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

乙醇诱导细胞色素 P450 2E1 (CYP2E1) 的调节和生物学作用

• 批准号: 6288632
• 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/6288632
• 关键词: DNA damage; acetaldehyde; adduct; alcoholic hepatitis; animal food; aromatic hydrocarbon receptor; carbon tetrachloride poisoning; cytochrome P450; disease /disorder model; drug withdrawal; enzyme inhibitors; ethanol; gene induction /repression; genetic regulatory element; genetic transcription; immunocytochemistry; laboratory rat; nutrition related tag; oxidative stress; protein degradation; ubiquitin

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"

我们之前已经证明了乙醇诱导细胞色素P450 2E1 （CYP2E1）的多种调控机制：通过转录诱导、mRNA稳定、mRNA翻译激活和蛋白质稳定、转录抑制、mRNA降解和蛋白质降解。我们最近报道了一种外源化合物YH439对CYP2E1基因的转录抑制。在本财政年度，我们研究了CYP2E1在长期酒精治疗的大鼠乙醛-蛋白加合物形成中的生物学作用。在酒精处理的大鼠中检测到37 kDa的乙醛加合物蛋白，而在配对喂养的对照动物中则不存在。在乙醇处理过程中，CYP2E1活性水平显著升高，而参与酒精或乙醛代谢的其他酶如酒精或醛脱氢酶等没有变化。YH439与乙醇共处理显著降低了37 kDa加合物的水平，这表明该加合物很可能是通过CYP2E1依赖机制产生的。此外，我们还检测了YH439对对乙酰氨基酚（AAP, Tylenol）等CYP2E1底物引起的C6胶质瘤细胞凋亡的保护作用。C6胶质瘤细胞相对于其他已建立的细胞系的主要优势是，我们不需要在使用之前转染CYP2E1 cDNA并选择稳定的转化子，因为CYP2E1已知在C6胶质瘤细胞中表达，尽管表达水平非常低。经经典DNA断裂证实，AAP或其他CYP2E1底物（包括乙醇）处理可引起C6细胞的时间和剂量依赖性凋亡。在C6细胞中，AAP处理后，c-jun N端蛋白激酶活性（JNK）被选择性地短暂激活。p-38蛋白激酶或丝裂原活化蛋白激酶的活性保持不变。AAP选择性激活JNK通路的机制类似于4-羟基壬烯醛（HNE）或四氯化碳（CYP2E1的另一底物）引起细胞死亡的机制。此外，YH439 （10 uM）预处理C6细胞不仅降低了CYP2E1水平，而且还阻止了aap处理后18和36小时的凋亡。与体外实验数据一致，在AAP或四氯化碳处理的小鼠肝脏中，JNK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处理CYP2E1底物（包括乙醇和花生四烯酸）时的作用。此外，我们还利用高效液相色谱法测定了CYP2E1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、细胞与分子生物学、酒精、代谢、细胞凋亡"