INVESTIGATION OF THE MECHANISTIC BASIS OF N,N-DIMETHYLFORMAMIDE TOXICITY - METABOLISM OF N,N-DIMETHYLFORMAMIDE AND ITS DEUTERATED ISOTOPOMERS BY CYTOCHROME-P450-2E1

Dimethylformamide (DMF) is an industrial solvent with hepatotoxic properties. The toxicity of DMF has been associated with its metabolism to S-(N-methylcarbamoyl)glutathione (SMG). The major urinary metabolite of DMF is N-(hydroxymethyl)-N-methylformamide (HMMF). HMMF undergoes oxidation in the formyl moiety, possibly via the intermediacy of its hydrolysis product N-methylformamide (NMF), and the reactive intermediate thus generated reacts with glutathione to yield SMG. Experiments were conducted to elucidate enzymatic details of the metabolism of DMF. Generation of HMMF from DMF in microsomes from rats which had received acetone, an inducer of cytochrome P450 2E1, was increased by 175% over that observed in control microsomes. In liver microsomes from 4 humans the metabolism of DMF to HMMF was inhibited by a monospecific antibody against rat liver P450 2E1, and the metabolic rates were correlated with those of NMF to SMG, a process known to be mediated via P450 2E1. DMF was also metabolized by purified rat liver P450 2E1. The kinetic parameters which characterize the metabolism of DMF or its deuterated isotopomers to the respective HMMF isotopomers, of HMMF to SMG and of NMF to SMG in liver microsomes, were computed from Eadie-Hofstee plots. The affinity of DMF for the metabolizing enzyme in rat liver microsomes is considerably higher (apparent K(m) = 0.20 mM) than that of NMF (K(m) = 4.28 mM) or of HMMF (K(m) = 2.52 mM). The respective values observed with human microsomes are very similar. The apparent K(m) values for the N-methyl oxidation of N,N-dimethyldeuterioformamide ([H-2(1)DMF) and N,N-bis(trideuteriomethyl)formamide([H-2(6)]DMF) in rat microsomes are 0.14 and 0.21 mM, respectively. The apparent V(max) for the oxidation of [H-2(1)]DMF is similar to that computed for DMF, and the V(max) for [H-2(6)]DMF is less than half of that computed for DMF. The kinetic deuterium isotope effect (KDIE) on DMF metabolism was determined in incubations with rat microsomes in three ways: (i) the noncompetitive intermolecular KDIE by the ratio of V(max)/K(m) for DMF to V(max)/K(m) for [H-2(6)]DMF, (ii) the competitive intermolecular KDIE as the quotient of metabolic products HMMF to N-(hydroxydideuteriomethyl)-N-(trideuteriomethyl) formamide in incubations of DMF together with [H-2(6)]DMF, and (iii) the intramolecular KDIE as the quotient of the ratio of N-(hydroxymethyl)-N-(trideuteriomethyl)formamide to N-(hydroxydideuteriomethyl)-N-methylformamide generated from N-(trideuteriomethyl)-N-methylformamide ([H-2(3)]DMF). The respective values were found to be (i) 2.4, (ii) 5.0, and (iii) 5.2. DMF inhibited the oxidation of NMF or HMMF to SMG. Deuterium substitution of the DMF methyl hydrogens did not affect the apparent K(i) for the inhibition of the oxidation of NMF to SMG. Among a series of 8 formamides and acetamides structurally related to DMF only N,N-diethylformamide and N,N-dimethylacetamide were equally effective with DMF as inhibitors. The results suggest that (i) hepatic P450 2E1 is an important catalyst of the metabolism of DMF and related low-molecular-weight amides, (ii) DMF inhibits its own metabolic toxification, and (iii) there is a marked KDIE on the metabolic oxidation of DMF.