Xenobiotic Metabolism - American Chemical Society

4 The Influence of Fermentable Dietary Fiber on the Disposition and Toxicity of Xenobiotics J. DONALD DEBETHIZY1 and ROBIN S. GOLDSTEIN2 Rohm and Haas Company, Toxicology Department, Spring House, PA 19477 Smith, Kline, and French Laboratories, L-66, Philadelphia, PA 19101

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Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch004

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Fermentable dietary fiber may modulate chemical toxicity by altering the microfloral metabolism of xenobiotics. A series of studies were conducted to assess the influence of fermentable fiber on the toxicity of xenobiotics that require microfloral metabolism to express their toxicity. The hepatic macromolecular covalent binding of 2,6-dinitrotoluenederived radioactivity and nitrobenzene-induced methemoglobinemia were enhanced in rats fed pectin supplemented purified diets to levels comparable to rats fed cereal-based diets. The increased toxicity of these xenobiotics was associated with a 2- to 3-fold increase in the number of cecal anaerobic bacteria in rats fed the pectin diets. The number of cecal anaerobic bacteria in cereal-based diet-fed rats was similar to rats fed the purified diet supplemented with pectin. Following a single oral dose of Amaranth, the peak plasma concentration of naphthionic acid, a microfloral metabolite of Amaranth, was 5-fold higher in rats fed a pectin-supplemented, purified diet. These studies indicated that feeding diets containing fermentable fibers such as pectin can enhance the toxicity of nitroaromatics by increasing the number of cecal anaerobic bacteria that are required for the microfloral metabolism of these xenobiotics to proximate toxicants. Dietary fiber has been suggested to play a protective role against chemically-induced toxicity (1) and against colon cancer (2). However, the mechanism(s) by which dietary fiber modulates chemical toxicity or colon cancer has not been well studied. The fiber fraction of the diet is resistant to mammalian digestive enzymes and consequently dietary fiber is not absorbed from the small intestine (3). However, certain types of dietary fiber; specifically fermentable fibers, including the pectic substances and hemicelluloses, are readily digested by the intestinal microflora (4,5). Pectic 0097-6156/85/0277-O037$06.00/0 © 1985 American Chemical Society

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


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

substances are a family of galacturonic acid polymers which are methoxylated t o varying degrees depending on the plant source (6). Hemicelluloses are primarily polymers of the pentose, xylose, with varying amounts of arabinose branching (7). Although hemicellulose constitutes the majority of the fermentable dietary f i b e r derived from most plant sources, i t has not been well studied because i t i s d i f f i c u l t to extract and i s o l a t e from plant c e l l walls (7). On the other hand, pectin, which i s the major component of pectic substances, i s e a s i l y isolated from apple and c i t r u s f r u i t s as a byproduct of the f r u i t j u i c e industry (8). Therefore, most studies on fermentable f i b e r have employed pectin as a model fermentable f i b e r . Animal feeds as well as human d i e t s vary considerably i n the type and quantity of dietary f i b e r . Wise and G i l b e r t (9) using modified detergent methods analyzed fourteen commercial rodent d i e t s and found that the t o t a l dietary f i b e r content varied from 8.3 to 22.4%. In f a c t , i t i s not unusual f o r commercially available cereal-based rodent diets to contain 20% dietary f i b e r on a dry weight basis (10). In general, the fermentable f i b e r s constitute more than h a l f of the t o t a l dietary f i b e r ; the remainder composed of the f i b e r s more resistant t o fermentation, such as c e l l u l o s e and l i g n i n (9). Thus, a s i g n i f i c a n t portion of rodent diets has the p o t e n t i a l to be fermented i n the i n t e s t i n a l t r a c t . One way i n which fermentable f i b e r could influence chemical t o x i c i t y i s by a l t e r i n g the m i c r o f l o r a l metabolism of xenobiotics. I t has been suggested that the fermentable components of dietary f i b e r s i g n i f i c a n t l y influence the i n t e s t i n a l m i c r o f l o r a l metabolism of xenobiotics by providing a p o t e n t i a l source of energy f o r microb i a l growth and a c t i v i t y (11). Using pectin as a model fermentable f i b e r , Bauer et a l . (12) demonstrated that there was a higher i n c i dence of dimethylhydrazine(DMH)-induced tumors of the colon i n Sprague-Dawley rats fed pectin-containing p u r i f i e d diets than i n rats fed a p u r i f i e d d i e t alone. These investigators speculated that pectin enhanced the metabolic a c t i v a t i o n of DMH as suggested by the concommitant elevation of m i c r o f l o r a l $-glucuronidase a c t i v i t y i n the pectin-fed animals. However, the r e l a t i o n s h i p between microfloral 3-glucuronidase a c t i v i t y and DMH-tumorigenicity has been questioned since there i s c o n f l i c t i n g evidence that hydrolysis of a glucuronide conjugate of DMH i s e s s e n t i a l f o r the a c t i v a t i o n of DMH to a proximate carcinogen (13). Amaranth Metabolism Our f i r s t i n d i c a t i o n that fermentable f i b e r could a l t e r m i c r o f l o r a l metabolism was based on studies assessing the influence of dietary f i b e r types on the d i s p o s i t i o n of model xenobiotics using pharmacok i n e t i c analysis (14). Amaranth was selected as a model xenobiotic for these studies because i t was absorbed only a f t e r reduction by gut microflora (15). In these studies adult, male Wistar rats were fed p u r i f i e d hydrated g e l a t i n d i e t s containing either no f i b e r or 15% c e l l u l o s e , l i g n i n , metamucil, or pectin (16). After 28 days on the d i e t the animals received a single o r a l dose of Amaranth (1 mmoleAg). Blood samples were c o l l e c t e d at various times and the plasma concentration of naphthionic acid (NA), the major m i c r o f l o r a l metabolite of



4. DEBETHIZY A N D GOLDSTEIN

Influence of Fermentable Dietary Fiber

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Amaranth, was determined by high pressure l i q u i d chromatography (HPLC) (14). The fermentable f i b e r , pectin, elevated the peak plasma concent r a t i o n of NA 5-fold over the other dietary groups (Figure 1) (17). To determine i f the higher plasma concentration of NA was due to enhanced absorption of NA or increased m i c r o f l o r a l metabolism of Amaranth, the i n v i t r o metabolism of Amaranth by c e c a l contents from rats fed the various diets was examined i n a prelimary experiment. Cecal contents from animals fed the fiber-containing diets were incubated anaerobically with Amaranth and the amount of NA produced per gram of cecal contents determined by HPLC. Although the specif i c a c t i v i t y (on a per gram of cecal contents basis) of m i c r o f l o r a l Amaranth-azoreductase was lower i n pectin-fed animals, the t o t a l amount of NA produced per cecum was elevated 2-fold over rats fed the other diets (data not shown). These r e s u l t s suggested that feeding c i t r u s pectin to rats elevated the m i c r o f l o r a l metabolism of Amaranth r e s u l t i n g i n greater amounts of NA i n the plasma. These findings l e d us to believe that the capacity f o r m i c r o f l o r a l metabolism of xenobiotics i s enhanced by feeding pectin.

Dinitrotoluene Hepatotoxicity Based on the Amaranth studies, i t was hypothesized that those chemic a l s requiring m i c r o f l o r a l metabolism to express t h e i r t o x i c i t y may be more toxic to animals consuming fermentable f i b e r . 2,6-Dinitrotoluene (DNT) i s a hepatocarcinogen i n Fischer-344 rats (18) and i s genotoxic i n the i n v i v o / i n v i t r o hepatocyte DNA repair assay (19). The hepatic genotoxic i t y of DNT was found to be dependent upon the presence of gut microflora (20). Long and Rickert (21) demonstrated that DNT i s excreted i n the b i l e of male rats as the 2,6-dinitrobenzylalcohol glucuronide. This glucuronide conjugate i s hydrolyzed by m i c r o f l o r a l ^-glucuronidase, permitting DNT to undergo enterohepatic c i r c u l a t i o n . Evidence also indicated that similar to the hepatic genotoxicity of DNT, hepatic macromolecular covalent binding (CVB) was also dependent upon the presence of gut microflora (22). CVB therefore was used as an endpoint to test the hypothesis that pectin-containing diets could enhance the t o x i c i t y of xenobiotics by elevating m i c r o f l o r a l metabolism. In these experiments adult, male Fischer-344 rats were fed a p u r i f i e d d i e t , AIN-76A, containing 5 or 10% c i t r u s pectin replacing cornstarch or one of two c e r e a l based d i e t s , Purina Rodent Chow 5002 and NIH-07. After 28 days of dietary treatment rats were given a single o r a l dose of t r i t i a t e d DNT (10 or 75 mgAg). Twelve hours a f t e r dosing, animals were k i l l e d and CVB was determined by exhaust i v e extraction. The cecum was also excised from these animals and microflora characterized by anaerobic culture techniques (10). The CVB of DNT-derived r a d i o a c t i v i t y to hepatic macromolecules was independent of the d i e t at a dose of 10 mg DNTAg (Table I ) . However, at a dose of 75 mgAg, CVB was increased 40% and 90% by supplementing 5% and 10% pectin to the p u r i f i e d d i e t s , respectively. Livers of animals fed Purina 5002 and NIH-07 exhibited s i g n i f i c a n t l y greater CVB than animals fed the p u r i f i e d d i e t with or without pectin supplementation. CVB was increased approximately s i x f o l d when the dose of DNT was increased from 10 to 75 mgAg i n animals


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

7.00

EFFECTS

control col I u I ooo I Ign I n


ao t u u o I I poet In

6

12

18 24 30 TIME

36 42 48 (hour.)

54

60

66 72

Figure 1. Concentration of naphthionic acid i n plasma from Wis tar rats given a single o r a l dose of Amaranth (1 mmole/kg) following feeding p u r i f i e d d i e t s containing no f i b e r (control) or 15% (w/w) c e l l u l o s e , l i g n i n , metamucil, or pectin f o r 30 days. Each point represents the mean of s i x r a t s .


4.

DEBETHIZY A N D GOLDSTEIN

Influence of Fermentable Dietary Fiber

41

fed NIH-07, Purina 5002, and p u r i f i e d d i e t plus 10% pectin, but increased only f o u r f o l d i n animals fed the p u r i f i e d d i e t or p u r i f i e d d i e t supplemented with 5% pectin.

Table I .

E f f e c t of Diet on the Hepatic Macromolecular Covalent Binding of DNT


Hepatic Covalent Binding (nmol equivalents/g l i v e r ) A f t e r a DNT Dose of 10 mgAg 75 mgAg AIN-76A AIN-76A plus 5% pectin AIN-76A plus 10% pectin NIH-07 Purina 5002

1.03 1.21 1.11 1.40 1.47

+ + + + +

0.19 0.23 0.17 0.13 0.16

3.75 5.21 7.06 9.29 8.82

+ + + + +

0.22** 0.43 0.71

Xenobiotic Metabolism - American Chemical Society

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