Johansson Fredrik, Allkvist Annika, Erixon Klaus, Malmvärn Anna, Nilsson Robert, Bergman Ake, Helleday Thomas, Jenssen Dag
Department of Genetics, Microbiology and Toxicology, Stockholm University, SE-106 91, Sweden.
Mutat Res. 2004 Sep 12;563(1):35-47. doi: 10.1016/j.mrgentox.2004.05.017.
The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited.
DRAG试验是一种快速高通量筛选检测方法，通过抑制具有不同DNA修复缺陷的中国仓鼠卵巢细胞（CHO）生长来检测可修复加合物。与野生型细胞（AA8）相比，某种DNA反应性物质在修复缺陷细胞系（EM9、UV4和UV5）中引起更明显的生长抑制，这被解释为它们无法修复诱导的DNA损伤的结果。因此，在DRAG试验中使用此类细胞系可能会提供某种遗传毒性物质诱导的DNA损伤类型的信息。为了选择最佳试验条件，并为解释结果提供机制基础，使用了模型化合物苯并(a)芘-7,8-二氢二醇-9,10-环氧化物（BPDE）、甲基磺酸乙酯（EMS）、丝裂霉素C（MMC）和过氧化氢（H2O2）。这些试剂可分别诱导大分子加合物、烷基加合物、交联和氧化损伤。DRAG试验的特异性是其在更广泛背景下实际应用的重要前提。为了评估这一方面，我们研究了多氯联苯（PCB）和多溴二苯醚（PBDE）的一系列代谢物和异构体以及其他一些卤代化合物的遗传毒性和细胞毒性特性。所有这些化合物都已在外部环境中被检测为污染物，而且对于它们中的大多数，传统检测方法没有令人信服的致突变性证据。正如从它们的作用方式所预测的那样，与野生型细胞相比，BPDE、MMC和EMS在修复缺陷细胞系中均被发现毒性更大。H2O2的结果尚无定论，PCB代谢物4,4'-二羟基-CB80仅表现出临界活性，而所有其他卤代化合物或其代谢物均无活性。总之，DRAG试验在筛选大量潜在遗传毒性试剂时可提供一个强大而有用的工具，同时还能提供机制信息。然而，所选细胞系检测氧化损伤的有用性可能有限。
