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UBC Theses and Dissertations

The protective effect of beta-carotene and vitamin A on genotoxicity and cytotoxicity of dimethylnitrosamine… Yee, Gina 1986

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THE PROTECTIVE EFFECT OF BETA-CAROTENE AND VITAMIN A ON GENOTOXICITY AND CYTOTOXICITY OF DIMETHYLNITROSAMINE IN VITAMIN A-DEFICIENT RATS By GINA YEE B.Sc, University of Manitoba, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1986 © Gina Y e e , ]986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of DE-6 (3 /8 'n ABSTRACT E p i d e m i o l o g i c a l evidence and chemopreventive t r i a l s p o i n t t o an antimutagenic and a n t i c a r c i n o g e n i c r o l e f o r r e t i n o i d s and c a r o t e n o i d s . S t u d i e s on human p o p u l a t i o n s a re l o g i s t i c a l l y d i f f i c u l t and o f t e n e t h i c a l l y u nacceptable. T h e r e f o r e , t o g a i n i n f o r m a t i o n on the p o t e n t i a l chemopreventive b e n e f i t s o f d i e t a r y v i t a m i n A and beta - c a r o t e n e , the f o l l o w i n g experimental model was used. Rats were made v i t a m i n A ( r e t i n o l ) d e f i c i e n t , then exposed t o the l i v e r c a r c i n o g e n d i m e t h y l n i t r o s a m i n e (DMN), p a r t i a l l y hepatectomized, and t h e r e a f t e r , examined f o r the exte n t o f l i v e r n e c r o s i s ( c y t o t o x i c i t y ) and the frequency o f anaphase and t e l o p h a s e c e l l s w i t h chromosomal fragments, chromatin b r i d g e s and m i c r o n u c l e i ( g e n o t o x i c i t y ) . V i t a m i n A - d e f i c i e n t r a t s (serum r e t i n o l l e v e l s <2 0 ng/ml and l i v e r r e t i n o l l e v e l s <43 ng/g t i s s u e ) showed i n c r e a s e d s e n s i t i v i t y towards the c y t o t o x i c and gen o t o x i c a c t i o n o f DMN as compared t o v i t a m i n A - f e d d i e t a r y c o n t r o l r a t s . T h i s e l e v a t e d s e n s i t i v i t y was a b o l i s h e d by supplementation o f the v i t a m i n A - d e f i c i e n t d i e t w i t h beta-carotene (10 mg/kg d i e t ) o r w i t h v i t a m i n A (2 mg/kg d i e t ) p r i o r t o DMN exposure. However, v i t a m i n A f e e d i n g a t h i g h e r doses (16 mg/kg d i e t ) , which l e d t o an e x c e s s i v e accumulation of r e t i n o l i n the l i v e r (>25,000 ng/g t i s s u e ) , r e s u l t e d i n an a c c e n t u a t i o n o f the n e c r o t i c and i g e n o t o x i c a c t i o n o f DMN when h i g h doses (35 mg/kg body weight) were a d m i n i s t e r e d . The r e s u l t s show t h a t v i t a m i n A d e f i c i e n c y l e a d s t o an i n c r e a s e d s u s c e p t a b i l i t y t o DMN-induced damage. D i e t a r y supplementation w i t h beta-carotene and w i t h v i t a m i n A (at adequate l e v e l s ) p r o t e c t e d a g a i n s t the d e l e t e r i o u s e f f e c t s o f DMN, but supplementation w i t h v i t a m i n A a t e x c e s s i v e l e v e l s p o t e n t i a t e d the a c t i o n o f the ca r c i n o g e n . T h i s d i v e r g e n t , dose-dependent a c t i o n o f v i t a m i n A c a l l s f o r c a u t i o n i n the d e s i g n o f i n t e r v e n t i o n s t r a t e g i e s . i i TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS i i LIST OF FIGURES i i i LIST OF TABLES i v 1. INTRODUCTION 1.1 The Cancer Problem 1 1.2 Chemoprevention o f Cancer 2 1.3 O b j e c t i v e 4 1.4 The Carcinogen 5 1.5 The Model System 7 2. MATERIALS AND METHODS 2.1 Animals 10 2.2 D e s c r i p t i o n o f D i e t 10 2.3 D i e t a r y Regimen 11 2.4 Exposure o f Animals t o the Carcinogen 12 2.5 P a r t i a l Hepatectomy o f Animals 12 2.6 A n a l y s i s o f Serum and L i v e r R e t i n o l L e v e l s ... 13 2.7 P r e p a r a t i o n o f L i v e r T i s s u e S e c t i o n s 14 3. RESULTS 3.1 R e t i n o l D e f i c i e n c y 16 3.2 R e v e r s a l o f R e t i n o l D e f i c i e n c y by D i e t a r y Supplementation w i t h V i t a m i n A 19 3.3 R e v e r s a l of R e t i n o l D e f i c i e n c y by D i e t a r y Supplementation w i t h Beta-carotene 20 3.4 P r o t e c t i v e E f f e c t o f R e t i n o l 3.4.1 C y t o t o x i c i t y 21 3.4.2 M i t o t i c I n h i b i t i o n 23 3.4.3 G e n o t o x i c i t y 25 3.5 P r o t e c t i v e E f f e c t o f Beta-carotene 3.5.1 C y t o t o x i c i t y 29 3.5.2 M i t o t i c I n h i b i t i o n 31 3.5.3 G e n o t o x i c i t y 32 4. DISCUSSION 58 5. SUMMARY 75 6. CONCLUDING REMARKS 79 BIBLIOGRAPHY 81 i i i LIST OF FIGURES PAGE FIGURE 1. Schematic I l l u s t r a t i o n of Dietary Regimens and Experimental Design 34 FIGURE 2. Growth Curves of Rats Maintained on Different Dietary Regimens 35 FIGURE 3. Liver Section of a Vitamin A-deficient Rat Exposed to DMN (to t a l dose of 3 0 mg/kg body weight) 3.1 x 10 2 mag'n 3 6 FIGURE 4. Liver Section of a Vitamin A-deficient Rat Exposed to DMSO (control) 3.1 x 10 2 mag'n .. 36 FIGURE 5. Liver Section of a Vitamin A-deficient Rat Exposed to DMN (to t a l dose of 30 mg/kg body weight) 1.0 x 10 3 mag'n 37 FIGURE 6. Liver Section of a Vitamin A-deficient Rat Exposed to DMSO (control) 1.0 x 10 3 mag'n .. 3 7 FIGURE 7. Liver Section of a Vitamin A-deficient Rat Exposed to DMN (to t a l dose of 3 0 mg/kg body weight) 3.1 x 10 2 mag'n 38 FIGURE 8. Tracing of Liver Section of a Vitamin A-de f i c i e n t Rat (in Figure 7) with Necrotic Areas of Tissue Circumscribed 38 FIGURE 9. Normal Anatelophase C e l l 3.1 x 10 3 mag'n ... 39 FIGURE 10. Anatelophase C e l l with a Chromosomal Fragment 3.1 x 10 mag'n 39 FIGURE 11. Anatelophase C e l l with a Chromatin Bridge 3.1 x 10 3 mag'n 4 0 FIGURE 12. Anatelophase C e l l with Micronuclei 3.1 x 10 3 mag'n 40 FIGURE 13. Anatelophase C e l l with a Chromatin Bridge and Micronuclei 3.1 x 10 3 mag'n 41 i v LIST OF TABLES PAGE TABLE 1. Composition of V i t a m i n A - f r e e D i e t 42 TABLE 2. Dimethylnitrosamine Treatment Schedule 43 TABLE 3. R e t i n o l L e v e l s i n Serum of Rats on a Vi t a m i n A-adequate (A+) or a V i t a m i n A-f r e e (A-) D i e t f o r 9 t o 10 Weeks 44 TABLE 4. R e t i n o l L e v e l s i n Serum and L i v e r a t Time o f S a c r i f i c e o f Rats on V i t a m i n A-adequate (A+) o r V i t a m i n A - f r e e (A-) D i e t f o r 10 t o 11 Weeks 45 TABLE 5. R e t i n o l L e v e l s i n Serum and L i v e r o f V i t a m i n A - d e f i c i e n t Rats f o l l o w i n g Supplementation of the D i e t w i t h V i t a m i n A a t Adequate L e v e l s (A~VA) or a t Boosted L e v e l s (A"8VA) f o r 17 t o 24 Days 46 TABLE 6. R e t i n o l l e v e l s i n Serum and L i v e r o f V i t a m i n A - d e f i c i e n t Rats f o l l o w i n g Supplementation of the D i e t w i t h Beta-carotene (A~BC) f o r 17 t o 18 Days 47 TABLE 7. P r o t e c t i v e E f f e c t o f D i e t a r y R e t i n o l Supplementation on the C y t o t o x i c A c t i o n o f DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 48 TABLE 8. M i t o t i c Index i n L i v e r s o f P a r t i a l Hepatectomized Rats 49 TABLE 9a. P r o t e c t i v e E f f e c t o f D i e t a r y V i t a m i n A Supplementation on the Genotoxic A c t i o n of DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 50 TABLE 9b. P r o t e c t i v e E f f e c t o f D i e t a r y V i t a m i n A Supplementation on the Genotoxic A c t i o n of DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 51 TABLE 10. P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the C y t o t o x i c A c t i o n o f DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 52 V TABLE 11a. P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the G e n t o t x i c A c t i o n of DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 53 TABLE l i b . P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the Genotoxic A c t i o n of DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats 54 TABLE 12. Chemopreventive I n t e r v e n t i o n S t u d i e s Supported by the U.S. N a t i o n a l Cancer I n s t i t u t e 55 TABLE 13. R e p r e s e n t a t i v e Types of C a r o t e n o i d s and Apo-carotenoids w i t h P r o v i t a m i n A A c t i v i t y .. 57 v i 1. INTRODUCTION 1.1 THE CANCER PROBLEM The World H e a l t h O r g a n i z a t i o n (WHO) has estimated t h a t , on a worldwide b a s i s , approximately e i g h t m i l l i o n people w i l l develop cancer each year (World h e a l t h s t a t i s t i c s , 1980). In Canada, one o f every t h r e e persons w i l l s u f f e r from some type of cancer some time i n h i s or her l i f e t i m e . About e i g h t y p e r c e n t o f a l l cancers are b e l i e v e d t o be caused p r i m a r i l y by environmental determinants over which an i n d i v i d u a l can e x e r c i s e some c o n t r o l (Greenwald and C u l l e n , 1985) . These, i n p r i n c i p l e , are p r e v e n t a b l e . The removal o f cancer c a u s a t i v e agents i s the most d e s i r a b l e way of r e d u c i n g the impact o f cancer on man. U n f o r t u n a t e l y , d i f f i c u l t i e s a r i s e because many o f the e x t r i n s i c f a c t o r s c a u s a l l y a s s o c i a t e d w i t h the most common human m a l i g n a n c i e s — s o l a r UV w i t h s k i n n e o p l a s i a , smoking wi t h l u n g and o r a l cancer, and d i e t w i t h b r e a s t , stomach and c o l o n c a r c i n o m a s — a r e so c l o s e l y t i e d t o c u l t u r a l p r a c t i c e s and p e r s o n a l h a b i t s t h a t t h e i r e l i m i n a t i o n from s o c i e t y i n the f o r e s e e a b l e f u t u r e i s h i g h l y u n l i k e l y without the i n s t i l l m e n t of major l i f e s t y l e changes (Paterson and Gentner, 1984). In a d d i t i o n , many carcinogens and suspected c a r c i n o g e n s are u b i q u i t o u s i n foods and i n the environment, making t h e i r removal v i r t u a l l y i m p o s s i b l e . T h e r e f o r e , w h i l e the avoidance of exposure t o cancer c a u s i n g agents remains the primary 1 s t r a t e g y f o r p r e v e n t i n g cancer, the development of a second l i n e o f p r e v e n t i o n , based on chemoprevention, i s needed when the former i s not p o s s i b l e or p r a c t i c a l (Wattenburg, 1985). 1.2 CHEMOPREVENTION OF CANCER Chemoprevention i s d e f i n e d by Wattenburg (1985) as "the means o f cancer c o n t r o l i n which the occurrence of t h i s d i s e a s e i s prevented by the a d m i n i s t r a t i o n of one or s e v e r a l chemical compounds". The s e a r c h f o r cancer p r e v e n t a t i v e agents t h a t can be p r e s c r i b e d i s an a t t r a c t i v e a l t e r n a t i v e t o the i d e n t i f i c a t i o n o f cancer c a u s i n g substances t h a t must be avoided and p r o s c r i b e d , f o r people may be more a c c e p t i n g of p r e s c r i p t i o n than of p r o s c r i p t i o n (Peto e t a l . , 1981). A c o n s i d e r a b l e number of compounds, both s y n t h e t i c and n a t u r a l l y - o c c u r r i n g , b e l o n g i n g t o over twenty d i f f e r e n t c h e m i c a l c l a s s e s , demonstrate a chemopreventative c a p a c i t y (reviewed by S l a g a and Digiovanne, 1984). I t i s noteworthy t h a t many of these cancer i n h i b i t o r s are c o n s t i t u e n t s o f common food p r o d u c t s . V i t a m i n A ( r e t i n o l ) and i t s d i e t a r y p r e c u r s o r , beta-carotene, f o r example, are p r o t e c t i v e agents t h a t are w i d e l y d i s t r i b u t e d i n f r u i t s , v e g e t a b l e s and animal p r o d u c t s . The presence o f a n t i c a r c i n o g e n i c substances i n food p r o d u c t s suggests t h a t cancer may be p r e v e n t a b l e through d i e t a r y m o d i f i c a t i o n . T h i s then p r o v i d e s a n u t r i t i v e b a s i s f o r cancer chemoprevention. 2 E p i d e m i o l o g i c a l s t u d i e s i n d i c a t e t h a t the i n c i d e n c e o f cancer i n humans may be lower among i n d i v i d u a l s who i n c l u d e f r e s h f r u i t s and v e g e t a b l e s i n t h e i r d i e t . Whether t h i s r e d u c t i o n i n cancer r i s k may be a t t r i b u t e d t o v i t a m i n A, bet a - c a r o t e n e , o r some oth e r p l a n t - d e r i v e d a n t i c a r c i n o g e n , cannot be d e l i n e a t e d from these s t u d i e s . R e t r o s p e c t i v e c a s e - c o n t r o l s t u d i e s suggest a p r o p h y l a c t i c e f f e c t o f d i e t a r y r e t i n o l and/or carotenes a g a i n s t subsequent development of c e r t a i n tumours, i n c l u d i n g carcinoma of the lu n g ( Z i e g l e r e t a l . , 1984; Mac Lennan e t a l . , 1977), stomach (Correa, 1985), l a r y n x (Graham e t a l . , 1984) and t o some degree, cancers o f the b r e a s t and ovary (Byers e t a l . , 1984). P r o s p e c t i v e s t u d i e s show t h a t low serum r e t i n o l l e v e l s are c o r r e l a t e d w i t h i n c r e a s e d r i s k f o r cancers o f the lu n g ( S h e k e l l e e t a l . , 1981; Wald e t a l . , 1980; Hirayama, 1979; B j e l k e , 1975), oesophagous (Yang e t a l . , 1984), p r o s t r a t e , s k i n , leukaemias, and Hodgkin's d i s e a s e (Kark e t a l . , 1982), as w e l l as tumours of the female r e p r o d u c t i v e t r a c t and the g a s t r o i n t e s t i n a l system (Kark e t a l . , 1981). Other r e p o r t s however, do not c o n f i r m these f i n d i n g s (Normura e t a l . , 1985; W i l l e t t e t a l . , 1984). Many q u e s t i o n s o f academic and p r a c t i c a l importance r e g a r d i n g cancer chemoprevention cannot be r e a d i l y answered by s t u d y i n g human p o p u l a t i o n s . L o g i s t i c a l l y , i n t e r v e n t i o n t r i a l s u s i n g the appearance of cancer as an endpoint are d i f f i c u l t t o conduct because of the l a r g e number of i n d i v i d u a l s r e q u i r e d , 3 the l o n g timespan and the e x c e s s i v e c o s t . As w e l l , e t h i c a l q u e s t i o n s a r i s e when c o n s i d e r i n g l a r g e s c a l e i n t e r v e n t i o n t r i a l s i n human p o p u l a t i o n s w i t h substances and dosages of un d e f i n e d a c t i v i t y . I t i s , t h e r e f o r e , necessary t o d e v i s e an animal model system which s i m u l a t e s as c l o s e l y as p o s s i b l e the c o n d i t i o n s p r e v a i l i n g i n man, and t o use t h i s model system t o t e s t the chemopreventative b e n e f i t s o f v i t a m i n A and bet a - c a r o t e n e f o r h i g h r i s k p o p u l a t i o n s and f o r the p o p u l a t i o n a t l a r g e . 1.3 OBJECTIVE The s p e c i f i c o b j e c t i v e of t h i s p r o j e c t was t o d e s i g n a s e n s i t i v e and economical, s h o r t term i n v i v o assay system t o determine the i n h i b i t o r y e f f i c a c y o f the d i e t a r y chemopreventive agents, v i t a m i n A and beta-carotene, a g a i n s t the p r o d u c t i o n o f carc i n o g e n - i n d u c e d damage t o the genome d u r i n g cancer i n d u c t i o n . More s p e c i f i c a l l y , w i t h t h i s t e s t system the f o l l o w i n g q u e s t i o n s were addressed: (1) Does v i t a m i n A d e f i c i e n c y i n c r e a s e the s e n s i t i v i t y t o exposure t o a chemical carcinogen? (2) Can a h y p e r s e n s i t i v e s t a t e which i s due t o v i t a m i n A d e f i c i e n c y be a b o l i s h e d by supplementation o f the d e f i c i e n t d i e t w i t h v i t a m i n A? 4 (3) Can supplementation of the d e f i c i e n t d i e t w i t h v i t a m i n A a t boosted amounts (8 x recommended d i e t a r y l e v e l s ) p r o v i d e g r e a t e r p r o t e c t i o n a g a i n s t a chemical carcinogen? (4) Can the h y p e r s e n s i t i v e s t a t e be a b o l i s h e d by supplementation of the d e f i c i e n t d i e t w i t h beta-carotene, which may be m e t a b o l i c a l l y c o n v e r t e d i n t o r e t i n o l and may have a n t i c a r c i n o g e n i c a c t i v i t i e s i n h e r e n t i n i t s e l f ? The experimental d e s i g n f o l l o w e d t h r e e b a s i c s t e p s . F i r s t , v i t a m i n A d e f i c i e n c y was induced i n r a t s by d i e t a r y d e p r i v a t i o n o f v i t a m i n A. Second, the v i t a m i n A - d e f i c i e n t s t a t e was r e v e r s e d i n some animals, p r i o r t o c a r c i n o g e n exposure, by d i e t a r y supplementation w i t h v i t a m i n A a t adequate o r a t boosted l e v e l s , o r by d i e t a r y supplementation w i t h b e t a - c a r o t e n e . F i n a l l y , the chemopreventive b e n e f i t s of v i t a m i n A and beta-carotene were assessed by measuring the r e l a t i v e e x t e n t of c y t o t o x i c and g e n o t o x i c damage induced by the c a r c i n o g e n i n animals from the d i f f e r e n t d i e t a r y treatment groups. 1.4 THE CARCINOGEN N-nitroso-dimethylamine [ d i m e t h y l n i t r o s a m i n e , (DMN)] i s a member o f the f a m i l y o f compounds known as N - n i t r o s o compounds. The d e t e c t i o n of N - n i t r o s o compounds i n tobacco smoke from tobacco amines and n i t r o u s gases, f i r s t d e s c r i b e d 5 by Druckrey e t a l . , (1963), was the e a r l i e s t example of the environmental importance of t h i s group of c a r c i n o g e n s . N - n i t r o s o compounds are a l s o formed endogenously by r e a c t i o n s between n i t r o s a t a b l e amino compounds (present i n foods, drugs, cosmetics, and the l i k e ) and from n i t r o s a t i n g agents such as n i t r i t e and n i t r o g e n oxides (NOx). DMN i s o f p a r t i c u l a r i n t e r e s t because of i t s wide d i s t r i b u t i o n i n beer, f i s h , meat and meat pro d u c t s , cheese and d a i r y p r o d u c t s , tobacco smoke, cosmetics, d r i n k i n g water and a i r (Preussmann and Eisenbrand, 1984) . A methyl carbonium i o n , generated by the m e t a b o l i c a c t i v a t i o n o f DMN by P-450 dependant microsomal h y d r o x y l a s e s i n t h e l i v e r , i s b e l i e v e d t o be the u l t i m a t e c a r c i n o g e n . T h i s h i g h l y r e a c t i v e e l e c t r o p h i l i c s p e c i e s a t t a c k s c e l l u l a r c o n s t i t u e n t s , s p e c i f i c a l l y p r o t e i n s and n u c l e i c a c i d s . In some i n s t a n c e s , t h i s r e s u l t s i n c e l l death ( c e l l n e c r o s i s , CN), and i n o t h e r i n s t a n c e s , t h i s l e a d s t o breaks and rearrangements i n the DNA. Breakage events t h a t produce chromosome and chromatid i n t e r c h a n g e s may be v i s u a l i z e d by the f o r m a t i o n of chromatin b r i d g e s (CB) d u r i n g l a t e anaphase. Breakage events t h a t generate a c e n t r i c chromosomal fragments (CF) and s p i n d l e a b n o r m a l i t i e s t h a t produce l a g g i n g chromosomes may l e a d t o the f o r m a t i o n of m i c r o n u c l e i (MN) i n the l a t e anaphase and t e l o p h a s e c e l l cytoplasm. Thus, DMN-induced damage t o the genome can be q u a n t i t a t e d by the appearance of CF, CB and MN i n anaphase and t e l o p h a s e c e l l s 6 f o l l o w i n g t h e s e events. The l i v e r i s the s p e c i f i c t a r g e t t i s s u e f o r t h i s h e p a t o t o x i n and hepatocarcinogen i n r a t s . An advantage t o u s i n g DMN i n t h i s study i s t h a t f u t u r e a n a l y s i s o f the l i v e r s o f DMN-exposed r a t s f o r p e r s i s t i n g methylated p r o d u c t s (such as DNA-adducts) may be c a r r i e d out, thereby d i r e c t l y r e l a t i n g the g e n e r a t i o n of DMN-induced damage w i t h DMN-products. 1.5 THE MODEL SYSTEM To a c h i e v e the purpose d e s c r i b e d i n s e c t i o n 1.3, a r a t l i v e r t e s t system was developed t o p r o v i d e a r a p i d and powerful assay f o r the e v a l u a t i o n of the chemopreventive b e n e f i t s o f d i e t a r y v i t a m i n A and b e t a - c a r o t e n e . The extent of CN and the appearance of CF, CB and MN i n anaphase and t e l o p h a s e ( 1 a n a t e l o p h a s e 1 ) c e l l s i n the r a t l i v e r , were asses s e d as endpoint i n d i c a t o r s f o r the c y t o t o x i c and g e n o t o x i c e f f e c t s of the complete c a r c i n o g e n , DMN. These n u c l e a r a b e r r a t i o n s have been p r e v i o u s l y d e s c r i b e d i n r e l a t i v e d e t a i l (Ashby and B e i j e , 1985; Dulout and O l i v e r a , 1984; Den Engelse e t a l . , 1983) and have been shown t o c o r r e l a t e w e l l w i t h tumour development (De Kok e t a l . , 1985; P a r o d i e t a l . , 1981; L u t z , 1979). The advantage o f s c o r i n g chromosomal anomalies i n anatelophases was t h a t the whole c e l l can be a s s e s s e d as a s i n g l e u n i t (Kocan e t a l . , 1982). Chromosomal s t u d i e s i n metaphase c e l l s , on the o t h e r hand, 7 r e q u i r e the a n a l y s i s o f each chromosome s e p a r a t e l y . Through the m a n i p u l a t i o n o f the d i e t a r y regimen o f r a t s , the chemopreventative p o t e n t i a l o f v i t a m i n A and of beta-carotene, a g a i n s t the g e n e r a t i o n o f these endpoints by DMN, was i n v e s t i g a t e d . Advantages t o u s i n g the r a t l i v e r as a t e s t system have been d e s c r i b e d p r e v i o u s l y (Williams 1981; Tates e t a l . , 1980; Barbason e t a l . , 1975) and are: (1) the l i v e r i s the most a c t i v e d r u g - m e t a b o l i z i n g organ and i s t h e r e f o r e w e l l s u i t e d f o r the study o f the e f f e c t s of i n d i r e c t mutagens and car c i n o g e n s such as DMN; (2) the l i v e r i s a l s o the major s t o r a g e s i t e and an important t a r g e t t i s s u e f o r v i t a m i n A and b e t a - c a r o t e n e — t h e degree of v i t a m i n A d e f i c i e n c y can be r e a d i l y q u a n t i t a t e d by measuring r e t i n o l l e v e l s i n l i v e r t i s s u e samples (and serum samples) from r a t s by h i g h p r e s s u r e l i q u i d chromatography (HPLC) a n a l y s i s ; (3) l i v e r s p e c i f i c damage i s induced by DMN without confounding damage t o o t h e r t i s s u e s — i t i s l o g i c a l l y a t t r a c t i v e t o i n v e s t i g a t e i n the same t i s s u e the a c t i v a t i o n of the p r o c a r c i n o g e n , the a c t i o n of the r e s u l t i n g c a r c i n o g e n and the i n t e r v e n t i o n o f the a n t i c a r c i n o g e n ; (4) m i t o s i s , which i s e s s e n t i a l f o r the e x p r e s s i o n o f the c l a s t o g e n i c endpoints (CF, CB and MN), can be s t i m u l a t e d i n t he q u i e s c e n t a d u l t r a t l i v e r by s u r g i c a l removal of two-t h i r d s o f the l i v e r [ i . e . , p a r t i a l hepatectomy (PH)]. 8 V e r s a t i l i t y i n the t e s t system a l l o w s f o r f u t u r e e x p l o r a t i o n o f a d d i t i o n a l q u e s t i o n s t h a t are important t o cancer p r e v e n t i o n , among them: (1) What are the cancer i n h i b i t o r y e f f i c a c i e s o f d i e t a r y chemopreventive agents b e s i d e s v i t a m i n A and beta-carotene, such as s t e r o l s , p h e n o l i c s , f l a v o n o i d s and o t h e r c a r o t e n o i d s ? (2) Are v i t a m i n A, beta-carotene and o t h e r chemopreventive agents e q u a l l y e f f e c t i v e a g a i n s t a v a r i e t y of environmental and chemical carcinogens? (3) What i n f l u e n c e s do o t h e r d i e t a r y f a c t o r s , i n c l u d i n g f a t s , f i b r e , a l c o h o l and n u t r i t i o n a l d e f i c i e n c i e s o f v i t a m i n E, selenium and r i b o f l a v i n , have on c a r c i n o g e n s e n s i t i v i t y ? (4) I f e f f e c t i v e chemopreventive agents are i d e n t i f i e d w i t h t h i s t e s t system, how might t h i s p r o t e c t i o n be brought about e f f i c i e n t l y f o r humans? The broader aim of t h i s p r o j e c t was t o develop a g e n e r a l t e s t system w i t h which a wide range of i n f o r m a t i o n r e l e v a n t t o cancer c o n t r o l c o u l d be c o l l e c t e d . Based upon the s e data, e f f e c t i v e chemopreventative s t r a t e g i e s c o u l d be designed and a p p l i e d t o human p o p u l a t i o n groups t h a t are a t an e l e v a t e d r i s k f o r the development of c e r t a i n types o f cancer and t o the g e n e r a l p o p u l a t i o n . 9 2. MATERIALS AND METHODS 2.1 ANIMALS Male weanling (21 day old) W i s t a r r a t s were o b t a i n e d from C h a r l e s R i v e r Canada Inc., S t . Constant, Quebec and were housed i n suspended wire mesh bottom cages i n c l i m a t e c o n t r o l l e d f a c i l i t i e s w i t h a l t e r n a t i n g 12 hour l i g h t - d a r k p e r i o d s and tap water s u p p l i e d ad l i b i t u m . 2.2 DESCRIPTION OF DIET The composition of the v i t a m i n A - f r e e s e m i s y n t h e t i c d i e t has been d e s c r i b e d p r e v i o u s l y by Suphakarn e t a l . , (1983) and i s p r e s e n t e d i n T a b l e 1. The v i t a m i n A - d e p r i v e d r a t s were f e d the v i t a m i n A - f r e e d i e t s . The v i t a m i n A-adequate d i e t (as d e f i n e d by the Ad Hoc Comittee, 1977) was prepared by supplementing the v i t a m i n A - f r e e d i e t w i t h 2 mg r e t i n y l a c e t a t e (Sigma Chemical Co.) per kg d i e t . The v i t a m i n A-boosted d i e t was prepared by supplementing the v i t a m i n A - f r e e d i e t w i t h 16 mg r e t i n y l a c e t a t e per kg d i e t (8 x adequate v i t a m i n A l e v e l s ) . The beta-carotene d i e t was prepared by supplementing the v i t a m i n A - f r e e d i e t w i t h 10 mg beta-carotene (Hoffman-La Roche Co.) per kg d i e t . R e t i n y l a c e t a t e or b e t a - c a r o t e n e was added i n t o the c o r n o i l , a l o n g w i t h a l l of the o t h e r v i t a m i n s , a t the time of d i e t p r e p a r a t i o n . A l l d i e t s were mixed w i t h 3% agar s o l u t i o n s and were made i n t o 10 g e l s t h a t c o n t a i n e d 50% water. D i e t s were prepared under y e l l o w l i g h t , t w i ce weekly and s t o r e d a t 4°C r e f r i g e r a t i o n temperature. 2.3 DIETARY REGIMEN Rats were f e d ad l i b i t u m a d i e t c o n t a i n i n g adequate v i t a m i n A or no added v i t a m i n A. A f t e r 9 t o 10 weeks, the v i t a m i n A - d e p r i v e d r a t s began t o show symptoms of v i t a m i n A d e f i c i e n c y . C o n f i r m a t i o n o f the v i t a m i n A - d e f i c i e n t s t a t e i n these r a t s was c a r r i e d out by HPLC a n a l y s i s o f t h e i r serum r e t i n o l l e v e l s from a b l o o d sample o b t a i n e d from the t a i l v e i n (Table 3 ) . One week p r i o r t o c a r c i n o g e n exposure, groups o f no l e s s than 10 d e f i c i e n t r a t s were switched from the v i t a m i n A - f r e e d i e t t o the v i t a m i n A-adequate d i e t (A~VA), the v i t a m i n A-boosted d i e t (A~8VA) or the beta-carotene d i e t (A~BC), w h i l e the remaining r a t s were l e f t on the v i t a m i n A - f r e e d i e t (A~). Rats were then maintained on the switched d i e t s f o r the remainder of the experiment. Rats i n i t i a l l y g i v e n v i t a m i n A-adequate d i e t (A+) were mai n t a i n e d on t h i s d i e t f o r the e n t i r e l e n g t h of the experiment. T h i s treatment group served as v i t a m i n A - f e d d i e t a r y c o n t r o l s . A schematic i l l u s t r a t i o n o f the g e n e r a l experimental d e s i g n i s g i v e n i n F i g u r e 1. 11 2.4 EXPOSURE OF ANIMALS TO THE CARCINOGEN N-nitrosodimethylamine (DMN) was o b t a i n e d from A l d r i c h Chemical Company. In a l l experiments r a t s were g i v e n i n t r a p e r i t o n e a l i n j e c t i o n s o f f r e s h l y prepared s o l u t i o n s o f DMN i n di m e t h y l s u l f o x i d e (DMSO) from Sigma Chemical Company f o r t h r e e s u c c e s s i v e days. The DMN doses and the d o s i n g schedule a r e d e s c r i b e d i n Tab l e 2. For example, a t o t a l DMN dose o f 3 0 mg/kg body weight was d e l i v e r e d i n t h r e e d a i l y i n j e c t i o n s o f 10 mg/kg body weight c o n c e n t r a t i o n . In Experiment A, some r a t s were g i v e n a t o t a l DMN dose o f 35 mg/kg which was d e l i v e r e d as 3 d a i l y i n j e c t i o n s o f 5 mg/kg body weight f o l l o w e d 5 days l a t e r by 2 more d a i l y i n j e c t i o n s o f 10 mg/kg body weight. 2.5 PARTIAL HEPATECTOMY OF ANIMALS S u r g i c a l removal of t w o - t h i r d s o f the l i v e r was c a r r i e d out 5 days f o l l o w i n g the l a s t DMN i n j e c t i o n , a c c o r d i n g t o p u b l i s h e d procedures (Higgins and Anderson, 1931), except t h a t i n c i s i o n s were c l o s e d w i t h 14 o r 18 mm wound c l i p s (100 M i c h e l ) . A l l s u r g i c a l procedures were c a r r i e d out between 8:30 am and 12:00 pm. Animals were k i l l e d by c a r d i a c puncture 48 hours f o l l o w i n g p a r t i a l hepatectomy. Serum and l i v e r samples were c o l l e c t e d from each r a t f o r r e t i n o l a n a l y s i s and f o r t i s s u e s e c t i o n s . 12 2.6 ANALYSIS OF SERUM AND LIVER RETINOL LEVELS Serum r e t i n o l l e v e l s were measured by i s o c r a t i c h i g h -p r e s s u r e l i q u i d chromatography (HPLC) a c c o r d i n g t o the procedure p r e v i o u s l y d e s c r i b e d by S t i c h e t a l . , (1985). 200 u l o f 95% e t h a n o l was added t o 200 u l o f serum i n a 1.5 ml m i c r o c e n t r i f u g e tube. Samples were v o r t e x e d f o r 2 minutes and heated f o r 1 hour i n a 50°C h e a t i n g b l o c k . A f t e r a l l o w i n g the samples t o c o o l , 400 u l o f hexane was added. The samples were v o r t e x e d a g a i n f o r 2 minutes and then c e n t r i f u g e d a t 3,500g f o r 5 minutes. A 300 u l a l i q u o t o f the upper-organic l a y e r was t r a n s f e r r e d t o a second m i c r o c e n t r i f u g e tube. Hexane i n the a l i q u o t e d samples was completely d r i e d o f f i n a Savant s p i n e v a p o r a t o r and the samples were resuspended i n 200 u l methanol. 50 u l of t h i s p r e p a r a t i o n was used f o r HPLC a n a l y s i s . A l l procedures were c a r r i e d out i n y e l l o w l i g h t . HPLC a n a l y s i s was done u s i n g a Vydac 201 TP reverse-phase column, i n t e r n a l diameter 3.2 mm, l e n g t h 25 cm, 10 micron p a r t i c l e s . The running s o l v e n t c o n s i s t e d of methanol p l u s 1% water, a t a flow r a t e o f 0.5 ml/minute. Peak d e t e c t i o n was made u s i n g a Perkin-Elmer LC-55 Spectrophotometer w i t h a tun g s t e n lamp s e t a t 325 nm. C a l i b r a t i o n was made w i t h a sta n d a r d o f 20 ng r e t i n o l (Sigma Chemical Co.) i n 50 u l methanol and determined s p e c t r o p h o t o m e t r i c a l l y a t 325 nm. L i v e r r e t i n o l l e v e l s were measured from r a t l i v e r t i s s u e samples (200 mg) t h a t were s a p o n i f i e d w i t h 5% KOH i n 95% et h a n o l (200 u l ) and incubated a t 50°C f o r 1 hour i n a 13 m i c r o c e n t r i f u g e tube. A f t e r a l l o w i n g the samples t o c o o l , 400 u l o f hexane was added t o each of the samples and r e t i n o l a n a l y s i s was c a r r i e d out i n the same manner as d e s c r i b e d above f o r serum samples. The mean l i v e r and serum r e t i n o l l e v e l s of r a t s from the d i f f e r e n t d i e t a r y groups were determined and were compared t o each o t h e r u s i n g the Mann-Whitney U s t a t i s t i c a l t e s t . The P-values a r e r e f e r r e d t o i n the a p p r o p r i a t e s e c t i o n s of the t e x t . Groups were c o n s i d e r e d s i g n i f i c a n t l y d i f f e r e n t i f P<0.05. 2.7 PREPARATION OF LIVER TISSUE SECTIONS At the time o f s a c r i f i c e , f o u r s e l e c t e d p o r t i o n s of the r a t l i v e r were f i x e d i n e t h a n o l - g l a c i a l a c e t i c a c i d (3:1) f o r 4 hours. T i s s u e p i e c e s were then removed and p l a c e d i n 70% e t h a n o l o v e r n i g h t . The l i v e r p i e c e s were pr o c e s s e d f o r 1 hour i n each o f t h e f o l l o w i n g s o l u t i o n s s e q u e n t i a l l y : 80% e t h a n o l , 95% e t h a n o l , two changes of b u t a n o l , two changes o f xylene and xylene/wax (1:1). T i s s u e p i e c e s were then embedded i n P a r a p l a s t t i s s u e embedding medium (Lancer, S t . L o u i s , MO.). S e c t i o n s o f t i s s u e were cu t a t 10 microns t h i c k n e s s onto a microscope s l i d e , and were s t a i n e d by the Feulgen method (Feulgen and Rossenbeck, 1924), which s p e c i f i c a l l y s t a i n s f o r DNA, and then c o u n t e r s t a i n e d w i t h F a s t green f o r t h e v i s u a l i z a t i o n o f the cytoplasm and the c e l l boundary. 14 B r i e f l y , the s t a i n i n g procedure i n v o l v e d p r o c e s s i n g the t i s s u e s e c t i o n s f o r 2 minutes i n each of 95% e t h a n o l , 70% e t h a n o l , d i s t i l l e d water and IN HC1 a t room temperature, p r i o r t o h e a t i n g t h e s e c t i o n s f o r 10 minutes i n IN HC1 a t 60°C. S e c t i o n s were r e t u r n e d t o the IN HC1 s o l u t i o n a t room temperature f o r 2 minutes and then p l a c e d i n t o S c h i f f ' s s t a i n f o r 1 hour. S e c t i o n s were removed from t h e S c h i f f ' s s t a i n , r i n s e d i n c o l d t a p water f o r 15 minutes, and then c o u n t e r s t a i n e d f o r a few seconds w i t h F a s t green ( F i s h e r S c i e n t i f i c , F a i r l a w n , NJ) d i s s o l v e d i n 95% e t h a n o l . By t h i s method, the p u r p l e CF, CB and MN i n the green cytoplasm were taken t o t r u l y r e p r e s e n t m a t e r i a l o f chromosomal o r i g i n (Heddle, 1973; Tates e t a l . , 1980). The mean l e v e l o f n e c r o s i s i n the r a t l i v e r and the average frequency of anomalies i n anaphase and t e l o p h a s e l i v e r c e l l s were determined f o r r a t s from the d i f f e r e n t d i e t a r y groups and were s u b j e c t e d t o Mann-Whitney U s t a t i s t i c a l a n a l y s i s . P-values are r e f e r r e d t o i n the a p p r o p r i a t e s e c t i o n s o f the t e x t . 15 3. RESULTS 3.1 RETINOL DEFICIENCY Rats f e d the v i t a m i n A - f r e e s e m i s y n t h e t i c d i e t f o r 9 t o 10 weeks d i s p l a y e d o v e r t s i g n s of v i t a m i n A d e f i c i e n c y , i n c l u d i n g growth p l a t e a u , disarrangement o f h a i r c o a t , and s e c r e t i o n s about the eyes of some o f the r a t s . F i g u r e 2 shows the p a t t e r n of weight g a i n of animals s u b j e c t e d t o the d i f f e r e n t d i e t a r y regimens through the course of t h e experiments. Supplementation of the v i t a m i n A - d e f i c i e n t d i e t w i t h v i t a m i n A or w i t h beta-carotene was marked by the resumption of growth. Some i n v e s t i g a t o r s r e g a r d the onset of the weight p l a t e a u as synonymous w i t h v i t a m i n A - d e f i c i e n c y (Dowling e t a l . , 1960; Sherman, 1961; Underwood, 1984). C o n f i r m a t i o n o f the d e f i c i e n t s t a t u s o f the v i t a m i n A - d e p r i v e d r a t s i n t h i s study was c a r r i e d out by HPLC a n a l y s i s o f serum r e t i n o l l e v e l s i n b l o o d samples o b t a i n e d from the t a i l v e i n . The r e s u l t s , p r e s e n t e d i n T a b l e 3, r e p r e s e n t the serum r e t i n o l l e v e l s o f r a t s a f t e r 9 t o 10 weeks on the s p e c i f i e d d i e t s and 1 week p r i o r t o c a r c i n o g e n exposure. Under normal circ u m s t a n c e s , serum r e t i n o l l e v e l s i n the r a t are maintained at homeostatic l e v e l s w i t h l i t t l e v a r i a n c e (Underwood, 1984). The s i g n i f i c a n t d e p r e s s i o n i n serum r e t i n o l l e v e l s of the v i t a m i n A - d e p r i v e d r a t s i n t h i s study, i n comparison t o serum r e t i n o l l e v e l s of the ' d i e t a r y ' c o n t r o l v i t a m i n A - f e d r a t s , 16 was i n t e r p r e t a t e d as a d e p l e t i o n o f r e t i n o l r e s e r v e s i n the v i t a m i n A - d e p r i v e d r a t s . S u r v i v a l o f r a t s a f t e r c a r c i n o g e n exposure and PH was re a s o n a b l y good (70% s u r v i v a l i n Experiment A), and improved as p a r t i a l hepatectomy s k i l l s improved (>95% s u r v i v a l i n Experiment G). Moribund r a t s were o f t e n c a n n i b a l i z e d b e f o r e p a t h o l o g i c a l examinations c o u l d be c a r r i e d out. T h e r e f o r e , i t c o u l d not be determined i f death was caused by s u r g i c a l m a n i p u l a t i o n s , c a r c i n o g e n exposure, acute v i t a m i n A - d e f i c i e n c y or secondary f a c t o r s such as i n f e c t i o n . T a b l e 4 g i v e s the serum and l i v e r r e t i n o l l e v e l s a t the time of s a c r i f i c e o f the r a t s maintained on v i t a m i n A-adequate or v i t a m i n A - f r e e d i e t s f o r the 10 t o 11 week d u r a t i o n o f the experiment and d u r i n g c a r c i n o g e n exposure. Rats t h a t were kept on v i t a m i n A-adequate d i e t s o r v i t a m i n A - d e f i c i e n t d i e t s , but were i n j e c t e d w i t h the s o l v e n t DMSO r a t h e r than the c a r c i n o g e n DMN, r e p r e s e n t e d c o n t r o l s (Experiment group 'Y'). C o n s i d e r a b l e v a r i a t i o n i n the d i s t r i b u t i o n o f the v i t a m i n between l o b e s o f the l i v e r and between s i t e s w i t h i n a l i v e r l o b e have been r e p o r t e d (Mc Laren e t a l . , 1978). Samples taken from the c e n t r a l p o r t i o n o f the r i g h t lobe are r e p o r t e d not t o be s i g n i f i c i a n t l y d i f f e r e n t from the o v e r a l l mean l i v e r r e t i n o l v a l u e , g e n e r a l l y f a l l i n g t o w i t h i n 15% (Olson e t a l . , 1979) . In t h i s study, a l l l i v e r r e t i n o l d e t e r m i n a t i o n s were c a r r i e d out u s i n g the c e n t r a l p o r t i o n o f the r i g h t l i v e r l obe. 17 Serum r e t i n o l l e v e l s o f the v i t a m i n A - d e p r i v e d r a t s a t the time of s a c r i f i c e were markedly depressed (<22 ng/ml serum) as were l i v e r r e t i n o l l e v e l s (<53 ng/g t i s s u e ) , r e g a r d l e s s o f exposure t o the c a r c i n o g e n DMN or DAB, and r e g a r d l e s s o f the c a r c i n o g e n dose a p p l i e d . Serum and l i v e r r e t i n o l l e v e l s i n v i t a m i n A - f e d r a t s were c o n s i d e r a b l y h i g h e r . V i t a m i n A d e p l e t i o n i n the v i t a m i n A - d e p r i v e d r a t s was v i r t u a l l y complete. No data were o b t a i n e d f o r serum r e t i n o l l e v e l s o f the v i t a m i n A - f e d r a t s i n Experiment E. Improper s t o r a g e o f the s e p a r t i c u l a r samples may have l e d t o p a r t i a l r e t i n o l d e g r a d a t i o n . Thus the r e s u l t s were deemed u n r e l i a b l e and were not i n c l u d e d . The l i v e r r e t i n o l l e v e l s o f two v i t a m i n A - f e d r a t s i n j e c t e d w i t h a t o t a l dose of 30 mg/kg body weight i n Experiment E were unexpectedly depressed (383 + 165 ng/g t i s s u e ) . T h i s may be accounted f o r , a t l e a s t i n p a r t , by e x c e s s i v e l i v e r c e l l death caused by the h i g h dosage o f DMN a d m i n i s t e r e d (see Table 10). N e c r o t i c parenchymal c e l l s may be unable t o m a i n t a i n t h e i r v i t a m i n A s t o r a g e f u n c t i o n . Release o f r e t i n o l from the n e c r o t i c areas o f t i s s u e would l e a d t o a decreased r e t i n o l measurement determined f o r the whole l i v e r , s i n c e v i a b l e areas of l i v e r t i s s u e were not d i s t i n g u i s h e d o r analyzed s e p a r a t e l y from dead or d y i n g p o r t i o n s o f t i s s u e . Moreover, v i t a m i n A uptake may be compromised i n carcinogen-damaged t i s s u e , a g a i n b r i n g i n g about an o v e r a l l d e p r e s s i o n of the l i v e r r e t i n o l l e v e l s . 18 3.2 REVERSAL OF RETINOL DEFICIENCY BY DIETARY  SUPPLEMENTATION WITH VITAMIN A V i t a m i n A d e f i c i e n c y , induced i n the r a t s m aintained on a v i t a m i n A - f r e e d i e t f o r 8 t o 10 weeks, was r e v e r s e d by s w i t c h i n g the r a t s t o a v i t a m i n A-supplemented d i e t f o r 1 week p r i o r t o DMN exposure and f o r the r e s t o f the experiment. D e f i c i e n t r a t s were switched t o d i e t s c o n t a i n i n g adequate l e v e l s o f v i t a m i n A (A~VA) or boosted l e v e l s o f v i t a m i n A (A""8VA) , as d e s c r i b e d i n the p r o t o c o l F i g u r e s l c and Id r e s p e c t i v e l y . T a b l e 5 g i v e s the r e t i n o l l e v e l s i n the serum and l i v e r o f v i t a m i n A - d e f i c i e n t r a t s a t the time of s a c r i f i c e , f o l l o w i n g 17 or 24 days of r e t i n o l supplementation. Serum r e t i n o l l e v e l s o f the r a t s i n both o f the v i t a m i n A-supplemented groups (A~VA and A~8VA) were no l o n g e r depressed but were r e v e r s e d t o 'normal 1 l e v e l s t h a t a re comparable t o serum r e t i n o l l e v e l found i n the d i e t a r y c o n t r o l r a t s f e d v i t a m i n A - c o n t a i n i n g d i e t s throughout the experiment. L i v e r r e t i n o l l e v e l s o f the r a t s from t h e A*~VA d i e t a r y group were e l e v a t e d w e l l above l i v e r r e t i n o l l e v e l s o f the v i t a m i n A-d e f i c i e n t r a t s , and were s i m i l a r t o r e t i n o l l e v e l s found f o r the v i t a m i n A - f e d r a t s . T h i s i n d i c a t e d t h a t s t o r e s o f v i t a m i n A i n the l i v e r o f the A~VA r a t s had been r e p l e n i s h e d . The v i t a m i n A - d e f i c i e n t r a t s t h a t r e c i e v e d boosted amounts (8 x adequate) of supplementary v i t a m i n A i n t h e i r d i e t s (A~8VA) had l i v e r r e t i n o l l e v e l s i n excess of 25,000 ng/g 19 t i s s u e . R e t i n o l s a t u r a t i o n i n the r a t occurs when l i v e r r e t i n o l c o n c e n t r a t i o n s reach approximately 10,000 ng/g t i s s u e (Olson, 1982). The r a t s i n the A~8VA d i e t a r y group may have r e t i n o l - s a t u r a t e d l i v e r s , although o v e r t p h y s i c a l symptoms of h y p e r v i t a m i n o s i s A were not observed. 3.3 REVERSAL OF RETINOL DEFICIENCY BY DIETARY SUPPLEMENTATION WITH BETA-CAROTENE The v i t a m i n A d e f i c i e n t r a t s g i v e n a beta - c a r o t e n e c o n t a i n i n g d i e t (A~BC) a t 10 mg/kg d i e t f o r 17 t o 18 days, were r e v e r s e d from t h e i r d e f i c i e n t s t a t e . The serum and l i v e r r e t i n o l l e v e l s a t the time o f s a c r i f i c e o f these r a t s are pre s e n t e d i n Tabl e 6. Supplemented A~BC r a t s t h a t were exposed t o DMSO ( c o n t r o l s ) o r t o DMN (from 6 mg/kg t o 3 0 mg/kg body we i g h t ) , had serum r e t i n o l l e v e l s g r e a t e r than 9 6 ng/ml, which i s comparable t o the serum r e t i n o l l e v e l s determined f o r the v i t a m i n A - f e d r a t s . Average l i v e r r e t i n o l l e v e l s o f the A~BC r a t s were g r e a t e r than 322 + 57 ng/g t i s s u e , and were e l e v a t e d s i g n i f i c a n t l y above average l i v e r r e t i n o l l e v e l s determined f o r t he v i t a m i n A - d e f i c i e n t r a t s t h a t were not g i v e n supplementary beta-carotene i n t h e i r d i e t s , which were l e s s than 53 ± 4 7 ng/g t i s s u e f o r r a t s exposed t o DMSO and l e s s than 4 + 6 ng/g t i s s u e f o r r a t s exposed t o DMN. T h i s 20 demonstrates t h a t the l i v e r s t o r e s of r e t i n o l were b e i n g r e p l e n i s h e d as a r e s u l t o f d i e t a r y supplementation w i t h b e t a - c a r o t e n e i n the A~BC r a t s . 3.4 PROTECTIVE EFFECT OF RETINOL 3.4.1 CYTOTOXICITY DMN, l i k e almost a l l hepatocarcinogens, i s a l s o a h e p a t o t o x i n and causes c y t o t o x i c i t y i n the l i v e r when ad m i n i s t e r e d a t h i g h doses. While the r e l a t i o n s h i p between c e l l death and c e l l t r a n s f o r m a t i o n i s not y e t understood, a s i n g l e DMN-derivative a c t i n g through a common or r e l a t e d mechanism may i n s t i g a t e both of these events. In t h i s study, the e x t e n t of c e l l u l a r n e c r o s i s (CN) was used as a measure of c y t o t o x i c i t y i n the l i v e r t i s s u e o f r a t s . DMN-induced h e p a t o n e c r o s i s , demonstrated i n F i g u r e 3, comprises areas of amorphous hepatocytes w i t h condensed, p y c n o t i c n u c l e i . In c o n t r a s t , the ' h e a l t h y 1 l i v e r t i s s u e o f a DMSO-exposed c o n t r o l r a t i s d e p i c t e d i n F i g u r e 4. No h e p a t o n e c r o s i s i s e v i d e n t . These same l i v e r s e c t i o n s are shown a t a h i g h e r m a g n i f i c a t i o n i n F i g u r e s 5 and 6 r e s p e c t i v e l y . The numerous m i t o t i c f i g u r e s are i n d i c a t i v e of a c t i v e c e l l u l a r p r o l i f e r a t i o n i n t h i s t i s s u e induced by PH. E s t i m a t i o n s of the extent o f l i v e r n e c r o s i s were made w i t h the a i d o f a V i d e o p l a n computerized image-analyzer 21 ( C a r l Z e i s s Inc., Don M i l l s , Ont.). N e c r o t i c areas of a p r o j e c t e d m i c r o s c o p i c image of a l i v e r s e c t i o n (67x mag'n) were c i r c u m s c r i b e d on a g r a p h i c a l measuring t a b l e t w i t h a drawing attachment. The p r o j e c t e d image and the c i r c u m s c r i b e d drawing are d i a g r a m a t i c a l l y shown i n F i g u r e s 7 and 8 r e s p e c t i v e l y . The extent of n e c r o s i s was measured as a percentage o f the t o t a l a rea of the p r o j e c t e d image. F i v e s e q u e n t i a l areas of each of f o u r l i v e r s e c t i o n s were examined f o r each r a t . T a b l e 7 summarizes the mean percentage of n e c r o s i s and standa r d d e v i a t i o n i n the l i v e r s o f r a t s from the d i e t a r y groups A+, A", A"VA and A~8VA. V i t a m i n A d e f i c i e n c y , i t s e l f , d i d not cause c y t o p a t h o l o g y i n the l i v e r s o f the r a t s maintained on the v i t a m i n A-d e f i c i e n t d i e t throughout the experiment. The s i x v i t a m i n A-d e f i c i e n t , DMSO-treated r a t s from the Experiment group Y had 'healthy' l i v e r s t h a t were s i m i l a r i n appearance t o the l i v e r s o f t he f i v e v i t a m i n A-fed, DMSO-treated r a t s i n the same experiment group ( F i g u r e 4 ) . N e c r o s i s was absent from the l i v e r s o f these r a t s . In Experiment A, l i v e r n e c r o s i s was observed i n the f o u r r a t s from the A~8VA d i e t a r y treatment group f o l l o w i n g exposure of t h e s e r a t s t o DMN a t 35 mg/kg body weight, the h i g h e s t DMN dose examined. A mean percentage of 3.3 + 4.3 o f the l i v e r s of t h e s e r a t s was n e c r o t i c . N e c r o s i s was not p r e s e n t i n l i v e r s o f r a t s i n the treatment groups A+ o r A~VA i n Experiment A, nor was i t observed i n the l i v e r s o f A~8VA r a t s 22 t h a t were i n j e c t e d w i t h a lower DMN dose of 15 mg/kg body weight. An average of 0.1 + 0.4 p e r c e n t of the l i v e r s o f seven v i t a m i n A - d e f i c i e n t r a t s exposed t o DMN a t a dose of 35 mg/kg body weight was found t o be n e c r o t i c , but t h i s was not s i g n i f i c a n t (P=0.51). T h e r e f o r e , exposure t o h i g h doses o f DMN w i t h e x c e s s i v e d i e t a r y r e t i n o l supplementation was found t o r e s u l t i n an a c c e n t u a t i o n o f the h e p a t o t o x i c a c t i o n o f DMN i n r a t s . T h i s f i n d i n g i s of p a r t i c u l a r importance and i t s i m p l i c a t i o n s i n t h e d e s i g n of v i t a m i n A i n t e r v e n t i o n t r i a l s w i l l be d i s c u s s e d l a t e r . 3.4.2 MITOTIC INHIBITION The g e n e r a l t o x i c e f f e c t of DMN and i t s p o s s i b l e enhancement by v i t a m i n A - d e f i c i e n c y can be estimated from the i n c i d e n c e o f chromatin b r i d g e s (CB), chromosomal fragments (CF) and m i c r o n u c l e i (MN) i n the l i v e r ( d e p i c t e d i n F i g u r e s 9-13). C e l l u l a r p r o l i f e r a t i o n i s necessary f o r the v i s u a l i z a t i o n of these g e n o t o x i c l e s i o n s . T h e r e f o r e , t o a v o i d erroneous c o n c l u s i o n s , i t i s important t o g a i n i n f o r m a t i o n on whether DMN, as w e l l as v i t a m i n A d e f i c i e n c y , a f f e c t s the m i t o t i c index. The number o f d i v i d i n g c e l l s per 2000 hepatocytes was counted f o r i n d i v i d u a l animals, and the average m i t o t i c index and s t a n d a r d d e v i a t i o n was determined f o r animals i n the same treatment group. The r e s u l t s are p r e s e n t e d i n T a b l e 8. 23 V i t a m i n A d e f i c i e n c y d i d not appear t o a f f e c t m i t o t i c a c t i v i t y i n the l i v e r , nor d i d the r e v e r s i o n o f the v i t a m i n A-d e f i c i e n t s t a t e by d i e t a r y v i t a m i n A supplementation. The average m i t o t i c index of f i v e DMSO exposed, v i t a m i n A - f e d r a t s was 5.2 + 2.2, and was not s i g n i f i c a n t l y d i f f e r e n t (P= 0.66) from the average m i t o t i c index o f the t e n DMSO exposed v i t a m i n A - d e f i c i e n t r a t s , which was 4.6 + 2.8. Rats from the d i e t a r y groups A+, A-, A~VA and A~8VA i n Experiment A t h a t were exposed t o DMN a t a dose o f 15 mg/kg body weight, e x h i b i t e d s i m i l a r m i t o t i c i n d i c e s a t 2.3 + 1.5, 2 . 3 + 2 . 7 , 2.2 + 1.1 and 2.3 + 2.2 r e s p e c t i v e l y . Thus, m a n i p u l a t i o n o f the d i e t a r y regimen d i d not appear t o s i g n i f i c a n t l y a l t e r c e l l u l a r p r o l i f e r a t i o n i n the l i v e r . Exposure t o the carc i n o g e n , DMN, had a s l i g h t i n h i b i t o r y i n f l u e n c e on c e l l u l a r p r o l i f e r a t i o n i n the l i v e r . F or example, t h e m i t o t i c i n d i c e s determined f o r t h r e e v i t a m i n A-f e d r a t s i n j e c t e d w i t h DMN a t 35 mg/kg body weight and f o r f i v e v i t a m i n A - f e d r a t s i n j e c t e d w i t h DMN a t 15 mg/kg body weight were 0.3 + 0.6 and 2.3 +1.5 r e s p e c t i v e l y . The m i t o t i c index o f f i v e v i t a m i n A - f e d c o n t r o l r a t s i n j e c t e d w i t h DMSO was 5.2 + 2.2, and was s i g n i f i c a n t l y d i f f e r e n t from the m i t o t i c index o f the r a t s exposed t o the h i g h e r dose o f DMN (P=0.02), but not from the m i t o t i c index o f the r a t s exposed t o t he lower dose o f DMN (P=0.48). R e l a t i v e l y h i g h doses o f ca r c i n o g e n may depress the m i t o t i c a c t i v i t y i n the PH l i v e r s o f r a t s t o a s m a l l degree. 24 There are no extreme changes i n the m i t o t i c index o f the l i v e r t h a t can be a t t r i b u t e d t o the e f f e c t s of DMN exposure or v i t a m i n A d e f i c i e n c y . T h i s i s not t o say t h a t these f a c t o r s are without i n f l u e n c e on c e l l u l a r p r o l i f e r a t i o n . More l i k e l y , s t i m u l a t i o n o f l i v e r c e l l s t o d i v i d e , induced by removal of 70% of the l i v e r , c o u l d c o n c e i v a b l y mask a weaker s t i m u l a t i n g or i n h i b i t i n g e f f e c t of the c a r c i n o g e n or the v i t a m i n A s t a t u s . 3.4.3 GENOTOXICITY Many chemicals, i n c l u d i n g DMN, induce chromosomal a b e r r a t i o n s . A r e l a t i o n s h i p between chromosomal a b e r r a t i o n s and cancer i s suggested by the f o l l o w i n g l i n e s of evidence: (1) many cancers show anomalous karyotypes; (2) i n d i v i d u a l s s u f f e r i n g from g e n e t i c a l l y i n h e r i t a b l e chromosomal i n s t a b i l i t y syndromes, are 'predisposed* t o the development o f c e r t a i n t y p e s o f c a n c e r s ; (3) many carcinogens are c l a s t o g e n i c , mutagenic or both; and (4) many c l a s t o g e n s ( i . e . , DNA-1 b r e a k i n g ' agents) have been shown t o be c a r c i n o g e n i c ( B r a m b i l l a e t a l . , 1985). I t i s d o u b t f u l t h a t t h e r e are any c l a s t o g e n s t h a t are not a l s o c a r c i n o g e n s , although i n s u f f i c i e n t evidence e x i s t s t o e s t a b l i s h t h i s as a c e r t a i n t y (Heddle e t a l . , 1983). At l e a s t 100 anatelophase c e l l s were examined i n the l i v e r o f each r a t . The r e s u l t s are p r e s e n t e d i n T a b l e 9a as the average p e r c e n t of anatelophase c e l l s a n a l y z e d t h a t 25 c a r r i e d a t l e a s t one chromosomal anomaly, and i n T a b l e 9b as the average number or frequency of n u c l e a r anomalies per anatelophase c e l l . The l a t t e r was determined as the t o t a l number o f observed anomalies d i v i d e d by the t o t a l number of anatelophase c e l l s analyzed, t o g i v e an i n d i c a t i o n of the e x t e n t of chromosomal damage on a per c e l l b a s i s i n the l i v e r . Abnormal anatelophase s t r u c t u r e s were seen v e r y i n f r e q u e n t l y i n the l i v e r c e l l s o f the e l e v e n DMSO-exposed c o n t r o l r a t s maintained on v i t a m i n A-adequate or v i t a m i n A-d e f i c i e n t d i e t s (Experiment group Y). The average percentage of anatelophase c e l l s w i t h a chromosomal anomaly was l e s s than 2% f o r the r a t s i n both of these d i e t a r y groups. None of the anatelophase c e l l s examined i n the l i v e r s o f these animals c a r r i e d more than one chromosomal a b e r r a t i o n . V i t a m i n A-d e f i c i e n c y , t h e r e f o r e , does not i n i t s e l f c o n t r i b u t e t o the g e n e r a t i o n of chromosomal a b e r r a t i o n s . E l e v a t i o n i n the percentage of abnormal anatelophases above t h i s 2% l e v e l i n the l i v e r s o f DMN-exposed r a t s was a t t r i b u t e d t o the genotoxic a c t i o n s o f the c a r c i n o g e n . In the t h r e e v i t a m i n A - f e d r a t s , i n j e c t e d w i t h DMN a t a t o t a l dose o f 35 mg/kg body weight, chromosomal anomalies were p r e s e n t i n an average of 10.3 + 2.1 p e r c e n t o f the examined anatelophase l i v e r c e l l s (Table 9a), and a t an average frequency o f 0.28 + 0.06 anomalies per anatelophase c e l l (Table 9b). In the same experiment, the s i x v i t a m i n A-d e f i c i e n t r a t s , g i v e n the same dosage of the c a r c i n o g e n , 26 showed a much h i g h e r l e v e l o f n u c l e a r anomalies (P=0.02). In these animals, an average o f 23.7 + 5.4 p e r c e n t o f the examined anatelophase c e l l s i n the l i v e r c o n t a i n e d chromosomal a b e r r a t i o n s . Moreover, an average frequency o f 0.70 + 0.32 anomalies p e r anatelophase c e l l was observed, i n d i c a t i n g t h a t t h e e x t e n t o f chromosomal damage on a per c e l l b a s i s was a l s o s i g n i f i c a n t l y e l e v a t e d i n the v i t a m i n A - d e f i c i e n t r a t s over t h a t found i n the v i t a m i n A - f e d r a t s (P=0.02). At a lower DMN dose of 15 mg/kg body weight, an average of o n l y 2.3 + 2.1 per c e n t o f the anatelophases examined i n the f i v e v i t a m i n A - f e d r a t s c o n t a i n e d chromosomal anomalies, and a t an average frequency o f 0.03 + 0.02 a b e r r a t i o n s p e r anatelophase c e l l . In c o n t r a s t , an average o f 22.8 + 5.3 pe r c e n t o f the anatelophases examined i n the l i v e r s o f the seven v i t a m i n A - d e f i c i e n t r a t s were anomalous, and a t an average frequency o f 0.3 6 ± 0.11 a b e r r a t i o n s p e r anatelophase c e l l . These r e s u l t s s t r o n g l y suggest t h a t the ge n o t o x i c a c t i o n o f DMN was s i g n i f i c a n t l y heightened (P=0.01) i n the v i t a m i n A - d e f i c i e n t r a t s as compared t o the v i t a m i n A - f e d r a t s . The f o u r r a t s from the A"VA d i e t a r y treatment group, i n j e c t e d w i t h DMN a t a t o t a l dose of 35 mg/kg body weight, showed anomalies i n t h e i r anatelophase l i v e r c e l l s a t an average o f 9.3 + 2.6 percen t , and a t an average frequency o f 0.15 + 0.03 anomalies per anatelophase c e l l . D i e t a r y v i t a m i n A supplementation a t adequate l e v e l s p r i o r t o 27 c a r c i n o g e n exposure was b e n e f i c i a l f o r the A~VA r a t s as they were not as s e n s i t i v e t o the gen o t o x i c e f f e c t s o f DMN as the v i t a m i n A - d e f i c i e n t r a t s . The extent o f DMN-induced genomic damage i n the l i v e r s o f the A~VA r a t s was not s i g n i f i c a n t l y d i f f e r e n t (P=0.59) from the extent o f damage i n t h e l i v e r s of the v i t a m i n A - f e d d i e t a r y c o n t r o l r a t s i n j e c t e d w i t h comparable doses o f DMN. The f o u r r a t s from the A"8VA d i e t a r y treatment group, exposed t o DMN a t the t o t a l dose of 35 mg/kg body weight, c a r r i e d chromosomal anomalies i n an average o f 29.8 + 6.6 p e r c e n t o f the anatelophases analyzed. T h i s was s i g n i f i c a n t l y e l e v a t e d (P=0.03) above the average percentage o f anomalous anatelophase c e l l s observed i n the v i t a m i n A - f e d d i e t a r y c o n t r o l r a t s , and was comparable t o t h a t observed i n the v i t a m i n A - d e f i c i e n t r a t s i n j e c t e d w i t h the same DMN dosage. Furthermore, a 4 - f o l d i n c r e a s e i n the ext e n t o f chromosomal damage per anatelophase c e l l was found f o r the A"~8VA animals as compared t o the extent o f chromosomal damage found i n v i t a m i n A - f e d c o n t r o l animals. An average frequency o f 1.25 + 0.82 n u c l e a r anomalies per anatelophase c e l l was observed i n the A~8VA animals. E x c e s s i v e genomic damage was not observed i n the A~8VA r a t s exposed t o DMN a t t o t a l dosages of 15 mg/kg body weight o r l e s s . At these lower dosage l e v e l s , the ext e n t o f DMN-induced g e n o t o x i c i t y i n the A""8VA r a t s was not s i g n i f i c a n t l y d i f f e r e n t (P=0.07) from the extent o f g e n o t o x i c i t y found i n the v i t a m i n A - f e d c o n t r o l r a t s . 28 T h e r e f o r e , d i e t a r y v i t a m i n A supplementation a t e x c e s s i v e l e v e l s was not p r o t e c t i v e , but r a t h e r i t appeared t o enhance the e x t e n t o f DMN-induced ge n o t o x i c damage i n l i v e r c e l l s o f the A~8VA r a t s when r e l a t i v e l y h i g h doses o f DMN were a d m i n i s t e r e d . 3.5 PROTECTIVE EFFECT OF BETA-CAROTENE 3.5.1 CYTOTOXICITY The p r o t e c t i v e e f f e c t of d i e t a r y b e t a - c a r o t e n e supplementation on the c y t o t o x i c a c t i o n o f DMN i n the l i v e r s of v i t a m i n A - d e f i c i e n t r a t s i s pr e s e n t e d i n Ta b l e 10. C y t o t o x i c l e s i o n s were not found i n the l i v e r s o f the DMSO-exposed c o n t r o l r a t s t h a t were d e f i c i e n t i n v i t a m i n A, nor were they observed i n the l i v e r o f the r a t s t h a t were d e f i c i e n t i n v i t a m i n A and then g i v e n b e t a - c a r o t e n e a t 10 mg/kg d i e t (A""BC) f o r one week p r i o r t o DMSO exposure. The l i v e r s o f the animals i n both of these d i e t a r y treatment groups resemble the l i v e r s o f the DMSO-exposed, v i t a m i n A - f e d d i e t a r y c o n t r o l animals (see F i g u r e 4 ). T h e r e f o r e , the ma n i p u l a t i o n o f d i e t a r y f a c t o r s alone, without the a p p l i c a t i o n of t he ca r c i n o g e n , was not found t o induce c y t o t o x i c i t y i n the l i v e r s o f r a t s i n t h i s study. The n i n e v i t a m i n A - d e f i c i e n t r a t s , g i v e n i n j e c t i o n s o f DMN a t a dose l e v e l o f 15 mg/kg body weight (Experiment F ) , 29 showed an average o f 11.6 + 7.7 p e r c e n t of t h e i r l i v e r s t o be n e c r o t i c . The e i g h t r a t s from the v i t a m i n A - f e d d i e t a r y group and the t e n r a t s from the A~BC d i e t a r y group d i d not show n e c r o s i s i n t h e i r l i v e r s a f t e r exposure t o DMN a t t h i s same dose l e v e l . V i t a m i n A d e f i c i e n c y , t h e r e f o r e , appeared t o i n c r e a s e the s e n s i t i v i t y o f r a t s t o the h e p a t o t o x i c e f f e c t s of DMN. H y p e r s e n s i t i v i t y t o DMN, a s s o c i a t e d w i t h v i t a m i n A d e f i c i e n c y i n r a t s , was even more pronounced when h i g h e r doses o f DMN were a d m i n i s t e r e d . A DMN dose of 22.5 mg/kg body weight r e s u l t e d i n the p r o d u c t i o n of an average of 22.3 + 12.2 p e r c e n t n e c r o s i s i n the l i v e r t i s s u e of the v i t a m i n A-d e f i c i e n t r a t s . These d i s e a s e d l i v e r s appeared as parenchymal patchworks of degenerating, haemorrhagic l e s i o n s and r e g e n e r a t i n g , h y p e r p l a s t i c r e g i o n s of l i v e r t i s s u e ( F i g u r e 3 and 5 ) . DMN-induced c y t o x i c i t y was l e s s e x t e n s i v e i n the l i v e r s o f the v i t a m i n A - f e d r a t s . An average of o n l y 10.7 + 8.5 p e r c e n t of t h e i r l i v e r s was found t o be n e c r o t i c when they were exposed t o DMN a t t h i s same dosage (P=0.05). The h y p e r s e n s i t i v e s t a t e was a b o l i s h e d by supplementation of the v i t a m i n A - d e f i c i e n t d i e t w i t h beta-carotene p r i o r t o DMN exposure a t 22.5 mg/kg body weight. A s i g n i f i c a n t decrease i n the e x t e n t of l i v e r n e c r o s i s (P=0.002), t o an average of l e s s than 1 p e r c e n t of the t i s s u e examined, was found i n the r a t s from the A~BC treatment group,. 30 A t a DMN dose o f 30 mg/kg body weight, e x t e n s i v e areas o f he p a t o n e c r o s i s were observed i n the two v i t a m i n A - d e f i c i e n t r a t s . DMN produced 45.0 + 7.1 pe r c e n t n e c r o s i s i n the l i v e r t i s s u e o f these r a t s , but produced o n l y 5.7 + 13.2 pe r c e n t n e c r o s i s i n the l i v e r t i s s u e of the t e n r a t s from the A"BC d i e t a r y treatment group (P=0.05). The same DMN dose produced 37.3 + 4.6 p e r c e n t n e c r o s i s i n the l i v e r t i s s u e o f seven v i t a m i n A - f e d r a t s . T h e r e f o r e , r e v e r s a l o f the v i t a m i n A-d e f i c i e n t s t a t e by d i e t a r y beta-carotene supplementation was p r o t e c t i v e a g a i n s t the c y t o t o x i c e f f e c t s o f DMN i n the l i v e r s o f r a t s i n t h i s study. In a d d i t i o n , the p r o t e c t i v e e f f e c t c o n f e r r e d by d i e t a r y beta-carotene supplementation appeared g r e a t e r than t h a t due t o d i e t a r y v i t a m i n A. 3.5.2 MITOTIC INHIBITION The m i t o t i c i n d i c e s o f the l i v e r s o f r a t s from the d i e t a r y treatment group A~BC are p r e s e n t e d i n Tabl e 8. D i e t a r y supplementation w i t h beta-carotene d i d not appear t o s i g n i f i c a n t l y i n f l u e n c e the m i t o t i c a c t i v i t y o f the l i v e r . The average m i t o t i c index of the l i v e r s of the v i t a m i n A-d e f i c i e n t r a t s exposed t o DMSO was 4.6 — 2.8 d i v i d i n g c e l l s p e r 1000 hepatocytes, w h i l e the average m i t o t i c index o f the l i v e r o f A~BC animals exposed t o DMSO was 6.3 + 3.8 d i v i d i n g c e l l s p e r 1000 hepatocytes (P=0.43). The average m i t o t i c index o f the l i v e r s o f A~BC r a t s exposed t o DMN a t a t o t a l 31 dose o f 30 mg/kg, was 4.1 + 2.7 d i v i d i n g c e l l s per 1000 hepat o c y t e s , and was not s i g n i f i c a n t l y d i f f e r e n t from t h a t observed i n the DMSO-exposed c o n t r o l r a t s (P=0.68). T h e r e f o r e , exposure o f the A~BC r a t s t o the ca r c i n o g e n , even a t r e l a t i v e l y h i g h doses, d i d not appear t o a f f e c t the p r o l i f e r a t i v e a c t i v i t y o f the l i v e r . 3.5.3 GENOTOXICITY The g e n o t o x i c e f f e c t s o f DMN i n the l i v e r s o f the v i t a m i n A - d e f i c i e n t r a t s and the A~BC r a t s a re p r e s e n t e d i n T a b l e s 11a and l i b . In the two s u r v i v i n g v i t a m i n A - d e f i c i e n t r a t s i n j e c t e d w i t h DMN a t a t o t a l dose of 30 mg/kg body weight, an average o f 18.0 p e r c e n t o f the examined anatelophases i n t h e i r l i v e r s were a b e r r a n t . An average frequency o f 0.35 anomalies per anatelophase c e l l was observed. In the same experiment, the t e n r a t s from the A~BC d i e t a r y treatment group t h a t r e c e i v e d the same t o t a l dose of DMN, had an average of o n l y 10.2 + 4.4 p e r c e n t o f anomalous anatelophases i n t h e i r l i v e r s , and a t an average frequency o f 0.17 + 0.12 anomalies per anatelophase c e l l . Again, i n t h e same experiment and a t the same DMN dosage, an average o f 16.3 + 3.1 p e r c e n t o f the examined anatelophase c e l l s c a r r i e d a b e r r a t i o n s , and a t a frequency o f 0.32 + 0.04 a b e r r a t i o n s per anatelophase, i n seven v i t a m i n A - f e d r a t s . The g e n o t o x i c damage induced by DMN was s i g n i f i c a n t l y lower (P=0.04) i n A~BC 32 r a t s than i n v i t a m i n A - d e f i c i e n t or v i t a m i n A - f e d r a t s . Beta-carotene supplementation of the v i t a m i n A - d e f i c i e n t d i e t , t h e r e f o r e appeared p r o t e c t i v e a g a i n s t the g e n o t o x i c a c t i o n of DMN i n the r a t l i v e r , and may c o n f e r a g r e a t e r p r o t e c t i o n than t h a t observed w i t h d i e t a r y v i t a m i n A. 33 Figure 1: Schematic i l l u s t r a t i o n of dietary regimens and experimental design lc ) A - V A regimen 8 to 9 weeks carcinogen exposure _ , PH 1 week v T 5 days s a c r i f i c e 48rn v V v v 3 V V V A A A A I A A A / A A A A V V V V 2Z3 Id) A-8VA regimen 8 to 9 weeks carcinogen _„ saci , exposure _ , PH 1 week _ T 5 days - 48h T r i f i c e Y Y Y l e ) A _BC regimen 8 to 9 weeks 1 week _ c a r c i n o g e n exposure ^ 5 days • HOU. PH T s a c r i f i c e 48h, vitamin A-adequate diet (2mg/kg diet) v i t a m i n A - d e f i c i e n t d i e t — v | v i t a m i n A-supplemented d i e t —^I w i t h adequate l e v e l s o f v i t a m i n A (2mg/kg d i e t ) — ^ v i t a m i n A-supplemented d i e t A w i t h b o o s t e d l e v e l s o f v i t a m i n A (16mg/kg d i e t ) • j b e t a - c a r o t e n e - s u p p l e m e n t e d d i e t (lOmg/kg d i e t ) 34 Figure 2: Growth curves of ra ts maintained on d i f f e r e n t d i e t a r y regimens, a) • v i t a m i n A-adequate r a t s ; b) • v i tamin A-d e f i c i e n t r a t s ; c) A v i t a m i n A-supplemented rats wi th adequate l e v e l s of v i t a m i n A; d) • v i tamin A-supplemented ra t s w i th boosted l e v e l s of v i t amin A; e) • beta-carotene supplemented r a t s . 350n 300-250-S, 200-S3 a 150-100-50-0- — r 4 -r-7 —r— 10 —i 11 T-0 2 - r -3 T" 8 T WEEKS 35 F i gure 3: L iver S e c t i o n of a Vitamin A - d e f i c i e n t Rat E x p o s e d to DMN £ t o t a _ L d o s e of 30 mg/kg body weight) 3.1 x 1 0 2 mag 'n i'fc v * V \ * * 4 rare Figure 4: Liver S e c t i o n of a Vitamin A - d e f i c i e n t Rat E x p o s e d to D M S O (contro l ) 3.1 x 102 mag 'n M 36 F i g u r e 5 : L i v e r S e c t i o n of a V i t a m i n A - d e f i c i e n t Ra t E x p o s e d to D M N ( t o t a l d o s e of 3 0 m g / k g body w e i g h t ) 1.0 x 1 0 3 mag 'n Figure 6 : L i v e r S e c t i o n of a V i t a m i n A - d e f i c i e n t Rat E x p o s e d to D M S O ( c o n t r o l ) 1.0 x 1 0 3 mag 'n 37 F i gure 7: L i ver S e c t i o n of a V i tamin A - d e f i c i e n t Rat E x p o s e d to DMN ( to ta l d o s e of 3 0 mg/kg body weight) 3.1 x 1 0 2 mag 'n Figure 8: T rac ing of L iver S e c t i o n of a Vitamin A - d e f i c i e n t Rat (in F i gu re 7) with N e c r o t i c A r e a s of T i s sue C i r c u m s c r i b e d 38 Figure 9: Normal A n a t e l o p h a s e Ce l l 4 0 Figure 1 3 : A n a t e l o p h a s e C e l l w i th a C h r o m a t i n B r i d g e and M i c r o n u c l e i 3.1 x 1 0 3 m a g ' n 4 1 T a b l e 1: Composition of V i t a m i n A - f r e e D i e t I n g r e d i e n t s Amount (per kg d i e t ) c a s e i n 200g sucrose 210g dextro s e 210g d e x t r i n 210g cor n o i l f * lOOg s a l t mix" 50g c h o l i n e c l o r i d e 3g i n o s i t o l 250mg t o c o p h e r y l a c e t a t e 150mg n i a c i n 50mg pa n t o t h e n i c a c i d 20mg f o l i c a c i d lOmg thiamine 8mg p y r o x i d i n e HCL 8mg r i b o f l a v i n 4mg v i t a m i n K l lmg v i t a m i n B12 0.05mg v i t a m i n D2 1500IU a Mazola o i l ; Corn Products Inc., Waltham M.A. b Rogers-Harper's M i n e r a l Mix; T e k l a d 42 T a b l e 2: D i m e t h y l n i t r o s a m i n e T r e a t m e n t S c h e d u l e E x p t . Code T o t a l Dose P e r I n j e c t i o n A 15 35 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t 3 x 5 3 X 5 + 2 X 1 0 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t E 30 mg/kg b o d y w e i g h t 22.5 mg/kg b o d y w e i g h t 3 X 1 0 3 X 7 . 5 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t F 2 2 . 5 15 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t 3 X 7 . 5 3 X 5 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t G 12 6 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t 3X4 3 X 2 mg/kg b o d y w e i g h t mg/kg b o d y w e i g h t Y 0 mg/kg b o d y w e i g h t 3 X DMSO ( c o n t r o l s ) 43 T a b l e 3: R e t i n o l L e v e l s i n Serum of Rats on a V i t a m i n A-adequate (A+) or a V i t a m i n A - f r e e (A~) D i e t f o r 9 t o 10 weeks Experiment Number of Serum R e t i n o l L e v e l s (ng/ml) Code Rats A+ A" A 9 93 + 22 56 + 9 B 22 122 + 3 7 17 + 8 C 48 147 ± 5 7 30 + 14 E 10 201 ± 6 4 56 ± 22 F 10 90 + 38 23 ± 10 G 8 175 + 5 3 35 + 30 44 T a b l e 4: R e t i n o l L e v e l s i n Serum and L i v e r a t Time of S a c r i f i c e o f Rats on V i t a m i n A-adequate (A+) a or V i t a m i n A - f r e e (A~) D i e t f o r 10 t o 11 Weeks R e t i n o l L e v e l s Treatment cpt. )de Agent Dose* (mg/kg) No. £ Rats 1 3f Serum (ncf/ml) L i v e r (ncf/cf) 3 A+ A" A+ A" A DMN 35 11 167+46 14±11 5821+ 129 9+ 8 15 9 119±12 9± 6 5465+2839 43+19 B DMN 22.5 28 127+20 2 0± 6 5397±1738 8+20 c DAB C 30 15 108+27 4+ 4 2886+1275 0 d D DAB 40 29 111+24 19± 8 5343+ 770 17+16 E DMN 30 5 NA e 0 383+ 165 4+ 6 22.5 14 NA 0 3138+1535 1± 1 F DMN 22.5 14 107+32 0 3072+1394 0 15 17 98+23 0 4428+2896 0 G DMN 12 12 107+ 8 2+ 5 4012+1743 0 6 13 140+26 0 5547+2468 0 Y f — _ 15 177+66 22±18 5465+2459 53+47 a T o t a l Dose per kg body weight (given i n 3 i n j e c t i o n s ) Number of s u r v i v i n g r a t s a f t e r c a r c i n o g e n exposure and p a r t i a l hepatectomy ° DAB = Dimethylaminoazobenzene d R e t i n o l l e v e l s below t h a t d e t e c t a b l e by HPLC ® NA = Data not a v a i l a b l e f Y = c o n t r o l s (exposed t o DMSO) 45 T a b l e 5: R e t i n o l L e v e l s i n Serum and L i v e r o f V i t a m i n A - d e f i c i e n t Rats f o l l o w i n g Supplementation o f the V i t a m i n A - f r e e D i e t w i t h V i t a m i n A a t Adequate L e v e l s (A~VA) a or a t Boosted L e v e l s (A~8VA) t o f o r 17 o r 24 Days R e t i n o l L e v e l s Treatment Expt. DMN #Rats- Serum fng/ml) L i v e r (ncf/cf) Code (mg/kg) A" A~VA A"8VA A" A"VA A"8VA A 35 8 d 14+11 135+31 141+40 9+ 8 4857+1375 25794+6302 15 9 e 9+ 6 86+19 126+47 43+19 6502+3959 28254+9061 a 2 mg r e t i n y l a c e t a t e / k g d i e t " 16 mg r e t i n y l a c e t a t e / k g d i e t ° number o f r a t s s a c r i f i c e d d v i t a m i n A supplementation f o r 24 days e v i t a m i n A supplementation f o r 17 days 46 T a b l e 6 : R e t i n o l L e v e l s i n S e r u m a n d L i v e r o f V i t a m i n A - d e f i c i e n t R a t s f o l l o w i n g S u p p l e m e n t a t i o n o f t h e V i t a m i n A - f r e e D i e t w i t h B e t a - c a r o t e n e ( A " B C ) a f o r 1 7 t o 18 d a y s R e t i n o l L e v e l s T r e a t m e n t E x p t . DMN # R a t s b S e r u m ( n q / m l ) L i v e r ( n q / q ) C o d e ( m g / k g ) A ~ A " B C A " A ~ B C 3 0 2 2 . 5 12 1 6 9 6 + 7 0 1 1 2 + 2 7 4 + 6 1+1 4 9 6 + 2 2 7 3 2 6 + 8 1 2 2 . 5 1 5 1 5 1 9 0 1+2 1 0 7 + 2 5 1 4 3 + 2 6 0 0 3 2 8 + 98 5 8 6 + 5 2 3 12 6 1 5 1 6 1+4 0 1 0 2 + 2 4 1 0 0 + 2 1 0 0 4 8 7 + 2 3 0 5 7 4 + 2 6 4 1 5 2 2 + 1 8 8 3 + 8 5 3 + 4 7 3 2 2 + 5 7 f_ 1 0 mg b e t a - c a r o t e n e / k g d i e t ( H o f f m a n - L a R o c h e C o . ) n u m b e r o f r a t s s a c r i f i c e d ^ R e t i n o l l e v e l s b e l o w t h a t d e t e c t a b l e b y H P L C Y = c o n t r o l s ( e x p o s e d t o DMSO) 4 7 T a b l e 7: P r o t e c t i v e E f f e c t o f D i e t a r y V i t a m i n A Supplementation on the C y t o t o x i c A c t i o n o f DMN i n the L i v e r s of V i t a m i n A - d e f i c i e n t Rats Treatment Average Percentage o f Rat L i v e r Expt. DMN dose No. of T i s s u e t h a t i s N e c r o t i c Code (mg/kg) R a t s a A+ A" A"VA A~8VA 35 15 18 19 0 0 0.2+0.4 0 0 0 3.3+4.3 0 11 ND ND a number of r a t s s a c r i f i c e d Y = c o n t r o l s (exposed t o DMSO) c ND = not done 48 T a b l e 8: M i t o t i c Index i n L i v e r s o f P a r t i a l Hepatectomized Rats Expt. DMN No. of M i t o t i c Index p e r 1000 C e l l s Code (mg/kg) R a t s a A+ A" A"VA A~8VA A~BC A 35 18 0.3+0.6 2.4+2.6 1. 3+1.3 1 .8±2 .2 ND 15 23 2.3+1.5 2.3+2.7 2. 2±1.1 2 . 3±2 .2 ND E 30 15 1.3+1.5 1.0+1.4 ND b ND 4.1±2.7 22.5 24 2.6+1.9 3.0+2.0 ND ND 3.0+1.4 F 22.5 21 1.8±2.4 3.3+2.2 ND ND 3.6+2.6 15 27 2.9+2.4 3.4+2.1 ND ND 3.4+2.1 G 12 20 3.6+1.5 2.3+2.1 ND ND 3.6+2.6 6 21 1.8±1.6 2.6+2.1 ND ND 4.3+2.5 Y c 0 19 5.2+2.2 4.6+2.8 ND ND 6.3+3.8 f- number o f r a t s s a c r i f i c e d ND = not done c y = c o n t r o l s (exposed t o DMSO) 49 T a b l e 9a: P r o t e c t i v e E f f e c t of D i e t a r y V i t a m i n A Supplementation on the Genotoxic A c t i o n of DMN i n the L i v e r s of V i t a m i n A - d e f i c i e n t Rats Treatment Average Percentage o f Anatelophase C e l l s Expt. DMN dose No. of w i t h Anomalies i n the L i v e r s of Rats Code (mg/kg) R a t s a A+ A" A"VA A~8VA A 35 18 10.3+2.1 23.7+5.4 9.3±2.6 29.8±6.6 15 19 2.3+2.1 22.8±5.3 7.8+1.3 10.0+3.6 Y b 0 11 0 1.0+1.3 ND C ND a number of r a t s s a c r i f i c e d Y = c o n t r o l s (exposed t o DMSO) c ND = not done 50 T a b l e 9b: P r o t e c t i v e E f f e c t o f D i e t a r y V i t a m i n A Supplementation on the Genotoxic A c t i o n o f DMN i n L i v e r s of V i t a m i n A - d e f i c i e n t Rats Treatment Average Frequency o f N u c l e a r Anomalies per Expt. DMN No. of Anatelophase C e l l i n the L i v e r s o f Rats Code (mg/kg) R a t s a A+ A~ A"VA A~8VA 35 18 0.28+0.06 0.70+0.32 0.15+0.03 1.25±0.82 15 19 0.03+0.02 0.36+0.11 0.14+0.04 0.15+0.07 11 0.01+0.01 ND ND f- number of r a t s s a c r i f i c e d Y = c o n t r o l s (exposed t o DMSO) c ND = not done 51 T a b l e 10: P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the C y t o t o x i c A c t i o n o f DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats Treatment Average Percentage o f Rat L i v e r Expt. DMN dose No. of T i s s u e t h a t i s N e c r o t i c Code (mg/kg) R a t s a A+ A" A"BC E 30 15 37.3+4.6 45.0+ 7.1 5.7+13.2 22.5 24 0.1+0.4 22.2+18.5 0.5+ 1.6 F 22.5 21 10.7+ 8.5 22.3+12.2 0 15 27 0 11.6+ 7.7 0 G 12 20 0 0 0 6 21 0 0 0 Y b 0 11 0 0 0 a number o f r a t s s a c r i f i c e d " Y = c o n t r o l s (exposed t o DMSO) 52 T a b l e 11a: P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the Genotoxic A c t i o n o f DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats Treatment Average Percentage o f Anatelophase C e l l s DMN No. o f w i t h Anomalies i n the L i v e r s o f Rats Code (mg/kg) R a t s a A+ A" A~BC E 30 15 16.3+3.1 18.0±0.0 10.2±4.4 22.5 24 7.9±3.3 12.5+3.8 4.4+1.8 F 22.5 21 5.8+3.3 17.9+2.9 3.6+1.8 15 27 6.5+0.9 17.8+5.7 7.5+1.4 G 12 20 8.0+3.9 15.3+7.2 7.6+3.3 6 21 5.4+3.3 14.0+5.3 6.3+3.2 Y b 0 19 0 1.1±1.1 0.5+1.0 a number of r a t s s a c r i f i c e d Y = c o n t r o l s (exposed t o DMSO) 53 T a b l e l i b : P r o t e c t i v e E f f e c t o f D i e t a r y Beta-carotene Supplementation on the Genotoxic A c t i o n o f DMN i n the L i v e r s o f V i t a m i n A - d e f i c i e n t Rats Treatment Average Frequency o f Nucl e a r Anomalies per Code (mg/kg) R a t s a A+ A" A~BC E 30 15 0.32+0.04 0.35+0.01 0.17+0.12 22.5 24 0.10±0.04 0.18+0.06 0.05+0.03 F 22.5 21 0.08+0.05 0.27+0.04 0.06+0.03 15 27 0.10±0.02 0.31+0.15 0.10+0.04 G 12 20 0.12+0.04 0.28+0.10 0.09+0.05 6 21 0.08+0.04 0.22+0.07 0.08+0.04 yb 0 11 0 0.01+0.01 0.01+0.01 a number of r a t s s a c r i f i c e d Y = c o n t r o l s (exposed t o DMSO) 54 T a b l e 12: Chemopreventive I n t e r v e n t i o n S t u d i e s Supported by the U.S. N a t i o n a l Cancer I n s t i t u t e T a r g e t S i t e / Organ C e r v i x C e r v i x Colon Colon Colon Colon Colon Colon T a r g e t / R i s k Group C e r v i c a l d y s p l a s i a C e r v i c a l d y s p l a s i a F a m i l i a l p o l y p o s i s F a m i l i a l p o l y p o s i s Adenomatous p o l y p s Adenomatous pol y p s Normal v o l u n t e e r s F a m i l i a l p o l y p o s i s I n h i b i t o r y Agents R e t i n o i c a c i d F o l i c a c i d V i t a m i n s C+E wheat bran 1 3 - c i s r e t i n o i c a c i d Beta-carotene Beta-carotene, v i t a m i n s C+E Name/Location E. Surwit, U n i v e r s i t y o f A r i z o n a , Tucson, AZ J . Chu, Fr e d Hutchinson Cancer Centre, S e a t t l e , WA L. DeCosse, Sloan K e t t e r i n g Cancer Centre, New York D. Quinlan, U n i v e r s i t y o f West V i r g i n i a , Morgantown P. Bowen, U n i v e r s i t y o f I l l i n o i s , Chicago, I L E. R. Greenberg, N o r r i s Cotton Cancer Centre, Hanover, NH Vita m i n s C+E C o l a c c h i o , Dartmouth, NH Esophagus D y s p l a s i a p a t i e n t s Esophagus General p o p u l a t i o n from h i g h r i s k area Lung Lung Lung Ch r o n i c smokers Asbestos C i g a r e t t e smokers Calcium carbonate Beta-carotene, centrum M u l t i p l e v i t a m i n s and m i n e r a l s V i t a m i n B12, f o l i c a c i d Beta-carotene, r e t i n o l Beta-carotene, r e t i n o l M. L i p k i n , S l o a n K e t t e r i n g Cancer Centre, New York T a y l o r , NCI, Bethesda, MD Chinese Academy, B e i j i n g T a y l o r , NCI, Bethesda, MD Chinese Academy, B e i j i n g D. Heimburger, U n i v e r s i t y of Alabama, Birmingham, AL G. Omenn, Fred Hutchinson Cancer Centre, S e a t t l e , WA E. Goodman, Fred Hutchinson Cancer Centre, S e a t t l e , WA 55 Lung Lung Lung S k i n S k i n S k i n S k i n S k i n S k i n A l l s i t e s A l l s i t e s Middle-age smoking males Smoking males Asbestos A l b i n o B a s a l c e l l carcinoma B a s a l c e l l carcinoma A c t i n i c k e r a t o s e s B a s a l c e l l carcinoma B a s a l c e l l carcinoma P h y s i c i a n s D e n t i s t s / nurses Beta-carotene, Albanes, NCI, Bethesda, MD v i t a m i n E F i n l a n d Beta-carotene L.H. K u l l e r , U n i v e r s i t y of P i t t s b u r g h , PA Beta-carotene, J . McLarty, U n i v e r s i t y of r e t i n o l Texas, T y l e r , TX Beta-carotene J . Luande, Muh i m b i l i M e d i c a l Centre, Dar-es-Salaam, Tanzania, A f r i c a Beta-carotene, B. S a f a i , S l o an K e t t e r i n g v i t a m i n s C+E Cancer Centre, New York Beta-carotene E.R. Greenberg, N o r r i s Cotton Cancer Centre, Hanover, NH R e t i n o l R e t i n o l , 1 3 - c i s r e t i n o i c a c i d 1 3 - c i s r e t i n o i c a c i d T.E. Moon, U n i v e r s i t y of A r i z o n a , Tucson, AZ F. Meyskens, U n i v e r s i t y of A r i z o n a , Tucson, AZ Tangrea, NCI, Bethesda, MD Beta-carotene, C. Hennekens, P e t e r Bent a s p i r i n Brigham, MA R e t i n y l C. Hennekens, P e t e r Bent palmetate, Brigham, MA sodium s e l e n i t e v i t a m i n s B6, E 56 T a b l e 13: R e p r e s e n t a t i v e Types of Car o t e n o i d s and Apo-carotenoids w i t h P r o v i t a m i n A A c t i v i t y C a r o t e n o i d s A c t i v i t y . (%) beta - c a r o t e n e 100 neo-beta-carotene U 38 alpha-c a r o t e n e 50-54 neo-alpha-carotene U 13 3,4,-dehydro-beta-carotene 7 5 3,4,3 1,4'-bisdehydro-beta-carotene 38 gamma-carotene 42-50 7,8'-dihydro-gamma-carotene 20-40 beta-carotene-5 1,6'-epoxide 21 alpha-carotene-5,6-epoxide 25 b e t a - c a r o t e n e - 5 , 6 , 5 1 , 6 1 - d i e p o x i d e a c t i v e 3-keto-beta-carotene 52 3- hydroxy-beta-carotene 50-60 4- hydroxy-beta-carotene 48 beta-apo-2'-carotenal a c t i v e b e t a - a p o - 8 1 - c a r o t e n a l 72 b e t a - a p o - 1 0 1 - c a r o t e n a l a c t i v e beta-apo-12'-carotenal 120 lycopene i n a c t i v e l u t e i n i n a c t i v e 57 4. DISCUSSION D i e t a r y d e f i c i e n c i e s o f v i t a m i n A, be t a - c a r o t e n e and ot h e r m i c r o - n u t r i e n t s are r e p o r t e d t o be important c o n t r i b u t i n g f a c t o r s t o the h i g h i n c i d e n c e o f c e r t a i n types o f cancers observed i n c e r t a i n p o p u l a t i o n s i n Northern China, the Northwest T e r r i t o r i e s , South A f r i c a and the P h i l l i p i n e s ( S t i c h e t a l . , 1985; Yang e t a l . , 1984; S t i c h and Rosin, 1984; S t i c h e t a l . , 1984). The v i t a m i n A d e f i c i e n t s t a t e may b r i n g about an i n c r e a s e d s e n s i t i v i t y t o environmental c a n c e r - c a u s i n g agents t o which these p o p u l a t i o n s are exposed. Short-term p i l o t s t u d i e s , as w e l l as long-term l a r g e s c a l e i n t e r v e n t i o n t r i a l s w i t h v i t a m i n A and/or beta-carotene have been i n i t i a t e d i n some human p o p u l a t i o n groups (Table 12). A l l of these endeavours, however, are plagued by a l a c k o f a t l e a s t t h r e e b a s i c p i e c e s o f i n f o r m a t i o n . F i r s t , the e f f i c a c y o f the doses of the chemopreventive agent(s) a d m i n i s t e r e d i s not known. Second, l i t t l e , i f anything, i s known about whether a p a r t i c u l a r treatment regimen a c t u a l l y causes a s i g n i f i c a n t e l e v a t i o n o f r e t i n o i d o r beta-carotene l e v e l s i n the t a r g e t t i s s u e s . T h i r d , o n l y sketchy i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g the nature o f i n i t i a t i n g and/or promoting agents a g a i n s t which a chemopreventive p r o t o c o l has been designed. C o n s i d e r i n g t h a t i n t e r v e n t i o n t r i a l s are b e i n g conducted i n the absence o f t h i s i n f o r m a t i o n , i t should be expected t h a t i n many cases the chosen doses and the s p a c i n g of a d m i n i s t r a t i o n of the chemopreventive agent(s) w i l l be l e s s than o p t i m a l . 58 The purpose o f the pr e s e n t i n v e s t i g a t i o n , t h e r e f o r e , was t o d e s i g n a v e r s a t i l e s h o r t - t e r m animal model system which c o u l d be used t o gather i n f o r m a t i o n and d e f i n e some of these parameters. In t h e s e experiments, v i t a m i n A d e f i c i e n c y was induced i n r a t s m aintained on a v i t a m i n A - f r e e d i e t f o r 9 t o 10 weeks. At the time o f s a c r i f i c e o f the r a t s (10 t o 11 weeks), r e t i n o l d e p l e t i o n from the serum and the l i v e r o f many o f the v i t a m i n A - d e p r i v e d r a t s was v i r t u a l l y complete. V i t a m i n A-d e f i c i e n t animals were h y p e r s e n s i t i v e t o the c y t o t o x i c and gen o t o x i c a c t i o n s o f the car c i n o g e n , DMN. L i v e r n e c r o s i s was e x t e n s i v e , c o m p r i s i n g up t o an average o f 45% of the l i v e r s of v i t a m i n A - d e f i c i e n t r a t s i n j e c t e d w i t h a t o t a l dose of DMN of 30 mg/kg body weight. At a lower dose of 15 mg/kg body weight, DMN was a b l e t o produce h e p a t o n e c r o s i s i n an average of 12% of the l i v e r t i s s u e from v i t a m i n A - d e f i c i e n t animals, though i t was unable t o produce l i v e r n e c r o s i s i n r a t s from the v i t a m i n A - f e d d i e t a r y c o n t r o l group, o r from the d i e t a r y supplemented groups A~VA, A~8VA and A~BC. C l a s t o g e n i c damage was enhanced a t l e a s t 2 - f o l d i n the l i v e r s o f v i t a m i n A - d e f i c i e n t r a t s over t h a t found i n v i t a m i n A - f e d r a t s a t a l l DMN doses examined (6 mg/kg t o 35 mg/kg body weight). T h i s was evidenced by an i n c r e a s e d p r o d u c t i o n o f the gen o t o x i c endpoints CF, CB and MN, l e a d i n g t o a d o u b l i n g o f the average percentage of abnormal anatelophase c e l l s i n the l i v e r c a r r y i n g these anomalies. 59 Furthermore, a g r e a t e r than 2 - f o l d i n c r e a s e i n the average frequency of these endpoints per anatelophase l i v e r c e l l was found i n v i t a m i n A - d e f i c i e n t r a t s over t h a t observed i n v i t a m i n A - f e d r a t s , s u g g e s t i n g s t r o n g l y t h a t the e x t e n t of DMN-induced damage was heightened by the v i t a m i n A - d e f i c i e n t s t a t e . The f i n d i n g s of the c u r r e n t i n v e s t i g a t i o n concur w e l l w i t h p r e v i o u s r e p o r t s . S e v e r a l i n v e s t i g a t o r s have shown t h a t e x p e r i m e n t a l animals f e d d i e t s d e f i c i e n t i n v i t a m i n A are more s e n s i t i v e t o c a r c i n o g e n exposure and develop a g r e a t e r than normal i n c i d e n c e of cancers and t h e i r p u t a t i v e p r e c u r s o r s (Longnecker e t a l . , 1983; Cohen e t a l . , 1976; Genta e t a l . , 1974). Wolbach and Howe, i n t h e i r o r i g i n a l c l a s s i c paper i n 1925, noted c e l l u l a r changes i n the mucus-secreting e p i t h e l i a l t i s s u e s of v i t a m i n A - d e f i c i e n t r a t s . During v i t a m i n A d e f i c i e n c y , proper d i f f e r e n t i a t i o n of stem c e l l s i n t o mature e p i t h e l i a l c e l l s o f t e n f a i l s t o occur, and abnormal c e l l u l a r d i f f e r e n t i a t i o n l e a d i n g t o h y p e r k e r a t i n i z a t i o n and squamous m e t a p l a s i a occurs f r e q u e n t l y . Furthermore, they noted e x c e s s i v e c e l l u l a r p r o l i f e r a t i o n i n many of the e p i t h e l i a of the v i t a m i n A - d e f i c i e n t animals. Wolbach and Howe concluded t h a t an adequate l e v e l of r e t i n o i d i s necessary f o r the c o n t r o l o f normal c e l l u l a r d i f f e r e n t i a t i o n and p r o l i f e r a t i o n . V i t a m i n A imbalance may permit c a r c i n o g e n - i n i t i a t e d c e l l s , which are normally h e l d i n check by homeostatic f a c t o r s , t o undergo c e l l p r o l i f e r a t i o n and form p r e n e o p l a s t i c 60 l e s i o n s . A d e f i c i e n c y i n v i t a m i n A (or a d e f i c i e n c y of i t s e s s e n t i a l f u n c t i o n ) may f o s t e r c o n d i t i o n s conducive t o the e s t a b l i s h m e n t of an a l t e r e d c e l l p o p u l a t i o n t h a t can e x i s t i n a more s e l f - s u f f i c i e n t s t a t e , t h a t i s , i n a s t a t e i n which v i t a m i n A i s no l o n g e r e s s e n t i a l f o r i t s s u r v i v a l . The c o n d i t i o n o f ' e s s e n t i a l f u n c t i o n d e f i c i e n c y ' i s the premise u n d e r l y i n g t h e e s t a b l i s h m e n t of b i o l o g i c a l a u t a rchy of tumour c e l l s , as put f o r t h by De Luca (1983). Under t h i s model, a d e f i c i e n c y of v i t a m i n A (the p h y s i o l o g i c a l 'antipromotor'), i n i t s e l f , may a l l o w tumour development, and a l o c a l i z e d d e f i c i e n c y of v i t a m i n A may be s u f f i c i e n t . In t h i s study, the v i t a m i n A - f e d r a t s i n j e c t e d w i t h a r e l a t i v e l y h i g h dose of DMN (30 mg/kg body weight) d i s p l a y e d depressed l i v e r r e t i n o l l e v e l s and e l e v a t e d l e v e l s of the c y t o t o x i c and g e n o t o x i c endpoints. Whether the l o s s of r e t i n o l and the p r o d u c t i o n of DMN-induced l e s i o n s occur i n the same areas of the l i v e r t i s s u e remains t o be i n v e s t i g a t e d . Nonetheless, w h i l e a 'cause and e f f e c t ' r e l a t i o n s h i p cannot be demonstrated from the r e s u l t s of t h i s study, the c o i n c i d e n c e o f t h e s e two events i s noteworthy. In an i n v i v o study of nine d i f f e r e n t types of M o r r i s hepatomas i n r a t s , Buchsel and R e u t t e r (1982) r e p o r t e d a l i n e a r c o r r e l a t i o n between l o g a r i t h m i c i n c r e a s e s i n growth r a t e s (which p a r a l l e l a g g r e s s i v e n e s s and l o s s of d i f f e r e n t i a t i o n ) of the tumours and decreases i n uptake r a t e s o f [ 3 H ] r e t i n o l . They concluded t h a t the decreased uptake of 61 r e t i n o l by M o r r i s hepatomas i s r e l a t e d t o malignant t r a n s f o r m a t i o n and not simply due t o i n c r e a s e d growth. The r a p i d growth of the l i v e r d u r i n g f e t a l development or a f t e r p a r t i a l hepatectomy i s a s s o c i a t e d w i t h an enhanced r e t i n o l b i n d i n g and uptake and an i n c r e a s e i n c e l l u l a r r e t i n o l b i n d i n g p r o t e i n s (CRBP). S i m i l a r l y , decreased b i n d i n g o f [ 3 H ] r e t i n o l i n v i t r o o ccurs i n p a r a l l e l w i t h a l o s s o f d i f f e r e n t i a t i o n i n d y s p l a s t i c human c e r v i x u t e r i t i s s u e (Palan and Romney, 1979). C o n s i s t e n t w i t h these r e p o r t s are the o b s e r v a t i o n s o f a 2 0 - f o l d decrease i n the c o n c e n t r a t i o n o f r e t i n o l i n r a t i n t e s t i n a l carcinoma as compared t o normal r a t i n t e s t i n a l t i s s u e (Sundaresan and De Luca, 1977), and a r e d u c t i o n t o l e s s than 1% of normal l i v e r r e t i n o l l e v e l s , which i s a s s o c i a t e d w i t h t h e e x p r e s s i o n of a no v e l o n c o f e t a l c e l l u l a r r e t i n o l b i n d i n g p r o t e i n [CRBP(F)] i n human hepatoma t i s s u e , i n comparison w i t h a d j a c e n t normal l i v e r t i s s u e (Muto and Omori, 1981). The s i g n i f i c a n c e o f t h i s new CRBP(F) i s not y e t known. Ong e t a l . , (1978), i n c o n t r a s t , observed an i n c r e a s e i n [ 3 H ] r e t i n o l b i n d i n g i n c o l o r e c t a l adenocarcinoma of r a t s . A n h y d r o r e t i n o l , an i n a c t i v e m e t a b o l i t e o f r e t i n o l , i s d e t e c t e d c o n c u r r e n t l y w i t h the d e p r e s s i o n o f l i v e r r e t i n o l l e v e l s i n hepatomas of humans and mice (Muto and Moriwaki, 1984). In spontaneously transformed mouse f i b r o b l a s t s (Balb/c 3T12-3 c e l l s ) , a 90% c o n v e r s i o n o f [ 1 4 C ] r e t i n o l t o [ 1 4 C ] a n h y d r o r e t i n o l i s observed (Bhat e t a l . , 1979). Thus, a l o c a l i z e d d e f i c i e n c y o f v i t a m i n A may be brought about by a 62 l o c a l i z e d increase i n conversion of r e t i n o l to anhydroretinol during hepatocarcinogenesis. In the current study, analysis for the presence of anhydroretinol was c a r r i e d out i n the l i v e r s of DMN-exposed, vitamin A-deficient rats, vitamin A-fed r a t s and vitamin A-d e f i c i e n t rats that received supplementary beta-carotene i n t h e i r d i e t s (data not shown). A r e l a t i o n s h i p between the l e v e l s of l i v e r r e t i n o l or l i v e r damage, and the l e v e l s of anhydroretinol i n the l i v e r was not observed. There are several possible explanations for t h i s . F i r s t , areas of the l i v e r containing putative preneoplastic lesions were not analyzed separately from the remaining normal l i v e r t i s s u e , thus a l o c a l i z e d increase i n anhydroretinol l e v e l s may have escaped detection. Second, the presence of necrotic c e l l s i n some samples may have masked an increased conversion of r e t i n o l to anhydroretinol i n a few v i a b l e c e l l s . F i n a l l y , the conversion of r e t i n o l to anhydroretinol may be an event associated with the l a t e r stages of tumour promotion and, therefore, would not be detected i n the l i v e r s of the rats i n t h i s short-term model system. Further investigations are required to elucidate the possible roles of anhydroretinol and CRBP(F) i n the production of l i v e r r e t i n o l deficiency, and i n the process of l i v e r carcinogenesis. Vitamin A deficiency i n man i s a major n u t r i t i o n a l problem throughout the world, second only i n incidence and prevalence to p r o t e i n - c a l o r i e malnutrition. Vitamin A 6 3 d e f i c i e n c y i s of p a r t i c u l a r concern i n t r o p i c a l and sub-t r o p i c a l r e g i o n s o f the globe, such as Southeast A s i a , China, A f r i c a , I n d i a and C e n t r a l America. The western world however, i s not spared. Although the p r e v a l e n c e i s lower, the d i s e a s e does occur and o f t e n goes unrecognized. A Canadian n u t r i t i o n a l survey and a t e n - s t a t e American n u t r i t i o n a l survey r e v e a l e d t h a t v i t a m i n A, i r o n and c a l c i u m are e s s e n t i a l n u t r i e n t s o f t e n p r e s e n t i n the d i e t i n marginal amounts. B l a c k s and H i s p a n i c groups are the most s e v e r e l y l a c k i n g i n v i t a m i n A ( f o r review see: Nut. Rev., 1983; US Dept. of He a l t h , 1972). These m a r g i n a l l y d e f i c i e n t p o p u l a t i o n groups may have an i n c r e a s e d s u s c e p t i b i l i t y t o environmental c a r c i n o g e n s t o which they may be exposed, and thus, they are c o n s i d e r e d t o be a t an e l e v a t e d r i s k f o r the development of cancer. In t h i s study, the r e v e r s a l o f v i t a m i n A - d e f i c i e n c y i n r a t s by the supplementation o f the v i t a m i n A - f r e e d i e t w i t h v i t a m i n A ( r e t i n y l a c e t a t e ) , l e d t o an a b o l i t i o n o f the h y p e r s e n s i t i v i t y o f these r a t s t o the d e l e t e r i o u s e f f e c t s o f DMN. Serum and l i v e r r e t i n o l l e v e l s o f the A~VA r a t s were r e s t o r e d t o l e v e l s found i n the v i t a m i n A - f e d d i e t a r y c o n t r o l r a t s , and concomi t a n t l y , the extent o f g e n o t o x i c damage i n the l i v e r s o f A~VA r a t s was comparable t o the ext e n t o f damage found i n the l i v e r s o f v i t a m i n A - f e d r a t s . 64 These e f f e c t s o f v i t a m i n A replacement are c o n s i s t e n t w i t h o t h e r s t u d i e s which show t h a t the a d m i n i s t r a t i o n o f r e t i n o i d s can suppress the pro c e s s o f c a r c i n o g e n e s i s i n v i v o (Kroes e t a l . , 1986; Aylsworth e t a l . , 1985; Alam e t a l . , 1984; Moon e t a l . , 1983; B e c c i e t a l . , 1979), and the development of the transformed phenotype i n v i t r o (Mass e t a l . , 1984; Huang e t a l . , 1982). In a d d i t i o n , a few s t u d i e s demonstrate t h a t r e t i n o i d s can e x e r t e f f e c t s on c e r t a i n f u l l y transformed, i n v a s i v e , n e o p l a s t i c c e l l s , l e a d i n g , i n c e r t a i n i n s t a n c e s , t o a s u p p r e s s i o n o f p r o l i f e r a t i o n (Fraker e t a l . , 1984) and m e t a s t a s i s (Morre, 1980; Lotan, 1980), and i n o t h e r i n s t a n c e s , t o the t e r m i n a l d i f f e r e n t i a t i o n and the p r o d u c t i o n o f a more benign, non-transformed phenotype i n these c e l l s ( S t r i c k l a n d e t a l . , 1980; Breitman e t a l . , 1980). Some i n v e s t i g a t o r s have found, i n some cases, no e f f e c t (Hard and Ogiu, 1984; Beems, 1984; Alam e t a l . , 1984), and i n oth e r cases, an enhancement i n precancerous endpoints due t o v i t a m i n A a d m i n i s t r a t i o n (Daoud and G r i f f i n , 1985; Longnecker e t a l . , 1983; L e v i j and P o l l i a c k , 1968). T h i s d i s c r e p a n c y may be r e l a t e d t o the time of treatment w i t h the r e t i n o i d r e l a t i v e t o the time of exposure t o t h e c a r c i n o g e n or t o the r e l a t i v e doses o f r e t i n o i d and c a r c i n o g e n a p p l i e d . The m a j o r i t y o f s t u d i e s i n d i c a t e a b e n e f i c i a l e f f e c t , and these r e s u l t s a re the b a s i s o f c u r r e n t attempts t o use r e t i n o i d s f o r cancer chemoprevention i n humans. 65 The u s e f u l n e s s o f v i t a m i n A supplementation i n the p r e v e n t i o n o f p r e n e o p l a s i a and n e o p l a s i a i n man may be l i m i t e d because of t o x i c s i d e e f f e c t s a s s o c i a t e d w i t h i t s i n g e s t i o n a t ph a r m a c o l o g i c a l doses (Underwood, 1984) . In t h i s study, the supplementation o f the v i t a m i n A - d e f i c i e n t r a t d i e t w i t h v i t a m i n A a t boosted l e v e l s (8 x adequate l e v e l s ) r e s u l t e d i n an a c c e n t u a t i o n o f the c y t o t o x i c i t y and g e n o t o x i c i t y induced i n t he l i v e r by DMN. T h i s enhancement was observed when r e l a t i v e l y h i g h doses of DMN (35 mg/kg body weight) were a d m i n i s t e r e d w i t h ensuing l i v e r t o x i c i t y . E x c e s s i v e r e t i n o l supplementation, i n the presence of l i v e r damage and n e c r o s i s , may p o t e n t i a t e the development of acute l i v e r d y s f u n c t i o n as w e l l as l i v e r c a r c i n o g e n e s i s . T h i s o b s e r v a t i o n deserves re-emphasis as i t has important i m p l i c a t i o n s f o r the d e s i g n o f i n t e r v e n t i o n p r o t o c o l s . The a d m i n i s t r a t i o n o f e x c e s s i v e amounts of v i t a m i n A t o human p o p u l a t i o n groups s u f f e r i n g from v i r a l - h e p a t i t i s i n f e c t i o n ( i . e . , some South A f r i c a n p o p u l a t i o n groups), l i v e r c i r r h o s i s ( i . e . , a l c o h o l i c s ) , l i v e r f l u k e s ( i . e . , some southern Chinese groups) or o t h e r form of l i v e r damage, maybe p o t e n t i a l l y dangerous t o the i n d i v i d u a l s o f these groups and t h e r e f o r e s h o u l d be a d v i s e d a g a i n s t . DMN e l i c i t e d v a r y i n g amounts of c y t o t o x i c i t y and g e n o t o x i c i t y i n the l i v e r s o f animals f e d v i t a m i n A-adequate d i e t s i n t h i s study, a l b e i t h i g h doses of DMN were necessary. S u s c e p t i b i l i t y t o c a r c i n o g e n exposure may be dependent on the 66 r e l a t i v e l e v e l s of exposure t o c a r c i n o g e n i c and a n t i c a r c i n o g e n i c substances (Newberne and Connor, 1986). D i e t a r y supplementation w i t h v i t a m i n A a t i n t e r m e d i a t e l e v e l s ( i . e . , g r e a t e r than adequate l e v e l s , but below l e v e l s t h a t produce l i v e r r e t i n o l s a t u r a t i o n ) should be examined i n r a t s f o r p o s s i b l e b e n e f i c i a l e f f e c t s . Supplementation of the r a t d i e t w i t h r e t i n o i c a c i d and w i t h s y n t h e t i c r e t i n o i d which are l e s s t o x i c and p o s s i b l y more e f f e c t i v e than r e t i n y l a c e t a t e , should a l s o be i n v e s t i g a t e d . L i t t l e i s known about the chemoprotective b e n e f i t s of b e t a - c a r o t e n e i n man. Most s t u d i e s i n d i c a t e a r e a s o n a b l y c o n s i s t e n t but not v e r y dramatic p r o t e c t i v e e f f e c t (Nomura, 1985; Winn e t a l . , 1984; Peto and D o l l , 1981). In the c u r r e n t i n v e s t i g a t i o n , b e ta-carotene supplementation of the v i t a m i n A-d e f i c i e n t d i e t was p r o t e c t i v e f o r r a t s a g a i n s t the noxious e f f e c t s of exposure t o DMN. L i v e r c e l l n e c r o s i s was d e t e c t e d i n the l i v e r s o f the A~BC r a t s o n l y a f t e r DMN a d m i n i s t r a t i o n a t r e l a t i v e l y h i g h doses (30 mg/kg body weight), and then a t a 7 - f o l d reduced l e v e l as compared t o the e x t e n t of n e c r o s i s observed i n the l i v e r s of v i t a m i n A - d e f i c i e n t r a t s . Furthermore, g e n o t o x i c damage i n the l i v e r s o f A~BC r a t s was reduced by 2 - f o l d as compared t o v i t a m i n A - d e f i c i e n t r a t s . D i e t a r y supplementation w i t h beta-carotene was p r o t e c t i v e a g a i n s t the c y t o t o x i c and g e n o t o x i c e f f e c t s o f DMN exposure. DMN-induced damage was l e s s e x t e n s i v e i n the l i v e r s o f the A~BC r a t s than i n the l i v e r s of the d i e t a r y c o n t r o l 67 v i t a m i n A - f e d r a t s when exposed t o s i m i l a r doses o f DMN. T h i s f i n d i n g suggests t h a t i n t h i s study, d i e t a r y b e t a - c a r o t e n e supplementation c o n f e r r e d a g r e a t e r p r o t e c t i v e e f f e c t than d i e t a r y v i t a m i n A. These experiments should be extended t o examine supplementation o f the r a t d i e t w i t h b e t a - c a r o t e n e a t h i g h e r doses. R e t t u r a e t a l . , (1983) found a s t r i k i n g p r o t e c t i v e e f f e c t i n mice when beta-carotene was supplemented a t a c o n c e n t r a t i o n o f 90 mg/kg d i e t , which i s s e v e r a l times h i g h e r than the average d a i l y content i n human d i e t s . Other i n v e s t i g a t o r s , u s i n g lower doses o f beta-carotene, d i d not f i n d b e t a - c a r o t e n e t o be p r o t e c t i v e ( L o p r i n z i and Verma, 1985; Alam and Alam, 1983) . Thus, d i e t a r y b e ta-carotene supplementation a t v a r i o u s doses should be t e s t e d i n order t o determine the dose f o r op t i m a l p r o t e c t i o n . The time and s p a c i n g o f beta-carotene treatments i n r e l a t i o n t o DMN exposure should a l s o be examined. In t h i s way an e f f e c t i v e and e f f i c i e n t treatment schedule f o r d i e t a r y b e t a - c a r o t e n e supplementation can be designed. U n l i k e r e t i n o i d s , c a r o t e n o i d s do not produce the adverse symptoms of h y p e r v i t a m i n o s i s A, even when i n g e s t e d i n l a r g e q u a n t i t i e s . While not the most abundant carotene, b e t a - c a r o t e n e i s , n e v e r t h e l e s s , widespread i n nature. Common food sources o f beta-carotene i n c l u d e c a r r o t s , b r o c c o l i , s p i n a c h , p a r s l e y , sweet potatoes and a v a r i e t y o f f r u i t s . The i n c l u s i o n o f these foods i n the d a i l y d i e t s o f humans i s a 68 n a t u r a l and n u t r i t i v e means t o b e t a c a r o t e n e supplementation. For the g e n e r a l p o p u l a t i o n t h a t i s not n e c e s s a r i l y a t an e l e v a t e d r i s k f o r cancer development, t h i s may be an a c c e p t a b l e avenue f o r chemoprevention. The s i g n i f i c a n c e of d i e t a r y c a r o t e n o i d s o t h e r than b e t a - c a r o t e n e has not y e t been assessed. A t t e n t i o n has p r i m a r i l y been focused on beta-carotene because of i t s p r o v i t a m i n A r o l e . Both h a l v e s of the symmetrical b e t a - c a r o t e n e molecule are r e l a t e d t o r e t i n o l , thus t h i s compound possesses maximal p r o v i t a m i n A a c t i v i t y . Other c a r o t e n o i d s have v a r y i n g amounts of p r o v i t a m i n A a c t i v i t y and some c a r o t e n o i d s are i n a c t i v e (Table 13; Simpson, 1983). I n v e s t i g a t i o n s i n t o the p r o t e c t i v e e f f i c a c y of o t h e r c a r o t e n o i d s u s i n g the r a t - l i v e r t e s t system would be o f c o n s i d e r a b l e i n t e r e s t as they would r e s o l v e the i s s u e as t o whether the p r o t e c t i v e e f f e c t of beta-carotene, and c a r o t e n o i d s i n g e n e r a l , i s a v i t a m i n A - r e l a t e d a c t i v i t y or i s i n h e r e n t i n the c a r o t e n o i d s themselves. M e c h a n i s t i c s t u d i e s on the m o l e c u l a r a c t i o n s of r e t i n o i d s and c a r o t e n o i d s are necessary b e f o r e the a n t i n e o p l a s t i c e f f e c t s of t h e s e agents can be understood. Numerous hypotheses have been proposed, but few have stood up t o experime n t a l t e s t s . R e t i n o l and r e t i n o i c a c i d demonstrate an a b i l i t y t o enhance T - c e l l - m e d i a t e d c y t o t o x i c i t y a g a i n s t tumour s p e c i f i c a n t i g e n s i n a l l o g e n i c and syngeneic h o s t s (Dennert e t a l . , 69 1979), and t o i n c r e a s e the n a t u r a l k i l l e r (NK) c e l l - m e d i a t e d ( n o n - s p e c i f i c ) c y t o t o x i c i t y of human lymphocytes i n v i t r o (Abb e t a l . , 1982). I t i s t h e r e f o r e suggested t h a t v i t a m i n A and v i t a m i n A - r e l a t e d substances may suppress cancer growth by augmenting an immunological response a g a i n s t tumour c e l l s i n the e a r l y stages of c a r c i n o g e n e s i s . However, i n a study by F r a k e r e t a l . , (1984), the i n h i b i t i o n of i n v i t r o growth of a human b r e a s t carcinoma c e l l l i n e (MDA-MB-231) i n athymic mice, appears t o be a d i r e c t r a t h e r than a immune-mediated e f f e c t of r e t i n o l . The absence of T - c e l l s i n athymic mice and the i n a b i l i t y o f r e t i n o l t o i n c r e a s e NK c e l l a c t i v i t y i n t h i s host make i t improbable t h a t antitumour immunity i s enhanced by r e t i n o l i n t h i s i n v i v o system, or t h a t augmentation o f an antitumour immune response i s the primary mechanism by which r e t i n o i d s a c t . An important b i o l o g i c a l f u n c t i o n of r e t i n o i d s i s t o p a r t i c i p a t e i n the g l y c o s y l a t i o n of g l y c o p r o t e i n s by means of the i n t e r m e d i a t e , r e t i n y l phosphate mannose, which i s a m e t a b o l i t e of r e t i n o l . V i t a m i n A d e f i c i e n c y i n r a t s r e s u l t s i n a decreased i n c o r p o r a t i o n of mannose i n t o l i v e r g l y c o p r o t e i n s i n v a r i o u s e p i t h e l i a l t i s s u e s . De Luca e t a l . , (1984) found t h a t p o s t n u c l e a r membranes from r a t hepatoma t i s s u e are i n a s t a t e of v i t a m i n A and r e t i n y l phosphate d e p l e t i o n , w h i l e the d o l i c h o l phosphate content i s s i m i l a r t o t h a t i n a d j a c e n t normal l i v e r membrane. R e t i n o i c a c i d and many s y n t h e t i c r e t i n o i d s do not c o n t r i b u t e t o r e t i n y l 70 phosphate s y n t h e s i s , but are a b l e t o suppress the n e o p l a s t i c t r a n s f o r m a t i o n ( o f t e n much more e f f e c t i v e l y than r e t i n o l ) . T h e r e f o r e , i t i s unreasonable t o b e l i e v e t h a t r e t i n y l phosphate mannose p l a y s a u n i v e r s a l and c r i t i c a l r o l e i n the c o n t r o l o f d i f f e r e n t i a t i o n , although f u r t h e r i n v e s t i g a t i o n s i n t h i s area are warranted. A m o l e c u l a r h y p o t h e s i s f o r r e t i n o i d s ' mechanism of a c t i o n t h a t i s compatable w i t h most experimental d a t a i s t h a t r e t i n o i d s modify gene e x p r e s s i o n . C h y t i l and Ong (1979) have demonstrated the presence o f n u c l e a r b i n d i n g s i t e s l o c a t e d on the chromatin t h a t are s p e c i f i c f o r r e t i n o l . In v i t a m i n A-d e f i c i e n t animals, a s t r i k i n g i n c r e a s e i n the s p e c i f i c b i n d i n g of r e t i n o l i n h e p a t i c n u c l e i i s found i n comparison w i t h v i t a m i n A - f e d c o n t r o l animals. In a manner s i m i l a r t o s t e r o i d hormones, the i n t e r a c t i o n o f v i t a m i n A w i t h the s p e c i f i c n u c l e a r b i n d i n g s i t e s p r o b a b l y modulates gene e x p r e s s i o n . Which genes are c o n t r o l l e d by r e t i n o i d s ? Westin e t a l . , (1982) have made an important o b s e r v a t i o n i n a human p r o m y e l o c y t i c leukemia (HL60) c e l l l i n e . R e t i n o i c a c i d , a t p h y s i o l o g i c a l l e v e l s , induced the d i f f e r e n t i a t i o n o f HL60 c e l l s t o g r a n u l o c y t e s , w i t h a concomitant s u p p r e s s i o n of the e x p r e s s i o n of the myc oncogene. More r e c e n t l y , G r e i p and De Luca (1985) have shown an analogous s u p p r e s s i o n of c-myc gene e x p r e s s i o n i n F9 murine t e r a t o c a r c i n o m a c e l l s upon r e t i n o i c a c i d - i n d u c e d d i f f e r e n t i a t i o n o f these c e l l s t o p a r i e t a l endoderm. T h i e l e e t a l . , (1985) have demonstrated an 71 85% decrease i n N-mvc gene e x p r e s s i o n , but not i n c-myc gene e x p r e s s i o n , c o n c u r r e n t w i t h the i n d u c t i o n o f d i f f e r e n t i a t i o n o f SMS-KCNR neuroblastoma c e l l s by r e t i n o i c a c i d . Oncogenes are, by d e f i n i t i o n , i m p l i c a t e d i n the process of n e o p l a s t i c t r a n s f o r m a t i o n . Many s t u d i e s demonstrate the a c t i v a t i o n o f c e l l u l a r proto-oncogenes by a m p l i f i c a t i o n , t r a n s l o c a t i o n o r p o i n t mutation ( f o r review see; Weinberg, 1983) . DMN-induced damage t o the DNA of hepatocytes o f r a t s ( e v i d e n t by the r e s u l t i n g chromosome fragments, chromatin b r i d g e s and m i c r o n u c l e i ) may have l e d t o an a c t i v a t i o n o f these genes. A c t i v a t i o n o f a c e l l u l a r proto-oncogene may l e a d t o an enhancement of gene e x p r e s s i o n o r t o the p r o d u c t i o n o f an a l t e r e d gene product, and through t h i s , e v e n t u a l l y l e a d t o the i n d u c t i o n o r promotion o f cancer. An e l e v a t i o n i n the e x p r e s s i o n o f the c-myc gene i s observed i n M o r r i s hepatoma c e l l l i n e s (Cote e t a l . , 1985) and i n c a r c i n o g e n - i n d u c e d primary l i v e r tumours, but not i n the adj a c e n t non-tumorous l i v e r t i s s u e , nor i n the r e g e n e r a t i n g l i v e r t i s s u e o f p a r t i a l l y hepatectomized r a t s (Makino e t a l . , 1984) . The e x p r e s s i o n o f the c-myc gene appears t o be s e l e c t i v e l y a s s o c i a t e d w i t h t u m o r i g e n e s i s i n the l i v e r and not simply w i t h i n c r e a s e d hepatocyte p r o l i f e r a t i o n . I t i s not known whether the c-myc gene i n these tumours i s mutated. As w e l l , the m o l e c u l a r f u n c t i o n s o f the mvc gene product remain t o be e l u c i d a t e d . 7 2 The r a t l i v e r t e s t system d e s c r i b e d i n t h i s study c o u l d be m o d i f i e d t o accommodate i n v e s t i g a t i o n s on the a c t i v a t i o n of the c-myc gene o r o t h e r c e l l u l a r oncogenes (c-onc) i n the l i v e r s o f DMN-exposed r a t s a t v e r y e a r l y stages i n h e p a t o c a r c i n o g e n e s i s . E x t r a c t i o n s o f RNA and DNA from hepatocytes of t r e a t e d animals c o u l d be a n a l y z e d f o r e l e v a t e d c-onc t r a n s c r i p t i o n and f o r a l t e r a t i o n s (mutations) i n c-onc genes. P o s s i b l e modulating i n f l u e n c e s on the e x p r e s s i o n of c-onc genes, due t o v i t a m i n A - d e f i c i e n c y or the r e v e r s i o n of the v i t a m i n A - d e f i c i e n t s t a t e i n r a t s (by d i e t a r y supplementation w i t h v i t a m i n A or beta-carotene) c o u l d be examined, thereby r e l a t i n g v i t a m i n A - s t a t u s , c a r c i n o g e n s e n s i t i v i t y and proto-oncogene a c t i v a t i o n t o g e t h e r i n an i n v i v o t e s t system. M o l e c u l a r mechanisms u n d e r l y i n g the chemopreventive a c t i o n s of d i e t a r y b e ta-carotene are not w e l l understood. A p r o t e c t i v e r o l e f o r beta-carotene and the o t h e r c a r o t e n o i d s has l o n g been e s t a b l i s h e d i n green p l a n t s , and i s due t o t h e i r e f f i c i e n t a b i l i t y t o quench the e x c i t a t i o n energy of s i n g l e t oxygen molecules and t r a p c e r t a i n f r e e r a d i c a l s p e c i e s t h a t are produced as by-products of p l a n t metabolism. More r e c e n t l y , Burton and I n g o l d (1984) showed t h a t b eta-carotene i s e f f e c t i v e i n t r a p p i n g f r e e r a d i c a l s a t oxygen p a r t i a l p r e s s u r e s s i m i l a r t o p h y s i o l o g i c a l c o n d i t i o n i n s i d e human t i s s u e s . D e a c t i v a t i o n of p r o o x i d a n t chemical s p e c i e s i n b i o l o g i c a l systems i s i m p e r a t i v e as they would otherwise 73 i n i t i a t e harmful r e a c t i o n s , such as l i p i d p e r o x i d a t i o n , and may, through t h i s p r o c e s s , e v e n t u a l l y induce cancer. S e v e r a l a n t i o x i d a n t systems are pr e s e n t i n the body, i n c l u d i n g v i t a m i n E and s e l e n i u m - c o n t a i n i n g g l u t a t h i o n i n e enzymes, thus the r e l a t i v e importance o f beta-carotene i n t h i s c a p a c i t y i n humans i s not known. Us i n g the r a t l i v e r t e s t system, d i e t a r y m a n i p u l a t i o n s i n r a t c o u l d be c a r r i e d out t o determine the p r o t e c t i v e e f f e c t s of v i t a m i n E, selenium, beta-carotene and o t h e r d i e t a r y f a c t o r s . For example, the i n d u c t i o n of v i t a m i n E and/or selenium d e f i c i e n c y i n r a t s , f o l l o w e d by d i e t a r y supplementation w i t h beta-carotene p r i o r t o exposure o f these r a t s t o c a r c i n o g e n s , may l e a d t o a c l a r i f i c a t i o n o f be t a - c a r o t e n e ' s c o n t r i b u t i o n as an a n t i o x i d a n t i n v i v o . A d d i t i o n a l s t u d i e s should be conducted t o e l u c i d a t e o t h e r p h y s i o l o g i c a l f u n c t i o n s o f beta-carotene t h a t may or may not be r e l e v a n t t o cancer p r e v e n t i o n . Of course what u l t i m a t e l y matters from a p u b l i c h e a l t h p o i n t o f view i s not so much the 'mechanisms' whereby d i e t a r y f a c t o r s may a f f e c t cancer, but r a t h e r the 'nature' o f the d i e t a r y f a c t o r s t h a t a re important determinants f o r cancer c o n t r o l . By analogy, i t i s more important t o know t h a t c i g a r e t t e s cause lung cancer than t o know the mechanisms of t h i s e f f e c t , although the g r e a t advantage o f understanding the u n d e r l y i n g mechanisms i s t h a t i t may l e a d t o the d i s c o v e r y of agents which may modulate these a c t i o n s ( D o l l and Peto, 1981). 74 5. SUMMARY In t h i s study, a r a t l i v e r model system was d e v i s e d as a sh o r t - t e r m assay f o r the chemopreventive b e n e f i t s o f d i e t a r y v i t a m i n A and beta-carotene supplementation a g a i n s t exposure t o the chemical c a r c i n o g e n , DMN. The system was a l s o designed t o be adaptable t o f u t u r e i n v e s t i g a t i o n s on o t h e r determinants t h a t may be a b l e t o m i t i g a t e the c a r c i n o g e n e s i s p r o c e s s . In t h i s t e s t system, v i t a m i n A - d e f i c i e n c y produced h y p e r s e n s i t i v i t y t o subsequent exposure DMN. T h i s was e v i d e n t by an i n c r e a s e i n the number of c y t o t o x i c (CN) and gen o t o x i c (CB, CF and MN) l e s i o n s generated by DMN. T h i s h y p e r s e n s i t i v i t y was a b o l i s h e d by supplementation of the v i t a m i n A - d e f i c i e n t d i e t w i t h v i t a m i n A or bet a - c a r o t e n e one week p r i o r t o DMN a p p l i c a t i o n . D i e t a r y v i t a m i n A a t adequate l e v e l s p r o v i d e d a r e l a t i v e p r o t e c t i v e e f f e c t f o r A""VA r a t s switched t o t h i s d i e t p r i o r t o DMN exposure and f o r v i t a m i n A-f e d d i e t a r y c o n t r o l r a t s maintained on t h i s d i e t f o r the e n t i r e d u r a t i o n o f the experiment. D i e t a r y b e t a - c a r o t e n e supplementation c o n f e r r e d a g r e a t e r p r o t e c t i o n f o r A~BC r a t s than was expected on the b a s i s of i t s c o n v e r s i o n t o r e t i n o l . F i n a l l y , d i e t a r y supplementation w i t h boosted amounts of v i t a m i n A l e d t o an a c c e n t u a t i o n of DMN-induced damage, when h i g h doses of DMN were a d m i n i s t e r e d w i t h ensuing l i v e r n e c r o s i s . In view of these o b s e r v a t i o n s , i n t e r v e n t i o n t r i a l s w i t h v i t a m i n A i n human p o p u l a t i o n s should be c a r r i e d out wi t h 75 extreme c a u t i o n , e s p e c i a l l y i n p o p u l a t i o n s where l i v e r d i s e a s e i s p r e v a l e n t . Although the chemopreventive e f f i c a c y of beta-carotene and v i t a m i n A (at a p p r o p r i a t e doses) i s apparent, s e v e r a l q u e s t i o n s remain t o be answered. (1) I s d i e t a r y v i t a m i n A or beta-carotene supplementation e f f e c t i v e a g a i n s t the c a r c i n o g e n i c a c t i o n s of a wide v a r i e t y of chemicals? Of p a r t i c u l a r i n t e r e s t would be the e f f i c i e n c y o f these agents a g a i n s t a f l a t o x i n , which i s a common mycotoxin contaminant of s p o i l e d foods and i s suspected of b e i n g an important e t i o l o g i c a l f a c t o r i n the p r e v a l e n c e of human primary h e p a t o c e l l u l a r cancer i n South A f r i c a and the F a r E a s t . (2) Are v i t a m i n A and beta-carotene e f f e c t i v e s u p p r e s s o r s of cancer development i n t i s s u e s o t h e r than the l i v e r ? S e v e r a l d i f f e r e n t endpoints should be examined s i m u l t a n e o u s l y a t the time of s a c r i f i c e o f the r a t s . For example, m i c r o n u c l e i frequency may be monitored i n the hepatocytes and the lymphocytes from a s i n g l e animal. A l t e r n a t e endpoints, i n c l u d i n g the presence of a n h y d r o r e t i n o l and CRBP(F) and the a c t i v a t i o n of c e l l u l a r oncogenes c o u l d be examined. In a d d i t i o n , by p r o l o n g i n g the experiment, the time course f o r development of h y p e r p l a s t i c f o c i , h y p e r p l a s t i c nodules and the e v e n t u a l development of h e p a t o c e l l u l a r carcinoma can be determined. 76 (3) A t what stage(s) o f the c a r c i n o g e n e s i s does v i t a m i n A and bet a - c a r o t e n e e x e r t t h e i r i n h i b i t o r y e f f e c t s ? Furthermore, what might be an a p p r o p r i a t e treatment schedule f o r e f f e c t i v e a p p l i c a t i o n o f v i t a m i n A and b e t a - c a r o t e n e a g a i n s t c h r o n i c o r acute exposure t o carci n o g e n s ? (4) To what ext e n t do o t h e r d i e t a r y f a c t o r s , i n c l u d i n g v i t a m i n E, selenium, r i b o f l a v i n , f l a v o n o i d s , p h e n o l i c s , f a t s , f i b r e and a l c o h o l , e x e r t i n h i b i t o r y o r enhancing i n f l u n c e s on cancer development? Do they a c t s y n e r g i s t i c a l l y o r a n t a g o n i s t i c a l l y w i t h v i t a m i n A and beta-carotene? (5) Q u a l i t a t i v e d i f f e r e n c e s between the i n v i v o pharmacodynamics of beta-carotene and v i t a m i n A are e v i d e n t from our experiments and c o r r o b o r a t e numerous o t h e r r e p o r t s . What i s the b a s i s f o r these d i f f e r e n c e s and what i s t h e i r r e l e v a n c e t o chemoprevention? Beta-carotene, w i t h the ot h e r c a r o t e n o i d s , deserves s p e c i a l c o n s i d e r a t i o n i n the d e s i g n of i n t e r v e n t i o n t r i a l s because i t possesses v i t a m i n A - a c t i v i t y without the dangers of v i t a m i n A - t o x i c i t y . The r e l a t i o n s h i p between cancer and be t a - c a r o t e n e i s e l e g a n t l y reviewed by Peto e t a l . , (1981). These authors concluded t h a t , "Although the evidence thus f a r a v a i l a b l e i s not c o m p e l l i n g t h a t b eta-carotene i s t r u l y p r o t e c t i v e a g a i n s t cancer (or a f o r t i o r i a g a i n s t t o t a l m o r t a l i t y ) t h i s i s not a good reason t o d e l a y s t a r t i n g c o n t r o l l e d t r i a l s because even i f , as seems probable, a few 77 t r u l y p r o t e c t i v e agents do await d i s c o v e r y among the dozen or two d i e t a r y f a c t o r s o f c u r r e n t i n t e r e s t t o the r e s e a r c h community, c o m p e l l i n g evidence may take decades t o emerge wit h o u t c o n t r o l l e d t r i a l s " . In a d d i t i o n t o c o n t r o l l e d i n t e r v e n t i o n t r i a l s i n human p o p u l a t i o n groups t h a t are a t h i g h r i s k f o r cancer development, e f f o r t s s h o u l d be made t o advocate t o the g e n e r a l p u b l i c the p o s s i b l e cancer p r e v e n t i v e b e n e f i t s o f e n r i c h i n g t h e i r d a i l y d i e t s w i t h foods c o n t a i n i n g c a r o t e n o i d s . 78 6. CONCLUDING REMARKS The p u b l i c i s much more aware of environmental cancer r i s k s . There i s l e g i s l a t i o n and r e g u l a t i o n a t the p r o v i n c i a l and f e d e r a l l e v e l s and i n i n t e r n a t i o n a l agreements t o c o n t r o l p o s s i b l e causes o f environmental cancer. N e v e r t h e l e s s , the p u b l i c , w h i l e concerned w i t h the i s s u e s , i s not w e l l informed. Many people f e e l t h a t man-made chemicals are the l a r g e s t cancer t h r e a t t o the g e n e r a l p o p u l a t i o n . T h i s i s not the case. Although manufactured chemicals are a s i g n i f i c a n t f a c t o r i n o c c u p a t i o n a l cancer, t h e i r impact on the g e n e r a l p u b l i c i s f a r l e s s than t h a t o f p e r s o n a l l i f e s t y l e f a c t o r s , such as c i g a r e t t e smoking, e x c e s s i v e sunbathing and c e r t a i n d i e t a r y components. Avoidance of exposure t o thes e cancer c a u s a t i v e agents i s the b e s t means f o r cancer p r e v e n t i o n . Thus, p u b l i c e d u c a t i o n campaigns and o t h e r s e r v i c e s s h o u l d be p r o v i d e d t o h e l p people modify p e r s o n a l h a b i t s t h a t are d e t r i m e n t a l t o h e a l t h . Exposure, however, cannot r e a l i s t i c a l l y be reduced t o zero. T h e r e f o r e , i t i s necessary t h a t t h e s e e f f o r t s be augmented w i t h cancer chemoprevention. Chemopreventive agents such as v i t a m i n A and bet a - c a r o t e n e , t h a t may be ad m i n i s t e r e d not o n l y t o 'high r i s k ' p o p u l a t i o n groups, but a l s o t o the g e n e r a l p o p u l a t i o n , deserve p a r t i c u l a r l y r i g o r o u s e v a l u a t i o n . The r a t l i v e r t e s t system used i n t h i s study was designed f o r t h i s purpose and the f o l l o w i n g r e s u l t s were obt a i n e d : 79 1. v i t a m i n A - d e f i c i e n c y l e d t o an i n c r e a s e d s u s c e p t i b i l i t y t o DMN-induced damage; 2. adequate l e v e l s o f v i t a m i n A c o n f e r r e d a p r o t e c t i v e e f f e c t a g a i n s t DMN exposure; 3. v i t a m i n A excess l e d t o an enhancement of DMN-induced damage when r e l a t i v e l y h i g h doses of DMN were a d m i n i s t e r e d ; 4. bet a - c a r o t e n e supplementation c o n f e r r e d a p r o t e c t i v e e f f e c t t h a t was g r e a t e r than the e f f e c t due t o i t s c o n v e r s i o n t o r e t i n o l alone. E x c e s s i v e d i e t a r y v i t a m i n A p o t e n t i a t e d c a r c i n o g e n -induced damage under some c o n d i t i o n s . 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