Hincks J R, Coulombe R A
Graduate Programs in Toxicology, Utah State University, Logan.
Environ Mol Mutagen. 1989;13(3):211-7. doi: 10.1002/em.2850130304.
Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN2), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents.
碱性洗脱是一种灵敏且常用的技术，用于检测以DNA链断裂和DNA交联形式存在的细胞DNA损伤。传统的碱性洗脱程序对设备要求广泛，且操作繁琐。我们实验室最近提出了一种对碱性洗脱技术的快速、简化且灵敏的改进方法，用于检测致癌物诱导的DNA链断裂。在本研究中，我们进一步改进了该技术，以能够快速表征培养的上皮细胞中化学诱导的DNA链间交联和DNA-蛋白质相关交联。将细胞暴露于三种已知的DNA交联剂，即氮芥（HN2）、丝裂霉素C（MMC）或紫外线照射（UV）。以0.25、1.0和4.0微摩尔浓度的HN2或20、40和60微摩尔浓度的MMC进行1小时暴露，这些试剂可产生剂量依赖性的总DNA交联诱导。用蛋白酶K消化显示，HN2和MMC诱导了DNA-蛋白质交联和DNA链间交联。紫外线照射诱导了DNA交联和DNA链断裂，后者与蛋白质或非蛋白质相关。结果表明，重力流碱性洗脱是一种灵敏且准确的表征DNA交联分子事件的方法。使用该程序，从处理过的细胞中洗脱DNA在1小时内完成，且每个样品仅分析三个组分。该方法可作为一种快速的遗传毒性筛选测定方法和/或作为其他潜在诱变剂或致癌剂预测测定方法的辅助手段。
