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

Since hypoxia-inducible factor-1alpha (HIF-1alpha) 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 pmCyp1a1-Luc. Hepa-I cells were transfected with pmCyp1a1-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 microM cobalt chloride, 1-100 microM picolinic acid, and 1-100 microM desferrioxamine. Luciferase activity of the reporter gene was measured from pmCyp1a1-Luc-transfected Hepa-I cell lysate which contains 2 microgram 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 microM ferrous sulfate with 1-100 microM desferrioxamine or 1-100 microM 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-1alpha and the AhR for the Arnt in Hepa-I cells.