Effect of ethanol on retinoid metabolism and signaling in zebrafish embryos

描述（由申请人提供）：这是一项R21探索性/开发资助申请，旨在开发量化人体组织（肝脏和食道）中酒精代谢酶及其变体（最初是酒精脱氢酶，最终是醛脱氢酶和微体乙醇氧化CYP同工酶）含量的方法。我们相信该应用符合R21指南，因为我们将使用新开发的质谱蛋白质组学方法来解决特定的目标。该应用也响应了PA-05-074，酒精诱导的组织损伤机制，因为它提供了重要的蛋白质表达信息，以支持酒精代谢酶基因型的研究，这些基因型赋予对酒精诱导的细胞/组织损伤的易感性或抗性。我们的中心假设是酒精代谢酶和变异的表达/含量决定了乙醇和乙醛在组织中的药代动力学和细胞毒理学。在目标#1中，我们将开发蛋白质组学方法，通过三重四极线性离子阱来量化组织中七种乙醇脱氢酶（ADH）同工酶的表达

DESCRIPTION (provided by applicant): This is an R21 exploratory/development grant application to develop methods to quantify the content of alcohol metabolizing enzymes and variants (initially alcohol dehydrogenase and eventually aldehyde dehydrogenase and microsomal ethanol oxidizing CYP isoenzymes) in human tissues (liver and esophagus). We believe that the application fits the R21 guidelines because we will use newly developed mass spectrometry proteomics methods to address the specific aims. The application is also in response to PA-05-074, Mechanisms of Alcohol-Induced Tissue Injury, because it provides important protein expression information to support the investigation of alcohol metabolizing enzyme genotypes that confer susceptibility or resistance to ethanol-induced cell/tissue damage. Our central hypothesis is that the expression/content of alcohol metabolizing enzymes and variants determine the pharmacokinetics and cellular toxicology of ethanol and acetaldehyde in tissues. In Aim #1, we will develop proteomics methods to quantify the expression of seven alcohol dehydrogenase (ADH) isoenzymes in tissues by triple quadrupole linear ion trap LC/MS/MS and multiple reactions monitoring analysis with appropriate peptide/protein internal standards. We will assess the feasibility of developing methods to look at the whole set of ADH, ALDH and microsomal ethanol oxidizing CYP isoenzymes involved in alcohol metabolism simultaneously. In Aim #2, we will collect and genotype human liver samples and matched normal esophagus and squamous cell carcinoma tissue samples from a local population in Indianapolis for the pilot proteomics study. The tissue samples will be genotyped at ADH and other alcohol metabolizing enzyme loci. In Aim #3, we will perform a preliminary proteomics study to look at the variability of expression/content of the seven ADH isoenzymes in the liver and esophagus tissue samples using the proteomics methods developed in Aim #1. We will identify whether there is a correlation between ADH polymorphisms and protein expression in tissues. We will estimate the individual contribution of the seven ADHs to alcohol metabolic capacity in tissue samples and the contribution of polymorphism to differences in alcohol metabolic capacity. We will assess the feasibility of performing targeted proteomics studies with tissues from other populations that express different SNPs in alcohol metabolizing enzymes.