Zwolak Iwona
Department of Cell Biology, Institute of Environmental Protection, John Paul II Catholic University of Lublin, Lublin, Poland
Toxicol Ind Health. 2015 Aug;31(8):677-90. doi: 10.1177/0748233713483199. Epub 2013 Mar 22.
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 (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) 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 NaVO(3) dose (=50 µM). Also, NaVO(3) 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 NaVO(3) 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 NaVO(3) 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 NaVO(3) 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 NaVO(3)-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 NaVO(3) 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 NaVO(3) cytotoxicity assessment.
本研究旨在比较五种不同的体外细胞毒性试验，以评估它们揭示钒对中国仓鼠卵巢（CHO）-K1细胞毒性的能力。将细胞暴露于10 - 1000 μM的偏钒酸钠（NaVO₃）中24小时，然后通过比色法体外试验测量NaVO₃的细胞毒性：中性红（NR）试验、2,3-双[2-甲氧基-4-硝基-5-磺基苯基]-2H-四唑-5-羧基苯胺内盐（XTT）试验、刃天青试验、磺基罗丹明B（SR-B）试验，并使用台盼蓝（TB）染色法通过显微镜评估细胞活力。在所使用的试验中，NR试验最敏感，因为它在最低的NaVO₃剂量（=50 μM）时就揭示了偏钒酸盐的细胞毒性。此外，以抑制浓度（IC）表示的NaVO₃细胞毒性在NR试验中显示出最低值。其他三种试验XTT、刃天青和SR-B试验显示出中等敏感性，在100 μM时揭示了NaVO₃的细胞毒性。为XTT、刃天青和SR-B试验计算的相应IC10和IC50值相似。TB染色法最不敏感，因为它在测试的最高NaVO₃浓度（=600 μM）时记录到了偏钒酸盐的细胞毒性。基于上述试验测量到的细胞毒性终点，可以得出结论，溶酶体/高尔基体损伤（通过NR试验测量）可能是NaVO₃对CHO-K1细胞的主要作用。线粒体的解体（通过XTT和刃天青试验评估）可能继发于溶酶体损伤。质膜通透性（用TB染色）发生在NaVO₃诱导的CHO-K1细胞毒性的后期。本研究工作获得的结果表明，NR试验可被推荐为评估CHO-K1细胞培养模型中NaVO₃细胞毒性的非常敏感的试验。考虑到试验操作的便利性以及足够的敏感性，XTT和刃天青试验也可用于NaVO₃细胞毒性评估。
