Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI 48674, USA.
Regul Toxicol Pharmacol. 2010 Jul-Aug;57(2-3):129-35. doi: 10.1016/j.yrtph.2010.01.003. Epub 2010 Jan 22.
This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with (14)C-EB (0.22 mM) in the presence of 1mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4+/-102.2 pmol/mg protein)>>rat (57.0+/-3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7+/-1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0+/-38.5 pmol/mg protein) followed by rat (148.3+/-14.7 pmol/mg protein) and human (89.8+/-3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed approximately 2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse=85% vs. 40%; human=59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.
本研究旨在确定在存在 NADPH 的情况下，来自小鼠、大鼠和人肝脏和肺微粒体的反应性代谢物与乙基苯（EB）形成的共价结合的物种差异。这些数据进一步了解了 EB 导致小鼠特异性肺毒性的机制，并且是对我们之前报告的选择性升高的后续研究，尽管幅度较小，但在小鼠肺微粒体中升高了环状氧化的反应性代谢物（Saghir 等人，2009 年）。还进行了结合测定，有无 5-苯基-1-戊炔（5P1P），CYP 2F2 的抑制剂和二乙基二硫代氨基甲酸盐（DDTC），CYP 2E1 的抑制剂，以评估它们在相关反应性代谢物形成中的作用。在生理条件下，将（14）C-EB（0.22mM）与 1mM NADPH 一起孵育 60 分钟，以肝和肺微粒体。在肺微粒体中，结合活性的顺序为小鼠（812.4+/-102.2 pmol/mg 蛋白）>大鼠（57.0+/-3.2 pmol/mg 蛋白）。人肺微粒体的结合活性很低（15.7+/-1.4 pmol/mg 蛋白），与无 NADPH 对照（9.9-16.7 pmol/mg 蛋白）相当。在肝微粒体中，小鼠的活性最高（469.0+/-38.5 pmol/mg 蛋白），其次是大鼠（148.3+/-14.7 pmol/mg 蛋白）和人（89.8+/-3.0 pmol/mg 蛋白）。5P1P 或 DDTC 的存在降低了物种和组织之间的结合。然而，在小鼠肺微粒体中观察到的抑制作用高得多（86％[DDTC]和 89％[5P1P]）比大鼠（56％[DDTC]和 59％[5P1P]）肺微粒体。DDTC 对小鼠和人肝微粒体中结合的抑制作用分别是 5P1P 的两倍（小鼠=85％比 40％；人=59％比 36％）。DDTC 对大鼠肝微粒体中结合的抑制作用比 5P1P 高 10 倍（72％比 7％）。这些结果表明 EB 反应性代谢物的形成存在物种，组织和酶差异。在大鼠和小鼠肺微粒体中，CYP2E1 和 CYP2F2 似乎都有助于 EB 反应性代谢物的形成。相比之下，CYP2E1 似乎是肝中 EB 反应性代谢物的主要 CYP 同工酶。
