Both oxidative stress-dependent and independent effects of amyloid beta 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 (Abeta) toxicity. However, the potency of Abeta 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 Abeta detected by MTT reduction assay was re-examined in detail by using cultured rat hippocampal neurons. Micromolar concentrations (>10 microM) of Abeta caused a decrease in cell viability, which resulted in a decrease in MTT reduction per well regardless of assay time. The micromolar Abeta-induced decrease of cellular MTT reduction was significantly attenuated by antioxidants (catalase, propyl gallate or Trolox). On the other hand, nanomolar Abeta 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 time-dependent, nanomolar Abeta-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 Abeta on MTT reduction. These results indicate that micromolar Abeta-induced, oxidative cell death is detected by MTT assay regardless of assay time, whereas nanomolar Abeta-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.
Abe
K
K
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. kazuhoab@mol.f.u-tokyo.ac.jp
Saito
H
H
D016229:Amyloid beta-Peptides / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004396:Coloring Agents; D005089:Exocytosis / Q000187:drug effects; D005562:Formazans; D009474:Neurons / Q000187:drug effects; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013778:Tetrazolium Salts; D013844:Thiazoles
D016229:Amyloid beta-Peptides; D004396:Coloring Agents; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; C079782:MTT formazan; C022616:thiazolyl blue