Evaluation of drug-induced hepatotoxicity by chemiluminescence and the role of MRP2 in the hepatotoxicity

氧化应激与多种疾病和药物副作用有关。本研究采用超弱化学发光技术评价肝脏药物代谢引起的氧化应激。谷胱甘肽（GSH）通过多药耐药相关蛋白2 （MKP2）分泌，提示MRP2参与氧化应激。因此，本研究通过体外、原位和体内实验系统研究了MRP2在氧化应激中的作用。脂质过氧化是由乙酸在肝脏中的氧化代谢引起的。这表明EA诱导的氧化应激不仅是由于EA'SG偶联物的形成引起细胞内GSH的减少，而且还与EA的氧化代谢有关。水杨酸和氯丙胺在氧化代谢过程中检测到超弱化学发光，从而通过氧化代谢诱导氧化应激。4-羟基壬烯醚（HNE）是脂质过氧化的有毒产物。HNE-SG是HNK的主要代谢物，研究了HNE-SG在mep2ht转染细胞和MRP2缺陷动物（EHKR）中的转运，以阐明MRP2在其胆道排泄中的作用。结果表明，MRP2在HNE-SG的胆汁排泄中起重要作用。我们利用MRP2转染的细胞进一步研究了MRP2是否降低了hne诱导的细胞毒性，有趣的是，MRP2的转染增强了hne诱导的细胞毒性，这可能是由于MRP2介导的GSH和HNE-SG的排泄引起细胞内GSH的减少。进一步，通过EA原位肝灌注研究MRP2在氧化应激下的细胞毒性。结果表明，与正常大鼠相比，EHBR中EA诱导的肝毒性和脂质过氧化的发生明显减少。因此，MRP2似乎增强了氧化应激诱导的细胞毒性。少

The oxidative stress is related to various diseases and drug-induced side effects. In this study, the oxidative stress caused by the drug metabolism in liver was evaluated by ultraweak chemiluminescence. Glutathione (GSH) is excreted through multidrug resistance-associated protein 2 (MKP2), suggesting the involvement of MRP2 in the oxidative stress. Therefore, the present study investigated the role of MRP2 under the oxidative stress, using in vitro, in situ and in vivo experimental systems.Lipid peroxidation was shown to occur by the oxidative metabolism of ethacrynic acid (EA) in liver. This indicated that the EA-induced oxidative stress was caused not only by the decrease of intracellular GSH induced by the formation of EA'SG conjugate but also by the oxidative metabolism of EA. Salicylic acid and chlorpropamide were found to induce the oxidative stress by their oxidative metabolism, since the ultraweak chemiluminescence was detected during their oxidative metabolism.4-Hydroxvnonena … More l (HNE) is a toxic product of lipid peroxidation. HNE-SG is a major metabolite of HNK The transport of HNE-SG in MEP2HTansfected cells and MRP2-defitient animals (EHKR) was investigated in order to clarify the contribution of MRP2 in its biliary excretion. It was shown that MRP2 played an important role in the biliary excretion of HNE-SG. We further investigated whether the HNE-induced cytotoxicity was reduced by MRP2 using MRP2-iaansfected cells, interestingly, the transfection of MRP2 enhanced the HNE-induced cytotoxicity This may possibly be due to the decrease of intracellular GSH provoked by the MKP2-mediated excretion of GSH and HNE-SG. Further, the cytotoxicity mediated by MRP2 under the oxidative stress was investigated using the in situ liver perfusion of EA. It was shown that the EA-induced hepatotoxicity and the occurrence of lipid peroxidation were markedly reduced in EHBR, compared with those of normal rats. Thus, the oxidative stress-induced cytotoxicity seems to be enhanced by MRP2. Less