Requirement of glutathione and cysteine in guanine-specific oxidation of DNA by carcinogenic potassium bromate

Potassium bromate (KBrO3), a food additive, induces renal-cell tumors in ra ts. KBrO3 induced 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human leukemia cell line HL-60 as well as in its H2O2-resistant clone, H P100, suggesting no involvement of H2O2. Depletion of GSH by buthionine sul foximine (BSO) had a little inhibitory effect on KBrO3-induced 8-oxodG form ation. However, the amount of 8-oxodG was still significantly higher than t hat in control, suggesting that intracellular Cys can affect KBrO3 to oxidi ze DNA, when GSH decreased. KBrO3 caused 8-oxodG in isolated DNA in the pre sence of GSH (tripeptide; gamma -GluCysGly), gamma -GluCys, CysGly, or Cys. Methional completely inhibited 8-oxodG formation induced by KBrO3 plus GSH , but typical hydroxyl radical scavengers, SOD and catalase, had little or no inhibitory effects. When bromine solution (BrO-) was used instead of BrO 3-, similar scavenger effects were observed. Experiments with P-32-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-H a-ras-l protooncogene suggested that KBrO3 induced 8-oxodG formation at 5'- site guanine of CTC and GGG sequences of double-stranded DNA in the presenc e of GSH and that treatment of formamidopyrimidine-DNA glycosylase led to c hain cleavages at the guanine residues. ESR spin-trapping studies showed th at 1:2:2:1 quarter DMPO (5,5-dimethyl-1-pyrroline N-oxide) spectrum similar to DMPO/hydroxy radical (. OH) adduct, but the signals were not inhibited by ethanol. Therefore, the signal seemed not to be due to OH but byproduct due to oxidation of DMPO by the reactive species. The signals were suppress ed by the addition of dGMP, but not by other mononucleotides, suggesting th e specific reactivity with guanine. On the basis of our results and previou s literature, it is speculated that reduction of KBrO3 by SH compounds in r enal proximal tubular cells yields bromine oxides and bromine radicals, whi ch are the reactive species that cause guanine oxidation, leading to renal carcinogenesis of KBrO3.