Regulation /Role--Ethanol Inducible Cytochrome P450 2e1

调节/作用--乙醇诱导细胞色素P450 2e1

• 批准号: 6530259
• 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/6530259
• 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. It is now known that CYP2E1 can metabolize more than 70 substrates of different chemical structures. These CYP2E1 substrates include: ethanol, acetaldehyde, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, long chain fatty acid including arachidonic acid, and nitrosamines. Increased CYP2E1 leads to elevated production of acetaldehyde, reactive oxygen species, free radical metabolites and lipid peroxides while reducing cellular anti-oxidants such as glutathione. Therefore, cells or tissues with increased CYP2E1 become more susceptible to damage or cell death, especially in the presence of an additional challenge. In the past, we have cloned the genes for human and rat CYP2E1 and demonstrated multiple regulatory mechanisms. Although other scientists already demonstrated cell damage (apoptosis and necrosis) by alcohol and acetaminophen using in vitro models, the molecular signaling mechanisms for their toxicities were not shown. During the last two or three years, we have been studying the role of elevated CYP2E1 in cell damage caused by alcohol and other CYP2E1 substrates and their mechanisms. Unlike other investigators who used CYP2E1-transfected HepG2 cells, we used C6 glioma cells since these cells contain CYP2E1, albeit in a small quantity, and undergo apoptosis upon exposure to CYP2E1 substrates. Therefore, we investigated our initial hypotheses 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 cell death pathway while they would suppress the enzymes involved in the cell survival pathway. In addition, inhibition of CYP2E1 and JNK elevated during apoptosis effectively prevents cell death caused by CYP2E1 substrates. Our results showed that APAP caused time and dose-dependent apoptosis of C6 glioma cells. Activities of JNK and its immediate upstream kinase SEK-1 were rapidly increased 15 min after APAP exposure and this effect lasted up to 4 h. However, APAP did not activate the following enzymes: p38 MAP kinase, extracellular-signal regulated protein kinase (ERK), phosphatidylinositol 3-kinase, and Akt protein serine/threonine kinase. APAP-induced cell death was preceded by elevation of cytochrome c release and activation of caspase 3, a critical enzyme in executing apoptosis. We then demonstrated the critical role of the selective JNK activation in APAP-induced apoptosis by three key experiments: 1) transient transfection of the cDNA for JNK wild type or the dominant negative JNK KR mutant followed by cell death rate measurement; 2) differential effects of cytotoxic APAP and its non-toxic analog 3-hydroxyacetanilide on JNK activation and cell death rate; and 3) efficient blockade of JNK activation and cell death by pretreatment of C6 cells with the CYP2E1 inhibitor, YH439, which effectively suppressed the levels of CYP2E1 activity and protein content. These data indicate the critical role of CYP2E1-dependent metabolism and JNK activation during apoptosis. Our result of the selective JNK activation, therefore, is in contrast with other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor 1 alpha and interleukin 1 beta, all of which activate p38 MAP kinase along with the JNK in a coordinated fashion. Our in vitro results were also observed in in vivo models where APAP and carbon tetrachloride selectively and transiently increased the activities of JNK and SEK-1. Because of the critical role of selective activation of JNK-related pathway in cell damage caused by the three CYP2E1 substrates (APAP, 4-hydroxynonenal, and carbon tetrachloride), we are investigating whether other CYP2E1 substrates (including ethanol and arachidonic acid) cause cell damage by a similar mechanism.

饮酒和某些病理生理状况（例如禁食和糖尿病）增加了人类和动物模型中乙醇诱导的细胞色素P450 2E1（CYP2E1）和其他P450酶的水平。现在众所周知，CYP2E1可以代谢70多个不同化学结构的底物。这些CYP2E1底物包括：乙醇，乙醛，对乙酰氨基酚（APAP），4-羟基苯烷，四氯化碳，包括蛛网膜烯酸和硝基胺的长链脂肪酸。 CYP2E1增加导致乙醛，活性氧，自由基代谢产物和脂质过氧化物的产生升高，同时还原细胞抗氧化剂（如谷胱甘肽）。因此，CYP2E1增加的细胞或组织变得更容易受到损伤或细胞死亡，尤其是在存在附加挑战的情况下。过去，我们克隆了人类和大鼠CYP2E1的基因，并证明了多种调节机制。尽管其他科学家已经使用体外模型证明了酒精和对乙酰氨基酚的细胞损伤（凋亡和坏死），但未显示其毒性的分子信号传导机制。在过去的两三年中，我们一直在研究CYP2E1升高在酒精和其他CYP2E1底物引起的细胞损伤中的作用。与其他使用CYP2E1转染HEPG2细胞的研究者不同，我们使用了C6胶质瘤细胞，因为这些细胞含有CYP2E1，尽管少量数量，并且在暴露于CYP2E1底物时会凋亡。因此，我们研究了我们的最初假设，即CYP2E1底物及其代谢产物将激活C-JUN N末端蛋白激酶（JNK）和p38有丝分裂原激活蛋白（MAP）激活蛋白（MAP）与细胞死亡途径相关的激酶，而它们会抑制涉及细胞存活途径的酶。另外，凋亡过程中CYP2E1和JNK升高的抑制有效地防止了CYP2E1底物引起的细胞死亡。我们的结果表明，APAP导致C6神经胶质瘤细胞的时间和剂量依赖性凋亡。 JNK及其直接上游激酶SEK-1的活性在APAP暴露后15分钟迅速增加，这种作用持续到4小时。但是，APAP并未激活以下酶：p38 MAP激酶，细胞外调节的蛋白激酶（ERK），磷脂酰肌醇3-激酶和Akt蛋白丝氨酸/苏氨酸激酶。在APAP诱导的细胞死亡之前是细胞色素C释放的升高和caspase 3的激活，Caspase 3是执行细胞凋亡的关键酶。然后，我们通过三个关键实验证明了选择性JNK激活在APAP诱导的凋亡中的关键作用：1）JNK野生型cDNA的瞬时转染或主要的负阴性JNK kr突变体，然后进行细胞死亡率测量； 2）细胞毒性APAP及其无毒类似物3-羟基乙酰苯胺对JNK激活和细胞死亡率的差异影响； 3）通过用CYP2E1抑制剂YH439对C6细胞预处理JNK激活和细胞死亡的有效阻断，这有效地抑制了CYP2E1活性和蛋白质含量的水平。这些数据表明CYP2E1依赖性代谢和JNK激活在凋亡过程中的关键作用。因此，我们选择性JNK激活的结果与其他凋亡刺激（例如过氧化氢，紫外线和X射线辐射）以及包括肿瘤坏死因子1 alpha和interleukin 1 beta在内的促炎细胞因子（包括p38 map kinkinase in Coildession Actional Feneses in Coordession）。在体内模型中也观察到了我们的体外结果，在体内模型中，APAP和四氯化碳选择性地，瞬时增加了JNK和SEK-1的活性。由于选择性激活JNK相关途径在由三种CYP2E1底物（APAP，4-羟基烯烯和四氯化碳）引起的细胞损伤中的关键作用，因此我们正在研究其他CYP2E1底物（包括乙醇和芳基酸）是否会导致细胞损伤受到类似机构的损害。