ATLA::Alternatives to Laboratory Animals

Volume 26, Number 3

Direct determination of glutathione S-transferase and glucose-6-phosphate dehydrogenase activities in cells cultured in microtitre plates as biomarkers for oxidative stress.

ATLA 26, 321-330, May/June 1998

Concepción García-Alfonso^1,2, Guillermo Repetto¹, Pilar Sanz,¹ Manuel Repetto¹, and Juan López-Barea²

¹National Institute of Toxicology, P.O. Box 863, 41080 Seville, Spain; ²Departamento de Bioquímica u Biología Molecular, Universidad de Córdova, Avda. de Medina Azahara s/n. 14071 Córdoba, Spain

SUMMARY

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 1-3 mM GSH and 1-3 mM 1-chloro-2,4-dinitrobenzene, while G-6PDH activity was measured at 7.5-37.5 mM glucose-6-phosphate and 55-275 mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 x 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. Speck GST activity increased to 145% and 171% compared to the controls in cells treated with 5 mM paraquat and 5 mM iron (II) chloride, respectively, but was inhibited after exposure to 25 mM iron (III) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5 mM paraquat, but was inhibited in cells exposed to 5 mM iron (II) chloride and 25 mM iron (III) chloride.

Keywords: molecular biomarkers, oxidative stress, direct in vitro assay, glutathione S-transferase, glucose-6-phosphate dehydrogenase, Vero cells.