REGULATION AND BIOLOGICAL ROLE OF ETHANOL INDUCIBLE CYTOCHROME P450 2E1 (CYP2E1)

在人类和动物模型中，乙醇诱导的细胞色素P450 2 E1（CYP 2 E1）被酒精诱导。 在过去，我们已经克隆了人类和大鼠CYP 2 E1的基因，并证明了多种调节机制：出生后通过转录诱导，在酮化状态下mRNA的稳定，CYP 2 E1底物激活mRNA翻译和蛋白质稳定，YH 439通过转录抑制，四氯化碳降解mRNA和蛋白质。 慢性饮酒或某些病理状态后CYP 2 E1增加导致乙醛、活性氧、自由基代谢物和脂质过氧化物的产生增加，同时减少细胞抗氧化剂如谷胱甘肽。因此，CYP 2 E1增加的细胞或组织变得更容易受到损伤或细胞死亡。事实上，其他研究人员已经证明，在暴露于各种CYP 2 E1底物（如乙醇、对乙酰氨基酚（AAP）、四氯化碳和花生四烯酸）后，转染CYP 2 E1 cDNA的肝癌HepG 2细胞或PC 12细胞对细胞死亡的敏感性远远高于未转染的对应细胞。尽管对CYP 2 E1底物引起的细胞死亡进行了大量研究，但这些CYP 2 E1底物发挥其毒理学作用的信号传导机制尚不清楚。 因此，我们继续研究各种CYP 2 E1底物引起的细胞凋亡过程中的信号转导机制。我们一直特别感兴趣的有丝分裂原活化蛋白（MAP）激酶参与细胞凋亡过程中的早期信号转导途径和参与细胞存活途径的酶的潜在作用。 这些酶包括：c-Jun N-末端蛋白激酶（JNK）、p38 MAP激酶、细胞外信号调节蛋白激酶（ERK）、磷脂酰肌醇3-激酶、Akt蛋白丝氨酸/苏氨酸激酶及其上游激酶或下游靶蛋白，包括参与细胞凋亡/存活过程的各种半胱天冬酶和bax/bcl-2蛋白。我们最初的假设是：CYP 2 E1底物及其代谢产物会激活JNK和p38 MAP激酶，同时抑制参与细胞存活途径的酶。此外，细胞凋亡过程中升高的CYP 2 E1和其他酶的抑制剂可有效防止CYP 2 E1底物引起的细胞死亡。我们的研究结果表明，AAP或其他CYP 2 E1底物的靶细胞（C6胶质瘤和PC 12细胞）的治疗引起这些细胞的时间和浓度依赖性的凋亡细胞的DNA片段和荧光染色的凋亡细胞证明。 在这些细胞中，c-jun N-末端蛋白激酶活性（JNK）被选择性和瞬时激活后，AAP或4-羟基壬烯醛（HNE），细胞毒性脂质醛处理。 出乎意料的是，这些有毒化合物不激活p38 MAP激酶或影响ERK活性。JNK在靶细胞中的选择性和瞬时激活对于它们的凋亡是至关重要的，因为通过阻断JNK或SEK-1的显性负突变体的cDNA来阻断JNK途径显著地阻断了凋亡。此外，我们的研究结果表明，10 μ M YH 439，一种CYP 2 E1基因的转录抑制剂，不仅降低CYP 2 E1和JNK活性，而且抑制由CYP 2 E1底物引起的凋亡，表明CYP 2 E1依赖的代谢在凋亡过程中的关键作用。p38 MAP激酶的选择性抑制剂SB 203580的使用进一步证实了p38 MAP激酶在细胞凋亡中的不参与。因此，我们的研究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和白细胞介素1-β，所有这些都可以激活P38 MAP激酶沿着JNK。 我们从体外培养的细胞中得到的结果在体内模型中得到了复制，其中AAP和四氯化碳选择性地和瞬时地激活JNK及其上游激酶SEK-1和MEK。 由于JNK的选择性激活CYP 2 E1底物，我们正在研究潜在的激活磷酸蛋白磷酸酶，特异性去磷酸化磷酸-P38 MAP激酶，处理后与CYP 2 E1底物。

It is well established that ethanol-inducible cytochrome P450 2E1 (CYP2E1) is induced by alcohol drinking in humans and animal models. In the past, we have cloned the genes for human and rat CYP2E1 and demonstrated multiple regulatory mechanisms: induction via transcription after birth, mRNA stabilization in ketotic states, activation of mRNA translation and protein stabilization by CYP2E1 substrates, suppression via transcription by YH439, mRNA degradation and protein degradation by carbon tetrachloride. Increased CYP2E1 after chronic alcohol consumption or certain pathological states 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. In fact, other investigators have already demonstrated that hepatoma HepG2 cells or PC12 cells transfected with the CYP2E1 cDNA are far more sensitive to cell death than the non-transfected counterparts after exposure to various CYP2E1 substrates such as ethanol, acetaminophen (AAP), carbon tetrachloride, and arachidonic acid. Despite numerous studies on cell death by CYP2E1 substrates, the signaling mechanisms by which these CYP2E1 substrates exert their toxicological effects are unknown. Therefore, we continued to investigate the signaling mechanisms during apoptosis caused by various CYP2E1 substrates. We have been particularly interested in the potential role of mitogen activated protein (MAP) kinases involved in the early signal transduction pathway during apoptosis and the enzymes involved in the cell survival pathway. These enzymes include: c-Jun N-terminal protein kinase (JNK), p38 MAP kinase, extracellular-signal regulated protein kinase (ERK), phophatidylinositol 3-kinase, Akt protein serine/threonine kinase and their upstream kinases or downstream target proteins including various caspases and bax/bcl-2 proteins involved in cell apoptosis/survival processes. Our initial hypotheses were: CYP2E1 substrates and their metabolites would activate the JNK and p38 MAP kinase while suppressing the enzymes involved in the cell survival pathway. In addition, inhibitors of CYP2E1 and other enzymes elevated during apoptosis effectively prevent cell death caused by CYP2E1 substrates. Our results showed that treatment of target cells (C6 glioma and PC12 cells) with AAP or other CYP2E1 substrates caused time- and concentration-dependent apoptosis of these cells as evidenced by DNA fragmentation and fluorescent staining of the apoptotic cells. In these cells, c-jun N-terminal protein kinase activity (JNK) was selectively and transiently activated after treatment with AAP or 4-hydoxynonenal (HNE), a cytotoxic lipid aldehyde. Unexpectedly, these toxic compounds did not activate p38 MAP kinase or affect the ERK activity. The selective and transient activation of the JNK in target cells was critical for their apoptosis since blockade of the JNK pathway by transfecting the cDNA of a dominant negative mutant of JNK or SEK-1 significantly blocked the apoptosis. In addition, our results showed that 10 uM YH439, a transcriptional inhibitor of the CYP2E1 gene, not only reduced the CYP2E1 and JNK activities but also suppressed the apoptosis caused by CYP2E1 substrates, indicating the critical role of CYP2E1-dependent metabolism during apoptosis. The non-involvement of p38 MAP kinase in the apoptosis was further confirmed by the use of its selective inhibitor, SB203580. Our results, therefore, are in contrast with other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor alpha and interleukin 1-beta, all of which can activate P38 MAP kinase along with the JNK. Our results from in vitro cultured cells were replicated in in vivo models where AAP and carbon tetrachloride selectively and transiently activated the JNK and its upstream kinases, SEK-1 and MEK. Because of the selective activation of JNK by CYP2E1 substrates, we are investigating the potential activation of a phospho-protein phosphatase, which specifically dephosphorylates phopho-P38 MAP kinase, after treatment with CYP2E1 substrates.