Inhibition of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-stimulated Cyp1a1 promoter activity by hypoxic agents

Abstract

Since hypoxia-inducible factor-1α (HIF-1α) and the arylhydrocarbon receptor (AhR) shared the AhR nuclear translocator (Arnt) for hypoxia- and AhR-mediated signaling, respectively, it was possible to establish the hypothesis that hypoxia could regulate cytochrome P450 1a1 ( Cyp1a1) expression. In order to test this hypothesis, we undertook to examine the effect of hypoxia on Cyp1a1 transcription in Hepa-I cells. Mouse Cyp1a1 5′-flanking DNA, 1.6 kb was cloned into pGL3 expression vector in order to construct pm Cyp1a1-Luc. Hepa-I cells were transfected with pm Cyp1a1-Luc and treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 1–100 μM cobalt chloride, 1–100 μM picolinic acid, and 1–100 μM desferrioxamine. Luciferase activity of the reporter gene was measured from pm Cyp1a1-Luc-transfected Hepa-I cell lysate which contains 2 μg total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 1-nM TCDD treatment in a dose-and time-dependent manner. Concomitant treatment of 150 μM ferrous sulfate with 1–100 μM desferrioxamine or 1–100 μM picolinic acid recovered luciferase activity from that inhibited by hypoxic agents or induced by TCDD. These data demonstrated that iron-chelating and hypoxic agents inhibited dioxin-induced Cyp1a1 transcription in Hepa-I cells. Thus, we might suggest that hypoxia inhibits TCDD-induced Cyp1a1 expression due to the competition between HIF-1α and the AhR for the Arnt in Hepa-I cells.