Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants.
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.
Department of Genetics, Microbiology and Toxicology, Stockholm University, SE-106 91, Sweden.
Fredrik
F
Johansson
Annika
A
Allkvist
Klaus
K
Erixon
Anna
A
Malmvärn
Robert
R
Nilsson
Ake
A
Bergman
Thomas
T
Helleday
Dag
D
Jenssen
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D000477:Alkylating Agents / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D002460:Cell Line; D006224:Cricetinae; D018736:DNA Adducts; D004260:DNA Repair; D004785:Environmental Pollutants / Q000378:metabolism / Q000633:toxicity*; D005020:Ethyl Methanesulfonate / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity; D008297:Male; D016685:Mitomycin / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D015394:Molecular Structure; D009152:Mutagenicity Tests; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D016877:Oxidants / Q000378:metabolism / Q000633:toxicity
D000477:Alkylating Agents; D002273:Carcinogens; D018736:DNA Adducts; D004785:Environmental Pollutants; D009153:Mutagens; D016877:Oxidants; D016685:Mitomycin; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D005020:Ethyl Methanesulfonate; D006861:Hydrogen Peroxide