Microsomal Oxidation of N,N-Diethylformamide and Its Effect on P450-Dependent Monooxygenases in Rat Liver

Abstract

N,N-Diethylformamide (DEF) is a hepatotoxic polar solvent in which metabolism has not been investigated. In this study we examined the following: (a) the oxidative metabolism of DEF using both liver microsomes from rats pretreated with selected P450 inducers and purified P450 enzyme (2B1, 2E1, 2C11); and (b) the effect of administration of DEF and its metabolite, the monoethylformamide (MEF), on induction and/or inhibition of the P450 isoforms in rats. DEF was deethylated by microsomal P450-dependent oxidation forming acetaldehyde and MEF according to Michaelis-Menten kinetic parameters. Microsomes from rats pretreated with acetone and pyrazole (selective P4502E1 inducers) or rats pretreated with dexamethasone and 200 mg/kg DEF were able to deethylate DEF in a biphasic manner, showing a low Km component with a Vmax of about 0.2 nmol/(min.mg of protein) and a Km between 70 microM and 250 microM. The low Km component was not present in control microsomes or in microsomes from rats treated with phenobarbital, beta-naphthoflavone, or clofibrate, where linear Kinetics were observed. The use of purified P4502E1 and 2C11 in a reconstituted system showed that 2E1, which oxidized DEF with a Vmax of 4.5 nmol/(min.nmol of P450) and a Km of 0.7 mM, can partially account for the low Km DEF deethylase, whereas 2C11, which oxidized DEF with a Vmax of 4.8 nmol/(min.nmol of P450) and a Km of 17 mM, might be the high Km deethylase. The purified 2B1 was barely able to deethylate DEF. A confirmation of the role of 2E1 in DEF metabolism was obtained by using various selective inhibitors of P450 isoforms and immunoprecipitation experiments with anti P4502E1 IgG. The low Km component of DEF deethylation in acetoneor pyrazole-induced microsomes was strongly inhibited (approximately 90%) by diethyldithiocarbamate, 4-methylpyrazole, and anti-2E1 IgG, but in 200 mg/kg DEF-induced microsomes the inhibition was partial, suggesting that other P450(s) may be involved. Administration of DEF 200 mg/kg ip for 4 days induced hepatic microsomal P4502E1-dependent aniline hydroxylase, P4502B1/2-linked pentoxyresorufin O-depentylase, 16 beta-testosterone hydroxylase P4503A1/2-associated erythromycin N-demethylase, and 6 beta-testosterone hydroxylase. Alternatively, the same dose regimen of MEF induced only the aniline hydroxylase and depressed the 3A1/2-linked activities. Immunoblot experiments verified these data. These findings indicate that DEF, at low concentrations, is predominantly oxidized by P4502E1 and that this enzyme may be induced in rodents by repeated MEF or DEF treatment, thereby increasing their own metabolism and potentially their cytotoxicity through the formation of ethyl isocyanate.

Effects of dioxane on cytochrome P450 enzymes in liver, kidney, lung and nasal mucosa of rat

“…Erythromycin N-demethylase (ErD) activity, a marker for CYP3A1/2, was determined as described by Tu and Yang (1983). Testosterone hydroxylase activity was determined by separating the hydroxylated metabolites using a Waters 1525 high-performance liquid chromatography apparatus equipped with a Supelco LC-18 column (25x4.6 mm) as previously reported (Amato et al 1996). KCN-insensitive palmitoyl CoA oxidation activity was determined in the whole hepatic homogenate as described by Bronfman et al (1979).…”

“…On the other hand, the ErD activity, dependent on the CYP3A isoform (Arlotto et al 1987), was induced (about 2-fold) only by the gavage treatment. To further investigate the effects of dioxane, the microsomal metabolism of testosterone, an endogenous substrate selectively oxidised by many CYPs, (Amato et al 1996) was studied (Fig. 2).…”

Cloning and expression of rat CYP2E1 inSaccharomyces cerevisiae: Detection of genotoxicity ofN-alkylformamides

“…After transfer, membranes were blocked for 1 hr in 5% nonfat milk in saturation buffer TBS (20 mM Tris base, 0.5 M NaCl, 0.15% Tween 20). The heterologous protein was immunoassayed using anti-rat P450 2E1 polyclonal antibodies previously prepared [Amato et al, 1996] and colored with 4-chloro naphthol as substrate.…”

“…After 1 hr of preincubation at room temperature, the reaction was started with 1 mM NADPH and carried out at 37°C for 30 min. AnH activity was determined as described earlier, whereas the production of formaldehyde and acetaldehyde, as the reaction products of NMF and NEF incubations, was determined as previously reported [Amato et al, 1996].…”

“…NMF, the main metabolite of DMF, is further metabolized by CYP2E1, producing methylisocyanate (MIC), a potent electrophilic agent [Mraz et al, 1993], probably responsible for the toxicity [Hyland et al, 1992]. NEF, the main metabolite of DEF [Amato et al, 1996], might be metabolized to ethylisocyanate (EIC) by 2E1, but it has never been demonstrated. In spite of the reactive intermediates formed during their metabolism, both NMF and NEF, which are very polar, have never been found to be mutagenic in short assay in vitro [Kennedy, 1985;IARC, 1989].…”