Transporter Expression in Response to Hepatotoxicants

描述（由申请人提供）：尽管对乙酰氨基酚（APAP）肝毒性的认识在过去三十年中取得了显著进展，但确切的毒性机制仍然未知。对一种称为自保护的现象知之甚少，其中肝脏适应亚毒性APAP暴露，导致对随后的毒性APAP剂量产生抗性。我们的长期目标是了解MRP（ABCC）转运蛋白在APAP诱导的肝毒性中的作用。在当前的资助周期中，我们确定外排转运蛋白Mrp 4是APAP肝毒性期间最显著诱导的MRP。我们的数据还显示了肝脏Mrp 4诱导和对APAP毒性的抗性发展之间的密切关系。因此，这种竞争性更新的假设是“Mrp 4诱导是对药物诱导的肝损伤的代偿反应，其赋予对随后的毒物攻击的抗性”。为了验证这一假设，提出了三个具体的研究目标。首先，我们将通过检查Mrp 4-/-小鼠对APAP的易感性来分析Mrp 4在APAP肝毒性中的作用。一种补充的体外方法是检查稳定转染的过表达Mrp 4细胞（hMRP 4-HC 04）的永生化人肝细胞对APAP细胞毒性的易感性。然后，我们将使用代谢组学方法来确定MRP 4诱导的功能后果，以确定由MRP 4转运的内源性分子，其具有促进增强的细胞防御和补偿性细胞增殖的旁分泌功能。最后，将使用分子生物学方法和生物信息学检查响应于氧化应激的MRP 4基因表达的转录调控。将对小鼠和人Mrp 4/MRP 4基因启动子及其近端区域的转录活性进行表征。其他研究将确定两种转录因子（NFR 1和HES-1）在MRP 4基因表达中的作用。在药物诱导的肝损伤的恢复和再生期间，改变的肝脏MRP 4表达可能对细胞存活至关重要。这种适应可能介导细胞毒性中间体的更有效的肝胆排泄和/或增强用于旁分泌信号传导的内源性底物向邻近肝细胞和非实质细胞的输出，以促进存活和组织修复。对MRP 4调节的了解增加，预计将导致更好地治疗特定的肝脏疾病，并降低药物性肝损伤的风险。! 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期间肝脏药物转运蛋白如何调节及其功能的了解，可以设计用于药物诱导的肝病的治疗和恢复的新治疗方法。本申请中提出的研究是重要的，因为它有望提供这方面的知识。本申请的目的是更好地确定MRP 4在药物诱导的急性肝毒性过程中的作用，并进一步表征MRP 4在氧化应激过程中的转录调控和功能。对APAP暴露的这种代偿反应的研究完全在NIDDK药物诱导肝损伤网络（DILIN）的使命和目标范围内。

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).