Regulation of Drug Metabolizing Enzyme Ontogeny

描述（由申请人提供）：在人类发育过程中，生理和分子参数发生显著变化，影响对环境毒物和治疗药物的不同反应。我们实验室先前的研究表征了几种主要的I相和II相酶在体外的人类肝脏发育表达模式。在分子水平上，在肝脏I相异生物质代谢酶中，细胞色素P4503 A（CYP 3A）和含黄素单加氧酶（FMO）基因家族中观察到最显著的变化。在这两种情况下，观察到在出生时或出生前后发生的胎儿和成人酶形式之间的过渡。然而，在这两种情况下，观察到相当大的差异。尽管有记录，但对调节这些发育过程的机制知之甚少，但意外的药物不良事件（例如，响应西沙必利治疗时对室性心动过速的敏感性）。拟议研究的总体目标是更好地了解个体发育期间调控CYP 3A和FMO转换的特定因素，并开始探索这些因素如何影响人类健康。这一目标将通过以下目的实现：使用多个互补实验系统确定调节人肝CYP 3A和FMO个体发生的分子机制，包括HepG 2细胞培养物、原代胎肝细胞和成体肝细胞培养物以及新型小鼠移植模型，其中已证明人胎肝细胞移植到小鼠宿主中后会经历时间依赖性分化和成熟为成体表型。实验将使用siRNA来调节细胞培养模型中的可疑转录因子，体外DNA结合测定来探索转录因子结合的时间变化，用先前表征的胎儿和成人组织样品进行亚硫酸氢盐测序来确定DNA甲基化的变化是否有助于表达变化，和染色质免疫沉淀，以验证体内转录因子结合，并确定染色质结构变化在控制所观察到的表达模式转变中的作用。最后，雷尼替丁将用作探针药物，以：a）确定体内人FMO 3的个体发育特征; B）确定与成人相比，大龄青少年体内人FMO代谢能力是否较低;以及c）确定先前表征的FMO 3遗传变异体在体内的功能影响。将使用高灵敏度LC/MS/MS测定法对患者志愿者的生物样本中的雷尼替丁及其N-氧化代谢物进行定量。这些研究的完成将对我们了解这两个基因家族做出重大贡献，包括它们对药物代谢、药物不良事件和个体毒物敏感性的贡献，特别是在儿科人群中。这些知识将是非常宝贵的修改风险政策，以尽量减少/避免不良药物事件和毒性，由于环境暴露的儿童。在人类发育过程中，生理和分子参数都发生了巨大变化，这些变化影响着对环境毒物和治疗药物的不同反应。公共卫生相关性：完成拟议的研究将作出重大贡献，我们的知识的两个酶的家庭重要的药物和毒物处置，已知在发展过程中经历显着的表达模式的转变。这些知识将是非常宝贵的修改风险政策，以尽量减少/避免不良药物事件和毒性，由于环境暴露的儿童。

DESCRIPTION (provided by applicant): Dramatic changes occur in both physiological and molecular parameters during human development that influence differential responses to environmental toxicants and therapeutics. Previous studies in our laboratory characterized the human hepatic developmental expression pattern of several major phase I and phase II enzymes in vitro. At the molecular level and among the hepatic phase I xenobiotic metabolizing enzymes, the most dramatic changes are observed in the cytochrome P4503A (CYP3A) and flavin-containing monooxygenases (FMO) gene families. In both, a transition is observed between fetal and adult enzyme forms that occur at or around birth. However, in both instances, considerable variability is observed. Little is known regarding the mechanisms regulating these developmental processes despite documented, but unexpected adverse drug events (e.g., sensitivity to ventricular tachycardia in response cisapride therapy) that have resulted from these temporal-specific transitions. The overall objective of the proposed studies is to better understand specific factors regulating the CYP3A and FMO transition during ontogeny and begin exploring how these factors affect human health. This objective will be achieved with the following aims: Determine molecular mechanisms regulating human hepatic CYP3A and FMO ontogeny using multiple, complementary experimental systems, including HepG2 cell culture, primary fetal and adult hepatocyte cultures, and a novel mouse transplantation model in which human fetal hepatocytes transplanted into a mouse host have been shown to undergo a time-dependent differentiation and maturation to an adult phenotype. Experiments will use siRNA to modulate suspected transcription factors in the cell culture models, in vitro DNA binding assays to explore temporal changes in transcription factor binding, bisulfite sequencing with previously characterized fetal and adult tissue samples to determine whether or not changes in DNA methylation contribute to expression changes, and chromatin immunoprecipitation to verify transcription factor binding in vivo and to determine what role chromatin structural changes have in controlling the observed transition in expression patterns. Finally, ranitidine will be used as a probe drug to: a) determine the ontogenic profile of human FMO3 in vivo; b) determine whether in vivo human FMO metabolic ability is lower in older adolescents compared to adults; and c) determine the functional impact of previously characterized FMO3 genetic variants in vivo. Ranitidine and its N-oxide metabolite will be quantified in biological specimens from patient volunteers using a highly sensitive, LC/MS/MS assay. Completion of these studies will make a significant contribution to our knowledge of these two gene families, including their contribution to drug metabolism, adverse drug events, and individual toxicant susceptibility, particularly in the pediatric population. This knowledge will be invaluable in modifying risk policies to minimize/avoid adverse drug events and toxicity due to environmental exposures in children. Dramatic changes occur in both physiological and molecular parameters during human development that influence differential responses to environmental toxicants and therapeutics. Public Health Relevance: Completion of the proposed studies will make a significant contribution to our knowledge of two enzyme families important for drug and toxicant disposition that are known to undergo significant transitions in expression patterns during development. This knowledge will be invaluable in modifying risk policies to minimize/avoid adverse drug events and toxicity due to environmental exposures in children.