Effects of the chrysotherapeutic agents auranofin and gold sodium thiomalate on hepatic and renal drug metabolism and heme metabolism.

These studies were designed to investigate the effects of the chrysotherapeutic agents auranofin and myochrysine (GST) on hepatic and renal drug-metabolizing enzymes and heme metabolism. Male Sprague-Dawley rats were either administered a single dose of auranofin (17, 34, or 68 mg/kg, p.o.) or administered daily doses of auranofin (0.2, 0.6, 2, 9, or 40 mg/kg/day, p.o.) or GST (1.2 or 5.8 mg/kg/day, i.p.) for 3 or 14 days. Rats were killed 24 h after the final treatment, and subcellular fractions of liver and kidney were prepared. Cytochrome P-450 (P-450) content and ethoxycoumarin-O-deethylase (ECOD), benzphetamine-N-demethylase (BPND), delta-aminolevulinic acid (ALA) synthetase, and heme oxygenase activities were determined. Twenty-four hours following single doses of auranofin, no effects on hepatic P-450, ECOD, or BPND were observed. Treatment with the positive control compounds, CoCl2 (60 mg/kg) and Co-protophorphyrin IX (33 mg/kg), produced decreases in all three variables at 24 hr. Auranofin, at 2 mg/kg, and GST treatment, at both doses, reduced hepatic P-450 and ECOD activity at 3 days. This effect was reversed with continued treatment for 14 days. BPND activity was unaffected at 3 days but was decreased at 14 days. Heme oxygenase activity was enhanced at 3 days and had returned to control activity at 14 days, while ALA synthetase was unaffected. With the exception of heme oxygenase, which was increased, renal variables were unaltered at 3 days. At 14 days, renal P-450 content was decreased in the high-dose auranofin group, heme oxygenase activity was increased in all groups, and ALA synthetase activity was elevated in high-dose auranofin animals. These data indicate that, at doses twenty times the human dose, auranofin and GST administration produced reversible decreases in hepatic and renal P-450 which may be the result of altered heme metabolism.

Characteristics of cytochrome P-450 and mixed function oxidase enzymes following treatment with PBBs.

The mixture of PBBs in FireMaster BP-6 has been demonstrated to constitute potent inducers of hepatic and extrahepatic mixed function oxidase (MFO) enzymes. Chronic dietary administration of PBBs to mature female rats results in a "mixed" pattern of induction, with increases in both cytochrome P-450 and P1-450 associated enzymes. Acute administration of PBBs (150 mg/kg IP) to mature female rats resulted in a time-dependent induction of MFO activities; the P-450-dependent enzymes were simulated early (24-48 hr after administration) while the P1-450 dependent enzymes reached maximal activities at later time points. However, studies of the kinetics and patterns of inhibition of the induced enzymes along with gel electrophoresis studies of the microsomal proteins indicate that PBBs may induce different proteins from those induced by the classical P-450 and P1-450 inducers, phenobarbital and 3-methylcholanthrene. In addition, the pattern of enzyme induction caused by PBBs in developing rats differs from that in adults, in that the P1-450-associated enzymes are stimulated prior to the P-450-associated enzymes. The overall pattern of enzyme induction in extrahepatic tissue differs from that seen in the liver and sex differences in enzyme induction have also been demonstrated. As modifications of MFO activity may alter the toxicity of chemicals, these findings suggest that the toxicity of chemicals may be altered in animals exposed to PBBs and that these toxicities may exhibit age, sex, and organ specificities different from those seen in control animals.

Distribution of polybrominated biphenyls after dietary exposure in pregnant and lactating rats and their offspring.

Female rats were fed PBBs in the diet (50 ppm) from day 8 of gestation to day 21 of gestation, from day 1 postpartum to day 14 postpartum or from day 8 of gestation through day 14 postpartum. Levels of PBBs were measured in various tissues. Small concentrations of PBBs (less than 5 microgram/g) were found in the brain, heart, lung, liver, small intestine, placenta, and gravid uterus. Larger concentrations (less than 30 microgram/g) were found in kidneys, the nongravid uterus, skin, mammary tissue, and fat. Lactation did not significantly alter the concentrations of PBBs found in tissues other than mammary tissue. Offspring were subjected to several exposure regimens by cross-fostering. Concentrations of PBBs in the neonatal livers were higher than in the adults nursing them. Transfer of PBBs via the milk appears to be much more important to appearance of PBBs in newborns than does placental transfer.

A new method for measuring covalent binding of chemicals to cellular macromolecules.

A new method has been developed for measuring the total covalent binding of metabolically activated compounds to cellular macromolecules. This method employs equilibrium dialysis, in the presence of the detergent sodium dodecyl sulfate (SDS), to remove unbound radiolabeled compound and its metabolites from cellular macromolecules. [14C] Bromobenzene (80 microM), [14C]aflatoxin B1 (5 microM) or 3-[14C]methylcholanthrene (100 microM) was incubated (37 degrees C) with primary hepatocytes or liver microsomes isolated from Fischer-344 rats. The covalent binding of 14C-radiolabel to hepatic or microsomal macromolecules was measured by SDS-equilibrium dialysis and compared with that measured by exhaustive extraction. After 1 h of incubation with hepatocytes or microsomes, 2--7 times more covalent binding was detected by SDS-equilibrium dialysis, than by exhaustive extraction. The radioactivity associated with these hepatic or microsomal macromolecules migrated to discrete positions on SDS-polyacrylamide disc gels. The non-dialysable radioactivity from incubations with [14C] bromobenzene could not be extracted with diethyl ether even after treatment of the dialysin with beta-glucuronidase-sulfatase or dilute acid. This was taken to indicate that the radioactivity in the dialysin did not include free bromobenzene or its metabolites, a conclusion supported by thin-layer chromatography analysis of the dialysin. The lower amount of covalent binding detected by exhaustive extraction may be related to the inability of trichloroacetic acid to quantitatively precipitate small molecular weight macromolecules. SDS-equilibrium dialysis is an easy, rapid and non-destructive technique for measuring covalent binding. The macromolecular integrity of the sample is maintained and allows further studies concerning the specificity of the covalent interactions.

Biliary excretion and enterohepatic circulation of 2,4-dinitrotoluene metabolites in Fischer-344 rats.

Technical grade dinitrotoluene (DNT) is hepatocarcinogenic when fed to rats. DNT is oxidatively metabolized by hepatic enzymes and reductively metabolized by rat intestinal microflora in vitro. The objectives of the present studies were to determine the importance of bile as a route of excretion for DNT metabolites and to investigate the role of enterohepatic circulation in the metabolism of DNT. The common bile ducts of male and female F-344 rats were cannulated with an uninterrupted cannula at the hepatic and ileal ends. After 24 hr, male rats were given a po dose of 35, 63, or 100 mg 2,4-[14C]DNT/kg; female rats received 35 mg 2,4-[14C]DNT/kg. Immediately prior to dosing, the cannula was snipped and bile was allowed to collect in a glass reservoir, surgically implanted in the peritoneal cavity, which could be sampled externally. In males, excretion of 14C in bile was linearly related to dose. From 9.2 to 29.2 mumol eq of [14C]DNT (approximately 25% of the dose) appeared in bile within 24 hr. Females dosed with 35 mg/kg excreted only 18% of the dose in the bile. Over 90% of the radioactivity in the bile was the glucuronide conjugate of 2,4-dinitrobenzyl alcohol (DNBAlc-G). In comparison to control rats, in which bile flow to the small intestine was uninterrupted, collection of bile decreased the amount of 14C excreted in urine. In both males and females most of the 2,4-DNT dose excreted in the urine was in the form of the oxidized metabolites DNBAlc-G and 2,4-dinitrobenzoic acid. These results indicate that bile is an important route of excretion for 2,4-DNT metabolites and that metabolites excreted in the bile can be reabsorbed from the gut.

Elevation of hepatic microsomal epoxide hydrolase activity by 2-acetylaminofluorene: role of metabolism.

Brief exposure of rats to hepatocarcinogenic agents causes a rapid elevation in hepatic microsomal epoxide hydrolase (EH) activity. Previous studies have demonstrated that animals which are resistant to the hepatocarcinogenic effects of 2-acetylaminofluorene (AAF) are also resistant to EH induction by this compound. The studies described here were designed to examine the role of several individual metabolic pathways on the induction of EH by AAF. EH was increased 4-fold in male Fischer 344 (F-344) or Sprague-Dawley (SD) rats treated with AAF; in female rats, deficient in N-hydroxylase and sulfotransferase activities, the activity was increased only 2-fold. Pretreatment of male F-344 rats with inducers of cytochrome P-448 activity caused a reduction in the EH response to AAF, probably due to a greater increase in ring-hydroxylation than N-hydroxylation of the AAF. Although AAF elicited only a small EH elevation in female F-344 rats, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) caused a large increase in these animals. The N-OH-AAF-induced increase was partially blocked by pretreatment with pentachlorophenol, an inhibitor of sulfotransferase activity, in both male and female F-344s. In female SD rats, possessing minimal sulfotransferase activity, N-OH-AAF treatment caused only a slight elevation of EH activity. Pretreatment of male F-344 rats with inhibitors of deacetylase activity had no effect on N-OH-AAF-dependent EH induction. These observations are consistent with the suggestion that formation of the sulfate conjugate of N-OH-AAF is necessary for elevation of EH by this compound.

Effect of hepatocarcinogens on epoxide hydrolase and other xenobiotic metabolizing enzymes.

Hepatocarcinogens cause marked biochemical changes in the liver at short intervals after administration. The studies described were designed to investigate the effects of hepatocarcinogens and hepatotoxicants on the microsomal mixed function oxidase system. DT-diaphorase and epoxide hydrolase. Following 5 day p.o. treatment of male F-344 rats with aflatoxin B1 (AFB), 2-acetylaminofluorene (AAF), technical grade dinitrotoluene (DNT), or 2,4-diaminotoluene, microsomal cytochrome P450 dependent enzyme activities were depressed while epoxide hydrolase activity was markedly elevated (3-8 times control). Diethylnitrosamine (DEN) given at 5 mg/kg/day and DL-ethionine at 1000 mg/kg/day failed to increase epoxide hydrolase. 3-Methylcholanthrene, methylnitrosourea, carbon tetrachloride, bromobenzene and vinyl chloride all failed to increase epoxide hydrolase activity. Using 3 daily i.p. injections, dose-response relationships for increases in epoxide hydrolase were generated for the hepatocarcinogens. With the exception of p-dimethylaminoazobenzene (DAB) and DEN, the carcinogens studied produced log-linear dose response curves for increase in epoxide hydrolase. Both DEN and DAB caused increases in epoxide hydrolase but classical sigmoidal dose-response curves were not obtained. The order of potency for increasing epoxide hydrolase was AFB greater than AAF greater than 2,6-dinitrotoluene greater than 3'-methyl-N,N-dimethyl-4-aminoazobenzene greater than DNT greater than 2, 4-dinitrotoluene. The slopes of the linear portions of the log dose-response curves were not statistically different from the slope of the dose-response curve obtained with AAF suggesting that structurally diverse carcinogens elicit increases in epoxide hydrolase by a common mechanism.

Errors in oxygen tension measurements caused by halothane.

Halothane, independent of oxygen tension, increased the signal of the gold/silver-silver chloride microelectrode. The output of microelectrodes at different oxygen tensions in the presence of increasing amounts of halothane has been measured. Halothane causes an increase in the electrode signal which is proportional to its concentration. This effect results from the polarographic reduction of halothane. It is concluded that the gold/silver-silver chloride microelectrode cannot be used to measure oxygen tension during halothane anaesthesia.

Elevation of hepatic microsomal epoxide hydrolase activity by 2-acetylaminofluorene: strain and species differences.

Hepatocarcinogens have been shown to cause marked elevation of hepatic microsomal epoxide hydrolyase activity in the rat at short intervals after administration. The present studies were designed to characterize 2-acetylaminofluorene (AAF) mediated epoxide hydrolase elevation and to investigate the relationship between epoxide hydrolase increases, AAF metabolism, and hepatocarcinogenicity. Oral or i.p. administration of AAF to F-344 rats produced log-linear dose-response curves for epoxide hydrolase elevation, measured with either benzo[a]pyrene-4,5-oxide or styrene oxide substrate. Following a single dose of AAF (35 mg/kg), epoxide hydrolase activity was maximally increased (560% of control) within 48 h, and the activity declined slowly, with a half-life of 17.5 days. Co-treatment with actinomycin D effectively blocked the AAF dependent increase in epoxide hydrolase, suggesting that de novo protein synthesis is associated with the increase in enzyme activity. Dose-response curves for epoxide hydrolase induction by AAF, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and 2-aminofluorene were compared, and the potencies for increasing epoxide hydrolase activity reflected the relative hepatocarcinogenic potentials of these agents. In mice, which are resistant to the hepatocarcinogenic action of AAF and deficient in AAF-N-hydroxylase activity, AAF caused no significant increase in hepatic microsomal epoxide hydrolase activity. Similarly, in Cotton rats and guinea pigs, which are lacking in ability to form the sulfate conjugate of N-OH-AAF, neither i.p. nor dietary administration of AAF elicited increases in epoxide hydrolase activity at doses which were maximally effective in F-344 rats. These results support the hypothesis that the ability of compounds to increase epoxide hydrolase activity is related to their carcinogenic potency. Furthermore, the results suggest that increases in epoxide hydrolase activity are associated with metabolism of AAF to the putative proximate carcinogen N-OH-AAF, and the subsequent conversion of this compound to the N-O-sulfate conjugate.

Effects of H2 receptor antagonists on the hepatotoxicity of various chemicals.

H2 receptor antagonist-hepatotoxicant interactions were evaluated in male Fischer-344 rats. The H2 receptor antagonists, cimetidine, ranitidine, oxmetidine, and 2-[2-(2-dimethyl-aminomethyl-5-furanylmethyl-thio)-ethylamino]-5-( 6-methyl- 3-picolyl)-4-pyrimidine trihydrohydrochloride (SK&F 93479) were administered (p.o.) at a dose of 0.143 mMoles/kg 30 minutes prior to hepatotoxicant treatment. Submaximal hepatotoxic doses (p.o.) of carbon tetrachloride (795 mg/kg), bromobenzene (748 mg/kg), chloroform (1,190 mg/kg), allyl alcohol (60 mg/kg), galactosamine (200 mg/kg, i.p.), and acetaminophen (1000 mg/kg) were employed. Hepatotoxicity was evaluated by determining serum alanine aminotransferase activity (ALT). Pretreatment with the H2 receptor antagonists did not significantly alter carbon tetrachloride or allyl alcohol hepatotoxicity. Bromobenzene and chloroform toxicities were unaffected by cimetidine, ranitidine, and oxmetidine pretreatment but were potentiated by SK&F 93479. Cimetidine and ranitidine decreased galactosamine mediated hepatotoxicity. Acetaminophen hepatotoxicity was markedly potentiated by ranitidine pretreatment but was unaltered by the other three H2 receptor antagonists. The mechanisms of hepatotoxicity potentiation or protection have not been determined, however, the lack of consistent H2 receptor antagonists effects indicates that it is unlikely that alterations in G.I. pH account for the effects observed. H2 receptor antagonist mediated changes in hepatotoxicant metabolism provide a more plausible mechanism of action, particularly in the cases of SK&F 93479 potentiation of bromobenzene and chloroform and ranitidine potentiation of acetaminophen hepatotoxicity.

Macromolecular weight specificity in covalent binding of bromobenzene.

Bromobenzene is a hepatotoxicant that causes centrilobular necrosis. Pretreatment of animals with 3-methylcholanthrene decreases and phenobarbital pretreatment enhances the hepatotoxic action of this compound. We have investigated the macromolecular weight specificity of the covalent interactions of bromobenzene with liver macromolecules following incubation of [14C]bromobenzene in isolated hepatocytes. Hepatocytes were prepared from Fischer-344 rats treated for 3 days with 3-methylcholanthrene, phenobarbital, or normal saline. After a 1-hr incubation, total covalent binding, as measured by sodium dodecyl sulfate-equilibrium dialysis, was twofold less in hepatocytes from 3-methylcholanthrene-treated rats and sixfold greater in hepatocytes from phenobarbital-treated rats, as compared to hepatocytes from control animals. Analysis of the arylated macromolecules by electrophoresis on 15% sodium dodecyl sulfate-polyacrylamide disc gels indicated that in the first 1 to 3 min of incubation substantial amounts of covalently bound radiolabel were associated with macromolecules of between 20,000 and 40,000. The amount of radioactivity associated with these macromolecules rapidly diminished in hepatocytes from control and 3-methylcholanthrene-treated animals. In hepatocytes from phenobarbital-treated animals, the amount of radioactivity associated with macromolecules, 20,000, increased throughout the incubation. The amount of radiolabel associated with macromolecules, 20,000, increased in all incubations. When nontoxic doses of phenylmethylsulfonyl fluoride, a specific inhibitor of serine proteases, were added to control hepatocytes incubated with [14C]-bromobenzene, the decrease in radioactivity associated with larger (greater than 20,000) macromolecules was inhibited and a corresponding lack of increase in radioactivity associated with smaller macromolecules was observed. In hepatocytes from phenobarbital-treated rats, either the rate of adduct formation with higher molecular weight macromolecules greatly exceeded the rate of their breakdown or the phenobarbital treatment compromised the degradation process. The toxicity induced by bromobenzene may result from the covalently bound material altering the biological function of macromolecules. The result of this study suggest that cellular degradation of the arylated macromolecules may be one mechanism of detoxification. Persistence of the arylated macromolecules within the cell may be associated with the toxic action of bromobenzene.

Ranitidine-acetaminophen interaction: effects on acetaminophen-induced hepatotoxicity in Fischer 344 rats.

Cimetidine has been shown to protect against acetaminophen-mediated hepatotoxicity in both rats and mice. In contrast to cimetidine, ranitidine recently has been determined to potentiate the hepatotoxic action of acetaminophen in Fischer 344 rats. The present studies were designed to characterize this ranitidine-acetaminophen interaction. Acetaminophen administration (750 mg per kg, p.o.) to F344 rats produced maximal hepatic necrosis, 24 hr after treatment, as assessed by SGPT activity and histopathology. Ranitidine pretreatment 30 min prior to acetaminophen treatment increased the toxicity but did not alter its course. Ranitidine administration (50 mg per kg) enhanced acetaminophen hepatotoxicity throughout the toxic dose range of acetaminophen (600 to 1,000 mg per kg) and potentiation of acetaminophen hepatotoxicity by ranitidine was dose-dependent. Maximal increases were observed at 50 mg per kg ranitidine whereas, doses of ranitidine greater than 100 mg per kg inhibited acetaminophen toxicity. SGPT data were corroborated by histopathologic evaluation. Ranitidine was not hepatotoxic when administered alone (500 mg per kg), or following glutathione depletion, or after induction of hepatic mixed-function oxidase activity. The results obtained in these studies support the suggestion that, at high doses (greater than 100 mg per kg), ranitidine reduces acetaminophen hepatotoxicity by reducing metabolic activation, while at lower doses ranitidine potentiates acetaminophen hepatotoxicity. Inhibition by ranitidine of acetaminophen conjugation is proposed as a possible mechanism of this potentiation.

Effects of chronic administration of polybrominated biphenyls on parameters associated with hepatic drug metabolism.

Parameters relating to hepatic microsomal drug metabolism were measured in vitro following 2 weeks exposure of rats to polybrominated biphenyls (PBB) as a dietary supplement at 4.69, 18.75, 75 and 300 ppm. The mixture of PBB was a potent inducer of hepatic microsomal drug metabolism. The pattern of induction seen after PBB exposure was compared with that produced by phenobarbital and 3-methyl-cholanthrene and the pattern of induction observed shared similarities with both of these agents.

Chronic toxicity and oncogenicity bioassay of inhaled ethylene in Fischer-344 rats.

The toxicity and oncogenicity of inhaled ethylene was determined in Fischer-344 rats. Nine hundred and sixty animals were randomly divided into four groups of one hundred twenty animals of each sex and were exposed 6 hr/day, 5 days/week, for up to 24 months to concentrations of ethylene in air of 0, 300, 1000, or 3000 ppm. The maximum tolerated dose was not used as concentrations above 3000 ppm were considered hazardous because of the risks associated with ethylene's explosive properties. The calculated time-weighted average concentrations for the 24 months of exposure were 0.0, 301, 1003, and 3003 ppm, respectively. Randomly selected animals were necropsied and examined after 6, 12, and 18 months of exposure. All surviving rats were necropsied at 24 months. A complete selection of tissues and organs from all animals in the control and 3000-ppm groups were examined for microscopic lesions. All animals were examined for clinical changes throughout the course of the study and selected animals were used to determine ophthalmologic or hematologic effects and for clinical blood chemistry or urinalysis effects. There were 151 unscheduled deaths (15.7% of 960 animals). There was no difference in mortality between groups during the 2-year study. Gross examination of rats dying during the study, or of those that were sacrificed as scheduled, did not reveal any lesions attributable to ethylene exposure. Histologically, a variety of proliferative, degenerative, and inflammatory lesions were observed in both the control and 3000-ppm groups. These lesions were typical of those seen in this strain of animal and were considered unrelated to ethylene exposure.(ABSTRACT TRUNCATED AT 250 WORDS)