Doerenkamp-Zbinden Chair for In Vitro Toxicology and Biomedicine, University of Konstanz, Universitätsstr. 10, Box 657, 78457, Constance, Germany,
Arch Toxicol. 2013 Dec;87(12):2215-31. doi: 10.1007/s00204-013-1072-y. Epub 2013 May 14.
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.
器官特异性体外毒性测定通常具有较高的灵敏度，但特异性不足。本文评估了可能影响测试特异性的测定特征实例，并就如何定义复杂生物测定中的阳性结果提出了一些一般性程序。分化的人 LUHMES 细胞被用作发育神经毒性测试的潜在模型。筛选了 40 种候选毒性物质，并获得了几个阳性结果并进行了确认。尽管这些细胞具有明确的神经元表型，但在这些培养物中使用一般的细胞死亡终点并不能特异性识别神经毒性物质。作为替代方法，测量神经突生长作为器官特异性功能终点。我们发现，各种化合物（如秋水仙碱、长春新碱、纳曲西嗪、鱼藤酮、环己酰亚胺或二氯喹啉）可特异性抑制正在发育的 LUHMES 细胞的神经突生长。这些化合物在不损害细胞活力的浓度下降低神经突生长，并且神经突生长受到的影响比成熟神经元发育中的神经突完整性更为强烈。神经突生长抑制和细胞死亡的 EC50 值之比>4 为与发育神经毒性危害相关的化合物提供了稳健的分类器。在测试系统中筛选非特异性毒性物质时，比值总是<4。该测定还鉴定了加速神经突生长的化合物，如 rho 激酶途径调节剂 blebbistatin 或 thiazovivin。rho 激酶抑制剂完全抑制秋水仙碱或鱼藤酮的负面作用。总之，我们建议使用功能终点（神经突生长）的测定可以特异性识别和表征（发育）神经毒性物质。
