Transporter Expression in Response to Hepatotoxicants

摘要 虽然对乙酰氨基酚（APAP）肝毒性的认识已经取得了进展， 值得注意的是，在过去的三十年里，毒性的确切机制仍然存在， 未知人们对一种被称为自动保护的现象知之甚少， 肝脏适应亚毒性APAP暴露，导致对随后的毒性 APAP剂量。我们的长期目标是了解MRP（ABCC）转运蛋白的作用 APAP诱导的肝毒性。在当前的融资周期中，我们确定， 外排转运蛋白Mrp 4是APAP过程中最显著的MRP诱导蛋白 肝毒性我们的数据还显示了肝脏Mrp 4诱导与 以及对APAP毒性的抗性的发展。因此，这一假设 竞争性更新是“Mrp 4诱导是对药物诱导的代偿反应， 肝损伤，赋予对随后的毒物攻击的抗性”。为了验证这一 假设，三个具体的研究目标已经制定。首先，我们将分析 Mrp 4在APAP肝毒性中的作用，通过检查Mrp 4-/-小鼠对 APAP。一种补充的体外方法是检查稳定的 转染过表达Mrp 4细胞的永生化人肝细胞（hMRP 4-HC 04） APAP细胞毒性。然后，我们将确定Mrp 4的功能后果 使用代谢组学方法鉴定转运的内源性分子的诱导 通过MRP 4与旁分泌功能促进细胞防御增强， 代偿性细胞增殖MRP 4基因的转录调控 将使用分子生物学方法检测响应于氧化应激的表达。 方法和生物信息学。小鼠转录活性的表征 和人Mrp 4/MRP 4基因启动子及其近端区域。 进一步的研究将确定两种转录因子（NFR 1和NFR 2）的作用。 HES-1）对MRP 4基因表达的影响。改变的肝脏MRP 4表达可能是至关重要的， 在药物诱导的肝损伤的恢复和再生期间的细胞存活。 这种适应可能介导更有效的肝胆排泄细胞毒性 中间体和/或增强旁分泌的内源性底物的输出 向邻近肝细胞和非实质细胞发出信号以促进存活， 组织修复对MRP 4调节的了解增加预计将导致更好的 治疗特定的肝脏疾病，降低药物性肝损伤的风险。 !

ABSTRACT Although knowledge on acetaminophen (APAP) hepatotoxicity has advanced significantly during the last three decades, the precise mechanism of toxicity remains unknown. Even less is known about a phenomenon known as autoprotection, in which the liver adapts to sub-toxic APAP exposure, resulting in resistance to subsequent toxic APAP dosing. Our long-term goal is to understand the role of MRP (ABCC) transporters in APAP-induced hepatotoxicity. During the current funding cycle we determined that the efflux transporter Mrp4 is the most significantly induced MRP during APAP hepatotoxicity. Our data also show a strong relationship between liver Mrp4 induction and development of resistance to APAP toxicity. Therefore, the hypothesis for this competing renewal is that "Mrp4 induction is a compensatory response to drug-induced liver injury that confers resistance to subsequent toxicant challenge". To test this hypothesis, three specific research aims have been formulated. First, we will analyze the role of Mrp4 in APAP hepatotoxicity by examining the susceptibility of Mrp4-/- mice to APAP. A complementary in vitro approach is to examine the susceptibility of stably transfected immortalized human hepatocytes overexpresing Mrp4 cells (hMRP4-HC04) to APAP cytotoxicity. Then, we will determine the functional consequences of Mrp4 induction using metabolomic approaches to identify endogenous molecules transported by MRP4 with paracrine functions promoting heightened cellular defenses and compensatory cell proliferation. Lastly, transcriptional regulation of MRP4 gene expression in response to oxidative stress will be examined using molecular biological approaches and bioinformatics. Characterization of the transcription activity of mouse and human Mrp4/MRP4 gene promoter and its proximal regions will be carried out. Additional investigations will determine the effects of two transcription factors (NFR1 and HES-1) in MRP4 gene expression. Altered liver MRP4 expression may be essential for cell survival during periods of recovery and regeneration from drug-induced liver injury. This adaptation may mediate more efficient hepatobiliary excretion of cytotoxic intermediates and/or enhance the export of endogenous substrates for paracrine signaling to adjacent hepatocytes and non-parenchymal cells to promote survival and tissue repair. An increased knowledge of MRP4 regulation is expected to lead to better treatments of specific liver disorders and reduce the risk of drug liver injury. !