Alcohol-Induced Fatty Liver and Injury in Humanized CYP2E1 Knockin Mice.

描述(申请人提供)：酒精导致细胞损伤的机制(S)尚不清楚。其中一个重要的机制是脂质过氧化和氧化应激在酒精毒性中的作用，这是相当多研究的焦点。许多途径被认为在乙醇诱导“氧化应激”的过程中起着关键作用。我们假设，体内诱导的氧化应激增加会使肝细胞对慢性乙醇诱导的肝毒性和脂肪变性敏感。我们认为，过氧亚硝酸盐等氧化剂、JNK和/或p38MAPK等MAP激酶的激活、NF-：B的激活和生存因子的合成减少以及线粒体功能障碍是CYP2E1增强肝毒性的下游介质。我们认为，在乙醇诱导的TNF1、同型半胱氨酸和内质网应激引起的TNF1、同型半胱氨酸和内质网应激的共同作用下，这些下游介体激活了成脂转录因子和酶，同时降低了脂解转录因子和酶的活性，从而导致脂肪肝。支持这些假说的初步数据是，乙醇诱导的脂肪肝在CYP2E1基因敲除小鼠中钝化，并在人源化的CYP2E1基因敲除小鼠中恢复。慢性酒精喂养后高水平人细胞色素P450_2E_1敲击小鼠的肝脏毒性和氧化/亚硝化应激增加。乙醇喂养的CYP2E1敲门小鼠似乎是一种有效的酒精性肝毒性的口服模型。野生型、基因敲除小鼠和敲除小鼠分别饲喂高脂饲料1~6周。对照组将与葡萄糖配对喂养。AIM1旨在评估CYP2E1在乙醇喂养的敲击小鼠中的高表达导致肝脏毒性的机制，并将包括氧化/亚硝化应激、线粒体功能障碍、CYP2E1、iNOS、TNF1、MAPK和NF：B的检测。AIM2旨在评估CYP2E1促进酒精诱导脂肪肝的途径，包括检测SREBP-1c和PPAR1等转录因子的水平，关键和限速脂肪生成和脂解酶的含量；将在喂饲乙醇或葡萄糖的野生型、CYP2E1基因敲除和CYP2E1敲除小鼠中测定脂肪酸氧化速率。相对于乙醇诱导的脂肪变性和肝毒性，将确定下游CYP2E1效应器的变化时间进程。在野生型和敲击型小鼠中，我们将测定细胞色素P450_2E_1、诱导型一氧化氮合酶、氧化/亚硝化应激、MAPK、线粒体功能障碍、肿瘤坏死因子1的合成对酒精性脂肪变性和肝损伤的影响。我们相信，这些实验将提供CYP2E1促进乙醇诱导的肝损伤和脂肪肝的分子机制，并可能进一步开发新的治疗设计，以治疗或最大限度地减少乙醇诱导的肝损伤的进展，这是RFA-AA-006的既定目标。 与公共卫生相关：酒精引起的肝毒性和脂肪肝的机制还不完全清楚。随着细胞色素P450基因敲除小鼠和人源化的细胞色素P450基因敲除小鼠的新用途，已经明确了细胞色素P4502基因在酒精毒性作用中的作用。确定酒精/CYP2E1作用的下游效应分子将对开发治疗酒精性肝损伤和脂肪肝的干预措施非常有用。

DESCRIPTION (provided by applicant): The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces "oxidative stress". We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to chronic ethanol induced hepatotoxicity and steatosis. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or p38 MAPK, a decline in activation of NF-:B and synthesis of survival factors, and mitochondrial dysfunction are downstream mediators of the CYP2E1 potentiated hepatotoxicity. We propose that such downstream mediators from CYP2E1 in association with ethanol-induced elevation of TNF1, homocysteine and ER stress activate lipogenic transcription factors and enzymes while decreasing activation of lipolytic transcription factors and enzymes, thereby resulting in fatty liver. Preliminary data in support of these hypotheses are the findings that ethanol-induced fatty liver is blunted in CYP2E1 knockout mice and restored in humanized CYP2E1 knockin mice. Hepatotoxicity and elevated oxidative/nitrosative stress in found in the CYP2E1 knockin mice with high levels of human CYP2E1 after chronic ethanol feeding. The ethanol-fed CYP2E1 knockin mice appear to be an effective ORAL model of alcohol-induced hepatotoxicity. Wild type, CYP2E1 knockout and CYP2E1 knockin mice will be fed the high fat Lieber-DeCarli diet for varying times, e.g. 1 to 6 weeks. Controls will be pair-fed with dextrose. AIM1 is designed to evaluate mechanisms by which elevated expression of CYP2E1 causes hepatotoxicity in the ethanol-fed CYP2E1 knockin mice and will include assays of oxidative/nitrosative stress, mitochondrial dysfunction, CYP2E1, iNOS, TNF1 , MAPK and NF:B. AIM 2 is designed to evaluate pathways by which CYP2E1 contributes to alcohol-induced fatty liver, including assays of levels of transcription factors such as SREBP-1c and PPAR1, content of key and rate- limiting lipogenic and lipolytic enzymes; rates of fatty acid oxidation will be determined in the wild type, CYP2E1 knockout and CYP2E1 knockin mice fed ethanol or dextrose. The time course of changes in the downstream CYP2E1 effectors will be determined relative to that for ethanol-induced steatosis and hepatotoxicity. The effect of inhibitors of CYP2E1, iNOS, oxidative/nitrosative stress, MAPK, mitochondrial dysfunction, TNF1 production on ethanol-induced steatosis and liver injury in wild type and CYP2E1 knockin mice will be determined. We believe these experiments will provide molecular mechanisms by which CYP2E1 promotes ethanol-induced liver injury and fatty liver and may further the development of new therapeutic designs to treat or minimize progression of ethanol-induced liver injury, a stated goal of RFA-AA-006. PUBLIC HEALTH RELEVANCE: Mechanisms responsible for alcohol-induced hepatotoxicity and fatty liver are not fully understood. With the novel use of CYP2E1 knockout mice and humanized CYP2E1 knockin mice, a role for CYP2E1 in these toxic actions of alcohol has been defined. Identification of downstream effectors of alcohol/CYP2E1 actions would be very informative in developing therapeutic interventions against alcohol-induced liver injury and fatty liver.