3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals
3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals
复制标题
DOI：
10.1002/em.22312
复制
发表时间：
2019-07-26
影响因子：
2.8
通讯作者：
Bemis, Jeffrey C.
中科院分区：
文献类型：
--
作者：
Dertinger, Stephen D.;Avlasevich, Svetlana L.;Bemis, Jeffrey C.
监管指导文件强调在评估化学品体内遗传毒性潜力时，评估多种组织中最合适的终点的价值。然而，进行几个独立的研究来评估多个终点和/或组织区室是资源密集的。此外，当依赖于视觉检测时，传统的遗传毒性终点评分方法可能是缓慢、乏味的，而且比理想的方法更不客观。为了解决当前实践中的这些问题，我们试图(1)设计出与肝脏和血液微核终点以及Pig-a基因突变试验兼容的资源节约治疗和收获计划，以及(2)利用基于流式细胞术的方法对每种遗传毒性生物标志物进行评分。用4周龄的雄性和雌性Crl:CD（SD）大鼠暴露于马兜铃酸I/II、苯并[a]芘、顺铂、环磷酰胺、二乙基亚硝胺、1,2-二甲基肼、二甲基亚硝胺、2,6-二硝基甲苯、羟基脲、美伐兰、替莫唑胺、喹啉或长春花碱中进行原理验证实验。这13种化学物质分别在两种治疗方案中进行测试：一个3天暴露周期和三个3天暴露周期。每次暴露、采血和肝脏采集均在标准的周一至周五工作周内完成。主要发现是，即使是这些经过充分研究的、相对有效的基因毒物在两种组织和所有检测中都没有活性（实际上只有顺铂在所有三种检测中都明显呈阳性）；尽管猪-a试验的敏感性明显受益于三个治疗周期而不是一个治疗周期，但微核试验在两种研究设计中产生的定性结果相似。总的来说，这些结果表明，通过结合流式细胞术评分方法同时评估三个终点和两个重要的组织区室，可以显著减少动物和其他资源的需求，同时改善体内遗传毒性潜力的评估。环绕。Mol. Mutagen, 2019。(c) 2019 Wiley期刊公司
Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen. 2019. (c) 2019 Wiley Periodicals, Inc.