8-hydroxyguanine and Leukemia-L5178

The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to detect oxidative DNA damage. In the present study, FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for the ability to modify the sensitivity of the comet assay. Mouse lymphoma L5178Y cells were treated with dimethyl sulphoxide (DMSO) as a standard solvent or reference agents known to induce oxidative damage (gamma irradiation and potassium bromate) or alkylation (methyl methanesulfonate, MMS; ethylnitrosurea, ENU). Using DMSO even up to toxic concentrations, no increase in breaks was seen with FPG, ENDOIII or hOGG1. With gamma irradiation (1-10 Gy), dose-related increases in breaks were seen with all three enzymes. FPG and hOGG1 gave similar increases in breaks after potassium bromate treatment between 0.25 and 2.5 mmol/l, but ENDOIII showed an increase only at the highest concentration, 2.5 mmol/l. Following MMS treatment (5-23 micromol/l), FPG induced a dramatic increase in breaks compared with control levels and ENDOIII also showed a significant but smaller increase; in marked contrast, hOGG1 gave no increase. With ENU (0.5-2.0 mmol/l), increases in breaks were seen with FPG and ENDOIII at 1 and 2 mmol/l but, again, no increase was observed with hOGG1. These data indicate that all three endonucleases recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage. Therefore, caution should be taken when using FPG and ENDOIII in the comet assay with an agent that has an unknown mode of action since any additional strand breaks induced by either enzyme cannot necessarily be ascribed to oxidative damage. The use of hOGG1 in the modified comet assay offers a useful alternative to FPG and is apparently more specific for 8-oxoguanine and methyl-fapy-guanine.

Topics: Animals; Bromates; Comet Assay; Deoxyribonuclease (Pyrimidine Dimer); Dimethyl Sulfoxide; DNA Damage; DNA Glycosylases; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Ethylnitrosourea; Gamma Rays; Guanine; Humans; Leukemia L5178; Methyl Methanesulfonate; Mice; Oxidation-Reduction; Sensitivity and Specificity

We recently showed that treatment of V79 cells with hyperbaric oxygen (HBO) efficiently induced DNA effects in the comet assay and chromosomal damage in the micronucleus test (MNT), but did not lead to gene mutations at the hprt locus. Using the comet assay in conjunction with bacterial formamidopyrimidine DNA glycosylase (FPG protein), we now provide indirect evidence that the same treatment leads to the induction of 8-oxoguanine, a premutagenic oxidative DNA base modification in V79 and mouse lymphoma (L5178Y) cells. We also demonstrate that HBO efficiently induces mutations in the mouse lymphoma assay (MLA). Exposure of L5178Y cells to HBO (98% O(2); 3bar) for 2h caused a clear mutagenic effect in the MLA, which was further enhanced after a 3h exposure. As this mutagenic effect was solely due to the strong increase of small colony (SC) mutants, we suggest that HBO causes mutations by induction of chromosomal alterations. Molecular characterization of induced SC mutants by loss of heterozygosity (LOH) analysis showed an extensive loss of functional tk sequences similar to the pattern found in spontaneous SC mutants. This finding confirmed that the majority of HBO-induced mutants is actually produced by a clastogenic mechanism. The induction of point mutations as a consequence of induced oxidative DNA base damage seems to be of minor importance.

Topics: Animals; Cell Line; Comet Assay; Cricetinae; Cricetulus; DNA Damage; DNA-Formamidopyrimidine Glycosylase; DNA, Neoplasm; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Guanine; Hyperbaric Oxygenation; Leukemia L5178; Loss of Heterozygosity; Mutagenicity Tests; Mutagens; N-Glycosyl Hydrolases; Oxygen; Point Mutation; Polymerase Chain Reaction; Thymidine Kinase