Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide.

S Kawanishi,S Inoue,S Sano

doi:10.1016/s0021-9258(17)38476-4

Abstract

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.

Highlights

Reactivities of chromium compoundswith DNA were mium(II1)compounds do not produce them [3]
Mechanism was investigated by ESR spectroscopy.In- Recent studies provided a hypothesis that theanionic hexcubation of double-stranded DNA with sodium chro- avalent form of chromium can cross the cell membranes mate(V1) plus hydrogen peroxide or potassium tetra- [7, 8], and it is reduced inside the cell to its trivalent form, peroxochromate(V)led to the cleavage at theposition of every base, of guanine
ESRstudies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during thereaction of sodium chromate(V1) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl and this may strongly react with DNA resulting in DNA

Summary

RESULTS

Reactions of DNA Fragments with Chromium Compounds in the Presence of Hydrogen Peroxide or GSH-32P-labeled DNA fragments were incubated with chromium compounds in the presence and theabsence of hydrogen peroxide or GSH. No oligonucleotides were produced by the incubation of DNA fragments with chromium(V1) compounds or hydrogen peroxide and subsequent piperidine treatment. The electrophoretic mobilities of oligonucleotidesproduced by reaction of sodium chromate(V1) plus hydrogen peroxide with 32P5'-end-labeled DNA showed that cleavage occurred at positions of every base residue, but cleavage at the positions of guanine was more dominant than those of other threebases with piperidine treatment. The g-value was identical with that of potassium tetraperoxochromate(V) in a buffer solution This ESR feature is characteristic for chromium(V) complex [31,32]. Mixture of sodium chromate(V1) and GSH showed an ESR spectrum characteristic for Cr(V) complex, but the ESRparameters (g = 1.9866, @,= = 18.2 G) were different from those of potassium tetraperoxochromate(V). Effecb of OH radical scavengers on the formation of the hydroxyl radical spin adduct of spin traps during the reaction of sodium chromatefVI) with hydrogen peroxide

The Production of Hydroxyl Radical during the Reaction of DMPO

Inhibition of formation of hydroxyl radical spin adduct

DISCUSSION

Inhibition of reducedby superoxideand thenoxidized by hydrogepneroxide