ro-19-8022 and potassium-bromate

Cells are continuously exposed to damaging reactive oxygen species (ROS), which are produced from both endogenous and exogenous sources. 8-Oxodeoxyguanosine (8-oxodG) is an abundant base lesion formed during oxidative stress which, if not repaired, can give rise to G:C-->T:A transversions in DNA. The 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated base excision repair (BER) pathway operates to remove 8-oxodG lesions. Ogg1 deletion and polymorphism may result in a hypermutator phenotype and susceptibility to oxidative pathologies including cancer. Limited and conflicting evidence exists regarding the repair capacity of a prevalent human OGG1 (hOGG1) polymorphism, the Cys326-hOGG1 variant. The formamidopyrimidine DNA glycosylase (FPG)-modified comet assay was used to investigate the ability of sodium dichromate, potassium bromate and Ro19-8022 (+light) to induce DNA damage in mogg1(-/-) null (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs) and to assess hOGG1 variant-initiated BER capacities under conditions of oxidative stress. Treatment of WT MEFs with these pro-oxidant agents induced direct DNA strand breaks in a concentration-dependent manner, whereas, identical treatment of KO MEFs produced no effect. In contrast, KO MEFs accumulated significantly more FPG-sensitive sites than WT MEFs. Expression of hOGG1 in KO MEFs restored the WT phenotype in response to all pro-oxidants tested. The results suggest OGG1-initiated BER generates direct DNA strand breaks detected by the conventional comet assay, thus it is important that researchers do not interpret these as direct damage per se but rather a reflection of the repair process. The data also indicate Cys326-hOGG1-initiated BER is transiently impaired with respect to Ser326-hOGG1 (wild-type)- and Gly326-hOGG1 (artificial)-initiated BER following pro-oxidant treatment, possibly via hOGG1 cysteine 326 oxidation. This finding suggests the homozygous cys326/cys326 genotype may be classified as a biomarker of disease susceptibility, which is in support of a growing body of epidemiological evidence.

Topics: Animals; Bromates; Cell Line; Chromates; Comet Assay; Deoxyadenosines; DNA Breaks; DNA Glycosylases; DNA Repair; Glutathione; Humans; Light; Mice; Mutagens; Oxidants; Oxidative Stress; Polymorphism, Genetic; Pyrrolidines; Quinolizines; Reactive Oxygen Species

Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.

Topics: Animals; Bromates; Cell Line; Cricetinae; Cricetulus; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Enzyme Induction; Female; Genes, Bacterial; Genes, Reporter; Mutagenesis; N-Glycosyl Hydrolases; Ovary; Oxidants; Oxidative Stress; Photochemistry; Photosensitizing Agents; Polymerase Chain Reaction; Pyrrolidines; Quinolizines; Transfection