Turini A, Amato G, Longo V, Gervasi P G
Laboratory of Genetic and Biochemical Toxicology, Istituto di Mutagenesi e Differenziamento, C.N.R., Pisa, Italy.
Arch Toxicol. 1998 Mar;72(4):207-14. doi: 10.1007/s002040050490.
Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16beta-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i.p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C1 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established.
甲基叔丁基醚（MTBE）和乙基叔丁基醚（ETBE）常用于无铅汽油中，以增加燃料的含氧量并减少机动车的一氧化碳排放。本研究旨在调查：（1）给大鼠施用ETBE及其代谢产物叔丁醇对肝P450同工酶的诱导和/或抑制作用；（2）用选定的P450诱导剂和纯化的大鼠P450（2B1、2E1、2C11、1A1）预处理的大鼠肝微粒体对MTBE和ETBE的氧化代谢。经口灌胃给予ETBE，剂量为2 ml/kg，持续2天，可诱导肝微粒体中与P4502E1相关的对硝基苯酚羟化酶以及与P4502B1/2相关的PROD和16β-睾酮羟化酶，免疫印迹实验证实了这一点。腹腔注射剂量为200和400 mg/kg的叔丁醇，持续4天，未改变任何肝微粒体单加氧酶。MTBE和ETBE都是大鼠肝微粒体的底物，分别被氧化脱烷基生成甲醛和乙醛。在苯巴比妥（PB）处理的大鼠中，MTBE和ETBE的脱烷基速率均增加了约四倍。在用吡唑（一种2E1诱导剂）预处理的大鼠中，仅MTBE的去甲基化增加了（约两倍）。当在重组系统中用纯化的P450研究MTBE和ETBE的氧化时，发现P4502B1对这两种溶剂的活性最高，而1A1和2C1活性较低；P4502E1对MTBE有明显活性，但对ETBE无活性。美替拉酮是一种有效的P450 2B抑制剂，始终抑制PB处理大鼠微粒体中MTBE和ETBE的脱烷基反应。此外，4-甲基吡唑（一种2E1的探针抑制剂）和抗P4502E1 IgG仅对MTBE的去甲基化有适度抑制作用，而对ETBE的脱乙基反应无抑制作用。抑制实验还表明，大鼠2A1可能在MTBE和ETBE的氧化中发挥重要作用。综上所述，这些结果表明，P4502B1表达时是参与这两种溶剂氧化的主要酶，而P4502E1可能在MTBE去甲基化中发挥作用，尽管作用较小。这些数据对MTBE和ETBE毒性的影响仍有待确定。
