Regulation /Role--Ethanol Inducible Cytochrome P450 2e1

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。现已知，细胞色素P450-2E_1能代谢70多种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-羟基壬烯醛、四氯化碳、包括花生四烯酸在内的长链脂肪酸和亚硝胺。细胞色素P450-2的增加导致乙醛、活性氧、自由基代谢物和过氧化脂质的产生增加，同时减少了细胞抗氧化剂，如谷胱甘肽。因此，具有升高的细胞或组织的细胞更容易受到损伤或细胞死亡，特别是在存在额外挑战的情况下。在过去，我们已经克隆了人和大鼠的CYP2E1基因，并展示了多种调控机制。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对细胞的损伤(细胞凋亡和坏死)，但它们毒性的分子信号机制尚未显示。在过去的两三年里，我们一直在研究升高的CYP2E1在酒精和其他CYP2E1底物引起的细胞损伤中的作用及其机制。与其他研究人员不同的是，我们使用了C6胶质瘤细胞，因为这些细胞含有CYP2E1，尽管数量很少，但在暴露于CYP2E1底物时会发生凋亡。因此，我们调查了我们最初的假设，即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。此外，在细胞凋亡过程中，对细胞色素P450-2E1和JNK的抑制程度升高，有效地阻止了由细胞色素P450-2E1底物引起的细胞死亡。结果表明，APAP可诱导C6胶质瘤细胞发生时间和剂量依赖性的凋亡。APAP作用15min后，JNK及其上游即刻激酶SEK-1活性迅速升高，并持续至4h，但APAP不能激活p38MAP、细胞外信号调节蛋白激酶(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，细胞色素c的释放增加，caspase3激活，caspase3是执行凋亡的关键酶。然后，我们通过三个关键实验证明了选择性JNK激活在APAP诱导的细胞凋亡中的关键作用：1)瞬时转染JNK野生型或显性阴性JNK KR突变体的cDNA，然后测量细胞死亡率；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不同影响；3)用CYP2E1抑制剂YH439预处理C6细胞，有效地阻断JNK的激活和细胞死亡，有效地抑制CYP2E1的活性和蛋白含量。这些数据表明，细胞色素P450-2依赖的代谢和JNK激活在细胞凋亡过程中起着关键作用。因此，我们选择性激活JNK的结果与其他凋亡刺激，如过氧化氢、紫外线和X射线照射，以及包括肿瘤坏死因子1α和白介素1β在内的促炎细胞因子形成鲜明对比，所有这些刺激都以协调的方式激活p38 MAPK和JNK。我们的体外结果也在体内模型中观察到，其中APAP和四氯化碳选择性和瞬时地增加JNK和SEK-1的活性。由于JNK相关途径的选择性激活在三种底物(APAP、4-羟基壬烯醛和四氯化碳)引起的细胞损伤中起着关键作用，我们正在研究其他底物(包括乙醇和花生四烯酸)是否通过类似的机制引起细胞损伤。

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.