Modulation of Mammary Tumor Incidence by Dietary Fat and

11 Modulation of Mammary Tumor Incidence by Dietary Fat and Antioxidants

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A Mechanistic Approach M. MARGARET KING1, JUNJI TERAO1,3, GEMMA BRUEGGEMANN1, PAUL B. McCAY1, and ROBERT A. MAGARIAN2 Biomembrane Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 2 Division of Medicinal Chemistry and Pharmacodynamics, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 1

Investigations on the effect of dietary fats and antioxidant compounds on the incidence of mammary tumors in female rats have provided clear evidence for modification of the tumorigenic process by these dietary components. High fat diets, particularly diets rich in polyunsaturated fats, markedly enhance the incidence of DMBA-induced mammary tumors in comparison with rats fed diets which are low in fat. Polyunsaturated fats easily undergo peroxidation unless the latter is prevented by the presence of antioxidant substances. Because antioxidant substances had been reported to inhibit carcenogenesis in some tumor models, we undertook to investigate the effect of antioxidants with little or no known toxicity supplemented in the diets of animals fed the different types of fat. The purpose was to determine if the antioxidants were exerting their protective effect on tumorigenesis primarily in animals fed diets rich in polyunsaturated fat. The results tend to support this concept excepting that some antioxidants are effective tumor inhibitors and some are not under these experimental conditions. Experiments were performed to determine if a differential uptake and turnover of antioxidant by the mammary gland could explain this inconsistency. The results presented in this report suggest that the most effective inhibitor of DMBA-induced mammary tumor is one that accumulated in mammary gland in the largest amount for the longest time. Over the past ten years the role of various levels and types of dietary fat and dietary antioxidants in modifying chemically induced cancer has been actively investigated in this laboratory with most of the work centering on mammary cancer. Current address: Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

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0097-6156/85/0277-0131$06.00/0 © 1985 American Chemical Society Finley and Schwass; Xenobiotic Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

XENOBIOTIC M E T A B O L I S M : N U T R I T I O N A L E F F E C T S


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I t has become v e r y w e l l - e s t a b l i s h e d t h a t the i n t a k e o f e l e v a t e d l e v e l s o f d i e t a r y f a t , e s p e c i a l l y p o l y u n s a t u r a t e d f a t , must be accompanied by an e l e v a t e d l e v e l o f a n t i o x i d a n t i f t i s s u e damage i s t o be a v o i d e d ( 1 ) . In the absence o f an a n t i o x i d a n t , animals consumi n g a h i g h p o l y u n s a t u r a t e d f a t d i e t d e v e l o p v a r i o u s p a t h o l o g i c a l and somewhat s p e c i e s - s p e c i f i c t i s s u e damage (2). The h i g h e r the p o l y u n s a t u r a t e d f a t t y a c i d i n t a k e , the h i g h e r the a n t i o x i d a n t r e q u i r e m e n t becomes i n o r d e r to p r e v e n t t h i s damage. I t was demonstrated by B i e r i _e_t j i l . t h a t some s p e c i e s fed v e r y low l e v e l s o f d i e t a r y f a t had no apparent r e q u i r e m e n t f o r an a n t i o x i d a n t ( 3 ) . From such s t u d i e s i t was found t h a t a - t o c o p h e r o l as v i t a m i n E was an e f f e c t i v e n a t u r a l l y o c c u r r i n g a n t i o x i d a n t which p r o v i d e d p r o t e c t i o n to t i s s u e s from the damaging e f f e c t o f p o l y u n s a t u r a t e d f a t - f e e d i n g ( 4 ) . I t was soon found t h a t o t h e r s t r u c t u r a l l y u n r e l a t e d , n a t u r a l l y o c c u r r i n g and even s y n t h e t i c a n t i o x i d a n t s c o u l d p r o v i d e s i m i l a r p r o t e c t i o n a g a i n s t the i n j u r i o u s e f f e c t s o f u n s a t u r a t e d f a t consumption (_5). A low l e v e l o f d i e t a r y p o l y u n s a t u r a t e d f a t i s e s s e n t i a l f o r l i f e i n h i g h e r organisms s i n c e the t o t a l p o l y u n s a t u r a t e d f a t t y a c i d s t r u c t u r e r e q u i r e d f o r p r o s t a g l a n d i n p r o d u c t i o n cannot be s y n t h e s i z e d i n these a n i m a l s . Thus, the requirement f o r l i n o l e i c a c i d t o be p r o v i d e d i n the d i e t ( 6 ) . L i n o l e i c a c i d i s found i n the food c h a i n i n a v a r i e t y o f p l a n t s o u r c e s ( 7 ) . An i n t a k e o f l i n o l e i c a c i d at t h e average d i e t a r y l e v e l o f most Americans would cause s i g n i f i c a n t t i s s u e damage t o c e r t a i n organs o f v a r i o u s l a b o r a t o r y a n i m a l s , e s p e c i a l l y young r a p i d l y growing a n i m a l s , u n l e s s i t was accompanied by an adequate i n t a k e o f d i e t a r y a n t i o x i d a n t . In s p i t e o f e x t e n s i v e i n v e s t i g a t i o n s to d e t e r m i n e the mechanism o f t h i s s p e c i f i c and r e p r o d u c i b l e e f f e c t , the e x a c t mechanism o f t i s s u e i n j u r y i s s t i l l unknown. P e r o x i d a t i v e a l t e r a t i o n s o f membrane l i p i d s have been strongly implicated (8). Many i n v e s t i g a t i o n s have i n d i c a t e d t h a t d i e t a r y f a t , e s p e c i a l l y p o l y u n s a t u r a t e d f a t , enhances t u m o r i g e n e s i s i n b o t h humans and l a b o r a t o r y animals. The importance o f u n d e r s t a n d i n g the r o l e o f these d i e t a r y f a c t o r s i n c a r c i n o g e n e s i s i s perhaps b e s t i n d i c a t e d by world-wide e p i d e m i o l o g i c a l surveys which have g e n e r a t e d data showing a s t r o n g c o r r e l a t i o n between d i e t a r y f a t i n t a k e and b r e a s t c a n c e r i n women (9) ( F i g u r e 1 ) . A s i m i l a r h i g h degree o f c o r r e l a t i o n was found f o r e p i d e m i o l o g i c a l e v i d e n c e o f c o l o n c a n c e r i n b o t h men and women (9^). The U n i t e d S t a t e s has one o f the h i g h e s t per c a p i t a consumption l e v e l s o f f a t i n the world and a l s o ranks among the h i g h e s t i n the i n c i d e n c e o f c a n c e r o f the c o l o n and b r e a s t . In l a b o r a t o r y a n i m a l s , i t i s w e l l - e s t a b l i s h e d t h a t h i g h e r l e v e l s o f d i e t a r y f a t , e s p e c i a l l y p o l y u n s a t u r a t e d f a t , i n c r e a s e tumor i n c i dence i n those t r e a t e d w i t h c e r t a i n c a r c i n o g e n s . The e a r l y s t u d i e s o f Tannenbaum i n the 1940 s (11) demonstrated t h a t d i e t a r y f a t enhances mammary tumor i n c i d e n c e . T h i s p o i n t has now been shown by many i n v e s t i g a t o r s , i n c l u d i n g o u r s e l v e s (11, 12, 13). That t h i s i s a s p e c i f i c d i e t a r y f a t e f f e c t and s e p a r a t e from a g e n e r a l i n f l u e n c e o f a h i g h c a l o r i c i n t a k e , has been shown many times as e a r l y as the 1940 s (14-18) and e x t e n d i n g through our own work b e i n g p r e s e n t e d here. S i m i l a r l y , i t has been demonstrated t h a t v a r i o u s a n t i o x i d a n t s i n h i b i t c a r c i n o g e n e s i s induced by a wide v a r i e t y o f compounds and i n s e v e r a l organ s i t e s (19, r e v i e w ) . The antagonism between d i e t a r y f a t s and a n t i o x i d a n t s on c a r c i n o g e n e s i s s u g g e s t s t h a t a common f a c t o r f

f

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

K I N G ET A L .

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Modulation of Mammary Tumor Incidence

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may be i n v o l v e d i n the b a l a n c e between d i e t a r y u n s a t u r a t e d f a t and the a n t i o x i d a n t c o n t e n t r e q u i r e d to m a i n t a i n t i s s u e i n t e g r i t y . Experimental

Design


I n i t i a l l y a model was e s t a b l i s h e d and t e s t e d many times t h a t r e p r o d u c i b l e r e s u l t s were o b t a i n e d .

to i n s u r e

Diets. Three b a s i c d i e t s were u t i l i z e d ( T a b l e I ) , a 2% low f a t d i e t (2% l i n o l e i c a c i d methyl e s t e r s ) , a 20% p o l y u n s a t u r a t e d f a t d i e t c o n t a i n i n g 20% s t r i p p e d c o r n o i l , and a h i g h s a t u r a t e d f a t d i e t c o n t a i n i n g 18% coconut o i l and 2% l i n o l e i c a c i d methyl e s t e r s to p r e v e n t an e s s e n t i a l f a t t y a c i d d e f i c i e n c y (6). A l l d i e t s were p r e p a r e d to our s p e c i f i c a t i o n s by ICN L i f e S c i e n c e s ( C l e v e l a n d , OH) and a n a l y z e d both by ICN and our l a b o r a t o r y f o r f a t t y a c i d s , a n t i o x i d a n t s and some trace minerals. They are r o u t i n e l y s t o r e d i n s e a l e d p l a s t i c cont a i n e r s at 4 ° . A n t i o x i d a n t s when added (see F i g u r e 2) were s u p p l e mented j u s t p r i o r to f e e d i n g and at 0.2% o r 0.3% of the d i e t by weight as s p e c i f i e d i n each e x p e r i m e n t .

Table

I.

High Polyunsaturated Fat D i e t

e

23 20

High Saturated Fat D i e t

Low

Fat D i e t

%

%

% Casein Fat Sucrose S a l t mixture** A l p h a c e l (nonn u t r i e n t bulk) Vitamin mixture

Diets

a

20

b

23 2

46 4

46 4

64 6

6

6

6

1

1

1

C

a

S t r i p p e d corn o i l . ^ S t r i p p e d , hydrogenated coconut o i l , 18% + l i n o l e i c a c i d , 2%. L i n o l e i c acid. S a l t mixture: H u b b e l l et a l . , J . Nutr. 14: 273-285, 1937 (modif i e d to c o n t a i n 0.03% z i n c c h l o r i d e ) . V i t a m i n mixture ( v i t a m i n f o r t i f i c a t i o n mixture (tocopherol d e l e t e d ) of ICN L i f e S c i e n c e s Co., C l e v e l a n d , OH 44128). c

d

e

A m o d i f i c a t i o n o f the Huggins model was u t i l i z e d i n which 50 d a y - o l d female Sprague-Dawley r a t s (Sasco I n c . , ftnaha, NE) r e c e i v i n g 10 mg 7 , 1 2 - d i m e t h y l b e n z ( a ) a n t h r a c e n e (Sigma Chemical Co., S t . L o u i s , MO) i . g . i n 1 ml s t r i p p e d c o r n o i l at 50 ±1 day o f age f o l l o w i n g a 24 hr f a s t . Unless o t h e r w i s e n o t e d , a l l animals were s t a r t e d on t h e i r r e s p e c t i v e d i e t s a t weaning (21 days o l d ) , the time they were r e c e i v e d i n t o the F a c i l i t y . They were weighed upon a r r i v a l and randomly d i v i d e d i n t o e x p e r i m e n t a l groups o f 30 and housed 5, 10 o r 15 per cage. At 5-6 wks o f age, each animal was g i v e n a s t a i n l e s s


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KING ET A L .


135

s t e e l e a r t a g w i t h a d i s t i n c t i v e and s e q u e n t i a l number f o r i d e n t i f i cation. A l l animals were housed i n an AAALAC-accredited animal c a r e f a c i l i t y w i t h a c o n t r o l l e d temperature (68-72°) and h u m i d i t y (50-60%) and a 12 h r l i g h t / d a r k c y c l e . A l l e x p e r i m e n t a l animals were weighed weekly from the time o f a r r i v a l and p a l p a t e d weekly f o r tumors b e g i n n i n g t h r e e weeks a f t e r c a r c i n o g e n t r e a t m e n t . Only tumors v e r i f i e d h i s t o l o g i c a l l y as mammary adenocarcinomas were i n c l u d e d i n incidence data. Animals used f o r s t u d y i n g drug m e t a b o l i s m and measuring v a r i o u s microsomal parameters were p l a c e d on d i e t s , weighed and t r e a t e d i n e x a c t l y the same manner as those b e i n g used f o r mammary tumor i n c i d e n c e s t u d i e s , except t h a t they were k i l l e d a t 50 days o f age, the time f o r exposure o f r a t s i n the o t h e r e x p e r i m e n t a l group t o the carcinogen. L i v e r s and/or mammary g l a n d s were h a r v e s t e d f o r p r e p a r a t i o n o f s u b c e l l u l a r f r a c t i o n s and drug m e t a b o l i s m s t u d i e s as p r e v i ously d e s c r i b e d (28). A n t i o x i d a n t measurements i n t i s s u e s were made u s i n g s i m i l a r animals t h a t had been f e d the b a s i c d i e t s supplemented w i t h a n t i o x i d a n t s f o r the s p e c i f i c times as i n d i c a t e d f o r each experiment. R e s u l t s and D i s c u s s i o n Over the p a s t s e v e r a l y e a r s the DMBA-induced r a t mammary tumor model has been t e s t e d many times w i t h v a r i o u s s p e c i f i c changes i n the e x p e r i m e n t a l parameters b e i n g t e s t e d . The v e r y f i r s t i n c i d e n c e s t u d y u t i l i z e d the same t h r e e b a s i c d i e t s ( i n c o r p o r a t i n g the H u b b e l l s a l t mix) w i t h and without 0.7% BHT s u p p l e m e n t a t i o n ( 1 3 ) . I t was found i n t h i s study t h a t BHT g i v e n a t t h i s l e v e l , a l t h o u g h an e f f e c t i v e tumor i n h i b i t o r , was a l s o s l i g h t l y t o x i c , r e s u l t i n g i n a s l i g h t d e c r e a s e i n weight g a i n and o c c a s i o n a l and i n t e r m i t t e n t h a i r l o s s . T h i s study was r e p e a t e d u s i n g the same d i e t s , b u t w i t h o n l y 0.3% BHT supplementation. Almost i d e n t i c a l tumor i n c i d e n c e and tumor numbers were o b t a i n e d u s i n g t h i s l e v e l o f BHT, but weight g a i n s were now the same as i n non-BHT-supplemented animals and no e v i d e n c e was seen o f any toxic side e f f e c t s . The same p r o t o c o l was employed f o r t e s t i n g each o f the f o l l o w i n g : BHA, a - t o c o p h e r o l (as a - t o c o p h e r y l a c e t a t e ) and p r o p y l g a l l a t e (PrG) as mammary tumor i n h i b i t o r s . Weight g a i n s i n a l l groups ± a n t i o x i d a n t s were e s s e n t i a l l y i d e n t i c a l and no t o x i c s i d e e f f e c t s were o b s e r v e d . F i g u r e 3 demonstrates the tumor i n c i d e n c e r e s u l t s o b t a i n e d i n these s t u d i e s when j u s t the t h r e e d i e t a r y regimens were compared, i . e . LF ( 2 % ) , HPF (20% s t r i p p e d c o r n o i l ) and HSF (18% s t r i p p e d c o r n o i l ± 2% l i n o l e i c a c i d ) . As c a n be seen i n t h i s f i g u r e , the animals consuming the HPF d i e t r o u t i n e l y d e v e l o p a 97-100% tumor i n c i d e n c e . Those on the HSF d i e t d e v e l o p a 55-70% i n c i d e n c e and those on the L F d e v e l o p o n l y a 20-30% i n c i d e n c e o f mammary tumors. These v a l u e s r e p r e s e n t t h r e e s e t s o f experiments w i t h 30 r a t s p e r group o r 90 t o t a l r a t s per each d i e t r e p r e s e n t e d . There i s a 10-15% v a r i a b i l i t y i n the i n c i d e n c e r a t e s between experiments performed under i d e n t i c a l c o n d i t i o n s whether done c o n c u r r e n t l y o r i n a sequence o f t i m e s . These r e s u l t s have been used as the s t a n d a r d f o r comparison o f a d d i t i o n a l experiments, and those b e i n g performed most r e c e n t l y compare w e l l with the e a r l i e r experiments. Thus, t h i s seems t o be a s a t i s f a c t o r i l y r e p r o d u c i b l e model.



136


F o r v a r i o u s reasons over the past t e n y e a r s , p r i m a r i l y i n attempt to improve the b i o l o g i c a l d e s i g n , changes have been made i n the o r i g i n a l b a s i c d i e t s . One o f the f i r s t changes was from the use of H u b b e l l S a l t Mix to the AIN 76 S a l t Mix. The changes caused no d e t e c t a b l e d i f f e r e n c e i n the r e s u l t s w i t h r e g a r d to tumor i n c i d e n c e or tumor numbers per t u m o r - b e a r i n g r a t . In some e x p e r i m e n t s , d i e t s have been i s o c a l o r i c a l l y p a i r - f e d to the LF d i e t , and more r e c e n t l y , a d j u s t e d so t h a t a l l d i e t s have e q u i v a l e n t n u t r i e n t - t o - c a l o r i e ratios. The l a s t adjustment was made ( T a b l e I I ) to compensate f o r the f a c t t h a t the animals which are f e d the LF d i e t s consume more d i e t ( i n g) to o b t a i n the same number c a l o r i e s a v a i l a b e to the HSF and HPF-fed a n i m a l s i n a much s m a l l e r amount o f d i e t . Through t h i s adjustment, a l l animals i n the v a r i o u s d i e t a r y groups w i l l r e c e i v e e s s e n t i a l l y the same amount o f t r a c e elements and v i t a m i n s . Our more r e c e n t s t u d i e s have been c a r r i e d out u s i n g these a d j u s t e d d i e t s ( T a b l e I I ) . When the BHT and BHA s t u d i e s were r e p e a t e d w i t h the v a r i o u s d i e t s , s i m i l a r r e s u l t s were o b t a i n e d compared to the e a r l i e r d i e t s ( T a b l e I and r e f e r e n c e 13). These r e s u l t s , a l o n g w i t h the companion r e s u l t s t h a t s i m i l a r tumor number and i n c i d e n c e were o b t a i n e d , support the h y p o t h e s i s t h a t the i n f l u e n c e o f d i e t on t h i s tumor model i s an e f f e c t o f the type and q u a n t i t y o f f a t f e d , and does not appear t o be a c a l o r i c e f f e c t . T h i s p o i n t has been r e p e a t e d l y s t r e s s e d i n both formal and i n f o r m a l s c i e n t i f i c exchanges o f v i e w s , and our work as w e l l as the work o f o t h e r s c o n t i n u e s to show t h a t c a l o r i c d e n s i t y per se i s not r e s p o n s i b l e f o r the enhancement o f t u m o r i g e n e s i s seen i n t h i s model.

Table I I . E x p e r i m e n t a l D i e t s (Equivalent N u t r i e n t : C a l o r i e Ratios) High Polyunsaturated Fat D i e t % by Casein Fat Sucrose Salt Mixture Alphacel (NonN u t r i e n t Bulk) Vitamin Mixture d

e

a

b

c

d

e

wt.

28.20 20.00 38.77 4.90 6.93 1.20

a

Low Fat Diet

High Saturated Fat Diet % by

wt.

28.20 20.00 38.77 4.90

% by

b

6.93 1.20

wt.

23.0 2.0 64.0 4.0

C

6.0 1.0

S t r i p p e d corn o i l . 1 8 % coconut o i l + 2% l i n o l e i c a c i d methyl e s t e r s . 2 % l i n o l e i c a c i d methyl e s t e r s . AIN-76 m i n e r a l m i x t u r e . AIN-76 v i t a m i n m i x t u r e .

With the a d d i t i o n o f the d i f f e r e n t a n t i o x i d a n t s to the b a s i c s e m i p u r i f i e d d i e t s d i f f e r i n g i n f a t c o n t e n t , v a r i e d r e s u l t s have been obtained. Both BHT and PrG have r o u t i n e l y g i v e n good p r o t e c t i o n when



11.

K I N G ET A L .


137

added t o the d i e t a t a l e v e l o f 0.3% (w/w). BHA and a - t o c o p h e r o l have shown o n l y m a r g i n a l o r no s i g n i f i c a n t p r o t e c t i o n a g a i n s t mammary adenocarcinomas ( F i g u r e 4 ) . The f a c t t h a t BHA showed no s i g n i f i c a n t p r o t e c t i o n a g a i n s t mammary t u m o r i g e n e s i s i n t h i s model was o f p a r t i c u l a r i n t e r e s t because o f i t s s i m i l a r i t y t o BHT, and because i t had been r e p o r t e d t o be an e f f e c t i v e tumor i n h i b i t o r i n o t h e r models o f c a r c i n o g e n e s i s (19) . Q u i t e a v a r i e t y o f a n t i o x i d a n t s have been t e s t e d and found t o be e f f e c t i v e i n s e v e r a l d i f f e r e n t models and t h e e f f e c t i s f a r from c o n s i s t e n t . A l l a n t i o x i d a n t s do not i n h i b i t any one tumor model and some tumor models do n o t appear t o be i n h i b i t e d by any a n t i o x i d a n t (20-22). I m p l i c i t i n many o f these s t u d i e s has been t h e assumption t h a t i t i s t h e a n t i o x i d a n t p r o p e r t i e s o f t h i s c l a s s o f p h e n o l i c compounds t h a t i s r e s p o n s i b l e f o r t h e i r tumor inhibitory capabilities. Certainly i n s u f f i c i e n t information i s a v a i l a b l e p r e s e n t l y t o make a d e c i s i o n one way o r a n o t h e r i n t h i s regard. This research area r e q u i r e s f u r t h e r i n v e s t i g a t i o n i n order to g a i n i n s i g h t s which may suggest the mechanisms i n v o l v e d i n r e g u l a t i n g tumor growth r a t h e r than approach t h e problem t o f i n d a c t u a l and p r a c t i c a l means o f c a n c e r p r e v e n t i o n . More r e c e n t l y , we have focused on t h e s t r a t e g y o f i n v e s t i g a t i n g the mechanism o f i n h i b i t i o n o f c a r c i n o g e n e s i s by BHT i n terms o f t h e a l t e r a t i o n s i n s p e c i f i c organ b i o c h e m i s t r y t h a t r e s u l t from f e e d i n g t h i s and o t h e r a n t i o x i d a n t s . In t h i s v e i n , we have been f o c u s i n g on the f o l l o w i n g experiments d e s i g n e d t o p r o v i d e i n f o r m a t i o n as t o t h e mechanisms o f tumor i n c i d e n c e / g r o w t h m o d i f i c a t i o n by d i e t a r y f a t and antioxidants: 1) i n v e s t i g a t i o n s t o determine how t h e l e v e l o f u n s a t u r a t i o n p e r se i n f l u e n c e s t h e e f f e c t i v e n e s s o f a n t i o x i d a n t s as i n h i b i t o r s o f mammary g l a n d t u m o r i g e n e s i s ; 2) i n v e s t i g a t i o n s t o d e t e r m i n e the e f f e c t o f time d u r i n g which t h e a n t i o x i d a n t t o be t e s t e d was s u p p l i e d t o t h e animal w i t h r e s p e c t t o t h e time o f exposure to the c a r c i n o g e n , DMBA; and 3) d e t e r m i n a t i o n s o f the a c t u a l t i s s u e c o n c e n t r a t i o n s o f the v a r i o u s a n t i o x i d a n t s with respect t o time o f f e e d i n g , and amount and type d i e t a r y f a t b e i n g f e d . The degree o f u n s a t u r a t i o n o f animal t i s s u e s i s a f u n c t i o n o f the l e v e l o f u n s a t u r a t e d f a t consumed i n the d i e t . Hence, a b a l a n c e between d i e t a r y f a c t o r s which e i t h e r f a c i l i t a t e s o r i n h i b i t s c a r c i n o gen metabolism may have an important e f f e c t on the e v e n t u a l outcome o f c h r o n i c c a r c i n o g e n exposure. One p o s s i b l e e f f e c t o f d i e t a r y l i p i d s and a n t i o x i d a n t s on c h e m i c a l c a r c i n o g e n e s i s t h a t must be cons i d e r e d i s t h e i n f l u e n c e o f these d i e t a r y components on t h e metabol i s m o f chemical carcinogens. A f t e r PAH's e n t e r t h e body, they a r e o x i d i z e d i n t h e endoplasmic r e t i c u l u m o f many t i s s u e s ( l i v e r , s k i n , mammary, e t c . ) t o h y d r o x y l a t e d p r o d u c t s which, b e i n g w a t e r - s o l u b l e , are more r e a d i l y e x c r e t e d ( F i g u r e 5 ) . U n f o r t u n a t e l y , i n t h i s p r o c e s s which u s u a l l y r e s u l t s i n d e t o x i f i c a t i o n , some compounds a r e c o n v e r t e d to r e a c t i v e a l k y l a t i n g agents which a r e c a r c i n o g e n i c . Benson o b s e r v e d t h a t mammary t i s s u e from animals on a h i g h f a t d i e t appeared more m e t a b o l i c a l l y a c t i v e when compared t o t i s s u e from animals b e i n g fed a low f a t d i e t ( 2 8 ) . More r e c e n t l y , Dao's l a b o r a t o r y has demons t r a t e d t h a t mammary g l a n d t i s s u e p e r se i s c a p a b l e o f m e t a b o l i z i n g DMBA, and that t h i s m e t a b o l i s m c a n be a f f e c t e d by known i n d u c e r s o f the h e p a t i c monooxygenase system such as 3 - m e t h y l c h o l a n t h r e n e ( 3 0 , 31). V i r g i n mammary glands from female Sprague-Dawley r a t s have been shown t o c o n t a i n an i n d u c i b l e cytochrome P-450 i n t h e m i c r o s o m a l monooxygenase system ( 2 9 ) . I t i s r e a s o n a b l e t o assume t h a t d i e t a r y



138


AGE

(weeks)

F i g u r e 3. I n c i d e n c e o f mammary tumors i n female Sprague-Dawley r a t s f e d d i e t s c o n t a i n i n g d i f f e r e n t amounts and types o f f a t . [•] 20% c o r n o i l f a t ; [A] 20% coconut o i l ; [•] 2% m e t h y l linoleate. See E x p e r i m e n t a l D e s i g n f o r d e t a i l s o f these d i e t s .

F i g u r e 4. D i e t a r y f a t and a n t i o x i d a n t mammary c a r c i n o g e n e s i s .

i n f l u e n c e s on M B A - i n d u c e d


11.

KING ET A L .

Modulation of Mammary Tumor Incidence CARCINOGEN

*

139

DETOXIFICATION

(PARENT COMPOUND)

ACTIVATION (AAH?)

?


[~PROXIMATE CARCINOGEN] [_

(INTERMEDIATE)

(EH)

^ DETOXIFICATION

_ J OR (GSH)

CELLULAR TARGET(S) FURTHER METABOLISM

ULTIMATE CARCINOGEN

•DETOXIFICATION OR (GSH)

Rx WITH CELLULAR TARGET(S)

Figure

5.

Scheme o f c a r c i n o g e n m e t a b o l i s m i n mammalian t a r g e

tissues.

f a t c a n a f f e c t c a r c i n o g e n metabolism i n the mammary g l a n d , s i n c e i t has been shown t o a f f e c t m i c r o s o m a l m e t a b o l i c a c t i v i t i e s i n o t h e r extra-hepatic tissues. F o r example, AHH a c t i v i t y has been shown t o i n c r e a s e i n the r a t k i d n e y as d i e t a r y f a t i s i n c r e a s e d above 4% i n the d i e t , r e a c h i n g a maximum a f t e r the d i e t s have been f e d f o r seven days ( 2 3 ) . A n o t h e r parameter o f the m e t a b o l i c p r o c e s s which c o u l d p o t e n t i a l l y be a f f e c t e d by d i e t a r y f a t s and a n t i o x i d a n t s i s a major component o f the endoplasmic r e t i c u l a r monooxygenase system, c y t o c h r o m e ( s ) P-450. These cytochromes a l o n g w i t h t h e i r a s s o c i a t e d enzyme NADPHcytochrome P-450 r e d u c t a s e , a r e a l s o l i k e l y t o be i n v o l v e d i n the oxidation of p o t e n t i a l l y carcinogenic substrates. This reductase i s a f l a v o p r o t e i n which t r a n s f e r s e l e c t r o n s from NADPH t o cytochrome P450 i n two s t e p s . The s e l e c t i v e i n d u c t i o n o f s e v e r a l l i v e r monooxygenases by BHT i s w e l l - e s t a b l i s h e d (25_, 3 4 ) . D i e t a r y BHT i n male r a t s f e d a chow d i e t a p p a r e n t l y produces a change i n t h e r e l a t i v e p r o p o r t i o n o f i n d i v i d u a l forms o f cytochrome P-450 w i t h o u t a l t e r i n g the t o t a l c o n c e n t r a t i o n o f cytochromes ( 2 6 ) . S i m i l a r l y , i n male r a t s fed e i t h e r the HPF o r HSF d i e t s , the s p e c i f i c c o n t e n t o f cytochrome P-450 i s s i g n i f i c a n t l y h i g h e r i n l i v e r microsomes from r a t s f e d t h e s e d i e t s compared t o the l o w - f a t f e d animals ( 2 9 ) . Whether the f a t was s a t u r a t e d o r p o l y u n s a t u r a t e d d i d not a f f e c t t h e s e m i c r o s o m a l components. With female r a t s , however, BHT produces an i n d u c t i o n o f l i v e r microsomal cytochrome P-450 c o n t e n t ( T a b l e I I I ) i n a n i m a l s consuming semipurified diets high i n either polyunsaturated or saturated f a t (27). Marked d e c r e a s e s i n NADPH cytochrome P-450 r e d u c t a s e a c t i v i t y were c o n s i s t e n t l y seen when BHT was i n c l u d e d i n t h e d i e t o f male r a t s (24) but no s i g n i f i c a n t e f f e c t s were noted i n female r a t s ( 2 7 ) .


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


140

Reductase a c t i v i t i e s were o n l y 30-35% o f those i n microsomes from unsupplemented male a n i m a l s , the most profound d e c r e a s e o c c u r r i n g i n the l o w - f a t - f e d a n i m a l s . Reductase i s g e n e r a l l y c o n s i d e r e d t o be t h e r a t e - l i m i t i n g component o f the microsomal monooxygenase system and t h i s d e c r e a s e i n a c t i v i t y c o u l d have profound e f f e c t s on the metabol i s m o f these f o r e i g n compounds. Mammary AHH has been shown i n v i r g i n r a t s and found to v a r y as a f u n c t i o n o f the age o f the a n i m a l , b e i n g h i g h e s t i n younger a n i m a l s (30-60 days o l d ) (32, 3 3 ) . I n d u c t i o n o f t h i s enzyme i s a l s o agedependent, b e i n g h i g h l y i n d u c i b l e between 40 and 60 days o f age. AHH a c t i v i t y i s important from the s t a n d p o i n t t h a t t h i s type o f a c t i v i t y r e s u l t s i n the f o r m a t i o n o f h i g h l y r e a c t i v e e l e c t r o p h o l i c i n t e r m e d i a t e s , arene o x i d e s , from r e l a t i v e l y i n e r t p r e c u r s o r s . P r e l i m i n a r y d a t a from o u r l a b o r a t o r y s u g g e s t s t h a t AHH a c t i v i t y i s s p e c i f i c a l l y i n f l u e n c e d by both the l e v e l and type o f d i e t a r y f a t . As can be seen from T a b l e IV, h e p a t i c AHH a c t i v i t y measured as b e n z ( a ) p y r e n e h y d r o x y l a s e and read a g a i n s t s t a n d a r d 3-0H-benzpyrene, i s h i g h e s t i n the HPF d i e t a r y group f o l l o w e d by the HSF and lowest i n the L F - f e d group, and the lowest v a l u e f o r LF i s s i g n i f i c a n t l y induced by HPF (P < 0.05). A d d i t i o n o f BHT t o the d i e t a t a l e v e l o f 0.3% as b e f o r e , r e s u l t e d i n a p p r o x i m a t e l y a f o u r - t o f i v e f o l d i n c r e a s e i n AHH a c t i v i t y (Table IV). Thus, d i e t a l s o i n f l u e n c e s t h e AHH l e v e l w i t h the HPF v a l u e s i g n i f i c a n t l y (P < 0.05) induced above LF ( s t u d e n t ' s t test). T a b l e I I I . E f f e c t s o f D i e t a r y F a t and A n t i o x i d a n t s on H e p a t i c Microsomal Cytochrome P. L e v e l s i n Female Sprague-Dawley Rats 5

Q

T o t a l Cytochrome P ^ Q (n moles/mg MS P r o t e i n )

Diet High P o l y u n s a t u r a t e d Fat •BHT •BHA •a-Toc •PrG

0.79 1.18 0.87 0.79 0.80

± + ± + +

0.04t 0.05* 0.06 0.05 0.04

High S a t u r a t e d F a t •BHT •BHA •a-Toc •PrG

0.85 1.10 0.73 0.74 0.77

+ + ± ± +

0.06 0.08* 0.07 0.06 0.03

Low F a t •BHT •BHA •a-Toc •PrG

0.81 0.92 0.76 0.78 0.74

+ ± ± ± ±

0.04 0.07 0.06 0.06 0.03

tMean ± SEM. * D i f f e r s i g n i f i c a n t l y from t h a t f o r the group on the c o r r e s p o n d i n g non-supplemented d i e t (P < 0.05 by s t u d e n t ' s t t e s t ) . BHT = b u t y l a t e d h y d r o x y t o l u e n e (0.3% by weight) BHA = b u t y l a t e d h y d r o x y a n i s o l e (0.3%) a-Toc = a - t o c o p h e r y l a c e t a t e (0.2%) PrG = p r o p y l g a l l a t e (0.3%)


11.

KING ET A L .


141

T a b l e IV. A r y l Hydrocarbon Hydroxylase A c t i v i t y i n H e p a t i c Microsomes from Animals Consuming V a r i o u s L e v e l s and Types o f F a t ± 0 . 3 % BHT Polyunsaturated Fat


-BHT +BHT

6.3 ± 0.4*t 26.5 ± 1.5

Saturated Fat 4.0 21.6

± 0.2 ± 1.0

tMean ± S.E. o f 10-12 d e t e r m i n a t i o n s . *AHH a c t i v i t y measured as AOD/min/mg microsomal

Low 3.4 17.3

Fat ± 0.1 ± 0.9

protein.

Thus, i t c a n be e a s i l y t h e o r i z e d t h a t t h e s h i f t seen i n t h e microsomal enzyme and cytochrome p a t t e r n s caused by d i e t a r y f a t and a n t i o x i d a n t s may be r e l a t e d t o the i n h i b i t o r y and enhancing e f f e c t s o f these compounds on c h e m i c a l c a r c i n o g e n e s i s . F u r t h e r s t u d i e s a r e i n d i c a t e d t o l o o k a t s p e c i f i c c a r c i n o g e n m e t a b o l i t e s produced by t h e drug m e t a b o l i z i n g systems o f animals consuming the v a r i o u s d i e t s , and to attempt t o c o r r e l a t e t h e c o r r e s p o n d i n g cytochrome and enzyme l e v e l s w i t h these m e t a b o l i t e s and u l t i m a t e l y w i t h r e s u l t a n t tumor i n c i d e n c e s and tumor numbers. C r i t i c a l Time o f BHT E x p o s u r e . Two complimentary s t u d i e s were p e r formed w i t h t h e s p e c i f i c o b j e c t i v e o f d e t e r m i n i n g t h e c r i t i c a l time d u r i n g which BHT must be f e d t o r e a l i z e p r o t e c t i o n a g a i n s t DMBAi n d u c e d mammary c a r c i n o g e n e s i s . These s t u d i e s have been r e p o r t e d elsewhere (35) and w i l l o n l y be b r i e f l y summarized h e r e . The r e s u l t s were comparable i n both t h e HPF and HSF-supplemented groups. In t h e f i r s t study 0.3% BHT was added t o the HPF and HSF d i e t s a t v a r i o u s times from weaning (21 days o f age) t o one week b e f o r e c a r c i n o g e n a d m i n i s t r a t i o n (42 days o f a g e ) , a t 50 days o f age, t h e time o f DMBA t r e a t m e n t , o r one, two o r t h r e e weeks a f t e r DMBA, and i n c l u d e d i n t h e d i e t from t h a t time throughout the e x p e r i m e n t a l p e r i o d o f 30 weeks. In a s i m i l a r experiment, BHT was added t o t h e d i e t f o r o n l y s h o r t p e r i o d s o f time, one week s t a r t i n g a t weaning (21 days o l d ) then removed from t h e d i e t , o r added f o r two, t h r e e , f o u r , f i v e o r s i x weeks from weaning and then removed. T h i s encompasses the time from weaning up t o and i n c l u d i n g t h r e e weeks p o s t - c a r c i n o g e n treatment a f t e r each o f the r e s p e c t i v e BHT f e e d i n g p e r i o d s , the a n t i o x i d a n t was removed from t h e d i e t s and the animals c o n t i n u e d f o r t h e remainder o f the e x p e r i m e n t a l p e r i o d on t h e i r r e s p e c t i v e HPF o r HSF d i e t s . When mammary tumor i n c i d e n c e r a t e s were compared w i t h non-BHT t r e a t e d animals i t was found from the f i r s t experiment t h a t BHT was most e f f e c t i v e i f added t o the d i e t a t t h e time o f weaning o r up t o t h e time o f c a r c i n o g e n t r e a t m e n t . I f one w a i t e d u n t i l t h r e e weeks p o s t c a r c i n o g e n t o supplement t h e d i e t s w i t h BHT, e s s e n t i a l l y no p r o t e c t i o n was seen, i . e . the r e s u l t s were comparable t o those when no BHT was added t o t h e d i e t throughout the e x p e r i m e n t a l f e e d i n g time. In the s h o r t - t e r m BHT s u p p l e m e n t a t i o n experiment, i t was found t h a t no p r o t e c t i o n was o b t a i n e d i f BHT was added f o r o n l y 1 o r 2 weeks and then removed from the d i e t (2 weeks p r e - c a r c i n o g e n t r e a t ment). I f i t was c o n t i n u e d f o r t h r e e weeks ( t o 1 week p r e - c a r c i n o gen) , o n l y minimal p r o t e c t i o n was seen. I f f e d up t o the time o f DMBA-treatment o r f o r one, two o r t h r e e weeks p o s t - c a r c i n o g e n then removed, maximal p r o t e c t i o n was seen, comparable t o the group t h a t consumed BHT throughout t h e e n t i r e e x p e r i m e n t a l p e r i o d . These two



142

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

EFFECTS

s t u d i e s i n d i c a t e t h a t BHT needs t o be p r e s e n t i n t h e d i e t f o r twot h r e e weeks b e f o r e and a f t e r the time o f c a r c i n o g e n exposure t o be effective. S t o p p i n g i t too soon o r s t a r t i n g i t too l a t e a f t e r DMBA exposure does not a l l o w i t t o be e f f e c t i v e as a tumor i n h i b i t o r . These r e s u l t s i m p l i c a t e BHT as h a v i n g a r o l e i n the a c t i v a t i o n , i n i t i a t i o n o r e a r l y p r o m o t i o n a l phase o f DMBA-induced c h e m i c a l carcinogenesis. A d e f i n i t e p o s s i b i l i t y i s t h a t i t may r e q u i r e t h i s p e r i o d o f time t o a l l o w f o r the i n d u c t i o n o f s p e c i f i c m e t a b o l i c enzymes i n v o l v e d i n the h a n d l i n g o f DMBA. T h i s time may be r e q u i r e d f o r the a t t a i n m e n t o f s u f f i c i e n t l e v e l s i n the t i s s u e s t o a l l o w e f f e c t i v e n e s s as an i n h i b i t o r . F u r t h e r s t u d i e s a r e c e r t a i n l y needed to narrow the wide range o f p o s s i b i l i t i e s . Many q u e s t i o n s i n the i n v e s t i g a t i o n o f a p o s s i b l e mechanism f o r a n t i o x i d a n t i n h i b i t i o n o f mammary c a r c i n o g e n e s i s have l e d us t o t h e need t o be a b l e t o measure a c t u a l t i s s u e c o n c e n t r a t i o n s o f these compounds. Since BHT was found t o be a v e r y e f f e c t i v e mammary tumor i n h i b i t o r i n t h i s model, and BHA t o have no measurable e f f e c t under the same e x p e r i m e n t a l c o n d i t i o n s , one has t o a s k t h e q u e s t i o n as t o whether o r not both a r e absorbed and taken up by the t i s s u e s , s p e c i f i c a l l y mammary t i s s u e , s i n c e t h i s i s the t a r g e t t i s s u e . BHT was not e f f e c t i v e as an i n h i b i t o r when f e d f o r t h r e e weeks from weaning, then stopped one week p r i o r t o c a r c i n o g e n treatment ( 3 5 ) . BHT was e f f e c t i v e i n r e d u c i n g tumor development when added t o the d i e t two-three weeks b e f o r e and up t o the time o f c a r c i n o g e n expos u r e , and m a i n t a i n e d i n the d i e t from t h a t time on through the 30 week e x p e r i m e n t a l time. The e f f e c t i v e n e s s o f BHT i n i n h i b i t i n g t u m o r i g e n e s i s d e c r e a s e d as the time o f i n i t i a t i n g BHT f e e d i n g a f t e r c a r c i n o g e n treatment i n c r e a s e d , w i t h s i m i l a r r e s u l t s b e i n g o b t a i n e d i n both HPF and HSF-fed a n i m a l s . I t was o f i n t e r e s t t o study how BHT p r o t e c t s a g a i n s t mammary tumors, w h i l e BHA does n o t (13, 36). A l l t h e s e o b s e r v a t i o n s l e a d one t o ask "what a r e the a c t u a l t i s s u e c o n c e n t r a t i o n s o f BHT and BHA a f t e r b e i n g consumed at 0.3% o f the d i e t by weight f o r v a r i o u s p e r i o d s o f time? Are t h e y taken up e q u a l l y w e l l , o r at a l l by the mammary t i s s u e and/or the l i v e r ? 11

I n i t i a l l y , a s e n s i t i v e and r e p r o d u c i b l e method o f d e t e c t i o n had t o be d e v e l o p e d . A h i g h performance l i q u i d chromatographic (HPLC) procedure was found t o be a v e r y s e n s i t i v e and e x a c t i n g way o f measuring these a n t i o x i d a n t s a l l o w i n g f o r d e t e c t i o n o f as l i t t l e as 0.1 ug/ml o f a n t i o x i d a n t . Standard c u r v e s were e s t a b l i s h e d r e l a t i n g c o n c e n t r a t i o n o f a n t i o x i d a n t t o peak h e i g h t f o r both BHA and BHT and the r e s u l t s were found t o be b o t h l i n e a r and r e p r o d u c i b l e . A t y p i c a l chromatogram i s shown f o r BHT ( F i g u r e 6) and BHA ( F i g u r e 7) w i t h absorbance b e i n g measured a t * = 280 nm. m

a

x

Samples o f mammary g l a n d , serum and l i v e r from 50 d a y - o l d female Sprague-Dawley r a t s consuming n o n - a n t i o x i d a n t - c o n t a i n i n g s e m i p u r i f i e d d i e t s were o b t a i n e d . To each o f these was added a known amount o f an a n t i o x i d a n t and an e x t r a c t i o n procedure d e v e l o p e d . P r i o r to t h i s time, no simple method has been r e p o r t e d f o r d e t e r m i n i n g t i s s u e conc e n t r a t i o n s o f these compounds, s h o r t o f steam d i s t i l l a t i o n s (37, 38). A m o d i f i c a t i o n o f the B l i g h and Dyer (39) method f o r e x t r a c t i n g l i p i d s was u t i l i z e d f o r the f i r s t s t e p . D e t a i l s o f t h i s procedure are r e p o r t e d i n Terao ^ t a^. ( 4 0 ) . A b r i e f summary i s g i v e n f o r BHA from mammary g l a n d i n F i g u r e 8 (the same procedure i s used f o r BHT but v a r i e s s l i g h t l y f o r serum and l i v e r [ 4 0 ] ) . When s t a n d a r d c u r v e s f o r each o f these compounds were p l o t t e d f o l l o w i n g e x t r a c t i o n , i t was found t h a t average r e c o v e r i e s f o r t h r e e known amounts o f a n t i o x i d a n t s



11.

KING ET A L .


143

i n samples o f mammary t i s s u e were 98.7% and 99.7% f o r BHT ( F i g u r e 9) and BHA ( F i g u r e 10), r e s p e c t i v e l y . The s t a n d a r d c u r v e s i n d i c a t e d a good c o r r e l a t i o n between peak h e i g h t and a n t i o x i d a n t c o n c e n t r a t i o n , and i t was f e l t t h a t an a c c u r a t e and r e p r o d u c i b l e method f o r measuri n g t i s s u e and serum c o n c e n t r a t i o n s had been d e f i n e d . The a c t u a l experiments began by f e e d i n g female Sprague-Dawley r a t s 0.3% BHT i n a low f a t d i e t f o r v a r i o u s p e r i o d s o f time. At t h e end o f the f e e d i n g p e r i o d , the animals were e u t h a n i z e d i n a chamb e r , mammary t i s s u e removed, p l a c e d i n an i c e b a t h , and trimmed f r e e o f extraneous f a t and lymph g l a n d s . The t i s s u e was e x t r a c t e d and a n a l y z e d by HPLC as d e s c r i b e d . I t was found t h a t BHT i s not r e t a i n e d i n any d e t e c t a b l e amount (< 0.1 ug/ml) i n the serum o r serum l i p i d s o f animals f e d t h e a n t i o x i d a n t f o r two o r t h r e e weeks, o r f o r seventeen weeks. The t o t a l mean l i p i d c o n t e n t i n the serum o f s i x animals f e d BHT f o r two and t h r e e weeks was found to be 2.0 ± 0.7 mg/ml and 1.7 ± 0.1 mg/ml, respectively. Co-chromatograms o f a BHT s t a n d a r d and e x t r a c t s o f l i v e r and mammary t i s s u e c o n f i r m e d t h e presence o f BHT i n b o t h ( F i g u r e 6; mammary, l i v e r not shown). Other than the p r e s e n c e o f the BHT peak,

Is

I 0

1 2

I

i 4

I

I 6

I

I 8

I

I 10

1

1 12

1

L 14

F i g u r e 6. HPLC a n a l y s i s o f mammary t i s s u e e x t r a c t s . (A) Rat f e d low f a t d i e t c o n t a i n i n g 0.3% BHT f o r 2 weeks. (B) C o n t r o l r a t f e d low f a t d i e t o n l y . * The arrow shows t h e p o s i t i o n o f t h e BHT peak.



144

Figure a diet

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

7. HPLC a n a l y s i s o f mammary t i s s u e e x t r a c t s from r a t s f e d c o n t a i n i n g b u t y l a t e d h y d r o x y a n i s o l e (BHA).

t h e r e were no d i f f e r e n c e s i n the chromatograms between the c o n t r o l and e x p e r i m e n t a l . Animals f e d BHT f o r two weeks had g r e a t e r c o n c e n t r a t i o n s (0.64 ± 0.26 ug/g) than those f e d the a n t i o x i d a n t f o r t h r e e weeks (0.35 ± 0.09 ug/g o f 1 i v e r ) . BHT l e v e l s found i n the l i v e r l i p i d (33.3 ± 9.7 ug/g) o f animals f e d the a n t i o x i d a n t f o r two weeks was 15.22 ± 2.10 ug/g l i p i d compared t o t h e amount from l i v e r s o f animals on BHT f o r t h r e e weeks o f 10.80 ± 2.71 ug/g l i p i d . BHT l e v e l s found i n mammary t i s s u e and mammary l i p i d a r e shown i n T a b l e V. A h i g h c o n c e n t r a t i o n o f BHT was found i n mammary t i s s u e , a l o n g w i t h s e v e r a l s u s p e c t e d m e t a b o l i t e peaks. The BHT peak was c o l l e c t e d , a n a l y z e d by gas chromatography/mass s p e c t r o s c o p y and conf i r m e d by comparison w i t h an a u t h e n t i c sample.


11.


KINGETAL.

145

TISSUE

MAMMARY

—

MINCED

—

WEIGH T H E AMOUNTS ( 1 G )

• B L I G H & DYER SOLVENT (MEOH:CHC1 :H 0 = 2:1:0.8) 7.6 ML 3

2

" M I X I N G WITH VORTEX MIXER FOR 1 M I N -HOMOGENIZE WITH POLYTRON HOMOGENIZER FOR 1 MIN.


-WASHED WITH B & D SOLVENT

I N AN I C E BATH

7.0 ML

- I N C U B A T E ^ H E A T I N G B L O C K , AT 40'C FOR 1 HOUR -ADD C H C I 3 -ADD H 0

2.0 ML 2.0 ML

2

- M I X I N G WITH VORTEX M I X E R FOR 1 M I N - C E N T R I F U G E AT 2500 RPM FOR 10 M I N

LOWER LAYER~| (CHC1 ) 7

I UPPER L A Y E R ] (ME0H-H 0)

REEXTRACTIQN

2

-ADD C H C I 3

2.0 ML

- M I X I N G 1 M I N (VORTEX M I X E R ) - C E N T R I F U G E AT 2500 RPM FOR 10 M I N

1 LOWER LAYER

UPPER

LAYER

-EVAPORATE BY No GAS AT 35°

U0°C

-DRY BY D E S S I C A T O R FOR 2 - 5 HR. - E X T R A C T BY ME0H -EVAPORATE BY N

2

1 ML ( 3 T I M E S ) GAS AT 35°

-ADD 1 ML M O B I L E PHASE HPLC

F i g u r e 8. E x t r a c t i o n p r o c e d u r e used i n t h e d e t e r m i n a t i o n o f BHA i n mammary g l a n d i n r a t s f e d a d i e t c o n t a i n i n g t h i s a n t i o x i d a n t .

BHT l e v e l s i n mammary t i s s u e and mammary l i p i d s o f a n i m a l s p l a c e d on d i e t a r y BHT a t d i f f e r e n t ages a r e shown i n T a b l e V. Group I a n i m a l s a t 91 days o f age were d i v i d e d i n t o t h r e e groups (10/group) and f e d 0.3% BHT i n a LF d i e t f o r 1, 2 and 3 weeks. Group I I a n i m a l s were f e d BHT as above b e g i n n i n g a t 35 days o f age, but f o r 2 o r 17 weeks. BHT was s i g n i f i c a n t l y i n c r e a s e d i n mammary t i s s u e o f Group I fed t h i s a n t i o x i d a n t f o r 2 weeks compared to 1 week, and t h i s i n c r e a s e was seen on a p e r t i s s u e weight o r a p e r l i p i d weight b a s i s . In the Group I I a n i m a l s , BHT l e v e l s were s i g n i f i c a n t l y lower than those i n Group I animals f e d BHT f o r 2 weeks i n d i c a t i n g a d e f i n i t e d i f f e r e n c e i n t i s s u e uptake i n a n i m a l s o f d i f f e r e n t ages. One f a c t o r t h a t might be r e s p o n s i b l e i s t h a t the whole mammary t i s s u e weight i n younger r a t s (Group I I ) was o n l y about o n e - h a l f (1.10 ± 0.19 g) t h a t of the o l d e r r a t s i n Group I (2.45 ± 0.67 g ) .


XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

146

y

=

80.79x + 1.59


-.b

A

i

i

0

i

1 1 0.5

i

i

i

I i

i

1.0

i

i

1 i i 1.5

concentration of BHT (pg/ml) F i g u r e 9. Standard curve f o r a n a l y s i s o f BHT. S e n s i t i v i t y i n c r e a s e d 8 times h i g h e r f o r the c o n c e n t r a t i o n range o f 0.1 t o 1.0 ug/ml o f BHT. d e g r e s s i o n l i n e y = mx + b, where y = peak h e i g h t and x = c o n c e n t r a t i o n o f BHT. ^ C o r r e l a t i o n c o e f f i c i e n t .

Y = 0.9995 y = 190.82x-2.667

0.2

Figure

10.

0.4 0.6 0.8 [BHA] ug/ml

Standard curve

1.0

f o r the d e t e r m i n a t i o n

o f BHA by HPLC.


11.

KING ET AL.


T3 TJ

Modulation of Mammary Tumor Incidence by Dietary Fat and

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