The influence of nickel and cobalt on putative members of the oxygen-sensing pathway of erythropoietin-producing HepG2 cells.
Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 microM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 microM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The .OH levels decreased in cobalt-treated (to 81%) as well as in nickel-treated (to 67%) HepG2 cells, accompanied by an increase of EPO expression to 167% and 150%, respectively. Our results underline the importance of .OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins.
Porwol
T
T
Max Planck Institut für molekulare Physiologie, Dortmund, Germany.
Ehleben
W
W
Zierold
K
K
Fandrey
J
J
Acker
H
H
D006528:Carcinoma, Hepatocellular; D002451:Cell Compartmentation; D003035:Cobalt / Q000494:pharmacology*; D004347:Drug Interactions; D004577:Electron Probe Microanalysis; D004921:Erythropoietin / Q000096:biosynthesis*; D016166:Free Radical Scavengers; D006420:Hemeproteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019216:Metals, Heavy / Q000302:isolation & purification; D018613:Microscopy, Confocal; D009532:Nickel / Q000494:pharmacology*; D010100:Oxygen / Q000494:pharmacology*; D010101:Oxygen Consumption; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013053:Spectrophotometry; D014407:Tumor Cells, Cultured
C045076:Fenton's reagent; D016166:Free Radical Scavengers; D006420:Hemeproteins; D019216:Metals, Heavy; D017382:Reactive Oxygen Species; D004921:Erythropoietin; D003035:Cobalt; D009532:Nickel; D006861:Hydrogen Peroxide; D007501:Iron; D010100:Oxygen