title: Biological significance of DNA adducts: comparison of increments over background for various biomarkers of genotoxicity in L5178Y tk(+/-) mouse lymphoma cells treated with hydrogen peroxide and cumene hydroperoxide.
abstract: DNA is affected by background damage of the order of one lesion per one hundred thousand nucleotides, with depurination and oxidative damage accounting for a major part. This damage contributes to spontaneous mutation and cancer. DNA adducts can be measured with high sensitivity, with limits of detection lower than one adduct per one billion nucleotides. Minute exposures to an exogenous DNA-reactive agent may therefore result in measurable adduct formation, although, as an increment over total DNA damage, a small increment in mutation cannot be measured and would be considered negligible. Here, we investigated whether this discrepancy also holds for adducts that are present as background induced by oxidative stress. L5178Y tk(+/-) mouse lymphoma cells were incubated for 4h with hydrogen peroxide (0, 0.8, 4, 20, 100, 500muM) or cumene hydroperoxide (0, 0.37, 1.1, 3.3, 10muM). Five endpoints of genotoxicity were measured in parallel from aliquots of three replicates of large batches of cells: Two DNA adducts, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 1,N(6)-etheno-2'-deoxyadenosine (varepsilondAdo) measured by LC-MS/MS, as well as strand breaks assessed with the comet assay and in vitro micronucleus test, and gene mutation as assessed using the thymidine kinase gene mutation assay. Background measures of 8-oxodGuo and varepsilondAdo were 500-1000 and 50-90 adducts per 10(9) nucleotides. Upon treatment, neither hydrogen peroxide nor cumene hydroperoxide significantly increased the DNA adduct levels above control. In contrast, dose-related increases above background were observed with both oxidants in the comet assay, the micronucleus test and the gene mutation assay. Differences in sensitivity of the assays were quantified by estimating the concentration of oxidant that resulted in a doubling of the background measure. We conclude that the increase in DNA breakage and mutation induced by hydrogen peroxide and cumene hydroperoxide observed in our in vitro experimental set-up was no direct consequence of the measured DNA adducts. In comparison with data obtained with the methylating agent methyl methanesulfonate we further conclude that the assumption of DNA adducts being oversensitive biomarkers is adduct-specific.
authors.affiliation: Department of Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Brink
authors.forename: Ingrid
authors.initials: I
authors.lastname: Richter
authors.forename: Ursula
authors.initials: U
authors.lastname: Lutz
authors.forename: Paul
authors.initials: P
authors.lastname: Wanek
authors.forename: Helga
authors.initials: H
authors.lastname: Stopper
authors.forename: Werner K
authors.initials: WK
authors.lastname: Lutz
mesh_terms: D000818:Animals; D001555:Benzene Derivatives / Q000633:toxicity; D015415:Biomarkers / Q000032:analysis; D018736:DNA Adducts; D006861:Hydrogen Peroxide / Q000633:toxicity; D007940:Leukemia L5178; D051379:Mice; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D018384:Oxidative Stress
chemical_list: D001555:Benzene Derivatives; D015415:Biomarkers; D018736:DNA Adducts; D009153:Mutagens; D006861:Hydrogen Peroxide; C007164:cumene hydroperoxide