The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO₂, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO₂ nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO₂ nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO₂ concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365 nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO₂ concentrations generally higher than 50 μg/ml), induced by the TiO₂–MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.