Direct Determination of Glutathione S-transferase and Glucose-6-phosphate Dehydrogenase Activities in Cells Cultured in Microtitre Plates as Biomarkers for Oxidative Stress
Concepción García-Alfonso, Guillermo Repetto, Pilar Sanz, Manuel Repetto and Juan López-Barea
The enzymes glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G-6PDH) are implicated in the defence against oxidative stress. GST is mainly involved in the conjugation of electrophilic compounds with glutathione (GSH), although some of its isoenzymes display peroxidase activity. G-6PDH and glutathione reductase regenerate NADPH and GSH, respectively, to restore the reduced intracellular redox status following oxidative stress. Enzymatic assays for GST and G-6PDH were adapted and optimised to permit the direct in vitro determination of the effects of toxicants which induce oxidative stress in cells on microtitre plates, thereby avoiding the need to prepare cell-free extracts. To optimise the conditions of the enzymatic assays, GST activity was measured at substrate concentrations of 1–3mM GSH and 1–3mM 1-chloro-2,4-dinitrobenzene, while G-6PDH activity was measured at 7.5–37.5mM glucose-6-phosphate and 55–275mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 × 105 cells/well. The effects on GST and G-6PDH activities of three toxicants which induce oxidative stress — paraquat, iron (II) chloride and iron (III) chloride — were compared in cultured Vero cells to validate the new assays. Specific GST activity increased to 145% and 171% compared to the controls in cells treated with 5mM paraquat and 5mM iron (II) chloride, respectively, but was inhibited after exposure to 25mM iron (III) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5mM paraquat, but was inhibited in cells exposed to 5mM iron (II) chloride and 25mM iron (III) chloride.