Tingle M D, Pirmohamed M, Templeton E, Wilson A S, Madden S, Kitteringham N R, Park B K
Department of Pharmacology and Therapeutics, University of Liverpool, U.K.
Biochem Pharmacol. 1993 Nov 2;46(9):1529-38. doi: 10.1016/0006-2952(93)90319-r.
Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.
具有化学反应性的环氧化物代谢物与多种形式的药物和化学毒性有关。萘可代谢生成1,2 - 环氧化物，在本研究中被用作模型化合物，以研究解毒机制的扰动对人肝微粒体存在时环氧化物体外毒性的影响。萘（100微摩尔）被人肝微粒体代谢为具有细胞毒性、与蛋白质反应且稳定但无基因毒性的代谢物。在苯巴比妥诱导的小鼠肝微粒体存在下，萘的代谢依赖性细胞毒性和与蛋白质的共价结合显著高于人肝微粒体。人肝微粒体和诱导的小鼠微粒体的反式 - 1,2 - 二氢二醇与1 - 萘酚的比率分别为8.6和0.4。萘对人外周血单核白细胞的代谢依赖性毒性不受共孵育细胞谷胱甘肽转移酶μ状态的影响。环氧三氯丙烷（TCPO；30微摩尔），一种环氧化物水解酶抑制剂，增加了萘（100微摩尔）的人肝微粒体依赖性细胞毒性（19.6±0.9%对28.7±1.0%；P = 0.02）和与蛋白质的共价结合（1.4±0.3%对2.8±0.2%；P = 0.03），并将1,2 - 二氢二醇与1 - 萘酚的比率从6.6（无TCPO）分别在TCPO浓度为30、100和500微摩尔时逆转至2.6、0.6和0.1。增加人肝微粒体蛋白浓度降低了萘的细胞毒性，同时增加了其与蛋白质的共价结合以及1,2 - 二氢二醇代谢物的形成。与谷胱甘肽（5毫摩尔）共孵育分别使萘的细胞毒性和与蛋白质的共价结合降低了68%和64%。孕二烯酮也抑制了与蛋白质的共价结合，而孕二烯酮（250微摩尔）和依诺沙星（250微摩尔）降低了稳定代谢物的形成。该研究表明，人肝细胞色素P450酶将萘代谢为一种具有细胞毒性和与蛋白质反应但无基因毒性的代谢物，该代谢物可能是一种环氧化物。这被微粒体环氧化物水解酶迅速解毒，其效率可通过测量稳定代谢物萘1,2 - 二氢二醇和1 - 萘酚的比率轻松确定。
