The role of metabolism and covalent binding in the cytotoxicity of a nitrogen-containing steroid toward rat hepatocytes.

The nitrogen-containing steroid N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (I) causes concentration- and time-dependent cytotoxicity toward freshly isolated F-344 rat hepatocytes. Because hepatocytes extensively metabolize I to both stable and reactive products, the role of metabolism and covalent binding in the cytotoxicity of I was investigated. Concentration-dependent covalent binding of I-related material to hepatocyte macromolecules was detected. Treatment of rats with phenobarbital increased the rate and extent of hepatocyte metabolism of I, increased the covalent binding of I-related material to hepatocyte macromolecules, but decreased the cytotoxicity of I. Addition of testosterone to incubations of hepatocytes and I inhibited the metabolism of I, decreased the covalent binding of I-related material to hepatocyte macromolecules, but potentiated the cytotoxicity of I. These results indicate that the covalent binding of reactive metabolites is not involved in the cytotoxicity of I. Moreover, the two major metabolites of I, the 4-carbinolamide and monoethyl analog, were much less cytotoxic toward hepatocytes than I. These data suggest that the metabolism of I represents detoxication and that the parent compound is the cytotoxicant.

Metabolism of a nitrogen-containing steroid by rat hepatocytes and hepatic subcellular fractions.

Freshly isolated rat hepatocytes metabolized the nitrogen-containing steroid N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- carboxamide (I) to six products analyzed by reverse phase HPLC. The major metabolite, which could account for greater than 50% of the total I-related material, exhibited chromatographic, NMR, and mass spectral properties identical to those of the authentic 4-carbinolamide of I. Thus, the major biotransformation pathway in hepatocytes was hydroxylation of the N-methyl group of I to form a stable carbinolamide intermediate of N-demethylation. Desmethyl-I was observed as a minor metabolite. Another metabolite which accounted for approximately 10% of the total I-related material had chromatographic and spectral properties identical to those of the authentic monoethyl analog of I. The other three metabolites were formed in variable quantities and were unstable when isolated. Mass spectral data suggested that one metabolite was the carbinolamide intermediate of N-deethylation. Treatment of rats with phenobarbital or dexamethasone increased the formation of the monoethyl metabolite of I in hepatocytes but had no effect upon the formation of the 4-carbinolamide metabolite. Rat hepatic microsomes catalyzed the NADPH-dependent metabolism of I to the same metabolites in the same relative amounts as observed with intact hepatocytes. Studies with alternative substrates and inhibitors demonstrated that microsomal cytochrome P-450 was responsible for the metabolism of I. Dog and human hepatic microsomes metabolized I to the same products as rat hepatic microsomes, but monoethyl I was the major metabolite.

Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation.

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.

Metabolism of lovastatin by rat and human liver microsomes in vitro.

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.

Studies with nitrogen-containing steroids and freshly isolated rat hepatocytes: role of cytochrome P-450 in detoxication.

Several nitrogen-containing steroids produced concentration- and time-dependent decreases in the viability of freshly isolated F-344 rat hepatocytes. N,N-Diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (I) was not cytotoxic at or below 0.3 mM but produced decreases in cell viability at higher concentrations. In contrast, the desmethyl analog of I was essentially nontoxic, demonstrating that relatively small structural changes result in substantial differences in cytotoxicity. Testosterone and other steroids specifically potentiated the cytotoxicity of I in a concentration-dependent manner, while having no effect upon the toxicity of other chemical agents. Pargyline and methimazole had no effect upon the cytotoxicity of I, suggesting that monoamine oxidase and flavin-containing monooxygenase are not involved. The cytochrome P-450 inhibitors octylamine and metyrapone potentiated the cytotoxicity of I. Induction of cytochrome P-450 isozymes by phenobarbital and beta-naphthoflavone treatment protected the cells against the cytotoxicity of I, while acetone or dexamethasone treatment had no effect. The initial rates of hepatocyte metabolism of the six nitrogen-containing steroids investigated did not correlate with cytotoxicity. Dithiothreitol and other thiol compounds had no effect upon the cytotoxicity of I, suggesting that sulfhydryl oxidation is not involved. Galactosamine and sulfate-free media had no effect upon the cytotoxicity of I. These results suggest that cytochrome P-450 is involved in the detoxication of I by rat hepatocytes while conjugative metabolism does not play a significant role.

Mechanism of reductive activation of a 5-nitroimidazole by flavoproteins: model studies with dithionite.

The flavoprotein nitroreductases NADPH:cytochrome P-450 reductase and xanthine oxidase catalyzed the cofactor-dependent anaerobic nitro group reduction and covalent binding to protein sulfhydryl groups of the 5-nitroimidazole substrate ronidazole [1-methyl-5-nitroimidazole-2-yl)-methyl carbamate). Studies with variously radiolabeled ronidazole molecules demonstrated that the imidazole ring was intact while greater than 80% of the C-4 3H and 2-carbamoyl group were lost from the covalently bound product. The stoichiometry of cofactor consumption during the enzyme-catalyzed reduction of the substrate could not be determined, so a model nitroreductase system which utilized dithionite as the reductant and agarose-immobilized cysteine as the target for alkylation was developed. Two moles of dithionite was consumed per mole of substrate for maximal reduction of uv absorbance due to the nitro group, for maximal release of C-4 3H, and for maximal covalent binding to agarose-immobilized cysteine. These results indicate that four electrons are required for the reductive activation of the substrate, consistent with formation of a hydroxylamine reactive intermediate. Covalent binding of variously radiolabeled substrate molecules after dithionite reduction exhibited the same labeling pattern as flavoprotein-catalyzed covalent binding, suggesting that covalent binding is mediated by the same species in both chemical and biological systems. The data are consistent with a mechanism where the substrate undergoes four-electron reduction to form a hydroxylamine, which is susceptible to nucleophilic attack at C-4. When water attacks C-4, the 2-carbamoyl group can eliminate to form a Michael-like acceptor which adds thiols at the 2-methylene position.