Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1promoter activity by hypoxic agents

Since hypoxia-inducible factor-1 alpha (HIF-1 alpha) and the arglhydrocarbo n receptor (AhR) shared the AhR nuclear translocator (Arnt) for hypoxia- an d AhR-mediated signaling, respectively, it was possible to establish the hy pothesis that hypoxia could regulate cytochrome P450 1a1 (Cyp1a1) expressio n. 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 D NA, 1.6 kb was cloned into pet? expression vector in order to construct pmC yp1a1-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 v arious hypoxic agents such as 1-100 mu M cobalt chloride, 1-100 mu M picoli nic acid, and 1-100 mu M desferrioxamine. Luciferase activity of the report er gene was measured from pmCyp1a1-Luc-transfected Hepa-I cell lysate which contains 2 mu g total protein using luciferin as a substrate. Hypoxic agen ts such as cobalt chloride, picolinic acid, and desferrioxamine showed inhi bition of luciferase activity that was induced by 1-nM TCDD treatment in a dose-and time-dependent manner. Concomitant treatment of 150 mu M ferrous s ulfate with 1-100 mu M desferrioxamine or 1-100 mu M picolinic acid recover ed luciferase activity from that inhibited by hypoxic agents or induced by TCDD. These data demonstrated that iron-chelating and hypoxic agents inhibi ted dioxin-induced Cyp1a1 transcription in Hepa-I cells. Thus, we might sug gest that hypoxia inhibits TCDD-induced Cyp1a1 expression due to the compet ition between HIF-1 alpha and the AhR for the Arnt in Hepa-I cells. (C) 200 0 Elsevier Science Inc.