Mráz J, Jheeta P, Gescher A, Hyland R, Thummel K, Threadgill M D
Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham, U.K.
Chem Res Toxicol. 1993 Mar-Apr;6(2):197-207. doi: 10.1021/tx00032a009.
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)
二甲基甲酰胺（DMF）是一种具有肝毒性的工业溶剂。DMF的毒性与其代谢生成S-（N-甲基氨基甲酰基）谷胱甘肽（SMG）有关。DMF的主要尿代谢产物是N-（羟甲基）-N-甲基甲酰胺（HMMF）。HMMF在甲酰基部分发生氧化，可能通过其水解产物N-甲基甲酰胺（NMF）作为中间体，由此产生的反应性中间体与谷胱甘肽反应生成SMG。进行了实验以阐明DMF代谢的酶学细节。在接受细胞色素P450 2E1诱导剂丙酮处理的大鼠微粒体中，DMF生成HMMF的量比对照微粒体中观察到的增加了175%。在4名人类的肝脏微粒体中，DMF向HMMF的代谢受到针对大鼠肝脏P450 2E1的单特异性抗体的抑制，并且代谢率与NMF向SMG的代谢率相关，已知该过程是通过P450 2E1介导的。DMF也可被纯化的大鼠肝脏P450 2E1代谢。从伊迪-霍夫斯蒂图计算出肝脏微粒体中DMF或其氘代同位素异构体代谢为各自的HMMF同位素异构体、HMMF代谢为SMG以及NMF代谢为SMG的动力学参数。DMF对大鼠肝脏微粒体中代谢酶的亲和力（表观Km = 0.20 mM）远高于NMF（Km = 4.28 mM）或HMMF（Km = 2.52 mM）。在人类微粒体中观察到的相应值非常相似。大鼠微粒体中N，N-二甲基氘代甲酰胺（[2H1]DMF）和N，N-双（三氘代甲基）甲酰胺（[2H6]DMF）的N-甲基氧化的表观Km值分别为0.14和0.21 mM。[2H1]DMF氧化的表观Vmax与DMF计算值相似，[2H6]DMF的Vmax小于DMF计算值的一半。用大鼠微粒体孵育以三种方式测定了DMF代谢的动力学氘同位素效应（KDIE）：（i）通过DMF的Vmax/Km与[2H6]DMF的Vmax/Km之比计算非竞争性分子间KDIE，（ii）作为DMF与[2H6]DMF共同孵育时代谢产物HMMF与N-（羟二氘代甲基）-N-（三氘代甲基）甲酰胺的商计算竞争性分子间KDIE，（iii）作为由N-（三氘代甲基）-N-甲基甲酰胺（[2H3]DMF）生成的N-（羟甲基）-N-（三氘代甲基）甲酰胺与N-（羟二氘代甲基）-N-甲基甲酰胺之比的商计算分子内KDIE。相应的值分别为（i）2.4，（ii）5.0和（iii）5.2。DMF抑制NMF或HMMF氧化为SMG。（摘要截断于400字）
