High-speciticity and high-sensitivity genotoxicity assessment in a human cell line:: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay
High-speciticity and high-sensitivity genotoxicity assessment in a human cell line:: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay
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DOI：
10.1016/j.mrgentox.2006.04.011
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发表时间：
2006-09-05
影响因子：
1.9
通讯作者：
Walmsley, Richard M.
中科院分区：
文献类型：
--
作者：
Hastwell, Paul W.;Chai, Li-Leng;Walmsley, Richard M.
大多数监管机构要求进行的遗传毒性试验包括细菌和哺乳动物细胞试验，并可识别几乎所有的遗传毒性致癌物。然而，沙门氏菌致突变试验（艾姆斯试验）相对较高的特异性被已建立的哺乳动物细胞试验的低特异性所抵消，这导致难以解释结果的生物学相关性。本文描述了一种新的高通量检测方法，该方法将人GADD 45 a基因的调控与绿色荧光蛋白（GFP）的产生联系起来。对75种具有不同DNA损伤诱导机制（包括非整倍体）的良好表征的遗传毒性和非遗传毒性化合物的研究表明，该检测方法对所有类型的遗传毒性损伤均具有高特异性和高灵敏度。当前微孔试验形式不包括代谢活化，但单独的低通量方案证明了使用模型前诱变剂环磷酰胺进行S9代谢活化试验的成功原理验证。该试验应作为选择候选化合物进行进一步开发的工具（由于体外试验组的信息相互矛盾，可能需要额外的数据）或在将停止使用动物的产品开发领域中具有价值。然而，作为微孔板检测，它具有高通量和低化合物使用的品质，这将有助于其在遗传毒性易感性的早期筛查中的应用。(c)2006 Elsevier B.V.保留所有权利。
The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity assay (Ames test) is offset by the low specificity of the established mammalian cell assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and nongenotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability. (c) 2006 Elsevier B.V. All rights reserved.