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Genotoxicity of chewing tobacco samples Woolcock, Bruce Wayne 1985

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GENOTOXICITY  OF CHEWING TOBACCO SAMPLES By  BRUCE WAYNE WOOLCOCK B.Sc,  The U n i v e r s i t y  o f V i c t o r i a , 1983  A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE  STUDIES  (Department o f P a t h o l o g y )  We a c c e p t t h i s  thesis  to the required  as conforming standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1985 ©BRUCE  WAYNE  WOOLCOCK, 1 9 8 5  In p r e s e n t i n g  this thesis  r e q u i r e m e n t s f o r an of  British  it  freely available  agree t h a t for  Library  s h a l l make  for reference  and  study.  I  for extensive copying of  h i s or  be  her  g r a n t e d by  shall  not  be  P a t h o l o g y  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  DE-6  (3/81)  of  further this  Columbia  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department of  the  representatives.  copying or p u b l i c a t i o n  f i n a n c i a l gain  University  the  s c h o l a r l y p u r p o s e s may  understood that  the  the  I agree that  permission  by  f u l f i l m e n t of  advanced degree a t  Columbia,  department or  for  in partial  written  ABSTRACT  The an  intra-oral  use o f t o b a c c o - c o n t a i n i n g m i x t u r e s p l a y s  important a e t i o l o g i c a l  role  i n the occurrence of  cancers.  In v i t r o genotoxicity  the rapid  evaluation of factors contributing to or  modulating t h i s essential  p r o v i d e means f o r  form o f t o b a c c o c a r c i n o g e n e s i s .  requirement  capability  a s s a y s may  f o r an e f f e c t i v e t e s t  to detect the genotoxic e f f e c t s  of a variety  tobacco, Khaini tobacco  systems: cells,  f o r genotoxic a c t i v i t y  micronuclei DNA  synthesis  DNA  ( U z b e k i s t a n , USSR),  i n three different  i n Chinese hamster  i n human f i b r o b l a s t s .  were found t o c o n t a i n d i r e c t  Chinese hamster ovary c e l l s .  The  ovary  and A  A l l four tobacco  acting  DNA  extracts  agents capable of  i n d u c i n g chromosome a b e r r a t i o n s and m i c r o n u c l e i  implicating  test  i n c l u d e d a s a complement t o t h e  synthesis assay.  the clastogenic a c t i v i t y  four  "chewing"  i n Chinese hamster ovary c e l l s  i n h i b i t i o n t e s t was  unscheduled  s n u f f and  ( I n d i a ) and n a s s  chromosome a b e r r a t i o n s  unscheduled repair  available  of  i n chemical  F r e s h l y p r e p a r e d aqueous e x t r a c t s o f  tobacco mixtures, l o c a l l y  were a s s a y e d  An  system i s the  tobacco mixtures which are expected t o d i f f e r composition.  oral  C a t a l a s e was  formation i n  found t o  o f t h e c h e w i n g and K h a i n i  suppress tobaccos,  H 0 - m e d i a t e d p r o d u c t i o n o f chromosome damage. 2  2  genotoxic a c t i v i t i e s  o f s n u f f and  ii  nass d i d not appear to  be  d e p e n d e n t on t h e g e n e r a t i o n o f H 0 . 2  tobacco  initiated  fibroblasts. initiated exerted the  unscheduled  snuff,  Khaini  consequence  of this  i t was  on DNA  t o b a c c o and  DNA  synthesis  reduced  synthesis,  inhibitory effect  unscheduled  results  DNA  was  Only the  synthesis  A l l tobacco extracts  unscheduled  an  DNA  2  i n human t h e l e v e l s o f UV  i n d i c a t i n g the repair.  The  nass t o induce a  On  the basis  failure  of these  c o n c l u d e d t h a t t h e chromosome a b e r r a t i o n  unscheduled  systems  DNA  synthesis,  appear  t o be  i i i  but  and not  s u i t a b l e as t e s t  f o r the study of f a c t o r s i n f l u e n c i n g o r a l  carcinogenicity.  of  a  m i c r o n u c l e u s t e s t s i n Chinese hamster ovary c e l l s , the  extracts  demonstrable  i n t e r p r e t e d t o be  inhibition.  chewing  tobacco  TABLE OF  CONTENTS PAGE  ABSTRACT  i i  TABLE OF CONTENTS  ..  iv  L I S T OF TABLES L I S T OF  v i i  FIGURES  viii  INTRODUCTION 1.  1  O r a l c a n c e r and t h e i n t r a - o r a l u s e of tobacco  1  2.  Extent  4  3.  Factors modifying the r i s k of tobacco-related o r a l cancer  4.  5.  o f problem  6  Model systems f o r t h e study o f o r a l tobacco c a r c i n o g e n i c i t y  8  Objective  9  METHODS AND MATERIALS  13  1.  Preparation  2.  Chemicals  13  3.  Tissue  13  3.1  of extracts  13  culture  Culture  media  13  4.  Growth o f s t o c k  cultures  5.  Chromosome a b e r r a t i o n 5.1  Preparation  5.2  Exposure t o t e s t  14  test  of c e l l  cultures  extract  14 14 15  5.3  Chromosome p r e p a r a t i o n s  15  5.4  A n a l y s i s o f metaphase p l a t e s f o r chromosome a b e r r a t i o n s  16  iv  CONTENTS  6.  7.  (continued)  PAGE  Micronucleus assay  16  6.1  Test  16  6.2  Analysis of interphase micronuclei  procedure cells for  16  DNA r e p a i r s t u d i e s  17  7.1  Preparation  of cell  7.2  Ultraviolet  irradiation  7.3  Exposure t o t e s t e x t r a c t and r a d i o a c t i v e l y - l a b e l l e d thymidine  18  F i x a t i o n and p r e p a r a t i o n f o r autoradiography  18  7.5  A u t o r a d i o g r a p h y and s t a i n i n g  19  7.6  A n a l y s i s o f autoradiograms  19  7.4  cultures  17  RESULTS 1.  2.  3.  21 Chromosme d a m a g i n g c a p a c i t y o f a q u e o u s tobacco extracts  21  Induction of micronuclei tobacco extracts  23  Induction by  by aqueous  o f u n s c h e d u l e d DNA  synthesis  aqueous t o b a c c o e x t r a c t s  4.  I n h i b i t i o n o f DNA-repair s y n t h e s i s  5.  Exploration of the active  6.  17  26 28  genotoxic  components o f t h e e x t r a c t s  33  Other t e s t  33  systems e x p l o r e d  6.1  UDS i n r a t o r a l m u c o s a l c e l l s  33  6.2  Micronuclei  35  i n r o d e n t o r a l mucosa  v  CONTENTS  (continued)  DISCUSSION . CONCLUSION . BIBLIOGRAPHY  L I S T OF TABLES PAGE TABLE I  TABLE I I  TABLE I I I  TABLE I V  CLASTOGENIC A C T I V I T Y OF CRUDE AQUEOUS TOBACCO EXTRACTS I N CHINESE HAMSTER OVARY CELLS  22  CLASTOGENIC A C T I V I T Y OF AQUEOUS TOBACCO EXTRACTS - DAMAGE BY TYPES  24  MICRONUCLEI FORMATION I N CHINESE HAMSTER OVARY CELLS EXPOSED TO AQUEOUS TOBACCO EXTRACTS  25  EFFECT OF CATALASE ON THE CLASTOGENIC A C T I V I T Y OF AQUEOUS TOBACCO EXTRACTS I N CHINESE HAMSTER OVARY CELLS  34  vii  L I S T OF FIGURES PAGE FIGURE 1.  FIGURE 2.  FIGURE 3.  FIGURE 4.  FIGURE 5.  U n s c h e d u l e d DNA s y n t h e s i s i n human f i b r o b l a s t s e x p o s e d t o a c h e w i n g t o b a c c o ...  27  I n h i b i t o r y e f f e c t o f a chewing t o b a c c o e x t r a c t o n U V - i n d u c e d DNA r e p a i r i n human f i b r o b l a s t s  29  U n s c h e d u l e d DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o K h a i n i t o b a c c o  30  U n s c h e d u l e d DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o nass  31  U n s c h e d u l e d DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o s n u f f  32  viii  INTRODUCTION  1.  Oral  Cancer  Oral  (WHO, 1 9 8 4 ) .  I t accounts  cancers  f o r more t h a n  one-third  a l l c a n c e r s i n some A s i a n c o u n t r i e s ( B i n n i e a n d R a n k i n ,  1984)  w i t h more t h a n a h u n d r e d  cancer occurring The  Use o f Tobacco  c a n c e r i s one o f t h e t e n most common  worldwide of  and t h e I n t r a - o r a l  relative  Jaffna  thousand  i n South-East A s i a  frequency o f o r a l  ( S r i Lanka),  Mainpuri  new c a s e s o f o r a l  each y e a r  (WHO,  1984).  cancer t o a l l cancers i n  ( n o r t h e r n India) and Neyyur  ( s o u t h e r n I n d i a ) a r e r e p o r t e d t o b e 66.1%. 67.9%, a n d 75.9%, repectively accounts nations  1979).  In contrast,  oral  f o r o n l y 3-5% o f m a l i g n a n c i e s i n most (Pindborg,  The varies  (Hirayama,  cancer  western  1980).  incidence rates  approximately  for oral  fifteen-fold  cancer around (McMichael,  the world  1984).  The  p r e v a l e n c e o f tobacco chewing h a b i t s i n a r e a s w i t h e l e v a t e d frequencies of oral connection. mixtures  c a n c e r s u g g e s t s an a e t i o l o g i c a l  I n I n d i a , where c h e w i n g o f t o b a c c o - c o n t a i n i n g  i s a s common a s s m o k i n g i s i n W e s t e r n  o v e r 70% o f t h e c a n c e r s o f t h e o r a l l a r y n x have been a t t r i b u t e d  pharynx,  and  t o t h e s e p a r a t e and combined  effects  of oral  1977).  A causal relationship  and  cavity,  societies,  t o b a c c o u s e and smoking  between o r a l  t h e e l e v a t e d frequency o f o r a l  1  (Jayant e t a l . , tobacco h a b i t s  cancer i n Asia i s  supported  by t h r e e l i n e s  of evidence.  greater prevalence of oral  as opposed t o t h o s e w i t h o u t  1968;  J u s s a w a l l a and  F o r e x a m p l e , Wahi oral  and  India,  1984;  (1968),  oropharyngeal  found  (Hirayama,  Deshpande, 1971; WHO,  there i s a  c a n c e r among i n d i v i d u a l s w i t h  habit  G u p t a e t a l . , 1980;  First,  Jayant  1966;  e t a l . , 1977;  J u s s a w a l l a e t a l . , 1985).  cancers  compared w i t h noh-chewers.  elevation  in risk  S e c o n d , an a e t i o l o g i c a l  arising  i n t h e s i t e where t h e t o b a c c o  1971).  1966;  Hirayama  Wahi, 1968;  (1966),  of  f o r chewers  i s s u p p o r t e d by t h e s t r o n g a s s o c i a t i o n  (Hirayama,  of  i n the Mainpuri d i s t r i c t  tobacco  held  Wahi,  i n an e p i d e m i o l o g i c a l s u r v e y  an e i g h t - f o l d  the  of  role for  cancer  quid i s habitually  J u s s a w a l l a and  a s an example, o b s e r v e d  Deshpande, that  78%  of  c a n c e r s a r o s e on t h e same s i d e o f t h e mouth a s t h e s i d e i n w h i c h t h e q u i d was distributed  on b o t h  h e l d , w h i l e c a n c e r s were e v e n l y s i d e s o f t h e mouth among p a t i e n t s  kept the quid i n both  s i d e s o f t h e mouth.  Finally,  cancers are a s s o c i a t e d with frequent, prolonged i n d u l g e n c e o f t o b a c c o w h i c h c a n be for  a dose-effect relationship.  Hirayama  (1976) h a v e shown t h e r i s k o f o r a l increase t h o s e who six-fold awake and  i n the  frequency  and  c o n s i d e r e d as  or  early  evidence Wahi  cancer t o r i s e with  r e t a i n the quid during t h e i r g r e a t e r r i s k t h a n t h o s e who  an  e a c h day.  s l e e p , Wahi n o t e d  chew o n l y when t h e y  a 3 6 - f o l d g r e a t e r r i s k t h a n non-chewers.  2  oral  (1966) and  d u r a t i o n of use  who  For a are  Hirayama  f o u n d t h e r i s k r o s e a s h i g h a s 63 t i m e s t h a t  f o r non-  chewers. It  h a s b e e n more d i f f i c u l t  t o e s t a b l i s h an a s s o c i a t i o n  between t h e u s e o f s n u f f o r chewing t o b a c c o and o r a l in  t h e West b e c a u s e o f t h e r e l a t i v e l y i n f r e q u e n t u s e o f  these products.  Nevertheless, a s t r i k i n g sex difference i n  the geographic d i s t r i b u t i o n  of oral  cancer i n t h e United  S t a t e s s u g g e s t s a r e l a t i o n s h i p between o r a l snuff dipping.  Oral cancer m o r t a l i t y  c a r c i n o m a and  rates areelevated  among women i n t h e s o u t h e a s t e r n U n i t e d S t a t e s .  This i s  consistent with the r e l a t i v e l y frequent use o f snuff f e m a l e s who h a v e o r a l  cancer  Several retrospective  s t u d i e s conducted  United  1983). fold  1977).  i n the southeastern  ( V o l g e r e t a l . , 1962;  F o r i n s t a n c e , Winn e t a l . (1981) e s t i m a t e d a 4.2-  s n u f f - d i p p i n g women. cancers a r i s i n g  attributed  and pharyngeal  a n d b u c c a l mucosa o f l o n g -  50 t i m e s t h a t  f o r non-users and  87% o f t h e c a n c e r s a r i s i n g  a t these s i t e s t o  Most o f t h e s e s t u d i e s have o b s e r v e d  cancer a r i s i n g (1983),  c a n c e r among  They a l s o e s t i m a t e d t h e r i s k f o r  i n the gingival  term u s e r s approached  McGuirt  among  1963; Winn e t a l . , 1981; M c G u i r t ,  increased risk of oral  snuff.  ( B l o t and Fraumeni,  States support t h i s hypothesis  R o s e n f e l d and Callaway,  of  cancer  an a s s o c i a t i o n  a t t h e s i t e where t h e s n u f f was h e l d .  f o r example, r e p o r t e d 8 9 % o f t h e p r i m a r y  t u m o u r s a r o s e a t t h e s i t e where t h e q u i d o f s n u f f was h e l d and  c o n c l u d e d t h a t t h i s was s u f f i c i e n t i n i t s e l f t o  3  implicate  smokeless t o b a c c o as  carcinomas i n the  the  causal  s n u f f - d i p p i n g group.  smokeless t o b a c c o use all.  and  oral  S m i t h and  The  cancers,  by  not  able  t o v a l i d a t e an  and  oral  cancer i n a ten year prospective  c a n n o t be  h i s colleagues  carcinogenic.  explained  by  a very  long  appearance of snuff-induced Pindborg,  2.  1973;  Extent The  of  McGuirt,  latency period carcinomas  Islands  by  million  for  tobacco-containing  the and  custom but  betel leaf,  a m i x t u r e o f t o b a c c o and  4  (1985)  mixtures:  450  composition  t o b a c c o and  place  r e p u b l i c s of the  Rosin  u s u a l l y consists of  nations  a p p r o x i m a t e l y 20  is  P h i l i p p i n e s and  the  o r more i n h a b i t a n t s o f I n d i a and  central Asian  the  i n d i v i d u a l s chew  million  gum;  on  however, m i g h t  S t i c h and  chew b e t e l q u i d ,  local  o r b e t e l nut,  c h e e k and  America  (Roed-Petersen  o r more i n d i v i d u a l s i n A s i a , t h e  which v a r i e s with areca  cancer,  snuff  and  Negative reports  oral  been e s t i m a t e d  carcinogenic  South P a c i f i c  of  1983).  t h a t g l o b a l l y more t h a n 600  million  study  of tobacco throughout the world  I t has  potentially  use  were  Problem  o r a l use  extensive.  not  (1970, 1975)  a s s o c i a t i o n between t h e  a s s o c i a t i o n o f s n u f f u s a g e and be  a s s o c i a t i o n of  type of snuff used i n North  considered  of  however, i s  accepted  concluded that the  agent  million  spices;  of the  100  several African  l i m e between natives  S o v i e t Union,  of  Iran  the the  and  the  A f g a n i s t a n put nass,  a complex m i x t u r e  of tobacco, slaked  lime,  ash w i t h o i l o r water under t h e tongue o r between  lower  l i p and  o r chewing  gum;  and  30 m i l l i o n N o r t h A m e r i c a n s  tobacco 1971 1983)  tobacco chewing h a b i t s a r e  i n North America.  Production of  i n t h e U n i t e d S t a t e s r o s e from  t o a n e s t i m a t e d 134 and  (Christen,  tobacco use  a t t r i b u t a b l e t o an tobacco habits, smokeless  increasing  o f t h e Northwest  o f t h e few  in  of  oral  Surveys  of  13.7%  t h a t h a v e b e e n done a r e c a u s e  26%  T e r r i t o r i e s have used are current users  indulge  1984),  (Poulson e t a l . ,  in the greater Atlanta  i n the l a s t  1985).  (Millar, teenage  and  13%  1984),  Only  (Pearce,  almost  o f 14 y e a r o l d m a l e s  1.4%  5  basis  o f a l l the boys This  reversal  a l s o been r e p o r t e d f o r  1985).  or  males i n Colorado  s t u d y smoked c i g a r e t t e s .  o f t o b a c c o usage p r e f e r e n c e has i n Oregon  chewing tobacco  a r e a chew o r d i p on a r e g u l a r  ( O f f e n b a c h and W e a t h e r s ,  for  o f t h e male s t u d e n t p o p u l a t i o n  and u r b a n  teenagers  11  be  acceptance  p a r t i c u l a r l y by y o u n g m a l e s .  one-quarter of the r u r a l  included  of  dramatic r i s e  d e c a d e may  social  (McGuirt,  at the rate The  over the l a s t  Approximately  s n u f f and  98 m i l l i o n p o u n d s i n  t o b a c c o u s e by t e e n a g e r s h a v e n o t b e e n e x t e n s i v e  but the r e s u l t s concern.  1980) .  rapidly  smokeless  m i l l i o n p o u n d s i n 1980  s a l e s have been i n c r e a s i n g  percent per year smokeless  snuff  tobacco.  S n u f f d i p p i n g and increasing  use  the  3.  Factors Modifying  the Risk of Tobacco-related  Oral  Cancer Variations  i n the prevalence  e x i s t between d i f f e r e n t f a c t o r s w h i c h c o u l d be differences  r i s k of o r a l  chewing p o p u l a t i o n s . responsible f o r the  Of  lesions  t h e many  observed  i t i s u n c e r t a i n w h i c h a r e most i m p o r t a n t .  t h e r e a r e numerous ways t o b a c c o s use,  and  are prepared  for  Since  intra-oral  i t w o u l d be m i s l e a d i n g t o assume t h a t a l l c h e w i n g  mixtures  will  variations  have t h e  in botantical,  characteristics species,  same c a r c i n o g e n i c p o t e n t i a l . chemical,  of l e a f tobacco  types, v a r i e t i e s ,  and p h y s i c a l  are  found  s t r a i n s and  t o t h e g e n e t i c makeup, e n v i r o n m e n t a l mineral  nutrition,  soil  among t h e  grades.  factors,  p r o p e r t i e s , moisture  t e m p e r a t u r e and  light  composition  p h y s i c a l p r o p e r t i e s of the  and  intensity,  Furthermore, v a r i a t i o n s  affect  the  i n curing, aging,  changes i n the tobacco.  substances could It  such  as  lime or b e t e l  including supply,  chemical leaf.  fermentation  enhance o r d e p r e s s i s probable  chewing mixture  Finally, nut  the  the a d d i t i o n of  frequency  o f use,  c h e w i n g v a r y g r e a t l y f r o m one  6  mixture  tobacco.  that the carcinogenic p o t e n t i a l  of  a  t h e manner o f c h e w i n g .  s i t e where t h e q u i d i s p l a c e d , t h e retained,  of  and  different  t o the chewing  the a c t i v i t y  i s i n f l u e n c e d by  various  In a d d i t i o n  o t h e r p o s t - h a r v e s t h a n d l i n g p r a c t i c e s b r i n g about chemical  Wide  and  l e n g t h of time  i t is  t h e a c t u a l amount  population to  another.  of  The  The o c c u r r e n c e o f s e c o n d a r y r i s k  factors  c a r c i n o m a w i t h i n t h e same p o p u l a t i o n may risk  of oral  lesions.  consumption  and K e l l e r ,  Graham e t a l . ,  1977;  Herity et a l . ,  (Wynder e t a l . , 1977;  1981; M a s h b e r g  h e a l t h and d e n t i t i o n  (Graham  1981), and p o o r d i e t  and n u t r i t i o n  Winn e t a l . ,  F o r example,  may  be  cavity  1984).  inversely  also affect  the  These i n c l u d e heavy t o b a c c o smoking  and heavy a l c o h o l 1972;  for oral  Wynder a n d  et a l . ,  et a l . ,  1957;  1977;  Stellman,  1981), p o o r Herity et  (Marshal e t a l . ,  1982;  tobacco chewing p o p u l a t i o n s ,  Winn e t a l . , subnormal  al., 1982;  1984).  levels  of  oral Among  retinol  ( v i t a m i n A) h a v e b e e n o b s e r v e d i n U z b e k i s ( Z a r i d z e e t 1 9 8 5 ) , P a k i s t a n i s and I n d i a n s al.,  1 9 7 7 ) , and t h e h i l l  Philippines  (Wahi e t a l . ,  tribes  (Stich et a l . ,  h a v e b e e n o b s e r v e d among I n u i t hand,  may  Normal  a vitamin A precursor  have a p r o t e c t i v e e f f e c t  A activity  (Stich et a l . ,  retinol  snuff dippers.  t h e s e t o b a c c o u s e r s have r e l a t i v e l y  beta-carotene,  1965:  o f n o r t h e r n Luzon,  1985).  al.,  Ibrahim et the levels  On t h e o t h e r  low l e v e l s  found i n p l a n t s  of which  independent of i t s pro-vitamin  1985).  P r e s u m a b l y , most N o r t h  A m e r i c a n a n d E u r o p e a n s n u f f d i p p e r s and t o b a c c o c h e w e r s not  suffer  currently  f r o m any n u t r i t i o n a l available  determine whether result  information  deficiencies.  Based  i t i s not p o s s i b l e  the observed d i f f e r e n c e s  from d i f f e r e n c e s  oral  i n t a k e o f v i t a m i n s A and C  associated with carcinomas i n the  (Marshal e t a l . ,  Rothman  i n oral  do  on  to lesions  i n t h e c o m p o s i t i o n o f t h e chewing  7  mixture,  t h e type o f chewing p a t t e r n ,  of the population  4.  the nutritional  o r the presence of other  risk  Model Systems f o r S t u d y i n g O r a l Tobacco The  studies  factors.  Carcinogenicity.  problem o f o r a l cancer i s extremely  Epidemiological  status  complex.  are not s u f f i c i e n t l y  refined to  ascertain the individual or interactive effects of the various due be  f a c t o r s w h i c h may m o d u l a t e t o b a c c o  carcinogenesis  t o t h e i n t e r f e r e n c e by thousands o f f a c t o r s which properly  system  controlled.  cannot  The u s e o f a t u m o u r - i n d u c t i o n t e s t  i n experimental animals i s i m p r a c t i c a l because o f the  relatively  long  time  (up t o two y e a r s ) a n d t h e v a s t  o f a n i m a l s needed t o t e s t even a s m a l l  number  number o f t h e f a c t o r s  t o which chewers a r e exposed and t o a s c e r t a i n  significant  differences  Furthermore,  topical  i n the various  treatment groups.  a p p l i c a t i o n o f unburned tobacco a l o n e has f a i l e d t o  produce o r a l carcinomas i n rodents Dunham a n d H e r r r o l d , 1971;  and T h i l a n d e r ,  1981;  e t a l . , 1983; S k l a r e t a l , 1 9 8 5 ) , o r h a s done s o o n l y  a very  al.,  1962; Dunham e t a l . , 1966; Homburger,  K a n d a r k a a n d S i r u t , 1977; H i r s c h  Hirsch at  (Peacock e t a l . , I960;  low frequency  1984).  In v i t r o  the  contrary,  are  economical,  correlation revealed  are well  ( R a n d i v e e t a l . , 1979; H i r s c h e t  short-term  tests f o rgenotoxicity,  s u i t e d f o r such s t u d i e s because  r a p i d and q u a n t i t a t i v e .  8  they  An e x c e l l e n t  e x i s t s between t h e g e n o t o x i c i t y  b y many o f t h e s e s h o r t - t e r m  on  o f a c h e m i c a l as  t e s t s and t h e  carcinogenicity term  in vitro  that  experiments  i n animals  (Hollstein et a l . ,  t e s t s a l s o h a v e an a d d i t i o n a l  advantage  in  materials.  Objective  The vitro  o b j e c t i v e o f t h i s t h e s i s has been t o s e l e c t  test  system  f o r the rapid  c o n t r i b u t i n g t o or modulating implication,  An  evaluation of  variety  t o b a c c o g e n o t o x i c i t y and,  requirement  of tobacco mixtures.  expected t o d i f f e r  f o r an e f f e c t i v e  test  Different  carcinogens.  systems d e t e c t d i f f e r e n t  of a are  therefore,  Since short-term  test  classes of carcinogens with varying  any p a r t i c u l a r  in vitro  test  system  may  enough t o d e t e c t t h e g e n o t o x i c e f f e c t s  tobacco mixtures.  system  tobacco mixtures  i n c h e m i c a l c o m p o s i t i o n and,  contain different  sensitive  by  carcinogenicity.  essential  efficiency,  an i n  factors  i s the c a p a b i l i t y t o detect the genotoxic e f f e c t s  may  Short-  c a n be d e s i g n e d t o r e v e a l t h e mechanisms o f  the genotoxic action of the t e s t  5.  1979).  Therefore, several  g e n o t o x i c e n d - p o i n t s were c h o s e n  9  not  of a l l  tobacco mixtures  f o r examination.  be  and  The variety  f o l l o w i n g four tobaccos, o f chewing m i x t u r e s  screened  f o r genotoxic  (a) S n u f f . used  i n North  A  found  finely  gum  used throughout  c u t o r powdered, f e r m e n t e d  and  sucked.  Chewing t o b a c c o .  i n t h a t the "quid" or use  areas with  difficult  leaves are  i n t h e West.  "chaw" o f t o b a c c o has  s m a l l and  the  and  oral  cancer  States. i n the  form  from s n u f f s  a s e x t e n s i v e and  is actively  of  also  These d i f f e r  fermentation p e r i o d i s not  o f chewing tobacco  rural  of  prepared  tobacco  Winn e t a l . (1981) h a v e  Smokeless tobacco  leaves or blocks of pressed  commercially  were  Europe which i s p l a c e d between  among women i n t h e s o u t h e a s t e r n U n i t e d  loose  the world,  a s t r o n g a s s o c i a t i o n between s n u f f use  (b)  the  activity:  A m e r i c a and  c h e e k o r l i p and  r e p r e s e n t i n g some o f  chewed.  the  Because  the  t r a d i t i o n a l l y been r e s t r i c t e d dispersed populations  to assess the carcinogenic p o t e n t i a l  i t has  of t h i s  to been  form  tobacco. (c) Nass,.  A mixture  of tobacco,  ash  and  lime  with  cotton-seed  o i l or water used i n c e n t r a l A s i a .  amounts a r e  i n s e r t e d under t h e tongue o r between t h e  and  after  gum  and  potential  some m i n u t e s s p a t o u t .  o f nass has  Milievskaya,  been w e l l documented  lips  carcinogenic  (Paches  and  1980).  (d) K h a i n i t o b a c c o . i n parts of India. the g i n g i v a l  The  Small  groove.  A powdered t o b a c c o  I t i s mixed w i t h  l i m e and  In c o n t r a s t with nass,  10  commonly  used  placed within the  lime i s  mixed w i t h t h e tobacco j u s t b e f o r e use. between u s e o f t h i s t o b a c c o and o r a l established  (Hirayama,  Genotoxic  A strong  link  c a n c e r has been  1966).  endpoints studied  were:  (a) Chromosome a b e r r a t i o n s i n C h i n e s e h a m s t e r (CHO) c e l l s . potential to  T h i s i s a standard screening assay f o r  c a r c i n o g e n s because  I s h i d a t e a n d Odashima,  Preston et a l . ,  agents  1981).  The p r e s e n c e  chromosome s t r u c t u r e (b) M i c r o n u c l e i  breakage  are carcinogenic  cells.  which,  lead t o  when n o t i n c o r p o r a t e d  1983).  i n v i v o t o animals  The  and i s  i n t h e s c r e e n i n g o f human  ( S t i c h and R o s i n ,  Unscheduled  fibroblasts.  will  (Heddle e t a l . ,  c a n be e x t e n d e d  currently being applied populations  cells  Chromosomal  as a measure o f g e n o t o x i c i t y has been  o n o v e r 150 c h e m i c a l s  micronucleus t e s t  alterations  nucleus, a r e d e t e c t a b l e as m i c r o n u c l e i .  micronucleus t e s t  (c)  i n metaphase  i n any p o p u l a t i o n o f d i v i d i n g  i n t o t h e daughter  validated  o f chromosome damage  as morphological  fragments  (Evans,  e t a l . , 1979;  f o r m a t i o n i n CHO c e l l s .  the loss of acentric  The  that  1977; H o l l s t e i n  can be d e t e c t e d c y t o l o g i c a l l y in  ovary  mammals a r e p o t e n t i n d u c e r s o f chromosome damage  1974;  firmly  1984).  DNA S y n t h e s i s (UDS) i n c u l t u r e d  human  The g e n o t o x i c e f f e c t s  o f c h e m i c a l s c a n be  d e t e c t e d b y t h e DNA r e p a i r e l i c i t e d  b y DNA b a s e damage,  s t r a n d breakage  DNA r e p a i r t e s t s a r e  reliable  and c r o s s - l i n k a g e .  indicators  f o rpotential  11  carcinogens  (Stich et a l . ,  1971;  San and S t i c h ,  1975; W i l l i a m s ,  M a r t i n e t a l , 1978; H o l l s t e i n 1983).  UDS  excision  s y n t h e s i z e d DNA  can  DNA of  repair  and i n c o r p o r a t i o n  (unscheduled s y n t h e s i s as opposed t o t h e UDS  be measured by a u t o r a d i o g r a p h i c d e t e c t i o n o f u n s c h e d u l e d  DNA,  that  repair silver  of thymidine,  one o f t h e f o u r b a s i c  has been l a b e l l e d w i t h t r i t i u m .  ( o r damage)  i s estimated  A dose-related  increase  damage.  The UDS  DNA  i n vivo or i n vitro to cells  isolated repair  demonstrable r e p a i r  from t e s t  a n i m a l s o r human  inhibition.  c o m p l i m e n t t o t h e UDS  This  s u b s t a n c e o n DNA r e p a i r . studies  cells have  biopsies.  i s i n c l u d e d as a  s y n t h e s i s as measured by  damage o r a s a n i n h i b i t o r y  inhibition  that  o f DNA  a s s a y b e c a u s e t h e a b s e n c e o f any  t h y m i d i n e u p t a k e c a n be i n t e r p r e t e d DNA  indication  assay i s not r e s t r i c t e d t o c u l t u r e d  c a n be a p p l i e d  (d)  The l e v e l o f number  g r a i n s over each nucleus.  been f r e s h l y  components  from t h e average  count i s considered as a p o s i t i v e  no  involving  o f newly  in grain  but  et a l . ,  s y n t h e s i s d u r i n g chromosome r e p l i c a t i o n ) .  incorporation of  e t a l . , 1979; M i t c h e l l  r e p r e s e n t s a f o r m o f DNA  o f damaged DNA  n o r m a l DNA  1976, 1977, 1978;  as e i t h e r  effect  the result of  e x e r t e d by t h e t e s t  T h e t e s t p r o c e d u r e f o r DNA  i s identical to that  with the exception that  tritiated  the c e l l s  s t a n d a r d dose o f U V - l i g h t p r i o r substance.  12  f o r t h e UDS  repair assay,  are i r r a d i a t e d with a  t o exposure t o t h e t e s t  METHODS AND  1.  MATERIALS  Preparation of Extracts S t o c k p r e p a r a t i o n s o f t o b a c c o e x t r a c t were  prepared in  f o r each experiment by m a c e r a t i n g t o b a c c o  tissue  without  freshly  c u l t u r e media  fetal  2.5% f e t a l  calf  calf  samples  (Eagle's Minimal E s s e n t i a l  Medium  serum o r A r g i n i n e d e f i c i e n t medium  serum) w i t h a p e s t l e a n d m o r t a r  with  for five  minutes.  E x t r a c t s were c e n t r i f u g e d a t 2600 rpm f o r t e n  minutes.  S u p e r n a t a n t s were removed, t h e pH a d j u s t e d t o  a p p r o x i m a t e l y 7.4 a n d s e r i a l testing.  d i l u t i o n s were p r e p a r e d f o r  The c o n c e n t r a t i o n s used  calculated  f o r a l l experiments  were  f r o m t h e amount o f t o b a c c o u s e d t o p r e p a r e t h e  stock.  2.  Chemicals N-methyl-N'-nitro-N-nitrosoguanidine  obtained  form A l d r i c h  (MNNG) was  C h e m i c a l Co., M i l w a u k e e , W i s .  C a t a l a s e : p u r i f i e d powder f r o m b o v i n e l i v e r ,  1100 Sigma  u n i t s p e r mg p r o t e i n was o b t a i n e d f r o m Sigma C h e m i c a l Co., St.  Louis,  Mo.  3.  Tissue  Culture  3.1  Culture  Media  E a g l e s ' s M i n i m a l E s s e n t i a l Medium Biological  (MEM, G r a n d  Company, B e r k e l e y , C a l i f o r n i a )  13  Island  was r e c o n s t i t u t e d  from  a powder w i t h d i s t i l l e d  through  a Millipore  Millipore medium minimal  Filter  filter  water and s t e r i l i z e d  (pore s i z e :  0.22  C o r p o r a t i o n , Mass.).  (ADM), b a s e d  e s s e n t i a l medium, was p r e p a r e d (1982).  kanamycin, media.  100 jug/ml  Sodium b i c a r b o n a t e 100 u n i t s / m l  s t r e p t o m y c i n s u l p h a t e , 100  a n d 2.5 jug/ml was  supplemented w i t h  w h e r e a s ADM  was  s u p p l e m e n t e d w i t h 2.5% FCS.  10% f e t a l  calf  serum (FCS)  a n d human  fibroblasts  Growth o f S t o c k C u l t u r e s Chinese hamster ovary  w e r e grown i n MEM The  (CHO) c e l l s  s u p p l e m e n t e d w i t h 10% FCS a n d  s t o c k c u l t u r e s were m a i n t a i n e d  flasks  a t 37 C i n a w a t e r - s a t u r a t e d C 0  2  5.1  Preparation of Cell  Cultures  For each  chromosome  aberration  approximately  60,000 CHO  c e l l s were s e e d e d  onto  d i s h e s and k e p t  i n MEM  i n 35 mm  petri  37 C f o r two d a y s .  Experiments  c u l t u r e s were a p p r o x i m a t e l y  experiment, 22  2  mm  ( 1 0 % FCS)  were b e g u n when t h e  60% c o n f l u e n t .  14  culture  (5%) i n c u b a t o r .  Chromosome A b e r r a t i o n T e s t  coverslips  antibiotics.  i n 260 m l p l a s t i c  5.  at  tiq/rnl  f u n g i z o n e were a d d e d t o t h e c u l t u r e  MEM  4.  f o r Eagle's  i n t h e manner  (1 mg/ml) a n d t h e f o l l o w i n g a n t i b i o t i c s , penicillin,  microns;  Arginine deficient  on t h e s t a n d a r d f o r m u l a  d e s c r i b e d b y San a n d S t i c h  by passage  5.2  Exposure t o Test The  dishes  Extract  t i s s u e c u l t u r e medium  and r e p l a c e d  was  suctioned  w i t h 1 ml o f t h e t e s t e x t r a c t .  experiments i n v o l v i n g the influence  of catalase  clastogenic  activity  of the test extract,  of  prepared  i n MEM  catalase  concentration prior  a 0.5 ml  aliquot  ( w i t h o u t serum) t o g i v e  a  final  dish  t o t h e a d d i t i o n o f 0.5 m l o f t h e t e s t e x t r a c t .  carcinogenic  period,  cells  i n MEM.  the test material  t w i c e w i t h MEM  Following  incubation  ( w i t h o u t serum) a n d 2 ml o f f r e s h MEM  Chromosome  (10%  dish.  Preparations  estimating  0.2 m l o f c o l c h i c i n e  t h e f r e q u e n c y o f chromosome ( 0 . 0 1 % i n MEM)  post-exposure t o the extracts were t h e n t r e a t e d  aberrations,  was a d d e d a t 16 h o u r s  and l e f t  f o r 3.5 h o u r s .  f o r 20 m i n u t e s w i t h a h y p o t o n i c  (1% sodium c i t r a t e ) t o s w e l l  the c e l l s  well-spread  metaphase p l a t e s .  immediately  followed  with  v / v ) f o r 20 m i n u t e s .  w i t h 2% o r c e i n  a 3 hour  cultures  was removed, t h e c o v e r s l i p s washed  FCS) were a d d e d t o e a c h p e t r i  For  The  t o a known g e n o t o x i c a n d  a g e n t was d e t e r m i n e d b y e x p o s i n g c e l l  t o MNNG d i s s o l v e d  (3:1,  For  on t h e  o f 0.1 mg/ml was a d d e d t o e a c h p e t r i  r e s p o n s e o f t h e CHO  5.3  from t h e p e t r i  This  solution  i n order to  treatment  obtain  was  fixation i n ethanol:acetic A i r - d r i e d s l i d e s were  i n 50% a c e t i c a c i d / w a t e r  d e h y d r a t e d a n d mounted.  15  Cells  acid  stained  (5-8 m i n u t e s ) ,  5.4  A n a l y s i s o f Metaphase P l a t e s S l i d e s were r a n d o m i z e d  For each  sample, a t l e a s t  analysed  f o r c h r o m a t i d gaps,  unstained  f o r Chromosome A b e r r a t i o n s  and coded  before being scored.  50 w e l l - s p r e a d m e t a p h a s e s w e r e breaks  and exchanges.  l e s i o n s along the length of a chromatid  c o n s i d e r e d gaps i f t h e d i s t a l dislocated  chromatid  o r i f t h e l e s i o n was  fragment  were was n o t  l e s s than the width  chromatid.  Only d i s l o c a t e d  as b r e a k s .  A l ltypes o f a b e r r a t i o n s which r e q u i r e  chromatid  fragments  were p l a c e d i n t o t h e c a t e g o r y e n t i t l e d ,  6.  Micronucleus  6.1  Test The  Small  of a  were  counted  rejoining  " Exchanges."  Assay  Procedure procedure  t h e chromosome exceptions: cells/dish,  for this  a s s a y was  identical  to that f o r  aberration test with the following  CHO  c e l l s were s e e d e d  a t a d e n s i t y o f 3 0,000  p o s t - t r e a t m e n t i n c u b a t i o n was  f o r 24 h o u r s ,  no  c o l c h i c i n e was a d d e d , a n d s o d i u m c i t r a t e t r e a t m e n t was f o r 4-5  6.2  minutes.  A n a l y s i s of Interphase C e l l s M i c r o n u c l e i a r e formed  fragments left  o r chromosomes  behind  nuclei.  f o r Micronuclei  when a c e n t r i c  chromosome  l a c k i n g a s p i n d l e attachment  are  a t anaphase and a r e e x c l u d e d from t h e daughter  M i c r o n u c l e i a r e d e t e c t a b l e as s m a l l b o d i e s  16  resembling 1000  t h e main nucleus  intact  interphase  micronuclei.  was  not  nucleus  s o t h a t t h e r e w o u l d be  blebs.  A  considered scored.  For each dose,  f o r the presence  scored  or  no  (2) t o u c h e d t h e  confusion with  w i t h many m i c r o n u c l e i and  was  a to  main  nuclear  no m a i n n u c l e u s  t o h a v e u n d e r g o n e k a r y o r r h e x i s and  A l l s l i d e s were r a n d o m i z e d and  of  i f i t (1) had  o n e - t h i r d t h a t o f the main nucleus  including binucleated c e l l s  cell  in size.  c e l l s were s c o r e d  A micronucleus  diameter g r e a t e r than avoid  except  was  not  coded b e f o r e  being  scored.  7.  DNA  Repair  Studies  7.1  Preparation of C e l l  Cultures  Human f i b r o b l a s t s were s e e d e d o n t o 22 35 and  mm  petri  covered  later,  cell  dishes  w i t h MEM  then  ADM  (2.5%  i n e a c h o f two  t r a n s f e r r e d t o new FCS).  A f t e r 5-6  coverslips in  30,000 c e l l s p e r  supplemented w i t h  c u l t u r e s were washed b y  about seven times and  at approximately  2  mm  10%  FCS.  dish  5-7  days  dipping the c o v e r s l i p s  b e a k e r s o f ADM  culture dishes d a y s , more t h a n  (no  serum)  containing 2 90%  of the  ml  cells  were a r r e s t e d a t  7.2  Ultraviolet The  tissue  c u l t u r e s t o be sterile  Irradiation c u l t u r e medium was  irradiated  and  the  removed by  s u c t i o n from  c u l t u r e s were r i n s e d w i t h  p h o s p h a t e b u f f e r e d s a l i n e t o remove any  17  remaining  c u l t u r e medium w h i c h irradiated  c a n a b s o r b UV  light.  C u l t u r e s were 2  f o r 2 0 seconds  w i t h a dose  o f 4 ergs/mm /s  m e a s u r e d by a B l a k - r a y UV  l i g h t meter  (Ultraviolet  Inc.,  San G a b r i e l ,  i r r a d i a t i o n was no.  California).  The  source of  a General E l e c t r i c germicidal  as  Products,  UV lamp  (model  G1RT8).  7.3  Exposure  to Test Extract  and  Radioactively-labelled  Thymidine Tritiated  thymidine  o b t a i n e d as t h y m i d i n e New  3  (methyl- H)  activity  20  Ci/mmol)  i n aqueous s o l u t i o n  E n g l a n d N u c l e a r and d i l u t e d t o a c o n c e n t r a t i o n o f  juCi/ml w i t h ADM  (2.5% FCS).  removed from t h e t e s t of  (specific  T i s s u e c u l t u r e medium  cultures,  and  3 h o u r s w i t h a c o m b i n a t i o n o f 0.5  t h y m i d i n e - c o n t a i n i n g medium and  7.4  Fixation  and  exposure,  ml  of  t r e a t e d w i t h sodium  citrate  ethanol/acetic acid  (3:1)  dry.  coverslips,  n e x t day,  was  for a  duration  tritiated  of the t e s t  o f ADM  f o r 15 m i n u t e s  of p a r a f f i n .  (no  f o r 15 m i n u t e s ,  extract.  with the c e l l  The  18  by  serum),  fixed  and  u p w a r d s , were a t t a c h e d t o g l a s s m i c r o s c o p e small quantity  20  t h e c o v e r s l i p s w e r e washed  5 times i n each o f 3 beakers  The  ml  from  Preparation f o r Autoradiography  Following test dipping  0.5  replaced  was  in  allowed to a i r monolayer  s l i d e s with a  s l i d e s were p a s s e d  through  a graded  series of alcohols  distilled  7.5  water  ( 9 5 % , 70%, a n d 20% e t h a n o l ) a n d  (10 m i n u t e s  Autoradiography  each) a n d a i r - d r i e d .  and S t a i n i n g  S l i d e s were i n d i v i d u a l l y d i p p e d i n Kodak NTB-3 n u c l e a r t r a c k emulsion and  diluted  1:1 w i t h d i s t i l l e d  were a i r - d r i e d for  t h a t h a d b e e n t h a w e d a t 43 C i n a  i n a vertical  14 d a y s i n l i g h t  s l i d e s were b r o u g h t  water.  The c o a t e d  position,  t i g h t boxes.  then stored  b a c k t o room t e m p e r a t u r e .  (10 m i n u t e s ) .  Processing  (3 m i n u t e s )  t h e s l i d e s were s t a i n e d  w i t h 2% a c e t o - o r c e i n , d e h y d r a t e d  superimposing  at 4 c  and  After rinsing the slides i n  r u n n i n g w a t e r f o r 30 m i n u t e s , minutes  slides  Before developing, the  was d o n e a t 18 C i n Kodak D-19 d e v e l o p e r Kodak f i x e r  water-bath  another c o v e r s l i p  f o r 20  a n d mounted b y  over that bearing t h e c e l l  monolayer.  7.6  Analysis The  by  of  Autoradiograms  amount o f u n s c h e d u l e d  DNA s y n t h e s i s was e s t i m a t e d  c o u n t i n g t h e number o f g r a i n s o v e r e a c h n u c l e u s m i n u s t h e  background each  count  nucleus.  automatic  C o u n t i n g was done w i t h a n A r t e k M o d e l 880  grain counter.  interphase nuclei cells  i n an a r e a e q u a l i n s i z e and a d j a c e n t t o  G r a i n c o u n t s were made on s m a l l  o f comparable s i z e  would be i n c l u d e d .  so t h a t  only  diploid  T h e mean g r a i n c o u n t p e r n u c l e u s  19  was  determined  locations  by  on e a c h  scoring  at least  slide.  20  30 n u c l e i  a t random  RESULTS  1.  Chromosome Damaging C a p a c i t y o f A q u e o u s T o b a c c o Crude  a q u e o u s e x t r a c t s were p r e p a r e d f r o m  commonly u s e d s m o k e l e s s clastogenic were exposed  activity.  t o b a c c o samples Chinese hamster  Extracts  several  and t e s t e d f o r ovary c e l l  cultures  f o r t h r e e hours t o the e x t r a c t s and c o l c h i c i n e -  a r r e s t e d m e t a p h a s e p l a t e s were a n a l y s e d f o r t h e p r e s e n c e o f chromosome a b e r r a t i o n s . for  a North American  and  snuff.  independent  Results  T a b l e I shows r e p r e s e n t a t i v e  chewing  tobacco, Khaini tobacco,  f o r each e x t r a c t  data nass  a r e f r o m two  e x p e r i m e n t s conducted on d i f f e r e n t  days.  The  e x t e n t o f chromosome damage i s e x p r e s s e d a s p e r c e n t a g e o f metaphase p l a t e s w i t h a t l e a s t  one c h r o m a t i d b r e a k o r  e x c h a n g e a n d a s t h e a v e r a g e number o f e x c h a n g e s p e r metaphase p l a t e .  T y p i c a l background  frequencies o f  metaphases w i t h chromatid a b e r r a t i o n s run  i n t h e absence  1%.  o f the t e s t  i n the control  positive  r e s p o n s e o f CHO c e l l s  carcinogenic  c u l t u r e s o f any experiment.  experiment,  to a 10  - 4  a f r e q u e n c y o f 69% m e t a p h a s e  M s o l u t i o n o f MNNG.  21  A  by exposing the c e l l s t o  p l a t e s w i t h c h r o m a t i d a b e r r a t i o n s was o b s e r v e d exposure  were  t o a known g e n o t o x i c a n d  a g e n t was d e m o n s t r a t e d  In a typical  samples  e x t r a c t s were b e t w e e n 0 a n d  O n l y s i n g l e gaps o r b r e a k s and never exchanges  observed  MNNG.  i nconcurrent  after  TABLE I  TOBACCO  CHEWING TOBACCO Exp. 1  Exp. 2  Khaini Tobacco Exp. 1  EXP- 2  Nass Exp. 1  Exp. 2  Snuff Exp. 1  Exp. 2  CLASTOGENIC ACTIVITY; OF CRUDE AQUEOUS TOBACCO EXTRACTS IN CHINESE HAMSTER OVARY PETTfi CONCENTRATION (mg equiv/ml)  TOTAL # METAPHASES ANALYSED  % METAPHASES WITH CHROMATID BREAKS AND EXCHANGES  AVERAGE # EXCHANGES PER METAPHASE  15 10 0  100 100 100  37.0 2.0 0.0  1.37 0.01 0.00  8 6 4 0  100 100 100 100  22.0 16.0 2.0 1.0  0.86 0.60 0.02 0.00  12 6 3 0  100 100 100 100  34.0 24.0 6.0 0.0  1.08 0.83 0.23 0.00  10 8 6 0  100 100 100 100  26.0 18.0 9.0 0.0  0.80 0.83 0.30 0.00  80 60 40 0  100 100 100 100  34.0 7.0 2.0 0.0  0.53 0.01 0.00 0.00  60 40 20 0  100 100 100 100  79.0 10.0 1.0 0.0  2.13 0.12 0.00 0.00  80 0  51 100  35.3 0.0  0.96 0.00  80 70 0  64 100 100  21.9 2.0 0.0  0.69 0.00 0.00  22  The extracts  data presented exhibited  i n T a b l e I show t h a t  potent  clastogenic  demonstrated by t h e g r e a t l y chromosome of  aberrations.  the tobacco extracts,  narrow c o n c e n t r a t i o n the  various  aberrations  h o w e v e r , was  activity  only  detectable  in a  that  varied  The a c t i v e d o s e s f o r t h e s n u f f  greatly.  Khaini  induced  of  tobacco extracts  and  chromosome  than those  f o r t h e chewing  tobaccos.  t y p e o f chromosome i n Table I I .  aberrations  induced are  Chromatid gaps, c h r o m a t i d  isochromatid  b r e a k s and s i n g l e and m u l t i p l e  were r e a d i l y  observed  of  f o r most  The r a n g e o f c o n c e n t r a t i o n s  n a s s w e r e many t i m e s h i g h e r  quantified  as  frequency of  The c l a s t o g e n i c  and  The  activity  increased  range.  a l l tobacco  i n treated  CHO  breaks,  exchange  cultures.  damage p e r m e t a p h a s e p l a t e w i t h a b e r r a t i o n s  The  figures extent  varied  widely  f r o m a s i n g l e gap, b r e a k o r e x c h a n g e t o c o m p l e t e f r a g m e n t a t i o n o f chromosomes the  e n t i r e chromosome  2.  Induction  tobacco, and  harvested  results data  Khaini  CHO  by Aqueous Tobacco  c e l l s were e x p o s e d t o s n u f f ,  t o b a c c o and n a s s e x t r a c t s  24 h o u r s p o s t - t r e a t m e n t .  obtained  exchanges  involving  complement o f t h e c e l l .  of Micronuclei  Culturestof  or multiple  f o r each o f t h e four  f r o m two r e p l i c a t e  frequency of micronuclei  experiments.  chewing  f o r three  hours  T a b l e I I I shows t h e extracts,  providing  The b a c k g r o u n d  i n control cultures  23  Extracts  ranged  from  0.4  TABLE I I  CLASTOGENIC ACTIVITY OF CRUDE AQUEOUS TOBACCO EXTRACTS DAMAGE BY TYPES  TOBACCOCONCENTRATION (mg eguiv/ml)  CHEWING TOBACCO Exp. 1  Exp. 2  KHAINI TOBACCO Exp. 1  Exp. 2  NASS Exp. 1  Exp. 2  SNUFF Exp.l  Exp. 2  TOTAL # METAPHASES ANALYSED  CHROMATID BREAK EXCHANGE  GAP  ~ 15 10 0  100 100 100  2 0 0  129 2 0  137 1 0  8 6 4 0  100 100 100 100  4 3 3 0  69 83 1 1  86 60 2 0  12 6 3 0  100 100 100 100  2 2 1 1  68 79 3 0  108 83 23 0  10 8 6 0  100 100 100 100  6 3 1 0  170 81 49 0  80 83 30 0  80 60 40 0  100 100 100 100  10 4 3 0  64 10 2 0  53 1 0 0  60 40 20 0  100 100 100 100  8 4 3 0  194 25 1 0  213 12 0 0  80 0  51 100  5 0  53 0  49 0  80 70 0  64 100 100  0 4 1  19 2  39 0 0  24  0  TABLE I I I  MICRONUCLEI FORMATION IN CHINESE HAMSTER OVARY fRT.Tfi EXPOSED TO AQUEOUS TOBACCO EXTRACTS  TOBACCO  CHEWING TOBACCO Exp. 1  Exp. 2  KHAINI TOBACCO Exp. 1  Exp. 2  NASS Exp. 1  Exp. 2  SNUFF Exp. 1  Exp. 2  CONCENTRATION (mg equivalents/ml)  PERCENT CELLS WITH MICRONUCLEI  10 6 0  13.6 9.0 0.6  10 6 0  10.7 3.1 0.6  6 3 0  9.5 4.7 0.9  8 6 0  13.9 9.6 0.7  60 40 20 0  11.3 9.1 2.0 0.6  80 60 40 0  7.5 6.5 2.9 0.4  80 60 0  12.1 3.6 0.6  70 60 0  3.9 1.3 0.5  25  to  0.9%  (averaging  0.6%).  In a t y p i c a l  f r e q u e n c y o f m i c r o n u c l e a t e d CHO a  10~  4  M s o l u t i o n o f MNNG was  e x t r a c t s produced s i g n i f i c a n t  cells  12.3%.  experiment, t h e  f o l l o w i n g exposure t o A l l of the tobacco  increases  i n micronuclei  frequency over doses s i m i l a r t o those which chromosome  3.  aberrations.  Induction  Tobacco  o f U n s c h e d u l e d DNA  Synthesis  by Aqueous  Extracts  Cultured  human f i b r o b l a s t s were k e p t  d e f i c i e n t medium  synthesis  without  w i t h chromosome simultaneously thymidine  replication.  These c u l t u r e s  hours.  associated  were  and  tritiated  The t o b a c c o s a m p l e s were  the highest  doses p o s s i b l e and  several times greater  p r o d u c e chromosome  entering  DNA  synthesis  exposed t o tobacco e x t r a c t s  prepared t o y i e l d  induction  o f unscheduled  i n t e r f e r e n c e b y DNA  f o r three  concentrations  i n arginine-  f o r 5-6 d a y s t o p r e v e n t them f r o m  S-phase and p e r m i t t h e d e t e c t i o n  the  induced  aberrations  than those required t o  i n CHO  o f u n s c h e d u l e d DNA  included  cells.  synthesis  Figure  1 shows  i n fibroblasts  exposed t o t h e N o r t h American chewing t o b a c c o e x t r a c t as revealed  by t h e l e v e l s  detected  by a u t o r a d i o g r a p h y .  thymidine of  thymidine  The l e v e l  doses.  increasing sharply,  Tritiated  thymidine  incorporation  of t r i t i a t e d  i n c o r p o r a t i o n was d e p e n d e n t on t h e  e x t r a c t used,  higher  of t r i t i a t e d  concentration  and t h e n d e c l i n i n g w i t h  i n c o r p o r a t i o n was n o t  TOBACCO CONCENTRATION (mg e q u i v a l e n t s per ml) F i g u r e 1. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed to a chewing tobacco e x t r a c t . Values i n d i c a t e d a r e g r a i n s p e r nucleus ( c o r r e c t e d f o r background) ± S.E.  27  detectable snuff,  i n fibroblasts  K h a i n i tobacco o r nass e x t r a c t .  t o b a c c o s were t e s t e d by  e x p o s e d t o any c o n c e n t r a t i o n o f  to the levels  the presence o f small n u c l e i  These c e l l s  heavily  absence o f t h e t e s t  4.  Inhibition  of DNA-repair  run i n  Synthesis as measured by  o f no DNA damage o r a s a n i n h i b i t o r y  procedure t o that  DNA-damaging  cultures  t h y m i d i n e u p t a k e c a n be i n t e r p r e t e d  To t e s t  orcein.  extracts.  a b s e n c e o f a n y d e m o n s t r a b l e UDS  tritiated  with  o f dead o r  ( a known  i n concurrent control  the  repair.  stained  I r r a d i a t i o n w i t h UV l i g h t  a g e n t ) i n i t i a t e d UDS  result  o f t o x i c i t y as r e v e a l e d  probably represent pycnotic nuclei  dying c e l l s .  The  A l l of these  effect  f o r t h i s second p o s s i b i l i t y described  as e i t h e r t h e on  DNA  an i d e n t i c a l  f o r the unscheduled  DNA  s y n t h e s i s t e s t was u s e d w i t h t h e e x c e p t i o n t h a t t h e fibroblasts  were e x p o s e d t o u l t r a v i o l e t r a d i a t i o n  exposure t o the tobacco e x t r a c t s . Figures  2-5.  R e s u l t s a r e shown i n  A l l examined t o b a c c o s produced  DNA  repair  per  nucleus i n tobacco-exposed  a s shown b y a r e d u c t i o n  fibroblasts  receiving  showed t h a t  the fibroblasts  concentrations that incorporation  compared t o  Trypan blue  exclusion  exposed t o tobacco  completely  were s t i l l  i n h i b i t i o n of  i n t h e number o f g r a i n s  fibroblasts  UV a l o n e .  prior to  inhibited  viable.  28  tritiated  These r e s u l t s  thymidine  indicate  ^oo-l  80-  3 60LU _J O  z or iu a.  40-  CO 2  < o  20-  no extract  i  —i—  10  20  TOBACCO CONCENTRATION (mg e q u i v a l e n t s per ml)  F i g u r e 2. I n h i b i t o r y e f f e c t o f a chewing tobacco e x t r a c t on induced DNA r e p a i r i n human f i b r o b l a s t s . E r r o r b a r s - S.E.  29  60  no  10  5  15  TOBACCO CONCENTRATION (mg e q u i v a l e n t s per ml)  20  F i g u r e 3. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed to K h a i n i tobacco: (a) Tobacco alone (•); (b) I n h i b i t o r y e f f e c t on UV-induced DNA r e p a i r (•); E r r o r bars = S.E.  30  100-1  F i g u r e 4. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed nass: (a) Nass alone (•); (b) I n h i b i t o r y e f f e c t on UV-induced DNA r e p a i r (•); E r r o r bars - S.E.  31  80 -  CO LU _J  o  or LU Q.  CO 2 < CC  O  no extract TOBACCO CONCENTRATION (mg e q u i v a l e n t s p e r m l ) F i g u r e 5. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed to s n u f f : (a) Snuff alone (•); (b) I n h i b i t o r y e f f e c t on UV-induced DNA r e p a i r (•); E r r o r bars - S.E.  32  that  the  effect  5.  tobacco extracts  on  DNA  r e p a i r and  Exploration  of the  are are  likely not  exerting  an i n h i b i t o r y  merely k i l l i n g  the  cells.  A c t i v e G e n o t o x i c Components o f  the  Extracts The dietary  modifying e f f e c t s of quid and  other  carcinogenicity  f a c t o r s on  components,  tobacco genotoxicity  require mechanistic  studies.  The  hydrogen peroxide i n the  clastogenic activity  e x t r a c t s was  e x p o s i n g CHO  e x a m i n e d by  t o t o b a c c o e x t r a c t s w i t h and are  displayed  i n T a b l e IV.  snuff  6. 6.1  or  no  protected  of the  c h e w i n g and  Systems  UDS  i n Rat  The  original  synthesis  hours  results  against  the  Khaini  clastogenic action  of  Explored  O r a l Mucosal C e l l s e f f o r t s of t h i s  towards the  adaptation  a s s a y t o m o n i t o r DNA  o r a l and  permitted  the  The  of  tobacco  for three  Catalase  e f f e c t on  role  nass.  Other Test  directed  rat  had  cells  and  of the  without catalase.  chromosome d a m a g i n g a c t i v i t y tobaccos but  alcohol,  the  of the  detection the  T h i s would  have  organ-specific actions  damage by  putative  inhibition  of  of  anticarcinogens.  r e s u l t s of these e f f o r t s  33  DNA  repair in freshly isolated  i n v e s t i g a t i o n of the  c a r c i n o g e n - i n d u c e d DNA the  of the  p r o j e c t were  unscheduled  oesophageal mucosal c e l l s .  c a r c i n o g e n s and  Unfortunately,  research  TABLE IV EFFECT OF CATALASE ON THE CLASTOGENIC ACTIVITY OF AQUEOUS TOBACCO EXTRACTS IN CHINESE HAMSTER OVARY CELLS TOBACCO  CHEWING TOBACCO Exp. 1  Exp. 2  KHAINI TOBACCO Exp. 1  Exp. 2  NASS Exp. 1  Exp. 2  SNUFF Exp. 1  Exp. 2  CONCENTRATION (mg equivalents/ml)  PERCENT METAPHASES WITH CHROMATID BREAKS AND EXCHANGES ALONE  + CATAIASE  20 15 10  MI 39 4  2 0 0  20 10 6  MI 24 11  1 0 0  20 10 8 6  MI 26 18 9  3 2 0 0  20 10 5  MI 34 5  7 1 0  60 40 20  72 17 0  69 14 1  80 60 40  36 9 4  32 2 1  80 70  22 2  27 1  80 70  18 1  22 7  f* Concentration o f catalase: 0.01 mg/ml MI. m i t o t i c i n h i b i t i o n : l e s s than 1 metaphase p l a t e among 6000 c e l l s .  b  34  a  were d i s a p p o i n t i n g .  Because the i s o l a t e d mucosal  c e l l s were a t numerous s t a g e s o f d i f f e r e n t i a t i o n v a r y i n g degrees variation  o f k e r a t i n i z a t i o n t h e r e was  i n g r a i n counts.  T h i s was  with  significant  most l i k e l y  s e l f - a b s o r p t i o n of t r i t i u m beta-decay  epithelial  due  by k e r a t i n which would  then prevent the i n t e r a c t i o n of b e t a - p a r t i c l e s with photographic emulsion. o t h e r systems  T h i s problem  (Lake e t a l . ,  1978).  to  has  the  been encountered  Furthermore,  in  when  s u s p e n s i o n s o f v i a b l e m u c o s a l c e l l s were o b t a i n e d t h r o u g h protease d i g e s t i o n procedure, of  10  o r more c e l l s .  limited  The  the c e l l s  clustering  the a n a l y s i s of experiments  n u c l e i within the c l u s t e r s  and  of c e l l s due  to isolate  procedure attempts  resulted  s i n g l e c e l l s by  severely  to the overlap of  cells  the  2  These t e c h n i c a l  r e n d e r t h e s t u d y o f UDS  o r a l mucosa  6.2  difficulties  and  even w i t h  c o n c e n t r a t i o n s as h i g h as 1 0 ~  were n o t  All  isolation  loss of v i a b i l i t y  S-phase w i t h h y d r o x y u r e a , M,  spillage  i n S-phase.  altering  i n an u n a c c e p t a b l e  to inhibit  i n clusters  i n t e r f e r e n c e by t h e  o f g r a i n s from v e r y h e a v i l y - l a b e l l i n g attempts  remained  a  successful. in rat  unfeasible.  Micronuclei  In p r i n c i p l e , v i v o t o animals.  i n Rodent O r a l  Mucosa  the micronucleus t e s t I t was  c a n be  h o p e d t h a t t h e o r a l mucosa  r o d e n t s c o u l d be  screened f o r the presence  exfoliated  o b t a i n e d by  cells  extended of  of micronuclei i n  swabbing o r s c r a p i n g t h e  35  in  oral  mucosa f o l l o w i n g a p r o c e d u r e s i m i l a r t o t h a t u s e d b y and  Rosin  (1984) i n t h e  This technique  s c r e e n i n g o f human p o p u l a t i o n s .  would have p r o v i d e d  chemoprevention regimes i n tobacco Regrettably,  sufficient  c o u l d n o t be  obtained  an  opportunity  treated  numbers o f i n t a c t  from the  oral  cavity  these  animals.  36  to  monitor  animals. cells  for analysis  of e i t h e r r a t s or  mice because o f t h e heavy k e r a t i n i z a t i o n o f t h e in  Stich  oral  mucosae  DISCUSSION  The use  a s s o c i a t i o n between o r a l  c a n c e r and  of tobacco-containing mixtures  established. reasons  the  intra-oral  i s already well  I t i s i m p o r t a n t , however, t o u n d e r s t a n d  for variations  p r e n e o p l a s t i c and  i n t h e p r e v a l e n c e and  neoplastic oral  risk  l e s i o n s among  the  of  different  tobacco chewing p o p u l a t i o n s t o h e l p i n t h e d e s i g n o f prevention strategies. tests  Short-term,  are p o t e n t i a l l y useful  w h i c h may b u t an  test  genotoxicity  i n the i d e n t i f i c a t i o n  c o n t r i b u t e t o or modify  in vitro  in vitro  system w i l l  of  agents  tobacco carcinogenesis, o n l y be u s e f u l  i n such  a  study i f i t i s capable of d e t e c t i n g the genotoxic e f f e c t s many d i f f e r e n t  types of tobacco mixtures.  The  composition of the v a r i o u s tobacco mixtures differ,  h e n c e t h e g e n o t o x i c and  also vary.  I t was  essential,  of tobacco mixtures different test  i n several  i s expected  to test  short-term assays  genotoxic end-points i n order to s e l e c t  a  mixtures possess  may  variety  with a  suitable  i n d i c a t e t h a t unburned  tobacco  substantial genotoxic a c t i v i t y ,  to the necessity of using a v a r i e t y  yet  also  o f e n d p o i n t s when  a s s e s s i n g t h e g e n o t o x i c i t y o f a compound o r m i x t u r e . crude  to  system. These experiments  point  chemical  c a r c i n o g e n i c components  therefore,  of  aqueous e x t r a c t s o f a l l t o b a c c o m i x t u r e s  produced  significant  assayed  c h r o m o s o m a l damage i n c u l t u r e d  37  The  Chinese  hamster ovary c e l l s gaps,  breaks  and  as r e v e a l e d by t h e p r e s e n c e  here  and  (micronucleus t e s t ) . concerning the  action  micronuclei i n interphase  Furthermore,  the data presented  i n f l u e n c e o f c a t a l a s e on t h e  of tobacco extracts  i n d i c a t e t h e way  the genotoxic e f f e c t s  t h e o t h e r h a n d , DNA thymidine uptake be d e m o n s t r a t e d  clastogenic  such model  s y s t e m s c a n be u s e d t o e x p l o r e t h e n a t u r e and of  modification  o f complex t o b a c c o m i x t u r e s .  damage a s m e a s u r e d b y  as a r e s u l t f o r o n l y one  o f DNA  tests,  assay, would appear  repair  synthesis could  of the tested tobaccos.  study of f a c t o r s The  s u i t a b l e as t e s t  i n f l u e n c i n g tobacco  be  justified  the  synthesis  systems f o r the  carcinogenicity.  f o r the examination  c a r c i n o g e n i c i t y may  of  because  and  tobacco most a g e n t s  that  c a r c i n o g e n i c i n animals are a l s o potent inducers of  chromosome damage  (Evans,  Hollstein  1979;  et a l . ,  even though involved  1974;  I s h i d a t e and  Preston et a l . ,  Odishima,  1981).  the precise nature of the genotoxic  1977;  Therefore, lesions  i n c a r c i n o g e n e s i s i s unknown, c h r o m o s o m a l  a b e r r a t i o n s and m i c r o n u c l e i may for  DNA  On  and  e x p l o i t a t i o n o f t h e chromosome a b e r r a t i o n  micronucleus tests  are  but not the unscheduled t o be  On  tritiated  b a s i s o f t h e s e r e s u l t s t h e chromosome a b e r r a t i o n micronucleus  chromatid  e x c h a n g e s i n m e t a p h a s e chromosomes  (chromosome a b e r r a t i o n t e s t ) cells  of  these lesions.  intrinsically  r e p r e s e n t c o n v e n i e n t markers  Alternatively,  involved  c h r o m o s o m a l damage may  i n the process of neoplasia.  38  be  Chromosomal a n o m a l i e s a r e carcinogenesis tissues, in  as  i n d y s p l a s i a s and  associated with  of diverse o r i g i n  s e g m e n t s may initiated.  be  t h e mechanism by  1983).  d e r e g u l a t i o n and  (De  expression  Klein,  1982;  should  result be  are under  of c e l l u l a r  growth  1983;  from the  l o s s of  measured i n  but  aspect  genome.  Thus,  f a c t o r s which can  from those tests.  genotoxic  be  identified  o f t h e u n s c h e d u l e d DNA  of the  The  be  UDS  by  a c t i o n of the  these  only  test  systems. differed  micronucleus  successful i n demonstrating  39  cancer  i . e . , damage  synthesis assay  chewing tobacco,  these  by i n f l u e n c i n g  chromosome a b e r r a t i o n and  a s s a y was  of  remembered t h a t  modulate c a r c i n o g e n e s i s  chromosome damage c a n Results  of carcinogenesis,  i t should  genetic  mutation.  themselves, d i r e c t precursors  to the  and  Rowley,  experiments are not, m e a s u r e o n l y one  in  (onco-)  Gilbert,  endpoints  two  different  result  of d e l e t i o n or g e n e t i c  noted that the  are  bring together  r e g u l a t i o n of c e l l  Cancers could a l s o a r i s e  f u n c t i o n as t h e  and  has  chromosomal  T h i s t r a n s p o s i t i o n may  aberrant  (1981)  w h i c h common c a n c e r s  f a r apart  genes f u n c t i o n i n g i n the differentiation  Non-random  Cairns  Chromosome r e a r r a n g e m e n t s may  regulatory control.  It  (Yunis,  and  a number o f human  t r a n s p o s i t i o n of major  genes t h a t are n o r m a l l y  1984).  other preneoplastic states,  o f most n e o p l a s i a s .  chromosome c h a n g e s a r e  proposed that the  of  e a r l y changes i n c a r c i n o g e n - e x p o s e d  the malignant c e l l s  cancers  observed throughout the process  only.  The  a  failure  of the other interpreted or  extracts to e l i c i t  a detectable  a s e i t h e r t h e r e s u l t o f a l a c k o f a n y DNA  i n h i b i t i o n o f DNA damage r e p a i r .  these extracts incubation  UDS c a n be damage  I t i s unlikely that  l a c k DNA d a m a g i n g a g e n t s ,  require  t i m e s f o r i n t e r a c t i o n w i t h DNA o r n e e d  longer metabolic  a c t i v a t i o n t o p r o d u c e r e a c t i v e compounds s i n c e chromosome aberrations cells  and m i c r o n u c l e i  formation  treated with these extracts  time.  believed Stetka,  t o be t h e b a s i s  double strand  (Brewen a n d  l e v e l s o f U V - i n i t i a t e d UDS w e r e s u b s t a n t i a l l y l o w e r  f i b r o b l a s t s c u l t u r e s exposed t o tobacco e x t r a c t s than i n  likely  exerting  indicating that the extracts  a n i n h i b i t o r y e f f e c t on DNA r e p a i r .  chewing tobacco, which d i d e l i c i t r e d u c e d U V - i n d u c e d DNA r e p a i r .  a m e a s u r a b l e UDS,  This  The  also  r e s u l t suggests  that  DNA damage i n d u c e d b y t h e c h e w i n g t o b a c c o i s p r o b a b l y  much more e x t e n s i v e synthesis  assay.  detectable  than revealed  exerts.  DNA  induces a  UDS p r o b a b l y d e p e n d s upon t h e b a l a n c e o f DNA  Therefore,  of repair inhibition i t  the ineffectiveness of the Khaini  t o b a c c o , nass and s n u f f should  by t h e u n s c h e d u l e d  Whether a t o b a c c o e x t r a c t  damage i t i n d u c e s a n d t h e l e v e l  UDS  breaks are  o f chromosome b r e a k s  f i b r o b l a s t s r e c e i v i n g UV a l o n e ,  the  can indeed  1982).  The  are  i n CHO  f o r t h e same p e r i o d o f  These r e s u l t s s i g n i f y t h a t these e x t r a c t s  damage DNA b e c a u s e u n r e p a i r e d  in  were p r o d u c e d  extracts to i n i t i a t e  n o t be i n t e r p r e t e d a s f a i l u r e  40  a demonstrable  of the extracts to  i n d u c e DNA damage b u t may b e a t t r i b u t e d t o a n o v e r r i d i n g inhibitory  effect  on DNA  repair.  Although t h e unscheduled  DNA s y n t h e s i s a s s a y s h a v e b e e n  v a l i d a t e d w i t h many s i n g l e compounds San  and S t i c h ,  al.,  1975; W i l l i a m s , 1976, 1977, 1978; M a r t i n e t  1978; H o l l s t e i n  the f a i l u r e  ( S t i c h e t a l . , 1971;  et al.,  1979; M i t c h e l l  to obtain positive  et al.,  r e s u l t s w i t h most o f t h e  t o b a c c o e x t r a c t s r e v e a l s an i n h e r e n t l i a b i l i t y when c o m p l e x m i x t u r e s a r e b e i n g a s s a y e d .  of this  strong  even though  DNA d a m a g i n g a g e n t s  i n h i b i t o r o f DNA r e p a i r  mixtures  s u c h a s human f a e c a l  effluents results  (Stich,  et al.,  previously with  e x t r a c t s a n d some  1981b).  they  This complex industrial  These m i x t u r e s  produce  i n d u c e chromosome a b e r r a t i o n s i n CHO  y e t do n o t i n d u c e u n s c h e d u l e d fibroblasts. i n d u c e d DNA  cells  DNA s y n t h e s i s i n human  T h e s e m i x t u r e s were shown t o i n h i b i t  UV-  repair.  c o n t r a s t i n g r e s u l t s based  o n chromosome a b e r r a t i o n s  o r m i c r o n u c l e i a n d UDS s u g g e s t a p o s s i b l e chromosome damage a n d t h e i n h i b i t i o n is  are present, i fa  s i m i l a r t o those obtained here with t h e tobacco  extracts:  The  DNA  i s also present.  phenomenon h a s b e e n e n c o u n t e r e d  test  A mixture o f  compounds may n o t t r i g g e r d e t e c t a b l e u n s c h e d u l e d synthesis,  1983),  link  between  o f DNA r e p a i r .  As i t  g e n e r a l l y b e l i e v e d t h a t chromosomal a b e r r a t i o n s a r e  caused by l e s i o n s  i n DNA a n d t h a t b r e a k s  when t h e mechanisms f o r r e p a i r  41  fail  and exchanges  o r make m i s t a k e s ,  arise i t is  r e a s o n a b l e t o assume t h a t by  a tobacco extract  an i n h i b i t o r y a c t i o n  may h a v e a p o t e n t i a t i n g  i n d u c e d chromosome damage. synthesis  Many i n h i b i t i o r s  and r e p a i r have a l r e a d y  Natarajan,  e f f e c t on o f DNA  i n d u c e d b y p h y s i c a l and  ( B r y a n t a n d I k i a k i s , 1984; K i h l m a n a n d  1984; K i h l m a n a n d A n d e r s o n ,  1985).  A  b e t w e e n chromosome damage a n d d e f i c i e n t r e p a i r s u p p o r t e d by t h e e x i s t e n c e between g r e a t e r increased  of a quantitative  link  i s also  relationship  l e v e l s o f DNA r e p a i r d e f i c i e n c y a n d  sensitivity  t o t h e chromosome d a m a g i n g a c 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 i n Xeroderma pigmentosum c e l l s al.,  i n h i b i t i o n o f DNA r e p a i r s y s t e m s b y o r a l  tobaccos  may p l a y  an important r o l e i n t h e i r c a r c i n o g e n i c  allowing  m i s - o r n o n - r e p a i r o f DNA damage l e a d i n g t o  mutation.  as  I t i s notable that  neutral  fraction of cigarette  inhibit  for  Cigarette  DNA r e p a i r  a l . , 1981).  by  smoke c o n d e n s a t e , a s w e l l by Gaudin e t a l .  i n h i b i t U V - s t i m u l a t e d DNA r e p a i r  lymphocytes.  capacity  dimethylsulphoxide extracts of  a l l other co-carcinogens tested  1972),  et  (San e t  1977). The  the  repair  b e e n shown t o i n c r e a s e t h e  f r e q u e n c y o f chromosome a b e r r a t i o n s chemical agents  o n DNA  i n normal  human  smoke h a s a l s o b e e n r e p o r t e d  i n t h e lungs o f exposed mice  (1971,  to  (Rasmussen  W h i l e t h e s e e x p e r i m e n t s do n o t p r o v e a r o l e  i n h i b i t e d DNA r e p a i r  i n tobacco carcinogenesis,  f o r a p r e v e n t i v e r o l e f o r DNA r e p a i r  i n carcinogenesis  p r o v i d e d b y t h e o c c u r r e n c e o f human h e r e d i t a r y  42  evidence is  diseases,  such  a s X e r o d e r m a pigmentosum and  ataxia telangiectasia,  in  which there i s a p r e d i s p o s i t i o n t o cancers of v a r i o u s types and  a b n o r m a l i t i e s i n DNA  repair activities  (Paterson et a l . ,  1984). In the experiments were a c t i v e w i t h o u t activating rat  reported here the tobacco  t h e a d d i t i o n o f an e x t e r n a l s o u r c e  enzymes, s u c h a s t h e 9000 x g s u p e r n a t a n t  l i v e r homogenate, i n d i c a t i n g t h a t t h e  substances with the -tobaccos that  are d i r e c t - a c t i n g .  few  (S9)  In v i t r o  consistent  s t u d i e s h a v e shown  damage chromosomes o f  ( P a t n a i k e t a l . , 1984), cause  i n d u c t i o n of s i s t e r chromatid  significant  exchanges i n v i r a l l y  transformed  and  lymphocytes  (Umezawa e t a l . , 1 9 8 1 ) , a r e m u t a g e n i c t o V79  cells  phytohaemagglutinin-stimulated  ( S h i r n a m e e t a l . , 1984)  other betel  and  human  are mutagenic  quid ingredients) to Salmonella  (Shirname e t a l . , 1983).  Indian tobacco  (mixed  (Umezawa e t a l . , 1978,  1981).  extracts  Western tobacco  (Whong e t a l . , 1 9 8 4 ) .  e x t r a c t s were a c t i v e w i t h o u t cases the e f f e c t s  S9  A l l of these activation,  also  43  cells  i f prepared  tobacco  though i n a  o f t h e e x t r a c t s were e n h a n c e d by  preparation.  with  snuff  e x t r a c t s are mutagenic t o Salmonella typhimurium low pH  CHO  typhimurium  i n d u c e m o r p h o l o g i c a l t r a n s f o r m a t i o n o f h a m s t e r embryo  at  of  r e p o r t s a v a i l a b l e on t h e g e n o t o x i c i t y o f  intended f o r o r a l use.  cepa  of  genotoxic  These f i n d i n g s a r e  e x t r a c t s of Indian-type tobaccos  Allium  extracts  an  S9  few  Very  few  g e n o t o x i c o r c a r c i n o g e n i c components  smokeless tobaccos  have y e t been i d e n t i f i e d .  of  S n u f f s have  b e e n shown t o c o n t a i n t h e c a r c i n o g e n i c t o b a c c o - s p e c i f i c 1  n i t r o s a m i n e s N - n i t r o s o n o r n i c o t i n e (NNN)  and  nitrososmethylamino)-1-(3-pyridyl)-1-butone relatively direct  high quantities  c o r r e l a t i o n has  1985).  and  oral  (Winn e t a l . , 1981;  i n I n d i a n chewing p r e p a r a t i o n s  and  i n the s a l i v a and  al.,  In s i t u  1984).  of both  Indian tobacco  to  cancer  among  Hoffman e t a l . ,  u s e r s a l s o o c c u r s and  additional  source  Adams, 1981;  ( H o f f m a n and  snuff dippers chewers  of exposure  may  (Hecht  resides  and  i n any  s i n g l e component. 2500 c h e m i c a l s  several genotoxic  released  from  i n the  constitute  variety  saliva  an  e t a l . , 1975;  Hoffman  and  i s not  activity  of modulating  entire  of smokeless  Non-pyrolysed (Hoffman and  tobacco tobaccos  Hecht,  c y t o t o x i c compounds a r e  the chewing mixture.  genotoxic potential the genotoxic  and  S i p a h i m a l a n i e t a l . , 1984;).  carcinogenic activity  c o n t a i n more t h a n  Hecht,  (Sipahimalani et  I t w o u l d be m i s l e a d i n g t o assume t h a t t h e genotoxic  been  (Hoffman  formation of nitrosamines  of tobacco  and  A  been i n f e r r e d between exposure  snuff dippers  Adams, 1981)  and  in  These t o b a c c o - s p e c i f i c n i t r o s a m i n e s have a l s o  detected 1985)  (NNK)  (Hoffman, e t a l . , 1 9 8 5 ) .  these n i c o t i n e - d e r i v e d N-nitrosamines long-term  4-(N-  likely  Furthermore, t o be  probably the  component.  which c o u l d enhance o r  44  total  a simple product  of each i n d i v i d u a l agents  1985)  of  A suppress  the  genotoxicity  influencing o r by  the  o f a m i x t u r e , t h r o u g h s u c h mechanisms  cellular  a c t i v a t i o n and  inhibitors  Finally,  i n the  sensitivity  of the  the  i n the  Copeland,  1985)  and  are  cigarette  target  1983;  f r e e r a d i c a l s may  Kensler  converts  i n the  this  2  2  to non-reactive  c h e w i n g and  Khaini  DNA  e t a l . , 1979;  mutations 2  2  has  1981)  and  (Freese,  H 0 2  and  Levin  has  b e e n p r o v e n t o be  1981).  45  of  i n the  the  Since  of  has  genotoxic catalase  observation  that  the  H 0 -mediated 2  2  2  has  b e e n shown t o  double-strand  Erickson,  1981) In  ( H i r o t a and  carcinogenic  breaks  (Speit  (Stich et a l . ,  e t a l . , 1982).  tumour p r o m o t i n g c a p a c i t y  Ames,  1985), a t t e n t i o n  chromatid exchanges  Bradley  1971;  a  s i n g l e - and  1971), s i s t e r  some  Cerutti,  clastogenic activity  1 9 8 2 ) , chromosome a b e r r a t i o n s  Bradley  H 0  species,  o f chromosome damage.  (Freese,  al.,  1984;  involvement  tobaccos implies  induce base d e s t r u c t i o n ,  play  clastogenic activity  (Nakayama e t a l . , 1984,  enzyme s u p r e s s e s t h e  production  Trush,  of these o r a l tobacco e x t r a c t s .  H 0  the  ( N a g a t a e t a l . , 1982;  and  been d i r e c t e d t o t h e i r p o s s i b l e activities  repair  cells.  o f tumours  involved  smoke  probably  t o b a c c o s must c e r t a i n l y a f f e c t  induction  1983;  are  p r e s e n c e o f DNA  B e c a u s e a c t i v e o x y g e n and role  d e t o x i f i c a t i o n systems,  d i r e c t i n t e r a c t i o n s of chemicals,  also released.  as  in  et  1978; and addition,  Yokayama,  (Ito et a l . ,  H 0 2  produced by t h e chewing and K h a i n i  2  extracts  i s most p r o b a b l y  phenolic  compounds.  i n tobacco leaves  formed by t h e a u t o o x i d a t i o n o f  Large groups o f p h e n o l i c s  1981a; S t i c h a n d P o w r i e , that H 0 2  genotoxic 1983).  2  (Brown, 1980; S t i c h e t a l . ,  1982; R o s i n ,  i s responsible,  activity  of phenolics  compounds, t h e m s e l v e s , o r t h e i r (e.g.  Oxidation  I t appears  i n part,  for the  (Hanham e t a l . , 1983; R o s i n , exists that the phenolic o x i d a t i o n and  quinone r a d i c a l s ) ,  degradation  may a l s o b e  genotoxic.  o f polyphenols a l s o produces a v a r i e t y o f products  w h i c h may c o n d e n s e w i t h  a l k a l o i d s a n d amino a c i d s i n  "browning" and analogous r e a c t i o n s could  1983).  at least  However, t h e p o s s i b i l i t y  products  are present  (Snook e t a l . , 1981) a n d many o f t h e s e  have been found t o be g e n o t o x i c  likely  tobacco  (Stedman,  1968) w h i c h  contribute t o the genotoxicity of the tobacco  mixtures. If the oxidation of phenolics genotoxic  and c a r c i n o g e n i c  i s important t o t h e  activity  o f tobacco,  one w o u l d  expect c o n d i t i o n s which aggravate t h e r a t e o f o x i d a t i o n o f t h e s e compounds t o e n h a n c e b o t h t h e g e n o t o x i c carcinogenic genotoxic  created  (Rosin,  1983).  pH l e v e l s  c a n enhance t h e  by c a t a l y s i n g t h e i r S u c h c o n d i t i o n s may a c t u a l l y  i n t h e mouths o f c e r t a i n t o b a c c o c h e w e r s .  widespread habit, lime  Elevated  effects of phenolics  autooxidation be  potential.  and  i n A s i a , o f adding various  from t h e s h e l l s  of snails,  46  molluscs,  The  quantities of  c o r a l s o r rocks t o  the  c h e w i n g m i x t u r e s c a n l e a d t o a n a l k a l i n e pH w i t h i n t h e  mouth o f a c h e w e r betel  quid  ( S t i c h and Rosin,  and g e n e r a t i o n  importance o f H 0 2  t h e balance  2  generation  between i t s r a t e o f g e n e r a t i o n  s a l i v a r y peroxidase 2  2  damage  (Tennovuo a n d P r u i t t , 2  2  will  defence systems a r e d e p l e t e d . term, low l e v e l  generation  e x p o s u r e may b e s u f f i c i e n t  1984). occur  Khaini tobaccos,  genotoxic  enzyme s y s t e m s .  Thus, a  of H 0 2  from c h r o n i c  2  from  these long-  tobacco  t o produce a genotoxic  the genotoxic  A  gentoxic  only a f t e r  effect.  made on t h e c h e w i n g a n d  activities  o f s n u f f and nass of H 0 . 2  2  p h e n o l i c s have been suggested t o be t h e source o f activity  o f nass  ( Z a r i d z e e t a l . , 1985).  c o n t r a s t t o b e t e l q u i d and K h a i n i t o b a c c o , mixing tobacco with i s used.  lime  use.  and o t h e r  and l o s s o f H 0 2  Perhaps, t h e long  snuff also results  Nass, i n  i s prepared  ingredients long  This p r a c t i c e probably  of tobacco phenolics before  depend  and t h e r a t e  n o t appear t o be dependent on t h e g e n e r a t i o n  Oxidized  of  will  I t i s possible that  In c o n t r a s t t o t h e observations  it  actually  2  s y s t e m may a l s o p r o t e c t human c e l l s  action attributable to H 0  did  2  i n vivo  a t which i t c a n be d e t o x i f i e d by c e l l u l a r  H 0  of H 0  i n the saliva.  The on  Lime i s added t o  and K h a i n i t o b a c c o j u s t p r i o r t o u s e so t h a t  oxidation of the phenolics occurs  1985).  results 2  capacity  and p r o c e s s i n g  i n the loss of the capacity t o  47  before  i n the oxidation  generation  fermentation  by  generate H 0 . 2  2  Whether p h e n o l i c  genotoxic a c t i v i t y  of snuff  compounds c o n t r i b u t e  remains  48  t o be  determined.  t o the  CONCLUSION  The cells  chromosome a b e r r a t i o n and  a p p e a r t o be  influencing studies,  oral  these  a p p l i c a b l e f o r the  t e s t s c o u l d be  utilized  as  adding lime,  to  the  c u r i n g and  areca  nuts,  chewing mixture.  to  genotoxic  b e t e l leaves  r e f u s e t o g i v e up  approach i n the prevention  design  be  beta-carotene  e t a l . , 1981)  frequency  and  of micronuclei  t o b a c c o chewers  and  the  processing customs  such  substances  the  chewing m i x t u r e s which still  acceptable  a p p l i e d towards a  of tobacco-related  effects gained  may  to  habit.  of the  second  oral-cancers. 1  target  cell s  chewing mixtures  which c o u l d help  of chemoprevention s t r a t e g i e s .  v i t a m i n A and (Peto  genotoxic can  of  or other  examining f a c t o r s which c o u l d modify the  information  of  of  This kind of i n v e s t i g a t i o n could  T h e s e t e s t s c o u l d a l s o be  valuable  effects  study  fermentation,  them l e s s h a z a r d o u s b u t  r e s i s t a n c e to the  future  chewing m i x t u r e s which l e a d  i n d e v i s i n g modifications to the  would render  By  In  of the various tobacco mixtures,  such as  c h e w e r s who  of factors  to  T h i s would i n v o l v e a s y s t e m a t i c  composition  aid  as  CHO  rapid bioassays  a r e d u c t i o n o r enhancement o f t h e  practices  study  tobacco c a r c i n o g e n i c i t y .  i d e n t i f y m o d i f i c a t i o n s of the  tobacco.  micronucleus t e s t s i n  For  example,  have a n t i c a n c e r  in  the  both  properties  have been found t o reduce  the  i n e x f o l i a t e d o r a l mucosa c e l l s  of  ( S t i c h e t a l . , 1984a; S t i c h e t a l . , 1984b;  49  Stich  e t a l . , 1985).  Whether b e t a - c a r o t e n e p r o v i d e s  unique p r o t e c t i v e e f f e c t A w i t h i n the  or  body i s u n c l e a r .  problems a s s o c i a t e d with these i n v i t r o question vitamin  to conversion  Provided  be  because beta-carotene w i l l A  tobacco users  vitamin  solubility be  overcome,  a b l e t o answer  not  be  this  converted  to  has  lend themselves t o the  in vitro.  simulation  S a l i v a c o l l e c t e d from  b e e n shown t o be  c l a s t o g e n i c t o CHO  from chewing m i x t u r e s i n v i v o .  o f t h e s e t e s t s can  i n c o r p o r a t i o n of s a l i v a  be  increased  o f chewers i n t h e  Thus,  by  of  Khaini cells  S t i c h , 1982), d e m o n s t r a t i n g t h a t g e n o t o x i c  released  relevancy  to  in vitro.  in vivo conditions  ( S t i c h and  the  b e t a - c a r o t e n e can  t e s t s sytems s h o u l d  These t e s t s e a s i l y  are  i s due  a  agents  the  the  study  of  modulating f a c t o r s . Finally, oral can  tobacco genotoxicity be  extrapolated  relevance is  after identifying  of the  s u p p o r t e d by  in vitro,  to the  in vitro reports  in vivo  situation,  to the  (Gosh and  induction of o r a l  cancers,  occurrence of micronucleated i n d i v i d u a l s who  use  snuff  from I n d i a n  Gosh, 1984)  cells  there  and, i s an  i n the  here oral  chromatid  tobacco more germane  increased  o r a l mucosa  ( S t i c h e t a l . , 1985)  50  the  e f f e c t s of  frequency of s i s t e r  exchanges i n p e r i p h e r a l lymphocytes chewers i s e l e v a t e d  for  studies  presented  i n vivo genotoxic The  modulate  these laboratory  genotoxicity data  of  t o b a c c o u s a g e i n humans.  f a c t o r s which can  nass  of  (Zaridze  et  a l . , 1984), t o b a c c o / b e t e l q u i d  Khaini tobacco tobacco  e t a l . , 1982).  c h e w e r s c a n be  m i c r o n u c l e i by 1984).  easily  non-invasive  T h u s , one  directly with  (Stich  will  i n which o r a l  screened  be p r o v i d e d w i t h  l i n k the r e s u l t s  e t a l . , 1984a),  The  techniques  of a n a l y t i c a l  i n v i v o g e n o t o x i c i t y data  tissue  (Stich  obtained  o r a l mucosa  and  of  Rosin,  the opportunity in vitro  studies  from t h e  actual  tobacco-related cancers w i l l  51  of  f o r the presence (Stich  and  arise.  to  BIBLIOGRAPHY  Ames BN  (1983) Dietary carcinogens S c i e n c e 221:1256-1264  B i n n i e WH  B l o t WJ  and  anticarcinogens.  and KV R a n k i n (1984) E p i d e m i o l o g i c a l and d i a g n o s t i c a s p e c t s o f o r a l squamous c e l l carcinoma. 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