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Modulation of the risk to oral cancer Hornby, Antony Paul 1989

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MODULATION OF THE RISK TO ORAL CANCER  by  Antony P a u l  Hornby  . B . S c , U n i v e r s i t y o f Wales, 1978 M.Sc,  University  o f B r i t i s h Columbia, 1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Pathology We a c c e p t t h i s  t h e s i s as conforming t o the  required  standard  The U n i v e r s i t y o f B r i t i s h  June, 1989  Antony P a u l  Hornby  Columbia  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  of  P/4rtfOt-0  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  J^W  <T,  /<tS<T  I  I further  purposes  gain  the  shall  requirements  agree  that  agree  may  representatives.  financial  permission.  Department  study.  of  be  It not  that  the  be  Library  an  advanced  shall  permission for  granted  is  for  by  understood allowed  the that  without  make  it  extensive  head  of  copying my  my or  written  ABSTRACT  M i l l i o n s o f people use smokeless  tobacco, such as s n u f f , chewing tobacco,  nass or nasswar (a mixture o f tobacco, tobacco nut,  s l a k e d lime, ash and o i l ) ,  Khaini  ( t o b a c c o and s l a k e d l i m e ) , or as p a r t o f a simple b e t e l q u i d ( a r e c a  tobacco,  l i m e , b e t e l l e a f ) or a complex "pan"  seeds, perfumes and s i l v e r  foils).  ( b e t e l q u i d w i t h catechu,  These h a b i t s which are i n v o l v e d i n the  e t i o l o g y o f o r a l cancer, have been o f concern s i n c e s n u f f d i p p i n g i s becoming p o p u l a r among teenagers o f Canada and the U n i t e d S t a t e s . To prevent development o f o r a l cancer among s n u f f d i p p e r s , i t appeared the e t i o l o g i c a l  factors,  the  n e c e s s a r y to t r a c e  to develop markers which would i d e n t i f y i n d i v i d u a l s a t  e l e v a t e d r i s k f o r o r a l cancer, and to t e s t the u s e f u l n e s s o f these " i n t e r m e d i a t e e n d p o i n t s " i n f o l l o w i n g the response a d m i n i s t r a t i o n o f chemopreventive The p o p u l a t i o n groups chosen  agents. f o r t h i s study i n c l u d e d I n u i t ,  Indians and E a s t I n d i a n s . Approximately (Northwest  57.0%  T e r r i t o r i e s ) used s n u f f , 62.6%  (Saskatchewan) d i p p e d s n u f f d a i l y ,  8.03  Canadian  o f I n u i t males from Gjoa Haven  o f n a t i v e Indians o f La Loche  and 54.0%  of East Indian fisherman  a l o n g the c o a s t o f K e r a l a ( I n d i a ) chewed tobacco The  o f the o r a l mucosa to the  from  i n the form o f a b e t e l q u i d .  chewing p a t t e r n s were as f o l l o w s : number o f d i p s per day f o r the I n u i t were a t 25.2  min p e r d i p , f o r the n a t i v e Indians 9.1  per d i p and f o r the E a s t I n i a n s 17.2  d i p s per day a t 20.3  chews per day a t 15.2  min  min per chew.  N - N i t r o s o compounds were found i n the s a l i v a o f s n u f f d i p p e r s and q u i d chewers. They are c o n s i d e r e d to be the most p r o b a b l e e t i o l o g i c a l  betel factors  i n the development o f o r a l c a n c e r s , s i n c e they are the o n l y known c a r c i n o g e n s p r e s e n t i n mg/kg q u a n t i t i e s i n the v a r i o u s tobacco  mixtures.  iii  High l e v e l s o f n i t r i t e ,  a precursor  to n i t r o s a m i n e s ,  were found i n tobaccos  used by the Canadian n a t i v e s . Up t o 1040 mg/kg n i t r i t e was d e t e c t e d  i n tobacco  samples used by t h i s p o p u l a t i o n . High l e v e l s o f n i t r i t e were a l s o d e t e c t e d i n the s a l i v a o f I n u i t and Canadian I n d i a n nitrite  s n u f f d i p p e r s : up t o 0.25 mg/ml o f  appeared i n the s a l i v a w i t h i n 5 t o 10 min o f a s n u f f d i p p i n g  N i t r i t e was a l s o d e t e c t e d averaging  session.  i n the s a l i v a o f E a s t I n d i a n b e t e l q u i d chewers,  36.27 pg/ral.  S i n c e n i t r i t e c a n serve  as a p r e c u r s o r  to n i t r o a m i n e  r e a c t i o n s , the i n  v i t r o n i t r o s a t i o n c a p a c i t y o f s a l i v a from a s n u f f d i p p e r was t e s t e d . A f t e r the a d d i t i o n o f 200 mg p r o l i n e t o the s a l i v a o f a s n u f f d i p p e r increase  a t pH 2.5, an 1 8 f o l d  i n n i t r o s o p r o l i n e (NPRO) was observed over c o n t r o l l e v e l s .  N,PR0 was o b s e r v e d i n the u r i n e o f chewers a t a f i v e f o l d  Moreover,  i n c r e a s e d l e v e l over  non-chewers. These r e s u l t s i n d i c a t e an e l e v a t e d l e v e l o f n i t r o s a t i o n w i t h i n i n d i v i d u a l s who d i p s n u f f . T h i s i n c r e a s e d endogenous n i t r o s a t i o n r e a c t i o n i n snuff dippers Snuff  c a n l e a d t o the f o r m a t i o n  dippers  nitrosamines  of carcinogenic  N-nitrosamines.  and tobacco chewers a r e a l s o exposed t o t o b a c c o - s p e c i f i c  (TSNA). L e v e l s  from 3200 ppb f o r N - n i t r o s o n o r n i c o t i n e  170,000 ppb f o r N - n i t r o s o a n a t a b i n e  (NNK) to  (NAT) were found i n commonly used brands o f  s n u f f which were c o m m e r c i a l l y a v a i l a b l e i n the Northwest T e r r i t o r i e s . I n a d d i t i o n , r e l a t i v e l y h i g h l e v e l s o f these c a r c i n o g e n i c detected The  nitrosamines  were  i n the s a l i v a o f chewers (up t o 980 Mg/ml o f s a l i v a ) . second o b j e c t i v e o f t h i s study was the development o f markers which  i n d i c a t e e a r l y changes i n a human t i s s u e exposed to c a r c i n o g e n s . were used t o d e t a i l  Two markers  the damage o c c u r r i n g i n the o r a l mucosa o f u s e r s o f  smokeless tobacco. The f i r s t was m i c r o n u c l e i which was a p p l i e d to e x f o l i a t e d c e l l s from the o r a l mucosa.  iv  This  assay  frequency snuff  i s a quantitative indicator of micronucleated  dippers, n a t i v e Canadian  containing betel quids, use  smokeless  lesion two  stage,  The  oral  study  (East  either  explored  reduced  of Inuit  cells  of using  the l e v e l  snuff dippers  the t r i a l  i n the o r a l  Indians)  the remission o f newly  significant  these  the preneoplastic  at elevated  risk for  t h e above-mentioned markers to  to the a d m i n i s t r a t i o n o f beta( 1 8 0 mg/week)  of micronucleated who c o n t i n u e d  period. Similarly,  cells  were s i g n i f i c a n t l y  for  i n the o r a l  t o use t h e i r  usual  the levels o f  mucosa o f chewers o f t o b a c c o - c o n t a i n i n g reduced a f t e r  three  betel  months on a regime  o r 180 mg/week o f b e t a - c a r o t e n e  plus  A.  reduction of micronucleated  chemopreventive  who d i d n o t  c h e w e r s . By u s i n g  damage d u r i n g  tobacco users  180 mg/week o f b e t a - c a r o t e n e  inhibition  tobacco-  leukoplakia, a preneoplastic  individuals  the p o s s i b i l i t y  o f smokeless  100,000 I U o f v i t a m i n  placebo.  to i d e n t i f y  of  individuals  a n d v i t a m i n A. The a d m i n i s t r a t i o n o f b e t a - c a r o t e n e  micronucleated  receiving  chewers  i n t h e o r a l mucosa o f b e t e l q u i d  amount o f s n u f f d u r i n g  than  Indian  mucosa o f I n u i t  cancer.  mucosa o f a group  The  and East  T h e s e c o n d m a r k e r was o r a l  t e n weeks s i g n i f i c a n t l y  of  Indians  i n the oral  as compared t o c o r r e s p o n d i n g  feasible  the response  carotene  was o b s e r v e d  to quantify carcinogen-induced  i t appeared  developing  follow  tobacco.  commonly f o u n d  markers  quids  cells  o f chromosomal b r e a k a g e . An e l e v a t e d  oral  mucosal c e l l s  o f l e u k o p l a k i a i n the East  Indian  occurred  more  rapidly  group as d i d t h e  f o r m e d l e u k o p l a k i a f o l l o w i n g t h e a d m i n i s t r a t i o n o f t h e two  agents.  The r e m i s s i o n  (P = 0.004) o n l y  beta-carotene  after  of established oral  s i x months  l e u k o p l a k i a was  i n the b e t e l quid  p l u s v i t a m i n A compared t o t h e group  chewers  receiving a  V  The  t r e a t e d group a l s o showed a s i g n i f i c a n t r e d u c t i o n i n the appearance of  oral leukoplakia  (P = 0.08). The  a d m i n i s t r a t i o n of v i t a m i n A alone  (200,000  IU/week) to b e t e l q u i d chewers produced a h i g h l y s i g n i f i c a n t r e m i s s i o n established leukoplakia new  leukoplakia  continued  (P = 0.0000089) p l u s an i n h i b i t i o n o f the  (P = 0.024) as compared to the p l a c e b o group. The  to chew b e t e l quids  new  of  formation East  of  Indians  throughout the a d m i n i s t r a t i o n o f chemopreventive  agents. Many d i f f e r e n t  scientific  d i s c i p l i n e s are n e c e s s a r y to  obtain  u n d e r s t a n d i n g o f the e t i o l o g i c a l f a c t o r s i n v o l v e d i n the development o f a p a r t i c u l a r cancer, carcinogens.  and  to r e c o g n i z e  e a r l y changes i n the  t a r g e t t i s s u e to  Only a p r o f o u n d u n d e r s t a n d i n g o f these events w i l l h e l p  e a r l y i d e n t i f i c a t i o n o f i n d i v i d u a l s a t e l e v a t e d r i s k to cancer, l a r g e s c a l e chemopreventive t r i a l s , chemopreventive agents.  and  i n the  the d e s i g n  the s e l e c t i o n of the most e f f e c t i v e  of  vi  CONTENTS  Page ABSTRACT  ii  TABLE OF CONTENTS  vi x  UST OF TABLES  xii  LIST O F F I G U R E S  xiv  LIST O F A B B R E V I A T I O N S  xv  ACKNOWLEDGEMENTS  INTRODUCTION  1  1. H i s t o r y o f Tobacco Usage  1  2. T o b a c c o - S p e c i f i c N - N i t r o s o Compounds  6  3. Precancerous  7  O r a l L e s i o n s and Cancer  4. M i c r o n u c l e a t e d C e l l s  9  5. Chemoprevention  12  6. O b j e c t i v e s  14  MATERIALS AND M E T H O D S  15  1. Use o f Smokeless Tobacco 1.1 I n u i t  i n the Northwest T e r r i t o r i e s  15 15  1.2 N a t i v e Canadian Indians i n Saskatchewan  15  1.3 E a s t Indians i n K e r a l a , I n d i a  16  2. N i t r i t e i n Smokeless Tobacco Samples  17  2.1 S n u f f and Chewing Tobacco  17  2.2 B e t e l Quid  17  vii  Page 2.3.  Determination  3. N i t r i t e  4.  i n the  of N i t r i t e Saliva  18  of Users  o f Smokeless Tobacco  3.1  Snuff Dippers  21  3.2  Betel Quid  21  Nitrosation  Chewers  Capacity of Saliva  Samples  21  5. N - N i t r o s o p r o l i n e i n t h e U r i n e o f S n u f f D i p p e r s 5.1 6.  Determination  Snuff  Brands  i n the  Saliva  of  Dippers  Frequency Dippers  29 of Nicotine  and  Cotinine  29  o f M i c r o n u c l e i i n the O r a l Mucosa o f  and  Precancerous i n Users  11.  in Different  26  8. D e t e r m i n a t i o n  10.  22  Tobacco  7. 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  9.  21  of Nitrosoproline  Tobacco-Specific Nitrosamines of  21  Tobacco  Snuff  Chewers  L e s i o n s and  Cancer  30 o f the O r a l  Cavity  o f Smokeless Tobacco  32  10.1  Leukoderma  32  10.2  Leukoplakia  33  Intervention  Strategies  35  11.1  Nitrite  11.2  A d m i n i s t r a t i o n of Beta-carotene  11.3  Inuit:  11.4  East  11.5  Trapping Agents  35 and  Vitamin A  Beta-carotene  Indians: Beta-carotene  37 37  and  Beta-carotene  plus Vitamin A  37  East  38  Indians: Vitamin A  viii  Page 12. D e t e r m i n a t i o n o f B e t a - c a r o t e n e and R e t i n o l  39  13. Q u e s t i o n n a i r e  42  14. S t a t i s t i c a l A n a l y s i s  45  RESULTS  46  1. Use o f Smokeless Tobacco  46  2. N i t r i t e  i n Smokeless Tobacco Samples  51  2.1 S n u f f and Chewing Tobacco Brands  51  2.2 E a s t I n d i a n Tobacco  54  3. N i t r i t e  i n the S a l i v a o f Users o f Smokeless Tobacco  54  3.1 S n u f f D i p p e r s  54  3.2 B e t e l Q u i d Chewers  54  4. N i t r o s a t i o n C a p a c i t y o f , S a l i v a Samples  57  5. N - N i t r o s o p r o l i n e i n t h e U r i n e o f S n u f f D i p p e r s  59  6. 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 i n D i f f e r e n t Brands o f Tobacco  59  7. 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 i n the S a l i v a o f S n u f f Dippers  62  7.1 Appearence o f TSNA i n the S a l i v a  62  7.2 V a r i a t i o n s i n S a l i v a r y TSNA o f S n u f f D i p p e r s  64  8. Frequency o f M i c r o n u c l e i i n the O r a l Mucosa o f S n u f f D i p p e r s and Tobacco Chewers  66  ix Page 9. P r e c a n c e r o u s L e s i o n s and Cancer o f the O r a l C a v i t y i n U s e r s o f Smokeless Tobacco  66  9.1 P r e c a n c e r o u s L e s i o n s  67  9.2 O r a l Cancer  69  10. I n t e r v e n t i o n S t r a t e g i e s  72  10.1 N i t r i t e T r a p p i n g Agents  73  10.2 I n u i t S n u f f D i p p e r s : B e t a - c a r o t e n e  77  10.3 E a s t I n d i a n Tobacco Chewers: B e t a - c a r o t e n e and Beta-carotene plus Vitamin A 10.4 E a s t I n d i a n Tobacco Chewers: V i t a m i n A DISCUSSION  84 91 94  1. Use o f Smokeless Tobacco  94  2. P r e c u r s o r s o f N i t r o s a m i n e s C o n t a i n e d i n Smokeless Tobacco Samples and the S a l i v a o f T h e i r Users  95  3. Smokeless Tobacco and I n g e s t i o n o f N - N i t r o s a m i n e s  98  4. The U n f i n i s h e d S e a r c h f o r t h e F a c t o r s I n v o l v e d i n Oral Carcinogenesis 5. E x p l o r i n g P r e v e n t i v e Measures 5.1 Mechanism o f A c t i o n o f Chemopreventive Agents 6. O u t l o o k  99 102 102 107  SUMMARY  109  REFERENCES  111  LIST OF TABLES Page Table I  A n a l y s i s o f Three o f the Same Tobacco Samples f o r TSNA  Table II  Frequency o f M i c r o n u c l e i i n Human T i s s u e s a t E l e v a t e d  28  R i s k f o r Cancer  31  Table I I I  C l a s s i f i c a t i o n o f O r a l Precancerous L e s i o n s  34  T a b l e IV  Q u e s t i o n n a i r e Used i n the Northwest T e r r i t o r i e s and Saskatchewan  43  Table V  Q u e s t i o n n a i r e Used i n K e r a l a , I n d i a  44  T a b l e VI  S n u f f D i p p i n g , Tobacco Chewing,  C i g a r e t t e Smoking,  D r i n k i n g o f A l c o h o l i c Beverages by N a t i v e I n u i t and E a s t Table VII  Indians, 48  Indians  Means and Ranges o f V a r i o u s S n u f f - R e l a t e d F a c t o r s Among I n u i t and N a t i v e  Table VIII  and  Indians  N i t r i t e Content i n Aqueous  50  E x t r a c t s o f Smokeless  Tobacco  Samples  53  T a b l e IX  Nitrite  i n the S a l i v a o f Smokeless Tobacco Chewers  56  Table X  I n V i t r o N i t r o s a t i o n C a p a c i t y o f S a l i v a i n a User o f Three D i f f e r e n t Brands o f Smokeless Tobacco ( S n u f f )  T a b l e XI  NPRO i n the U r i n e o f Two  Snuff Dipping Populations 60  ( I n u i t and I n d i a n ) Table XII  TSNA i n V a r i o u s Samples o f Canadian and F o r e i g n Tobacco 61  Brands Table X I I I  58  TSNA i n the S a l i v a o f Two and A f t e r S n u f f D i p p i n g  I n d i v i d u a l s P r i o r t o , During 63  xi  Page T a b l e XIV  TSNA i n the S a l i v a o f S n u f f D i p p i n g I n u i t  (Gjoa Haven) 65  T a b l e XV  P r e v a l e n c e o f O r a l L e s i o n s Among S n u f f D i p p e r s and Tobacco Chewers  Table XVI  68  E f f e c t o f the D a i l y Number o f B e t e l Q u i d Chews on the R e l a t i v e R i s k f o r O r a l Cancer i n I n d i a and K e r a l a  T a b l e XVII  70  E f f e c t o f S n u f f D i p p i n g on the R e l a t i v e R i s k f o r O r a l Cancer i n the Southern U n i t e d S t a t e s  Table X V I I I  I n h i b i t o r y E f f e c t of Ascorbate Formation  Table XIX  and C a f f e i c A c i d on the  o f NPRO i n a S n u f f D i p p i n g V o l u n t e e r  Serum L e v e l s o f R e t i n o l and Beta-carotene Inuit Prior to I n i t i t i a t i o n  T a b l e XX  71  76  i n Male  o f the P r e v e n t i o n T r i a l  Frequency (%) o f M i c r o n u c l e a t e d C e l l s  78  i n Oral  L e u k o p l a k i a s and i n Normal Mucosa o f T o b a c c o / B e t e l  Quid  Chewers B e f o r e and A f t e r the A d m i n i s t r a t i o n o f Chemopreventive Agents f o r 3 Months Table XXI  Response o f O r a l L e u k o p l a k i a s of for  Table XXII  Beta-carotene  for  to the A d m i n i s t r a t i o n  o r Beta-carotene  plus Vitamin A  3 Months  89  Response o f O r a l L e u k o p l a k i a s of  Beta-carotene  87  t o the A d m i n i s t r a t i o n  o r Beta-carotene  plus Vitamin A  6 Months  T a b l e XXIII Response o f O r a l L e u k o p l a k i a s o f V i t a m i n A f o r 6 Months  90 t o the A d m i n i s t r a t i o n 93  xii  LIST O F F I G U R E S  Page Figure 1  A t y p i c a l b e t e l q u i d showing b e t e l  leaf,  a p o r t i o n o f s u n d r i e d tobacco, and a q u a r t e r o f a b e t e l n u t . Lime i s shown on t h e r i g h t f i n g e r t i p .  5  Figure 2  A Homogenous L e u k o p l a k i a i n t h e R i g h t B u c c a l Mucosa  Figure 3  Procedure f o r N i t r i t e D e t e r m i n a t i o n  20  Figure 4  Procedure  f o r N - n i t r o s o p r o l i n e (NPRO) D e t e r m i n a t i o n  25  Figure 5  E x p e r i m e n t a l P r o t o c o l f o r Endogenous NPRO F o r m a t i o n  36  Figure 6  D e t e r m i n a t i o n o f Serum B e t a - c a r o t e n e and R e t i n o l  41  Figure 7  N i t r i t e i n t h e S a l i v a o f Seven I n u i t S n u f f D i p p e r s  55  Figure 8  The Appearence o f NPRO i n t h e U r i n e o f a V o l u n t e e r Who Dipped S n u f f and,Ingested P r o l i n e and A s c o r b a t e  Figure 9  75  E f f e c t o f B e t a - c a r o t e n e Treatment on MNC i n the O r a l Mucosa  F i g u r e 11  74  The Appearence o f NPRO i n t h e U r i n e o f a V o l u n t e e r Who Dipped S n u f f and I n g e s t e d P r o l i n e and A s c o r b a t e  F i g u r e 10  9  81  Changes i n t h e Frequency o f MNC a t t h e O r a l S i t e Where t h e Tobacco i s L o c a t e d Over a P e r i o d P r e c e e d i n g the P i l o t T r i a l and A f t e r a 10-Week A d m i n i s t r a t i o n of Beta-carotene  F i g u r e 12  82  Frequency o f A n u c l e a t e d E x f o l i a t e d C e l l s from the O r a l S i t e Where t h e Tobacco i s L o c a t e d B e f o r e and A f t e r a 10-Week A d m i n i s t r a t i o n o f B e t a - c a r o t e n e  83  xiii  Page F i g u r e 13  Response o f MNC  i n Areas o f L e u k o p l a k i a to a 3-Month  A d m i n i s t r a t i o n o f B e t a - c a r o t e n e and B e t a - c a r o t e n e P l u s V i t a m i n A v s . Placebo  86  xiv  LIST OF ABBREVIATIONS  DMNM  Nitroso-cis-2,6-dimethylmorpholine  MNC  Micronucleated C e l l  MNNG  N-Methyl-N'-nitro-nitrosoguanadine  NAB  N-Nitrosoanabasine  NAT  N-Nitrosoanatabine  NIC  Nicotine  NNK  4-(Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone  NNN  N'-Nitrosonornicotine  NPIC  N-Nitrosopiperidine-2-carboxylic acid  NPRO  Nitrosoproline  TSNA  Tobacco-specific nitrosamines  Other abbreviations which are widely used without d e f i n i t i o n i n the biochemical literature  (eg. DNA, IU) are not s p e c i f i c a l l y defined here.  XV  ACKNOWLEDGEMENTS I would l i k e  t o e x p r e s s my  suggestions, discussions, investigations for  excellent  research  team  reported  involved  Cancer Research  Trivandrum,  t o D r . H.F. S t i c h f o r g u i d a n c e ,  encouragement d u r i n g  t h e s i s . I would a l s o  I n s t i t u t e f o r Disease o f TSNA  in saliva  f o r scoring  like  the course  o f the  t o thank Dr. Bruce  India,  like  moral  f o r diagnosis  to dedicate  support  of  this  Dunn  i n s t r u c t i o n , a n d t h e f o l l o w i n g members o f t h e  i n the chemopreventive  Dana  Centre,  gratitude  trials  for their  Prevention,  and s n u f f  assistance:  American  D r . K.D.  Health  s a m p l e s ; M r s . H.F. S t i c h , B.C.  of exfoliated buccal  mucosal c e l l s f o r  D r . B a b u Mathew a n d D r . R. S a n k a r a n a r a y a n a n , R e g i o n a l C a n c e r  I would unfailing  i n this  for analysis  micronuclei;  and u n f a i l i n g  t e c h n i c a l and s c i e n t i f i c  Brunneraann, N a y l o r Foundation,  sincere  Centre,  leukoplakia.  thesis  t o my w i f e  and encouragement throughout  D a r l e n e , who h a s o f f e r e d t h e c o u r s e o f my  studies.  1 INTRODUCTION 1. History of Tobacco Usage Tobacco has been chewed f o r as l o n g as i t has been smoked. But because  partly  i t i s n o t a v e r y v i s i b l e h a b i t , t h i s o r a l use i s seldom r e c o g n i z e d as a  common p r a c t i c e o r as a h e a l t h h a z a r d .  A rough e s t i m a t e p u t s the number o f  people i n the w o r l d u s i n g tobacco o r a l l y on a d a i l y b a s i s a t more than million  (IARC,  600  1985) .  Tobacco use can be t r a c e d back more than 7000 y e a r s , when i t appears to have been f i r s t  cultivated.  I t was  p r o b a b l y used d u r i n g t h i s p e r i o d f o r such  purposes as a l l e v i a t i n g hunger and w h i t e n i n g the t e e t h , and as a d i s i n f e c t a n t when the j u i c e was  e x p e c t o r a t e d onto wounds. The  and s n u f f tobacco were p r o b a b l y American as those o f Amerigo V e s p u c c i i n 1499, the  c o a s t o f V e n e z u e l a , chewing  t h e i r necks. V e s p u c c i f e l t t h i r s t , because chewing  f i r s t people to smoke, chew,  I n d i a n s . Reports from the 1400s, such  speak o f I n d i a n s on M a r g a r i t a I s l a n d , o f f  a green herb t h a t was  c a r r i e d i n gourds  t h a t the green herb, tobacco, was  i n c l u d i n g Samuel de Champlain,  Quebec, and Columbus, mention  t h a t the use o f chewing  In  the Americas  as t w i s t s . Of the 348  the founder o f  tobacco by n a t i v e s  tobacco chewing became p o p u l a r among the n o n - n a t i v e chewed e i t h e r i n the form o f a p l u g or  tobacco f a c t o r i e s l i s t e d  and N o r t h C a r o l i n a , o n l y seven manufactured chewing  from  t h i r s t and f a t i g u e among i t s u s e r s .  p o p u l a t i o n . D u r i n g the 1860s tobacco was  Tobacco  that  a number o f r e p o r t s  explorers during this period,  reduced hunger,  used to quench  o f the s c a r c i t y o f water on the i s l a n d and the f a c t  the l e a f i n c r e a s e d s a l i v a r y flow. Indeed,  around  i n the 1860  Census f o r V i r g i n i a  smoking p r o d u c t s (Heimann,  reached an a l l - t i m e h i g h i n America by 1890,  pounds (about 1.5  kg) o f p l u g , t w i s t or f i n e - c u t chewing  1960).  when some three  tobacco were chewed  a n n u a l l y p e r c a p i t a i n the U n i t e d S t a t e s (Heimann, 1960). T h i s remained  the  2  dominant form of tobacco usage u n t i l the expansion of the cigarette industry i n 1918  (Maxwell, 1980). Its decline was hastened by the "germ theory of disease"  which made the habit of s p i t t i n g i n public places s o c i a l l y unacceptable. Antis p i t t i n g laws were passed i n New York and Philadelphia i n 1896 and i n Toronto in 1904  (Kozlowski, 1981).  During the l a t t e r h a l f of the 1960s and through the 1970s, there was a resurgence i n the use of smokeless tobacco i n North America. This renaissance was probably due to an increased awareness of the harmful e f f e c t s of tobacco smoking coupled with a lack of awareness of those r e s u l t i n g from i t s oral use. It has become p a r t i c u l a r l y popular among adolescent males, promoted by sports figures and the Western "macho" image. I t can be practised i n areas where smoking i s hazardous, such as i n the s t e e l , coal or petroleum industries, and i s often said to be cheaper than smoking. In addition, people who require the use of t h e i r hands while working or playing can chew tobacco much more conveniently than they can smoke. Since the 1960s, the U.S. production of smokeless tobacco has increased by approximately 60%. The t o t a l U.S.  consumption of such products was  approximately 132 m i l l i o n pounds (60 m i l l i o n kg) per year during the period from 1980 to 1982  (Tobacco I n s t i t u t e , 1981, 1982,  1983). On 1 January  1982,  some types of chewing tobacco were r e c l a s s i f i e d as snuff (U.S. Department of Agriculture, 1983). Under this c l a s s i f i c a t i o n , consumption  of chewing tobacco  in 1982 was 88 m i l l i o n pounds (40 m i l l i o n kg) (Tobacco I n s t i t u t e ,  1983).  In other parts of the world tobacco i s often chewed i n combination with other ingredients, i n p a r t i c u l a r with areca nut, b e t e l l e a f and slaked lime, as i n India and Southeast Asia. I t i s estimated that more than 450 m i l l i o n people practise this habit i n these regions.  3  The  chewing o f b e t e l q u i d w i t h o u t tobacco i s a h a b i t o f g r e a t B.C.,  when i t was  antiquity.  The  f i r s t mention o f b e t e l q u i d dates from 504  recorded  the  "Mahawamsa," a r e g i s t e r o f events i n S r i Lanka w r i t t e n i n P a l i ,  p r i n c e s s made a g i f t o f b e t e l to her nurse (Krenger, 1942). The a r e c a nut  l e a f o f the b e t e l v i n e , "tambuli,"  and  B.C.  (Raghavan and  and  Persian  Baruah, 1958)  believed  S a n s k r i t name f o r  "tambula," p e r s i s t s i n modern H i n d i  i s unchanged i n A r a b i c  Marco Polo  near Benares. The  a  chewing o f  i s a l s o mentioned i n S a n s k r i t m a n u s c r i p t s , Sushruta Samhita,  to have been w r i t t e n around 600  1298,  that  in  (Gode, 1961)  as  ( N a i r and K i r k , 1960).  wrote i n h i s t r a v e l o g u e s  the  In  that  "the  people o f I n d i a have a h a b i t o f k e e p i n g i n t h e i r mouth a c e r t a i n l e a f c a l l e d the  "tembuli"  (Krenger,  1942).  A l t h o u g h the chewing o f b e t e l q u i d i s p r a c t i s e d i n a number o f d i f f e r e n t ways i n v a r i o u s These  countries,  the major components are r e l a t i v e l y  consistent.  are: A r e c a nut  ( b e t e l nut)  a s m a l l genus c o m p r i s i n g f a m i l y . The Pacific varies  i s the f r u i t o f the A r e c a c a t e c h u L.  about 20 s p e c i e s  a r e c a palm i s n a t i v e  i s l a n d s . The i n s i z e and  fruit  o f s l e n d e r palms i n the  to South and  Southeast A s i a and  The  to  several  f i b r o u s p e r i c a r p o f the f r u i t  i s generally  i s separated  seed or endosperm which i s then used f r e s h or a f t e r s u n - d r y i n g or (Arjungi,  Palmaceae  grows i n l a r g e bunches a t the base o f the l e a v e s  shape. I t i s orange-yellow i n c o l o u r and  s i z e o f a s m a l l egg.  tree. Areca i s  from  and  the the  curing  1976).  B e t e l l e a f ( P i p e r b e t l e L.) are c u l t i v a t e d i n hot  been used s i n c e a n c i e n t  and humid c l i m a t i c c o n d i t i o n s  India, Indonesia, Malaysia  and  Lime, known c o l l o q u i a l l y from the c a l c a r e o u s  has  times. B e t e l  i n d i f f e r e n t parts  vines  of  S r i Lanka. i n I n d i a as chuna or chunam, i s p r e p a r e d e i t h e r  or s i l i c l o u s  covering  o f marine i n v e r t e b r a t e s  (sea s h e l l s )  harvested along the coastline of India, or from quarried stone i n central India. I t i s manufactured on an i n d u s t r i a l scale and i s sold as a paste mixed with water i n order to release calcium  hydroxide.  Tobacco i s often added to the abovementioned ingredients. The tobacco i s usually only sun-dried, cut into s t r i p s and chewed with no further processing. A t y p i c a l b e t e l quid i s shown i n Figure 1.  5  F i g u r e 1: A t y p i c a l b e t e l q u i d showing b e t e l l e a f , a p o r t i o n o f s u n - d r i e d tobacco and  a q u a r t e r o f a b e t e l nut.  Lime i s shown on the r i g h t  fingertip.  6 2. Tobacco-Specific N-Nitroso Compounds Since  tobacco  constituents  o f any  compounds, t h i s oral the  cancer  and  during  nicotine  of  the  thesis  the  and  the  other  and  I t has tobacco  compounds o v e r  other  TSNA i s much h i g h e r average  smoker p e r  of  exposure  day,  rise  of n i t r a t e  (NAT); p l u s  chewing  mixtures, shown  tobacco,  exceeds by  consumer  tobaccos  at  products.  include  the  4-(N-nitrosomethylamino)-1-(3-  the v o l a t i l e  from packaging  N-nitrosoanabasine  nitrosamines,  materials  N-  i n some s n u f f s ) ,  N-  i n some s n u f f s ) a n d N - n i t r o s o p y r r o l i d i n e  ( N a t i o n a l R e s e a r c h C o u n c i l , 1981)  to a  twentyfold  i n chewing tobacco 10  tobacco  to  of  N-nitrosamines  f a r i n other  ( T S N A ) , s u c h as  causes  s t u d i e s have  nitrosamines  i n s n u f f s and  consumer p r o d u c t s .  to N-nitroso  In a d d i t i o n  these  carcinogenic  i n chewing  processing of  to c a r c i n o g e n i c  f o u n d so  to  most l i k e l y  l a r g e number o f and  known  snuffs).  chewer consumes  a  levels  contained  (NDMA) ( f o u n d  been estimated  smoke g i v e s  rise  concentrations  (NMOR) ( f o r m e d  i n some  rise  the  ( N N K ) , N ' - n i t r o s o n o r n i c o t i n e (NNN),  nitrosodimethylamine (found  other. A  concentration of  N-nitrosoanatabine  nitrosomorpholine  (NPYR)  give  nitrosamines  pyridyl)-1-butanone (NAB)  the  only  give  components as  curing, fermentation  known c a r c i n o g e n s  tobacco-specific  these  which can  the  c a r c i n o g e n i c compounds f o u n d  alkaloids  a hundredfold The  on  f a r o u t w e i g h any  ageing,  components a r e  above m i x t u r e s  ( H o f f m a n n , e t a l . 1 9 8 4 ) . The least  a r e c a nut  focuses  i n u s e r s . Of  N-nitrosamines  that  a few  the  g of  tobacco  increase  chewing appears compounds  N-nitroso  in cigarette  versus  less  t o be  (Hoffmann and  the  than  concentration  smoke, and 1 g of  may  U.S.,  N-nitroso  since  of the  tar inhaled  g r e a t e s t exogenous  Hecht,  compounds, t o b a c c o  w h i c h , when c o n v e r t e d  i n the  i n consumption of  However, s i n c e the than  that  by  source  1985). often contain  to n i t r i t e through  ageing  or  high  reduction  7  i n the s a l i v a ,  c a n supply the p r e c u r s o r elements f o r n i t r o s a t i o n r e a c t i o n s .  T h i s i s important when c o n s i d e r i n g the a l k a l o i d c o n t e n t o f the a r e c a n u t . In v i t r o experiments w i t h a r e c o l i n e and n i t r i t e have shown t h a t t h i s nut a l k a l o i d can g i v e r i s e t o a t l e a s t f o u r N - n i t r o s a m i n e s : nitrosoguvacoline, propionaldehyde  areca  N-  3 - ( m e t h y l n i t r o s a m i n o ) - p r o p i o n i t r i l e , 3-(methylnitrosamino)-  (Wenke and Hoffmann, 1983) and N - N i t r o s o g u v a c i n e  (Nair e t a l . ,  1985) . N - n i t r o s o g u v a c o l i n e has been d e t e c t e d i n the s a l i v a o f b e t e l chewers t o g e t h e r w i t h TSNA when the q u i d c o n t a i n s tobacco R e c e n t l y , N - n i t r o s o g u v a c o l i n e and N - n i t r o s o g u v a c i n e  quid  (Wenke e t a l . , 1984).  have been found  together i n  the s a l i v a o f b e t e l q u i d chewers ( N a i r e t a l . , 1985). A f u r t h e r important  c o n s i d e r a t i o n i s the i n t e r a c t i o n o f a l c o h o l w i t h N-  n i t r o s a m i n e s . Of p a r t i c u l a r r e l e v a n c e to the c h e w i n g / a l c o h o l  problem i s the  o b s e r v a t i o n t h a t a l c o h o l changes the d i s t r i b u t i o n p a t t e r n o f c a r c i n o g e n i c nitrosamines liver  (Swann, 1982). I n r a t s , a l c o h o l d i v e r t e d n i t r o s a m i n e s  from the  t o o t h e r organs (e.g., the esophagus) which a r e more s e n s i t i v e t o these  c a r c i n o g e n s . T h i s change i n p h a r m a c o k i n e t i c s i n esophageal  may e x p l a i n the t h r e e f o l d i n c r e a s e  carcinomas when d i e t h y l n i t r o s a m i n e and e t h a n o l a r e a d m i n i s t e r e d ,  compared t o d i e t h y l n i t r o s a m i n e p l u s d r i n k i n g water ( G i b e l , important  1967). E q u a l l y  appears to be the o b s e r v a t i o n t h a t N-methyl-N'-nitro-N-  nitrosoguanidine  (MNNG) induces  a h i g h frequency  o f esophageal  cancer when  a p p l i e d c o n c u r r e n t l y w i t h a l c o h o l ( Y i o r i s e t a l . , 1984). Normally  MNNG i s  c a r c i n o g e n i c o n l y i n the stomach o f r a t s .  3.  Precancerous Oral Lesions and Cancer Many r e p o r t s o f case s e r i e s have emphasized the r e l a t i v e l y h i g h  o f tobacco  frequency  chewing and s n u f f use among o r a l cancer p a t i e n t s (IARC, 1985). The  c l i n i c a l c h a r a c t e r i s t i c s o f cancer p a t i e n t s who use smokeless tobacco  products  8  have a l s o been d e s c r i b e d cancers  to occur  (IARC, 1985), e s p e c i a l l y the p r o p e n s i t y o f these  i n the presence o f l e u k o p l a k i a , to o f t e n have a  verrucous  appearance, and to be slow-growing, w e l l - d i f f e r e n t i a t e d , squamous-cell carcinomas. P a t i e n t s w i t h cancer f r e q u e n t l y d e s c r i b e d as h a v i n g  and w i t h a chewing tobacco o r s n u f f h a b i t are  cancer  a t the s i t e o r on the s i d e where the q u i d  i s most f r e q u e n t l y p l a c e d . Much e p i d e m i o l o g i c a l evidence significant quid  i n c r e a s e i n o r a l cancer  among chewers, v e r s u s  indicates a highly non-chewers, o f b e t e l  (IARC, 1985). Areas o f I n d i a and Southeast A s i a , where the chewing o f  b e t e l q u i d i s common, have the h i g h e s t  i n c i d e n c e o f o r a l cancer  and o r a l  p r e n e o p l a s t i c l e s i o n s ( l e u k o p l a k i a ) o f any r e g i o n i n the world. In K e r a l a , I n d i a , where some o f the s t u d i e s p r e s e n t e d  i n t h i s t h e s i s were c a r r i e d out,  o r a l l e u k o p l a k i a (see F i g u r e 2) among the C h r i s t i a n f i s h e r m e n approximately prevalence cancer  40% o f chewers o f b e t e l q u i d p l u s tobacco,  o f 0.2% i n people w i t h o u t  o f the mouth i s the most p r e v a l e n t cancer  males, c a n c e r s  compared to a  (IARC, 1985). I n a d d i t i o n , observed i n t h i s area. I n  o f the mouth, tongue and oropharynx were r e s p o n s i b l e f o r 27.2%  of a l l o c c u r r i n g c a n c e r s ; The  such a h a b i t  occurred i n  these  g r e a t m a j o r i t y were cancers  s i t e s accounted f o r 14.8% o f tumors i n females. o f the b u c c a l mucosa. G e n e r a l l y ,  the i n c i d e n c e  o f o r a l cancer  i s much h i g h e r where tobacco i s added t o the q u i d or where q u i d  chewing o c c u r s  c o n c u r r e n t l y w i t h smoking (IARC, 1985).  10  4.  Micronucleated Cells Beyond p r e n e o p l a s t i c l e s i o n s ,  the c e l l u l a r  the s e a r c h f o r a marker to d e t e c t damage a t  l e v e l l e d to the development o f the m i c r o n u c l e u s  t e s t . When  a p p l i e d t o e x f o l i a t e d c e l l s o f the b u c c a l mucosa, t h i s t e s t p e r m i t s  the  i d e t e c t i o n o f l o c a l i z e d g e n o t o x i c damage to t h a t t i s s u e .  I t i s based  on  the  h y p o t h e s i s t h a t an i n c r e a s e o f m i c r o n u c l e i , which r e s u l t from chromosome and chromatid  fragments,  s h o u l d be  found  Three o b s e r v a t i o n s appear to suggest  i n the e a r l y stages o f c a r c i n o g e n e s i s . this.  Firstly,  most c h e m i c a l  carcinogens  are a c t i v e c l a s t o g e n s and s h o u l d induce a v a r i e t y o f chromosome a b e r r a t i o n s i n human t i s s u e exposed to c a r c i n o g e n i c m i x t u r e s . carcinogens  Secondly,  many, i f not  seem to produce a l t e r e d chromosome numbers and  rearrangements,  chromosomal  some of which i n v o l v e p a r t i c u l a r r e g i o n s (Rowley,  Mitelman, 1985). These abnormal k a r y o t y p e s  1983;  i n t r a n s f o r m e d c e l l s must evolve  from chromosomal a b e r r a t i o n s d u r i n g the p r e n e o p l a s t i c s t a g e . The i s based  all,  on a more t h e o r e t i c a l c o n s i d e r a t i o n . A m p l i f i c a t i o n and  t h i r d argument transposition  o f oncogenes seem to be mechanisms i n v o l v e d i n n e o p l a s t i c t r a n s f o r m a t i o n and i n p r o g r e s s i o n of a malignant c h r o m a t i d breaks  phenotype. These oncogene changes r e s u l t  and exchanges t h a t can be  i n d i r e c t l y d e t e c t e d by  from  the  appearance o f m i c r o n u c l e a t e d c e l l s a t p r e i n v a s i v e s t a g e s . With t h i s working h y p o t h e s i s i n mind, we  a p p l i e d the m i c r o n u c l e u s  e x f o l i a t e d c e l l s o f i n d i v i d u a l s a t e l e v a t e d r i s k o f cancer of the o r a l (Stich,  et a l . ,  t e s t to cavity  1982a,b; S t i c h and R o s i n , 1983b), esophagus ( Z a r i d z e e t a l . ,  1985), u r i n a r y b l a d d e r  (Raafat et a l . ,  1984)  and c e r v i x  (Stich,  H.F.,  u n p u b l i s h e d d a t a ) . A s i g n i f i c a n t i n c r e a s e i n the f r e q u e n c y o f m i c r o n u c l e i i n e x f o l i a t e d human c e l l s  ( m i c r o n u c l e a t e d c e l l s or MNC)  i n d i v i d u a l s . Moreover, an e l e v a t e d frequency o f MNC  was was  found  in virtually a l l  a l s o observed  in  v a r i o u s t i s s u e s o f i n d i v i d u a l s a f f l i c t e d w i t h a c a n c e r - p r e d i s p o s i n g syndrome  11  such as Bloom's syndrome o r a t a x i a - t e l a n g i e c t a s i a  ( R o s i n and German,  1985;  R o s i n and Ochs, 1986). There are c o n s i d e r a b l e advantages to a p p l y i n g the m i c r o n u c l e u s  test  d i r e c t l y t o t i s s u e s o f i n d i v i d u a l s exposed to c a r c i n o g e n s . 1. ) The  f r e q u e n c y o f MNC  exfoliated cells  or i n c e l l  treatment w i t h pronase  can be r e a d i l y e s t i m a t e d i n smear p r e p a r a t i o n s  suspensions  o b t a i n e d from b i o p s i e s f o l l o w i n g  and/or c o l l a g e n a s e . Thus g e n o t o x i c damage can  actually  be measured i n t i s s u e s which are the t a r g e t s o f c a r c i n o g e n s . 2. ) E x f o l i a t e d c e l l s and b i o p s i e s can a l s o be used of  r e t i n o l , beta-carotene,  to e s t i m a t e the  or a number o f o t h e r chemopreventive agents.  level Thus  l o c a l i z e d d e f i c i e n c y i n p r o t e c t i v e agents, which c o u l d i n c r e a s e s e n s i t i v i t y towards the a c t i o n o f c a r c i n o g e n s , can be e s t i m a t e d i n t i s s u e s i n which carcinogen-induced  g e n o t o x i c damage can a l s o be  quantified.  3. ) S i n c e e x f o l i a t e d c e l l s can be sampled from s m a l l a r e a s , d i s t r i b u t i o n o f MNC to  w i t h i n a t i s s u e can be mapped. Thus i t s h o u l d be p o s s i b l e  l i n k f o c i w i t h h i g h f r e q u e n c i e s o f MNC  lesions,  carcinoma  4. ) The  in situ,  s c o r i n g o f MNC,  or carcinomas  to r e g i o n s i n which p r e n e o p l a s t i c preferentially  develop.  which i s c u r r e n t l y a l a b o u r - i n t e n s i v e and  consuming t a s k , lends i t s e l f to automation, s c r e e n e d w i t h i n minutes f o r the presence Since both  the  time-  so t h a t thousands o f c e l l s can be  of micronuclei.  the i n c i d e n c e o f p r e n e o p l a s t i c l e s i o n s and the i n c i d e n c e o f  m i c r o n u c l e a t e d c e l l s are e l e v a t e d among chewers o f tobacco and/or b e t e l q u i d , these two  f a c t o r s were used  to a s s e s s c a r c i n o g e n i c r i s k i n the p o p u l a t i o n s  s t u d i e d i n t h i s t h e s i s , and to determine chemopreventive  agents.  the p o p u l a t i o n s ' response  to  12  5.  Chemoprevention Having p e o p l e  o b v i o u s l y be  g i v e up chewing v a r i o u s t o b a c c o - c o n t a i n i n g mixtures  the most e f f e c t i v e way  T h i s i s not an easy  to p r e v e n t  the o r a l cancers  tobacco  produces.  t a s k , however. In I n d i a , the p r a c t i c e o f chewing b e t e l q u i d  has been around f o r many thousands o f y e a r s and culture.  this  would  i s deeply  i n t e g r a t e d i n t o Hindu  In a d d i t i o n , i n the c o u n t r i e s where these h a b i t s are p r a c t i s e d , i s o f t e n a major cash crop t h a t cannot simply be r e p l a c e d by  another  commodity. A l s o , the a d d i c t i o n to n i c o t i n e can o f t e n l e a d to the more dangerous h a b i t o f c i g a r e t t e smoking i f an i n d i v i d u a l i s encouraged to g i v e up  the  chewing h a b i t . Other means o f p r e v e n t i o n are c u r r e n t l y needed. C o n s i d e r a b l e a t t e n t i o n i s c u r r e n t l y b e i n g g i v e n to the use o f r e t i n o i d s and c a r o t e n o i d s as chemopreventive agents (Peto, 1983; There are s e v e r a l reasons  for this.  Peto e t a l . , 1981).  E p i d e m i o l o g i c a l evidence  p o i n t s to an  i n v e r s e r e l a t i o n s h i p between the i n t a k e o f b e t a - c a r o t e n e - c o n t a i n i n g green yellow vegetables 1981;  and  the i n c i d e n c e o f c a n c e r s a t v a r i o u s s i t e s  K v a l e e t a l . , 1983;  M a r s h a l l e t a l . , 1982;  e t a l . , 1986), i n c l u d i n g o r a l cancer beta-carotene  or  (Hirayama,  Z i e g l e r e t a l . , 1984;  Menkes  (Winn e t a l . , 1984). Animal s t u d i e s show  and v i t a m i n A to have a marked p r o t e c t i v e e f f e c t a g a i n s t a  v a r i e t y of carcinogens  (Santamaria  e t a l . , 1983;  v i t r o experiments have r e v e a l e d an antimutagenic a n t i t r a n s f o r m a t i o n (Som e f f e c t s of beta-carotene  e t a l . , 1984) are due  ( K r i n s k y and Deneke, 1982;  Mathews-Roth, 1982), and i n ( B e l i s a r i o e t a l . , 1985)  and  a c t i v i t y . Whether these p r o t e c t i v e  to i t s p o t e n t i a l f o r scavenging  radicals  Burton and I n g o l d , 1984), to i t s a b i l i t y  i n t e r f e r e w i t h the a c t i v a t i o n o f c a r c i n o g e n s ,  to  o r to i t s c o n v e r s i o n i n t o v i t a m i n  A, which i s the a c t u a l chemopreventive agent, i s a t p r e s e n t d i f f i c u l t  to  a s s e s s . S e v e r a l l a r g e - s c a l e i n t e r v e n t i o n t r i a l s have been i n i t i a t e d to prove or  13  d i s p r o v e the u s e f u l n e s s o f b e t a - c a r o t e n e  and v i t a m i n A i n p r e v e n t i n g  development o f carcinomas. However, c l i n i c a l are expensive, a l o n g time.  T e s t s t h a t c o u l d p r o v i d e more r a p i d r e s u l t s would be protocols before  are i n i t i a t e d .  precancerous  The  two  long-term,  "intermediate  l e s i o n s and m i c r o n u c l e a t e d  Preneoplastic lesions, al.,  as an  r e q u i r e a r e l a t i v e l y l a r g e number o f p a r t i c i p a n t s , and  a s s e s s i n g treatment trials  t r i a l s u s i n g cancer  the  last for  invaluable i n  manpower-intensive i n t e r v e n t i o n  endpoints"  cells,  endpoint  may  be  discussed  earlier,  the answer to t h i s need.  i n c l u d i n g d y s p l a s i a ( C a i , 1982), p o l y p s  (DeCosse e t  1975), l e u k o p l a k i a ( R y s s e l e t a l . , 1971), and e s o p h a g i t i s (Munoz e t a l . ,  1985) , have been s u c c e s s f u l l y a p p l i e d to t r a c k the response towards chemopreventive agents.  In our p i l o t  the e f f e c t o f b e t a - c a r o t e n e ,  t r i a l on b e t e l q u i d chewers, we  beta-carotene  p l u s v i t a m i n A,  on the r e m i s s i o n o f e s t a b l i s h e d o r a l l e u k o p l a k i a s and  examined  and v i t a m i n A  the development o f  alone new  ones. The  usefulness of micronucleated  t r i a l s has b een e x p l o r e d  c e l l s as markers i n chemoprevention  ( S t i c h e t a l . , 1984a,b, 1985) . M i c r o n u c l e a t e d  appear to be good i n d i c a t o r s o f c a r c i n o g e n - i n d u c e d complements ( S t i c h and R o s i n , c e l l s were observed  1985). I n c r e a s e d  cells  i n j u r i e s to chromosome  frequencies of  micronucleated  i n t i s s u e s a t e l e v a t e d r i s k o f d e v e l o p i n g cancer, i n c l u d i n g  the o r a l mucosa o f b e t e l q u i d chewers ( S t i c h e t a l . , 1982a), s n u f f d i p p e r s ( S t i c h e t a l . , 1985), u s e r s o f v a r i o u s 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 , K h a i n i tobacco  ( M i r v i s h , 1982)  smokers ( S t i c h and R o s i n ,  and  "nass"  1983a; S t i c h ,  ( Z a r i d z e e t a l . , 1985), and c i g a r e t t e  1987). Since m i c r o n u c l e i r e v e a l the  " p a t h o b i o l o g i c a l l y e f f e c t i v e dose," which i s more i n d i c a t i v e "exposure dose," they s h o u l d p r o v i d e chemopreventive  agents.  including  than a mere  i n f o r m a t i o n on the p r o t e c t i v e a c t i o n o f  6. Objectives The objectives of this thesis were f i v e f o l d : 1) To determine the snuff dipping and chewing patterns i n three communities (Inuit i n the Northwest T e r r i t o r i e s , native Canadian Indians i n northern Saskatchewan and East Indians i n Kerala, India). 2) To determine the amounts of nitrosamine precursors ( n i t r i t e ) and tobacco-specific nitrosamines i n samples of smokeless tobacco used by members of the abovementioned communities. 3) To determine the amounts of preformed and endogenously formed nitrosamines i n users of smokeless tobacco. 4) To determine the pattern of oral lesions (micronucleated leukoderma and precancerous  cells,  leukoplakia) i n the three population  groups, which d i f f e r i n their chewing patterns. 5) To explore the e f f e c t s of beta-carotene the frequency of precancerous  and vitamin A on reducing  o r a l lesions.  15  MATERIALS AND METHODS  1. USE OF SMOKELESS TOBACCO 1.1 Inuit in the Northwest Territories The community o f Gjoa Haven was percentage o f i n d i v i d u a l s who  chosen f o r study because o f the l a r g e  d i p s n u f f and because o f the low consumption o f  a l c o h o l r e s u l t i n g from l i q u o r p r o h i b i t i o n s i n c e  1978.  By the 1984/1985 census (Northwest T e r r i t o r i e s Data Book 1984  - 85)  this  community has a p o p u l a t i o n o f 523, o f which 95% are I n u i t . The sex d i s t r i b u t i o n i s 50% male and 50% female, w i t h an age d i s t r i b u t i o n o f : 0-4, 15-64, 48%; 65 and over, 2%. The major economic a c t i v i t i e s h u n t i n g and f i s h i n g ,  17%; 5-14,  33%;  are t r a p p i n g ,  the l a s t two a l s o b e i n g the major s o u r c e s o f food. The  community has two s m a l l s t o r e s which s u p p l y canned and packaged foods p l u s i n t e r m i t t e n t s u p p l i e s o f f r e s h f r u i t and v e g e t a b l e s . The s n u f f d i p p i n g h a b i t s o f i n d i v i d u a l s i n t h i s community were examined by door t o door q u e s t i o n n a i r e . No o r a l carcinomas were seen among the 180 c a n c e r s d i a g n o s e d between and 1974  i n the I n u i t o f the Canadian A r c t i c  (Schaefer et a l . ,  1949  1975).  1.2 Native Canadian Indians in Saskatchewan La Loche was  chosen as a study s i t e because o f i t s l a r g e number o f s n u f f  d i p p e r s and because o f a r e l a t i v e l y h i g h consumption o f a l c o h o l . By the 1983/1984 census c a r r i e d out by the l o c a l band o f f i c e of L a Loche has a p o p u l a t i o n o f 1847, 90% o f which are Chippewan,  the community Metis  I n d i a n s . La Loche has a sex d i s t r i b u t i o n o f 53% male and 47% female, and an age d i s t r i b u t i o n o f : 0-4,  15%; 5-14,  25%; 15-64, 57%; 65 and over, 3%. The major  16  economic a c t i v i t i e s  include trapping, hunting,  Loche has a m o d e r a t e - s i z e d g r o c e r y f r u i t and v e g e t a b l e s  frequency  store, plus smaller outlets providing fresh  and packaged and canned f o o d p r o d u c t s  government l i q u o r s t o r e i s a l s o The  f i s h i n g and some m i n i n g . L a  y e a r round; a s m a l l  present.  o f o r a l cancer  i s the same as t h a t found i n the r e s t o f the  Canadian p o p u l a t i o n t h a t do n o t use s n u f f . The  brands o f tobacco used by the Canadian n a t i v e s were checked by  r e q u e s t i n g to see the cans o f s n u f f o r bags o f chewing tobacco.  The weight o f  tobacco was determined u s i n g a s m a l l s c a l e and immediately weighing the s n u f f a f t e r i t had been "pinched"  1.3  by the i n d i v i d u a l b e i n g  interviewed.  East Indians in Kerala. India  Fishermen l i v i n g a l o n g a c o a s t a l s t r i p near Trivandrum, K e r a l a ,  India,  were chosen f o r study because o f the l a r g e numbers o f b e t e l q u i d chewers, the r e l a t i v e l y h i g h consumption o f a l c o h o l and the f r e q u e n t l y observed precancerous lesions  ( l e u k o p l a k i a ) and o r a l c a n c e r s .  the most p r e v a l e n t cancer.  In t h i s area,  I n males, cancers  o f tumors i n females.  mucosa. G e n e r a l l y ,  these  The g r e a t m a j o r i t y were cancers  the o r a l cancer  o f the mouth i s  o f the mouth, tongue and  oropharynx were r e s p o n s i b l e f o r 27.2% o f a l l c a n c e r s ; 14.8%  cancer  s i t e s accounted f o r o f the b u c c a l  i n c i d e n c e i s much h i g h e r where tobacco i s  added to the q u i d o r where chewing occurs  concurrently with  smoking (IARC,  1985). No seeds, s p i c e s , sugar or f l a v o u r i n g s , which a r e i n g r e d i e n t s used i n b e t e l q u i d o f n o r t h e r n fishermen.  commonly  I n d i a , were added to the quids used by the  Weights o f q u i d i n g r e d i e n t s were o b t a i n e d by a s k i n g  i n d i v i d u a l s to  g i v e a sample o f t h e i r u s u a l q u i d and l a t e r weighing each p o r t i o n on an analytical  balance.  17  Demographic d a t a was u n a v a i l a b l e f o r . t h e f i s h e r m e n o f K e r a l a a t the time of w r i t i n g t h i s  thesis.  2. Nitrite in Smokeless Tobacco Samples 2.1 Snuff and Chewing Tobacco The  f o l l o w i n g smokeless  d i f f e r e n t commercial  brands  tobacco samples were used i n t h i s study: 5 o f s n u f f (packed i n Canada),  and K (4 samples each); one "chewing" tobacco  l a b e l l e d C, B, SL, SW  (U.S. b r a n d ) , l a b e l l e d R;  homemade "nass" from Samarkand, the S o v i e t Union  (4 samples);  and K h a i n i  tobacco from Bengal, I n d i a (4 samples). The s n u f f , chewing tobacco and K h a i n i tobacco c o n t a i n e r s o r boxes were f r e s h l y opened p r i o r t o c h e m i c a l a n a l y s i s and p r i o r t o b e i n g used by the v o l u n t e e r s . D u r i n g i n t e r v i e w s we took p a r t i c u l a r c a r e t o d i s t i n g u i s h s n u f f (commercially a v a i l a b l e , chewing tobacco  f i n e l y ground  tobacco)  from  ( c o m m e r c i a l l y a v a i l a b l e s t r a n d s o f t o b a c c o ) . We checked the  brands by r e q u e s t i n g t o see the cans o f s n u f f o r bags o f chewing tobacco  used  by the v o l u n t e e r s . The weight o f tobacco was determined w i t h a s m a l l s c a l e . The s n u f f was weighed immediately a f t e r i t had been " p i n c h e d " by the i n d i v i d u a l being interviewed.  2.2 Betel Quid The  E a s t I n d i a n f i s h e r m e n surveyed i n t h i s study a l l chewed a simple b e t e l  q u i d c o n s i s t i n g o f a r e c a n u t (Areca c a t e c h u L . ) , tobacco stems  (Nicotiana  tabacum L . ) , s l a k e d l i m e ( c a l c i u m h y d r o x i d e ) from marine s h e l l s ,  and b e t e l  ( P i p e r b e t l e L . ) . Only the tobacco p o r t i o n o f the q u i d was a n a l y z e d f o r nitrite.  leaf  18  2.3 Determination of Nitrite Water e x t r a c t s o f tobacco were p r e p a r e d by suspending  1 g o f tobacco i n 10  ml o f d i s t i l l e d water and s h a k i n g v i g o r o u s l y i n a 37°C water b a t h f o r 30 T h i s was  f o l l o w e d by c e n t r i f u g a t i o n  (2000 rpm  f o r 2 min)  min.  to remove tobacco  particles. The n i t r i t e  c o n c e n t r a t i o n o f b o t h s a l i v a and water e x t r a c t s o f tobacco  determined by the same procedure. C o l o u r reagent was of  sulfanilic  a c i d and 0.1  100 ml o f 20% a c e t i c a c i d ;  was  p r e p a r e d by d i s s o l v i n g 1 g  g of N-l-naphthylethylenediamine dihydrochloride i n 25 nl  o f e i t h e r s a l i v a or water e x t r a c t o f tobacco  were added to 2 ml o f the c o l o u r reagent i n a Nalgene t e s t tube and a l l o w e d to r e a c t f o r 15 min.  Absorbance  was  r e a d a t 540 nm  on a Bausch and Lomb m i n i  S p e c t r o n i c 20 a g a i n s t a b l a n k o f u n r e a c t e d c o l o u r r e a g e n t . C o n c e n t r a t i o n s o f n i t r i t e were determined was  from a s t a n d a r d c u r v e . A known amount o f sodium  c a r e f u l l y weighed out on an a n a l y t i c a l b a l a n c e and d i s s o l v e d i n a p r e c i s e  volume o f d i s t i l l e d water. was  nitrite  S e r i a l d i l u t i o n s were made and the s t a n d a r d curve  p r e p a r e d by the procedure d e s c r i b e d above. The  average was  recovery of n i t r i t e  95%.  added t o s a l i v a a t l e v e l s r a n g i n g from  to  30 ppm  Large v a r i a t i o n s were seen i n the n i t r i t e  of  56 E a s t I n d i a n s a l i v a samples gave a mean o f 36.3  ppm  samples:  10  analysis  with a standard  d e v i a t i o n o f 35.06 and a c o e f f i c i e n t o f v a r i a t i o n o f 96.7%. These v a r i a t i o n s c o u l d have been due  to the v a r y i n g d i e t a r y i n t a k e o f n i t r a t e and to the o r a l  hygiene o f the i n d i v i d u a l s . S n u f f samples purchased  i n different locations  showed l a r g e v a r i a t i o n s  in nitrite  s n u f f samples purchased  i n d i f f e r e n t areas o f Canada (NWT,  Vancouver) had a mean n i t r i t e d e v i a t i o n o f 235  also  c o n c e n t r a t i o n . For example, 4 Copenhagen  c o n c e n t r a t i o n o f 735 ppm  and a c o e f f i c i e n t o f v a r i a t i o n o f 32%.  N. Saskatchewan and  with a standard These v a r i a t i o n s were  19  most p r o b a b l y due t o d i f f e r e n t s t o r a g e times. However, determinations o f n i t r i t e  10 i n d i v i d u a l  from the same sample o f tobacco  gave a mean  nitrite  c o n c e n t r a t i o n o f 434 ppm, a s t a n d a r d d e v i a t i o n o f 71.4 and a c o e f f i c i e n t o f variation  o f 16%. A l l n i t r i t e  for n i t r i t e  d e t e r m i n a t i o n s were done by m y s e l f . The procedure  d e t e r m i n a t i o n i s o u t l i n e d i n F i g u r e 3.  20  F i g u r e 3: Procedure  25  f o r N i t r i t e Determination  u l water e x t r a c t o f tobacco o r s a l i v a  i n t o 2 ml c o l o u r reagent  (1 gm s u l f a n i l i c a c i d + 0.1 gm N - l - n a p h t h y l e t h y l e n e diamine d i h y d r o c h l o r i d e i n 100 ml 20% a c e t i c a c i d )  determine absorbance a t 540 nm (mini S p e c t r o n i c 20) a g a i n s t u n r e a c t e d c o l o u r reagent as blank  •  determine  n i t r i t e c o n c e n t r a t i o n from standard curve  21  3. Nitrite in the Saliva of Users of Smokeless Tobacco 3.1  Snuff Dippers S a l i v a was c o l l e c t e d f o r n i t r i t e d e t e r m i n a t i o n i n the Canadian p o p u l a t i o n s  (64 I n u i t and 20 I n d i a n ) by h a v i n g i n d i v i d u a l s  e x p e c t o r a t e 2-4 ml o f s a l i v a  i n t o Nalgene t e s t tubes. The samples were a n a l y z e d immediately  after.  Saliva  samples were n o t c o l l e c t e d d u r i n g a chewing p e r i o d . For n i t r i t e  d e t e r m i n a t i o n d u r i n g chewing, seven s n u f f d i p p i n g I n u i t males  i n Gjoa Haven, r a n g i n g from 14 to 20 y e a r s o f age, donated  3.2  their saliva.  Betel Quid Chewers S a l i v a samples were c o l l e c t e d by the same procedure  as t h a t d e s c r i b e d  above from 50 d i f f e r e n t b e t e l q u i d chewers i n K e r a l a , b u t n o t d u r i n g a chewing period.  4. Nitrosation Capacity of Saliva Samples For e s t i m a t i n g the n i t r o s a t i o n c a p a c i t y , s a l i v a was c o l l e c t e d from one v o l u n t e e r u s i n g 1 g o f common North American brands o f s n u f f (Copenhagen, Skoal or B i g Horn).  D u r i n g a 20-min p e r i o d o f k e e p i n g t h e tobacco  g i n g i v a l groove,  i n t h e lower  t h e v o l u n t e e r e x p e c t o r a t e d the s a l i v a i n t o a Nalgene t e s t  tube. The  f o r m a t i o n o f NPRO was q u a n t i f i e d by mixing,  the s n u f f u s e r w i t h p r o l i n e  i nvitro,  the s a l i v a o f  (200 mg), and i n c u b a t i n g the mixture  f o r 30 min a t  37°C.  5. N-Nitrosoproline in the Urine of Snuff Dippers B e f o r e , d u r i n g and a f t e r the use o f s n u f f , u r i n e was c o l l e c t e d i n b o t t l e s c o n t a i n i n g 300 mg o f s o l i d NaOH. The u r i n e was s t o r e d a t -20°C u n t i l  22  nitrosoproline  (NPRO) a n a l y s i s , which took p l a c e w i t h i n one day o f sampling.  Where u r i n e samples were c o l l e c t e d from  i n d i v i d u a l s i n a p o p u l a t i o n , the same  method o f s t o r a g e and a n a l y s i s was used. In the I n u i t s e t t l e m e n t o f Gjoa Haven, u r i n e samples were c o l l e c t e d  from  14 r e g u l a r s n u f f u s e r s and 15 non-users who were matched by sex and age. I n a d d i t i o n , u r i n e samples from Indians i n the community o f L a Loche were c o l l e c t e d from 47 s n u f f u s e r s and 27 non-users o f comparable age and sex.  5.1 Determination of Nitrosoproline Nitrosoproline  (NPRO) and N - n i t r o s o p i p e r i d i n e - 2 - c a r b o x y l i c a c i d  were s y n t h e s i z e d and p u r i f i e d u s i n g p u b l i s h e d procedures  (NPIC)  (Lijinsky et a l . ,  1970). E x t r a c t i o n columns, u n b u f f e r e d , s t y l e TE 3020, were o b t a i n e d from F i s h e r S c i e n t i f i c . A l l o t h e r c h e m i c a l s were reagent o r e q u i v a l e n t grade. Samples o f u r i n e were p r e s e r v e d by the a d d i t i o n o f 300 mg s o l i d NaOH p e r 100 ml, and were s t o r e d f r o z e n u n t i l a n a l y s i s . Samples were a n a l y z e d by the procedure  o f Sen e t a l . (1983) w i t h minor m o d i f i c a t i o n s . Samples o f u r i n e  (15 ml) were a c i d i f i e d w i t h 3 ml 20% ammonium s u l f a m a t e  i n 1.8 M ^ S O ^ , and 10  /L*1 i n t e r n a l s t a n d a r d s o l u t i o n i n methanol c o n t a i n i n g 150 ng NPIC was added. U r i n e samples were l o a d e d onto 20-ml c a p a c i t y d i s p o s a b l e e x t r a c t i o n columns (Extube, A n a l y t i c h e m  I n t e r n a t i o n a l ) , and a l l o w e d t o absorb.  Columns were then  e l u t e d w i t h 4 x 20 ml e t h y l a c e t a t e , which was d r i e d over 20 g anhydrous NaSO^, p r i o r t o use. The o r g a n i c e x t r a c t s were decanted w i t h washings through wool p l u g i n a f u n n e l i n t o round-bottomed f l a s k s , u s i n g a r o t a r y e v a p o r a t o r . Residues  a cotton  and e v a p o r a t e d u n t i l d r y  i n the f l a s k s were d i s s o l v e d i n a t o t a l o f  4 ml methanol, and t r a n s f e r r e d t o 5-ml c o n i c a l - b o t t o m e d sample v i a l s . The methanol was e v a p o r a t e d  t o a volume o f a p p r o x i m a t e l y  0.1 ml u s i n g a  h e a t i n g b l o c k a t 50°C and a stream o f n i t r o g e n ; 0.5 ml BF3 reagent  23  (14% methanol) was added, and the samples were capped and i n c u b a t e d f o r e i t h e r 4 h r a t 50°C o r o v e r n i g h t a t room temperature. Dichloromethane (0.5 ml) and 2 ml water were added, and the samples were shaken v i g o r o u s l y t o e x t r a c t the e s t e r i f i e d n i t r o s a m i n e s i n t o the c h l o r i n a t e d s o l v e n t . C a l i b r a t i o n samples were p r e p a r e d by e s t e r i f y i n g 150 ng NPIC o r NPRO i n 0.5 ml BF^ methanol i n a s i m i l a r manner. Gas-chromatograhic (GC) a n a l y s i s o f 10-^1 a l i q u o t s o f the dichloromethane s o l u t i o n was performed on a P e r k i n Elmer 3920 gas chromatograph, equipped w i t h a n i c k e l column Chromosorb  (2 m x 2 mm i . d . ) packed w i t h 10% Carbowax 20 M on 80-100 mesh  W. Chromatographic c o n d i t i o n s were: i n j e c t o r 250°C, column  190°C,  c a r r i e r h e l i u m a t 50 ml/min. Under these c o n d i t i o n s , NPIC and NPRO r e t e n t i o n times o f 3.5 and 4.2 min, r e s p e c t i v e l y , were a c h i e v e d and were n e a r l y b a s e l i n e resolved. N i t r o s a m i n e s were d e t e c t e d u s i n g a thermal energy a n a l y z e r  (TEA) (Model  502A; Thermal E l e c t r o n Corp., Waltham, MA) o p e r a t i n g a t a chamber p r e s s u r e o f 0.55 t o r r ,  and u s i n g a t r a p c o o l e d w i t h d r y i c e . NPRO was q u a n t i f i e d u s i n g  d a i l y c a l i b r a t i o n samples and an i n t e r n a l s t a n d a r d program on a S p e c t r a P h y s i c s SP4100 computing i n t e g r a t o r  ( S p e c t r a P h y s i c s Corp., Santa C l a r a , CA). P r a c t i c a l  d e t e c t i o n l i m i t s were i n the o r d e r o f 0.1 ng NPRO p e r i n j e c t i o n ,  corresponding  to a p p r o x i m a t e l y 0.5 ng NPRO/ml u r i n e . R e c o v e r i e s from samples s p i k e d w i t h s t a n d a r d NPRO were g e n e r a l l y i n the 95-100% range. A n a l y s i s o f 9 s e p a r a t e samples t a k e n over a 17-hr p e r i o d from t h e same i n d i v i d u a l produced a mean NPRO v a l u e o f 16.09 ng/ml, w i t h a s t a n d a r d d e v i a t i o n o f 2.31 and a c o e f f i c i e n t o f v a r i a t i o n o f 14.4%.  24  In o r d e r  t o determine the r e p r o d u c i b i l i t y and the r a t e o f e l i m i n a t i o n o f  NPRO from the human body i n i n v i v o experiments, u r i n e samples were f o r NPRO a f t e r i n g e s t i o n o f v a r i a b l e amounts o f n i t r a t e . 65,  In f i v e  130, 195, 260 and 325 mg n i t r a t e were i n g e s t e d t o g e t h e r  p r o l i n e . I n each case,  with  experiments, 500 mg  a s i m i l a r p a t t e r n o f NPRO e x c r e t i o n was o b t a i n e d , and  the amount o f NPRO r e t u r n e d  to the background l e v e l w i t h i n 24 h r , i n d i c a t i n g  t h a t NPRO was t o t a l l y e l i m i n a t e d i n t o the u r i n e w i t h i n t h a t p e r i o d . the NPRO a n a l y z e d  analyzed  Therefore  i n u r i n e c o l l e c t e d over 24 h r a f t e r d o s i n g was an i n d i c a t o r  o f d a i l y n i t r o s a t i o n i n v i v o . N i t r o s a m i n e s i n s a l i v a and e x t r a c t s were determined i n a manner s i m i l a r to t h a t s t a t e d above by s c a l i n g down e x t r a c t i o n volumes. The p r o c e d u r e f o r NPRO d e t e r m i n a t i o n  \  i s o u t l i n e d i n F i g u r e 4.  F i g u r e 4: Procedure f o r N - n i t r o s o p r o l i n e  (NPRO) D e t e r m i n a t i o n  Urine preserved with 300 mg s o i i d NaOH/100 ml 15 ml urine Acidify with 3 ml 20% ammonium sulfamate i n 1.8 M F^SO^ add 10 u l (150 ng NPIC) internal standard i n MeOH •  Load onto 20-ml capacity extraction columns elute 4 x 20 ml ethyl acetate (dried over 20 gm anhydrous NaSO.) 4 Collect organic extracts through cottonwoo 1 plug into round-bottomed flask  Evaporate to dryness with rotary evaporator  Redissolve residue in 4 ml MeOH and transfer to 5-ml conicalbottomed v i a l •  Evaporate to 0.1 ml using heating block at 50°C-under N„ Add 0.5 ml BF 3 (14% MeOH) •  Cap and incubate for 4 hr at 50°C or overnight at room temperature  1  Add 0.5 ml dichloromethane and 2 ml water, and shake vigorously Gas chromatographic/ Thermal energy analyzer analysis  26  6. Tobacco-Specific Nitrosamines in Different Brands of Tobacco Two  brands  o f s n u f f (Copenhagen and Skoal) commonly chewed by  Canadian  n a t i v e s were a n a l y z e d f o r the presence o f TSNA. Water e x t r a c t s were p r e p a r e d by suspending 1 g o f tobacco i n 10 ml o f d i s t i l l e d water and s h a k i n g v i g o r o u s l y i n a 37°C water b a t h f o r 30 min.  0.5  ml o f the e x t r a c t was  used f o r TSNA a n a l y s i s .  A f t e r adding i n t e r n a l standards (N-nitrosodimethylaminen i t r o s o n o r n i c o t i n e - ' ' " ^ C) , each sample was  C and  N'-  e x t r a c t e d f o u r times w i t h 20 ml  ethyl  a c e t a t e , u s i n g a prewetted p r e t u b e . The o r g a n i c f r a c t i o n s were p o o l e d and c o n c e n t r a t e d t o a p p r o x i m a t e l y 3 ml, and then s u b j e c t e d t o column chromatography on 65 g b a s i c alumina. A f t e r e l u t i o n w i t h 200 ml f r e s h l y dichloromethane  distilled  ( f r a c t i o n I, c o n t a i n i n g v o l a t i l e n i t r o s a m i n e ) , the samples were  chromatographed w i t h 200 ml dichloromethane-acetone  (4:1)  c o n t a i n i n g the TSNA). A f t e r b e i n g c o n c e n t r a t e d t o 1 ml,  (fraction II,  the samples were  a n a l y z e d by GC-TEA. For the v o l a t i l e n i t r o s a m i n e s , a 12 f t x 1/4  i n (2 mm  i.d.)  g l a s s column packed w i t h 10% Carbowax 20 M on Chromosorb WNAW (100-120 mesh) a t an oven temperature  o f 175°C was  used. For the TSNA, a 12 f t x 1/4  i . d . ) g l a s s column packed w i t h 10% UCW-98 on Gas oven temperature f r a c t i o n s was  o f 200°C was  (Adams e t a l . ,  mm  Chrom Q (80-100 mesh) a t an 1983).  used f o r s c i n t i l l a t i o n c o u n t i n g to determine  S e v e r a l b l a n k samples, was  used  i n (2  An a l i q u o t o f b o t h the r e c o v e r y r a t e .  u s i n g 5 ml water, were a l s o assayed to ensure t h a t t h e r e  no c o n t a m i n a t i o n d u r i n g a n a l y s i s ,  s i n c e the l a r g e s t sources o f e r r o r i n a l l  n i t r o s a m i n e a n a l y s e s r e s u l t from c o n t a m i n a t i o n by n i t r o s a m i n e s o r p r e c u r s o r s from o t h e r s o u r c e s . D e t e c t i o n l i m i t s f o r TSNA are 2.4 o f DMNM are those below 0.9%.  ppb  and a c c e p t a b l e l e v e l s  R e c o v e r i e s from s a l i v a s p i k e d w i t h TSNA were  g e n e r a l l y 80-90%. T a b l e I g i v e s the means and p r e c i s i o n o f t h r e e a n a l y s e s .  27  The f o l l o w i n g f o u r TSNA were e s t i m a t e d . i n the s a l i v a o f the s n u f f N'-nitrosonornicotine  dippers:  (NNN), N - n i t r o s o a n a t a b i n e (NAT), N - n i t r o s o a n a b a s i n e  (NAB), and 4 - ( m e t h y l - N - n i t r o s a m i n o ) - 1 - ( 3 - p y r i d y l ) - 1 - b u t a n o n e  (NNK).  T a b l e I A n a l y s i s o f t h r e e o f the same tobacco samples f o r TSNA  Sample  Compound  Mean (/ig/g) and P r e c i s i o n  Tobacco  N - N i t r o s o n o r n i c o t i n e (NNN)  45.6 ± 7%  4-(Methyl-N-nitrosamino)-1(3-pyridyl)-1-butanone N-Nitrosoanatabine  (NNK)  (NAT)  N - N i t r o s o a n a b a s i n e (NAB)  7.4 ± 8% 47.7 ± 8% 1.8 ± 12%  (%)  29  7. Tobacco-Specific Nitrosamines in the Saliva of Snuff Dippers S u b j e c t s from whom s a l i v a samples were c o l l e c t e d were asked t o take  their  u s u a l q u a n t i t y o f s n u f f and t o e x p e c t o r a t e i n t o a tube a t i n t e r v a l s when they would n o r m a l l y s p i t . C o l l e c t i o n o f s a l i v a took p l a c e over a p e r i o d o f 15 minutes ( b e f o r e , d u r i n g and a f t e r the chewing p e r i o d ) . One ml o f c i t r a t e phosphate b u f f e r  (pH 4.5) c o n t a i n i n g 3.25 mg NaNj ( n i t r o s a t i o n i n h i b i t o r ) and  1.00 mg c i s - 2 , 6 - d i m e t h y l m o r p h o l i n e  ( s e r v i n g as monitor amine) was added to a l l  s a l i v a samples, which were then f r o z e n . The amount o f s a l i v a c o l l e c t e d v a r i e d between 3.0 and 6.5 ml. A l l p a r t i c i p a n t s i n t h i s study used t h e same brand o f s n u f f (made i n t h e U.S.A. f o r N a t i o n a l Tobacco Co., L t d . , M o n t r e a l , Canada) and none were smokers. A f t e r t h e s a l i v a samples had been a l l o w e d t o thaw, 0.5 ml was removed f o r the radioimmunoassay o f n i c o t i n e and c o t i n i n e . The remainder was used f o r the a n a l y s i s o f TSNA and n i t r o s o - c i s - 2 , 6 - d i m e t h y l m o r p h o l i n e  (DMNM) by the method  d e s c r i b e d above.  8. Determination of Nicotine and Cotinine N i c o t i n e and c o t i n i n e were determined from 0.5-ml thawed s a l i v a samples by radioimmunoassay a c c o r d i n g t o the method o f Langone e t a l .  (1973) w i t h  specific  a n t i s e r a produced by i n j e c t i o n i n t o r a b b i t s o f t r a n s - 3 - s u c c i n y l m e t h y l n i c o t i n e and t r a n s - 4 - c a r b o x v c o t i n i n e bound t o albumin. R e c o v e r i e s o f 98-104% r e l a t i v e t o the i n t e r n a l s t a n d a r d were o b t a i n e d on s t a n d a r d samples. By s u i t a b l e d i l u t i o n o f a s t a n d a r d s o l u t i o n the minimum d e t e c t a b l e l e v e l was found t o be 5 ng, i . e . , 300 n m o l e / l i t r e .  equivalent to a concentration o f  30  R e p l i c a t e a n a l y s e s o f a smoker's u r i n e showed a c o e f f i c i e n t o f v a r i a t i o n o f 7.3%  f o r n i c o t i n e and 9.0%  f o r c o t i n i n e . The  c a l c u l a t e d n i c o t i n e : c o t i n i n e r a t i o s was  c o e f f i c i e n t o f v a r i a t i o n o f the  11.1%.  9. Frequency of Micronuclei in the Oral Mucosa of Snuff Dippers and Tobacco Chewers E x f o l i a t e d c e l l s o f the o r a l mucosa were o b t a i n e d by swabbing w i t h a moistened slide,  wooden tongue d e p r e s s o r ,  a i r - d r y i n g , and  fixing  smearing  the c e l l s onto a c l e a n  microscope  i n 80-85% e t h a n o l . I f e x f o l i a t e d c e l l s  from  s m a l l e r areas of the g i n g i v a l groove were d e s i r e d , s p l i t wooden p o p s i c l e s t i c k s were used.  The  f i x e d c e l l p r e p a r a t i o n s were s t a i n e d w i t h Feulgen  reaction  f o l l o w i n g a 10 min h y d r o l y s i s w i t h N-HC1, and c o u n t e r s t a i n e d w i t h f a s t The  F e u l g e n - s t a i n e d s l i d e s were s c r e e n e d f o r v a r i o u s n u c l e a r  green.  anomalies,  i n c l u d i n g m i c r o n u c l e i , whose s i z e can v a r y from s m a l l F e u l g e n - p o s i t i v e b o d i e s to l a r g e r , n u c l e u s - l i k e s t r u c t u r e s w i t h membranes and c l e a r l y chromatin. (Stich,  visible  The p r o s and cons o f t h i s technique have been r e c e n t l y  reviewed  1987). M i c r o n u c l e i were s c o r e d o n l y i n i n t a c t e p i t h e l i a l c e l l s .  We  f o l l o w e d w e l l - e s t a b l i s h e d c r i t e r i a f o r e s t i m a t i n g the frequency of human e x f o l i a t e d c e l l s w i t h m i c r o n u c l e i ( S t i c h and R o s i n , 1983b, 1984a). usefulness of t h i s technique agents  i n f o l l o w i n g the response  i s d i s c u s s e d by S t i c h and R o s i n  m i c r o n u c l e a t e d c e l l s was r i s k of developing cancer determined  The  to chemopreventive  (1985). An e l e v a t e d f r e q u e n c y  of  found to be a simple marker i n d i c a t i n g an e l e v a t e d (Table I I ) . The m i c r o n u c l e a t e d c e l l f r e q u e n c i e s were  i n t h i s study by Mrs.  H.F.  S t i c h , an e x p e r t i n t h i s  field.  A n u c l e a t e d e x f o l i a t e d c e l l s are common i n the p a l a t e , but r a r e i n the b u c c a l mucosa o f non-chewers o f tobacco and non-smokers. At l e a s t 300 c e l l s sample were counted  to e s t i m a t e the percentage  s t a i n e d smear p r e p a r a t i o n s were used.  of anucleated c e l l s .  Feulgen-  per  31  TABLE II Frequency o f M i c r o n u c l e i i n Human T i s s u e s a t E l e v a t e d Risk  Tissue  Suspected Carcinogenic Factors  Elevation of Micronucleated C e l l s (Fold)  f o r Cancer  Reference  O r a l mucosa: G i n g i v a l groove Inner l i p F l o o r o f mouth B u c c a l mucosa  Esophagus  Snuff K h a i n i tobacco Nass Betel quid Smoking and d r i n k i n g  4.2 4.4 8.2 9.4 4.6  Smoking Curry  3.4 1.5  Unknown ( e s o p h a g i t i s )  3.4  Smoking  a  S t i c h e t a l . (1985) S t i c h e t a l . (1982b) S t i c h (1987) S t i c h e t a l . (1982a) S t i c h and Rosin (1983a) Fontham e t a l . (1986) P i c k e r and Fox (1986) S t i c h and Z a r i d z e (unpublished data) Mandard e t a l . (1987)  Urinary bladder  Schistosoma haematobium 13.5 Smoking 6.0 Smoking and c o f f e e d r i n k i n g 9.4 Smoking Shift to higher values  Raafat e t a l . (1984) Fontham e t a l . (1986) S t i c h (1987) R e a l i e t a l . (1987)  Bronchial epithelium  Smoking Smoking  2.2  Fontham e t a l . (1986) Stich (unpublished data)  Cervical epithelium  Smoking Unknown  5.1 3.0  Fontham e t a l . (1986) S t i c h (1987)  Blood lymphocytes  Smoking Styrene Styrene Styrene  2.6  (dysplasia)  X-ray c o n t r a s t media  1.6-4.3  Hflgstedt e t a l . (1983a) Hogstedt e t a l . (1983b) Meretoja e t a l . (1978) Nordenson and Beckman (1984) Parvez e t a l . (1987)  Bone marrow  Antileukemia  >34.0  Abe e t a l . (1984)  Spermatids  Smoking  1.5 9.0 4.0  agents  1.4  Lahdetie  E l e v a t e d f r e q u e n c i e s were observed i n o n l y 5% o f p a t i e n t s examined.  (1986)  32  10. Precancerous Lesions and Cancer of the Oral Cavity of Users of Smokeless Tobacco  10.1 Leukoderma O r a l l e s i o n s i n users according  o f s n u f f and chewing tobacco have been c l a s s i f i e d ,  t o t h e i r appearance, i n t o f o u r groups by A x e l l e t a l . (1976) and i n t o  t h r e e c a t e g o r i e s by Greer and P o u l s o n (1983). s u b d i v i d i n g these l e s i o n s , we r e c o r d e d  To a v o i d any s u b j e c t i v e b i a s i n  o n l y two types o f anomalies: (1) w h i t i s h  to y e l l o w i s h w r i n k l e d patches o f the mucosa w i t h (corresponding  o r w i t h o u t furrows  t o c a t e g o r i e s 2 and 3 o f Greer and Poulson  [1983]),  which a r e  the most commonly observed anomalies, and (2) d e f i n i t e l e u k o p l a k i a s , as d e s c r i b e d by t h e World H e a l t h O r g a n i z a t i o n  (1980). There i s a c e r t a i n ambiguity  i n p r o p e r l y d e f i n i n g the many w h i t i s h l e s i o n s o f the o r a l mucosa. The w h i t i s h wrinkled  areas which we observed a t the s i t e s where the s n u f f o r chewing  tobacco came i n c l o s e c o n t a c t w i t h preleukoplakias,  leukoplakias  chewers (IARC, 1985) o r u s e r s and  the mucosa a r e n o t comparable t o the  o r e r y t h r o p l a k i a s so commonly seen i n b e t e l q u i d o f "nass"  (a mixture o f tobacco, s l a k e d lime, o i l  ash) ( Z a r i d z e e t a l . , 1986). The w h i t i s h areas can d i s a p p e a r  weeks, whereas r e m i s s i o n o f o t h e r mucosal a b n o r m a l i t i e s f o l l o w i n g the c e s s a t i o n o f s n u f f o r b e t e l q u i d use. l e u k o p l a k i a s , p a r t i c u l a r l y those w i t h r e d d i s h areas, whereas t h e r e dippers  i s no evidence t o c o n s i d e r  a p r e n e o p l a s t i c stage.  were r e c o r d e d  w i t h i n days or  r e q u i r e s s e v e r a l months  The p r e c a n c e r o u s s t a t e o f appears w e l l - e s t a b l i s h e d ,  the w h i t i s h w r i n k l e d patches o f s n u f f  The presence o r absence o f these l a t t e r l e s i o n s  f o r a l l the Canadian I n u i t and I n d i a n s  surveyed.  33  Leukoplakia  10.2  A p r e c a n c e r o u s l e s i o n i s d e f i n e d as a m o r p h o l o g i c a l l y which cancer i s more l i k e l y  to o c c u r than i n i t s a p p a r e n t l y  This condition i s , therefore, significantly  increased  Precancerous L e s i o n s , l e s i o n s are  l e u k o p l a k i a and  include sideropenic The  a generalized  r i s k o f cancer (WHO  1978;  altered tissue i n normal  counterpart.  state associated with a C o l l a b o r a t i n g Centre f o r O r a l  A x e l l e t a l . , 1984). Examples o f o r a l precancerous e r y t h r o p l a k i a ; o r a l precancerous l e s i o n s also  dysphagia, submucous f i b r o s i s ,  and  p o s s i b l y l i c h e n planus.  concept o f l e u k o p l a k i a as a precancerous l e s i o n i s based on  findings  t h a t a s i g n i f i c a n t number o f o r a l carcinomas are a s s o c i a t e d w i t h p r e - e x i s t i n g leukoplakia,  and  transformation  t h a t some l e u k o p l a k i a s  (Pindborg  e t a l . , 1975;  appear to undergo malignant S i l v e r m a n e t a l . , 1984). However,  s t u d i e s o f o r a l l e u k o p l a k i a are o f t e n d i f f i c u l t uniformity  i n the h i s t o l o g i c a l d e f i n i t i o n o f  to compare because o f a l a c k of  leukoplakia.  I n t h i s t h e s i s l e u k o p l a k i a are c l a s s i f i e d a c c o r d i n g C o l l a b o r a t i n g C e n t r e f o r O r a l Precancerous L e s i o n s III.  to the  WHO  (1978), as o u t l i n e d i n Table  Classification  o f O r a l Precancerous  Lesions  (WHO C o l l a b o r a t i n g Centre  f o r O r a l Precancerous  Lesions,  1978)  Predisposition to Cancer  Condition  Description of Lesion  Histopathology  Leukoplakia  White, w h i t i s h / y e l l o w , o r grey patch a f f e c t i n g v e r y s m a l l t o l a r g e areas o f the mucosa; smooth o r w r i n k l e d s u r f a c e often with cracks; may p r e s e n t as homogeneous, nodular o r s p e c k l e d  Hyperorthokeratosis ; hyperparakeratosis; atrophy; d i f f u s e c h r o n i c inflammation i n lamina p r o p r i a (lymphocyte and plasma c e l l s ) ; some d y s p l a s i a i n nodular type  Erythroplakia  B r i g h t r e d , v e l v e t y plaques; irregular, well-defined outline; o c c a s i o n a l nodular s u r f a c e w i t h white o r y e l l o w spots  Marked e p i t h e l i a l atrophy; variable epithelial dysplasia; heavy c h r o n i c inflammation i n s u b e p i t h e l i a l connective t i s s u e (mainly plasma c e l l s )  Leukoderma  Whitish to yellowish wrinkled patches; w i t h o r w i t h o u t furrows  Epithelium hyperplastic; None known m i c r o b i a l c o l o n i z a t i o n of irregular surface; s u p e r f i c i a l e p i t h e l i a l c e l l s l a r g e and angular, p y k n o t i c n u c l e i , cytoplasm l i g h t l y s t a i n e d o r empty  6-10% become malignant  G r e a t e r than 10% become malignant  35 11. Intervention Strategies  11.1 Nitrite Trapping Agents The  i n h i b i t o r y e f f e c t o f a s c o r b a t e and c a f f e i c a c i d on n i t r o s a m i n e  f o r m a t i o n was s t u d i e d u s i n g NPRO as a model i n b o t h i n v i t r o and i n v i v o ( a s c o r b a t e o n l y ) systems. The  i n v i t r o method was done by h a v i n g a v o l u n t e e r (myself) chew 1 g o f  t h r e e d i f f e r e n t brands o f s n u f f and e x p e c t o r a t e the s a l i v a  (10 ml) i n t o a  Nalgene t e s t tube. To the s a l i v a 200 mg p r o l i n e was added e i t h e r  alone  ( c o n t r o l ) o r w i t h e i t h e r 200 mg a s c o r b a t e o r 200 mg c a f f e i c a c i d . The s o l u t i o n s were then taken t o pH 2.5 u s i n g s m a l l amounts o f normal H C l and a l l o w e d to i n c u b a t e a t 37°C f o r 30 min.  NPRO a n a l y s i s was then made by the procedure  d e s c r i b e d above. For the i n v i v o d e t e r m i n a t i o n o f endogenously formed NPRO a v o l u n t e e r (myself) chewed 1 g o f s n u f f (2 times a t 60 min i n t e r v a l s ) p l u s i n g e s t e d s i m u l t a n e o u s l y 200 mg o f p r o l i n e  ( c o n t r o l ) . T h i s experiment  was r e p e a t e d w i t h  i n g e s t i o n o f 500 mg o f chewable a s c o r b i c a c i d 30 min p r i o r t o chewing the s n u f f and  i n g e s t i n g p r o l i n e and 30 min f o l l o w i n g s n u f f chewing and p r o l i n e  The  same experiment  was r e p e a t e d w i t h a l a r g e r i n g e s t i o n o f a s c o r b a t e a t 30 min  p r i o r t o t h e s n u f f and p r o l i n e , min a f t e r t h e f i n a l over the next  ingestion.  30 min a f t e r and a g a i n 30 min,  60 min and 90  i n g e s t i o n o f p r o l i n e and s n u f f chewing. A l l u r i n e passed  17 h r was c o l l e c t e d f o r NPRO a n a l y s i s . The e x p e r i m e n t a l p r o t o c o l  i s shown i n F i g u r e 5.  36  Figure  5: Experimental Protocol  @  SNUFF + PROLINE  SNUFF + PROLINE  4  •i,  Formation  -rASC  r  ©  f o r E n d o g e n o u s NPRO  ASC  I  -30  SNUFF + PROLINE  ASC  4,  I  SNUFF + PROLINE  ASC  I  1  I  1  30  SNUFF + PROLINE  4> 1 SNUFF + PROLINE  —T  -  60  -fh  ASC  120  ASC  180  ASC  1  240  MIN  37  11.2 Administration of Beta-carotene and Vitamin A Beta-carotene  was s u p p l i e d by Hoffmann-La Roche as w a t e r - d i s p e r s a l  beadlets c o n s i s t i n g o f approximately 10-11% b e t a - c a r o t e n e ,  20% s t a r c h , 20% d e x t r o s e , 30-50% g e l a t i n ,  and t r a c e amounts o f a l p h a - t o c o p h e r o l and a s c o r b a t e  p a l m i t a t e . The 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 h y d r o x y t o l u e n e some forms o f b e t a - c a r o t e n e were absent V i t a m i n A c a p s u l e s were purchased  from our p r e p a r a t i o n s . from S t a n l e y Drug Company L t d . ,  Vancouver. Each c a p s u l e c o n t a i n e d v i t a m i n A d e r i v e d from f i s h l i v e r e q u i v a l e n t t o 15 mg  present i n  oils  retinol.  Placebo c a p s u l e s s u p p l i e d by Hoffmann-La Roche c o n t a i n e d d e x t r o s e .  11.3 Inuit: Beta-carotene Three b e t a - c a r o t e n e  capsules  (30 mg b e t a - c a r o t e n e  p e r c a p s u l e ) were  a d m i n i s t e r e d twice weekly f o r 10 weeks under the s t r i c t  s u p e r v i s i o n o f myself  and an I n u i t i n t e r p r e t e r who spent the e n t i r e t r i a l p e r i o d i n Gjoa Haven. Treatment w i t h b e t a - c a r o t e n e was i n i t i a t e d w i t h 27 s n u f f d i p p i n g i n d i v i d u a l s , p l u s 10 r e c e i v i n g p l a c e b o .  I n d i v i d u a l s were s e l e c t e d who took more than 6 s n u f f  d i p s p e r day. Three boys l o s t i n t e r e s t i n c o o p e r a t i n g . received beta-carotene,  p l a c e b o c a p s u l e s o r no treatment  The p a r t i c i p a n t s  who  (18 i n d i v i d u a l s ) were  chosen from the s u b j e c t s p r e v i o u s l y i n t e r v i e w e d . To the b e s t o f our knowledge, n e i t h e r the number o f tobacco p l u g s , the s i t e , nor the l e n g t h o f time t h a t the tobacco was kept i n the mouth changed d u r i n g the 10-week t r i a l p e r i o d .  11.4 East Indians : Beta-carotene and Beta-carotene plus Vitamin A A t o t a l o f 130 b e t e l q u i d chewers w i t h o r a l l e u k o p l a k i a s were d i v i d e d t h r e e groups.  into  One (35 p a r t i c i p a n t s ) r e c e i v e d p l a c e b o c a p s u l e s , a second (35  p a r t i c i p a n t s ) r e c e i v e d beta-carotene,  and a t h i r d  (60 p a r t i c i p a n t s ) r e c e i v e d  38  b e t a - c a r o t e n e p l u s v i t a m i n A. I n d i v i d u a l s were s e l e c t e d on the b a s i s o f h a v i n g e s t a b l i s h e d l e u k o p l a k i a p l u s the l o c a t i o n i n which they l i v e d t o make the d i s t r i b u t i o n o f c a p s u l e s e a s i e r t o people all  i n the same v i c i n i t y .  t r i a l p a r t i c i p a n t s r e c e i v e d the d e s i r e d doses,  the p l a c e b o ,  To ensure  that  beta-carotene  and v i t a m i n A c a p s u l e s were a d m i n i s t e r e d twice weekly f o r 6 months under the strict  s u p e r v i s i o n o f myself,  Cancer Centre, The  a l o c a l nurse and a s t a f f member o f the R e g i o n a l  Trivandrum.  f o l l o w i n g were g i v e n twice weekly t o each person,  group: (1) 3 b e t a - c a r o t e n e beta-carotene  depending on h i s  c a p s u l e s , each c o n t a i n i n g 30 mg b e t a - c a r o t e n e ;  c a p s u l e s p l u s one v i t a m i n A c a p s u l e  (2) 3  (50,000 IU), and (3) 3  p l a c e b o c a p s u l e s c o n t a i n i n g d e x t r o s e and w a t e r - d i s p e r s a l b e a d l e t s . Of the original participants, due  t o death,  5 i n Group 1, 4 i n Group 2, and 9 i n Group 3 were l o s t  i l l n e s s o r e m i g r a t i o n . Endpoints were examined a t 3 months and  a g a i n a t 6 months.  11.5 East Indians : Vitamin A B e t e l q u i d chewers w i t h w e l l - e s t a b l i s h e d l e u k o p l a k i a s (diagnosed by Drs. Mathew and Sankaranarayanan a c c o r d i n g to the WHO C o l l a b o r a t i n g Centre Precancerous of  Lesions  [1978]) were d i s t r i b u t e d i n t o two groups based  for Oral  on the area  the beach i n which they l i v e d : one group o f 35 p a r t i c i p a n t s r e c e i v e d placebo  capsules  ( d e x t r o s e ) , and the second  group o f 30 p a r t i c i p a n t s r e c e i v e d v i t a m i n A  (200,000 IU/week), g i v e n twice weekly as 2 c a p s u l e s o f 50,000 IU o f v i t a m i n A each.  I n the p l a c e b o  group, 33 o u t o f 35 chewers completed  the t r i a l ,  and i n  the v i t a m i n A - t r e a t e d group, 21 o u t o f 30 chewers remained on the v i t a m i n A regime. I n a l l the i n t e r v e n t i o n s t u d i e s c a r e f u l a t t e n t i o n was taken by myself or  a s t a f f member o f the R e g i o n a l Cancer Centre, Trivandrum  the p i l l s  were a c t u a l l y swallowed by each o f the t r i a l  t o make sure t h a t  participants.  39  12. Determination of Beta-carotene and Retinol  To determine  the e x i s t i n g l e v e l s o f b e t a - c a r o t e n e and r e t i n o l  p o p u l a t i o n s t u d i e d , serum samples non-users  were c o l l e c t e d from 110  i n the I n u i t  subjects including  70  o f s n u f f and 40 u s e r s .  B l o o d samples vacutainers  were c o l l e c t e d i n the community o f Gjoa Haven u s i n g  (No. 6421)  c o n t a i n i n g a c l o t a c t i v a t o r . Samples were s t o r e d on i c e  f o r approximately 1 hr a f t e r c o l l e c t i o n , (700 g f o r 5 min). Serum was  then spun i n a benchtop  centrifuge  c o l l e c t e d with a Pasteur p i p e t i n t o  c r y o t u b e s , and s t o r e d i n l i q u i d n i t r o g e n u n t i l  2-ml  analysis.  A l i q u o t s o f serum (200 /il) were p l a c e d i n 1.5-ml p o l y p r o p y l e n e m i c r o c e n t r i f u g e tubes, and 200 /il a l i q u o t s o f 5% KOH  i n methanol  were added.  Samples were d i g e s t e d f o r 1 h r a t 50°C, and then 600 /xl o f hexane were added. Samples were e x t r a c t e d by v o r t e x m i x i n g f o r 1 min,  and the hexane was  separated  from the sample by c e n t r i f u g a t i o n . A l i q u o t s o f hexane (400 /il) were removed from the samples,  and d r i e d under reduced p r e s s u r e i n 1.5-ml p o l y p r o p y l e n e  m i c r o c e n t r i f u g e tubes i n a c e n t r i f u g a l e v a p o r a t o r (Speed Vac Model SVC100H, Savant Instruments, H i c k s v i l l e , NY). Residues were r e d i s s o l v e d i n 20 methylene  c h l o r i d e , and then d i l u t e d to a t o t a l o f 200 pi  with  pi  methanol.  R e t i n o l and c a r o t e n e were measured by i s o c r a t i c h i g h - p r e s s u r e l i q u i d chromatography  (HPLC) ( M i l l e r e t a l . ,  1984).  For r e t i n o l ,  50-jul a l i q u o t s o f  sample were i n j e c t e d i n t o a Brownlee S p h e r i 5 reversed-phase column (5 micron p a c k i n g , 2.1 mm  i n t e r n a l diameter x 22 cm)  c o u p l e d to a 2.1 mm  internal  diameter x 3 cm guard column w i t h the same p a c k i n g . S o l v e n t c o n s i s t e d o f 99% methanol  and 1% water  a t a f l o w r a t e o f 0.5  ml/min. For c a r o t e n e ,  50-^1  a l i q u o t s o f sample were i n j e c t e d i n t o a Vydac 201 TP reversed-phase column (10 m i c r o n p a c k i n g , 3.2 mm  i n t e r n a l diameter x 25 cm).  S o l v e n t c o n s i s t e d o f 95%  40  methanol and 5% methylene c h l o r i d e a t a f l o w r a t e o f 1.0 ml/min. A l l s o l v e n t s c o n t a i n e d 0.1% p h o s p h o r i c a c i d t o p r o t e c t a g a i n s t p o s s i b l e column d e g r a d a t i o n by a l k a l i n e  samples.  R e t i n o l and c a r o t e n e were d e t e c t e d by a v a r i a b l e wavelength absorbance d e t e c t o r u s i n g a t u n g s t e n lamp, which was  UV/VIS HPLC  s e t a t 325 nm f o r r e t i n o l  and t h e n s w i t c h e d t o 450 nm f o r c a r o t e n e . Samples were q u a n t i f i e d by an e l e c t r o n i c computing i n t e g r a t o r which had been c a l i b r a t e d by i n j e c t i o n o f r e t i n o l and c a r o t e n e s t a n d a r d s i n methanol. C a l i b r a t i o n s t a n d a r d s were p r e p a r e d by d i s s o l v i n g c r y s t a l l i n e  r e t i n o l or c a r o t e n e (Sigma, S t . L o u i s , MO)  in  methanol, and d e t e r m i n i n g the c o n c e n t r a t i o n o f s o l u t i o n s by t h e i r absorbance, u s i n g molar e x t i n c t i o n c o e f f i c i e n t s i n methanol o f 52,500 ( r e t i n o l , 130,000 ( c a r o t e n e , 450  325 nm)  and  nm).  W i t h i n - d a y p r e c i s i o n was  5.6%  f o r b e t a - c a r o t e n e i n 10 samples from a  p o o l e d s i n g l e specimen o f normal serum to-day p r e c i s i o n d u r i n g 27 days was  (mean = 57 ng/ml;  5.9%  SD = 3.2 ng/ml).  (mean = 57 ng/ml,  Day-  SD = 3.4 ng/ml) f o r  the same p o o l . The e f f i c i e n c y o f e x t r a c t i o n o f b e t a - c a r o t e n e from serum appeared to exceed 95%: the absorbance a t 450 nm a f t e r a second e x t r a c t i o n l e s s than 3% o f t h a t f o r the o r i g i n a l  was  extract.  R e t i n o l a n a l y s i s o f the same plasma on 10 c o n s e c u t i v e days gave a mean v a l u e o f 450 ng/ml (SD = 19.8 ng/ml), and a c o e f f i c i e n t o f v a r i a t i o n o f  4.4%.  P r e c i s i o n f o r the same-day a n a l y s i s o f 10 a l i q u o t s o f a second plasma gave a mean o f 440 ng/ml (SD = 18.0 ng/ml), and a c o e f f i c i e n t o f v a r i a t i o n o f 4.1%. The procedure f o r the d e t e r m i n a t i o n o f serum r e t i n o l and b e t a - c a r o t e n e i s outlined i n Figure  6.  41 F i g u r e 6: D e t e r m i n a t i o n of Serum Beta-carotene and R e t i n o l  Serum (200 ul)  i n t o 1.5-ml p o l y p r o p y l e n e m i c r o c e n t r i f u g e tubes  r add 200-ul  of 5% KOH  aliquots  i n MeOH digest  f o r 1 h r a t 50°C  •  add 600 u l hexane  e x t r a c t by v o r t e x mixing f o r 1 min  s e p a r a t e hexane from by c e n t r i f u g a t i o n  sample  remove 400-ul a l i q u o t s o f hexane and d r y under reduced p r e s s u r e in 1.5-ml p o l y p r o p y l e n e m i c r o c e n t r i f u g e tubes in c e n t r i f u a a l evaoorator  I I  r e d i s s o i v e i n 20 jjl methylene c h l o r i d e and d i l u t e t o 200 ul with MeOH  inject  50 u l i n t o HPLC  42  13. Questionnaire The  I n u i t surveys were conducted d u r i n g the summer o f 1986  communities o f Gjoa Haven, NWT,  and  the n a t i v e I n d i a n surveys  of 1987  i n the community o f La Loche, N.  Indians  and  for  lesions.  200  sampling  d u r i n g the summer  Saskatchewan. A t o t a l o f 300  I n u i t were i n t e r v i e w e d , and  In a s e t t l e m e n t ,  i n the  was  t h e i r o r a l c a v i t i e s were c a r r i e d out  native screened  i n houses, s c h o o l s ,  s p o r t s grounds and n a t i v e band o f f i c e s . The  q u e s t i o n n a i r e used (Table IV)  d i p p i n g or tobacco tobacco age  included questions  on p a t t e r n s of s n u f f  chewing, smoking and a l c o h o l consumption; l o c a l i z a t i o n of  w i t h i n the o r a l c a v i t y ;  and band a f f i l i a t i o n .  i n g e s t i o n o f s o f t d r i n k s , c o f f e e or tea;  I c a r r i e d out the i n t e r v i e w s accompanied by a n a t i v e  s o c i a l worker, n u r s e o r t r a n s l a t o r . of i n d i v i d u a l p a r t i c i p a n t s  sex,  To a v o i d any peer p r e s s u r e ,  were conducted i n the absence o f any  the  interviews  interfering  onlookers. A similar questionnaire consumption was  (Table V) about chewing h a b i t s and a l c o h o l  a d m i n i s t e r e d house to house among the E a s t I n d i a n fishermen  K e r a l a . In a l l more than 500  fishermen were i n t e r v i e w e d .  in  43 TABLE IV Questionnaire  Used  i n the Northwest T e r r i t o r i e s  and Saskatchewan  Study code:  Date o f i n t e r v i e w :  Document c o d e :  Interviewer:  Name:  Place  o f interview:  Age: Sex: Current Ethnic Place  address: origin:  of birth:  Chewing  Pattern  Smoking  Pattern  Type o f t o b a c c o :  Type  Number p e r d a y :  Brand:  Weight o f p i n c h :  Number p e r d a y :  Location  Years o f smoking:  i n mouth:  T i m e p e r chew:  (cigarette,  cigar,  Inhale:  Years o f chewing:  A l c o h o l Use Type Beer  Wine:  Whiskey  Other:  Bottles  Home brew:  Tea:  Cups/day:  Coffee:  Cups/day:  Vitamins Type:  Dose:  p e r week:  etc.):  44  TABLE V Questionnaire  Interview  Used  i n Kerala,  India  no.:  P a t i e n t no.:  Date: Smoking No. o f b i d i  p e r day:  No. o f c i g a r e t t e s p e r d a y : Alcohol  (ml p e r d a y )  Toddy:  Local:  Chewing No. o f q u i d s  Duration  p e r day:  o f chew:  Nut+leaf+tobacco+lime:  Nut+leaf+lime:  Use  Use  tobacco  leaf:  Appearance o f 1.  No  2.  New  3.  Complete  4.  Partial  5.  change: lesion: remission: regression: i n lesion  size:  P a t i e n t has m u l t i p l e l e s i o n s  c) Change  in lesion  d)  changes c l a s s i f i c a t i o n :  Lesion  Progression  of  appearance:  increases  b)  changes c l a s s i f i c a t i o n :  Lesion  Smears: Brushings:  Only  some  Specify: Specify:  lesion:  a) L e s i o n  Photos:  stem:  Lesion  a) D e c r e a s e b)  tobacco  Overnight:  i n size: Specify:  disappear:  45  14. Statistical Analysis Tests f o r s i g n i f i c a n t Student's  d i f f e r e n c e i n outcome were performed  either  by  T - t e s t o r by F i s h e r ' s e x a c t t e s t . A P v a l u e o f < 0.05 was c o n s i d e r e d  significant.  F i s h e r ' s e x a c t t e s t was performed  S p i n n e l l i , Department o f Epidemiology, Cancer Research  Centre.  Biometry  under the d i r e c t i o n  o f Dr. J .  and O c c u p a t i o n a l Oncology, B.C.  46  RESULTS 1. Use of Smokeless Tobacco The  o b j e c t i v e s o f t h i s s e c t i o n were to examine by q u e s t i o n n a i r e  prevalence  o f o r a l tobacco  use  ( I n u i t and  Indian)  E a s t I n d i a n community ( K e r a l a , I n d i a ) , to r e c o r d  and  one  o t h e r o r a l h a b i t s which may c i g a r e t t e smoking and  i n two  n o r t h e r n Canadian n a t i v e  the  exert a modifying  In the two cut tobacco tobacco  Canadian s e t t l e m e n t s  commercially  plugs  and  e f f e c t on c a r c i n o g e n e s i s , such as  the d r i n k i n g o f a l c o h o l i c beverages, and  v a r i o u s p a t t e r n s o f s n u f f i n g and chewing  settlements  to examine the  tobacco.  examined, the d i p p i n g o f s n u f f  (finely  a v a i l a b l e i n cans) f a r exceeds the a c t u a l chewing of  t w i s t s . The  placement o f f i n e l y ground tobacco  c a v i t y i s d e s i g n a t e d by n a t i v e peoples  i n t o the  oral  with a v a r i e t y of labels, including  " s n u f f d i p p i n g , " " u s i n g snoose," "chewing snoose," " t a k i n g a p i n c h , " or " t a k i n g a chew." The betel  East Indian fishermen  use  chewing tobacco  o n l y as p a r t o f  the  quid. I n u i t , Indians  and  E a s t Indians use  smokeless tobacco  i n conjunction with  o t h e r o r a l h a b i t s , i n c l u d i n g c i g a r e t t e smoking and a l c o h o l d r i n k i n g . Two these h a b i t s can take p l a c e c o n c u r r e n t l y ,  (e.g., s n u f f d i p p i n g p l u s a l c o h o l  d r i n k i n g , or s n u f f d i p p i n g p l u s c i g a r e t t e smoking). The i n d i v i d u a l s w i t h one  examined r e p r e s e n t approximately  The  I n u i t examined r e p r e s e n t a p p r o x i m a t e l y  Gjoa Haven. The approximately India.  300  10%  prevalence  o r more h a b i t s i s shown i n T a b l e V I . The  Indians 152  of  10%  114  of snuff dipping  o f the t o t a l La Loche p o p u l a t i o n . 15%  o f the n a t i v e p o p u l a t i o n of  E a s t I n d i a n b e t e l q u i d chewers examined r e p r e s e n t  o f the p o p u l a t i o n o f f i s h e r m e n  l i v i n g c l o s e to Trivandrum,  47  The  a l c o h o l i c beverages consumed by Indians i n La Loche are m a i n l y  and wine. The  E a s t I n d i a n f i s h e r m e n o f K e r a l a d r i n k fermented,  from palm t r e e s ( a p p r o x i m a t e l y 40%  living  K e r a l a ) has  r e c e n t l y been completed  (Gupta,  area  1984). Canadian Indians  i n n o r t h e r n Saskatchewan d i d not r e p o r t the d r i n k i n g o f L y s o l ,  p o l i s h or Chinese  sap  e t h a n o l ) . A more d e t a i l e d e v a l u a t i o n o f the  d r i n k i n g h a b i t s o f the E a s t I n d i a n p o p u l a t i o n i n a n e i g h b o u r i n g (Ernakulam,  distilled  beer  shoe  c o o k i n g wine. These types of a l c o h o l i c items, however, are  consumed by some I n d i a n s and non-Indians  living  i n Vancouver.  TABLE VI Snuff Dipping, Tobacco Chewing, Cigarette Smoking, and Drinking of Alcoholic Beverages by Native Indians, Inuit and East Indians  Snuff Dipping (%)  Tobacco Chewing (%)  Cigarette Smoking (%)  Alcohol Drinking (%)  Location  Sex  Number Examined  La Loche (Indian)  M F  55 59  62.6 32.2  0.0 0.0  27.2 50.8  47.2 44.1  Gjoa Haven (Inuit)  M F  107 45  57.0 0.0  10.3 0.0  32.7 91.1  0.0 0.0  Kerala (East Indian)  M F  120 110  0.0 0.0  54.0 39.0  14.6 0.0  *  36.6 0.0  49  Information  on the l e n g t h o f time t h a t s n u f f u s e r s  tobacco i n t h e i r mouth p r o v e d to be more d i f f i c u l t because o f a g e n e r a l  l a c k o f i n t e r e s t i n and  r e s u l t s shown i n T a b l e V I I were o b t a i n e d ,  than 15 min. periods by  p a r t l y , by  interviews  and,  p a r t l y , by  a r u l e , younger s n u f f  They s t a t e d t h a t l a r g e r q u a n t i t i e s o f s n u f f h e l d f o r  the n a t i v e I n d i a n s  were Copenhagen, Skoal  brands o f s n u f f and  dippers  o f Saskatchewan and  the I n u i t d u r i n g  longer  chewing tobacco used the study p e r i o d  and Redman.  amount o f tobacco i n a " p i n c h " o f s n u f f v a r i e s a c c o r d i n g  o f a person's f i n g e r s and t i n of s n u f f w i l l  The  a s m a l l e r amount o f s n u f f and keep i t i n the mouth f o r l e s s  o f time made them " d i z z y . " The  The  to o b t a i n than a n t i c i p a t e d ,  l a c k o f f e e l i n g f o r "time."  a c t u a l l y measuring the s n u f f d i p p i n g p e r i o d . As u s u a l l y s t a r t with  or chewers r e t a i n  to the  the number o f f i n g e r s used. Knowledge o f how  l a s t an i n d i v i d u a l can be m i s l e a d i n g  amount o f tobacco used d a i l y ,  size  long a  when c a l c u l a t i n g the  s i n c e v i r t u a l l y a l l n a t i v e Indians  and  Inuit  share t h e i r s n u f f . These d i f f i c u l t i e s were overcome by weighing s n u f f p i n c h e s taken by d i f f e r e n t I n u i t was 1.3  0.61  g chewed by  s n u f f d i p p e r s . The  g compared to an average o f 1.5 Indian  Indians  i n one  was  g (SD = 0.63).  5.9  average weight o f tobacco chewed by  females. The  b e t e l q u i d was  1.1  g chewed by  51 I n d i a n males  amount o f chewing tobacco used by  g (SD = 0.6).  60 and  East  The weight o f the e n t i r e q u i d  TABLE VII Means and Ranges of Various Snuff-Related Factors Among Inuit and Native Indians  Number o f Dips p e r Day  Length of Dip (min)  Number of Years of Use  Snuff Kept in Mouth per Day (min)  Weight of Tobacco per Pinch (gm)  Sex  Number Examined  Gjoa Haven (Inuit)  M  60  8.03 (1-20.0)  25.2 (1-180.0)  4.1 (0.5-20.0)  208.8 (3-1140.0)  0.61 (0.45-0.83)  La Loche (Indian)  M  51  9.1 (1-30.0)  20.3 (3-60.0)  8.5 (1-25.0)  174.3 (10-600.0)  1.5 (0.3-3.5)  24  6.2 (1-25.0)  11.9 (3-30.0)  5.7 (1-20.0)  57.8 (3-150.0)  1.3 (0.5-2.6)  Location  O  51  Approximately  90% o f the 114 a d u l t male and female s n u f f d i p p e r s examined  i n L a Loche p l a c e the tobacco i n t o the lower g i n g i v a l groove and h o l d i t e i t h e r to  the l e f t o r the r i g h t o f c e n t r e . Less than 5% o f I n d i a n s keep i t i n the  c e n t r e b e h i n d the lower l i p d u r i n g a d i p p i n g s e s s i o n . I t was s t a t e d r e p e a t e d l y t h a t they f e e l p a i n i n the frenulum when the s n u f f i s kept i n the c e n t r e f o r a l o n g time. A d o l e s c e n t males and younger a d u l t females attempt  to conceal t h e i r  s n u f f t a k i n g by p l a c i n g the tobacco f a r back i n t h e i r cheeks. V i r t u a l l y a l l s n u f f u s e r s change the l o c a t i o n o f the s n u f f each week due t o i r r i t a t i o n i f they keep the tobacco too l o n g i n the same p l a c e . Some ( a p p r o x i m a t e l y 6%) o f s n u f f d i p p e r s a c t i v e l y move the s n u f f around  the o r a l c a v i t y w i t h the tongue.  2. Nitrite in Smokeless Tobacco Samples Tobacco has been found i n a l l chewing m i x t u r e s t h a t i n c r e a s e the r i s k o f d e v e l o p i n g o r a l c a n c e r (IARC, 1985).  I t has been c o n c l u d e d t h a t  smokeless  tobacco must c o n t a i n e i t h e r p r e c u r s o r s o f c a r c i n o g e n s o r c a r c i n o g e n i c agents themselves.  S i n c e s n u f f and chewing tobacco a r e n o t exposed  t o e x c e s s i v e heat,  they do n o t c o n t a i n c a r c i n o g e n i c p y r o l y s i s p r o d u c t s , u n l i k e c i g a r e t t e s and c i g a r s . T h i s s e c t i o n f o c u s e s on the c a p a c i t y o f smokeless nitrite,  tobacco t o supply  which c o u l d l e a d t o the f o r m a t i o n o f c a r c i n o g e n i c N - n i t r o s a m i n e s i n  s n u f f d i p p e r s and tobacco chewers.  2.1  Snuff and Chewing Tobacco Brands  N i t r i t e was e s t i m a t e d i n aqueous e x t r a c t s o f s e v e r a l tobacco i n c l u d i n g f i v e d i f f e r e n t brands Canada, f o u r "nass"  samples,  o f s n u f f which were c o m m e r c i a l l y a v a i l a b l e i n  (tobacco mixed w i t h s l a k e d l i m e , ash and o i l ) samples from  Samarkand i n the S o v i e t Union,  and f o u r K h a i n i tobacco specimens from Bengal,  I n d i a . Each tobacco sample was a n a l y z e d t h r e e times. The f i g u r e s g i v e n i n Table  52  V I I I r e p r e s e n t t h e i r averages. four d i f f e r e n t  cans or boxes.  Tobacco samples 1 to 4 i n T a b l e V I I I  represent  TABLE VIII Nitrite  Content  i n Aqueous E x t r a c t s  o f S m o k e l e s s T o b a c c o Samples  Nitrite  (mg/kg)  Individual  Samples  a Tobacco Type  Brand  Origin  Snuff  C  Canada  1040  Snuff  B  Canada  Snuff  SL  Snuff  1  2  3  4  800  550  550  500  400  305  95  Canada  820  750  500  SW  Canada  75  18  5  70 !b  Snuff  K  Canada  ND  ND  ND  ND  Chewing  R  U.S.  ND  ND  ND  ND  Nass  Uzbekistan  20  10  Khaini, dry  India  ND  ND  ND  ND  Khaini, dry  India  . ND  ND  ND  ND  8  ND  7  Chewing  Parijat  India  ND  ND  ND  ND  Chewing  Gundi  India  ND  ND  ND  ND  Chewing  Sun-dried  India  ND  ND  ND  ND  a C, Copenhagen K, K o d i a k ; R, ND,  B, B i g Horn; Redman  not d e t e c t a b l e  SL, Skoal;  SW,  Skoal Wintergreen;  54  East Indian Tobacco  2.2 The quids, tobacco  tobacco  used by the East I n d i a n f i s h e r m e n  i s a sun-dried variety.  The n i t r i t e  3.  l e v e l s i n the samples o f t h i s  were t o o low t o be d e t e c t e d by the a n a l y t i c a l method used (Table  Two o t h e r E a s t I n d i a n chewing tobaccos also  i n K e r a l a , t o make b e t e l  examined, w i t h s i m i l a r n e g a t i v e  VIII).  which a r e used to make a b e t e l q u i d were  results  (Table  VIII).  Nitrite in the Saliva of Users of Smokeless Tobacco  3.1 Snuff Dippers S a l i v a was c o l l e c t e d from seven I n u i t v o l u n t e e r s p r i o r t o s n u f f d i p p i n g and  a f t e r they had p l a c e d 0.5 g o f tobacco  (Copenhagen) i n t o the lower  groove. S a l i v a was c o l l e c t e d a t 5 min i n t e r v a l s f o r the next nitrite  determined i n these  s a l i v a samples. C o n s i d e r a b l e  30 min, and the  quantities  were r e l e a s e d from the s n u f f i n t o the s a l i v a ( F i g u r e 7 ) . No a c t i v e the tobacco  o c c u r r e d d u r i n g the e x p e r i m e n t a l  from the o r a l c a v i t y ,  the n i t r i t e  l e v e l s w i t h i n approximately  gingival  of n i t r i t e "chewing" o f  p e r i o d . Once the s n u f f was removed  l e v e l s o f the s a l i v a approached  10 min. The mean n i t r i t e  control  l e v e l s a r e g i v e n i n Table  IX f o r i n d i v i d u a l s n o t d u r i n g a chewing p e r i o d .  3.2 Betel Quid Chewers S a l i v a samples were c o l l e c t e d from 50 d i f f e r e n t b e t e l  q u i d chewers i n  K e r a l a , b u t n o t d u r i n g a chewing p e r i o d . The r e s u l t s a r e shown i n Table IX.  F i g u r e 6: N i t r i t e i n the S a l i v a o f Seven  I n u i t Snuff  Dippers  O-30-i  0-25-  2  0-20-  •—  > 0-151 j <  o-ioOJ  I i2  0-05-  15  20  25  TIME (MINI PERIOD PRIOR  Brackets  TQ  OF S N U F F SNUFF  DIPPING  DIPPING  r e p r e s e n t the Standard  D e v i a t i o n o f the Mean  30  T A B L E IX Nitrite  i n the Saliva  Population  o f Smokeless  Number Examined  Tobacco  Tobacco Type  Chewers  Mean N i t r i t e (ppm)  Inuit (Gjoa Haven)  64  Snuff  5.02 (SD=7.07 CV=140%)  Indian (La Loche)  20  Snuff  6.87 (SD=3.68 CV=53%)  East Indian (Kerala)  56  Sun-dried  36.27 (SD=35.06 CV=96.7%)  57  4.  Nitrosation Capacity of Saliva Samples The o b j e c t i v e o f t h i s p a r t o f the study was t o show whether t h e n i t r i t e  r e l e a s e d from tobacco i n t o the s a l i v a o f s n u f f d i p p e r s n i t r o s a t i o n . The f o r m a t i o n  of N-nitrosoproline  experiment was c a r r i e d o u t on one v o l u n t e e r  c a n a c t u a l l y l e a d to  (NPRO) was used as an assay. The  h o l d i n g 1 g o f s n u f f i n t h e lower  g i n g i v a l groove. S a l i v a was c o l l e c t e d over a 20-min p e r i o d . The f o r m a t i o n o f NPRO was q u a n t i f i e d by m i x i n g , i n v i t r o , proline  (200 mg), and i n c u b a t i n g  the mixture f o r 30 min a t 37°C.  amounts o f NPRO were formed d u r i n g The use o f Copenhagen, Skoal  the s a l i v a o f the s n u f f u s e r w i t h  the i n c u b a t i o n p e r i o d a t pH 2.5 (Table X ) .  or B i g Horn brands o f s n u f f , r e s u l t e d i n s a l i v a  samples t h a t showed comparable l e v e l s o f NPRO f o r m a t i o n system.  Considerable  i n this  i nvitro  test  58  TABLE  X  In Vitro Nitrosation Capacity of Saliva i n a User of Three Different Brands of Smokeless Tobacco (Snuff) NPRO (ng/ml) No Tobacco  Snuff C a  Snuff SL a  Snuff B a  Incubation Mixture  pH  Saliva  7 .5  <1  771  647  283  7 .5  <1  672  821  149  2 .5  32  791  506  334  2 .5  60  14,476  11,797  13,496  Saliva + 200  mg  proline  Saliva Saliva + 200  mg  C, Copenhagen;  proline  SL, Skoal;  B, Big Horn  59  5.  N-Nitrosoproline in the Urine of Snuff Dippers The  increased intake of n i t r i t e  from s n u f f c o u l d l e a d t o an e l e v a t e d  o f n i t r o s a t i o n i n s n u f f d i p p e r s . T h i s i s s u e was a n a l y z e d by e s t i m a t i n g  level  levels  o f NPRO i n t h e u r i n e o f s n u f f d i p p e r s and o f i n d i v i d u a l s who do n o t use smokeless tobacco.  The r e s u l t s a r e shown i n Table XI. The NPRO i n the u r i n e o f  I n u i t s n u f f u s e r s was f i v e times g r e a t e r than f o r non-users, and f o r the Indian snuff users  the NPRO l e v e l was 2.9 times g r e a t e r than i n non-users. The r e s u l t s  i n d i c a t e t h a t exposure t o h i g h l e v e l s o f n i t r i t e tobacco and t h a t t h e n i t r i t e  occurs  c a n r e a c t t o form N - n i t r o s o  i n users  o f smokeless  compounds  endogenously.  6.  Tobacco-Specific Nitrosamines in Different Brands of Tobacco The  amounts o f TSNA found i n samples o f smokeless tobacco purchased i n the  Northwest T e r r i t o r i e s a r e shown i n T a b l e XII.  Compared t o smokeless tobaccos  from o t h e r c o u n t r i e s , the l e v e l s o f TSNA measured i n t h e two m o i s t brands o f Canadian s n u f f a r e by f a r t h e h i g h e s t observed. T h i s r e s u l t c o u l d be due to continuous  n i t r o s a t i o n d u r i n g the extended s t o r a g e  s m a l l shops o f t h e n o r t h e r n  communities.  o f t h e s n u f f samples i n the  TABLE XI NPRO i n t h e U r i n e o f Two  Snuff  Dipping  Number Examined  Populations  ( I n u i t and  Mean Number o f D i p s p e r Day  Indian)  Mean L e n g t h of Dip (min)  Snuff (gm/Chew)  28.6 ± 14.8  1.010.7  7.5 ± 4.9  19.5 ± 16.7  1.4 + 0.8  4.3 ± 4.7 (4.3)  Mean  Habit  Population  Snuff users  Inuit  14  G.4 ± 3.4  Snuff  Indian  47  7.2  Nonusers  Inuit  15  1.5 ± 2.2 (1.4)  Nonusers  Indian  27  1.5 ± 1.3 (1.5)  ±  5.2  users  F o r t h e c a l c u l a t i o n o f m e a n s , two v a l u e s f o r " n o t d e t e c t a b l e " were a s s u m e d : maximum n o n - d e t e c t a b l e v a l u e ; 0.0 n g / m l , l o w e s t p o s s i b l e v a l u e ( f i g u r e s i n Inuit snuff users v s . Inuit non-users: Indian snuff users v s . Indian non-users: T o t a l snuff users v s . t o t a l non-users: N o n - p a r a m e t r i c by Mann-Whitney U Inuit  snuff users  vs. Inuit  P<0.001 P<0.001 P<0.001  test:  non-users:  Indian snuff users v s . Indian non-users: T o t a l snuff users v s . t o t a l non-users:  P=0.0001 P=0.001 P=0.00001  ng/ml  0.2 n g / m l , parentheses)  TABLE X I I TSNA  i n Various  Samples  o f Canadian and F o r e i g n Tobacco Brands  TSNA Origin  Tobacco Product  Canada  Plug  Canada  Snuff  79,000  Canada  Snuff  50,400  USAb  Chewing Tobacco b  Chewing Tobacco  India b USA J  b  Sweden . b Bavaria ,b Denmark  (ppb)a  NNN  NAT  NAB  NNK  2090  1580  100  240  152,000  4000  5800  170,000  4800  3200  3500-8200  2400  500-7000  -  Snuff  800-89,000  200-4000  Snuff  2000-6700  900-2400  Snuff  6000-6800  3900-4400  Snuff  4500-8000  2600-6200  _  100-3000  200-8300 600-1500 1500-1600 1400-7000  NNN, N ' - n i t r o s o n o r m c o t i n e ; NAT, N - n i t r o s o a n a t a b i n e ; NAB, N-nitrosoanabasine; NNK, 4 - ( N - n i t r o s o m e t h y l a m i n o ) - 1 - ( 3 - p y r i d y l ) - l butanone From H o f f m a n n a n d Adams  (1981)  62  7.  Tobacco-Specific Nitrosamines in the Saliva ol Snuff Dippers  7.1  Appearance of TSNA in the Saliva  S a l i v a was c o l l e c t e d from two i n d i v i d u a l s ( I n u i t G21 and G60) p r i o r t o , d u r i n g and a f t e r s n u f f d i p p i n g . The r e s u l t s on s u b j e c t G60 show a steady i n c r e a s e i n NNN and NAT+NAB w i t h  the l e n g t h o f time t h a t the s n u f f was kept i n  the g i n g i v a l groove (Table X I I I ) . I t i s i n t e r e s t i n g to note t h a t t h e s a l i v a o f the  two d i p p e r s  contained  measurable amounts o f NNN p r i o r t o t a k i n g s n u f f ,  a l t h o u g h they had r i n s e d t h e i r mouth, and t h a t the NNN and NAT+NAB do not disappear  completely  w i t h i n 5 min a f t e r removal o f the tobacco from the o r a l  c a v i t y . These r e s u l t s i n d i c a t e t h a t a s n u f f d i p p e r l o n g e r p e r i o d than t h e a c t u a l use o f tobacco  i s exposed t o TSNA f o r a  itself.  TABLE X I I I TSNA i n the S a l i v a o f Two I n d i v i d u a l s Dipping  P r i o r t o , During and A f t e r  Snuff  TSNA (ppb) Nicotine (ppm)  Cotinine (ppm)  14.7  0.21  64.9  228.0  1.50  959.0  1160.0  19.3  0.30  67.2  33.4  2.3  0.55  30.6  ND  44.8  0.69  216.0  10 min i n t o d i p  132.0  1.66  15 min i n t o d i p  164.0 30.4  Code No.  Time o f Sampling  G21  Prior to dip 15 min i n t o d i p After  G60  dip  Prior to dip 5 min i n t o d i p  After  dip  NNN  NAT+NAB  ND  NNK  DMNM (%)  ND  0.004  46.5  0.118  ND  0.028  ND  0.006  357.0  ND  0.010  499.0  624.0  17.4  0.012  1.89  650.0  943.0  ND  0.010  0.76  129.0  130.0  ND  0.025  ND, not d e t e c t a b l e C o n t r o l s (non-chewers, non-smokers) had ND s a l i v a (n=60)  l e v e l s o f TSNA i n t h e i r  64  7 .2 Variations in Salivary TSNA of Snuff Dippers To l e a r n about the e x t e n t o f v a r i a t i o n o f amounts o f TSNA i n the s a l i v a o f s n u f f u s e r s , 20 d i p p e r s were asked v a r i e d between a p p r o x i m a t e l y  to take t h e i r r e g u l a r amount o f s n u f f , which  0.5 and 1.5 g p e r d i p , and keep i t i n the mouth i n  t h e i r u s u a l manner f o r 15 min. S a l i v a samples were then c o l l e c t e d , l e v e l s o f n i c o t i n e , c o t i n i n e and TSNA were determined The  r e s u l t s show c o n s i d e r a b l e v a r i a t i o n s  and the  (Table XIV).  i n the l e v e l s o f TSNA d e s p i t e the  f a c t t h a t the chewing times were kept c o n s t a n t . The t h r e e f o l d  difference  between the amounts o f s n u f f used can h a r d l y e x p l a i n the a p p r o x i m a t e l y d i f f e r e n c e i n the amount o f NNN between the two extreme cases  22-fold  o r the 3 7 - f o l d d i f f e r e n c e f o r NAT+NAB found  (G4 and G9). In a d d i t i o n , the s a l i v a was  analyzed  f o r n i t r o s o - c i s - 2 , 6 - d i m e t h y l m o r p h o l i n e (DMNM)( T a b l e X I V ) , i n o r d e r to monitor f o r unintended  n i t r o s a t i o n d u r i n g h a n d l i n g and work-up. I n a l l examined cases,  the amount o f DMNM was even lower than the u s u a l l y a c c e p t a b l e amounts. The  r e s u l t s show t h a t exposure t o known c a r c i n o g e n i c agents  i n smokeless tobaccos  (TSNA) p r e s e n t  o c c u r s a t r e l a t i v e l y h i g h l e v e l s . The exposure o f the  o r a l mucosa t o the c a r c i n o g e n i c and mutagenic agents  c o n t a i n e d i n these  tobaccos may r e s u l t i n the i n c r e a s e d l e v e l s o f m i c r o n u c l e a t e d c e l l s seen i n the o r a l mucosa o f s n u f f d i p p e r s .  TABLE XIV TSNA i n the S a l i v a  of Snuff Dipping  Inuit  (Gjoa Haven)  TSNA (ppb) Code No.  G4 G8 G9 G13 G20 G21 G23x G23y G24 G26 G31 G 3 ?  Nicotine (ppm)  b  b  b  G42x G42y G46 G47 G48 G49 G50 G60 G61 JER  b  Cotinine (ppm)  512 566 320 808 123 171 802 202 695 255 869 350 170 330 482 1300 1150 223 239 142  70 450  2.90 2.08 0.84 3.94 1.61 1.19 3.00 1.16 2.33 1.02 3.11 1.64 1.83 2.75 2.14 3.55 3.66 1.19 1.28 1.27 0.54 2.04  NNN  NAT+NAB  2610 1730 115 2570 491 610 2200 683 295 363 607 443 407 855 2150 357 2260 515 423 272 407 1210  4560 2100 123 4030 731 840 3070 . 1130 253 617 670 569 437 795 2570 377 2890 697 618 418 527 896  a  ND,  b  x and y, s a l i v a samples taken a t two d i f f e r e n t day from the same s n u f f d i p p e r  NNK  201.0 116.0 NDa 150.0 2.4 ND 159.0 25.7 29.0 ND 35.2 19.9 ND 20.4 151.0 24.5 166.0 31.3 20.5 ND 14.9 59.5  DMNM (%)  0.013 0.026 0.008 0.033 0.016 ND 0.028 0.123 0.014 0.021 0.025 0.014 0.010 0.019 0.056 0.032 0.058 0.010 ND 0.020 0.027 0.017  not d e t e c t a b l e  C o n t r o l s (non-chewers, non-smokers) had ND s a l i v a (n=60)  times d u r i n g the same  l e v e l s o f TSNA i n t h e i r  66  8.  Frequency of Micronuclei in the Oral Mucosa of Snuff Dippers and Tobacco Chewers An i n c r e a s e d 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 c e l l s  has been observed  (MNC)  i n the o r a l mucosa  i n i n d i v i d u a l s a t e l e v a t e d r i s k f o r o r a l cancer,  including  smokers and a l c o h o l d r i n k e r s , K h a i n i tobacco u s e r s from I n d i a , and b e t e l q u i d chewers o f O r i s s a and Meghalaya, I n d i a (reviewed i n T a b l e I I ) . In t h i s we compared the MNC  f r e q u e n c y i n the o r a l mucosa o f s n u f f d i p p i n g I n u i t  Haven) w i t h t h a t o f s n u f f d i p p i n g Indians  study, (Gjoa  (La Loche) and E a s t I n d i a n tobacco  chewers ( K e r a l a ) . The e x f o l i a t e d c e l l s were c o l l e c t e d from areas o f the mucosa a t which the tobacco had been kept. The r e s u l t s shown i n T a b l e I I i n d i c a t e an e l e v a t e d f r e q u e n c y o f MNC  9.  i n the group o f tobacco chewers and s n u f f d i p p e r s .  Precancerous Lesions and Cancer of the Oral Cavity in Users of Smokeless Tobacco Two  types o f anomalies  o f the o r a l mucosa were r e c o r d e d : (a) w h i t i s h to  y e l l o w i s h w r i n k l e d patches w i t h or w i t h o u t furrows, c o r r e s p o n d i n g to c a t e g o r i e s 2 and 3 i n the c l a s s i f i c a t i o n o f Greer and Poulson l e u k o p l a k i a s , as d e s c r i b e d by the WHO Precancerous  Lesions  (1978)(Tablelll).  (1983),  and (b) d e f i n i t e  C o l l a b o r a t i n g Centre f o r O r a l  67  9.1 P r e c a n c e r o u s L e s i o n s Of the 50 smokeless  tobacco u s e r s examined i n Gjoa Haven, 26% showed  w h i t i s h t o y e l l o w i s h w r i n k l e d patches  (Table XV). A p p r o x i m a t e l y o n e - t h i r d a l s o  had one o r more furrows i n the w h i t i s h areas o f the mucosa. The l e s i o n s were l o c a t e d i n the lower g i n g i v a l groove and a t the f r o n t o f the mouth, where the tobacco i s kept. No anomalies were d e t e c t e d i n r e g i o n s o f the o r a l c a v i t y n o t d i r e c t l y exposed (leukoderma)  t o tobacco. The occurence o f the w h i t i s h t o y e l l o w i s h patches  i n the o r a l c a v i t y o f s n u f f d i p p i n g I n d i a n s (La Loche) was h i g h e r  (Table XV) than among the I n u i t . I n t h i s c o n n e c t i o n , i t may be o f i n t e r e s t to note t h a t the I n d i a n s n u f f d i p p e r s a r e r e l a t i v e l y heavy d r i n k e r s o f a l c o h o l i c beverages. No d e f i n i t e l e u k o p l a k i a s , which a r e b e l i e v e d to be precancerous l e s i o n s , were n o t e d among the examined s n u f f d i p p e r s . However, i t s h o u l d be k e p t i n mind t h a t a h i g h percentage o f the s u b j e c t s used i n the study were teenagers who had d i p p e d s n u f f f o r too s h o r t a time t o have a l r e a d y developed precancerous o r a l  lesions.  L e u k o p l a k i a s , which r e p r e s e n t precancerous  l e s i o n s , were observed i n a  r e l a t i v e l y h i g h f r e q u e n c y i n the b e t e l q u i d chewers examined i n K e r a l a (Table XV). L e u k o p l a k i a s were d i a g n o s e d by m y s e l f and c o n f i r m e d by two e x p e r t s , B. Mathew, M.D.,  and R.Sankaranarayanan, M.D.  A l l l e u k o p l a k i a s were  and the r e c o r d s s t o r e d a t the B.C. Cancer Research Centre,  photographed  Vancouver.  68  TABLE XV Prevalence  o f O r a l L e s i o n s Among S n u f f D i p p e r s  Population  Location  Inuit  Gjoa  Indians  Individuals with Whitish Patches  I  Chewers  Individuals with Oral Leukoplakia  %  M  50  26.0  0  La Loche  M  49  46.9  0  Indians  La Loche  F  23  52.2  0  East  Kerala  M  325  Indians  Haven  Sex  Number o f Tobacco Users  and Tobacco  0  59.7  69  Oral Cancer  9.2  T h i s study  focused  on t h r e e r e s t r i c t e d p o p u l a t i o n groups w i t h  p a t t e r n s o f smokeless tobacco use.  No  attempt was  made to e v a l u a t e  different the  b i o l o g i c a l e f f e c t s o f s n u f f d i p p i n g or tobacco chewing on a l a r g e r p o p u l a t i o n group. I n f o r m a t i o n  on the  tobacco chewers can,  i n c i d e n c e o f o r a l cancer  however, be  r e l a t i v e r i s k o f o r a l cancer  c o n t a i n i n g b e t e l quids (1933) was  XVII).  chewed per day  examined i n t h i s increased with  the number o f  i n I n d i a (Table XVI).  The  tobacco-  study by  carcinomas o f the o r a l c a v i t y and pharynx f a c e d by  i n the s o u t h e r n Reports o f the  United States i s also considerably  i n c i d e n c e o f o r a l cancer  r e p o r t e d f o r E a s t I n d i a n chewers and  U n i t e d S t a t e s . No  o r a l cancers  e t a l . , 1975). The  increased  Orr The  snuff (Table  among Canadian I n u i t and  d i f f e r from those  (Schaefer  thesis.  c a r r i e d out i n K e r a l a , the l o c a t i o n o f our r e s e a r c h p r o j e c t .  r i s k of developing dippers  and  found i n s e v e r a l e p i d e m i o l o g i c a l s t u d i e s of  p o p u l a t i o n groups comparable to those The  among s n u f f d i p p e r s  Indians  f o r snuff dippers  i n the  were r e p o r t e d i n the I n u i t over a 20-year p e r i o d  i n c i d e n c e o f o r a l cancer  i n snuff dipping  Indians  i s comparable to t h a t found i n the Canadian p o p u l a t i o n not engaging i n t h i s habit  ( N u t r i t i o n Canada, 1975b).  TABLE X V I Effect Oral  o f t h e D a i l y Number o f B e t e l Q u i d Chews on t h e R e l a t i v e R i s k f o r  Cancer  i n India  ( H i r a y a m a , 1966) a n d K e r a l a  Relative  Risk  ( O r r , 1933)  f o rOral  Cancer  No. B e t e l Q u i d s per  Day  India  Kerala  0  1.0  1.0  <2  8.5  8.1  3-5  14.5  12.9  >6  18.1  12.8  63.7  62.9  Retaining quid during sleep  TABLE  XVII  E f f e c t of Snuff  Dipping  Southern  States  Site  Gum  United  o f Cancer  and b u c c a l  O t h e r mouth  mucosa  and pharynx  on t h e R e l a t i v e R i s k (from  f o r O r a l Cancer  W i n n e t a l . , 1981)  Years o f S n u f f Use  Relative Risk  0  1.0  1-24  13.8  25-49  12.6  >50  47.5  0  1.0  1-24  1.7  25-49  3.8  >50  1.3  i n the  72  10. Intervention Strategies 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 can be o b t a i n e d by u s i n g anti-promoting,  anti-initiating,  or a n t i - p r o l i f e r a t i n g agents. A b e t t e r u n d e r s t a n d i n g  precursors of carcinogens, procarcinogens,  carcinogens  o f the  and promoters i n v o l v e d  i n n e o p l a s t i c t r a n s f o r m a t i o n s h o u l d h e l p i n the s e l e c t i o n o f chemopreventive agents. Our  r e s u l t s and  s t u d i e s from o t h e r l a b o r a t o r i e s p o i n t to the  n i t r o s a m i n e s as the main c a r c i n o g e n i c agents r e l e a s e d from smokeless They may  induce g e n e t i c anomalies i n the o r a l mucosa, and  development o f o r a l precancerous  they may  l e s i o n s and o r a l carcinomas  Ntobacco.  initiate  (IARC, 1985).  Based on these assumptions, the e f f i c a c y of f o u r chemopreventive agents examined: a s c o r b i c a c i d and c a f f e i c a c i d as n i t r i t e a f r e e r a d i c a l scavenger,  was  trappers, beta-carotene  and v i t a m i n A as an agent w i t h scavenging  and the c a p a c i t y to r e g u l a t e gene f u n c t i o n i n e p i t h e l i a l  the  cells.  as  potential  73  10.1 Nitrite Trapping Agents R e l a t i v e l y l a r g e amounts o f NPRO appeared  i n the u r i n e o f a v o l u n t e e r who  d i p p e d s n u f f and i n g e s t e d p r o l i n e s i m u l t a n e o u s l y ( F i g u r e 7 and 8; T a b l e X V I I I ) . The  e x c r e t e d NPRO c o u l d be formed endogenously, o r c o u l d be preformed and  r e l e a s e d from t h e tobacco. The s n u f f brand used had an NPRO c o n t e n t o f 14.0 /ig/g tobacco  (wet w e i g h t ) . To g a i n i n f o r m a t i o n on the amount o f NPRO formed  endogenously, the experiment  was r e p e a t e d w i t h the a d d i t i o n a l i n g e s t i o n o f  a s c o r b i c a c i d . The e x p e r i m e n t a l d e s i g n i s shown i n F i g u r e 5. The NPRO r e l e a s e d i n t h e u r i n e o f a s n u f f u s e r over an 11 h r p e r i o d was 26.1 ng when no ascorbate was used,  and t h i s was reduced  t o 7.8 ng when a s c o r b a t e was p r e s e n t a t a t o t a l  dose o f 1.5 g ( F i g u r e 8 ) . With a reduced  i n g e s t i o n o f ascorbate  the amount o f NPRO e x c r e t e d i n t h e u r i n e was reduced 14.9  (2 x 500 mg),  from 27.3 n& i n 11 h r s t o  ng ( F i g u r e 9 ) . The e f f i c a c y o f a s c o r b a t e and c a f f e i c a c i d i n r e d u c i n g the  endogenous f o r m a t i o n o f NPRO i s shown i n T a b l e  XVIII.  74 F i g u r e 7: The Appearence o f NPRO i n the U r i n e o f a V o l u n t e e r Who Dipped Snuff and Ingested P r o l i n e and Ascorbate ( f o r d e t a i l e d o u t l i n e , see F i g u r e 5C ) .  3-6n WITHOUT  ASCORBATE  3-2-  2-81 I 2-4H  (J  - 2-CH • rx  n  Z 1-6H WITH  1-2-  ASCORBATE  0-8I 0-4-  T"  0  T"  2  -r 8  4  6  TIME  ( HRS )  10  F i g u r e 8: The Appearence o f NPRO i n the U r i n e o f a Volunteer Who Dipped Snuff and Ingested P r o l i n e and Ascorbate ( f o r d e t a i l e d o u t l i n e , see F i g u r e 5 B ) .  "~1  WITHOUT ASCORBATE  WITH ASCORBATE  -r 2  T"  T"  6  8  T I M E CHPS)  10  76  TABLE XVIII Inhibitory E f f e c t of Ascorbate and Caffeic Acid on the Formation of i n a Snuff Dipping Volunteer  NPRO  NPRO (ng/ml)  Incubation Mixture  P  H  Saliva + 200 mg proline  2.5  Saliva + 200 mg proline + 200 mg ascorbate Saliva + 200 mg proline + 200 mg c a f f e i c acid  C, Copenhagen;  No Tobacco  Snuff SL a  Snuff B a  14 ,476  11,797  13,496  2.5  5,497  3,237  442  2.5  910  667  145  SL, Skoal;  60  Snuff C a  B, Big Horn  77 10.2 Inuit Snuff Dippers :Beta-carotene Chemopreventive  trials  on t o b a c c o - a r e c a nut chewers i n the P h i l i p p i n e s  have shown the c a p a c i t y o f b e t a - c a r o t e n e t o reduce the 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 c e l l s which were used as i n d i c a t o r s o f g e n e t i c damage i n the b u c c a l mucosa ( S t i c h e t a l . , p o p u l a t i o n group  1984a,b). A v i t a m i n A d e f i c i e n c y p r e v a i l e d i n the  of this intervention t r i a l ,  as was  e v i d e n t from a r e l a t i v e l y  h i g h i n c i d e n c e o f n i g h t b l i n d n e s s and x e r o p h t h a l m i a . The q u e s t i o n was whether I n u i t s n u f f d i p p e r s o f Gjoa Haven, who r e t i n o l , would respond i n the same way  have "normal"  raised  serum l e v e l s o f  to the o r a l a d m i n i s t r a t i o n o f b e t a -  carotene . Serum samples from 110 male I n u i t were a n a l y z e d f o r r e t i n o l and b e t a c a r o t e n e . S u b j e c t s were d i v i d e d i n t o those who  used smokeless  i n d i v i d u a l s ) and those who  (70 i n d i v i d u a l s ) . No  d i d not use tobacco  d i f f e r e n c e i n serum r e t i n o l or b e t a - c a r o t e n e was groups  ( T a b l e X I X ) . The  tobacco  (40'  significant  observed between the  two  r e t i n o l l e v e l s o f the I n u i t males examined were  comparable t o those found f o r Canadian males consuming a "normal"  Western-type  diet  fall  ( N u t r i t i o n Canada, 1975a). The observed b e t a - c a r o t e n e l e v e l s  s u b s t a n t i a l l y below those observed i n a Canadian p o p u l a t i o n . The serum b e t a - c a r o t e n e may  r e s u l t from an i n s u f f i c i e n t  low l e v e l s o f  intake of green-yellow  v e g e t a b l e s by the I n u i t o f Gjoa Haven. Serum r e t i n o l l e v e l s are i n the range due  t o the r e l a t i v e l y l a r g e i n t a k e o f s e a l and c a r i b o u meat and  "normal" liver.  TABLE  XIX  Serum L e v e l s o f R e t i n o l a n d B e t a - c a r o t e n e I n i t i a t i o n of the P r e v e n t i o n T r i a l  Habit  i n Male  Inuit  Prior  to  Number o f  Retinol  Beta-carotene  Subjects  (ng/ml)  (ng/ml)  Non-users  70  447  ± 104  57.4  ± 36.2  Tobacco users  40  463  ± 127  47.0  ± 27.6  Standard  deviations  P values carotene  calculated (P>0.40)  (±) are by  given  Student's  f o r number o f t-test  subjects  for retinol  and  shown beta-  79  S i n c e the d i s t r i b u t i o n p a t t e r n o f MNC w i t h i n the o r a l c a v i t y o f s n u f f d i p p e r s i s unequal MNC  (Stich,  1987), i t i s paramount f o r any comparative  f r e q u e n c i e s t o sample c e l l s  study o f  from p r e c i s e l y the same l o c a t i o n s w i t h i n the  mouth. I n t h i s study, e x f o l i a t e d c e l l s were c o l l e c t e d from the a r e a o f the mucosa a t the s i t e where the tobacco was kept. The r e s u l t s o f a 10-week a d m i n i s t r a t i o n o f b e t a - c a r o t e n e a r e shown i n F i g u r e 10, where the frequency o f MNC  b e f o r e treatment  i s p l o t t e d a g a i n s t t h a t f o l l o w i n g a d m i n i s t r a t i o n o f the  chemopreventive agent. The r e s u l t s i n d i c a t e an i n h i b i t o r y e f f e c t o f b e t a c a r o t e n e t r e a t m e n t . The average c a r o t e n e was 1.87% ± 0.92% a f t e r treatment MNC  frequency o f MNC  p r i o r t o treatment w i t h beta-  (n = 24), and d e c r e a s e d s i g n i f i c a n t l y  (P < 0.001)  t o 0.74% ± 0.42%. No s i g n i f i c a n t r e d u c t i o n i n the frequency o f  o c c u r r e d i n i n d i v i d u a l s who r e c e i v e d no treatment o r who were g i v e n a  placebo  ( F i g u r e 10). The average  week p e r i o d were 2.0% ± 0.94 MNC  f r e q u e n c i e s o f MNC  p r i o r t o and a f t e r the 10-  (n = 31) and 1.99% ± 0.80,  f r e q u e n c i e s were determined  respectively.  i n the o r a l mucosa o f 5 s n u f f u s e r s 8  months and 1 month p r i o r t o , a t the onset o f , and a f t e r treatment w i t h b e t a carotene  ( F i g u r e 11). The r e s u l t s i n d i c a t e t h a t the f r e q u e n c i e s o f MNC i n  i n d i v i d u a l s who use s n u f f d a i l y a r e r e l a t i v e l y s t a b l e . A f t e r a 10-week a d m i n i s t r a t i o n o f b e t a - c a r o t e n e , the frequency o f MNC  d e c r e a s e d i n the mucosa  o f the s n u f f d i p p e r s . A l l the s n u f f u s e r s c o n t i n u e d t h e i r u s u a l h a b i t throughout  the c o u r s e o f the study.  B e s i d e s the m i c r o n u c l e i , some e x f o l i a t e d c e l l s from the o r a l mucosa o f s n u f f u s e r s show a l o s s o f n u c l e i . The frequency o f a n u c l e a t e d c e l l s b e f o r e treatment was compared t o t h a t seen a f t e r treatment w i t h b e t a - c a r o t e n e . As can be seen from F i g u r e 12, no s i g n i f i c a n t changes o c c u r r e d .  80  The  r e s u l t s show t h a t b e t a - c a r o t e n e appears t o be an e f f i c i e n t i n h i b i t o r  of micronuclei,  which i n d i c a t e g e n e t i c  who do n o t s u f f e r from a v i t a m i n  damage i n the o r a l mucosa o f s n u f f  A deficiency.  users  81 F i g u r e 10: E f f e c t o f B e t a - c a r o t e n e Treatment on MNC (A) Frequency o f MNC  i n the O r a l Mucosa.  a t the o r a l s i t e where the tobacco i s l o c a t e d :  b e f o r e and a f t e r a 10-week i n g e s t i o n o f p l a c e b o c a p s u l e s : O, b e f o r e and a f t e r the 10-week t r i a l p e r i o d  (no t r e a t m e n t ) . (B) Changes i n the frequency  of MNC b e f o r e and a f t e r a 10-week a d m i n i s t r a t i o n o f b e t a - c a r o t e n e (180 mg/week).  %  B E F O R E  T « E A T M 6 I S J T  MtCnOMUCLEATeO CELLS  82  Figure  11:  Changes i n the Frequency of MNC a t the O r a l S i t e Where the Tobacco i s Located Over a P e r i o d P r e c e d i n g the P i l o t T r i a l and A f t e r a 10-Week A d m i n i s t r a t i o n of B e t a - c a r o t e n e (5 d i f f e r e n t I n u i t males u s i n g s n u f f )  —  i  5  FEB  i  i  l MAY  i  i  i AUG  MONTH  i  i  i NOV  i  83  F i g u r e 12: Frequency  of Anucleated E x f o l i a t e d  C e l l s from the O r a l  Where the Tobacco i s Located B e f o r e and A f t e r a 10-Week Administration  o f Beta-carotene  (180 mg/week).  Site  84  10.3 East Indian Tobacco Chewers: Beta-carotene and Beta-carotene plus Vitamin A Because o f the c l o s e a s s o c i a t i o n o f MNC  f r e q u e n c i e s , precancerous  oral  l e s i o n s and o r a l c a n c e r w i t h the h a b i t o f chewing t o b a c c o - c o n t a i n i n g b e t e l quids, p i l o t  i n t e r v e n t i o n t r i a l s were attempted  on a group o f E a s t I n d i a n  f i s h e r m e n . The o b j e c t i v e was t o i n v e s t i g a t e the p r o t e c t i v e e f f e c t o f b e t a c a r o t e n e and b e t a - c a r o t e n e p l u s v i t a m i n A on the l e v e l s o f m i c r o n u c l e a t e d o r a l mucosal c e l l s and on the i n c i d e n c e o f o r a l l e u k o p l a k i a s found i n t h i s The  group.  frequency o f MNC was e l e v a t e d i n areas o f o r a l l e u k o p l a k i a s and i n  normal-appearing  mucosa o f b e t e l q u i d chewers who p a r t i c i p a t e d i n the  intervention t r i a l  ( T a b l e XX). There were no s i g n i f i c a n t d i f f e r e n c e s i n MNC  f r e q u e n c i e s between samples o f e x f o l i a t e d c e l l s  taken from  normal-appearing  areas o f the b u c c a l mucosa and those c o l l e c t e d from r e g i o n s w i t h w e l l e s t a b l i s h e d l e u k o p l a k i a s . Swabs o f normal mucosa were taken a d j a c e n t to areas w i t h l e u k o p l a k i a . T h i s c l o s e sampling p a t t e r n was n e c e s s a r y s i n c e the frequency o f m i c r o n u c l e a t e d c e l l s can d i f f e r a t v a r i o u s l o c a t i o n s o f the o r a l mucosa. I t has been shown t h a t the frequency o f MNC  i s h i g h e s t i n areas where the b e t e l  q u i d o r tobacco p l u g comes i n c l o s e c o n t a c t w i t h the mucosa ( S t i c h e t a l . , 1982a,b; S t i c h and R o s i n , In the p l a c e b o group,  1985). the frequency o f MNC  d i d n o t change s i g n i f i c a n t l y  over the 3-month study p e r i o d ( F i g u r e 13; T a b l e XX). F o l l o w i n g the twice-weekly a d m i n i s t r a t i o n o f b e t a - c a r o t e n e and b e t a - c a r o t e n e p l u s v i t a m i n A, the frequency o f MNC  was found to be s i g n i f i c a n t l y reduced  areas o f normal-appearing  i n areas w i t h l e u k o p l a k i a and  mucosa ( T a b l e XX). The degree  l e u k o p l a k i a s and normal-appearing  o f MNC  reduction i n  mucosa was comparable. The f r e q u e n c i e s o f  m i c r o n u c l e i i n each i n d i v i d u a l b e f o r e and a f t e r b e t a - c a r o t e n e o r p l a c e b o a d m i n i s t r a t i o n were p a i r e d and compared i n a paired-sample The r e d u c t i o n i n MNC  Student's  t-test.  f o l l o w i n g the a d m i n i s t r a t i o n o f b e t a - c a r o t e n e and b e t a -  85  carotene  p l u s v i t a m i n A i n b o t h l e u k o p l a k i a s and n o r m a l - a p p e a r i n g mucosa i s  s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.001). The e f f e c t o f the treatment on the frequency  o f MNC  i n areas  p a r t i c i p a n t i s shown i n F i g u r e 13. The MNC tobacco/betel beta-carotene.  beta-carotene  o f l e u k o p l a k i a o f each frequency  trial  o f a t l e a s t one  q u i d chewer d i d n o t respond t o the 3-month a d m i n i s t r a t i o n o f Comparable r e s u l t s were o b t a i n e d when b e t a - c a r o t e n e  A were a d m i n i s t e r e d  f o r 3 months (Table XX; F i g u r e 13).  and v i t a m i n  F i g u r e 13:  Response of MNC i n Areas of L e u k o p l a k i a to a 3-Month A d m i n i s t r a t i o n of B e t a - c a r o t e n e and Beta-carotene P l u s V i t a m i n A v s . Placebo  87  TABLE  XX  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 C e l l s i n O r a l L e u k o p l a k i a s a n d i n Normal Mucosa o f T o b a c c o / B e t e l Q u i d Chewers B e f o r e and A f t e r t h e A d m i n i s t r a t i o n o f C h e m o p r e v e n t i v e A g e n t s f o r 3 Months  Leukoplakia Number o f Individuals  Treatment  After  Before  Normal Mucosa Before  After  Placebo  30  3.60±1.22  4.00±1.32  4.10±1.54  3.83±1.23  Beta-carotene  31  4.09±1.01  1.18±0.77  4.1111.48  1.01±0.71  51  4.01±1.05  1.16±0.94  4.18±0.78  1.2210.88  Beta-carotene vitamin A  Significant  +  a t P<0.01 w i t h S t u d e n t ' s  t-test:  Beta-carotene treatment: micronucleus frequencies before v s . a f t e r s u p p l e m e n t a t i o n f o r a r e a s w i t h l e u k o p l a k i a , and f r e q u e n c i e s b e f o r e v s . a f t e r s u p p l e m e n t a t i o n f o r a r e a s o f normal mucosa; beta-carotene + vitamin A treatment: before v s . a f t e r supplementation, values f o r both l e u k o p l a k i a s and normal mucosa  88  A p p r o x i m a t e l y 48%  o f a l l examined a d u l t chewers who  i n h a b i t the c o a s t  K e r a l a , near Trivandrum, South I n d i a , had w e l l - e s t a b l i s h e d l e u k o p l a k i a s , d e f i n e d by The  the WHO  C o l l a b o r a t i n g Centre f o r O r a l Precancerous L e s i o n s  i n t r a o r a l d i s t r i b u t i o n of leukoplakias  among 160  8.4%;  l i p , 4.4%;  according  others,  1.4%.  to t h e i r appearance was  l e u k o p l a k i a , 2.9%;  erythematous a r e a s .  cannot be  associated with  e n t i r e treatment p e r i o d . As  at  taken, the  and  i t is difficult  are due  chemopreventive agents caused by  also occurred (Tables XXI  leukoplakias  recording of  t h e r e a f t e r each month d u r i n g  l o c a t i o n with  the  of b e t e l  time (Gupta et  to d e c i d e whether these  to changes i n the h o l d i n g o f the b e t e l q u i d  seasonal  of o r a l leukoplakias  i n g e s t i o n of  dietary variations.  and  the development o f new  among the t r i a l p a r t i c i p a n t s who  r e c e i v e d placebo  and X X I I ) . A f t e r t h r e e months o f b e t a - c a r o t e n e and  plus vitamin A administration,  recorded  leukoplakias  described previously, oral leukoplakias  1986). A t p r e s e n t ,  remission  speckled  leukoplakia  d i f f e r e n t s i t e s w i t h i n the o r a l c a v i t y , or to changes i n the  The  tongue,  appearance o f each o r a l l e u k o p l a k i a were  q u i d chewers have a tendency to change i n s i z e and  "spontaneous" r e g r e s s i o n s  as  a p a r t i c u l a r histopathological pattern.  l o c a t i o n , s i z e and  1980,  Only one  S i n c e b i o p s i e s were not  done p r i o r to the o n s e t o f the t r i a l ,  al.,  (1978).  leukoplakias  : homogeneous l e u k o p l a k i a , 83%;  by marking them on a c h a r t and by photographs. The was  9.3%;  frequency d i s t r i b u t i o n o f 160  and v e r r u c o u s l e u k o p l a k i a , 5.7%.  contained  The  The  as  chewers i n K e r a l a was  f o l l o w s : b u c c a l mucosa, 53.4%; commissure, 23.3%; r e t r o m o l a r ,  of  the percentage o f l e u k o p l a k i a s  leukoplakias  capsules beta-carotene  that  regressed  was  comparable to t h a t found i n the p l a c e b o group, whereas the development of  new  l e u k o p l a k i a s was  already  s i g n i f i c a n t l y reduced. A f t e r a 6-month treatment  p e r i o d , a s i g n i f i c a n t d i f f e r e n c e between the two new  leukoplakias)  and  t r e a t e d groups ( r e m i s s i o n  the p l a c e b o group became e v i d e n t  (Table  XXII).  and  89  TABLE XXI  Response of Oral Leukoplakias to the Administration of Beta-carotene or Beta-carotene plus Vitamin A for 3 Months  Individuals with  Treatment  Number of Individuals with Oral Leukoplakia  Remission Number (%)  No Change Number (%)  New Leukoplakia Number  (%)  Placebo  33  1  3.0  25 75.8  7  21.2  Beta-carotene (180 mg/week)  35  3  8.6  25 71.4  7  20.0  Beta-carotene (180 mg/week) + vitamin A (100,000 IU/week)  51  4  7.8  46 90.2  1  2.0  Test f o r s i g n i f i c a n t difference i n outcome between treatment groups after 3 months of supplementation: chi-square = 10.03; df = 4; P = 0.04  90  TABLE  XXII  Response o f O r a l L e u k o p l a k i a s t o t h e A d m i n i s t r a t i o n o f B e t a - c a r o t e n e B e t a - c a r o t e n e p l u s V i t a m i n A f o r 6 Months  Individuals  Treatment  Number o f Individuals with Oral Leukoplakia  Remission Number  No C h a n g e  (%)  Number  (%)  or  with New  Leukoplakia . Number (%)  Placebo  33  1  3.0  25  75.8  7  21.2  Beta-carotene (180 mg/week)  27  4  14.8  19  70.4  4  14.8  Beta-carotene (180 mg/week) + vitamin A (100,000 IU/week)  51  14  27.5  33  64.7  4  7.8  T e s t f o r s i g n i f i c a n t d i f f e r e n c e i n outcome between t r e a t m e n t groups a f t e r 6 months o f s u p p l e m e n t a t i o n : c h i - s q u a r e =10.1; d f = 4; P = 0.38 P v a l u e s (2-sided) by F i s h e r ' s e x a c t t e s t : Placebo v s . beta-carotene: r e m i s s i o n , P=0.16; new l e u k o p l a k i a s , P=0.53. Placebo v s . beta-carotene + v i t a m i n A: r e m i s s i o n , P=0.004; new l e u k o p l a k i a s , P=0.08. Beta-carotene v s . b e t a - c a r o t e n e + v i t a m i n A: r e m i s s i o n , P=0.26; new l e u k o p l a k i a s ,  P=0.44  91  P r i o r t o , once d u r i n g , and a t the end o f the t r i a l , number o f t o b a c c o - c o n t a i n i n g b e t e l quids chewed p e r day,  we  examined the  the l e n g t h o f the  chewing p e r i o d , and the a l c o h o l consumption and smoking p a t t e r n o f a l l p a r t i c i p a n t s . These o r a l h a b i t s d i d n o t change d u r i n g the c o u r s e o f the It  trial.  can t h e r e f o r e be assumed t h a t a l l the p a r t i c i p a n t s were exposed to  a p p r o x i m a t e l y the same types and l e v e l s o f c a r c i n o g e n i c and mutagenic  agents  r e l e a s e d from tobacco and o t h e r b e t e l q u i d i n g r e d i e n t s .  10.4 East Indian Tobacco Chewers: Vitamin A The  c a p a c i t y o f v i t a m i n A to reduce  the 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 b u c c a l  mucosal c e l l s o f tobacco chewers has been p r e v i o u s l y r e p o r t e d ( S t i c h and Rosin, 1984a). The  o b j e c t i v e o f t h i s study was  to determine  the e f f e c t s o f v i t a m i n A  on the r e m i s s i o n o f o r a l l e u k o p l a k i a s . T h i s s h o r t - t e r m p i l o t  t r i a l was  carried  out on f i s h e r m e n l i v i n g a l o n g the c o a s t near Trivandrum,  Kerala. Questionnaires  on chewing, smoking and d r i n k i n g p a t t e r n s were completed  a t the onset o f the  trial,  a f t e r t h r e e months, and a t the end o f the six-month  treatment p e r i o d .  The  r e s u l t s p e r m i t t e d us t o draw the c o n c l u s i o n t h a t the v a r i o u s o r a l h a b i t s  did  not change throughout  study chewed an average Each q u i d was  the course o f the study. The p a r t i c i p a n t s  o f 13.1  (SD =9.1)  kept i n the mouth f o r 26.1  t o b a c c o - c o n t a i n i n g quids per (SD = 25.4)  min.  Exposure  and a r e c a n u t - r e l a t e d c a r c i n o g e n s amounted to a p p r o x i m a t e l y To ensure  i n this  to tobacco-  s i x hours  each  t h a t a l l the t r i a l p a r t i c i p a n t s r e c e i v e d the d e s i r e d doses,  p l a c e b o and v i t a m i n A c a p s u l e s were a d m i n i s t e r e d twice weekly under the s u p e r v i s i o n o f a l o c a l nurse and a s t a f f member o f the R e g i o n a l Cancer i n Trivandrum Due  o r myself, who  to t h i s approach,  regime was  day.  checked  day. the  strict Centre  t h a t the c a p s u l e s were a c t u a l l y swallowed.  an e x c e l l e n t compliance  w i t h the v i t a m i n a d m i n i s t r a t i o n  a c h i e v e d . Only 4 o f 39 v i t a m i n A o r p l a c e b o doses  ( s i x months) were  92  m i s s e d among 6% o f the p a r t i c i p a n t s , and 2 of.39 doses were missed by 14% o f all  t r i a l participants.  Despite  the continuous  exposure t o c a r c i n o g e n s , the  a d m i n i s t r a t i o n o f 200,000 IU o f v i t a m i n A, r e s u l t e d i n a pronounced of o r a l leukoplakias any  remission  (Table X X I I I ) . T h i s amount o f v i t a m i n A d i d n o t produce  d e t e c t a b l e adverse e f f e c t s d u r i n g the t r i a l p e r i o d , such as dryness o f the  l i p s and mouth, changes i n the s k i n ( s c a l i n g o f palms), headaches o r d i z z i n e s s . New o r a l l e u k o p l a k i a s developed throughout the t r i a l of tobacco-containing  b e t e l quids.  I t was t h e r e f o r e p o s s i b l e t o examine the  e f f e c t o f v i t a m i n A treatment on the f o r m a t i o n group r e c e i v i n g the p l a c e b o ,  p e r i o d among chewers  o f new l e u k o p l a k i a s . I n the  7 (21%) new l e u k o p l a k i a s were formed, whereas no  new l e u k o p l a k i a s developed i n the group r e c e i v i n g v i t a m i n A over the six-month t r i a l p e r i o d (Table  XXIII).  9,3  TABLE  XXIII  Response o f O r a l L e u k o p l a k i a s  to the Administration of Vitamin A f o r  6 Months  Number o f Individuals with Treatment  Oral Leukoplakia  Remission Number  Individuals  with  No C h a n g e  New  Leukoplakia  (%)  Number  (%)  Number  (%)  Placebo  33  1  3.0  25  75.8  7  21.2  Vitamin A  21  12  57.1  9  42.9  0  0.0  • (200,000 I U / week)  T e s t f o r s i g n i f i c a n t d i f f e r e n c e i n outcome between t r e a t m e n t groups a f t e r 6 months o f s u p p l e m e n t a t i o n : c h i - s q u a r e =22.27; d f = 3; P = 0.000015 P v a l u e s (2-sided) by F i s h e r ' s e x a c t t e s t : P l a c e b o v s . v i t a m i n A: r e m i s s i o n , P=0.0000089. P l a c e b o v s . v i t a m i n A: new l e u k o p l a k i a , P=0.024  94  DISCUSSION The o b j e c t i v e s o f t h i s r e s e a r c h were t o compare the usage o f tobacco among t h r e e communities a n a l y z e smokeless  tobacco samples,  etiological factors, may  1.  ( I n u i t , Canadian  smokeless  I n d i a n and E a s t I n d i a n ) , to  and s a l i v a and u r i n e o f chewers f o r v a r i o u s  and t o i n v e s t i g a t e p o s s i b l e chemopreventive  agents which  reduce the o r a l l e s i o n s caused by the use o f s n u f f or chewing tobacco.  U s e of S m o k e l e s s T o b a c c o Although a s t r i c t  the d a t a accumulated each group: daily,  random sampling o f each p o p u l a t i o n was  n o t c a r r i e d out  do i n d i c a t e a l a r g e p r e v a l e n c e o f smokeless  86% o f the male Canadian  tobacco use by  I n d i a n s examined s a i d they used  as d i d 50% o f the females. In the I n u i t p o p u l a t i o n , 70% o f the males  responded  t h a t they used s n u f f d a i l y ,  as d i d 4.2%  o f females, a l t h o u g h t h i s  h a b i t i s s o c i a l l y u n a c c e p t a b l e f o r t h i s l a t t e r group.  In the E a s t I n d i a n  p o p u l a t i o n where b e t e l q u i d chewing i s a c c e p t a b l e f o r b o t h males and r o u g h l y 57% o f those q u e s t i o n e d responded In  t h i s study we  h a b i t s which may  a l s o attempted  males and 10.4%  t h a t they p r a c t i s e d t h i s  to determine  exert a carcinogenic effect,  c i g a r e t t e smoking. In the Canadian  1979),  the p r e v a l e n c e o f o t h e r o r a l  such as a l c o h o l d r i n k i n g  and of  chew b e t e l q u i d . C o n s i d e r i n g the  the p o s s i b i l i t y  and c i g a r e t t e smoking (Walters  t h a t a s i m i l a r e f f e c t may  and s n u f f o r chewing tobacco cannot be e x c l u d e d . The d i p p i n g , a l c o h o l d r i n k i n g and c i g a r e t t e smoking was males and 21.5%  habit.  o f females combine the d r i n k i n g o f a l c o h o l w i t h s n u f f d i p p i n g ,  s y n e r g i s t i c a c t i o n between a l c o h o l consumption al.,  females,  I n d i a n p o p u l a t i o n , a p p r o x i m a t e l y 26.3%  as do 37% o f the E a s t I n d i a n males who  et  snuff  o f females among the Canadian  o c c u r between a l c o h o l  combination o f s n u f f found to o c c u r i n 35.1%  I n d i a n s s t u d i e d . The  of  influence of  95  smoking can be  seen from the o b s e r v a t i o n t h a t the h i g h e s t p r e v a l e n c e  l e u k o p l a k i a occurs  among i n d i v i d u a l s who  smoking c i g a r e t t e s ( S t i c h and R o s i n ,  chewed by each group: 0.61  Indians  and  1.1  are c o n c u r r e n t l y u s i n g "nass"  g by  g by the E a s t Indians,  the r e l a t i v e l y c o n s i s t e n t weight o f the I n u i t , suggesting  1.5  g by  the Canadian  that other modifying  must be a t work which i n f l u e n c e the d i f f e r e n t o r a l cancer these  and  1984).  A l s o o f i n t e r e s t i n t h i s study was tobacco  of  effects  r a t e s seen amongst  groups.  2. Precursors of Nitrosamines Contained in Smokeless Tobacco Samples and the Saliva of Their Users To  i n v e s t i g a t e the p r o b a b l e  tobacco,  we  e t i o l o g i c a l factors contained  f o c u s e d on the N - n i t r o s o compounds and  t h e i r precursors since  are the o n l y known c a r c i n o g e n i c compounds c o n t a i n e d mixtures  chewed throughout the w o r l d  Nitrite  i n smokeless  tobacco  l e v e l s were examined i n a v a r i e t y o f smokeless tobacco  (U.S.),  f o u r "nass"  samples ( mixtures  o i l p l a c e d under the tongue by  samples,  common b r a n d o f chewing  o f tobacco,  inhabitants i n southern  Union, A f g h a n i s t a n and P a k i s t a n ) , f o u r K h a i n i tobacco  s l a k e d lime, ash  p r e p a r a t i o n s w i d e l y used and  used i n the p r e p a r a t i o n o f b e t e l q u i d s . R e l a t i v e l y h i g h  of n i t r i t e were found  East  levels  i n t h r e e p o p u l a r brands o f s n u f f commonly used by  n o r t h e r n Canadian n a t i v e s . The  amounts o f n i t r i t e  samples, which v a r i e d from 95 to 1,040 Samarkand ( U z b e k i s t a n , g r e a t l y exceed those  and  r e g i o n s o f the S o v i e t  i n s e v e r a l e a s t e r n s t a t e s o f I n d i a (e.g., B i h a r , O r i s s a and B e n g a l ) , I n d i a n tobacco  these  (IARC, 1985).  i n c l u d i n g f i v e common brands o f s n u f f (Canada), one tobacco  i n smokeless  S o v i e t Union),  found  mg/kg, and  i n the t h r e e Canadian s n u f f i n f o u r "nass"  samples from  which v a r i e d from 70 to 200 mg/kg,  i n s e v e r a l food products  such as n i t r i t e - c u r e d bacon  96  (12-42 mg/kg), n i t r i t e - c u r e d ham (16-37 mg/kg), smoked sausages (18-31 mg/kg) (Kawabata e t a l . , 1984), o r Chinese cabbage (Walters  e t a l . , 1979).  To a r r i v e a t an e s t i m a t i o n o f n i t r i t e which c o u l d c o n c e i v a b l y  e n t e r the  d i g e s t i v e system o f s n u f f o r nass u s e r s , we c a n assume a s a l i v a f l o w o f 16 ml per 20 min (Anderson and K r i n s k y ,  1973) and a c o n c e n t r a t i o n o f 0.2 mg n i t r i t e  per ml o f s a l i v a . Based on these assumptions, an e s t i m a t e d will  3.2 mg o f n i t r i t e  e n t e r the stomach d u r i n g a 20-min p e r i o d o f tobacco use i f the s n u f f  d i p p e r swallows r a t h e r than e x p e c t o r a t e s  the s a l i v a . Furthermore, assuming t h a t  the tobacco i s k e p t w i t h i n the mouth f o r 3 h r s p e r day, nitrite  approximately  29 mg o f  would e n t e r the stomach i n a 24-hr p e r i o d . T h i s amount i s c o n s i d e r a b l y  l a r g e r than the 4.2 mg o f n i t r i t e food p r o d u c t s  and beverages  Considerable  i n g e s t e d by an i n d i v i d u a l p e r day from  solid  (Kawabata e t a l . , 1984).  c o n c e r n has been expressed  e f f e c t s of tobacco-related nitrosamines  about the p o s s i b l e c a r c i n o g e n i c  (Hoffmann and Hecht, 1985) which are  formed d u r i n g tobacco p r o c e s s i n g  (Andersen and Kasperbauer, 1984). The  p o s s i b i l i t y cannot be c o m p l e t e l y  i g n o r e d t h a t the r e l a t i v e l y l a r g e doses o f  nitrite  r e l e a s e d i n t o t h e s a l i v a o f s n u f f d i p p e r s c o u l d pose an even g r e a t e r  hazard.  By u s i n g l e v e l s o f e x c r e t e d NPRO, i n f o r m a t i o n about the n i t r o s a t i o n  a c t i v i t y w i t h i n man c a n be o b t a i n e d 1982). U s i n g  (Ohshima and B a r t s c h ,  1981; Ohshima e t a l . ,  t h i s method, r e l a t i v e l y h i g h l e v e l s o f NPRO were found i n the  urine o f s e v e r a l snuff dippers, suggesting  an i n c r e a s e d endogenous n i t r o s a t i o n  c a p a c i t y . Thus the d a i l y use o f n i t r i t e - c o n t a i n i n g s n u f f c o u l d l e a d to the endogenous n i t r o s a t i o n o f a g r e a t many i n g e s t e d d i e t a r y compounds. s t u d i e s have shown t h e f o r m a t i o n  o f s t r o n g l y mutagenic, and by i m p l i c a t i o n  c a r c i n o g e n i c compounds f o l l o w i n g the n i t r o s a t i o n o f meat p r o d u c t s al.,  Several  (Marquardt e t  1977; Tomita e t a l . , 1984), beverages ( L i n and T a i , 1980), v e g e t a b l e s (Van  Der Hoeven e t a l . , 1984) and s p i c e s (Namiki e t a l . , 1983). The t o b a c c o - r e l a t e d  97  nitrosamines  (Hecht e t a l . , 1984; Hoffmann and Hecht, 1985) and p o s s i b l y  tobacco-derived N-nitroso containing  mutagens (Whong e t a l . , 1985) may r e p r e s e n t  o n l y one group o f  compounds which c o u l d c o n c e i v a b l y be formed i n u s e r s  f a c t o r a f f e c t i n g t h e endogenous f o r m a t i o n  of N-nitroso  l e v e l s may be o n l y one compounds ( M i r v i s h ,  1985) , and t h a t a n e g l e c t o f t h e i n t e r a c t i o n s between n i t r i t e s c a v e n g i n g c h e m i c a l s c o u l d l e a d t o erroneous c o n c l u s i o n s T h i s argument i s undoubtedly a p p l i c a b l e to users expected, the a d d i t i o n o f a s c o r b a t e  snuff dipper volunteer  of nitrite-  tobacco.  I t was r e c e n t l y p o i n t e d o u t t h a t s a l i v a r y n i t r i t e  As  other  i n h i b i t e d the i n v i t r o  and numerous  (Forman e t a l . , 1985).  of n i t r i t e - c o n t a i n i n g snuff.  t o a mixture o f p r o l i n e and s a l i v a o f a  formation  o f NPRO. A s c o r b a t e taken by a  b o t h c o n c u r r e n t l y and s h o r t l y a f t e r the use o f s n u f f and p r o l i n e  reduces the e x c r e t i o n o f NPRO. I t c o u l d be s p e c u l a t e d  that the observed  d i f f e r e n c e s i n u r i n a r y NPRO l e v e l s between I n u i t (Northwest T e r r i t o r i e s ) and Indian  (Saskatchewan) s n u f f u s e r s  vegetables  and f r u i t s ,  scavenging phenolics al.,  1984c). T h i s  i s due t o d i f f e r e n c e s i n t h e i n t a k e o f  which a r e the major source o f a s c o r b i c a c i d and n i t r i t e -  ( P i g n a t e l l i e t a l . , 1982; S t i c h and R o s i n ,  i s s u p p o r t e d by the o b s e r v a t i o n  1984b; S t i c h e t  t h a t the p e r c e n t a g e o f I n u i t  males i n the 10-54 y e a r age range who show severe v i t a m i n C d e f i c i e n c i e s i s s i g n i f i c a n t l y higher  than t h a t o f Indians  o f comparable age ( N u t r i t i o n Canada,  1975b) . Whether the w i d e s p r e a d use o f smokeless tobacco among i n h a b i t a n t s o f A r c t i c regions,  combined w i t h  a d e f i c i e n t i n t a k e o f n i t r i t e - t r a p p i n g compounds,  makes them more prone t o t h e development o f o r a l cancer c a n o n l y be a s c e r t a i n e d by a d e t a i l e d e p i d e m i o l o g i c a l  study.  98  3. Smokeless Tobacco and Ingestion of N-Nitrosamines  R e l a t i v e l y high  l e v e l s o f TSNA, i n c l u d i n g NNN,  NAT+NAB and NNK,  were found  i n the s a l i v a o f I n u i t d u r i n g a 15-min s n u f f t a k i n g p e r i o d . An e s t i m a t e , on the r e s u l t s p r e s e n t e d , a snuff dipper TSNA per day,  1 ml o f s a l i v a o f the I n u i t s n u f f d i p p e r , per d i p p i n g p e r i o d , and  then the amount o f NNN  i n g e s t e d s h o u l d be  day.  The  XIV)  shows an i n t a k e o f a p p r o x i m a t e l y 899  r e s u l t i n g repeated  must be  added to the  the amount o f n i t r o s a m i n e s  ng NNN.  The  e r  day),  nitrosamines  can be  r e s u l t s of bioassays  (G13,  185  ng o f NNN,  i n g e s t e d d a i l y by  Table  ng P ^ e  absorbed from the s a l i v a  ng o f NAT+NAB and  making a t o t a l o f 444 snuff dippers day),  ng P  e r  ng-  exceeds by  Thus  e a t i n g c u r e d meat p r o d u c t s day)(Kawabata e t a l . , 1984).  e t a l . , 1984;  Nand  the  Hecht e t a l . ,  the c o n t r i b u t i o n o f N - n i t r o s a m i n e s to  f o r o r a l cancer.  10  f a r that  (Hoffmann and Adams, 1981)  f o r c a r c i n o g e n i c i t y (Peto  make i t p o s s i b l e to estimate  do  extreme  r e c o g n i t i o n o f the f a c t t h a t c a r c i n o g e n i c  increased r i s k of snuff dippers  per  g a i n a more complete  a p p r o x i m a t e l y 249  or u s i n g cosmetics (0.41  a n a l y t i c a l data,  ng  o f 185  per  Most o f the s a l i v a produced  i n s n u f f d i p p e r s . To  swallowed through d r i n k i n g beer (0.34  1983)  i n the o r d e r  ml  s p i t t i n g which i s so common among A s i a t i c  p i c t u r e o f the amount o f TSNA i n g e s t e d ,  ng P  ng  per  swallowed, s i n c e most I n u i t s n u f f d i p p e r s  b e t e l q u i d chewers does not occur  ng o f NNK  dips  a s a l i v a f l o w o f 5.83  or o n l y o c c a s i o n a l l y , s p i t d u r i n g a d i p p i n g s e s s i o n . The  s a l i v a t i o n and  (0.17  an average o f 6.5  same c a l c u l a t i o n a p p l i e d to a more i n t e n s i v e s n u f f u s e r  during snuff taking i s probably not,  swallowed by  are by no means n e g l i g i b l e . Assuming an average l e v e l o f 980  an average o f 25 min  5 min,  r e v e a l s t h a t the amounts o f n i t r o s a m i n e s  based  the  Peto e t a l . (1984) showed t h a t  1 mg/kg N - n i t r o s o d i m e t h y l a m i n e (NDMA) or N - n i t r o s o d i e t h y l a m i n e  (NDEA)  99  administered  i n d r i n k i n g water caused l i v e r neoplasms i n 23% and 42% o f r a t s ,  r e s p e c t i v e l y . These i n c i d e n c e r a t e s were s i g n i f i c a n t l y h i g h e r  than those  i n the  c o n t r o l s . I n a d d i t i o n , 1 mg/kg NDEA caused eosophageal tumors i n 27% o f male r a t s and none i n c o n t r o l animals.  The t o t a l dose a d m i n i s t e r e d  t o the animals  d u r i n g t h e i r l i f e t i m e was about 37 mg/kg f o r males and 64 mg/kg f o r females. T h i s study  s u g g e s t e d the e x i s t e n c e o f a l i n e a r dose-response r e l a t i o n s h i p i n  the dose range o f 0.033 t o 1 mg/kg. S i n c e NNK l e v e l s  i n m o i s t s n u f f o f the f o u r  Canadian brands average g r e a t e r than 2.5 Mg/kg (Table X I I ) , 30 y e a r s o f exposure f o r average s n u f f d i p p e r s , who consume about 10 gm o f s n u f f d a i l y , amounts to 270 mg NNK o r about 3.9 mg/kg. I n a d d i t i o n , s n u f f d i p p e r s a r e exposed t o about 900 mg NNN (±13 mg/kg), 800 mg NAT (±11.4mg/kg) and 56 mg NAB (±0.8  mg/kg) d u r i n g a 30-year span. These e s t i m a t e s ,  response study probably  together with  the dose-  i n r a t s c i t e d above and the f a c t t h a t N - n i t r o s a m i n e s a r e  formed d u r i n g s n u f f d i p p i n g , s t r o n g l y suggest t h a t N - n i t r o s a m i n e s p l a y  a major r o l e i n the i n c r e a s e d o r a l cancer hypothesis  i s supported  of 1 mg NNK induces  r i s k of snuff dippers.  This  by the r e c e n t o b s e r v a t i o n t h a t a s i n g l e a d m i n i s t r a t i o n  a s i g n i f i c a n t number o f tumors i n S y r i a n golden hamsters  (Hecht e t a l . , 1983).  4. The Unfinished Search for the Factors Involved in Oral Carcinogenesis  The  comparative study  o f tobacco u s e r s  among I n u i t , I n d i a n and E a s t  Indian  p o p u l a t i o n groups was i n i t i a t e d i n the hope o f r e v e a l i n g the main c a r c i n o g e n i c f a c t o r s i n v o l v e d i n the development o f o r a l precancerous l e s i o n s and o r a l cancer.  However, the c o m p l e x i t y  prevented  us from f i n d i n g simple  of carcinogenesis answers.  and i t s e t i o l o g i c a l f a c t o r s  100  The  examined groups a t e l e v a t e d r i s k f o r o r a l cancer  engage i n a h a b i t  t h a t l e a d s t o an i n c r e a s e d i n g e s t i o n o f TSNA. I n a l l these groups the frequency of micronucleated  cells  i s a l s o i n c r e a s e d . These u s e r s o f smokeless  i n c l u d e s n u f f d i p p e r s i n the U n i t e d S t a t e s and Canada, "nass" S o v i e t Union, and tobacco  tobacco  chewers i n the  chewers i n the U n i t e d S t a t e s and I n d i a . Moreover,  TSNA a l s o appear i n the s a l i v a o f c i g a r e t t e smokers and " r e v e r s e " smokers, who h o l d the b u r n i n g  end o f the c i g a r e t t e i n the mouth. A d i f f i c u l t y a r i s e s when  q u a n t i t a t i v e r e l a t i o n s h i p s between TSNA l e v e l s and o r a l precancerous o r a l cancer differ  l e s i o n s or  a r e b e i n g sought. F o r example, v a r i o u s groups o f s n u f f d i p p e r s can  significantly  i n t h e i r TSNA l e v e l s . L e v e l s o f NNN found  among I n u i t  s n u f f d i p p e r s s i g n i f i c a n t l y exceed those d e t e c t e d i n the s a l i v a o f s n u f f d i p p i n g American women (Hoffmann and Adams, 1981). Approximately I n u i t exceeded 420 ppb NNN, which was the h i g h e s t l e v e l found the American d i p p e r s , and 22% o f the I n u i t reached  65% o f the  i n the s a l i v a o f  l e v e l s exceeding  2,100 ppb.  L e v e l s o f NNN i n the s a l i v a o f s n u f f d i p p i n g I n u i t were a l s o h i g h e r than the 16.5  t o 59.7 M g / l found m  i n I n d i a n tobacco  chewers ( N a i r e t a l . , 1985), o r the  1.2 t o 31.0 /ig/ml and 1.6 t o 14.7 Mg/™- i n u s e r s o f tobacco quids  containing betel  ( N a i r e t a l . , 1985; Wenke e t a l . , 1984). The i n c i d e n c e o f o r a l  cancer,  however, does n o t f o l l o w t h i s p a t t e r n . The h i g h e s t i n c i d e n c e i s among chewers of tobacco-containing b e t e l quids southern United States  (IARC, 1985) f o l l o w e d by s n u f f d i p p e r s i n the  (IARC, 1985). The i n c i d e n c e o f o r a l cancer  s n u f f d i p p e r s who have the h i g h e s t l e v e l s o f TSNA i n t h e i r tobacco  among I n u i t samples and  t h e i r s a l i v a must be v e r y low s i n c e no o r a l carcinomas were seen among the 180 cancers  diagnosed  between 1949 and 1974 i n the I n u i t o f the Canadian A r c t i c  ( S c h a e f e r e t a l . , 1975). Moreover, the frequency cells  of micronucleated  i n the s n u f f d i p p i n g I n u i t i s lower (1.8%) than i n o t h e r  groups: 4.4% i n the f l o o r o f the mouth o f "nass"  users  o r a l mucosal  tobacco-using  i n Uzbekistan,  8.4% i n  101  the b u c c a l mucosa o f t o b a c c o / b e t e l q u i d chewers o f O r i s s a , I n d i a ( S t i c h e t a l . , 1982a,b), 4.8%  i n t o b a c c o / b e t e l q u i d chewers o f the P h i l i p p i n e s  1984a,b), and 2.8% tobacco  (Mirvish,  The  (Stich et a l . ,  i n the mucosa o f the lower groove o f I n d i a n u s e r s o f K h a i n i 1982).  l a c k o f any simple q u a n t i t a t i v e r e l a t i o n s h i p between the amount o f  c a r c i n o g e n s i n g e s t e d by a tobacco u s e r and the f r e q u e n c y o f g e n e t i c l e s i o n s (MNC), l e u k o p l a k i a and cancer i n the t a r g e t t i s s u e p o i n t s to the involvement o f m o d u l a t i n g f a c t o r s . One l e s i o n s are due  c o u l d s p e c u l a t e t h a t the observed d i f f e r e n c e s i n o r a l  to d i f f e r e n t i n t a k e s o f d i e t a r y v i t a m i n A and/or b e t a - c a r o t e n e .  Many A s i a t i c p o p u l a t i o n s are a f f l i c t e d w i t h a v i t a m i n A d e f i c i e n c y ,  as  suggested by the o c c u r r e n c e o f x e r o p h t h a l m i a and n i g h t b l i n d n e s s . Subnormal l e v e l s o f r e t i n o l were a c t u a l l y observed i n Uzbekis  (Zardize et a l . ,  1985),  and  i n P a k i s t a n i s and I n d i a n s a t e l e v a t e d r i s k f o r o r a l cancer (Ibrahim e t a l . , 1977;  Wahi e t a l . ,  1965). Another p o s s i b i l i t y worth c o n s i d e r i n g i s t h a t the  a f o r e m e n t i o n e d d i f f e r e n c e s i n the f r e q u e n c y o f p r e n e o p l a s t i c changes or carcinomas may  r e s u l t from d i f f e r e n c e s i n the c o m p o s i t i o n o f the chewing  m i x t u r e s . Recent  s t u d i e s i n our l a b o r a t o r y have shown t h a t aqueous e x t r a c t s o f  a r e c a nut enhance the f o r m a t i o n o f t r a n s f o r m e d f o c i DNA  t r a n s f o r m a t i o n assay ( S t i c h and Tsang,  s t r a i n of p l a t y f i s h  ( S t i c h and Anders,  1989),  1989)  i n a bovine p a p i l l o m a v i r u s  and induce melanomas i n a  which responds  to tumor  promoters  r a t h e r than t o tumor i n i t i a t o r s . The promoting  a c t i v i t y o f the a r e c a nut  e x t r a c t was  DNA  by no means n e g l i g i b l e .  In the BPV  comparable t o the a c t i v i t y o f a p p r o x i m a t e l y 0.5 promoter.  T h i s promoting  t r a n s f o r m a t i o n assay, i t was  mg/ml mezerein,  a c t i v i t y o f a r e c a nut compounds may  the e x c e p t i o n a l l y h i g h f r e q u e n c y o f MNC,  a  second-stage  be r e s p o n s i b l e f o r  l e u k o p l a k i a and o r a l carcinomas  b e t e l q u i d chewers. T h i s p r o m o t i o n a l stage appears  in  to be l a c k i n g i n i n d i v i d u a l s  102  who  use o n l y s n u f f or chewing tobacco, which show o n l y a s m a l l a c t i v i t y i n  s e v e r a l assays f o r cancer  promoters.  5. Exploring Preventive Measures  5.1 Mechanism of Action of Chemopreventive Agents The  ICRDB ( B o u t w e l l , 1979)  papers concerned w i t h r e t i n o l , c a r o t e n o i d s . Together,  has compiled summaries o f 324 cancer r e s e a r c h o t h e r r e t i n o i d s , b e t a - c a r o t e n e or o t h e r  these show t h a t v a r i o u s r e t i n o i d s can  reversibly  suppress the m a l i g n a n t b e h a v i o u r o f c u l t u r e d c e l l s t h a t have been transformed by v i r u s e s , c h e m i c a l s or i o n i z i n g r a d i a t i o n ;  they can d e l a y or p r e v e n t the  onset o f c a n c e r i n e x p e r i m e n t a l animals p r e v i o u s l y t r e a t e d w i t h DNA-binding c a r c i n o g e n s ; they can cause human s k i n k e r a t o s e s o r o r a l l e u k o p l a k i a s to r e g r e s s ; they can p r e v e n t "tumor promoters"  from e l i c i t i n g p a p i l l o m a s on mouse  s k i n ; and they can i n h i b i t some o f the v e r y s p e c i f i c b i o c h e m i c a l e f f e c t s o f tumor promoters r e t i n o i d s may  on c e l l s  i n v i v o or i n c u l t u r e . A l t h o u g h i n a p p r o p r i a t e use o f  do more harm than good (Schroder and B l a c k , 1980),  r e a s o n a b l e t o hope t h a t exposure  i t seems  o f human t i s s u e s t o r e t i n o i d - l i k e a c t i v i t y  be m a n i p u l a t e d to reduce cancer r i s k s  can  (Sporn and Newton, 1979) .  I t has been suggested t h a t r e t i n o i d a p p l i c a t i o n can i n d e e d promote tumor development i n a few model systems (Schroder and B l a c k , 1980). term s t u d i e s r e p o r t e d here no i n c r e a s e i n l e u k o p l a k i a was E a s t I n d i a n f i s h e r m e n g i v e n v i t a m i n A;  i n fact,  In the s h o r t -  seen i n any o f the  the o p p o s i t e was  observed.  The  o n l y h a r m f u l e f f e c t o f v i t a m i n A a d m i n i s t r a t i o n i s h y p e r v i t a m i n o s i s A, but no symtoms o f t h i s were observed i n any o f the s u b j e c t s . I t can be c o n c l u d e d t h a t the doses o f v i t a m i n A g i v e n to people i n these s t u d i e s produced no h a r m f u l side-effects  i n the time p e r i o d d u r i n g which i t was  administered.  103  N e o p l a s t i c t r a n s f o r m a t i o n c a n o f t e n be s u b d i v i d e d i n t o " s t a g e s , " the e a r l y stage i n v o l v i n g c o n v e r s i o n o f a normal c e l l , and the l a t e r stage conversion of a p a r t i a l l y altered c e l l effective,  into a f u l l y  involving  n e o p l a s t i c c e l l . To be  the n e o p l a s t i c c e l l must n o t be e l i m i n a t e d , b u t must p r o l i f e r a t e  i n t o a p a t h o l o g i c a l tumor. E a r l y - and l a t e - s t a g e changes a r e b o t h  necessary  ( h a l v i n g e i t h e r may thus be an e q u a l l y e f f e c t i v e way o f h a l v i n g cancer b u t t y p i c a l l y have d i f f e r e n t causes protective effects of retinol  (Peto, 1977, 1979). The  (and perhaps  demonstrated  b e t a - c a r o t e n e ) suggest t h a t i t i s  the l a t e r s t a g e s o f n e o p l a s t i c p r o g r e s s i o n which a r e c h i e f l y a f f e c t e d , perhaps  w i t h the f i n a l  risks),  together  p r o c e s s o f tumor growth. There may o f t e n be a l a t e n t  p e r i o d o f a few decades b e f o r e a d o u b l i n g o r h a l v i n g o f the e a r l y p r o c e s s has any e f f e c t on human cancer r i s k w i t h i n about final  process  5 y e a r s , w h i l e e f f e c t s on the  ( p r o l i f e r a t i o n ) may be even more r a p i d l y e v i d e n t . Thus human  cancer r i s k s may be n o t i c e a b l y i n f l u e n c e d by the average r e t i n o l w i t h i n a p e r i o d o f as l i t t l e In  c o n t r a s t t o the r e t i n o i d s ,  levels of blood  as 5 o r 10 y e a r s .  the q u e s t i o n o f whether d i e t a r y b e t a -  c a r o t e n e o r some o t h e r c a r o t e n o i d s can a f f e c t cancer r i s k has n o t r e c e i v e d n e a r l y as much e x p e r i m e n t a l a t t e n t i o n . P o s s i b l e mechanisms t o c o n s i d e r i n c l u d e (1) a d i r e c t r e t i n o i d - l i k e e f f e c t o f some c a r o t e n o i d s on c e l l u l a r d i f f e r e n t i a t i o n i n the t a r g e t t i s s u e s ,  (2) c o n v e r s i o n i n the t a r g e t t i s s u e o f  some c a r o t e n o i d s i n t o molecules w i t h such r e t i n o i d - l i k e e f f e c t s , o r (3) p r o t e c t i o n by c a r o t e n o i d s o f the t a r g e t t i s s u e s v i a mechanisms n o t i n c o n t r o l of  cellular differentiation,  ( R e t t u r a , 1975; F e l i x e t a l . ,  f o r example, by a f f e c t i n g immunological 1976), o r by quenching  function  s i n g l e t oxygen, a h i g h l y  r e a c t i v e , e x c i t e d m o l e c u l a r s p e c i e s which o c c u r s as a t o x i c by-product normal p r o c e s s e s i n b o t h animals and p l a n t s .  o f many  104  In  the s t u d i e s p r e s e n t e d here,  the twice weekly o r a l a d m i n i s t r a t i o n o f  b e t a - c a r o t e n e , b e t a - c a r o t e n e p l u s v i t a m i n A or v i t a m i n A a f f e c t e d o r a l mucosal l e s i o n s at three d i f f e r e n t l e v e l s :  the frequency o f m i c r o n u c l e a t e d c e l l s  was  reduced, w e l l - e s t a b l i s h e d l e u k o p l a k i a s r e g r e s s e d , and the development o f  new  l e u k o p l a k i a s was First,  i n h i b i t e d . The r e s u l t s are noteworthy  the e f f e c t i v e doses  for several  reasons.  are c o n s i d e r a b l y lower than the 1 t o 2 mg/kg body  weight/day o f 1 3 - c i s - r e t i n o i c a c i d s u c c e s s f u l l y g i v e n to p a t i e n t s w i t h leukoplakias  (Cordero e t a l . ,  1981;  Koch, 1981;  Hong e t a l . ,  oral  1986). I f the  recommended c o n v e r s i o n f a c t o r f o r b e t a - c a r o t e n e and v i t a m i n A i s a p p l i e d (FAO/WHO, 1967;  Polacchi,  a p p r o x i m a t e l y 0.14  1980), then our dose o f r e t i n o l e q u i v a l e n t s was  mg/kg body weight/day (0.7 mg  from v i t a m i n A,  and 0.7  mg  from b e t a - c a r o t e n e ) . Second, the a d m i n i s t r a t i o n o f b e t a - c a r o t e n e alone had a s i g n i f i c a n t e f f e c t on the frequency o f m i c r o n u c l e a t e d c e l l s , b u t a l e s s e r on the r e m i s s i o n o f l e u k o p l a k i a s . Among the 31 chewers who c a r o t e n e a l o n e , o n l y one  the t r i a l .  r e c e i v e d beta-  (3%) f a i l e d t o show a r e d u c t i o n i n m i c r o n u c l e a t e d  c e l l s , whereas no r e m i s s i o n was completed  one  seen i n 23  (85%) o f 27 p a r t i c i p a n t s  who  T h i r d , m i c r o n u c l e a t e d c e l l s respond much more r a p i d l y  than  l e u k o p l a k i a t o b e t a - c a r o t e n e o r to b e t a - c a r o t e n e p l u s v i t a m i n A. F o u r t h , a l l p a r t i c i p a n t s i n the i n t e r v e n t i o n t r i a l c o n t i n u e d t o chew t o b a c c o - c o n t a i n i n g b e t e l quids i n t h e i r accustomed manner and a t t h e i r u s u a l r a t e . Thus r e m i s s i o n of  o r a l l e u k o p l a k i a s , reduced development o f new  l e u k o p l a k i a s , and r e d u c t i o n o f  m i c r o n u c l e a t e d c e l l s o c c u r r e d d u r i n g d a i l y exposure  to v a r i o u s c a r c i n o g e n s  r e l e a s e d from b e t e l q u i d i n g r e d i e n t s . I n t h i s r e s p e c t , the t r i a l s  with  r e t i n o i d s on b e t e l q u i d chewers d i f f e r from those conducted by Koch  (1981),  Shah e t a l . (1983) or Hong e t a l . (1986) on p a t i e n t s w i t h o r a l l e u k o p l a k i a , who may  n o t have been exposed to c a r c i n o g e n s d u r i n g the treatment p e r i o d . F i f t h ,  the doses o f b e t a - c a r o t e n e and v i t a m i n A a d m i n i s t e r e d d i d n o t cause  any  105  detectable side-effects,  such as dryness o f t h e l i p s and mouth, s c a l i n g and  p e e l i n g o f the s k i n , o r c o n j u n c t i v i t i s . The  q u e s t i o n must be r a i s e d whether t h e r e a r e ways t o reduce the dose o f  beta-carotene  and v i t a m i n A t o l e v e l s t h a t can be r e a d i l y a d m i n i s t e r e d  produce no u n d e s i r a b l e The  and t h a t  side-effects.  development o f new o r a l l e u k o p l a k i a s was i n h i b i t e d ,  e s t a b l i s h e d l e u k o p l a k i a s induced,  and r e m i s s i o n o f  by the six-month o r a l a d m i n i s t r a t i o n o f  v i t a m i n A a t a dose o f 0.14 mg/kg body weight p e r day. T h i s p r e v e n t i v e  dose was  about 1 3 - f o l d h i g h e r  t h a n the FAO/WHO (1967) recommended i n t a k e o f r e t i n o i d s ,  and  than the 0.02 mg/kg body weight p e r day o f d i e t a r y v i t a m i n  sevenfold higher  A which, a c c o r d i n g  t o e p i d e m i o l o g i c a l evidence,  conveys a p r o t e c t i v e e f f e c t  ( M a r s h a l l e t a l . , 1982). The dose o f v i t a m i n A was c o n s i d e r a b l y lower than those  r e p o r t e d p r e v i o u s l y : approximately  Gupta e t a l . , 1980); 1 t o 2 mg/kg/day when a d m i n i s t e r e d  as a lozenge  1 mg/kg/day  (Cordero  e t a l . , 1981;  (Hong e t a l . , 1986); 3, 5 o r 10 mg/kg/day  (Wahi e t a l . , 1965); 10 mg/kg/day  (Nair et a l . ,  1980); and 20 t o 100 mg/kg/day f o r s e v e r a l weeks ( R y s s e l e t a l . , 1971). (To make the r e t i n o i d l e v e l s comparable, they were r e c a l c u l a t e d from p u b l i s h e d r e s u l t s , assuming a body weight o f 70 kg f o r an a d u l t ) . One way t o reduce the t o t a l amount o f v i t a m i n A would be t o a t f i r s t administer  f o r s e v e r a l months heavy doses o f v i t a m i n A t o reduce the frequency  of m i c r o n u c l e i and l e u k o p l a k i a , and t h e n f o l l o w t h i s treatment w i t h the nontoxic beta-carotene.  Preliminary r e s u l t s obtained  on the t o b a c c o / b e t e l  quid  chewers i n K e r a l a r e v e a l e d t h a t the p r o t e c t i v e e f f e c t o f v i t a m i n A a d m i n i s t r a t i o n can be m a i n t a i n e d carotene. approach .  f o r a t l e a s t 5 a d d i t i o n a l months w i t h  S e v e r a l o t h e r experiments can be mentioned i n support  of this  beta-  106  Following a r e l a t i v e l y short-term retinoids  (0.6 mg/kg/day), the p r o t e c t i v e e f f e c t was  i n the range o f 0.3  mg  etretinate/kg/day  chemopreventive e f f e c t induced by was  a d m i n i s t r a t i o n o f h i g h e r doses o f maintained  by  ( S l o b e r g e t a l . , 1983).  1 mg/kg/day of Ro  lower doses  The  10-9359 f o r 2 to 10 weeks  similar. Another approach worth c o n s i d e r i n g c o n s i s t s o f combining  a d m i n i s t r a t i o n o f the somewhat t o x i c r e t i n o i d s w i t h carotene.  In animal models, b e t a - c a r o t e n e  carcinogenesis, populations,  the n o n - t o x i c  is effective  beta-carotene  has  i n preventing  a l s o e x e r t e d a p r o t e c t i v e e f f e c t , as shown by  of micronucleated  b e t e l q u i d chewers ( S t i c h ,  1987;  ( S t i c h e t a l . , 1985). The  cells  S t i c h e t a l . , 1984a,b) and  snuff  t h a t induced by  (100,000 IU/ week) and  100,000 IU r e t i n o l ) . The  pilot  v i t a m i n A t r e a t m e n t induced participants  200,000 IU g i v e n i n p a r t  i n p a r t , as b e t a - c a r o t e n e  (n = 51)  (Stich,  treatments may  to the f a c t t h a t o n l y a f r a c t i o n o f b e t a - c a r o t e n e  as  (amount equal  to  T h e o r e t i c a l l y , doses o f the n o n - t o x i c  beta-carotene  of  administration  1987). T h i s d i f f e r e n c e i n  o n l y be apparent, m a i n l y i s converted  treatment p r o t o c o l s are not s t r i c t l y c o u l d be  l e v e l s matching t h a t o f 100,000 IU o f v i t a m i n A u s i n g one f a c t o r s (Ibrahim  200,000  the r e m i s s i o n o f o r a l l e u k o p l a k i a s i n 57.1%  i n o n l y 27.5%  Thus the doses o f the two  beta-  t r i a l s on b e t e l q u i d chewers r e v e a l e d t h a t  p r o t e c t i v e a c t i v i t y between the two  conversion  induced by  (n = 21), whereas the v i t a m i n A p l u s b e t a - c a r o t e n e  resulted i n remission  dippers  e f f i c a c y o f r e p l a c i n g doses o f v i t a m i n A w i t h  IU o f v i t a m i n A per week w i t h  a  i n the b u c c a l mucosa o f  can be j u d g e d by comparing the e x t e n t o f r e m i s s i o n  vitamin A  beta-  i n c l u d i n g t h a t of the o r a l c a v i t y (Suda e t a l . , 1986). In human  r e d u c t i o n i n the frequency  carotene  the  e t a l . , 1977). However, the  into vitamin  due A.  comparable.  i n c r e a s e d to  o f the  current  implementation o f  this  107  i d e a c o u l d meet w i t h o b j e c t i o n s because o f the l a r g e number o f b e t a - c a r o t e n e capsules  to be  swallowed by  the t r i a l p a r t i c i p a n t s .  6. Outlook  A l t h o u g h the r i s k o f o r a l cancer i s not h i g h amongst these p e o p l e s I n u i t and has  Indian),  the use  o f o r a l tobacco i s a r e l a t i v e l y new  (both  phenomenon which  i n c r e a s e d d r a m a t i c a l l y s i n c e the 1960s (IARC, 1985), and which i s p r a c t i s e d  m a i n l y by young  adolescents.  In the U n i t e d  States,  the use  y e a r l y . Given t h i s s i t u a t i o n , expect to see  o f smokeless tobacco i n c r e a s e s by  the q u e s t i o n must be  i n the near f u t u r e the h i g h  tobacco-containing regions United  b e t e l quids  associated with  of o r a l leukoplakias  In the l a t t e r case,  and  o f "nass" i n southern  or s n u f f d i p p e r s  s n u f f d i p p i n g was  o f s n u f f was  i n the  found to be  c a n c e r s o f the o r a l c a v i t y , pharynx and  e t a l (1984) showed t h a t the use  can  t h a t i s so common among chewers o f  i n Asian countries, users  o f the S o v i e t U n i o n (IARC, 1985) States.  r a i s e d as to whether one  incidence  o r a l c a n c e r s among Canadian s n u f f d i p p e r s  10-11%  southeastern strongly  l a r y n x . A study by Winn  associated with a f o u r f o l d  i n c r e a s e i n the r i s k o f o r a l and p h a r y n g e a l c a n c e r s ,  and  t h a t the r e l a t i v e  i n c r e a s e d markedly to almost f i f t y f o l d f o r those who  had  d i p p e d s n u f f f o r 50  years  risk  or more. The  d i s t r i b u t i o n o f b e t a - c a r o t e n e or v i t a m i n A to a l a r g e  remains an u n r e s o l v e d difficulties capsules,  i s s u e . Because o f the expenses and  population  logistical  i n v o l v e d i n a l a r g e - s c a l e i n t e r v e n t i o n program u s i n g p i l l s  o t h e r methods must be  sought and  implemented. The  or  f o l l o w i n g examples  are p r a c t i c a l l y f e a s i b l e and warrant c l o s e r c o n s i d e r a t i o n . Red  palm o i l p r o v i d e s  a v e r y r i c h source o f b e t a - c a r o t e n e ,  i n t r o d u c t i o n o f t h i s substance i n t o the d i e t c o u l d p r o v i d e  and  the  the p r o t e c t i o n  108  needed by i n c r e a s i n g b o t h b e t a - c a r o t e n e and v i t a m i n A l e v e l s i n i t s consumers. In N o r t h America,  an i n c r e a s e d i n t a k e o f g r e e n / y e l l o w v e g e t a b l e s and e d u c a t i o n  on the importance  o f these p r o d u c t s i n the d i e t may  l e a d to a change i n the  r e s u l t i n g serum l e v e l s o f b e t a - c a r o t e n e and v i t a m i n A, and e v e n t u a l l y  reduce  the r i s k n o t o n l y f o r o r a l but a l s o f o r o t h e r e p i t h e l i a l c a n c e r s . In Taiwan, sweet p o t a t o s t r a i n s which are r i c h i n b e t a - c a r o t e n e are b e i n g used as p o t a t o chips,  f r e n c h f r i e s and f l o u r . In t h i s way,  the d i e t o f a p o p u l a t i o n can be  e n r i c h e d w i t h b e t a - c a r o t e n e w i t h o u t any major changes i n e a t i n g h a b i t s .  109  SUMMARY The  o n l y known c a r c i n o g e n s  contained  i n any tobacco  mixtures  used  o r a l l y throughout the w o r l d a r e the N - n i t r o s o compounds. An a n a l y s i s of the l e v e l s o f these  agents,  plus t h e i r precursors,  i n the tobaccos  used and i n the s a l i v a o f t h e i r u s e r s was conducted i n three communities. High l e v e l s o f n i t r i t e were found used by Canadian I n u i t  (Gjoa Haven) and Indians  s a l i v a o f s n u f f d i p p e r s . These h i g h n i t r i t e precursors  i n the s n u f f  different tobacco  (La Loche) and i n the  l e v e l s can a c t as  t o n i t r o s a t i o n r e a c t i o n s , as e v i d e n c e d  by the h i g h e r  levels  o f NPRO d e t e c t e d i n t h e u r i n e o f s n u f f u s e r s v e r s u s non-users. I n addition, r e l a t i v e l y high l e v e l s of t o b a c c o - s p e c i f i c nitrosamines d e t e c t e d i n tobacco The  genotoxic  samples and i n the s a l i v a o f s n u f f u s e r s . damage induced by exposure t o these c a r c i n o g e n s  from t h e e l e v a t e d l e v e l s o f m i c r o n u c l e a t e d s n u f f d i p p e r s . Although,  a t present  c e l l s found  was e v i d e n t  i n the o r a l mucosa o f  t h e I n u i t and I n d i a n communities s t u d i e d ,  do n o t have an e l e v a t e d i n c i d e n c e o f o r a l cancer, genome,  were  these a b n o r m a l i t i e s o f the  t o g e t h e r w i t h t h e w h i t i s h / y e l l o w i s h w r i n k l e d patches observed  mucosa, i n d i c a t e a r i s k o f d e v e l o p i n g precancerous  i n the  l e s i o n s and/or cancer  over a  l o n g enough p e r i o d o f time. The E a s t I n d i a n p o p u l a t i o n s t u d i e d shows a s u b s t a n t i a l l y i n c r e a s e d r i s k f o r o r a l cancer,  high micronucleated  cell  counts,  plus a l a r g e percentage o f i n d i v i d u a l s with p r e n e o p l a s t i c o r a l l e s i o n s (leukoplakia). In the I n u i t p o p u l a t i o n s t u d i e d , i t was shown t h a t the MNC l e v e l c o u l d be reduced  by a s h o r t - t e r m  beta-carotene.  intervention t r i a l  (10 weeks) u s i n g 180 mg/week o f  I n the E a s t I n d i a n group, i t was p o s s i b l e t o reduce the MNC  l e v e l and t h e frequency  o f l e u k o p l a k i a using beta-carotene  (180 mg/week) p l u s  110  vitamin A  (100,000 IU/week) over a six-month p e r i o d . An even more marked  r e d u c t i o n i n l e s i o n frequency was v i t a m i n A alone I t now  appears c o n c l u s i v e t h a t exposure to the c a r c i n o g e n i c  N-nitroso  i n smokeless tobacco i s i n v o l v e d i n the e t i o l o g y o f  t h a t o f the r i s k can be reduced by  i n t a k e o f these compounds.  oral  i n c r e a s i n g serum l e v e l s of beta-  c a r o t e n e and v i t a m i n A v i a s u p p l e m e n t a t i o n w i t h p i l l s to i n c r e a s e  of  (200,000 IU/week).  compounds c o n t a i n e d cancer and  observed a f t e r a six-month a d m i n i s t r a t i o n  or by a d j u s t i n g the  diet  Ill  REFERENCES Abe,  T., Isemura, T. and K i k u c h i , Y. (1984) M i c r o n u c l e i i n human bone-marrow  cells:  e v a l u a t i o n o f the m i c r o n u c l e u s t e s t u s i n g human leukemia p a t i e n t s  treated with  antileukemic  agents. M u t a t i o n Res., 130. 113-120.  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