Regulation And Biological Role Of Ethanol Inducible Cyto

在人类和动物模型中，饮酒和某些病理生理条件，如禁食和糖尿病，会增加乙醇诱导的细胞色素P450 2E1(CYP2E1)和其他P450酶的水平。其底物包括：乙醇、乙醛、对乙酰氨基酚(APAP)、4-羟基壬烯醛、四氯化碳、亚硝胺和长链脂肪酸，如花生四烯酸(AA)和二十二碳六烯酸(DHA)。增加的细胞色素P450-2，导致更多的活性代谢产物的产生，同时减少细胞抗氧化剂，如谷胱甘肽。活性代谢物包括：乙醛、活性氧、自由基代谢物和过氧化脂质。因此，细胞或组织中的细胞或组织中的CYP2E1水平升高，特别是在其他因素协同作用的情况下，更容易受到损伤或细胞死亡的影响。然而，对于不同底物引起的毒性的分子信号机制还没有进行详细的研究。在过去的三四年里，我们一直在研究酒精和其他CYP2E1底物对细胞损伤的机制。我们最初的假设是，CYP2E1底物及其代谢产物将激活与细胞死亡途径相关的c-jun氨基末端蛋白激酶(JNK)和p38丝裂原激活蛋白(MAP)激酶，而它们将抑制细胞生存途径中涉及的酶。因此，我们专门研究了暴露于细胞色素P450_2E_1底物后JNK和p38激酶的时间依赖性激活及其在细胞死亡中的作用。本实验室最近的研究结果表明，三种细胞色素P450-2E1底物(APAP、4-羟基壬烯醛和四氯化碳)通过选择性地激活JNK相关的细胞死亡途径而导致细胞凋亡。在过去的一年里，我们继续调查其他CYP2E1底物，如DHA和乙醇是否以类似的机制导致细胞损伤。结果表明，DHA可诱导转染细胞色素P421基因的人肝癌细胞株(E47)发生时间和剂量依赖性的凋亡。相比之下，DHA对不含CYP2E1cDNA的对照HepG2细胞(C34)的损伤明显较小。DHA诱导的E47 HepG2细胞损伤后，细胞色素c的释放和执行细胞凋亡的关键酶caspase3的激活。分别用氯甲唑和槲皮素抑制细胞色素P4502E1和JNK，可显著降低DHA对E47细胞的损伤，提示细胞色素P4502E1DHA代谢和JNK活化在DHA介导的细胞死亡中起重要作用。此外，细胞死亡率与E47肝癌细胞中积累的过氧化脂质水平无关。因此，我们的结果表明，在导致细胞死亡方面，早期的信号事件可能比稳态的过氧化脂质水平更重要。此外，我们还对乙醇引起细胞死亡的机制进行了研究。乙醇暴露通过激活HCT-116细胞和SK-N-SH神经母细胞瘤细胞中的JNK和p38蛋白而导致细胞死亡。我们的初步结果显示，选择性地抑制JNK或p38-激酶可显著抑制乙醇诱导的结肠细胞损伤，提示联合激活JNK和p38-K介导的细胞损伤的重要性。这一结果类似于过氧化氢、紫外线和X射线照射等其他凋亡刺激以及包括肿瘤坏死因子α在内的促炎细胞因子导致的细胞死亡机制，所有这些因素都以协同方式激活p38激酶和JNK。因此，不同的靶细胞类型和不同的CYP2E1底物对细胞损伤的机制不同。

Alcohol drinking and certain pathophysiological conditions such as fasting and diabetes increase the levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and other P450 enzymes in humans and animal models. The substrates of CYP2E1 include: ethanol, acetaldehyde, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, nitrosamines, and long chain fatty acid such as arachidonic acid (AA) and docosahexaenoic acid (DHA). Increased CYP2E1 leads to the production of more reactive metabolites of CYP2E1 substrates while reducing cellular anti-oxidants such as glutathione. The reactive metabolites include: acetaldehyde, reactive oxygen species, free radical metabolites and lipid peroxides. Therefore, cells or tissues with increased CYP2E1 become more susceptible to damage or cell death, especially in the presence of an additional factor working in a synergistic manner. However, the molecular signaling mechanisms for the toxicities caused by various CYP2E1 substrates have not been studied in detail. During the last three or four years, we have been studying the mechanism of cell damage caused by alcohol and other CYP2E1 substrates. Our initial hypotheses were that CYP2E1 substrates and their metabolites would activate the c-Jun N-terminal protein kinase (JNK) and p38 mitogen activated protein (MAP) kinase associated with the cell death pathway while they would suppress the enzymes involved in the cell survival pathway. Therefore, we specifically investigated the time-dependent activation and the role of the JNK and p38 kinase in cell death after exposure to CYP2E1 substrates. Recent results from this laboratory showed that three CYP2E1 substrates (APAP, 4-hydroxynonenal, and carbon tetrachloride) cause apoptosis through selective activation of the JNK-related cell death pathway. During this past year, we continued to investigate whether other CYP2E1 substrates such as DHA and ethanol cause cell damage in a similar mechanism. Our results showed that DHA causes time and dose-dependent apoptosis of HepG2 hepatoma cells (E47) transfected with CYP2E1 cDNA. In contrast, DHA causes significantly less damage to control HepG2 cells (C34) without CYP2E1 cDNA. DHA-induced damage to E47 HepG2 cells was followed by cytochrome c release and activation of caspase 3, a critical enzyme in executing apoptosis. Inhibition of CYP2E1 and JNK by chlormethiazole and quercetin, respectively, markedly reduce the rate of DHA-mediated damage to E47 cells, suggesting the important role of CYP2E1 metabolism of DHA and JNK activation in DHA-mediated cell death. Furthermore, the cell death rate did not correlate with the levels of lipid peroxides accumulated in E47 hepatoma cells. Our results, therefore, demonstrate that the early signaling event may be more important than the steady state levels of lipid peroxides in causing the cell death. In addition, we studied the mechanism of cell death caused by ethanol. Ethanol exposure caused cell death in HCT-116 colon cells and SK-N-SH neuroblastoma cells by activating the JNK and p38 kinases in both cells. Our preliminary results showed that selective inhibition of JNK or p38 kinase by their respective inhibitor markedly suppressed the rate of ethanol-induced cell damage in the colon cells, suggesting the importance of co-activation of JNK and p38-kinase mediated cell damage. This result is similar to the mechanism of cell death caused by other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor alpha, all of which activate p38 kinase along with the JNK in a coordinate fashion. Therefore, the mechanism of cell damage caused by various CYP2E1 substrates differ, depending on the type of target cells and each CYP2E1 substrate.