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

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INHIBITION OF DNA REPAIR BY SODIUM ASCORBATE IN VITRO AND IN VIVO  by  DONALD JAMES KOROPATNICK B.Sc,  The U n i v e r s i t y of B r i t i s h Columbia, 1974  M.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE  FACULTY OF GRADUATE STUDIES Department o f G e n e t i c s  We a c c e p t t h i s  t h e s i s as conforming  to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1981 Donald  James K o r o p a t n i c k , 1981  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  in p a r t i a l  f u l f i l m e n t o f the  the U n i v e r s i t y  s h a l l make i t  freely  of B r i t i s h  available  for  requirements f o r  Columbia,  I agree  reference and study.  f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  for  thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  that  representatives.  It  this thesis for financial  written  University  Graduate S t u d i e s of B r i t i s h  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  gain s h a l l  not be allowed without my  permission.  Department of The  i s understood that copying or p u b l i c a t i o n  Ptprf&lflM  Columbia  (Genetics)  ABSTRACT  S e v e r a l s h o r t - t e r m assays are In use to a s s e s s the c a r c i n o g e n i c hazard o f c h e m i c a l s . events t h a t may  While  the a b i l i t y  to induce  initiating  l e a d to c a r c i n o g e n e s i s i s measured, compounds and  c o n d i t i o n s t h a t might modify  the a b i l i t y o f chemicals to cause  those i n i t i a t i n g events a r e not assessed by such t e s t s .  In a d d i t i o n ,  compounds t h a t a f f e c t the a b i l i t y of c e l l s to r e a c t i n a normal f a s h i o n to the damaging a c t i o n of c a r c i n o g e n s are not d e t e c t e d by these methods. S h i f t s i n a l k a l i n e s u c r o s e g r a d i e n t p r o f i l e s of c e n t r i f u g e d DNA  (as an i n d i c a t i o n of DNA  and a l k y l DNA  adducts  fragmentation)  and  (as an i n d i c a t i o n of DNA  f o r m a t i o n of a r y l m o d i f i c a t i o n ) have  been used as s h o r t - t e r m assays f o r c a r c i n o g e n i c and mutagenic potential.  R e p a i r of DNA  damage has been measured by r e s t o r a t i o n of  n e a r - c o n t r o l s e d i m e n t a t i o n p r o f i l e s of DNA a l k y l adducts  and the l o s s of a r y l  over time a f t e r damage or m o d i f i c a t i o n of DNA  and  by  c a r c i n o g e n s and mutagens. In t h i s study, the a b i l i t y of sodium a s c o r b a t e to modify the DNA  fragmenting  and adductf.forming a c t i o n of the c a r c i n o g e n s  N-methyl-N'-nitro-N-nitrosoguanidine was  investigated.  (MNNG) and benzo(a)pyrene  I n a d d i t i o n , the a b i l i t y of c e l l s In  and In v i t r o to r e p a i r DNA  (BP)  vivo  i n the presence of sodium a s c o r b a t e  was  a s s e s s e d by the two methods d e s c r i b e d above. I t was and i n v i t r o . DNA  found t h a t sodium a s c o r b a t e i n h i b i t e d r e p a i r i_n v i v o In a d d i t i o n , sodium a s c o r b a t e was  found to  fragment  ija v i v o and i n v i t r o i n the presence o f copper, and t o i n h i b i t  the a c t i o n of c a r c i n o g e n s _in v i v o and  i n v i t r o by  nucleophilic  -iii-  scavenging  of e l e c t r o p h i l i c  carcinogens.  Sodium a s c o r b a t e was a l s o found t o i n h i b i t  the b i n d i n g  of BP to DNA jLn v i v o and In v i t r o .  On the o t h e r hand, o t h e r  reducing  agents had other e f f e c t s .  Propyl gallate  reducing  compound) i n h i b i t e d b i n d i n g o f BP to DNA i n v i t r o , but  enhanced b i n d i n g o f BP t o DNA ixi v i v o .  (a s u l p h y d r y l  The s u l p h y d r y l  reducing  agent g l u t a t h i o n e enhanced b i n d i n g of BP to DNA i n v i v o and i n vitro. A l k a l i n e sucrose  g r a d i e n t a n a l y s i s of DNA damage and r e c o v e r y  from t h a t damage, and BP adduct f o r m a t i o n  i n DNA and disappearance  over time, appear to be s u i t a b l e methods f o r assessment of the modifying  p r o p e r t i e s o f compounds and c o n d i t i o n s on t h e i n i t i a t i n g  events t h a t may l e a d to mutation or c a r c i n o g e n e s i s .  -iv-  TABLE OF CONTENTS  PAGE Abstract  i i  T a b l e o f Contents List  iv  of T a b l e s  v i i  L i s t of F i g u r e s  viii  Acknowledgements  xi  Introduction  1  Chemical  Carcinogenesis  1  I n t e r a c t i o n o f Carcinogens w i t h I n f o r m a t i o n a l C e l l u l a r Macromolecules I n V i v o  3  M e t a b o l i c A c t i v a t i o n to R e a c t i v e E l e c t r o p h i l e s  3  S i t e s of E l e c t r o p h i l i c Attack In Vivo  5  Carcinogenic Process  6  DNA R e p a i r  9  DNA R e p a i r I n h i b i t o r s  11  Measurement o f DNA Damage and R e p a i r  13  1)  A l k a l i n e sucrose gradient sedimentation  13  2)  E x c i s i o n o f a r y l and a l k y l adducts  14  F a c t o r s A f f e c t i n g Chemical 1) 2)  3)  from DNA ..  Carcinogenesis  F a c t o r s a f f e c t i n g the i n i t i a t i n g a c t i o n of c a r c i n o g e n s I n h i b i t i o n of non-enzymatic f o r m a t i o n of u l t i m a t e c a r c i n o g e n s  15  16 20  I n h i b i t i o n o f a c t i o n of u l t i m a t e carcinogens  20  Sodium A s c o r b a t e  21  Types o f Carcinogens  22  1)  Primary  carcinogens  23  -v-  PAGE 2) P r e c a r c i n o g e n s  23  3) Cocarcinogens  24  S p e c i f i c Carcinogens  25  1) N i t r o g e n compounds  25  2) D i a l k y l n i t r o s a m i n e s  25  3) P o l y c y c l i c a r o m a t i c hydrocarbons  (PAH)  O u t l i n e o f the Problem  28 31  M a t e r i a l s and Methods  33  Chemicals  33  Radionuclides  33  Chemical Carcinogens  33  Reducing  33  Agents  Metal Salt Solutions  34  N i t r o s a t i o n of Methylguanidine  34  E x p e r i m e n t a l Animals  .  34  C e l l Cultures  35  P r e p a r a t i o n of S9 A c t i v a t i o n M i x t u r e  37  A d m i n i s t r a t i o n o f Chemicals  37  Dimethylnitrosamine  37  N i t r o s a t i o n products of methylguanidine  38  MNNG  38  Sodium a s c o r b a t e  39  Benzo(a)pyrene  39  A l k a l i n e s u c r o s e g r a d i e n t a n a l y s i s o f DNA damage and r e p a i r A l k a l i n e sucrose gradients  40 41  -vi-  PAGE BP Adduct Measurement o f DNA  42  DNA i s o l a t i o n DNA c o n c e n t r a t i o n  42 determination  43  Measurement o f r a d i o a c t i v i t y  41  Results  45  I n h i b i t i o n o f DNA r e p a i r by sodium a s c o r b a t e 1) A l k a l i n e s u c r o s e g r a d i e n t  45  analysis  of DNA damage and r e p a i r a)  45  In v i t r o  b) - -In v i v o 2) BP-DNA adduct a n a l y s i s o f DNA r e p a i r  45 •••  56 63  a)  T_n v i t r o  63  b)  In vivo  75  Other e f f e c t s of sodium a s c o r b a t e  84  E f f e c t o f sodium a s c o r b a t e and o t h e r agents on b i n d i n g  o f BP t o DNA  Discussion  107 125  Summary  136  Perspectives  137  Literature Cited  140  -vii-  LIST OF TABLES TABLE 1  PAGE I n h i b i t i o n o f C a r c i n o g e n e s i s by I n d u c t i o n o f Microsomal Enzyme A c t i v i t y  17  2  Carcinogenicity  30  3  Recovery o f R a d i o a c t i v i t y from A l k a l i n e Sucrose G r a d i e n t s  o f Some Chemicals  55  -viii-  LIST OF FIGURES FIGURE 1  PAGE Proposed  i n t e r a c t i o n of c e l l d i f f e r e n t i a t i o n  and n e o p l a s i a  8  2  DNA r e p a i r  pathways  10  3  Activation  of n i t r o s o  4  Activation  of dimethylnitrosamine  5  Metabolism o f benzo(a)pyrene  6  Sedimentation p r o f i l e of cultured fibroblasts  7  8  compounds  S e d i m e n t a t i o n p r o f i l e s of c u l t u r e d f i b r o b l a s t s : r e p a i r over 30 h r  26 27 29 human 47 human 49  S e d i m e n t a t i o n p r o f i l e s o f c u l t u r e d human f i b r o b l a s t s : i n h i b i t i o n o f r e p a i r by sodium a s c o r b a t e  51  S e d i m e n t a t i o n p r o f i l e s o f c u l t u r e d human f i b r o b l a s t s : resumption of r e p a i r a f t e r removal o f sodium a s c o r b a t e  54  S e d i m e n t a t i o n p r o f i l e o f c u l t u r e d human f i b r o b l a s t s : l a c k o f f r a g m e n t a t i o n by sodium ascorbate alone  58  S e d i m e n t a t i o n p r o f i l e s o f mouse g a s t r i c c e l l s : r e p a i r over 30 h r  60  S e d i m e n t a t i o n p r o f i l e o f mouse g a s t r i c c e l l s : i n h i b i t i o n o f r e p a i r by sodium ascorbate  62  S e d i m e n t a t i o n p r o f i l e s o f mouse g a s t r i c c e l l s : A) l a c k o f f r a g m e n t a t i o n by sodium a s c o r b a t e a l o n e , and B) resumption o f r e p a i r a f t e r c e s s a t i o n o f sodium a s c o r b a t e treatment  65  14  A v a i l a b i l i t y o f BP i n s o l u t i o n  67  15  E f f e c t of BP c o n c e n t r a t i o n on BP adduct  9  10  11  12  13  f o r m a t i o n i n CHO c e l l s  69  16  R e p a i r course of BP adducts i n CHO c e l l s  72  17  I n h i b i t i o n o f BP adduct e x c i s i o n i n CHO c e l l s by sodium a s c o r b a t e -  74  -ix-  FIGURE 18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  PAGE BP adduct e x c i s i o n i n CHO c e l l s : l a c k o f i n h i b i t i o n by c y s t e i n e  77  E f f e c t o f BP c o n c e n t r a t i o n on BP adduct f o r m a t i o n i n mouse g a s t r i c c e l l s i n v i v o  79  R e p a i r c o u r s e o f BP adducts gastric c e l l s i n vivo  81  i n mouse  I n h i b i t i o n o f BP adduct e x c i s i o n i n mouse g a s t r i c c e l l s t r e a t e d i n v i v o w i t h sodium a s c o r b a t e  83  DNA f r a g m e n t a t i o n by sodium a s c o r b a t e and copper i n v i t r o  86  DNA f r a g m e n t a t i o n by sodium a s c o r b a t e and copper i n v i v o  88  S e d i m e n t a t i o n p r o f i l e o f c u l t u r e d human f i b r o b l a s t s t r e a t e d w i t h DMN  90  I n h i b i t i o n o f DNA f r a g m e n t a t i o n by d i m e t h y l n i t r o s a m i n e i n the presence.of sodium a s c o r b a t e  92  I n h i b i t i o n o f non-enzymatic a c t i v a t i o n of m e t h y l g u a n i d i n e by r e a c t i o n w i t h n i t r o u s a c i d i n the presence o f a s c o r b a t e  95  I n h i b i t i o n o f DNA f r a g m e n t a t i o n by MNNG i n v i t r o by i n c u b a t i o n w i t h sodium ascorbate  97  I n h i b i t i o n o f DNA f r a g m e n t a t i o n by MNNG i n v i v o by i n c u b a t i o n w i t h sodium ascorbate  100  Enhancement o f DNA f r a g m e n t a t i o n by MNNG i n v i t r o by c o - a d m i n i s t r a t i o n w i t h sodium ascorbate  102  Enhancement o f DNA f r a g m e n t a t i o n by MNNG i n v i v o by c o - a d m i n i s t r a t i o n w i t h sodium ascorbate  104  Lack o f f r a g m e n t a t i o n o f DNA by sodium a s c o r b a t e a l o n e i n v i t r o and i n v i v o  106  I n h i b i t i o n o f BP adduct f o r m a t i o n i n DNA i n the presence o f sodium a s c o r b a t e i n v i t r o  ...  109  -x-  FIGURE 33  34  35  36  37  38  39  PAGE E f f e c t o f sodium a s c o r b a t e on BP adduct f o r m a t i o n i n mouse g a s t r i c c e l l s t r e a t e d in vivo  112  I n h i b i t i o n o f BP adduct f o r m a t i o n i n DNA i n the presence o f p r o p y l g a l l a t e in vitro  114  Enhancement of BP adduct f o r m a t i o n i n DNA o f mouse g a s t r i c mucosal c e l l s treated with propyl g a l l a t e i n vivo  116  Enhancement of BP adduct f o r m a t i o n i n DNA i n the presence o f g l u t a t h i o n e i n v i t r o  118  Enhancement of BP adduct f o r m a t i o n in. DNA o f mouse g a s t r i c mucosal c e l l s treated with glutathione i n vivo  120  E f f e c t o f harman on BP adduct f o r m a t i o n i n DNA o f c e l l s t r e a t e d i n v i t r o  122  E f f e c t o f norharman on BP adduct i n DNA o f c e l l s t r e a t e d i n v i t r o  124  formation  -xi-  ACKNOWLEDGEMENTS  I thank my r e s e a r c h s u p e r v i s o r , Dr. Hans S t i c h , f o r h i s and  enthusiasm i n the r e s e a r c h t h a t l e d t o t h i s t h e s i s .  and  a i d w i l l always be a p p r e c i a t e d . Thanks a l s o t o f e l l o w students  Urs, Bob, Lan, M i r i a m  interest  H i s example  and r e s e a r c h e r s Anne, Bruce,  and S i n g , a l l o f whom p r o v i d e d happy arm-  waving d i s c u s s i o n s and p r a c t i c a l h e l p . I thank Bruce Dunn and Anne Hanham, who h e l p e d w i t h adduct  the DNA  experiments. S p e c i a l thanks go t o Jane, who not only l i v e d w i t h  but made the supreme s a c r i f i c e o f h e l p i n g w i t h  the w r i t e r ,  the t y p i n g .  The h e l p o f the N a t i o n a l Cancer I n s t i t u t e o f Canada and t h e N a t i o n a l S c i e n c e and E n g i n e e r i n g Research C o u n c i l , i m t h e of g r a n t s  t o Dr. H.F. S t i c h , i s g r a t e f u l l y acknowledged.  form  -1-  INTRODUCTION  CHEMICAL CARCINOGENESIS  A l t h o u g h c h e m i c a l c a r c i n o g e n e s i s was f i r s t  d i s c o v e r e d i n humans  over 200 y e a r s ago ( M i l l e r , 1978) t h e r e a r e , today, o n l y about  20  compounds o r m i x t u r e s t h a t a r e known to i n c r e a s e the r i s k of cancer i n s e v e r a l organ s i t e s i n v a r i o u s s u b p o p u l a t i o n s d e f i n e d by t h e i r i n i n d u s t r i a l , medical or s o c i e t a l s i t u a t i o n s . the causes o f the major burden o f cancer primary bronchogenic While  carcinoma  exposure  However, these a r e not  ( a s i d e from the i n d u c t i o n o f  caused by c i g a r e t t e s m o k e ) ( D o l l , 1977).  the c a r c i n o g e n s r e s p o n s i b l e f o r the g r e a t e r p a r t o f human cancers  are unknown, much i n d i r e c t evidence i n d i c a t e s t h a t environmental  factors  (very p r o b a b l y c h e m i c a l s f o r the most p a r t ) a r e i n v o l v e d i n the g e n e s i s of these neoplasms ( D o l l , 1977).  A l a r g e number o f c h e m i c a l s can cause  cancer i n v a r i o u s t i s s u e s of e x p e r i m e n t a l a n i m a l s , and humans a r e exposed to some o f these c a r c i n o g e n s sporadic administration).  ( g e n e r a l l y a t much lower c o n c e n t r a t i o n and  G e n e r a l l y , the concept  i s t h a t a l a r g e number  of cancers i n the g e n e r a l p o p u l a t i o n may be p o t e n t i a l l y p r e v e n t a b l e by the i d e n t i f i c a t i o n and e l i m i n a t i o n o f these c a r c i n o g e n s . In r e c e n t y e a r s the number o f c h e m i c a l s r e c o r d e d i n the l i t e r a t u r e has been a measure o f the v a s t i n c r e a s e i n c h e m i c a l knowledge (Maugh, 1978) , and t h e g r e a t m a j o r i t y o f the a p p r o x i m a t e l y 4 m i l l i o n known c h e m i c a l compounds a r e s y n t h e t i c l a b o r a t o r y p r o d u c t s 1979) .  A c e r t a i n number a r e u s e f u l enough to be used  have appeared  i n the human environment,  ( M i l l e r and M i l l e r , to the extent  and a p p r o x i m a t e l y  e s t i m a t e d to be i n "common use" i n the U.S. (Maugh, 1978).  they  60,000 a r e The exact  number may be d i s p u t e d , but i t i s c l e a r t h a t a t l e a s t s e v e r a l  thousand  -2-  compounds a r e brought amounts d a i l y .  i n t o c o n t a c t w i t h the human p o p u l a t i o n i n s m a l l  A major source of c o n t a c t of humans w i t h n a t u r a l l y -  o c c u r r i n g chemicals  i s i n t h e i r d a i l y i n t a k e of n a t u r a l foods, which  c o n t a i n s e v e r a l thousand  low m o l e c u l a r weight,  non-nutritive organic  compounds among which a r e c a r c i n o g e n s and mutagens ( M i l l e r and 1979).  may  Among these low m o l e c u l a r weight s y n t h e t i c and  Miller,  naturally-  o c c u r r i n g compounds e x i s t i n g i n food, a i r , water, c l o t h i n g ,  comsetics,  e t c . , t h e r e e x i s t s a wide v a r i e t y of s t r u c t u r e s which can produce a l a r g e range of e f f e c t s i n l i v i n g t o x i c a t s u f f i c i e n t dose, and  systems.  t o x i c and pharmacologic  i n l a r g e q u a n t i t y on these compounds. tedium,  and  A l l of the compounds are  However, due  expense of s t u d i e s n e c e s s a r y  information exists  to the  complexity,  to produce the i n f o r m a t i o n ,  knowledge of the mechanism of a c t i o n i s s e v e r e l y l i m i t e d .  Most i n f o r m a t i o n  c o n s i s t s s o l e l y of g r o s s data on the amount of c h e m i c a l r e q u i r e d to produce g r o s s While non-covalent molecules  effects.  the m a j o r i t y of t o x i c and p h a r m a c o l o g i c a l e f f e c t s a r e due (and, t h e r e f o r e , r e v e r s i b l e ) i n t e r a c t i o n s w i t h  ( G o l d s t e i n , et^ a l . ,  cellular  1974), r e c e n t y e a r s have shown t h a t the  t o x i c e f f e c t of c h e m i c a l c a r c i n o g e n s , many mutagens, some a l l e r g e n s , a few drugs a r e due  to c o v a l e n t i n t e r a c t i o n s of these compounds and  m e t a b o l i t e s ±n v i v o w i t h c r i t i c a l The  to  cellular  and their  molecules.  g r e a t m a j o r i t y of c h e m i c a l c a r c i n o g e n s a r e s m a l l o r g a n i c compounds  (of m o l e c u l a r weight l e s s than 500)  and a r e g e n e r a l l y l i p i d - s o l u b l e  not v e r y water s o l u b l e , a l t h o u g h e x c e p t i o n s e x i s t . c a r c i n o g e n s , i n c l u d i n g metals  There are some i n o r g a n i c  ( b e r y l l i u m , c o b a l t , cadmium, chromium, and  n i c k e l compounds) as w e l l as c i s - p l a t i n u m ( I I ) d i a m i n e d i c h l o r i d e et a l . , 1970).  and  (Leopold,  -3-  INTERACTION OF CARCINOGENS WITH INFORMATIONAL CELLULAR MACROMOLECULES IN VIVO  The  t r a n s f o r m a t i o n of normal c e l l s to tumour c e l l s appears  to need,  a t l e a s t , a h e r i t a b l e , quasi-permanent a l t e r a t i o n i n phenotype t h a t i n v o l v e s c o n t r o l of m i t o s i s .  T h i s may  be due  to 1) h e r i t a b l e changes i n DNA,  2) quasi-permanent changes i n genome t r a n s c r i p t i o n  (analogous  d i f f e r e n t i a l e x p r e s s i o n s of genomes of normal somatic case i n f o r m a t i o n a l m o l e c u l e s  a r e i n v o l v e d , and  cells).  or  to the In e i t h e r  c h e m i c a l c a r c i n o g e n s must  i n t e r a c t d i r e c t l y or i n d i r e c t l y w i t h one or more i n f o r m a t i o n a l macromolecules (DNA,  RNA  or p r o t e i n ) t h a t have some e f f e c t on c e l l m i t o s i s .  evidence has been p r o v i d e d f o r a l i n k between DNA e s i s by UV  light  (Hart, eit al_., 1977).  Direct  a l t e r a t i o n s and  However, f o r c h e m i c a l  carcinogen-  carcinogens,  the c r i t i c a l m o d i f i c a t i o n i n any one o f the macromolecules has not been demonstrated as the mechanism of a c t i o n of any On the o t h e r hand, a l l a d e q u a t e l y  c h e m i c a l or v i r a l  studied chemical carcinogens  carcinogen.  form  c o v a l e n t l y - b o u n d d e r i v a t i v e s w i t h c e l l u l a r macromolecules i n v i v o (with the e x c e p t i o n of a d r i a m y c i n , which b i n d s t i g h t l y (Marquardt,  e t a l . , 1977)  macromolecules; and  and  generates  i n vivo non-covalently  f r e e r a d i c a l s , but does not b i n d o t h e r  2,3,7,8-tetrachlorodibenzo-p-dioxin  i n d u c e r of m i x e d - f u n c t i o n o x i d a s e s i n l i v e r may  to DNA  (TCDD), which i s an  (Poland and Kende, 1977)  p o t e n t i a t e the a c t i o n of o t h e r c a r c i n o g e n s .  and  so  G e n e r a l l y speaking, some  b i n d i n g i n t e r a c t i o n o c c u r s between c a r c i n o g e n s and  t a r g e t macromolecules i n  c e l l s to i n i t i a t e c a r c i n o g e n e s i s ( M i l l e r and M i l l e r ,  1979).  METABOLIC ACTIVATION TO REACTIVE ELECTROPHILES  The m a j o r i t y of c h e m i c a l c a r c i n o g e n s r e q u i r e a c t i v a t i o n - metabolism to r e a c t i v e forms - to r e a c t c o v a l e n t l y w i t h n u c l e i c a c i d s and p r o t e i n s i n v i v o .  -4-  The c o v a l e n t i n t e r a c t i o n i s by means o f non-enzymatic n u c l e o p h i l i c  substitution.  The c a r c i n o g e n s a r e termed p r e c a r c i n o g e n s , and must be c o n v e r t e d to t h e i r f i n a l r e a c t i v e form - u l t i m a t e c a r c i n o g e n s - i n a c o n v e r s i o n t h a t i s u s u a l l y c a t a l y z e d by enzymes, and c e r t a i n i n t e r m e d i a t e m e t a b o l i t e s c a r c i n o g e n s ) may be generated  (proximate  i n the process.  The u l t i m a t e c a r c i n o g e n i c form o f most, i f not a l l ,  chemical  carcinogens  i s a s t r o n g e l e c t r o p h i l i c r e a c t a n t (Poland and Kende, 1977; M i l l e r , 1970) which i s a b l e t o a c q u i r e e l e c t r o n s from n u c l e o p h i l i c atoms i n c e l l u l a r components ( e s p e c i a l l y t h e i n f o r m a t i o n a l macromolecules such as n u c l e i c a c i d s and p r o t e i n s ) . forms of substances  T h i s i s i n c o n t r a s t to t h e f a c t t h a t the p r e c a r c i n o g e n i c a b l e to induce cancers may have v e r y l i t t l e  i n t h e way o f s t r u c t u r a l f e a t u r e s .  i n common  The common f a c t o r i s t h e s t r o n g  e l e c t r o p h i l i c i t y o f t h e u l t i m a t e r e a c t a n t to which they a r e c o n v e r t e d . Some c a r c i n o g e n i c a l k y l a t i n g and a c y l a t i n g agents  do not r e q u i r e  m e t a b o l i c a c t i v a t i o n , s i n c e they a r e s t r o n g e l e c t r o p h i l i c r e a c t a n t s as such, and r e q u i r e o n l y d i s s o l u t i o n i n water to produce c a r c i n o g e n i c species.  Some c h e m i c a l s do n o t g i v e r i s e to u l t i m a t e e l e c t r o p h i l i c  d e r i v a t i v e s , but appear t o be a b l e to cause development o f tumours i n e x p e r i m e n t a l animals n o n e t h e l e s s . of tumours i n a s t r i c t i n i t i a t i o n events  These compounds may n o t be i n i t i a t o r s  sense, but m o d i f i e r s o f secondary  (promotion,  immune response)  of tumours from p r e v i o u s l y i n i t i a t e d  responses to  t h a t permit t h e development  cells.  In a d d i t i o n to t h e common u l t i m a t e e l e c t r o p h i l i c i t y o f t h e d i v e r s e pantheon o f c h e m i c a l c a r c i n o g e n s , i t has been shown t h a t whenever t h e r e a c t i v e form o f t h e c a r c i n o g e n can be brought mutagenicity  i n t o c o n t a c t w i t h DNA i n  t e s t systems, they show mutagenic a c t i v i t y .  a c t i v a t i o n i s taken i n t o c o n s i d e r a t i o n , a p p r o x i m a t e l y carcinogens e x h i b i t mutagenicity  When m e t a b o l i c  90-95% o f c h e m i c a l  i n a v a r i e t y o f t e s t systems ( H o l l a e n d e r ,  -5-  1978;  McCann, e t a l . , 1975;  Sugimura, et a l . , 1976). have l i t t l e  McCann and Ames, 1976;  Purchase,  Most n o n - c a r c i n o g e n i c analogs and  o r no m u t a g e n i c i t y , and  et a l . ,  1978;  metabolites  thus t h e r e i s a s t r o n g l y f o r m a l p o s i t i v e  r e l a t i o n s h i p between c h e m i c a l c a r c i n o g e n i c i t y and  mutagenicity.  SITES OF ELECTROPHILIC ATTACK IN VIVO  One  g o a l of cancer r e s e a r c h i s to determine  what the macromolecular  t a r g e t of c h e m i c a l c a r c i n o g e n s i s w i t h r e s p e c t to the c e l l .  Because of the  s t r o n g r e l a t i o n s h i p between c a r c i n o g e n i c i t y and m u t a g e n i c i t y , step i s g e n e r a l l y regarded as i n v o l v i n g a mutation these mutations  may  be prevented,  • r e p a i r mechanisms.  or they may  or mutations  s t a t e , the r o l e of DNA  However, c h e m i c a l c a r c i n o g e n s b i n d to RNA so t h a t e p i g e n e t i c mechanisms of i n i t i a t i o n  ( M i n t z , 1978)).  critical  site  The  i n DNA,  and  DNA  step and  the  attractive.  and p r o t e i n i n v i v o as w e l l ,  can be invoked  (i.e_. , s t a b l e ,  precursor lesions i n  t h a t RNA  and protein-bound  (Berenblum, 1974;  carcinogen  derivatives  S c r i b n e r and B o u t w e l l , 1972).  t h e r e i s no e x p e r i m e n t a l evidence to i n d i c a t e t h a t t h i s i s t r u e ,  p h o r b o l m y r i s t a t e a c e t a t e (a potent promoter) does not b i n d to macromolecules ±n v i v o ( W e i n s t e i n , e_t a l . , 1979). data to e x p l a i n the p l e i o t r o p i c responsed cell  and  s c e n a r i o i s t h a t a c a r c i n o g e n c o u l d b i n d to a  c o u l d then induce promotion While  i n DNA,  i s especially  h e r i t a b l e s t a t e s of a b e r r a n t c e l l behaviour without DNA  initiation  be " f i x e d " by v a r i o u s  Because of the r a p i d i t y of the i n i t i a t i o n  p e r s i s t e n c e of the i n i t i a t e d  the  c u l t u r e s , and  There i s v e r y  to t h i s agent  the mechanisms of promotion.  carcinogenesis.  little  i n mouse s k i n or  T h e r e f o r e , both g e n e t i c and  e p i g e n e t i c mechanisms (with the p o s s i b l e p a r t i c i p a t i o n of v i r a l must be c o n s i d e r e d i n s t u d i e s on i n i t i a t i o n  cellular  and promotion  information)  i n chemical  -6-  In the case of n u c l e i c a c i d s , many n u c l e o p h i l i c s i t e s are open to a t t a c k by may  carcinogens i n vivo.  The  most n u c l e o p h i l i c base i s guanine,  be e l e c t r o p h i l i c a l l y s u b s t i t u t e d at the N-3,  Adenine, a l t h o u g h l e s s p r e f e r e n t i a l a s i t e , N-7,  and  c y t o s i n e a t N-3  and  4 0 .  N-7,  N  i s attacked  Thymine may  be  2  , 0  6  and  C-8  atoms.  at N - l , N-3  s u b s t i t u t e d at 0  and  and  2  and  4 0 .  V i r t u a l l y any  bone may  be  s i t e i s open to a t t a c k ,  and  even the n u c l e i c a c i d back-  s u b s t i t u t e d a t phosphate oxygen atoms.  In each case, a  bond i s formed between the e l e c t r o p h i l i c carbon or n i t r o g e n i n the u l t i m a t e  c a r c i n o g e n and  In p r o t e i n s , nitrogen by  the  carcinogenic  r a d i c a l present  the n u c l e o p h i l i c s i t e i n the n u c l e i c a c i d .  s u l p h u r atoms of methionine and  of h i s t i d i n e , and  covalent  tyrosine  (at two  cysteine,  p o s i t i o n s ) may  e l e c t r o p h i l e s (reviewed by M i l l e r and  be  Miller,  the  ring  substituted 1979).  CARCINOGENIC PROCESS  The has  i n d u c t i o n of malignant tumours i s a m u l t i f a c t o r i a l p r o c e s s  a multi-step  reactions i n man  and  and  evolution.  There i s a p r o g r e s s i o n  of complex i n d i v i d u a l  p r o c e s s e s t h a t are thought to l e a d to the  animals.  Each of these r e a c t i o n s  and  that  f i n a l overt  conditions  may  be  cancer subject  to c o n t r o l by a number of p o s s i b l e m o d i f y i n g f a c t o r s . 1)  A chemical carcinogen introduced  a c t i v a t e d to an u l t i m a t e enzyme systems. and  elimination 2) The  A l t h o u g h RNA  i n t o an  i n vivo  system may  c a r c i n o g e n , e i t h e r n o n - s p e c i f i c a l l y or by  T h i s r e a c t i o n may  be m o d i f i e d  be  specific  by b i o c h e m i c a l d e t o x i f i c a t i o n  reactions.  ultimate and  t h a t the r e l e v a n t  c a r c i n o g e n may  react with targets  p r o t e i n t a r g e t s have not target  stereochemical conditions  i s DNA. and  This  i n the  been r u l e d out,  i t is  i n t e r a c t i o n i s subject  competitive  cell. postulated  to  i n h i b i t i o n t h a t are not  yet  -7-  well-def ined.  The  be r e p a i r e d and e r r o r may  a l t e r e d r e l e v a n t macromolecule (e.j*., DNA)  may  then  r e s t o r e d by r e p a i r enzyme systems whose s u s c e p t i b i l i t y  p l a y a s i g n i f i c a n t p a r t i n a l t e r i n g the c e l l ' s c o n v e r s i o n  to  to  the malignant s t a t e . 3)  The  subsequently 4)  a l t e r e d carcinogen  receptor  i s d u p l i c a t e d so t h a t i t becomes  immune to the o p e r a t i o n of r e p a i r systems.  C e l l s c o n t a i n i n g the abnormal r e c e p t o r w i l l d i v i d e to form  extremely malignant tumours, or benign tumours, depending on the c e l l u l a r t a r g e t s of the c a r c i n o g e n i c s t i m u l u s . a r e the t a r g e t of c a r c i n o g e n i c s t i m u l i , (i..e_., m e t a s t a s i z i n g tissue) w i l l  tumours, r e l a t i v e l y  result.  stimulus,  I f p r i m o r d i a l stem c e l l s  then r e l a t i v e l y malignant tumours independent of c o n t r o l by  If r e l a t i v e l y well-differentiated c e l l s  o n l y the f i n a l stages of m a t u r a t i o n  initial  are subjected  to the  undergoing  carcinogenic  then r e l a t i v e l y benign tumours (1.e_., n o n - m e t a s t a s i z i n g ,  d i v i d i n g tumours r e l a t i v e l y dependent on the i n f l u e n c e of tissues) w i l l l i k e l y result. less differentiated  t a r g e t e d by the c a r c i n o g e n i c s t i m u l u s ) .  i f c e l l s of  levels  intermediate  (Pierce,  1974;  1).  Benign tumours would tend s i n c e , b e i n g more d i f f e r e n t i a t e d  to appear e a r l i e r than malignant tumours, than stem c e l l s ,  " f o r e i g n " to the t i s s u e i n which they appear, and  they would not be  so  a t h r e s h o l d number would  not need to accumulate to produce a tumour ( G r o b s t e i n and  a prolonged  cells  t i s s u e (1. e_., t h a t t i s s u e  d i f f e r e n t i a t i o n were the t a r g e t s of oncogenic s t i m u l u s  P i e r c e , et a l . , 1978).  surrounding  Tumours of i n t e r m e d i a t e  of d i f f e r e n t i a t i o n would be the expected r e s u l t  P i e r c e , et a l . , 1 9 7 8 ) ( F i g .  slow-  These benign tumours would c o n t a i n no  than t h e i r normal c o u n t e r p a r t  surrounding  Zwilling,  1953;  U n d i f f e r e n t i a t e d malignant stem c e l l s would have  l a t e n t p e r i o d , but would be l a r g e l y r e f r a c t o r y to e n v i r o n m e n t a l  s t i m u l u s when a c r i t i c a l number had  been produced.  -8-  Figure  D i f f e r e n t i a t i o n and N e o p l a s i a  1  mitotically active —^postmitotic differentiated malignant cells- benign cells Q ^ r ^ I A 1 , ^ grade 3 or 4 * L^~pL^ malignancy x  i  '  Q  CO CO  0  c  "0 CD  o C U  o  0 I  c  0  CD  O C  \j  a u  stem cell  CO CO  0  c  0  CD  i  i  O  -0-3  o u  C -H  C U  L-  grade 2 malignancy  r C T Q ) benign tumour  CD 3  tissue renewal  O co  o  normal senescent differentiated cell - from P i e r c e ,  et a l . , 1978  -9DNA-chemical i n t e r a c t i o n i s , as shown here, f i n a l o b s e r v a t i o n of g r o s s  tumours.  f a r removed from  the  The model system developed to e x p l a i n  l i v e r c a r c i n o g e n e s i s proposed t h a t the " i n i t i a t i o n " p r o c e s s  (or e a r l y ,  r a p i d events o c c u r r i n g i n hours or days) l e a d s to the appearance of c e l l populations.  T h i s model system has been r e f e r r e d to as " n e o p l a s t i c  c e l l u l a r e v o l u t i o n " ( F a r b e r , e t a l , , 1974). l i v e r carcinogen  diethylnitrosamine  (DEN)  A s i n g l e dose o f the  administered  d i v i s i o n f o l l o w i n g p a r t i a l hepatectomy w i l l not l e a d i n g to p r o g r e s s i v e c e l l p o p u l a t i o n s , s e v e r a l weeks f o l l o w i n g i n i t i a l et a l . , 1979;  Fiala,  and  induce  of the tumour, DNA  has  Farber,  L a i s h e s , e t a l . , 1978).  carcinogens  produce a n o n - l e t h a l , h e r i t a b l e DNA  modify c e l l u l a r DNA change expressed  when such c e l l s a r e viewed as a group  DNA  Since a final  Ogawa, link  appearance  ( F o u l d s , 1969;  carcinogenesis  i n such a way  as  to  u l t i m a t e l y as a tumour Weinstein,  at a l . , 1979).  REPAIR  When c h e m i c a l chemical  carcinogens  i n t e r a c t w i t h DNA  T h i s may  t h e r e may  be a d i r e c t  d e p u r i n a t i o n , e i t h e r spontaneous or enzyme-mediated, t h a t l e a d s  d i s r u p t i o n of the sugar-phosphate backbone and  and  1977;  until  been proposed as the r e l e v a n t c e l l u l a r t a r g e t of  In g e n e r a l , the somatic m u t a t i o n theory of  proposes t h a t c h e m i c a l  cell  cellular alterations  hence to l i v e r cancer,  c e l l damage ( S o l t and  et a l . , 1972;  potent  during l i v e r  must be found between the i n i t i a t e d damage to the c e l l and  carcinogens.  new  to  thus s i n g l e - s t r a n d breaks.  occur a f t e r treatment w i t h a l k y l a t i n g agents t h a t a l t e r  covalent  hydrophobic b i n d i n g c h a r a c t e r i s t i c s of bases (e.£., n i t r o g e n mustard).  For the most p a r t , p u r i n e bases a r e a l k y l a t e d , a l t h o u g h a l k y l a t e d to a l e s s e r extent  p y r i m i d i n e s may  (Lawley, 1976).  However, t h i s "spontaneous" h y d r o l y s i s of DNA  strands  i s not  the  be  -10-  FIGURE 2 DNA duplex containing d i s t o r t i o n s caused by chemicals  \ A.  Excision  repair:  Incision (damage-specific endonuclease  B.  Post-replication Replication  repair:  and gap formation  Repair r e p l i c a t i o n (DNA polymerase) Recombination (Nuclease(s) + ?)  E x c i s i o n and r e j o i n i n g (5'-<-3' exonuclease + polynucleotide ligase) Repair r e p l i c a t i o n and r e j o i n i n g (DNA polymerase + l i g a s e )  Replication Replication  -v^^w^— +  -11-  only method f o r s t r a n d bases from DNA  has  breakage.  a l s o been proposed  mediated e x c i s i o n of damaged DNA of DNA  r e p a i r - an attempt by  immediate use t h e r e are  and  Enzyme-mediated removal of  t h r e e p r i n c i p a l mechanisms f o r the  reactivation repair requires thymine dimers.  whereby e r r o r s i n DNA place  and  c e l l to m a i n t a i n t r u e DNA  to daughter c e l l s .  r e a c t i v a t i o n , e x c i s i o n r e p a i r and  cleave  (Ikegami, et a l . , 1970).  copies for  r e p a i r of DNA  post-replication repair and  P o s t - r e p l i c a t i o n repair includes  damage:  recombination  taken  the r e p l i c a t i o n The  process  be bypassed and  (Hanawalt, 1975).  replacing  i t w i t h the  correct  "left  Excision repair i s  an enzyme-mediated p r o c e s s by which the c e l l r e p a i r s damage to DNA the a f f e c t e d p o r t i o n and  Photo-  a l l those p r o c e s s e s  as to l e a v e gaps i n daughter s t r a n d s . Damage may  photo-  photocatalytically  s t r a n d s have been bypassed by  i s l a r g e l y t h e o r e t i c a l i n mammalian c e l l s .  cells  ( F i g . 2).  t h a t p e r s i s t a f t e r n u c l e a r r e p l i c a t i o n has  behind"by means of DNA  cell-  In most eukaryote  a s i n g l e enzyme to b i n d  damaged a r e a s i n DNA  enzymes i n such a way  This  segments i s thought to be p a r t of a p r o c e s s  the  transmission  alkylated  structure  by  removing  before  undergoing d i v i s i o n . The  covalent  or n o n - c o v a l e n t b i n d i n g  l o c a l d i s t o r t i o n that  can  of c h e m i c a l to DNA  s e r v e as a s i t e f o r endonuclease a t t a c k  p o s i t i o n f o r "spontaneous" h y d r o l y t i c f i s s i o n of the DNA 1971).  However, where c o i n c i d e n t  f a i t h f u l reconstruction absence of an  may  single-strand  to  or as  a  ( P a u l , et a l . ,  breaks take p l a c e ,  of the o r i g i n a l s t r u c t u r e  i n t a c t complementary s t r a n d  chain  lead  i s u n l i k e l y , due  a c t i n g as a template f o r  a to  the  poly-  merase a c t i v i t y .  DNA  REPAIR INHIBITORS  The  p r o c e s s of i n h i b i t i o n of DNA  r e p a i r i n mammalian c e l l s i s  still  -12r a t h e r p o o r l y understood.  A c c o r d i n g to C l e a v e r  (1974) c a f f e i n e i s an  out-  s t a n d i n g i n h i b i t o r of e x c i s i o n r e p a i r i n b a c t e r i a , a l o n g w i t h c h l o r o q u i n e , quinacrine, chloramphenicol is  due  and a c r i f l a v i n e .  i m p l i c a t e d , and  polymerases,  endonucleases  and  exonucleases  (reviewed by Kihlman, 1977).  c a f f e i n e i s not i n c l u d e d among those agents r e p a i r r e p l i c a t i o n o r e x c i s i o n of UV  this  as w e l l as has been  some c e l l - m e d i a t e d b i n d i n g of the i n h i b i t o r to DNA  the n e c e s s a r y p r e r e q u i s i t e  not  The p r e c i s e method by which  done i s unknown, a l t h o u g h b i n d i n g to s i n g l e - s t r a n d e d DNA  i n h i b i t i o n of DNA  its  this  to i n h i b i t i o n of r e p a i r r e p l i c a t i o n a t c o n c e n t r a t i o n s t h a t do  a f f e c t normal s e m i - c o n s e r v a t i v e r e p a i r . is  In the b a c t e r i a l case,  may  be  Interestingly,  t h a t a r e a b l e to i n h i b i t  dimers i n mammalian c e l l s  DNA  (although  e f f e c t on the e x c i s i o n of o t h e r types of c h e m i c a l l e s i o n s i n not known).  Compounds observed  to i n h i b i t  r e p a i r r e p l i c a t i o n i n mammalian c e l l s  also i n h i b i t  s e m i - c o n s e r v a t i v e DNA  acriflavine,  crystal violet,  replication.  a c t i n o m y c i n D,  These compounds a r e  c h l o r o q u i n e and  i o d o a c e t a t e , and  they appear to a c t by some mechanism t h a t i n c l u d e s c o v a l e n t or b i n d i n g t o DNA  (Kihlman,  will  non-covalent  1977).  However, c a f f e i n e has been shown to i n h i b i t  the g a p - f i l l i n g a s s o c i a t e d  w i t h p o s t - r e p l i c a t i o n r e p a i r i n rodent c e l l s a t c o n c e n t r a t i o n s t h a t have little 1969;  e f f e c t on s e m i - c o n s e r v a t i v e DNA F u j i w a r a , 1975;  has l i t t l e filling  replication  N i l s s o n and Lehmann, 1975).  ( C l e a v e r and Thomas, In human c e l l s ,  caffeine  e f f e c t on normal human f i b r o b l a s t s , but s t r o n g l y i n h i b i t s  (Buhl and Regan, 1975;  s y n t h e s i z e DNA  Lehmann, e t a l . ,  1975)  and  segments of normal s i z e a t l o n g times a f t e r  (Buhl and Regan, 1974)  gap-  the a b i l i t y  to  irradiation  i n c u l t u r e d xeroderma pigmentosum s k i n  fibroblasts.  -13-  MEASUREMENT OF DNA DAMAGE AND REPAIR  The o n l y t r u e t e s t f o r c a r c i n o g e n i c i t y of chemicals i s t h e i n d u c t i o n of tumours i n whole a n i m a l s .  Because o f t h e h i g h c o s t i n time and e f f o r t  i n v o l v e d i n a s s a y s of t h i s type, s h o r t - t e r m i n v i v o and i n v i t r o b i o a s s a y s have been developed  to a s s e s s c a r c i n o g e n i c r i s k by measuring t h e  a b i l i t y o f compounds to induce primary events i n c e l l s where those  primary  events can be l i n k e d t o t h e c a u s a l , i n i t i a t i n g events o f c a r c i n o g e n e s i s . DNA damage and i t s subsequent events  ( M i l l e r and M i l l e r ,  r e p a i r have been i m p l i c a t e d as j u s t  1969; Kihlman,  such  1977; W e i n s t e i n , e t a l . , 1979;  F o u l d s , 1969).  1) A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n :  The  f i n a l DNA l i g a t i o n and chromosomal r e o r g a n i z a t i o n s t e p s o f DNA  r e p a i r , as w e l l as t h e o r i g i n a l c a r c i n o g e n o r enzyme-mediated s t e p , can be monitored  by examining  cleavage  the s i n g l e - s t r a n d molecular  weight  d i s t r i b u t i o n o f DNA i n a l k a l i n e s u c r o s e g r a d i e n t zone s e d i m e n t a t i o n p r o f i l e s . Originally  adapted  ionizing radiation  t o d e t e c t DNA f r a g m e n t a t i o n i n c u l t u r e d c e l l s by (Lett, et a l . ,  1967; McGrath and W i l l i a m s , 1966) i t  has been m o d i f i e d t o assay c h e m i c a l - i n d u c e d f r a g m e n t a t i o n i n c u l t u r e d  cells  ( L a i s h e s and S t i c h , 1973; S t i c h and L a i s h e s , 1973; Andoh and Ide, 1972; Coyle and S t r a u s s , 1970) and mammalian c e l l s i n v i v o (Cox, et a l . , 1973; Laishes, et a l . ,  1975; K o r o p a t n i c k and S t i c h , 1976; Abanobi,  T h i s procedure w i l l r e g i s t e r breaks  i n the a l k a l i - l a b i l e  backbone o f t h e DNA s t r u c t u r e , a l t h o u g h DNA i s s t a b i l i z e d due t o a l a c k o f 2'-hydroxyl groups on t h e r i b o s e . developed  a system  et a l , ,  1977).  l i n k a g e s i n the i n t h i s respect  We have r e c e n t l y  f o r jLn v i v o d e t e r m i n a t i o n o f DNA damage and r e p a i r by  -14-  c h e m i c a l agents u s i n g mouse g a s t r i c e p i t h e l i a l c e l l s as a t e s t ( K o r o p a t n i c k and S t i c h , 1976;  S t i c h and K o r o p a t n i c k , 1977).  the terms " s i n g l e - s t r a n d break" and " f r a g m e n t a t i o n of DNA" a t i o n a l l y and r e f e r to DNA DNA  sedimented under  of DNA  In t h i s method a r e used  i d e n t i c a l conditions  (Abanobi, e t a l . ,  1977).  f r a g m e n t a t i o n to those a p p r o x i m a t i n g p r o f i l e peaks  administration.  i n the p e r i o d f o l l o w i n g c h e m i c a l  T h i s r e p a i r i n v o l v e s , not o n l y the r e j o i n i n g of s i n g l e  s t r a n d s , but a l s o r e p a i r - t y p e p r o c e s s e s t h a t i n v o l v e c o n s i d e r a b l y segments than those concerned w i t h s i n g l e - s t r a n d r e j o i n i n g ,  r e o r g a n i z a t i o n of chromatin substances t h a t may p r o f i l e s ) may simple  Repair  i n s e d i m e n t a t i o n p r o f i l e peaks, from r e g i o n s  d e r i v e d from u n t r e a t e d c o n t r o l DNA,  DNA  oper-  t h a t sediments s l o w l y i n comparison w i t h c o n t r o l  i s measured by a s h i f t  i n d i c a t i n g DNA  system  ( E l k i n d and Kamper, 1970).  be shown to i n h i b i t r e p a i r  inhibit  DNA  larger  possibly  Therefore,  (as monitored on  gradient  complex chromatin r e o r g a n i z a t i o n a l s t e p s as w e l l as  ligation.  2) E x c i s i o n of a r y l and a l k y l adducts from  DNA:  A r y l a t i o n o r a l k y l a t i o n of macromolecular  targets i n c e l l s occurs  w i t h almost a l l c a r c i n o g e n i c s p e c i e s (see above). a r y l a t i n g agents w i l l modify DNA  (Feldman,  p r o c e s s e s w i t h i n the  adducts a r e e x c i s e d from DNA  hamster embryo c e l l s et a l . ,  ( I v a n o v i c , jst a l . ,  1978)  and  to produce m o d i f i e d bases, which may  be e x c i s e d by enzymatic or non-enzymatic Benzo(a)pyrene  Both a l k y l a t i n g  then  cell.  i n v i v o i n the case of primary  1978), human l u n g c e l l  and mouse embryo f i b r o b l a s t s  In a d d i t i o n , m e t h y l a t e d s i t e s i n mammalian DNA  cultures  (Brown, e t a l , ,  1979).  produced by a v a r i e t y of  c h e m i c a l agents a d m i n i s t e r e d i n v i v o have been shown to be e x c i s e d over a p e r i o d of 24 to 72 hours, a l t h o u g h a s i g n i f i c a n t amount (10-30% of  -15-  o r i g i n a l methylated  s i t e s ) appears  Montesano, e^t a l . , 1979; 1976;  Swann and Mace, 1980;  Goth and Rajewsky, 1974;  1980).  to be r e s i s t a n t  Nicoll,  to e x c i s i o n  (Pegg,  1978;  K l e i h u e s and Margison,  1974,  e t a l . , 1977;  In g e n e r a l , c u l t u r e d c e l l s a r e t r e a t e d w i t h  T h o r g i e r s s o n , et a l . , radioactively-labelled  a r y l or a l k y l a t i n g agents by i n c u b a t i o n i n medium f o r from 12 to 24 hours ( I v a n o v i c , et a l . , 1978)  and mammals are i n j e c t e d subcutaneously  or  i n t r a p e r i t o n e a l l y and a l l o w e d to remain u n t r e a t e d f o r 12 to 18 hours ( T h o r g e i r s s o n , e_t a l . , 1980).  DNA  i s . i s o l a t e d from c e l l s o r t i s s u e s of  i n t e r e s t a t up to 72 hours f o l l o w i n g the procedures  d e s c r i b e d , and  amount of r a d i o a c t i v e l a b e l c o v a l e n t l y bound to the DNA  i s used  e s t i m a t e the l e v e l of a r y l or a l k y l a t i o n of DNA.  S i n c e 0-6  guanines  i n mutagenic  may  be more important  carcinogenic effects  than o t h e r adducts  ( L o v e l e s s , 1969;  ment of t h i s procedure  s e p a r a t e p u r i n e bases on Sephadex G-10 0-6  methylation s p e c i f i c a l l y  to  a r y l and  alkyl  and  Gerchman and Ludlum, 1973)  has been to h y d r o l y s e DNA  the  a refine-  by a c i d o r enzymes, and  columns to determine  the extent of  ( K l e i h u e s and Magee, 1973).  FACTORS AFFECTING CHEMICAL CARCINOGENESIS  In view of the m u l t i - s t e p n a t u r e of mammalian i n d u c t i o n of  cancer,  m o d i f i c a t i o n of the a b i l i t y of c h e m i c a l c a r c i n o g e n s to cause tumours take p l a c e a t two affect  the primary  different  levels.  environmental  factors  may  " i n i t i a t i o n " steps i n c a r c i n o g e n e s i s t h a t take p l a c e  d u r i n g the p e r i o d immediately s e v e r a l days) and,  First,  may  second,  f o l l o w i n g exposure to c a r c i n o g e n s  they may  affect  the behaviour  (up to  of c e l l s t h a t  a l r e a d y c a r r y the c r i t i c a l change t h a t p l a c e s them on the path toward becoming tumour c e l l s . The  factors f a l l i n g  i n t o the f i r s t  group i n c l u d e those t h a t a f f e c t  the  -16a c t i v a t i o n o f p r e c a r c i n o g e n s , the i n t e r a c t i o n o f u l t i m a t e c a r c i n o g e n s macromolecular t a r g e t s i n c e l l s , to i n t e r a c t i o n o f c a r c i n o g e n s  and those t h a t a l t e r the c e l l u l a r  (e.£. , c e l l d i v i s i o n , DNA r e p a i r ) .  f a c t o r s t h a t a r e i n c l u d e d i n t h e second  1979) o r i n h i b i t  a host o f i l l - d e f i n e d "age  at f i r s t  response The  group a r e those t h a t modulate  programs o f gene e x p r e s s i o n t o induce c l o n a l growth of i n i t i a t e d (Weinstein, et^ a l . ,  with  cells  t h e i r growth ( H i g g i n s o n , 1979) and  factors, including sociological characteristics  (e.j*.,  i n t e r c o u r s e " o r "age a t f i r s t m a r r i a g e " w i t h r e s p e c t t o t h e  i n c i d e n c e o f a d u l t mammary tumours) t h a t have a complex e f f e c t on tumour induction  (reviewed by H i g g i n s o n ,  1979).  1) F a c t o r s a f f e c t i n g the i n i t i a t i n g a c t i o n of c a r c i n o g e n s  A number o f s t u d i e s have demonstrated t h a t i t i s p o s s i b l e to p r o t e c t a g a i n s t t h e c a r c i n o g e n i c e f f e c t s of c h e m i c a l c a r c i n o g e n s by i n d u c i n g i n c r e a s e d mixed f u n c t i o n oxygenase a c t i v i t y .  Polycyclic  hydrocarbons  ( e i t h e r c a r c i n o g e n i c or n o n - c a r c i n o g e n i c ) have been shown to i n h i b i t t h e o c c u r r e n c e o f h e p a t i c cancer r e s u l t i n g from the f e e d i n g o f 3'-methyl-4dimethylaminobenzene (Richardson, .et a l . , 1952; M i l l e r , e t a l . , 1958). A l s o , p o l y a r o m a t i c hydrocarbon  (PAH) i n d u c e r s o f mixed f u n c t i o n oxygenases  have been shown to markedly reduce  the i n c i d e n c e o f tumours o f the l i v e r ,  mammary g l a n d , e a r duct, and s m a l l i n t e s t i n e i n r a t s f e d 2-acetylaminof l u o r e n e or 7 - f l u o r o - 2 - a c e t y l a m i n o f l u o r e n e ( M i l l e r , e t a l . ,  1958).  Recently,  i t has been shown t h a t i t i s p o s s i b l e to p r o t e c t a g a i n s t the c a r c i n o g e n i c e f f e c t s o f a number of o t h e r c a r c i n o g e n s , i n c l u d i n g urethane, anthracene  (DMBA), a f l a t o x i n , bracken  f e r n , and benzo(a)pyrene  In each case, t h e m o d i f y i n g compounds used a r e potent f u n c t i o n oxygenase a c t i v i t y .  dimethylbenz(a) (Table 1 ) .  i n d u c e r s of mixed  H  user M (t>  I n h i b i t i o n o f carcinogenesis by Induction of microsomal enzyme a c t i v i t y  References  Carcinogen  Inducer  Species Organ  3'-methyl-4-dimethylaminoazobenzene  P o l y c y c l i c hydrocarbons a-benzene hydrochloride  rat  Urethane  g-naphthoflavone  mouse . lung  Yamamoto, e_t a l . , 1971  7,12-dimethylbenz(a)anthracene  P o l y c y c l i c hydrocarbons  rat  breast  Huggins, e t a l . , 1964  Aflatoxin  Phenobarbltol  rat  breast  McLean and M a r s h a l l , 1971  Phenothiazine  rat  small i n t e s t i n e bladder  Pamukcu, e t a l . , 1971  g-naphthoflavone  mouse  lung,  Wattenberg and Leong, 1970  • Bracken fern  Benzo(a)pyrene  carcinogen  M i l l e r , e_t a l . , 1958 Richardson, e t a l . , 1972  liver  skin  -18-  The  paradoxic  carcinogenesis  nature  of t h i s i n d u c t i o n e f f e c t r e s u l t i n g i n reduced  r e q u i r e s some e x p l a n a t i o n .  I t can be argued t h a t i f a  compound i s a c t i v a t e d by an enzyme system to a c a r c i n o g e n i c form, then enhancement of t h a t system would r e s u l t T h i s would be  i n g r e a t e r i n d u c t i o n of  the case i n s i t u a t i o n s i n v o l v i n g an e f f e c t  cancers.  i n which  there  i s a s u b s t a n t i a l " t h r e s h o l d l e v e l " f o r the c a r c i n o g e n i c agent, below which tumours would not be  initiated,  i r r e s p e c t i v e of l e n g t h of exposure.  However, t h e r e appears, i n the case of chemical no  t h r e s h o l d or a v e r y  low  threshold  carcinogens,  ( D i p a o l o , e t a l . , 1971).  i t might be expected t h a t slow a c t i v a t i o n would r e s u l t effect  than r a p i d a c t i v a t i o n .  must be a p p l i e d a t a c r i t i c a l  to be e i t h e r  I t c o u l d be  Therefore,  i n greater  t h a t the c a r c i n o g e n i c  time or times i n the c e l l  i s l e s s l i k e l i h o o d of l o s s of a c t i v a t e d s p e c i e s  (due  w i t h n o n - s p e c i f i c c e l l u l a r t a r g e t s ) which might be  cycle.  carcinogenic species  Also,  to c a r c i n o g e n  interaction  expected to occur when  an excess amount of c a r c i n o g e n i c s p e c i e s i s produced over t h a t most for  the number of c r i t i c a l b i n d i n g s i t e s  there  (Wattenberg, 1974).  effective  In a d d i t i o n ,  the u l t i m a t e l y r e a c t i v e c a r c i n o g e n i c s p e c i e s are o n l y t r a n s i e n t compounds i n a pathway employed by the c e l l  to d e t o x i f y those  i n d u c e r s of the mixed f u n c t i o n oxygenases induce by r i n g h y d r o x y l a t i o n )  compounds.  Thus,  detoxification (usually  as w e l l as a c t i v a t i o n ( u s u a l l y by n i t r o g e n  as i s the case f o r aromatic  amines ( M i l l e r and M i l l e r ,  hydroxylation)  1969).  In a d d i t i o n to enhancement of mixed f u n c t i o n oxygenase a c t i v i t y , c e r t a i n c o n d i t i o n s may some c h e m i c a l s  decrease enzyme a c t i v i t y .  depresses enzyme a c t i o n ( d i e t h y l d i t h i o c a r b a m a t e  reduce metabolism of d i m e t h y l n i t r o s a m i n e coumarin or a - a n g e l i c a l a c t o n e and  A d m i n i s t r a t i o n of  (Abanobi, e_t a l . , 1977)  to and  to reduce metabolism of benzo(a)pyrene  benzo(a)pyrene-induced n e o p l a s i a of forestomach (Wattenberg, et a l . ,  1979).  A l s o , n u t r i t i o n a l s t a t e s may  a l s o decrease enzyme a c t i v i t y  -  -19s t a r v a t i o n of Sprague-Dawley r a t s f o r 24 hours r e s u l t s i n almost l o s s of mixed f u n c t i o n oxygenase a c t i v i t y 1971)  i n small i n t e s t i n e  as i s the case f o r r a t s f e d a f a t - f r e e d i e t  (Wattenberg, 1974).  (Wattenberg,  (Wattenberg, 1974).  P u r i f i e d d i e t s a l s o r e s u l t e d i n l o s s of a c t i v a t i o n a b i l i t y intestine  total  Thus, most, i f not a l l ,  i n small  of the mixed  f u n c t i o n oxygenase a c t i v i t y of the s m a l l i n t e s t i n e i s caused by an exogenous i n d u c e r or The  inducers. ability  to enhance the p r o d u c t i o n of u l t i m a t e  carcinogens  or to s t i m u l a t e t h e i r d e a c t i v a t i o n i s not n e c e s s a r i l y c o n f i n e d to modifying  compounds.  The  p y r o l y s i s products  (9H-pyrido-(3,4-b)-indole), are capable  harman, and  pathway of BP, i n c l u d i n g  to u l t i m a t e c a r c i n o g e n i c form, and  of water s o l u b l e m e t a b o l i t e s ( F u j i n o , est a l . , 1978;  -norharman  3-methyl i n d o l e ( s k a t o l e ) -  of i n h i b i t i n g the whole m e t a b o l i c  both the c o n v e r s i o n of BP  in vitro  of tryptophan  separate  production  from the u l t i m a t e c a r c i n o g e n i c s p e c i e s  Matsumoto, e_t a l . , 1977).  On  the other hand, i n  systems employing an a r t i f i c i a l a c t i v a t i o n mixture  with  S-9  microsomes d e r i v e d from r a t l i v e r , h i g h c o n c e n t r a t i o n s of harman or norharman r e s u l t  i n a decrease  i n mutagenic p o t e n t i a l , w h i l e low  c e n t r a t i o n s produce an i n c r e a s e i n mutagenic p o t e n t i a l 1978).  T h i s i s due  con-  ( F u j i n o , et a l . ,  to a d i f f e r e n t i a l i n h i b i t i o n of a c t i v a t i n g or d e a c t i v a t i n g  enzyme systems, s i n c e harman and norharman a r e more a c t i v e i n d e p r e s s i n g the d e a c t i v a t i n g a b i l i t y of enzymes t h a t c o n v e r t hydrophobic to h y d r o p h i l i c ones (R^)(Matsumoto, et a l . , 1977) a c t i v a t i n g a b i l i t y of enzymes t h a t c o n v e r t oxygenated i n t e r m e d i a t e exert p a r t i a l  (R-?)•  i n h i b i t i o n of R^  i n accumulation  L°  w  and  of c a r c i n o g e n i c BP*  metabolites  than i n d e p r e s s i n g  the parent  compound to  c o n c e n t r a t i o n s of i n h i b i t o r may complete i n h i b i t i o n of R^, intermediates:  BP  conjugates  the  the therefore  resulting  -20-  2) I n h i b i t i o n of non-enzymatic f o r m a t i o n  The  a t low pH  M e t h y l g u a n i d i n e may  to produce mutagenic and  methylnitrosourea  n i t r o s a t i o n by  the n i t r o u s a c i d ( M i r v i s h , e t a l . , 1972;  i n a r e a c t i o n t h a t i s w e l l understood  nitro-  be r e a c t e d  with  c a r c i n o g e n i c compounds, i n c l u d i n g  and N-methyl-N'-nitro- N - n i t r o s o g u a n i d i n e  A s c o r b i c a c i d i s a b l e to prevent  reducing  the non-enzymatic  of c a r c i n o g e n i c s p e c i e s i s the case of n i t r o s a t i o n of  s a t a b l e compounds i n a c i d c o n d i t i o n s .  1978).  carcinogens:  best known example of a compound p r e v e n t i n g  production  nitrite  of u l t i m a t e  (Lo and  Stich,  preferentially  Synnot, e t a l . , 1975)  (Dahn, e_t a l . , 1960).  Other  reducing  agents, i n c l u d i n g c y s t e i n e , cysteamine, and p r o p y l g a l l a t e are a l s o a b l e to  inhibit  the f o r m a t i o n  of these n i t r o s a t i o n p r o d u c t s ,  which have been  i m p l i c a t e d as b e i n g b i o l o g i c a l l y a v a i l a b l e by r e a c t i o n of n i t r i t e v a r i o u s compounds i n the a c i d c o n d i t i o n s of the stomach (Lo and  with  Stich,  1978).  3) I n h i b i t i o n of a c t i o n of u l t i m a t e  carcinogens:  Once the r e a c t i v e form of a c a r c i n o g e n i c s p e c i e s has agents t h a t a l t e r the a c t i v a t i o n of p r e c a r c i n o g e n s w i l l have l i t t l e  may  I f the r e a c t i v e s p e c i e s  (such as water and  e x e r t a p r o t e c t i v e e f f e c t by a c t i n g as n o n - c r i t i c a l " t a r g e t s "  and M i l l e r , be  by enzymatic means  e f f e c t on c a r c i n o g e n i c p o t e n t i a l .  i s s u f f i c i e n t l y e l e c t r o p h i l i c , weak n u c l e o p h i l e s  been c r e a t e d ,  inhibited  1979).  However, c a r c i n o g e n s  protein) (Miller  of l e s s e r e l e c t r o p o t e n t i a l may  i n t h e i r a c t i o n by " t r a p p i n g " of the r e a c t i v e e l e c t r o p h i l i c  s p e c i e s through a d m i n i s t r a t i o n of agents t h a t a c a r c i n o g e n  reduces p r e f e r -  e n t i a l l y over c r i t i c a l c e l l u l a r macromolecules ( B a r t s c h , et a l . , 1972  1973;  -21M i l l e r and M i l l e r , 1973) .  Reducing  hydroxytoluene  agents  1969;  1977), cysteamine 1972)  1974,  .1976; M i l l e r ,  1970;  S c r i b n e r and Naimy,  such as b u t y l a t e d h y d r o x y a n i s o l e and b u t y l a t e d  (Grantham, e t a l . ,  P i p k i n , et a l . ,  Kruger,  1969,  1973), a s c o r b i c a c i d  S c h l e g e l , et a l . ,  (Marguardt,  et a l . ,  (Guttenplan,  1969), selenium  1974)  1977;  (Jacobs, e t a l . ,  and d i s u l f i r a m  (Schmall  and  have a l s o been shown to i n h i b i t c a r c i n o g e n e s i s i n animal  test  systems, or mutagenesis i n b a c t e r i a l a s s e y s , i f they a r e a p p l i e d c o n c u r r e n t l y with carcinogens.  Chemical  c a r c i n o g e n s of every type capable of p r o d u c i n g  r e a c t i v e e l e c t r o p h i l e s a r e s u s c e p t i b l e ( R o s i n and S t i c h ,  1978).  Sodium A s c o r b a t e  While  s y n t h e t i c c h e m i c a l s and  those found  i n e x o t i c foods  most a t t e n t i o n when c a r c i n o g e n i c and mutagenic hazards  of  environmental  agents a r e a s s e s s e d , c h e m i c a l s which a r e an i n t e g r a l p a r t of  cellular  metabolism or a r e of v i t a l n u t r i t i o n a l v a l u e a r e o f t e n passed Ascorbic acid (Lewin, regarded  1976;  P a u l i n g , 1970).  1975,  I t has been w i d e l y used, and  as s a f e , as a food a d d i t i v e to prevent  1974;  1976).  phage (Murata,  over.  ( v i t a m i n C) i s consumed i n l a r g e doses by humans  n i t r o s a t i o n p r o d u c t s , and et a l . ,  i s generally  f o r m a t i o n of c a r c i n o g e n i c  t o p r e v e n t "browning" by a i r o x i d a t i o n ( Cardesa,  F i d d l e r , et a l . ,  1978;  Lo, e t a l . ,  1978;  M i r v i s h , et a l . ,  Sodium a s c o r b a t e has a c a p a c i t y f o r i n a c t i v a t i n g 1975)  attract  bacterio-  and mammalian v i r u s e s (Jungeblut, 1935,1939), p o s s i b l y  by the l i b e r a t i o n of p e r o x i d e from o x i d a t i o n of a s c o r b a t e s i n c e c a t a l a s e p r o v i d e s p r o t e c t i o n (Schwerdt and  Schwerdt, 1975;  Wong, e t a l . ,  or by the a c t i o n of monodehydroascorbate r a d i c a l produced oxidation process shown to i n h i b i t  (Bielski,  est: ' a l . , 1975).  1974),  d u r i n g the  Sodium a s c o r b a t e has been  the f o r m a t i o n of u l t i m a t e c a r c i n o g e n s by a c t i n g as a  -22p r e f e r r e d s i t e of o x i d a t i o n i n the o x i d a t i o n - r e d u c t i o n r e a c t i o n t h a t reduces peroxide 1976)  by the a c t i o n of p e r o x i d a s e  and  an e l e c t r o n donor ( F l o y d , et a l . ,  as w e l l as a c t i n g as a " t r a p p i n g " agent by p r e f e r e n t i a l r e d u c t i o n  u l t i m a t e e l e c t r o p h i l e s (see a b o v e ) .  I n a d d i t i o n , sodium a s c o r b a t e  been shown to have a n t i m u t a g e n i c p r o p e r t i e s (Guttenplan, S t i c h , 1979; and  Marquardt, e t a l . , 1977)  Campbell j 1974;  and  P i p k i n , e_t 'al;-,. 1969;  However, a s c o r b i c a c i d has  has  Rosin  anti-cancer properties  and  (Cameron  R a i n e r l - a n d ' W e i s b u r g e t , 1975) .  been r e p o r t e d  the p r o t e c t i v e ones o u t l i n e d above.  1978;  of  to e x e r t e f f e c t s other  Sodium a s c o r b a t e  has  than  been shown to  i n c r e a s e the f r e q u e n c y of s i s t e r chromatid exchanges (a s e n s i t i v e i n d i c a t o r of DNA and  damage) ( S p e i t , et a l . , 1980).  I t has been shown t o fragment  induce chromosome a b e r r a t i o n s i n c u l t u r e d c e l l s  as w e l l as m u t a t i o n s i n S. Typhimurium as w e l l as M n ( l l ) and e f f e c t of a s c o r b a t e H2O2,  F e ( l l ) and  ( S t i c h e_t a l . ,  ( S t i c h , e t a l . , 1978).  DNA 1976)  Copper(II)  ( I I I ) enhance the chromosome-damaging  by c a t a l y z i n g the a u t o x i d a t i o n of a s c o r b a t e  to form  s i n c e c a t a l a s e reduces or a b o l i s h e s the e f f e c t s ( S t i c h , et a l . ,  1979).  Ascorbate w i l l also i n h i b i t m i t o s i s Sodium a s c o r b a t e  has  a l s o been shown t o convey an i n c r e a s e d  v i t y to c e l l s to treatment w i t h c a r c i n o g e n s d e a t h and  required  and m u t a t i o n c o n c e n t r a t i o n s  ( S t i c h , e t a l . , 1978,  enhance the m u t a g e n i c i t y  sensiti-  and mutagens, c a u s i n g  decreased i n c o r p o r a t i o n of thymidine by unscheduled  s y n t h e s i s a t lower c a r c i n o g e n normally  ( S t i c h , et a l . , 1979).  1979).  cell  DNA  than those  Sodium a s c o r b a t e  of N — h y d r o x y - 2 - a c e t y l a m i n o f l u o r e n e  will  > also  (rhorgeirsson,  et a l . , 1980).'  TYPES OF CARCINOGENS  Chemical • c a r c i n o g e n s  may  be d i v i d e d i n t o three c l a s s e s :  :  primary  or  u l t i m a t e c a r c i n o g e n s , secondary  (promoting  1)  Primary  o r p r e c a r c i n o g e n s , and c o c a r c i n o g e n s  agents and f a c t o r s ) .  carcinogens:  These c h e m i c a l s a r e b i o l o g i c a l l y a c t i v e c a r c i n o g e n s without to m e t a b o l i c a c t i v a t i o n .  recourse  They may i n t e r a c t d i r e c t l y w i t h t i s s u e s and a l l  components t o y i e l d m o d i f i e d macromolecules t h a t may produce p r e n e o p l a s t i c cells.  Primary c a r c i n o g e n s i n c l u d e v a r i o u s types of a l k y l a t i n g  agents,  such as n i t r o g e n and sulphur mustards, s u l p h o n i c e s t e r s and s u l f o n e s , e t h y l e n e imines and imides, s t r a i n e d or  a, g-unsaturated  p e r o x i d e s and c h l o r o a l k y l e t h e r s ( M i l l e r , 1978) .  l a c t o n e s , epoxides,  They a r e i n t h e i r  r e a c t i v e form as a d m i n i s t e r e d , and take p a r t i n S^2  final  (substitution,  n u c l e o p h i l i c , b i m o l e c u l a r ) r e a c t i o n s w i t h c r i t i c a l macromolecules i n v i v o ( P r i c e , e t a l . , 1969; Ross, 1962; S h a p i r o , 1969).  They a r e not g e n e r a l l y  s t r o n g c a r c i n o g e n s - due, presumably, t o the i n t e r f e r e n c e of n o n - c r i t i c a l n u c l e o p h i l e s (water, p r o t e i n s ) i n v i v o w h i c h makes i t n e c e s s a r y  to a d m i n i -  s t e r l a r g e doses a t l o c a l t i s s u e s i t e s t o induce tumours ( M i l l e r ,  1978).  Much of the e l e c t r o p h i l e may be d i s p o s e d of b e f o r e e n t r y i n t o t a r g e t cells. Most of the i n o r g a n i c c a r c i n o g e n s in their  2)  (e.g., metals)  are electrophiles  i o n i c forms ( F o r s t and Haro, 1969).  Precarcinogens:  Most c a r c i n o g e n i c c h e m i c a l s , s y n t h e t i c or n a t u r a l , f a l l class,  so s t r u c t u r e s d i f f e r w i d e l y .  into  this  They a r e u s u a l l y , c h e m i c a l l y and b i o l o g i -  c a l l y i n e r t w i t h r e s p e c t to t a r g e t macromolecules, and r e q u i r e spontaneous  -24-  or  host-mediated  and c o n t r o l l e d a c t i v a t i o n r e a c t i o n s to c o n v e r t them t o  their ultimate reactive species. to p r i m a r y c a r c i n o g e n s range of organs and their  Where they are spontaneously  by h y d r o l y s i s , they may  s p e c i e s , due  converted  be a c t i v e f o r a broad  to the s i m p l i c i t y and u n i v e r s a l i t y of  activation. However, when s p e c i f i c h o s t - c o n t r o l l e d b i o c h e m i c a l a c t i v a t i o n i s  r e q u i r e d , t h e r e can be g r e a t d i v e r s i t y i n a c t i v i t y from organ t o organ, i n d i v i d u a l to i n d i v i d u a l or s p e c i e s to s p e c i e s . s p e c i f i c enzyme systems (Kasper, 1974; 1974).  T h i s i s a p o s s i b l e r e a s o n why  or mutagenic i n one organism  3)  system may  Mukhtar, e t a l . , 1979; a c h e m i c a l which may  or system where the r e q u i r e d a c t i v a t i n g  be c a r c i n o g e n i c  system  i s absent.  Cocarcinogens:  p o t e n t i a t e the tumour-inducing Complex m i x t u r e s  to induce tumours, but  will  a c t i o n of u l t i m a t e or p r e c a r c i n o g e n s .  such as tobacco smoke a r e thought  to c o n t a i n l a r g e amounts  c o c a r c i n o g e n s , but s m a l l r e l a t i v e amounts or p r e c a r c i n o g e n s (Wynder  and Hoffman, 1967; oil  Grover, e t a l .  be n o n — c a r c i n o g e n i c or mutagenic i n an  These a r e agents t h a t cannot be used  of  A c t i v a t i o n depends on  Van Duurnen, e t a l . , 1973;  Saffiotti,  1969).  i s the b e s t known c o c a r c i n o g e n (and e x t r a c t of c r o t o n r e s i n ) .  Croton It  promotes s k i n tumour f o r m a t i o n a f t e r the a p p l i c a t i o n of a c a r c i n o g e n i c p o l y c y c l i c aromatic hydrocarbon, 1974;  such as 2-methylcholanthrene  S i v a k and Van Duurnen, 1971).  (Boutwell,  -25SPECIFIC CARCINOGENS  1)  N i t r o g e n compounds:  Two sourea  compounds t h a t a r e p o t e n t i a l a l k y l a t i n g agents a r e m e t h y l n i t r o -  (MNU)  and N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e  potent c a r c i n o g e n s  (Table 2 ) .  (MNNG).  Both a r e  As a c l a s s , the a l k y l n i t r o s a m i d e s are  i m p r e s s i v e because of the wide v a r i e t y of t i s s u e s i n which they;' induce tumours, and by the s u s c e p t i b i l i t y to low doses of most of the s p e c i e s i n which they have been t e s t e d (Lowry and R i c h a r d s o n , 1976) .  T h i s i s presumably due  r e a c t i o n i n the a c i d c o n d i t i o n s found Heath, 1962;  critical  Both of these compounds a r e found among  the n i t r o s a t i o n p r o d u c t s of m e t h y l g u a n i d i n e  1968;  t h i o l s , amino groups) to be  i n t o e l e c t r o p h i l e s t h a t can s u c c e s s f u l l y a l k y l a t e  c e l l u l a r nucleophiles ( F i g . 3).  Magee, e t a l . ,  to the f a c t t h a t b o t h r e q u i r e o n l y r e a c t i o n  w i t h c e r t a i n u b i q u i t o u s n u c l e o p h i l e s (water, converted  1976;  which may  be produced  by  i n human stomach (Hedler and Marquardt,  Montesarro and Magee, 1970)  or a t n e u t r a l pH  by  a l i m e n t a r y b a c t e r i a (Badger, 1962). Because of the u b i q u i t o u s n a t u r e  of  these n i t r o s a t a b l e compounds, they have been i m p l i c a t e d i n the p r o d u c t i o n of human carcinomas ( K o t i n and F a l k , 1963;  2)  L r j i n s k y and Ross, 1967).  Dialkylnitrosamines:  The N-nitrosod-ialkylamine's'comprise .a' l a r g e c l a s s of compounds, some of w h i c h a r e r o u t i n e l y d e t e c t a b l e i n the environment Marquardt, 1968). compounds.  Dimethylnitrosamine  (DMN)  (Hedler  i s the s i m p l e s t of  and  these  They a r e l e s s v e r s a t i l e than the n i t r o s o a m i d e s because enzymic  N-demethylation  i s the f i r s t  step i n p r o d u c t i o n of the u l t i m a t e c a r c i n o g e n i c  -26-  FIGURE 3  O CH -N-C-NH NO methyl nitrosourea 3  2  NH CH -N-C-NHN0 NO N-methyl-N-nitroN-nitrosoguanidine 3  2  R-SH CH3-NH-NO CH3-N2 "OH  alkylation of target macromolecules  -27-  RGURLi  CH\  N  Chk> /  DMN  NO enzymic oxidation hydrolysis CH N^ 3  N-OH  J  CH3N2  1 CHJ  alkylation of target macromolecules  ^  -28-  s p e c i e s , and t h e s e compounds a r e , t h e r e f o r e , p r e c a r c i n o g e n s (Heath, 1962) . The e n z y m a t i c a l l y produced m e t a b o l i t e s of DMN s y n t h e t i c a l l y , and a r e too s h o r t - l i v e d  a r e d i f f i c u l t <bo p r e p a r e  to s t o r e f o r a n a l y s i s and  testing.  The exact n a t u r e of the a l k y l a t i n g s p e c i e s i s unknown, but a mechanism o f a c t i o n has been h y p o t h e s i z e d  ( F i g . 4)  (Heath, 1962).  DMN  i s a potent  - '> l i v e r c a r c i n o g e n i n the r a t (Table 2) and i s m e t a b o l i z e d i n human l i v e r s l i c e s a t c l o s e to the same r a t e as i n r a t l i v e r  slices  (Montesano and  Magee, 1970).  3)  P o l y c y c l i c a r o m a t i c hydrocarbons (PAH)  PAH a r e formed d u r i n g the incomplete combustion o f o r g a n i c m a t t e r (e.g., f o s s i l f u e l s ) and man ways (Badger, 1962). and food  i s t h e r e f o r e exposed to them i n a v a r i e t y of  PAH have been d e t e c t e d i n a i r , water, s o i l ,  ( K o t i n and F a l k , 1963; L y j i n s k i and Ross, 1967;  1971;. anon., 1973).  Benzo(a)pyrene (BP) i s an example  shown to be a p o t e n t c a r c i n o g e n (Table 2).  BP was  vegetation  Shabad, et a l . ,  t h a t has been  once thought to be  c a r c i n o g e n i c as such, s i n c e i t was commonly a b l e to i n d u c e tumours a t the s i t e of a p p l i c a t i o n . by the low aqueous  However, t h i s o b s e r v a t i o n might b e t t e r be e x p l a i n e d  s o l u b i l i t y of the compound which makes a s i n g l e  t i o n i n t o a c h r o n i c exposure.  applica-  The metabolism of BP, as w i t h other PAH, i s  c a r r i e d out by "mixed f u n c t i o n oxygenases", enzymes t h a t a r e NADPHdependent and c a t a l y z e the i n c o r p o r a t i o n of m o l e c u l a r oxygen i n t o the s u b s t r a t e m o l e c u l e s (Holtzman, e t a l . , forms of the cytochrome P-450 ( F i g . 5 ) . a r y l hydrocarbon h y d r o x y l a s e (AHH) epoxides a t the 4,5:7,8: and 9,10  1967) and which  containmultiple'  The oxygenases a r e a l s o  termed  and serve to induce the f o r m a t i o n of positions.  A l t h o u g h the 4,5-epoxide  i s the most s t a b l e , the 9,10-epoxide i s c o n s i d e r e d to be the p r e c u r s o r most  1-  -29-  FIHIRE 5  Benzo(a)pyrene  u  water soluble conjugates  u  BP-O-SG  diol- epoxides  -30-  TABLF.2 C a r c i n o g e n i c i t y of Some Chemicals  Carcinogen  Animal  S i t e s of tumour formation  MNNG  rat  glandular stomach, forestomach, i n t e s t i n e , subcutanepus ( s i t e of i n j e c t i o n i n t e s t i n e , forestomach, s k i n ( s i t e of i n j e c t i o n ) glandular stomach, i n t e s t i n e lung stomach, i n t e s t i n e  mouse S.G. hamster rabbit dog  MNU  r a t , mouse  c e n t r a l and p e r i p h e r a l nervous system, i n t e s t i n e , kidney, forestomach, glandular stomach, s k i n and annexes, jaw, b l a d d e r , uterus, vagina, lung, l i v e r , pharynx, esophagus, trachea, b r o n c h i , o r a l c a v i t y , pancreas, e a r ducts  DMN  rat mouse S.G. hamster rabbit  l i v e r , kidney, n a s a l c a v i t i e s l i v e r , lung, kidney l i v e r , nasal cavities liver  B(a)P  mouse  c e r v i x , hematological tumours, s k i n , r e s p i r a t o r y system, mammary tumours, l i v e r , lung, forestomach (when f e d B(a)P) subcutaneous, mammary, lung, o v a r i e s subcutaneous, stomach, trachea, s k i n skin subcutaneous  rats hamsters rabbits newts  •from "Survey of Compounds Which Have Been Tested For C a r c i n o g e n i c i t y DHEW P u b l i c a t i o n No. (NIH) 73-35, P u b l i c Health S e r v i c e P u b l i c a t i o n No. 149, U.S. Dept. of Health, Education and Welfare, U.S.A. (1969)  important f o r c a r c i n o g e n i c i t y and m u t a g e n i c i t y ( B a i r d , et^ a l . , r e a c t i o n s to form these epoxides go on i n the endoplasmic cytochrome P-450's  1975).  reticulum, although  and epoxide h y d r a t a s e s a l s o occur a t s i m i l a r  a c t i v i t i e s i n the n u c l e a r membrane (Kasper, 1974;  The  specific  Mukhtar, e t al.,'. 1979) .  A l t h o u g h t h i s membrane i s much s m a l l e r than the endoplasmic  r e t i c u l u m (and  so a b s o l u t e amounts of enzyme a r e small) the p r o x i m i t y of the membrane to DNA  might make the metabolism  important. (Oesch,  of PAH  t h a t goes on t h e r e d i s p r o p o r t i o n a t e l y  Epoxide h y d r a t a s e s do not r e q u i r e NADPH or o t h e r . c o f a c t o r s  et a l . ,  1971)  and have the c a p a c i t y t o c o n v e r t BP-epoxides t o  dihydrodiol-s ( K a p i t U l n i k , et - a l . ,  1977).  G l u t a t h i o n e - S - t r a n s f e r a s e (GSHT) ,  a s o l u b l e f r a c t i o n enzyme, c o n v e r t s epoxides to water s o l u b l e (Nemata and G e l b o i n , 1975) has been demonstrated  conjugates  a l t h o u g h non-enzymatic c o n j u g a t i o n of g l u t a t h i o n e  (Mukhtar and B r e s n i c k , 1976).  been d e s c r i b e d which may  conjugates  Conjugases  i n c o r p o r a t e BP—epoxides i n t o water  have a l s o  soluble  (Nemata and Takayama, 1977).  OUTLINE OF THE  PROBLEM  A wide v a r i e t y of t e s t s have been d i v i s e d to assay the a b i l i t y environmental compounds to induce c a n c e r . and  These t e s t s are both i n v i v o  i n v i t r o , as w e l l as s h o r t - t e r m (to measure pre-cancerous  and long-term  (to measure the i n d u c t i o n of r e s u l t a n t tumours).  pure compounds and While  of  complex mixtures have been and are to be  lesions) Both  tested.  these t e s t s a r e u s e f u l i n i n v e s t i g a t i n g c h e m i c a l s t h a t  may  cause cancer i n d e p e n d e n t l y , they are not g e n e r a l l y aimed a t d e t e c t i n g those c h e m i c a l s or c o n d i t i o n s which may At p r e s e n t , o n l y those agents  modify  important  the i n d u c t i o n of c a n c e r . i n promoting  the p r o d u c t i o n  -32-  of  tumours a f t e r the i n i t i a t i n g  are b e i n g w i d e l y i n v e s t i g a t e d .  events  The aim o f t h i s t h e s i s i s t o demonstrate  t h a t m o d i f i c a t i o n o f two proposed (DNA  i n c a r c i n o g e n e s i s a r e complete  initiation  steps i n carcinogenesis  damage by f r a g m e n t a t i o n o r carcinogen-adduct  r e p a i r ) may be brought  about by the a d d i t i o n o f c h e m i c a l r e d u c i n g  (sodium a s c o r b a t e , p r o p y l g a l l a t e , g l u t a t h i o n e ) . c h e m i c a l s or c o n d i t i o n s to modify i n s h o r t and long-term I t becomes n e c e s s a r y  f o r m a t i o n , and DNA  The a b i l i t y  agents  of c e r t a i n  DNA damage and r e p a i r w i l l go undetected  c a r c i n o g e n e s i s assays as they a r e g e n e r a l l y a p p l i e d .  f o r these t e s t s t o address  themselves  to such  compounds and c o n d i t i o n s by s e t t i n g s t a n d a r d c a r c i n o g e n e s i s c o n d i t i o n s (i..e. , MNNG f r a g m e n t a t i o n of DNA, BP-adduct f o r m a t i o n i n DNA) and t e s t i n g compounds f o r t h e i r a b i l i t y  t o modify  them.  In t h i s way, a b e t t e r  c o r r e l a t i o n between cancer i n c i d e n c e and environmental be  achieved.  cause may u l t i m a t e l y  -33-  MATERIALS AND METHODS  CHEMICALS  Sucrose, EDTA ( e t h y l e n e d i a m l n e t e t r a a c e t i c a c i d , sodium c h l o r i d e , sodium h y d r o x i d e and o t h e r common r e a g e n t s and s o l v e n t s were o b t a i n e d from t h e F i s h e r Chemical Co., Vancouver,  B.C.  RADIONUCLIDES  3 Thymidine-methyl- H (1 mCi/ml, s p e c i f i c a c t i v i t y : 20 Ci/mmole) was 3 o b t a i n e d from the New England N u c l e a r C o r p o r a t i o n , D o r v a l , P.Q. benzo(a)pyrene  (5 mCi/ml, s p e c i f i c a c t i v i t y :  from Amersham Corp., L t d . , O a k v i l l e ,  (G- H)  37 Ci/mmole) was o b t a i n e d  Ontario.  CHEMICAL CARCINOGENS  D i m e t h y l n i t r o s a m i n e (DMN) was purchased from K and K L a b o r a t o r i e s , P l a i n v i e w , N.Y.  N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) was purchased  from the A l d r i c h Chemical Comapny, Milwaukee, W i s c o n s i n .  REDUCING AGENTS  L-ascorbic acid  (sodium s a l t ) ,  glutathione  (reduced) and p r o p y l -  g a l l a t e were o b t a i n e d from the Sigma Chemical Company, S t . L o u i s , and were s t o r e d i n a d e s i c c a t o r a t 4° C to minimize a i r o x i d a t i o n .  MD,  _34METAL SALT SOLUTIONS  A s o l u t i o n of 0.1 as was  M c u p r i c s u l p h a t e was  g l y c i n e a t a c o n c e n t r a t i o n of 0.5  prepared  M.  in distilled  water,  Stock s o l u t i o n s of g l y c i n e  complexed copper were made by mixing a p p r o p r i a t e amounts of g l y c i n e w i t h metal  s t o c k , f o l l o w e d by d i l u t i o n w i t h 2.5%  (MEM  p l u s 2.5%  was  1:10.  f e t a l c a l f serum).  minimal  stock  e s s e n t i a l medium  The molar r a t i o of copper  to g l y c i n e  NITROSATION OF METHYLGUANIDINE  The n i t r o s a t i o n of 1-methyl-guanidine 1974)  w i t h m o d i f i c a t i o n s made by Lo and  A 1 ml r e a c t i o n m i x t u r e Chemical  Co.), 0.6  the volume brought w i t h MEM  without  Stich  pH was  0.6  (Endo and  (1978) was  (0.2 M 1-methyl-guanidine  M NaN0£ and  one hour a t 37° C.  sulphate  followed.  sulphate  M sodium a s c o r b a t e ) was  a d j u s t e d to 7.4  used  (Sigma incubated f o r  w i t h 2 N sodium carbonate  to 2 ml by a d d i t i o n of d i s t i l l e d water.  f e t a l c a l f serum was  Takahashi,  to lower  Serial  and  dilution  the c o n c e n t r a t i o n  -4 of  the o r i g i n a l m e t h y l g u a n i d i n e Alternatively,  of  to 5 X 10  the above procedure  sodium a s c o r b a t e i n the r e a c t i o n  M. was  c a r r i e d out w i t h the o m i s s i o n  mixture.  EXPERIMENTAL ANIMALS  Outbred,  4 month o l d , male Swiss mice were o b t a i n e d from the Animal  U n i t , F a c u l t y of M e d i c i n e , U n i v e r s i t y of B r i t i s h Columbia Connaught L a b o r a t o r i e s , W i l l o w d a l e , O n t a r i o ) .  (origin:  They were m a i n t a i n e d  e x p e r i m e n t a t i o n on a d i e t of Standard P u r i n a Lab  Chow and water ad  during libitum,  -35-  and were s u b j e c t e d to a 12 hr l i g h t Food, but not water, was  cycle.  removed from cages a t 5:00  p.m.  on  the  day b e f o r e c a r c i n o g e n a d m i n i s t r a t i o n . In the case of d e n s i t y g r a d i e n t s e d i m e n t a t i o n a n a l y s i s of f r a g m e n t a t i o n , mice were i n j e c t e d  DNA  i n t r a p e r i t o n e a l l y w i t h 5 X 10 ^ C i  3 (0.05 ml) use,  of  ( H)TdR i n d i s t i l l e d water a t 72, 24 and  16 h r s p r i o r to  to l a b e l mucosal c e l l s of the stomach ( K o r o p a t n i c k and  Stich,  1976).  CELL CULTURES  C u l t u r e d human f i b r o b l a s t s were grown from s k i n punch b i o p s i e s taken from the forearm of a 22 year o l d Caucasian was  female.  teased i n t o minute fragments w i t h s y r i n g e n e e d l e s and  sandwiched between g l a s s c o v e r s l i p s and medium) w i t h 12-20% f e t a l c a l f incubator.  Growth medium was  began to m i g r a t e  i n c u b a t e d i n MEM  The  skin  the p i e c e s (minimal  essential  serum f o r 2 to 3 weeks a t 37° C i n a CO2 changed every t h i r d day.  When f i b r o b l a s t s  from the t i s s u e fragments the c o v e r s l i p s were opened  and g r o s s t i s s u e fragments removed, l e a v i n g a p a r t i a l monolayer of b l a s t s on the c o v e r s l i p s .  These were i n c u b a t e d as above u n t i l  fibro-  the  f i b r o b l a s t s became a complete monolayer, a t which p o i n t the c e l l s were s u b c u l t u r e d by s t a n d a r d  techniques.  C u l t u r e s were m a i n t a i n e d in p l a s t i c P e t r i dishes. experiments.  The  i n p l a t e a u phase a t 37° C i n a CO2 i n c u b a t o r  T r a n s f e r passages 3 to 6 were used  c u l t u r e s were r o u t i n e l y m a i n t a i n e d  supplemented w i t h 15% p e n i c i l l i n / m l , 4 ug  in a l l  i n Eagle's  f e t a l c a l f serum and a n t i b i o t i c s  (200  MEM,  units  streptomycin/ml).  A l i n e of Chinese hamster c e l l s l a b o r a t o r y of Dr. L. Skarsgard  (CHO)(kindly s u p p l i e d by  ( P a l c i c and  Skarsgard,  1978)  the  were grown i n  -36-  MEM  (Grand  I s l a n d B i o l o g i c a l Co.) supplemented w i t h 15% f e t a l  serum, a n t i b i o t i c s  (streptomicin sulfate  (29.6 ^ig/ml), p e n i c i l l i n G  (204 ^ig/ml) , kanamycin (100 yug/ml) , and f u n g i z o n e and  7.5% sodium b i c a r b o n a t e (10 ml/800 ml medium).  c u l t u r e s were m a i n t a i n e d  calf  (2.5  pg/ml))  The s t o c k  i n 250 ml p l a s t i c c u l t u r e f l a s k s ( F a l c o n  p l a s t i c s ) and kept i n MEM w i t h 15% f e t a l c a l f  serum a t 37° C i n a  w a t e r - s a t u r a t e d CO2 i n c u b a t o r . F o r experiments  where damage to DNA,  damage, was to be determined  but not r e p a i r of t h a t  i n human f i b r o b l a s t s o r CHO  a p p r o x i m a t e l y 1.6 X 10~* c e l l s were seeded  cells,  i n t o 55 mm p l a s t i c d i s h e s  ( F a l c o n p l a s t i c s ) and kept i n MEM w i t h 15% f e t a l c a l f serum a t 37° C f o r 2 to 3 days.  Experiments  were begun when c e l l s were 70-80%  confluent. Where DNA r e p a i r was to be determined  i n human or CHO  cells,  f i b r o b l a s t s were grown i n the p l a s t i c d i s h e s i n 15% MEM u n t i l 50% c o n f l u e n t .  In o r d e r to i n h i b i t  about  c e l l d i v i s i o n a f t e r r e a c h i n g 70-80%  c o n f l u e n c e , the c e l l s were then t r a n s f e r r e d to a r g i n i n e d e f i c i e n t medium (ADM) supplemented w i t h 2.5% f e t a l c a l f i n c u b a t e d f o r another  3 to 4 days, a t which p o i n t they were 70-80%  c o n f l u e n t and ready f o r use. scheduled DNA  serum and  S i n c e these c e l l s were not undergoing  s y n t h e s i s , t h i s p r o c e s s was not confused w i t h r e p a i r  s y n t h e s i s of DNA. In the case o f d e n s i t y g r a d i e n t s e d i m e n t a t i o n a n a l y s i s of DNA f r a g m e n t a t i o n , DNA of c u l t u r e d c e l l s was p r e l a b e l l e d by i n c u b a t i o n  3 w i t h 5 ml of 10% MEM 24 h r s immediately  supplemented w i t h  p r i o r to use.  H-TdR ( O ^ ^ C i / m l ) f o r  -37-  PREPARATION OF S9 ACTIVATION MIXTURE  A d u l t male Swiss mice were k i l l e d by d e c a p i t a t i o n and b l e e d i n g . The l i v e r s were q u i c k l y removed and minced w i t h i c e - c o l d phosphate-buffered  saline  Dulbecco's  (PBS) w i t h 0.25 M s u c r o s e , homogenized by  a S o r v a l l t e f l o n p e s t l e t o r s i o n homogenizer a t 1000 rpm and c e n t r i f u g e d a t 9000 X £ f o r 10 min a t 4° C (Garner and Hanson, 1971). The r e s u l t i n g p o s t m i t o c h o n d r i a l supernatant f r a c t i o n was f r o z e n i n l i q u i d n i t r o g e n and s t o r e d i n a Revco f r e e z e r a t -71° C. The a c t i v a t i o n mixture c o n s i s t e d o f 4 umoles NADPH, 25 nmoles M g C l , 20 p i o l e s glucose-6-phosphate, 2  0.1 ml NaOH (0.2 N) and 0.4  ml o f p o s t m i t o c h o n d r i a l l i v e r supernatant a p p r o x i m a t e l y 240 mg o f organ wet-weight  fraction  (which c o n t a i n s  ( L a i s h e s and S t i c h ,  1973)).  I n g r e d i e n t s were mixed and 0.25 ml o f S9 a c t i v a t i o n m i x t u r e added to c e l l c u l t u r e s w i t h i n 5 min.  ADMINISTRATION OF CHEMICALS  Dimethylnitrosamine: To each p l a t e o f c u l t u r e d human f i b r o b l a s t s was added 1) 4.5 ml o f MEM w i t h o u t f e t a l c a l f MEM w i t h o u t f e t a l c a l f  serum, 2) 0.25 ml o f DMN  serum (0.1 M ) ( s u f f i c i e n t  dissolved i n  to produce  a final  _3 DMN  c o n c e n t r a t i o n o f 5 X 10  activation  M on t h e p l a t e ) , and 3) 0.5 ml o f S9  mixture.  In the case o f sodium a s c o r b a t e i n h i b i t i o n o f n i t r o s a t i o n formation, s u f f i c i e n t  sodium a s c o r b a t e  c a l f serum) was added t o produce  ( d i s s o l v e d i n MEM without -2  a c o n c e n t r a t i o n o f 1 X 10  p r i o r to a d d i t i o n o f S9 a c t i v a t i o n  product  mixture.  M  fetal  immediately  -38-  C e l l s were exposed a t 37° C f o r 1 h r and washed t h r e e times w i t h PBS s o l u t i o n  (8.0 g NaCl, 0.2 g KC1, 1.15 g N a H P 0 2  4  and 0.2 g K H P 0  i n 1000 ml o f d i s t i l l e d water: pH 7.45) and used f o r a l k a l i n e gradient  2  4  sucrose  analysis.  N i t r o s a t i o n products of methylguanidine:  S e r i a l d i l u t i o n o f the 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 w i t h MEM without f e t a l c a l f serum was made t o reduce t h e s t a r t i n g c o n c e n t r a t i o n -4 of m e t h y l g u a n i d i n e  t o 5 X 10  M.  C e l l s were t r e a t e d f o r 1 h r and  prepared f o r a l k a l i n e s u c r o s e g r a d i e n t (ASG) a n a l y s i s i n the manner d e s c r i b e d above. MNNG:  1) I n v i t r o : (DMSO) and d i l u t e d  MNNG was d i s s o l v e d i n 1 ml o f d i m e t h y l s u l p h o x i d e t o the a p p r o p r i a t e c o n c e n t r a t i o n by a d d i t i o n o f  99 ml o f MEM w i t h 2.5% f e t a l c a l f serum. hr a t 37° C i n a C 0  2  C e l l s were t r e a t e d f o r 0.5  i n c u b a t o r f o l l o w e d by p r e p a r a t i o n f o r ASG a n a l y s i s  i n the manner d e s c r i b e d above. 2) I n v i v o :  MNNG was f o r c e - f e d t o e x p e r i m e n t a l animals under  e t h e r a n a e s t h e s i a by esophageal ml.  i n t u b a t i o n i n a t o t a l volume o f 0.5  The 0.5 ml c o n t a i n e d 0.1 ml DMSO; 0.4 ml d i s t i l l e d water; MNNG.  A f t e r 4 h r the animals were k i l l e d by c e r v i c a l d i s l o c a t i o n and b l e e d i n g and t i s s u e samples  taken.  -39-  Sodium a s c o r b a t e :  Sodium a s c o r b a t e was d i s s o l v e d a t twice the d e s i r e d c o n c e n t r a t i o n i n MEM w i t h o u t  fetal calf  serum.  Glycine/CuSO^'SI^O s o l u t i o n s were  made as d e s c r i b e d above a t twice the d e s i r e d c o n c e n t r a t i o n .  The two  s o l u t i o n s were mixed i n equal volumes and added t o c e l l s w i t h i n 10 s e c . In  cases where sodium a s c o r b a t e o r CuSO^ SH^O/glycine were -  a d m i n i s t e r e d a l o n e , the c o n c e n t r a t e d s o l u t i o n s were mixed w i t h MEM (without f e t a l c a l f Cells 37° C.  serum) alone and added t o c e l l s .  were i n c u b a t e d i n v i t r o w i t h m i x t u r e s  Cells  were washed w i t h PBS and used  f o r 0.5 min a t  i n ASG a n a l y s i s of DNA  damage. Mice under l i g h t mixtures and  e t h e r a n a e s t h e s i a were f o r c e - f e d 0.5 ml o f  of the d e s i r e d concentration.  A f t e r 4 hr the mice were k i l l e d  t i s s u e s e x c i s e d f o r ASG a n a l y s i s of DNA damage.  Benzo(a)pyrene:  1) I n v i t r o :  3 H-BP i n t o l u e n e was p l a c e d i n a 1 l i t r e  Sufficient  g l a s s b o t t l e t o produce 4.16 X 10 ^ M BP when d i l u t e d t o s t o c k volume. 3 2 ml o f DMSO was added t o the H-BP and a i r blown over the s u r f a c e o f the f l u i d w i t h an a i r pump and P a s t e u r p i p e t f o r 15 min under the fume hood t o remove v o l a t i l e  toluene.  serum was added to the m i x t u r e , b e f o r e use.  498 ml o f MEM w i t h 5% f e t a l  calf  shaken, and a l l o w e d t o s i t 10 min  The m i x t u r e was used w i t h i n 1 h r .  Sodium a s c o r b a t e , p r o p y l g a l l a t e o r g l u t a t h i o n e was made up i n MEM w i t h o u t  fetal calf  serum and used w i t h i n 10 min o f m i x i n g .  -40-  The  pH o f g l u t a t h i o n e m i x t u r e s was a d j u s t e d  NaOH.  t o 7.0 w i t h  concentrated  A l l c o n c e n t r a t i o n s were 2.083 times g r e a t e r than t h a t r e q u i r e d  i n the f i n a l mixture, 2.4  s i n c e d i l u t i o n took p l a c e i n c e l l c u l t u r e d i s h e s .  ml o f r e d u c i n g  agent, 2.4 ml o f BP m i x t u r e and 0.2 ml o f  S9 a c t i v a t i o n m i x t u r e were added t o the c e l l s , C e l l s were i n c u b a t e d  i n that  a t 37° C i n a CO2 i n c u b a t o r  c e l l s were then washed 2 times w i t h  order. f o r 2 hr.  The  5 ml o f i c e - c o l d PBS and the DNA  i s o l a t e d f o r BP adduct a n a l y s i s . 3 2) I n v i v o :  H-BP i n t o l u e n e was added t o DMSO i n the r a t i o o f  1 ^ug BP t o 0.1 ml DMSO. as d e s c r i b e d above. to produce a f i n a l  3  Toluene was v o l a t i l i z e d  i n the same way  Enough MEM w i t h 2.5% f e t a l c a l f serum was added —6 H-BP c o n c e n t r a t i o n o f 1 X 10 g/ml ( s u f f i c i e n t t o  d e l i v e r 100 ng t o each mouse). Mice which had been d e p r i v e d 5:00  o f food, b u t not water, a t  p.m. the n i g h t b e f o r e were f o r c e - f e d 0.5 ml o f the BP m i x t u r e  (500 ng) 1:00  under l i g h t  ether anaesthesia  p.m. t h e f o l l o w i n g day.  between 10:00  Mice were k i l l e d  a.m. and  and stomachs e x c i s e d  a t v a r y i n g times f o l l o w i n g t h i s prodedure, the DNA e x t r a c t e d , and BP adduct a n a l y s i s c a r r i e d  A l k a l i n e sucrose  1) I n v i v o :  out.  g r a d i e n t a n a l y s i s o f DNA damage and r e p a i r :  The method o f Cox,  a l l of the e s s e n t i a l s .  e t a l . (1973) was f o l l o w e d i n  The animal was k i l l e d by c e r v i c a l  and  bleeding.  and  d i s t a l t o t h e esophagus, opened a l o n g  twice  The stomach was e x c i s e d p r o x i m a l  i n i c e - c o l d PBS.  dislocation  t o the p y l o r i c  i t s a n t e r i o r aspect  sphincter and r i n s e d  G a s t r i c s u r f a c e mucosa was removed by s c r a p i n g  w i t h a c o l d g l a s s microscope s l i d e .  The s c r a p i n g s were mixed w i t h  -413.0 ml of i c e - c o l d PBS f o r 3 min  to sediment  and spun a t 1000 cells.  i n a Dynac c l i n i c a l  The c e l l s were mixed w i t h 3.0 ml  spun down a g a i n , and resuspended a l i q u o t was  rpm  i n 0.5 ml of i c e - c o l d PBS.  PBS,  A 25  ulitre  l a y e r e d on the g r a d i e n t .  2) In v i t r o : i c e - c o l d PBS.  0.5  C h e m i c a l l y t r e a t e d c e l l s were r i n s e d 3 times w i t h ml of c o l d PBS  was  added and the c e l l s were scrubbed  away from the d i s h w i t h a rubber policeman. was  centrifuge  The c e l l  suspension (0.5  p l a c e d i n a 3 ml c e n t r i f u g e tube and spun a t 2600 rpm  c e n t r i f u g e f o r 5 min.  The c e l l - f r e e supernatant was  100 u l i t r e s of c o l d PBS  was  in a  ml)  clinical  removed and d i s c a r d e d .  added and the c e l l s were kept on an i c e - b e d  i n p r e p a r a t i o n f o r l a y e r i n g on g r a d i e n t s .  A l k a l i n e sucrose g r a d i e n t s :  G r a d i e n t s were p r e p a r e d an hour b e f o r e use, a c c o r d i n g to the method of Cox,  et a l . ,  (Beckman Instrument  (1973).  Co.,  Into n i t r o c e l l u l o s e c e n t r i f u g e  Vancouver,  1 ml o f 2.3 M s u c r o s e ; 5-20%  0.5%  was  laid,  i n succession:  a l k a l i n e s u c r o s e g r a d i e n t (0.9 M NaCl,  0.3 M NaOH); 0.3 ml l y s i n g s o l u t i o n 0.1 M t r i s - H C l ,  B.C.)  tubes  (0.3 M NaCl, 0.03  sodium d o d e c y l s u l p h a t e (SDS);  M EDTA,  1 X  10 5  5 X 10^ c e l l s o r i n t a c t c e l l n u c l e i i n a volume not exceeding 50 j j l i t r e s ; 0.3 ml l y s i n g s o l u t i o n ; i s o - o c t a n e to w i t h i n 0.5 tube.  cm of the top of the  G r a d i e n t s were p l a c e d i n the buckets of a Beckman SW  40  u l t r a c e n t r i f u g e r o t o r and spun a t 77,561 X j> a t an average r a d i u s of 11.10  cm  (25,000 rpm)  f o r 30 min a t 20° C w i t h the brake o f f i n  a Beckman L2B u l t r a c e n t r i f u g e .  F i f t e e n s e q u e n t i a l f r a c t i o n s were taken  from the bottoms o f the p i e r c e d tubes, p r e c i p i t a t e d w i t h  8-10%  t r i c h l o r o a c e t i c a c i d and c o l l e c t e d on n i t r o c e l l u l o s e membrane  filters.  -42-  A c i d s o l u b l e r a d i o a c t i v i t y was  removed by washing the f i l t e r s w i t h  8-10%  ethanol.  was  t r i c h l o r o a c e t i c a c i d and  Acid insoluble radioactivity  counted by immersing the d r i e d f i l t e r s i n t o l u e n e  f l u i d and liquid  counting  f o r 10 min  scintillation  per v i a l on the S e a r l e D e l t a  300  counter.  BP adduct measurement i n  DNA  scintillation  DNA:  i s o l a t i o n prodedure was  a m o d i f i c a t i o n of t h a t used  by  Diamond, et a l . (1967). 1) T i s s u e s :  Mouse stomach was  a n a l y s i s , r i n s e d by h o l d i n g w i t h of i c e - c o l d PBS,  and  e x c i s e d as d e s c r i b e d f o r  f o r c e p s and  plunging  the whole stomach p l a c e d  g l a s s t e s t tube w i t h 5 ml of 1% sodium d o d e c y l The  stomach was  t r a n s f e r r e d to a 15 ml CHO  sec.  teflon-capped  cells  t r e a t e d w i t h a c t i v a t e d BP  C e l l s from two  teflon-capped  Pyrex  sulphate/25 mM  EDTA.  t i s s u e s o l u t i o n was  g l a s s c e n t r i f u g e tube. 6 X 10  c e l l s per p l a t e ) were  f o r 2 hr as d e s c r i b e d above and were r i n s e d  added to each p l a t e and  policeman.  The  (approximately  2 times w i t h 5 ml a l i q u o t s of PBS. was  i n a 25 ml  washes  d i s p e r s e d u s i n g a P o l y t r o n t i s s u e homogenizer at  s e t t i n g 5, number 2 head, f o r 20  2) C e l l s :  i n two  ASG  2.5  the c e l l s  ml of 1% SDS/25 mM  scraped  EDTA  o f f w i t h a rubber  p l a t e s were t r a n s f e r r e d to a 15  ml  g l a s s c e n t r i f u g e tube.  DNA  isolation:  1)  S o l u t i o n s were e x t r a c t e d 4 times w i t h 5 ml  phenol (90% phenol,  tris-equilibrated  e q u i l i b r a t e d w i t h e q u a l volumes of 0.5  M  tris-  -43-  HC1  ( a d j u s t e d to pH 8.0  2)  S o l u t i o n s were then e x t r a c t e d 2 times w i t h 5 ml e t h y l e t h e r .  3)  1 ml of beef p a n c r e a t i c RNAase (200 yug/ml, Sigma Chemical Corp.)  was  added and  4)  1 ml of 0.02%  s o l u t i o n s incubated  1 hr at 37°  o v e r n i g h t at 37°  C. added  and  C.  S o l u t i o n s were e x t r a c t e d 4 times w i t h 5 ml of  isoamyl a l c o h o l (24:1 6)  HC1)).  pronase (Sigma Chemical Corp.) was  s o l u t i o n s incubated 5)  with concentrated  chloroform/  v/v).  S o l u t i o n s were e x t r a c t e d t h r e e times w i t h 5 ml e t h e r , and  the  l a s t e t h e r t r a c e s were b o i l e d o f f by p l a c i n g the open tubes i n warm tap water (approximately  60° C) f o r 15  7)  6 ml of 2% sodium a c e t a t e i n 99%  and  the tubes l e f t  8)  DNA  was  s e t t i n g 100 9)  f o r 20 min  (approximately  d i s c a r d e d and  2000  the DNA  rpm). p e l l e t d r i e d and  resuspended  M sodium a c e t a t e .  concentration  determination:  Purified calf  thymus DNA  (Sigma Chemical Corp.) was  M sodium a c e t a t e s o l u t i o n and  curve of DNA  added to p r e c i p i t a t e  at -4° C o v e r n i g h t .  Supernatant was  i n a 0.03  e t h a n o l was  p r e c i p i t a t e d by s p i n n i n g i n a Dynac c l i n i c a l c e n t r i f u g e at  i n 4 ml of 0.03  DNA  min.  concentration versus  serially diluted  absorbance at 260  nm.  dissolved to produce a I t was  found  that: A  260  „„ = DNA  A  . concentration  , .-1. tug*ml )  0.024  All  samples were measured i n 1 ml quartz c u v e t t e s i n a Bausch and Lomb  DNA,  -44-  S p e c t r o n i c 21 UV  spectrophotometer.  DNA  s o l u t i o n s were d i l u t e d , i f  n e c e s s a r y , to ensure t h a t absorbance r e a d i n g s f e l l between 0.1 0.8  absorbance u n i t s  (the range i n which the graph was  The absorbance o f each sample at 260 and 280 nm was samples w i t h A „ , _ / A _ ZbU  ZoU 00  of each sample was  most  used f o r DNA  linear).  taken and  f a l l i n g below 2.00 were d i s c a r d e d .  and  any  1.0  ml  concentration determination.  Measurement o f r a d i o a c t i v i t y :  2.5 ml of DNA  s o l u t i o n was  p l a c e d i n a 25 ml  plastic  s c i n t i l l a t i o n v i a l and 15 ml of PCS w a t e r - m i s c i b l e s c i n t i l l a t i o n fluid  (Amersham Corp.) was  added and mixed by s h a k i n g .  The  tubes  were kept i n the dark a t room temperature f o r 24 hr to minimize  .  chemiluminescence. 3 Samples were counted f o r D e l t a 300 l i q u i d  H r a d i o a c t i v i t y on the S e a r l e  s c i n t i l l a t i o n c o u n t e r f o r a s u f f i c i e n t p e r i o d to  a l l o w no more than 1% e r r o r i n c o u n t - r e a d i n g .  D i s i n t e g r a t i o n s per  minute were c a l c u l a t e d f o r each sample u s i n g c h a n n e l s - r a t i o quench correction. U s i n g the s p e c i f i c a c t i v i t y of the BP, i n the DNA  s o l u t i o n was  the BP  concentration  c a l c u l a t e d , and the r e s u l t expressed as  "ng of benzo(a)pyrene per g of  DNA".  -45-  RESULTS  I n h i b i t i o n o f DNA r e p a i r by sodium a s c o r b a t e  1) A l k a l i n e s u c r o s e g r a d i e n t a n a l y s i s o f DNA damage and r e p a i r :  a)  In v i t r o :  DNA from c u l t u r e d human f i b r o b l a s t s , when  r e l e a s e d by c e l l l y s i s on a l k a l i n e s u c r o s e g r a d i e n t s , has been shown t o sediment t o the s a t u r a t e d s u c r o s e c u s h i o n (Stich, et a l . ,  1979,a,b).  t r e a t e d w i t h MNNG (1 X 10  ( F i g . 6)  When c u l t u r e d human f i b r o b l a s t s were M) f o r 30 min,  and sampled  immediately  (1. e_., p l a c e d on i c e w i t h i n 5 min and then i n t o l y s i n g w i t h i n 20 min)  a shift  solution  i n the peak o f DNA c o n c e n t r a t i o n from  the  r e g i o n s o c c u p i e d by f a s t - s e d i m e n t i n g DNA t o slow-sedimenting DNA  was seen, between f r a c t i o n s 10 and 15 ( F i g . 7A).  t r e a t e d i d e n t i c a l l y , but switched a f t e r treatment  t o MEM w i t h 5% f e t a l c a l f  serum  w i t h MNNG and then allowed t o i n c u b a t e f o r 30 h r a t  37° showed DNA sedimenting shift  However, c e l l s  a t c l o s e t o c o n t r o l r e g i o n s ( F i g . 7B).  This  i n DNA p r o f i l e s from damage t o r e p a i r r e g i o n s was taken t o i n d i c a t e  repair. On  the o t h e r hand, c e l l s t r e a t e d w i t h MNNG f o r 0.5 h r ( F i g . 8A) _3  but which have a s o l u t i o n o f sodium a s c o r b a t e  (1 X 10  M) i n the  5% MEM b a t h i n g medium i n which they r e p a i r e d d u r i n g the f o l l o w i n g 30 hr showed decreased 30 h r ( F i g .  ability  t o approach c o n t r o l DNA p r o f i l e s  after  8B).  When c e l l s were t r e a t e d w i t h MNNG as above, but were allowed t o _3 r e p a i r i n 5% MEM supplemented w i t h sodium a s c o r b a t e  (1 X 10  which was renewed every 4 h r up t o 20 h r a f t e r MNNG  treatment  M)  -46-  Figure 6 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of from c u l t u r e d human  fibroblasts.  H-DNA  -47-  0  5  10  15  <- S E D I M E N T A T I O N  -48-  F i g u r e 7A 3 A l k a l i n e sucrose  gradient  sedimentation  p r o f i l e o f H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 f o r 0.5 h r .  F i g u r e 7B 3 A l k a l i n e sucrose  gradient sedimentation  p r o f i l e o f H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 f o r 0.5 h r f o l l o w e d by i n c u b a t i o n i n 5% MEM f o r 30 h r at 37° C.  -49-  -50-  F i g u r e 8A 3 A l k a l i n e sucrose g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 ~* f o r 0.5 h r .  F i g u r e 8B 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 f o r 0.5 h r f o l l o w e d by i n c u b a t i o n i n 5% MEM supplemented w i t h sodium a s c o r b a t e  (1 X 1 0 ~ M) f o r 30 h r a t 37° C. 3  -51-  0  5 10 15 -*—SEDIMENTATION  -52-  (5 medium changes i n a l l ) , was  even more i n h i b i t e d  then r e s t o r a t i o n o f c o n t r o l DNA p r o f i l e s  ( F i g . 9A).  This i n d i c a t e d that continued  i n c u b a t i o n o f sodium a s c o r b a t e may i n h i b i t  i t s repair-inhibiting  p r o p e r t i e s , e i t h e r by a u t o x i d a t i o n t o o t h e r p r o d u c t s , o r by c e l l u l a r i n a c t i v a t i o n o f the r e p a i r - i n h i b i t i n g s p e c i e s . Because apparent  l a c k o f r e p a i r on a l k a l i n e sucrose g r a d i e n t s  might be due t o c e l l death,  i t was n e c e s s a r y  sodium a s c o r b a t e had not i n f l i c t e d c e l l s t o prevent  sufficient  t o demonstrate t h a t i n j u r y on the  them from b e i n g a b l e t o r e c o v e r .  fibroblast  T h e r e f o r e , c e l l s were  t r e a t e d w i t h MNNG, then w i t h 5 doses o f sodium a s c o r b a t e over a 20 h r p e r i o d , and then a l l o w e d t o r e p a i r i n a medium c o n t a i n i n g 5% MEM up t o 72 h r f o l l o w i n g MNNG treatment DNA  from these c e l l s once a g a i n sedimented  (Fig.  9B).  i n a manner t h a t approached  t h a t o f c o n t r o l DNA. I t might be argued cell killing  t h a t sodium a s c o r b a t e c o n t r i b u t e s t o  such t h a t c e r t a i n c e l l s d i e w h i l e o t h e r s a r e a b l e  to go on t o r e p a i r DNA t o r e s t o r e i t s f a s t - s e d i m e n t i n g a b i l i t y . A measure o f t h i s was the amount o f r a d i o a c t i v e from the g r a d i e n t s .  3  H l a b e l recoverable  C e l l s t h a t have undergone r e p a i r should have 3  decreased  recovery of H l a b e l  some c e l l s had been unable  (a measure o f the amount o f DNA) i f  t o r e c o v e r and had been l y s e d .  shows r e s u l t s o b t a i n e d from the experiment The r e s u l t s o f t h r e e s e p a r a t e experiments two  came from the f i r s t  experiment,  from the t h i r d experiment.  illustrated a r e shown.  Table 3  i n F i g . 9. Of the 5 r e s u l t s ,  two from the second,  and one sample  I n t h i s case, c e l l s t h a t had undergone r e p a i r  a f t e r MNNG and sodium a s c o r b a t e treatment  still  r e t a i n e d 70-93% o f t h e  3 H l a b e l r e c o v e r a b l e from c o n t r o l c e l l s u n t r e a t e d w i t h MNNG o r sodium ascorbate.  I f h i g h e r c o n c e n t r a t i o n s o f sodium a s c o r b a t e were used,  -53-  F i g u r e 9A 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 for  M)  0.5 h r f o l l o w e d by i n c u b a t i o n i n 5% MEM supplemented  w i t h sodium a s c o r b a t e  (1 X 10  M) f o r 30 h r a t 37° C.  The MEM  w i t h sodium a s c o r b a t e was r e p l a c e d w i t h f r e s h l y - m i x e d sodium a s c o r b a t e s o l u t i o n every 4 h r f o r the f i r s t a f t e r MNNG  20 h r  treatment.  F i g u r e 9B 3 Alkaline  s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o d i l e o f H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 for  M)  0.5 h r f o l l o w e d by i n c u b a t i o n i n 5% MEM supplemented  w i t h sodium a s c o r b a t e  (1 X 1 0 ~ M) f o r 30 h r a t 37° C. The 3  MEM w i t h sodium a s c o r b a t e was r e p l a c e d w i t h f r e s h l y - m i x e d sodium a s c o r b a t e s o l u t i o n every 4 h r f o r the f i r s t a f t e r MNNG treatment.  20 h r  A t 3 h r post-MNNG treatment, t h e  sodium a s c o r b a t e s o l u t i o n was removed and r e p l a c e d w i t h 5% MEM without  sodium a s c o r b a t e , i n c u b a t e d f o r a f u r t h e r  72 h r a t 37° C, and sampled.  -54-  O  5 10 15 <—SEDIMENTATION  -55-  TABLE 3 Recovery of r a d i o a c t i v i t y from a l k a l i n e sucrose gradients  Source of DNA  c.p.m. (mean of 5 samples + standard e r r o r )  % of radioactivity in control c e l l s recoverable from gradients  1392 + 320  100%  MNNG treatment followed by i n c u b a t i o n with 5 sodium ascorbate treatments over 20 hours  1580 + 107  114%  MNNG treatment followed by sodium ascorbate treatment followed by r e p a i r i n MEM without sodium ascorbate up to 72 hours f o l l o w i n g MNNG treatment  1220 + 66  88%  3 H-treated c o n t r o l (200,000 c e l l s )  cells  -563 the r e c o v e r a b i l i t y o f H counts tended t o decrease, shape o f the a l k a l i n e sucrose Therefore,  sodium a s c o r b a t e  although the  g r a d i e n t p r o f i l e s was not  c o n c e n t r a t i o n s f o r treatment  found  t o change.  of c e l l s  were chosen such t h a t they were as h i g h as p o s s i b l e without 3 decreasing  H r e c o v e r a b i l i t y t o any g r e a t e x t e n t .  MNNG c o n c e n t r a t i o n s 3  chosen f o r use were those g r e a t enough t o cause the mass o f H l a b e l t o accumulate between f r a c t i o n s 9 and 13, but no g r e a t e r . In t h i s way,  the lowest  p o s s i b l e MNNG c o n c e n t r a t i o n s  (those  just  c a u s i n g enough damage t o observe r e p a i r ) and the h i g h e s t p o s s i b l e sodium a s c o r b a t e  concentrations  (those t h a t d i d not c o n t r i b u t e t o  enhanced c e l l k i l l i n g ) were used. Sodium a s c o r b a t e (Fig.  treatment  a l o n e d i d not  fragment DNA  10).  b)  In v i v o :  DNA from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d  MNNG (40 mg/kg body weight) waas shown t o be damaged 4 h r a f t e r f o r c e feeding  ( F i g . 11A).  c o n t r o l sedimentation  However, r e p a i r o c c u r r e d  t h a t r e t u r n s DNA t o near  p r o f i l e s by 30 h r f o l l o w i n g f o r c e - f e e d i n g ( F i g . 11B).  When, on the o t h e r hand, MNNG was f o r c e - f e d t o mice i n the same c o n c e n t r a t i o n ( F i g . 12A) f o l l o w e d by f o r c e - f e e d i n g sodium (20 mg/kg body weight a t 15 min,  4 h r , 8 h r , 12 h r , 16 hr, 20 h r and  24 h r f o l l o w i n g MNNG treatment) no evidence by 30 h r and s e d i m e n t a t i o n  ascorbate  o f r e p a i r was apparent  o f DNA d i d not approach n e a r - c o n t r o l p r o f i l e s ,  but remained slow-sedimenting  ( F i g . 12B).  a l o n e d i d not fragment DNA ( F i g . 1 0 ,  Sodium a s c o r b a t e  treatment  .  In the same manner as c u l t u r e d human f i b r o b l a s t s ±n v i t r o , g a s t r i c mucosal c e l l s JLn v i v o r e t a i n e d the a b i l i t y measured by t h e i r a b i l i t y  t o r e p a i r DNA (as  t o r e s t o r e f a s t - s e d i m e n t i n g p r o p e r t i e s t o DNA)  -57-  Figure  10 3  A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o sodium a s c o r b a t e _3  (1 X 10  M) f o r 20 h r .  The sodium a s c o r b a t e  solution  was r e p l a c e d w i t h f r e s h l y mixed s o l u t i o n every 4 h r f o r the f i r s t 20 h r a f t e r t h e b e g i n n i n g o f t r e a t m e n t .  -58-  CO  l_ 1  z O u 20 ZD  I  ro _J  TOT  < 10  LL  O  o  o  0 5 10 15 SEDIMENTATION  -59-  F i g u r e 11A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) a t 0 h r and sampled a t 4 h r .  F i g u r e 11B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) a t 0 h r and sampled a t 30 h r .  -60-  20 00  8  1  0  u  IE  ro _j  ,0-0'  0  LL  O 20  /  10  /  "\  \  o  \  /  0 0  O-r  0-0  5 10 15 * — SEDIMENTATION  -61-  F i g u r e 12A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) a t 0 h r and sampled a t 4 h r .  F i g u r e 12B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) a t 0 h r f o l l o w e d by f o r c e - f e e d i n g of  sodium a s c o r b a t e  (20 mg p e r kg body weight) a t 15 min,  4 h r , 8 h r , 12 h r , 16 h r , 20 h r and 24 h r , and sampled a t 30 h r .  -62-  0 5 10 15 * — SEDIMENTATION  -63s i n c e f a s t s e d i m e n t a t i o n was r e s t o r e d when mice t h a t had been f o r c e - f e d MNNG, 7 doses o f sodium a s c o r b a t e allowed t o r e p a i r without  (as i n F i g . 12) and were  f u r t h e r sodium a s c o r b a t e  treatment  up t o 72 h r a f t e r MNNG a d m i n i s t r a t i o n ( F i g . 13B).  2)  BP-DNA adduct  a n a l y s i s o f DNA r e p a i r :  a) I n v i t r o : i)  One o f the problems a s s o c i a t e d w i t h the a d m i n i s t r a t i o n  of p o l y c y c l i c a r o m a t i c hydrocarbons  t o c u l t u r e d c e l l s o r mammalian  systems i s the h y d r o p h o b i c i t y a s s o c i a t e d w i t h them.  BP tends  to l e a v e water s o l u t i o n s by attachment t o the g l a s s o r p l a s t i c c o n t a i n e r , o r by s e p a r a t i o n o f the non-polar BP from the water s o l u t i o n .  s o l v e n t c a r r y i n g the  I n o r d e r t o s o l v e t h i s problem,  3 H-BP  i n t o l u e n e was d i s s o l v e d i n DMSO, the t o l u e n e blown o f f by  a stream o f a i r , and the r e s u l t i n g w a t e r - m i s c i b l e DMSO/BP s o l u t i o n d i s s o l v e d i n MEM supplemented w i t h 5% f e t a l c a l f solution  (presumably  serum.  BP i n t h i s  bound t o the p r o t e i n component o f f e t a l c a l f  remains a v a i l a b l e i n s o l u t i o n f o r a t l e a s t  serum)  70 min f o l l o w i n g s o l u t i o n ,  w h i l e BP t r e a t e d i d e n t i c a l l y , but d i s s o l v e d i n MEM without FCS, r a p i d l y becomes u n a v a i l a b l e i n the water s o l u t i o n  ( F i g . 14).  BP i n  s o l u t i o n was measured by s c i n t i l l a t i o n c o u n t i n g o f an a l i q u o t o f s o l u t i o n . ii)  BP b i n d i n g t o DNA was determined  for 2 hr incubations  of c o n c e n t r a t i o n s r a n g i n g from 0.1 X 10 ^ M t o 3 X 10 ^ M produced by d i l u t i o n o f a s i n g l e p a r e n t BP s t o c k w i t h MEM w i t h 5% f e t a l serum ( F i g . 15).  S i n c e the 2 X 10 ^ M c o n c e n t r a t i o n f a l l s  calf  i n a region  of the curve w i t h c o n s t a n t s l o p e , i t was deemed an a p p r o p r i a t e c o n c e n t r a t i o n to use r o u t i n e l y f o r b i n d i n g and r e p a i r s t u d i e s .  -64-  F i g u r e 13A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  of  H-DNA  from g a s t r i c mucosal c e l l s of mice f o r c e - f e d sodium a s c o r b a t e (20 mg p e r kg body weight) a t 15 min, 4 h r , 8 h r , 12 h r , 16 h r , and 20 h r and sampled a t 24 h r .  F i g u r e 13B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  of  H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) a t 0 h r f o l l o w e d by f o r c e - f e e d i n g of fed  sodium a s c o r b a t e  (20 mg p e r kg body weight) f o r c e -  a t 15 min, 4 h r , 8 h r , 12 h r , 16 h r , 20 h r and 24 h r  and sampled a t 72 h r .  -66-  F i g u r e 14 3 H-BP  a v a i l a b l e i n s o l u t i o n a t v a r i o u s times a f t e r mixing  w i t h MEM  without  5% f e t a l c a l f  fetal  calf  serum ( O ) .  3  serum (• ) o r w i t h MEM  H-BP  i n a 0.5 ml a l i q u o t was measured  by s c i n t i l l a t i o n c o u n t i n g i n w a t e r - m i s c i b l e f l u i d a t the times i n d i c a t e d . for  each time.  with  scintillation  A s i n g l e sample i s p l o t t e d  -67-  "D O  T 3  100  CQ  "D <D c_  <D > O u  <D L.  Q_ CD  0  10  30  50  time after mixing (minutes)  70  -68-  Figure  15  B i n d i n g of  3  H-BP  to DNA  of c u l t u r e d CHO 3  to v a r i o u s c o n c e n t r a t i o n s of  H-BP  cells  exposed  i n combination  an S9 a c t i v a t i o n system f o r 2 hr and  sampled  with  immediately.  The mean of 10 samples i s p l o t t e d f o r each c o n c e n t r a t i o n ± standard e r r o r .  -69-  B P concentration (M x 1 0 0  -70-  iii) 3  H-BP  To f o l l o w  from DNA,  CHO  the disappearance of c o v a l e n t l y  c e l l s t h a t had been i n h i b i t e d i n the G.^ stage of  c e l l d i v i s i o n by i n c u b a t i o n 1970)  were t r e a t e d  w i t h BP  i n a r g i n i n e - d e f i c i e n t medium(ADM)(Stich and ( i n 5% ADM),  rinsed  twice w i t h ADM  f e t a l c a l f serum, and then a l l o w e d to r e p a i r i n 5% ADM CHO  c e l l s rather  rates  bound  BP b i n d i n g  without  without  than human f i b r o b l a s t s were employed because  o f c e l l d i v i s i o n a l l o w e d l a r g e r numbers of c e l l s to be to DNA  was  to 72 hr ( F i g . 16).  BP. greater  used.  determined a t v a r i o u s times a f t e r r e a c t i o n , There was  San,  up  a s l i g h t r i s e i n t o t a l BP adducts between  3 0 and 1.5  hr a f t e r  H-BP  r e a c t i o n , which may  be due to b i n d i n g  t h a t had not been c o m p l e t e l y removed from the system 1978).  of BP  (Ivanovic,  et a l . ,  Thereafter there i s a rapid decline  at which time about  30% of the i n i t i a l  i n adducts up to 12 h r , 3 bound H l a b e l remained. Up to  72 h r , the r a t e of decrease i n bound BP was  much l e s s and about  70%  3 of the bound iv)  H l a b e l a t 24 h r remained When CHO  c e l l s were t r e a t e d  a t 72 h r . i d e n t i c a l l y to those  above, but were s u b j e c t e d to sodium a s c o r b a t e (1-5 X 10 i n the post-BP inhibited was  incubation  ( F i g . 17).  l o s t altogether.  to 24 h r , a t which  medium, l o s s of bound  At 5 X 10 DNA-bound  -3 3  M,  3  _3  M)  described dissolved  H l a b e l was s i g n i f i c a n t l y  the r a p i d decrease i n bound BP  H l a b e l remained  r e l a t i v e l y c o n s t a n t up  time i t began to drop o f f to c o n t r o l l e v e l s .  sodium a s c o r b a t e induced l e s s r a p i d  initial  1 X 10  -3  M  3 l o s s of bound H l a b e l .  3 By 24 h r the bound  H l a b e l reached the same l e v e l as c o n t r o l  taken a t the same time.  I t appears t h a t  sodium a s c o r b a t e i n h i b i t s the  e a r l y , r a p i d e x c i s i o n of bound BP p r o d u c t s to DNA. rapid autoxidation might  explain  samples  However, the r e l a t i v e l y  of sodium a s c o r b a t e to dehydroascorbate i n s o l u t i o n  the r e t u r n  to normal e x c i s i o n  l e v e l s by 24 to 48 h r .  -71-  Figure  16  B i n d i n g of  3  H-BP  c e l l s exposed  to the DNA o f n o n - d i v i d i n g c u l t u r e d CHO 3 -7 to H-BP (2 X 10 M) i n combination w i t h  an S9 a c t i v a t i o n system f o r 2 hr f o l l o w e d by r i n s i n g w i t h PBS  and a d d i t i o n of 5% ADM. 3  72 hr a f t e r exposure to p l o t t e d f o r each time.  H-BP.  Samples were taken a t up to Three i d e n t i c a l runs are  -72-  < z  120 h"  o  8°  a  -  E  o o) 8 0  -  o o o o o  0 8  j_  cu  o o  Q.  o  8  o oo  Q_  £ 40 m o  o  o o o  O)  o c o c  8 o  0  •  0  1  24  48  o o -  72  time after BP administration (hours)  -73-  Figure  17  B i n d i n g of  3  H-BP  to the DNA of n o n - d i v i d i n g c u l t u r e d CHO 3 -7 c e l l s exposed to H-BP (2 X 10 M) i n combination w i t h an S9 a c t i v a t i o n system f o r 2 hr f o l l o w e d by r i n s i n g PBS  and a d d i t i o n of 5% MEM -3 a s c o r b a t e , 5 X 10 M ( O )  MEM  without  with  supplemented w i t h sodium -3 or 1 X 10 M (-*-), or 5%  sodium a s c o r b a t e  (•).  The mean of 5  samples i s p l o t t e d f o r each time, ± s t a n d a r d  error.  -74-  time after BP administration (hours)  -75-  v)  A l t h o u g h sodium a s c o r b a t e  the s u l p h y d r y l r e d u c i n g  c o u l d be used to i n h i b i t r e p a i r ,  agent c y s t e i n e d i d not a p p a r e n t l y  inhibit  r e p a i r of BP adducts ( F i g . 18). b) In v i v o : i)  BP b i n d i n g to mouse g a s t r i c mucosal c e l l DNA  was  determined  3 for  v a r i o u s amounts of  as the mean of 8-10  H-BP  trials  f o r c e - f e d to mice, and ± standard  i s r e l a t i v e l y l i n e a r from 100  a steeper for  r i s e up  to 1000  e r r o r ( F i g . 19).  ng per mouse to 750  ng per mouse.  f u r t h e r experiments t h a t f e l l  The  curve  ng per mouse, w i t h 3  In o r d e r  to use a  H-BP  concentration  i n t o the r e g i o n of the graph where  s l o p e v a r i e d the l e a s t , a c o n c e n t r a t i o n of 500 as a s t a n d a r d  the r e s u l t s p l o t t e d  ng per mouse was  chosen  f o r f u r t h e r experiments. 3  ii)  In o r d e r  to determine the time course  to mouse g a s t r i c c e l l DNA samples were taken 1.5 of BP bound to DNA  a f t e r f o r c e - f e e d i n g 500  of  H-BP 3  ng of  H-BP,  to 72 hr a f t e r a d m i n i s t r a t i o n and  c a l c u l a t e d ( F i g . 20).  binding  stomach  the amount  BP b i n d i n g i n c r e a s e d up  12 h r , f o l l o w e d by a r e l a t i v e l y r a p i d decrease to approximately of the 12 hr b i n d i n g by 48 h r . reduced by  72  iii)  The  48 hr b i n d i n g was  not  to  50%  significantly  hr. In o r d e r  to determine whether sodium a s c o r b a t e  had  an  3 e f f e c t on the l o s s of of  3  H-BP  H-BP  at 0 hr f o l l o w e d by 100 mg  ( i n a t o t a l volume of 0.5 (Fig.  l a b e l jm v i v o , mice were f o r c e - f e d 500  21).  3  ml of MEM)  at 15 min,  4,  8,  per g body weight  12,  24 and  36  hr  H l a b e l remained constant  c o n t r o l mice l o s t approximately  50%  f o r c e - f e e d i n g of sodium a s c o r b a t e , to  of sodium a s c o r b a t e  ng  72 hr, by which time i t had  3  from 12 to 36 hr w h i l e DNA from 3 of H label. F o l l o w i n g c e s s a t i o n of  H label f e l l  r a p i d l y from 40  hr  reached the same l e v e l as t h a t observed  i n c o n t r o l mice sampled 72 hr a f t e r BP  force-feeding.  -76-  Figure  18  B i n d i n g of  3  H-BP  to the DNA of n o n - d i v i d i n g c u l t u r e d CHO 3 -7 c e l l s exposed to H-BP (2 X 10 M) i n combination w i t h  an S9 a c t i v a t i o n system f o r 2 hr f o l l o w e d by  rinsing  w i t h PBS  and a d d i t i o n of 5% MEM supplemented w i t h _3  cysteine  (5 X 10  M)(o)  or 5% MEM without  cysteine  (•).  The mean of 5 samples i s p l o t t e d f o r each time, ± standard error.  -77-  0  12  24  36  48  time a f t e r BP administration (hours)  -78-  F i g u r e 19 3 B i n d i n g of  H-BP  to DNA  from g a s t r i c c e l l s of mice  f o r c e - f e d BP o f v a r i o u s c o n c e n t r a t i o n s sampled a t 18 h r .  The mean of v a l u e s  i s p l o t t e d f o r each c o n c e n t r a t i o n ,  a t 0 hr and from 10 mice  ± standard  error  -79-  VN Q  Eo  c_ O)  20 15  <D  CL  am  Q_ CQ  t_  o c D C  10 5 0 0  200 400  6 0 0 8 0 0 1000  BP administered (nanograms)  -80-  Figure  20  Binding of 3 H-BP  3 H-BP to DNA from g a s t r i c c e l l s o f mice  force-fed  (500 ng p e r mouse) a t 0 h r and sampled a t  various  times t h e r e a f t e r .  The mean o f v a l u e s from 10  mice i s p l o t t e d f o r each time, ± s t a n d a r d  error.  -81-  _J  0  I  I  24  48  L.  72  time after BP administration (hours)  -82-  F i g u r e 21 3 B i n d i n g o f H-BP t o DNA from g a s t r i c c e l l s o f mice 3 force-fed  H-BP  (500 ng p e r mouse) a t 0 h r f o l l o w e d  by f o r c e - f e e d i n g sodium a s c o r b a t e (100 mg per kg mouse) a t 15 min, 4, 8, 12, 24 and 36 h r ( O ) o r by no f u r t h e r treatment  (•).  The mean o f v a l u e s from 10 mice i s  p l o t t e d f o r each time, ± s t a n d a r d e r r o r .  -83-  time after BP administration (hours)  -84Other e f f e c t s o f sodium a s c o r b a t e  While  sodium a s c o r b a t e was  damage i n f l i c t e d on DNA  a b l e to i n h i b i t  by MNNG and BP,  i t was  the r e p a i r of  a b l e to  produce  o t h e r c l a s t o g e n i c o r p r o t e c t i v e e f f e c t s both ±n v i t r o and 1)  Sodium a s c o r b a t e fragmented  b l a s t s i n the presence of copper  of c u l t u r e d human f i b r o -  ( F i g . 22B), w h i l e CuSO^'5^0  a l o n e e x e r t e d no d e t e c t a b l e e f f e c t sodium a s c o r b a t e  DNA  i n vivo.  (0.5 ml of 0.15  ( F i g . 22A).  In a s i m i l a r  way,  M s o l u t i o n ) or CuSO^•SH^O/glycine  complex f o r c e - f e d to mice d i d not, i n i s o l a t i o n from each o t h e r , fragment  g a s t r i c mucosal c e l l DNA  ( F i g . 23A).  However, when  i d e n t i c a l amounts of sodium a s c o r b a t e and c o p p e r / g l y c i n e complex were f o r c e - f e d t o g e t h e r , DNA apparent, by 4 hr r e p a i r DNA  ( F i g . 23B).  Sodium a s c o r b a t e has been used as a " t r a p p i n g " agent  demonstrated  (see i n t r o d u c t i o n ) .  This  ( F i g . 24)  to  was  by the a b i l i t y of sodium a s c o r b a t e to i n h i b i t  a c t i o n of a c t i v a t e d DMN. alone  a t n e a r - c o n t r o l l e v e l s by 48 hr  ( F i g . 23B).  scavenge r e a c t i v e e l e c t r o p h i l e s  DMN  was  These damaged c e l l s were a b l e to  so t h a t i t sedimented  post-treatment 2)  fragmentation i n g a s t r i c c e l l s  the  C u l t u r e d human f i b r o b l a s t s exposed to  showed no DNA  fragmentation, while a d d i t i o n  of S9 a c t i v a t i o n system  induced c o n s i d e r a b l e breakage ( F i g . 25A).  Only 12% of r e c o v e r a b l e  3 H counts were found i n f r a c t i o n s 1-5  i n t o which 80-90% of undamaged DNA  normally f a l l s ) .  (the r e g i o n  When sodium a s c o r b a t e  _2 (1 X 10  M) was  added to c e l l s  a c t i v a t i o n system,  immediately  l e s s f r a g m e n t a t i o n o f DNA  p r i o r to a d d i t i o n of the S9 was  observed.  Approximately  3 40% of r e c o v e r a b l e  H counts were found i n f r a c t i o n s 1-5  The decrease i n f r a g m e n t a t i o n was  p r o b a b l y not due  ( F i g . 25B).  to i n h i b i t i o n of the  -85-  F i g u r e 22A A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of  3 H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o CuSO,'5H„0 -5 (1.8 X 10 M) f o r 0.5 h r . 4  2  F i g u r e 22B 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of  H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o sodium a s c o r b a t e (1.8 X 10~ M) i n t h e presence o f CuSO '5H 0 (1.8 X 1 0 ~ M) 3  5  4  f o r 0.5 h r .  2  -86-  5  10  ^-SEDIMENTATION  15  -87-  F i g u r e 23A A l k a l i n e sucrose g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f  3 H-DNA  from g a s t r i c mucosal c e l l s of mice f o r c e - f e d CuSO '5H-0 -5 -4 (5 X 10 M) p l u s g l y c i n e (5 X 10 M ) ( O ) , o r 0.5 ml of  sodium a s c o r b a t e  (0.15 M ) ( # ) a t 0 h r and sampled a t 4 h r .  F i g u r e 23B 3 A l k a l i n e sucrose g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from g a s t r i c mucosal c e l l s of mice f o r c e - f e d CuSO *5H 0 -5 -5 (5 X 10 M) p l u s g l y c i n e (5 X 10 M) p l u s sodium 9  ascorbate  (0.15 M) i n a t o t a l volume o f 0.5 ml a t  0 h r and sampled a t 4 h r ( O )  o r 48 h r ( • ) .  -89-  Figure  24  Alkaline  s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of  3 H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed to d i m e t h y l n i t r o s a m i n e _3 (5 X 10 M) without S9 a c t i v a t i o n system f o r 0.5 h r .  -90-  LO h-  Z  ZD  O u 25-  oo _J  ;< i  LL  O o  20151050-  5 10 15 SEDIMENTATION  -91-  F i g u r e 25A Alkaline  sucrose gradient sedimentation p r o f i l e of  3 H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o d i m e t h y l n i t r o s a m i n e _3 (5 X 10  M) i n t h e presence o f an S9 a c t i v a t i o n  system  f o r 0.5 h r .  \  F i g u r e 25B Alkaline  sucrose gradient sedimentation p r o f i l e of  3 H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o d i m e t h y l n i t r o s a m i n e _3 (5 X 10 M) i n t h e presence o f an S9 a c t i v a t i o n system _2 and sodium a s c o r b a t e (1 X 10 M) f o r 0.5 h r .  -92-  i  1  1  r  _1  I  I  |  2520 -  0  <  5 10 15 SEDIMENTATION  -93a c t i v a t i o n system, s i n c e t h e p r e c a r c i n o g e n s t e r i g m a t o c y s t i n was n o t inhibited  i ni t s ability  t o induce DNA r e p a i r i n the presence  -2 o f 1 X 10  M sodium a s c o r b a t e and S9 a c t i v a t i o n system (Lo and S t i c h , 1978).  Sterig-  m a t o c y s t i n r e q u i r e s e p o x i d a t i o n t o form c a r c i n o g e n i c s p e c i e s , i n a manner analogous  t o BP (Wogan, e t a l . ,  1979).  Therefore, i n h i b i t i o n of activated  DMN i s i m p l i c a t e d (although some c e l l u l a r i n t e r a c t i o n o f sodium a s c o r b a t e cannot  be r u l e d out i n t h i s case.  no fragmenting 3.)  e f f e c t on c u l t u r e d human f i b r o b l a s t DNA ( L a i s h e s , 1974).  While  sodium a s c o r b a t e may i n h i b i t  c a r c i n o g e n s , i t may a l s o i n h i b i t carcinogens. presence  S9 a c t i v a t i o n system a l o n e e x e r t s  the a c t i o n o f a c t i v a t e d  the non-enzymatic a c t i v a t i o n o f those  When c u l t u r e d human f i b r o b l a s t s were i n c u b a t e d i n t h e  o f the n i t r o s a t i o n products of methylguanidine  f o r 1 h r , DNA -3  fragmentation r e s u l t e d  ( F i g . 26A). However, when 1.5 X 10  M sodium  a s c o r b a t e was added t o the r e a c t i o n v e s s e l where n i t r o u s a c i d was used to n i t r o s a t e the m e t h y l g u a n i d i n e , were unable  the r e s u l t i n g n i t r o s a t i o n  t o e x e r t as g r e a t a f r a g m e n t a t i o n e f f e c t  products  ( F i g . 26B). The  i m p l i c a t i o n i s t h a t the f o r m a t i o n o f n i t r o s a t i o n p r o d u c t s was i n h i b i t e d by the presence 4)  o f sodium a s c o r b a t e .  As demonstrated above, sodium a s c o r b a t e may i n h i b i t the  a c t i o n of a c t i v a t i o n of precarcinogens.  In addition,  u l t i m a t e c a r c i n o g e n s may be a f f e c t e d as w e l l .  direct-acting,  When equal volumes o f MEM  c o n t a i n i n g sodium a s c o r b a t e and MNNG were added t o c e l l c u l t u r e s , i n -3 t h a t o r d e r , such t h a t the f i n a l c o n c e n t r a t i o n s a r e 1 X 10 1 X 10  and  M, r e s p e c t i v e l y , c o n s i d e r a b l e f r a g m e n t a t i o n o f DNA r e s u l t e d  ( F i g . 21k).  However, when the sodium a s c o r b a t e and MNNG were i n c u b a t e d  f o r 30 min a t 37° C i n a c l o s e d v e s s e l p r i o r t o a d d i t i o n t o c e l l s , much l e s s f r a g m e n t a t i o n o c c u r r e d exists ^n vivo.  ( F i g . 27B).  The same s i t u a t i o n  When MNNG (20 mg per kg body weight) and sodium  -94-  F i g u r e 26A 3 Alkaline  s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of  H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o t h e n i t r o s a t i o n products of methylguanidine ( s t a r t i n g c o n c e n t r a t i o n of -4 m e t h y l g u a n i d i n e was 5 X 10 M) f o r 1 h r .  F i g u r e 26B 3 Alkaline  s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e of  H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o t h e n i t r o s a t i o n products o f methylguanidine ( s t a r t i n g concentration o f -4 m e t h y l g u a n i d i n e was 5 X 10 M) where n i t r o s a t i o n had taken _3 p l a c e i n t h e presence o f sodium a s c o r b a t e Exposure was f o r 1 h r .  (1.5 X 10  M).  -95-  40  30  to i —  20  z: O  10  u n  I  _J  o-o-o._/-o'°''Vo 0  0  0 0  0  £ 401 O  30 20  \ 10  o o  0 0  < —  /  5 10 15 SEDIMENTATION  -96-  F i g u r e 27A 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 M) _3 i n t h e presence o f sodium a s c o r b a t e (1 X 10 M) f o r 0.5 h r .  F i g u r e 27B Alkaline  3 s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 "* M) _3 i n t h e presence o f sodium a s c o r b a t e  (1 X 10  M)  t h a t was i n c u b a t e d a t 37° C f o r 0.5 h r p r i o r t o a d d i t i o n to c e l l s .  Exposure  was f o r 0.5 h r .  -98-  ascorbate  (100 mg p e r kg body weight) were mixed and immediately  ( w i t h i n 30 sec) f o r c e - f e d t o mice i n a volume o f 0.5 ml, damage t o DNA results  ( F i g . 28A).  On the o t h e r hand, i n c u b a t i o n o f t h e  mixture  f o r 0.5 h r a t 37° C b e f o r e f o r c e - f e e d i n g r e s u l t e d i n decreased  DNA f r a g m e n t a t i o n o f mouse g a s t r i c mucosal c e l l s c o n c e n t r a t i o n s used here those used weight). minimize  ( F i g . 28B). The  (100 mg p e r kg body weight) were h i g h e r  t o i n h i b i t DNA r e p a i r  than  ( c f . F i g . 10,11)(40 mg p e r kg body  The low a s c o r b a t e c o n c e n t r a t i o n s were used p r e v i o u s l y t o long-term  t o x i c i t y to c e l l s being t e s t e d f o r t h e i r a b i l i t y to  r e p a i r damage over 72 h r , w h i l e the h i g h c o n c e n t r a t i o n s ( F i g . 27) were used  t o maximize the observed  i n h i b i t i o n o f damage over  short-term  4 h r treatments. 5)  Although  i n c u b a t i o n o f sodium a s c o r b a t e w i t h some c a r c i n o g e n s  (e_.j*. , DMN) decreased  t h e i r DNA-f ragmen t i n g a b i l i t y ,  sodium a s c o r b a t e  may a l s o enhance the DNA-fragmenting a b i l i t y o f o t h e r s .  MNNG (1 X 10  when a d m i n i s t e r e d t o c u l t u r e d human f i b r o b l a s t s , fragmented DNA ( F i g . 29A).  When e q u a l volumes o f MEM c o n t a i n i n g sodium a s c o r b a t e and MNNG  were added t o c e l l c u l t u r e s , i n t h a t o r d e r , such t h a t the f i n a l -3 -5 c o n c e n t r a t i o n s were 1 X 10 M and 1 X 10 M, r e s p e c t i v e l y , c o n s i d e r a b l y more f r a g m e n t a t i o n o f DNA r e s u l t e d  ( F i g . 29B).  In v i v o ,  when MNNG (40 mg p e r kg body weight) was f o r c e - f e d t o mice, fragmentation r e s u l t e d  ( F i g . 30A). When MNNG (40 mg per kg body  weight) was mixed w i t h sodium a s c o r b a t e and  immediately  ( w i t h i n 10 sec) f o r c e - f e d , i n c r e a s e d f r a g m e n t a t i o n  of g a s t r i c mucosal c e l l s r e s u l t e d The  (100 mg per kg body weight)  ( F i g . 30B).  i n c r e a s e d damage t o DNA was n o t due t o t h e independent  action  of sodium a s c o r b a t e , s i n c e sodium a s c o r b a t e a l o n e d i d n o t fragment DNA i n v i v o ( F i g . 31A) o r i n v i t r o  ( F i g . 31B).  M) ,  -99-  F i g u r e 28A 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from g a s t r i c mucosal  c e l l s o f mice f o r c e - f e d a m i x t u r e  of MNNG (20 mg p e r kg body weight) and sodium a s c o r b a t e (100 mg p e r kg body weight)  immediately a f t e r m i x i n g .  Samples were taken 4 h r a f t e r  force-feeding.  F i g u r e 28B 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from g a s t r i c mucosal  c e l l s o f mice f o r c e - f e d a m i x t u r e o f  MNNG (20 mg p e r kg body weight) and sodium a s c o r b a t e (100 mg p e r kg body weight) a f t e r a 0.5 h r i n c u b a t i o n of the m i x t u r e a t 37°. force-feeding.  Samples were taken 4 h r a f t e r  -100-  0 <  5 10 15 SEDIMENTATION  -101-  F i g u r e 29A 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10  M)  f o r 0.5 h r .  F i g u r e 29B 3 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e o f H-DNA from c u l t u r e d human f i b r o b l a s t s exposed t o MNNG (1 X 10 _3 i n t h e presence o f sodium a s c o r b a t e (1 X 10 0.5 h r .  M) f o r  M)  -102-  0  5 10 15 ^ — SEDIMENTATION  -103-  F i g u r e 30A 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40  mg p e r kg body weight) a t 0 h r and sampled a t 4 h r .  F i g u r e 30B 3 A l k a l i n e sucrose gradient sedimentation p r o f i l e  o f H-DNA  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d MNNG (40 mg p e r kg body weight) i n t h e presence o f sodium a s c o r b a t e (100 mg p e r kg body weight) a t 0 h r and sampled a t 4 hr.  -105-  F i g u r e 31A 3 Alkaline  sucrose gradient sedimentation p r o f i l e of  from g a s t r i c mucosal c e l l s o f mice f o r c e - f e d ascorbate  H-DNA  sodium  (100 mg p e r kg body weight) a t 0 h r and  sampled a t 4 h r .  F i g u r e 3IB 3 Alkaline  sucrose gradient sedimentation p r o f i l e of  H-DNA  from c u l t u r e d human f i b r o b l a s t s exposed t o sodium a s c o r b a t e (5 X 1 0 " M) f o r 0.5 h r . 3  -106-  20 h  ?.  CO  § 10 O (J CO  0~ -0 0  I  0  O 20 '  o  / \  10  \ \  0  0  o-o'°-o.  5 10 15 <— SEDIMENTATION  -107-  6)  I t might be argued  a b i l i t y o f MNNG observed  t h a t the decrease  i n fragmenting  a f t e r i n c u b a t i o n w i t h sodium a s c o r b a t e might  have been due t o l o s s o f sodium a s c o r b a t e from t h e system by o x i d a t i o n , b i n d i n g t o p r o t e i n i n s o l u t i o n , e t c . , d u r i n g the i n c u b a t i o n p e r i o d . By t h i s argument, sodium a s c o r b a t e would have o n l y the a b i l i t y t o enhance f r a g m e n t a t i o n  i n t h i s system, and t h e decrease  i n fragmentation  seen a f t e r i n c u b a t i o n would be due t o l o s s o f enhancement o n l y . However, when sodium a s c o r b a t e was i n c u b a t e d f o r 0.5 h r w i t h MNNG and f o r c e - f e d t o mice ( F i g . 28B) f r a g m e n t a t i o n o f DNA was l e s s than t h a t observed when MNNG a l o n e was f o r c e - f e d t o mice, even when t h e MNNG f o r c e - f e d a l o n e was a t a dose 25% h i g h e r than t h a t f o r c e - f e d w i t h sodium a s c o r b a t e damaging a b i l i t y  ( F i g . 30A). Thus, t h e decrease  i n DNA-  o f MNNG a f t e r i n c u b a t i o n w i t h sodium a s c o r b a t e  was l i k e l y due t o t h e a c t i o n o f a c o r b a t e o r dehydroascorbate r a t h e r than merely  t h e l o s s o f sodium a s c o r b a t e from the system.  E f f e c t o f sodium a s c o r b a t e and o t h e r agents  1)  on MNNG,  on b i n d i n g o f BP t o DNA:  S i n c e sodium a s c o r b a t e may i n h i b i t damage t o DNA by scavenging  e l e c t r o p h i l i c c a r c i n o g e n i c and mutagenic s p e c i e s , the a b i l i t y  o f sodium  a s c o r b a t e t o prevent b i n d i n g o f BP t o DNA i n v i t r o and i n v i v o was investigated.  When sodium a s c o r b a t e was i n c u b a t e d w i t h BP and S9  a c t i v a t i o n mixture over CHO c e l l s ,  i n c r e a s i n g c o n c e n t r a t i o n s decreased  3 the amount o f H-BP l a b e l a s s o c i a t e d w i t h p u r i f i e d DNA ( F i g . 3 2 ) . When BP (500 ng p e r mouse) was mixed w i t h sodium a s c o r b a t e o f v a r i o u s 3 c o n c e n t r a t i o n s and f o r c e - f e d t o mice,  H-BP l a b e l a s s o c i a t e d w i t h  DNA decreased w i t h i n c r e a s i n g sodium a s c o r b a t e c o n c e n t r a t i o n . a low c o n c e n t r a t i o n o f sodium a s c o r b a t e  (15 mM) produced  However,  an i n c r e a s e d  -108-  Figure  32  3 B i n d i n g of H-BP to DNA from c u l t u r e d CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and v a r i o u s c o n c e n t r a t i o n s of sodium a s c o r b a t e for 2 hr.  i n the presence of an S9 a c t i v a t i o n  P l a t e s were r i n s e d w i t h PBS  immediately.  and DNA  system  isolated  The mean of 5 samples i s p l o t t e d f o r each  c o n c e n t r a t i o n , ± standard e r r o r .  -109-^  500 < Q  E  o c_  D) £_ <D Q.  300 r  Q_ DQ C/)  E  o c_  D) O  c o c  100  0  50  100  Sodium a s c o r b a t e  150 (mM)  -110-  l e v e l o f bound BP ( F i g . 33) i n two s e p a r a t e 2)  Other r e d u c i n g  BP b i n d i n g to DNA.  agents b e s i d e s  experiments.  sodium a s c o r b a t e may  affect  When the r e d u c i n g agent p r o p y l g a l l a t e was  i n c u b a t e d w i t h BP and S9 a c t i v a t i o n system over CHO c e l l s ,  reduced  b i n d i n g of BP r e s u l t e d ( F i g . 34) i n a manner s i m i l a r to t h a t observed w i t h sodium a s c o r b a t e . and  However, when p r o p y l g a l l a t e was mixed w i t h BP  f o r c e - f e d to mice, b i n d i n g t o g a s t r i c c e l l DNA  increasing propyl gallate concentration  ( F i g . 35).  increased  with  Due to the l i m i t e d  s o l u b i l i t y of p r o p y l g a l l a t e , o n l y about h a l f the molar c o n c e n t r a t i o n of sodium a s c o r b a t e  used to e x e r t e f f e c t  S u l f h y d r y l as w e l l as h y d r o x y l e f f e c t on b i n d i n g o f BP to DNA. g l u t a t h i o n e , when incubated CHO c e l l s ,  i n the case o f p r o p y l reducing  agents may e x e r t an  The s u l f h y d r y l r e d u c i n g  agent  w i t h BP and S9 a c t i v a t i o n system over  i n c r e a s e d the b i n d i n g of BP to DNA by over 3 f o l d  ( F i g . 36).  When g l u t a t h i o n e was mixed w i t h BP and f o r c e - f e d to mice,  the same i n c r e a s e i n b i n d i n g of BP to DNA o c c u r r e d case,  gallate.  ( F i g . 37).  In t h i s  the extreme a c i d i t y of g l u t a t h i o n e s o l u t i o n s allowed molar  of o n l y about o n e - s i x t h t h a t of sodium a s c o r b a t e  to be used.  s o l u t i o n s were n e u t r a l i z e d by the a d d i t i o n of c o n c e n t r a t e d 3)  S i n c e a l t e r a t i o n i n b i n d i n g of BP by r e d u c i n g  concentrations  These  10 M NaOH.  agents may  be due to d i f f e r e n t i a l i n h i b i t i o n o f a c t i v a t i n g and d e a c t i v a t i n g enzyme systems, the e f f e c t o f two compounds known to e x e r t e f f e c t - harman and norharman - was i n v e s t i g a t e d i n t h i s Incubation  this  system.  of harman w i t h BP and S9 a c t i v a t i o n system over  CHO  c e l l s e x e r t e d no e f f e c t on BP b i n d i n g over the c o n c e n t r a t i o n studied  ( F i g . 38).  norharman ( F i g . 39).  The same l a c k o f e f f e c t was observed  with  range  -111-  F i g u r e 33 3 B i n d i n g o f H-BP t o DNA from g a s t r i c c e l l s of mice 3 force-fed  H-BP  (500 ng p e r mouse) i n the presence  of v a r i o u s c o n c e n t r a t i o n s o f sodium a s c o r b a t e . t o t a l volume o f 0.5 ml was a d m i n i s t e r e d  A  to each mouse.  Samples were taken a t 12 h r a f t e r f o r c e - f e e d i n g .  The  mean o f v a l u e s from 10 mice i s p l o t t e d f o r each c o n c e n t r a t i o n , ± standard e r r o r . The  c o n c e n t r a t i o n s i n d i c a t e d a r e those o f the f o r c e - f e d  s o l u t i o n s , not stomach c o n t e n t s  after force-feeding.  discussion f o r further consideration.  See  -112-  12 [  Sodium ascorbate (mM)  -113-  Figure  34  3 B i n d i n g of H-BP to DNA from c u l t u r e d CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and v a r i o u s c o n c e n t r a t i o n s of p r o p y l g a l l a t e i n the presence of an S9 a c t i v a t i o n system f o r 2 hr.  P l a t e s were r i n s e d w i t h PBS  immediately.  and DNA  isolated  The mean of 10 samples i s p l o t t e d f o r each  c o n c e n t r a t i o n , ± standard  error.  -114-  500  < z Q  E o c_  U) t_  300  Q.  CL CD  to  E o t_  D) O  o c  100 h  Propyl gallate (mM)  -115-  F i g u r e 35 Binding  3 o f H-BP to DNA from g a s t r i c c e l l s o f mice 3  force-fed  H-BP  (500 ng p e r mouse) i n the presence  of v a r i o u s c o n c e n t r a t i o n s volume  of propyl g a l l a t e . A t o t a l  o f 0.5 ml was a d m i n i s t e r e d  to each mouse.  Samples were taken 12 h r a f t e r f o r c e - f e e d i n g . of v a l u e s ± standard The  from 10 mice i s p l o t t e d f o r each  The mean  concentration,  error. concentrations  i n d i c a t e d a r e those o f f o r c e - f e d  s o l u t i o n s , not stomach c o n t e n t s  after force-feeding.  discussion f o r further consideration.  See  -116-  U)  o  c o c  2  -  •  -•  0 1.4 Propyl gallate (mM)  >-  14.1  -117-  Figure  36  3 B i n d i n g of H-BP to DNA from c u l t u r e d CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and v a r i o u s c o n c e n t r a t i o n s of reduced g l u t a t h i o n e i n the presence of an S9 a c t i v a t i o n system f o r 2 h r .  P l a t e s were r i n s e d w i t h PBS  i s o l a t e d immediately.  and  DNA  R e s u l t s from d u p l i c a t e p l a t e s  at each c o n c e n t r a t i o n a r e p l o t t e d .  -118-  500r  300  100h  Glutathione (mM)  -119-  F i g u r e 37 Binding  3 o f H-BP to DNA from g a s t r i c c e l l s o f mice 3  force-fed  H-BP  (500 ng per mouse) i n the presence  of v a r i o u s c o n c e n t r a t i o n s  o f reduced  glutathione.  A t o t a l volume o f 0.5 ml was a d m i n i s t e r e d  to each mouse.  Samples were taken a t 12 h r a f t e r f o r c e - f e e d i n g . Results  from d u p l i c a t e samples a t each  concentration  are p l o t t e d The  concentrations  i n d i c a t e d a r e those o f the f o r c e -  f e d s o l u t i o n s , not stomach c o n t e n t s See  after force-feeding.  discussion f o r further consideration.  -120-  < Q E  20 r  o i_ CD  <L> Q. Q_ CD  cn  E  o t_ cn O c o c  Glutathione (mM)  -121-  Figure 38 3 B i n d i n g of H-BP to DNA from c u l t u r e d CHO c e l l s exposed 3 -7 to H-BP (2 X 1 0 M) and v a r i o u s c o n c e n t r a t i o n s of harman i n the presence of an S9 a c t i v a t i o n system f o r 2 hr.  P l a t e s were r i n s e d and DNA  R e s u l t s from t r i p l i c a t e are p l o t t e d .  i s o l a t e d immediately.  samples f o r each c o n c e n t r a t i o n  -122-  <  z 300 Q E o  CD c_  <D  Q.  200  CL CD 10  E o  100 h  L. CD  o C O  c  0  100 HARMAN (uM)  200  -123-  Figure  39  3 B i n d i n g of H-BP to DNA from c u l t u r e d CHO c e l l s exposed 3 -7 to H-BP (2 X 10 M) and v a r i o u s c o n c e n t r a t i o n s of norharman i n the presence of an S9 a c t i v a t i o n system for 2 hr.  P l a t e s were r i n s e d and DNA  i s o l a t e d immediately.  R e s u l t s from d u p l i c a t e samples f o r each are p l o t t e d .  concentration  -124-  o  100 NORHARMAN (uM)  200  -125DISCUSSION  S t u d i e s aimed a t i d e n t i f i c a t i o n o f c h e m i c a l c a r c i n o g e n s have h i s t o r i c a l l y f o c u s s e d on two  a s p e c t s of the problem.  First,  the  investigation  of substances t h a t a r e c h e m i c a l l y c o n s t r u c t e d f o r t h e i r a b i l i t y to induce cancer, and which are v i r t u a l l y absent  from the  environment,  have been u s e f u l because of the i n f o r m a t i o n they have p r o v i d e d on the p r o c e s s of cancer i n i t i a t i o n benz(a)anthracene  and 4 - n i t r o q u i n o l i n e - l - o x i d e ) .  r e l a t i v e l y e x o t i c chemicals made by man or domestic  purpose  substances.  (e.j*.,  Secondly,  those  f o r some i n d u s t r i a l ,  have proved a f r u i t f u l  Their reactivity  7,12-dimethyl  medical  source o f c a r c i n o g e n i c  (the source o f t h e i r u s e f u l n e s s i n the  above s i t u a t i o n s ) makes them good c a n d i d a t e s f o r the p o t e n t i a l  electro-  p h i l i c r e a c t a n t s t h a t have been i m p l i c a t e d as u l t i m a t e c a r c i n o g e n s mutagens.  and  Only r e c e n t l y have the g r e a t mass of u b i q u i t o u s , n a t u r a l l y -  o c c u r r i n g substances come under the eye of i n v e s t i g a t o r s .  Realization  of the u l t i m a t e e l e c t r o p h i l i c n a t u r e of c a r c i n o g e n s has made i t p o s s i b l e to " s c r e e n " the p r e v i o u s l y i n t i m i d a t i n g number of compounds so t h a t p a r t i c u l a r l y p r o m i s i n g c a n d i d a t e s a r e r e c o g n i z a b l e by t h e i r carcinogenic nature  (capable of b e i n g r e a l i z e d by c e l l - m e d i a t e d  or n o n - c e l l u l a r c h e m i c a l m o d i f i c a t i o n ) r a t h e r than by t h e i r unmetabolized  initial,  reactivity.  In o r d e r to r e c o g n i z e a l l p o s s i b l e c a r c i n o g e n s i n the a v a r i e t y of s c r e e n i n g t e s t s have been proposed range  potential  environment,  to i d e n t i f y them.  These  from a c t u a l i n d u c t i o n of tumours i n rodents and o t h e r mammals to  the d e t e c t i o n of s h o r t - t e r m c e l l u l a r and t i s s u e changes t h a t have been c a u s a l l y or e m p i r i c a l l y a s s o c i a t e d w i t h i n d u c t i o n of c a n c e r . group i n c l u d e s s h o r t - t e r m t e s t s o f t h r e e t y p e s :  1) those t h a t  T h i s second assay  -126-  for  changes induced by chemicals i n i s o l a t e d DNA o r chromatin ( e . ,  unwinding  of b a c t e r i o p h a g e DNA c l e a v e d by chemicals  ( K u h n l e i n , 1980),  2) those t h a t assay f o r c h e m i c a l or m o r p h o l o g i c a l changes i n c u l t u r e d mammalian o r b a c t e r i a l c e l l s  ( i n d u c t i o n o f DNA r e p a i r , chromosome a b e r -  r a t i o n s , DNA a l t e r a t i o n s , germ c e l l anomalies, DNA-carcinogen  adducts,  e t c . ( r e v i e w e d by S t i c h and San, 1980)), and 3) those t h a t assay f o r c h e m i c a l or m o r p h o l o g i c a l changes i n mammalian c e l l s  i n vivo.  These  i n c l u d e many o f the t e s t s a v a i l a b l e f o r c u l t u r e d c e l l s , as w e l l as those p o s s i b l e o n l y i n systems c o n t a i n i n g organs c a p a b l e of e x h i b i t i n g ment of p r e n e o p l a s t i c t i s s u e mutations  develop-  ( S o l t and F a r b e r , 1977) o r s p e c i f i c l o c u s  (Maier and Zbinden,  1980).  T h i s a r r a y o f t e s t s i s a u s e f u l one f o r d e t e c t i o n o f c h e m i c a l s which may induce cancer, but i t which may modify  cannot be used to d e t e c t c h e m i c a l s  the i n d u c t i o n o f cancer by c h e m i c a l o r o t h e r agents.  I t would b e h i g h l y d e s i r a b l e ' to -have' an--assay -system t h a t ^ c o u l d * be' used ;  for  the d e t e c t i o n o f such m o d i f y i n g c h e m i c a l s , f o r use both ^ n v i t r o  and jin v i v o , and one o f the aims o f t h i s work was to e v a l u a t e the usefulness  o f the a l k a l i n e s u c r o s e g r a d i e n t and BP-DNA adduct  tests  f o r DNA damage and r e p a i r as s u i t a b l e c a n d i d a t e s f o r such an a p p l i c a t i o n . As an a p p l i c a t i o n of these t e s t s , both were used to assay the a b i l i t y o f sodium a s c o r b a t e t o modify the c a p a c i t y o f both c u l t u r e d c e l l s and mammalian e p i t h e l i a l  cells  i n s i t u to r e p a i r  damage  to DNA caused by d i r e c t - a c t i n g c a r c i n o g e n (MNNG) o r a p r e c a r c i n o g e n (BP) a p p l i e d i n the presence of an a p p r o p r i a t e a c t i v a t i n g  system.  I t was found t h a t sodium a s c o r b a t e , s u p p l i e d t o c e l l s the a p p l i c a t i o n o f MNNG to fragment  DNA,  r e s t o r e the c a p a c i t y of DNA to sediment gradients.  inhibited  after  t h e i r a b i l i t y to  q u i c k l y i n a l k a l i n e sucrose  T h i s r e s t o r a t i o n n o r m a l l y takes p l a c e by 30 hours  after  -127c a r c i n o g e n treatment  (Koropatnick, 1978).  I n h i b i t i o n of r e p a i r  p l a c e f o r the p e r i o d t h a t u n o x i d i z e d sodium a s c o r b a t e was  present,  and  r e p a i r c a p a c i t y was  and  g a s t r i c c e l l s i n v i v o , when sodium a s c o r b a t e treatments  stopped.  r e s t o r e d , both to c u l t u r e d c e l l s  Sodium a s c o r b a t e  e f f e c t on DNA.  treatment  a l o n e had no  In a d d i t i o n , sodium a s c o r b a t e  a f t e r the a p p o i c a t i o n of BP  t h e i r a b i l i t y to e x c i s e those adducts.  I t was  absence of sodium a s c o r b a t e , c u l t u r e d CHO x i m a t e l y 60% of bound treatment,  3 H-BP  in vitro were  DNA-fragmenting  s u p p l i e d to  to form c o v a l e n t DNA  took  adducts  found  cells inhibited  t h a t , i n the  cells rapidly lost  appro-  12 hours a f t e r BP 3 t h e r e a f t e r l o s t o n l y v e r y s m a l l amounts of H-BP  and  d u r i n g the f i r s t  up to 72 hours p o s t - t r e a t m e n t .  label  T h i s i s s i m i l a r to r e s u l t s a b t a i n e d  3 when r a t s were i n j e c t e d w i t h  H-BP  a r y l s u b s t i t u t i o n a t the 0-6  and  specific  s i t e of guanine i n l i v e r was  (P. K l e i h u e s , p e r s o n a l communication). in  the extent of  The presence  determined  of sodium  ascorbate  the medium p r e s e n t d u r i n g the r e p a i r p e r i o d s t r o n g l y i n h i b i t e d  the e x c i s i o n d u r i n g the f i r s t 12 hours, but, a t low c o n c e n t r a t i o n s -3 3 of sodium a s c o r b a t e (1 X 10 M), H-BP b i n d i n g r e t u r n e d to n e a r - c o n t r o l l e v e l s by 24 hours a f t e r BP  treatment.  At h i g h c o n c e n t r a t i o n  _3 (5 X 10  M),  c o n t r o l l e v e l s were not reached  u n t i l 36-48 hours  post-treatment. The  same s i t u a t i o n was  Bound BP reached 3  H-BP.  3  H-binding  observed  cells.  a peak i n these c e l l s 12 hours a f t e r f o r c e - f e e d i n g fell  to 50% of t h i s h i g h l e v e l i n the f i r s t  hours a f t e r peak b i n d i n g , and to  i n v i v o i n mouse g a s t r i c  36  then d i d not s i g n i f i c a n t l y change up  48 hours a f t e r peak b i n d i n g .  In the presence  of sodium a s c o r b a t e ,  3 f o r c e - f e d a f t e r the peak of H-binding was reached, v i r t u a l l y no 3 i n c r e a s e i n bound H-BP l e v e l s o c c u r r e d . W i t h i n f o u r hours a f t e r  -128-  3  the end of sodium a s c o r b a t e treatments, bound  H-BP  levels  fell  r a p i d l y , and normal b i n d i n g l e v e l s were reached between 12 and hours a f t e r c e s s a t i o n of sodium a s c o r b a t e One  DNA  treatment.  of the q u e s t i o n s r a i s e d by the observed  r e s t o r a t i o n of normal DNA  is:  of DNA,  the e x c i s i o n of BP adducts  i t appears  i n h i b i t i o n of  a t which stage of r e s t o r a t i o n of  does sodium a s c o r b a t e e x e r t i t s e f f e c t ?  inhibited  36  S i n c e sodium a s c o r b a t e  and r e s t o r a t i o n of f a s t  t h a t the i n h i b i t o r y e f f e c t  sedimentation  i s exerted at a very  e a r l y p o i n t i n r e p a i r , a t or b e f o r e the e x c i s i o n stage of damaged r e g i o n s of DNA DNA  (assuming  a common r e p a i r mechanism f o r BP and MNNG-induced  r e p a i r ) . S i n c e MNNG may  a l k y l a t e DNA  to produce a p u r i n i c s i t e s t h a t  a r e c o n v e r t e d to the s i n g l e - s t r a n d breaks  t h a t a r e observed  on  s u c r o s e g r a d i e n t s , such e x c i s i o n of r e p a i r a b l e r e g i o n s of DNA  alkaline i s a necessary  p r e r e q u i s i t e i n the r e p a i r of MNNG-induced damage, as w e l l as r e p a i r of BP-adducts.  In a d d i t i o n , r e s t o r a t i o n of f a s t s e d i m e n t a t i o n to  DNA  on a l k a l i n e s u c r o s e g r a d i e n t s i s dependent upon l i g a t i o n of  single-  s t r a n d breaks, as w e l l as e x c i s i o n of damaged r e g i o n s .  inhibition  of r e p a i r of DNA f o r e be due  observed  i n the presence  to i n h i b i t i o n of DNA  p r o c e s s e s l e a d i n g up to i t .  The  of sodium a s c o r b a t e might t h e r e -  l i g a t i o n or i n h i b i t i o n of any of the  Thus, sodium a s c o r b a t e may  inhibit  repair  a t one o r more s t e p s . A second  q u e s t i o n thay may  be asked  is:  a s c o r b a t e a l o n e i n h i b i t e x c i s i o n of BP adducts s e d i m e n t a t i o n of DNA,  does the sodium and r e s t o r a t i o n of  or are the o x i d a t i v e or o t h e r m e t a b o l i c  of sodium a s c o r b a t e r e s p o n s i b l e f o r t h i s e f f e c t ? i s o x i d i z e d i n v i v o to dehydroascorbate dehydroascorbate The  fast  products  Sodium a s c o r b a t e  by a r e v e r s i b l e p r o c e s s ,  and  i s i r r e v e r s i b l y o x i d i z e d to d i k e t o g u l o n a t e and o x a l a t e .  i n i t i a l o x i d a t i o n step may  be c a t a l y z e d by sodium a s c o r b a t e  itself,  -129e s p e c i a l l y i f metal i o n s are added 1973).  Thus, because of the  oxidation  Foyer, 1976;  Michelson,  i r r e v e r s i b l e n a t u r e of l a t e r o x i d a t i o n  sodium a s c o r b a t e c o n c e n t r a t i o n f a v o u r of i n c r e a s i n g  ( H a l l i w e l l and  i n a solution w i l l quickly decline  concentration  of i t s o x i d a t i o n  p r o d u c t s were r e s p o n s i b l e  products.  steps,  in  I f these  f o r r e p a i r i n h i b i t i o n , then  older  sodium a s c o r b a t e s o l u t i o n s would be more e f f i c i e n t r e p a i r i n h i b i t o r s than f r e s h s o l u t i o n s . was  However, when a s i n g l e s o l u t i o n of sodium a s c o r b a t e  added to c u l t u r e d human f i b r o b l a s t s r e c o v e r i n g ,  30 hours, from damage i n f l i c t e d by MNNG, r e p a i r was  over a p e r i o d  of  less inhibited  than when f r e s h s o l u t i o n s of sodium a s c o r b a t e were added every 4 hours up  to 20 hours.  by  i t i n the  DNA  repair.  initial  Third, r e t a r d DNA  Thus, e i t h e r sodium a s c o r b a t e , or some substance produced oxidation  step,  are n e c e s s a r y f o r i n h i b i t i o n of  the q u e s t i o n of r e l e v a n c e of the  i n these experiments must be  c o n c e n t r a t i o n s used  raised.  The  to  concentrations  of sodium a s c o r b a t e used to i n h i b i t r e p a i r i n c u l t u r e d  cells  ranged  _3 from 1 X 10 DNA  M to i n h i b i t r e s t o r a t i o n of f a s t s e d i m e n t a t i o n to -3 from c u l t u r e d human f i b r o b l a s t s to 5 X 10 M to i n h i b i t e x c i s i o n -3  of BP  adducts from c u l t u r e d  ascorbate could  CHO  be used to i n h i b i t  12 hours a f t e r a d m i n i s t r a t i o n concentration feeding,  (although 1 X 10  e x c i s i o n of BP  of BP).  however, are more d i f f i c u l t , easier.  c e l l s i n mice f o r c e - f e d  the  adducts i n the  although empirical  There are  two  sodium a s c o r b a t e :  sodium a s c o r b a t e i s d i l u t e d e q u a l l y 2) by assuming t h a t  sodium first  C a l c u l a t i o n of the e f f e c t i v e  e f f e c t i v e sodium a s c o r b a t e c o n c e n t r a t i o n  volume, and  M  of sodium a s c o r b a t e i n experiments i n v o l v i n g  to human consumption are the  cells  ways to  force-  comparisons consider  on g a s t r i c mucosal 1) by assuming  the  throughout the whole body  sodium a s c o r b a t e i s d i l u t e d only  to  the  -130volume of the stomach.  Because complete d i s s e m i n a t i o n of chemicals  the body r e q u i r e s a l e n g t h y  time p e r i o d , the f i r s t method i s b e t t e r  s u i t e d to o b s e r v a t i o n of long-term e f f e c t s of a d m i n i s t e r e d S i n c e r e s t o r a t i o n of f a s t - s e d i m e n t a t i o n of DNA b l a s t s on a l k a l i n e sucrose  (e.j>., e x c i s i o n of DNA  the f i r s t i s not  chemical  a d m i n i s t r a t i o n , long-term  the whole-body d i l u t i o n  g a s t r i c mucosa l i n e the stomach, and  Because of food p r e s e n t  t e s t c e l l s of  to be  Concentrations  to c u l t u r e d  of sodium  cells.  Variation  0.002 to 0.005 l i t r e s ) may  also  as a  supply by b i o s y n t h e s i s from  U n l i k e humans, they a l s o possess an a s c o r b i c a c i d  enzyme t h a t has  is available in sufficient  the a b i l i t y to m e t a b o l i z e  ascorbic acid  Reynolds, 1963).  and  I f t h i s enzyme  l e v e l s i n g a s t r i c mucosal c e l l s , r a p i d meta-  b o l i s m c o u l d reduce the l e n g t h of time over which the sodium effective.  chemicals  A l s o , mice do not r e q u i r e sodium a s c o r b a t e  remove i t from the system (Brown and  is  the  to b a t h i n g  decrease the e f f e c t i v e c o n c e n t r a t i o n .  d i e t a r y supplement, and produce t h e i r own glucuronic acid.  by  i n the stomach, n o n - s p e c i f i c b i n d i n g or o x i d a t i o n  i n mouse stomach volume (estimated a f f e c t concentration.  The  stream.  d i f f e r here from those a d m i n i s t e r e d  not  system.  so are s u b j e c t e d  b e f o r e they a r e absorbed i n t o the b l o o d  reductase  i n s t a b i l i t y , are  second method - c a l c u l a t i n g e f f e c t i v e c o n c e n t r a t i o n  of a s c o r b i c a c i d may  incubation  A l s o , i n c r e a s e or decrease of c o n c e n t r a t i o n of  stomach volume - seems more a p p r o p r i a t e here.  ascorbate  hours  p r e r e q u i s i t e metabolic  by e x c r e t i o n , d e t o x i f i c a t i o n , or chemical  taken i n t o account by The  and  18  adducts) occur at t h e i r g r e a t e s t r a t e i n  24 hours a f t e r c a r c i n o g e n  s u i t a b l e here.  1978)  chemicals.  from c u l t u r e d human f i b r o -  g r a d i e n t s o c c u r s between 12 and  a f t e r MNNG a d m i n i s t r a t i o n (Koropatnick, steps  in  F i n a l l y , movement of stomach c o n t e n t s  i n t o the  ascorbate small  i n t e s t i n e w i l l a l s o reduce the e f f e c t i v e time of a c t i o n of sodium  ascorbate.  -131With t h i s i n mind, the c o n c e n t r a t i o n of sodium a s c o r b a t e to which g a s t r i c mucosal c e l l s were exposed, based mouse stomach, was  1.4  X 10  -2  M to 3.5  s u c r o s e g r a d i e n t a n a l y s i s of DNA i n the case of BP-DNA adduct o u t l i n e d above, these may  s o l e l y on the volume of  X 10  r e p a i r and  -2  M i n the case of a l k a l i n e  3.5  a n a l y s i s of DNA  X 10  -2  repair.  M to 8.75 For  reasons  and  2.0  In o r d e r to a c h i e v e sodium a s c o r b a t e c o n c e n t r a t i o n s i n the range  e f f e c t i v e i n r e d u c i n g r e p a i r i n mice, doses of a p p r o x i m a t e l y grams per treatment,  1-35  a d m i n i s t e r e d p e r i o d i c a l l y over the p e r i o d of  r e p a i r of mucosal c e l l DNA,  would have to be g i v e n .  This i s a high  l e v e l i n comparison w i t h t h a t r e q u i r e d f o r normal h e a l t h (30-40 mg d a y ) ( P e t t , 1955).  treatment  of c a n c e r s  are w i t h i n t h i s While  (Cameron, e_t .al. , 1975;  Stone,  1972)  Cameron and P a u l i n g ,  1978)  range.  sodium a s c o r b a t e was  a b l e to i n h i b i t r e p a i r as measured by  a l k a l i n e s u c r o s e g r a d i e n t s and e x c i s i o n of BP adducts in vitro,  per  However, some recommended l e v e l s of sodium a s c o r b a t e  a d m i n i s t r a t i o n f o r the p r e v e n t i o n of c o l d s ( P a u l i n g , 1976;  and  removal of sodium a s c o r b a t e  to DNA  i n vivo  ( i n the case of a l k a l i n e  sucrose  g r a d i e n t a n a l y s i s of r e p a i r i n c u l t u r e d human f i b r o b l a s t s ) and nonrenewal  M  cells.  In humans, stomach volume v a r i e s r o u g h l y between 0.5  and  -2  be c o n s i d e r e d the upper l i m i t s of a s c o r b a t e  c o n c e n t r a t i o n s a c t u a l l y a p p l i e d to the  litres.  X 10  of sodium a s c o r b a t e treatments  ( i n the case of ASG  r e p a i r i n mouse g a s t r i c mucosal c e l l s , and BP adduct i n both c u l t u r e d CHO  ascorbate.  a n a l y s i s of r e p a i r  c e l l s and mouse g a s t r i c mucosal c e l l s ) r e s u l t e d i n  the e v e n t u a l r e t u r n of the DNA r e p a i r t h a t was  a n a l y s i s of  of the c e l l s i n q u e s t i o n to a s t a t e of  c l o s e to t h a t observed  i n c e l l s u n t r e a t e d w i t h sodium  T h i s i s an i n d i c a t i o n t h a t a) the i n h i b i t o r y e f f e c t i s  r e v e r s i b l e , and b) t h a t sodium a s c o r b a t e i s r a p i d l y decomposed i n the  -132-  system  t o remove i t s e f f e c t ,  presence  of theascorbate.  normal l e v e l s cultured  cells  treatments repair  of excision after  removal from  took  of  from  suggestion  i n c u l t u r e d human  that rapid  that  be invoked  observed promotion  sodium a s c o r b a t e  inhibits  to e x p l a i n theobserved  i s removed  retention  mucosal  presence  from  which  treated with  o f sodium a s c o r b a t e and  repair  systems  loss of  t o the presence  systems appear repair,  effects.  One s u g g e s t i o n  by carcinogens  copper  the  effect  theeffect  o f sodium a s c o r b a t e  When s o d i u m a s c o r b a t e  left  behind  fibroblasts  b y MNNG.  such  that,  by  added,  but rather incomplete  a n d mouse i n the  t o fragment t h e  a n d mouse g a s t r i c t h a t enhancement  o f t h eobserved  on i n i t i a l  i nv i t r o  Sodium a s c o r b a t e  c o u l d be used  Because o f t h e p o s s i b i l i t y  t h e cause r a t h e r than  i s that  t h a t s o d i u m a s c o r b a t e may e n h a n c e  f i b r o b l a s t s jin v i t r o  was  ascorbate  carcinogen  c e l l s jin v i v o , caused  an i n i t i a l  explanations  systems and sodium a s c o r b a t e  o f DNA i n c u l t u r e d human  of glycine-complexed  c e l l s jin v i v o .  other  to favour the  o f damage i s n o t d u e t o l a c k o f r e p a i r ,  I t was o b s e r v e d  o f c u l t u r e d human  feeding,  cell  o f damage b y r e s i d u a l  fragmentation  gastric  DNA  and f r e s h  over  i s responsible f o r i t s eventual  adaptation of cellular  removal and r i n s i n g . the  fibroblasts  decomposition  than  treatments,  i n c r e a s e d t h e l e n g t h o f time  t h e o b s e r v a t i o n s made i n b o t h  when c a r c i n o g e n  to  returned to  DNA more q u i c k l y  s o d i u m a s c o r b a t e may p o t e n t i a t e damage c a u s e d  the  ascorbate  ascorbate. While  can  place  t h e system  r a t h e r than  force-fed  o f BP a d d u c t s  o f sodium a s c o r b a t e  inhibition  effect,  Since mice  becomes a c c l i m a t i z e d t o t h e  c e s s a t i o n o f sodium a s c o r b a t e  MNNG, i t seems l i k e l y its  or thec e l l  mucosal  o f DNA damage  retention  o f damage,  damage t o DNA was m e a s u r e d .  a n d BP w e r e a d m i n i s t e r e d  t o mice by f o r c e -  i n c r e a s e i n b i n d i n g o f BP was o b s e r v e d  c o n c e n t r a t i o n s , f o l l o w e d by a decrease  a t low sodium  i n BP b i n d i n g  with  -133i n c r e a s i n g sodium a s c o r b a t e c o n c e n t r a t i o n .  T h i s s m a l l but s t a t i s t i c a l l y  s i g n i f i c a n t and r e p r o d u c i b l e i n c r e a s e i n b i n d i n g was observed  at close  to the same c o n c e n t r a t i o n (100 mg sodium a s c o r b a t e per kg body weight) t h a t was used  t o cause the maintenance o f h i g h l e v e l s o f BP adducts and  slow-sedimenting  DNA c h a r a c t e r i s t i c s  (40 mg p e r kg body weight) o f mouse  g a s t r i c mucosal c e l l s t r e a t e d i n v i v o .  However, sodium a s c o r b a t e  admini-  s t e r e d to c u l t u r e d c e l l s i n combination  w i t h BP d i d not induce any i n c r e a s e  i n BP b i n d i n g t o DNA a t a s c o r b a t e c o n c e n t r a t i o n s as h i g h o r h i g h e r those used  than  to i n h i b i t r e p a i r o f DNA f r a g m e n t a t i o n o r BP adducts i n  c u l t u r e d CHO c e l l s o r human f i b r o b l a s t s . Thus, even when BP i s a d m i n i s t e r e d w i t h sodium a s c o r b a t e i n c o n c e n t r a t i o n s much h i g h e r than those l i k e l y incomplete observed,  to be l e f t behind by  r i n s i n g , o n l y a v e r y s m a l l amount o f i n c r e a s e d b i n d i n g i s and then o n l y jLn v i v o .  to be enough to account i n DNA observed  f o r the r e t e n t i o n o f h i g h l e v e l s o f BP  i n the presence  of r e p a i r i n g c e l l s .  T h i s s m a l l i n c r e a s e does not appear adducts  o f sodium a s c o r b a t e i n the b a t h i n g medium  T h e r e f o r e , i t seems l i k e l y  t h a t the r e t e n t i o n o f  MNNG and BP-induced DNA m o d i f i c a t i o n seen i n the presence  o f sodium  a s c o r b a t e i s due t o an e f f e c t on r e p a i r o f DNA r a t h e r than on the i n i t i a l DNA-damaging While  events. sodium a s c o r b a t e c o u l d be used  f r a g m e n t a t i o n and BP adducts, may a l s o be used DNA fragmenting  to i n h i b i t  to i n h i b i t  t h e r e p a i r o f DNA  the "scavenging" a b i l i t y of sodium a s c o r b a t e  the a c t i o n o f s e v e r a l c a r c i n o g e n s .  The  p r o p e r t y o f S 9 - a c t i v a t e d DMN was i n h i b i t e d by the presence  of sodium a s c o r b a t e , presumably by i n h i b i t i o n o f a c t i o n o f the c a r c i n o g e n r a t h e r than by i n h i b i t i o n o f the a c t i v a t i o n system (Lo and S t i c h ,  1978).  Sodium a s c o r b a t e i n h i b i t e d the non-enzymatic f o r m a t i o n o f n i t r o s a t i o n products of methylguanidine,  as shown by the decreased  DNA-fragmenting  -134a b i l i t y of methylguanidine reacted Sodium a s c o r b a t e ,  when incubated  i n the presence o f a s c o r b a t e .  w i t h MNNG f o r 30 minutes p r i o r t o t r e a t -  ment i f c u l t u r e d c e l l s o r mouse g a s t r i c mucosal c e l l s , f r a g m e n t i n g a c t i o n o f the MNNG. of MNNG f r a g m e n t a t i o n  inhibited  the DNA-  T h i s was i n c o n t r a s t t o the enhancement  seen when mixed MNNG and sodium a s c o r b a t e  a p p l i e d i n v i t r o o r i n v i v o without the 30 minute i n c u b a t i o n .  were This  enhancement was due, presumably, t o i n h i b i t i o n o f r e p a i r o f DNA. the r e d u c i n g  p o t e n t i a l o f sodium a s c o r b a t e  This,  c o u l d be employed t o "scavenge"  e l e c t r o p h i l e s and prevent t h e i r a c t i o n . However, sodium a s c o r b a t e co-administered  c o u l d be used t o fragment DNA when  w i t h copper, both i n v i t r o and i n v i v o .  This  fragmentation  was r e p a i r a b l e , s i n c e mouse g a s t r i c mucosal c e l l s t r e a t e d i n v i v o were able to restore near-control 48 hours a f t e r treatment.  sedimentation  T h i s fragmenting a b i l i t y was due, presumably,  to DNA damage caused by hydrogen p e r o x i d e sodium a s c o r b a t e  p r o p e r t i e s t o t h e i r DNA by  produced by a u t o x i d a t i o n o f  i n the presence o f t r a n s i t i o n metals ( S t i c h , e t a l . , 1979).  Since the b i o l o g i c a l reducing  agent sodium a s c o r b a t e  had an  e f f e c t on scavenging r e a c t i v e e l e c t r o p h i l e s , an attempt was made to measure the scavenging a b i l i t y o f two o t h e r p r o p y l g a l l a t e and g l u t a t h i o n e . to prevent o x i d a t i o n  reducing  agents -  P r o p y l g a l l a t e , a food a d d i t i v e used  (as i s sodium a s c o r b a t e ) , was found t o i n h i b i t  the b i n d i n g o f a c t i v a t e d BP i n c u l t u r e d CHO c e l l s , as expected from data  gathered u s i n g sodium a s c o r b a t e .  However, when p r o p y l g a l l a t e  was f o r c e - f e d t o mice i n the presence o f S9 a c t i v a t i o n system, an i n c r e a s e i n bound BP i n r e l a t i o n t o p r o p y l g a l l a t e c o n c e n t r a t i o n was found.  I n a d d i t i o n , reduced g l u t a t h i o n e was found t o i n c r e a s e  b i n d i n g o f BP, both i n v i t r o and i n v i v o .  Because o f the l i m i t e d  of p r o p y l g a l l a t e , o n l y h a l f the molar c o n c e n t r a t i o n  o f sodium  solubility  ascorbate  -135r e q u i r e d to a l t e r BP-adduct f o r m a t i o n was r e l a t i v e l y low  concentrations  used.  of g l u t a t h i o n e  (approximately  of the molar c o n c e n t r a t i o n of sodium a s c o r b a t e were used. be due  In a d d i t i o n , o n l y  required for effect)  In view of t h i s d i s p a r i t y , the d i f f e r e n c e i n e f f e c t might  to c o n c e n t r a t i o n r a t h e r than q u a l i t a t i v e e f f e c t s .  initial  one-sixth  i n c r e a s e i n BP-binding to DNA  o f sodium a s c o r b a t e  In f a c t ,  i n v i v o observed i n the presence  o c c u r r e d a t c l o s e to the same molar c o n c e n t r a t i o n  p r o p y l g a l l a t e t h a t caused a t w o - f o l d  i n d i c a t e t h a t , at h i g h e r p r o p y l g a l l a t e and  t i o n s in  v i v o , the same i n h i b i t i o n of BP-binding to DNA  i n the presence of sodium a s c o r b a t e  might be  glutathione  seen. (inhibiting  enhancing BP b i n d i n g jin v i v o ) , the  tryptophan  harman and norharman ( a l r e a d y shown to e i t h e r  enhance or i n h i b i t BP m u t a g e n i c i t y were a d m i n i s t e r e d  concentra-  as t h a t observed  Because of the d i f f e r e n t i a l e f f e c t of p r o p y l g a l l a t e BP b i n d i n g jin v i t r o but  to CHO  such an e f f e c t was  i n b a c t e r i a , depending on  c e l l s i n the presence of BP  observable  i n t h i s system.  i n h i b i t i o n or enhancement of BP b i n d i n g was  No  concentration)  to observe whether  significant  observed over the range of  c o n c e n t r a t i o n s where i n h i b i t i o n or enhancement of BP m u t a g e n i c i t y b a c t e r i a was  seen ( F u j i n o , e t a l . , 1978).  Therefore,  u n l i k e l y t h a t such a d i f f e r e n t i a l e f f e c t , due degree of i n h i b i t i o n of a c t i v a t i n g and a s s o c i a t e d w i t h BP, propyl gallate  i s at work here.  may  be  invoked  to d i f f e r e n c e s i n  i n a c t i v a t i n g enzyme systems While the r e d u c i n g  p r o p e r t i e s of  to e x p l a i n decreased  i s i n t r i g u i n g to s p e c u l a t e t h a t the i n c r e a s e i n bound BP be due  in  i t seems  (as the d r i v i n g f o r c e behing a "scavenging"  r e a c t i v e e l e c t r o p h i l e s ) may it  of  increase i n binding i n vivo.  T h i s may  p y r o l y s i s products  the  to i n h i b i t i o n of r e p a i r mechanisms.  effect BP  binding,  observed  However, i t must  kept i n mind t h a t d i f f e r e n t i a l enzyme i n h i b i t i o n may  still  on  be  be a f a c t o r  -136-  in vivo,  as  difficulties  and  time of  the  enzymes r e s p o n s i b l e  may  differ  On  the  with  exposure  from  other  the  hand,  i n determination  i n the  f o r BP  liver  glutathione  inhibition  increased  of  binding  will  of  BP  increase  well  i n the  be  gallate  concentration  i n g a s t r i c mucosal  i n the  S9  e x p o s u r e and  r e p a i r may  propyl  o u t l i n e d above, e x i s t .  metabolism  enzymes u s e d  controlled glutathione  Thus,  stomach, as  of  S9  of  cells  a c t i v a t i o n system.  binding  of  BP  in  a c t i v a t i o n , as  a reasonable  presence  Also,  vitro  well  as  mechanism to  in vivo.  explain  glutathione.  Summary  In lesions early  general,  sodium a s c o r b a t e  introduced  lesion  i n t o DNA  e x c i s i o n steps  reorganization  steps  human f i b r o b l a s t s , mice  treated with While  by  do  not  of  BP  of  DNA  and  late  observed  DNA  ligation  in cultured  r e s p e c t i v e l y , as  well  as  r e a c t i v e e l e c t r o p h i l e s , and  to  be  responsible  f r o m DNA  or  the  a l k a l i n e sucrose the  w e r e shown t o  enhanced  also  enhanced  BP  that  inhibition  DNA  or  cultured  in vivo. inhibit also  copper,  observed  of  DNA  damage  fragment  DNA  these e f f e c t s  r e t a r d a t i o n of  fast-sedimenting  excision  ability  gradients.  inhibit  BP-binding  of  the  with  the  chromatin  cells  could  r e s t o r a t i o n of  reducing  binding,  for  to  of  both  in gastric cells  demonstrated  when c o - a d m i n i s t e r e d  and  CHO  was  jin v i t r o  repair  I n h i b i t i o n of  sodium a s c o r b a t e  In a d d i t i o n ,  gallate  and  BP.  sodium a s c o r b a t e  adducts  ascorbate  MNNG o r  inhibit  and  appear  on  was  shown t o  carcinogen  "scavenging"  in vivo  by  was  agents propyl BP  binding  ±n v i v o .  both  The  in vivo  r e p a i r may  be  and  g a l l a t e and  in vitro, reducing jm  agent  vitro.  responsible  but  It  f o r the  sodium propyl glutathione is possible observed  -137i n c r e a s e i n DNA  lesions.  Perspectives  While  the r e d u c i n g agent  i n r e d u c i n g the e x t e n t o f DNA c a r c i n o g e n s , i t remains  sodium a s c o r b a t e has been  implicated  r e p a i r o f damage caused by  chemical  to be seen what r e l e v a n c e t h i s o b s e r v a t i o n  has to the e f f e c t of v i t a m i n C l e v e l s i n human h e a l t h . l i n k of reduced r e p a i r c a p a c i t y and  The b e s t  i n d u c t i o n of c a n c e r s comes from  data gleaned from s t u d i e s of human p a t i e n t s .  S u f f e r e r s of xeroderma  pigmentosum, L o u i s - B a r syndrome, Fanconi's anaemia, f a m i l i a l p o l y p o s i s , and Cockayne's syndrome (German, 1977; German, 1978) reduced  Paterson,  rectal 1977;  a l l show p r e d i l e c t i o n f o r cancer i n a s s o c i a t i o n w i t h  excision repair capacity.  However, t h e r e are some i n d i v i d u a l s  s u f f e r i n g from these d i s e a s e s t h a t e x h i b i t normal r e p a i r c a p a c i t y . T h e r e f o r e , the p o s s i b i l i t y e x i s t s t h a t enhanced to  susceptibility  cancer depends upon the q u a l i t y of r e p a i r of damage, and not o n l y on  reduced q u a n t i t y of r e p a i r .  I t seems r e a s o n a b l e to suppose t h a t  a decrease i n DNA  l e a d to i n c r e a s e d c e l l  r e p a i r may  than i n i t i a t i o n of tumours (Kihlman,  1977).  death r a t h e r  Sodium a s c o r b a t e  has been shown to p o t e n t i a t e the t o x i c i t y of n i t r o - a r o m a t i c compounds (Koch,  et a l . ,  1979)  and a s c o r b i c a c i d has been shown to  inhibit  the e x p r e s s i o n o f c u l t u r e d mouse c e l l f o c i transformed by 3-methyl cholanthrene  (Benedict, et a l . ,  1980).  Sodium a s c o r b a t e has  also  been shown to promote m i t o t i c i n h i b i t i o n i n c e l l s t r e a t e d w i t h carcinogenic chemicals  (Stich, et a l . ,  i n h i b i t i o n of the extent of DNA  1979).  In the l i g h t of  this,  r e p a i r a f t e r c a r c i n o g e n treatment  prevent tumour i n i t i a t i o n i n mammals by promoting  may  the death of a f f e c t e d  -138-  I t i s d e s i r a b l e t h a t jiLn v i v o experiments be done i n which  cells.  sodium a s c o r b a t e  i s a p p l i e d to animal t e s t s u b j e c t s a f t e r  a d m i n i s t r a t i o n to i n h i b i t or days.  The  the s h o r t term r e p a i r t h a t o c c u r s  e f f e c t on the p r o d u c t i o n  t i s s u e would then be observed. scavenging e f f e c t and v i t a m i n C may DNA  of tumours or  In t h i s way,  anti-cancer  be excluded, and  r e p a i r events observed.  preneoplastic  the e l e c t r o p h i l e -  o n l y the e f f e c t of i n h i b i t i o n  A l s o , the e f f e c t of sodium  This  sodium  as w e l l as a v o i d the d i f f i c u l t i e s a s s o c i a t e d w i t h  presence of r e s i d u a l c a r c i n o g e n  a f t e r treatment of  In the case of p r o p y l g a l l a t e and e f f e c t of i n c r e a s e d DNA remains unknown.  I t has  in their  been observed t h a t , w h i l e  ultimate  presence  sodium  ascorbate  compounds, s u l p h y d r y l  agents such as g l u t a t h i o n e , c y s t e i n e , cysteamine  mercaptoethanol i n h i b i t  the  cells.  g l u t a t h i o n e , the  damage by c a r c i n o g e n s  enhances c y t o t o x i c i t y of n i t r o - a r o m a t i c  1979).  of  ascorbate  would be d e s i r a b l e .  would exclude the e f f e c t of e l e c t r o p h i l e - s c a v e n g i n g by  reducing  i n hours  c e l l properties implicated for  on r e p a i r of UV-induced l e s i o n s i n DNA  ascorbate,  carcinogen  and  c y t o t o x i c i t y of these compounds (Koch, e_t al_. ,  While the mechanism of these phenomena remains unknown, f u r t h e r  i n v e s t i g a t i o n i s c e r t a i n l y warranted. of g l u t a t h i o n e and t h e r e i s a chemical  sodium a s c o r b a t e competition  Experiments u s i n g  in vitro,  combinations  to determine whether  between the e f f e c t s , would be a  fruitful  avenue of i n v e s t i g a t i o n . As may  g e n e r a l methods f o r o b s e r v i n g  modify the s h o r t - t e r m  adduct and ASG The most w i d e l y  the e f f e c t of compounds t h a t  e f f e c t s of c a r c i n o g e n i c  sedimentation  a n a l y s i s of DNA  used s h o r t - t e r m  compounds,  BP  appear to be w e l l - s u i t e d .  assay f o r DNA  r e p a i r employs  3 unscheduled i n c o r p o r a t i o n of  H-TdR ( S t i c h and  San,  1980).  This  -139assay cannot be used t o d i f f e r e n t i a t e between decreased damage and  3 decreased r e p a i r , so t h a t  i n h i b i t i o n of incorporation  observed i n the presence o f some m o d i f y i n g compound cither effect. observe u s i n g or jin v i v o .  Also,  r e p a i r jin v i v o  t h i s method. In addition,  of  could  H-TdR be due t o  i s t e c h n i c a l l y d i f f i c u l t to  The BP adduct assay may be used i n v i t r o the ASG s e d i m e n t a t i o n assay may a l s o be  used t o d i s t i n g u i s h between both DNA damage and DNA r e p a i r , both i n . v i v o and ±n v i t r o . occurring  I t may a l s o be used t o measure the l a t e -  r e p a i r events, up to and i n c l u d i n g chromatin  reorganization,  3 t h a t a r e not addressed by measurement o f unscheduled or BP adduct  e x c i s i o n from  DNA.  H-TdR  uptake  -140-  LITERATURE  CITED  Abanobiy S.E., Popp, J.A., Chang, S.K., H a r r i n g t o n , G.W., Lotlikar, P.D., H a d j i o l o v , D . , L e v i t t , M . , R a j a l a k s h i n , S., and Sarma, D.S.R., J . 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