ALCOHOL/CYP2E1- INDUCED LIVER MITOCHONDRIAL DNA DAMAGE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 乙醇导致肝细胞损伤的机制尚不清楚。在最近的研究中，氧化应激在酒精毒性中的作用是一个重要的途径，也是相当多研究的焦点。乙醇诱导细胞色素P450的2E1亚型可能是乙醇产生氧化应激状态的核心机制之一。我们以前已经证明，由CYP2E1衍生的氧化剂对线粒体的损伤是细胞内CYP2E1依赖的毒性整个途径中的一个早期事件。然而，目前还不清楚CYP2E1过度表达、乙醇代谢和线粒体损伤之间的机制联系。 该方案的中心假设是，乙醇暴露下的肝细胞线粒体DNA(MtDNA)损伤是由CYP2E1衍生的氧化剂引起的，这种mtDNA损伤导致线粒体功能障碍。我们将通过追求以下两个具体目标来验证我们的中心假设： 具体目的：1.体外检测线粒体DNA是否是细胞色素P450_2E_1衍生氧化剂的靶标。我们推测，细胞色素P450-2E_1过度表达的一个后果是线粒体DNA损伤，这种损伤是通过产生更多的活性氧造成的。研究将使用过表达CYP2E1的基因工程肝细胞和体外乙醇孵育的对照(亲代)肝细胞进行。我们预计在远距离定量聚合酶链式反应中，高表达的肝细胞比非高表达的肝细胞发生更多的线粒体DNA损伤，并且抗氧化剂可以防止线粒体DNA损伤。 具体目的2.确定乙醇诱导的线粒体DNA损伤是否由细胞色素P450 2E 1引起。基因工程小鼠(CYP2E1基因敲除)和野生型小鼠将接受乙醇治疗(急性和慢性模型)。我们假设，CYP2E1基因敲除的小鼠将受到保护，免受乙醇诱导的线粒体DNA损伤。我们预计，从酒精喂养的野生型小鼠中分离出的肝脏mtDNA将显示出比酒精喂养的CYP2E1基因敲除小鼠更高的损伤水平。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanisms by which ethanol causes liver cell injury are not clear. A major pathway that is a focus of considerable research in recent studies is the role of oxidative stress in alcohol toxicity. Induction of the 2E1 isoform of cytochrome P450 (CYP2E1) by ethanol appears to be one of the central mechanisms by which ethanol generates a state of oxidative stress. We have previously shown that damage to mitochondria by CYP2E1-derived oxidants is an early event in the overall pathway of cellular CYP2E1-dependent toxicity. However, the mechanistic link between CYP2E1 overexpression, ethanol metabolism and mitochondrial damage is currently unknown. The central hypothesis of this proposal is that CYP2E1-derived oxidants induce mitochondrial DNA (mtDNA) damage in liver cells exposed to ethanol, and this mtDNA damage causes mitochondrial dysfunction. We will test our central hypothesis by pursuing the following two specific aims: Specific Aim 1. To test whether mtDNA is a target of CYP2E1-derived oxidant species in vitro. We hypothesize that one consequence of CYP2E1 overexpression is mtDNA damage via increased generation of reactive oxygen species. Studies will be conducted using genetically engineered liver cells that overexpress CYP2E1, and control (parental) liver cells incubated with ethanol in vitro. We expect that CYP2E1 overexpressing liver cells will develop more mtDNA lesions evaluated with long distance qPCR than non-CYP2E1 expressing cells, and that mtDNA damage will be prevented by antioxidants. Specific Aim 2. To determine if CYP2E1 causes the mtDNA damage by ethanol in vivo. Genetically engineered mice (CYP2E1 knock-out) and wild type mice will be treated with ethanol (acute and chronic models). We hypothesize that CYP2E1 knock-out mice will be protected from ethanol-induced mtDNA damage. We expect that liver mtDNA isolated from alcohol-fed wild-type mice will show higher levels of damage than alcohol-fed CYP2E1-knockout mice.