Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2.

The reduction of the tetrazolium salts, MTT and XTT, is used to estimate cell viability and proliferation. However, superoxide can also reduce tetrazolium salts to produce the absorbant formazan end products. Evidence indicates that nano-TiO2 induces superoxide formation in different mammalian cells. Therefore, studies investigating the cytological effects of nano-TiO2 may encounter misleading results when using MTT/XTT to measure viability or proliferation. In this study, cell viabilities of Chinese hamster ovary cells were assayed using MTT, XTT and the trypan blue exclusion assay following exposure to nano-TiO2. In comparison to the trypan blue exclusion assay, the MTT and XTT assays inaccurately predicted cell toxicity or overestimated cell viability respectively. XTT, in particular, appears more sensitive to superoxide than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective than that of MTT, indicating that XTT is more reactive with O2- than MTT. Therefore, using XTT or MTT for measuring cell viability or proliferation may yield inaccurate results when conditions in cultured cell increase superoxide formation.