Houk V S, Schalkowsky S, Claxton L D
Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
Mutat Res. 1989 May;223(1):49-64. doi: 10.1016/0165-1218(89)90062-1.
Since its development by Dr. Bruce Ames and his colleagues more than a decade ago, the Salmonella/mammalian microsome mutagenicity assay has become a widely accepted tool to assist in the identification of chemicals with mutagenic and carcinogenic potential. Several automated approaches to Salmonella testing have been proposed in recent years but have failed to gain acceptance in the scientific community due to poor performance or lack of demonstrated usefulness. In this paper we report on an automated system that successfully generates dose-response data and, moreover, reduces the labor, materials, and sample mass required to obtain such information. In the standard plate-incorporation assay, dose-response relationships are defined by testing discrete doses of the test agent on a series of agar plates. In contrast, the spiral Salmonella assay generates dose-response data from a continuous concentration gradient on a single agar plate. Upon analysis, each spiral plate yields a dose-response curve consisting of 13 data points that span a concentration range of about 15:1, which is equivalent to 5 two-fold serial dilutions. The performance of the spiral Salmonella assay was compared to that of the conventional plate-incorporation assay using 13 mutagens and 7 nonmutagens selected from a variety of chemical classes. Concordant qualitative responses were obtained for all compounds tested, and comparable dose-response relationships were generated by all mutagens with the exception of sodium azide and cyclophosphamide, which are highly water-soluble and, thus, are unable to maintain a well-defined concentration gradient on a spiral plate due to rapid diffusion. In general, toxicity was expressed at a lower dose in the spiral assay, and the mutagenic potencies (slopes of the dose-response curves) were greater in the spiral assay relative to the plate-incorporation assay. These differences will be discussed, as will the applicability of the spiral plating technique to routine screening and its relevancy to future mutagenesis testing.
自十多年前布鲁斯·艾姆斯博士及其同事开发沙门氏菌/哺乳动物微粒体诱变性试验以来，该试验已成为一种广泛接受的工具，用于协助鉴定具有诱变和致癌潜力的化学物质。近年来，已经提出了几种沙门氏菌检测的自动化方法，但由于性能不佳或缺乏实用性证明，未能在科学界得到认可。在本文中，我们报告了一种自动化系统，该系统成功生成剂量-反应数据，而且减少了获取此类信息所需的劳动力、材料和样品量。在标准平板掺入试验中，通过在一系列琼脂平板上测试受试物的离散剂量来确定剂量-反应关系。相比之下，螺旋沙门氏菌试验在单个琼脂平板上从连续浓度梯度生成剂量-反应数据。分析时，每个螺旋平板产生一条由13个数据点组成的剂量-反应曲线，这些数据点跨越约15:1的浓度范围，相当于5次两倍连续稀释。使用从各种化学类别中选出的13种诱变剂和7种非诱变剂，将螺旋沙门氏菌试验的性能与传统平板掺入试验进行了比较。对所有测试化合物均获得了一致的定性反应，除叠氮化钠和环磷酰胺外，所有诱变剂产生了可比的剂量-反应关系，叠氮化钠和环磷酰胺高度水溶性，因此由于快速扩散而无法在螺旋平板上维持明确的浓度梯度。一般来说，螺旋试验中在较低剂量下表现出毒性，相对于平板掺入试验，螺旋试验中的诱变效力（剂量-反应曲线的斜率）更大。将讨论这些差异，以及螺旋平板技术在常规筛选中的适用性及其与未来诱变试验的相关性。
