Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen and Schmallenberg, Germany.
Neurotoxicol Teratol. 2012 Jul;34(4):413-24. doi: 10.1016/j.ntt.2012.04.006. Epub 2012 Apr 24.
Rodents are widely used to test the developmental neurotoxicity potential of chemical substances. The regulatory test procedures are elaborate and the requirement of numerous animals is ethically disputable. Therefore, non-animal alternatives are highly desirable, but appropriate test systems that meet regulatory demands are not yet available. Hence, we have developed a new developmental neurotoxicity assay based on specific whole-mount immunostainings of primary and secondary motor neurons (using the monoclonal antibodies znp1 and zn8) in zebrafish embryos. By classifying the motor neuron defects, we evaluated the severity of the neurotoxic damage to individual primary and secondary motor neurons caused by chemical exposure and determined the corresponding effect concentration values (EC₅₀). In a proof-of-principle study, we investigated the effects of three model compounds thiocyclam, cartap and disulfiram, which show some neurotoxicity-indicating effects in vertebrates, and the positive controls ethanol and nicotine and the negative controls 3,4-dichloroaniline (3,4-DCA) and triclosan. As a quantitative measure of the neurotoxic potential of the test compounds, we calculated the ratios of the EC₅₀ values for motor neuron defects and the cumulative malformations, as determined in a zebrafish embryo toxicity test (zFET). Based on this index, disulfiram was classified as the most potent and thiocyclam as the least potent developmental neurotoxin. The index also confirmed the control compounds as positive and negative neurotoxicants. Our findings demonstrate that this index can be used to reliably distinguish between neurotoxic and non-neurotoxic chemicals and provide a sound estimate for the neurodevelopmental hazard potential of a chemical. The demonstrated method can be a feasible approach to reduce the number of animals used in developmental neurotoxicity evaluation procedures.
啮齿动物被广泛用于测试化学物质的发育神经毒性潜力。监管测试程序非常详细，需要大量动物，这在伦理上存在争议。因此，非常需要非动物替代品，但符合监管要求的适当测试系统尚未可用。因此，我们基于斑马鱼胚胎中初级和次级运动神经元的特定全组织免疫染色（使用单克隆抗体 znp1 和 zn8）开发了一种新的发育神经毒性测定方法。通过对运动神经元缺陷进行分类，我们评估了化学暴露对单个初级和次级运动神经元的神经毒性损伤的严重程度，并确定了相应的效应浓度值（EC₅₀）。在一项原理验证研究中，我们研究了三种模型化合物噻虫环胺、杀螟丹和双硫仑的影响，这些化合物在脊椎动物中表现出一些神经毒性作用，阳性对照物乙醇和尼古丁以及阴性对照物 3,4-二氯苯胺（3,4-DCA）和三氯生。作为测试化合物神经毒性潜力的定量指标，我们计算了运动神经元缺陷的 EC₅₀ 值与斑马鱼胚胎毒性测试（zFET）中确定的累积畸形的比值。基于该指数，双硫仑被归类为最有效的发育神经毒素，噻虫环胺为最无效的发育神经毒素。该指数还证实了对照化合物为阳性和阴性神经毒物。我们的研究结果表明，该指数可用于可靠地区分神经毒性和非神经毒性化学物质，并为化学物质的神经发育危害潜力提供可靠估计。所证明的方法可以成为减少发育神经毒性评估程序中使用动物数量的可行方法。
