Transporter Expression in Response to Hepatotoxicants

描述(由申请人提供)：尽管在过去的三十年中，关于对乙酰氨基酚(APAP)肝脏毒性的知识有了很大的进步，但其确切的毒性机制仍然不清楚。对一种被称为自身保护的现象更是知之甚少，在这种现象中，肝脏适应亚毒性APAP暴露，导致对后续毒性APAP剂量的抵抗。我们的长期目标是了解MRP(ABCC)转运体在APAP诱导的肝毒性中的作用。在目前的资金周期中，我们确定外排转运蛋白MRP4是APAP肝毒性过程中最显着诱发的MRP。我们的数据还表明，肝脏MRP4的诱导和对APAP毒性的抵抗力之间存在很强的关系。因此，这种竞争性更新的假设是“MRP4诱导是对药物诱导的肝损伤的代偿反应，使其对随后的毒物挑战具有抵抗力”。为了验证这一假设，我们制定了三个具体的研究目标。首先，我们将通过检测MRP4-/-小鼠对APAP的易感性来分析MRP4在APAP肝毒性中的作用。另一种辅助的体外方法是检测稳定表达MRP4细胞的永生化人肝细胞(hMRP4-HC04)对APAP细胞毒性的敏感性。然后，我们将使用代谢组学方法确定MRP4诱导的功能后果，以确定MRP4转运的内源分子具有旁分泌功能，促进细胞防御和代偿性细胞增殖。最后，将使用分子生物学和生物信息学的方法来研究MRP4基因表达在氧化应激反应中的转录调控。将对小鼠和人类MRP4/MRP4基因启动子及其近端区域的转录活性进行表征。进一步的研究将确定两种转录因子(NFR1和HES-1)对MRP4基因表达的影响。肝脏MRP4表达的改变可能是药物诱导的肝损伤恢复和再生期间细胞存活所必需的。这种适应可能介导更有效的细胞毒性中间体的肝胆排泄和/或促进内源性底物的输出，从而将旁分泌信号传递给邻近的肝细胞和非实质细胞，以促进生存和组织修复。对MRP4调节知识的增加有望导致对特定肝病的更好治疗，并降低药物肝损伤的风险。好了！ 公共卫生相关性：药物性肝损伤(DILI)是一个非常严重的人类健康问题。通过增加对DILI过程中肝脏药物转运体的调节及其功能的了解，可能会设计出治疗和恢复药物所致肝病的新的治疗方法。本申请中提出的研究具有重要意义，因为它有望提供这种知识。这项应用的目的是更好地确定MRP4在药物诱导的急性肝毒性中的作用，并进一步表征MRP4在氧化应激过程中的转录调节和功能。对APAP暴露的这种代偿反应的研究完全符合NIDDK药物诱导肝损伤网络(DILIN)的任务和目标。

DESCRIPTION (provided by applicant): 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. ! PUBLIC HEALTH RELEVANCE: Drug-induced liver injury (DILI) is a very serious human health problem. By increasing the knowledge on how hepatic drug transporters are regulated during DILI and their function , new therapeutic approaches for treatment and recovery from liver diseases induced by drugs may be designed. The research proposed in this application is significant because it is expected to provide this knowledge. The goal of this application is to better define the role of MRP4 during drug-induced acute hepatotoxicity and to further characterize the transcriptional regulation and function of MRP4 during oxidative stress. The study of this compensatory response to APAP exposure is well within the mission and goals of the NIDDK Drug Induced Liver Injury Network (DILIN).