John F. Brady

Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity.

The levels and activities of cytochrome P450 enzymes are influenced by a variety of factors, including the diet. In this article, the effects of selected non‐nutritive dietary chemicals, macronutrients, micronutrients, and ethanol on cytochromes P450 and xenobiotic metabolism are reviewed in the light of our current understanding of the multiplicity and substrate specificity of cytochrome P450 enzymes. Although the mechanisms of action of several dietary chemicals on specific cytochrome P450 isozymes have been established, those for macro‐ and micronutrients are largely unknown. It is known, however, that specific nutrients may have varied effects on different cytochrome P450 forms and thus may affect the metabolism of various drugs differently. Nutritional deficiencies generally cause lowered rates of xenobiotic metabolism. In certain cases, such as thiamin deficiency and mild riboflavin deficiency, however, enhanced rates of metabolism of xenobiotics were observed. The effects of dietary modulation of xenobiotic metabolism on chemical toxicity and carcinogenicity are discussed.—Yang, C. S.; Brady, J. F.; Hong, J.‐Y. Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity. FASEB J. 6: 737‐744; 1992.

Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in rats☆

Disulfiram, widely used in avoidance therapy for alcohol abuse, has been shown to have protective effects against chemically induced toxicity and carcinogenesis. The purpose of this work was to elucidate the biochemical mechanisms of this protective action by examining its effects on cytochrome P450IIE1 and other related microsomal enzyme activities. When a dose of disulfiram was given intragastrically to rats, a very rapid decrease of N-nitrosodimethylamine (NDMA) demethylase activity, possibly due to the inactivation of P450IIE1, was seen. The loss of P450IIE1 protein from the microsomal membrane was observed at 18 hr after receiving disulfiram, but not within the first 5 hr after the treatment. P450IIB1, on the other hand, was induced markedly between 15 and 72 hr after the disulfiram treatment. The treatment, however, caused only moderate changes in some other P450 isozymes. Carbon disulfide, a putative metabolite of disulfiram, produced similar effects on P450IIE1, but with shorter duration. Carbon disulfide, however, did not induce P450IIB1. Diethyldithiocarbamate, a reductive product of disulfiram, was an inhibitor of P450IIE1 activity in vitro, and upon preincubation with microsomes, it produced an NADPH-dependent inactivation of NDMA demethylase activity. The results suggest that this or other metabolites of disulfiram are inhibitors of P450IIE1 and are responsible for the inactivation of P450IIE1 in vivo. Hepatotoxicity of NDMA or CCI4 in rats was blocked by pretreatment with disulfiram. The present work demonstrates that P450IIE1 was inhibited and inactivated by disulfiram, and this mechanism can account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis.

Modulation of rat hepatic microsomal monooxygenase enzymes and cytotoxicity by diallyl sulfide

Diallyl sulfide (DAS) and other organosulfur compounds inhibit chemically induced carcinogenic and toxic responses in rodent model systems. A possible mechanism of action is the inhibition of the hepatic cytochrome P450IIE1-dependent bioactivation of the procarcinogens and protoxicants. Previous work showed competitive inhibition by DAS of N-nitrosodimethylamine (NDMA) demethylase activity in vitro, and a reduction in the microsomal level of P450IIE1 after in vivo treatment with DAS. The present studies demonstrated a time- and dose-dependent decrease of hepatic microsomal P450IIE1 activity, induction of P450IIB1 and pentoxyresorufin dealkylase activity, and moderate induction of ethoxyresorufin dealkylase activity by oral DAS treatment. DAS treatment elevated P450IIB1 mRNA but had no effect on P450IIE1 mRNA. Treatment with putative metabolites of DAS, diallyl sulfoxide and diallyl sulfone, led to similar modulations in monooxygenase activities, but the decrease of P450IIE1 activity by the sulfone occurred more rapidly. In studies in vitro, diallyl sulfone caused a metabolism-dependent inactivation of P450IIE1, but such inactivation was not observed with DAS or diallyl sulfoxide. The profile of microsomal testosterone metabolism after DAS treatment indicated an enhancement of P450IIB1-dependent 16 beta-hydroxylase activity, and a decrease in 6 beta-hydroxytestosterone production possibly related to a lower level of P450IIIA1 or IIIA2. When rats were subjected to a 48-hr fast and DAS treatment, the starvation-induced microsomal P450IIE1 level was decreased by DAS. Inhibition of hepatotoxicity due to exposure to P450IIE1 substrates, CCl4 and NDMA, by DAS was observed under a variety of treatment schedules.

Effect of phenethyl isothiocyanate on microsomal N-nitrosodimethylamine metabolism and other monooxygenase activities

1. Phenethyl isothiocyanate (PEITC), a dietary compound derived from cruciferous vegetables, has previously been shown to decrease N-nitrosodimethylamine (NDMA)-induced methylation of hepatic DNA, apparently by inhibition of microsomal activation of the procarcinogen. 2. Using hepatic microsomes from acetone-treated rats, PEITC exhibited competitive inhibition of NDMA demethylase activity with an apparent Ki of 1 microM. In studies using a two-stage incubation protocol, the inhibition by PEITC was time- and metabolism-dependent. 3. Using control rat liver microsomes, PEITC selectively inhibited P450 IIE1-mediated NDMA-demethylase activity as compared to the demethylation of benzphetamine and ethylmorphine. 4. Pretreatment of rats with a single oral dose of PEITC (1 mmol/kg body wt) 24 h before killing caused a marked decrease in hepatic NDMA demethylase activity, but an 11-fold increase in 7-pentoxyresorufin O-dealkylase activity. These trends agreed with immunoblot analysis which indicated that PEITC was a suppressor of P450 IIE1 but an inducer of P450 IIB1. 5. The selective inhibition of P450 IIE1 activity and suppression of its level in microsomes indicates a role for PEITC as a chemopreventive agent against toxic or carcinogenic metabolites of this isozyme.

Metabolism of methyltertiary-butyl ether by rat hepatic microsomes

Exposure to methyltertiary-butyl ether (MTBE), a commonly used octane booster in gasoline, has previously been shown to alter various muscle, kidney, and liver metabolic activities. In the present study, the metabolism of MTBE by liver microsomes from acetoneor phenobarbital-treated Sprague-Dawley rats was studied at concentrations of up to 5 mM MTBE. Equimolar amounts oftertiary-butanol, as measured by head-space gas chromatography, and formaldehyde were formed. TheVmax for the demethylation increased by 4-fold and 5.5-fold after acetone and phenobarbital treatments, respectively. The apparentKm value of 0.70 mM using control microsomes was decreased slightly after acetone treatment, but was increased by 2-fold after phenobarbital treatment. The metabolism of MTBE (1 mM) was inhibited by 35% by monoclonal antibodies against P450IIE1, the acetone/ethanol inducible form of cytochrome P450, suggesting a partial contribution by this isozyme. A single 18-h pretreatment of rats with 1 or 5 ml/kg MTBE (i. p.) resulted in a 50-fold induction of liver microsomal pentoxyresorufin dealkylase activity but no change inN-nitrosodimethylamine demethylase activity. These trends in activity agreed with immunoblot analysis which showed an elevation in P450IIB1 but no change in P450IIE1 levels.

Induction of cytochromes P450IIE1 and P450IIB1 by secondary ketones and the role of P450IIE1 in chloroform metabolism.

It has been shown previously that the potentiation of chloroform-induced hepatotoxicity by linear secondary ketones increases with the carbon-chain length. The present work examines the possibility that this potentiation is due to the induction of P450IIE1. The metabolism of chloroform, as measured using headspace gas chromatography, in the presence of microsomes from acetone-treated rats was elevated threefold compared to controls. Inclusion of monoclonal antibody against P450IIE1 inhibited the metabolism by 81%. Alternate substrates of P450IIE1 were also inhibitory. Chloroform metabolism was observed using purified, reconstituted P450IIE1 plus cytochrome b5, but was not detected using P450IIB1. The inductive effect of 18-hr oral pretreatment (15 mmol/kg body wt) with each of three secondary ketones on two isozymes of rat liver microsomal cytochrome P450, P450IIE1, and P450IIB1 was studied. The content of total microsomal P450 and NADPH-dependent cytochrome c reductase, the rates of oxidation of N-nitrosodimethylamine, benzphetamine, and pentoxyresorufin, as well as levels of immunoreactive protein for both of the isozymes were elevated by the pretreatments in the rank order of acetone less than or equal to 2-butanone less than 2-hexanone, in agreement with other trends noted by previous investigators. The results provide further evidence for the role of P450IIE1 induction in the potentiation phenomenon.

Alteration of rat liver microsomal monooxygenase activities by gasoline treatment.

Previous work has shown an increase in rat liver enzyme activities after chronic exposure to gasoline vapor. In the present study, male Sprague-Dawley rats were pretreated with unleaded gasoline at 1 and 5 ml/kg, i.p., and selected hepatic microsomal monooxygenase activities were determined at 18, 48, and 72 h. At 18 h, moderate increases were observed in P450 content (1.3-fold), cytochromec-reductase activity (1.25-fold), and inN-nitrosodimethylamine demethylation rate (1.25- to 1.6-fold). Pentoxyresorufin dealkylase activity (an activity displayed primarily by P450IIB1) was significantly elevated at 18 and 48 h (30- to 60-fold), and ethoxyresorufin dealkylase activity (an activity displayed by P450 IA1) was elevated (2- to 4-fold). Immunoblot analysis revealed no change in P450IIE1 at these time points, but an elevation in P450IIB1 in agreement with the pentoxyresorufin dealkylase activity measurements.

A BASIC program for the estimation of Michaelis-Menten parameters by the direct linear plot

The use of manual graphical methods for the estimation of Michaelis-Menten kinetic parameters as recommended by Eisenthal and Cornish-Bowden (Biochem. J. 139 (1974) 715-720) was found to be impractical for large (n greater than 5) sample numbers. A BASIC program providing the rank ordered coordinates of intersections, which correspond to estimates of Km and Vmax in the direct linear plot method, is described.

Effect of 1,3-butanediol on rat liver microsomal NDMA demethylation and other monooxygenase activities.

The administration of 1,3-butanediol (BD) previously has been shown to elevate blood concentrations of ketone bodies, to potentiate carbon tetrachloride hepatotoxicity, and to increase the hepatic microsomal content of cytochrome P450 and the activity of aniline hydroxylase. In the present study, oral treatment (10 g/kg) with racemic BD and each of its enantiomers (R-BD and S-BD) induced NDMA demethylase activity by approx. 1.5-fold in rat hepatic microsomes obtained 12 h later, suggesting an induction of P450IIE1, the acetone/ethanol-inducible form of P450. The results agreed with an immunochemically determined increase in the levels of this isozyme. No change in P450 content, NADPH-cytochrome-c reductase, or in pentoxyresorufin dealkylase activity were detected. Blood levels of acetone were determined during a 10-h period after BD administration and showed a higher initial rate of increase by R-BD, possibly due to steroselective metabolic oxidative metabolism. However, no difference in the induction of NDMA demethylase activity by the enantiomers could be detected. Induction of P450IIE1 probably contributes to the previously described potentiation of haloalkane-induced hepatotoxicity by BD administration.