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

我们以前已经证明了乙醇诱导的细胞色素P450 2 E1（CYP 2 E1）的多种调节机制：通过转录诱导，mRNA稳定，mRNA翻译和蛋白质稳定的激活，通过转录抑制，mRNA降解和蛋白质降解。我们最近报道了外源化合物YH 439对CYP 2 E1基因的转录抑制作用。在本财政年度，我们研究了CYP 2 E1在长期接受酒精治疗的大鼠中乙醛-蛋白质加合物形成中的生物学作用。37 kDa的乙醛加合物蛋白在酒精处理的大鼠中被检测到，而在成对喂养的对照动物中不存在。在乙醇处理期间，CYP 2 E1活性水平显著升高，而参与乙醇或乙醛代谢的其他酶如乙醇或乙醛脱氢酶则无变化。YH 439与乙醇的共处理显著降低了37 kDa加合物的水平，表明该加合物最有可能通过CYP 2 E1依赖性机制产生。此外，我们测试了YH 439对醋氨酚（AAP，Tylenol）和其他CYP 2 E1底物引起的C6胶质瘤细胞凋亡的保护作用。C6神经胶质瘤细胞相对于其他已建立的细胞系的主要优点是，我们不需要在使用前检测CYP 2 E1 cDNA并选择稳定的转化体，因为已知CYP 2 E1在C6神经胶质瘤细胞中表达，尽管表达水平非常低。AAP或其他CYP 2 E1底物（包括乙醇）的处理可引起C6细胞的时间和剂量依赖性凋亡，这一点可通过经典的DNA片段化得到证明。在C6细胞中，c-jun N-末端蛋白激酶活性（JNK）被选择性地和瞬时激活后AAP处理。p-38蛋白激酶或丝裂原活化蛋白激酶的活性保持不变。AAP对JNK通路的选择性激活与CYP 2 E1的另一种底物4-羟基壬烯醛（HNE）或四氯化碳引起的细胞死亡机制相似。此外，YH 439（10 μ M）预处理C6细胞不仅降低了CYP 2 E1水平，而且还阻止了AAP处理后18和36小时观察到的细胞凋亡。与体外数据一致，JNK在用AAP或四氯化碳处理的小鼠肝脏中被选择性激活。为了进一步阐明c-jun激酶激活与培养细胞凋亡之间的关系，我们正在研究各种半胱天冬酶在包括乙醇和花生四烯酸在内的CYP 2 E1底物处理后的作用。此外，通过HPLC测定CYP 2 E1转染细胞和C6胶质瘤细胞中DNA加合物水平的变化。- 神经科学、健康与行为、分子遗传学、肝硬化、肝病学、细胞和分子生物学、酒精、代谢、细胞凋亡”

We have previously demonstrated multiple regulatory mechanisms for ethanol inducible cytochrome P450 2E1 (CYP2E1): induction via transcription, mRNA stabilization, activation of mRNA translation and protein stabilization, suppression via transcription, mRNA degradation and protein degradation. We recently reported a transcriptional suppression of CYP2E1 gene by an exogenous compound, YH439. During this fiscal year, we studied the biological role of CYP2E1 in acetaldehyde- protein adduct formation in rats chronically treated with alcohol. The 37 kDa acetaldehyde adduct protein was detected in alcohol treated rats while it is absent in pair-fed control animals. During the ethanol treatment, the level of CYP2E1 activity was significantly elevated while other enzymes such as alcohol or aldehyde dehydrogenase involved in alcohol or acetaldehyde metabolism were unchanged. Co-treatment of YH439 with ethanol markedly reduced the level of the 37 kDa adduct, suggesting that this adduct is most likely produced by a CYP2E1- dependent mechanism. In addition, we tested the protective effect of YH439 on the apoptosis of C6 glioma cells caused by acetaminophen (AAP, Tylenol) and other CYP2E1 substrates. The major advantage of C6 glioma cells over other established cell lines is that we dont need to transfect CYP2E1 cDNA and select the stable transformants prior to use, since CYP2E1 is known to be expressed in C6 glioma cells despite very low level of expression. Treatment of AAP or other CYP2E1 substrates including ethanol caused time and dose-dependent apoptosis of C6 cells as evidenced by classical DNA fragmentation. In C6 cells, c-jun N- terminal protein kinase activity (JNK) was selectively and transiently activated after AAP treatment. The activity of p-38 protein kinase or mitogen activated protein kinase remained unchanged. The selective activation of the JNK pathway by AAP is similar to the mechanisms of cell death caused by 4-hydroxynonenal (HNE) or carbon tetrachloride, another substrates of CYP2E1. Furthemore, pretreatment of YH439 (10 uM) of C6 cells not only reduced the CYP2E1 level but also prevented the apoptosis observed at 18 and 36 hr post-AAP treatment. Consistent with the in vitro data, JNK was selectively activated in the mouse liver treated with AAP or carbon tetrachloride. To further elucidate the relationship between the c-jun kinase activation and apoptosis of cultured cells, we are studying the role of various caspases upon treatment of CYP2E1 substrates including ethanol and arachidonic acid. In addition, changes in the level of DNA-adducts in the CYP2E1- transfected cells and C6 glioma cells are being determined by HPLC. - neurosciences, health & behavior, molecular genetics, cirrhosis, hepatology, cell and molecular biology, alcohol, metabolism, apoptosis"