Investigation of the mechanistic basis of N,N-dimethylformamide toxicity. Metabolism of N,N-dimethylformamide and its deuterated isotopomers by cytochrome P450 2E1

Abstract

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 Km = 0.20 mM) than that of NMF (Km = 4.28 mM) or of HMMF (Km = 2.52 mM). The respective values observed with human microsomes are very similar. The apparent Km values for the N-methyl oxidation of N,N-dimethyldeuterioformamide ([2H1]DMF) and N,N-bis(trideuteriomethyl)formamide ([2H6]DMF) in rat microsomes are 0.14 and 0.21 mM, respectively. The apparent Vmax for the oxidation of [2H1]DMF is similar to that computed for DMF, and the Vmax for [2H6]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 Vmax/Km for DMF to Vmax/Km for [2H6]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 [2H6]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 ([2H3]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.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of the Developmental Toxicity, Metabolism, and Placental Transfer of N,N-Dimethylformamide Administered to Pregnant Rats

“…In all mammalian species studied, DMF is metabolized through an oxidative pathway that leads to HMMF, NMF, and F (Gescher, 1993). An alternative metabolic pathway involves glutathione, forming ultimately AMCC (Mraz et al, 1989(Mraz et al, , 1993Mraz et Nohova, 1992).…”

“…This observation also suggests a minimal metabolic contribution, if any, of the placenta and/or the embryo or fetus to the generation of HMMF, NMF, F, and/ or AMCC. Oxidation of DMF is mediated by the P450 system in various tissues of adult animals, and cytochrome P450 2E] is believed to play an important role (Mraz et al, 1993;Chieli et ai, 1995). Hitherto, there is little evidence for the expression of P450 2E r related isoforms in mammalian conceptus tissues, although several other P450 isozymes are known to be expressed prenatally in rodents and humans (Nau and Neubert, 1978;Jones et al, 1992;Juchau et al, 1992;Borlakoglu et al, 1993;Raucy and Carpenter, 1993;Cederbaum, 1993a,b, 1996;Casazza et al, 1994;Hakkola et al, 1994;Pasanen and Pelkonen, 1994;Miller etal, 1996).…”

N,N-DIMETHYLFORMAMIDE (CAS No. 68-12-2)

“…Na avaliação de pessoas expostas ao DMF é observada necrose hepática, e também ocorre a degeneração do órgão e hipertrofia hepatocelular; além de alterações em valores séricos das enzimas Aspartato aminotransferase (AST) e Alanina aminotransferase (ALT). 39 Quanto à função cardíaca, existem divergências na literatura. A maioria dos estudos não relata dano cardíaco aos trabalhadores expostos ao DMF, porém, algumas pesquisas indicam a ocorrência de taquicardia e aumento da pressão sanguínea.…”

“…Um dos produtos da biotransformação do DMF em humanos (Esquema 25), 39 a Nmetilformamida (NMF), pode ser utilizado como o biomarcador para indicar os níveis de contaminação por DMF. 3 A Norma Regulamentadora N • 7, do Ministério do Trabalho, estabelece que a urina é o material biológico utilizado para pesquisa de Nmetilformamida, contudo, não está estabelecido o valor de referência para dosagem deste metabólito, sendo o Índice Biológico Máximo Permitido de 40 mg/g de creatinina.…”

“…40 É importante citar que a meiavida biológica da NMF urinária é de 4,75 ± 1,63 horas após exposição cutânea ao DMF, sendo mais curta, caso ocorra exposição aos seus vapores (2,42 ± 0,63 horas). 39 …”