nitroarginine and picolinic-acid

Since it is known that hypoxia increases inducible nitric oxide synthase (iNOS) gene expression through the hypoxia responsive element, it was hypothesized that nitric oxide could be a mediator of hypoxia to inhibit Cyp1a1 promoter activity. In order to test this hypothesis, we have undertaken a study to examine the effects of hypoxia and nitric oxide on Cyp1a1 promoter activity in Hepa I cells. Mouse Cyp1a1 5' flanking DNA, 1.6kb, was cloned into pGL3 expression vector in order to construct pmCyp1a1-Luc. Hepa I cells were transfected with pmCyp1a1-Luc and were treated with 10(-9)M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 10(-6)-10(-4) cobalt chloride or 10(-6)-10(-4)M picolinic acid or 10(-6)-10(-4) M desferrioxamine. The luciferase activity of the reporter gene was measured from pmCyp1a1-Luc transfected Hepa I cell lysate which contains 2 microg 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 10(-9) M TCDD treatment in a dose dependent manner. Concomitant treatment of 1 mM N(G)-nitro-l-arginine with 10(-6)-10(-4) M cobalt chloride or 10(-6)-10(-4) M desferrioxamine or 10(-6)-10(-4) M picolinic acid or 10(-6)-10(-4) M sodium nitroprusside recovered luciferase activity from the TCDD induced luciferase activity that was inhibited by hypoxic agents. These data demonstrated that nitric oxide might be a mediator of iron chelating agents and hypoxic agents to inhibit dioxin induced Cyp1a1 promoter activity in Hepa I cells.

Topics: Animals; Arginine; Cells, Cultured; Cobalt; Cytochrome P-450 CYP1A1; Deferoxamine; Dioxins; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Reporter; Lipopolysaccharides; Luciferases; Mice; Nitric Oxide; Nitroarginine; Nitroprusside; Picolinic Acids; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Teratogens; Transfection