Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, B105-06, Research Triangle Park, NC 27711, USA.
Toxicology. 2010 Apr 11;270(2-3):121-30. doi: 10.1016/j.tox.2010.02.004. Epub 2010 Feb 10.
In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation.
体外测试方法可快速筛选大量化学物质，以确定其产生毒性（危害识别）的潜力。为了识别潜在的发育神经毒性剂，已经提出了一系列用于神经发育过程的体外测试，如细胞增殖、分化、生长和突触发生。为了开发毒性测试的体外方法，需要选择与关注终点相适应的模型系统。本研究比较了几种细胞系作为神经元增殖模型的适用性。使用高通量筛选系统评估了三种物种（PC12、大鼠；N1E-115、小鼠；SH-SY5Y、人）来源的神经元细胞系对已知影响细胞增殖的化学物质的敏感性。在优化了 96 孔板中细胞生长条件后，通过 BrdU 掺入复制 DNA 来测量增殖，BrdU 是在 S 期进行复制的。通过免疫细胞化学检测 BrdU 标记的细胞，并使用自动图像采集和分析获得细胞计数。在 BrdU 脉冲 4 小时后，三种细胞系的 S 期细胞约占 30-40%。在 BrdU 脉冲前 20 小时用 DNA 聚合酶抑制剂阿霉素处理可显著降低三种细胞系的增殖。通过暴露于已知影响增殖的八种化学物质（阳性对照）并确定抑制增殖 50%的浓度（I（50））来比较细胞系的敏感性。PC12 细胞对化学物质最敏感；在测试浓度下，8 种化学物质中的 6 种（阿霉素、镉、胞嘧啶阿拉伯糖苷、地塞米松、5-氟尿嘧啶和甲基汞）抑制了增殖。SH-SY5Y 细胞对化学物质的敏感性稍低，8 种化学物质中有 5 种抑制增殖；地塞米松没有作用，而镉仅在降低细胞活力的浓度下抑制增殖。N1E-115 细胞系的数据在实验之间差异极大，只有 8 种化学物质中的 4 种导致增殖抑制。先前未显示改变增殖的化学物质（阴性对照）不会影响任何细胞系的增殖或细胞活力。结果表明，高通量筛选可用于快速评估化学物质对增殖的影响。三种神经元细胞系对化学物质对该终点的影响表现出不同的敏感性，PC12 细胞对增殖抑制最敏感。
