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

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MODULATION OF THE RISK TO ORAL CANCER by Antony Paul Hornby . B.Sc, Un i v e r s i t y of Wales, 1978 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Pathology We accept t h i s thesis as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia June, 1989 Antony Paul Hornby In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P/4rtfOt-0 The University of British Columbia Vancouver, Canada Date J ^ W <T, /<tS<T DE-6 (2/88) ABSTRACT M i l l i o n s of people use smokeless tobacco, such as snuff, chewing tobacco, nass or nasswar (a mixture of tobacco, slaked lime, ash and o i l ) , Khaini tobacco (tobacco and slaked lime), or as part of a simple betel quid (areca nut, tobacco, lime, b e t e l leaf) or a complex "pan" (betel quid with catechu, seeds, perfumes and s i l v e r f o i l s ) . These habits which are involved i n the etiology of o r a l cancer, have been of concern since snuff dipping i s becoming popular among teenagers of Canada and the United States. To prevent the development of o r a l cancer among snuff dippers, i t appeared necessary to trace the e t i o l o g i c a l f a c t o r s , to develop markers which would i d e n t i f y individuals at elevated r i s k f o r o r a l cancer, and to test the usefulness of these "intermediate endpoints" i n following the response of the o r a l mucosa to the administration of chemopreventive agents. The population groups chosen for t h i s study included Inuit, Canadian Indians and East Indians. Approximately 57.0% of Inuit males from Gjoa Haven (Northwest T e r r i t o r i e s ) used snuff, 62.6% of native Indians of La Loche (Saskatchewan) dipped snuff d a i l y , and 54.0% of East Indian fisherman from along the coast of Kerala (India) chewed tobacco i n the form of a b e t e l quid. The chewing patterns were as follows: number of dips per day for the Inuit were 8.03 at 25.2 min per dip, for the native Indians 9.1 dips per day at 20.3 min per dip and f o r the East Inians 17.2 chews per day at 15.2 min per chew. N-Nitroso compounds were found i n the s a l i v a of snuff dippers and b e t e l quid chewers. They are considered to be the most probable e t i o l o g i c a l factors i n the development of o r a l cancers, since they are the only known carcinogens present i n mg/kg quantities i n the various tobacco mixtures. i i i High l e v e l s of n i t r i t e , a precursor to nitrosamines, were found i n tobaccos used by the Canadian natives. Up to 1040 mg/kg n i t r i t e was detected i n tobacco samples used by t h i s population. High l e v e l s of n i t r i t e were also detected i n the s a l i v a of I n u i t and Canadian Indian snuff dippers: up to 0.25 mg/ml of n i t r i t e appeared i n the s a l i v a within 5 to 10 min of a snuff dipping session. N i t r i t e was also detected i n the s a l i v a of East Indian b e t e l quid chewers, averaging 36.27 pg/ral. Since n i t r i t e can serve as a precursor to nitroamine reactions, the i n v i t r o n i t r o s a t i o n capacity of s a l i v a from a snuff dipper was tested. A f t e r the addit i o n of 200 mg p r o l i n e to the s a l i v a of a snuff dipper at pH 2.5, an 18fold increase i n n i t r o s o p r o l i n e (NPRO) was observed over c o n t r o l l e v e l s . Moreover, N,PR0 was observed i n the urine of chewers at a f i v e f o l d increased l e v e l over non-chewers. These r e s u l t s indicate an elevated l e v e l of n i t r o s a t i o n within i n d i v i d u a l s who dip snuff. This increased endogenous n i t r o s a t i o n reaction i n snuff dippers can lead to the formation of carcinogenic N-nitrosamines. Snuff dippers and tobacco chewers are also exposed to tobacco-specific nitrosamines (TSNA). Levels from 3200 ppb for N-nitrosonornicotine (NNK) to 170,000 ppb f o r N-nitrosoanatabine (NAT) were found i n commonly used brands of snuff which were commercially a v a i l a b l e i n the Northwest T e r r i t o r i e s . In addition, r e l a t i v e l y high l e v e l s of these carcinogenic nitrosamines were detected i n the s a l i v a of chewers (up to 980 Mg/ml of s a l i v a ) . The second objective of t h i s study was the development of markers which indicate e a r l y changes i n a human tissue exposed to carcinogens. Two markers were used to d e t a i l the damage occurring i n the o r a l mucosa of users of smokeless tobacco. The f i r s t was micronuclei which was applied to e x f o l i a t e d c e l l s from the o r a l mucosa. i v T h i s a s s a y i s a q u a n t i t a t i v e i n d i c a t o r o f chromosomal b r e a k a g e . An e l e v a t e d f r e q u e n c y o f m i c r o n u c l e a t e d c e l l s was o b s e r v e d i n the o r a l mucosa o f I n u i t s n u f f d i p p e r s , n a t i v e C a nadian I n d i a n s and E a s t I n d i a n chewers o f tobacco-c o n t a i n i n g b e t e l q u i d s , as compared to c o r r e s p o n d i n g i n d i v i d u a l s who d i d not use smokeless t o b a c c o . The second marker was o r a l l e u k o p l a k i a , a p r e n e o p l a s t i c l e s i o n commonly found i n the o r a l mucosa o f b e t e l q u i d chewers. By u s i n g these two markers t o q u a n t i f y c a r c i n o g e n - i n d u c e d damage d u r i n g the p r e n e o p l a s t i c s t a g e , i t appeared f e a s i b l e to i d e n t i f y i n d i v i d u a l s a t e l e v a t e d r i s k f o r d e v e l o p i n g o r a l c a n c e r . The s t u d y e x p l o r e d the p o s s i b i l i t y o f u s i n g the above-mentioned markers to f o l l o w the response o f smokeless tobacco u s e r s t o the a d m i n i s t r a t i o n o f be t a -c a r o t e n e and v i t a m i n A. The a d m i n i s t r a t i o n o f b e t a - c a r o t e n e (180 mg/week) f o r t e n weeks s i g n i f i c a n t l y r e d u c e d the l e v e l 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 o f a group o f I n u i t s n u f f d i p p e r s who c o n t i n u e d t o use t h e i r u s u a l amount o f s n u f f d u r i n g the t r i a l p e r i o d . S i m i l a r l y , the l e v e l s o f m i c r o n u c l e a t e d c e l l s i n the o r a l mucosa o f chewers o f t o b a c c o - c o n t a i n i n g b e t e l q u i d s ( E a s t I n d i a n s ) were s i g n i f i c a n t l y r e d u c e d a f t e r t h r e e months on a regime o f e i t h e r 180 mg/week o f b e t a - c a r o t e n e o r 180 mg/week o f b e t a - c a r o t e n e p l u s 100,000 IU o f v i t a m i n A. The r e d u c t i o n o f m i c r o n u c l e a t e d o r a l mucosal c e l l s o c c u r r e d more r a p i d l y t h a n the r e m i s s i o n o f l e u k o p l a k i a i n the E a s t I n d i a n group as d i d the i n h i b i t i o n o f newly formed l e u k o p l a k i a f o l l o w i n g the a d m i n i s t r a t i o n o f the two chemopreventive a g e n t s . The r e m i s s i o n o f e s t a b l i s h e d o r a l l e u k o p l a k i a was s i g n i f i c a n t (P = 0.004) o n l y a f t e r s i x months i n the b e t e l q u i d chewers r e c e i v i n g b e t a - c a r o t e n e p l u s v i t a m i n A compared to the group r e c e i v i n g a p l a c e b o . V The treated group also showed a s i g n i f i c a n t reduction i n the appearance of new o r a l leukoplakia (P = 0.08). The administration of vitamin A alone (200,000 IU/week) to b e t e l quid chewers produced a highly s i g n i f i c a n t remission of established leukoplakia (P = 0.0000089) plus an i n h i b i t i o n of the formation of new leukoplakia (P = 0.024) as compared to the placebo group. The East Indians continued to chew b e t e l quids throughout the administration of chemopreventive agents. Many d i f f e r e n t s c i e n t i f i c d i s c i p l i n e s are necessary to obtain understanding of the e t i o l o g i c a l factors involved i n the development of a p a r t i c u l a r cancer, and to recognize early changes i n the target tissue to carcinogens. Only a profound understanding of these events w i l l help i n the e a r l y i d e n t i f i c a t i o n of i n d i v i d u a l s at elevated r i s k to cancer, the design of largescale chemopreventive t r i a l s , and the s e l e c t i o n of the most e f f e c t i v e chemopreventive agents. v i ABSTRACT TABLE OF CONTENTS U S T OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS INTRODUCTION 1 1. History of Tobacco Usage 1 2. Tobacco-Specific N-Nitroso Compounds 6 3. Precancerous Oral Lesions and Cancer 7 4. Micronucleated C e l l s 9 5. Chemoprevention 12 6. Objectives 14 MATERIALS AND METHODS 15 1. Use of Smokeless Tobacco 15 1.1 I n u i t i n the Northwest T e r r i t o r i e s 15 1.2 Native Canadian Indians i n Saskatchewan 15 1.3 East Indians i n Kerala, India 16 2. N i t r i t e i n Smokeless Tobacco Samples 17 2.1 Snuff and Chewing Tobacco 17 2.2 Bete l Quid 17 CONTENTS Page i i v i x x i i x i v xv v i i Page 2.3. D e t e r m i n a t i o n o f N i t r i t e 18 3. N i t r i t e i n the S a l i v a o f Users o f Smokeless Tobacco 21 3.1 S n u f f D i p p e r s 21 3.2 B e t e l Q u i d Chewers 21 4. N i t r o s a t i o n C a p a c i t y o f S a l i v a Samples 21 5. N - N i t r o s o p r o l i n e i n the U r i n e o f S n u f f D i p p e r s 21 5.1 D e t e r m i n a t i o n o f N i t r o s o p r o l i n e 22 6. T o b a c c o - S p e c i f i c N i t r o s a m i n e s i n D i f f e r e n t Brands o f Tobacco 26 7. T o b a c c o - S p e c i f i c N i t r o s a m i n e s i n the S a l i v a o f S n u f f D i p p e r s 29 8. D e t e r m i n a t i o n o f N i c o t i n e and C o t i n i n e 29 9. Frequency o f M i c r o n u c l e i i n the O r a l Mucosa o f S n u f f D i p p e r s and Tobacco Chewers 30 10. P r e c a n c e r o u s L e s i o n s and Cancer o f the O r a l C a v i t y i n U s e r s o f Smokeless Tobacco 32 10.1 Leukoderma 32 10.2 L e u k o p l a k i a 33 11. I n t e r v e n t i o n S t r a t e g i e s 35 11.1 N i t r i t e T r a p p i n g Agents 35 11.2 A d m i n i s t r a t i o n o f B e t a - c a r o t e n e and V i t a m i n A 37 11.3 I n u i t : B e t a - c a r o t e n e 37 11.4 E a s t I n d i a n s : B e t a - c a r o t e n e and B e t a - c a r o t e n e p l u s V i t a m i n A 37 11.5 E a s t I n d i a n s : V i t a m i n A 38 v i i i Page 12. Determination of Beta-carotene and R e t i n o l 39 13. Questionnaire 42 14. S t a t i s t i c a l A n a l y s i s 45 RESULTS 46 1. Use of Smokeless Tobacco 46 2. N i t r i t e i n Smokeless Tobacco Samples 51 2.1 Snuff and Chewing Tobacco Brands 51 2.2 East Indian Tobacco 54 3. N i t r i t e i n the S a l i v a of Users of Smokeless Tobacco 54 3.1 Snuff Dippers 54 3.2 B e t e l Quid Chewers 54 4. N i t r o s a t i o n Capacity o f , S a l i v a Samples 57 5. N - N i t r o s o p r o l i n e i n the Urine of Snuff Dippers 59 6. Tobacco-Specific Nitrosamines i n D i f f e r e n t Brands of Tobacco 59 7. Tobacco-Specific Nitrosamines i n the S a l i v a o f Snuff Dippers 62 7.1 Appearence of TSNA i n the S a l i v a 62 7.2 V a r i a t i o n s i n S a l i v a r y TSNA of Snuff Dippers 64 8. Frequency of M i c r o n u c l e i i n the O r a l Mucosa of Snuff Dippers and Tobacco Chewers 66 i x Page 9. Precancerous Lesions and Cancer of the Ora l C a v i t y i n Users of Smokeless Tobacco 66 9.1 Precancerous Lesions 67 9.2 O r a l Cancer 69 10. I n t e r v e n t i o n S t r a t e g i e s 72 10.1 N i t r i t e Trapping Agents 73 10.2 I n u i t Snuff Dippers: Beta-carotene 77 10.3 East Indian Tobacco Chewers: Beta-carotene and Beta-carotene plus Vitamin A 84 10.4 East Indian Tobacco Chewers: Vitamin A 91 DISCUSSION 94 1. Use of Smokeless Tobacco 94 2. Precursors of Nitrosamines Contained i n Smokeless Tobacco Samples and the S a l i v a of Their Users 95 3. Smokeless Tobacco and I n g e s t i o n of N-Nitrosamines 98 4. The U n f i n i s h e d Search f o r the Factors Involved i n O r a l Carcinogenesis 99 5. E x p l o r i n g Preventive Measures 102 5.1 Mechanism of A c t i o n of Chemopreventive Agents 102 6. Outlook 107 SUMMARY 109 REFERENCES 111 LIST OF TABLES Table I Analysis of Three of the Same Tobacco Samples for TSNA Table II Frequency of Micronuclei i n Human Tissues at Elevated Risk f o r Cancer Table III C l a s s i f i c a t i o n of Oral Precancerous Lesions Table IV Questionnaire Used i n the Northwest T e r r i t o r i e s and Saskatchewan Table V Questionnaire Used i n Kerala, India Table VI Snuff Dipping, Tobacco Chewing, Cigarette Smoking, and Drinking of A l c o h o l i c Beverages by Native Indians, I n u i t and East Indians Table VII Means and Ranges of Various Snuff-Related Factors Among In u i t and Native Indians Table VIII N i t r i t e Content i n Aqueous Extracts of Smokeless Tobacco Samples Table IX N i t r i t e i n the S a l i v a of Smokeless Tobacco Chewers Table X In V i t r o N i t r o s a t i o n Capacity of S a l i v a i n a User of Three D i f f e r e n t Brands of Smokeless Tobacco (Snuff) Table XI NPRO i n the Urine of Two Snuff Dipping Populations (Inuit and Indian) Table XII TSNA i n Various Samples of Canadian and Foreign Tobacco Brands Table XIII TSNA i n the S a l i v a of Two Individuals P r i o r to, During and A f t e r Snuff Dipping Page 28 31 34 43 44 48 50 53 56 58 60 61 63 x i Page Table XIV TSNA i n the S a l i v a of Snuff Dipping Inuit (Gjoa Haven) 65 Table XV Prevalence of Oral Lesions Among Snuff Dippers and Tobacco Chewers 68 Table XVI E f f e c t of the Daily Number of Betel Quid Chews on the Relati v e Risk f o r Oral Cancer i n India and Kerala 70 Table XVII E f f e c t of Snuff Dipping on the Relative Risk f o r Oral Cancer i n the Southern United States 71 Table XVIII I n h i b i t o r y E f f e c t of Ascorbate and C a f f e i c Acid on the Formation of NPRO i n a Snuff Dipping Volunteer 76 Table XIX Serum Levels of Retinol and Beta-carotene i n Male I n u i t P r i o r to I n i t i t i a t i o n of the Prevention T r i a l 78 Table XX Frequency (%) of Micronucleated C e l l s i n Oral Leukoplakias and i n Normal Mucosa of Tobacco/Betel Quid Chewers Before and A f t e r the Administration of Chemopreventive Agents f o r 3 Months 87 Table XXI Response of Oral Leukoplakias to the Administration of Beta-carotene or Beta-carotene plus Vitamin A for 3 Months 89 Table XXII Response of Oral Leukoplakias to the Administration of Beta-carotene or Beta-carotene plus Vitamin A for 6 Months 90 Table XXIII Response of Oral Leukoplakias to the Administration of Vitamin A for 6 Months 93 x i i LIST OF FIGURES Page Figure 1 A t y p i c a l b e t e l q u id showing b e t e l l e a f , a p o r t i o n of sundried tobacco, and a quarter of a b e t e l nut. Lime i s shown on the r i g h t f i n g e r t i p . 5 Figure 2 A Homogenous Leukoplakia i n the Right Buccal Mucosa 9 Figure 3 Procedure f o r N i t r i t e Determination 20 Figure 4 Procedure f o r N - n i t r o s o p r o l i n e (NPRO) Determination 25 Figure 5 Experimental P r o t o c o l f o r Endogenous NPRO Formation 36 Figure 6 Determination of Serum Beta-carotene and R e t i n o l 41 Figure 7 N i t r i t e i n the S a l i v a of Seven I n u i t Snuff Dippers 55 Figure 8 The Appearence of NPRO i n the Urine of a Volunteer Who Dipped Snuff and,Ingested P r o l i n e and Ascorbate 74 Figure 9 The Appearence of NPRO i n the Urine of a Volunteer Who Dipped Snuff and Ingested P r o l i n e and Ascorbate 75 Figure 10 E f f e c t of Beta-carotene Treatment on MNC i n the O r a l Mucosa 81 Figure 11 Changes i n the Frequency of MNC at the O r a l S i t e Where the Tobacco i s Located Over a P e r i o d Preceeding the P i l o t T r i a l and A f t e r a 10-Week A d m i n i s t r a t i o n of Beta-carotene 82 Figure 12 Frequency of Anucleated E x f o l i a t e d C e l l s from the Or a l S i t e Where the Tobacco i s Located Before and A f t e r a 10-Week A d m i n i s t r a t i o n of Beta-carotene 83 x i i i Page Figure 13 Response of MNC i n Areas of Leukoplakia to a 3-Month Administration of Beta-carotene and Beta-carotene Plus Vitamin A vs. Placebo 86 xiv LIST OF ABBREVIATIONS DMNM Nitroso-cis-2,6-dimethylmorpholine MNC Micronucleated Cell MNNG N-Methyl-N'-nitro-nitrosoguanadine NAB N-Nitrosoanabasine NAT N-Nitrosoanatabine NIC Nicotine NNK 4-(Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone NNN N'-Nitrosonornicotine NPIC N-Nitrosopiperidine-2-carboxylic acid NPRO Nitrosoproline TSNA Tobacco-specific nitrosamines Other abbreviations which are widely used without definition in the biochemical literature (eg. DNA, IU) are not specifically defined here. XV ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e to Dr. H.F. S t i c h f o r guidance, s u g g e s t i o n s , d i s c u s s i o n s , and u n f a i l i n g encouragement d u r i n g the cou r s e o f the i n v e s t i g a t i o n s r e p o r t e d i n t h i s t h e s i s . I would a l s o l i k e to thank Dr. Bruce Dunn f o r e x c e l l e n t t e c h n i c a l and s c i e n t i f i c i n s t r u c t i o n , and the f o l l o w i n g members o f the r e s e a r c h team i n v o l v e d i n the chemopreventive t r i a l s f o r t h e i r a s s i s t a n c e : Dr. K.D. Brunneraann, N a y l o r Dana I n s t i t u t e f o r D i s e a s e P r e v e n t i o n , American H e a l t h F o u n d a t i o n , f o r a n a l y s i s o f TSNA i n s a l i v a and s n u f f samples; Mrs. H.F. S t i c h , B.C. Cancer R e s e a r c h C e n t r e , f o r s c o r i n g o f e x f o l i a t e d b u c c a l mucosal c e l l s f o r m i c r o n u c l e i ; Dr. Babu Mathew and Dr. R. Sankaranarayanan, R e g i o n a l Cancer C e n t r e , T r i v a n d r u m , I n d i a , f o r d i a g n o s i s o f l e u k o p l a k i a . I would l i k e t o d e d i c a t e t h i s t h e s i s to my w i f e D a r l e n e , who has o f f e r e d u n f a i l i n g m o r al s u p p o r t and encouragement throughout the c o u r s e o f my s t u d i e s . 1 INTRODUCTION 1. History of Tobacco Usage Tobacco has been chewed for as long as i t has been smoked. But p a r t l y because i t i s not a very v i s i b l e habit, t h i s o r a l use i s seldom recognized as a common p r a c t i c e or as a health hazard. A rough estimate puts the number of people i n the world using tobacco o r a l l y on a d a i l y basis at more than 600 m i l l i o n (IARC, 1985) . Tobacco use can be traced back more than 7000 years, when i t appears to have been f i r s t c u l t i v a t e d . I t was probably used during t h i s period f or such purposes as a l l e v i a t i n g hunger and whitening the teeth, and as a d i s i n f e c t a n t when the j u i c e was expectorated onto wounds. The f i r s t people to smoke, chew, and snuff tobacco were probably American Indians. Reports from the 1400s, such as those of Amerigo Vespucci i n 1499, speak of Indians on Margarita Island, o f f the coast of Venezuela, chewing a green herb that was c a r r i e d i n gourds around t h e i r necks. Vespucci f e l t that the green herb, tobacco, was used to quench t h i r s t , because of the s c a r c i t y of water on the i s l a n d and the f a c t that chewing the l e a f increased s a l i v a r y flow. Indeed, a number of reports from explorers during t h i s period, including Samuel de Champlain, the founder of Quebec, and Columbus, mention that the use of chewing tobacco by natives reduced hunger, t h i r s t and fatigue among i t s users. In the Americas tobacco chewing became popular among the non-native population. During the 1860s tobacco was chewed e i t h e r i n the form of a plug or as twists. Of the 348 tobacco f a c t o r i e s l i s t e d i n the 1860 Census for V i r g i n i a and North Carolina, only seven manufactured smoking products (Heimann, 1960). Tobacco chewing reached an a l l - t i m e high i n America by 1890, when some three pounds (about 1.5 kg) of plug, twist or fine-c u t chewing tobacco were chewed annually per c a p i t a i n the United States (Heimann, 1960). This remained the 2 dominant form of tobacco usage unt i l the expansion of the cigarette industry in 1918 (Maxwell, 1980). Its decline was hastened by the "germ theory of disease" which made the habit of spitting in public places socially unacceptable. Anti-spitting laws were passed in New York and Philadelphia in 1896 and in Toronto in 1904 (Kozlowski, 1981). During the latter half of the 1960s and through the 1970s, there was a resurgence in the use of smokeless tobacco in North America. This renaissance was probably due to an increased awareness of the harmful effects of tobacco smoking coupled with a lack of awareness of those resulting from i t s oral use. It has become particularly popular among adolescent males, promoted by sports figures and the Western "macho" image. It can be practised in areas where smoking is hazardous, such as in the steel, coal or petroleum industries, and is often said to be cheaper than smoking. In addition, people who require the use of their hands while working or playing can chew tobacco much more conveniently than they can smoke. Since the 1960s, the U.S. production of smokeless tobacco has increased by approximately 60%. The total U.S. consumption of such products was approximately 132 million pounds (60 million kg) per year during the period from 1980 to 1982 (Tobacco Institute, 1981, 1982, 1983). On 1 January 1982, some types of chewing tobacco were reclassified as snuff (U.S. Department of Agriculture, 1983). Under this classification, consumption of chewing tobacco in 1982 was 88 million pounds (40 million kg) (Tobacco Institute, 1983). In other parts of the world tobacco is often chewed in combination with other ingredients, in particular with areca nut, betel leaf and slaked lime, as in India and Southeast Asia. It is estimated that more than 450 million people practise this habit in these regions. 3 The chewing of b e t e l quid without tobacco i s a habit of great a n t i q u i t y . The f i r s t mention of b e t e l quid dates from 504 B.C., when i t was recorded i n the "Mahawamsa," a r e g i s t e r of events i n S r i Lanka written i n P a l i , that a princess made a g i f t of b e t e l to her nurse (Krenger, 1942). The chewing of areca nut i s also mentioned i n Sanskrit manuscripts, Sushruta Samhita, believed to have been wr i t t e n around 600 B.C. near Benares. The Sanskrit name for the l e a f of the b e t e l vine, "tambula," p e r s i s t s i n modern Hindi (Gode, 1961) as "tambuli," and i s unchanged i n Arabic and Persian (Nair and Kirk, 1960). In 1298, Marco Polo (Raghavan and Baruah, 1958) wrote i n h i s travelogues that "the people of India have a habit of keeping i n t h e i r mouth a c e r t a i n l e a f c a l l e d the "tembuli" (Krenger, 1942). Although the chewing of b e t e l quid i s p r a c t i s e d i n a number of d i f f e r e n t ways i n various countries, the major components are r e l a t i v e l y consistent. These are: Areca nut (betel nut) i s the f r u i t of the Areca catechu L. tree. Areca i s a small genus comprising about 20 species of slender palms i n the Palmaceae family. The areca palm i s native to South and Southeast A s i a and to several P a c i f i c i s l a n d s . The f r u i t grows i n large bunches at the base of the leaves and v a r i e s i n s i z e and shape. I t i s orange-yellow i n colour and i s generally the s i z e of a small egg. The fibrous pericarp of the f r u i t i s separated from the seed or endosperm which i s then used fresh or a f t e r sun-drying or curing (Arjungi, 1976). Betel l e a f (Piper betle L.) has been used since ancient times. Betel vines are c u l t i v a t e d i n hot and humid c l i m a t i c conditions i n d i f f e r e n t parts of India, Indonesia, Malaysia and S r i Lanka. Lime, known c o l l o q u i a l l y i n India as chuna or chunam, i s prepared either from the calcareous or s i l i c l o u s covering of marine invertebrates (sea s h e l l s ) harvested along the coastline of India, or from quarried stone in central India. It is manufactured on an industrial scale and is sold as a paste mixed with water in order to release calcium hydroxide. Tobacco is often added to the abovementioned ingredients. The tobacco is usually only sun-dried, cut into strips and chewed with no further processing. A typical betel quid is shown in Figure 1. 5 Figure 1: A t y p i c a l b e t e l quid showing b e t e l l e a f , a p o r t i o n of sun-dried tobacco and a quarter of a b e t e l nut. Lime i s shown on the r i g h t f i n g e r t i p . 6 2. Tobacco-Specific N-Nitroso Compounds S i n c e t o b a c c o and a few a r e c a nut components are the o n l y known c o n s t i t u e n t s o f any o f the above m i x t u r e s which can g i v e r i s e t o c a r c i n o g e n i c compounds, t h i s t h e s i s f o c u s e s on these components as the most l i k e l y causes of o r a l c a n c e r i n u s e r s . Of the c a r c i n o g e n i c compounds found i n chewing m i x t u r e s , the N - n i t r o s a m i n e s f a r outweigh any o t h e r . A l a r g e number o f s t u d i e s have shown t h a t d u r i n g the a g e i n g , c u r i 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 t o b a c c o , n i c o t i n e and o t h e r a l k a l o i d s g i v e r i s e t o c a r c i n o g e n i c N - n i t r o s a m i n e s (Hoffmann,et a l . 1984). The c o n c e n t r a t i o n o f these n i t r o s a m i n e s exceeds by a t l e a s t a h u n d r e d f o l d the c o n c e n t r a t i o n s found so f a r i n o t h e r consumer p r o d u c t s . The known c a r c i n o g e n s c o n t a i n e d i n s n u f f s and chewing t o b a c c o s i n c l u d e the 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 (TSNA), such as 4 - ( N - n i t r o s o m e t h y l a m i n o ) - 1 - ( 3 -p y r i d y l ) - 1 - b u t a n o n e (NNK), N ' - n i t r o s o n o r n i c o t i n e (NNN), N - n i t r o s o a n a b a s i n e (NAB) and N - n i t r o s o a n a t a b i n e (NAT); p l u s the v o l a t i l e n i t r o s a m i n e s , N-n i t r o s o m o r p h o l i n e (NMOR) (formed from p a c k a g i n g m a t e r i a l s i n some s n u f f s ) , N-n i t r o s o d i m e t h y l a m i n e (NDMA) (f o u n d i n some s n u f f s ) and N - n i t r o s o p y r r o l i d i n e (NPYR) ( f o u n d i n some s n u f f s ) . I t has been e s t i m a t e d ( N a t i o n a l R e s e a r c h C o u n c i l , 1981) t h a t i n the U.S., t o b a c c o smoke g i v e s r i s e t o a t w e n t y f o l d i n c r e a s e i n consumption o f N - n i t r o s o compounds ove r o t h e r consumer p r o d u c t s . However, s i n c e the c o n c e n t r a t i o n o f TSNA i s much h i g h e r i n chewing tob a c c o than i n c i g a r e t t e smoke, and s i n c e the average chewer consumes 10 g o f tobacco v e r s u s l e s s t h a n 1 g o f t a r i n h a l e d by a smoker p e r day, t o b a c c o chewing appears to be the g r e a t e s t exogenous sou r c e o f exposure t o N - n i t r o s o compounds (Hoffmann and H echt, 1985). I n a d d i t i o n t o the N - n i t r o s o compounds, tobacco may o f t e n c o n t a i n h i g h l e v e l s o f n i t r a t e w h i c h , when c o n v e r t e d t o n i t r i t e t h r o u g h a g e i n g o r r e d u c t i o n 7 i n the s a l i v a , can supply the precursor elements for n i t r o s a t i o n reactions. This i s important when considering the a l k a l o i d content of the areca nut. In v i t r o experiments with arecoline and n i t r i t e have shown that t h i s areca nut a l k a l o i d can give r i s e to at le a s t four N-nitrosamines: N-nitrosoguvacoline, 3-(methylnitrosamino)-propionitrile, 3-(methylnitrosamino)-propionaldehyde (Wenke and Hoffmann, 1983) and N-Nitrosoguvacine (Nair et a l . , 1985) . N-nitrosoguvacoline has been detected i n the s a l i v a of b e t e l quid chewers together with TSNA when the quid contains tobacco (Wenke et a l . , 1984). Recently, N-nitrosoguvacoline and N-nitrosoguvacine have been found together i n the s a l i v a of b e t e l quid chewers (Nair et a l . , 1985). A fu r t h e r important consideration i s the i n t e r a c t i o n of alcohol with N-nitrosamines. Of p a r t i c u l a r relevance to the chewing/alcohol problem i s the observation that alcohol changes the d i s t r i b u t i o n pattern of carcinogenic nitrosamines (Swann, 1982). In ra t s , alcohol diverted nitrosamines from the l i v e r to other organs (e.g., the esophagus) which are more s e n s i t i v e to these carcinogens. This change i n pharmacokinetics may explain the th r e e f o l d increase i n esophageal carcinomas when diethylnitrosamine and ethanol are administered, compared to diethylnitrosamine plus drinking water (Gibel, 1967). Equally important appears to be the observation that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a high frequency of esophageal cancer when applied concurrently with alcohol ( Y i o r i s et a l . , 1984). Normally MNNG i s carcinogenic only i n the stomach of r a t s . 3. Precancerous Oral Lesions and Cancer Many reports of case ser i e s have emphasized the r e l a t i v e l y high frequency of tobacco chewing and snuff use among o r a l cancer patients (IARC, 1985). The c l i n i c a l c h a r a c t e r i s t i c s of cancer patients who use smokeless tobacco products 8 have also been described (IARC, 1985), e s p e c i a l l y the propensity of these cancers to occur i n the presence of leukoplakia, to often have a verrucous appearance, and to be slow-growing, w e l l - d i f f e r e n t i a t e d , squamous-cell carcinomas. Patients with cancer and with a chewing tobacco or snuff habit are frequently described as having cancer at the s i t e or on the side where the quid i s most frequently placed. Much epidemiological evidence indicates a highly s i g n i f i c a n t increase i n o r a l cancer among chewers, versus non-chewers, of betel quid (IARC, 1985). Areas of India and Southeast Asia, where the chewing of bet e l quid i s common, have the highest incidence of o r a l cancer and ora l preneoplastic lesions (leukoplakia) of any region i n the world. In Kerala, India, where some of the studies presented i n th i s thesis were c a r r i e d out, o r a l leukoplakia (see Figure 2) among the C h r i s t i a n fishermen occurred i n approximately 40% of chewers of b e t e l quid plus tobacco, compared to a prevalence of 0.2% i n people without such a habit (IARC, 1985). In addition, cancer of the mouth i s the most prevalent cancer observed i n th i s area. In males, cancers of the mouth, tongue and oropharynx were responsible for 27.2% of a l l occurring cancers; these s i t e s accounted f o r 14.8% of tumors i n females. The great majority were cancers of the buccal mucosa. Generally, the incidence of o r a l cancer i s much higher where tobacco i s added to the quid or where quid chewing occurs concurrently with smoking (IARC, 1985). 10 4. Micronucleated Cells Beyond preneoplastic l e s i o n s , the search for a marker to detect damage at the c e l l u l a r l e v e l l e d to the development of the micronucleus t e s t . When applied to e x f o l i a t e d c e l l s of the buccal mucosa, t h i s t e s t permits the i d etection of l o c a l i z e d genotoxic damage to that t i s s u e . I t i s based on the hypothesis that an increase of micronuclei, which r e s u l t from chromosome and chromatid fragments, should be found i n the early stages of carcinogenesis. Three observations appear to suggest t h i s . F i r s t l y , most chemical carcinogens are a c t i v e clastogens and should induce a v a r i e t y of chromosome aberrations i n human tissue exposed to carcinogenic mixtures. Secondly, many, i f not a l l , carcinogens seem to produce a l t e r e d chromosome numbers and chromosomal rearrangements, some of which involve p a r t i c u l a r regions (Rowley, 1983; Mitelman, 1985). These abnormal karyotypes i n transformed c e l l s must evolve from chromosomal aberrations during the preneoplastic stage. The t h i r d argument i s based on a more t h e o r e t i c a l consideration. A m p l i f i c a t i o n and transposition of oncogenes seem to be mechanisms involved i n neoplastic transformation and i n progression of a malignant phenotype. These oncogene changes r e s u l t from chromatid breaks and exchanges that can be i n d i r e c t l y detected by the appearance of micronucleated c e l l s at preinvasive stages. With t h i s working hypothesis i n mind, we applied the micronucleus test to e x f o l i a t e d c e l l s of i n d i v i d u a l s at elevated r i s k of cancer of the o r a l cavity (Stich, et a l . , 1982a,b; S t i c h and Rosin, 1983b), esophagus (Zaridze et a l . , 1985), urinary bladder (Raafat et a l . , 1984) and cervix (Stich, H.F., unpublished data). A s i g n i f i c a n t increase i n the frequency of micronuclei i n e x f o l i a t e d human c e l l s (micronucleated c e l l s or MNC) was found i n v i r t u a l l y a l l i n d i v i d u a l s . Moreover, an elevated frequency of MNC was also observed i n various tissues of i n d i v i d u a l s a f f l i c t e d with a cancer-predisposing syndrome 11 such as Bloom's syndrome or a t a x i a - t e l a n g i e c t a s i a (Rosin and German, 1985; Rosin and Ochs, 1986). There are considerable advantages to applying the micronucleus t e s t d i r e c t l y to tissues of i n d i v i d u a l s exposed to carcinogens. 1. ) The frequency of MNC can be r e a d i l y estimated i n smear preparations e x f o l i a t e d c e l l s or i n c e l l suspensions obtained from biopsies following treatment with pronase and/or collagenase. Thus genotoxic damage can a c t u a l l y be measured i n tissues which are the targets of carcinogens. 2. ) E x f o l i a t e d c e l l s and biopsies can also be used to estimate the l e v e l of r e t i n o l , beta-carotene, or a number of other chemopreventive agents. Thus l o c a l i z e d d e f i c i e n c y i n protective agents, which could increase s e n s i t i v i t y towards the a c t i o n of carcinogens, can be estimated i n tissues i n which carcinogen-induced genotoxic damage can also be qua n t i f i e d . 3. ) Since e x f o l i a t e d c e l l s can be sampled from small areas, the d i s t r i b u t i o n of MNC within a tissue can be mapped. Thus i t should be possible to l i n k f o c i with high frequencies of MNC to regions i n which preneoplastic l e s i o n s , carcinoma i n s i t u , or carcinomas p r e f e r e n t i a l l y develop. 4. ) The scoring of MNC, which i s c u r r e n t l y a labour-intensive and time-consuming task, lends i t s e l f to automation, so that thousands of c e l l s can be screened w i t h i n minutes for the presence of micronuclei. Since both the incidence of preneoplastic lesions and the incidence of micronucleated c e l l s are elevated among chewers of tobacco and/or b e t e l quid, these two f a c t o r s were used to assess carcinogenic r i s k i n the populations studied i n t h i s t h e s i s , and to determine the populations' response to chemopreventive agents. 12 5. Chemoprevention Having people give up chewing various tobacco-containing mixtures would obviously be the most e f f e c t i v e way to prevent the o r a l cancers t h i s produces. This i s not an easy task, however. In India, the p r a c t i c e of chewing b e t e l quid has been around for many thousands of years and i s deeply integrated into Hindu cult u r e . In addition, i n the countries where these habits are practised, tobacco i s often a major cash crop that cannot simply be replaced by another commodity. Also, the addiction to n i c o t i n e can often lead to the more dangerous habit of c i g a r e t t e smoking i f an i n d i v i d u a l i s encouraged to give up the chewing habit. Other means of prevention are c u r r e n t l y needed. Considerable a t t e n t i o n i s c u r r e n t l y being given to the use of r e t i n o i d s and carotenoids as chemopreventive agents (Peto, 1983; Peto et a l . , 1981). There are several reasons for t h i s . Epidemiological evidence points to an inverse r e l a t i o n s h i p between the intake of beta-carotene-containing green or yellow vegetables and the incidence of cancers at various s i t e s (Hirayama, 1981; Kvale et a l . , 1983; Marshall et a l . , 1982; Z i e g l e r et a l . , 1984; Menkes et a l . , 1986), inc l u d i n g o r a l cancer (Winn et a l . , 1984). Animal studies show beta-carotene and vitamin A to have a marked pr o t e c t i v e e f f e c t against a v a r i e t y of carcinogens (Santamaria et a l . , 1983; Mathews-Roth, 1982), and i n v i t r o experiments have revealed an antimutagenic ( B e l i s a r i o et a l . , 1985) and antitransformation (Som et a l . , 1984) a c t i v i t y . Whether these protective e f f e c t s of beta-carotene are due to i t s p o t e n t i a l f or scavenging r a d i c a l s (Krinsky and Deneke, 1982; Burton and Ingold, 1984), to i t s a b i l i t y to i n t e r f e r e with the a c t i v a t i o n of carcinogens, or to i t s conversion into vitamin A, which i s the actual chemopreventive agent, i s at present d i f f i c u l t to assess. Several large-scale intervention t r i a l s have been i n i t i a t e d to prove or 13 disprove the usefulness of beta-carotene and vitamin A i n preventing the development of carcinomas. However, c l i n i c a l t r i a l s using cancer as an endpoint are expensive, require a r e l a t i v e l y large number of p a r t i c i p a n t s , and l a s t for a long time. Tests that could provide more rapid r e s u l t s would be invaluable i n assessing treatment protocols before long-term, manpower-intensive intervention t r i a l s are i n i t i a t e d . The two "intermediate endpoints" discussed e a r l i e r , precancerous l e s i o n s and micronucleated c e l l s , may be the answer to t h i s need. Preneoplastic l e s i o n s , including dysplasia (Cai, 1982), polyps (DeCosse et a l . , 1975), leukoplakia (Ryssel et a l . , 1971), and esophagitis (Munoz et a l . , 1985) , have been s u c c e s s f u l l y applied to track the response towards chemopreventive agents. In our p i l o t t r i a l on b e t e l quid chewers, we examined the e f f e c t of beta-carotene, beta-carotene plus vitamin A, and vitamin A alone on the remission of established o r a l leukoplakias and the development of new ones. The usefulness of micronucleated c e l l s as markers i n chemoprevention t r i a l s has b een explored ( S t i c h et a l . , 1984a,b, 1985) . Micronucleated c e l l s appear to be good i n d i c a t o r s of carcinogen-induced i n j u r i e s to chromosome complements ( S t i c h and Rosin, 1985). Increased frequencies of micronucleated c e l l s were observed i n tissues at elevated r i s k of developing cancer, including the o r a l mucosa of b e t e l quid chewers (Stich et a l . , 1982a), snuff dippers (Stich et a l . , 1985), users of various tobacco-containing mixtures, including Khaini tobacco (Mirvish, 1982) and "nass" (Zaridze et a l . , 1985), and cigarette smokers ( S t i c h and Rosin, 1983a; Stich, 1987). Since micronuclei reveal the " p a t h o b i o l o g i c a l l y e f f e c t i v e dose," which i s more i n d i c a t i v e than a mere "exposure dose," they should provide information on the protective action of chemopreventive agents. 6. Objectives The objectives of this thesis were fivefold: 1) To determine the snuff dipping and chewing patterns in three communities (Inuit in the Northwest Territories, native Canadian Indians in northern Saskatchewan and East Indians in Kerala, India). 2) To determine the amounts of nitrosamine precursors (nitrite) and tobacco-specific nitrosamines in samples of smokeless tobacco used by members of the abovementioned communities. 3) To determine the amounts of preformed and endogenously formed nitrosamines in users of smokeless tobacco. 4) To determine the pattern of oral lesions (micronucleated c e l l s , leukoderma and precancerous leukoplakia) in the three population groups, which differ in their chewing patterns. 5) To explore the effects of beta-carotene and vitamin A on reducing the frequency of precancerous oral lesions. 15 MATERIALS AND METHODS 1. USE OF SMOKELESS TOBACCO 1.1 Inuit in the Northwest Territories The community of Gjoa Haven was chosen for study because of the large percentage of i n d i v i d u a l s who dip snuff and because of the low consumption of alcohol r e s u l t i n g from l i q u o r p r o h i b i t i o n since 1978. By the 1984/1985 census (Northwest T e r r i t o r i e s Data Book 1984 - 85) this community has a population of 523, of which 95% are Inuit . The sex d i s t r i b u t i o n i s 50% male and 50% female, with an age d i s t r i b u t i o n of: 0-4, 17%; 5-14, 33%; 15-64, 48%; 65 and over, 2%. The major economic a c t i v i t i e s are trapping, hunting and f i s h i n g , the l a s t two also being the major sources of food. The community has two small stores which supply canned and packaged foods plus intermittent supplies of fresh f r u i t and vegetables. The snuff dipping habits of i n d i v i d u a l s i n t h i s community were examined by door to door questionnaire. No o r a l carcinomas were seen among the 180 cancers diagnosed between 1949 and 1974 i n the Inui t of the Canadian A r c t i c (Schaefer et a l . , 1975). 1.2 Native Canadian Indians in Saskatchewan La Loche was chosen as a study s i t e because of i t s large number of snuff dippers and because of a r e l a t i v e l y high consumption of alcohol. By the 1983/1984 census c a r r i e d out by the l o c a l band o f f i c e the community of La Loche has a population of 1847, 90% of which are Chippewan, Metis Indians. La Loche has a sex d i s t r i b u t i o n of 53% male and 47% female, and an age d i s t r i b u t i o n of: 0-4, 15%; 5-14, 25%; 15-64, 57%; 65 and over, 3%. The major 16 economic a c t i v i t i e s include trapping, hunting, f i s h i n g and some mining. La Loche has a moderate-sized grocery store, plus smaller outlets providing fresh f r u i t and vegetables and packaged and canned food products year round; a small government l i q u o r store i s also present. The frequency of o r a l cancer i s the same as that found i n the re s t of the Canadian population that do not use snuff. The brands of tobacco used by the Canadian natives were checked by requesting to see the cans of snuff or bags of chewing tobacco. The weight of tobacco was determined using a small scale and immediately weighing the snuff a f t e r i t had been "pinched" by the i n d i v i d u a l being interviewed. 1.3 East Indians in Kerala. India Fishermen l i v i n g along a coastal s t r i p near Trivandrum, Kerala, India, were chosen f o r study because of the large numbers of b e t e l quid chewers, the r e l a t i v e l y high consumption of alcohol and the frequently observed precancerous lesions (leukoplakia) and o r a l cancers. In t h i s area, cancer of the mouth i s the most prevalent cancer. In males, cancers of the mouth, tongue and oropharynx were responsible f o r 27.2% of a l l cancers; these s i t e s accounted for 14.8% of tumors i n females. The great majority were cancers of the buccal mucosa. Generally, the o r a l cancer incidence i s much higher where tobacco i s added to the quid or where chewing occurs concurrently with smoking (IARC, 1985). No seeds, spices, sugar or flavourings, which are ingredients commonly used i n b e t e l quid of northern India, were added to the quids used by the fishermen. Weights of quid ingredients were obtained by asking i n d i v i d u a l s to give a sample of t h e i r usual quid and l a t e r weighing each portion on an a n a l y t i c a l balance. 17 Demographic data was unavailable for.the fishermen of Kerala at the time of w r i t i n g t h i s t h e s i s . 2. Nitrite in Smokeless Tobacco Samples 2.1 Snuff and Chewing Tobacco The following smokeless tobacco samples were used i n t h i s study: 5 d i f f e r e n t commercial brands of snuff (packed i n Canada), l a b e l l e d C, B, SL, SW and K (4 samples each); one "chewing" tobacco (U.S. brand), l a b e l l e d R; homemade "nass" from Samarkand, the Soviet Union (4 samples); and Khaini tobacco from Bengal, India (4 samples). The snuff, chewing tobacco and Khaini tobacco containers or boxes were f r e s h l y opened p r i o r to chemical analysis and p r i o r to being used by the volunteers. During interviews we took p a r t i c u l a r care to d i s t i n g u i s h snuff (commercially a v a i l a b l e , f i n e l y ground tobacco) from chewing tobacco (commercially a v a i l a b l e strands of tobacco). We checked the brands by requesting to see the cans of snuff or bags of chewing tobacco used by the volunteers. The weight of tobacco was determined with a small scale. The snuff was weighed immediately a f t e r i t had been "pinched" by the i n d i v i d u a l being interviewed. 2.2 Betel Quid The East Indian fishermen surveyed i n t h i s study a l l chewed a simple b e t e l quid c o n s i s t i n g of areca nut (Areca catechu L.), tobacco stems (Nicotiana  tabacum L.), slaked lime (calcium hydroxide) from marine s h e l l s , and b e t e l l e a f (Piper b e t l e L.). Only the tobacco portion of the quid was analyzed f o r n i t r i t e . 18 2.3 Determination of Nitrite Water extracts of tobacco were prepared by suspending 1 g of tobacco i n 10 ml of d i s t i l l e d water and shaking vigorously i n a 37°C water bath f o r 30 min. This was followed by c e n t r i f u g a t i o n (2000 rpm for 2 min) to remove tobacco p a r t i c l e s . The n i t r i t e concentration of both s a l i v a and water extracts of tobacco was determined by the same procedure. Colour reagent was prepared by d i s s o l v i n g 1 g of s u l f a n i l i c a c i d and 0.1 g of N-l-naphthylethylenediamine dihydrochloride i n 100 ml of 20% a c e t i c acid; 25 nl of ei t h e r s a l i v a or water extract of tobacco were added to 2 ml of the colour reagent i n a Nalgene te s t tube and allowed to react f or 15 min. Absorbance was read at 540 nm on a Bausch and Lomb mini Spectronic 20 against a blank of unreacted colour reagent. Concentrations of n i t r i t e were determined from a standard curve. A known amount of sodium n i t r i t e was c a r e f u l l y weighed out on an a n a l y t i c a l balance and dissolved i n a precise volume of d i s t i l l e d water. S e r i a l d i l u t i o n s were made and the standard curve was prepared by the procedure described above. The average recovery of n i t r i t e added to s a l i v a at l e v e l s ranging from 10 to 30 ppm was 95%. Large v a r i a t i o n s were seen i n the n i t r i t e samples: analysis of 56 East Indian s a l i v a samples gave a mean of 36.3 ppm with a standard deviation of 35.06 and a c o e f f i c i e n t of v a r i a t i o n of 96.7%. These v a r i a t i o n s could have been due to the varying dietary intake of n i t r a t e and to the o r a l hygiene of the i n d i v i d u a l s . Snuff samples purchased i n d i f f e r e n t locations also showed large v a r i a t i o n s i n n i t r i t e concentration. For example, 4 Copenhagen snuff samples purchased i n d i f f e r e n t areas of Canada (NWT, N. Saskatchewan and Vancouver) had a mean n i t r i t e concentration of 735 ppm with a standard deviation of 235 and a c o e f f i c i e n t of v a r i a t i o n of 32%. These v a r i a t i o n s were 19 most probably due to d i f f e r e n t storage times. However, 10 i n d i v i d u a l determinations of n i t r i t e from the same sample of tobacco gave a mean n i t r i t e concentration of 434 ppm, a standard deviation of 71.4 and a c o e f f i c i e n t of v a r i a t i o n of 16%. A l l n i t r i t e determinations were done by myself. The procedure for n i t r i t e determination i s outlined i n Figure 3. 20 Figure 3: Procedure for N i t r i t e Determination 25 ul water extract of tobacco or s a l i v a into 2 ml colour reagent (1 gm s u l f a n i l i c a c i d + 0.1 gm N-l-naphthylethylene-diamine dihydrochloride in 100 ml 20% a c e t i c acid) determine absorbance at 540 nm (mini Spectronic 20) against unreacted colour reagent as blank determine • n i t r i t e concentration from standard curve 21 3. Nitrite in the Saliva of Users of Smokeless Tobacco 3.1 Snuff Dippers S a l i v a was c o l l e c t e d f o r n i t r i t e determination i n the Canadian populations (64 Inuit and 20 Indian) by having i n d i v i d u a l s expectorate 2-4 ml of s a l i v a into Nalgene t e s t tubes. The samples were analyzed immediately a f t e r . S a l i v a samples were not c o l l e c t e d during a chewing period. For n i t r i t e determination during chewing, seven snuff dipping Inuit males i n Gjoa Haven, ranging from 14 to 20 years of age, donated t h e i r s a l i v a . 3.2 Betel Quid Chewers S a l i v a samples were c o l l e c t e d by the same procedure as that described above from 50 d i f f e r e n t b e t e l quid chewers i n Kerala, but not during a chewing period. 4. Nitrosation Capacity of Saliva Samples For estimating the n i t r o s a t i o n capacity, s a l i v a was c o l l e c t e d from one volunteer using 1 g of common North American brands of snuff (Copenhagen, Skoal or Big Horn). During a 20-min period of keeping the tobacco i n the lower g i n g i v a l groove, the volunteer expectorated the s a l i v a into a Nalgene te s t tube. The formation of NPRO was qu a n t i f i e d by mixing, i n v i t r o , the s a l i v a of the snuff user with p r o l i n e (200 mg), and incubating the mixture f o r 30 min at 37°C. 5. N-Nitrosoproline in the Urine of Snuff Dippers Before, during and a f t e r the use of snuff, urine was c o l l e c t e d i n bo t t l e s containing 300 mg of s o l i d NaOH. The urine was stored at -20°C u n t i l 22 ni t r o s o p r o l i n e (NPRO) analysis, which took place within one day of sampling. Where urine samples were c o l l e c t e d from i n d i v i d u a l s i n a population, the same method of storage and analysis was used. In the Inui t settlement of Gjoa Haven, urine samples were c o l l e c t e d from 14 regular snuff users and 15 non-users who were matched by sex and age. In addition, urine samples from Indians i n the community of La Loche were c o l l e c t e d from 47 snuff users and 27 non-users of comparable age and sex. 5.1 Determination of Nitrosoproline N i t r o s o p r o l i n e (NPRO) and N-nitrosopiperidine-2-carboxylic a c i d (NPIC) were synthesized and p u r i f i e d using published procedures ( L i j i n s k y et a l . , 1970). Ex t r a c t i o n columns, unbuffered, s t y l e TE 3020, were obtained from Fisher S c i e n t i f i c . A l l other chemicals were reagent or equivalent grade. Samples of urine were preserved by the ad d i t i o n of 300 mg s o l i d NaOH per 100 ml, and were stored frozen u n t i l a n a l y sis. Samples were analyzed by the procedure of Sen et a l . (1983) with minor modifications. Samples of urine (15 ml) were a c i d i f i e d with 3 ml 20% ammonium sulfamate i n 1.8 M ^SO^, and 10 /L*1 i n t e r n a l standard s o l u t i o n i n methanol containing 150 ng NPIC was added. Urine samples were loaded onto 20-ml capacity disposable e x t r a c t i o n columns (Extube, Analytichem I n t e r n a t i o n a l ) , and allowed to absorb. Columns were then eluted with 4 x 20 ml eth y l acetate, which was dr i e d over 20 g anhydrous NaSO^, p r i o r to use. The organic extracts were decanted with washings through a cotton wool plug i n a funnel into round-bottomed f l a s k s , and evaporated u n t i l dry using a rotary evaporator. Residues i n the fl a s k s were dissolved i n a t o t a l of 4 ml methanol, and transferred to 5-ml conical-bottomed sample v i a l s . The methanol was evaporated to a volume of approximately 0.1 ml using a heating block at 50°C and a stream of nitrogen; 0.5 ml BF3 reagent 23 (14% methanol) was added, and the samples were capped and incubated for either 4 hr at 50°C or overnight at room temperature. Dichloromethane (0.5 ml) and 2 ml water were added, and the samples were shaken vigorously to extract the e s t e r i f i e d nitrosamines into the chlorinated solvent. C a l i b r a t i o n samples were prepared by e s t e r i f y i n g 150 ng NPIC or NPRO i n 0.5 ml BF^ methanol i n a s i m i l a r manner. Gas-chromatograhic (GC) analysis of 10-^1 aliquots of the dichloromethane s o l u t i o n was performed on a Perkin Elmer 3920 gas chromatograph, equipped with a n i c k e l column (2 m x 2 mm i.d.) packed with 10% Carbowax 20 M on 80-100 mesh Chromosorb W. Chromatographic conditions were: i n j e c t o r 250°C, column 190°C, c a r r i e r helium at 50 ml/min. Under these conditions, NPIC and NPRO retention times of 3.5 and 4.2 min, re s p e c t i v e l y , were achieved and were nearly baseline resolved. Nitrosamines were detected using a thermal energy analyzer (TEA) (Model 502A; Thermal E l e c t r o n Corp., Waltham, MA) operating at a chamber pressure of 0.55 t o r r , and using a trap cooled with dry i c e . NPRO was qu a n t i f i e d using d a i l y c a l i b r a t i o n samples and an i n t e r n a l standard program on a Spectra Physics SP4100 computing integrator (Spectra Physics Corp., Santa Clara, CA). P r a c t i c a l detection l i m i t s were i n the order of 0.1 ng NPRO per i n j e c t i o n , corresponding to approximately 0.5 ng NPRO/ml urine. Recoveries from samples spiked with standard NPRO were generally i n the 95-100% range. Analysis of 9 separate samples taken over a 17-hr period from the same i n d i v i d u a l produced a mean NPRO value of 16.09 ng/ml, with a standard deviation of 2.31 and a c o e f f i c i e n t of v a r i a t i o n of 14.4%. 24 In order to determine the r e p r o d u c i b i l i t y and the rate of elim i n a t i o n of NPRO from the human body i n i n vivo experiments, urine samples were analyzed for NPRO a f t e r ingestion of v a r i a b l e amounts of n i t r a t e . In f i v e experiments, 65, 130, 195, 260 and 325 mg n i t r a t e were ingested together with 500 mg pr o l i n e . In each case, a s i m i l a r pattern of NPRO excretion was obtained, and the amount of NPRO returned to the background l e v e l within 24 hr, i n d i c a t i n g that NPRO was t o t a l l y eliminated into the urine within that period. Therefore the NPRO analyzed i n urine c o l l e c t e d over 24 hr a f t e r dosing was an ind i c a t o r of d a i l y n i t r o s a t i o n i n vivo. Nitrosamines i n s a l i v a and extracts were determined i n a manner s i m i l a r to that stated above by s c a l i n g down extraction volumes. The procedure f o r NPRO determination i s out l i n e d i n Figure 4. \ Figure 4: Procedure for N-nitrosoproline (NPRO) Determination Urine preserved with 300 mg soiid NaOH/100 ml 15 ml urine Acidify with 3 ml 20% ammonium sulfamate in 1.8 M F^SO^ add 10 u l (150 ng NPIC) internal standard in MeOH • Load onto 20-ml capacity extraction columns elute 4 x 20 ml ethyl acetate (dried over 20 gm anhydrous NaSO.) 4 Collect organic extracts through cottonwoo 1 plug into round-bottomed flask Evaporate to dryness with rotary evaporator Redissolve residue in 4 ml MeOH and transfer to 5-ml conical-bottomed v i a l • Evaporate to 0.1 ml using heating block at 50°C-under N„ Add 0.5 ml BF3 (14% MeOH) • Cap and incubate for 4 hr at 50°C or overnight at room temperature 1 Add 0.5 ml dichloromethane and 2 ml water, and shake vigorously Gas chromatographic/ Thermal energy analyzer analysis 26 6. Tobacco-Specific Nitrosamines in Different Brands of Tobacco Two brands of snuff (Copenhagen and Skoal) commonly chewed by Canadian natives were analyzed for the presence of TSNA. Water extracts were prepared by suspending 1 g of tobacco i n 10 ml of d i s t i l l e d water and shaking vigorously i n a 37°C water bath for 30 min. 0.5 ml of the extract was used for TSNA analysis. A f t e r adding i n t e r n a l standards (N-nitrosodimethylamine- C and N'-nitrosonornicotine-''"^ C) , each sample was extracted four times with 20 ml ethyl acetate, using a prewetted pretube. The organic f r a c t i o n s were pooled and concentrated to approximately 3 ml, and then subjected to column chromatography on 65 g basic alumina. A f t e r e l u t i o n with 200 ml f r e s h l y d i s t i l l e d dichloromethane ( f r a c t i o n I, containing v o l a t i l e nitrosamine), the samples were chromatographed with 200 ml dichloromethane-acetone (4:1) ( f r a c t i o n I I , containing the TSNA). Aft e r being concentrated to 1 ml, the samples were analyzed by GC-TEA. For the v o l a t i l e nitrosamines, a 12 f t x 1/4 i n (2 mm i.d.) glass column packed with 10% Carbowax 20 M on Chromosorb WNAW (100-120 mesh) at an oven temperature of 175°C was used. For the TSNA, a 12 f t x 1/4 i n (2 mm i.d.) glass column packed with 10% UCW-98 on Gas Chrom Q (80-100 mesh) at an oven temperature of 200°C was used (Adams et a l . , 1983). An a l i q u o t of both f r a c t i o n s was used for s c i n t i l l a t i o n counting to determine the recovery rate. Several blank samples, using 5 ml water, were also assayed to ensure that there was no contamination during analysis, since the l a r g e s t sources of error i n a l l nitrosamine analyses r e s u l t from contamination by nitrosamines or precursors from other sources. Detection l i m i t s f o r TSNA are 2.4 ppb and acceptable l e v e l s of DMNM are those below 0.9%. Recoveries from s a l i v a spiked with TSNA were generally 80-90%. Table I gives the means and p r e c i s i o n of three analyses. 27 The following four TSNA were estimated.in the s a l i v a of the snuff dippers: N'-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Table I Analysis of three of the same tobacco samples f o r TSNA Sample Compound Mean (/ig/g) and P r e c i s i o n (%) Tobacco N-Nitrosonornicotine (NNN) 45.6 ± 7% 4-(Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) 7.4 ± 8% N-Nitrosoanatabine (NAT) 47.7 ± 8% N-Nitrosoanabasine (NAB) 1.8 ± 12% 29 7. Tobacco-Specific Nitrosamines in the Saliva of Snuff Dippers Subjects from whom s a l i v a samples were c o l l e c t e d were asked to take t h e i r usual quantity of snuff and to expectorate into a tube at i n t e r v a l s when they would normally s p i t . C o l l e c t i o n of s a l i v a took place over a period of 15 minutes (before, during and a f t e r the chewing period). One ml of c i t r a t e -phosphate b u f f e r (pH 4.5) containing 3.25 mg NaNj ( n i t r o s a t i o n i n h i b i t o r ) and 1.00 mg cis-2,6-dimethylmorpholine (serving as monitor amine) was added to a l l s a l i v a samples, which were then frozen. The amount of s a l i v a c o l l e c t e d v a r i e d between 3.0 and 6.5 ml. A l l p a r t i c i p a n t s i n t h i s study used the same brand of snuff (made i n the U.S.A. for National Tobacco Co., Ltd., Montreal, Canada) and none were smokers. A f t e r the s a l i v a samples had been allowed to thaw, 0.5 ml was removed for the radioimmunoassay of ni c o t i n e and cot i n i n e . The remainder was used f or the analysis of TSNA and nitroso-cis-2,6-dimethylmorpholine (DMNM) by the method described above. 8. Determination of Nicotine and Cotinine Nicotine and cot i n i n e were determined from 0.5-ml thawed s a l i v a samples by radioimmunoassay according to the method of Langone et a l . (1973) with s p e c i f i c a n t i s e r a produced by i n j e c t i o n into rabbits of trans - 3 -succinylmethylnicotine and trans-4-carboxvcotinine bound to albumin. Recoveries of 98-104% r e l a t i v e to the i n t e r n a l standard were obtained on standard samples. By su i t a b l e d i l u t i o n of a standard s o l u t i o n the minimum detectable l e v e l was found to be 5 ng, i . e . , equivalent to a concentration of 300 nmole/litre. 30 Replicate analyses of a smoker's urine showed a c o e f f i c i e n t of v a r i a t i o n of 7.3% for n i c o t i n e and 9.0% for c o t i n i n e . The c o e f f i c i e n t of v a r i a t i o n of the c a l c u l a t e d n i c o t i n e : c o t i n i n e r a t i o s was 11.1%. 9. Frequency of Micronuclei in the Oral Mucosa of Snuff Dippers and Tobacco Chewers E x f o l i a t e d c e l l s of the o r a l mucosa were obtained by swabbing with a moistened wooden tongue depressor, smearing the c e l l s onto a clean microscope s l i d e , a i r - d r y i n g , and f i x i n g i n 80-85% ethanol. I f e x f o l i a t e d c e l l s from smaller areas of the g i n g i v a l groove were desired, s p l i t wooden popsicle s t i c k s were used. The f i x e d c e l l preparations were stained with Feulgen reaction following a 10 min h y d r o l y s i s with N-HC1, and counterstained with f a s t green. The Feulgen-stained s l i d e s were screened for various nuclear anomalies, inc l u d i n g micronuclei, whose s i z e can vary from small Feulgen-positive bodies to larger, n ucleus-like structures with membranes and c l e a r l y v i s i b l e chromatin. The pros and cons of t h i s technique have been recently reviewed (Sti c h , 1987). Micronuclei were scored only i n i n t a c t e p i t h e l i a l c e l l s . We followed w e l l - e s t a b l i s h e d c r i t e r i a f o r estimating the frequency of human e x f o l i a t e d c e l l s with micronuclei ( S t i c h and Rosin, 1983b, 1984a). The usefulness of t h i s technique i n following the response to chemopreventive agents i s discussed by S t i c h and Rosin (1985). An elevated frequency of micronucleated c e l l s was found to be a simple marker i n d i c a t i n g an elevated r i s k of developing cancer (Table I I ) . The micronucleated c e l l frequencies were determined i n t h i s study by Mrs. H.F. Stic h , an expert i n t h i s f i e l d . Anucleated e x f o l i a t e d c e l l s are common i n the palate, but rare i n the buccal mucosa of non-chewers of tobacco and non-smokers. At l e a s t 300 c e l l s per sample were counted to estimate the percentage of anucleated c e l l s . Feulgen-stained smear preparations were used. 31 TABLE II Frequency of Micronuclei i n Human Tissues at Elevated Risk for Cancer Tissue Suspected Carcinogenic Factors Elevation of Micronucleated C e l l s (Fold) Reference Oral mucosa: Gingival groove Snuff 4.2 Inner l i p Khaini tobacco 4.4 Floor of mouth Nass 8.2 Buccal mucosa Betel quid 9.4 Smoking and drinking 4.6 Smoking 3.4 Curry 1.5 Unknown (esophagitis) 3.4 Smoking a Schistosoma haematobium Smoking Smoking and coffee drinking Smoking Esophagus Urinary bladder Bronchial epithelium C e r v i c a l epithelium Blood lymphocytes Bone marrow Spermatids Smoking Smoking Smoking Unknown (dysplasia) Smoking Styrene Styrene Styrene X-ray contrast media Antileukemia agents Smoking 13.5 6.0 9.4 S h i f t to higher values 2.6 2.2 5.1 3.0 1.5 9.0 4.0 1.6-4.3 >34.0 1.4 Sti c h et a l . (1985) S t i c h et a l . (1982b) St i c h (1987) St i c h et a l . (1982a) St i c h and Rosin (1983a) Fontham et a l . (1986) Picker and Fox (1986) St i c h and Zaridze (unpublished data) Mandard et a l . (1987) Raafat et a l . (1984) Fontham et a l . (1986) S t i c h (1987) Reali et a l . (1987) Fontham et a l . (1986) St i c h (unpublished data) Fontham et a l . (1986) Stich (1987) Hflgstedt et a l . (1983a) Hogstedt et a l . (1983b) Meretoja et a l . (1978) Nordenson and Beckman (1984) Parvez et a l . (1987) Abe et a l . (1984) Lahdetie (1986) Elevated frequencies were observed i n only 5% of patients examined. 32 10. Precancerous Lesions and Cancer of the Oral Cavity of Users of Smokeless Tobacco 10.1 Leukoderma Oral l e s i o n s i n users of snuff and chewing tobacco have been c l a s s i f i e d , according to t h e i r appearance, into four groups by A x e l l et a l . (1976) and into three categories by Greer and Poulson (1983). To avoid any subjective bias i n subdividing these l e s i o n s , we recorded only two types of anomalies: (1) whitish to yellowish wrinkled patches of the mucosa with or without furrows (corresponding to categories 2 and 3 of Greer and Poulson [1983]), which are the most commonly observed anomalies, and (2) d e f i n i t e leukoplakias, as described by the World Health Organization (1980). There i s a c e r t a i n ambiguity i n properly d e f i n i n g the many whitish lesions of the o r a l mucosa. The whitish wrinkled areas which we observed at the s i t e s where the snuff or chewing tobacco came i n close contact with the mucosa are not comparable to the preleukoplakias, leukoplakias or erythroplakias so commonly seen i n be t e l quid chewers (IARC, 1985) or users of "nass" (a mixture of tobacco, slaked lime, o i l and ash) (Zaridze et a l . , 1986). The whitish areas can disappear within days or weeks, whereas remission of other mucosal abnormalities requires several months following the cessation of snuff or b e t e l quid use. The precancerous state of leukoplakias, p a r t i c u l a r l y those with reddish areas, appears well-established, whereas there i s no evidence to consider the whitish wrinkled patches of snuff dippers a preneoplastic stage. The presence or absence of these l a t t e r lesions were recorded f o r a l l the Canadian Inuit and Indians surveyed. 33 10.2 Leukoplakia A precancerous l e s i o n i s defined as a morphologically a l t e r e d tissue i n which cancer i s more l i k e l y to occur than i n i t s apparently normal counterpart. This c o n d i t i o n i s , therefore, a generalized state associated with a s i g n i f i c a n t l y increased r i s k of cancer (WHO Collaborating Centre for Oral Precancerous Lesions, 1978; A x e l l et a l . , 1984). Examples of o r a l precancerous lesions are leukoplakia and erythroplakia; o r a l precancerous lesions also include sideropenic dysphagia, submucous f i b r o s i s , and p o s s i b l y l i c h e n planus. The concept of leukoplakia as a precancerous l e s i o n i s based on findings that a s i g n i f i c a n t number of o r a l carcinomas are associated with pr e - e x i s t i n g leukoplakia, and that some leukoplakias appear to undergo malignant transformation (Pindborg et a l . , 1975; Silverman et a l . , 1984). However, studies of o r a l leukoplakia are often d i f f i c u l t to compare because of a lack of uniformity i n the h i s t o l o g i c a l d e f i n i t i o n of leukoplakia. In t h i s t h e s i s leukoplakia are c l a s s i f i e d according to the WHO Collaborating Centre f o r Oral Precancerous Lesions (1978), as o u t l i n e d i n Table I I I . C l a s s i f i c a t i o n of Oral Precancerous Lesions (WHO Collaborating Centre for Oral Precancerous Lesions, 1978) Condition Description of Lesion Histopathology Predisposition to Cancer Leukoplakia White, whitish/yellow, or grey patch a f f e c t i n g very small to large areas of the mucosa; smooth or wrinkled surface often with cracks; may present as homogeneous, nodular or speckled Hyperorthokeratosis ; hyperparakeratosis; atrophy; d i f f u s e chronic inflammation i n lamina propria (lymphocyte and plasma c e l l s ) ; some dysplasia i n nodular type 6-10% become malignant Erythroplakia Bright red, velvety plaques; i r r e g u l a r , well-defined outl i n e ; occasional nodular surface with white or yellow spots Marked e p i t h e l i a l atrophy; variable e p i t h e l i a l dysplasia; heavy chronic inflammation i n sub e p i t h e l i a l connective ti s s u e (mainly plasma c e l l s ) Greater than 10% become malignant Leukoderma Whitish to yellowish wrinkled patches; with or without furrows Epithelium hyperplastic; None known microbial colonization of i r r e g u l a r surface; s u p e r f i c i a l e p i t h e l i a l c e l l s large and angular, pyknotic n u c l e i , cytoplasm l i g h t l y stained or empty 35 11. Intervention Strategies 11.1 Nitrite Trapping Agents The i n h i b i t o r y e f f e c t of ascorbate and c a f f e i c a c i d on nitrosamine formation was studied using NPRO as a model i n both i n v i t r o and i n vivo (ascorbate only) systems. The i n v i t r o method was done by having a volunteer (myself) chew 1 g of three d i f f e r e n t brands of snuff and expectorate the s a l i v a (10 ml) into a Nalgene t e s t tube. To the s a l i v a 200 mg pr o l i n e was added e i t h e r alone (control) or with e i t h e r 200 mg ascorbate or 200 mg c a f f e i c acid. The solutions were then taken to pH 2.5 using small amounts of normal HCl and allowed to incubate at 37°C f o r 30 min. NPRO analysis was then made by the procedure described above. For the i n vivo determination of endogenously formed NPRO a volunteer (myself) chewed 1 g of snuff (2 times at 60 min i n t e r v a l s ) plus ingested simultaneously 200 mg of p r o l i n e ( c o n t r o l ) . This experiment was repeated with ingestion of 500 mg of chewable ascorbic a c i d 30 min p r i o r to chewing the snuff and ingesting p r o l i n e and 30 min following snuff chewing and p r o l i n e ingestion. The same experiment was repeated with a larger ingestion of ascorbate at 30 min p r i o r to the snuff and pr o l i n e , 30 min a f t e r and again 30 min, 60 min and 90 min a f t e r the f i n a l ingestion of pr o l i n e and snuff chewing. A l l urine passed over the next 17 hr was c o l l e c t e d f or NPRO analysis. The experimental protocol i s shown i n Figure 5. 36 F i g u r e 5 : E x p e r i m e n t a l P r o t o c o l f o r Endogenous NPRO Fo r m a t i o n @ © A S C r A S C I -30 S N U F F + PROLINE 4 - r -S N U F F + PROLINE 4, A S C I I S N U F F + PROLINE 1 A S C I 1 30 S N U F F + PROLINE •i, S N U F F + PROLINE 4> 1 SNUFF + PROLINE -fh — T -60 A S C 120 A S C 180 A S C 1 240 MIN 37 11.2 Administration of Beta-carotene and Vitamin A Beta-carotene was supplied by Hoffmann-La Roche as water-dispersal beadlets c o n s i s t i n g of approximately 20% starch, 20% dextrose, 30-50% g e l a t i n , 10-11% beta-carotene, and trace amounts of alpha-tocopherol and ascorbate palmitate. The butylated hydroxyanisole and butylated hydroxytoluene present i n some forms of beta-carotene were absent from our preparations. Vitamin A capsules were purchased from Stanley Drug Company Ltd., Vancouver. Each capsule contained vitamin A derived from f i s h l i v e r o i l s equivalent to 15 mg r e t i n o l . Placebo capsules supplied by Hoffmann-La Roche contained dextrose. 11.3 Inuit: Beta-carotene Three beta-carotene capsules (30 mg beta-carotene per capsule) were administered twice weekly f or 10 weeks under the s t r i c t supervision of myself and an Inui t i n t e r p r e t e r who spent the e n t i r e t r i a l period i n Gjoa Haven. Treatment with beta-carotene was i n i t i a t e d with 27 snuff dipping i n d i v i d u a l s , plus 10 r e c e i v i n g placebo. Individuals were selected who took more than 6 snuff dips per day. Three boys l o s t i n t e r e s t i n cooperating. The p a r t i c i p a n t s who received beta-carotene, placebo capsules or no treatment (18 in d i v i d u a l s ) were chosen from the subjects previously interviewed. To the best of our knowledge, neither the number of tobacco plugs, the s i t e , nor the length of time that the tobacco was kept i n the mouth changed during the 10-week t r i a l period. 11.4 East Indians : Beta-carotene and Beta-carotene plus Vitamin A A t o t a l of 130 b e t e l quid chewers with o r a l leukoplakias were divided into three groups. One (35 p a r t i c i p a n t s ) received placebo capsules, a second (35 pa r t i c i p a n t s ) received beta-carotene, and a t h i r d (60 p a r t i c i p a n t s ) received 38 beta-carotene plus vitamin A. Individuals were selected on the basis of having established leukoplakia plus the l o c a t i o n i n which they l i v e d to make the d i s t r i b u t i o n of capsules easier to people i n the same v i c i n i t y . To ensure that a l l t r i a l p a r t i c i p a n t s received the desired doses, the placebo, beta-carotene and vitamin A capsules were administered twice weekly f o r 6 months under the s t r i c t supervision of myself, a l o c a l nurse and a s t a f f member of the Regional Cancer Centre, Trivandrum. The following were given twice weekly to each person, depending on h i s group: (1) 3 beta-carotene capsules, each containing 30 mg beta-carotene; (2) 3 beta-carotene capsules plus one vitamin A capsule (50,000 IU), and (3) 3 placebo capsules containing dextrose and water-dispersal beadlets. Of the o r i g i n a l p a r t i c i p a n t s , 5 i n Group 1, 4 i n Group 2, and 9 i n Group 3 were l o s t due to death, i l l n e s s or emigration. Endpoints were examined at 3 months and again at 6 months. 11.5 East Indians : Vitamin A Betel quid chewers with well-established leukoplakias (diagnosed by Drs. Mathew and Sankaranarayanan according to the WHO Collaborating Centre for Oral Precancerous Lesions [1978]) were d i s t r i b u t e d into two groups based on the area of the beach i n which they l i v e d : one group of 35 p a r t i c i p a n t s received placebo capsules (dextrose), and the second group of 30 p a r t i c i p a n t s received vitamin A (200,000 IU/week), given twice weekly as 2 capsules of 50,000 IU of vitamin A each. In the placebo group, 33 out of 35 chewers completed the t r i a l , and i n the vitamin A-treated group, 21 out of 30 chewers remained on the vitamin A regime. In a l l the intervention studies c a r e f u l a t t e n t i o n was taken by myself or a s t a f f member of the Regional Cancer Centre, Trivandrum to make sure that the p i l l s were a c t u a l l y swallowed by each of the t r i a l p a r t i c i p a n t s . 39 12. Determination of Beta-carotene and Retinol To determine the e x i s t i n g l e v e l s of beta-carotene and r e t i n o l i n the Inuit population studied, serum samples were c o l l e c t e d from 110 subjects including 70 non-users of snuff and 40 users. Blood samples were c o l l e c t e d i n the community of Gjoa Haven using vacutainers (No. 6421) containing a c l o t a c t i v a t o r . Samples were stored on ice for approximately 1 hr a f t e r c o l l e c t i o n , then spun i n a benchtop centrifuge (700 g for 5 min). Serum was c o l l e c t e d with a Pasteur pipet into 2-ml cryotubes, and stored i n l i q u i d nitrogen u n t i l a n a l y sis. Aliquots of serum (200 /il) were placed i n 1.5-ml polypropylene microcentrifuge tubes, and 200 /il aliquots of 5% KOH i n methanol were added. Samples were digested f or 1 hr at 50°C, and then 600 /xl of hexane were added. Samples were extracted by vortex mixing for 1 min, and the hexane was separated from the sample by c e n t r i f u g a t i o n . Aliquots of hexane (400 /il) were removed from the samples, and d r i e d under reduced pressure i n 1.5-ml polypropylene microcentrifuge tubes i n a c e n t r i f u g a l evaporator (Speed Vac Model SVC100H, Savant Instruments, H i c k s v i l l e , NY). Residues were redissolved i n 20 pi methylene c h l o r i d e , and then d i l u t e d to a t o t a l of 200 pi with methanol. Ret i n o l and carotene were measured by i s o c r a t i c high-pressure l i q u i d chromatography (HPLC) ( M i l l e r et a l . , 1984). For r e t i n o l , 50-jul aliquots of sample were i n j e c t e d into a Brownlee Spheri 5 reversed-phase column (5 micron packing, 2.1 mm i n t e r n a l diameter x 22 cm) coupled to a 2.1 mm i n t e r n a l diameter x 3 cm guard column with the same packing. Solvent consisted of 99% methanol and 1% water at a flow rate of 0.5 ml/min. For carotene, 50-^1 aliquots of sample were i n j e c t e d into a Vydac 201 TP reversed-phase column (10 micron packing, 3.2 mm i n t e r n a l diameter x 25 cm). Solvent consisted of 95% 40 methanol and 5% methylene chloride at a flow rate of 1.0 ml/min. A l l solvents contained 0.1% phosphoric a c i d to protect against possible column degradation by a l k a l i n e samples. Re t i n o l and carotene were detected by a v a r i a b l e wavelength UV/VIS HPLC absorbance detector using a tungsten lamp, which was set at 325 nm for r e t i n o l and then switched to 450 nm for carotene. Samples were q u a n t i f i e d by an e l e c t r o n i c computing integrator which had been c a l i b r a t e d by i n j e c t i o n of r e t i n o l and carotene standards i n methanol. C a l i b r a t i o n standards were prepared by d i s s o l v i n g c r y s t a l l i n e r e t i n o l or carotene (Sigma, St. Louis, MO) i n methanol, and determining the concentration of solutions by t h e i r absorbance, using molar e x t i n c t i o n c o e f f i c i e n t s i n methanol of 52,500 ( r e t i n o l , 325 nm) and 130,000 (carotene, 450 nm). Within-day p r e c i s i o n was 5.6% for beta-carotene i n 10 samples from a pooled si n g l e specimen of normal serum (mean = 57 ng/ml; SD = 3.2 ng/ml). Day-to-day p r e c i s i o n during 27 days was 5.9% (mean = 57 ng/ml, SD = 3.4 ng/ml) for the same pool. The e f f i c i e n c y of ex t r a c t i o n of beta-carotene from serum appeared to exceed 95%: the absorbance at 450 nm a f t e r a second extraction was less than 3% of that f o r the o r i g i n a l extract. R e t i n o l analysis of the same plasma on 10 consecutive days gave a mean value of 450 ng/ml (SD = 19.8 ng/ml), and a c o e f f i c i e n t of v a r i a t i o n of 4.4%. P r e c i s i o n f o r the same-day analysis of 10 aliquots of a second plasma gave a mean of 440 ng/ml (SD = 18.0 ng/ml), and a c o e f f i c i e n t of v a r i a t i o n of 4.1%. The procedure for the determination of serum r e t i n o l and beta-carotene i s out l i n e d i n Figure 6. 41 Figure 6: Determination of Serum Beta-carotene and Retinol Serum (200 ul) into 1.5-ml polypropylene microcentrifuge tubes r add 200-ul aliquots of 5% KOH i n MeOH digest for 1 hr at 50°C • add 600 ul hexane extract by vortex mixing for 1 min separate hexane from sample by c e n t r i f u g a t i o n remove 400-ul aliquots of hexane and dry under reduced pressure in 1.5-ml polypropylene microcentrifuge tubes in c e n t r i f u a a l evaoorator I redissoive in 20 jjl methylene chloride and d i l u t e to 200 ul with MeOH I i n j e c t 50 u l into HPLC 42 13. Questionnaire The In u i t surveys were conducted during the summer of 1986 i n the communities of Gjoa Haven, NWT, and the native Indian surveys during the summer of 1987 i n the community of La Loche, N. Saskatchewan. A t o t a l of 300 native Indians and 200 I n u i t were interviewed, and t h e i r o r a l c a v i t i e s were screened for l e s i o n s . In a settlement, sampling was c a r r i e d out i n houses, schools, sports grounds and native band o f f i c e s . The questionnaire used (Table IV) included questions on patterns of snuff dipping or tobacco chewing, smoking and alcohol consumption; l o c a l i z a t i o n of tobacco within the o r a l cavity; ingestion of s o f t drinks, coffee or tea; sex, age and band a f f i l i a t i o n . I c a r r i e d out the interviews accompanied by a native s o c i a l worker, nurse or t r a n s l a t o r . To avoid any peer pressure, the interviews of i n d i v i d u a l p a r t i c i p a n t s were conducted i n the absence of any i n t e r f e r i n g onlookers. A s i m i l a r questionnaire (Table V) about chewing habits and alcohol consumption was administered house to house among the East Indian fishermen i n Kerala. In a l l more than 500 fishermen were interviewed. 43 TABLE IV Q u e s t i o n n a i r e Used i n the Northwest T e r r i t o r i e s and Saskatchewan Study code: Date o f i n t e r v i e w : Document code: I n t e r v i e w e r : Name: P l a c e o f i n t e r v i e w : Age: Sex: C u r r e n t a d d r e s s : E t h n i c o r i g i n : P l a c e o f b i r t h : Chewing P a t t e r n Smoking P a t t e r n Type o f t o b a c c o : Type ( c i g a r e t t e , c i g a r , e t c . ) : Number per day: Brand: Weight o f p i n c h : Number per day: L o c a t i o n i n mouth: Years o f smoking: Time per chew: I n h a l e : Years o f chewing: A l c o h o l Use  Type Beer Wine: B o t t l e s per week: Whiskey Other: Home brew: Tea: Cups/day: C o f f e e : Cups/day: V i t a m i n s Type: Dose: 44 TABLE V Q u e s t i o n n a i r e Used i n K e r a l a , I n d i a I n t e r v i e w no.: Date: Smoking No. o f b i d i p e r day: No. o f c i g a r e t t e s p e r day: A l c o h o l (ml p e r day) Toddy: L o c a l : Chewing No. o f q u i d s p e r day: Nut+leaf+tobacco+lime: Use t o b a c c o l e a f : Appearance o f L e s i o n P a t i e n t no.: D u r a t i o n o f chew: Nut+leaf+lime: Use to b a c c o stem: O v e r n i g h t : 1. No change: 2. New l e s i o n : 3. Complete r e m i s s i o n : 4. P a r t i a l r e g r e s s i o n : a) Decrease i n l e s i o n s i z e : b) P a t i e n t has m u l t i p l e l e s i o n s c) Change i n l e s i o n appearance: d) L e s i o n changes c l a s s i f i c a t i o n : 5. P r o g r e s s i o n o f l e s i o n : a) L e s i o n i n c r e a s e s i n s i z e : b) L e s i o n changes c l a s s i f i c a t i o n : Smears: B r u s h i n g s : Photos: Only some d i s a p p e a r : S p e c i f y : S p e c i f y : S p e c i f y : 45 14. Statistical Analysis Tests f o r s i g n i f i c a n t difference i n outcome were performed e i t h e r by Student's T-test or by Fisher's exact t e s t . A P value of < 0.05 was considered s i g n i f i c a n t . Fisher's exact test was performed under the d i r e c t i o n of Dr. J. S p i n n e l l i , Department of Epidemiology, Biometry and Occupational Oncology, B.C. Cancer Research Centre. 46 RESULTS 1. Use of Smokeless Tobacco The objectives of t h i s s e ction were to examine by questionnaire the prevalence of o r a l tobacco use i n two northern Canadian native settlements (Inuit and Indian) and one East Indian community (Kerala, India), to record other o r a l habits which may exert a modifying e f f e c t on carcinogenesis, such as c i g a r e t t e smoking and the drinking of a l c o h o l i c beverages, and to examine the various patterns of snuffing and chewing tobacco. In the two Canadian settlements examined, the dipping of snuff ( f i n e l y cut tobacco commercially a v a i l a b l e i n cans) f a r exceeds the actual chewing of tobacco plugs and twists. The placement of f i n e l y ground tobacco into the o r a l c a v i t y i s designated by native peoples with a v a r i e t y of l a b e l s , including "snuff dipping," "using snoose," "chewing snoose," "taking a pinch," or "taking a chew." The East Indian fishermen use chewing tobacco only as part of the b e t e l quid. Inuit, Indians and East Indians use smokeless tobacco i n conjunction with other o r a l habits, i n c l u d i n g c i g a r e t t e smoking and alcohol drinking. Two of these habits can take place concurrently, (e.g., snuff dipping plus alcohol drinking, or snuff dipping plus c i g a r e t t e smoking). The prevalence of i n d i v i d u a l s with one or more habits i s shown i n Table VI. The 114 snuff dipping Indians examined represent approximately 10% of the t o t a l La Loche population. The 152 Inuit examined represent approximately 15% of the native population of Gjoa Haven. The 300 East Indian b e t e l quid chewers examined represent approximately 10% of the population of fishermen l i v i n g close to Trivandrum, India. 47 The a l c o h o l i c beverages consumed by Indians i n La Loche are mainly beer and wine. The East Indian fishermen of Kerala drink fermented, d i s t i l l e d sap from palm trees (approximately 40% ethanol). A more d e t a i l e d evaluation of the drinking habits of the East Indian population i n a neighbouring area (Ernakulam, Kerala) has recently been completed (Gupta, 1984). Canadian Indians l i v i n g i n northern Saskatchewan d i d not report the drinking of Lysol, shoe p o l i s h or Chinese cooking wine. These types of a l c o h o l i c items, however, are consumed by some Indians and non-Indians l i v i n g i n Vancouver. TABLE VI Snuff Dipping, Tobacco Chewing, Cigarette Smoking, and Drinking of Alcoholic Beverages by Native Indians, Inuit and East Indians Snuff Tobacco Cigarette Alcohol Number Dipping Chewing Smoking Drinking Location Sex Examined (%) (%) (%) (%) La Loche M 55 62.6 0.0 27.2 47.2 (Indian) F 59 32.2 0.0 50.8 44.1 Gjoa Haven M 107 57.0 10.3 32.7 0.0 (Inuit) F 45 0.0 0.0 91.1 0.0 Kerala M 120 0.0 54.0 14.6 36.6 (East Indian) F 110 0.0 39.0 0.0 * 0.0 49 Information on the length of time that snuff users or chewers r e t a i n tobacco i n t h e i r mouth proved to be more d i f f i c u l t to obtain than anticipated, because of a general lack of i n t e r e s t i n and lack of f e e l i n g f o r "time." The r e s u l t s shown i n Table VII were obtained, p a r t l y , by interviews and, p a r t l y , by a c t u a l l y measuring the snuff dipping period. As a r u l e , younger snuff dippers u s u a l l y s t a r t with a smaller amount of snuff and keep i t i n the mouth for less than 15 min. They stated that larger quantities of snuff held for longer periods of time made them "dizzy." The brands of snuff and chewing tobacco used by the native Indians of Saskatchewan and the Inuit during the study period were Copenhagen, Skoal and Redman. The amount of tobacco i n a "pinch" of snuff v a r i e s according to the size of a person's f i n g e r s and the number of fingers used. Knowledge of how long a t i n of snuff w i l l l a s t an i n d i v i d u a l can be misleading when c a l c u l a t i n g the amount of tobacco used d a i l y , since v i r t u a l l y a l l native Indians and Inuit share t h e i r snuff. These d i f f i c u l t i e s were overcome by weighing snuff pinches taken by d i f f e r e n t snuff dippers. The average weight of tobacco chewed by 60 Inu i t was 0.61 g compared to an average of 1.5 g chewed by 51 Indian males and 1.3 g chewed by Indian females. The amount of chewing tobacco used by East Indians i n one b e t e l quid was 1.1 g (SD = 0.6). The weight of the e n t i r e quid was 5.9 g (SD = 0.63). TABLE VII Means and Ranges of Various Snuff-Related Factors Among Inuit and Native Indians Location Sex Number Examined Number of Dips per Day Length of Dip (min) Number of Years of Use Snuff Kept in Mouth per Day (min) Weight of Tobacco per Pinch (gm) Gjoa Haven (Inuit) M 60 8.03 25.2 (1-20.0) (1-180.0) 4.1 208.8 0.61 (0.5-20.0) (3-1140.0) (0.45-0.83) La Loche (Indian) M 51 24 9.1 20.3 (1-30.0) (3-60.0) 6.2 11.9 (1-25.0) (3-30.0) 8.5 (1-25.0) 5.7 (1-20.0) 174.3 (10-600.0) 57.8 (3-150.0) 1.5 (0.3-3.5) 1.3 (0.5-2.6) O 51 Approximately 90% of the 114 adult male and female snuff dippers examined i n La Loche place the tobacco into the lower g i n g i v a l groove and hold i t either to the l e f t or the r i g h t of centre. Less than 5% of Indians keep i t i n the centre behind the lower l i p during a dipping session. I t was stated repeatedly that they f e e l pain i n the frenulum when the snuff i s kept i n the centre for a long time. Adolescent males and younger adult females attempt to conceal t h e i r snuff taking by pl a c i n g the tobacco f a r back i n t h e i r cheeks. V i r t u a l l y a l l snuff users change the l o c a t i o n of the snuff each week due to i r r i t a t i o n i f they keep the tobacco too long i n the same place. Some (approximately 6%) of snuff dippers a c t i v e l y move the snuff around the o r a l c a v i t y with the tongue. 2. Nitrite in Smokeless Tobacco Samples Tobacco has been found i n a l l chewing mixtures that increase the r i s k of developing o r a l cancer (IARC, 1985). I t has been concluded that smokeless tobacco must contain e i t h e r precursors of carcinogens or carcinogenic agents themselves. Since snuff and chewing tobacco are not exposed to excessive heat, they do not contain carcinogenic p y r o l y s i s products, unlike c i g a r e t t e s and cig a r s . This s e c t i o n focuses on the capacity of smokeless tobacco to supply n i t r i t e , which could lead to the formation of carcinogenic N-nitrosamines i n snuff dippers and tobacco chewers. 2.1 Snuff and Chewing Tobacco Brands N i t r i t e was estimated i n aqueous extracts of several tobacco samples, including f i v e d i f f e r e n t brands of snuff which were commercially a v a i l a b l e i n Canada, four "nass" (tobacco mixed with slaked lime, ash and o i l ) samples from Samarkand i n the Soviet Union, and four Khaini tobacco specimens from Bengal, India. Each tobacco sample was analyzed three times. The figures given i n Table 52 VIII represent t h e i r averages. Tobacco samples 1 to 4 i n Table VIII represent four d i f f e r e n t cans or boxes. TABLE VIII N i t r i t e C o n t e n t i n Aqueous E x t r a c t s o f Smokeless Tobacco Samples N i t r i t e (mg/kg) I n d i v i d u a l Samples a Tobacco Type Brand O r i g i n 1 2 3 4 S n u f f C Canada 1040 800 550 550 S n u f f B Canada 500 400 305 95 S n u f f SL Canada 820 750 500 70 Snuff SW Canada 75 18 5 ND! Snuff K Canada ND ND ND ND Chewing R U.S. ND ND ND ND Nass U z b e k i s t a n 20 10 8 7 K h a i n i , dry I n d i a ND ND ND ND K h a i n i , dry I n d i a . ND ND ND ND Chewing P a r i j a t I n d i a ND ND ND ND Chewing Gundi I n d i a ND ND ND ND Chewing S u n - d r i e d I n d i a ND ND ND ND a C, Copenhagen B, B i g Horn; S L , S k o a l ; SW, S k o a l Wintergreen; b K, Kodiak; R, Redman ND, not d e t e c t a b l e 54 2 . 2 East Indian Tobacco The tobacco used by the East Indian fishermen i n Kerala, to make be t e l quids, i s a sun-dried v a r i e t y . The n i t r i t e l e v e l s i n the samples of t h i s tobacco were too low to be detected by the a n a l y t i c a l method used (Table VIII). Two other East Indian chewing tobaccos which are used to make a b e t e l quid were also examined, with s i m i l a r negative r e s u l t s (Table V I I I ) . 3. Nitrite in the Saliva of Users of Smokeless Tobacco 3.1 Snuff Dippers S a l i v a was c o l l e c t e d from seven Inu i t volunteers p r i o r to snuff dipping and a f t e r they had placed 0.5 g of tobacco (Copenhagen) into the lower g i n g i v a l groove. S a l i v a was c o l l e c t e d at 5 min i n t e r v a l s f o r the next 30 min, and the n i t r i t e determined i n these s a l i v a samples. Considerable quantities of n i t r i t e were released from the snuff into the s a l i v a (Figure 7). No active "chewing" of the tobacco occurred during the experimental period. Once the snuff was removed from the o r a l c a v i t y , the n i t r i t e l e v e l s of the s a l i v a approached control l e v e l s within approximately 10 min. The mean n i t r i t e l e v e l s are given i n Table IX for i n d i v i d u a l s not during a chewing period. 3.2 Betel Quid Chewers S a l i v a samples were c o l l e c t e d from 50 d i f f e r e n t b e t e l quid chewers i n Kerala, but not during a chewing period. The r e s u l t s are shown i n Table IX. Figure 6: N i t r i t e i n the S a l i v a of Seven Inuit Snuff Dippers O-30-i 0-25-2 0-20-•— > 0-151 j < o-io-OJ I i-2 0-05-15 20 25 TIME (MINI PERIOD OF S N U F F DIPPING PRIOR TQ S N U F F DIPPING 30 Brackets represent the Standard Deviation of the Mean TABLE IX N i t r i t e i n the S a l i v a o f Smokeless Tobacco Chewers Number Mean N i t r i t e P o p u l a t i o n Examined Tobacco Type (ppm) Inuit 64 Snuff 5.02 (Gjoa Haven) (SD=7.07 CV=140%) Indian 20 Snuff 6.87 (La Loche) (SD=3.68 CV=53%) East Indian (Kerala) 56 S u n - d r i e d 36.27 (SD=35.06 CV=96.7%) 57 4. Nitrosation Capacity of Saliva Samples The objective of t h i s part of the study was to show whether the n i t r i t e released from tobacco into the s a l i v a of snuff dippers can a c t u a l l y lead to n i t r o s a t i o n . The formation of N-nitrosoproline (NPRO) was used as an assay. The experiment was c a r r i e d out on one volunteer holding 1 g of snuff i n the lower g i n g i v a l groove. S a l i v a was c o l l e c t e d over a 20-min period. The formation of NPRO was q u a n t i f i e d by mixing, i n v i t r o , the s a l i v a of the snuff user with p r o l i n e (200 mg), and incubating the mixture f o r 30 min at 37°C. Considerable amounts of NPRO were formed during the incubation period at pH 2.5 (Table X). The use of Copenhagen, Skoal or Big Horn brands of snuff, r e s u l t e d i n s a l i v a samples that showed comparable l e v e l s of NPRO formation i n t h i s i n v i t r o test system. 58 TABLE X In Vitro Nitrosation Capacity of Saliva in a User of Three Different Brands of Smokeless Tobacco (Snuff) NPRO (ng/ml) No Incubation Mixture pH Tobacco Snuff Ca Snuff SLa Snuff Ba Saliva 7 .5 <1 771 647 283 Saliva + 200 mg proline 7 .5 <1 672 821 149 Saliva 2 .5 32 791 506 334 Saliva + 200 mg proline 2 .5 60 14,476 11,797 13,496 C, Copenhagen; SL, Skoal; B, Big Horn 59 5. N-Nitrosoproline in the Urine of Snuff Dippers The increased intake of n i t r i t e from snuff could lead to an elevated l e v e l of n i t r o s a t i o n i n snuff dippers. This issue was analyzed by estimating levels of NPRO i n the urine of snuff dippers and of i n d i v i d u a l s who do not use smokeless tobacco. The r e s u l t s are shown i n Table XI. The NPRO i n the urine of Inuit snuff users was f i v e times greater than f o r non-users, and for the Indian snuff users the NPRO l e v e l was 2.9 times greater than i n non-users. The results indicate that exposure to high l e v e l s of n i t r i t e occurs i n users of smokeless tobacco and that the n i t r i t e can react to form N-nitroso compounds endogenously. 6 . Tobacco-Specific Nitrosamines in Different Brands of Tobacco The amounts of TSNA found i n samples of smokeless tobacco purchased i n the Northwest T e r r i t o r i e s are shown i n Table XII. Compared to smokeless tobaccos from other countries, the l e v e l s of TSNA measured i n the two moist brands of Canadian snuff are by f a r the highest observed. This r e s u l t could be due to continuous n i t r o s a t i o n during the extended storage of the snuff samples i n the small shops of the northern communities. TABLE XI NPRO i n the U r i n e o f Two S n u f f D i p p i n g P o p u l a t i o n s ( I n u i t and Indian) H a b i t Number Mean Number o f P o p u l a t i o n Examined D i p s per Day Mean Length o f Dip (min) S n u f f (gm/Chew) Mean ng/ml S n u f f u s e r s I n u i t 14 G.4 ± 3.4 28.6 ± 14.8 1.010.7 7.5 ± 4.9 S n u f f u s e r s I n d i a n 47 7.2 ± 5.2 19.5 ± 16.7 1.4 + 0.8 4.3 ± 4.7 (4.3) Non-u s e r s I n u i t 15 1.5 ± 2.2 (1.4) Non-u s e r s I n d i a n 27 1.5 ± 1.3 (1.5) For the c a l c u l a t i o n o f means, two v a l u e s f o r "not d e t e c t a b l e " were assumed: 0.2 ng/ml, maximum n o n - d e t e c t a b l e v a l u e ; 0.0 ng/ml, lowest p o s s i b l e v a l u e ( f i g u r e s i n parentheses) I n u i t s n u f f u s e r s v s . I n u i t n o n - u s e r s : P<0.001 I n d i a n s n u f f u s e r s v s . I n d i a n non-users: P<0.001 T o t a l s n u f f u s e r s v s . t o t a l n o n - u s e r s : P<0.001 Non-parametric by Mann-Whitney U t e s t : I n u i t s n u f f u s e r s v s . I n u i t n o n - u s e r s : P=0.0001 I n d i a n s n u f f u s e r s v s . I n d i a n non-users: P=0.001 T o t a l s n u f f u s e r s v s . t o t a l n o n - u s e r s : P=0.00001 TABLE X I I TSNA i n V a r i o u s Samples o f Canadian and F o r e i g n Tobacco Brands TSNA ( p p b )a O r i g i n Tobacco P r o d u c t NNN NAT NAB NNK Canada P l u g 2090 1580 100 240 Canada S n u f f 79,000 152,000 4000 5800 Canada S n u f f 50,400 170,000 4800 3200 USAb Chewing Tobacco 3500-8200 500-7000 - 100-3000 b I n d i a Chewing Tobacco 2400 - - -b USA S n u f f 800-89,000 200-4000 - 200-8300 J b Sweden S n u f f 2000-6700 900-2400 - 600-1500 . b B a v a r i a S n u f f 6000-6800 3900-4400 - 1500-1600 , b Denmark S n u f f 4500-8000 2600-6200 _ 1400-7000 NNN, N ' - n i t r o s o n o r m c o t i n e ; NAT, N - n i t r o s o a n a t a b i n e ; NAB, N - n i t r o s o a n a b a s i n e ; NNK, 4 - ( N - n i t r o s o m e t h y l a m i n o ) - 1 - ( 3 - p y r i d y l ) - l -butanone From Hoffmann and Adams (1981) 62 7. Tobacco-Specific Nitrosamines in the Saliva ol Snuff Dippers 7.1 Appearance of TSNA in the Saliva S a l i v a was c o l l e c t e d from two i n d i v i d u a l s ( I n u i t G21 and G60) p r i o r to, during and a f t e r snuff dipping. The r e s u l t s on subject G60 show a steady increase i n NNN and NAT+NAB with the length of time that the snuff was kept i n the g i n g i v a l groove (Table X I I I ) . I t i s i n t e r e s t i n g to note that the s a l i v a of the two dippers contained measurable amounts of NNN p r i o r to taking snuff, although they had r insed t h e i r mouth, and that the NNN and NAT+NAB do not disappear completely within 5 min a f t e r removal of the tobacco from the ora l c a v i t y . These r e s u l t s i n d i c a t e that a snuff dipper i s exposed to TSNA for a longer period than the actual use of tobacco i t s e l f . TABLE XIII TSNA i n the S a l i v a o f Two Individuals P r i o r to, During and After Snuff Dipping Code Time of No. Sampling Nicotine (ppm) Cotinine (ppm) TSNA (ppb) NNN NAT+NAB NNK DMNM (%) G21 P r i o r to dip 14.7 15 min into dip 228.0 After dip 19.3 G60 Prior to dip 2.3 5 min into dip 44.8 10 min into dip 132.0 15 min into dip 164.0 After dip 30.4 0.21 1.50 0.30 0.55 0.69 1.66 1.89 0.76 64.9 ND ND 0.004 959.0 1160.0 46.5 0.118 67.2 30.6 216.0 499.0 650.0 129.0 33.4 ND ND 357.0 624.0 943.0 130.0 ND ND 0.028 0.006 0.010 17.4 0.012 ND 0.010 ND 0.025 ND, not detectable Controls (non-chewers, non-smokers) had ND l e v e l s of TSNA in t h e i r s a l i v a (n=60) 64 7 .2 Variations in Salivary TSNA of Snuff Dippers To l e a r n about the extent of v a r i a t i o n of amounts of TSNA i n the s a l i v a of snuff users, 20 dippers were asked to take t h e i r regular amount of snuff, which v a r i e d between approximately 0.5 and 1.5 g per dip, and keep i t i n the mouth i n t h e i r usual manner f o r 15 min. S a l i v a samples were then c o l l e c t e d , and the l e v e l s of n i c o t i n e , cotinine and TSNA were determined (Table XIV). The r e s u l t s show considerable v a r i a t i o n s i n the l e v e l s of TSNA despite the fact that the chewing times were kept constant. The t h r e e f o l d difference between the amounts of snuff used can hardly explain the approximately 22-fold diffe r e n c e i n the amount of NNN or the 37-fold diff e r e n c e f o r NAT+NAB found between the two extreme cases (G4 and G9). In addition, the s a l i v a was analyzed for nitroso-cis-2,6-dimethylmorpholine (DMNM)( Table XIV), i n order to monitor for unintended n i t r o s a t i o n during handling and work-up. In a l l examined cases, the amount of DMNM was even lower than the us u a l l y acceptable amounts. The r e s u l t s show that exposure to known carcinogenic agents (TSNA) present i n smokeless tobaccos occurs at r e l a t i v e l y high l e v e l s . The exposure of the o r a l mucosa to the carcinogenic and mutagenic agents contained i n these tobaccos may r e s u l t i n the increased l e v e l s of micronucleated c e l l s seen i n the o r a l mucosa of snuff dippers. TABLE XIV TSNA in the S a l i v a of Snuff Dipping Inuit (Gjoa Haven) TSNA (ppb) Code Nicotine Cotinine DMNM No. (ppm) (ppm) NNN NAT+NAB NNK (%) G4 512 2.90 2610 4560 201.0 0.013 G8 566 2.08 1730 2100 116.0 0.026 G9 320 0.84 115 123 NDa 0.008 G13 808 3.94 2570 4030 150.0 0.033 G20 123 1.61 491 731 2.4 0.016 G21 171 1.19 610 840 ND ND G23xb 802 3.00 2200 3070 159.0 0.028 G23y b 202 1.16 683 . 1130 25.7 0.123 G24 695 2.33 295 253 29.0 0.014 G26 255 1.02 363 617 ND 0.021 G31 869 3.11 607 670 35.2 0.025 G 3 ? b 350 1.64 443 569 19.9 0.014 G42x 170 1.83 407 437 ND 0.010 G42y b 330 2.75 855 795 20.4 0.019 G46 482 2.14 2150 2570 151.0 0.056 G47 1300 3.55 357 377 24.5 0.032 G48 1150 3.66 2260 2890 166.0 0.058 G49 223 1.19 515 697 31.3 0.010 G50 239 1.28 423 618 20.5 ND G60 142 1.27 272 418 ND 0.020 G61 70 0.54 407 527 14.9 0.027 JER 450 2.04 1210 896 59.5 0.017 aND, not detectable b x and y, s a l i v a samples taken at two d i f f e r e n t times during the same day from the same snuff dipper Controls (non-chewers, non-smokers) had ND l e v e l s of TSNA in the i r s a l i v a (n=60) 66 8 . Frequency of Micronuclei in the Oral Mucosa of Snuff Dippers and Tobacco Chewers An increased frequency of micronucleated c e l l s (MNC) i n the o r a l mucosa has been observed i n i n d i v i d u a l s at elevated r i s k f o r o r a l cancer, including smokers and alcohol drinkers, Khaini tobacco users from India, and b e t e l quid chewers of Orissa and Meghalaya, India (reviewed i n Table I I ) . In t h i s study, we compared the MNC frequency i n the o r a l mucosa of snuff dipping Inuit (Gjoa Haven) with that of snuff dipping Indians (La Loche) and East Indian tobacco chewers (Kerala). The e x f o l i a t e d c e l l s were c o l l e c t e d from areas of the mucosa at which the tobacco had been kept. The r e s u l t s shown i n Table II indicate an elevated frequency of MNC i n the group of tobacco chewers and snuff dippers. 9. Precancerous Lesions and Cancer of the Oral Cavity in Users of Smokeless Tobacco Two types of anomalies of the o r a l mucosa were recorded: (a) whitish to yellowish wrinkled patches with or without furrows, corresponding to categories 2 and 3 i n the c l a s s i f i c a t i o n of Greer and Poulson (1983), and (b) d e f i n i t e leukoplakias, as described by the WHO Collaborating Centre f or Oral Precancerous Lesions ( 1 9 7 8 ) ( T a b l e l l l ) . 67 9.1 P r e c a n c e r o u s L e s i o n s Of the 50 smokeless tobacco users examined i n Gjoa Haven, 26% showed whitish to yellowish wrinkled patches (Table XV). Approximately one-third also had one or more furrows i n the whitish areas of the mucosa. The lesions were located i n the lower g i n g i v a l groove and at the front of the mouth, where the tobacco i s kept. No anomalies were detected i n regions of the o r a l c a v i t y not d i r e c t l y exposed to tobacco. The occurence of the whitish to yellowish patches (leukoderma) i n the o r a l c a v i t y of snuff dipping Indians (La Loche) was higher (Table XV) than among the Inuit. In t h i s connection, i t may be of i n t e r e s t to note that the Indian snuff dippers are r e l a t i v e l y heavy drinkers of a l c o h o l i c beverages. No d e f i n i t e leukoplakias, which are believed to be precancerous les i o n s , were noted among the examined snuff dippers. However, i t should be kept i n mind that a high percentage of the subjects used i n the study were teenagers who had dipped snuff f o r too short a time to have already developed precancerous o r a l l e s i o n s . Leukoplakias, which represent precancerous le s i o n s , were observed i n a r e l a t i v e l y high frequency i n the b e t e l quid chewers examined i n Kerala (Table XV). Leukoplakias were diagnosed by myself and confirmed by two experts, B. Mathew, M.D., and R.Sankaranarayanan, M.D. A l l leukoplakias were photographed and the records stored at the B.C. Cancer Research Centre, Vancouver. 68 TABLE XV P r e v a l e n c e o f O r a l L e s i o n s Among S n u f f D i p p e r s and Tobacco Chewers Number o f I n d i v i d u a l s I n d i v i d u a l s Tobacco w i t h W h i t i s h with O r a l P o p u l a t i o n L o c a t i o n Sex Users Patches L e u k o p l a k i a I % I n u i t G joa Haven M 50 26.0 0 I n d i a n s La Loche M 49 46.9 0 I n d i a n s La Loche F 23 52.2 0 Ea s t I n d i a n s K e r a l a M 325 0 59.7 69 9.2 Oral Cancer This study focused on three r e s t r i c t e d population groups with d i f f e r e n t patterns of smokeless tobacco use. No attempt was made to evaluate the b i o l o g i c a l e f f e c t s of snuff dipping or tobacco chewing on a larger population group. Information on the incidence of o r a l cancer among snuff dippers and tobacco chewers can, however, be found i n several epidemiological studies of population groups comparable to those examined i n t h i s t h e s i s . The r e l a t i v e r i s k of o r a l cancer increased with the number of tobacco-containing b e t e l quids chewed per day i n India (Table XVI). The study by Orr (1933) was c a r r i e d out i n Kerala, the l o c a t i o n of our research project. The r i s k of developing carcinomas of the o r a l c a v i t y and pharynx faced by snuff dippers i n the southern United States i s also considerably increased (Table XVII). Reports of the incidence of o r a l cancer among Canadian Inuit and Indians d i f f e r from those reported for East Indian chewers and for snuff dippers i n the United States. No o r a l cancers were reported i n the I n u i t over a 20-year period (Schaefer et a l . , 1975). The incidence of o r a l cancer i n snuff dipping Indians i s comparable to that found i n the Canadian population not engaging i n t h i s h a b it ( N u t r i t i o n Canada, 1975b). TABLE XVI E f f e c t o f the D a i l y Number o f B e t e l Q u i d Chews on the R e l a t i v e R i s k f o r O r a l Cancer i n I n d i a (Hirayama, 1966) and K e r a l a ( O r r , 1933) R e l a t i v e R i s k f o r O r a l Cancer No. B e t e l Quids per Day I n d i a K e r a l a 0 1.0 1.0 <2 8.5 8.1 3-5 14.5 12.9 >6 18.1 12.8 R e t a i n i n g q u i d 63.7 62.9 d u r i n g s l e e p TABLE XVII E f f e c t o f S n u f f D i p p i n g on the R e l a t i v e R i s k f o r O r a l Cancer i n the Southern U n i t e d S t a t e s (from Winn e t a l . , 1981) Years o f R e l a t i v e S i t e o f Cancer S n u f f Use Ri s k Gum and b u c c a l mucosa 0 1.0 1-24 13.8 25-49 12.6 >50 47.5 Other mouth and pharynx 0 1.0 1-24 1.7 25-49 3.8 >50 1.3 72 10. Intervention Strategies I n h i b i t i o n of carcinogenesis can be obtained by using a n t i - i n i t i a t i n g , anti-promoting, or a n t i - p r o l i f e r a t i n g agents. A better understanding of the precursors of carcinogens, procarcinogens, carcinogens and promoters involved i n neoplastic transformation should help i n the s e l e c t i o n of chemopreventive agents. Our r e s u l t s and studies from other l a b o r a t o r i e s point to the N-nitrosamines as the main carcinogenic agents released from smokeless tobacco. They may induce genetic anomalies i n the o r a l mucosa, and they may i n i t i a t e the development of o r a l precancerous lesions and o r a l carcinomas (IARC, 1985). Based on these assumptions, the e f f i c a c y of four chemopreventive agents was examined: ascorbic a c i d and c a f f e i c a c i d as n i t r i t e trappers, beta-carotene as a free r a d i c a l scavenger, and vitamin A as an agent with scavenging p o t e n t i a l and the capacity to regulate gene function i n e p i t h e l i a l c e l l s . 73 10.1 Nitrite Trapping Agents R e l a t i v e l y large amounts of NPRO appeared i n the urine of a volunteer who dipped snuff and ingested p r o l i n e simultaneously (Figure 7 and 8; Table XVIII). The excreted NPRO could be formed endogenously, or could be preformed and released from the tobacco. The snuff brand used had an NPRO content of 14.0 /ig/g tobacco (wet weight). To gain information on the amount of NPRO formed endogenously, the experiment was repeated with the a d d i t i o n a l ingestion of ascorbic acid. The experimental design i s shown i n Figure 5. The NPRO released i n the urine of a snuff user over an 11 hr period was 26.1 ng when no ascorbate was used, and t h i s was reduced to 7.8 ng when ascorbate was present at a t o t a l dose of 1.5 g (Figure 8). With a reduced ingestion of ascorbate (2 x 500 mg), the amount of NPRO excreted i n the urine was reduced from 27.3 n& i n 11 hrs to 14.9 ng (Figure 9). The e f f i c a c y of ascorbate and c a f f e i c a c i d i n reducing the endogenous formation of NPRO i s shown i n Table XVIII. 74 Figure 7: The Appearence of NPRO i n the Urine of a Volunteer Who Dipped Snuff and Ingested P r o l i n e and Ascorbate (for d e t a i l e d o u t l i n e , see Figure 5C ) . 3-6n 3-2-2-8-1 I 2-4H (J - 2-CH • rx n Z 1-6H 1-2-0-8-I WITHOUT A S C O R B A T E WITH ASCORBATE 0 -4 -T" 0 T" 2 4 6 -r 8 10 T I M E ( H R S ) Figure 8: The Appearence of NPRO i n the Urine of a Volunteer Who Dipped Snuff and Ingested Proline and Ascorbate (for d e t a i l e d o u t l i n e , see Figure 5 B ) . -r 2 "~1 WITHOUT ASCORBATE WITH A S C O R B A T E T" 6 T" 8 10 T I M E C HPS) 76 TABLE XVIII Inhibitory Effect of Ascorbate and Caffeic Acid on the Formation of NPRO in a Snuff Dipping Volunteer Incubation Mixture PH No Tobacco NPRO (ng/ml) Snuff Ca Snuff SLa Snuff Ba Saliva + 200 mg proline 2.5 60 14 ,476 11,797 13,496 Saliva + 200 mg proline + 200 mg ascorbate 2.5 5,497 3,237 442 Saliva + 200 mg proline + 200 mg caffeic acid 2.5 910 667 145 C, Copenhagen; SL, Skoal; B, Big Horn 77 10.2 Inuit Snuff Dippers :Beta-carotene Chemopreventive t r i a l s on tobacco-areca nut chewers i n the P h i l i p p i n e s have shown the capacity of beta-carotene to reduce the frequency of micronucleated c e l l s which were used as i n d i c a t o r s of genetic damage i n the buccal mucosa ( S t i c h et a l . , 1984a,b). A vitamin A d e f i c i e n c y p r e v a i l e d i n the population group of t h i s i n tervention t r i a l , as was evident from a r e l a t i v e l y high incidence of night blindness and xerophthalmia. The question was r a i s e d whether Inui t snuff dippers of Gjoa Haven, who have "normal" serum l e v e l s of r e t i n o l , would respond i n the same way to the o r a l administration of beta-carotene . Serum samples from 110 male Inui t were analyzed for r e t i n o l and beta-carotene. Subjects were divided into those who used smokeless tobacco (40' i n d i v i d u a l s ) and those who d i d not use tobacco (70 i n d i v i d u a l s ) . No s i g n i f i c a n t difference i n serum r e t i n o l or beta-carotene was observed between the two groups (Table XIX). The r e t i n o l l e v e l s of the Inu i t males examined were comparable to those found for Canadian males consuming a "normal" Western-type d i e t ( N u t r i t i o n Canada, 1975a). The observed beta-carotene l e v e l s f a l l s u b s t a n t i a l l y below those observed i n a Canadian population. The low l e v e l s of serum beta-carotene may r e s u l t from an i n s u f f i c i e n t intake of green-yellow vegetables by the Inu i t of Gjoa Haven. Serum r e t i n o l l e v e l s are i n the "normal" range due to the r e l a t i v e l y large intake of seal and caribou meat and l i v e r . TABLE XIX Serum L e v e l s o f R e t i n o l and B e t a - c a r o t e n e i n Male I n u i t P r i o r t o I n i t i a t i o n o f the P r e v e n t i o n T r i a l Number o f R e t i n o l B e t a - c a r o t e n e H a b i t S u b j e c t s (ng/ml) (ng/ml) Non-users 70 447 ± 104 57.4 ± 36.2 Tobacco u s e r s 40 463 ± 127 47.0 ± 27.6 Standard d e v i a t i o n s ( ± ) a r e g i v e n f o r number of s u b j e c t s shown P v a l u e s c a l c u l a t e d by S t u d e n t ' s t - t e s t f o r r e t i n o l and b e t a -c a r o t e n e (P>0.40) 79 Since the d i s t r i b u t i o n pattern of MNC within the o r a l c a v i t y of snuff dippers i s unequal (St i c h , 1987), i t i s paramount f o r any comparative study of MNC frequencies to sample c e l l s from p r e c i s e l y the same locations within the mouth. In t h i s study, e x f o l i a t e d c e l l s were c o l l e c t e d from the area of the mucosa at the s i t e where the tobacco was kept. The r e s u l t s of a 10-week administration of beta-carotene are shown i n Figure 10, where the frequency of MNC before treatment i s p l o t t e d against that following administration of the chemopreventive agent. The r e s u l t s indicate an i n h i b i t o r y e f f e c t of beta-carotene treatment. The average frequency of MNC p r i o r to treatment with beta-carotene was 1.87% ± 0.92% (n = 24), and decreased s i g n i f i c a n t l y (P < 0.001) a f t e r treatment to 0.74% ± 0.42%. No s i g n i f i c a n t reduction i n the frequency of MNC occurred i n i n d i v i d u a l s who received no treatment or who were given a placebo (Figure 10). The average frequencies of MNC p r i o r to and a f t e r the 10-week period were 2.0% ± 0.94 (n = 31) and 1.99% ± 0.80, re s p e c t i v e l y . MNC frequencies were determined i n the o r a l mucosa of 5 snuff users 8 months and 1 month p r i o r to, at the onset of, and a f t e r treatment with beta-carotene (Figure 11). The r e s u l t s indicate that the frequencies of MNC i n in d i v i d u a l s who use snuff d a i l y are r e l a t i v e l y stable. A f t e r a 10-week administration of beta-carotene, the frequency of MNC decreased i n the mucosa of the snuff dippers. A l l the snuff users continued t h e i r usual habit throughout the course of the study. Besides the micronuclei, some e x f o l i a t e d c e l l s from the o r a l mucosa of snuff users show a loss of n u c l e i . The frequency of anucleated c e l l s before treatment was compared to that seen a f t e r treatment with beta-carotene. As can be seen from Figure 12, no s i g n i f i c a n t changes occurred. 80 The r e s u l t s show that beta-carotene appears to be an e f f i c i e n t i n h i b i t o r of micronuclei, which indicate genetic damage i n the o r a l mucosa of snuff users who do not s u f f e r from a vitamin A deficiency. 81 Figure 10: E f f e c t of Beta-carotene Treatment on MNC i n the Oral Mucosa. (A) Frequency of MNC at the o r a l s i t e where the tobacco i s located: before and a f t e r a 10-week ingestion of placebo capsules: O, before and a f t e r the 10-week t r i a l period (no treatment). (B) Changes i n the frequency of MNC before and a f t e r a 10-week administration of beta-carotene (180 mg/week). % B E F O R E T « E A T M 6 I S J T MtCnOMUCLEATeO CELLS 82 Figure 11: Changes i n the Frequency of MNC at the Oral Site Where the Tobacco i s Located Over a Period Preceding the P i l o t T r i a l and A f t e r a 10-Week Administration of Beta-carotene (5 d i f f e r e n t Inuit males using snuff) — i i i l i i i i i i i 5 FEB MAY AUG NOV M O N T H 83 Figure 12: Frequency of Anucleated E x f o l i a t e d C e l l s from the Oral Site Where the Tobacco i s Located Before and Aft e r a 10-Week Administration of Beta-carotene (180 mg/week). 84 10.3 East Indian Tobacco Chewers: Beta-carotene and Beta-carotene plus Vitamin A Because of the close a s s o c i a t i o n of MNC frequencies, precancerous o r a l l e s i o n s and o r a l cancer with the habit of chewing tobacco-containing b e t e l quids, p i l o t i n t e r v e n t i o n t r i a l s were attempted on a group of East Indian fishermen. The objective was to investigate the pro t e c t i v e e f f e c t of beta-carotene and beta-carotene plus vitamin A on the l e v e l s of micronucleated o r a l mucosal c e l l s and on the incidence of o r a l leukoplakias found i n t h i s group. The frequency of MNC was elevated i n areas of o r a l leukoplakias and i n normal-appearing mucosa of b e t e l quid chewers who p a r t i c i p a t e d i n the int e r v e n t i o n t r i a l (Table XX). There were no s i g n i f i c a n t differences i n MNC frequencies between samples of e x f o l i a t e d c e l l s taken from normal-appearing areas of the buccal mucosa and those c o l l e c t e d from regions with well-established leukoplakias. Swabs of normal mucosa were taken adjacent to areas with leukoplakia. This close sampling pattern was necessary since the frequency of micronucleated c e l l s can d i f f e r at various locations of the o r a l mucosa. I t has been shown that the frequency of MNC i s highest i n areas where the b e t e l quid or tobacco plug comes i n close contact with the mucosa (S t i c h et a l . , 1982a,b; S t i c h and Rosin, 1985). In the placebo group, the frequency of MNC d i d not change s i g n i f i c a n t l y over the 3-month study period (Figure 13; Table XX). Following the twice-weekly administration of beta-carotene and beta-carotene plus vitamin A, the frequency of MNC was found to be s i g n i f i c a n t l y reduced i n areas with leukoplakia and areas of normal-appearing mucosa (Table XX). The degree of MNC reduction i n leukoplakias and normal-appearing mucosa was comparable. The frequencies of micronuclei i n each i n d i v i d u a l before and a f t e r beta-carotene or placebo administration were paired and compared i n a paired-sample Student's t - t e s t . The reduction i n MNC following the administration of beta-carotene and beta-85 carotene plus vitamin A i n both leukoplakias and normal-appearing mucosa i s s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.001). The e f f e c t of the beta-carotene treatment on the frequency of MNC i n areas of leukoplakia of each t r i a l p a r t i c i p a n t i s shown i n Figure 13. The MNC frequency of at l e a s t one tobacco/betel quid chewer did not respond to the 3-month administration of beta-carotene. Comparable r e s u l t s were obtained when beta-carotene and vitamin A were administered f o r 3 months (Table XX; Figure 13). Figure 13: Response of MNC i n Areas of Leukoplakia to a 3-Month Administration of Beta-carotene and Beta-carotene Plus Vitamin A vs. Placebo 87 TABLE XX Frequency (%) o f M i c r o n u c l e a t e d C e l l s i n O r a l L e u k o p l a k i a s and i n Normal Mucosa o f T o b a c c o / B e t e l Quid Chewers B e f o r e and A f t e r the A d m i n i s t r a t i o n o f Chemopreventive Agents f o r 3 Months L e u k o p l a k i a Treatment Number o f I n d i v i d u a l s B e f o r e A f t e r Normal Mucosa B e f o r e A f t e r P lacebo 30 B e t a - c a r o t e n e 31 B e t a - c a r o t e n e + 51 v i t a m i n A 3.60±1.22 4.00±1.32 4.09±1.01 1.18±0.77 4.01±1.05 1.16±0.94 4.10±1.54 3.83±1.23 4.1111.48 1.01±0.71 4.18±0.78 1.2210.88 S i g n i f i c a n t a t P<0.01 w i t h S t u d e n t ' s t - t e s t : B e t a - c a r o t e n e t r e a t m e n t : m i c r o n u c l e u s f r e q u e n c i e s b e f o r e v s . a f t e r s u p p l e m e n t a t i o n f o r a r e a s w i t h l e u k o p l a k i a , and f r e q u e n c i e s b e f o r e v s . a f t e r s u p p l e m e n t a t i o n f o r a r e a s o f normal mucosa; b e t a - c a r o t e n e + v i t a m i n A t r e a t m e n t : b e f o r e v s . a f t e r s u p p l e m e n t a t i o n , v a l u e s f o r both l e u k o p l a k i a s and normal mucosa 88 Approximately 48% of a l l examined adult chewers who inhabit the coast of Kerala, near Trivandrum, South India, had w e l l - e s t a b l i s h e d leukoplakias, as defined by the WHO Collaborating Centre f o r Oral Precancerous Lesions (1978). The i n t r a o r a l d i s t r i b u t i o n of leukoplakias among 160 chewers i n Kerala was as follows: buccal mucosa, 53.4%; commissure, 23.3%; retromolar, 9.3%; tongue, 8.4%; l i p , 4.4%; others, 1.4%. The frequency d i s t r i b u t i o n of 160 leukoplakias according to t h e i r appearance was : homogeneous leukoplakia, 83%; speckled leukoplakia, 2.9%; and verrucous leukoplakia, 5.7%. Only one leukoplakia contained erythematous areas. Since biopsies were not taken, the leukoplakias cannot be associated with a p a r t i c u l a r h i s t o p a t h o l o g i c a l pattern. The l o c a t i o n , s i z e and appearance of each o r a l leukoplakia were recorded by marking them on a chart and by photographs. The recording of leukoplakias was done p r i o r to the onset of the t r i a l , and thereafter each month during the e n t i r e treatment period. As described previously, o r a l leukoplakias of betel quid chewers have a tendency to change i n si z e and l o c a t i o n with time (Gupta et a l . , 1980, 1986). At present, i t i s d i f f i c u l t to decide whether these "spontaneous" regressions are due to changes i n the holding of the b e t e l quid at d i f f e r e n t s i t e s within the o r a l cavity, or to changes i n the ingestion of chemopreventive agents caused by seasonal dietary v a r i a t i o n s . The remission of o r a l leukoplakias and the development of new leukoplakias also occurred among the t r i a l p a r t i c i p a n t s who received placebo capsules (Tables XXI and XXII). A f t e r three months of beta-carotene and beta-carotene plus vitamin A administration, the percentage of leukoplakias that regressed was comparable to that found i n the placebo group, whereas the development of new leukoplakias was already s i g n i f i c a n t l y reduced. A f t e r a 6-month treatment period, a s i g n i f i c a n t difference between the two treated groups (remission and new leukoplakias) and the placebo group became evident (Table XXII). 89 TABLE XXI Response of Oral Leukoplakias to the Administration of Beta-carotene or Beta-carotene plus Vitamin A for 3 Months Individuals with Number of Remission No Change New Leukoplakia Individuals with Treatment Oral Leukoplakia Number (%) Number (%) Number (%) Placebo 33 1 3.0 25 75.8 7 21.2 Beta-carotene 35 3 8.6 25 71.4 7 20.0 (180 mg/week) Beta-carotene 51 4 7.8 46 90.2 1 2.0 (180 mg/week) + vitamin A (100,000 IU/week) Test for significant difference in outcome between treatment groups after 3 months of supplementation: chi-square = 10.03; df = 4; P = 0.04 90 TABLE XXII Response o f O r a l L e u k o p l a k i a s t o the A d m i n i s t r a t i o n o f B e t a - c a r o t e n e o r B e t a - c a r o t e n e p l u s V i t a m i n A f o r 6 Months I n d i v i d u a l s w i t h Number o f Re m i s s i o n No Change New L e u k o p l a k i a I n d i v i d u a l s w i t h . Treatment O r a l L e u k o p l a k i a Number (%) Number (%) Number (%) Placebo 33 1 3.0 25 75.8 7 21.2 B e t a - c a r o t e n e 27 4 14.8 19 70.4 4 14.8 (180 mg/week) B e t a - c a r o t e n e 51 14 27.5 33 64.7 4 7.8 (180 mg/week) + v i t a m i n A (100,000 IU/week) T e s t f o r s i g n i f i c a n t d i f f e r e n c e i n outcome between treatment groups a f t e r 6 months o f s u p p l e m e n t a t i o n : c h i - s q u a r e =10.1; d f = 4; P = 0.38 P v a l u e s (2-sided) by F i s h e r ' s e x a c t t e s t : P l a c e b o v s . b e t a - c a r o t e n e : r e m i s s i o n , P=0.16; new l e u k o p l a k i a s , P=0.53. Placebo v s . b e t a - c a r o t e n e + v i t a m i n A: r e m i s s i o n , P=0.004; new l e u k o p l a k i a s , P=0.08. B e t a - c a r o t e n e v s . b e t a - c a r o t e n e + v i t a m i n A: r e m i s s i o n , P=0.26; new l e u k o p l a k i a s , P=0.44 91 P r i o r to, once during, and at the end of the t r i a l , we examined the number of tobacco-containing b e t e l quids chewed per day, the length of the chewing period, and the alcohol consumption and smoking pattern of a l l p a r t i c i p a n t s . These o r a l habits d i d not change during the course of the t r i a l . I t can therefore be assumed that a l l the p a r t i c i p a n t s were exposed to approximately the same types and l e v e l s of carcinogenic and mutagenic agents released from tobacco and other b e t e l quid ingredients. 10.4 East Indian Tobacco Chewers: Vitamin A The capacity of vitamin A to reduce the frequency of micronucleated buccal mucosal c e l l s of tobacco chewers has been previously reported (Stich and Rosin, 1984a). The objective of t h i s study was to determine the e f f e c t s of vitamin A on the remission of o r a l leukoplakias. This short-term p i l o t t r i a l was c a r r i e d out on fishermen l i v i n g along the coast near Trivandrum, Kerala. Questionnaires on chewing, smoking and drinking patterns were completed at the onset of the t r i a l , a f t e r three months, and at the end of the six-month treatment period. The r e s u l t s permitted us to draw the conclusion that the various o r a l habits did not change throughout the course of the study. The p a r t i c i p a n t s i n this study chewed an average of 13.1 (SD =9.1) tobacco-containing quids per day. Each quid was kept i n the mouth for 26.1 (SD = 25.4) min. Exposure to tobacco-and areca nut-related carcinogens amounted to approximately s i x hours each day. To ensure that a l l the t r i a l p a r t i c i p a n t s received the desired doses, the placebo and vitamin A capsules were administered twice weekly under the s t r i c t supervision of a l o c a l nurse and a s t a f f member of the Regional Cancer Centre i n Trivandrum or myself, who checked that the capsules were a c t u a l l y swallowed. Due to t h i s approach, an excellent compliance with the vitamin administration regime was achieved. Only 4 of 39 vitamin A or placebo doses ( s i x months) were 92 missed among 6% of the p a r t i c i p a n t s , and 2 of.39 doses were missed by 14% of a l l t r i a l p a r t i c i p a n t s . Despite the continuous exposure to carcinogens, the administration of 200,000 IU of vitamin A, re s u l t e d i n a pronounced remission of o r a l leukoplakias (Table XXIII). This amount of vitamin A d i d not produce any detectable adverse e f f e c t s during the t r i a l period, such as dryness of the l i p s and mouth, changes i n the skin ( s c a l i n g of palms), headaches or dizziness. New o r a l leukoplakias developed throughout the t r i a l period among chewers of tobacco-containing b e t e l quids. I t was therefore possible to examine the e f f e c t of vitamin A treatment on the formation of new leukoplakias. In the group r e c e i v i n g the placebo, 7 (21%) new leukoplakias were formed, whereas no new leukoplakias developed i n the group r e c e i v i n g vitamin A over the six-month t r i a l period (Table XXIII). 9,3 TABLE X X I I I Response o f O r a l L e u k o p l a k i a s t o the A d m i n i s t r a t i o n o f V i t a m i n A f o r 6 Months I n d i v i d u a l s w i t h Number o f R e m i s s i o n No Change New L e u k o p l a k i a I n d i v i d u a l s w i t h Treatment O r a l L e u k o p l a k i a Number (%) Number (%) Number (%) Placebo 33 1 3.0 25 75.8 7 21.2 V i t a m i n A 21 12 57.1 9 42.9 0 0.0 • (200,000 IU/ week) T e s t f o r s i g n i f i c a n t d i f f e r e n c e i n outcome between treat m e n t groups a f t e r 6 months o f s u p p l e m e n t a t i o n : c h i - s q u a r e =22.27; d f = 3; P = 0.000015 P v a l u e s (2-sided) by F i s h e r ' s e x a c t t e s t : P l a c e b o v s . v i t a m i n A: r e m i s s i o n , P=0.0000089. P l a c e b o v s . v i t a m i n A: new l e u k o p l a k i a , P=0.024 94 D I S C U S S I O N The objectives of t h i s research were to compare the usage of smokeless tobacco among three communities (Inuit, Canadian Indian and East Indian), to analyze smokeless tobacco samples, and s a l i v a and urine of chewers for various e t i o l o g i c a l f a c t o r s , and to investigate possible chemopreventive agents which may reduce the o r a l l e s i o n s caused by the use of snuff or chewing tobacco. 1. U s e of S m o k e l e s s T o b a c c o Although a s t r i c t random sampling of each population was not c a r r i e d out the data accumulated do ind i c a t e a large prevalence of smokeless tobacco use by each group: 86% of the male Canadian Indians examined sa i d they used snuff d a i l y , as d i d 50% of the females. In the Inu i t population, 70% of the males responded that they used snuff d a i l y , as d i d 4.2% of females, although t h i s habit i s s o c i a l l y unacceptable for t h i s l a t t e r group. In the East Indian population where b e t e l quid chewing i s acceptable for both males and females, roughly 57% of those questioned responded that they p r a c t i s e d t h i s habit. In t h i s study we also attempted to determine the prevalence of other o r a l habits which may exert a carcinogenic e f f e c t , such as alcohol drinking and c i g a r e t t e smoking. In the Canadian Indian population, approximately 26.3% of males and 10.4% of females combine the drinking of alcohol with snuff dipping, as do 37% of the East Indian males who chew b e t e l quid. Considering the s y n e r g i s t i c a c t i o n between alcohol consumption and c i g a r e t t e smoking (Walters et a l . , 1979), the p o s s i b i l i t y that a s i m i l a r e f f e c t may occur between alcohol and snuff or chewing tobacco cannot be excluded. The combination of snuff dipping, alcohol drinking and c i g a r e t t e smoking was found to occur i n 35.1% of males and 21.5% of females among the Canadian Indians studied. The influence of 95 smoking can be seen from the observation that the highest prevalence of leukoplakia occurs among i n d i v i d u a l s who are concurrently using "nass" and smoking c i g a r e t t e s ( S t i c h and Rosin, 1984). Also of i n t e r e s t i n t h i s study was the r e l a t i v e l y consistent weight of tobacco chewed by each group: 0.61 g by the Inuit, 1.5 g by the Canadian Indians and 1.1 g by the East Indians, suggesting that other modifying e f f e c t s must be at work which influence the d i f f e r e n t o r a l cancer rates seen amongst these groups. 2. Precursors of Nitrosamines Contained in Smokeless Tobacco Samples  and the Saliva of Their Users To investigate the probable e t i o l o g i c a l factors contained i n smokeless tobacco, we focused on the N-nitroso compounds and t h e i r precursors since these are the only known carcinogenic compounds contained i n smokeless tobacco mixtures chewed throughout the world (IARC, 1985). N i t r i t e l e v e l s were examined i n a v a r i e t y of smokeless tobacco samples, incl u d i n g f i v e common brands of snuff (Canada), one common brand of chewing tobacco (U.S.), four "nass" samples ( mixtures of tobacco, slaked lime, ash and o i l placed under the tongue by inhabitants i n southern regions of the Soviet Union, Afghanistan and Pakistan), four Khaini tobacco preparations widely used i n several eastern states of India (e.g., Bihar, Orissa and Bengal), and East Indian tobacco used i n the preparation of b e t e l quids. R e l a t i v e l y high l e v e l s of n i t r i t e were found i n three popular brands of snuff commonly used by northern Canadian natives. The amounts of n i t r i t e i n the three Canadian snuff samples, which v a r i e d from 95 to 1,040 mg/kg, and i n four "nass" samples from Samarkand (Uzbekistan, Soviet Union), which v a r i e d from 70 to 200 mg/kg, greatly exceed those found i n several food products such as n i t r i t e - c u r e d bacon 96 (12-42 mg/kg), n i t r i t e - c u r e d ham (16-37 mg/kg), smoked sausages (18-31 mg/kg) (Kawabata et a l . , 1984), or Chinese cabbage (Walters et a l . , 1979). To a r r i v e at an estimation of n i t r i t e which could conceivably enter the digest i v e system of snuff or nass users, we can assume a s a l i v a flow of 16 ml per 20 min (Anderson and Krinsky, 1973) and a concentration of 0.2 mg n i t r i t e per ml of s a l i v a . Based on these assumptions, an estimated 3.2 mg of n i t r i t e w i l l enter the stomach during a 20-min period of tobacco use i f the snuff dipper swallows rather than expectorates the s a l i v a . Furthermore, assuming that the tobacco i s kept within the mouth for 3 hrs per day, approximately 29 mg of n i t r i t e would enter the stomach i n a 24-hr period. This amount i s considerably larger than the 4.2 mg of n i t r i t e ingested by an i n d i v i d u a l per day from s o l i d food products and beverages (Kawabata et a l . , 1984). Considerable concern has been expressed about the possible carcinogenic e f f e c t s of tobacco-related nitrosamines (Hoffmann and Hecht, 1985) which are formed during tobacco processing (Andersen and Kasperbauer, 1984). The p o s s i b i l i t y cannot be completely ignored that the r e l a t i v e l y large doses of n i t r i t e released into the s a l i v a of snuff dippers could pose an even greater hazard. By using l e v e l s of excreted NPRO, information about the n i t r o s a t i o n a c t i v i t y within man can be obtained (Ohshima and Bartsch, 1981; Ohshima et a l . , 1982). Using t h i s method, r e l a t i v e l y high l e v e l s of NPRO were found i n the urine of several snuff dippers, suggesting an increased endogenous n i t r o s a t i o n capacity. Thus the d a i l y use of n i t r i t e - c o n t a i n i n g snuff could lead to the endogenous n i t r o s a t i o n of a great many ingested dietary compounds. Several studies have shown the formation of strongly mutagenic, and by implication carcinogenic compounds following the n i t r o s a t i o n of meat products (Marquardt et a l . , 1977; Tomita et a l . , 1984), beverages ( L i n and Tai , 1980), vegetables (Van Der Hoeven et a l . , 1984) and spices (Namiki et a l . , 1983). The tobacco-related 97 nitrosamines (Hecht et a l . , 1984; Hoffmann and Hecht, 1985) and pos s i b l y other tobacco-derived mutagens (Whong et a l . , 1985) may represent only one group of N-nitroso compounds which could conceivably be formed i n users of n i t r i t e -containing tobacco. I t was recently pointed out that s a l i v a r y n i t r i t e l e v e l s may be only one fact o r a f f e c t i n g the endogenous formation of N-nitroso compounds (Mirvish, 1985) , and that a neglect of the inte r a c t i o n s between n i t r i t e and numerous scavenging chemicals could lead to erroneous conclusions (Forman et a l . , 1985). This argument i s undoubtedly applicable to users of n i t r i t e - c o n t a i n i n g snuff. As expected, the ad d i t i o n of ascorbate to a mixture of p r o l i n e and s a l i v a of a snuff dipper i n h i b i t e d the i n v i t r o formation of NPRO. Ascorbate taken by a volunteer both concurrently and sh o r t l y a f t e r the use of snuff and pr o l i n e reduces the excretion of NPRO. I t could be speculated that the observed differences i n urinary NPRO l e v e l s between Inuit (Northwest T e r r i t o r i e s ) and Indian (Saskatchewan) snuff users i s due to differences i n the intake of vegetables and f r u i t s , which are the major source of ascorbic a c i d and n i t r i t e -scavenging phenolics ( P i g n a t e l l i et a l . , 1982; St i c h and Rosin, 1984b; Stich et a l . , 1984c). This i s supported by the observation that the percentage of Inuit males i n the 10-54 year age range who show severe vitamin C d e f i c i e n c i e s i s s i g n i f i c a n t l y higher than that of Indians of comparable age ( N u t r i t i o n Canada, 1975b) . Whether the widespread use of smokeless tobacco among inhabitants of A r c t i c regions, combined with a d e f i c i e n t intake of n i t r i t e - t r a p p i n g compounds, makes them more prone to the development of o r a l cancer can only be ascertained by a d e t a i l e d epidemiological study. 98 3. Smokeless Tobacco and Ingestion of N-Nitrosamines R e l a t i v e l y high l e v e l s of TSNA, including NNN, NAT+NAB and NNK, were found i n the s a l i v a of I n u i t during a 15-min snuff taking period. An estimate, based on the r e s u l t s presented, reveals that the amounts of nitrosamines swallowed by a snuff dipper are by no means n e g l i g i b l e . Assuming an average l e v e l of 980 ng TSNA per 1 ml of s a l i v a of the Inuit snuff dipper, an average of 6.5 dips per day, an average of 25 min per dipping period, and a s a l i v a flow of 5.83 ml per 5 min, then the amount of NNN ingested should be i n the order of 185 ng per day. The same c a l c u l a t i o n applied to a more intensive snuff user (G13, Table XIV) shows an intake of approximately 899 ng NNN. Most of the s a l i v a produced during snuff taking i s probably swallowed, since most Inui t snuff dippers do not, or only occasionally, s p i t during a dipping session. The extreme s a l i v a t i o n and r e s u l t i n g repeated s p i t t i n g which i s so common among A s i a t i c b e t e l quid chewers does not occur i n snuff dippers. To gain a more complete p i c t u r e of the amount of TSNA ingested, approximately 249 ng of NAT+NAB and 10 ng of NNK must be added to the 185 ng of NNN, making a t o t a l of 444 ng- Thus the amount of nitrosamines ingested d a i l y by snuff dippers exceeds by far that swallowed through drinking beer (0.34 ng P e^ day), eating cured meat products (0.17 ng P e r day), or using cosmetics (0.41 ng P e r day)(Kawabata et a l . , 1984). The a n a l y t i c a l data, recognition of the f a c t that carcinogenic N-nitrosamines can be absorbed from the s a l i v a (Hoffmann and Adams, 1981) and the r e s u l t s of bioassays for c a r c i n o g e n i c i t y (Peto et a l . , 1984; Hecht et a l . , 1983) make i t possible to estimate the c o n t r i b u t i o n of N-nitrosamines to the increased r i s k of snuff dippers for o r a l cancer. Peto et a l . (1984) showed that 1 mg/kg N-nitrosodimethylamine (NDMA) or N-nitrosodiethylamine (NDEA) 99 administered i n drinking water caused l i v e r neoplasms i n 23% and 42% of rats, r e s p e c t i v e l y . These incidence rates were s i g n i f i c a n t l y higher than those i n the controls. In addition, 1 mg/kg NDEA caused eosophageal tumors i n 27% of male rats and none i n c o n t r o l animals. The t o t a l dose administered to the animals during t h e i r l i f e t i m e was about 37 mg/kg for males and 64 mg/kg for females. This study suggested the existence of a l i n e a r dose-response r e l a t i o n s h i p i n the dose range of 0.033 to 1 mg/kg. Since NNK l e v e l s i n moist snuff of the four Canadian brands average greater than 2.5 Mg/kg (Table XII), 30 years of exposure f o r average snuff dippers, who consume about 10 gm of snuff d a i l y , amounts to 270 mg NNK or about 3.9 mg/kg. In addition, snuff dippers are exposed to about 900 mg NNN (±13 mg/kg), 800 mg NAT (±11.4mg/kg) and 56 mg NAB (±0.8 mg/kg) during a 30-year span. These estimates, together with the dose-response study i n rats c i t e d above and the f a c t that N-nitrosamines are probably formed during snuff dipping, strongly suggest that N-nitrosamines play a major r o l e i n the increased o r a l cancer r i s k of snuff dippers. This hypothesis i s supported by the recent observation that a single administration of 1 mg NNK induces a s i g n i f i c a n t number of tumors i n Syrian golden hamsters (Hecht et a l . , 1983). 4. The Unfinished Search for the Factors Involved in Oral Carcinogenesis The comparative study of tobacco users among Inuit, Indian and East Indian population groups was i n i t i a t e d i n the hope of revealing the main carcinogenic factors involved i n the development of o r a l precancerous lesions and o r a l cancer. However, the complexity of carcinogenesis and i t s e t i o l o g i c a l factors prevented us from f i n d i n g simple answers. 100 The examined groups at elevated r i s k f o r o r a l cancer engage i n a habit that leads to an increased ingestion of TSNA. In a l l these groups the frequency of micronucleated c e l l s i s also increased. These users of smokeless tobacco include snuff dippers i n the United States and Canada, "nass" chewers i n the Soviet Union, and tobacco chewers i n the United States and India. Moreover, TSNA also appear i n the s a l i v a of c i g a r e t t e smokers and "reverse" smokers, who hold the burning end of the c i g a r e t t e i n the mouth. A d i f f i c u l t y a r ises when quantitative r e l a t i o n s h i p s between TSNA l e v e l s and o r a l precancerous lesions or o r a l cancer are being sought. For example, various groups of snuff dippers can d i f f e r s i g n i f i c a n t l y i n t h e i r TSNA l e v e l s . Levels of NNN found among Inuit snuff dippers s i g n i f i c a n t l y exceed those detected i n the s a l i v a of snuff dipping American women (Hoffmann and Adams, 1981). Approximately 65% of the Inuit exceeded 420 ppb NNN, which was the highest l e v e l found i n the s a l i v a of the American dippers, and 22% of the Inuit reached l e v e l s exceeding 2,100 ppb. Levels of NNN i n the s a l i v a of snuff dipping In u i t were also higher than the 16.5 to 59.7 Mg/ ml found i n Indian tobacco chewers (Nair et a l . , 1985), or the 1.2 to 31.0 /ig/ml and 1.6 to 14.7 Mg/™- i n users of tobacco containing b e t e l quids (Nair et a l . , 1985; Wenke et a l . , 1984). The incidence of o r a l cancer, however, does not follow t h i s pattern. The highest incidence i s among chewers of tobacco-containing b e t e l quids (IARC, 1985) followed by snuff dippers i n the southern United States (IARC, 1985). The incidence of o r a l cancer among Inuit snuff dippers who have the highest l e v e l s of TSNA i n t h e i r tobacco samples and t h e i r s a l i v a must be very low since no o r a l carcinomas were seen among the 180 cancers diagnosed between 1949 and 1974 i n the Inuit of the Canadian A r c t i c (Schaefer et a l . , 1975). Moreover, the frequency of micronucleated o r a l mucosal c e l l s i n the snuff dipping In u i t i s lower (1.8%) than i n other tobacco-using groups: 4.4% i n the f l o o r of the mouth of "nass" users i n Uzbekistan, 8.4% i n 101 the buccal mucosa of tobacco/betel quid chewers of Orissa, India ( S t i c h et a l . , 1982a,b), 4.8% i n tobacco/betel quid chewers of the P h i l i p p i n e s ( S t i c h et a l . , 1984a,b), and 2.8% i n the mucosa of the lower groove of Indian users of Khaini tobacco (Mirvish, 1982). The lack of any simple quantitative r e l a t i o n s h i p between the amount of carcinogens ingested by a tobacco user and the frequency of genetic lesions (MNC), leukoplakia and cancer i n the target t i s s u e points to the involvement of modulating f a c t o r s . One could speculate that the observed differences i n o r a l l e s i o n s are due to d i f f e r e n t intakes of d i e t a r y vitamin A and/or beta-carotene. Many A s i a t i c populations are a f f l i c t e d with a vitamin A deficiency, as suggested by the occurrence of xerophthalmia and nightblindness. Subnormal l e v e l s of r e t i n o l were a c t u a l l y observed i n Uzbekis (Zardize et a l . , 1985), and i n Pakistanis and Indians at elevated r i s k f o r o r a l cancer (Ibrahim et a l . , 1977; Wahi et a l . , 1965). Another p o s s i b i l i t y worth considering i s that the aforementioned differences i n the frequency of preneoplastic changes or carcinomas may r e s u l t from differences i n the composition of the chewing mixtures. Recent studies i n our laboratory have shown that aqueous extracts of areca nut enhance the formation of transformed f o c i i n a bovine papilloma v i r u s DNA transformation assay ( S t i c h and Tsang, 1989), and induce melanomas i n a s t r a i n of p l a t y f i s h ( S t i c h and Anders, 1989) which responds to tumor promoters rather than to tumor i n i t i a t o r s . The promoting a c t i v i t y of the areca nut extract was by no means n e g l i g i b l e . In the BPV DNA transformation assay, i t was comparable to the a c t i v i t y of approximately 0.5 mg/ml mezerein, a second-stage promoter. This promoting a c t i v i t y of areca nut compounds may be responsible for the exceptionally high frequency of MNC, leukoplakia and o r a l carcinomas i n b e t e l quid chewers. This promotional stage appears to be lacking i n individuals 102 who use only snuff or chewing tobacco, which show only a small a c t i v i t y i n several assays f o r cancer promoters. 5. Exploring Preventive Measures 5.1 Mechanism of Action of Chemopreventive Agents The ICRDB (Boutwell, 1979) has compiled summaries of 324 cancer research papers concerned with r e t i n o l , other r e t i n o i d s , beta-carotene or other carotenoids. Together, these show that various r e t i n o i d s can r e v e r s i b l y suppress the malignant behaviour of cultured c e l l s that have been transformed by vi r u s e s , chemicals or i o n i z i n g r a d i a t i o n ; they can delay or prevent the onset of cancer i n experimental animals previously treated with DNA-binding carcinogens; they can cause human skin keratoses or o r a l leukoplakias to regress; they can prevent "tumor promoters" from e l i c i t i n g papillomas on mouse skin; and they can i n h i b i t some of the very s p e c i f i c biochemical e f f e c t s of tumor promoters on c e l l s i n vivo or i n culture. Although inappropriate use of r e t i n o i d s may do more harm than good (Schroder and Black, 1980), i t seems reasonable to hope that exposure of human tissues to r e t i n o i d - l i k e a c t i v i t y can be manipulated to reduce cancer r i s k s (Sporn and Newton, 1979) . I t has been suggested that r e t i n o i d a p p l i c a t i o n can indeed promote tumor development i n a few model systems (Schroder and Black, 1980). In the short-term studies reported here no increase i n leukoplakia was seen i n any of the East Indian fishermen given vitamin A; i n f a c t , the opposite was observed. The only harmful e f f e c t of vitamin A administration i s hypervitaminosis A, but no symtoms of t h i s were observed i n any of the subjects. I t can be concluded that the doses of vitamin A given to people i n these studies produced no harmful s i d e - e f f e c t s i n the time period during which i t was administered. 103 Neoplastic transformation can often be subdivided into "stages," the early stage i n v o l v i n g conversion of a normal c e l l , and the l a t e r stage involving conversion of a p a r t i a l l y a l t e r e d c e l l into a f u l l y n eoplastic c e l l . To be e f f e c t i v e , the neoplastic c e l l must not be eliminated, but must p r o l i f e r a t e into a p a t h o l o g i c a l tumor. Early- and late-stage changes are both necessary (halving e i t h e r may thus be an equally e f f e c t i v e way of ha l v i n g cancer r i s k s ) , but t y p i c a l l y have d i f f e r e n t causes (Peto, 1977, 1979). The demonstrated protective e f f e c t s of r e t i n o l (and perhaps beta-carotene) suggest that i t i s the l a t e r stages of neoplastic progression which are c h i e f l y affected, together perhaps with the f i n a l process of tumor growth. There may often be a latent period of a few decades before a doubling or halving of the ea r l y process has any e f f e c t on human cancer r i s k within about 5 years, while e f f e c t s on the f i n a l process ( p r o l i f e r a t i o n ) may be even more r a p i d l y evident. Thus human cancer r i s k s may be noticeably influenced by the average l e v e l s of blood r e t i n o l within a period of as l i t t l e as 5 or 10 years. In contrast to the r e t i n o i d s , the question of whether dietary beta-carotene or some other carotenoids can a f f e c t cancer r i s k has not received nearly as much experimental attention. Possible mechanisms to consider include (1) a d i r e c t r e t i n o i d - l i k e e f f e c t of some carotenoids on c e l l u l a r d i f f e r e n t i a t i o n i n the target tissues, (2) conversion i n the target tissue of some carotenoids into molecules with such r e t i n o i d - l i k e e f f e c t s , or (3) protection by carotenoids of the target tissues v i a mechanisms not i n control of c e l l u l a r d i f f e r e n t i a t i o n , f o r example, by a f f e c t i n g immunological function (Rettura, 1975; F e l i x et a l . , 1976), or by quenching s i n g l e t oxygen, a highly reactive, excited molecular species which occurs as a toxic by-product of many normal processes i n both animals and plants. 104 In the studies presented here, the twice weekly o r a l administration of beta-carotene, beta-carotene plus vitamin A or vitamin A a f f e c t e d o r a l mucosal lesi o n s at three d i f f e r e n t l e v e l s : the frequency of micronucleated c e l l s was reduced, w e l l - e s t a b l i s h e d leukoplakias regressed, and the development of new leukoplakias was i n h i b i t e d . The r e s u l t s are noteworthy for several reasons. F i r s t , the e f f e c t i v e doses are considerably lower than the 1 to 2 mg/kg body weight/day of 1 3 - c i s - r e t i n o i c a c i d s u c c e s s f u l l y given to patients with o r a l leukoplakias (Cordero et a l . , 1981; Koch, 1981; Hong et a l . , 1986). I f the recommended conversion f a c t o r f or beta-carotene and vitamin A i s applied (FAO/WHO, 1967; Polacchi, 1980), then our dose of r e t i n o l equivalents was approximately 0.14 mg/kg body weight/day (0.7 mg from vitamin A, and 0.7 mg from beta-carotene). Second, the administration of beta-carotene alone had a s i g n i f i c a n t e f f e c t on the frequency of micronucleated c e l l s , but a l e s s e r one on the remission of leukoplakias. Among the 31 chewers who received beta-carotene alone, only one (3%) f a i l e d to show a reduction i n micronucleated c e l l s , whereas no remission was seen i n 23 (85%) of 27 p a r t i c i p a n t s who completed the t r i a l . Third, micronucleated c e l l s respond much more r a p i d l y than leukoplakia to beta-carotene or to beta-carotene plus vitamin A. Fourth, a l l p a r t i c i p a n t s i n the i n t e r v e n t i o n t r i a l continued to chew tobacco-containing b e t e l quids i n t h e i r accustomed manner and at t h e i r usual rate. Thus remission of o r a l leukoplakias, reduced development of new leukoplakias, and reduction of micronucleated c e l l s occurred during d a i l y exposure to various carcinogens released from b e t e l quid ingredients. In t h i s respect, the t r i a l s with r e t i n o i d s on b e t e l quid chewers d i f f e r from those conducted by Koch (1981), Shah et a l . (1983) or Hong et a l . (1986) on patients with o r a l leukoplakia, who may not have been exposed to carcinogens during the treatment period. F i f t h , the doses of beta-carotene and vitamin A administered d i d not cause any 105 detectable s i d e - e f f e c t s , such as dryness of the l i p s and mouth, s c a l i n g and peeling of the skin, or c o n j u n c t i v i t i s . The question must be r a i s e d whether there are ways to reduce the dose of beta-carotene and vitamin A to l e v e l s that can be r e a d i l y administered and that produce no undesirable s i d e - e f f e c t s . The development of new o r a l leukoplakias was i n h i b i t e d , and remission of established leukoplakias induced, by the six-month o r a l administration of vitamin A at a dose of 0.14 mg/kg body weight per day. This preventive dose was about 13-fold higher than the FAO/WHO (1967) recommended intake of r e t i n o i d s , and sevenfold higher than the 0.02 mg/kg body weight per day of dietary vitamin A which, according to epidemiological evidence, conveys a pro t e c t i v e e f f e c t (Marshall et a l . , 1982). The dose of vitamin A was considerably lower than those reported previously: approximately 1 mg/kg/day (Cordero et a l . , 1981; Gupta et a l . , 1980); 1 to 2 mg/kg/day (Hong et a l . , 1986); 3, 5 or 10 mg/kg/day when administered as a lozenge (Wahi et a l . , 1965); 10 mg/kg/day (Nair et a l . , 1980); and 20 to 100 mg/kg/day for several weeks (Ryssel et a l . , 1971). (To make the r e t i n o i d l e v e l s comparable, they were r e c a l c u l a t e d from published r e s u l t s , assuming a body weight of 70 kg for an a d u l t ) . One way to reduce the t o t a l amount of vitamin A would be to at f i r s t administer f o r several months heavy doses of vitamin A to reduce the frequency of micronuclei and leukoplakia, and then follow t h i s treatment with the non-toxic beta-carotene. Preliminary r e s u l t s obtained on the tobacco/betel quid chewers i n Kerala revealed that the pro t e c t i v e e f f e c t of vitamin A administration can be maintained f o r at l e a s t 5 a d d i t i o n a l months with beta-carotene. Several other experiments can be mentioned i n support of th i s approach . 106 Following a r e l a t i v e l y short-term administration of higher doses of r e t i n o i d s (0.6 mg/kg/day), the protective e f f e c t was maintained by lower doses i n the range of 0.3 mg etretinate/kg/day (Sloberg et a l . , 1983). The chemopreventive e f f e c t induced by 1 mg/kg/day of Ro 10-9359 for 2 to 10 weeks was s i m i l a r . Another approach worth considering consists of combining the administration of the somewhat toxic r e t i n o i d s with the non-toxic beta-carotene. In animal models, beta-carotene i s e f f e c t i v e i n preventing carcinogenesis, i n c l u d i n g that of the o r a l c a v i t y (Suda et a l . , 1986). In human populations, beta-carotene has also exerted a p r o t e c t i v e e f f e c t , as shown by a reduction i n the frequency of micronucleated c e l l s i n the buccal mucosa of b e t e l quid chewers (Sti c h , 1987; S t i c h et a l . , 1984a,b) and snuff dippers ( S t i c h et a l . , 1985). The e f f i c a c y of replacing doses of vitamin A with beta-carotene can be judged by comparing the extent of remission induced by 200,000 IU of vitamin A per week with that induced by 200,000 IU given i n part as vitamin A (100,000 IU/ week) and i n part, as beta-carotene (amount equal to 100,000 IU r e t i n o l ) . The p i l o t t r i a l s on b e t e l quid chewers revealed that vitamin A treatment induced the remission of o r a l leukoplakias i n 57.1% of p a r t i c i p a n t s (n = 21), whereas the vitamin A plus beta-carotene administration re s u l t e d i n remission i n only 27.5% (n = 51) (Stich, 1987). This difference i n protective a c t i v i t y between the two treatments may only be apparent, mainly due to the f a c t that only a f r a c t i o n of beta-carotene i s converted into vitamin A. Thus the doses of the two treatment protocols are not s t r i c t l y comparable. T h e o r e t i c a l l y , doses of the non-toxic beta-carotene could be increased to l e v e l s matching that of 100,000 IU of vitamin A using one of the current conversion factors (Ibrahim et a l . , 1977). However, the implementation of this 107 idea could meet with objections because of the large number of beta-carotene capsules to be swallowed by the t r i a l p a r t i c i p a n t s . 6. Outlook Although the r i s k of o r a l cancer i s not high amongst these peoples (both Inu i t and Indian), the use of o r a l tobacco i s a r e l a t i v e l y new phenomenon which has increased dramatically since the 1960s (IARC, 1985), and which i s practised mainly by young adolescents. In the United States, the use of smokeless tobacco increases by 10-11% yearly. Given t h i s s i t u a t i o n , the question must be r a i s e d as to whether one can expect to see i n the near future the high incidence of o r a l leukoplakias and o r a l cancers among Canadian snuff dippers that i s so common among chewers of tobacco-containing b e t e l quids i n Asian countries, users of "nass" i n southern regions of the Soviet Union (IARC, 1985) or snuff dippers i n the southeastern United States. In the l a t t e r case, snuff dipping was found to be strongly associated with cancers of the o r a l cavity, pharynx and larynx. A study by Winn et a l (1984) showed that the use of snuff was associated with a f o u r f o l d increase i n the r i s k of o r a l and pharyngeal cancers, and that the r e l a t i v e r i s k increased markedly to almost f i f t y f o l d f o r those who had dipped snuff for 50 years or more. The d i s t r i b u t i o n of beta-carotene or vitamin A to a large population remains an unresolved issue. Because of the expenses and l o g i s t i c a l d i f f i c u l t i e s involved i n a large-scale intervention program using p i l l s or capsules, other methods must be sought and implemented. The following examples are p r a c t i c a l l y f e a s i b l e and warrant c l o s e r consideration. Red palm o i l provides a very r i c h source of beta-carotene, and the introduction of t h i s substance into the d i e t could provide the protection 108 needed by increasing both beta-carotene and vitamin A l e v e l s i n i t s consumers. In North America, an increased intake of green/yellow vegetables and education on the importance of these products i n the d i e t may lead to a change i n the r e s u l t i n g serum l e v e l s of beta-carotene and vitamin A, and eventually reduce the r i s k not only f o r o r a l but also for other e p i t h e l i a l cancers. In Taiwan, sweet potato s t r a i n s which are r i c h i n beta-carotene are being used as potato chips, french f r i e s and f l o u r . In t h i s way, the d i e t of a population can be enriched with beta-carotene without any major changes i n eating habits. 109 SUMMARY The only known carcinogens contained i n any tobacco mixtures used o r a l l y throughout the world are the N-nitroso compounds. An analysis of the l e v e l s of these agents, plus t h e i r precursors, i n the tobaccos used and i n the s a l i v a of t h e i r users was conducted i n three d i f f e r e n t communities. High l e v e l s of n i t r i t e were found i n the snuff tobacco used by Canadian Inu i t (Gjoa Haven) and Indians (La Loche) and i n the s a l i v a of snuff dippers. These high n i t r i t e l e v e l s can act as precursors to n i t r o s a t i o n reactions, as evidenced by the higher l e v e l s of NPRO detected i n the urine of snuff users versus non-users. In addition, r e l a t i v e l y high l e v e l s of tobacco-specific nitrosamines were detected i n tobacco samples and i n the s a l i v a of snuff users. The genotoxic damage induced by exposure to these carcinogens was evident from the elevated l e v e l s of micronucleated c e l l s found i n the o r a l mucosa of snuff dippers. Although, at present the Inuit and Indian communities studied, do not have an elevated incidence of o r a l cancer, these abnormalities of the genome, together with the whitish/yellowish wrinkled patches observed i n the mucosa, indi c a t e a r i s k of developing precancerous lesions and/or cancer over a long enough perio d of time. The East Indian population studied shows a s u b s t a n t i a l l y increased r i s k f o r o r a l cancer, high micronucleated c e l l counts, plus a large percentage of i n d i v i d u a l s with preneoplastic o r a l lesions (leukoplakia). In the Inuit population studied, i t was shown that the MNC l e v e l could be reduced by a short-term intervention t r i a l (10 weeks) using 180 mg/week of beta-carotene. In the East Indian group, i t was possible to reduce the MNC l e v e l and the frequency of leukoplakia using beta-carotene (180 mg/week) plus 110 vitamin A (100,000 IU/week) over a six-month period. An even more marked reduction i n l e s i o n frequency was observed a f t e r a six-month administration of vitamin A alone (200,000 IU/week). I t now appears conclusive that exposure to the carcinogenic N-nitroso compounds contained i n smokeless tobacco i s involved i n the e t i o l o g y of o r a l cancer and that of the r i s k can be reduced by increasing serum l e v e l s of beta-carotene and vitamin A v i a supplementation with p i l l s or by adjusting the diet to increase intake of these compounds. I l l REFERENCES Abe, T., Isemura, T. and Kikuchi, Y. (1984) Micronuclei i n human bone-marrow c e l l s : evaluation of the micronucleus t e s t using human leukemia patients treated with antileukemic agents. Mutation Res., 130. 113-120. Adams, J.D., Brunnemann, K.D. and Hoffmann, D. (1983) Rapid method for the analysis of tobacco-specific N-nitrosamines by g a s - l i q u i d chromatography with a thermal energy analyser. J . Chromatogr., 2 5 6 . 347-351. Andersen, R.A. and Kasperbauer, M.J. (1984) Post-harvest treatment and the accumulation of n i t r i t e and N'-nitrosonornicotine i n burley tobacco. In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. O ' N e i l l , R.C. von B o r s t e l , C.T. M i l l e r , J . Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency f or Research on Cancer, Lyon, pp. 877-883. Anderson, S.M. and Krinsky, N.I. (1973) Protective a c t i o n of carotenoid pigments against photodynamic damage to liposomes. Photochem. Photobiol., 18. 403-408. Arjungi, K.M. (1976) Areca nut. A review. A r z n e i m i t t e l f o r s c h . 2 6 , 951-956. A x e l l , T., Mornstad, H. and Sundstrom, B. (1976) The r e l a t i o n of the c l i n i c a l p i c t u re to the histopathology of snuff dipper's lesions i n a Swedish population. J . Oral Pathol., 5, 229-236. 112 A x e l l , T., Holmstrup, P., Kramer, I.R.A., Pindborg, J . J . and Shear, M. (1984) International seminar on o r a l leukoplakia and associated lesions r e l a t e d to tobacco habits. Commun. Dent. Oral Epidemiol., _V2, 146-154. B e l i s a r i o , M.A. , Pecce, R. , B a t t i s t a , C , Panza, N.' and P a c i l i o , G. (1985) I n h i b i t i o n of cyclophosphamide mutagenicity by beta-carotene. Biomed. Pharmacother., 39, 445-448. Boutwell, R.K. (1979) (consulting reviewer) Selected Abstracts on Vitamin A i n Cancer Biology, International Cancer Research Data Bank, NCI, Bethesda, MD. Burton, G.W. and Ingold, K.U. (1984) Beta-carotene: an unusual type of l i p i d antioxidant. Science, 224. 569-573. Cai, H.Y. (1982) Eti o l o g y and prevention of esophageal cancer i n China. In: Carcinogens and Mutagens i n the Environment, Vol. I, Food Products, H.F. Stich (ed.), CRC Press, Boca Raton, FL, pp. 39-52. Cordero, A.A., A l l e v a t o , M.A.J., Barclay, C.A., T r a b a l l i , C.A. and Donatti, L.B. (1981) Treatment of l i c h e n planus and leukoplakia with the o r a l r e t i n o i d Ro 10-9359. In: Retinoids: Advances i n Basic Research and Therapy, C.E. Orfanos, 0. Braun-Falco, E.M. Farber, C. Grupper, M.K. Polano and R. Schuppli (eds.), Springer-Verlag, B e r l i n , Heidelberg, New York, pp. 273-278. DeCosse,•J.J., Adams, M.B., Kuzma, J.F., LoGerfo, P. and Condon, R.E. (1975) E f f e c t of ascorbic a c i d on r e c t a l polyps of patients with f a m i l i a l polyposis. Surgery, 78, 608. 113 FAO/WHO (1967) J o i n t Expert Committee on Food Additives, Requirements of vitamin A, thiamine, r i b o f l a v i n e and n i a c i n , Wld Health Org. Techn. Rep. Ser. No. 362, WHO, Geneva. F e l i x , E.L., Cohen, M.H. and Loyd, B.C. (1976) J . surg. Res. 21, 307-312. Fontham, E. ; Correa, P., Rodriguez, E. and L i n , Y. (1986) V a l i d a t i o n of smoking h i s t o r y with the micronuclei t e s t . In: Mechanisms i n Tobacco Carcinogenesis, D. Hoffmann and C.C. Harris(eds.), Banbury Report 23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 113-119. Forman, D., Al-Dabbagh, S. and D o l l , R. (1985) N i t r a t e s , n i t r i t e s and g a s t r i c cancer i n Great B r i t a i n . Nature (Lond.), 313. 620-625. Gibel, W. (1967) Arch. Geschwulstforsch., 30, 181-189. Gode, P.K. (1961) Studies i n Indian C u l t u r a l History, V ol. 1, Vishueshvaranand Vedic Research I n s t i t u t e , Hoshiarpar, India, pp. 113-114. Greer, R.O. J r and Poulson, T.C. (1983) Oral tissue a l t e r a t i o n s associated with the use of smokeless tobacco by teen-agers. Part 1. C l i n i c a l f indings. Oral Surg., 56, 275-284. Gupta, P.C. (1984) Epidemiologic study of the a s s o c i a t i o n between alcohol habits and o r a l leukoplakia. Commun. Dent. Oral Epidemiol., .12, 47-50. 114 Gupta, P.C., Mehta, F.S., Daftary, D.K., Pindborg, J . J . , Bhonsle, R.B., Jalnawalla, P.N., Sinor, P.N., Pitkar, V.K., Murti, P.R., I r a n i , R.R., Shah, H.T., Kadam, P.M., Iyer, K.S.S., Iyer, H.M., Hedge, A.K., Chandrashekar, G.K., Shorff, B.C., Sahiar, B.E. and Mehta, M.N. (1980) Incidence rates of o r a l cancer and natural h i s t o r y of o r a l precancerous lesions i n a 10-year follow-up study of Indian v i l l a g e r s . Commun. Dent. Oral Epidemiol., 8, 287-333. Gupta, P.C., Mehta, F.S., Pindborg, J . J . , Aghi, M.B., Bhonsle, R.B., Daftary, D.K., Murti, P.R., Shah, H.T. and Sinor, P.N. (1986) Intervention study for primary prevention of o r a l cancer among 36,000 Indian tobacco users. Lancet, J, 1235-1239. Hecht, S.S., Adams, J.D., Nunoto, S. and Hoffmann, D. (1983) Induction of r e s p i r a t o r y t r a c t tumors i n Syrian golden hamsters by a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the e f f e c t of smoke in h a l a t i o n . Carcinogenesis, 4, 1287-1290. Hecht, S.S., Castonguay, A., Chung, F.L. and Hoffmann, D. (1984) Carcinogenicity and metabolic a c t i v a t i o n of tobacco s p e c i f i c nitrosamines: current status and future prospects. In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. O ' N e i l l , R.C. von Borste l , C.T. M i l l e r , J . Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency f o r Research on Cancer, Lyon, pp. 763-778. 115 Heimann, R.K. (1960) Tobacco and Americans, McGraw-Hill, New York, pp. 8, 65, 118-119, 236. Hirayama, T. (1966) An epidemiological study of o r a l and pharyngeal cancer i n Central and South-East Asia. B u l l . Wld Health Org., 34, 41-69. Hirayama, T. (1981) A large-scale cohort study on the r e l a t i o n s h i p between di e t and selected cancers of digestive organs. In: G a s t r o i n t e s t i n a l Cancer: Endogenous Factors, W.R. Bruce, P. Correa, M. L i p k i n , S.R. Tannenbaum and T.D. Wilkins (eds.), Banbury Report 7, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 409-426. Hoffmann, D. and Adams, J.D. (1981) Carcinogenic tobacco-specific N-nitrosamines i n snuff and i n the s a l i v a of snuff dippers. Cancer Res., 41, 4305-4308. Hoffmann, D. and Hecht, S.S. (1985) Nicotine-derived N-nitrosamines and tobacco r e l a t e d cancer: current status and future d i r e c t i o n s . Cancer Res., 45, 935-944. Hoffmann, D., Brunnemann, K.D., Adams, J.D. and Hecht, S.S. (1984) Formation and analysis of N-nitrosamines i n tobacco products and t h e i r endogenous formation i n consumers. In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l Effects and Relevance to Human Cancer, I.K. O' N e i l l , R.C. von B o r s t e l , C.J. M i l l e r , J. Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency for Research on Cancer, Lyon, pp. 743-762. 116 Hogstedt, B., Akesson, B., A x e l l , K., Gullberg, B., Mitelman, F., Peto, R.W., Skerfving, S. and Welinder, H. (1983a) Increased frequency of lymphocyte micronuclei i n workers producing r e i n f o r c e d polyester r e s i n with low exposure to styrene. Scand. J . Work Environ. Health, 49, 271-276. Hogstedt, B., Gullberg, B., Hedner, K., Kolnig, A.M., Mitelman, F., Skerfving, S. and Widegren, B. (1983b) Chromosome aberrations and micronuclei i n bone marrow c e l l s and perip h e r a l blood lymphocytes i n humans exposed to ethylene oxide. Hereditas, 98, 105-113. Hong, W.K., Endicott, J . , I t r i , L.M., Doos, W., Batsakis, J.G., B e l l , R., Fofonoff, S., Byers, R. , Atkinson, E.N. , Vaughan, C , Toth, B.B. , Kramer, A., Dimery, I.W., Skipper, P. and Strong, S. (1986) 13-cis-Retinoic a c i d i n the treatment of o r a l leukoplakia. New Engl. J . Med., 315. 1501-1505. IARC (1985) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vo l . 37, Tobacco Habits Other than Smoking; Betel-Quid and Areca-Nut Chewing; and Some Related Nitrosamines, International Agency for Research on Cancer, Lyon. Ibrahim, K., Jafarey, N.A. and Zuberi, S.J. (1977) Plasma vitamin "A" carotene l e v e l s i n squamous c e l l carcinoma of o r a l c a v i t y and oro-pharynx. C l i n . Oncol., 3, 203-207. Kawabata, T., Matsui, M., Ishibashi, T. and Hamano, M. (1984) Analysis and occurrence of t o t a l N-nitroso compounds i n the Japanese d i e t . In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. 117 O ' N e i l l , R.C. von B o r s t e l , C.T. M i l l e r , J . Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency f o r Research on Cancer, Lyon, pp. 25-,31. Koch, H.F. (1981) E f f e c t of r e t i n o i d s on precancerous lesions of o r a l mucosa. In: Retinoids: Advances i n Basic Research and Therapy, C.E. Orfanos, 0. Braun-Falco, E.M. Farber, C. Grupper, M.K. Polano and R. Schuppli (eds.), Springer-Verlag, B e r l i n , Heidelberg, New York, pp. 307-312. Kozlowski, L.T. (1981) The determinants of tobacco use: c i g a r e t t e smoking i n the context of other forms of tobacco use. Can. J . Publ. Health, 72, 396-401. Krenger, W. (1942) History and culture of b e t e l chewing. Synthesis and preparation of b e t e l (Ger.). Ciba Z. , 84, 2922-2941. Krinsky, N.I. and Deneke, S.M. (1982) Interaction of oxygen and oxy-radicals with carotenoids. J . nat. Cancer Inst., 69, 205-209. Kvale, G., Bjelke, E. and Gart, J . J . (1983) Dietary habits and lung cancer r i s k . Int. J . Cancer, 31, 397-405. Lahdetie, J . (1986) Micronucleated spermatids i n the seminal f l u i d of smokers and nonsmokers. Mutation Res., 172. 255-263. Langone, J . J . , Gjika, H.B. and Van Vunakis, H. (1973) Radioimmunoassay for n i c o t i n e and c o t i n i n e . Biochemistry, J2, 5025-5030. 118 L i j i n s k y , W., Keefer, L. and Loo, J . (1970) The preparation and properties of some nitrosamino acids. Tetrahedron, 26, 5137-5153. Lin, Y.K. and T a i , M.W. (1980) Mutagenicity of Chinese wine treated with n i t r i t e . Food Cosmet. Tox i c o l . , .18, 241-243. Mandard, A.M., Duigou, F., Marnay, J . , Masson, P., Qiu, S.L., Y i , J.S., B a r r e l l i e r , P. and Lebigot, G. (1978) Analysis of the r e s u l t s of the micronucleus t e s t i n patients presenting upper digestive t r a c t cancers and i n non-cancerous subjects. Int. J . Cancer, 3_9, 442-444. Marquardt, H., Rufino, F. and Weisburger, J.H. (1977) On the aetiology of g a s t r i c cancer: mutagenicity of food extracts a f t e r incubation with n i t r i t e . Food Cosmet. T o x i c o l . , 15, 97-100. Marshall, J . , Graham, S., M e t t l i n , C , Shedd, D., and Swanson, M. (1982) Diet i n the epidemiology of o r a l cancer. N u t r i t . Cancer, 3, 145-149. Mathews-Roth, M.M. (1982) Antitumor a c t i v i t y of beta-carotene, canthaxanthin and phytoene. Oncology, 39, 33-37. Maxwell, J . C , J r (1980) Chewing, snuff i s growth segment. Tobacco Rep., 107. 32-35. Menkes, M.S., Comstock, G.W., Vuilleumier, J.P., Helsing, K.J., Rider, A.A. and Brookmeyer, R. (1986) Serum beta-carotene, vitamins A and E, selenium, and the r i s k of lung cancer. New Engl. J . Med., 315, 1250-1254. 119 Meretoja, T., Jarventaus, H., Sorsa, M. and Vainio, H. (1978) Chromosome aberrations i n lymphocytes of workers exposed to styrene. Scand. J . Work Environ. Health, 4 (Suppl. 2), 259-264. M i l l e r , K.W., Lorr, N.A. and Yang, C.S. (1984) Simultaneous determination of plasma r e t i n o l , alpha-tocopherol, lycopene, alpha-carotene and beta-carotene by high-performance l i q u i d chromatography. Anal. Biochem., 138. 340-345. Mirvish, S.S. (1982) In vivo formation of N-nitroso compounds: formation from n i t r i t e and nitrogen dioxide, and r e l a t i o n to g a s t r i c cancer. In: Nitrosamines and Human Cancer, N. Magee (ed.), Banbury Report 12, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 227-241. Mirvish, S.S. (1985) G a s t r i c cancer and s a l i v a r y n i t r a t e and n i t r i t e . Science, 315. 461-462. Mitelman, F. (1985). Catalog of Chromosome Aberrations i n Cancer, 2nd ed., Progress and Topics i n Cytogenetics, Vol. 5., Alan R. L i s s , New York. Muir, C.S. and Kirk, R. (1960) Betel, tobacco and cancer of the mouth. Br. J . Cancer, 14, 597-608. Munoz, N., Wahrendorf, J . , Lu, L.B., Crespi, M., Thurnham, D.I., Day, N.E., Zheng, H.J., L i , J.Y., Correa, P., O'Conor, G.T. and Bosch, X. (1985) No e f f e c t of r i b o f l a v i n e , r e t i n o l and zinc on prevalence of precancerous lesions of 120 oesophagus. Randomized double-blind intervention study i n h i g h - r i s k population of China. Lancet, 2, 111-114. Nair, J . , Ohshima, H., Friesen, M., Croisy, A., Bhide, S.V. and Bartsch, H. (1985) Tobacco-specific and b e t e l n u t - s p e c i f i c N-nitroso compounds: Occurence i n s a l i v a and urine of b e t e l quid chewers and formation i n v i t r o by n i t r o s a t i o n of b e t e l quid. Carcinogenesis, 6, 295-303. Nair, K.K., B a r t e l s , P.H., Mahon, D.C., Olson, G.B. a n d O l o f f s , P.C. (1980) Image analysis of hepatocyte n u c l e i from chlordane-treated r a t s . Anal. Quant. Cytol. J . , 2, 285-289. Namiki, K., Osawa, T. and Namiki, M. (1983) Mutagen formation by the n i t r i t e -spice r e a c t i o n and i t s i n h i b i t i o n . In: Carcinogens and Mutagens i n the Environment, V o l . I l l , N a t u rally Occurring Compounds: Epidemiology and D i s t r i b u t i o n , H.F. S t i c h (ed.), CRC Press, Boca Raton, FL, pp. 119-127. National Research Council (1981) The Health E f f e c t s of N i t r a t e , N i t r i t e and N-Nitroso Compounds, Assembly of L i f e Sciences, National Academy of Sciences, National Academy Press, Washington, D.C., pp. 7-35 - 7-36. Nordenson, I. and Beckman, L. (1984) Chromosomal aberrations i n lymphocytes of workers exposed to low l e v e l s of styrene. Hum. Hered., 34, 178-182. N u t r i t i o n Canada (1975a) The Eskimo Survey Report, Bureau of N u t r i t i o n a l Sciences, Department of National Health and Welfare, Ottawa. 121 N u t r i t i o n Canada (1975b) The Indian Survey Report, Bureau of N u t r i t i o n a l Sciences, Department of National Health and Welfare, Ottawa. Ohshima, H. and Bartsch, H. (1981) Quantitative estimation of endogenous n i t r o s a t i o n i n humans by monitoring N-nitrosoproline excreted i n the urine. Cancer Res., 41, 3658-3662. Ohshima, H., Bereziat, J.C. and Bartsch, H. (1982) Measurement of endogenous N-n i t r o s a t i o n i n rats and humans by monitoring urinary and f a e c a l excretion of N-nitrosamino acids. In: N-Nitroso Compounds: Occurrence and B i o l o g i c a l E f f e c t s . H. Bartsch, I.K. O ' N e i l l , M. Castegnaro and M. Okada (eds.), IARC S c i . Publ. No. 41, International Agency for Research on Cancer, Lyon, pp. 397-411. Orr, I.M. (1933) Oral cancer i n b e t e l nut chewers i n Travancore: i t s aetiology, pathology and treatment. Lancet, 2, 575-580. Parvez, Z., Kormano, M., Satokari, K., Moncada, R. and Eklund, R. (1987) Induction of m i t o t i c micronuclei by X-ray contrast media i n human peripheral lymphocytes. Mutation Res., 192. 145-149. Peto, R. (1977) Epidemiology, multistage models, and short-term mutagenicity t e s t s . In: Origins of Human Cancer (H.H. H i a t t , J.D. Watson, and J . Winsten, (eds.), Cold Spring Harbor Laboratory, New York, pp. 1403-1428. Peto, R. (1979) Proc. Roy. Soc. Lond. B, 205, 111-120. 122 Peto, R. (1983) The marked differences between carotenoids and r e t i n o i d s : methodological implications f or biochemical epidemiology. Cancer Surv., 2, 327-340. Peto, R., D o l l , R. , Buckley, J.D. and Sporn, M.B. (1981) Can die t a r y beta-carotene m a t e r i a l l y reduce human cancer rates? Nature (Lond.), 290. 201-208. Peto, R., Gray, R., Brantom, P. and Grasso, P. (1984) Nitrosamine carcinogenesis i n 5120 rodents: chronic administration of sixteen d i f f e r e n t concentrations of NDEA, NDMA, NPYR and NPIP i n the water of 4440 inbred rats, with p a r a l l e l studies on NDEA alone of the e f f e c t of age of s t a r t i n g (3, 6 or 20 weeks) and of species (rats, mice or hamsters). In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. O ' N e i l l , R.C. von B o r s t e l , C.T. M i l l e r , J . Long and H. Bartsch (eds), IARC S c i . Publ. No. 57, International Agency for Research on Cancer, Lyon, pp. 627-665. Picker, J.D. and Fox, D.P. (1986) Do c u r r i e d foods produce micronuclei i n buccal e p i t h e l i a l c e l l s ? Mutation Res., 171. 185-188. P i g n a t e l l i , B., Bereziat, J.C., Descotes, G. and Bartsch, H. (1982) Catal y s i s of n i t r o s a t i o n i n v i t r o and i n vivo i n rats by catechin and r e s o r c i n o l and i n h i b i t i o n by chlorogenic acid. Carcinogenesis, 3, 1045-1049. Pindborg, J . J . , Mehta, F.S. and Daftary, D.K. (1975) Incidence of o r a l cancer among 30,000 v i l l a g e r s i n India i n a 7 year follow-up study of o r a l precancerous l e s i o n s . Commun. Dent. Oral Epidemiol., 3, 86-88. 123 Polacchi, W. (1980) Quantitative expression of vitamin A. Food & N u t r i t i o n , 6, 44-45. Raafat, M., El-Gerzawi, S. and Stich, H.F. (1984) Detection of mutagenicity i n u r o t h e l i a l c e l l s of b i l h a r z i a l patients by "the micronucleus t e s t . " J . Egypt. Natl. Cancer Inst., 1, 63-73. Raghavan, V. and Baruah, H.K. (1958) Arecanut: India's popular masticatory -h i s t o r y , chemistry and u t i l i z a t i o n . Econ. Bot., J 2 , 315-325. R e a l i , D. (1987) Micronuclei i n e x f o l i a t e d u r o t h e l i a l c e l l s and urine mutagenicity i n smokers. Mutation Res., 192. 145-149. Rettura, G. (1975) 170th nat. Meet. Am. chem. Soc. Abstr. 59. Rosin, M.P. and German, J . (1985) Evidence f o r chromosome i n s t a b i l i t y i n vivo i n Bloom syndrome: increased numbers of micronuclei i n e x f o l i a t e d c e l l s . Hum. Genet., 71, 187-191. Rosin, M.P. and Ochs, H. (1986) In vivo chromosomal i n s t a b i l i t y i n ataxia-t e l a n g i e c t a s i a homozygotes and heterozygotes. Hum. Genet., 74, 335-340. Rowley, J.D. (1983) Human oncogene locations and chromosome aberrations. Nature (Lond.), 301, 290-291. 124 Ryssel, H.J., Brunner, K.W. and Bollag, W.. (1971) Die perorale Anwendung von Vitamin-A-Saure b e i Leukoplakien, Hyperkeratosen und Blattenepithelkarzinomen: Ergebnisse und V e r t r a g l i c h k e i t . Schweiz. Med. Wschr., .101, 1027-1030. Santamaria, L., Bianchi, A., Arnaboldi, A., Andreoni, L. and Bermond, P. (1983) Benzo(a)pyrene c a r c i n o g e n i c i t y and i t s prevention by carotenoids..In: Modulation and Mediation of Cancer by Vitamins, F.L. Meyskens and K.N. Prasad (eds.), Karger, Basel, pp. 81-88. Schaefer, 0., Hildes, J.A., Medd, L.M. and Cameron, D.G. (1975) The changing pattern of neoplastic disease i n Canadian Eskimos. Can. Med. Assoc. J . , 112. 1399-1404. Schroder, E.W. and Black, H.P. (1980) Retinoids: tumor preventers or tumor enhancers? J . nat. Cancer Inst., 65, 671-674. Sen, N.P., Tessier, L. and Seaman, S. (1983) Determination of N-nitrosoguvacoline and N-nitrososarcosine i n malt and beer. J . Agric. Food Chem., 31, 1033. Shah, J.P., Strong, E.W., DeCosse, J . J . , I t r i , L. and S e l l e r s , P. (1983) E f f e c t of r e t i n o i d s on o r a l leukoplakia. Am. J . Surg., 146. 466-470. Silverman, S., J r , Gorsky, M. and Lozada, F. (1984) Oral leukoplakia and malignant transformation. A follow-up study of 257 patients. Cancer, 53, 563-568. 125 Sloberg, K., Hersle, K. , Mobacken, H. and Thilander, H. (1983) Severe o r a l l i c h e n planus: remission and maintenance with vitamin A analogues. J . Oral Pathol., J2, 473-477. Som, S., Chatterjee, M. and Banerjee, M.R. (1984) Beta-carotene i n h i b i t i o n of 7,12-dimethylbenz(a)anthracene-induced transformation of murine mammary c e l l s i n v i t r o . Carcinogenesis, 5, 937-940. Sporn, M.B. and Newton, D.L. (1979) Chemoprevention of cancer with r e t i n o i d s Fed. P r o c , 38, 2528-2534. Sti c h , H.F. (1987) Micronucleated e x f o l i a t e d c e l l s as i n d i c a t o r s f o r genotoxic damage and as markers i n chemoprevention t r i a l s . J . N u t r i t . Growth Cancer, 4, 9-18. St i c h , H.F. and Anders, F. (1989) The involvement of reac t i v e oxygen species i n o r a l cancers of b e t e l quid/tobacco chewers. Mutation Res., i n press. Stich, H.F. and Rosin, M.P. (1983a). Quantitating the s y n e r g i s t i c e f f e c t of smoking and alcohol consumption with the raicronucleus t e s t on human buccal mucosa c e l l s . Int. J . Cancer, .31, 305-308. Stich, H.F. and Rosin, M.P. (1983b) Micronuclei i n e x f o l i a t e d human c e l l s as an i n t e r n a l dosimeter f o r exposures to carcinogens. In: Carcinogens and Mutagens i n the Environment, Vol. I I , Naturally Occurring Compounds: Endogenous Formation and Modulation, H.F. S t i c h (ed.), CRC Press, Boca Raton, FL, pp. 17-25. 126 Stich , H.F. and Rosin, M.P. (1984a) Micronuclei 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 studies i n cancer r i s k and cancer intervention. Cancer Lett., 22, 241-253. Stich , H.F. and Rosin, M.P. (1984b) Naturally occurring phenolics as antimutagenic and anticarcinogenic agents. In: N u t r i t i o n a l and T o x i c o l o g i c a l Aspects of Food Safety, M. Friedman (ed.), Plenum Press, New York, pp. 1-29. S t i c h , H.F. and Rosin, M.P. (1985) Towards a more comprehensive evaluation of a genotoxic hazard i n man. Mutation Res., 150. 43-50. St i c h , H.F. and Tsang, S.S. (1989) Promoting a c t i v i t y of b e t e l quid ingredients and t h e i r i n h i b i t i o n by r e t i n o l . Cancer Lett. , i n press. S t i c h , H.F., S t i c h W. and Parida, B.B. (1982a). Elevated frequency of micronucleated c e l l s i n the buccal mucosa of i n d i v i d u a l s at high r i s k f o r o r a l cancer: b e t e l quid chewers. Cancer Lett. , X7, 125-134. Stich, H.F., C u r t i s , J.R. and Parida, B.B. (1982b). A p p l i c a t i o n of the micronucleus t e s t to e x f o l i a t e d c e l l s of high cancer r i s k groups: tobacco chewers. Int. J . Cancer, 30, 553-559. Stich, H.F., Rosin, M.P. and V a l l e j e r a , M.O. (1984a) Reduction with vitamin A and beta-carotene administration of proportion of micronucleated buccal mucosal c e l l s i n Asian b e t e l nut and tobacco chewers. Lancet, 1_, 1204-1206. 127 Stich , H.F., St i c h , W., Rosin, M.P. and V a l l e j e r a , M.O. (1984b) Use of the micronucleus t e s t to monitor the e f f e c t of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of b e t e l nut/tobacco chewers. Int. J . Cancer, 34, 745-750. Stich, H.F., Dunn, B.P., P i g n a t e l l i , B., Ohshima, H. and Bartsch, H. (1982c) Dietary phenolics and b e t e l nut extracts as modifiers of N-nitrosation i n ra t and man. In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. O' N e i l l , R.C. von Bor s t e l , C.T. M i l l e r , J . Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency for Research on Cancer, Lyon, pp. 213-222. Sti c h , H.F., Hornby, A.P. and Dunn, B.P. (1985) A p i l o t beta-carotene i n t e r v e n t i o n t r i a l with Inuits using smokeless tobacco. Int. J . Cancer, 36. 321-327. Suda, D., Schwartz, J . and Shklar, G. (1986) I n h i b i t i o n of experimental o r a l carcinogenesis by t o p i c a l beta-carotene. Carcinogenesis, 7, 711-715. Swann, P.F. (1982) Metabolism of nitrosamines: observations on the e f f e c t of alcohol on nitrosamine metabolism and on human cancer. In: Nitrosamines and Human Cancer, P.N. Magee (ed.), Banbury Report 12, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 53-68. Tobacco I n s t i t u t e (1981) Tobacco Industry P r o f i l e 1981, Washington, D.C. Tobacco I n s t i t u t e (1982) Tobacco Industry P r o f i l e 1982, Washington, D.C. 128 Tobacco I n s t i t u t e (1983) Tobacco Industry P r o f i l e 1983, Washington, D.C. Tomita, I., Kinae, N. , Nakamura, Y., Takenaka, H., Kanamori, H., Hashizume, H. and Yokoyama, T. (1984) Mutagenicity of various Japanese foodstuffs treated with n i t r i t e . I I . D i r e c t l y - a c t i n g mutagens produced from N-containing compounds i n foodstuffs. In: N-Nitroso Compounds: Occurrence, B i o l o g i c a l E f f e c t s and Relevance to Human Cancer, I.K. O ' N e i l l , R.C. von B o r s t e l , C.T. M i l l e r , J . Long and H. Bartsch (eds.), IARC S c i . Publ. No. 57, International Agency for Research on Cancer, Lyon, pp. 33-41. U.S. Department of A g r i c u l t u r e (1983) Tobacco Outlook and S i t u a t i o n (TS-183), U.S. Department of A g r i c u l t u r e , Economic Research Service, Washington, D.C, March, pp. 5 - 8. Van Der Hoeven, J.C.M., Lagerweij, W.J., Van Gastel, A., Huitink, J . , De Dreu, R. and Van Broekhoven, L.W. (1984) I n t e r c u l t i v a r d i f f e r e n c e with respect to mutagenicity of fava beans ( V i c i a faba L.) a f t e r incubation with n i t r i t e . Mutation Res., 130, 391-394. Wahi, P.N., Kehar, U. and L a h i r i , B. (1965) Factors i n f l u e n c i n g o r a l and oropharyngeal cancer i n India. Br. J . Cancer, 19, 646-660. Walters, C.L., Carr, F.P.A., Dyke, C.S., Saxby, M.J., Smith, P.L.R. and Walker, R. (1979) N i t r i t e sources and nitrosamine formation i n v i t r o and i n vivo. Food Cosmet. T o x i c o l . , 17, 473-479. 129 Wenke, G. and Hoffmann, D. (1983) A study of b e t e l quid carcinogenesis. 1. On the i n v i t r o N -nitrosation of arecoline. Carcinogenesis, 4, 169-172. Wenke, G., Brunnemann, K.D., Hoffmann, D. and Bhide, S.V. (1984) A study of be t e l quid carcinogenesis. IV. Analysis of the s a l i v a of b e t e l chewers: a preliminary report. J . Cancer Res. C l i n . Oncol., 108. 110-113. WHO Collaborating Centre f o r Oral Precancerous Lesions (1978) D e f i n i t i o n of leukoplakia and r e l a t e d l e s i o n s : an a i d to studies on o r a l precancer. Oral Surg., 46, 518-539. Whong, W.Z., Stewart, J.D. and Ong, T. (1985) Formation of b a c t e r i a l mutagens from the r e a c t i o n of chewing tobacco with n i t r i t e . Mutation Res., 158. 105-110. Winn, D.M., Blot, W.J., Shy, CM., Pi c k l e , L.W., Toledo, M.A. and Fraumeni, J.F., J r (1981) Snuff dipping and o r a l cancer among women i n the southern United States. New Engl. J . Med., 304, 745-749. Winn, D.M., Zi e g l e r , R.G., P i c k l e , L.W., Gridley, G., Blot, W.J. and Hoover, R.N. (1984) Diet i n the et i o l o g y of o r a l and pharyngeal cancer among women from the southern United States. Cancer Res., 44, 1216-1222. World Health Organization (1980) Guide to epidemiology and diagnosis of o r a l mucosal diseases and conditions. Commun. Dent. Oral Epidemiol., 8, 1-26. Y i o r i s , N., Ivankovic, S. and Lehment, T. (1984) Oncology, 41, 36-38. 130 Zaridze, D.G., Blettner, M., Trapeznikov,. N.N., Kuvshinov, J.P., Matiakin, E.G., Poljakov, B.P., Poddubni, B.K., Parshikova, S.M., Rottenberg, V.I., Chamrakulov, F.S., Chodjaeva, M.C., Stic h , H.F., Rosin, M.P., Thurnham, D.I., Hoffmann, D. and Brunnemann, K.D. (1985) Survey of a population with a high incidence of o r a l and oesophageal cancer. Int. J . Cancer, 36, 153-158. Zaridze, D.G., Blettner, M., Matiakin, E.G., Poljakov, B.P., S t i c h , H.F., Rosin, M.P., Hoffmann, D. and Brunnemann, K.D. (1986) The e f f e c t of nass use and smoking on the r i s k of o r a l leukoplakia. Cancer Detect. Prev., 9, 435-440. Zi e g l e r , R., Mason, T., Stemhagen, A., Hoover, R., Schoenberg, J . , Gridley, G. and Fraumeni, J . , J r (1984) Carotene, f r u i t and vegetables, and lung cancer. Am. J . Epidemiol., 120, 499. 

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