Estradiol attenuation of beta-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17 beta -estradiol (beta E2) has been shown to attenuate the toxicity of be ta -amyloid peptides (A beta) in neuronal cultures with the effective conce ntration of beta E2 ranging from low nM to high muM. This study compares th e effective neuroprotective concentration of beta E2 against both A beta -m ediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective beta E2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum bet a E2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM beta E2 conferring significant protection in th e calcein AM assay but 1 muM beta E2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occu red at sub-toxic A beta concentrations and did not correlate with other mar kers of cellular viability including calcein fluorescence, dye exclusion (p ropidium iodide or trypan blue), cellular ATP levels, or reduction of anoth er tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3- (4-sulfophenyl) tetrazolium (MTS). By contrast, there was no different betw een the MTT and calcein AM assays with respect to H2O2 toxicity or the neur oprotective effectiveness of 10 nM beta E2 against H2O2 toxicity. These res ults indicate that low concentrations of beta E2 can attenuate A beta and H 2O2 toxicity in a human neuroblastoma cell line. Further, these results sug gest that the MTT assay is not an appropriate assay for the determination o f beta E2-mediated attenuation of A beta toxicity.