i d e r e s i d u e s , and carcinogens and  e t a l . , 1978;  suspected  t h i s s u s p i c i o n was nitrite in  involved i n  (Hirayama,  p r e s e n t i n the s a l t e d f i s h .  1966; was  The  reason  t h a t the s a l t used f o r p r e s e r v i n g the f i s h had  a high  concentration  (Dr C a i H a i - Y i n g , p e r s o n a l communication) and was  secondary amines (Gruger, Nitrosamines  be  The p o s s i b l e agent c a u s i n g cancer  t o be the n i t r o s a m i n e s  nitrite  Gruger, 1972).  the consumption of s a l t e d f i s h  Yang, 1980).  Moreover,  from i n g e s t e d p r e c u r s o r s such as  example used i n t h i s study t o show t h a t d i e t may  human c a r c i n o g e n e s i s was Ho  f r y i n g p r o c e s s e s , p e s t i c i d e and  secondary amines ( S p i e g e l h a l d e r e t a l . , 1976; One  c l a s t o g e n s , compounds  a h o s t o f v a r i o u s food a d d i t i v e s .  can be formed w i t h i n man  man  for  rich  1972).  have been i m p l i c a t e d i n the development o f nasopharyngeal  carcinomas among Cantonese  (Ho,  carcinomas i n h i g h r i s k areas  1971;  Huang e t a l . , 1978), of esophageal  such as L i n x i a n county  (Yang, 1980), of stomach  cancer among p o p u l a t i o n s consuming s a l t - p r e s e r v e d f i s h and of u r i n a r y b l a d d e r cancer Carcinogenic nitrosamines  (Hawksworth and H i l l ,  (Hirayama, 1966),  1974).  are c l a s s i c a l l y produced by the  r e a c t i o n between n i t r i t e o r n i t r o u s a c i d and  2  electrophilic  secondary o r t e r t i a r y amines  under a c i d c o n d i t i o n s s i m i l a r t o those i n the human stomach and i n animal experiments  ( M i r v i s h , 1978).  Ease o f n i t r o s a t i o n c h e m i c a l l y may  be  i n f l u e n c e d by many f a c t o r s such as the b a s i c i t y o f the amine, pH,  substrate  c o n c e n t r a t i o n and t h e p r e s e n c e o f some i n o r g a n i c i o n s , e.g., t h i o c y a n a t e (Wolff and Wasserman, 1972).  I t has been p o s t u l a t e d t h a t up t o 700 ug o f  N - n i t r o s o d i m e t h y l a m i n e i s formed d a i l y w i t h i n man  (Tannenbaum, 1979).  In  a d d i t i o n , n i t r o s a m i n e s can be formed i n food systems i f the c o n c e n t r a t i o n s of  nitrite  appropriate atmosphere  and n i t r o s a t a b l e amines a r e h i g h enough and the c o n d i t i o n s (Fan and Tannenbaum, 1973). (Fine e t a l . , 1977)  They have a l s o been d e t e c t e d i n the  and i n r i v e r water  (Fine e t a l . ,  1976).  N i t r i t e s have been used as f o o d a d d i t i v e s t o s t a b i l i z e t h e c o l o u r of c u r e d meats (Cho and B r a t z l e r , 1970) , t o c o n t r i b u t e f l a v o u r  (Wasserman and  T a l l e y , 1972)  (Duncan and  F o s t e r , 1968).  and t o p r o t e c t a g a i n s t the danger o f b o t u l i s m  N i t r i t e s can a l s o be produced by the b a c t e r i a l r e d u c t i o n o f  n i t r a t e s which a r e w i d e l y d i s t r i b u t e d i n v e g e t a b l e s such as s p i n a c h , b e e t s , c e l e r y and l e t t u c e fish  (Ashton, 1970).  (Gruger, 1972), v e g e t a b l e s  e t a l . , 1960).  Secondary amines have been r e p o r t e d i n  (Phillips,  1966)  and f r u i t j u i c e s (Stewart  In some i n s t a n c e s , a s i n g l e meal may  100 mg o f secondary amines  c o n t a i n as much as  (Sebranek and Cassens, 1973).  Thus the amount o f  n i t r i t e and secondary amines consumed by humans m e r i t s some a t t e n t i o n . The second model used i n t h i s study was  the chewing o f b e t e l nut.  c e n t u r i e s , numerous p l a n t p r o d u c t s have been chewed by many d i f f e r e n t l a t i o n groups.  Among t h e s e p r o d u c t s , the b e t e l nut (Areca catechu)  For popu-  has  become t h e b e s t known example because o f i t s suspected r o l e i n the e t i o l o g y of  o r a l and esophageal carcinomas ( J u s s a w a l l a , 1976;  Ramanathan and L a k s h i m i , 1976)  Muir and K i r k ,  1960;  and i t s i n d u c t i o n o f neoplasms i n e x p e r i m e n t a l  3  animals  (Bhide e t a l . ,  1979;  Ranadive e t a l . ,  1979).  In a d d i t i o n , the  s a l i v a o f b e t e l nut chewers has been shown t o c o n t a i n r e l a t i v e l y q u a n t i t i e s o f p h e n o l i c s and shows chromosome-damaging a c t i v i t y Stich,  large  ( S t i c h and  1982).  I t was  a l s o o f i n t e r e s t t o note t h a t t h e i n c i d e n c e o f o r a l c a n c e r i s  g r e a t l y i n c r e a s e d i n chewers o f l i m e - c o n t a i n i n g m i x t u r e s (H.F. S t i c h , p e r s o n a l communication).  However, no s i g n i f i c a n t i n c r e a s e i n o r a l c a n c e r  was observed among chewers.of l e a v e s which produce an a c i d i c o r n e u t r a l pH i n the s a l i v a ,  such as miang  (tea) l e a v e s  (Simarak e t a l . ,  1977).  The  a d d i t i o n o f l i m e - c o n t a i n i n g m i x t u r e s would i n c r e a s e the a l k a l i n i t y o f the s a l i v a o f b e t e l nut chewers, which would cause o x i d a t i o n o f p h e n o l i c s and g i v e r i s e t o H^O^, al.,  c o n c o m i t a n t l y enhancing i t s c l a s t o g e n i c i t y  (Hanham e t  1983). One o f t h e major components  i n b e t e l nut o t h e r than the a l k a l o i d  ( a r e c o l i n e ) , eugenol and q u e r c e t i n  (Stich et a l . ,  1981a) i s b e t e l t a n n i n .  In  t h i s study, t h e mutagenic and c l a s t o g e n i c e f f e c t of a water e x t r a c t of the b e t e l nut and b e t e l t a n n i n was examined. these components  In a d d i t i o n , l i m e was  t o s i m u l a t e the chewing h a b i t o f the chewers.  added t o T h i s would  i n c r e a s e t h e a l k a l i n i t y o f the m i x t u r e and hence p r o v i d e good c o n d i t i o n s f o r the o x i d a t i o n o f t h e components S. typhimurium  i n water e x t r a c t and b e t e l t a n n i n .  The  s t r a i n TA102 was used f o r the m u t a g e n i c i t y study s i n c e  t e s t e r s t r a i n was  shown t o d e t e c t o x i d a t i v e agents ( L e v i n e t a l . ,  new  this  1982).  Hydrogen p e r o x i d e a s s a y was c a r r i e d out under these c o n d i t i o n s t o determine i f B^O£ was  a c t u a l l y formed.  i n d i c a t o r f o r H„0„  C a t a l a s e was used as a n a t u r a l i n h i b i t o r and/or  formation.  4  C o n s i d e r i n g t h e l a r g e number o f p r o c a r c i n o g e n s o r c a r c i n o g e n s which are consumed d a i l y by man, t h e q u e s t i o n as t o t h e type and e f f i c i e n c y o f p r o t e c t i o n mechanisms a g a i n s t t h e s e damaging agents must be r a i s e d . Because o f t h e c o m p l e x i t y o f f o o d p r o d u c t s and t h e m u l t i t u d e o f p o s s i b l e c h e m i c a l i n t e r a c t i o n s , t h e r e i s a need f o r model systems t o study  factors  which can suppress o r enhance c a r c i n o g e n i c i t y o f v a r i o u s food components. S i n c e c h e m i c a l c a r c i n o g e n s can induce a v a r i e t y o f g e n e t i c damages and A c t o n , 1979), numerous s h o r t - t e r m in vitro  t e s t systems have been •  developed t o d e t e c t c h e m i c a l c a r c i n o g e n s as g e n o t o x i c s u b s t a n c e s . the g e n e t i c damages employed f o r t h i s purpose  t e s t s can a l s o be used f o r u n c o v e r i n g f a c t o r s w i t h enhancing e f f e c t s on g e n o t o x i c compounds (Rosin and S t i c h , 1979, 1980; 1981).  Some o f  i n c l u d e mutation, DNA damage,  chromosome a b e r r a t i o n and gene c o n v e r s i o n ( S t i c h and San, 1979).  al.,  (Stich  In t h i s study, t h e mutation on S. typhimurium  These  or i n h i b i t i n g Buening e t  and chromosomal  a b e r r a t i o n on CHO c e l l s were used t o examine t h e g e n o t o x i c and c l a s t o g e n i c e f f e c t o f s a l t e d f i s h and b e t e l n u t . S e v e r a l p o s s i b l e p r o t e c t i v e modulators reaction.  were c o n s i d e r e d i n t h e n i t r o s a t i o n  D u r i n g t h e consumption o f 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 o r enhance t h e n i t r o s a t i o n r e a c t i o n a r e mixed with s a l i v a . (Stich et a l . ,  Human s a l i v a has been shown t o i n h i b i t n i t r o s a t i o n o f methylurea 1982b).  The q u e s t i o n was t h e r e f o r e r a i s e d as t o t h e e f f e c t o f  s a l i v a on t h e f o r m a t i o n o f mutagenic n i t r o s o compounds i n t h e s a l t e d  fish  extract. S i n c e t h e presence  of n i t r i t e  i s a p r e r e q u i s i t e f o r n i t r o s o compound  f o r m a t i o n , any compound which r e a c t s r e a d i l y w i t h n i t r i t e w i l l , i n h i b i t amine n i t r o s a t i o n .  i n effect,  Examples o f such known compounds a r e a s c o r b i c  5  a c i d and a-tocopherol, with n i t r i t e  which can b l o c k t h e n i t r o s a t i o n r e a c t i o n by r e a c t i n g  ( M i r v i s h , 1981;  Newmark and Mergens, 1981).  Knowles (1974) showed t h a t n i t r i t e smoke phenols i n bacon d u r i n g polymeric phenolics  can i n t e r a c t w i t h a wide v a r i e t y o f  processing  w i t h secondary amines o r a l k y l u r e a s  lifestyle  and f r y i n g .  have been shown by M i r v i s h  of the c o n t r i b u t i o n of phenolics  In a d d i t i o n ,  Several  simple and  (1981) t o compete s u c c e s s f u l l y  for n i t r o s a t i n g species.  An u n d e r s t a n d i n g  t o the t o t a l g e n o t o x i c burden of d i e t and  f a c t o r s appears t o be o f s i g n i f i c a n c e because o f 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  ( S t i c h and Powrie,  1982) . The  phenolic  compounds i n v o l v e d  i n t h i s study i n c l u d e  which a r e p r e s e n t i n o u r d a i l y d i e t such as c h l o r o g e n i c pyrogallol, gallic during  a c i d and t a n n i c  The c o n d i t i o n s  consumption o f a meal c o n s i s t i n g o f f i s h ,  were a l s o s i m u l a t e d . be  acid.  jointly  ingested  This  acid,  catechin,  which may o c c u r  n i t r i t e , phenolics  i s because these p h e n o l i c  and t e a  compounds and t e a s may  w i t h f i s h p r o d u c t s and t h i s may l e a d t o t h e endogenous  formation o f carcinogenic  nitrosamines or alkylureas  There a r e a l s o s t u d i e s which i n d i c a t e t h a t could  some common ones  ( M i r v i s h e t a l . , 1978).  some p h e n o l i c  a c t as c a t a l y s t s on t h e n i t r o s a t i o n r e a c t i o n  compounds  (Davies and McWeeny, 1977;  Walker e t a l . , 1982).  Two c h e m i c a l a n a l y s e s were t h e r e f o r e  the c h e m i s t r y i n v o l v e d  i n the n i t r o s a t i o n r e a c t i o n i n the presence o f  phenolics: reaction.  (1) n i t r o s a t i o n o f p r o l i n e in vitro, These two t e s t s p r o v i d e i n f o r m a t i o n  the p h e n o l i c s  used t o study  and (2) t h e n i t r i t e  depletion  on whether t h e s t r u c t u r e o f  had any e f f e c t on t h e n i t r o s a t i o n r e a c t i o n and i f the p h e n o l i c s  competed w i t h t h e a v a i l a b l e  nitrite.  6  The  removal o f c a r c i n o g e n s  from man's environment would appear t o be  the simplest\.and most d i r e c t manner i n which t o 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 , mixture  i s part of a regular diet  provides pleasure  i f a carcinogenic  (e.g., s a l t e d f i s h i n Chinese p o p u l a t i o n s ) ,  (e.g., smoking), o r has a r i t u a l i s t i c  b e t e l n u t chewing among some A s i a n p o p u l a t i o n s ) .  significance  (e.g.,  In such c a s e s , chemo-  p r e v e n t i o n c o u l d be a p p l i e d w i t h t h e aim o f (1) t r a p p i n g t h e c a r c i n o g e n s (or promoters) b e f o r e they can induce c e l l u l a r changes,  (2) p r e v e n t i n g t h e  mutated c e l l s from d i v i d i n g , o r (3) i n h i b i t i n g the f o r m a t i o n o f c o l o n i e s o f mutated c e l l s  from which n e o p l a s t i c a l l y t r a n s f o r m e d  c e l l s could a r i s e .  i s t h e i n t e n t i o n o f t h i s t h e s i s t o e x p l o r e d i e t and l i f e s t y l e which may be i n v o l v e d i n human c a n c e r .  It  factors  I n a d d i t i o n , some p o s s i b l e chemo-  p r e v e n t i v e agents a r e s t u d i e d i n o r d e r t o p r e v e n t the f o r m a t i o n o f damaging agents.  7  MATERIALS AND  METHODS  Chemicals C h l o r o g e n i c a c i d , p y r o g a l l o l , c a t e c h i n , r e s o r c i n o l , sodium proline, sulfanilic a f l a t o x i n B^,  a c i d , ammonium molybdate,  S t L o u i s , MO.  L o u i s , MO.  C were purchased from Sigma Chemical  G a l l i c a c i d and t a n n i c a c i d were o b t a i n e d from A l d r i c h  Chemical Co., Milwaukee, WI. St  catalase,  sodium a z i d e , b e n z o ( a ) p y r e n e , 2 - n i t r o f l u o r e n e , N-methyl-N'-  n i t r o - N - n i t r o s o g u a n i d i n e and mitomycin Co.,  potassium i o d i d e ,  nitrite,  A s c o r b i c a c i d was  s u p p l i e d by M a l l i n c k r o d t I n c . ,  N-l-Naphthylethylenediamine dihydrochloride, starch  N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d were purchased BDH  Chemicals L t d . , Vancouver,  B.C.  and from  Other p h e n o l i c compounds, namely,  p-hydroxybenzoic a c i d , 2,3-dihydroxybenzoic a c i d , 2,4-dihydroxybenzoic 2,5-dihydroxybenzoic a c i d , 2,6-dihydroxybenzoic a c i d , and  3,5-dihydroxy-  b e n z o i c a c i d , were p r e p a r e d by Dr B. Bohm o f the Department o f U n i v e r s i t y of B r i t i s h  determined by n u c l e a r  resonance s p e c t r o s c o p y , c a r r i e d out a t the Department o f Chemistry,  U n 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. observed.  L i t t l e or no i m p u r i t i e s were  A l l o t h e r c h e m i c a l s were r e a g e n t grade.  P r e p a r a t i o n o f Chemical Stock  Solutions  The p h e n o l i c s and b e t e l t a n n i n were d i s s o l v e d i n phosphate (PBS)  buffer  solution  f o r the S a l m o n e l l a m u t a g e n i c i t y assay or i n "wash" medium f o r c l a s t o -  g e n i c a c t i v i t y i n CHO to  Botany,  Columbia.  The p u r i t y o f a l l the p h e n o l i c compounds was magnetic  acid,  cells.  The  help d i s s o l v e a l l chemicals.  s o l u t i o n s were warmed i n a hot water-bath' The f i n a l pH was  experimental design.  8  a d j u s t e d a c c o r d i n g t o the  Nitrosoproline  Synthesis  10 g o f L - p r o l i n e and 6 g o f NaNC^ were d i s s o l v e d i n 7.5 ml o f d o u b l e - d i s t i l l e d water.  The m i x t u r e was c o o l e d over i c e and 8.0 ml o f  c o n c e n t r a t e d HC1 was s l o w l y added w h i l e s t i r r i n g f o r a p e r i o d o f 4 h r . A l i g h t - y e l l o w s o l i d was o b t a i n e d which was f i l t e r e d and d r i e d under vacuum. T h i s 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 o f h o t c h l o r o f o r m .  The  s o l u t i o n was f i l t e r e d and l e f t t o c o o l o v e r n i g h t on an i c e - b a t h .  This  produced l i g h t - y e l l o w c r y s t a l s o f pure n i t r o s o p r o l i n e w i t h m.p. o f 98 ± 2°C which was v e r y c l o s e t o t h e l i t e r a t u r e v a l u e  (Lijinsky et a l . ,  1970).  Preparation of N i t r o s a t i o n Reaction o f P r o l i n e Mixture Reaction mixtures  c o n s i s t e d o f t h e f o l l o w i n g components:  ( f i n a l c o n c e n t r a t i o n , 30 mM), v a r i o u s doses o f modulators p h e n o l i c s o r s a l i v a ) , and sodium n i t r i t e  L-proline  ( t e a samples,  ( f i n a l c o n c e n t r a t i o n , 7 mM).  components were combined i n t h e o r d e r i n d i c a t e d .  The whole m i x t u r e s  a c i d i f i e d t o pH 2.00 ± 0.01 and i n c u b a t e d f o r 1 h r a t 37°C. were then a n a l y z e d f o r t h e f o r m a t i o n o f NPRO by h i g h p r e s s u r e  The were  The mixtures liquid  chromatography. Analysis of Nitrosoproline A 2.0 y l a l i q u o t o f t h e r e a c t i o n mixture HPLC equipped  with the f o l l o w i n g :  was used t o determine NPRO by  a Beckman 165 v a r i a b l e wavelength d e t e c t o r ,  a S p e c t r o - P h y s i c s SP 8700 s o l v e n t 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 l e n g t h , ODS-3) r e v e r s e phase column.  The  s o l v e n t was i n i t i a l i z e d a t 2% methanol i n d o u b l e - d i s t i l l e d water w i t h a g r a d i e n t o f 1.8% methanol/min.  The methanol c o n c e n t r a t i o n was r a i s e d t o 100%  a t 10 min and then h e l d a t 100% f o r 2 min l o n g e r b e f o r e r e s e t t i n g i t t o 2%.  9  The flow rate of the solvent was set a t 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, d i f f e r e n t phenolic compounds and s a l i v a employed i n t h i s study contained no detectable l e v e l s of preformed NPRO.  In addition, no i n t e r -  f e r i n g peaks appeared at these retention times. Determination of N i t r i t e 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 c l a s s i c a l Griess reaction with s u l f a n i l i c acid and NED under a c i d i c conditions.  The colour reagent was prepared by d i s s o l v i n g 0.1 g of NED  and 1.0 g of s u l f a n i l i c acid i n 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, s a l i v a or f i s h extract and 1.0 ml of g l a s s - d i s t i l l e d water. for 1 hr at 37°C, pH 2.00 ± 0.01.  This reaction mixture was incubated  A 25 u l 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 l i v e r microsomal mixture was prepared from l i v e r of aroclor 1254pretreated rats to induce r a t l i v e r enzymes f o r carcinogen a c t i v a t i o n , as described by Ames et a l .  (1975).  The aroclor 1254, a polychlorinated biphenyl  mixture, was d i l u t e d i n 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 a t a dose o f 500 mg/kg body weight t h r e e days b e f o r e sacrifice.  The r a t s were g i v e n d r i n k i n g water and P u r i n a L a b o r a t o r y Chow  up t o the day b e f o r e s a c r i f i c e . head and then d e c a p i t a t e d . i n t o Erlenmeyer f l a s k s ;  The r a t s were s a c r i f i c e d by a blow t o the  The l i v e r s were removed  from the r a t s and p u t  0.15 M KC1 a t 3 ml/g wet l i v e r was  homogenized w i t h a P o t t e r - E l v e j h e m homogenizer.  added and  The homogenate was  c e n t r i f u g e d f o r 10 min a t 9000 x g a t 4°C and the s u p e r n a t a n t saved. s u p e r n a t a n t was  then q u i c k l y f r o z e n down i n s m a l l p o r t i o n s and s t o r e d a t -80°C.  F o r e v e r y 3 ml o f s t a n d a r d S9 r e a c t i o n m i x t u r e t h e r e was 0.5 M M g C l , 0.013 2  The  ml o f 3.3 M KC1,  NADP, 0.293 ml o f 20 mM  0.132  ml o f 50 mM  G6P,  0.013  0.132  ml o f  ml o f 40  mM  HEPES, and 2.121 ml o f l x PBS o r "wash" medium.  T h i s s o l u t i o n was brought t o pH 7.4  and 0.3 ml o f l i v e r  s u p e r n a t a n t added.  The s o l u t i o n was p r e p a r e d f r e s h b e f o r e each experiment. P r e p a r a t i o n o f Sodium Phosphate B u f f e r The sodium phosphate b u f f e r 2.0 g KC1, 11.5 g Na HPC> 2  water. was  4  T h i s m i x t u r e was  (PBS) was p r e p a r e d by d i s s o l v i n g 80 g NaCl,  and 2.0 g KH PC> 2  4  i  n  t  o  i i±  ter  Q  a u t o c l a v e d a t 121°C f o r 30 min.  f double-distilled The pH of the PBS  7.4.  Preparation of S a l i v a  Samples  S a l i v a was o b t a i n e d from 5 non-smokers consumed a r e g u l a r "Western-type" d i e t . 10 and 11 a.m. s a l i v a was filter  (2 males and 3 females) who  The s a l i v a was  which was about 2-3 h r a f t e r b r e a k f a s t .  collected  P r i o r t o use, t h e  a d j u s t e d t o pH 2.0 and p a s s e d through a 20 um pore s i z e  (Stich et a l . ,  1982b).  11  between  Millipore  Preparation The local  of S a l t e d F i s h E x t r a c t  s a l t e d f i s h , c o m m e r c i a l l y known as  food s t o r e i n Chinatown, Vancouver.  by a m o d i f i c a t i o n o f the procedure of van The salt  "Pak The  Wik" , was  purchased from a  s a l t e d f i s h were  der Hoeven and  van  Leeuwen  s a l t e d f i s h were washed w i t h d i s t i l l e d water t o remove the c r y s t a l s p r i o r to extraction.  i n a blender, (3 ml/g  (3 ml/g  The  r e m a i n i n g m a t e r i a l was  of f i s h ) ,  d i s t i l l e d water e x t r a c t was  discarded.  extracted  Residual  of r e s i d u e ) .  The  three  superficial  times w i t h hexane  hexane was  then e x t r a c t e d  the methanol e v a p o r a t e d , and  (1 ml/g  (1980).  f i s h samples were minced, homogenized  f r e e z e - d r i e d f o r 48 h r , and  o f f i s h ) which was  drying.  The  extracted  three  removed by a i r -  times w i t h methanol  the r e s i d u e  reconstituted in  aqueous f r a c t i o n of the  used throughout the e n t i r e study  fish  (Appendix I ) .  N i t r o s a t i o n o f the S a l t e d F i s h E x t r a c t The  n i t r o s a t i o n m i x t u r e s c o n s i s t e d of the  concentration equivalent  sodium n i t r i t e  i n the o r d e r  0.01  indicated.  incubated and  (0.8 mg  The  o r 0.01  M).  whole m i x t u r e was  f o r 1 hr a t 37°C.  The  The  mg  (phenolics, teas  or  components were added  a c i d i f i e d t o pH  m i x t u r e was  (200-450  2.00  ±  0.01  then n e u t r a l i z e d t o pH  7.00  of B e t e l Nut  Extracts  b e t e l nuts were e x t r a c t e d by a m o d i f i c a t i o n o f the procedure of  Mathew and extracted  doses o f m o d u l a t i n g agents  (final  used i n the m u t a g e n i c i t y t e s t (Appendix I I ) .  Preparation The  aqueous f r a c t i o n o f the f i s h e x t r a c t  of f i s h ) , various  s a l i v a ) and  and  i n mg/ml):  f o l l o w i n g components  Govindarajan  (1964).  The  t w i c e w i t h hexane (1.5 ml/g  d r i e d t o remove the hexane.  The  b e t e l nuts were c r u s h e d i n t o powder of b e t e l n u t ) .  The  r e m a i n i n g m a t e r i a l was  12  r e s i d u e was  then e x t r a c t e d  and  airtwice  ±  w i t h water mixture was  (1.0 ml/g o f r e s i d u e ) , heated  i n a b o i l i n g water-bath, and t h e  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.  named "water e x t r a c t " .  n-butanol  The r e s i d u e was then e x t r a c t e d t h r e e times  (1.0 ml/g o f r e s i d u e ) and t h e n-butanol  evaporated.  c a f f e i n e was added t o t h i s water mixture  (83.4  mg t a n n i n / n u t )  with  The d r i e d  b u t a n o l e x t r a c t was taken up i n 200 ml o f g l a s s - d i s t i l l e d water; 1.5%  This  100 ml o f  t o p r e c i p i t a t e the tannin  (Appendix I I I ) .  P r e p a r a t i o n o f Tea Samples The  t h r e e t e a samples used i n c l u d e d a Chinese  a Japanese green (Bee b r a n d ) . tea leaves  t e a (Pu E r h Beeng b r a n d ) ,  t e a ( S h i n c h a - O h h a s h i r i brand) and a b l a c k Ceylanese t e a  The t e a samples were p r e p a r e d by adding b o i l i n g water t o t h e  (100 mg/ml w a t e r ) .  and t h e s u p e r n a t a n t s  decanted  A f t e r 5 min, t h e samples were c e n t r i f u g e d f o r t h e study.  S a l m o n e l l a M u t a g e n i c i t y Assay Salmonella  typhimurium  t e s t e r s t r a i n s TA98, TA100, TA102 and TA1535  were o b t a i n e d from Dr B.N. Ames o f t h e U n i v e r s i t y o f C a l i f o r n i a , The b a c t e r i a l s t r a i n s were grown and m a i n t a i n e d  Berkeley.  as d e s c r i b e d by Ames e t a l .  (1975). S t r a i n TA98 i s a h i s t i d i n e auxotroph factor  which c o n t a i n s a r e s i s t a n c e t r a n s f e r  (R f a c t o r ) and i s s e n s i t i v e t o f r a m e s h i f t mutagens.  TA100 i s a h i s t i d i n e auxotroph  Tester  strain  which c o n t a i n s an R f a c t o r and r e v e r t s t o  h i s t i d i n e independence by b a s e - p a i r s u b s t i t u t i o n .  T e s t e r s t r a i n TA153 5 can  a l s o be used t o d e t e c t mutagens c a u s i n g b a s e - p a i r s u b s t i t u t i o n s , b u t i t does not c o n t a i n t h e R f a c t o r A-T  (McCann e t a l . , 1975).  base p a i r s a t t h e s i t e o f m u t a t i o n  T e s t e r s t r a i n TA102 c o n t a i n s  i n contrast t o the other  t e s t e r s t r a i n s which d e t e c t mutagens damaging G'C base p a i r s .  13  Salmonella This  strain  d i f f e r s from p r e v i o u s t e s t e r s t r a i n s i n t h a t t h e mutation has been i n t r o d u c e d i n t o a m u l t i c o p y p l a s m i d , so t h a t a p p r o x i m a t e l y 30 c o p i e s o f t h e mutant gene a r e a v a i l a b l e f o r back mutation  (Levin e t a l . ,  1982).  P r e l i m i n a r y experiments were conducted u s i n g t h e p r e i n c u b a t i o n m o d i f i cation  (Nagao e t a l . ,  F r o z e n master  1977) o f t h e procedure developed by Ames e t a l . (1975).  s t o c k was i n n o c u l a t e d i n t o 5 ml o f D i f c o n u t r i e n t b r o t h and  grown o v e r n i g h t (14-16 hr) on a Labquake r o t a r y wheel a t 37°C i n an i n c u b a t o r . The treatment m i x t u r e s were composed o f t h e f o l l o w i n g which were added i n the o r d e r i n d i c a t e d : PBS  0.1 ml o f t h e o v e r n i g h t b a c t e r i a c u l t u r e , 0.5 ml o f  o r t h e s t a n d a r d S9 l i v e r microsomal m i x t u r e , and 0.1 ml o f t h e t e s t  samples.  The m i x t u r e s were then i n c u b a t e d a t 37°C i n a water-bath f o r 20 min;  2.0 ml o f molten t o p agar a t 49°C c o n t a i n i n g 0.455 mM h i s t i d i n e was added t o each o f t h e t r e a t m e n t m i x t u r e s and poured onto minimal g l u c o s e agar p l a t e s (Ames e t a l . ,  1975).  These p l a t e s were i n c u b a t e d a t 37°C f o r 2 days.  The  c o l o n i e s were counted on an A r t e k Model 880 automatic c o l o n y c o u n t e r (Farmingdale, N.Y.). histidine  The mutagenic  a c t i v i t y was e x p r e s s e d as t h e number o f  ( h i s ) revertants per p l a t e . +  To t e s t the e f f e c t o f i n c r e a s e d a l k a l i n i t y o f b e t e l nut water  extract  and b e t e l t a n n i n , 20 y l o f l i m e (10 mg/ml) was added t o t h e b e t e l n u t water e x t r a c t and b e t e l t a n n i n t o b r i n g t h e pH t o 10.00. i n c u b a t e d f o r 30 min a t 37°C.  These m i x t u r e s were  The pH was a d j u s t e d back t o 7.00 w i t h HC1 (1 M)  b e f o r e they were a p p l i e d t o t h e b a c t e r i a . The m u t a g e n i c i t y o f t h e n i t r o s a t i o n r e a c t i o n p r o d u c t s o f t h e aqueous e x t r a c t was assayed u s i n g a m o d i f i c a t i o n method o f Ames e t a l . (1975).  fish  (Rosin and S t i c h , 1979) o f t h e  T h i s procedure p r o v i d e s i n f o r m a t i o n on t h e  m u t a g e n i c i t y o f t e s t e d m i x t u r e s w h i l e a t t h e same time measuring  14  cell  survival  frequencies,  a f a c t o r o f major importance when examining t h e e f f e c t o f  m o d u l a t i n g agents on induced m u t a g e n i c i t y .  Logarithmically  growing  cultures  Q o f S. typhimurium of an o v e r n i g h t  (5-8 x 10 culture  c e l l s / m l ) were p r e p a r e d by r e i n n o c u l a t i n g  (14-16 hr) i n t o 5.0 ml o f f r e s h n u t r i e n t  broth.  These l a t t e r c u l t u r e s were grown on a Labquake r o t a r y wheel i n an f o r 4 h r a t 37°C. and  One ml o f t h i s c u l t u r e was p l a c e d  0.1 ml  incubator  into centrifuge  t h e 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 a t 3000 rpm f o r 5 min.  tubes The  p e l l e t s were then resuspended i n 0.5 ml o f t h e n i t r o s a t i o n r e a c t i o n p r o d u c t s . The  treatment d u r a t i o n  was 20 min a t 37°C.  The b a c t e r i a were p e l l e t e d and  washed by r e s u s p e n s i o n i n PBS and c e n t r i f u g a t i o n . 9 suspended i n 0.5 ml PBS a t c a . 10 saline  c e l l s / m l , and a l i q u o t s were d i l u t e d w i t h  (0.01 ml/10 ml x 0.01 ml/10 ml) and p l a t e d  plates f o r s u r v i v a l studies  (Ames e t a l . , 1975).  b a c t e r i a l suspensions were added t o l o w - h i s t i d i n e t h e agar o v e r l a y e d his  +  revertants.  The b a c t e r i a were then r e -  (0.3 ml) onto n u t r i e n t agar Aliquots  (0.1 ml) o f  (0.455 mM)  t o p agar and  on minimal agar p l a t e s i n o r d e r t o e s t i m a t e the number o f The p l a t e s were s c o r e d  a f t e r 48 h r i n c u b a t i o n  Mutagenic a c t i v i t y was c a l c u l a t e d i n terms o f t h e number o f h i s 7 per 10 s u r v i v i n g b a c t e r i a l c e l l s C o n t r o l s f o r M u t a g e n i c i t y Assay The  +  revertants  (Rosin and S t i c h , 1979).  a c t i v i t y o f t h e S9 a c t i v a t i o n m i x t u r e was assayed w i t h B ( a ) P on TA100  at a concentration was an e l e v a t e d B(a)P  a t 37°C.  o f 10 u g / p l a t e .  number o f r e v e r t a n t s  a t t h e same c o n c e n t r a t i o n  The S9 was deemed s a t i s f a c t o r y i f t h e r e p e r p l a t e from 100 ± 16 t o 760 ± 63.  w i t h o u t S9 p r e s e n t s h o u l d not e l e v a t e t h e  spontaneous frequency o f TA100 over t h a t found i n the u n t r e a t e d  control.  The  f o l l o w i n g c h e m i c a l s were used t o t e s t t h e a c t i v i t y o f t h e s t r a i n s by showing an e l e v a t e d  number o f r e v e r t a n t s  per p l a t e :  15  NaAz  (3 y g / p l a t e ) gave 1205 ± 13  r e v e r t a n t s / p l a t e from 100 from  26 ± 4 t o 887  ± 16 on TA100;  2NF  ± 13 r e v e r t a n t s / p l a t e ;  on TA102 gave an i n c r e a s e from 250  (5 y g / p l a t e ) gave an i n c r e a s e  and mitomycin C  ± 20 t o 3108  (0.5  yg/plate)  ± 174 r e v e r t a n t s / p l a t e .  Chromosome A b e r r a t i o n T e s t The method employed was CHO  c e l l s were grown i n MEM  biotics 100  t h a t p r e v i o u s l y d e s c r i b e d by S t i c h e t a l . (1979). supplemented w i t h 10%  ( s t r e p t o m y c i n s u l f a t e , 29.6  yg/ml;  and f u n g i z o n e , 2.5  yg/ml;  f e t a l c a l f serum, a n t i -  penicillin,  125  yg/ml;  yg/ml) and sodium b i c a r b o n a t e  kanamycin,  (1 mg/ml).  F o r the a n a l y s i s of chromosome a b e r r a t i o n s , a p p r o x i m a t e l y  140,000  CHO  2 c e l l s were seeded onto each 22 mm Experiments medium was mixture.  c o v e r s l i p i n 3.5  cm p l a s t i c d i s h e s ( F a l c o n ) .  were begun when c e l l s were 40-60% c o n f l u e n t .  The  tissue  removed from the p e t r i d i s h e s and r e p l a c e d w i t h 1 ml of F o r 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  culture  reaction  o r c a t a l a s e , 0.5  ml  t h e s e a l i q u o t s were added t o each p e t r i d i s h p r i o r t o the a d d i t i o n o f 0.5 of a double-strength t e s t chemical.  F o l l o w i n g a 3-hr  exposure t o the  m i x t u r e s , the c o v e r s l i p s were washed t w i c e w i t h "wash" MEM, MEM  w i t h 10%  f e t a l c a l f serum was  and  2.0 ml  of ml  test fresh  added t o the p e t r i d i s h e s .  To t e s t the e f f e c t o f lime on b e t e l nut water e x t r a c t and b e t e l t a n n i n , 20 y l o f l i m e  (10 mg/ml) was  added t o the b e t e l nut water e x t r a c t and  t a n n i n t o b r i n g t h e pH t o 10.00.  These m i x t u r e s were i n c u b a t e d f o r 30  a t 37°C and the pH  a d j u s t e d back t o 7.00  mixed w i t h the CHO  cells.  Chromosome a b e r r a t i o n f r e q u e n c y was  w i t h HC1  betel min  (1 M) b e f o r e they were  e s t i m a t e d by sampling  the c e l l s 20 h r  a f t e r c o m p l e t i o n o f treatment w i t h c h e m i c a l s ;  4 h r p r i o r t o sampling,  of c o l c h i c i n e  C e l l s were then t r e a t e d w i t h a  hypotonic  (0.1% i n "wash" MEM)  was  added.  s o l u t i o n of 1% sodium c i t r a t e f o r 20 min.  16  The  citrate  0.1  solution  ml  p e r m i t s the c e l l s t o s w e l l .  The c e l l s were then f i x e d w i t h Carnoy's  ( a c e t i c a c i d / e t h a n o l , 1:3)  f o r 20 min.  A i r - d r i e d s l i d e s were s t a i n e d w i t h  2% o r c e i n i n 50% a c e t i c a c i d / w a t e r , dehydrated  and mounted i n permount.  F o r each sample, a minimum o f 100 metaphase p l a t e s were a n a l y z e d f o r c h r o m a t i d breaks o r chromatid exchanges.  Exchanges s c o r e d i n c l u d e d chromatid  and chromosome exchanges, mono- and m u l t i - r a d i a l s , and C o n t r o l s f o r CHO The a c t i v i t y controls.  C e l l Conditions o f the S9 a c t i v a t i o n m i x t u r e was  A s t o c k s o l u t i o n o f 2 x 10  t o c o n c e n t r a t i o n s o f 2 x 10  -4  M,  0.5 ml o f t h e s e s o l u t i o n s was "wash" MEM  -2  2 x 10  The S9 was  assayed w i t h the  M aflatoxin -5  was  M and 1 x 10  then added w i t h 0.5  t o a f i n a l c o n c e n t r a t i o n of 1 x 10  a f l a t o x i n B^.  rings.  -4  M,  -5  following  serially  diluted  M i n "wash"  MEM;  ml o f the S9 m i x t u r e 1 x 10  -5  or  M and 5 x 10  -6  M  c o n s i d e r e d t o be s a t i s f a c t o r y i f a t l e a s t 80% o f  t h e metaphase p l a t e s had a minimum o f one chromosome break  at a concentration  -6 of  5 x 10  M aflatoxin B .  A f l a t o x i n B^ a t the same c o n c e n t r a t i o n , but  w i t h o u t S9, s h o u l d not e l e v a t e the frequency o f chromosome a b e r r a t i o n s over t h a t found i n n o n - t r e a t e d c o n t r o l c u l t u r e s . and c a t a l a s e were a l s o added t o the CHO  "Wash" MEM,  S9 r e a c t i o n  c e l l s t o check f o r any  mixture  increase i n  the spontaneous frequency o f chromosome b r e a k s . Assay  f o r Hydrogen P e r o x i d e 100 ml c o l o u r r e a g e n t was  potassium was  p r e p a r e d f r e s h each day and c o n t a i n e d 0.4  i o d i d e , 2 x 10 ^ M ammonium molybdate and 3% s t a r c h .  heated and s t i r r e d  c o n c e n t r a t i o n o f 0.2 p i p e t t e d i n t o two  M.  u n t i l dissolved. Aliquots  s e p a r a t e tubes.  A c e t i c a c i d was  (1.0 ml)  T h i s mixture  then added t o a f i n a l  of the sample t o be assayed were  C a t a l a s e (0.1 yg i n 10 y l ) was  17  M  added t o  one  sample,  and t h i s sample was a l l o w e d t o i n c u b a t e a t room temperature f o r a  minimum o f 10 min. each sample.  Aliquots  Absorbance  (1.0 ml) o f c o l o u r reagent were then added t o  was determined a t 575 nm i n a d u a l beak P e r k i n  Elmer Lambda 3 spectrophotometer, u s i n g t h e ( p e r o x i d e - f r e e ) sample as a b l a n k .  The r e a d i n g was taken a t t h e maximum absorbance.  l e v e l s were e s t i m a t e d from a s t a n d a r d curve p r e p a r e d u s i n g b u f f e r e d samples  catalase-treated  c o n t a i n i n g 0 t o 100 uM H^O^.  T h i s procedure was a  m o d i f i c a t i o n o f t h a t d e s c r i b e d by Wang and Nixon  18  phosphate-  (1978).  H  2^2  RESULTS  In t h i s study, a l l t h e d a t a shown a r e t h e average o f two s e t s o f experiments w i t h t r i p l i c a t e a n a l y s e s f o r each s e t ± t h e s t a n d a r d d e v i a t i o n . E f f e c t s o f Doses o f N i t r i t e , P r o l i n e , Time and pH on Formation  o f NPRO  N i t r o s a t i o n o f p r o l i n e was chosen as a model t o study t h e e f f e c t o f modulators on t h e n i t r o s a t i o n r e a c t i o n s i n c e i t p r o v i d e s a simple w i t h which t o work.  P r o l i n e a l s o has a chemical  Moreover, p r o l i n e i s w a t e r - s o l u b l e .  system  s t r u c t u r e o f an amine.  T h i s model may a l s o . s i m u l a t e , a t l e a s t  t o some e x t e n t , t h e s i t u a t i o n o f foods coming i n t o c o n t a c t w i t h  nitrosating  agents. The  f o u r v a r i a b l e s c o n s i d e r e d were (1) c o n c e n t r a t i o n o f NaN02, (2)  concentration of proline, r e a c t i o n mixture.  (3) time o f t h e r e a c t i o n , and (4) t h e pH o f t h e  The f o r m a t i o n o f NPRO in vitro  dependent on n i t r i t e c o n c e n t r a t i o n .  t o be s t r o n g l y  There was o n l y a s l i g h t i n c r e a s e i n t h e  f o r m a t i o n o f NPRO a t up t o 2 mM NaNO^. was  was found  However, when 3 t o 6 mM o f n i t r i t e  added, 2 t o 5 times more NPRO was formed.  The f o r m a t i o n o f NPRO  i n c r e a s e d e x p o n e n t i a l l y w i t h t h e dose o f n i t r i t e p r e s e n t  (Fig. 1).  The e f f e c t o f t h e amount o f p r o l i n e added on t h e f o r m a t i o n o f NPRO was a l s o s t u d i e d ( F i g . 2 ) . When d i f f e r e n t amounts o f p r o l i n e were added t o 6 mM NaN0  2  and i n c u b a t e d f o r 1 h r a t 37°C, a l i n e a r dose response  up t o 100 mM p r o l i n e . off.  was observed  Between 100 and 200 mM, t h e i n c r e a s e began t o l e v e l  Thus t h e f o r m a t i o n o f NPRO in vitro  dose o f p r o l i n e p r o v i d e d up t o 100 mM.  appeared t o be p r o p o r t i o n a l t o t h e The i n c r e a s e slowed down between  100 and 200 mM, p r o b a b l y due t o t h e l i m i t e d amount o f NaNO  19  available.  Figure  1. T h e a b s o r p t i o n varying  NaN0  absorption  Figure  2  peak a r e a s  concentrations.  units.  concentration  for  l h , a t 3 7 ° C a n d pH 2.  2. T h e a b s o r p t i o n proline  absorption  of proline  peak a r e a s  concentration  for  l h , a t 3 7 ° C a n d pH 2.  were from t h r e e  by t h e f o r m a t i o n  o f NaNO^ was 6mM.  20  experiments.  peak a r e a s  were from t h r e e T h e m i x t u r e was  with  were i n  incubated  o f NPRO a t 238nm  The a b s o r p t i o n  T h e x±S.D.- p l o t t e d  The  peak a r e a s  was lOOmM. T h e m i x t u r e was  concentrations.  units.  o f NPRO a t 230nm  The a b s o r p t i o n  The x±S.D. p l o t t e d  The  varying  by t h e formation  with  were i n  experiments. incubated  F i g u r e 3. 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 t i m e s . The x±S.D. p l o t t e d were from t h r e e experiments. 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 c o n c e n t r a t i o n s  o f NaNO^and p r o l i n e were 6mM and lOOmM  respectively.  F i g u r e 4. 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 pH l e v e l s . The a b s o r p t i o n peak areas were i n a b s o r p t i o n u n i t s . The c o n c e n t r a t i o n s o f NaNO^and p r o l i n e were 6mM and lOOmM r e s p e c t i v e l y . The m i x t u r e  was i n c u b a t e d a t 37°C, f o r l h . The x±S.D.  p l o t t e d were form t h r e e experiments.  21  Figure 3  Figure 4  21a  The studied.  c o n s i d e r a t i o n o f time and pH on t h e e f f i c a c y o f NPRO f o r m a t i o n was A l i n e a r i n c r e a s e was observed  p l a t e a u a f t e r 2 hr.  from 0 t o 30 min and i t reached a  A g a i n , t h i s may be due t o t h e amount o f NaN0 and 2  proline available f o r the reaction.  A f t e r 1 h r , 85% o f NPRO had been formed  (Fig. 3). The h i g h e s t response pH  t o NPRO f o r m a t i o n was observed  4 t h e f o r m a t i o n o f NPRO d e c r e a s e d d r a s t i c a l l y  the r e a c t i o n m i x t u r e  a t pH 3, whereas a t  ( F i g . 4 ) . The a c i d i t y of  t h e r e f o r e seems t o p l a y a major r o l e i n t h e n i t r o s a t i o n  reaction. Based on t h e s e e x p e r i m e n t a l d a t a , t h e c o n d i t i o n s f o r t h e f o l l o w i n g s t u d i e s on NPRO f o r m a t i o n were chosen a t 6 mM NaN0  2  and 30 mM p r o l i n e .  A  r e a c t i o n time o f 1 h r a t pH 2 and 37°C was chosen t o make t h e e x p e r i m e n t a l c o n d i t i o n s comparable t o t h e f i s h e x t r a c t m u t a g e n i c i t y s t u d i e s . E f f e c t s o f F i v e P h e n o l i c s , Teas and S a l i v a on NPRO Formation The  five phenolics, g a l l i c  a c i d , t a n n i c a c i d , p y r o g a l l o l , c a t e c h i n and  c h l o r o g e n i c a c i d , a l l i n h i b i t e d t h e f o r m a t i o n o f NPRO.  Their inhibitory  e f f e c t was v e r y s i m i l a r and they were a l l more e f f i c i e n t than a s c o r b i c a c i d ( F i g s 5 and 6 ) .  T h i s o b s e r v a t i o n was i n good agreement w i t h t h a t  observed  w i t h t h e i n h i b i t i o n by these p h e n o l i c s on s a l t e d f i s h e x t r a c t m u t a g e n i c i t y ( F i g s 17 and 1 8 ) . The  t h r e e t e a samples  (Chinese, Japanese and Ceylanese)  i n h i b i t e d t h e f o r m a t i o n o f NPRO. e f f e c t , whereas t h e Ceylanese  Chinese  studied also  t e a showed t h e g r e a t e s t i n h i b i t o r y  t e a was t h e l e a s t a c t i v e  ( F i g . 7 ) . T h i s may be  due t o t h e d i f f e r e n t components i n t h e t e a samples s i n c e t e a i n f u s i o n s were complex m i x t u r e s . S a l i v a a t 20% c o n c e n t r a t i o n reduced  t h e f o r m a t i o n o f NPRO by 50%.  This  showed t h a t s a l i v a as an i n h i b i t o r o f 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 e x t r a c t o r simple compounds such as p r o l i n e 22  ( F i g . 8.).  F i g u r e  5.  The  i n h i b i t o r y  t a n n i c  o f  was  a c i d  n i t r i t e  the  compared  F i g u r e  6.  ( •  w i t h  p r o l i n e  t h a t  The  i n h i b i t o r y  f o r m a t i o n :  from  absence  o f  %NPRO  o f  t h e s e  the  ( •  6mM  );  i n  on  g a l l i c  and  the  a c i d  30mM  i n  n i t r o s a t i o n  ( •  ).  The  r e s p e c t i v e l y .  the  p r e s e n c e  absence  o f  the  o f  o f  p r o l i n e :  c o n c e n t r a t i o n s  %NPRO  the  c o n t r o l  p h e n o l i c s  p h e n o l i c s .  P l o t t e d  e x p e r i m e n t s .  o f  a c i d  o f  p h e n o l i c s  o b t a i n e d  o b t a i n e d  e f f e c t  c o n c e n t r a t i o n s  p r e s e n c e  t h r e e  were  u n i t s  t h r e e  a s c o r b i c  r e s p e c t i v e l y .  o f  p y r o g a l l o l  absorbance  x±S.D.  the  );  and  a r e  The  e f f e c t  two  p h e n o l i c s  ( A ) ;  n i t r i t e  c o n t r o l  m o d u l a t o r s .  c a t e c h i n  and  was  m o d u l a t o r s  the  a s c o r b i c  ( • ) ;  p r o l i n e  were  a r e  w i t h  a c i d  on  c h l o r o g e n i c  a b s o r p t i o n  compared  P l o t t e d  23  and  6mM  peak  t h a t  x i S . D .  and  a c i d  ( # ) .  30mM  a r e a s  o b t a i n e d  from  NPRO  t h r e e  o b t a i n e d  i n  i n  the  e x p e r i m e n t s .  C h (D  00  *3  H-  C CVi  F i g u r e 7. The i n h i b i t o r y e f f e c t o f t h r e e t e a samples on NPRO f o r m a t i o n a t 6mM n i t r i t e and 30mM p r o l i n e : Chinese Ceylon tea  t e a (•) ; Japanese t e a (•) ;  ( • ). %NPRO c o n t r o l was t h e a b s o r p t i o n peak a r e a s  i n t h e presence  o f t h e t e a s compared w i t h t h a t observed  absence o f t h e t e a s . P l o t t e d a r e x±S.D. form t h r e e  obtained  i n the  experiments.  F i g u r e 8. The i n h i b i t o r y e f f e c t o f whole s a l i v a o f f i v e i n d i v i d u a l s on the f o r m a t i o n o f NPRO. The c o n c e n t r a t i o n s o f n i t r i t e  and p r o l i n e  were 6mM and 30mM r e s p e c t i v e l y . %NPRO c o n t r o l was t h e a b s o r p t i o n peak areas o b t a i n e d i n t h e presence t h a t observed from t h r e e  o f t h e s a l i v a compared w i t h  i n t h e absence o f t h e s a l i v a . The x±S.D. p l o t t e d were  experiments.  24  24a  E f f e c t s o f Dihydroxybenzoic A c i d With and Without R e s o r c i n o l Moiety on Nitrosation of Proline 2,4-  and 3,5-Dihydroxybenzoic  a c i d both e x h i b i t e d an enhancement o f  NPRO f o r m a t i o n w i t h i n a dose range. maximum a t 1 mM  2  a c i d gave a peak  c o n c e n t r a t i o n , whereas 3,5-dihydroxybenzoic a c i d  i t s maximum NPRO f o r m a t i o n a t 7.6 mM NaN0 .  2,4-Dihydroxybenzoic  c o n c e n t r a t i o n a t 30 mM  However, 2,4-dihydroxybenzoic a c i d demonstrated  on n i t r o s a t i o n a t 10 mM,  reached  p r o l i n e and 6  an i n h i b i t o r y - e f f e c t  whereas 3,5-dihydroxybenzoic a c i d d i d not  n i t r o s a t i o n even a t a c o n c e n t r a t i o n o f 19  mM  inhibit  mM.  R e s o r c i n o l showed the g r e a t e s t enhancement compared t o the above, but i t had a narrow range i n which r e s o r c i n o l a c t e d as a c a t a l y s t . c o n c e n t r a t i o n of 3 mM, proline  and NaN0 . 2  After  a  r e s o r c i n o l became an i n h i b i t o r a t the dose used f o r  p-Hydroxybenzoic  a c i d , which has been shown not t o  i n h i b i t n i t r o s a t i o n p r o d u c t f o r m a t i o n (H.F. S t i c h , p e r s o n a l and a s c o r b i c a c i d , which  communication),  i n h i b i t s n i t r o s a t i o n , are i n c l u d e d f o r comparison  (Fig. 9). 2,3-,  2,5- and 2,6-Dihydroxybenzoic  acids,  which do not have  resorcinol  moiety, d i d n o t show any enhancement o f NPRO f o r m a t i o n a t the dose of and NaN0  2  used  proline  ( F i g . 10).  E f f e c t s o f D i h y d r o x y b e n z o i c A c i d w i t h R e s o r c i n o l Moiety a t Low Nitrite When the n i t r i t e c o n c e n t r a t i o n was  C o n c e n t r a t i o n of  reduced from 6 t o 0.5 mM,  2,4-,  3,5-  d i h y d r o x y b e n z o i c a c i d 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 f o r m a t i o n . R e s o r c i n o l was 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  t h e 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 o f p r o l i n e  acid  ( F i g . 11).  These r e s u l t s were i n good agreement w i t h those o b t a i n e d as shown i n F i g . 9. These r e s u l t s i n d i c a t e t h a t t h e r e was  some o p t i m a l r a t i o between NaN0 , p r o l i n e 2  p h e n o l i c s w i t h r e s o r c i n o l moiety f o r the p h e n o l i c s t o a c t as c a t a l y s t s 25  and  or i n h i b i t o r s .  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  d i h y d r o x y b e n z o i c  a c i d  ( ® )  p r o l i n e  from  x±S.D.  F i g u r e  10.  o f  ( • )  were  f o r m a t i o n .  peak  t h a t  from  E f f e c t  2 , 6 -  t o  i n  t h r e e  t h r e e  o n  F i g u r e  11.  t h e  a c i d  compared  are  from  absence  peak  t o  t h r e e  e f f e c t  a n d 30mM  a r e a s  absence  t h r e e  i n  o f  o f  o f  t h e  a s c o r b i c  n i t r i t e  %NERO. c o n t r o l  p r e s e n c e  ( # ) ; 3 , 5 -  ( A" ) ;  o f  a n d  was  o b t a i n e d  m o d u l a t o r s  t h e m o d u l a t o r s .  ( * )  a c i d s :  I n c l u d e d  a n d a s c o r b i c  t h a t  2 , 3 -  ( • ) ;  T h e . c o n c e n t r a t i o n s  peak  i n  P l o t t e d  a r e  a r e a s  t h e  f o r  absence  ( A ) ; : .  NaNO^  a n d  comparison'  t h e  o f  p r e s e n c e  p r o l i n e  were  ( ® ) . %NPRO  a c i d  i n  o f  2 , 5 -  c o n t r o l  o f  t h e s e  t h e m o d u l a t o r s .  P l o t t e d  e x p e r i m e n t s .  o f  r e s o r c i n o l ( • ) ;  a n d 3 , 5 - d i h y d r o x y b e n z o i c  n i t r i t e  the  t h e  a b s o r p t i o n  m o d u l a t o r s  ( • )  i n  r e s p e c t i v e l y .  from  T h e i n h i b i t o r y  a r e a s  f o r m a t i o n .  6mM a n d 30mM  x±S.D.  r e s p e c t i v e l y .  a c i d  a c i d  T h e c o n c e n t r a t i o n s  d i h y d r o x y b e n z o i c  NPRO  o b t a i n e d  d i h y d r o x y b e n z o i c  e x p e r i m e n t s .  p - h y d r o x y b e n z o i c  was  2 , 4 -  ( A ) ; p - h y d r o x y b e n z o i c  6mM a n d 30mM  a b s o r p t i o n  compared  a c i d  o n NPRO  were  ( • ) ;  p r o l i n e .  t h e p r e s e n c e  a c i d  %NPRO  o f  t h e m o d u l a t o r s .  ( A )  c o n t r o l  t h e s e  2 , 4 - d i h y d r o x y b e n z o i c  o n NPRO  was  e x p e r i m e n t s .  26  o b t a i n e d  m o d u l a t o r s  T h e x±S.D.  f o r m a t i o n  shown  from  compared  were  t o  a t  a c i d  0.5mM  a b s o r p t i o n  t h a t  o b t a i n e d  i n  from  Clastogenic The  A c t i v i t y o f Aqueous and  N i t r i t e - T r e a t e d Aqueous F i s h  a b i l i t y o f aqueous f i s h e x t r a c t and  e x t r a c t t o induce chromosome damage was c e l l chromosome a b e r r a t i o n e x t r a c t and  assay.  The  clastogenic a c t i v i t y  increased  fish  monitored w i t h a c o n v e n t i o n a l  CHO  clastogenic a c t i v i t y  of the  induce chromosomal breakage.  S9  a t a dose o f 360  The However, the  l i v e r microsomal a c t i v a t i o n d i d not  e f f e c t on the c l a s t o g e n i c i t y . cells  fish  by a p p r o x i m a t e l y 10 times when exposed t o  n i t r i t e - t r e a t e d f i s h extract.  the CHO  n i t r i t e - t r e a t e d aqueous  the 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 i s shown i n T a b l e 1.  aqueous f i s h e x t r a c t a l o n e d i d not  any  Extract  The  mg  have  aqueous f i s h e x t r a c t became t o x i c t o  equivalent  o f s a l t e d f i s h per ml  mixture  ( i . e . , no metaphase o b s e r v e d ) . M u t a g e n i c i t y of Aqueous S a l t e d F i s h The  a b i l i t y o f the aqueous f r a c t i o n o f the  m u t a g e n i c i t y was 1977) 11a,  Extract  examined w i t h the p r e i n c u b a t i o n  o f the Ames S a l m o n e l l a m u t a g e n i c i t y assay 12a  and  13  show the  f i s h extract to modification  induce  (Nagao e t a l . ,  (Ames e t a l . , 1975).  Figs  e f f e c t o f adding t h i s aqueous f i s h e x t r a c t t o  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  the  mutagenic  activities  of the aqueous f i s h e x t r a c t were e x p r e s s e d as the number of h i s  revertants  per p l a t e .  The  concentrations  e x p r e s s e d i n terms o f gram e q u i v a l e n t  of the aqueous f i s h e x t r a c t were  o f s a l t e d f i s h per ml  r e s u l t s shown here were based on the average number of h i s p l a t e ± S.D. The TA98 and was  o f two  TA100 ( F i g s 12a  observed on  +  of e x t r a c t . revertants  s e t s of experiments, each w i t h t r i p l i c a t e  aqueous f i s h e x t r a c t was and  The per  plates.  s t r o n g l y mutagenic t o b o t h t e s t e r s t r a i n s  13a).  No  s i g n i f i c a n t enhancement of m u t a g e n i c i t y  s t r a i n TA1535 ( F i g . 14).  t o x i c a t a dose h i g h e r than 0.12  +  The  g equivalent  27  aqueous f i s h e x t r a c t became o f f i s h / m l e x t r a c t on  both  TABLE 1 CLASTOGENIC ACTIVITY OF AQUEOUS FISH EXTRACT AND NITRITE-TREATED FISH EXTRACT  P e r c e n t Metaphase P l a t e s w i t h Chromatid Breaks and/or Exchanges (±S.D.) Fish Extract H Equivalent of fish/ml  -S9  N i t r i t e - T r e a t e d F i s h Extract''" +S9  -S9  +S9  0.36  T  0.18  3±2  1±1  37±5  28±7  0.09  0  0  14±3  9±3  2  N i t r i t e a t 0.8 mg/ml m i x t u r e a l o n e = 0%. " ' " 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 was composed o f t h e i n d i c a t e d c o n c e n t r a t i o n o f f i s h e x t r a c t , 0.8 mg/ml m i x t u r e o f n i t r i t e , i n c u b a t e d f o r 1 h r a t 37°C, pH 2. 2 T = toxic.  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 m e t a b o l i c a c t i v a t i o n d i d not appear t o a f f e c t m u t a g e n i c i t y o f the aqueous f i s h e x t r a c t . number o f h i s for  +  the  Of the p o s i t i v e c o n t r o l s ,  r e v e r t a n t s per p l a t e can be seen i n T a b l e 2.  the  These r e s u l t s  the p o s i t i v e c o n t r o l s are i n good agreement w i t h those o b t a i n e d by  Environmental  C a r c i n o g e n e s i s U n i t of the B.C.  Cancer Research  Centre  the  (Table 3 ) .  Mutagenicity of 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 Nitrite-treated  (pH 2, 1 h r , 3 7°C)  f i s h e x t r a c t a g a i n showed an i n c r e a s e  i n the mutagenic e f f e c t on s t r a i n s TA98 and TA100. d i f f e r e n c e i n m u t a g e n i c i t y was  obtained i n both cases.  i n c r e a s e was of  observed  significant  observed between n i t r i t e - t r e a t e d and  n i t r i t e - t r e a t e d aqueous f i s h e x t r a c t ( F i g s 12b was  However, no  and 13b).  non-  A similar trend  In the case o f s t r a i n TA1535, a s i g n i f i c a n t  and no t o x i c e f f e c t was  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 used  seen when exposed t o the doses  ( F i g . 14).  Once a g a i n , S9  microsomal a c t i v a t i o n had no e f f e c t on the m u t a g e n i c i t y of the f i s h on any o f the above s t r a i n s .  S t r a i n TA1535 was  liver nitrite-treated  t h e r e f o r e used  throughout  the study o f n i t r o s a t i o n of s a l t e d f i s h e x t r a c t w i t h o u t S9 l i v e r microsomal activation. S i n c e the p r e i n c u b a t i o n t e s t p r o t o c o l does not p r o v i d e s u r v i v a l t h e r e was assay.  a need t o use another m o d i f i e d v e r s i o n of the S a l m o n e l l a  The  liquid  suspension t e s t  the i n h i b i t o r y e f f e c t of the modulators.  estimated;  used t o b e t t e r a s s e s s  The advantages o f u s i n g t h i s  (1) b o t h r e v e r s i o n f r e q u e n c i e s and  due  t o a t o x i c e f f e c t on the b a c t e r i a or an  e f f e c t e x e r t e d by t h e modulators;  and  the chemical i s b e t t e r c o n t r o l l e d .  29  assay  s u r v i v a l f r e q u e n c i e s can  (2) i t p r o v i d e s i n f o r m a t i o n on whether a r e d u c t i o n o f  r e v e r t a n t s / p l a t e was  mutagenicity  (Rosin and S t i c h , 1979), which i s another  m o d i f i c a t i o n o f the method of Ames e t a l . (1975), was  are t h r e e f o l d :  data,  his  +  inhibitory  (3) the exposure of the b a c t e r i a t o  be  F i g u r e  12a.  M u t a g e n i c i t y  s a l t e d  f i s h  o f  F i g u r e  12b.  M u t a g e n i c i t y  and  w i t h o u t  from  F i g u r e  13a.  two  two  F i g u r e  13b.  o f  S.  ( • )  S9  e f f e c t  o f  ( A )  e f f e c t  t y p h i m u r i u m  a r e  p l a t i n g  i n  from  o f  two  )  and  x+S.D.  (TA98)  s a l t e d  the  exposure  w i t h o u t  from  two  f i s h  f o l l o w i n g  aqueous  p r e - i n c u b a t i o n  t r i p l i c a t e  s a l t e d  f i s h  w i t h o u t  ( •  )  t o  S9  i n  the  e x p e r i m e n t s  w i t h  n i t r o s a t e d  w i t h  S9.  ( A )  p r e - i n c u b a t i o n  e a c h .  30  and  t e s t .  The  f i s h  on  S.  a r e  x±S.D.  ( • )  w i t h  t y p h i m u r i u m  shown  p l a t i n g  aqueous  e x p e r i m e n t s  ( A )  e a c h .  x±S.D.  w i t h o u t  to  w i t h  P l o t t e d  e x t r a c t  t r i p l i c a t e  s a l t e d  exposure  e x t r a c t  p l a t i n g  aqueous  ( • )  e x p e r i m e n t s  (TA100)  f o l l o w i n g  each.  w i t h  and  p r e - i n c u b a t i o n  shown  o f  ( •  a r e  t y p h i m u r i u m  p r o d u c t s  w i t h  M u t a g e n i c  S.  p l a t i n g  (TA98)  w i t h  P l o t t e d  e x p e r i m e n t s  M u t a g e n i c  (TA100)  e x t r a c t  t e s t .  t r i p l i c a t e  n i t r o s a t i o n  t y p h i m u r i u m  aqueous  p r e - i n c u b a t i o n  w i t h  S.  a r e  each.  e x t r a c t  S9.  from  The  on  x±S.D.  t r i p l i c a t e  30a  TABLE 2 POSITIVE CONTROLS FOR DIFFERENT TESTER STRAINS  TA98  TA100  yg/plate  -S9  +S9  -S9  B(a)P  NT  NT  93±8  (10)  NaAz (3)  2NF (5)  1  NT  795±33  NT  1310±43  TA1535 +S9  -S9  +S9  NT  NT  NT  NT  NT  NT  NT  510±21  NT  NT  NT  NT  MNNG (20)  NT  NT  NT  Solvent  24±3  23±5  96±3  NT.= n o t t e s t e d .  31  100±4  1310±43  10±2  NT  11±3  TABLE 3 HISTORICAL RANGES FOR MUTAGENICITY  IN EXPERIMENTAL  RUNS  Revertants per P l a t e TA98 Concentration (yg/plate)  Treatment  PBS  -S9  8-56  1  B(a)P  4  TA100 +S9  11-66  10  NT  MNNG  20  NT  NT  NaAz  3  NT  NT  1  2  2NF  1  5  1  Mitomycin C  3  From Environmental  0.5  NT  From L e v i n e t a l . (1982).  TA1535 +S9  TA102  -S9  +S9  10-18  11-19  -S9  +S9  74-197  73-215  71-194  201-702  NT  NT  NT  NT  NT  1310±43  NT  NT  NT  NT  NT  NT  NT  NT  NT 735-1606  233-275  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  2772  NT  C a r c i n o g e n e s i s U n i t , B.C. Cancer Research  'From Marquardt e t a l . (1977) .  NT = n o t t e s t e d .  238-942  NT  — -S9  Centre.  F i g u r e  14.  The  e f f e c t  s a l t e d  f i s h  (TA1535)  w i t h  e x t r a c t  F i g u r e  15.  w i t h  on  f o l l o w i n g  a  f i s h  a r e  e x t r a c t  and  two  M u t a g e n i c i t y  o f  s a l t e d  u s i n g  ( A )  b a s e d  o f  e x t r a c t  o b t a i n e d  on  aqueous  r e v e r s i o n  p r e - i n c u b a t i o n  w i t h o u t  ( y )  and  ( • )  and  s u r v i v a l  f i s h  t o  i n  from  two  ( • )  the  S9  f r e q u e n c i e s  o f  S.  t r e a t e d  t y p h i m u r i u m  aqueous  s a l t e d  p l a t i n g  e x t r a c t  f i s h  w i t h  o f  an  ( # ) ;  t e s t .  t r i p l i c a t e  aqueous  x±S.D.  e a c h .  t y p h i m u r i u m  a l o n e  s u s p e n s i o n  w i t h  S.  p l o t t e d  p r o d u c t s  e x t r a c t  e x p e r i m e n t s  33  a r e  n i t r i t e  f i s h  t r e a t e d  t r i p l i c a t e  l i q u i d  o f  S a l t e d  n i t r i t e  f i s h  and  f r e q u e n c y  n i t r o s a t i o n  e x t r a c t :  ( A )  w i t h  e x t r a c t  t e s t .  S9;  w i t h o u t  e x p e r i m e n t s  exposure  s a l t e d  f i s h  aqueous  (TA1535)  f r a c t i o n "  n i t r i t e  The  x±S.D.  p l a t i n g  t r e a t e d  shown  each.  A t t h e doses used f o r n i t r o s a t i o n , t h e f i s h e x t r a c t by i t s e l f no d e t e c t a b l e mutagenic a c t i v i t y  showed  ( F i g . 15). F o r t e s t i n g the i n h i b i t o r y o r  enhancing e f f e c t s o f p h e n o l i c s , s a l i v a and t e a s , t h e n i t r o s a t i o n r e a c t i o n m i x t u r e was chosen t o c o n s i s t o f 340 mg e q u i v a l e n t o f f i s h p e r ml and 0.8 mg sodium n i t r i t e .  These r e a c t i o n c o n d i t i o n s r e s u l t e d i n t h e p r o d u c t i o n o f  mutagenic p r o d u c t s revertants per 10  which induced 7  a r e v e r s i o n frequency  s u r v i v o r s (n = 8) (182 ± 6 S.D. h i s  o f 15.1 ± 0.1 h i s +  revertants/minimal  agar p l a t e ) and r e s u l t e d i n no t o x i c i t y i n t h e t r e a t e d b a c t e r i a . sodium n i t r i t e c o n c e n t r a t i o n used a l s o d i d n o t show any mutagenic The  spontaneous r e v e r s i o n frequency  +  The effect.  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 r e v e r t a n t s p e r 10 s u r v i v o r s (n = 8) o r 7 ± 0.8 S.D. his  +  revertants/minimal  agar p l a t e .  E f f e c t o f Human S a l i v a on M u t a g e n i c i t y Extract  R e s u l t i n g from N i t r i t e - T r e a t e d F i s h  Human s a l i v a was chosen s i n c e most c h e m i c a l s i n t e r a c t with s a l i v a .  I t i s t h e r e f o r e reasonable  human s a l i v a on 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 .  i n g e s t e d through food  will  t o look a t t h e e f f e c t o f The a d d i t i o n of human  s a l i v a t o t h e n i t r i t e and f i s h e x t r a c t m i x t u r e a t pH 2 reduced t h e f o r m a t i o n of r e a c t i o n products  which induce  his  +  r e v e r t a n t s i n S. typhimurium.  F i g . 16  shows t h e r e s u l t s o b t a i n e d w i t h 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,  mutagenicity  a l l non-smokers).  There was a d o s e - r e l a t e d r e d u c t i o n o f  as t h e c o n c e n t r a t i o n o f t h e s a l i v a i n c r e a s e d .  S a l i v a samples  d i l u t e d t o as low as 5% (v/v) o f t h e o r i g i n a l s a l i v a c o n c e n t r a t i o n  still  s i g n i f i c a n t l y reduced t h e mutagenic a c t i v i t y o f t h e n i t r o s a t i o n p r o d u c t s (percent m u t a g e n i c i t y ,  77.6 + 8.4, S.D., n = 5 ) .  34  F i g u r e  16.  The  e f f e c t  and  m u t a g e n i c i t y  aqueous  and  o f  s a l t e d  n i t r i t e  e q u i v a l e n t  x±S.D.  from  17.  o f  f i s h  u s e d  o f  i n d i v i d u a l .  F i g u r e  whole  two  t h a t  The  v a l u e s  The  i n h i b i t o r y  e x t r a c t  have  the  0.8mg  and  ( •  p l a t i n g  The  i n  i s  c o r r e c t e d  o f  t h r e e  n i t r i t e - t r e a t e d  );  g a l l i c  n i t r i t e  P l o t t e d  a c i d  u s e d  The  a r e  c o n c e n t r a t i o n s  o f  f i s h  whole  r e v e r s i o n  the  f o r  ( A ) .  340mg  v a l u e s  have  from  35  were  n i t r o s a t e d  spontaneous  on  o f  compared  m u t a g e n i c i t y  e q u i v a l e n t  two  n i t r o s a t e d  e x t r a c t  a c i d  c o n c e n t r a t i o n s  been  o f  o n l y .  r e v e r s i o n .  e x t r a c t : t a n n i c  The  a r e  each  s a l i v a  f i s h  t r e a t e d  340mg  f o r  f r e q u e n c y  s u r v i v a l  e x t r a c t  P l o t t e d  p l a t i n g  p r e s e n c e  p h e n o l i c s  f i s h  s a l i v a  r e s p e c t i v e l y .  t r i p l i c a t e  w i t h  e a c h .  c e l l  n i t r i t e  were  x±S.D.  on  to  the  i n  i n d i v i d u a l s  c o n c u r r e n t l y  w i t h  w i t h  b a c t e r i a  e f f e c t  f i v e  a n d . 0 . 8 m g  b a c t e r i a  been  r e s p e c t i v e l y .  r e v e r s i o n .  ml  % m u t a g e n i c i t y  w i t h  p y r o g a l l o l  exposed  p e r  e x t r a c t - t r e a t e d  o f  TA1535  e x p e r i m e n t s  o b s e r v e d  o f  c o m b i n a t i o n  f i s h  TA1535  ( • )  e x t r a c t .  i n  f i s h  s a l i v a  o f  c o r r e c t e d  e x p e r i m e n t s  on  ( •  o f  f i s h  f o r  w i t h  s t r a i n  );  f i s h  p e r  ml  .  and  spontaneous  t r i p l i c a t e  E f f e c t o f N a t u r a l l y O c c u r r i n g P h e n o l i c s on M u t a g e n i c i t y Fish Extract  of N i t r i t e - T r e a t e d  F i v e n a t u r a l l y o c c u r r i n g p h e n o l i c compounds were assayed f o r modulating  a c t i v i t y on t h e f o r m a t i o n o f mutagens r e s u l t i n g from t h e n i t r o -  sation of the f i s h extract.  These p h e n o l i c s a r e known t o be p r e s e n t a t  high concentrations i n teas, vegetables Powrie, 1982).  and e d i b l e f r u i t s  ( S t i c h and  F i g s 17 and 18 show the i n h i b i t o r y e f f e c t o f 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 , c a t e c h i n and c h l o r o g e n i c a c i d on t h e m u t a g e n i c i t y of the n i t r o s a t i o n r e a c t i o n p r o d u c t s .  A l l these p h e n o l i c s show  similar  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 r e a c t i o n f i s h products-induced reversion. reduced  Concentrations  the mutagenicity  his  +  o f t h e p h e n o l i c s as low as 1 mg/ml a l r e a d y  by a p p r o x i m a t e l y  50%.  F o r comparison, the e f f e c t  o f a s c o r b i c a c i d on t h i s r e a c t i o n was i n c l u d e d i n F i g . 18, s i n c e i t i s known t h a t t h i s compound can r e a c t w i t h t h e n i t r i t e .  The i n h i b i t o r y  of t h e f i v e p h e n o l i c s were comparable t o t h a t o f a s c o r b i c a c i d .  effects  A l l the  r e a c t i o n s were performed a t c o n c e n t r a t i o n s which d i d not a f f e c t t h e s u r v i v a l o f S. typhimurium  ((upper c u r v e s o f F i g s 17 and 18) .  E f f e c t o f Teas on M u t a g e n i c i t y  R e s u l t i n g from 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  S i n c e s i n g l e p h e n o l i c 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 t o whether complex m i x t u r e s  +  r e v e r s i o n , the q u e s t i o n was r a i s e d as  o f p h e n o l i c s a l s o behave i n t h e same manner.  Teas a r e p h e n o l i c compound-containing m i x t u r e s  ( S t i c h and Powrie, 1982)  which may be i n g e s t e d j o i n t l y w i t h f i s h p r o d u c t s  d u r i n g a meal.  The mutagenic a c t i v i t y 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 was markedly i n h i b i t e d i f t h e n i t r o s a t i o n r e a c t i o n proceeded i n the presence o f t e a infusions.  F i g . 19 shows the i n h i b i t o r y e f f e c t o f Chinese,  36  Japanese and  F i g u r e 18. The i n h i b i t o r y e f f e c t o f a s c o r b i c a c i d and two p h e n o l i c s on ;.v± m u t a g e n i c i t y on s t r a i n TA1535 o f the n i t r i t e t r e a t e d f i s h ascorbic acid  ( A ) ; catechin  ( • ); c h l o r o g e n i c a c i d  extract:  ( • ). The  c o n c e n t r a t i o n o f s a l t e d f i s h e x t r a c t and n i t r i t e used were 340mg e q u i v a l e n t p e r ml and 0.8mg r e s p e c t i v e l y . The v a l u e s have been c o r r e c t e d f o r spontaneous  r e v e r s i o n . The x+S.D. shown a r e o b t a i n e d  from two s e t o f experiments w i t h t r i p l i c a t e  F i g u r e 19. The e f f e c t o f t h r e e tea.samples on c e l l of  plating  each.  s u r v i v a l and m u t a g e n i c i t y  TA1535 t r e a t e d w i t h n i t r o s a t e d f i s h p r o d u c t s i n l i q u i d  :.  s u s p e n s i o n t e s t : Chinese t e a ( • ) ; Japanese t e a ( • ); Ceylon t e a ( A ) . P l o t t e d a r e x±S.D. from two experiments w i t h t r i p l i c a t e each. V a l u e s have been c o r r e c t e d f o r spontaneous  37  plating  reversion.  Ceylanese t e a s on t h e m u t a g e n i c i t y o f t h e t e s t system.  Once a g a i n , the  observed decrease i n mutagenic a c t i v i t y o f the n i t r o s a t i o n m i x t u r e s was not due t o a t o x i c e f f e c t on t h e t r e a t e d b a c t e r i a  (upper c u r v e o f F i g . 19).  Study o f t h e I n t e r a c t i o n o f Aqueous S a l t e d F i s h E x t r a c t w i t h NaNO^ by a C o l o r i m e t r i c Method Since 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 showed an enhanced mutagenic  activity  on b a c t e r i a l t e s t e r s t r a i n TA1535, t h e r e was a need t o e x p l o r e  whether the  f i s h e x t r a c t reacts with the a v a i l a b l e n i t r i t e .  involved  m i x i n g t h e sodium n i t r i t e ,  f i s h e x t r a c t and a c o l o u r r e a g e n t t o g e t h e r i n  the p r e s e n c e o f h y d r o c h l o r i c a c i d . to y i e l d nitrous a c i d  (HlsK^) .  compound w i t h s u l f a n i l i c azo  The procedure  The a c i d r e a c t e d w i t h t h e sodium n i t r i t e  T h i s n i t r o u s a c i d then formed a d i a z o  a c i d which i n t u r n c o u p l e d t o NED t o produce an  compound w i t h an a b s o r p t i o n  maximum a t 550 nm (Fan and Tannenbaum, 1971).  T h i s s e r i e s o f r e a c t i o n s i s o u t l i n e d i n F i g . 20. i n the production was i n c u b a t e d studies depleted  o f the azo compound r e s u l t e d when s a l t e d f i s h  with the n i t r i t e .  (340 mg e q u i v a l e n t  extract  A t the dose used f o r the m u t a g e n i c i t y  o f f i s h / m l ) , 50% o f t h e n i t r i t e c o n t e n t was  of the coloured  Interaction of Phenolics, f i v e phenolics  the c o n c e n t r a t i o n  azo compound.  Teas and S a l i v a w i t h NaNO^ by a C o l o r i m e t r i c Method  used a l l showed a r e d u c t i o n  of the phenolics  increased.  the n i t r i t e t h e b e s t among t h e f i v e p h e n o l i c s least n i t r i t e depletion a b i l i t y . All  reduction  ( F i g . 21). The aqueous s a l t e d f i s h e x t r a c t d i d n o t i n t e r f e r e w i t h  the a b s o r p t i o n  The  A significant  f i v e phenolics  Ascorbic  i n n i t r i t e c o n t e n t as  Chlorogenic tested.  a c i d reacted  Catechin  a c i d was i n c l u d e d  used c o n s i s t e n t l y showed a b e t t e r n i t r i t e  c a p a c i t y than a s c o r b i c a c i d  ( F i g s 22 and 23).  38  with  showed the  f o r comparison. depletion  F i g u r e 20: The r e a c t i o n pathways o f c o l o r e d compound f o r m a t i o n w i t h NaNO  (Fan and Tannenbaum, 1971).  F i g u r e 21. The n i t r i t e The i n i t i a l  d e p l e t i o n c a p a c i t y o f aqueous s a l t e d f i s h  extract.  c o n c e n t r a t i o n o f NaNO^ used was 0.01M i n t h e f i n a l  mixture. % n i t r i t e  remained was o b t a i n e d  from the absorbance  o f t h e c o l o r e d azo compound formed i n the presence o f f i s h  extract  compared w i t h t h a t o f t h e o r i g i n a l c o n c e n t r a t i o n o f n i t r i t e . The x±S.D. shown was;obtained from t h r e e  40'.  experiments.  HH  0 200 MG  1  300 E Q V OF F I S H / M L  F i g u r e 21  40a  H  400  Figure 22. The n i t r i t e depletion capacity of three phenolics: g a l l i c  acid(A);  p y r o g a l l o l ( • ); tannic acid ( • ) . The i n i t i a l concentration of n i t r i t e used was O.OlmM i n the finalmixture. % n i t r i t e  remained  was obtained from the absorbance of the color generated by the azo compound formed i n the presence of the phenolics 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.  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 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 modulators compared with that of the o r i g i n a l concentration of n i t r i t e . Plotted are x±S.D. from three experiments.  41  NITRITE  R E M A I N E D  C n  CD  to to  r o  | i i %  pc  n CD  2  r  M  NITRITE  R E M A I N E D  F i g u r e 24. The % n i t r i t e  remained i n t h e p r e s e n c e o f t h r e e t e a samples:  Chinese t e a ( • ); Japanese t e a ( • ) and C e y l o n t e a ( A ) . The concentrations of n i t r i t e % nitrite  initially  was O.OlmM i n t h e f i n a l m i x t u r e .  remained was t h e absorbance o f t h e c o l o u r e d azo compound  formed i n t h e presence o f t h e t e a samples compared w i t h t h a t o f the  o r i g i n a l concentration o f n i t r i t e without the teas.  are  x±S.D. from t h r e e experiments.  F i g u r e 25. The n i t r i t e The  initial  % nitrite  depletion capacity of the s a l i v a a t various  Plotted  concentrations.  c o n c e n t r a t i o n o f NaNO^used was O.OlmM i n t h e m i x t u r e .  remained was determined from t h e absorbance o f t h e c o l o u r e d  azo compound formed i n t h e presence o f t h e s a l i v a compared w i t h that o f the o r i g i n a l concentration o f n i t r i t e without the s a l i v a . The x±S.D. shown were o b t a i n e d from t h r e e experiments.  42  (D  Tea  i n f u s i o n s (Chinese,  for mutagenicity observed.  Japanese and C e y l a n e s e ) ,  s t u d i e s , a l l reacted with n i t r i t e .  a t t h e doses used A dose response was  A l l the t e a s had s i m i l a r r e a c t i v i t y towards t h e n i t r i t e  available.  A t a dose o f 10 mg e q u i v a l e n t o f t e a p e r ml, o n l y about 10%  o f t h e o r i g i n a l n i t r i t e remained  ( F i g . 24).  S a l i v a a l s o showed a r e d u c t i o n i n n i t r i t e c o n t e n t of s a l i v a increased. p h e n o l i c s and t e a s .  as the c o n c e n t r a t i o n  I t s e f f i c i e n c y seemed t o be lower than t h a t of However, i t i s n o t a b s o l u t e l y comparable s i n c e t h e  c o n c e n t r a t i o n s were expressed  differently  ( F i g . 25).  Under t h e c o n d i t i o n s used f o r m u t a g e n i c i t y e x t r a c t on S. typhimurium  studies of s a l t e d f i s h  s t r a i n TA1535, a l l these modulators r e a c t e d  with  more n i t r i t e than t h e s a l t e d f i s h e x t r a c t . Mutagenicity The  o f B e t e l Nut Water E x t r a c t a t pH 7.0 and 10.0  preincubation t e s t with three Salmonella  tester strains  (TA98,  TA100 and TA102) was used f o r t h i s study.  S t r a i n TA102 used used t o d e t e c t  i f any o x i d a t i v e mutagens were g e n e r a t e d .  A t pH 7.00 ± 0.01, no i n c r e a s e  in h i s  +  without  r e v e r t a n t s was observed i n t e s t e r s t r a i n s TA98 and TA100 w i t h o r S9 l i v e r microsomal mixture.  There was a s l i g h t i n c r e a s e i n h i s  +  r e v e r t a n t s on s t r a i n TA102 a t 108 mg e q u i v a l e n t o f b e t e l nut p e r p l a t e i n the absence o f S9.  A t t h i s dose, a s l i g h t r e d u c t i o n i n m u t a g e n i c i t y  observed i n t h e p r e s e n c e o f S9. be made based on t h i s r e s u l t  was  However, no d e f i n i t i v e c o n c l u s i o n c o u l d  (Table 4 ) .  A t pH 10.00 ± 0.01, a c o n d i t i o n which was f e a s i b l e f o r o x i d a t i o n o f the p h e n o l i c compounds i n the water e x t r a c t t o generate 2 ° 2 ' H  mutagenicity  was observed i n a l l t e s t e r s t r a i n s .  r e v e r s i o n frequency  n  °  n  c  r  e  a  s  e  i  No d i f f e r e n c e i n  was observed between t h e two pH l e v e l s  43  i  (Table 5 ) .  n  TABLE 4 MUTAGENIC EFFECT OF BETEL NUT WATER EXTRACT AT pH 7.00 ± 0.05  Average Number o f R e v e r t a n t s p e r P l a t e ± S.D. TA98 Concentration (mg/plate)  TA100  TA102  -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  -S9  +S9  TABLE 5 MUTAGENIC EFFECT OF BETEL NUT WATER EXTRACT WITH LIME AT pH 10.00 ± 0.05  Average Number o f R e v e r t a n t s p e r P l a t e ± S.D. TA98 Concentration (mg/plate)  TA100  -S9  +S9  108.0  24±3  86.4  TA102  -S9  +S9  -S9  +S9  23±1  103±5  113±8  290+6  292+9  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  24±3  26±2  94±3  101±5  249110  23718  Lime a l o n e 20 y l (10 mg/ml)  45  Mutagenicity No  o f B e t e l Tannin a t pH  7.0  enhancement i n m u t a g e n i c i t y  TA100 and TA102, was  observed.  e f f e c t on m u t a g e n i c i t y . between pH  7.00  ± 0.01  and  10.0  on a l l the t h r e e t e s t e r s t r a i n s , TA98,  S9 l i v e r microsomal a c t i v a t i o n showed no  No d i f f e r e n c e i n r e v e r s i o n frequency and  10.00  ± 0.01.  of Hydrogen  L e v i n e t a l . (1982), was  used i n t h i s study.  c o n c e n t r a t i o n as h i g h as 100  Assay f o r 2 ° 2 pH. Levels'. . H  G  e  n  e  r  a  S i n c e t h e r e was  t  e  d  b  Any  W  a  s  a  c  t  u  a  Hy  v  B e t e l Nut  produced.  t  a  mM  Water E x t r a c t and B e t e l Tannin a t  The  t e s t was  of  Two  on s t r a i n TA102 by the b e t e l  7 and  10,  a t e s t was  carried  out  o f p h e n o l i c compounds, namely, a c o l o r i m e t r i c a n a l y s i s performed  c o n t a i n i n g potassium i o d i d e , ammonium molybdate  s t a r c h w i t h the b e t e l nut water e x t r a c t and b e t e l t a n n i n .  c a t a l y z e d the f o r m a t i o n  a  c o n c e n t r a t i o n lower than 100  no e f f e c t on m u t a g e n i c i t y  by m i x i n g a c o l o u r reagent and  Reagent grade ^2^2  by  ( F i g . 26).  t o determine whether an o x i d a t i v e p r o d u c t 2°2'  shown t o respond t o  showed o n l y a d o u b l i n g e f f e c t i n the  nut water e x t r a c t and b e t e l t a n n i n a t pH  H  7).  e f f e c t o r o n l y a s l i g h t i n c r e a s e i n the number of  r e v e r t a n t s per p l a t e  +  mM  on t h i s t e s t e r s t r a i n .  showed e i t h e r a n e g a t i v e his  (Tables 6 and  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  mutagenicity  observed  A t a c o n c e n t r a t i o n of 5 mg/plate,  the b e t e l t a n n i n became t o x i c t o the b a c t e r i a s t r a i n s Mutagenicity  was  from p o t a s s i u m i o d i d e by  with starch to give a purple coloured.solution. maximum a b s o r p t i o n wavelength a t 575  nm.  The  The  molybdate  which r e a c t e d  T h i s p u r p l e c o l o u r had  b e t e l nut water e x t r a c t  b e t e l t a n n i n d i d not i n t e r f e r e w i t h the a b s o r p t i o n a t t h i s wavelength.  46  a  and  TABLE 6 MUTAGENIC EFFECT OF BETEL TANNIN AT pH 7.00 ± 0.05  Average Number of R e v e r t a n t s per P l a t e ± S.D. TA98 Concentration (mg/plate)  TA100  TA102  -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  +S9  TABLE 7 MUTAGENIC EFFECT OF BETEL TANNIN WITH LIME AT pH 10. 00 1 0. 05  Average Number o f R e v e r t a n t s per P l a t e 1 S.D. TA98 Concentration (mg/plate)  TA100  -S9  +S9  10  T  8  T  5  21±3  4  TA102  -S9  +S9  T  93±2  T  -S9  +S9  97+2  17319  17113  97±2  9216  17616  17715  19±1  10813  10419  25715  261111  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  24±5  23±3  9614  234110  25117  Lime alone 20 y l (10 mg/ml)  1  T = toxic  48  9915  A t pH 7.00, t h e b e t e l n u t water e x t r a c t produced 21.7 uM H^O^, whereas b e t e l t a n n i n generated 36.3 uM a t t h e doses used a s i g n i f i c a n t i n c r e a s e i n ^2^2 P ° d  The  b e t e l n u t water e x t r a c t and b e t e l t a n n i n produced 51.2 uM and 83.7 uM o f  H  2°2'  r e s  P  occurred  e c t :  '-  v e ]  -y•  u c t ;  o n  a  t P  However, t h e r e  was  r  >-  (Table 8 ) .  T h i s showed t h a t a t h i g h e r pH, t h e o x i d a t i o n  more r e a d i l y and generated more U^d^.  compared t o a s t a n d a r d c o l o u r reagent  10.00 by the two m i x t u r e s .  H  The v a l u e s  process  obtained  curve by a d d i n g a known amount o f pure ^2^2  were  t  ^  0  S  ( F i g . 27).  C l a s t o g e n i c E f f e c t o f B e t e l Nut Water E x t r a c t a t pH 7.0 and 10.0 Since the Salmonella  mutagenicity  assay showed a n e g a t i v e  response t o  b e t e l n u t water e x t r a c t and b e t e l t a n n i n , and s i n c e t h e ^2°2 c o l o r i m e t r i c a s s a y supported t h e i d e a t h a t ^2 ~ 2 ^ ^ ° l v e d , another b i o l o g i c a l (  system was r e q u i r e d .  >  ia<  test  e v  A study on c l a s t o g e n i c a c t i v i t y i n CHO c e l l s by these  m i x t u r e s was c a r r i e d o u t . The  frequencies  o f chromosome a b e r r a t i o n s  to the m i x t u r e s a r e shown i n T a b l e  9.  i n CHO c e l l s exposed f o r 3 h r  At a concentration  as low as 1 mg  e q u i v a l e n t o f b e t e l n u t p e r ml, t h e r e was a s i g n i f i c a n t i n c r e a s e o f 15% metaphase p l a t e s w i t h  a t l e a s t one c h r o m a t i d break o r exchange.  Exchanges  which were s c o r e d as p o s i t i v e i n c l u d e d c h r o m a t i d breaks and exchanges, monoor m u l t i - r a d i a l s .  C a t a l a s e , which changed ^2°2  as a p o s s i b l e i n d i c a t o r . concurrently  and  w  a  t  e  r  ^  anc  ox  Y9 > en  w  a  o f 0.1 mg c a t a l a s e / m l  s  used  added  t o t h e e x t r a c t , t h e t o x i c i t y by t h e b e t e l n u t water e x t r a c t a t  5 mg e q u i v a l e n t / m l concurrently  At a concentration  tC>  was reduced.  A 5% S9 microsomal a c t i v a t i o n m i x t u r e added  t o t h e b e t e l n u t water e x t r a c t showed a r e d u c t i o n i n t o x i c i t y  c l a s t o g e n i c i t y on the CHO c e l l s a t 1.0 t o 5.0 mg  49  equivalent/ml.  F i g u r e 26. M u t a g e n i c i t y o f S. typhimurium concentrations of  (TA102) f o l l o w i n g exposure o f v a r i o u s  (• ). The x±S.D. shown were o b t a i n e d from  two prer-iiicubation experiments w i t h t r i p l i c a t e p l a t i n g  F i g u r e 27. R e f e r e n c e curve f o r t h e d e t e r m i n a t i o n o f H^O^  each.  concentrations.  Known q u a n t i t i e s o f c o m m e r c i a l l y - a v a i l a b l e H^O^ were assayed w i t h the p o t a s s i u m i o d i d e - s t a r c h c o l o r r e a g e n t and p l o t t e d v s absorbance a t 575nm. T h i s assay was l i n e a r between 1 and lOOuM. P l o t t e d a r e x±S.D. o b t a i n e d from t h r e e s e t o f experiments.  'SO  J'J.  500H  200 H  F i g u r e 26  (above).  F i g u r e 27  (right).  2  O  2  Cm M  ]  H  50a  50  2  Q  2  C  M  M)  100  TABLE 8 AMOUNT OF H 0 (1.0 mg/ml) 2  2  GENERATED BY BETEL NUT WATER EXTRACT (10.8 mg/ml) AND BETEL TANNIN  H 0 2  pH = 7.00 ± 0.01  2  (UM) pH = 10.00 ± 0.01  B e t e l n u t water e x t r a c t  21.7 ± 3.1  51.2 ± 4.8  Betel  36.3 ± 4.5  83.7 ± 7.2  tannin  51  TABLE 9 CLASTOGENIC ACTIVITY OF BETEL NUT WATER EXTRACT AT pH 7.00 ± 0.05 Average P e r c e n t Metaphase P l a t e s w i t h Chromatid Breaks and/or Exchanges (±S.D.). B e t e l Nut Water E x t r a c t (mg e q u i v a l e n t / m l )  Wash Medium  +Catalase (0.1 mg/ml)  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  1±1  0  1 2  +S9 (5%)  T = toxic 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 p e r p l a t e  52  observed.  When t h e pH o f t h e b e t e l n u t water e x t r a c t was a d j u s t e d t o 10 by t h e a d d i t i o n o f l i m e , t h e c l a s t o g e n i c a c t i v i t y was i n c r e a s e d . S9 and c a t a l a s e decreased  t h e frequency  o f chromosome a b e r r a t i o n s .  appeared t o e x e r t a g r e a t e r r e d u c t i o n e f f e c t . a t 1.0 mg e q u i v a l e n t / m l was  e i n  S9  The i n c r e a s e i n c l a s t o g e n i c i t y  by r a i s i n g t h e pH o f t h e b e t e l n u t water e x t r a c t  n o t due t o t h e a d d i t i o n o f lime  t o more ^2°2 k  Once a g a i n , 5%  (Table 10).  T h i s i n c r e a s e may be due  9 " generated.  C l a s t o g e n i c E f f e c t o f B e t e l T a n n i n and Tannic  A c i d a t pH 7.0 and 10.0  B e t e l t a n n i n a t pH 7 showed a s i g n i f i c a n t i n c r e a s e i n chromosomal breakage a t 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 a l s o reduced t h e t o x i c i t y a p p l i e d by t h e b e t e l t a n n i n . destroyed  A 5% S9 l i v e r microsomal m i x t u r e  this clastogenic effect.  completely  In a d d i t i o n , S9 a l s o reduced t h e t o x i c  e f f e c t as observed i n t h e case o f t h e b e t e l n u t water e x t r a c t (Table 1 1 ) . When lime was added t o t h e b e t e l t a n n i n s o l u t i o n t o i n c r e a s e t h e a l k a l i n i t y t o pH 10, b o t h t h e t o x i c and c l a s t o g e n i c a c t i v i t y were r a i s e d compared t o t h a t a t n e u t r a l pH. i n c r e a s e d by approximately  The frequency  50% a t 0.015 mg/ml.  o f chromosome breakage was Catalase  (0.1 mg/ml) and 5%  S9 microsomal m i x t u r e added t o t h e b e t e l t a n n i n s o l u t i o n reduced both t h e t o x i c i t y and c l a s t o g e n i c i t y e x e r t e d by t h e b e t e l t a n n i n . not show any c l a s t o g e n i c a c t i v i t y Tannic  a c i d was a l s o a n a l y z e d  Again,  lime d i d  (Table 1 2 ) . f o r i t s c l a s t o g e n i c e f f e c t f o r comparison.  A t t h e same c o n c e n t r a t i o n , pure t a n n i c a c i d c o n s i s t e n t l y showed a s m a l l e r frequency was  o f chromosomal a b e r r a t i o n s than b e t e l t a n n i n  (Table 13).  When lime  added t o t a n n i c a c i d t o b r i n g t h e pH t o 10, an enhancement of t h e c l a s t o -  g e n i c e f f e c t was observed  (Table 14).  C a t a l a s e and an S9 m i x t u r e reduced both  53  TABLE 10 CLASTOGENIC ACTIVITY OF BETEL NUT WATER EXTRACT WITH LIME AT pH 10.00 ± 0.05  Average P e r c e n t Metaphase P l a t e s w i t h Chromatid Breaks and/or Exchanges (±S.D.) B e t e l Nut Water E x t r a c t (mg e q u i v a l e n t / m l )  +Catalase (0.1 mg/ml)  Wash Medium  5.0  T  3.0  T  2.0  MI  1.0  1  T MI  +S9 (5%)  9±2 2  5±2  12±2  4±1  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  1±1  0  0  Lime alone 20 u l (10 mg/ml)  T = toxic 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 p e r p l a t e  54  observed.  TABLE 11 CLASTOGENIC ACTIVITY OF BETEL TANNIN AT pH 7.00 ± 0.05  Average  P e r c e n t Metaphase P l a t e s w i t h Chromatid Breaks and/or Exchanges (±S.D.)  B e t e l Tannin (mg/ml)  Wash Medium  +Catalase (0.1 mg/ml)  0.50  T  T  0.25  T  MI  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  1  +S9 (5%)  8±2 2  6±2  T = toxic i  'MI = m i t o t i c  inhibition:  fewer than 40 d i p l o i d metaphases p e r p l a t e  55  observed.  TABLE 12 CLASTOGENIC ACTIVITY OF BETEL TANNIN WITH LIME AT pH 10.00 ± 0.05  Average  P e r c e n t Metaphase P l a t e s w i t h Chromatid and/or Exchanges (±S.D.)  Breaks  B e t e l Tannin (mg/ml)  Wash Medium  +Catalase (0.1 mg/ml)  +S9 (5%)  0.50  T  T  15±3  0.25  T  T  10±2  0.125  T  MI  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  1  2  0  0  1±1  1±1  Lime a l o n e 20 y l (10 mg/ml)  1±1  0  2±1  T = toxic >  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 p e r p l a t e  56  observed.  TABLE 13 CLASTOGENIC ACTIVITY OF TANNIC ACID AT pH 7.00 ± 0.05  Average  Tannic A c i d (mg/ml)  0.25  P e r c e n t Metaphase P l a t e s w i t h Chromatid and/or Exchanges (±S.D.) +Catalase (0.1 mg/ml)  Wash Medium  T  MI  1  Breaks  +S9 (5%)  2  13±2  12±2  8±1  0.125  MI  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 = toxic i 'MI = m i t o t i c  inhibition:  fewer than 40 d i p l o i d metaphases p e r p l a t e  57  observed.  TABLE 14 CLASTOGENIC ACTIVITY  OF TANNIC ACID WITH LIME AT pH 10.00 ± 0 . 0 5  Average P e r c e n t Metaphase P l a t e s w i t h Chromatid Breaks and/or Exchanges (±S.D.) Tannic A c i d (mg/ml)  0.25 0.125  +Catalase (0.1 mg/ml)  Wash Medium  T  1 MI  MI  2  +S9 (5%)  14±2  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 = toxic  •  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  58  p e r p l a t e observed.  the t o x i c and c l a s t o g e n i c a greater  e f f e c t a t both pH l e v e l s .  A g a i n , 5% S9  e f f e c t than c a t a l a s e a t 0.1 mg/ml on r e d u c t i o n  clastogenic effects.  59  exhibited  of the t o x i c and  DISCUSSION  Food and  Cancer  E p i d e m i o l o g i c a l i n v e s t i g a t i o n s have i m p l i c a t e d s e v e r a l a s p e c t s o f d i e t and  lifestyle  i n the e t i o l o g y o f cancer  (Table 15).  Peers e t 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 d i e t a r y l e v e l s of the c a r c i n o g e n i c a f l a t o x i n s and primary Nitrosamines  ( I s s e n b e r g , 1976)  c h e m i c a l components which may genesis.  hepato-  l i v e r cancer i n c e r t a i n a r e a s of A f r i c a .  and s a c c h a r i n (Reuber, 1978)  are o t h e r  be important c o n t r i b u t o r s t o c h e m i c a l c a r c i n o -  However, an i n c r e a s i n g number and d i v e r s i t y of compounds w i t h  the  c a p a c i t y t o i n h i b i t the o c c u r r e n c e o f n e o p l a s i a when a d m i n i s t e r e d p r i o r t o o r s i m u l t a n e o u s l y w i t h exposure t o c a n c e r - c a u s i n g agents For example, b u t y l a t e d h y d r o x y a n i s o l e  are b e i n g  identified.  (BHA), an a n t i o x i d a n t u t i l i z e d  to  i n c r e a s e s h e l f - l i f e , p r e v e n t s the i n d u c t i o n o f e x p e r i m e n t a l tumours i n rodents  (Wattenberg, 1975).  Other n a t u r a l l y o c c u r r i n g n o n - n u t r i e n t c o n s t i t u e n t s o f foods have been implicated i n modifying c a r c i n o g e n i c i t y .  C r u c i f e r o u s v e g e t a b l e s such  as  B r u s s e l s p r o u t s , cabbage and c a u l i f l o w e r , c o n t a i n i n d o l e s which have been shown t o i n c r e a s e the metabolism of c a r c i n o g e n i c p o l y c y c l i c aromatic carbons  (Pantuck  et a l . ,  1976;  hydro-  Wattenberg and Loub, 1978).  I t i s g e n e r a l l y b e l i e v e d t h a t most c h e m i c a l c a r c i n o g e n s are compounds which c o n t a i n h i g h l y r e a c t i v e e l e c t r o n - d e f i c i e n t r e g i o n s .  In a d d i t i o n ,  c h e m i c a l c a r c i n o g e n s can a l s o induce the mixed f u n c t i o n o x i d a s e s t o a c t i v a t e the c a r c i n o g e n s i n t o e l e c t r o p h i l i c s p e c i e s (Wattenberg e t a l . , can b i n d c o v a l e n t l y and n o n - e n z y m a t i c a l l y e l e c t r o n - r i c h s i t e s p r e s e n t i n DNA, ( M i l l e r and M i l l e r ,  RNA  1976).  t o the abundant n u c l e o p h i l i c o r  and p r o t e i n s i n t a r g e t t i s s u e s  1977).  60  They  TABLE 15 PROPORTION OF CANCER CASES ATTRIBUTED TO VARIOUS DIFFERENT FACTORS BY DIFFERENT AUTHORS  P e r c e n t o f a l l Cancer Cases i n : England, Birmingham Region, Based on H i g g i n s o n and Muir (1979) Factor or Class of Factors  Tobacco  Male  Female  United States, Based on Wynder and G o r i (1977)* Male  Female  30  7  28  8  Tobacco/alcohol  5  3  4  1  Diet  -  -  40  57  30  63  -  -  6  2  4  2  10  10 8  8  Ionizing radiation  1  1  Iatrogenic  1  1  -  -  Exogenous hormones  -  -  -  4  Congenital  2  2 16  20  15  11  Lifestyle Occupation Sunlight  Unknown  *Deduced from h i s t o g r a m s ; unknown. From D o l l and Peto  non-environmental f a c t o r s equated w i t h c o n g e n i t a l  (1981).  61  and  Plant Phenolics  and  T h e i r Role i n Human  Plant phenolics i n man's d a i l y d i e t . grams per day  Carcinogenesis  c o n s t i t u t e a major p o r t i o n of the components consumed The  e s t i m a t e d range i s between 600  (Maga, 1978).  p l a y i n human  Many d e l e t e r i o u s e f f e c t s o f p l a n t p h e n o l i c s  i s shown i n T a b l e 16  s t u d i e s on p h e n o l i c  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 v a r i e t y of chemical carcinogens  cinnamic a c i d , c a f f e i c a c i d and B(a)P-induced neoplasia S t i c h and  Rosin  under a v a r i e t y of  on t h e i r r o l e i n  carcinogenic 1979).  a c t i o n s of a wide In p a r t i c u l a r ,  d e r i v a t i v e s o f cinnamic a c i d  (o-hydroxy-  f e r u l i c a c i d ) were e f f e c t i v e i n  suppressing  i n the forestomach of r a t s  (1984) have a l s o r e p o r t e d  (Wattenberg e t a l . , 1980).  that phenolic  derivatives  i n h i b i t the mutagenic a c t i v i t i e s o f b o t h d i r e c t - a c t i n g c a r c i n o g e n s and c a r c i n o g e n s i n the presence of mixed f u n c t i o n o x i d a s e s . on t o demonstrate t h a t they are a b l e mutagenic and  carcinogenic  A  compounds have been c a r r i e d out  (Wattenberg, 1972,  three n a t u r a l l y occurring phenolic  carcinogenesis.  (Hanham, 1983).  However, c o n f l i c t i n g r e p o r t s have been p u b l i s h e d Extensive  several  have been r e p o r t e d .  composite l i s t o f g e n o t o x i c events caused by p h e n o l i c s  carcinogenesis.  and  T h e i r b i o l o g i c a l a c t i v i t y i s t h e r e f o r e o f some  i n t e r e s t i n d e t e r m i n i n g the r o l e which they may  test conditions  mg  t o reduce the in  n i t r o s o compounds.  In man,  The  vitro  pre-  r e p o r t a l s o goes formation  phenolics  of  were  observed t o reduce l e v e l s of n i t r o s o p r o l i n e i n u r i n e f o l l o w i n g  administration  of n i t r a t e , p r o l i n e and  nitrosation  phenolic  t e s t substances.  of p r o l i n e i n the presence o f p h e n o l i c s  The  in vitro  used i n t h i s study, as demonstrated  by HPLC a n a l y s i s , a l s o shows a c o r r e l a t i o n w i t h t h i s o b s e r v a t i o n To  f u r t h e r complicate  carcinogenesis,  other  the i s s u e of p h e n o l i c s  and  (Figs  t h e i r r o l e i n human  i n v e s t i g a t o r s have suggested t h a t some p h e n o l i c  62  5-7).  compounds  TABLE 16 GENOTOXIC EFFECTS MEASURED WITH PLANT PHENOLICS PHENOLIC GROUP  ASSAY  ORGANISM  REFERENCE  Simple Phenols : Phenol  S i s t e r chromatid exchange  Human lymphocytes  Morimoto and W o l f f  Catechol  P o i n t mutation M i t o t i c crossover S i s t e r chromatid exchange Nephrotoxicity Chromosome a b e r r a t i o n s  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  Kunz e t a l . (1980) Kunz e t a l . (1980) Morimoto and W o l f f (1980) C a l d e r e t a l . (1975) S t i c h e t a l . (1981a)  P o i n t mutation  _S. typhimurium  P o i n t mutation Micronuclei Chromosome a b e r r a t i o n s  E. c o l i Mice CHO c e l l s  DNA i n h i b i t i o n Micronuclei  Mice Mice  Pyrogallol  Resorcinol  Benzoic Gallic  Ben-Gurion (1979) Yamaguchi (1981) B i l i m o r i a (1975) M i t r a and Manna (1977) Gocke e t a l . (1981) S t i c h e t a l . (1981a) S e i l e r (1977) M i t r a and Manna  (1977)  Acids: acid  P o i n t mutation Chromosome a b e r r a t i o n s  Protocatechuic Vanillic Cinnamic  (1980)  acid  acid  S_. typhimurium CHO c e l l s  Yamaguchi (1981) S t i c h e t a l . (1981a)  Chromosome a b e r r a t i o n s  CHO  cells  S t i c h e t a l . (1981a)  Chromosome a b e r r a t i o n s  CHO  cells  S t i c h e t a l . (1981a)  Acids:  Caffeic acid  Gene c o n v e r s i o n Chromosome a b e r r a t i o n s  _S. c e r e v i s i a e CHO c e l l s  S t i c h and Powrie (1982) S t i c h e t a l . (1981a)  Chlorogenic a c i d  Gene c o n v e r s i o n Chromosome a b e r r a t i o n s  _S. c e r e v i s i a e CHO c e l l s  S t i c h e t a l . (1981a) S t i c h e t a l . (1981b)  From Hanham  (1983).  _  such as r e s o r c i n o l , p - n i t r o s o p h e n o l , and p h l o r o g l u c i n o l than an  compounds.  reaction with n i t r i t e ,  T h i s may  e x p l a i n the  c a r r y r e s o r c i n o l moiety  can  catalyze  n i t r i t e reduction  they may  A p o s s i b l e mechanism f o r the  increasing  D u r i n g an  i n forming the c a t a l y s t and (Fig.  of the  phenol  initial  This postulated  step,  nitrosating  (thought t o be  a  mechanism i m p l i e s  that  amounts of a c a t a l y t i c a l l y a c t i v e  Accordingly,  to  t h e r e would be  64  species. phenolic  a l a r g e excess of p h e n o l i c  n i t r o s a t i n g agent would be  11).  phenolic  intermediate,  o f the n i t r o s a t i n g  the optimum r a t i o f o r n i t r i t e  would i n h i b i t n i t r o s a t i o n s i n c e the  C-nitroso  compounds on n i t r o -  c o n c e n t r a t i o n s o f the C - n i t r o s o  shown i n F i g . 9.  on  f o r m a t i o n of  formed which f u r t h e r r e a c t w i t h the  T h i s mechanism would e x p l a i n  catalytic reaction  and  oxide.  c o n s e q u e n t l y t o a reduced c o n c e n t r a t i o n  compound, as  nitroso  i n h i b i t n i t r o s a t i o n by  t o generate a more p o w e r f u l n i t r o s a t i n g agent  compound l e a d t o i n c r e a s i n g  up  therefore  been proposed by Walker e t a l . (1982). be  the  c a t a l y s i s of p h e n o l i c  n i t r o s o q u i n o n e oxime d e r i v a t i v e ) .  and  e i t h e r by  Phenolics  coupled with o x i d a t i o n  r e d u c i n g the n i t r o s a t i n g agent t o n i t r i c  assay systems c o n t a i n i n g  f o r m a t i o n of  C a t a l y t i c E f f e c t by  t o NO,  t o guinones, a c t as scavengers;  d e r i v a t i v e s may  the  C-nitroso  (Fig. 9).  Those p h e n o l s which consume n i t r i t e ,  C-nitroso  r e a d i l y form  that  f o r m a t i o n of n i t r o s o p r o l i n e , m o l e c u l e s which  P o s s i b l e Mechanism o f I n h i b i t i o n and N i t r o s a t i o n Reactions  s a t i o n has  rather  o b s e r v e d c a t a l y t i c a c t i v i t i e s of 2,4-  3,5-dihydroxybenzoic a c i d on the  p h e n o l s or by  1982)  P i g n a t e l l i e t a l . (1982) a l s o i n d i c a t e d  compounds w i t h r e s o r c i n o l moiety, which can  d e r i v a t i v e s by  species  ( P i g n a t e l l i et a l . ,  (Walker e t a l . , 1982) , showed a c a t a l y t i c e f f e c t  inhibitory effect.  phenolic  catechin  compound  c o m p l e t e l y used  l e f t none t o c o n t i n u e  the  How  are t h e s e two  phenomena - i n h i b i t o r y e f f e c t and  applied to a p h y s i o l o g i c a l situation? found i n human s a l i v a i s 0.097 mM above, the several  amount o f p h e n o l i c s  grams per day.  would not be  The  catalytic effect -  average c o n c e n t r a t i o n  (Walters e t a l . , 1979)  and,  of  as mentioned  consumed by humans i s between 600  mg  Thus, p h y s i o l o g i c a l l y , i t would seem t h a t  enough n i t r i t e t o c o n t i n u e the  c a t a l y t i c e f f e c t o f n i t r o s a t i o n by p h e n o l i c However, p h y s i o l o g i c a l l y , i t i s o n l y  catalytic reaction.  and  there Hence  compounds i s c h e m i c a l l y  p r o b a b l e , but  nitrite  n o n e t h e l e s s an  the  feasible.  unreasonable  proposition. Relation  of S a l t e d F i s h t o Human C a r c i n o g e n e s i s  Salted 1971;  Ho  f i s h has  l o n g been thought t o be  e t a l . , 1978;  shown t h a t the u r i n e  Huang e t a l . , 1978;  (D.  There are a l s o r e p o r t s  H.F.  communication).  Huang e t a l . (1978) r e p o r t e d  g r e a t l y enhanced i t s mutagenic a c t i v i t y .  fish  the Ames S a l m o n e l l a  Mower, p e r s o n a l  mutagenic a c t i v i t i e s were observed i n both TA98 and  (Ho,  been  which i n d i c a t e t h a t t h e r e are more than one  of n i t r o s o compound i n s a l t e d f i s h .  were a type of  I t has  s a l t e d / d r i e d Hawaiian  d e t e c t e d by HPLC and  I c h i n o t s u b o and  i n human c a n c e r s  Yang, 1980).  e x c r e t e d from humans e a t i n g  c o n t a i n s mutagens which can be m u t a g e n i c i t y assay  involved  TA100, and  type  that  t h a t S9  mix  They proposed t h a t these mutagens  nitrosamine.  Other r e p o r t s  (Marquardt e t 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, p r o b a b l y nitrosamides.  In t h i s study, the r e s u l t s i n d i c a t e t h a t the mutagenic components  i n s a l t e d f i s h e x t r a c t a r e w a t e r - s o l u b l e and and  13).  In a d d i t i o n , t h e r e are  d i r e c t - a c t i n g compounds ( F i g s  components p r e s e n t which can  mutagenic n i t r o s o compounds w i t h the a d d i t i o n o f n i t r i t e  65  be  converted  ( F i g . 14;  Table  12 to 1).  However, the a c t u a l c h e m i c a l n a t u r e o f the mutagenic f r a c t i o n s w i l l have t o l be determined  by HPLC and/or gas chromatography combined w i t h a mass  spectrometer. N i t r o s a t i o n R e a c t i o n s Modulated  i n a Complex M i x t u r e  There has always been a concern as t o whether o r not the above c h e m i c a l s t u d i e s on p u r i f i e d compounds can be used t o p r e d i c t how a r e modulated i n a complex mixture t h e r e f o r e made t o s i m u l a t e in vitro w i t h i n the e n t i r e organism.  such as food p r o d u c t s .  nitrosation reactions Attempts  those c o n d i t i o n s which may  Such approaches  may  were  actually  occur  b r i d g e the gap between  e p i d e m i o l o g i c a l e v i d e n c e p o i n t i n g t o a l i n k between the consumption o f f o o d p r o d u c t s and an e l e v a t e d r i s k f o r g a s t r i c cancer and l e a d t o an of  the c h e m i s t r y o f n i t r o s a t i o n In  t h i s study, we  understanding  reactions.  t r i e d t o s i m u l a t e the events which may  o c c u r d u r i n g 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 , t e a s and p l a n t p h e n o l i c s which a r e p r e s e n t i n v e g e t a b l e s . f i s h was  An aqueous f r a c t i o n o f a Chinese  n i t r o s a t e d i n the absence or presence  p l a n t p h e n o l i c s , t e a s and human s a l i v a .  salted  of s e v e r a l n a t u r a l l y o c c u r r i n g  Catechin, chlorogenic acid,  gallic  a c i d , p y r o g a l l o l and t a n n i c a c i d , when added t o the n i t r o s a t i o n m i x t u r e s , e x e r t e d a s t r o n g i n h i b i t o r y e f f e c t on the f o r m a t i o n o f d i r e c t - a c t i n g mutagens which were d e t e c t e d by the Ames S a l m o n e l l a assay.  These r e s u l t s o b t a i n e d on  the complex m i x t u r e o f a f i s h e x t r a c t a r e i n good agreement w i t h the o b s e r v a t i o n t h a t t h e s e p h e n o l i c s i n h i b i t NPRO f o r m a t i o n , and i n o t h e r simple model systems such as the m u t a g e n i c i t y r e s u l t i n g from in vitro methylurea  ( S t i c h e t a l . , 1982c).  n i t r o s a t i o n of  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  a l s o observed w i t h the Chinese, Japanese and Ceylanese which a r e a c t u a l l y i n g e s t e d .  66  was  teas at concentrations  These o b s e r v a t i o n s p r o v i d e the b a s i s t h a t i n h i b i t i o n o f n i t r o s a t i o n n a t u r a l l y o c c u r r i n g p h e n o l i c s and t e a s on simple p u r i f i e d compounds can occur i n complex food m i x t u r e s as t o what may  occur i n  In o r d e r t o prove  in vitro.  Hence i t p r o v i d e s an  indication  t h a t p h e n o l i c s do compete w i t h the amines f o r the  t h a t a l l p h e n o l i c s , t e a s and  ( F i g s 22-25).  also  vivo.  n i t r o s a t i n g agents, the n i t r i t e d e p l e t i o n assay was observed  by  c a r r i e d out.  It  s a l i v a d e p l e t e d the n i t r i t e  was  available  In a d d i t i o n , s a l t e d f i s h aqueous e x t r a c t a l s o r e a c t e d w i t h  n i t r i t e t o generate  the azo compound  ( F i g . 21).  Thus the i n h i b i t i o n  n i t r o s a t i o n p r o d u c t f o r m a t i o n by p h e n o l i c s , t e a s and s a l i v a i s due a b i l i t y t o compete w i t h s a l t e d f i s h e x t r a c t and p r o l i n e f o r the  of  to t h e i r  available  nitrite. R e l a t i o n of B e t e l Nut Chewing t o Human C a r c i n o g e n e s i s B e t e l q u i d chewing has been a common p r a c t i c e f o r a l o n g time i n Southeast A s i a , C e n t r a l A s i a and c o u n t r i e s of the Western P a c i f i c .  The h i g h i n c i d e n c e  of o r a l cancer i n t h e s e a r e a s i s r e p o r t e d t o be a s s o c i a t e d w i t h the h a b i t of b e t e l q u i d chewing  (Muir and K i r k , 1960;  J u s s a w a l l a , 1976).  b e t e l q u i d induces neoplasms i n e x p e r i m e n t a l animals Ranadive e t a l . ,  In a d d i t i o n ,  (Bhide e t a l . ,  1979;  1979).  There are a l s o r e p o r t s on the c l a s t o g e n i c a c t i v i t y of the s a l i v a of b e t e l q u i d chewers ( S t i c h and S t i c h , 1982;  Stich et a l . ,  e l e v a t e d the frequency of m i c r o n u c l e a t e d c e l l s chromosome breakage i n CHO  cells.  1983a), i n t h a t i t  i n the b u c c a l mucosa and  caused  S h i v a p u r k a r e t a l . (1980) r e p o r t e d t h a t the  n i t r i t e and n i t r o s a m i n e l e v e l s i n the s a l i v a of b e t e l nut chewers were a l s o elevated.  However, b e t e l nut e x t r a c t has a l s o been shown t o i n h i b i t  endogenous n i t r o s a t i o n i n humans ( S t i c h e t a l . , i n r e g a r d t o human c a r c i n o g e n e s i s s t i l l  1983b).  the  Thus b e t e l nut chewing  remains an u n r e s o l v e d  issue.  In t h i s study, b e t e l nut water e x t r a c t and b e t e l t a n n i n major p h e n o l i c components o f b e t e l nut) the c l a s t o g e n i c a c t i v i t y on CHO  cells  (one of  the  showed a d o s e - r e l a t e d i n c r e a s e i n  (Tables 9 and  11).  These r e s u l t s : '  c o r r e l a t e w i t h the data on the s a l i v a of b e t e l q u i d chewers as shown by S t i c h and  Stich  (1982).  I t i s o f i n t e r e s t t o note t h a t the i n c i d e n c e o f o r a l cancer  i s greatly  i n c r e a s e d i n chewers of l i m e - c o n t a i n i n g m i x t u r e s which cause an a l k a l i n e (H.F.  S t i c h , personal  communication).  The  s a l i v a of b e t e l nut  contains r e l a t i v e l y large q u a n t i t i e s of phenolics. m i x t u r e enhanced o x i d a t i o n which gave r i s e t o ^2°2 e t a l . , 1983).  The  generation  r a d i c a l s concomitantly  o f 2 ° 2 ' superoxide, H  chewers  Lime added t o the an<  ^  ^  r e e  r a <  hydroxyl  3i  saliva  c a  and  ls  chewing  (Hanham  semiquinone  enhanced the c l a s t o g e n i c i t y of the p h e n o l i c s .  The  d a t a on t h e c l a s t o g e n i c e f f e c t o f b e t e l nut water e x t r a c t and b e t e l t a n n i n i n t h e presence o f lime supported assay f o r H^O^  t h i s p r o p o s i t i o n (Tables 10 and  a l s o showed t h a t a t a l k a l i n e pH  from b e t e l nut water e x t r a c t and b e t e l t a n n i n Salmonella  mutagenicity  were not p o s i t i v e .  T h i s was  l e v e l s more H^O^  12).  was  generated  (Table 8 ) .  t e s t s on t e s t e r s t r a i n s TA98, TA100 and probably  An  because these  t o the o x i d a t i v e mutagens a t the c o n c e n t r a t i o n used.  TA102  s t r a i n s were not s e n s i t i v e The  amount of  H^O^  r e q u i r e d f o r TA102 t o be p o s i t i v e i s much h i g h e r than the amount generated by b o t h b e t e l nut water e x t r a c t and b e t e l t a n n i n S a l i v a and During  i t s Role i n  (Table 8;  F i g . 26) .  Carcinogenesis  the consumption o f a meal, chemicals  beverages w i l l become mixed w i t h s a l i v a .  consumed through food  T h i s m i x i n g can  and  lead to a reduction  or enhancement of mutagenic dr c a r c i n o g e n i c a c t i v i t y o f the i n g e s t e d mutagens and c a r c i n o g e n s .  S a l i v a has proven t o be an a c t i v e i n h i b i t o r of the  68  mutagenicity  of s e v e r a l man-made ( N i s h i o k a e t a l . , and R o s i n , 1984) c a r c i n o g e n s .  1981) and n a t u r a l l y o c c u r r i n g ( S t i c h  The q u e s t i o n was t h e r e f o r e r a i s e d as t o the  e f f e c t o f s a l i v a on t h e f o r m a t i o n o f mutagenic compounds. The presence  o f n i t r i t e i n human s a l i v a i s well-known  The n i t r i t e appears  ( S a v o s t i a n o v , 1937).  t o be t h e p r o d u c t o f m i c r o b i a l r e d u c t i o n o f n i t r a t e ,  which c i r c u l a t e s through t h e s a l i v a r y glands i s t h e presence  of n i t r i t e  i n human s a l i v a  (Tannenbaum e t a l . ,  (Tannenbaum e t a l . ,  1974).  i n human s a l i v a which causes n i t r o s a m i n e 1974, 1978;  It  formation  Spiegelhalder et a l . ,  1976).  In a d d i t i o n , S h i v a p u r k a r e t a l . (1980) showed t h a t t h i s i n c r e a s e d n i t r i t e l e v e l may be r e l a t e d t o t h e development o f o r a l cancer i n I n d i a . However, i n t h i s study, s a l i v a e x e r t e d a r e l a t i v e l y s t r o n g s u p p r e s s i v e e f f e c t on t h e appearance o f mutagenic n i t r o s a t i o n p r o d u c t s when p r e s e n t d u r i n g n i t r o s a t i o n o f t h e f i s h e x t r a c t and NPRO f o r m a t i o n  ( F i g s 8 and 16).  This  o b s e r v a t i o n was i n good c o r r e l a t i o n w i t h t h e study on model n i t r o s a t i o n r e a c t i o n s i n t h e presence The  o f s a l i v a by S t i c h e t a l .  (1982b).  complex c o m p o s i t i o n o f s a l i v a w i l l make i t d i f f i c u l t t o i d e n t i f y  those c h e m i c a l s 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 w i t h  nitrite.  Moreover, whole s a l i v a i s n o t o n l y a s o l u t i o n o f c h e m i c a l s , b u t c o n t a i n s a wide a r r a y o f b a c t e r i a , e p i t h e l i a l c e l l s and v a r i o u s b l o o d c e l l s which can influence the r a t e of formation of nitrosamines  (Tannenbaum e t a l . ,  1978).  The q u e s t i o n must be r a i s e d as t o t h e amounts o f mutagens and c a r c i n o g e n s which would s a t u r a t e t h e i n h i b i t o r y c a p a c i t y o f s a l i v a . nut r e l e a s e s numerous c h e m i c a l s i n t o t h e s a l i v a .  The chewing o f b e t e l  I t appears  l i k e l y that  s a l i v a - b o r n e mutagens and c a r c i n o g e n s can then a f f e c t the mucosal t i s s u e s o f the o r a l c a v i t y , pharnyx and, on swallowing, S t i c h and S t i c h  the mucosa o f t h e esophagus.  (1982) demonstrated t h e appearance o f p o t e n t c l a s t o g e n i c ,  mutagenic and c o n v e r t o g e n i c agents  i n the s a l i v a o f v o l u n t e e r s who chewed b e t e l  69  nuts f o r a few  minutes.  the b e t e l n u t s ,  Despite  the i n t e n s i v e s a l i v a t i o n due  the s a l i v a o f chewers was  o f the r e l e a s e d g e n o t o x i c agents. Tannenbaum e t a l . (1981) who  not a b l e t o suppress the  A comparable s u g g e s t i o n  was  activities  made by  suggested t h a t the d e p l e t i o n of the  a n t i n i t r o s a t i o n f a c t o r s o f the g a s t r i c j u i c e was h i g h cancer  t o chewing of  protective  an e s s e n t i a l c o n d i t i o n f o r  risk.  B i o l o g i c a l P r o t e c t i v e Mechanism Organisms have o b v i o u s l y  evolved  mechanisms t o p r o t e c t themselves  the g e n o t o x i c e f f e c t s o f o x i d a t i o n b y - p r o d u c t s , p h e n o l i c s f a c t , an e a r l y e x p l a n a t i o n o b l i g a t e anaerobes was  and  t  o  ±  e  r  a  n  c  e  s  •  l a c k e d t h i s enzyme and were s u b s e q u e n t l y k i l l e d by ^2°2  Catalases  of aerobes  whereas anaerobes w  h  e  n  e x  P  o s e <  ^ t o oxygen  and  p e r o x i d a s e s c a t a l y z e the c o n v e r s i o n of 2 ° 2 H  of ^2^2  t  2°  t  0  0  H  a n c  ^  2 ° -'- 9 2 ° 2  2 H  us  n  H  a  °2" s  the e l e c t r o n donor i n the case o f c a t a l a s e s , or a v a r i e t y of r e d u c t a n t s the case o f p e r o x i d a s e s  a c t i v i t y i n the  observed t o e l i m i n a t e the  s o l u t i o n s of b e t e l tannin  Whether c a t a l a s e had  t r e a t e (  indeed removed a l l ^2^2  ^ s o l u t i o n was  A s i m i l a r degree o f a c t i v i t y was betel tannin. genic  The  a  s  ^^- ^  ver  e  with  clastogenic a c t i v i t y  a l s o e l i m i n a t e d by c a t a l a s e  the of  (Hanham, 1983). and  t o i n f e r t h a t some p o r t i o n of the c l a s t o -  a c t i v i t i e s o f the b e t e l nut water e x t r a c t and due  w  (Tables  o b s e r v e d i n the b e t e l nut water e x t r a c t  T h i s would p e r m i t one  or w i t h o u t lime) was  clastogenic  and b e t e l nut water e x t r a c t  potassium i o d i d e - s t a r c h c o l o r i m e t r i c assay. commercial 2 ° 2 ~  in  ( F r i d o v i c h , 1976) .  Commercially p r e p a r e d c a t a l a s e was  H  and  in air.  These enzymes c a t a l y z e the d i v a l e n t r e d u c t i o n  9-12) .  In  Thus aerobes were thought  t o c o n t a i n c a t a l a s e as a defence mechanism a g a i n s t E^O^,  l e v e l s present  nitrosamines.  f o r the d i f f e r e n t oxygen t o l e r a n c e s  based on ^2^2  against  t o the p r e s e n c e of hydrogen  70  betel tannin peroxide.  (treated with  There a r e enzymes which can m e t a b o l i z e or d e t o x i f y the mutagens or carcinogens.  F o r example, mixed f u n c t i o n o x i d a s e s are a c l a s s of enzymes  i n v o l v e d i n the b i o t r a n s f o r m a t i o n c a r c i n o g e n s and o t h e r  and  o x i d a t i v e a c t i v a t i o n of chemical  exogenous compounds (Autrup, 1982).  l o c a l i z e d p r i m a r i l y i n the microsomal membrane and various  forms o f cytochrome P-450 and  c o n t a i n one  the a s s o c i a t e d  enzymes such as NADPH cytochrome P-450 r e d u c t a s e ,  These enzymes are or more of  electron  the  transport  cytochrome b  r  and NADH  b  cytochrome b^ r e d u c t a s e .  S9 mix  i s one  type of these mixed f u n c t i o n o x i d a s e s  which have been shown t o enhance the c l a s t o g e n i c a c t i v i t y of s e v e r a l  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 a c i d ; S9 mix  a l s o shows no e f f e c t on o t h e r s  ( S t i c h e t a l . , 1981b). of o t h e r p h e n o l i c s fruits  such as r e s o r c i n o l and p y r o g a l l o l  However, S9 shows a decrease i n c l a s t o g e n i c  activity  such as c a t e c h o l , g a l l i c a c i d ( S t i c h e t a l . , 1981b), d r i e d  ( S t i c h e t a l . , 1981c) and  caramel  ( S t i c h e t a l . , 1981d).  In t h i s study, S9 showed no e f f e c t on the mutagenic and a c t i v i t i e s of s a l t e d f i s h aqueous e x t r a c t However, when S9 was mixture, i t exerted  ( F i g s 12,  13,  14;  clastogenic Table 1).  added t o the b e t e l nut water e x t r a c t and a great reduction  i n c l a s t o g e n i c and  the b e t e l nut water e x t r a c t and b e t e l t a n n i n  (Tables  betel  toxic activities  9-12).  These  again p o i n t t o the f a c t t h a t i t i s v e r y d i f f i c u l t t o d e f i n e the of a complex m i x t u r e such as  tannin of  observations  genotoxicity  food.  Unresolved Issues The  question  has been r a i s e d many times as t o whether one  the g e n o t o x i c i t y o f a complex m i x t u r e such as food and as smoking.  Numerous e p i d e m i o l o g i c a l  i s r e l a t e d t o human cancer  ( D o l l and  l i f e s t y l e f a c t o r s such  d a t a have supported the Peto, 1981;  71  can d e f i n e a t a l l  World H e a l t h  i d e a t h a t smoking Organization,  1975).  The  changed.  composition  The  of food, upon e n t e r i n g man,  w i l l become c o n t i n u o u s l y  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 w e l l as w i t h c e l l u l a r substances inumerable.  Some of these  i n t e r a c t i o n s may  while others  l e a d t o the s u p p r e s s i o n  are  c o n t r i b u t e t o an enhancement,  o f the net g e n o t o x i c  a c t i v i t y of  the  substances i n v o l v e d . With r e g a r d t o g e n o t o x i c i t y and spectrum of a p p a r e n t l y  contradictory effects.  e t a l . , 1981b) and mutagenic found t o have a r e l a t i v e l y genotoxic  species  (Brown, 1980).  strong genotoxic  a c t i v i t y o f p h e n o l i c s and  properties.  On  c a r c i n o g e n i c i t y , p h e n o l i c s can e x e r t a They can be c l a s t o g e n i c  (Stich  S i m i l a r l y , t e a i n f u s i o n s were effect  (Nagao e t a l . , 1979).  This  teas could i n d i c a t e p o t e n t i a l carcinogenic  the o t h e r hand, p h e n o l i c s have the c a p a c i t y t o t r a p n i t r o s a t i n g  ( M i r v i s h e t 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 and in vivo  amides i n vitro  ( P i g n a t e l l i e t a l . , 1976;  Walker e t a l . , 1975)  ( S t i c h e t a l . , 1983b).  Does e p i d e m i o l o g i c a l evidence m o r t a l i t y r a t e s due  support  There were h i g h  t o esophageal carcinomas i n Japanese p o p u l a t i o n  consuming l a r g e q u a n t i t i e s o f t e a g r u e l p h e n o l i c - c o n t a i n i n g b e t e l nuts  ( J u s s a w a l l a , 1976) , and  and B r a z i l  r a t e s f o r stomach cancer  a r e r e l a t i v e l y low  e.g.,  (Prudente,  1963).  among  However, the m o r t a l i t y  ( S e g i , 1975)  and b e t e l  1976).  There have a l s o been many arguments on whether d i e t and are the cause of cancer.  matte'-drinking  i n groups w i t h a h i g h i n c i d e n c e of  t e a gruel-consuming Japanese  quid-chewing G u j r a t i s ( J u s s a w a l l a ,  groups  (Hirayama, 1979), among chewers of  i n d i v i d u a l s i n Argentina  esophageal c a n c e r s ,  such a c o n c l u s i o n ?  E p s t e i n and  Swartz (1981) argued t h a t c a n c e r s  caused by e n v i r o n m e n t a l and o c c u p a t i o n a l They c l a i m t h a t the r o l e of l i f e s t y l e  l i f e s t y l e factors  exposure t o c a n c e r - c a u s i n g  f a c t o r s has  72  are  agents.  been exaggerated by those  with  an economic o r i n t e l l e c t u a l investment i n v o l u n t a r y exposures t o c a r c i n o g e n s carcinogens.  They supported  i n t h i s t h e o r y , l a r g e l y by  excluding  and m i n i m i z i n g the r o l e o f o c c u p a t i o n a l  t h e i r p o i n t of view by the r e l a t i v e r i s k s of  cancer o f t h e b l a d d e r and pancreas which were v a r i o u s l y c h a r a c t e r i z e d as r e l a t e d t o o r caused  by smoking (Cole e t a l . , 1971;  Wynder e t a l . , 1973).  However, the r e l a t i v e r i s k s f o r t h e s e c a n c e r s 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 a d d i t i o n , excess b l a d d e r cancer r a t e s had  been  i d e n t i f i e d i n s e v e r a l o c c u p a t i o n a l c a t e g o r i e s , i n c l u d i n g rubber, p a i n t manufacturing  and t e x t i l e - d y e i n g workers (Cole and Goldman, 1975).  They a l s o  c l a i m e d t h a t from an e p i d e m i o l o g i c a l s t a n d p o i n t , a c o r r e l a t i o n between a h i g h f a t d i e t and b r e a s t and c o l o n c a n c e r was with'.a h i g h f a t i n t a k e and To f u r t h e r c o m p l i c a t e of  human c a n c e r s  likely  weak s i n c e t h e r e were p o p u l a t i o n s  l i t t l e bowel cancer the i s s u e , C a i r n s  (Berg, 1975).  (1981) suggested  t h a t the  origin  i s not from c o n v e n t i o n a l mutagens but t h a t they are more  t o be the r e s u l t o f g e n e t i c t r a n s p o s i t i o n s . He c l a i m s t h a t the i s s u e  i s n o t simply whether c o n v e n t i o n a l mutagens are capable o f c a u s i n g cancer, whether they r e a c h our c e l l s  but  i n s u f f i c i e n t q u a n t i t y t o make a major c o n t r i b u t i o n  to our p r e s e n t n a t i o n a l i n c i d e n c e of c a n c e r .  I t i s a q u e s t i o n of the  balance  between the dose o f mutagens and the e f f i c i e n c y of our v a r i o u s pathways f o r DNA  repair. Cancer i s a d i s e a s e of m u l t i f a c t o r i a l e t i o l o g y .  lifestyle  There i s no doubt t h a t  and d i e t a r y f a c t o r s are i n v o l v e d , b u t these two  not the o n l y cause o f cancer. environmental  f a c t o r s are  certainly  Other f a c t o r s such as o c c u p a t i o n a l , g e n e t i c  f a c t o r s i n r e l a t i o n t o c a n c e r cannot be e l i m i n a t e d .  e p i d e m i o l o g i c a l d a t a t h a t most c a n c e r s a r e caused by l i f e s t y l e , o c c u p a t i o n and the environment, c a n c e r would thus seem t o be  73  Based on  diet,  preventable.  and  Following  t h e awareness o f t h e h e a l t h hazards o f a d e t e r i o r a t i n g  e x t e r n a l environment  (Higginson  and Muir, 1973;  f u r t h e r awareness i s c u r r e n t l y d e v e l o p i n g environment which we c a s u a l l y p l a c e  Wynder and G o r i , 1977) , a  t h a t the p a r t o f t h e man-made  i n t o our mouths and swallow has more  f a r - r e a c h i n g h e a l t h consequences than p r e v i o u s l y b e l i e v e d .  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Gann 12.' 451-454 (1981).  83  sodium  APPENDIX I  EXTRACTION PROCEDURE OF SALTED FISH  Salted  fish  Rinsed with d i s t i l l e d  water  V Freeze-dried  Extracted  3x w i t h hexane Centrifuged  Hexane  evaporated  Residue  air-dried  E x t r a c t e d 3x w i t h methanol  Residue r e c o n s t i t u t e d i n d i s t i l l e d water  84  Methanol evaporated  APPENDIX I I  NITROSATION OF SALTED FISH AQUEOUS EXTRACT  10 ml aqueous e x t r a c t 10 ml NaNO (3.0xl0~ M f i n a l ) 2  2  pH a d j u s t e d t o 2 w i t h HC1  Incubated f o r 1 h r a t 37°C  V  pH a d j u s t e d t o 7 w i t h NaOH  M u t a g e n i c i t y and c l a s t o g e n i c i t y t e s t e d  3-5  APPENDIX I I I  EXTRACTION PROCEDURE OF BETEL NUT  Betel nut  Crushed i n t o powder  \1/  E x t r a c t e d 2x w i t h hexane  V Hexane removed by a i r - d r y i n g , r e s i d u e e x t r a c t e d 2x w i t h 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 e x t r a c t )  Residue e x t r a c t e d 3x w i t h n - b u t a n o l  n-Butanol evaporated  V 1.5%  D i s s o l v e d i n d i s t i l l e d water, c a f f e i n e added t o p r e c i p i t a t e t a n n i n  86  APPENDIX IV  Resorcinol  OH  Pyrogallol  Gallic  OH  acid  COOH  Ascorbic acid  Benzoic  87  acid  

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