Both oxidative stress-dependent and independent effects of amyloid β protein are detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay has been widely used for evaluating amyloid beta protein (A beta) toxicity. However, the potency of A beta in inhibiting cellular MTT reduction and the underlying mechanism have been reported with some discrepancies among researchers. To understand what makes such discrepancies, the effect of A beta detected by MTT reduction assay was re-examined in detail by using cultured rat hippocampal neurons. Micromolar concentrations (> 10 mu M) of A beta caused a decrease in cell viability, which resulted in a decrease in MTT reduction per well regardless of assay time. The micromolar A beta-induced decrease of cellular MTT reduction was significantly attenuated by antioxidants (catalase, propyl gallate or Trolox). On the other hand, nanomolar A beta did not affect cellular MTT reduction activity at an initial stage of assay (<1 h), and decreased the total production of MTT formazan by accelerating the exocytosis of MTT formazan when MTT assay was performed for a longer time (> 2 h). The assay rime-dependent, nanomolar A beta-induced decrease of cellular MTT reduction was not at all affected by antioxidants. Furthermore, subtoxic concentration of H2O2 failed to mimic the effect of nanomolar A beta on MTT reduction. These results indicate that micromolar A beta-induced, oxidative cell death is detected by MTT assay regardless of assay time, whereas nanomolar A beta-induced acceleration of MTT formazan exocytosis is not mediated by oxidative stress and detected only when MTT assay is performed for a longer time. The time of MTT assay should be properly chosen depending on the purpose of the study. (C) 1999 Elsevier Science B.V. All rights reserved.