ETHANOL AND ACETALDEHYDE EFFECTS ON LIVER FUNCTION

乙醇和乙醛对肝功能的影响

• 批准号: 2682954
• 负责人: ARTHUR I CEDERBAUM
• 金额: $ 26.49万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-29 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2682954
• 关键词: NAD(H) phosphate; acetaldehyde; adduct; alcoholism /alcohol abuse; antioxidants; chelating agents; cytochrome P450; ethanol; free radical oxygen; hepatotoxin; iron; isozymes; laboratory rat; liver function; liver metabolism; metal complex; microsomes; mitochondria; nicotinamide adenine dinucleotide; oxidative stress; tissue /cell culture; unsaturated fatty acids; xanthine oxidase

The overall goal is to evaluate the role of reactive oxygen intermediates (ROl) in the molecular mechanisms by which ethanol is toxic. Major focus will be on the role of P4502E1, interaction with iron, and reactivity of NADH and NADPH as cofactors. Specific Aim 1 - Our laboratory developed a HepG2 cell line which stably expresses human P4502E1. Ethanol was toxic to these cells, but not to control cells. These cells appear to represent a novel model to assess the role of P4502E1 or ROl generated during the metabolism of ethanol by P4502E1 in the hepatotoxic actions of ethanol. Studies will be carried out to characterize ethanol toxicity, effect of P4502E1 inhibitors, antioxidants, iron chelators, enrichment of cell membranes with PUFA and the ability of ethanol, acting via P4502E1,to produce a state of oxidative stress: Lipid, protein, and DNA targets for ROl produced by ethanol will be studied. Acetaldehyde and lipid adducts will be immunochemically detected. A final study will be to assess whether ethanol toxicity involves an apoptotic mechanism; if so, protection by BCl-2 will be studied. Specific Aim 2 - The role of NADH-cyt b5 reductase, cyt b5, and cyt P450 in the NADH-dependent production of ROl by microsomes will be studied employing reconstituted systems containing the purified enzymes, and inhibitors and antibodies against these enzymes. Specific Aim 3 - The interaction of NADPH-P450 reductase and P4502E1 with various ferric complexes to produce ROl will be investigated to test the hypothesis that the ability of the reductase versus P450 to interact with iron is not only dependent upon the chelated form of the iron, but also the concentration of iron. Specific Aim 4 - The production of ROl by microsomes and mitochondria isolated from periportal and pericentral hepatocytes of control and ethanol-treated rats will be determined since P4502E1 is present at highest levels in the PC zone and ethanol injury originates here. Specific Aim 5 - Studies will be carried out to compare the production of ROl by human P4502E1 with other human P450 isozymes. Ethanol toxicity and the role of 2E1 and ROl will be studied in immortalized normal human liver cells. These studies will help define the role of P4502E1 and ROl in the mechanism by which ethanol is hepatotoxic, help to design therapeutic interventions to prevent or ameliorate this toxicity, and provide basic mechanistic information of value to the oxygen radical and P450 areas of research.

总体目标是评估活性氧中间体的作用 （ROL）在乙醇有毒的分子机制中。主要重点 将扮演P4502E1的角色，与铁的相互作用以及 NADH和NADPH作为辅助因子。 特定目标1-我们的实验室开发了HEPG2细胞系，稳定 表达人P4502E1。乙醇对这些细胞有毒，但对 控制细胞。这些细胞似乎代表了一种新型模型，以评估 P4502E1或在乙醇代谢过程中产生的P4502E1或ROL的作用 P4502E1在乙醇的肝毒性作用中。研究将进行 为了表征乙醇毒性，P4502E1抑制剂的作用， 抗氧化剂，铁螯合剂，用PUFA和PUFA的细胞膜富集 通过P4502E1作用的乙醇产生氧化状态的能力 压力：乙醇产生的ROL的脂质，蛋白质和DNA靶标 被研究。乙醛和脂质加合物将是免疫化学的 检测到。最终研究将是评估乙醇毒性是否 涉及凋亡机制；如果是这样，Bcl-2的保护将是 研究。 特定目标2- NADH -CYT B5还原酶，Cyt B5和Cyt P450的作用 在NADH依赖性生产中，将研究微粒体的ROL 采用包含纯化酶的重构系统，并 针对这些酶的抑制剂和抗体。 特定目标3- NADPH -P450还原酶和P4502E1的相互作用与 将研究各种生产ROL的铁配合物，以测试 假设还原酶与P450与P450相互作用的能力 铁不仅取决于铁的螯合形式，还取决于 铁的浓度。 特定目标4-微粒体和线粒体的ROL产生 从对照的周围和周围和腹膜肝细胞中分离出来 由于P4502E1存在于 PC区域和乙醇损伤的最高水平起源于此。 具体目标5-将进行研究以比较 人类P4502E1与其他人类P450同工酶。乙醇毒性和 2E1和ROL的作用将在永生的正常人肝脏中进行研究 细胞。 这些研究将有助于定义P4502E1和ROL在 乙醇具有肝毒性的机制，有助于设计治疗 预防或改善这种毒性并提供基本的干预措施 氧自由基和P450区域的有价值信息 研究。