RESUMO
OBJECTIVE: Although the pharmacokinetics of several drugs that are mainly eliminated by the CYP3A4 metabolism vary according to their dosing time, the mechanism of the variation remains poorly understood. In this study, we investigated how the 24-h oscillation in the expression of CYP3A4 mRNA was generated in hepatic cells. METHODS AND RESULTS: As brief exposure of HepG2 cells to 50% serum induced the 24-h oscillation in the expression of clock genes, serum-shocked HepG2 cells were employed as an in-vitro model to study the molecular mechanism underlying the circadian clock in the human liver. Both mRNA levels and metabolic activity of CYP3A4 in serum-shocked HepG2 cells fluctuated rhythmically with a period length of about 24 h. The oscillation in the expression of the CYP3A4 gene seemed to be the underlying cause of the rhythmic change in its metabolic activity. Luciferase reporter gene analysis and electrophoretic mobility shift assay revealed that the circadian transcriptional factor, D-site-binding protein (DBP), activated the transcription of the CYP3A4 gene by binding to the DNA sequence near the upstream of the transcriptional start site. The transactivation of the CYP3A4 gene by DBP was repressed by the E4 promoter-binding protein-4 (E4BP4), a negative component of the circadian clock. CONCLUSIONS: Results from this study suggest that DBP and E4BP4 might consist of a reciprocating mechanism in which DBP activates the transcription of the CYP3A4 gene during the time of day when DBP is abundant, and E4BP4 suppresses the transcription at other times of day. Our current findings provide a molecular link between the circadian clock and the xenobiotic metabolism.
Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Meios de Cultura , Citocromo P-450 CYP3A , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Hepatócitos/metabolismo , Humanos , Dados de Sequência Molecular , Farmacogenética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
[Structure: see text] A trichloroacetamide group was converted to an isocyanate, which was in situ captured by a variety of alcohols in the presence of CuCl and n-BuN4Cl to afford the corresponding carbamates. The scope and limitation of this transformation are also described.
