"Science, Faculty of"@en . "Zoology, Department of"@en . "DSpace"@en . "UBCV"@en . "Yee, Gina"@en . "2010-07-08T04:02:07Z"@en . "1986"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Epidemiological evidence and chemopreventive trials point to an antimutagenic and anticarcinogenic role for retinoids and carotenoids. Studies on human populations are logistically difficult and often ethically unacceptable. Therefore, to gain information on the potential chemopreventive benefits of dietary vitamin A and beta-carotene, the following experimental model was used. Rats were made vitamin A (retinol) deficient, then exposed to the liver carcinogen dimethylnitrosamine (DMN), partially hepatectomized, and thereafter, examined for the extent of liver necrosis (cytotoxicity) and the frequency of anaphase and telophase cells with chromosomal fragments, chromatin bridges and micronuclei (genotoxicity). Vitamin A-deficient rats (serum retinol levels <2 0 ng/ml and liver retinol levels <43 ng/g tissue) showed increased sensitivity towards the cytotoxic and genotoxic action of DMN as compared to vitamin A-fed dietary control rats. This elevated sensitivity was abolished by supplementation of the vitamin A-deficient diet with beta-carotene (10 mg/kg diet) or with vitamin A (2 mg/kg diet) prior to DMN exposure. However, vitamin A feeding at higher doses (16 mg/kg diet), which led to an excessive accumulation of retinol in the liver (>25,000 ng/g tissue), resulted in an accentuation of the necrotic and genotoxic action of DMN when high doses (35 mg/kg body weight) were administered. The results show that vitamin A deficiency leads to an increased susceptability to DMN-induced damage. Dietary supplementation with beta-carotene and with vitamin A (at adequate levels) protected against the deleterious effects of DMN, but supplementation with vitamin A at excessive levels potentiated the action of the carcinogen. This divergent, dose-dependent action of vitamin A calls for caution in the design of intervention strategies."@en . "https://circle.library.ubc.ca/rest/handle/2429/26211?expand=metadata"@en . "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 \u00C2\u00A9 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 \u00E2\u0080\u0094 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 \u00E2\u0080\u0094 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 \u00E2\u0080\u0094 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 \u00E2\u0080\u0094 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\u00C2\u00B0C 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\u00C2\u00B0C 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\u00C2\u00B0C 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\u00C2\u00B0C. 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 \u00C2\u00B1 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 \u00C2\u00B1 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 \u00E2\u0080\u0094 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 _\u00E2\u0080\u009E 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 \u00E2\u0080\u00A2 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 \u00E2\u0080\u0094 v | v i t a m i n A-supplemented d i e t \u00E2\u0080\u0094^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 ) \u00E2\u0080\u0094 ^ 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 ) \u00E2\u0080\u00A2 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) \u00E2\u0080\u00A2 v i t a m i n A-adequate r a t s ; b) \u00E2\u0080\u00A2 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) \u00E2\u0080\u00A2 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) \u00E2\u0080\u00A2 beta-carotene supplemented r a t s . 350n 300-250-S, 200-S3 a 150-100-50-0- \u00E2\u0080\u0094 r 4 -r-7 \u00E2\u0080\u0094r\u00E2\u0080\u0094 10 \u00E2\u0080\u0094i 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 \u00C2\u00A3 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 \u00C2\u00B1 5 7 30 + 14 E 10 201 \u00C2\u00B1 6 4 56 \u00C2\u00B1 22 F 10 90 + 38 23 \u00C2\u00B1 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. \u00C2\u00A3 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\u00C2\u00B111 5821+ 129 9+ 8 15 9 119\u00C2\u00B112 9\u00C2\u00B1 6 5465+2839 43+19 B DMN 22.5 28 127+20 2 0\u00C2\u00B1 6 5397\u00C2\u00B11738 8+20 c DAB C 30 15 108+27 4+ 4 2886+1275 0 d D DAB 40 29 111+24 19\u00C2\u00B1 8 5343+ 770 17+16 E DMN 30 5 NA e 0 383+ 165 4+ 6 22.5 14 NA 0 3138+1535 1\u00C2\u00B1 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 \u00E2\u0080\u0094 _ 15 177+66 22\u00C2\u00B118 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 \u00C2\u00B0 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 \u00C2\u00AE 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 \u00C2\u00B0 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\u00C2\u00B12 .2 ND 15 23 2.3+1.5 2.3+2.7 2. 2\u00C2\u00B11.1 2 . 3\u00C2\u00B12 .2 ND E 30 15 1.3+1.5 1.0+1.4 ND b ND 4.1\u00C2\u00B12.7 22.5 24 2.6+1.9 3.0+2.0 ND ND 3.0+1.4 F 22.5 21 1.8\u00C2\u00B12.4 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\u00C2\u00B11.6 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\u00C2\u00B12.6 29.8\u00C2\u00B16.6 15 19 2.3+2.1 22.8\u00C2\u00B15.3 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\u00C2\u00B10.82 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\u00C2\u00B10.0 10.2\u00C2\u00B14.4 22.5 24 7.9\u00C2\u00B13.3 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\u00C2\u00B11.1 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\u00C2\u00B10.04 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\u00C2\u00B10.02 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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