Inactivation of rat liver cytochrome P450 (P450) by N, N-dimethylformamide and N, N-dimethylacetamide

Abstract

N, N-dimethylformamide (DMF), an organic solvent widely used in industry, is bioactivated by cytochrome P450 (P450) to reactive metabolites which are believed to be responsible for the hepatotoxicity observed in animals and humans. A decrease of the activating enzyme has been reported in rats treated with DMF, although the specific P450 isoform(s) involved and the nature of the reactive species responsible for this and the other toxic effects are still being investigated. In the present work, the effect of DMF and of the structurally related N, N-dimethylacetamide (DMAc) on the activating enzyme and the nature of the reactive species involved in the mechanism of P450 inactivation by the two chemicals were investigated in vitro. Incubation of liver microsomes from pyridine-induced rats with either substrate resulted in a dose-dependent (0–20 mM) loss of P450 (up to 28 and 24% for DMF and DMAc, respectively), microsomal haem (up to 24 and 20% for DMF and DMAc, respectively), but not protoporphyrin IX content. Moreover, bubbling of CO through the incubation mixture gave almost complete protection against substrate-dependent P450 inactivation, and the spin trapping agent N- tert-butyl-α-phenylnitrone, but neither glutathione nor vitamin C, provided a significant protection against DMF- or DMAc-dependent haem loss. Finally, electron spin resonance analysis of microsomal incubations in presence of DMF or DMAc showed spectral evidence for a carbon centered radical intermediate. The results indicate, overall, that both compounds are metabolized in vitro by P450, probably CYP2E1, to free radical metabolites which attack the haem prosthetic group, leading to suicidal enzyme inactivation.