OXIDATIVE DNA-DAMAGE INDUCED BY POTASSIUM BROMATE UNDER CELL-FREE CONDITIONS AND IN MAMMALIAN-CELLS

The oxidative DNA damage induced by the renal carcinogen potassium bro mate (KBrO3) in cultured mammalian cells and in a cell-free system was characterized by means of various repair endonucleases. Under cell-fr ee conditions, no modifications were induced by KBrO3 alone, but exten sive DNA damage was observed in the presence of glutathione (GSH). The DNA damage was found to consist mostly of base modifications sensitiv e to Fpg protein (formamidopyrimidine-DNA glycosylase). HPLC analysis demonstrated that many of the modifications were 7,8-dihydro-8-oxoguan ine (8-hydroxyguanine) residues. Single-strand breaks, sites of base l oss (AP sites) and base modifications sensitive to endonuclease III (5 ,6-dihydropyrimidine derivatives) were formed in only low amounts. Thi s 'damage profile' and experiments with various scavengers (catalase, superoxide dismutase, deferoxamine, azide, tert-butanol) and D2O as so lvent excluded the involvement of hydroxyl radicals and singlet oxygen in the damage production, but were consistent with a radical mechanis m involving bromine radicals. In L1210 mouse leukemia cells and LLC-PK 1 porcine kidney cells, KBrO3 alone gave rise to a DNA damage profile similar to that observed after treatment of cell-free DNA with KBrO3 p lus GSH, i.e. base modifications sensitive to Fpg protein were formed in high excess of all other lesions quantified. In LLC-PK1 cells (deri ved from the target organ of KBrO3-induced carcinogenesis) the level o f DNA damage was twice that in the L1210 cells. DNA damage was partial ly prevented by depletion of intracellular GSH with diethylmaleate, in dicating that GSH played an activating role in the cells similar to th at seen under cell-free conditions. The Fpg-sensitive base modificatio ns induced by KBrO3 were repaired with only moderate efficiency (38 +/ - 10% of the lesions were still present after 18 h in full medium) und er conditions that did not influence cell proliferation.