Toxicology and Drug Disposition, Schering-Plough Research Institute, PO Box 20, 5342 CC Oss, The Netherlands.
Mutat Res. 2010 Feb;696(1):21-40. doi: 10.1016/j.mrgentox.2009.12.007. Epub 2009 Dec 16.
Four different mechanism-based high-throughput luciferase-reporter assays were developed in human HepG2 cells, which contain phase I and II metabolic activity and a functionally active p53 protein. The promoter regions of RAD51C and Cystatin A, as well as the responsive element of the p53 protein, were selected for the generation of the genotoxicity reporter assays. Moreover, a luciferase-based reporter assay was generated that measures the activation of the Nrf2 oxidative stress pathway. Validation with respect to the ECVAM compound list [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108] resulted in an overall sensitivity of the HepG2 genotoxicity reporter assays for genotoxicity of 85% (17/20). The specificity and predictivity were high with 81% (34/42) and 82% (51/62), respectively. Various compounds had a positive score although metabolic activation was needed. The HepG2 reporter data were also compared with the available data on bacterial mutagenicity (Ames test), in vitro clastogenicity and in vivo clastogenicity for an additional set of 192 compounds. The predictivity for mutagenicity results was 74% (sensitivity, 61%, 30/49; specificity, 80%, 77/96) and for in vitro clastogenicity 59% (sensitivity, 45%, 35/78; specificity 83%, 38/46). The correlation between results from the HepG2 genotoxicity reporter assays and in vivo clastogenicity was much higher with 77% (sensitivity, 74%, 28/38; specificity 81%, 26/32). Results from the Nrf2 reporter assay showed that a large number of genotoxic compounds activated the Nrf2 oxidative stress pathway. In conclusion, four high-throughput mechanism-based reporter assays in the HepG2 cell line were developed, which can be applied for screening in the early research phase of drug development. The use of these assays in combination with the previously validated Vitotox and RadarScreen assays will certainly reduce the attrition rate due to genotoxicity in the developmental phase of drug development.
四种不同的基于机制的高通量荧光素酶报告基因检测在含有一期和二期代谢活性和功能活跃的 p53 蛋白的人 HepG2 细胞中建立。RAD51C 和半胱氨酸蛋白酶抑制剂 A 的启动子区域以及 p53 蛋白的反应元件被选为遗传毒性报告基因检测的生成。此外，还生成了一种基于荧光素酶的报告基因检测方法，用于测量 Nrf2 氧化应激途径的激活。用 ECVAM 化合物清单 [D. Kirkland、P. Kasper、L. Muller、R. Corvi、G. Speit，用于评估新的或改进的遗传毒性测试性能的遗传毒性和非遗传毒性化学品的推荐清单：ECVAM 研讨会的后续行动，Mutagenesis Res. 653（2008）99-108] 进行验证，结果表明 HepG2 遗传毒性报告基因检测对遗传毒性的总体敏感性为 85%（17/20）。特异性和预测性分别为 81%（34/42）和 82%（51/62）。尽管需要代谢激活，但各种化合物仍具有阳性评分。还将 HepG2 报告数据与可用的细菌致突变性（Ames 试验）、体外细胞遗传毒性和另外 192 种化合物的体内细胞遗传毒性数据进行了比较。致突变性结果的预测性为 74%（敏感性，61%，30/49；特异性，80%，77/96），体外细胞遗传毒性为 59%（敏感性，45%，35/78；特异性 83%，38/46）。HepG2 遗传毒性报告基因检测结果与体内细胞遗传毒性的相关性更高，为 77%（敏感性，74%，28/38；特异性 81%，26/32）。Nrf2 报告基因检测结果表明，大量遗传毒性化合物激活了 Nrf2 氧化应激途径。总之，在 HepG2 细胞系中建立了四种高通量基于机制的报告基因检测方法，可用于药物开发的早期研究阶段的筛选。这些检测方法与之前验证的 Vitotox 和 RadarScreen 检测方法结合使用，肯定会降低药物开发过程中因遗传毒性而导致的淘汰率。
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