Mitochondrial Dysfunction and Drug Hepatotoxicity

描述（由申请人提供）：对乙酰氨基酚（AAP）过量是美国有毒药物摄入的第二大原因，也是急性肝衰竭的最常见原因。已经确定，导致AAP诱导的细胞死亡的事件序列是由反应性代谢物的形成引发的，该代谢物首先耗尽谷胱甘肽，然后使细胞内大分子芳基化。然而，蛋白质芳基化后导致细胞死亡的事件顺序还不完全清楚。基于已发表的数据和我们自己的初步调查，我们提出了以下新的假设，胞质钙蛋白酶激活是一个关键事件，导致进行性线粒体功能障碍，并最终打开膜通透性转换（MPT）孔，这引发了细胞的生物能量危机，导致细胞死亡。特别是，我们将通过研究4个具体目标来测试这一假设：首先，我们将表征钙蛋白酶的激活，并确定其对AAP过量后线粒体功能障碍、MPT和细胞死亡的意义。其次，我们将评估Bcl-2家族成员Bid和Bax易位到线粒体对线粒体功能障碍、MPT和细胞死亡的作用。第三，我们将评估MPT对线粒体释放核酸内切酶G的作用及其对DNA片段化和细胞死亡的功能意义。第四，我们将描述线粒体DMA耗尽和核DMA片段化和聚（ADP-核糖）聚合酶（PARP）激活AAP诱导的细胞死亡和再生的作用。该提议是创新的，因为它测试了肝细胞中坏死细胞死亡的细胞内信号级联的新概念。该研究将建立预防肝细胞死亡的关键干预点，远远超过该过程的开始，因此可能更适用于药物过量后的治疗干预。这种对AAP诱导的细胞死亡的信号传导机制的新见解有望建立新的治疗方法，用于预防AAP诱导的肝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.