Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants

Abstract

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.