Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line)

Abstract

This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO3) in the range of 10–1000 µM for 24 h and thereafter the cytotoxic effects of NaVO3 were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO3 dose (=50 µM). Also, NaVO3 cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO3 at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO3 concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO3 on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO3-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO3 cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity, the XTT and resazurin assays can also be advocated for NaVO3 cytotoxicity assessment.