Biphenyl Hydroxylase, Arylhydrocarbon Hydroxylase, and Epoxide

20 Biphenyl Hydroxylase, Arylhydrocarbon Hydroxylase, and Epoxide Hydrolase Activities in Intestinal and Liver Microsomes of Rats Fed Selected Types of Dietary Fibers J. C. O P D Y C K E

1

and J. C. S T R E E T

Utah State University, Department of Animal, Dairy, and Veterinary Science, Toxicology Program, Logan, UT 84322

Downloaded by UNIV OF LEEDS on October 18, 2016 | http://pubs.acs.org Publication Date: April 11, 1983 | doi: 10.1021/bk-1983-0214.ch020

"Dietary fiber" has been shown to be protective against induction of colon cancer by some chemical carcinogens. One possible mechanism for this protective action may be altered xenobiotic metabolism. The affects of diets containing different types of purified fiber on intestinal and liver microsomal metabolism of biphenyl, benzo(a)pyrene and epoxides were assessed. Adult, male Wistar rats were individually fed chemically defined, nutrient density balanced rations containing either no dietary fiber, or15%pectin, Metamucil, lignin or cellulose for 30 days;a similar group was fed commercial laboratory chow diet as reference. The microsomal hydroxylations of biphenyl and benzo(a)pyrene (BaP) were compared following incubation with appropriate preparations of liver and intestinal mucosa. The hydroxybiphenyl products were determined by fluorimetric HPLC, hydroxylated BaP was determined by fluorimetry, and epoxide hydrolase activity was determined using 3-(p-nitrophenoxy)-1 ,2-propene oxide as a substrate. Activities were higher with liver microsomes than the intestinal microsomal preparations for each assay. Fiber-fed rats showed significantly higher hepatic but not intestinal epoxide hydrolase levels than did control rats. Hepatic arylhydrocarbon hydroxylase levels were higher in synthetic diet-fed rats than lab chowfed animals. Variations in intestinal biphenyl hydroxylase activities were not significant with respect to fiber type regardless of the specific metabolite measured. Hepatic biphenyl hydroxylase activities were significantly higher with synthetic diet-fed rats. 1

Current address: Mobay Chemical Corporation, Kansas City, MO

64120

0097-6156/83/0214-0285$06.00/0 © 1983 American Chemical Society

Furda; Unconventional Sources of Dietary Fiber ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

UNCONVENTIONAL


286

SOURCES OF DIETARY FIBER

Some c u r r e n t hypotheses about the e t i o l o g i e s of colon cancer and c h o l e s t e r e m i c disease impute a s p e c i a l f u n c t i o n t o d i e t a r y f i b e r and to some o f i t s components; that f u n c t i o n i s g e n e r a l l y a s s o c i a t e d w i t h disease prevention. Evidence f o r the p r o t e c t i v e f u n c t i o n o f d i e t a r y f i b e r has been obtained both from epidemiol o g i c a l s t u d i e s ( 1 - 8 ) and from experiments w i t h animals. Animal experiments, i n p a r t i c u l a r , have been u s e f u l i n t e s t i n g v a r i o u s hypotheses about t h e r e l a t i v e e f f e c t i v e n e s s o f d i f f e r e n t kinds o f d i e t a r y f i b e r and about p o s s i b l e mechanisms o f f i b e r e f f e c t s . Thus, s t u d i e s o f b u l k i n g a c t i o n ( 9 - 1 0 ) , o f s e l e c t i v e secondary b i l e a c i d b i n d i n g ( 1 1 - 1 2 ) , o f increased i n t e s t i n a l t r a n s i t times ( J J 3 ) , o f a l t e r e d b a c t e r i a l a c t i v i t i e s and secondary b i l e a c i d production ( 1 4 - 1 5 ) , and s e l e c t i v e b i n d i n g o f c a r c i n o gens ( 1 6 - 1 9 ) have been c a r r i e d out i n l a b o r a t o r y animals. S e v e r a l r e l a t e d s t u d i e s have been conducted i n humans as w e l l . Among the l a t t e r are i n v e s t i g a t i o n s o f t h e r e l a t i o n s h i p between t h e b u l k f i b e r c o n t e n t o f d i e t s and f e c a l b i l e a c i d s o r o t h e r s t e r o i d s ( 4 , 2 0 - 2 1 ) .

Because o f t h e p o s s i b l e i n t e r p l a y o f many f a c t o r s and p r o c e s s e s t h a t m i g h t o c c u r among t h e d i f f e r e n t c h e m i c a l s cons t i t u t i n g d i e t a r y f i b e r i t s e l f and carcinogens or promoters, we have been c o n d u c t i n g a s y s t e m a t i c s t u d y o f s u c h i n t e r a c t i o n s u s i n g a model e x p e r i m e n t a l d e s i g n w i t h t h e r a t . Our model cons i s t s of feeding purified diets containing different purified f i b e r s , i n h y d r a t e d form, f o r a 3 0 day p e r i o d t o a l l o w f o r t h o r o u g h b a c t e r i a l a d a p t a t i o n ( 2 2 ) . A t t h a t t i m e each r a t was t r e a t e d per os w i t h a s i n g l e dose o f a x e n o b i o t i c chemical s e l e c ted to e x e m p l i f y a s p e c i f i c aspect o f x e n o b i o t i c metabolism. T o x i c o k i n e t i c analyses were then u t i l i z e d to q u a n t i f y the c r i t i c a l parameters, such as a b s o r p t i o n and e l i m i n a t i o n r a t e constants f o r t h e p a r e n t compounds, t h e i r b a c t e r i a l m e t a b o l i t e s , and m e t a b o l i t e s undergoing enterohepatic c i r c u l a t i o n . By r e p e t i t i v e study w i t h d i f f e r e n t x e n o b i o t i c s w i t h i n the f i x e d d i e t a r y design, a comprehensive understanding o f the f i b e r r a n i m a l : c a r c i n o g e n i n t e r a c t i o n s i s being developed ( 2 2 - 2 4 ) . In order to f u l l y i n t e r p r e t the t o x i c o k i n e t i c experiments, a d d i t i o n a l b a s a l d a t a a r e r e q u i r e d f r o m t h i s d i e t model; i t i s p a r t i c u l a r y necessary to e s t a b l i s h parameters f o r the h e p a t i c and i n t e s t i n a l x e n o b i o t i c m e t a b o l i s m i n r a t s on the standard f i b e r d i e t s . T h i s paper p r e s e n t s our f i n d i n g s o f the e f f e c t s o f d i f f e r e n t d i e t a r y f i b e r types on h e p a t i c and i n t e s t i n a l metabol i s m o f reference x e n o b i o t i c s . Previous Work R e l a t i n g to S e l e c t i o n o f Substrates C h a r a c t e r i s t i c s o f I n t e s t i n a l vs H e p a t i c X e n o b i o t i c M e t a b o l i s m . I n t e s t i n a l mucosa has been shown both to m e t a b o l i c a l l y a c t i v a t e and to d e a c t i v a t e x e n o b i o t i c s . The mixed f u n c t i o n oxidase (MFO)


20.

OPDYCKE AND STREET

Intestinal and Liver Microsome Activities 287


of i n t e s t i n a l mucosa responds to inducers and i n h i b i t o r s d i f f e r e n t l y than h e p a t i c MFO. I n t e s t i n a l MFO a l s o d i f f e r s from h e p a t i c MFO i n i t s c o n s t i t u a t i v e l e v e l s , m e t a b o l i c p a t t e r n s , and subs t r a t e s p e c i f i c i t y (25-41 )» B i p h e n y l H y d r o x y l a t i o n . F a c t o r s a f f e c t i n g 2-,3-, and 4-biphenyl hydroxylase enzymes are: age, sex, species, n u t r i t i o n a l s t a t u s , genetic background, x e n o b i o t i c preexposure, and t i s s u e type. The 2- and 4-hydroxy biphenyl hydroxylases have been demonstrated to be d i f f e r e n t on the b a s i s of s e l e c t i v e i n d u c t i o n , conducted both i n v i v o and in v i t r o , and on d i f f e r e n t species s p e c i f i c i t i e s ( 4 2 jTT). Toftgard et al.(51 ) reported that changes i n d i f f e r e n t forms of cytochrome P-450 give r i s e to a l t e r e d proportions of biphenyl metabolites. I t i s now c l e a r that biphenyl 2-hydroxylase a c t i v i t y i s conferred only by cytochrome P-448-type i n d u c t i o n . Rat h e p a t i c b i p h e n y l 4-hydroxylase i s maintained at higher c o n s t i t u a t i v e l e v e l s than 2-hydroxylase, and i s conferred by both cytochromes P-450 and P-448 (52). W i e b k i n e t al.(53-54) summarized advant a g e s f o r use o f b i p h e n y l as s u b s t r a t e i n t h a t i t s m e t a b o l i c p r o f i l e provides an i n d i c a t o r to the t o x i c i t y of substrate and p r o d u c t s , i t h i g h l i g h t s f u r t h e r m e t a b o l i s m o f p r i m a r y metabol i t e s , and i n d i c a t e s the r e l a t i o n s h i p s among d i f f e r e n t MFO inducers; f o r these reasons we chose to use biphenyl as an i n d i c a t o r s u b s t r a t e to a s s e s s o v e r a l l MFO m e t a b o l i s m , as s u g g e s t e d by B i l l i n g s and McMahon (55)* I n t e s t i n a l microsomal metabolism can be monitored v i a biphen y l h y d r o x y l a t i o n as w e l l . Biphenyl hydroxylase assays are more s e n s i t i v e than some other hydroxylase assays which has permitted measurements of the low c o n s t i t u a t i v e enzyme l e v e l s i n i n t e s t i n a l mucosa and some other extrahepatic t i s s u e s (56-59)» Arylhydrocarbon Hydroxylase. B e n z o ( a ) p y r e n e (BaP) i s c o n s i d e r e d an i d e a l model compound f o r m o n i t o r i n g arylhydrocarbon hydroxy l a s e (AAH) MFO l e v e l s i n r a t s . I t s m e t a b o l i s m has been w i d e l y i n v e s t i g a t e d i n both r a t l i v e r and e x t r a h e p a t i c t i s s u e s (4041.61 -70). Arylhydrocarbon h y d r o x y l a s e has been compared to b i phenyl hydroxylase i n the i n t e s t i n e s of r a t s (25.28). I n t e s t i n a l AAH has been shown to be induced up to 3 0 - f o l d w i t h cytochrome P448-type inducers, while being i n s e n s i t i v e to P-450-type induction. Therefore, we have used i n t e s t i n a l AAH i n conjunction with biphenyl hydroxylation, to evaluate the e f f e c t s of d i e t a r y f i b e r s on i n t e s t i n a l metabolism. E p o x i d e H y d r o l a s e . E p o x i d e h y d r o l a s e (EH) a c t i v i t y has been studied i n numerous t i s s u e s with many d i f f e r e n t substrates (71 72). Hepatic microsomal EH has been shown to be induced by polyc y c l i c aromatic hydrocarbons, but i s i n s e n s i t i v e to cytochrome P450 type inducers (73)» Epoxide hydrolase has been r e l a t e d to the a c t i v a t i o n of p o l y c y c l i c aromatic hydrocarbons i n t o r e a c t i v e carcinogens (70). Epoxide hydrolase a c t i v i t y has been determined


UNCONVENTIONAL SOURCES OF DIETARY FIBER

288

i n t h e stomach as w e l l as t h e i n t e s t i n e and o t h e r e x t r a h e p a t i c t i s s u e s (74-75). I n d i c a t i o n s o f the e f f e c t s o f s p e c i f i c d i e t f i b e r s on epoxide hydrolase a c t i v i t y could be d i r e c t l y a p p l i e d to s u b s t a n t i a t e or modify f i b e r : c a r c i n o g e n e s i s hypotheses.


Methods and M a t e r i a l s D i e t a ry F i b e r s . The c e l l u l o s e used i n t h e s e e x p e r i m e n t s was SolkaFlok, KS-1016, a c e l l u l o s e derived from s u l f i t e processing o f wood ( b i r c h , beech, and maple) h a v i n g a p a r t i c l e s i z e o f 290 m i c r o n s and c o n t a i n i n g 0.15-0.2$ l i g n i n and 8-9$ h e m i c e l l u l o s e (Brown C o . , B e r l i n , N.H.). The l i g n i n used was I n d u l i n AT, w h i c h i s a K r a f t l i g n i n described as being 99$ l i g n i n and 1.0$ dry ash, f r e e of simple sugars, i n s o l u b l e i n water and s o l u b l e i n a l k a l i (Westvaco, N o r t h C h a r l e s t o n , S.C.). The l i g n i n was S o x h l e t ext r a c t e d f o r 30 hours using methylene c h l o r i d e which was evaporated from the l i g n i n i n a fume hood. The extracted, d r i e d l i g n i n was t h e n suspended i n d e i o n i z e d w a t e r and f i l t e r e d t h r o u g h a Buchner funnel w i t h Whatman 1 f i l t e r paper. T h i s washing process was r e p e a t e d u n t i l the f i l t r a t e was l i g h t orange i n c o l o r . The h e m i c e l l u l o s e used was M e t a m u c i l ( S e a r l e L a b o r a t o r i e s , C h i c a go,111.), a p h a r m a c e u t i c a l p r e p a r a t i o n o f I s p a g h u l a husk f r o m P l a n t a g o o v a t a ( F o r s k ) w h i c h has been c h a r a c t e r i z e d (76). The p e c t i n used was an NF c i t r u s p e c t i n , f i n e l y ground to a powder with p a r t i c l e s i z e of 200 microns (Hercules,Inc., Wilmington,DL). Diet Preparations. The American I n s i t u t e of N u t r i t i o n (AIN) synt h e t i c d i e t f o r m u l a t i o n was used a l o n g w i t h g e l a t i n t o form h y d r a t e d g e l l e d d i e t s . The i n g r e d i e n t composition i n grams/100 grams d i e t was: d e x t r o s e 64.6, c a s e i n 16.2, d l - m e t h i o n i n e 0.2, AIN s a l t m i x t u r e 3·5, AIN v i t a m i n m i x t u r e 1.5, g e l a t i n 6.0, and l a r d 8.0 f o r the c o n t r o l d i e t ; f i b e r d i e t s contained 15$ l e s s of each i n g r e d i e n t plus 15g f i b e r . Dry i n g r e d i e n t s were premixed and the hydrated g e l l e d d i e t s were prepared weekly by adding water (I90ml/I00g) and l a r d t o the a p p r o p r i a t e amount o f p r e m i x , blending f o r 1 minute, l e t t i n g the d i e t s g e l f o r 2 hours at 5°C, and reblending the s e m i g e l l e d d i e t s to evenly d i s t r i b u t e i n s o l u b l e i n g r e d i e n t s . The p r e p a r e d d i e t s were s t o r e d a t 5°C i n t u b s w i t h p l a s t i c f i l m l a i d d i r e c t l y on the s u r f a c e o f the d i e t s to minimize evaporative l o s s . Animal Treatment. S i x groups of 11 , t h i r t y - d a y o l d , male W i s t a r r a t s (100-120g), were r a n d o m l y d i v i d e d i n t o i n d i v i d u a l cages. A f t e r f i v e days a c c l i m a t i o n the groups were fed: Purina Lab Chow ad l i b i t u m , c o n t r o l ( f i b e r - f r e e g e l l e d d i e t ) and the g e l l e d d i e t s which c o n t a i n e d 15$ p e c t i n , M e t a m u c i l , l i g n i n , and c e l l u l o s e , r e s p e c t i v e l y . Every second day each r a t was weighed, unused food was weighed and f r e s h d i e t placed i n feeders secured at the f r o n t o f each cage. A t t h e end o f 28 days, two r a t s from each group were s a c r i f i c e d each day f o r 5 consecutive days.


20.

OPDYCKE AND STREET

Intestinal and Liver Microsome

Activities

289


Enzyme P r e p a r a t i o n . Each r a t was weighed, k i l l e d by c e r v i c a l d i s l o c a t i o n , the l i v e r removed and 2g homogenized i n 10 ml 0.05M T r i s . , 0.15M KCL, pH 7.8 b u f f e r . I n t e s t i n a l m i c r o s o m e s were p r e p a r e d a c c o r d i n g to S t o h s et al.(28). The e n t i r e s m a l l i n t e s t i n e o f each r a t was removed, washed w i t h b u f f e r , measured f o r length, s p l i t l o n g i t u d i n a l l y w i t h s c i s s o r s , l a i d mucosal s i d e up on a g l a s s p l a t e and s c r a p e d w i t h a g l a s s s l i d e . The m u c o s a l c e l l s were weighed and homogenized i n 10ml b u f f e r w i t h 20$ g l y c e r o l , 25mg t r y p s i n i n h i b i t o r and 2 u n i t s h e p a r i n . H e p a t i c and i n t e s t i n a l homogenates were c e n t r i f u g e d at 600g f o r 2 minutes and then at 13000g f o r 20 minutes, with the microsomes sedimented at 105,000g f o r 60 minutes. A s s a y s . P r o t e i n was d e t e r m i n e d a c c o r d i n g to Lowry ejb a l ( 7 7 ) ) . Biphenyl hydroxylase a c t i v i t y was determined w i t h 2 mg microsomal p r o t e i n , 400 micromoles NADP, 4 micromoles glucose-6-phosphate, 3 micromoles magnesium c h l o r i d e , and 0.5 u n i t s glucose-6-phosphate dehydrogenase i n 1 ml t o t a l volume. A f t e r a 2 minute preincubat i o n , 1 mM b i p h e n y l i n 10 m i c r o l i t e r s o f a c e t o n e was added to s t a r t the 15 minute incubation. The r e a c t i o n s were stopped w i t h 0.5 ml 6N HCL, e x t r a c t e d w i t h i s o o c t a n e and c h r o m a t o g r a p h e d by HPLC on M i c r o p a c NHp-C-10 ( V a r i a n ) w i t h i s o o c t a n e i a c e t o n i t r i l e : i s o a m y l a l c o h o l (100:4:4)(59). D e t e c t i o n o f the 2-, 3-, and 4 - h y d r o x y b i p h e n y l p r o d u c t s was by f l u o r e s c e n c e d e t e c t i o n w i t h e x c i t a t i o n at 275nm and e m i s s i o n at 38nm. Q u a n t i t a t i o n of 2-, 3-, and 4-hydroxybiphenyls was accomplished with peak height comparisons to 1 .25, 2.50, and 3*75 m i c r o g r a m s o f each s t a n d a r d ; s t a n dards were incubated and extracted with each set of determinat i o n s . Epoxide hydrolase a c t i v i t y was determined by the method of G i u l i a n o ejt a l ( 7 5 ) u s i n g 3-(p-nitrophenoxy)-1 ,2-propene oxide as substrate w i t h 0.5mg microsomal p r o t e i n . The d i o l was separated u s i n g ODS r e v e r s e phase HPLC w i t h 40$ a c e t o n i t r i l e i n water. D e t e c t i o n was accomplished u s i n g UV absorbance at 315nm. A r y l h y drocarbon hydroxylase a c t i v i t y was determined by the assay procedure of Dehnen e_t al.(78) on an AmincoBowman s p e c t r o f l u o r o m e t e r a f t e r i n c u b a t i o n s w i t h 0.5mg microsomal p r o t e i n and NADPH genera t i n g system. One u n i t a c t i v i t y i s defined as picomoles/mg/minute as based on the f l u o r e s c e n c e of 20 ng 3-hydroxybenzo(a)pyrene. Results and D i s c u s s i o n Results of body, l i v e r and l i v e r / b o d y weight r a t i o s a f t e r 30 days on s p e c i f i e d d i e t s a r e l i s t e d i n T a b l e I. Each o f the treatment groups showed an i n i t i a l weight l o s s upon i n t r o d u c i n g the p u r i f i e d d i e t s . However, a l l but the l i g n i n - f e d a n i m a l s q u i c k l y recovered to steady weight gains throughout the r e s t of the experiment. Lab Chow-fed r a t s were s i g n i f i c a n t l y h e a v i e r than the p u r i f i e d d i e t - f e d r a t s . Body weights i n pectin-and Metamucilfed r a t s were s i g n i f i c a n t l y (0.05 l e v e l ) h i g h e r than c o n t r o l - ,



290

Table I.

Body and L i v e r Weights and L i v e r t o

Body Weight Ratios o f Fiber-Fed R a t s .


Diet Type

Control

(No F i b e r )

Body Weigjit

L i v e r Weight

a

L i v e r Wt./Body Wt.

g

g

g

182 + 12

6.90 + 1.38

0.037 + 0.006

Lab Chow

300

15% P e c t i n

b

28

11.14

220°

18

15% Metamucil

224°

15% L i g n i n

15% C e l l u l o s e

b

1.63

.037

.004

7.73

1.19

.034

.003

12

7.83

1.19

.034

.006

153

15

5.61

0.64

.036

.003

172

10

6.10

0.50

.035

.002

Values are means + S.D. f o r groups of 11 r a t s . b

S i g n i f i c a n t l y d i f f e r e n t from a l l other groups (P=.05) .

c S i g n i f i c a n t l y d i f f e r e n t from the other groups f e d p u r i f i e d d i e t s (P=.05),



20.

OPDYCKE AND

STREET


Activities

291

l i g n i n - o r c e l l u l o s e - f e d a n i m a l s . L i g n i n - f e d a n i m a l s c o u l d be considered to be under weight. L i g n i n - and c o n t r o l - f e d a n i m a l s ate s i g n i f i c a n t l y l e s s p u r i f i e d d i e t than other treatment groups. L i v e r weights i n Lab Chow-fed r a t s were a l s o s i g n i f i c a n t l y higher than i n r a t s fed p u r i f i e d d i e t s . The l i v e r weights of p e c t i n - and Metamucil-fed r a t s were s l i g h t l y higher (not s i g n i f i c a n t l y ) than c o n t r o l - , l i g n i n - or c e l l u l o s e - f e d r a t s . L i v e r : t o : b o d y r a t i o s were not s i g n i f i c a n t l y d i f f e r e n t i n any of the treatment groups. The i n f l u e n c e o f s p e c i f i c d i e t a r y f i b e r s on s m a l l i n t e s t i n e length, on the amount of mucosal m a t e r i a l scraped from the i n t e s t i n e , and on the r a t i o s o f grams mucosa/cm o r cm/g mucosa i s i n d i c a t e d by the d a t a i n T a b l e I I , w h i l e T a b l e I I I l i s t s the i n t e s t i n a l microsomal p r o t e i n (expressed as t o t a l or mg/cm s m a l l i n t e s t i n e ) . Lab Chow and d i e t s c o n t a i n i n g p e c t i n o r M e t a m u c i l produced i n t e s t i n e lengths that were s i g n i f i c a n t l y longer than i n the r a t s f e d the c o n t r o l , l i g n i n , o r c e l l u l o s e d i e t s . These r e s u l t s support s i m i l a r f i n d i n g s by deBethizy (23-24) and Rotenb e r g and J a k o b s e n (79). The s t a b l e g e l s formed by M e t a m u c i l and p e c t i n f i b e r s caused s i g n i f i c a n t i n t e s t i n a l t r a c t enlargement, even though f e d i n h y d r a t e d form. The r a t s f e d Lab Chow and the p e c t i n d i e t had the highest mucosal weights, which were s i g n i f i c a n t l y higher than i n c o n t r o l - , l i g n i n - , o r c e l l u l o s e - f e d animals. L i g n i n - f e d animals y i e l d e d the l e a s t amount of mucosal m a t e r i a l , which c o i n c i d e s w i t h t h e i r having the s h o r t e s t s m a l l i n t e s t i n e s and lowest body weights (Table IV). T o t a l i n t e s t i n a l microsomal p r o t e i n d i d not d i f f e r s i g n i f i c a n t l y (0.05 l e v e l ) among treatment groups, although p e c t i n d i e t and Lab Chow-fed r a t s had r e l a t i v e l y higher t o t a l microsomal p r o t e i n values. Both l i v e r and i n t e s t i n a l mucosa m i c r o s o m a l p r o t e i n v a l u e s a r e l i s t e d i n T a b l e IV. T h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t groups f o r e i t h e r tissue. L i s t e d i n T a b l e V a r e the v a l u e s o f 2 - , 3 - , 4 - b i p h e n y l hyd r o x y l a s e a c t i v i t i e s i n l i v e r microsomes of f i b e r - f e d r a t s . V a l u e s a r e e x p r e s s e d as nanomoles (2-, 3-,or 4 - h y d r o x y b i p h e n y l ) / m g / l 5 m i n u t e i n c u b a t i o n ; the l e a s t d e t e c t a b l e amount was 0.05 moles/mg. The h e p a t i c m i c r o s o m a l l e v e l s o f 4 - b i p h e n y l hyd r o x y l a s e were on the o r d e r o f 4 0 - f o l d h i g h e r t h a n the 2-or 3biphenyl hydroxylase a c t i v i t i e s . This difference i s s l i g h t l y g r e a t e r t h a n the h i g h e r c o n s t i t u a t i v e 4 - b i p h e n y l h y d r o x y l a s e l e v e l s r e p o r t e d by o t h e r s (46,50,80). The y i e l d o f 2 - h y d r o x y b i p h e n y l was s i g n i f i c a n t l y h i g h e r w i t h h e p a t i c m i c r o s o m e s from l i g n i n - f e d a n i m a l s t h a n w i t h m i c r o s o m e s from any o f the o t h e r treatment groups. The s l i g h t l y higher b i p h e n y l hydroxylase values f o r the p u r i f i e d d i e t - f e d r a t s i s notable. L i g n i n - f e d r a t s a l s o showed the highest 3-hydroxybiphenyl y i e l d s although t h i s e f f e c t was not s i g n i f i c a n t . The l e v e l s of h e p a t i c microsomal 4-hydroxyb i p h e n y l h y d r o x y l a s e were s i g n i f i c a n t l y l o w e r i n Lab Chow-fed animals than l i g n i n - f e d animals. There were no s i g n i f i c a n t d i f f e r e n c e s among the p u r i f i e d d i e t t r e a t m e n t groups, however, l i g n i n - f e d r a t s again had s l i g h t l y higher l e v e l s of a c t i v i t y .



4.95 6.39 6.74 6.48

34.70 39.86 48.01 41.06

.004 .004 .003 .004

.029 .025 .021 .026

6 6

108° e

I n t e s t i n e s i g n i f i c a n t l y longer

°

(P=.05).

(P=.05).

Mucosal weight s i g n i f i c a n t l y greater

^

f o r groups of 11 rats.

4

96

.35

2.30

15% Cellulose

Means + S.D.

6

93

.35

1.98

15% Lignin

108

.51

2.78

.39

2.88

e

31.84

III

.42

.004

36.79 + 6.92

.032

0.028 + 0.005

94 + 4

cm

cm/g Mucosa

8

g

cm

15% Metamucil

b

Mucosal Wt./cm

Small I n t e s t i n e

3.15

b

3.52

2.65 + 0.47

g

Mucosa

a

Small I n t e s t i n e Length, Mucosal Weight and t h e i r Ratio i n Fiber-Fed R a t s .

15% Pectin

Lab Chow

Control (No Fiber)

Diet Type

Table I I .


20.

OPDYCKE AND STREET


293

Activities

Table I I I . Microsomal P r o t e i n i n Small I n t e s t i n e


Mucosa of Fiber-Fed Rats.

Type

T o t a l mg

Control

(No F i b e r )

13.77 + 5.80

mg/cm Length

0.14 + 0.06

Lab Chow

14.81

7.88

.13

.07

15% P e c t i n

15.23

5.45

.14

.05

15% Metamucil

10.50

5.20

.19

.05

15% L i g n i n

10.09

6.74

.11

.08


11.27

6.42

.12

.07

a

Mean microsomal p r o t e i n + S.D. f o r groups o f 11 r a t s ; d i f f e r e n c e s were not s i g n i f i c a n t (P=.05).


294


Table IV.

Microsomal P r o t e i n i n L i v e r and I n t e s t i n a l


Preparations

Diet Type

from Fiber-Fed

mg P r o t e i n / g L i v e r

Rats.

a

mg P r o t e i n / g

Mucosa

C o n t r o l (No F i b e r )

11.8 + 6.0

5.59 + 3.48

Lab Chow

14.5

4.6

4.26

2.46

15% P e c t i n

12.4

5.4

4.97

1.87

9.4

3.7

3.71

1.94

15% L i g n i n

12.1

7.4

5.11

3.83


15.1

4.3

4.91

2.30

15% Metamucil

Values are mean + S.D. f o r groups of 11 r a t s . treatments were not s i g n i f i c a n t (P=.05).

D i f f e r e n c e s among


20.


OPDYCKE AND STREET

Table V.

295

Activities

Biphenyl Hydroxylase A c t i v i t i e s i n L i v e r Microsomes o f Fiber-Fed R a t s .

3

Nanomoles/mg/15 min


Diet Type

2-OH

3-0H

Control (No F i b e r ) 0.43 + 0.10

Lab Chow

,30

15% P e c t i n

b

4-OH

0.40 + 0.13

.06

.36

.44

.15

15% Metamucil

.41

15% L i g n i n


b

15.98 + 5.75

b

.07

9.77

1.94

.32

.09

12.46

3.04

.08

.29

.07

13.82

4.00

.81°

.35

.62

.18

20.03

5.65

.30

.06

.28

.07

11.43

2.57

a

Values are mean hydroxylase a c t i v i t y + SE (N=10) i n nanomoles/mg p r o t e i n / 1 5 min.; min. d e t e c t a b l e amount was 0.05 nmole.

b

D i f f e r e n c e from l i g n i n - t r e a t e d animals s i g n i f i c a n t

C

D i f f e r e n c e from c o n t r o l and a l l other groups s i g n i f i c a n t

(P=.05).


(P=.05).


296


I n t e s t i n a l microsomal biphenyl h y d r o x y l a t i o n f o r f i b e r - f e d r a t s i s l i s t e d i n T a b l e VI. Due to the v a r i a b i l i t y o f the d a t a t h e r e were no s i g n i f i c a n t (0.05 l e v e l ) d i f f e r e n c e s among the groups o f f i b e r - f e d r a t s . L i g n i n f e e d i n g l e d to the h i g h e s t i n t e s t i n a l 2-biphenyl hydroxylase a c t i v i t i e s , while a l l the r a t s f e d p u r i f i e d d i e t s had h i g h e r l e v e l s t h a n the Lab Chow group. Among the groups fed p u r i f i e d d i e t s , the p e c t i n - f e d r a t s had the lowest 2-biphenylhydroxylase l e v e l s . P e c t i n feeding a l s o gave the highest i n t e s t i n a l 3-biphenylhydroxylase although that d i f f e r e n c e o n l y approached s i g n i f i c a n c e a t the 0.05 l e v e l . Lab Chow-fed r a t s had the l o w e s t 3 - b i p h e n y l h y d r o x y l a s e l e v e l i n i n t e s t i n a l microsomes. I n t e s t i n a l microsomes from f i b e r - f e d r a t s d i d not d i f f e r s i g n i f i c a n t l y (0.05 l e v e l ) i n 4-biphenyl hydroxylase a c t i v i t y . C e l l u l o s e - f e d animals had the lowest l e v e l , with the cont r o l s and l i g n i n - f e d r a t s showing the highest i n t e s t i n a l microsomal 4-biphenylhydroxylase values. P e c t i n - and Metamucil-fed r a t s had r e l a t i v e l y low l e v e l s of 4-biphenyl hydroxylase a c t i v i t i e s . The r e s u l t s of a p r e l i m i n a r y i n t e s t i n a l microsomal biphenyl hydroxylase t r i a l are l i s t e d i n Table VII. These r a t s were on the same h y d r a t e d p u r i f i e d d i e t s c o n t a i n i n g 15$ f i b e r f o r 32 days before the microsomal enzyme determinations. One d i f f e r e n c e between these animals and those i n the primary experiment reported h e r e i n i s t h a t t h e y were s t a r t e d on the d i e t s a t 45 days o f age and weighed about 200g at the s t a r t . The biphenyl h y d r o x y l a t i o n i n c u b a t i o n was done with 5 mg i n t e s t i n a l microsomal p r o t e i n as compared to 2 mg p r o t e i n used i n the subsequent incubations. The b i p h e n y l h y d r o x y l a t i o n v a l u e s were not s i g n i f i c a n t l y d i f f e r e n t (0.05) i n any of the treatment groups although Lab Chow-fed r a t s had s l i g h t l y higher 4-hydroxy biphenylhydroxylase values. Benzo(a)pyrene hydroxylase a c t i v i t i e s i n h e p a t i c and i n t e s t i n a l microsomes of f i b e r - f e d r a t s are presented i n Table V I I I . There were no s i g n i f i c a n t d i f f e r e n c e s (0.05 l e v e l ) among any of the p u r i f i e d f i b e r treatment groups i n e i t h e r l i v e r or i n t e s t i n a l microsomes. Lab Chow-fed r a t s had the l o w e s t l i v e r m i c r o s o m a l benzo(a)pyrene h y d r o x y l a s e (AHH) l e v e l . The i n d i c a t i o n o f enhanced i n t e s t i n a l P-448-dependent h y d r o x y l a t i o n of biphenyl i n the p e c t i n - f e d r a t s (seen as elevated 3-hydroxybiphenyl formation i n microsomes, Table Vl) was not supported by these p r e l i m i n a r y data. Microsomal epoxide hydrolase a c t i v i t i e s are l i s t e d i n Table IX. There was s i g n i f i c a n t l y higher (0.05 l e v e l ) h e p a t i c microsomal e p o x i d e h y d r o l a s e a c t i v i t y i n p e c t i n - , M e t a m u c i l - , and c e l l u l o s e - t r e a t e d r a t s , as compared to c o n t r o l - , l i g n i n - and Lab Chow-fed animals. There was no s i g n i f i c a n t d i f f e r e n c e i n i n t e s t i n a l m i c r o s o m a l e p o x i d e h y d r o l a s e a c t i v i t i e s among any o f the treatment groups. Studies d i r e c t l y probing the i n t e r a c t i o n of d i e t a r y f i b e r s on f o r e i g n compound m e t a b o l i s m a r e l i m i t e d . Brown,_ejt al.(81 ) reported that r a t s fed b a s a l chow d i e t s had no l i v e r metabolism d i f f e r e n c e s compared to r a t s on d i e t s supplemented w i t h p e c t i n or


20.

OPDYCKE AND STREET

Table VI.


Activities

297

Biphenyl Hydroxylase A c t i v i t y i n I n t e s t i n a l Microsomes of F i b e r - F e d R a t s .

a


Nanomoles/mg/15 min Diet Type


2-OH

0.38 + 0.16

3-OH

4-OH

0.79 + 0.35

1.13 + 0.74

Lab Chow

.30

.09

.46

.14

.79

.24

15% P e c t i n

.19

.05

2.08

.74

.35

.09

15% Metamucil

.50

.01

.88

.13

.92

.45

15% L i g n i n

.68

.32

1.14

.26

1.32

1.06


.47

.07

.46

.13

.22

.08

a

Values are mean hydroxylase a c t i v i t y + SE f o r groups of 7 r a t s ; d i f f e r e n c e s among treatments were not s i g n i f i c a n t (P=.05).


Furda; Unconventional Sources of Dietary Fiber ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 3.22

S i g n i f i c a n t d i f f e r e n c e from c o n t r o l , P>.05.

5.73

C

0.62

Nanomoles/mg p r o t e i n i n mucosal microsomes/15 min.

2.86

0.25

6.39

4.41

1.24

0.50

0.64

1.40

.97

.97

.83

.41

0.95

n=2.

a

.21

.42

.15

.12

.73

.44

.85

.72

0.98

.12

.19

.11

.09

+ 0.09

Biphenyl Hydroxylase^ 3-OH

+ 0.22

2-OH

of 3 r a t s , except hydroxylase values where

5

2.56

0.27

^

103


7

2.55

0.70

7.28 + 0.90

Microsomal x) Protein (mg/g)

Values are means + S.D.

101

15% L i g n i n

8

2.51

2.30 + 0.45

Mucosal x) Weight (g)

Rats.

.73

.45

.33

1.19

0.77

and Biphenyl Hydroxylase A c t i v i t i e s

i n I t i t e s t i n a l Microsomes of Fiber-Fed

Small Intestine Parameters

3

119

15% Metamucil

6

100 + 3

108

(No Fiber)

I n t e s t i n e x) Length (cm)

Lab Chow

Control

Fiber Type

Table VII.


+

4-OH

.09

.07

.07

.56

0.11

20.

OPDYCKE AND STREET

Table V I I I .


Activities

299

Benzo(a)pyrene Hydroxylase A c t i v i t i e s i n

Hepatic and I n t e s t i n a l Mucosa Microsomes of Fiber-Fed

Rats.

a


Picomoles /mg/min Diet Type

Control

(No F i b e r )

Lab Chow

Liver

216 +

92

u


87.9

7.1

14.5 +

1.6

19.9

6.8

15% P e c t i n

193

81.1

17.9

4.7

15% Metamucil

263

98.1

18.7

5.3

15% L i g n i n

255

86.4

19.3

3.4


245

98.1

19.6

4.8

Values are means + SE f o r groups of 7. Q u a n t i t a t i o n was based on the fluorescence of 20ng 3-hydroxy-benzo(a)pyrene and s c a l e d to mg microsomal p r o t e i n . S i g n i f i c a n t d i f f e r e n c e from c o n t r o l s , P>.05.


300


Table IX.

Epoxide Hydrolase A c t i v i t i e s i n

L i v e r and I n t e s t i n a l Mucosa Microsomes of Fiber-Fed

Rats.


Nanomoles/mg/5 min Diet Type

Liver



38.0 4- 8.13

14.9 + 2.94

Lab Chow

45.0

7.24

13.1

3.28

15% P e c t i n

64.3

11.81

12.6

3.05

15%

62.0

13.32

16.3

3.24

15% L i g n i n

44.9

4.94

14.3

4.33


64.4

18.6

7.01

Metamucil

υ

U

22.1

Values are means + SE f o r groups o f seven s c a l e d to mg microsomal p r o t e i n f o r 3-(p-nitrophenoxy)-l,2-propene oxide as s u b s t r a t e . S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s , P>.05.



20.

OPDYCKE AND STREET


Activities

301

wheat bran as judged by a n t i p y r i n e and acetaminophen metabolism, Chadwick et al.(82), however, d e m o n s t r a t e d s i g n i f i c a n t enhancement o f l i n d a n e m e t a b o l i s m i n p e c t i n - f e d r a t s - P r o i a e t al.(83) r e c e n t l y reported that p e c t i n feeding lowered hepatic cytochrome P-450 l e v e l s w h i l e a t t h e same t i m e i n c r e a s i n g i n t e s t i n a l benzo(a)pyrene hydroxylase l e v e l s . I t remains to be evaluated to what e x t e n t t h e d i f f e r e n c e s o b s e r v e d i n d r u g o r o t h e r f o r e i g n compound _in v i v o metabolism i n r a t s f e d p e c t i n or other p u r i f i e d f i b e r s can be explained by d i f f e r e n c e s i n c o n s t i t u a t i v e l e v e l s of the relèvent enzymes i n l i v e r and i n t e s t i n a l mucosa. Our d a t a suggest that MFO h y d r o x y l a t i n g a c t i v i t i e s i n both t i s s u e s are not p a r t i c u l a r l y s e n s i t i v e to e f f e c t s o f feeding p u r i f i e d f i b e r s at high l e v e l s (the d i e t model used i n our work represents extremes i n f i b e r i n t a k e ) . The s i g n i f i c a n t e f f e c t on h e p a t i c e p o x i d e hydrolase seen with p e c t i n - , Metamucil-, and c e l l u l o s e - f e d r a t s (Table IX) may point toward a more important response i n r e l a t i o n to chemical carcinogenesis. Studies are i n progress i n our l a b o r a t o r y t o compare G S H - t r a n s f e r a s e a c t i v i t i e s and t h e t i s s u e g l u t a t h i o n e s t a t u s i n s p e c i f i c f i b e r - f e d r a t s . These s t u d i e s may enhance o u r a p p r e c i a t i o n o f t h e c o n t r i b u t i o n s o f t h e nono x i d a t i v e enzymes i n f o r e i g n compound metabolism to the r e l a t i o n ships between n u t r i t i o n a l s t a t e and chemical carcinogenesis. Acknowledgment s T h i s r e s e a r c h was s u p p o r t e d , i n p a r t , by a g r a n t f o r the study of "Diet and Colon Cancer i n Man: The E f f e c t o f F i b e r (CA25580)" and a g r a n t f o r t o x i c o l o g y t r a i n i n g (ES-7097) from the N a t i o n a l I n s t i t u t e s o f H e a l t h . F a c i l i t i e s and o t h e r s u p p o r t by the Utah A g r i c u l t u r a l Experiment S t a t i o n are a l s o acknowledged.

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