Abstract

Studies have been carried out to establish an experimental in vitro model system for routine testing of oxidative stress inducers through biochemical analysis using human breast carcinoma (MCF-7) cell line. Hydrogen peroxide (H₂O­₂) has been chosen as a test chemical oxidant to assess the level of induced glutathione (GSH), lipid peroxidation (LPO), superoxide dismutase (SOD), catalase, lactate dehydrogenase (LDH) release and cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays in MCF-7 cells. Cells were exposed with H₂O₂ in the range of 0.1 to 1.6 mM in MEM culture medium up to 24 h. The sensitivity of the system was examined by determining the dose response curve for induction of mitochondrial activity and growth inhibition. The concentrations of H₂O₂ above 0.5 mM were found to be cytotoxic, whereas, lower concentrations did not cause any significant decrease in cell viability. Results of the study showed a decrease in GSH level at 12 and 24 h (39 and 44% of control) and maximum increase (60% of control) in LPO at 24 h. In case of catalase and SOD, a concentration of 0.5 mM of H₂O₂ was found instantly effective and caused reduction in activity within 2 h, with which decreases significantly up to 24 h. The results indicate that the H₂O₂ induced oxidative stress mediated cytotoxicity in MCF-7 cells and usefulness of these cell types as sensitive biological system for routine testing of chemical oxidative stressors.  

Key words: Hydrogen peroxide (H₂O₂), MCF-7 cells, Cytotoxicity oxidative stress.

Abbreviation

Abbreviations: PD, Parkinson’s disease; ROS, reactive oxygen species; MEM,minimum essential medium; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NRU, neutral red uptake;PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; EDTA, ethylenediaminetetraacetic acid; DTNB, 5,5’-dithionitrobenzoic acid;LPO, lipid peroxidation; TBARS, thiobarbituric acid-reactive substances; TBA,thiobarbituric acid; SOD, superoxide dismutase; PMS, phenozine methosulphate;NBT, nitroblue tetrazolium; NADH, nicotinamide adenine dinucleotide; GSH,glutathione.