Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing
Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing
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DOI：
10.1002/(sici)1098-2280(2000)35:3
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发表时间：
2000-01-01
影响因子：
2.8
通讯作者：
Sasaki, YF
中科院分区：
文献类型：
--
作者：
Tice, RR;Agurell, E;Sasaki, YF
在1999年3月25日至26日在华盛顿举行的遗传毒性测试程序国际研讨会上，一个专家小组开会制定了在遗传毒理学中使用单细胞凝胶(SCG)/彗星试验的指导方针。专家小组达成共识，认为彗星试验用于鉴定具有遗传毒性活性的试剂的最佳版本是Singh等人开发的碱性(pH，13)版本。[1988]。PH>；13版本能够检测DNA单链断裂(SSB)、碱不稳定位点(ALS)、DNA-DNA/DNA蛋白质交联以及与不完全切除修复位点相关的SSB。与HER的遗传毒性测试相比，SCG测试的优势包括：对检测低水平的DNA损伤表现出的敏感性，每个样本只需少量细胞，其灵活性，低成本，易于应用，以及完成研究所需的时间较短。专家小组认为，碱性(pH和GT；13)彗星试验的任何单一版本都不是明显更好的。然而，讨论了分析中的关键技术步骤，并制定了以下方面的指南：用琼脂糖凝胶制备玻片、裂解细胞以释放DNA、将释放的DNA暴露于碱中以产生单链DNA并将ALS表达为单链DNA、在pH>；13碱性条件下对DNA进行电泳法、碱中和、DNA染色、彗星可视化和数据收集。根据目前的知识状况，专家小组制定了进行体外或体内彗星检测的指导方针。专家小组的目标是确定获得被认为适合监管机构提交的可重复和可靠的彗星数据的最低标准，在制定相应的体外和体内SCG分析指南期间，专家小组使用了经济合作与发展组织(经合组织)目前的体外和体内遗传毒理学研究指南。审议的指南主题包括初步考虑、测试方法的原则、测试方法的说明、程序、结果、数据分析和报告。专家小组特别考虑了与细胞毒性有关的DNA降解对解释彗星试验结果的潜在不利影响。专家小组还讨论了可能有助于解释阳性彗星数据的相关SCG方法，讨论的相关方法包括：(1)在电泳过程中使用不同的pH条件来区分DNA链断裂和ALS；(2)使用修复酶或抗体来检测特定类别的DNA损伤；(3)使用中性扩散试验来确定凋亡/坏死细胞；以及(4)使用无细胞SCG分析来评估测试物质与DNA直接相互作用的能力。专家小组制定的碱性(pH和GT；13)彗星分析指南代表了一项正在进行的工作。在对其在遗传毒理学中的效用进行批判性评估之前，还需要更多的信息。所需要的信息包括关于碱性(pH>；3)SCG分析固有的不同变异性来源(例如，细胞到细胞、凝胶到凝胶、Run到Run、培养到培养、动物到动物、实验到实验)的综合数据，基于使用这些指南的体外和体内测试的大型数据库的生成，以及适当设计的多实验室国际验证研究的结果。(C)2000 Wiley-Liss，Inc.
At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH, 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNAprotein cross-linking, and SSB associated with incomplete excision repair sites. Relative to at her genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, ifs ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission, The expert panel used the current Organization for Economic Go-operation and Development (OECD) guidelines Far in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data, The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies. (C) 2000 Wiley-Liss, Inc.