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巨大变化，影响对环境毒物和治疗的不同反应。我们实验室之前的研究描述了几种主要的I期和II期酶在体外的人类肝脏发育表达模式。在分子水平和肝脏I期外源代谢酶中，细胞色素P4503A （CYP3A）和含黄素单加氧酶（FMO）基因家族的变化最为显著。在这两种情况下，在出生时或出生前后观察到胎儿和成人酶形式之间的转变。然而，在这两种情况下，观察到相当大的变化。关于调节这些发育过程的机制知之甚少，尽管有文献记载，但意想不到的药物不良事件（例如，西沙比利治疗对室性心动过速的敏感性）是由这些时间特异性转变引起的。拟议研究的总体目标是更好地了解个体发育过程中调节CYP3A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。该目标将通过以下目标来实现：利用多种互补的实验系统，包括HepG2细胞培养、原代胎儿和成人肝细胞培养，以及一种新的小鼠移植模型，确定调节人类肝脏CYP3A和FMO个体发生的分子机制，在这种模型中，移植到小鼠宿主中的人类胎儿肝细胞已被证明经历了时间依赖性分化和成熟到成人表型。实验将在细胞培养模型中使用siRNA来调节可疑的转录因子，在体外DNA结合试验中探索转录因子结合的时间变化，亚硫酸盐测序与先前表征的胎儿和成人组织样本一起确定DNA甲基化的变化是否有助于表达变化。以及染色质免疫沉淀，以验证转录因子在体内的结合，并确定染色质结构变化在控制观察到的表达模式转变中所起的作用。最后，雷尼替丁将作为探针药物用于：a)确定人FMO3在体内的致癌性；b)确定大龄青少年体内FMO代谢能力是否低于成人；c)确定先前表征的FMO3遗传变异在体内的功能影响。雷尼替丁及其n -氧化物代谢物将在患者志愿者的生物标本中使用高灵敏度的LC/MS/MS测定法进行定量。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易感性的贡献，特别是在儿科人群中。这些知识在修改风险政策以减少/避免儿童环境暴露引起的药物不良事件和毒性方面将是无价的。在人类发育过程中，生理和分子参数都会发生巨大变化，从而影响对环境毒物和治疗方法的不同反应。公共卫生相关性：完成拟议的研究将对我们对药物和毒物处置重要的两个酶家族的认识做出重大贡献，这两个酶家族在发育过程中经历了显著的表达模式转变。这些知识在修改风险政策以减少/避免儿童环境暴露引起的药物不良事件和毒性方面将是无价的。

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.