In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2.
Copper, though essential, is highly toxic when present in excess, as in Wilson disease, a genetic disorder of hepatic copper metabolism. We hypothesized that mitochondria are a major target of copper-induced cytotoxicity in Wilson disease. We used the human hepatoma line Hep G2 to examine copper-mediated cytotoxicity and three different methods to assess organelle damage: MTT assay (mitochondria), neutral red (NR; lysosomes) and Trypan blue exclusion assay (TB; plasma membrane). For all assays, cells at approximately 60% confluence in microtitre plates were incubated with CuCl(2) (concentration range: 50-100-150-200 microM) for 24 or 48 h. Results were expressed as percent of untreated control. At 24 h, cytotoxicity as detected by NR assay was significantly higher at all concentrations of copper than for MTT or TB ( p<0.005 at all concentrations). Cytotoxicity as detected by MTT was higher than that detected by TB at all concentrations except at 200 microM (p<0.05 for 50 microM, p<0.005 for 100 microM, p = 0.001 for 150 microM). Results at 48 h were similar (NR versus others: p <0.001 MTT versus TB: NS except at 150 microM where p<0.01). We investigated reactive oxygen species (ROS) production in copper-associated hepatocytoxicity by incubating sub-confluent cells with 2('),7(')-dichlorodihydrofluorescein diacetate dye plus copper (concentration range: 0-200 microM) for 1-1.5 h. Copper, but not zinc, produced significant increases in ROS (p<0.001). In summary, Hep G2 lysosomes appeared more susceptible to Cu-mediated damage than mitochondria; the cell membrane was highly resistant to damage.
Seth
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Department of Paediatrics, University of Toronto, Room 8267, Black Wing, 555 University Avenue, Toronto, Ont., Canada M5G 1X8.
Yang
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Choi
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Sabean
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Roberts
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EA
D006528:Carcinoma, Hepatocellular / Q000473:pathology; D004396:Coloring Agents; D003300:Copper / Q000633:toxicity*; D006527:Hepatolenticular Degeneration / Q000503:physiopathology*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008247:Lysosomes / Q000473:pathology; D008928:Mitochondria / Q000473:pathology*; D009499:Neutral Red; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D014343:Trypan Blue; D014407:Tumor Cells, Cultured
D004396:Coloring Agents; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D009499:Neutral Red; D003300:Copper; C022616:thiazolyl blue; D014343:Trypan Blue