ATLA::Alternatives to Laboratory Animals

Volume 23, Number 4

Direct determination of glutathione reductase in cells cultured in microtitre plates as a biomarker for oxidative stress.

ATLA 23, 531-538, July/August 1995

Concepción García-Alfonso^1,3, Pilar Sanz¹, Guillermo Repetto¹ Manuel Repetto¹ and Juan López-Barea²

¹National Institute of Toxicology, P.O. Box 863, 41080 Seville, Spain; ²Departmento de Bioquímica y Biología Molecular, Universidad de Córdoba, Avda de Medina Azahara s/n 14071 Córdoba, Spain; ³Present address: Departamento de Bioquímica y Biología Molecular Universidad de Córdoba, Avda de Medina Azahara s/n, 14071 Córdoba, Spain

SUMMARY

A new method was developed for the direct determination of glutathione reductase (GOR) activity in Vero cells cultured in microtitre plates, avoiding cell-free extract preparation. The cells in each well were washed twice with phosphate-buffered saline, Iysed with Triton X-100, and assayed in 0.1 M potassium phosphate, pH 7.0. After subtracting oxidase activity which increased with NADPH concentration, the net GOR activity was similar at different oxidised glutathione (GSSG) and NADPH concentrations, thus confirming enzyme saturation. The optimised GOR assay used 2.5 mM GSSG and 0.12 mM NADPH; 5 mM EDTA was also added to prevent the enzyme from redox inactivation. The GOR activity was directly proportional to the number of cells per well for a wide range of cell densities, thus supporting the assay's validity for use with cultured cells.

The effects on GOR activity of three chemicals which induce oxidative stress, namely, paraquat iron (II) chloride and iron (III) chloride, were examined to validate the assay under experimental conditions. The specific enzymatic activity increased to 357% of untreated control activity in 5 mM paraquat-treated cells, and to 407% of control activity in cells exposed to 7.5 mM iron (II) chloride. By contrast, activity decreased to 56% of control activity in cells exposed to 5 mM iron (III) chloride. In conclusion, the changes in GOR activity detected in Vero cells confirm that the new assay is suitable for routine in vitro screening of toxicants capable of inducing oxidative stress.

Keywords: Vero cells, glutathione reductase assay, oxidative stress, paraquat, iron (II), iron (III), biomarker, toxicity