title: Effect of diquat on the antioxidant system and cell growth in human neuroblastoma cells.
abstract: Oxidative stress elicits an adaptive antioxidant response, which varies with tissue type. Diquat, a potent redox cycler that generates reactive oxygen species, has been used to study oxidative stress; however, its effect on the antioxidant system has not been characterized in neuronal cells. Accordingly, we measured antioxidant parameters and cell growth in human neuroblastoma SH-SY5Y cells cultured for 48 h in medium containing 5, 10, or 25 microM diquat dibromide or phosphate-buffered saline. Viable cells were assayed for glutathione (GSH) and activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPX), and glucose-6-phosphate dehydrogenase (GPDH). Mitochondrial function was evaluated by glutamate dehydrogenase (GDH) activity and MTT reduction. Diquat caused a marked concentration-related decrease in viable cell count ( by 26, 51, and 87% at 5, 10, and 25 microM diquat). Cell viability was only affected at 10 and 25 microM diquat and did not fully account for the decreased viable cell count. Concentration-related increases also occurred with GSH levels and a majority of antioxidant enzymes activities; however, the mode and magnitude varied with parameter. Increases in GSH, CAT, SOD, and GR were maximal at 25 microM diquat (to 3-, 6-, 2-, and 1.5-fold control values, respectively). GPDH activity was maximal at 10 microM diquat and then decreased to 86% of control activity at 25 microM diquat. GPX activity showed a concentration-related decrease (to 35% of control). Activity of the mitochondrial enzyme GDH increased 3-fold at 25 microM diquat, along with a lesser increase in MTT reduction. We conclude that diquat reduces cell growth in neuroblastoma cells and induces an adaptive antioxidant response, which are concentration dependent and occur at sublethal concentrations. At higher concentrations, diquat alters mitochondrial function and becomes increasingly toxic.
authors.affiliation: Safety Assessment, GlaxoSmithKline Pharmaceuticals, Park Road, Ware, Herts, SG12 ODP, United Kingdom.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Slaughter
authors.forename: Hansa
authors.initials: H
authors.lastname: Thakkar
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000975:Antioxidants / Q000378:metabolism*; D001932:Brain Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004178:Diquat / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D006540:Herbicides / Q000633:toxicity*; D006801:Humans; D007202:Indicators and Reagents; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
chemical_list: D000975:Antioxidants; D006540:Herbicides; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D004178:Diquat; C022616:thiazolyl blue; D005978:Glutathione