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Genotoxicity of chewing tobacco samples Woolcock, Bruce Wayne 1985

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GENOTOXICITY OF CHEWING TOBACCO SAMPLES By BRUCE WAYNE WOOLCOCK B . S c , The U n i v e r s i t y o f V i c t o r i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming t o t he r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA December 1985 © B R U C E WAYNE WOOLCOCK, 1985 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f P a t h o l o g y  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT The i n t r a - o r a l use o f t o b a c c o - c o n t a i n i n g mixtures p l a y s an important a e t i o l o g i c a l r o l e i n the o c c u r r e n c e of o r a l c a n c e r s . In v i t r o g e n o t o x i c i t y assays may p r o v i d e means f o r t h e r a p i d e v a l u a t i o n of f a c t o r s c o n t r i b u t i n g t o o r modulating t h i s form o f tobacco c a r c i n o g e n e s i s . An e s s e n t i a l requirement f o r an e f f e c t i v e t e s t system i s the c a p a b i l i t y t o d e t e c t the g e n o t o x i c e f f e c t s o f a v a r i e t y of tobacco mixtures which are expected t o d i f f e r i n chemical composition. F r e s h l y prepared aqueous e x t r a c t s o f f o u r tobacco mixtures, l o c a l l y a v a i l a b l e s n u f f and "chewing" tobacco, K h a i n i tobacco (India) and nass (Uzbekistan, USSR), were assayed f o r g e n o t o x i c a c t i v i t y i n t h r e e d i f f e r e n t t e s t systems: chromosome a b e r r a t i o n s i n Chinese hamster ovary c e l l s , m i c r o n u c l e i i n Chinese hamster ovary c e l l s and unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s . A DNA r e p a i r i n h i b i t i o n t e s t was i n c l u d e d as a complement t o the unscheduled DNA s y n t h e s i s assay. A l l f o u r tobacco e x t r a c t s were found t o c o n t a i n d i r e c t a c t i n g agents capable of i n d u c i n g chromosome a b e r r a t i o n s and m i c r o n u c l e i f o r m a t i o n i n Chinese hamster ovary c e l l s . C a t a l a s e was found t o suppress the c l a s t o g e n i c a c t i v i t y o f the chewing and K h a i n i tobaccos, i m p l i c a t i n g H 20 2-mediated p r o d u c t i o n o f chromosome damage. The g e n o t o x i c a c t i v i t i e s o f s n u f f and nass d i d not appear to i i be dependent on the g e n e r a t i o n of H 2 0 2 . Only the chewing tobacco i n i t i a t e d unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s . A l l tobacco e x t r a c t s reduced the l e v e l s of UV i n i t i a t e d unscheduled DNA s y n t h e s i s , i n d i c a t i n g the e x t r a c t s e x e r t e d an i n h i b i t o r y e f f e c t on DNA r e p a i r . The f a i l u r e o f the s n u f f , K h a i n i tobacco and nass t o induce a demonstrable unscheduled DNA s y n t h e s i s was i n t e r p r e t e d t o be a consequence o f t h i s i n h i b i t i o n . On the b a s i s o f these r e s u l t s i t was concluded t h a t the chromosome a b e r r a t i o n and micronucleus t e s t s i n Chinese hamster ovary c e l l s , but not the unscheduled DNA s y n t h e s i s , appear t o be s u i t a b l e as t e s t systems f o r the study of f a c t o r s i n f l u e n c i n g o r a l tobacco c a r c i n o g e n i c i t y . i i i TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS . . i v LIST OF TABLES v i i LIST OF FIGURES v i i i INTRODUCTION 1 1. O r a l cancer and the i n t r a - o r a l use of tobacco 1 2. Ex t e n t o f problem 4 3. F a c t o r s m o d i f y i n g the r i s k of t o b a c c o - r e l a t e d o r a l cancer 6 4. Model systems f o r the study o f o r a l tobacco c a r c i n o g e n i c i t y 8 5. O b j e c t i v e 9 METHODS AND MATERIALS 13 1. P r e p a r a t i o n o f e x t r a c t s 13 2. Chemicals 13 3. T i s s u e c u l t u r e 13 3.1 C u l t u r e media 13 4. Growth of s t o c k c u l t u r e s 14 5. Chromosome a b e r r a t i o n t e s t 14 5.1 P r e p a r a t i o n o f c e l l c u l t u r e s 14 5.2 Exposure t o t e s t e x t r a c t 15 5.3 Chromosome p r e p a r a t i o n s 15 5.4 A n a l y s i s of metaphase p l a t e s f o r chromosome a b e r r a t i o n s 16 i v CONTENTS (continued) PAGE 6. Mi c r o n u c l e u s assay 16 6.1 T e s t procedure 16 6.2 A n a l y s i s o f i n t e r p h a s e c e l l s f o r m i c r o n u c l e i 16 7. DNA r e p a i r s t u d i e s 17 7.1 P r e p a r a t i o n o f c e l l c u l t u r e s 17 7.2 U l t r a v i o l e t i r r a d i a t i o n 17 7.3 Exposure t o t e s t e x t r a c t and r a d i o a c t i v e l y - l a b e l l e d thymidine 18 7.4 F i x a t i o n and p r e p a r a t i o n f o r autoradiography 18 7.5 Autoradiography and s t a i n i n g 19 7.6 A n a l y s i s o f autoradiograms 19 RESULTS 21 1. Chromosme damaging c a p a c i t y o f aqueous tobacco e x t r a c t s 21 2. I n d u c t i o n o f m i c r o n u c l e i by aqueous tobacco e x t r a c t s 23 3. I n d u c t i o n o f unscheduled DNA s y n t h e s i s by aqueous tobacco e x t r a c t s 26 4. I n h i b i t i o n o f DNA-repair s y n t h e s i s 28 5. E x p l o r a t i o n o f the a c t i v e g e n o t o x i c components o f the e x t r a c t s 33 6. Other t e s t systems e x p l o r e d 33 6.1 UDS i n r a t o r a l mucosal c e l l s 33 6.2 M i c r o n u c l e i i n rodent o r a l mucosa 35 v CONTENTS (continued) DISCUSSION . CONCLUSION . BIBLIOGRAPHY L I S T OF TABLES PAGE TABLE I CLASTOGENIC A C T I V I T Y OF CRUDE AQUEOUS TOBACCO EXTRACTS I N CHINESE HAMSTER OVARY CELLS 22 TABLE I I CLASTOGENIC A C T I V I T Y OF AQUEOUS TOBACCO EXTRACTS - DAMAGE BY TYPES 24 TABLE I I I MICRONUCLEI FORMATION I N CHINESE HAMSTER OVARY CELLS EXPOSED TO AQUEOUS TOBACCO EXTRACTS 25 TABLE I V EFFECT OF CATALASE ON THE CLASTOGENIC A C T I V I T Y OF AQUEOUS TOBACCO EXTRACTS I N CHINESE HAMSTER OVARY CELLS 34 v i i LIST OF FIGURES PAGE FIGURE 1. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o a chewing tobacco ... 27 FIGURE 2. I n h i b i t o r y e f f e c t o f a chewing tobacco e x t r a c t on UV-induced DNA r e p a i r i n human f i b r o b l a s t s 29 FIGURE 3. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o K h a i n i tobacco 30 FIGURE 4. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o nass 31 FIGURE 5. Unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s exposed t o s n u f f 32 v i i i INTRODUCTION 1. O r a l Cancer and the I n t r a - o r a l Use of Tobacco O r a l cancer i s one of the t e n most common cancers worldwide (WHO, 1984). I t accounts f o r more than o n e - t h i r d o f a l l cancers i n some A s i a n c o u n t r i e s ( B i n n i e and Rankin, 1984) w i t h more than a hundred thousand new cases o f o r a l c ancer o c c u r r i n g i n South-East A s i a each y e a r (WHO, 1984). The r e l a t i v e frequency o f o r a l cancer t o a l l c a n c e r s i n J a f f n a ( S r i Lanka), M a i n p u r i (northern India) and Neyyur (southern India) are r e p o r t e d t o be 66.1%. 67.9%, and 75.9%, r e p e c t i v e l y (Hirayama, 1979). In c o n t r a s t , o r a l cancer accounts f o r o n l y 3-5% of ma l i g n a n c i e s i n most western n a t i o n s (Pindborg, 1980). The i n c i d e n c e r a t e s f o r o r a l cancer around the world v a r i e s approximately f i f t e e n - f o l d (McMichael, 1984). The p r e v a l e n c e o f tobacco chewing h a b i t s i n areas w i t h e l e v a t e d f r e q u e n c i e s o f o r a l cancer suggests an a e t i o l o g i c a l c o n n e c t i o n . In I n d i a , where chewing of 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 i s as common as smoking i s i n Western s o c i e t i e s , over 70% o f the cancers o f the o r a l c a v i t y , pharynx, and l a r y n x have been a t t r i b u t e d t o the sepa r a t e and combined e f f e c t s o f o r a l tobacco use and smoking (Jayant e t a l . , 1977). A c a u s a l r e l a t i o n s h i p between o r a l tobacco h a b i t s and t h e e l e v a t e d frequency o f o r a l cancer i n A s i a i s 1 supported by t h r e e l i n e s o f evidence. F i r s t , t h e r e i s a g r e a t e r p r e v a l e n c e o f o r a l cancer among i n d i v i d u a l s w i t h the h a b i t as opposed t o those without (Hirayama, 1966; Wahi, 1968; J u s s a w a l l a and Deshpande, 1971; Jayant e t a l . , 1977; Gupta e t a l . , 1980; WHO, 1984; J u s s a w a l l a e t a l . , 1985). Fo r example, Wahi (1968), i n an e p i d e m i o l o g i c a l survey of o r a l and oropharyngeal cancers i n the M a i n p u r i d i s t r i c t of I n d i a , found an e i g h t - f o l d e l e v a t i o n i n r i s k f o r chewers compared w i t h noh-chewers. Second, an a e t i o l o g i c a l r o l e f o r tobacco i s supported by the s t r o n g a s s o c i a t i o n o f cancer a r i s i n g i n the s i t e where the tobacco q u i d i s h a b i t u a l l y h e l d (Hirayama, 1966; Wahi, 1968; J u s s a w a l l a and Deshpande, 1971). Hirayama (1966), as an example, observed t h a t 78% of c a n c e r s arose on the same s i d e of the mouth as the s i d e i n which the q u i d was h e l d , w h i l e cancers were e v e n l y d i s t r i b u t e d on both s i d e s o f the mouth among p a t i e n t s who kept t h e q u i d i n both s i d e s of the mouth. F i n a l l y , o r a l c a n c e r s are a s s o c i a t e d w i t h f r e q u e n t , prolonged o r e a r l y i n d u l g e n c e of tobacco which can be c o n s i d e r e d as evidence f o r a d o s e - e f f e c t r e l a t i o n s h i p . Hirayama (1966) and Wahi (1976) have shown the r i s k o f o r a l cancer t o r i s e w i t h an i n c r e a s e i n the frequency and d u r a t i o n o f use each day. For those who r e t a i n the q u i d d u r i n g t h e i r s l e e p , Wahi noted a s i x - f o l d g r e a t e r r i s k than those who chew o n l y when they are awake and a 3 6 - f o l d g r e a t e r r i s k than non-chewers. Hirayama 2 found t h e r i s k r o s e as h i g h as 63 times t h a t f o r non-chewers. I t has been more d i f f i c u l t t o e s t a b l i s h an a s s o c i a t i o n between t h e use of s n u f f o r chewing tobacco and o r a l cancer i n t h e West because o f the r e l a t i v e l y i n f r e q u e n t use of th e s e p r o d u c t s . N e v e r t h e l e s s , a s t r i k i n g sex d i f f e r e n c e i n the geographic d i s t r i b u t i o n o f o r a l cancer i n the U n i t e d S t a t e s suggests a r e l a t i o n s h i p between o r a l carcinoma and s n u f f d i p p i n g . O r a l cancer m o r t a l i t y r a t e s a re e l e v a t e d among women i n the so u t h e a s t e r n U n i t e d S t a t e s . T h i s i s c o n s i s t e n t w i t h the r e l a t i v e l y f r e q u e n t use o f s n u f f among females who have o r a l cancer ( B l o t and Fraumeni, 1977). S e v e r a l r e t r o s p e c t i v e s t u d i e s conducted i n the s o u t h e a s t e r n U n i t e d S t a t e s support t h i s h y p o t h e s i s ( V o l g e r e t a l . , 1962; R o s e n f e l d and Callaway, 1963; Winn e t a l . , 1981; McGuirt, 1983). F o r i n s t a n c e , Winn e t a l . (1981) e s t i m a t e d a 4.2-f o l d i n c r e a s e d r i s k o f o r a l and pharyngeal cancer among s n u f f - d i p p i n g women. They a l s o e s t i m a t e d the r i s k f o r c a n c e r s a r i s i n g i n the g i n g i v a l and b u c c a l mucosa o f l o n g -term u s e r s approached 50 times t h a t f o r non-users and a t t r i b u t e d 87% o f the cancers a r i s i n g a t thes e s i t e s t o s n u f f . Most o f these s t u d i e s have observed an a s s o c i a t i o n o f c a ncer a r i s i n g a t the s i t e where the s n u f f was h e l d . McGuirt (1983), f o r example, r e p o r t e d 89% of the primary tumours arose a t the s i t e where the q u i d o f s n u f f was h e l d and concluded t h a t t h i s was s u f f i c i e n t i n i t s e l f t o 3 i m p l i c a t e smokeless tobacco as the c a u s a l agent o f carcinomas i n the s n u f f - d i p p i n g group. The a s s o c i a t i o n of smokeless tobacco use and o r a l cancers, however, i s not accepted by a l l . Smith and h i s c o l l e a g u e s (1970, 1975) were not a b l e t o v a l i d a t e an a s s o c i a t i o n between the use of s n u f f and o r a l cancer i n a t e n y e a r p r o s p e c t i v e study and concluded t h a t the type o f s n u f f used i n North America cannot be c o n s i d e r e d c a r c i n o g e n i c . Negative r e p o r t s on the a s s o c i a t i o n o f s n u f f usage and o r a l cancer, however, might be e x p l a i n e d by a v e r y l o n g l a t e n c y p e r i o d f o r the appearance o f s n u f f - i n d u c e d carcinomas (Roed-Petersen and Pindborg, 1973; McGuirt, 1983). 2. E x t e n t o f Problem The o r a l use o f tobacco throughout the w o r l d i s e x t e n s i v e . I t has been estimated by S t i c h and R o s i n (1985) t h a t g l o b a l l y more than 600 m i l l i o n i n d i v i d u a l s chew p o t e n t i a l l y c a r c i n o g e n i c 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 : 450 m i l l i o n o r more i n d i v i d u a l s i n A s i a , the P h i l i p p i n e s and the South P a c i f i c I s l a n d s chew b e t e l q u i d , the composition of which v a r i e s w i t h l o c a l custom but u s u a l l y c o n s i s t s o f the a r e c a o r b e t e l nut, b e t e l l e a f , tobacco and s p i c e s ; 100 m i l l i o n or more i n h a b i t a n t s of I n d i a and s e v e r a l A f r i c a n n a t i o n s p l a c e a mixture of tobacco and l i m e between the cheek and gum; approximately 20 m i l l i o n n a t i v e s o f the c e n t r a l A s i a n r e p u b l i c s o f the S o v i e t Union, I r a n and 4 A f g a n i s t a n put nass, a complex mixture o f tobacco, s l a k e d l i m e , ash w i t h o i l o r water under the tongue o r between the lower l i p and gum; and 30 m i l l i o n North Americans use s n u f f o r chewing tobacco. S n u f f d i p p i n g and tobacco chewing h a b i t s are r a p i d l y i n c r e a s i n g i n North America. P r o d u c t i o n o f smokeless tobacco i n t h e U n i t e d S t a t e s r o s e from 98 m i l l i o n pounds i n 1971 t o an e s t i m a t e d 134 m i l l i o n pounds i n 1980 (McGuirt, 1983) and s a l e s have been i n c r e a s i n g a t t h e r a t e of 11 p e r c e n t per y e a r ( C h r i s t e n , 1980) . The dramatic r i s e i n smokeless tobacco use over the l a s t decade may be a t t r i b u t a b l e t o an i n c r e a s i n g s o c i a l acceptance of o r a l tobacco h a b i t s , p a r t i c u l a r l y by young males. Surveys of smokeless tobacco use by teenagers have not been e x t e n s i v e but the r e s u l t s of the few t h a t have been done are cause f o r concern. Approximately 26% of the male student p o p u l a t i o n o f t h e Northwest T e r r i t o r i e s have used chewing tobacco or s n u f f and 13.7% are c u r r e n t u s e r s ( M i l l a r , 1984), almost one-quarter o f the r u r a l and urban teenage males i n Colorado i n d u l g e (Poulson e t a l . , 1984), and 13% o f 14 y e a r o l d males i n t h e g r e a t e r A t l a n t a area chew o r d i p on a r e g u l a r b a s i s (Offenbach and Weathers, 1985). Only 1.4% o f a l l the boys i n c l u d e d i n the l a s t study smoked c i g a r e t t e s . T h i s r e v e r s a l o f tobacco usage p r e f e r e n c e has a l s o been r e p o r t e d f o r teenagers i n Oregon (Pearce, 1985). 5 3. F a c t o r s M o d i f y i n g the R i s k of T o b a c c o - r e l a t e d O r a l  Cancer V a r i a t i o n s i n the p r e v a l e n c e and r i s k o f o r a l l e s i o n s e x i s t between d i f f e r e n t chewing p o p u l a t i o n s . Of the many f a c t o r s which c o u l d be r e s p o n s i b l e f o r the observed d i f f e r e n c e s i t i s u n c e r t a i n which are most important. Since t h e r e are numerous ways tobaccos are prepared f o r i n t r a - o r a l use, i t would be m i s l e a d i n g t o assume t h a t a l l chewing mixtures w i l l have the same c a r c i n o g e n i c p o t e n t i a l . Wide v a r i a t i o n s i n b o t a n t i c a l , chemical, and p h y s i c a l c h a r a c t e r i s t i c s o f l e a f tobacco are found among the v a r i o u s s p e c i e s , t y p e s , v a r i e t i e s , s t r a i n s and grades. In a d d i t i o n t o the g e n e t i c makeup, environmental f a c t o r s , i n c l u d i n g m i n e r a l n u t r i t i o n , s o i l p r o p e r t i e s , moisture supply, temperature and l i g h t i n t e n s i t y , a f f e c t the chemical c o m p o s i t i o n and p h y s i c a l p r o p e r t i e s o f the l e a f . Furthermore, v a r i a t i o n s i n c u r i n g , aging, f e r m e n t a t i o n and o t h e r p o s t - h a r v e s t h a n d l i n g p r a c t i c e s b r i n g about d i f f e r e n t c hemical changes i n the tobacco. F i n a l l y , the a d d i t i o n of substances such as lime or b e t e l nut t o the chewing mixture c o u l d enhance o r depress the a c t i v i t y o f tobacco. I t i s probable t h a t the c a r c i n o g e n i c p o t e n t i a l o f a chewing mixture i s i n f l u e n c e d by the manner o f chewing. The s i t e where the q u i d i s p l a c e d , the l e n g t h o f time i t i s r e t a i n e d , the frequency o f use, and the a c t u a l amount o f chewing v a r y g r e a t l y from one p o p u l a t i o n t o another. 6 The occurrence o f secondary r i s k f a c t o r s f o r o r a l carcinoma w i t h i n the same p o p u l a t i o n may a l s o a f f e c t the r i s k o f o r a l l e s i o n s . These i n c l u d e heavy tobacco smoking and heavy a l c o h o l consumption (Wynder e t a l . , 1957; Rothman and K e l l e r , 1972; Graham e t a l . , 1977; Wynder and Stellman, 1977; H e r i t y e t a l . , 1981; Mashberg e t a l . , 1981), poor o r a l h e a l t h and d e n t i t i o n (Graham e t a l . , 1977; H e r i t y e t a l . , 1981), and poor d i e t and n u t r i t i o n (Marshal e t a l . , 1982; Winn e t a l . , 1984). For example, i n t a k e of v i t a m i n s A and C may be i n v e r s e l y a s s o c i a t e d w i t h carcinomas i n t h e o r a l c a v i t y (Marshal e t a l . , 1982; Winn e t a l . , 1984). Among tobacco chewing p o p u l a t i o n s , subnormal l e v e l s o f r e t i n o l ( v i t a m i n A) have been observed i n Uzbekis ( Z a r i d z e e t a l . , 1985), P a k i s t a n i s and Indians (Wahi e t a l . , 1965: Ibrahim e t a l . , 1977), and the h i l l t r i b e s o f n o r t h e r n Luzon, the P h i l i p p i n e s ( S t i c h e t a l . , 1985). Normal r e t i n o l l e v e l s have been observed among I n u i t s n u f f d i p p e r s . On the o t h e r hand, t h e s e tobacco u s e r s have r e l a t i v e l y low l e v e l s o f b e t a - c a r o t e n e , a v i t a m i n A p r e c u r s o r found i n p l a n t s which may have a p r o t e c t i v e e f f e c t independent o f i t s p r o - v i t a m i n A a c t i v i t y ( S t i c h e t a l . , 1985). Presumably, most North American and European s n u f f d i p p e r s and tobacco chewers do not s u f f e r from any n u t r i t i o n a l d e f i c i e n c i e s . Based on c u r r e n t l y a v a i l a b l e i n f o r m a t i o n i t i s not p o s s i b l e t o determine whether the observed d i f f e r e n c e s i n o r a l l e s i o n s r e s u l t from d i f f e r e n c e s i n the composition o f the chewing 7 mixture, the type o f chewing p a t t e r n , the n u t r i t i o n a l s t a t u s o f the p o p u l a t i o n o r the presence o f o t h e r r i s k f a c t o r s . 4. Model Systems f o r Studying O r a l Tobacco C a r c i n o g e n i c i t y . The problem o f o r a l cancer i s extremely complex. E p i d e m i o l o g i c a l s t u d i e s are not s u f f i c i e n t l y r e f i n e d t o a s c e r t a i n the i n d i v i d u a l o r i n t e r a c t i v e e f f e c t s o f the v a r i o u s f a c t o r s which may modulate tobacco c a r c i n o g e n e s i s due t o the i n t e r f e r e n c e by thousands o f f a c t o r s which cannot be p r o p e r l y c o n t r o l l e d . The use o f a tumour-induction t e s t system i n experimental animals i s i m p r a c t i c a l because of the r e l a t i v e l y l o n g time (up t o two years) and the v a s t number of animals needed t o t e s t even a s m a l l number o f the f a c t o r s t o which chewers are exposed and t o a s c e r t a i n s i g n i f i c a n t d i f f e r e n c e s i n the v a r i o u s treatment groups. Furthermore, t o p i c a l a p p l i c a t i o n o f unburned tobacco alone has f a i l e d t o produce o r a l carcinomas i n rodents (Peacock e t a l . , I960; Dunham and H e r r r o l d , 1962; Dunham e t a l . , 1966; Homburger, 1971; Kandarka and S i r u t , 1977; H i r s c h and T h i l a n d e r , 1981; H i r s c h e t a l . , 1983; S k l a r e t a l , 1985), or has done so onl y a t a v e r y low frequency (Randive e t a l . , 1979; H i r s c h e t a l . , 1984). In v i t r o s h o r t - t e r m t e s t s f o r g e n o t o x i c i t y , on the c o n t r a r y , are w e l l s u i t e d f o r such s t u d i e s because they a r e economical, r a p i d and q u a n t i t a t i v e . An e x c e l l e n t c o r r e l a t i o n e x i s t s between the g e n o t o x i c i t y o f a chemical as r e v e a l e d by many of these s h o r t - t e r m t e s t s and the 8 c a r c i n o g e n i c i t y i n animals ( H o l l s t e i n e t a l . , 1979). Short-term i n v i t r o t e s t s a l s o have an a d d i t i o n a l advantage i n t h a t experiments can be designed t o r e v e a l the mechanisms of t h e g e n o t o x i c a c t i o n of the t e s t m a t e r i a l s . 5. O b j e c t i v e The o b j e c t i v e o f t h i s t h e s i s has been t o s e l e c t an i n  v i t r o t e s t system f o r the r a p i d e v a l u a t i o n o f f a c t o r s c o n t r i b u t i n g t o or modulating tobacco g e n o t o x i c i t y 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 i t y . An e s s e n t i a l requirement f o r an e f f e c t i v e t e s t system i s t h e c a p a b i l i t y t o d e t e c t the g e n o t o x i c e f f e c t s o f a v a r i e t y of tobacco mixtures. D i f f e r e n t tobacco mixtures are expected t o d i f f e r i n chemical composition and, t h e r e f o r e , may c o n t a i n d i f f e r e n t c a r c i n o g e n s . S i n c e s h o r t - t e r m t e s t systems d e t e c t d i f f e r e n t c l a s s e s o f c a r c i n o g e n s w i t h v a r y i n g e f f i c i e n c y , any p a r t i c u l a r i n v i t r o t e s t system may not be s e n s i t i v e enough t o d e t e c t the g e n o t o x i c e f f e c t s of a l l tobacco mixtures. T h e r e f o r e , s e v e r a l tobacco mixtures and g e n o t o x i c end-points were chosen f o r examination. 9 The f o l l o w i n g f o u r tobaccos, r e p r e s e n t i n g some of the v a r i e t y o f chewing mixtures used throughout the world, were screened f o r g e n o t o x i c a c t i v i t y : (a) S n u f f . A f i n e l y c u t o r powdered, fermented tobacco used i n North America and Europe which i s p l a c e d between the cheek o r l i p and gum and sucked. Winn e t a l . (1981) have found a s t r o n g a s s o c i a t i o n between s n u f f use and o r a l cancer among women i n the s o u t h e a s t e r n U n i t e d S t a t e s . (b) Chewing tobacco. Smokeless tobacco i n t h e form of l o o s e l e a v e s o r b l o c k s of p r e s s e d l e a v e s a r e a l s o commercially prepared i n the West. These d i f f e r from s n u f f s i n t h a t the f e r m e n t a t i o n p e r i o d i s not as e x t e n s i v e and the " q u i d " o r "chaw" o f tobacco i s a c t i v e l y chewed. Because the use o f chewing tobacco has t r a d i t i o n a l l y been r e s t r i c t e d t o r u r a l areas w i t h s m a l l and d i s p e r s e d p o p u l a t i o n s i t has been d i f f i c u l t t o assess the c a r c i n o g e n i c p o t e n t i a l o f t h i s form o f tobacco. (c) Nass,. A mixture of tobacco, ash and l i m e w i t h c o t t o n - s e e d o i l or water used i n c e n t r a l A s i a . Small amounts are i n s e r t e d under the tongue or between the l i p s and gum and a f t e r some minutes spat out. The c a r c i n o g e n i c p o t e n t i a l of nass has been w e l l documented (Paches and M i l i e v s k a y a , 1980). (d) K h a i n i tobacco. A powdered tobacco commonly used i n p a r t s o f I n d i a . I t i s mixed w i t h l i m e and p l a c e d w i t h i n t h e g i n g i v a l groove. In c o n t r a s t w i t h nass, the lime i s 10 mixed w i t h the tobacco j u s t b e f o r e use. A s t r o n g l i n k between use o f t h i s tobacco and o r a l cancer has been f i r m l y e s t a b l i s h e d (Hirayama, 1966). Genotoxic endpoints s t u d i e d were: (a) Chromosome a b e r r a t i o n s i n Chinese hamster ovary (CHO) c e l l s . T h i s i s a standard s c r e e n i n g assay f o r p o t e n t i a l c a r c i n o g e n s because agents t h a t a re c a r c i n o g e n i c t o mammals are po t e n t i n d u c e r s o f chromosome damage (Evans, 1974; I s h i d a t e and Odashima, 1977; H o l l s t e i n e t a l . , 1979; Pr e s t o n e t a l . , 1981). The presence o f chromosome damage can be d e t e c t e d c y t o l o g i c a l l y as m o r p h o l o g i c a l a l t e r a t i o n s i n chromosome s t r u c t u r e i n metaphase c e l l s . (b) M i c r o n u c l e i f o r m a t i o n i n CHO c e l l s . Chromosomal breakage i n any p o p u l a t i o n o f d i v i d i n g c e l l s w i l l l e a d t o the l o s s o f a c e n t r i c fragments which, when not i n c o r p o r a t e d i n t o the daughter nucleus, are d e t e c t a b l e as m i c r o n u c l e i . The micronucleus t e s t as a measure o f g e n o t o x i c i t y has been v a l i d a t e d on over 150 chemicals (Heddle e t a l . , 1983). The micr o n u c l e u s t e s t can be extended i n v i v o t o animals and i s c u r r e n t l y b e i n g a p p l i e d i n the s c r e e n i n g o f human p o p u l a t i o n s ( S t i c h and Rosin, 1984). (c) Unscheduled DNA S y n t h e s i s (UDS) i n c u l t u r e d human f i b r o b l a s t s . The g e n o t o x i c e f f e c t s o f chemicals can be d e t e c t e d by the DNA r e p a i r e l i c i t e d by DNA base damage, s t r a n d breakage and c r o s s - l i n k a g e . DNA r e p a i r t e s t s are r e l i a b l e i n d i c a t o r s f o r p o t e n t i a l c a r c i n o g e n s ( S t i c h e t a l . , 11 1971; San and S t i c h , 1975; W i l l i a m s , 1976, 1977, 1978; M a r t i n e t a l , 1978; H o l l s t e i n e t a l . , 1979; M i t c h e l l e t a l . , 1983). UDS r e p r e s e n t s a form of DNA r e p a i r i n v o l v i n g e x c i s i o n o f damaged DNA and i n c o r p o r a t i o n o f newly s y n t h e s i z e d DNA (unscheduled s y n t h e s i s as opposed t o the normal DNA s y n t h e s i s d u r i n g chromosome r e p l i c a t i o n ) . UDS can be measured by a u t o r a d i o g r a p h i c d e t e c t i o n o f unscheduled i n c o r p o r a t i o n o f thymidine, one of the f o u r b a s i c components of DNA, t h a t has been l a b e l l e d w i t h t r i t i u m . The l e v e l of DNA r e p a i r (or damage) i s es t i m a t e d from t h e average number of s i l v e r g r a i n s over each n u c l e u s . A d o s e - r e l a t e d i n c r e a s e i n g r a i n count i s c o n s i d e r e d as a p o s i t i v e i n d i c a t i o n o f DNA damage. The UDS assay i s not r e s t r i c t e d t o c u l t u r e d c e l l s but can be a p p l i e d i n v i v o o r i n v i t r o t o c e l l s t h a t have been f r e s h l y i s o l a t e d from t e s t animals o r human b i o p s i e s . (d) DNA r e p a i r i n h i b i t i o n . T h i s i s i n c l u d e d as a compliment t o the UDS assay because the absence o f any demonstrable r e p a i r s y n t h e s i s as measured by t r i t i a t e d t hymidine uptake can be i n t e r p r e t e d as e i t h e r the r e s u l t o f no DNA damage o r as an i n h i b i t o r y e f f e c t e x e r t e d by the t e s t substance on DNA r e p a i r . The t e s t procedure f o r DNA r e p a i r i n h i b i t i o n s t u d i e s i s i d e n t i c a l t o t h a t f o r the UDS assay, w i t h t h e e x c e p t i o n t h a t the c e l l s are i r r a d i a t e d w i t h a st a n d a r d dose of U V - l i g h t p r i o r t o exposure t o the t e s t substance. 12 METHODS AND MATERIALS 1. P r e p a r a t i o n o f E x t r a c t s Stock p r e p a r a t i o n s o f tobacco e x t r a c t were f r e s h l y p r e pared f o r each experiment by macerating tobacco samples i n t i s s u e c u l t u r e media (Eagle's Minimal E s s e n t i a l Medium wit h o u t f e t a l c a l f serum o r A r g i n i n e d e f i c i e n t medium w i t h 2.5% f e t a l c a l f serum) w i t h a p e s t l e and mortar f o r f i v e minutes. E x t r a c t s were c e n t r i f u g e d a t 2600 rpm f o r t e n minutes. Supernatants were removed, the pH a d j u s t e d t o approximately 7.4 and s e r i a l d i l u t i o n s were prepared f o r t e s t i n g . The c o n c e n t r a t i o n s used f o r a l l experiments were c a l c u l a t e d from the amount of tobacco used t o prepare the st o c k . 2. Chemicals N-met h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) was ob t a i n e d form A l d r i c h Chemical Co., Milwaukee, Wis. C a t a l a s e : p u r i f i e d powder from bovine l i v e r , 1100 Sigma u n i t s p e r mg p r o t e i n was o b t a i n e d from Sigma Chemical Co., St . L o u i s , Mo. 3. T i s s u e C u l t u r e 3.1 C u l t u r e Media E a g l e s ' s Minimal E s s e n t i a l Medium (MEM, Grand I s l a n d B i o l o g i c a l Company, Berkeley, C a l i f o r n i a ) was r e c o n s t i t u t e d 13 from a powder w i t h d i s t i l l e d water and s t e r i l i z e d by passage through a M i l l i p o r e f i l t e r (pore s i z e : 0.22 microns; M i l l i p o r e F i l t e r C o r p o r a t i o n , Mass.). A r g i n i n e d e f i c i e n t medium (ADM), based on the standa r d formula f o r E a g l e ' s minimal e s s e n t i a l medium, was prepared i n the manner d e s c r i b e d by San and S t i c h (1982). Sodium b i c a r b o n a t e (1 mg/ml) and the f o l l o w i n g a n t i b i o t i c s , 100 u n i t s / m l p e n i c i l l i n , 100 jug/ml s t r e p t o m y c i n s u l p h a t e , 100 tiq/rnl kanamycin, and 2.5 jug/ml fungizone were added t o the c u l t u r e media. MEM was supplemented w i t h 10% f e t a l c a l f serum (FCS) whereas ADM was supplemented w i t h 2.5% FCS. 4. Growth o f Stock C u l t u r e s Chinese hamster ovary (CHO) c e l l s and human f i b r o b l a s t s were grown i n MEM supplemented w i t h 10% FCS and a n t i b i o t i c s . The s t o c k c u l t u r e s were maintained i n 260 ml p l a s t i c c u l t u r e f l a s k s a t 37 C i n a w a t e r - s a t u r a t e d C0 2 (5%) i n c u b a t o r . 5. Chromosome A b e r r a t i o n T e s t 5.1 P r e p a r a t i o n o f C e l l C u l t u r e s F o r each chromosome a b e r r a t i o n experiment, approximately 60,000 CHO c e l l s were seeded onto 22 mm2 c o v e r s l i p s i n 35 mm p e t r i d i s h e s and kept i n MEM (10% FCS) a t 37 C f o r two days. Experiments were begun when the c u l t u r e s were approximately 60% c o n f l u e n t . 14 5.2 Exposure t o T e s t E x t r a c t The t i s s u e c u l t u r e medium was s u c t i o n e d from the p e t r i d i s h e s and r e p l a c e d w i t h 1 ml o f the t e s t e x t r a c t . For experiments i n v o l v i n g the i n f l u e n c e o f c a t a l a s e on the c l a s t o g e n i c a c t i v i t y o f the t e s t e x t r a c t , a 0.5 ml a l i q u o t o f c a t a l a s e prepared i n MEM (without serum) t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.1 mg/ml was added t o each p e t r i d i s h p r i o r t o the a d d i t i o n o f 0.5 ml of the t e s t e x t r a c t . The response o f the CHO c e l l s t o a known g e n o t o x i c and c a r c i n o g e n i c agent was determined by exposing c e l l c u l t u r e s t o MNNG d i s s o l v e d i n MEM. F o l l o w i n g a 3 hour i n c u b a t i o n p e r i o d , the t e s t m a t e r i a l was removed, the c o v e r s l i p s washed t w i c e w i t h MEM (without serum) and 2 ml o f f r e s h MEM (10% FCS) were added t o each p e t r i d i s h . 5.3 Chromosome P r e p a r a t i o n s For e s t i m a t i n g the frequency o f chromosome a b e r r a t i o n s , 0.2 ml o f c o l c h i c i n e (0.01% i n MEM) was added a t 16 hours post-exposure t o the e x t r a c t s and l e f t f o r 3.5 hours. C e l l s were then t r e a t e d f o r 20 minutes w i t h a h y p o t o n i c s o l u t i o n (1% sodium c i t r a t e ) t o s w e l l the c e l l s i n o r d e r t o o b t a i n w e l l - s p r e a d metaphase p l a t e s . T h i s treatment was immediately f o l l o w e d w i t h f i x a t i o n i n e t h a n o l : a c e t i c a c i d (3:1, v/v) f o r 20 minutes. A i r - d r i e d s l i d e s were s t a i n e d w i t h 2% o r c e i n i n 50% a c e t i c acid/water (5-8 minutes), dehydrated and mounted. 15 5.4 A n a l y s i s o f Metaphase P l a t e s f o r Chromosome A b e r r a t i o n s S l i d e s were randomized and coded b e f o r e b e i n g s c o r e d . F o r each sample, a t l e a s t 50 w e l l - s p r e a d metaphases were a n a l y s e d f o r chromatid gaps, breaks and exchanges. Small u n s t a i n e d l e s i o n s a l o n g the l e n g t h o f a chromatid were c o n s i d e r e d gaps i f the d i s t a l chromatid fragment was not d i s l o c a t e d o r i f the l e s i o n was l e s s than t h e width of a chromatid. Only d i s l o c a t e d chromatid fragments were counted as breaks. A l l types of a b e r r a t i o n s which r e q u i r e r e j o i n i n g were p l a c e d i n t o the c a t e g o r y e n t i t l e d , " Exchanges." 6. M i c r o n u c l e u s Assay 6.1 T e s t Procedure The procedure f o r t h i s assay was i d e n t i c a l t o t h a t f o r the chromosome a b e r r a t i o n t e s t w i t h the f o l l o w i n g e x c e p t i o n s : CHO c e l l s were seeded a t a d e n s i t y o f 3 0,000 c e l l s / d i s h , p ost-treatment i n c u b a t i o n was f o r 24 hours, no c o l c h i c i n e was added, and sodium c i t r a t e treatment was f o r 4-5 minutes. 6.2 A n a l y s i s o f Interphase C e l l s f o r M i c r o n u c l e i M i c r o n u c l e i are formed when a c e n t r i c chromosome fragments o r chromosomes l a c k i n g a s p i n d l e attachment are l e f t behind a t anaphase and are excluded from the daughter n u c l e i . M i c r o n u c l e i are d e t e c t a b l e as s m a l l b o d i e s 16 resembling t h e main nucleus except i n s i z e . For each dose, 1000 i n t a c t i n t e r p h a s e c e l l s were s c o r e d f o r the presence of m i c r o n u c l e i . A micronucleus was not s c o r e d i f i t (1) had a diameter g r e a t e r than o n e - t h i r d t h a t o f the main nucleus t o a v o i d i n c l u d i n g b i n u c l e a t e d c e l l s o r (2) touched the main nu c l e u s so t h a t t h e r e would be no c o n f u s i o n w i t h n u c l e a r b l e b s . A c e l l w i t h many m i c r o n u c l e i and no main nucleus was c o n s i d e r e d t o have undergone k a r y o r r h e x i s and was not s c o r e d . A l l s l i d e s were randomized and coded b e f o r e b e i n g s c o r e d . 7. DNA R e p a i r S t u d i e s 7.1 P r e p a r a t i o n of C e l l C u l t u r e s Human f i b r o b l a s t s were seeded onto 22 mm2 c o v e r s l i p s i n 35 mm p e t r i d i s h e s a t approximately 30,000 c e l l s p e r d i s h and covered w i t h MEM supplemented w i t h 10% FCS. 5-7 days l a t e r , c e l l c u l t u r e s were washed by d i p p i n g the c o v e r s l i p s about seven times i n each o f two beakers o f ADM (no serum) and then t r a n s f e r r e d t o new c u l t u r e d i s h e s c o n t a i n i n g 2 ml ADM (2.5% FCS). A f t e r 5-6 days, more than 90% of the c e l l s were a r r e s t e d a t 7.2 U l t r a v i o l e t I r r a d i a t i o n The t i s s u e c u l t u r e medium was removed by s u c t i o n from c u l t u r e s t o be i r r a d i a t e d and the c u l t u r e s were r i n s e d w i t h s t e r i l e phosphate b u f f e r e d s a l i n e t o remove any remaining 17 c u l t u r e medium which can absorb UV l i g h t . C u l t u r e s were i r r a d i a t e d f o r 2 0 seconds w i t h a dose of 4 ergs/mm 2/s as measured by a B l a k - r a y UV l i g h t meter ( U l t r a v i o l e t Products, Inc., San G a b r i e l , C a l i f o r n i a ) . The source o f UV i r r a d i a t i o n was a General E l e c t r i c g e r m i c i d a l lamp (model no. G1RT8). 7.3 Exposure t o T e s t E x t r a c t and R a d i o a c t i v e l y - l a b e l l e d  Thymidine T r i t i a t e d thymidine ( s p e c i f i c a c t i v i t y 20 Ci/mmol) was o b t a i n e d as thymidine (methyl- 3H) i n aqueous s o l u t i o n from New England N u c l e a r and d i l u t e d t o a c o n c e n t r a t i o n o f 20 juCi/ml w i t h ADM (2.5% FCS). T i s s u e c u l t u r e medium was removed from the t e s t c u l t u r e s , and r e p l a c e d f o r a d u r a t i o n o f 3 hours w i t h a combination of 0.5 ml o f t r i t i a t e d t h y m i d i n e - c o n t a i n i n g medium and 0.5 ml o f the t e s t e x t r a c t . 7.4 F i x a t i o n and P r e p a r a t i o n f o r Autoradiography F o l l o w i n g t e s t exposure, the c o v e r s l i p s were washed by d i p p i n g 5 times i n each o f 3 beakers o f ADM (no serum), t r e a t e d w i t h sodium c i t r a t e f o r 15 minutes, f i x e d i n e t h a n o l / a c e t i c a c i d (3:1) f o r 15 minutes and allowed t o a i r -dry . The next day, c o v e r s l i p s , w i t h the c e l l monolayer upwards, were a t t a c h e d t o g l a s s microscope s l i d e s w i t h a s m a l l q u a n t i t y of p a r a f f i n . The s l i d e s were passed through 18 a graded s e r i e s o f a l c o h o l s (95%, 70%, and 20% ethanol) and d i s t i l l e d water (10 minutes each) and a i r - d r i e d . 7.5 Autoradiography and S t a i n i n g S l i d e s were i n d i v i d u a l l y dipped i n Kodak NTB-3 n u c l e a r t r a c k emulsion t h a t had been thawed a t 43 C i n a water-bath and d i l u t e d 1:1 w i t h d i s t i l l e d water. The coated s l i d e s were a i r - d r i e d i n a v e r t i c a l p o s i t i o n , then s t o r e d a t 4 c f o r 14 days i n l i g h t t i g h t boxes. Before d e v e l o p i n g , the s l i d e s were brought back t o room temperature. P r o c e s s i n g was done a t 18 C i n Kodak D-19 deve l o p e r (3 minutes) and Kodak f i x e r (10 minutes). A f t e r r i n s i n g t he s l i d e s i n run n i n g water f o r 30 minutes, the s l i d e s were s t a i n e d f o r 20 minutes w i t h 2% a c e t o - o r c e i n , dehydrated and mounted by superimposing another c o v e r s l i p over t h a t b e a r i n g the c e l l monolayer. 7.6 A n a l y s i s o f Autoradiograms The amount o f unscheduled DNA s y n t h e s i s was estimated by c o u n t i n g the number o f g r a i n s over each nucleus minus the background count i n an area equal i n s i z e and a d j a c e n t t o each n u c l e u s . Counting was done w i t h an A r t e k Model 880 automatic g r a i n counter. G r a i n counts were made on s m a l l i n t e r p h a s e n u c l e i o f comparable s i z e so t h a t o n l y d i p l o i d c e l l s would be i n c l u d e d . The mean g r a i n count p e r nucleus 19 was determined by s c o r i n g a t l e a s t 30 n u c l e i a t random l o c a t i o n s on each s l i d e . 20 RESULTS 1. Chromosome Damaging C a p a c i t y o f Aqueous Tobacco E x t r a c t s Crude aqueous e x t r a c t s were prepared from s e v e r a l commonly used smokeless tobacco samples and t e s t e d f o r c l a s t o g e n i c a c t i v i t y . Chinese hamster ovary c e l l c u l t u r e s were exposed f o r t h r e e hours t o the e x t r a c t s and c o l c h i c i n e -a r r e s t e d metaphase p l a t e s were a n a l y s e d f o r the presence o f chromosome a b e r r a t i o n s . T a b l e I shows r e p r e s e n t a t i v e data f o r a North American chewing tobacco, K h a i n i tobacco, nass and s n u f f . R e s u l t s f o r each e x t r a c t are from two independent experiments conducted on d i f f e r e n t days. The ext e n t o f chromosome damage i s expressed as percentage o f metaphase p l a t e s w i t h a t l e a s t one chromatid break o r exchange and as the average number o f exchanges p e r metaphase p l a t e . T y p i c a l background f r e q u e n c i e s o f metaphases w i t h chromatid a b e r r a t i o n s i n c o n c u r r e n t samples run i n the absence o f the t e s t e x t r a c t s were between 0 and 1%. Only s i n g l e gaps o r breaks and never exchanges were observed i n the c o n t r o l c u l t u r e s o f any experiment. A p o s i t i v e response o f CHO c e l l s t o a known g e n o t o x i c and c a r c i n o g e n i c agent was demonstrated by exposing the c e l l s t o MNNG. I n a t y p i c a l experiment, a frequency o f 69% metaphase p l a t e s w i t h chromatid a b e r r a t i o n s was observed a f t e r exposure t o a 1 0 - 4 M s o l u t i o n o f MNNG. 21 TABLE I CLASTOGENIC ACTIVITY; OF CRUDE AQUEOUS TOBACCO EXTRACTS IN CHINESE HAMSTER OVARY PETTfi TOBACCO CONCENTRATION TOTAL # % METAPHASES WITH AVERAGE # (mg equiv/ml) METAPHASES CHROMATID BREAKS EXCHANGES ANALYSED AND EXCHANGES PER METAPHASE CHEWING TOBACCO Exp. 1 15 100 37.0 1.37 10 100 2.0 0.01 0 100 0.0 0.00 Exp. 2 8 100 22.0 0.86 6 100 16.0 0.60 4 100 2.0 0.02 0 100 1.0 0.00 Khaini Tobacco Exp. 1 12 100 34.0 1.08 6 100 24.0 0.83 3 100 6.0 0.23 0 100 0.0 0.00 EXP- 2 10 100 26.0 0.80 8 100 18.0 0.83 6 100 9.0 0.30 0 100 0.0 0.00 Nass Exp. 1 80 100 34.0 0.53 60 100 7.0 0.01 40 100 2.0 0.00 0 100 0.0 0.00 Exp. 2 60 100 79.0 2.13 40 100 10.0 0.12 20 100 1.0 0.00 0 100 0.0 0.00 Snuff Exp. 1 80 51 35.3 0.96 0 100 0.0 0.00 Exp. 2 80 64 21.9 0.69 70 100 2.0 0.00 0 100 0.0 0.00 22 The data p r e s e n t e d i n Ta b l e I show t h a t a l l tobacco e x t r a c t s e x h i b i t e d p otent c l a s t o g e n i c a c t i v i t y as demonstrated by the g r e a t l y i n c r e a s e d frequency o f chromosome a b e r r a t i o n s . The c l a s t o g e n i c a c t i v i t y f o r most of the tobacco e x t r a c t s , however, was o n l y d e t e c t a b l e i n a narrow c o n c e n t r a t i o n range. The range o f c o n c e n t r a t i o n s of the v a r i o u s tobacco e x t r a c t s t h a t induced chromosome a b e r r a t i o n s v a r i e d g r e a t l y . The a c t i v e doses f o r the s n u f f and nass were many times h i g h e r than those f o r the chewing and K h a i n i tobaccos. The type o f chromosome a b e r r a t i o n s induced a r e q u a n t i f i e d i n Ta b l e I I . Chromatid gaps, chromatid breaks, i s o c h r o m a t i d breaks and s i n g l e and m u l t i p l e exchange f i g u r e s were r e a d i l y observed i n t r e a t e d CHO c u l t u r e s . The ext e n t o f damage per metaphase p l a t e w i t h a b e r r a t i o n s v a r i e d w i d e l y from a s i n g l e gap, break or exchange t o complete f r a g m e n t a t i o n o f chromosomes or m u l t i p l e exchanges i n v o l v i n g t h e e n t i r e chromosome complement o f the c e l l . 2. I n d u c t i o n o f M i c r o n u c l e i by Aqueous Tobacco E x t r a c t s C u l t u r e s t o f CHO c e l l s were exposed t o s n u f f , chewing tobacco, K h a i n i tobacco and nass e x t r a c t s f o r t h r e e hours and h a r v e s t e d 24 hours post-treatment. T a b l e I I I shows the r e s u l t s o b t a i n e d f o r each of t h e f o u r e x t r a c t s , p r o v i d i n g d a t a from two r e p l i c a t e experiments. The background frequency o f m i c r o n u c l e i i n c o n t r o l c u l t u r e s ranged from 0.4 23 TABLE II CLASTOGENIC ACTIVITY OF CRUDE AQUEOUS TOBACCO EXTRACTS -DAMAGE BY TYPES TOBACCOCONCENTRATION TOTAL # (mg eguiv/ml) METAPHASES CHROMATID  ANALYSED GAP BREAK EXCHANGE CHEWING ~ TOBACCO Exp. 1 15 100 10 100 0 100 2 129 137 0 2 1 0 0 0 Exp. 2 8 6 4 0 100 100 100 100 4 3 3 0 69 83 1 1 86 60 2 0 KHAINI TOBACCO Exp. 1 Exp. 2 12 6 3 0 10 8 6 0 100 100 100 100 100 100 100 100 2 2 1 1 6 3 1 0 68 79 3 0 170 81 49 0 108 83 23 0 80 83 30 0 NASS Exp. 1 Exp. 2 80 60 40 0 60 40 20 0 100 100 100 100 100 100 100 100 10 4 3 0 8 4 3 0 64 10 2 0 194 25 1 0 53 1 0 0 213 12 0 0 SNUFF Exp.l 80 51 5 53 49 0 100 0 0 0 Exp. 2 80 64 0 19 39 70 100 4 2 0 0 100 1 0 0 24 TABLE III MICRONUCLEI FORMATION IN CHINESE HAMSTER OVARY fRT.Tfi EXPOSED TO AQUEOUS TOBACCO EXTRACTS TOBACCO CONCENTRATION PERCENT CELLS (mg equivalents/ml) WITH MICRONUCLEI CHEWING TOBACCO Exp. 1 10 13.6 6 9.0 0 0.6 Exp. 2 10 10.7 6 3.1 0 0.6 KHAINI TOBACCO Exp. 1 6 9.5 3 4.7 0 0.9 Exp. 2 8 13.9 6 9.6 0 0.7 NASS Exp. 1 60 11.3 40 9.1 20 2.0 0 0.6 Exp. 2 80 7.5 60 6.5 40 2.9 0 0.4 SNUFF Exp. 1 80 12.1 60 3.6 0 0.6 Exp. 2 70 3.9 60 1.3 0 0.5 25 t o 0.9% (averaging 0.6%). In a t y p i c a l experiment, the frequency o f m i c r o n u c l e a t e d CHO c e l l s f o l l o w i n g exposure t o a 10~ 4 M s o l u t i o n o f MNNG was 12.3%. A l l o f the tobacco e x t r a c t s produced s i g n i f i c a n t i n c r e a s e s i n m i c r o n u c l e i frequency over doses s i m i l a r t o those which induced chromosome a b e r r a t i o n s . 3. I n d u c t i o n o f Unscheduled DNA S y n t h e s i s by Aqueous  Tobacco E x t r a c t s C u l t u r e d human f i b r o b l a s t s were kept i n a r g i n i n e -d e f i c i e n t medium f o r 5-6 days t o prevent them from e n t e r i n g S-phase and permit the d e t e c t i o n o f unscheduled DNA s y n t h e s i s without i n t e r f e r e n c e by DNA s y n t h e s i s a s s o c i a t e d w i t h chromosome r e p l i c a t i o n . These c u l t u r e s were s i m u l t a n e o u s l y exposed t o tobacco e x t r a c t s and t r i t i a t e d thymidine f o r t h r e e hours. The tobacco samples were prepared t o y i e l d the h i g h e s t doses p o s s i b l e and i n c l u d e d c o n c e n t r a t i o n s s e v e r a l times g r e a t e r than those r e q u i r e d t o produce chromosome a b e r r a t i o n s i n CHO c e l l s . F i g u r e 1 shows the i n d u c t i o n o f unscheduled DNA s y n t h e s i s i n f i b r o b l a s t s exposed t o the North American chewing tobacco e x t r a c t as r e v e a l e d by the l e v e l s o f t r i t i a t e d thymidine i n c o r p o r a t i o n d e t e c t e d by autoradiography. The l e v e l o f t r i t i a t e d t hymidine i n c o r p o r a t i o n was dependent on the c o n c e n t r a t i o n o f e x t r a c t used, i n c r e a s i n g s h a r p l y , and then d e c l i n i n g w i t h h i g h e r doses. T r i t i a t e d thymidine i n c o r p o r a t i o n was not T O B A C C O C O N C E N T R A T I O N ( m g e q u i v a l e n t s p e r m l ) Figure 1. Unscheduled DNA synthesis i n human f i b r o b l a s t s exposed to a chewing tobacco extract. Values indicated are grains per nucleus (corrected for background) ± S.E. 2 7 d e t e c t a b l e i n f i b r o b l a s t s exposed t o any c o n c e n t r a t i o n of s n u f f , K h a i n i tobacco or nass e x t r a c t . A l l o f these tobaccos were t e s t e d t o the l e v e l s o f t o x i c i t y as r e v e a l e d by the presence o f s m a l l n u c l e i s t a i n e d h e a v i l y w i t h o r c e i n . These c e l l s p r o b a b l y r e p r e s e n t p y c n o t i c n u c l e i o f dead or d y i n g c e l l s . I r r a d i a t i o n w i t h UV l i g h t (a known DNA-damaging agent) i n i t i a t e d UDS i n c o n c u r r e n t c o n t r o l c u l t u r e s run i n the absence o f the t e s t e x t r a c t s . 4. I n h i b i t i o n of DNA-repair S y n t h e s i s The absence o f any demonstrable UDS as measured by t r i t i a t e d thymidine uptake can be i n t e r p r e t e d as e i t h e r the r e s u l t o f no DNA damage or as an i n h i b i t o r y e f f e c t on DNA r e p a i r . To t e s t f o r t h i s second p o s s i b i l i t y an i d e n t i c a l procedure t o t h a t d e s c r i b e d f o r t h e unscheduled DNA s y n t h e s i s t e s t was used w i t h the e x c e p t i o n t h a t the f i b r o b l a s t s were exposed t o u l t r a v i o l e t r a d i a t i o n p r i o r t o exposure t o the tobacco e x t r a c t s . R e s u l t s are shown i n F i g u r e s 2-5. A l l examined tobaccos produced i n h i b i t i o n of DNA r e p a i r as shown by a r e d u c t i o n i n the number of g r a i n s p e r n u c l e u s i n tobacco-exposed f i b r o b l a s t s compared t o f i b r o b l a s t s r e c e i v i n g UV alone. Trypan b l u e e x c l u s i o n showed t h a t t h e f i b r o b l a s t s exposed t o tobacco c o n c e n t r a t i o n s t h a t completely i n h i b i t e d t r i t i a t e d thymidine i n c o r p o r a t i o n were s t i l l v i a b l e . These r e s u l t s i n d i c a t e 28 ^oo-l 8 0 -3 6 0 -LU _J O z or iu a. CO 2 < o 4 0 -2 0 -no extract i 10 — i — 2 0 T O B A C C O C O N C E N T R A T I O N (mg equivalents per ml) Figure 2. Inhibitory e f f e c t of a chewing tobacco extract on induced DNA repair i n human f i b r o b l a s t s . Error bars - S.E. 29 60 no 5 10 15 2 0 TOBACCO C O N C E N T R A T I O N (mg e q u i v a l e n t s per ml) Figure 3. Unscheduled DNA synthesis i n human f i b r o b l a s t s exposed to Khaini tobacco: (a) Tobacco alone (•); (b) Inh i b i t o r y e f f e c t on UV-induced DNA repair (•); Error bars = S.E. 30 100-1 Figure 4. Unscheduled DNA synthesis i n human f i b r o b l a s t s exposed nass: (a) Nass alone (•); (b) Inhibitory e f f e c t on UV-induced DNA repair (•); Error bars - S.E. 31 CO LU _J o or LU Q. CO 2 < CC O 80 -no extract T O B A C C O C O N C E N T R A T I O N (mg e q u i v a l e n t s p e r m l ) Figure 5. Unscheduled DNA synthesis i n human f i b r o b l a s t s exposed to snuff: (a) Snuff alone (•); (b) Inhibitory e f f e c t on UV-induced DNA repair (•); Error bars - S.E. 32 t h a t the tobacco e x t r a c t s are l i k e l y e x e r t i n g an i n h i b i t o r y e f f e c t on DNA r e p a i r and are not merely k i l l i n g the c e l l s . 5. E x p l o r a t i o n o f the A c t i v e Genotoxic Components of the  E x t r a c t s The m o d i f y i n g e f f e c t s of q u i d components, a l c o h o l , d i e t a r y and o t h e r f a c t o r s on tobacco g e n o t o x i c i t y and c a r c i n o g e n i c i t y r e q u i r e m e c h a n i s t i c s t u d i e s . The r o l e of hydrogen p e r o x i d e i n the c l a s t o g e n i c a c t i v i t y of the tobacco e x t r a c t s was examined by exposing CHO c e l l s f o r t h r e e hours t o tobacco e x t r a c t s w i t h and without c a t a l a s e . The r e s u l t s are d i s p l a y e d i n T a b l e IV. C a t a l a s e p r o t e c t e d a g a i n s t the chromosome damaging a c t i v i t y o f the chewing and K h a i n i tobaccos but had no e f f e c t on the c l a s t o g e n i c a c t i o n of s n u f f o r nass. 6. Other T e s t Systems E x p l o r e d 6.1 UDS i n Rat O r a l Mucosal C e l l s The o r i g i n a l e f f o r t s o f t h i s r e s e a r c h p r o j e c t were d i r e c t e d towards the a d a p t a t i o n of the unscheduled DNA s y n t h e s i s assay t o monitor DNA r e p a i r i n f r e s h l y i s o l a t e d r a t o r a l and oesophageal mucosal c e l l s . T h i s would have p e r m i t t e d the d e t e c t i o n of the o r g a n - s p e c i f i c a c t i o n s of c a r c i n o g e n s and the i n v e s t i g a t i o n o f the i n h i b i t i o n of c a r c i n o g e n - i n d u c e d DNA damage by p u t a t i v e a n t i c a r c i n o g e n s . U n f o r t u n a t e l y , the r e s u l t s o f these e f f o r t s 33 TABLE IV EFFECT OF CATALASE ON THE CLASTOGENIC ACTIVITY OF AQUEOUS TOBACCO EXTRACTS IN CHINESE HAMSTER OVARY CELLS TOBACCO CONCENTRATION (mg equivalents/ml) PERCENT METAPHASES WITH CHROMATID BREAKS AND EXCHANGES + CATAIASEa ALONE CHEWING TOBACCO Exp. 1 20 MI 2 15 39 0 10 4 0 Exp. 2 20 MI 1 10 24 0 6 11 0 KHAINI TOBACCO Exp. 1 20 MI 3 10 26 2 8 18 0 6 9 0 Exp. 2 20 MI 7 10 34 1 5 5 0 NASS Exp. 1 60 72 69 40 17 14 20 0 1 Exp. 2 80 36 32 60 9 2 40 4 1 SNUFF Exp. 1 80 22 27 70 2 1 Exp. 2 80 18 22 70 1 7 f* Concentration of catalase: 0.01 mg/ml b MI. mitotic inhibition: less than 1 metaphase plate among 6000 c e l l s . 34 were d i s a p p o i n t i n g . Because the i s o l a t e d mucosal e p i t h e l i a l c e l l s were a t numerous stages of d i f f e r e n t i a t i o n w i t h v a r y i n g degrees of k e r a t i n i z a t i o n t h e r e was s i g n i f i c a n t v a r i a t i o n i n g r a i n counts. T h i s was most l i k e l y due t o s e l f - a b s o r p t i o n of t r i t i u m beta-decay by k e r a t i n which would then p r e v e n t the i n t e r a c t i o n o f b e t a - p a r t i c l e s w i t h the p h o t o g r a p h i c emulsion. T h i s problem has been encountered i n o t h e r systems (Lake e t a l . , 1978). Furthermore, when suspensions o f v i a b l e mucosal c e l l s were o b t a i n e d through a p r o t e a s e d i g e s t i o n procedure, the c e l l s remained i n c l u s t e r s o f 10 o r more c e l l s . The c l u s t e r i n g o f c e l l s s e v e r e l y l i m i t e d the a n a l y s i s o f experiments due t o the o v e r l a p o f n u c l e i w i t h i n the c l u s t e r s and i n t e r f e r e n c e by the s p i l l a g e o f g r a i n s from v e r y h e a v i l y - l a b e l l i n g c e l l s i n S-phase. A l l attempts t o i s o l a t e s i n g l e c e l l s by a l t e r i n g the i s o l a t i o n procedure r e s u l t e d i n an unacceptable l o s s o f v i a b i l i t y and attempts t o i n h i b i t S-phase w i t h hydroxyurea, even w i t h c o n c e n t r a t i o n s as h i g h as 10~ 2 M, were not s u c c e s s f u l . These t e c h n i c a l d i f f i c u l t i e s render the study o f UDS i n r a t o r a l mucosa u n f e a s i b l e . 6.2 M i c r o n u c l e i i n Rodent O r a l Mucosa In p r i n c i p l e , the micronucleus t e s t can be extended i n v i v o t o animals. I t was hoped t h a t the o r a l mucosa of rodents c o u l d be screened f o r the presence 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 c e l l s o b t a i n e d by swabbing o r s c r a p i n g the o r a l 35 mucosa f o l l o w i n g a procedure s i m i l a r t o t h a t used by S t i c h and R o s i n (1984) i n the s c r e e n i n g o f human p o p u l a t i o n s . T h i s technique would have p r o v i d e d an o p p o r t u n i t y t o monitor chemoprevention regimes i n tobacco t r e a t e d animals. R e g r e t t a b l y , s u f f i c i e n t numbers of i n t a c t c e l l s f o r a n a l y s i s c o u l d not be o b t a i n e d from the o r a l c a v i t y o f e i t h e r r a t s or mice because o f the heavy k e r a t i n i z a t i o n o f t h e o r a l mucosae i n t h e s e animals. 36 DISCUSSION The a s s o c i a t i o n between o r a l cancer and the i n t r a - o r a l use o f t o b a c c o - c o n t a i n i n g mixtures i s a l r e a d y w e l l e s t a b l i s h e d . I t i s important, however, t o understand the reasons f o r v a r i a t i o n s i n the p r e v a l e n c e and r i s k of p r e n e o p l a s t i c and n e o p l a s t i c o r a l l e s i o n s among d i f f e r e n t tobacco chewing p o p u l a t i o n s t o h e l p i n t h e d e s i g n of p r e v e n t i o n s t r a t e g i e s . Short-term, i n v i t r o g e n o t o x i c i t y t e s t s are p o t e n t i a l l y u s e f u l i n the i d e n t i f i c a t i o n o f agents which may c o n t r i b u t e t o o r modify tobacco c a r c i n o g e n e s i s , but an i n v i t r o t e s t system w i l l o n l y be u s e f u l i n such a study i f i t i s capable of d e t e c t i n g the g e n o t o x i c e f f e c t s of many d i f f e r e n t types o f tobacco mixtures. The chemical c o m p o s i t i o n o f t h e v a r i o u s tobacco mixtures i s expected t o d i f f e r , hence the g e n o t o x i c and c a r c i n o g e n i c components may a l s o v a r y . I t was e s s e n t i a l , t h e r e f o r e , t o t e s t a v a r i e t y o f tobacco mixtures i n s e v e r a l s h o r t - t e r m assays w i t h d i f f e r e n t g e n o t o x i c end-points i n o r d e r t o s e l e c t a s u i t a b l e t e s t system. These experiments i n d i c a t e t h a t unburned tobacco m i x t u r e s possess s u b s t a n t i a l g e n o t o x i c a c t i v i t y , y e t a l s o p o i n t t o the n e c e s s i t y o f u s i n g a v a r i e t y o f endpoints when a s s e s s i n g the g e n o t o x i c i t y o f a compound o r mixture. The crude aqueous e x t r a c t s o f a l l tobacco mixtures assayed produced s i g n i f i c a n t chromosomal damage i n c u l t u r e d Chinese 37 hamster ovary c e l l s as r e v e a l e d by the presence o f chromatid gaps, breaks and exchanges i n metaphase chromosomes (chromosome a b e r r a t i o n t e s t ) and m i c r o n u c l e i i n i n t e r p h a s e c e l l s (micronucleus t e s t ) . Furthermore, the data presented h e r e c o n c e r n i n g the i n f l u e n c e of c a t a l a s e on t h e c l a s t o g e n i c a c t i o n of tobacco e x t r a c t s i n d i c a t e the way such model systems can be used t o e x p l o r e the nature and m o d i f i c a t i o n o f the g e n o t o x i c e f f e c t s of complex tobacco mixtures. On the o t h e r hand, DNA damage as measured by t r i t i a t e d thymidine uptake as a r e s u l t o f DNA r e p a i r s y n t h e s i s c o u l d be demonstrated f o r o n l y one of the t e s t e d t o b a c c o s . On the b a s i s o f these r e s u l t s the chromosome a b e r r a t i o n and micronucleus t e s t s , but not the unscheduled DNA s y n t h e s i s assay, would appear t o be s u i t a b l e as t e s t systems f o r the study o f f a c t o r s i n f l u e n c i n g tobacco c a r c i n o g e n i c i t y . The e x p l o i t a t i o n of t h e chromosome a b e r r a t i o n and m i c r o n u c l e u s t e s t s f o r the examination o f tobacco c a r c i n o g e n i c i t y may be j u s t i f i e d because most agents t h a t ar e c a r c i n o g e n i c i n animals are a l s o p otent i n d u c e r s of chromosome damage (Evans, 1974; I s h i d a t e and Odishima, 1977; H o l l s t e i n e t a l . , 1979; P r e s t o n e t a l . , 1981). T h e r e f o r e , even though the p r e c i s e nature of the g e n o t o x i c l e s i o n s i n v o l v e d i n c a r c i n o g e n e s i s i s unknown, chromosomal a b e r r a t i o n s and m i c r o n u c l e i may r e p r e s e n t convenient markers f o r t h e s e l e s i o n s . A l t e r n a t i v e l y , chromosomal damage may be i n t r i n s i c a l l y i n v o l v e d i n the p r o c e s s o f n e o p l a s i a . 38 Chromosomal anomalies are observed throughout the p r o c e s s of c a r c i n o g e n e s i s as e a r l y changes i n carcinogen-exposed t i s s u e s , i n d y s p l a s i a s and o t h e r p r e n e o p l a s t i c s t a t e s , and i n the malignant c e l l s o f most n e o p l a s i a s . Non-random chromosome changes are a s s o c i a t e d w i t h a number of human can c e r s o f d i v e r s e o r i g i n (Yunis, 1983). C a i r n s (1981) has proposed t h a t the t r a n s p o s i t i o n o f major chromosomal segments may be the mechanism by which common cancers are i n i t i a t e d . Chromosome rearrangements may b r i n g t o g e t h e r two genes t h a t are normally f a r a p a r t and are under d i f f e r e n t r e g u l a t o r y c o n t r o l . T h i s t r a n s p o s i t i o n may r e s u l t i n d e r e g u l a t i o n and a b e r r a n t e x p r e s s i o n o f c e l l u l a r (onco-) genes f u n c t i o n i n g i n the r e g u l a t i o n of c e l l growth and d i f f e r e n t i a t i o n (De K l e i n , 1982; G i l b e r t , 1983; Rowley, 1984). Cancers c o u l d a l s o a r i s e from the l o s s o f g e n e t i c f u n c t i o n as t h e r e s u l t of d e l e t i o n o r g e n e t i c mutation. I t s h o u l d be noted t h a t the endpoints measured i n these experiments are not, themselves, d i r e c t p r e c u r s o r s o f cancer but measure o n l y one aspect o f c a r c i n o g e n e s i s , i . e . , damage t o t h e genome. Thus, i t s h o u l d be remembered t h a t o n l y f a c t o r s which can modulate c a r c i n o g e n e s i s by i n f l u e n c i n g chromosome damage can be i d e n t i f i e d by these t e s t systems. R e s u l t s o f the unscheduled DNA s y n t h e s i s assay d i f f e r e d from those of the chromosome a b e r r a t i o n and micronucleus t e s t s . The UDS assay was s u c c e s s f u l i n demonstrating a g e n o t o x i c a c t i o n o f the chewing tobacco, o n l y . The f a i l u r e 39 o f t h e o t h e r e x t r a c t s t o e l i c i t a d e t e c t a b l e UDS can be i n t e r p r e t e d as e i t h e r the r e s u l t o f a l a c k o f any DNA damage o r i n h i b i t i o n o f DNA damage r e p a i r . I t i s u n l i k e l y t h a t t h e s e e x t r a c t s l a c k DNA damaging agents, r e q u i r e l o n g e r i n c u b a t i o n times f o r i n t e r a c t i o n w i t h DNA o r need m e t a b o l i c a c t i v a t i o n t o produce r e a c t i v e compounds s i n c e chromosome a b e r r a t i o n s and m i c r o n u c l e i f o r m a t i o n were produced i n CHO c e l l s t r e a t e d w i t h these e x t r a c t s f o r the same p e r i o d o f time. These r e s u l t s s i g n i f y t h a t these e x t r a c t s can indeed damage DNA because u n r e p a i r e d double s t r a n d breaks are b e l i e v e d t o be the b a s i s o f chromosome breaks (Brewen and S t e t k a , 1982). The l e v e l s o f U V - i n i t i a t e d UDS were s u b s t a n t i a l l y lower i n f i b r o b l a s t s c u l t u r e s exposed t o tobacco e x t r a c t s than i n f i b r o b l a s t s r e c e i v i n g UV alone, i n d i c a t i n g t h a t the e x t r a c t s are l i k e l y e x e r t i n g an i n h i b i t o r y e f f e c t on DNA r e p a i r . The chewing tobacco, which d i d e l i c i t a measurable UDS, a l s o reduced UV-induced DNA r e p a i r . T h i s r e s u l t suggests t h a t t h e DNA damage induced by the chewing tobacco i s p r o b a b l y much more e x t e n s i v e than r e v e a l e d by the unscheduled DNA s y n t h e s i s assay. Whether a tobacco e x t r a c t induces a d e t e c t a b l e UDS pro b a b l y depends upon the balance o f DNA damage i t induces and the l e v e l o f r e p a i r i n h i b i t i o n i t e x e r t s . T h e r e f o r e , the i n e f f e c t i v e n e s s o f the K h a i n i tobacco, nass and s n u f f e x t r a c t s t o i n i t i a t e a demonstrable UDS sho u l d not be i n t e r p r e t e d as f a i l u r e o f the e x t r a c t s t o 40 induce DNA damage but may be a t t r i b u t e d t o an o v e r r i d i n g i n h i b i t o r y e f f e c t on DNA r e p a i r . Although the unscheduled DNA s y n t h e s i s assays have been v a l i d a t e d w i t h many s i n g l e compounds ( S t i c h e t a l . , 1971; San and S t i c h , 1975; W i l l i a m s , 1976, 1977, 1978; M a r t i n e t a l . , 1978; H o l l s t e i n e t a l . , 1979; M i t c h e l l e t a l . , 1983), t h e f a i l u r e t o o b t a i n p o s i t i v e r e s u l t s w i t h most o f the tobacco e x t r a c t s r e v e a l s an i n h e r e n t l i a b i l i t y o f t h i s t e s t when complex mixtures are b e i n g assayed. A mixture o f compounds may not t r i g g e r d e t e c t a b l e unscheduled DNA s y n t h e s i s , even though DNA damaging agents are p r e s e n t , i f a s t r o n g i n h i b i t o r o f DNA r e p a i r i s a l s o p r e s e n t . T h i s phenomenon has been encountered p r e v i o u s l y w i t h complex mixtures such as human f a e c a l e x t r a c t s and some i n d u s t r i a l e f f l u e n t s ( S t i c h , e t a l . , 1981b). These mixtures produce r e s u l t s s i m i l a r t o those o b t a i n e d here w i t h the tobacco e x t r a c t s : they induce chromosome a b e r r a t i o n s i n CHO c e l l s y e t do not induce unscheduled DNA s y n t h e s i s i n human f i b r o b l a s t s . These mixtures were shown t o i n h i b i t UV-induced DNA r e p a i r . The c o n t r a s t i n g r e s u l t s based on chromosome a b e r r a t i o n s o r m i c r o n u c l e i and UDS suggest a p o s s i b l e l i n k between chromosome damage and the i n h i b i t i o n o f DNA r e p a i r . As i t i s g e n e r a l l y b e l i e v e d t h a t chromosomal a b e r r a t i o n s are caused by l e s i o n s i n DNA and t h a t breaks and exchanges a r i s e when the mechanisms f o r r e p a i r f a i l o r make mistakes, i t i s 41 r e a s o n a b l e t o assume t h a t an i n h i b i t o r y a c t i o n on DNA r e p a i r by a tobacco e x t r a c t may have a p o t e n t i a t i n g e f f e c t on induced chromosome damage. Many i n h i b i t i o r s o f DNA s y n t h e s i s and r e p a i r have a l r e a d y been shown t o i n c r e a s e the frequency o f chromosome a b e r r a t i o n s induced by p h y s i c a l and chemic a l agents (Bryant and I k i a k i s , 1984; Kihlman and Na t a r a j a n , 1984; Kihlman and Anderson, 1985). A l i n k between chromosome damage and d e f i c i e n t r e p a i r i s a l s o supported by the e x i s t e n c e o f a 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 g r e a t e r l e v e l s o f DNA r e p a i r d e f i c i e n c y and i n c r e a s e d s e n s i t i v i t y t o the chromosome damaging a c t i o n o f chemic a l c a r c i n o g e n s i n Xeroderma pigmentosum c e l l s (San e t a l . , 1977). The i n h i b i t i o n o f DNA r e p a i r systems by o r a l tobaccos may p l a y an important r o l e i n t h e i r c a r c i n o g e n i c c a p a c i t y by a l l o w i n g mis- or n o n - r e p a i r o f DNA damage l e a d i n g t o mutation. I t i s n o t a b l e t h a t d i m e t h y l s u l p h o x i d e e x t r a c t s o f the n e u t r a l f r a c t i o n o f c i g a r e t t e smoke condensate, as w e l l as a l l o t h e r co-carcinogens t e s t e d by Gaudin e t a l . (1971, 1972), i n h i b i t UV-stimulated DNA r e p a i r i n normal human lymphocytes. C i g a r e t t e smoke has a l s o been r e p o r t e d t o i n h i b i t DNA r e p a i r i n the lungs o f exposed mice (Rasmussen e t a l . , 1981). While these experiments do not prove a r o l e f o r i n h i b i t e d DNA r e p a i r i n tobacco c a r c i n o g e n e s i s , evidence f o r a p r e v e n t i v e r o l e f o r DNA r e p a i r i n c a r c i n o g e n e s i s i s p r o v i d e d by the occurrence o f human h e r e d i t a r y d i s e a s e s , 42 such as Xeroderma pigmentosum and a t a x i a t e l a n g i e c t a s i a , i n which t h e r e i s a p r e d i s p o s i t i o n t o cancers of v a r i o u s types and a b n o r m a l i t i e s i n DNA r e p a i r a c t i v i t i e s (Paterson e t a l . , 1984). In the experiments r e p o r t e d here the tobacco e x t r a c t s were a c t i v e without the a d d i t i o n o f an e x t e r n a l source of a c t i v a t i n g enzymes, such as the 9000 x g supernatant (S9) of r a t l i v e r homogenate, i n d i c a t i n g t h a t the g e n o t o x i c substances are d i r e c t - a c t i n g . These f i n d i n g s are c o n s i s t e n t w i t h the few r e p o r t s a v a i l a b l e on the g e n o t o x i c i t y o f -tobaccos intended f o r o r a l use. In v i t r o s t u d i e s have shown t h a t e x t r a c t s of Indian-type tobaccos damage chromosomes of A l l i u m cepa (Patnaik e t a l . , 1984), cause s i g n i f i c a n t i n d u c t i o n o f s i s t e r chromatid exchanges i n v i r a l l y t r a n s f o r m e d and p h y t o h a e m a g g l u t i n i n - s t i m u l a t e d human lymphocytes (Umezawa e t a l . , 1981), are mutagenic t o V79 CHO c e l l s (Shirname e t a l . , 1984) and are mutagenic (mixed w i t h 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 ) t o S a l m o n e l l a typhimurium (Shirname e t a l . , 1983). I n d i a n tobacco e x t r a c t s a l s o induce m o r p h o l o g i c a l t r a n s f o r m a t i o n o f hamster embryo c e l l s (Umezawa e t a l . , 1978, 1981). Western tobacco s n u f f e x t r a c t s are mutagenic t o S a l m o n e l l a typhimurium i f prepared a t low pH (Whong e t a l . , 1984). A l l of t h e s e tobacco e x t r a c t s were a c t i v e without S9 a c t i v a t i o n , though i n a few cases t h e e f f e c t s o f the e x t r a c t s were enhanced by an S9 p r e p a r a t i o n . 43 Very few g e n o t o x i c o r c a r c i n o g e n i c components of smokeless tobaccos have y e t been i d e n t i f i e d . S n u f f s have been shown t o c o n t a i n the c a r c i n o g e n i c t o b a c c o - s p e c i f i c n i t r o s a m i n e s N 1 - n i t r o s o n o r n i c o t i n e (NNN) and 4-(N-n i t r o s o s m e t h y l a m i n o ) - 1 - ( 3 - p y r i d y l ) - 1 - b u t o n e (NNK) i n r e l a t i v e l y h i g h q u a n t i t i e s (Hoffman, e t a l . , 1985). A d i r e c t c o r r e l a t i o n has been i n f e r r e d between exposure t o t h e s e n i c o t i n e - d e r i v e d N-nitrosamines and o r a l cancer among long-term s n u f f d i p p e r s (Winn e t a l . , 1981; Hoffman e t a l . , 1985). These t o b a c c o - s p e c i f i c n i t r o s a m i n e s have a l s o been d e t e c t e d i n I n d i a n chewing p r e p a r a t i o n s (Hoffman and Hecht, 1985) and i n the s a l i v a of both s n u f f d i p p e r s (Hoffman and Adams, 1981) and I n d i a n tobacco chewers ( S i p a h i m a l a n i e t a l . , 1984). In s i t u f o r m a t i o n of n i t r o s a m i n e s i n the s a l i v a o f tobacco u s e r s a l s o o c c u r s and may c o n s t i t u t e an a d d i t i o n a l source o f exposure (Hecht e t a l . , 1975; Hoffman and Adams, 1981; S i p a h i m a l a n i e t a l . , 1984;). I t would be m i s l e a d i n g t o assume t h a t the e n t i r e g e n o t o x i c and c a r c i n o g e n i c a c t i v i t y of smokeless tobacco r e s i d e s i n any s i n g l e component. Non-pyrolysed tobaccos c o n t a i n more than 2500 chemicals (Hoffman and Hecht, 1985) and s e v e r a l g e n o t o x i c and c y t o t o x i c compounds are probably r e l e a s e d from the chewing mixture. Furthermore, the t o t a l g e n o t o x i c p o t e n t i a l i s not l i k e l y t o be a simple product of t h e g e n o t o x i c a c t i v i t y o f each i n d i v i d u a l component. A v a r i e t y o f modulating agents which c o u l d enhance o r suppress 44 t h e g e n o t o x i c i t y of a mixture, through such mechanisms as i n f l u e n c i n g c e l l u l a r a c t i v a t i o n and d e t o x i f i c a t i o n systems, o r by the d i r e c t i n t e r a c t i o n s of chemicals, a r e p r o b a b l y a l s o r e l e a s e d . F i n a l l y , the presence o f DNA r e p a i r i n h i b i t o r s i n the tobaccos must c e r t a i n l y a f f e c t the s e n s i t i v i t y o f the t a r g e t c e l l s . Because a c t i v e oxygen and f r e e r a d i c a l s may p l a y some r o l e i n t h e i n d u c t i o n o f tumours (Nagata e t a l . , 1982; Ames, 1983; Copeland, 1983; K e n s l e r and Trush, 1984; C e r u t t i , 1985) and are i n v o l v e d i n the c l a s t o g e n i c a c t i v i t y of c i g a r e t t e smoke (Nakayama e t a l . , 1984, 1985), a t t e n t i o n has been d i r e c t e d t o t h e i r p o s s i b l e involvement i n the g e n o t o x i c a c t i v i t i e s o f these o r a l tobacco e x t r a c t s . S i n c e c a t a l a s e c o n v e r t s H 2 0 2 t o n o n - r e a c t i v e s p e c i e s , the o b s e r v a t i o n t h a t t h i s enzyme supresses the c l a s t o g e n i c a c t i v i t y of the chewing and K h a i n i tobaccos i m p l i e s a H 20 2-mediated p r o d u c t i o n o f chromosome damage. H 2 0 2 has been shown t o induce base d e s t r u c t i o n , s i n g l e - and d o u b l e - s t r a n d breaks i n DNA (Freese, 1971), s i s t e r chromatid exchanges ( S p e i t e t a l . , 1982), chromosome a b e r r a t i o n s ( S t i c h e t a l . , 1978; B r a d l e y e t a l . , 1979; B r a d l e y and E r i c k s o n , 1981) and mutations (Freese, 1971; L e v i n e t a l . , 1982). In a d d i t i o n , H 2 0 2 has tumour promoting c a p a c i t y ( H i r o t a and Yokayama, 1981) and has been proven t o be c a r c i n o g e n i c ( I t o e t a l . , 1981). 45 H 2 0 2 produced by the chewing and K h a i n i tobacco e x t r a c t s i s most pro b a b l y formed by the a u t o o x i d a t i o n o f p h e n o l i c compounds. Large groups o f p h e n o l i c s a re p r e s e n t i n tobacco l e a v e s (Snook e t a l . , 1981) and many of these have been found t o be geno t o x i c (Brown, 1980; S t i c h e t a l . , 1981a; S t i c h and Powrie, 1982; Rosin, 1983). I t appears l i k e l y t h a t H 2 0 2 i s r e s p o n s i b l e , a t l e a s t i n p a r t , f o r the ge n o t o x i c a c t i v i t y o f p h e n o l i c s (Hanham e t a l . , 1983; Rosin, 1983). However, the p o s s i b i l i t y e x i s t s t h a t the p h e n o l i c compounds, themselves, o r t h e i r o x i d a t i o n and d e g r a d a t i o n p r o d u c t s (e.g. quinone r a d i c a l s ) , may a l s o be g e n o t o x i c . O x i d a t i o n o f polyphenols a l s o produces a v a r i e t y o f products which may condense w i t h a l k a l o i d s and amino a c i d s i n "browning" and analogous r e a c t i o n s (Stedman, 1968) which c o u l d c o n t r i b u t e t o the g e n o t o x i c i t y o f the tobacco m i x t u r e s . I f t he o x i d a t i o n o f p h e n o l i c s i s important t o the ge n o t o x i c and c a r c i n o g e n i c a c t i v i t y o f tobacco, one would expect c o n d i t i o n s which aggravate the r a t e o f o x i d a t i o n o f th e s e compounds t o enhance both the g e n o t o x i c and c a r c i n o g e n i c p o t e n t i a l . E l e v a t e d pH l e v e l s can enhance the ge n o t o x i c e f f e c t s o f p h e n o l i c s by c a t a l y s i n g t h e i r a u t o o x i d a t i o n (Rosin, 1983). Such c o n d i t i o n s may a c t u a l l y be c r e a t e d i n the mouths of c e r t a i n tobacco chewers. The widespread h a b i t , i n A s i a , o f adding v a r i o u s q u a n t i t i e s o f li m e from the s h e l l s o f s n a i l s , m o l l u s c s , c o r a l s o r rocks t o 46 t h e chewing mixtures can l e a d t o an a l k a l i n e pH w i t h i n the mouth o f a chewer ( S t i c h and Rosin, 1985). Lime i s added t o b e t e l q u i d and K h a i n i tobacco j u s t p r i o r t o use so t h a t o x i d a t i o n o f the p h e n o l i c s and g e n e r a t i o n o f H 2 0 2 a c t u a l l y o c c u r s i n the s a l i v a . The importance o f H 2 0 2 g e n e r a t i o n i n v i v o w i l l depend on t h e balance between i t s r a t e o f g e n e r a t i o n and the r a t e a t which i t can be d e t o x i f i e d by c e l l u l a r enzyme systems. A s a l i v a r y p e r o x i d a s e system may a l s o p r o t e c t human c e l l s from H 2 0 2 damage (Tennovuo and P r u i t t , 1984). Thus, a g e n t o x i c a c t i o n a t t r i b u t a b l e t o H 2 0 2 w i l l occur o n l y a f t e r these defence systems are d e p l e t e d . I t i s p o s s i b l e t h a t l o n g -term, low l e v e l g e n e r a t i o n o f H 2 0 2 from c h r o n i c tobacco exposure may be s u f f i c i e n t t o produce a g e n o t o x i c e f f e c t . In c o n t r a s t t o the o b s e r v a t i o n s made on the chewing and K h a i n i tobaccos, the gen o t o x i c a c t i v i t i e s o f s n u f f and nass d i d not appear t o be dependent on the g e n e r a t i o n o f H 2 0 2 . O x i d i z e d p h e n o l i c s have been suggested t o be the source o f ge n o t o x i c a c t i v i t y o f nass ( Z a r i d z e e t a l . , 1985). Nass, i n c o n t r a s t t o b e t e l q u i d and K h a i n i tobacco, i s prepared by mix i n g tobacco w i t h lime and o t h e r i n g r e d i e n t s l o n g b e f o r e i t i s used. T h i s p r a c t i c e p r o b a b l y r e s u l t s i n the o x i d a t i o n o f tobacco p h e n o l i c s and l o s s o f H 2 0 2 g e n e r a t i o n c a p a c i t y b e f o r e use. Perhaps, the lo n g f e r m e n t a t i o n and p r o c e s s i n g o f s n u f f a l s o r e s u l t s i n the l o s s o f the c a p a c i t y t o 47 generate H 2 0 2 . Whether p h e n o l i c compounds c o n t r i b u t e t o the g e n o t o x i c a c t i v i t y o f s n u f f remains t o be determined. 48 CONCLUSION The chromosome a b e r r a t i o n and micronucleus t e s t s i n CHO c e l l s appear t o be a p p l i c a b l e f o r the study o f f a c t o r s i n f l u e n c i n g o r a l tobacco c a r c i n o g e n i c i t y . In f u t u r e s t u d i e s , t h e s e t e s t s c o u l d be u t i l i z e d as r a p i d b i o a s s a y s t o i d e n t i f y m o d i f i c a t i o n s of the chewing mixtures which l e a d t o a r e d u c t i o n o r enhancement of the g e n o t o x i c e f f e c t s of tobacco. T h i s would i n v o l v e a s y s t e m a t i c study o f the c o m p o s i t i o n of the v a r i o u s tobacco mixtures, o f p r o c e s s i n g p r a c t i c e s such as c u r i n g and f e r m e n t a t i o n , and customs such as adding lime, a r e c a nuts, b e t e l l e a v e s o r o t h e r substances t o the chewing mixture. T h i s k i n d of i n v e s t i g a t i o n c o u l d a i d i n d e v i s i n g m o d i f i c a t i o n s t o the chewing mixtures which would render them l e s s hazardous but s t i l l a c c e p t a b l e t o chewers who r e f u s e t o g i v e up the h a b i t . These t e s t s c o u l d a l s o be a p p l i e d towards a second approach i n the p r e v e n t i o n o f t o b a c c o - r e l a t e d o r a l - c a n c e r s . By examining f a c t o r s which c o u l d modify the t a r g e t c e l l 1 s r e s i s t a n c e t o the g e n o t o x i c e f f e c t s o f the chewing mixtures v a l u a b l e i n f o r m a t i o n can be gained which c o u l d h e l p i n the d e s i g n o f chemoprevention s t r a t e g i e s . For example, both v i t a m i n A and beta-carotene may have a n t i c a n c e r p r o p e r t i e s (Peto e t a l . , 1981) and have been found t o reduce the frequency 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 o r a l mucosa c e l l s of tobacco chewers ( S t i c h e t a l . , 1984a; S t i c h e t a l . , 1984b; 49 S t i c h e t a l . , 1985). Whether beta-carotene p r o v i d e s a unique p r o t e c t i v e e f f e c t or i s due t o c o n v e r s i o n t o v i t a m i n A w i t h i n the body i s u n c l e a r . P r o v i d e d the s o l u b i l i t y problems a s s o c i a t e d w i t h b eta-carotene can be overcome, t h e s e i n v i t r o t e s t s sytems should be a b l e t o answer t h i s q u e s t i o n because beta-carotene w i l l not be c o n v e r t e d t o v i t a m i n A i n v i t r o . These t e s t s e a s i l y l e n d themselves t o the s i m u l a t i o n of i n v i v o c o n d i t i o n s i n v i t r o . S a l i v a c o l l e c t e d from K h a i n i tobacco u s e r s has been shown t o be c l a s t o g e n i c t o CHO c e l l s ( S t i c h and S t i c h , 1982), demonstrating t h a t g e n o t o x i c agents are r e l e a s e d from chewing mixtures i n v i v o . Thus, the r e l e v a n c y o f these t e s t s can be i n c r e a s e d by the i n c o r p o r a t i o n of s a l i v a o f chewers i n the study o f modulating f a c t o r s . F i n a l l y , a f t e r i d e n t i f y i n g f a c t o r s which can modulate o r a l tobacco g e n o t o x i c i t y i n v i t r o , these l a b o r a t o r y s t u d i e s can be e x t r a p o l a t e d t o the i n v i v o s i t u a t i o n , f o r the r e l e v a n c e of the i n v i t r o g e n o t o x i c i t y data p r e s e n t e d here i s supported by r e p o r t s of i n v i v o g e n o t o x i c e f f e c t s of o r a l t obacco usage i n humans. The frequency o f s i s t e r chromatid exchanges i n p e r i p h e r a l lymphocytes from I n d i a n tobacco chewers i s e l e v a t e d (Gosh and Gosh, 1984) and, more germane t o the i n d u c t i o n of o r a l cancers, t h e r e i s an i n c r e a s e d o c c u rrence o f m i c r o n u c l e a t e d c e l l s i n the o r a l mucosa of i n d i v i d u a l s who use s n u f f ( S t i c h e t a l . , 1985) nass ( Z a r i d z e 50 e t a l . , 1984), t o b a c c o / b e t e l q u i d ( S t i c h e t a l . , 1984a), and K h a i n i tobacco ( S t i c h e t a l . , 1982). The o r a l mucosa of tobacco chewers can be e a s i l y screened f o r the presence of m i c r o n u c l e i by n o n - i n v a s i v e t e c h n i q u e s ( S t i c h and Rosin, 1984). Thus, one w i l l be p r o v i d e d w i t h the o p p o r t u n i t y t o d i r e c t l y l i n k the r e s u l t s o f a n a l y t i c a l i n v i t r o s t u d i e s w i t h i n v i v o g e n o t o x i c i t y data o b t a i n e d from t h e a c t u a l t i s s u e i n which o r a l t o b a c c o - r e l a t e d cancers w i l l a r i s e . 51 BIBLIOGRAPHY Ames BN (1983) D i e t a r y c a r c i n o g e n s and a n t i c a r c i n o g e n s . S c i e n c e 221:1256-1264 B i n n i e WH and KV Rankin (1984) E p i d e m i o l o g i c a l and d i a g n o s t i c a s p e c t s o f o r a l squamous c e l l carcinoma. J O r a l P a t h o l 13:333-341 B l o t WJ and J F Fraumeni, J r (1977) Geographic p a t t e r n s of o r a l cancer i n the U n i t e d S t a t e s : e t i o l o g i c i m p l i c a t i o n s . J C h r o n i c D i s 30:745-757 B r a d l e y MO and LC E r i c k s o n (1981) Comparison of the e f f e c t s o f hydrogen p e r o x i d e and X-ray i r r a d i a t i o n on t o x i c i t y , mutation, and DNA damage/repair i n mammalian c e l l s . Biochem Biophys A c t a 654:135-141 B r a d l e y MO, IC Hsu and CC H a r r i s (1979) R e l a t i o n s h i p between s i s t e r chromatid exchange and m u t a g e n i c i t y , t o x i c i t y and DNA damage. Nature 282:318-320 Brewen JG and DG S t e t k a (1982) C y t o g e n e t i c events i n v i v o , i n JA Heddle (ed) " M u t a g e n i c i t y : New Horizons i n G e n e t i c T o x i c o l o g y " Academic p r e s s , Inc., Orlando, F l a . , pp351-384 Brown JP (1980) A review o f the g e n e t i c e f f e c t s of n a t u r a l l y o c c u r r i n g f l a v o n o i d s , anthraquinones and r e l a t e d compounds. Mutat Res 75:243-277 Bryant PE and G I l i a k i s (1984) P o s s i b l e c o r r e l a t i o n s between c e l l k i l l i n g , chromosome damage and DNA r e p a i r a f t e r X - i r r a d i a t i o n , i n A C o l l i n s , CS Downes and RT Johnson (eds) "DNA R e p a i r and i t s I n h i b i t i o n " IRL Press, Oxford, 1984 pp291-308 C a i r n s J (1981) The o r i g i n s of human cancer. Nature 289:353-357 52 C e r r u t i PA (1985) Prooxidant s t a t e s and tumour promotion. Sc i e n c e 227:375-381 C h r i s t e n AG (1980) The case a g a i n s t smokeless tobacco: f i v e f a c t s f o r the h e a l t h p r o f e s s i o n a l t o c o n s i d e r . JADA 101:464-469 C o p l e l a n d ES (1983) Free r a d i c a l s i n promotion - A Chemical Pathology Study S e c t i o n workshop. Cancer Res 43:5631-5637 De K l e i n A, A van K e s s e l , G Gr o s v e l d , e t a l . (1982) A c e l l u l a r oncogene i s t r a n s l o c a t e d t o t h e P h i l a d e l p h i a chromosome i n c h r o n i c m y e l o c y t i c leukaemia. Nature 300:765-767 Dunham U and KM H a r r o l d (1962) F a i l u r e t o produce tumours i n hamster cheek pouch by exposure t o the i n g r e d i e n t s o f b e t e l q u i d : h i s t o p a t h o l o g i c changes i n the pouch and ot h e r organs by exposure t o known ca r c i n o g e n s . J N a t l Cancer I n s t 29:1047-1068 Dunham L J , CS Muir, and JE Hammer,III (1966) E p i t h e l i a l a t y p i a i n hamster cheek pouches t r e a t e d r e p e a t e d l y w i t h c a l c i u m hydroxide. Br J Cancer 20:588-593 Evans HJ (1974) E f f e c t s o f i o n i z i n g r a d i a t i o n on mammalian chromosomes, i n German J (ed) "Chromosomes and Cancer" John Wiley and Sons, NY ppl91-237 Fre e s e E (1971) M o l e c u l a r mechanisms o f mutations, i n Ho e l l a n d e r A (ed) "Chemical Mutagens: P r i c i p l e s and Methods f o r t h e i r D e t e c t i o n " Plenum, NY pp46-48 Gaudin D, RS Gregg and KL Y i e l d i n g (1971) DNA r e p a i r i n h i b i t i o n : a p o s s i b l e mechanism o f a c t i o n o f co-car c i n o g e n s . Biochem Biophys Res Comm 45:63 0-636 Gaudin D, RS Gregg and KL Y i e l d i n g (1972) I n h i b i t i o n o f DNA r e p a i r by coc a r c i n o g e n s . Biochem Biophys Res Comm 48:945-949 53 G i l b e r t F (1983) Chromosome a b e r r a t i o n s and oncogenes. Nature 303:475 Gosh R and PK Gosh (1984) S i s t e r chromatid exchanges i n b e t e l and tobacco chewers. Mutat Res 139:79-81 Graham S, H Dayal, T Rohrer, e t a l . (1977) D e n t i t i o n , d i e t , tobacco and a l c o h o l i n the epidemiology of o r a l cancer. J N a t l Cancer I n s t 59:1611-1617 Gupta PC, FS Mehta, DK D a f t a r y , e t a l . (1980) Inc i d e n c e r a t e s of o r a l cancer and n a t u r a l h i s t o r y of o r a l precancerous l e s i o n s i n a 10-year foll o w - u p study of I n d i a n v i l l a g e r s . Comm Dent O r a l E p i d e m i o l 8:287-333 Hanham AF, BP Dunn and HF S t i c h (1983) C l a s t o g e n i c a c t i v i t y o f c a f f e i c a c i d and i t s r e l a t i o n s h i p t o hydrogen p e r o x i d e generated d u r i n g a u t o o x i d a t i o n . Mutat Res 116:333-339 Hecht SS, RM O r n a f f and D Hoffmann (1975) Chemical s t u d i e s on tobacco smoke XXXIII. N • - n i t r o s o n o r n i c o t i n e i n tobacco: a n a l y s i s of p o s s i b l e c o n t r i b u t i n g f a c t o r s and b i o l o g i c a l i m p l i c a t i o n s . J N a t l Cancer I n s t 54:1237-1244 Heddle JA, M H i t e , B K i r h a r t , e t a l . (1983) The i n d u c t i o n o f m i c r o n u c l e i as a measure o f g e n o t o x i c i t y . Mutat Res 123:61-118 H e r i t y B, M M o r i a r t y , GJ Bourke and L Daley (1981) A case-c o n t r o l study o f head and neck cancer i n the R e p u b l i c o f I r e l a n d . Br J Cancer 43: 177-182 Hirayama T (1966) An e p i d e m i o l o g i c a l study o f o r a l and pharyngeal cancer i n C e n t r a l and South-East A s i a . B u l l WHO 34:41-69 54 Hirayama T (1979) E p i d e m i o l o g i c a l e v a l u a t i o n o f n a t u r a l l y o c c u r r i n g c a r c i n o g e n s and modulators o f c a r c i n o g e n e s i s , i n M i l l e r EC, e t a l . (eds) " N a t u r a l l y O c c u r r i n g Carcinogens-mutagens and Modulators o f C a r c i n o g e n e s i s " U n i v e r s i t y Press, B a l t i m o r e pp359-380 H i r o t a N and T Yokayama (1981) Enhancing e f f e c t o f hydrogen p e r o x i d e upon duodenal and upper j e j u n a l c a r c i n o g e n e s i s i n r a t s . Gann 72:811-812 H i r s c h JM and SL Johansson (1983) E f f e c t of long-term a p p l i c a t i o n o f s n u f f on the o r a l mucosa: an e xperimental study i n the r a t . J O r a l P a t h o l 12:187-198 H i r s c h JM and H T h i l a n d e r (1981) S n u f f - i n d u c e d l e s i o n s of the o r a l mucosa - an experimental model i n the r a t . J O r a l P a t h o l 10:342-253 H i r s c h JM, B Svennerholm and A Vahle (1984) E f f e c t of s n u f f and Herpes simplex v i r u s 1 on r a t mucosa. P o s s i b l e a s s o c i a t i o n w i t h the development o f squamous c e l l carcinoma. J O r a l P a t h o l 13:52-62 Hoffman D and JD Adams (1981) C a r c i n o g e n i c t o b a c c o -s p e c i f i c N-Nitrosamines i n s n u f f and s a l i v a o f s n u f f d i p p e r s . Cancer Res 41:4305-4308 Hoffman D and SS Hecht (1985) N i c o t i n e - d e r i v e d N-n i t r o s a m i n e s and t o b a c c o - r e l a t e d cancer: Current s t a t u s and f u t u r e d i r e c t i o n s . Cancer Res 45:935-944 Hoffman D, E J L a v o i e and SS Hecht (1985) N i c o t i n e : a p r e c u r s o r f o r c a r c i n o g e n s . Cancer L e t t r 26:67-75 H o l l s t e i n MJ, J McCann, FA Angelosanto and WW N i c h o l s (1979) Short-term t e s t s f o r c a r c i n o g e n s and mutagens. Mutat Res 65:133-226 55 Homburger F (1971) Mechanical i r r i t a t i o n , p o l y c y c l i c hydrocarbons and s n u f f : E f f e c t s on f a c i a l s k i n , cheek pouch, and o r a l mucosa i n S y r i a n hamsters. Ar c h P a t h o l 91:411-417 Ibrahim K, NA J a f a r e y and SJ Z u b e r i (1977) Plasma v i t a m i n "A" and carotene l e v e l s i n squamous c e l l carcinomas o f o r a l c a v i t y and oro-pharynx. C l i n Oncol 3:203-207 I s h i d a t e M and S Odashima (1977) Chromosome t e s t s w i t h 134 compounds i n Chinese hamster c e l l i n v i t r o - a s c r e e n i n g f o r chemical c a r c i n o g e n s . Mutat Res 48:337-354 I t o A, H Wantanbe, M N a i t o and Y N a i t o (1981) I n d u c t i o n of duodenal tumours i n mice by o r a l a d m i n i s t r a t i o n of hydrogen p e r o x i d e . Gann 72:174-175 Jayant K, V B a l i s h k r i s h a n , LD Sanghvi e t a l . (1977) Q u a n t i f y i n g the r o l e o f smoking and chewing tobacco i n o r a l , pharyngeal and oesophageal ca n c e r s . Br J Cancer 35:232-235 J u s s a l w a l l a DJ and VA Deshpande (1971) E v a l u a t i o n of cancer r i s k i n tobacco chewers and smokers: an e p i d m i o l o g i c a l assessment. Cancer 28:244-252 J u s s a w a l l a DJ, BB Yeole and MV Nutekar (1985) Cancer i n I n d i a n Moslems. Cancer 55:1149-1158 Kandarkar SV and SM S i r s u t (1977) Changes i n v i t a m i n A c o n d i t i o n e d hamster cheek pouch e p i t h e l i u m on exposure t o commercial s h e l l l i m e ( c a l c i u m hydroxide) and tobacco I - o p t i c a l h i s t o p a t h o l o g y . J O r a l P a t h o l 6:191-202 K e n s l e r TW and MA Trush (1984) Role o f oxygen r a d i c a l s i n tumour promotion. E n v i r o n Mut 6:593-616 Kihlman BA and HC Anderson (1985) S y n e r g i s t i c enhancement o f the frequency of chromatid a b e r r a t i o n s i n c u l t u r e d human lymphocytes by combinations of i n h i b i t o r s o f DNA r e p a i r . Mutat Res 150:313-325 Khilman BA and AT N a t a r a j a n (1984) P o t e n t i a t i o n of chromosomal a l t e r a t i o n s by i n h i b i t o r s of DNA r e p a i r , i n C o l l i n s A, CS Downes and RT Johnson (ed) "DNA R e p a i r and i t s I n h i b i t i o n " IRL Press, Oxford 1984 pp319-339 Lake RS, ML Kropko, MR P e z z u t t i , e t a l . (1978) Chemical i n d u c t i o n of unscheduled DNA s y n t h e s i s i n human s k i n e p i t h e l i a l c e l l c u l t u r e s . Cancer Res 38:2091-2098 L e v i n DE, M H o l l s t e i n , MF Christman, e t a l . (1982) A new Samonella t e s t e r s t r a i n (TA102) w i t h AT base p a i r s a t the s i t e o f mutation d e t e c t s o x i d a t i v e mutagens. Proc N a t l Acad S c i USA 79:7445-7449 Masberg A, L G a r f u n k e l and S H a r r i s (1981) A l c o h o l as a primary r i s k f a c t o r i n o r a l squamous carcinoma. CA 31:146-155 Marshal J , S Graham, C M e t t t l i n , e t a l . (1982) D i e t i n the epidemiology o f o r a l cancer. Nutr Cancer 3:145-149 M a r t i n CN, AC McDermid and RC Gardner (1978) T e s t i n g known carc i n o g e n s and noncarcinogens f o r t h e i r a b i l i t y t o induce unscheduled DNA s y n t h e s i s i n HeLa c e l l s . Cancer Res 38:2621-2627 McGuirt WF (1983) Snuff d i p p e r ' s carcinoma. Arch O t o l a r y n g o l 109:757-760 McMichael AJ (1984) O r a l cancer i n the T h i r d World: Time f o r p r e v e n t i v e i n t e r v e n t i o n ? I n t J E p i d 13:403-405 57 M i t c h e l l AD, DA Casciano, ML Metz, e t a l . (1983) Unscheduled DNA s y n t h e s i s t e s t s . A r e p o r t o f the US Environmental P r o t e c t i o n Agency Gene-Tox Progam. Mutat Res 123:363-410 M i l l a r WJ (1984) Use of chewing tobacco and s n u f f by students i n the Northwest T e r r i t o r i e s 1982. Chron D i s Canada 4:54-56 Nagata C, M Kodama, Y I o k i and T Kimura (1982) Free r a d i c a l s produced from chemical c a r c i n o g e n s and t h e i r s i g n i f i c a n c e i n c a r c i n o g e n e s i s , i n F l o y d R (ed) "Free R a d i c a l s and Cancer" M a r c e l Decker, Inc., NY ppl-62 Nakayama T, M Kaneko,M Komada and C Nagata (1985) C i g a r e t t e smoke induces DNA s i n g l e s t r a n d breaks i n human c e l l s . Nature 314:462-464 Nakayama T, M Komada and C Nagata (1984) G e n e r a t i o n of hydrogen p e r o x i d e and superoxide a n i o n r a d i c a l from c i g a r e t t e smoke. Gann 75:95-98 Offenbacher S and DR Weathers (1985) E f f e c t s o f smokeless tobacco on the p e r i d o n t a l , mucosal and c a r i e s s t a t u s o f a d o l e s c e n t males. J O r a l P a t h o l 14:169-181 Paches AT and I L M i l i e v s k a y a (1980) E p i d e m i o l o g i c a l study of cancer o f the mucous membrane o f t h e o r a l c a v i t y i n the USSR. N a t l I n s t H e a l t h 80:177-184 Pat e r s o n MC, NE Gentner, MV M i d d l e s t a d t and M W i e n f e l d (1984) Cancer p r e d i s p o s i t i o n , c a r c i n o g e n h y p e r s e n s i t i v i t y , and a b e r r a n t DNA metabolism. J C e l l u l a r Phys s u p p l . 3:45-62 P a t n a i k S, BL Saran and SN P a t n a i k (1984) E f f e c t of Zarda (processed tobacco l e a f ) e x t r a c t on the chromosomes o f A l l i u m cepa L i n n . C y t o l o g i a 49:807-814 58 Peacock EE J r , BG Greenberg and BW Brawley (1960) The e f f e c t o f s n u f f and tobacco on the p r o d u c t i o n of o r a l carcinoma: an experimental and e p i d m i o l o g i c a l study. Ann Surg 151:542-550 Pearce F (1985) Tobacco suckers r i s k o r a l cancer. New S c i e n t i s t 105:6 Peto R, R D o l l , JD Buckley and MB Sporn (1981) Can d i e t a r y b e t a-carotene m a t e r i a l l y reduce human cancer r a t e s ? Nature 290:201-208 Pindborg J J (1980) O r a l cancer and Precancer. John Wright and Sons, L t d . B r i s t o l P r e s t o n RJ, W Au, MA Bender, e t a l . (1981) Mammalian i n v i v o and i n v i t r o c y t o g e n e t i c assays. A r e p o r t of the US EPA Gene-Tox Program. Mutat Res 87:143-188 Poulson TC, JE Lindenmuth and RO Greer (1984) A comparison o f the use o f smokeless tobacco i n r u r a l and urban teenagers. CA 34:248-261 Randive KJ, SN Randive, NM Shivapukar and AV Gothoskar (1979) B e t e l q u i d chewing and o r a l cancer: experimental s t u d i e s on hamsters. I n t J Cancer 24:835-843 Rasmussen RE, CH Boyd, DR Dansie, e t a l . (1981) DNA r e p l i c a t i o n and unscheduled DNA s y n t h e s i s i n lungs of mice exposed t o c i g a r e t t e smoke. Cancer Res 41:2583-1588 Roeg-Petersen B and J J Pindborg (1973) A study o f Danish s n u f f - i n d u c e d o r a l l e u k o p l a k i a s . J O r a l P a t h o l 2:301-313 R o s e n f e l d L and J Callaway (1963) S n u f f d i p p e r ' s cancer. Am J Surg 106:840-844 R o s i n MP (1983) The i n f l u e n c e of pH on the c o n v e r t o g e n i c a c t i v i t y of p l a n t p h e n o l i c s . Mutat Res 135:109-113 59 Rothman K and E K e l l e r (1972) The e f f e c t of j o i n t exposure t o a l c o h o l and tobacco on r i s k o f cancer o f the mouth and pharynx. J Chron D i s 25:711-716 Rowley JD (1984) B i o l o g i c a l i m p l i c a t i o n s o f c o n s i s t e n t chromosome rearrangements i n leukaemia and lymphoma. Cancer Res 44:3159-3168 San RHC and HF S t i c h (1975) DNA r e p a i r s y n t h e s i s of c u l t u r e d human c e l l s as a r a p i d b i o s a s s a y f o r chemical c a r c i n o g e n s . I n t J Cancer 16:284-291 San RHC and HF S t i c h (1982) Measurement o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s as a s h o r t - t e r m b i o a s s y f o r chemical c a r c i n o g e n s and c a r c i n o g e n i c mixtures, i n Hsu TC (ed) " C y t o g e n e t i c Assays of Environmental Mutagens" A l l a n h e l d , Osmun, Totowa, NJ pp237-248 San RHC, W S t i c h and HF S t i c h (1977) D i f f e r e n t i a l s e n s i t i v i t y of Xeroderma pigmentosum c e l l s o f d i f f e r e n t r e p a i r c a p a c i t i e s towards the chromosome b r e a k i n g a c t i o n o f c a r c i n o g e n s and mutagens. I n t J Cancer 20:181-187 Shirname LP, MM Menon and SV Bhide (1984) M u t a g e n i c i t y of b e t e l q u i d and i t s i n g r e d i e n t s u s i n g mammalian t e s t systems. C a r c i n o g e n e s i s 5:501-503 Shirname LP, MM Menon, J N a i r and SV Bhide (1983) C o r r e l a t i o n o f m u t a g e n i c i t y and t u m o r i g e n i c i t y of b e t e l q u i d and i t s i n g r e d i e n t s . Nutr Cancer 5:87-91 S i p h i m a l a n i AT, MS Chadra, SV Bhide, e t a l . (1984) D e t e c t i o n of N-nitrosamines i n the s a l i v a o f h a b i t u a l chewers of tobacco. Food Chem T o x i c o l 22:261-264 S k l a r G, K N i u k i a n , M Hassan and EG Herbosa (1985) E f f e c t s o f smokeless tobacco and s n u f f on o r a l mucosa of experimental animals. J O r a l M a x i l l o f a c Surg 43:80-86 60 Smith J F (1975) Snuff d i p p e r ' s l e s i o n : A t e n y e a r f o l l o w -up. A r c h O t o l a r y n g o l 101:276-277 Smith J F , HA Mincer, KP Hopkins, e t a l . (1970) S n u f f -d i p p e r ' s l e s i o n : A c y t o l o g i c a l and p a t h o l o g i c a l study of a l a r g e p o p u l a t i o n . A r c h O t o l a r y n g o l 92:459-456 Snook ME, PJ F o r s t o n and OT Chrtyk (1981) G e l chromatography f o r the i s o l a t i o n o f p h e n o l i c a c i d s from tobacco l e a f . Annal Chem 53:374-377 S p e i t G, W Vogel and M Wolf (1982) C h a r a c t e r i z a t i o n of s i s t e r chromatid exchanges - I n d u c t i o n by H 2 0 2 . E n v i r o n Mut 4:135-142 Stedman FL (1968) The chemical composition o f tobacco and tobacco smoke. Chem Rev 68:153-207 S t i c h HF, JR C u r t i s and BB P a r i d a (1982a) A p p l i c a t i o n of the micronucleus t e s t t o e x f o l i a t e d c e l l s o f h i g h cancer r i s k groups: tobacco chewers. I n t J Cancer 30:553-559 S t i c h HF, AP Horby and BP Dunn (1985) A p i l o t b e t a -carotene i n t e r v e n t i o n t r i a l on I n u i t s u s i n g smokeless tobacco. I n t J Cancer 36:321-327 S t i c h HF and WD Powrie (1982) P l a n t p h e n o l i c s as g e n o t o x i c agents and as modulators f o r the m u t a g e n i c i t y o f o t h e r components, i n S t i c h HF (ed) "Carcinogens and Mutagens i n the Environment, v o l . 1" CRC P r e s s , Inc. Boca Raton, F l a . ppl35-145 S t i c h HF and MP R o s i n (1984) 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 as a t o o l f o r s t u d i e s i n cancer r i s k and cancer i n t e r v e n t i o n . Cancer L e t t r 22:241-253 S t i c h HF and MP R o s i n (1985) Towards a more comprehensive e v a l u a t i o n o f a g e n o t o x i c hazard i n man. Mutat Res 150:43-50 61 S t i c h HF, MP R o s i n and MO V a l l e j e r a (1984a) Reduction w i t h v i t a m i n A and beta-carotene a d m i n i s t r a t i o n o f p r o p o r t i o n 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 i n A s i a n b e t e l nut and tobacco chewers. Lancet 1:1204-1206 S t i c h HF, MP Rosin, CH Wu and WD Powrie (1981a) A comparative g e n o t o x i c i t y study of c h l o r o g e n i c a c i d ( 3 - 0 - c a f f e o y l q u i n i c a c i d ) . Mutat Res 90:201-212 S t i c h HF, RCH San and HJ Freeman (1981b) DNA r e p a i r s y n t h e s i s (UDS) as an i n v i t r o b i o a s s a y t o d e t e c t p r e c a r c i n o g e n s , u l t i m a t e c a r c i n o g e n s , and o r g a n t r o p i c c a r c i n o g e n s , i n S t i c h HF and RCH San (eds) "Short-term T e s t s f o r Chemical Carcinogens" S p r i n g e r - V e r l a g , B e r l i n pp65-82 S t i c h HF, RCH San, and Y Kawazoe (1971) DNA r e p a i r s y n t h e s i s i n mammalian c e l l s exposed t o a s e r i e s o f oncogenic and nononcogenic d e r i v a t i v e s of 4-n i t r o q u i n o l i n e 1-oxide. Nature 229:416-419 S t i c h HF and W S t i c h (1982) Chromosome-damaging a c t i v i t y o f s a l i v a o f b e t e l nut and tobacco chewers. Cancer L e t t r 15:193-202 S t i c h HF, W S t i c h and BB P a r i d a (1982) E l e v a t e d frequency o f m i c r o n u c l e a t e d c e l l s on the b u c c a l mucosa o f i n d i v i d u a l s a t h i g h r i s k f o r o r a l cancer: b e t e l q u i d chewers. Cancer L e t t r 17:,'125-134 S t i c h HF, W S t i c h , MP R o s i n and MO V a l l e j e r a (1984b) Use o f the micronucleus t e s t t o monitor the e f f e c t of v i t a m i n A, beta-carotene and c a n t h a x a n t h i n on the b u c c a l mucosa of b e t e l nut/tobacco chewers. I n t J Cancer 34:745-750 S t i c h HF, L Wei and P Lam (1978) The need f o r a mammalian t e s t system f o r mutagens: a c t i o n o f some r e d u c i n g agents. Cancer L e t t r 5:199-204 62 Tenovuo J and KM P r u i t t (1984) R e l a t i o n s h i p o f t h e human s a l i v a r y p e r o x i d a s e system t o o r a l h e a l t h . J O r a l P a t h o l 13:573-584 Umezawa K, S F u j i e , M Sawamura, e t a l . (1981) M o r p h o l o g i c a l t r a n s f o r m a t i o n , s i s t e r chromatid exchanges and mutagenesis assay o f b e t e l c o n s t i t u e n t s . T o x i c o l L e t t r 8:17-22 Umezawa K, S Takayama, S F u j i e , e t a l . (1978) In v i t r o t r a n s f o r m a t i o n of hamster embryo c e l l s by b e t e l tobacco e x t r a c t s . T o x i c o l L e t t r 2:243-246 V o l g e r WR, NJ L l o y d and BK Milmore (1962) A r e t r o s p e c t i v e study o f e t i o l o g i c a l f a c t o r s i n cancer o f t h e mouth, pharynx and l a r y n x . Cancer 15:246-258 WHO (1984) C o n t r o l o f o r a l cancer i n d e v e l o p i n g c o u n t r i e s . B u l l WHO 62:817-830 Wahi PN (1968) The epidemiology of o r a l and oropharyngeal cancer: A r e p o r t o f the study i n M a i n p u r i D i s t r i c t , U t t a r Pradesh, I n d i a . B u l l WHO 38:495-521 Wahi PN (1976) O r a l and oropharyngeal tumours. Gann Monogr 18-19-26 Wahi PN, U Kehar and B L a h i r i (1965) F a c t o r s i n f l u e n c i n g o r a l and pharyngeal cancer i n I n d i a . Br J Cancer 19:646-660 Whong W, RG Ames and T Ong (1984) M u t a g e n i c i t y o f tobacco s n u f f : p o s s i b l e h e a l t h i m p l i c a t i o n s f o r c o a l miners. J T o x i c o l E n v i r o n H e a l t h 14:491-496 W i l l i a m s GM (1976) Carcinogen-induced DNA r e p a i r i n primary r a t l i v e r c e l l c u l t u r e s : a p o s s b l e s c r e e n f o r chemical c a r c i n o g e n s . Cancer L e t t 1:231-236 63 W i l l i a m s GM (1977) D e t e c t i o n o f chemical c a r c i n o g e n s by unscheduled DNA s y n t h e s i s i n primary r a t l i v e r c e l l c u l t u r e s . Cancer Res 37:1845-1851 W i l l i a m s GM (1978) F u r t h e r improvements i n the he p a t o t c y t e primary c u l t u r e DNA r e p a i r t e s t f o r c a r c i n o g e n s : d e t e c t i o n o f c a r c i n o g e n i c b i p h e n y l d e r i v a t i v e s . Cancer L e t t r 4:69-75 Winn D, WJ B l o t , CM Shy, e t a l . (1981) S n u f f - d i p p i n g and o r a l cancer among women i n the southern U n i t e d S t a t e s . N En g l J Med 304:745-749 Winn D, RG Z e i g l e r , LW P i c k e l , e t a l . (1984) D i e t i n the e t i o l o g y o f o r a l and pharyngeal cancer among women from the southern U n i t e d S t a t e s . Cancer Res 44:1216-1222 Wynder EL, I J Bross and RM Feldman (1957) A study o f the e t i o l o g y f a c t o r s i n cancer o f the mouth. Cancer 10:1300-1323 Wynder EL and SD Ste l l m a n (1977) Comparative epidemiology o f t o b a c c o - r e l a t e d c a n c e r s . Cancer Res 37:4608-4622 Yunis J J (1983) The chromosomal b a s i s o f human n e o p l a s i a . S c i e n c e 221:227-236 Z a r i d z e DG, M B l e t t n e r , NN Ira p e z n i k o v , e t a l . (1984) Precancerous l e s i o n s o f the o r a l mucosa and oesophagus i n U z b e k i s t a n (USSR), i n IARC Annual Report, 1984 IARC, Lyon, France pp54-56 Z a r i d z e DG, NN Trapenzikov, JP Kushinov, e t a l . (1985) Survey o f a p o p u l a t i o n w i t h a moderately h i g h i n c i d e n c e o f o r a l and oesophageal cancer. I n t J Cancer 36:153-158 64 

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