Promotion of Liver Carcinogenesis - ACS Symposium Series (ACS

25 Promotion of Liver Carcinogenesis Interactions of Barbiturates and a Choline-Deficient Diet H. SHINOZUKA, A. J. DEMETRIS, S. L. KATYAL, and M. I. R. PERERA

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Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 A choline deficient (CD) diet has been shown to exert a strong promoting effect on the emergence of foci of enzyme altered hepatocytes and on the induction of hepatomas in rats initiated with a carcinogen. Earlier, we demonstrated that inclusion of barbiturates in a CD diet resulted in a synergistic enhancement of tumor promotion, but inhibited the CD diet-induced stimulation of liver DNA synthesis and liver cell proliferation. In the present study, we examined how modifications of the quality of fat in a CD diet alters the promoting effect and the diet-induced lipid peroxidation in the liver. Feeding a CD diet for one week induced lipid peroxidation of liver microsomal membrane lipids as determined by diene conjugate formation. A CD diet with corn oil as the source of fat exerted a stronger promoting effect and induced lipid peroxidation earlier and more intensely than did a CD diet containing Primex and corn oil. Dietary administration of phenobarbital in a choline supplemented diet induced no lipid peroxidation and no evidence of enhanced lipid peroxidation was noted when phenobarbital was added to a CD diet. Thus, a CD diet and barbiturates exert their promoting effects through different mechanisms and the combination results in a synergistic enhancement. During the past ten years, the concept of initiation and promotion of chemical carcinogenesis, originally demonstrated in the skin of mice and rabbits, has been extended to several other organ systems including the liver (1,2). Thus, hepatocellular carcinomas can be induced in experimental animals by a relatively brief initiation by a carcinogen followed by prolonged imposition of a promoting stimulus. A number of promoters with diverse properties have been identified in liver tumor induction (1). Table I lists several known liver tumor promoters which can be arbitrarily divided into two broad categories; agents which are inducers of microsomal mono0097-6156/85/0277-0327$06.00/0 © 1985 American Chemical Society

Finley and Schwass; Xenobiotic Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

328

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

oxygenases and the ones which are n o t . I t i s evident that a large number of x e n o b i o t i c s which a c t as p r i m a r y h e p a t o t o x i c a n t s a r e promoters of l i v e r c a r c i n o g e n e s i s . The mechanisms by which d i f f e r ent agents o r c o n d i t i o n s e x e r t the common e f f e c t of l i v e r tumor p r o m o t i o n a r e not known.

TABLE I Promoting


Enzyme

Agents of

Hepatocarcinogenesis

Inducers

Non-Inducers

Estradiol-17-phenylpropionate

Phénobarbital (other b a r b i t u r a t e s )

Ethiny]

Dichlorodiphenyltrichloroethane (DDT)

P o l y c h l o r i n a t e d (brominated) biphenyl(s) 1

3-(3,5-di c h l o r o p h e n y l ) 5 , 5 dimethyloxazoline-1,4-dione

estradiol

Lithocholic

acid

Choline-deficient

diet

Methapyrilene

Orotic

acid

2,3,7,8-tetrachlorodibenzo-pd i o x i n (TCDD) Hexachlorocyclohexane

Microsomal

monoxygenases.

We have been i n v e s t i g a t i n g a few s e l e c t e d a s p e c t s of the mechanism of l i v e r tumor promotion by a d i e t d e v o i d of c h o l i n e , a c h o l i n e d e f i c i e n t (CD) d i e t . The d i e t i s an e f f e c t i v e promot e r of the emergence of e a r l y presumptive p r e n e o p l a s t i c f o c i o f γ-glutamyltranspeptidase (GGT)-positive hepatocytes as w e l l as of the p r o g r e s s i o n of G G T - p o s i t i v e f o c i t o hepatomas ( 3 , 4 ) . A CD d i e t w i t h a h i g h f a t c o n t e n t (14%) e x e r t e d a s t r o n g e r promo t i n g a c t i o n than a CD d i e t w i t h a low f a t c o n t e n t (5%) ( 5 ) . The addition of phénobarbital, another type of liver tumor promoter, t o a CD d i e t r e s u l t e d i n a marked s y n e r g i s t i c enhancement on l i v e r tumor promotion (6). I n both c a s e s , however, the e f f i c a c y of the promotion was not d i r e c t l y c o r r e l a t e d w i t h



SHINOZUKA ET AL.

Barbiturates, Choline-Deficient Diet, and Liver Cancer

the e x t e n t of l i v e r cell proliferation induced by the d i e t s (5,7). Thus, as i n the case of s k i n tumor p r o m o t i o n ( 8 , 9 ) , the stimulation of cell proliferation in target cells may be important but may not be s u f f i c i e n t c o n d i t i o n s f o r l i v e r tumor promotioru I t has been p o s t u l a t e d t h a t c e r t a i n promoters of carcinogenesis may act by generating oxygen radicals and resulting in lipid peroxidation (10,11). T h i s n o t i o n stemmed from the observation that several free radical generating compounds such as benzoyl peroxide, lauryl peroxide and c h l o r o p e r b e n z o i c a c i d have been shown to induce s k i n tumors i n mice (12,13). L i p i d p e r o x i d a t i o n may lead to s t r u c t u r a l and f u n c t i o n a l p e r t u r b a t i o n of the c e l l u l a r membranes and/or DNA, and such a l t e r a t i o n s may s e r v e as an u n d e r l y i n g mechanism of tumor p r o m o t i o n . Among promoters of l i v e r tumor i n d u c t i o n , phénobarbital, TCDD and a CD d i e t have been r e p o r t e d to induce lipid peroxidation in the liver under certain experimental c o n d i t i o n s (14,15,16,17). In t h i s paper, we w i l l p r e s e n t some of our r e c e n t d a t a on, m o d i f i c a t i o n of the CD-diet mediated p r o m o t i o n by c h a n g i n g the q u a l i t y of d i e t a r y f a t , g e n e r a t i o n of l i p i d p e r o x i d a t i o n by a CD d i e t and the e f f e c t s of phénobarbit a l on C D - d i e t - i n d u c e d l i p i d p e r o x i d a t i o n . Q u a l i t y of F a t as a M o d i f y i n g CD D i e t

F a c t o r of L i v e r Tumor Promotion by

a

Experimental^, d i e t a r y f a t s , both q u a n t i t y and quality, play important r o l e s i n the g e n e s i s of c a n c e r s of many organs (18,19). E a r l i e r , we demonstrated t h a t a h i g h f a t CD diet i s a more efficient liver tumor promoter than the d i e t w i t h a low f a t content (5) . The p r o p o r t i o n o f s a t u r a t e d and polyunsaturated f a t t y a c i d s i n a CD d i e t i s an important d e t e r m i n i n g f a c t o r f o r the s e v e r i t y of the d i e t - i n d u c e d f a t t y l i v e r (20,21). Saturated f a t t y a c i d s c o n t a i n i n g 14 to 18 carbons i n the d i e t appeared to i n c r e a s e the d e p o s i t i o n of f a t i n the l i v e r more so than u n s a t u r a ted f a t t y a c i d s (22). I t became of c o n s i d e r a b l e i n t e r e s t to d e t e r m i n e whether and how c h a n g i n g the q u a l i t y of f a t i n a CD d i e t m o d i f i e s the p r o m o t i n g e f f i c a c y of the d i e t . Male Sprague Dawley r a t s w e i g h i n g 180-200 gm were i n i t i a t e d w i t h a s i n g l e dose of d i e t h y l n i t r o s a m i n e (DEN) at 40 mg/KG 18 h o u r s a f t e r p a r t i a l hepatectomy. One week t h e r e a f t e r , groups of r a t s were f e d a c h o l i n e supplemented (CS) or CD d i e t i n which the degree of s a t u r a t i o n of f a t was o b t a i n e d by u s i n g hydrogenated v e g e t a b l e o i l ( s a t u r a t e d ) (Primex) and c o r n o i l ( p o l y u n s a t u r a t e d ) (CO). The r e g u l a r CS and CD d i e t s c o n t a i n e d 4% CO and 10% Primex w h i l e the CS and CD-CO d i e t c o n t a i n e d 14% CO and the CS, CD-primex d i e t s 14% Primex. A f t e r 7 weeks of promotion by the d i f f e r e n t d i e t a r y regimens, the a n i m a l s were k i l l e d and enzyme a l t e r e d f o c i (GGT) i n the l i v e r were q u a n t i t a t e d ( 3 ) . The r e s u l t s as shown i n T a b l e I I i n d i c a t e t h a t f e e d i n g the CS d i e t s of d i f f e r e n t f a t q u a l i t y showed no promoting a c t i v i t y . The number of G G T - p o s i t i v e f o c i d e v e l o p e d a f t e r 7 weeks of promotion by the CD d i e t w i t h p r e d o m i n a n t l y CO was 2.6 times h i g h e r than t h a t by the r e g u l a r CD diet.


329

M

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL E F F E C T S

TABLE I I Induction


Group

o f GGT(+) F o c i by V a r i o u s Regimens o f D i e t a r y P r o m o t i o n .

D i e t a r y Promotion

No. Foci/cm'

0.98 ± 0.3

1

CS

2

CS = Corn O i l

1.20 ± 0.5

3

CS

0.90 ± 0.4

4

CD = R e g u l a r

10.8

± 1.6

5

CD = Corn O i l

28.5

± 5.0

6

CD = Primex

Regular

Primex

***

4.9 ± 1.0

Initiation: Each animal r e c e i v e d a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n o f d i e t h y l n i t r o s a m i n e (PEN) (40 mg/KG) 18 hours a f t e r p a r t i a l hepatectomy. **Each f i g u r e r e p r e s e n t s t h e Mean ± S.E. o f 7-8 r a t s . D i f f e r e n c e s between t h e means were e v a l u a t e d s t a t i s t i c a l l y ***by S t u d e n t ' s t t e s t . Ρ 0 . 0 2 when compared w i t h group 5 and Ρ

Promotion of Liver Carcinogenesis - ACS Symposium Series (ACS

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