Sensitive neurotoxicity assessment of bisphenol A using double immunocytochemistry of DCX and MAP2

Bisphenol A (BPA) is an environmental toxin widely used in manufacturing industries. Studies conducted on the neurotoxicity of BPA demonstrated that at excessive, high concentrations (≥ 200 µM) adverse responses occurred which were not detectable using traditional toxicity tests at lower chemical quantities than 200 µM. Thus, a method capable of effectively detecting neurotoxicity at low concentrations (≤ 100 µM) was devised. Bisphenol A-mediated neurotoxicity was examined in primary cultured neurons using various methods, including Western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and reactive oxygen species assays. These methods confirmed BPA-induced toxicity at 200 μM, but no marked effect was observed at concentrations below 200 μM. However, when immunocytochemistry (ICC) was performed using a co-immunofluorescence assay of doublecortin (DCX) and microtubule-associated protein 2 (MAP2), BPA adversely affected neuronal maturation in neural progenitor cells at concentrations as low as 100 µM, at which the three traditional methods failed to detect any neurotoxic effect. Our DCX/MAP2 ICC findings indicate that low concentrations of BPA are toxic to developing neurons, and suggest that the devised double ICC technique might provide an effective means of assessing neurotoxic effects of environmental toxins at low concentrations.