MICROSOMAL OXIDATION OF N,N-DIETHYLFORMAMIDE AND ITS EFFECT ON P450-DEPENDENT MONOOXYGENASES IN RAT-LIVER

N,N-Diethylformamide (DEF) is a hepatotoxic polar solvent in which met abolism has not been investigated. In this study we examined the follo wing: (a) the oxidative metabolism of DEF using both Liver microsomes from rats pretreated with selected P450 inducers and purified P450 enz yme (2B1, 2E1, 2C11); and (b) the effect of administration of DEF and its metabolite, the monoethylformamide (MEF), on induction andlor inhi bition of the P450 isoforms in rats. DEF was deethylated by microsomal P450-dependent oxidation forming acetaldehyde and MEF according to Mi chaelis-Menten kinetic parameters. Microsomes from rats pretreated wit h acetone and pyrazole (selective P4502E1 inducers) or rats pretreated with dexamethasone and 200 mg/kg DEF mere able to deethylate DEF in a biphasic manner, showing a low K-m component with a V-max of about 0. 2 nmol/(min . mg of protein) and a K-m between 70 mu M and 250 mu M. T he low K-m component was not present in control microsomes or in micro somes from rats treated with phenobarbital, beta-naphthoflavone, or cl ofibrate, where linear kinetics were observed. The use of purified P45 02E1 and 2C11 in a reconstituted system showed that 2E1, which oxidize d DEF with a V-max of 4.5 nmol/(min . nmol of P450) and a K-m of 0.7 m M, can partially account for the low K-m DEF deethylase, whereas 2C11, which oxidized DEF with a V-max of 4.8 nmol/(min . nmol of P450) and a K-m of 17 mM, might be the high K-m deethylase. The purified 2B1 was barely able to deethylate DEF. A confirmation of the role of 2E1 in D EF metabolism was obtained by using various selective inhibitors of P4 50 isoforms and immunoprecipitation experiments with anti P4502E1 IgG. The low K-m component of DEF deethylation in acetone-or pyrazole-indu ced microsomes was strongly inhibited (similar to 90%) by diethyldithi ocarbamate, 4-methyIpyrazole, and anti-2E1 IgG, but in 200 mg/kg DEF-i nduced microsomes the inhibition was partial, suggesting that other P4 50(s) may be involved. Administration of DEF 200 mg/kg ip for 4 days i nduced hepatic microsomal P4502E1-dependent aniline hydroxylase, P4502 B1/2-linked pentoxyresorufin O-depentylase, 16 beta-testosterone hydro xylase P4503A1/2-associated erythromycin N-demethylase, and 6 beta-tes tosterone hydroxylase. Alternatively, the same dose regimen of MEF ind uced only the aniline hydroxylase and depressed the 3A1/2-linked activ ities. Immunoblot experiments verified these data. These findings indi cate that DEF, at low concentrations, is predominantly oxidized by P45 02E1 and that this enzyme may be induced in rodents by repeated MEF or DEF treatment, thereby increasing their own metabolism and potentiall y their cytotoxicity through the formation of ethyl isocyanate.