Mechanisms of DNA damage and insight into mutations by chromium(VI) in the presence of glutathione.
The mechanisms of hexavalent chromium(VI) induced DNA damage were unveiled by detecting products of single- and double-stranded DNA in the presence of glutathione. The absence of a detectable hydroxyl radical in the reactions indicates that DNA damage was exclusively by hypervalent chromium species. Polyacrylamide gel electrophoresis (PAGE) experiments with 32-mer single-stranded oligonucleotide and its complementary duplex revealed cleavages largely at purine bases with significant enhancement of such cleavages in the presence of a base. Quantitative estimations of bases released by HPLC before and after enzymatic digestion with exonucleases unequivocally established the excessive release of purine bases. This release was accompanied by the concomitant formation of phosphoglycolate as characterized by liquid chromatography-mass spectrometry (LC-MS). These data connote that the preponderance DNA damage is due to an oxidation specifically at H4' of the ribose moiety leading to the formation of apurinic sites. In addition to the oxidation at H4', DNA oxidation was also initiated through H5' site as evidenced by the identification of furfural. This pathway appears to be non-selective and more abundant for ssDNA as cleavages were observed at both purine and pyrimidine bases. Finally, the detection of guanidinohydantoin as a minor product points the involvement of an oxygen activated hypervalent chromium species, perhaps a peroxo-chromium species. Both major and minor pathways lead to cleavages at purine sites for ds-DNA and are consistent with the observation that DNA cleavage was enhanced in the presence of a base. In contrast, when hydrogen peroxide was added to the reactions, random DNA cleavages were apparent indicating involvement of multiple species including a hydroxyl radical. These data pinpoint mutation mechanisms induced by chromium(VI) in the presence of glutathione due to transversion either by inserting the wrong bases opposite to the apurinic sites during replication or by purine-purine mismatch.
Mazzer
Paula A
PA
Department of Chemistry and Biochemistry, Northern Illinois University, Faraday Hall, DeKalb, IL 60115, USA.
Maurmann
Leila
L
Bose
Rathindra N
RN
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002857:Chromium / Q000494:pharmacology*; D004249:DNA Damage; D005978:Glutathione / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction
D002857:Chromium; D005978:Glutathione