Rapid and sensitive neurotoxicity test based on the morphological changes of PC12 cells with simple computer-assisted...

In order to develop a rapid and sensitive bioassay for the screening of chemicals with possible neurotoxicity, a computer-assisted simple image-analysis system was developed to quantify small changes in the specific morphology of the cultured pheochromocytoma cell line, PC12. This cell forms a neuron-like microfibril network (neurites) in response to a nerve growth factor (NGF) stimulation in vitro. Dichrolvos (DDVP) and methylmercury chloride (MMC) were employed as model neurotoxicants. In DDVP treatment, there was no large difference in the ED50s (effective dose that reduces the morphological index by 50%) among the toxicities determined from various morphological indices, but they were significantly lower than those observed by whole-cell-area-based toxicity assay using the hepatoblastoma cell line, Hep G2. In contrast, in MMC treatment, neurite-length-based toxicity was observed as early as 2 h, and at 48 h this was lower by over three orders of magnitude compared with whole-cell-area-based one (2.06 x 10(-7) mM vs. 6.42 x 10(-4) mM). These results demonstrate that the developed bioassay using image analysis of nerve-tissue-derived cell morphology allows us to screen possible neurotoxic chemicals very rapidly with highly enhanced sensitivity, particularly for some chemicals that preferentially act on nerve fibers.