Butler Kimberly S, Casey Brendan J, Garborcauskas Garret V M, Dair Benita J, Elespuru Rosalie K
U.S. Food and Drug Administration, Office of Medical Products and Tobacco, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories, Division of Biology, Silver Spring, MD 20993, United States.
U.S. Food and Drug Administration, Office of Medical Products and Tobacco, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories, Division of Chemistry and Materials Science, Silver Spring, MD 20993, United States.
Mutat Res Genet Toxicol Environ Mutagen. 2014 Jul 1;768:14-22. doi: 10.1016/j.mrgentox.2014.04.008. Epub 2014 Apr 24.
Due to their unique properties, the use of nanoparticles (NPs) is expanding; these same properties may affect their potential risk to humans. However, standard methods for genotoxicity assessment may not be adequate for NPs; altered tests reported here have been developed to address perceived inadequacies. The bacterial reverse mutation assay is an essential part of the battery of tests to determine genotoxicity. The utility of this test for assessing NPs is currently questioned, due to negative results seemingly caused by failure of particle uptake. To probe uptake issues, we examined the physical state in different media, dose and time dependent association, uptake and mutagenicity of titanium dioxide (TiO2) NPs in Salmonella typhimurium and Escherichia coli. The NPs suspended in water were characterized using dynamic light scattering, NP tracking analysis and transmission electron microscopy. NP association with bacteria was assessed by flow cytometry. Association was found to be time and dose dependent, with maximal association by 60 min. Therefore mutagenicity was assessed after a 60 min pre-incubation in a miniaturized assay demonstrating enhanced sensitivity. To assess potential indirect effects on bacterial mutagenicity, the effect of TiO2 NPs on the action of standard mutagens or on DNA repair capability was also investigated. TiO2 NPs did not affect mutant yields in standard strains of S. typhimurium or E. coli, including those detecting oxidative damage, using the modified methods. Nor did TiO2 NPs affect the action of standard mutagens or DNA excision repair capability. Despite particle association with the bacteria, subsequent analysis using electron microscopy and energy dispersive x-ray spectroscopy indicated that the NPs were not internalized. This work demonstrates that additional studies, including flow cytometry, are valuable tools for understanding the action of NPs in biological systems.
由于其独特的性质，纳米颗粒（NPs）的应用正在不断扩大；而这些相同的性质可能会影响它们对人类的潜在风险。然而，标准的遗传毒性评估方法可能不适用于纳米颗粒；本文报道的经过改进的测试方法已被开发出来，以解决人们所认为的不足之处。细菌回复突变试验是确定遗传毒性的一系列测试中的重要组成部分。目前，由于颗粒摄取失败导致的阴性结果，该测试在评估纳米颗粒方面的效用受到质疑。为了探究摄取问题，我们研究了二氧化钛（TiO₂）纳米颗粒在鼠伤寒沙门氏菌和大肠杆菌中的不同介质中的物理状态、剂量和时间依赖性结合、摄取和致突变性。使用动态光散射、纳米颗粒跟踪分析和透射电子显微镜对悬浮在水中的纳米颗粒进行了表征。通过流式细胞术评估纳米颗粒与细菌的结合。发现结合具有时间和剂量依赖性，60分钟时结合达到最大值。因此，在小型化试验中预孵育60分钟后评估致突变性，结果显示灵敏度有所提高。为了评估对细菌致突变性的潜在间接影响，还研究了TiO₂纳米颗粒对标准诱变剂作用或DNA修复能力的影响。使用改进的方法，TiO₂纳米颗粒并未影响鼠伤寒沙门氏菌或大肠杆菌标准菌株中的突变体产量，包括那些检测氧化损伤的菌株。TiO₂纳米颗粒也未影响标准诱变剂的作用或DNA切除修复能力。尽管纳米颗粒与细菌有结合，但随后使用电子显微镜和能量色散X射线光谱进行的分析表明，纳米颗粒并未被内化。这项工作表明，包括流式细胞术在内的其他研究是理解纳米颗粒在生物系统中作用的有价值工具。
