Regulation & Biological Role Of Ethanol Inducible Cyp2e1

我们最近报道了c-Jun氨基末端蛋白激酶(JNK)在乙醇诱导的细胞色素P450 2E1(CYP2E1)的各种底物如对乙酰氨基酚(APAP)、4-羟基壬烯醛(HNE)、四氯化碳和长链脂肪酸以及非CYP2E1底物如曲格列酮引起的肝损伤中的重要作用。作为我们继续研究细胞凋亡机制的一部分，我们对乙醇致细胞死亡的机制进行了研究，因为乙醇对细胞或器官损伤的早期信号机制知之甚少。结果表明，乙醇可引起SK-N-SH神经母细胞瘤细胞和HCT116人结肠癌细胞死亡，并呈时间和剂量依赖性。乙醇以时间和浓度依赖的方式增加JNK和p38激酶的活性。然而，JNK和p38激酶的激活在乙醇诱导的细胞死亡中似乎都是重要的，因为分别用JNK或p38激酶的抑制剂预处理显著降低了这两种激酶的活性和乙醇诱导的细胞凋亡率。我们还观察到，乙醇增加了caspase8依赖的BID裂解水平，这是促进线粒体凋亡的关键因素。用选择性的caspase8抑制剂处理细胞后，这一事件被显著阻断。这些结果有力地表明，乙醇通过激活JNK和p38激酶以及促进Bid裂解来促进细胞凋亡。 大量饮酒会对各种细胞和组织的功能产生负面影响。这个 酒精的有害作用主要是通过酒精代谢改变氧化还原状态，产生活性醛，产生活性氧和氮物种，减少包括线粒体谷胱甘肽在内的保护性抗氧化剂，升高肿瘤坏死因子α和其他细胞毒性细胞因子，改变丝裂原活化蛋白激酶的活性等，这些酒精介导的事件导致氧化应激和损伤状态。然而，目前还不清楚哪些蛋白质对氧化修饰敏感，以及它们在酒精暴露后的氧化损伤中所起的作用。因此，我们试图确定氧化蛋白及其在乙醇介导的氧化损伤中的潜在作用。已知转导了CYP2E1的E47 HepG2肝癌细胞在乙醇和APAP等多种毒性底物作用下发生了凋亡。E47肝癌细胞线粒体经100 mM乙醇处理不同时间后，用生物素标记的碘乙酸酯处理，以检测所有蛋白质中氧化半胱氨酸残基。随后用链霉亲和素-辣根过氧化物酶检测蛋白质结合的生物素，然后增强化学发光。我们的结果显示，70、54和48 kDa的蛋白质在酒精暴露后4小时和8小时被修饰，远早于在酒精暴露后16或24小时观察到的实际细胞死亡。这些蛋白质在25、50和100 mM乙醇作用8h后也发生了修饰。由于酒精暴露后蛋白质修饰(早期观察到)和细胞凋亡(后期观察到)的时差，我们认为这些被氧化的蛋白质在乙醇介导的氧化损伤的细胞保护中可能是至关重要的。由于半胱氨酸的分子大小和活性部位相似(Cys302)，我们推测54 kDa的线粒体蛋白可能是乙醛脱氢酶2(ALDH2)。为了证实我们的假设，我们正在确定每种氧化蛋白的特性及其在酒精暴露后功能的潜在变化。我们还在分析乙醇处理的小鼠肝脏中的线粒体蛋白，以与体外结果相关联。这种方法将提供有关氧化修饰的蛋白质靶标及其在氧化损伤中的作用的新信息。

We have recently reported an important role of c-Jun N-terminal protein kinase (JNK) in apoptosis caused by various substrates of ethanol-inducible cytochrome P450 2E1 (CYP2E1) such as acetaminophen (APAP), 4-hydroxynonenal (HNE), carbon tetrachloride, and long chain fatty acids as well as a non-CYP2E1 substrate such as troglitazone, which causes hepatic damage. As a part of our continued studies on the apoptosis mechanism, we studied the mechanism of cell death by ethanol, because the early signaling mechanism of cell or organ damage by ethanol is poorly understood. Our results show that ethanol caused time- and dose-dependent cell death in SK-N-SH neuroblastoma cells and HCT116 human colon cancer cell used as models in our study. Ethanol increased the activities of JNK and p38 kinase in a time- and concentration dependent manner. However, activation of both JNK and p38 kinase seemed important in ethanol-induced cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rate of ethanol-induced apoptosis. We also observed that ethanol increases the level of caspase 8-dependent Bid cleavage, a critical factor for promoting mitochondrial apoptosis. This event was significantly blocked by pretreatment of cells with a selective inhibitor of caspase 8. These results strongly indicate that ethanol promotes apoptosis by activating the JNK and p38 kinase as well as promoting Bid cleavage. Heavy alcohol consumption negatively affects the functions of various cells and tissues. The detrimental effects of alcohol mainly result from: changes in redox state through alcohol metabolism, production of reactive aldehydes, generation of reactive oxygen and nitrogen species, reduction in protective anti-oxidants including mitochondrial glutathione, elevation of tumor necrosis factor alpha and other cytotoxic cytokines, changes in the activities of mitogen activated protein kinases, etc. These alcohol-mediated events lead to a state of oxidative stress and damage. However, it is still unknown which proteins are sensitive to oxidative modification and their roles in oxidative damage after alcohol exposure. Therefore, we sought to identify oxidized proteins and their potential role in ethanol-mediated oxidative damage. E47 HepG2 hepatoma cells with transduced CYP2E1 were known to undergo apoptosis upon exposure to various toxic substrates of CYP2E1 including ethanol and APAP. Mitochondrial fractions from E47 hepatoma cells, treated with 100 mM ethanol for different times, were treated with biotin-conjugated iodoacetate to detect oxidized cysteine residues in all proteins. Protein-bound biotin was subsequently detected by streptavidin-HRP followed by enhanced chemiluminescence. Our results showed that 70, 54, and 48 kDa proteins were modified at 4 and 8 h, long before the actual cell death observed at 16 or 24 h after ethanol exposure. Modification of these proteins also took place after exposure to 25, 50 and 100 mM ethanol for 8 h. Because of the time differences in protein modification (observed at early times) and apoptosis (at later times) after alcohol exposure, we believe that these oxidized proteins may be critically important in cellular protection against ethanol-mediated oxidative injury. Because of the similar molecular size and active site cysteine (Cys302), we speculate that the 54 kDa mitochondrial protein may be aldehyde dehydrogenase 2 (ALDH2). To confirm our hypothesis, the identity of each of the oxidized proteins and potential changes in their functions after ethanol exposure are being determined. We are also analyzing the mitochondrial proteins from ethanol-treated mouse liver to correlate with the in vitro results. This approach would provide new information about the protein targets of oxidative modification and their role in oxidative damage.