Regulation And Biological Role Of Ethanol Inducible Cyto

乙醇诱导细胞的调控及生物学作用

• 批准号: 6676953
• 负责人: BYOUNG-JOON SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6676953
• 关键词: DNA damage; JUN kinase; acetaldehyde; adduct; alcoholic hepatitis; animal food; apoptosis; arachidonate; aromatic hydrocarbon receptor; carbon tetrachloride poisoning; cytochrome P450; cytotoxicity; drug withdrawal; enzyme inhibitors; enzyme mechanism; enzyme substrate; ethanol; gene induction /repression; genetic regulatory element; genetic transcription; immunocytochemistry; laboratory rat; nutrition related tag; oxidative stress; protein degradation; ubiquitin

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.

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。CYP2E1的底物包括：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）。CYP2E1增加导致CYP2E1底物产生更多反应性代谢物，同时减少细胞抗氧化剂，如谷胱甘肽。反应性代谢物包括：乙醛、活性氧、自由基代谢物和脂质过氧化物。因此，CYP2E1增加的细胞或组织更容易受到损伤或细胞死亡，特别是在另一个以协同方式起作用的因子存在的情况下。然而，各种CYP2E1底物引起的毒性的分子信号机制尚未得到详细的研究。在过去的三四年里，我们一直在研究酒精和其他CYP2E1底物引起细胞损伤的机制。我们最初的假设是CYP2E1底物及其代谢物会激活与细胞死亡途径相关的c-Jun n端蛋白激酶（JNK）和p38有丝分裂原活化蛋白（MAP）激酶，而它们会抑制与细胞存活途径相关的酶。因此，我们专门研究了暴露于CYP2E1底物后JNK和p38激酶在细胞死亡中的时间依赖性激活和作用。该实验室最近的研究结果表明，三种CYP2E1底物（APAP、4-羟基壬烯醛和四氯化碳）通过选择性激活jnk相关的细胞死亡途径导致细胞凋亡。在过去的一年里，我们继续研究其他CYP2E1底物如DHA和乙醇是否以类似的机制引起细胞损伤。我们的研究结果表明，DHA可引起转染CYP2E1 cDNA的HepG2肝癌细胞（E47）的时间和剂量依赖性凋亡。相比之下，DHA对不含CYP2E1 cDNA的对照HepG2细胞（C34）的损伤要小得多。dha诱导的E47 HepG2细胞损伤后，细胞色素c释放，caspase 3激活，caspase 3是执行细胞凋亡的关键酶。氯甲基唑和槲皮素分别抑制CYP2E1和JNK可显著降低DHA介导的E47细胞损伤率，提示DHA的CYP2E1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肝癌细胞中积累的脂质过氧化物水平无关。因此，我们的研究结果表明，在导致细胞死亡的过程中，早期信号事件可能比稳态水平的脂质过氧化物更重要。此外，我们还研究了乙醇引起细胞死亡的机制。乙醇暴露通过激活HCT-116结肠细胞和SK-N-SH神经母细胞瘤细胞中的JNK和p38激酶而导致细胞死亡。我们的初步结果表明，JNK或p38激酶各自的抑制剂选择性抑制其在结肠细胞中显著抑制乙醇诱导的细胞损伤速率，这表明JNK和p38激酶共同激活介导的细胞损伤的重要性。这一结果与其他凋亡刺激（如过氧化氢、紫外线和x射线照射）和促炎细胞因子（包括肿瘤坏死因子α）引起的细胞死亡机制相似，它们都以协调的方式激活p38激酶和JNK。因此，不同CYP2E1底物引起细胞损伤的机制不同，这取决于靶细胞的类型和每种CYP2E1底物。