Effects of benzene metabolites on receptor-mediated phagocytosis and cytoskeletal integrity in mouse peritoneal macrophages.

Exposure to benzene can induce a number of hematotoxicities and decrease host resistance to microorganisms and tumors. Several studies have shown that metabolism of benzene to reactive intermediates is required for myelotoxicity. Since receptor-mediated phagocytosis by macrophages is an important host defense, we have examined the effects of benzene metabolites on receptor-mediated phagocytosis in cultured murine peritoneal macrophages. 1,4-Benzoquinone (BQ) was the most potent of the metabolites examined. Ten-minute exposures to a 12.5 microM concentration inhibited Fc and complement receptor-mediated phagocytosis by > or = 90%. Macrophage viability was largely unaffected by BQ treatment. Exposure to 50 and 100 microM 1,2,4-benzenetriol (BT) inhibited Fc receptor-mediated phagocytosis by 70 and 95%, respectively. Hydroquinone (HQ) elicited a major decrease (50%) only at 100 microM. The comparative inhibitory potencies of BT and HQ correlate with previously published data on their relative facility for autooxidation to quinones at physiological pH. Catechol had no effect at the concentrations employed. Macrophages treated with BQ and BT failed to recover their Fc receptor-mediated phagocytic capacity when incubated overnight in the absence of the xenobiotics. Only small differences in the inhibition of Fc receptor-mediated phagocytosis were observed between macrophages exposed to BQ at 4 versus 37 degrees C. BQ also had little effect on the Fc receptor binding of target cells. Fluorescent digital imaging microscopy demonstrated that BQ treatment markedly decreased the filamentous actin content of macrophages. However, BQ bound in low amounts to purified actin and did not affect its assembly. Our findings suggest that a mechanism for inhibition of Fc receptor-mediated phagocytosis by BQ is disruption of filamentous actin via an effect(s) other than the direct alkylation of actin by BQ.

Metabolism and bioactivation of 3-methylindole by Clara cells, alveolar macrophages, and subcellular fractions from rabbit lungs.

3-Methylindole (3MI), a fermentation product of tryptophan produced by intestinal and ruminal microflora, has been shown to cause pneumotoxicity in several species subsequent to cytochrome P450-mediated biotransformation. Among several species studied, rabbits are comparatively resistant to 3MI-induced pneumotoxicity, especially when compared to goats or mice. In this study, rabbit pulmonary cells and subcellular fractions were used to examine the metabolism and bioactivation of 3MI. A covalent-binding metabolite was produced in 3MI incubations by both Clara cells and macrophages. The addition of the cytochrome P450 inhibitor, 1-aminobenzotriazole, to these incubations inhibited the production of covalent-binding metabolite(s) by 94% in Clara cells and only 24% in macrophages. In incubations of Clara cells or macrophages with 3MI and N-acetylcysteine (NAC), a polar conjugate was detected and tentatively identified as an adduct of 3-hydroxy-3-methylindolenine (3H3MIN). Also identified were 3[(N-glutathione-S-yl)-methyl]-indole (3MI-GSH) and 3-methyloxindole (3MOI). In rabbit lung microsomal incubations with 3MI and glutathione (GSH), 3MI-GSH, 3MOI, indole-3-carbinol, and a GSH adduct of 3H3MIN were identified. The addition of cytosol to the microsomal incubations with GSH did not increase the rate of formation of the GSH adducts, indicating that cytosolic GSH-S-transferases are not essential in the formation of these metabolites. GSH significantly decreased the covalent binding of an electrophilic metabolite in microsomal incubations. These data suggest that GSH may be important in the mitigation of 3MI toxicity. Furthermore, the comparison of 3MI bioactivation to electrophilic intermediates in Clara cells and alveolar macrophages suggests that 3MI is metabolized by different oxidative pathways in the two different cell types, although the same metabolites were produced by the two cell types. This study shows that rabbit pulmonary enzymes are capable of bioactivating 3MI to reactive intermediates which become covalently bound to cellular macromolecules. This indicates that the relative resistance of rabbits to 3MI-induced pneumotoxicity is probably not due to differences in metabolic enzymes which convert 3MI to reactive intermediates.

N-benzylimidazole-mediated changes in hepatic drug-metabolizing enzyme activities in Ah-responsive and Ah-non-responsive mice.

1. The inductive effect of N-benzylimidazole (NBI) on hepatic microsomal and cytosolic drug-metabolizing enzyme activities in aryl hydrocarbon (Ah)-responsive C57BL/6N (B6) and Ah-non-responsive DBA/2N (D2) mouse strains was determined and compared with that caused by beta-naphthoflavone (BNF). 2. Relative Ah-responsiveness of the two strains was confirmed by measurement of BNF-induced ethoxyresorufin deethylase (EROD) activity and ELISA immunoquantification. BNF markedly induced EROD activity only in the Ah-responsive B6 mouse strain (65-fold increase). 3. NBI (150 mg/kg per day for 3 days) increased cytochrome P450 concentration similarly in both strains (40 and 60% in B6 and D2 strains, respectively). Compared with BNF treatment of the B6 strain, increases in EROD activity following NBI treatment were only minor. In addition, EROD activity increases were greater in the Ah-nonresponsive D2 strain (300%) than in the Ah-responsive B6 strain (100%) suggesting the possibility of an induction mechanism different from that of recognized Ah receptor agonists. 4. Induction of UDP-glucuronosyltransferase activity (p-nitrophenol acceptor) by BNF was greater in the Ah-responsive B6 strain than in the Ah-non-responsive D2 strain. NBI failed to induce this activity in either strain. 5. Induction of glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene following NBI treatment occurred to the same extent (25% increase) as that seen following BNF treatment, in the Ah-responsive B6 strain. Neither xenobiotic affected this activity in the Ah-non-responsive D2 strain. 6. Although NBI is a major inducer, possessing Ah-like inducing properties in rat, it caused only minor changes in murine drug metabolizing enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in conjugative enzyme activity and acetaminophen metabolism in young and senescent male F-344 rats following prolonged exposure to buthionine sulfoximine.

This study examined how advanced age affects glucuronide and sulfate conjugation of acetaminophen after prolonged exposure to L-buthionine-S,R-sulfoximine (BSO) in male Fischer 344 rats. Young (4-5 month) and senescent (21-22 month) rats received 11 doses of BSO (2 mmol/kg) at 12-h intervals via a gastric cannula. Hepatic metabolism was assessed in vivo by measuring the products of reactions mainly responsible for acetaminophen elimination, namely the formation of the glucuronide and sulfate conjugates. Selected drug-metabolizing enzyme activities were also determined in vitro. BSO treatment increased the partial clearance to acetaminophen glucuronide by 90% and 41% in young and old rats, respectively, and similarly, induced p-nitrophenol and 1-naphthol UDP-glucuronosyl transferase activities to a greater extent in young versus senescent animals. Thus, the induction of these UDP-glucuronosyl transferase activities by BSO is preserved in senescent animals. Although the partial clearance to acetaminophen sulfate was decreased in senescent control rats compared to young controls, BSO treatment decreased the in vivo rate of sulfation in both age groups. Similar to previous findings with the Sprague-Dawley strain, BSO treatment did not induce hepatic cytochrome P-450 content or activity or cytosolic p-nitrophenol sulfotransferase activity in young and senescent Fischer 344 rats.

Drug metabolizing enzyme changes after chronic buthionine sulfoximine exposure modify acetaminophen disposition in rats.

This study examined the effects of prolonged exposure to buthionine sulfoximine (BSO) on 1) the overall elimination pharmacokinetics of acetaminophen; 2) the sulfate and glucuronide conjugation processes primarily responsible for acetaminophen elimination; and 3) in vitro microsomal and cytoplasmic enzyme activities in rats. Rats imbibed drinking water containing 30 mM BSO for 6 days and then received an iv injection of acetaminophen, 150 mg/kg in a propylene glycol vehicle. Exposure to BSO, a specific inhibitor of gamma-glutamylcysteine synthetase, produced marked depletion of glutathione (GSH) and resulted in induction of hepatic UDP-glucuronosyltransferase and GSH-S-transferase enzyme activities, but not cytochrome P-450. BSO pretreatment had no effect on the total or renal clearance of acetaminophen in rats. However, BSO exposure increased the partial clearance of acetaminophen to acetaminophen glucuronide by 47% (1.29 +/- 0.08 vs. 1.90 +/- 0.23 ml/min/kg; p less than 0.01) and significantly (p less than 0.02) increased the percentage of the dose recovered as the glucuronide conjugate from 17.6 +/- 2.5 to 26.5 +/- 0.6 The partial clearance of acetaminophen to acetaminophen sulfate was decreased, although not significantly, from 4.46 +/- 0.62 to 3.39 +/- 0.82 ml/min/kg. BSO treatment increased microsomal UDP-glucuronosyltransferase activity toward three xenobiotic aglycones, p-nitrophenol, 1-naphthol, and morphine by 308, 61, and 66%, respectively (p less than 0.05), but not toward testosterone or estrone. Cytosolic GSH-S-transferase activity toward 1-chloro-2,4-dinitrobenzene was increased 52% by BSO, whereas p-nitrophenol sulfotransferase activity was not altered. Cytochrome P-450 concentration and monooxygenase activity were unchanged by BSO exposure.

Induction of rat UDP-glucuronosyltransferase and glutathione S-transferase activities by L-buthionine-S,R-sulfoximine without induction of cytochrome P-450.

The effect of prolonged exposure to buthionine sulfoximine (BSO) on rat hepatic Phase I and Phase II drug-metabolizing enzymes has been examined. Exposure to 30 mM BSO in drinking water for 7 days induced hepatic microsomal UDP-glucuronosyltransferase activity (detergent-activated) toward p-nitrophenol (250%), 1-naphthol (210%), morphine (130%) and testosterone (140%), but not estrone. Glucuronosyltransferase activities were also induced after exposure for as short as 3 and as long as 13 days. When rats were returned to unsupplemented drinking water for 1 day prior to sacrifice following 6 days on 30 mM BSO, comparable induction to that seen after 7 consecutive days on the BSO solution was observed despite liver glutathione concentration having rebounded to 127% of control. Daily ingestion of BSO was similar (1 mmol/rat/day) for all periods of 30 mM BSO-drinking water exposure, with a body weight-adjusted dose range of 3.2-6.3 mmol/kg/day. An analogous inductive response caused by drinking 30 mM BSO for 3 days was elicited for p-nitrophenol and morphine glucuronidation by 6 mmol/kg doses of BSO given as single daily intraperitoneal or intragastric injections for 3 days. Intraperitoneal, intragastric and all BSO-drinking water exposures also significantly induced (130-195%) cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Significant increases in UDP-glucuronosyltransferase and glutathione S-transferase activities were also observed following 3 days of exposure to BSO in the drinking water at a concentration as low as 5 mM. Cytosolic p-nitrophenol sulfotransferase activity, with one minor exception, was not enhanced by any BSO treatment regimen. Alterations in transferase activities were not accompanied by any major changes in either overall cytochrome P-450 concentration or oxidative reactions selective for two isozymes. Thus, in addition to its well-documented glutathione-depleting property, BSO also selectively induces several Phase II drug-metabolizing enzymes, an effect to be considered in studies employing extended BSO treatment.

Metabolism of 6-nitrochrysene by intestinal microflora.

Since bacterial nitroreduction may play a critical role in the activation of nitropolycyclic aromatic hydrocarbons, we have used batch and semicontinuous culture systems to determine the ability of intestinal microflora to metabolize the carcinogen 6-nitrochrysene (6-NC). 6-NC was metabolized by the intestinal microflora present in the semicontinuous culture system to 6-aminochrysene (6-AC), N-formyl-6-aminochrysene (6-FAC), and 6-nitrosochrysene (6-NOC). These metabolites were isolated and identified by high-performance liquid chromatography, mass spectrometry, and UV-visible spectrophotometry and compared with authentic compounds. Almost all of the 6-NC was metabolized after 10 days. Nitroreduction of 6-NC to 6-AC was rapid; the 6-AC concentration reached a maximum at 48 h. The ratio of the formation of 6-AC to 6-FAC to 6-NOC at 48 h was 93.4:6.3:0.3. Interestingly, compared with results in the semicontinuous culture system, the only metabolite detected in the batch studies was 6-AC. The rate of nitroreduction differed among human, rat, and mouse intestinal microflora, with human intestinal microflora metabolizing 6-NC to the greatest extent. Since 6-AC has been shown to be carcinogenic in mice and since nitroso derivatives of other nitropolycyclic aromatic hydrocarbons are biologically active, our results suggest that the intestinal microflora has the enzymatic capacity to generate genotoxic compounds and may play an important role in the carcinogenicity of 6-NC.

Biotransformation of 1-nitropyrene to 1-aminopyrene and N-formyl-1-aminopyrene by the human intestinal microbiota.

The nitropolycyclic aromatic hydrocarbon 1-nitropyrene (1-NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1-NP by the human intestinal microbiota was studied using a semicontinuous culture system that simulates the colonic lumen. [3H]-1-Nitropyrene was metabolized by the intestinal microbiota to 1-aminopyrene (1-AP) and N-formyl-1-aminopyrene (FAP) as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. Twenty-four hours after the addition of [3H]-1-NP, the formylated compound and 1-AP accounted for 20 and 80% of the total metabolism, respectively. This percentage increased to 66% for FAP after 24 h following 10 d of chronic exposure to unlabeled 1-NP, suggesting metabolic adaptation to 1-NP by the microbiota. Both 1-AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1-NP.

Conversion of 5-fluorocytosine to 5-fluorouracil by human intestinal microflora.

The mechanism of toxicity from 5-fluorocytosine chemotherapy is unclear. However, recent evidence suggests that the generation of 5-fluorouracil by a host may play an important role in the development of this toxicity. Using an in vitro semicontinuous culture system to mimic the intestinal microflora, we examined the capacity of this complex microbial community to convert 5-fluorocytosine to 5-fluorouracil. The system was dosed initially and after 2 weeks of chronic exposure to 5-fluorocytosine with radiolabeled 5-fluorocytosine. No detectable production of 5-fluorouracil was observed up to 8 h after the acute dose; however, at 24 h and at all time points thereafter, increasing levels of 5-fluorouracil were detected for 4 days. The chronic dose resulted in an increased rate of 5-fluorouracil production without the 8-h lag time. These findings suggest that the enzyme or enzymes responsible for the deamination of 5-fluorocytosine to 5-fluorouracil by the intestinal microflora can be induced by chronic exposure to 5-fluorocytosine and that this conversion may provide a mechanism through which 5-fluorocytosine toxicity is manifested.

Metabolism of the benzidine-based azo dye Direct Black 38 by human intestinal microbiota.

Benzidine-based azo dyes are proven mutagens and have been linked to bladder cancer. Previous studies have indicated that their initial reduction is the result of the azo reductase activity of the intestinal microbiota. Metabolism of the benzidine-based dye Direct Black 38 was examined by using a semicontinuous culture system that simulates the lumen of the human large intestine. The system was inoculated with freshly voided feces, and an active flora was maintained as evidenced by volatile fatty acid and gas production. Within 7 days after exposure to the dye, the following metabolites were isolated and identified by gas chromatography-mass spectrometry:benzidine, 4-aminobiphenyl, monoacetylbenzidine, and acetylaminobiphenyl. Benzidine reached its peak level after 24 h, accounting for 39.1% of the added dye. Its level began to decline, and by day 7 the predominant metabolite was acetylaminobiphenyl, which accounted for 51.1% of the parent compound. Formation of the deaminated and N-acetylated analogs of benzidine, which have enhanced mutagenicity and lipophilicity, previously has not been attributed to the intestinal microbiota.