Mitochondrial Dysfunction and Drug Hepatotoxicity

描述(由申请人提供)：醋氨酚(AAP)过量是美国有毒药物摄入的第二大原因，也是导致急性肝功能衰竭的最常见原因。众所周知，导致AAP诱导细胞死亡的一系列事件是由一种反应性代谢物的形成启动的，该代谢物首先耗尽谷胱甘肽，然后使细胞内大分子芳构化。然而，蛋白质芳基化后导致细胞死亡的事件序列还不完全清楚。基于已发表的数据和我们自己的初步研究，我们提出了以下新的假设，即胞浆Calain激活是导致进行性线粒体功能障碍和最终打开膜通透性转换(MPT)孔的关键事件，从而触发细胞生物能量危机，导致细胞死亡。特别是，我们将通过调查4个具体目标来验证这一假说：首先，我们将表征Calain的激活，并确定它们在AAP过量服药后线粒体功能障碍、MPT和细胞死亡中的意义。其次，我们将评估易位到Bid和Bax家族成员的线粒体在线粒体功能障碍、MPT和细胞死亡中的作用。第三，我们将评估MPT在线粒体释放核酸内切酶G中的作用及其在DNA断裂和细胞死亡中的功能意义。第四，我们将研究线粒体DNA耗竭、核DNA断裂和多聚腺苷二磷酸核糖聚合酶(PARP)激活在AAP诱导的细胞死亡和再生中的作用。这项提议的创新之处在于，它测试了肝细胞内坏死细胞死亡的细胞内信号级联的新概念。这项研究将建立关键的干预点，以防止肝细胞死亡远远超出这一过程的启动，因此可能更适用于药物过量后的治疗干预。这一对AAP诱导细胞死亡的信号机制的新见解有望建立新的治疗方法来预防AAP诱导的人类肝功能衰竭和潜在的其他形式的药物毒性。

DESCRIPTION (provided by applicant): Acetaminophen (AAP) overdose is the second leading cause of toxic drug ingestion and the most frequent cause of acute liver failure in the US. It is well established that the sequence of events leading to AAP-induced cell death is initiated by the formation of a reactive metabolite, which first depletes glutathione and then arylates intracellular macromolecules. However, the sequence of events leading to cell death after protein arylation are incompletely understood. Based on published data and our own preliminary investigations, we propose the following novel hypothesis that cytosolic calpain activation is a key event in causing progressive mitochondrial dysfunction and eventually opening of the membrane permeability transition (MPT) pore, which triggers a cellular bioenergetic crisis resulting in cell death. In particular, we will test this hypothesis by investigating 4 specific aims: First, we will characterize the activation of calpains and establish their significance for mitochondrial dysfunction, MPT and cell death after AAP overdose. Second, we will evaluate the role of translocation to the mitochondria of Bcl-2 family members Bid and Bax for mitochondrial dysfunction, MPT and cell death. Third, we will assess the role of MPT for mitochondrial release of endonuclease G and its functional significance for DMA fragmentation and cell death. Fourth, we will characterize the role of mitochondrial DMA depletion and nuclear DMA fragmentation and poly(ADP-ribose)polymerase (PARP) activation for AAP-induced cell death and regeneration. This proposal is innovative in that it tests a novel concept of an intracellular signaling cascade of necrotic cell death in liver cells. The investigation will establish critical intervention points for preventing liver cell death well beyond the initiation of the process and thus may be more applicable for therapeutic interventions after drug overdose. This new insight into the signaling mechanism of AAP-induced cell death holds the promise of establishing novel therapeutic approaches for preventing AAP-induced liver failure and potentially other forms of drug toxicity in humans.