Luijten Mirjam, Zwart Edwin P, Dollé Martijn E T, de Pooter Maaike, Cox Julie A, White Paul A, van Benthem Jan
Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
Environ Mol Mutagen. 2016 Dec;57(9):643-655. doi: 10.1002/em.22063. Epub 2016 Nov 9.
Assessment of genotoxic potential is an important step in the safety evaluation of chemical substances. Under most regulatory jurisdictions, the first tier of testing comprises a standard battery of in vitro genotoxicity tests in bacterial and mammalian cells. However, the mammalian cell tests commonly used exhibit a relatively high rate of misleading positive results, which may lead to unnecessary in vivo testing. We previously established a proof-of-concept for the LacZ reporter assay in proliferating primary hepatocytes as a promising alternative genotoxicity test. Here, cryopreserved instead of freshly isolated hepatocytes were used and the assay was evaluated in more detail. We examined the effect of cryopreservation on phenotype and metabolic capacity of the LacZ hepatocytes, and assessed the predictive performance of the assay by testing a set of substances comprising true positive, true negative, and misleading positive substances. Additionally, a historical negative control database was created and the type of mutations induced was analyzed for two of the substances tested. Our findings indicate that proliferating cryopreserved primary hepatocytes derived from LacZ plasmid mice retain their hepatocyte-specific characteristics and metabolic competence. Furthermore, we demonstrate that both gene mutations and genome rearrangements due to large deletions can be detected with the LacZ reporter assay. The assay seems to have a lower rate of misleading positive test results compared to the assays currently used. Together, our findings strongly support the use of the LacZ reporter assay in cryopreserved primary hepatocytes as follow-up to the standard in vitro test battery for genotoxicity testing. Environ. Mol. Mutagen. 57:643-655, 2016. © 2016 Wiley Periodicals, Inc.
遗传毒性潜力评估是化学物质安全性评价的重要步骤。在大多数监管辖区，第一层测试包括在细菌和哺乳动物细胞中进行的一系列标准体外遗传毒性测试。然而，常用的哺乳动物细胞测试显示出相对较高的误导性阳性结果发生率，这可能导致不必要的体内测试。我们之前在增殖的原代肝细胞中建立了LacZ报告基因检测的概念验证，作为一种有前景的替代遗传毒性测试。在此，使用的是冷冻保存而非新鲜分离的肝细胞，并对该检测进行了更详细的评估。我们研究了冷冻保存对LacZ肝细胞表型和代谢能力的影响，并通过测试一组包括真阳性、真阴性和误导性阳性物质的物质来评估该检测的预测性能。此外，创建了一个历史阴性对照数据库，并对两种测试物质诱导的突变类型进行了分析。我们的研究结果表明，源自LacZ质粒小鼠的增殖冷冻保存原代肝细胞保留了其肝细胞特异性特征和代谢能力。此外，我们证明LacZ报告基因检测可以检测到由于大片段缺失导致的基因突变和基因组重排。与目前使用的检测相比，该检测似乎具有较低的误导性阳性测试结果发生率。总之，我们的研究结果有力地支持了在冷冻保存的原代肝细胞中使用LacZ报告基因检测作为遗传毒性测试标准体外测试组合的后续检测。《环境与分子突变》57:643 - 655，2016年。© 2016威利期刊公司。
