Hu J J, Lee M J, Vapiwala M, Reuhl K, Thomas P E, Yang C S
Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789.
Toxicol Appl Pharmacol. 1993 Sep;122(1):16-26. doi: 10.1006/taap.1993.1167.
The objective of this study is to elucidate the role of cytochrome P450 2E1 in the metabolic activation of acetaminophen (APAP) in mouse kidneys. With the kidney microsomes from C3H/HeJ mice, a significant sex-related difference was observed in the NADPH-dependent formation of a reactive APAP metabolite which was trapped as a glutathione conjugate. The enzyme activity in male mouse kidney was about 35- and 50-fold higher than that in the females at substrate concentrations 50 and 500 microM, respectively, and treatment of female mice with testosterone significantly induced the enzyme activity in the mouse kidney. No sex-related difference in this enzyme activity, however, was observed in the livers. The oxidative metabolism of APAP in control male mouse kidney microsomes displayed an apparent low Km of 43-45 microM and an apparent high Km of 603-702 microM. Studies using monoclonal antibodies against P450 2E1 showed that P450 2E1 accounted for about half of the activity in the metabolic activation of APAP in both mouse liver and kidney. Furthermore, there was as strong correlation between the renal P450 2E1 content (measured by Western blot analysis) and the rate of APAP oxidation by renal microsomes. To test the hypothesis that the susceptibility of renal tissue to APAP toxicity is determined by the distribution and level of P450 2E1, toxicity studies were conducted. A time-dependent depletion of hepatic and renal cortical glutathione was observed in both male and female mice following a dose of 1200 mg/kg APAP. At 4 hr after APAP dosing, the level of renal cortical glutathione depletion in male mice was significantly greater than that in the females. The level of blood creatinine elevation in male mice was higher than that in the females 8 hr after APAP dosing. Histopathology studies by light and electron microscopic assessments demonstrated that renal damage by APAP was restricted mostly to the epithelial cells of the proximal convoluted tubules where P450 2E1 was localized. The renal proximal tubular necrosis induced by APAP was more severe in males than that in the females. Results from this study suggest that P450 2E1 plays an important role in the metabolic activation of APAP and is a key factor in determining the sex-related difference of APAP-mediated toxicity in the mouse kidney.
本研究的目的是阐明细胞色素P450 2E1在小鼠肾脏中对乙酰氨基酚（APAP）代谢活化的作用。利用C3H/HeJ小鼠的肾脏微粒体，在以谷胱甘肽共轭物形式捕获的APAP活性代谢物的NADPH依赖性形成过程中观察到显著的性别相关差异。在底物浓度为50和500 microM时，雄性小鼠肾脏中的酶活性分别比雌性高约35倍和50倍，用睾酮处理雌性小鼠可显著诱导小鼠肾脏中的酶活性。然而，在肝脏中未观察到该酶活性的性别相关差异。对照雄性小鼠肾脏微粒体中APAP的氧化代谢显示出明显较低的Km值为43 - 45 microM和明显较高的Km值为603 - 702 microM。使用针对P450 2E1的单克隆抗体进行的研究表明，P450 2E1在小鼠肝脏和肾脏中APAP代谢活化的活性中约占一半。此外，肾脏P450 2E1含量（通过蛋白质免疫印迹分析测定）与肾脏微粒体对APAP的氧化速率之间存在很强的相关性。为了检验肾脏组织对APAP毒性的易感性由P450 2E1的分布和水平决定这一假设，进行了毒性研究。在给予1200 mg/kg APAP剂量后，雄性和雌性小鼠的肝脏和肾皮质谷胱甘肽均出现时间依赖性消耗。在给予APAP后4小时，雄性小鼠肾皮质谷胱甘肽的消耗水平显著高于雌性。在给予APAP后8小时，雄性小鼠血液肌酐升高水平高于雌性。通过光镜和电镜评估的组织病理学研究表明，APAP引起的肾脏损伤主要局限于P450 2E1所在的近端曲管上皮细胞。APAP诱导的肾近端小管坏死在雄性中比雌性更严重。本研究结果表明，P450 2E1在APAP的代谢活化中起重要作用，并且是决定小鼠肾脏中APAP介导的毒性性别相关差异的关键因素。
