Tolando R, Zanovello A, Ferrara R, Iley J N, Manno M
Institute of Occupational Medicine, University of Padua, Via Facciolati, 71, Padua 35128, Italy.
Toxicol Lett. 2001 Oct 15;124(1-3):101-11. doi: 10.1016/s0378-4274(01)00384-8.
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-alpha-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.
N,N-二甲基甲酰胺（DMF）是一种在工业中广泛使用的有机溶剂，可被细胞色素P450（P450）生物活化生成反应性代谢产物，据信这些代谢产物是导致动物和人类肝毒性的原因。据报道，用DMF处理的大鼠体内活化酶减少，尽管所涉及的具体P450同工酶以及导致这种情况和其他毒性作用的反应性物种的性质仍在研究中。在本研究中，在体外研究了DMF和结构相关的N,N-二甲基乙酰胺（DMAc）对活化酶的影响以及这两种化学物质参与P450失活机制的反应性物种的性质。用这两种底物之一孵育吡啶诱导的大鼠的肝微粒体，导致P450呈剂量依赖性（0-20 mM）损失（DMF和DMAc分别高达28%和24%），微粒体血红素（DMF和DMAc分别高达24%和20%）损失，但原卟啉IX含量未变。此外，向孵育混合物中通入CO几乎完全保护了底物依赖性的P450失活，自旋捕获剂N-叔丁基-α-苯基硝酮可提供显著保护以防止DMF或DMAc依赖性的血红素损失，而谷胱甘肽和维生素C则不能。最后，在存在DMF或DMAc的情况下对微粒体孵育进行电子自旋共振分析，显示出以碳为中心的自由基中间体的光谱证据。总体而言，结果表明这两种化合物在体外均被P450（可能是CYP2E1）代谢为自由基代谢产物，这些代谢产物攻击血红素辅基，导致自杀性酶失活。
