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.
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