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. 2005 Jul;18(7):1140-9.
doi: 10.1021/tx050033y.

Guanine-specific oxidation of double-stranded DNA by Cr(VI) and ascorbic acid forms spiroiminodihydantoin and 8-oxo-2'-deoxyguanosine

Peter G Slade  1 , M Katie Hailer, Brooke D Martin, Kent D Sugden
Affiliations

Guanine-specific oxidation of double-stranded DNA by Cr(VI) and ascorbic acid forms spiroiminodihydantoin and 8-oxo-2'-deoxyguanosine

Peter G Slade et al. Chem Res Toxicol. 2005 Jul.

Abstract

7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular reductant such as ascorbate, can oxidize DNA. In this study, duplex DNA was reacted with Cr(VI) and ascorbate to identify and quantify the base lesions formed. Guanine bases were observed to be preferentially oxidized with 5' guanines within purine repeats showing enhanced oxidation. Trapping of the guanine lesions by the base excision repair enzymes hOGG1 and mNEIL2 showed nearly exclusive trapping by mNEIL2, suggesting that 8-oxoG was not the major lesion but rather a lesion recognized by mNEIL2 such as Sp. Formation of the Sp lesion in the Cr(VI)/Asc oxidation reaction with DNA was confirmed by LC-ESI-MS detection. HPLC-ECD was used to identify and quantify any 8-oxoG arising from Cr(VI)/Asc oxidation of DNA. Concentrations of Cr(VI) (3.1-50 microM) with a corresponding 1:10 ratio of Asc oxidized between 0.3% and 1.5% of all guanines within the duplex DNA strand to Sp. 8-oxoG was also identified but with the highest Cr(VI) concentration converting approximately 0.1% of all guanines to 8-oxoG. These results show that Sp was present in concentrations approximately 20 times greater than that of 8-oxoG in this system. The results indicate that 8-oxoG, while present, was not the major product of Cr(VI)/Asc oxidation of DNA and that Sp predominates under these conditions. These results further imply that Sp may be the lesion that accounts for the carcinogenicity of this metal in cellular systems.

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Figures

Figure 1
Figure 1
Further-oxidized lesions of guanine: spiroiminodi-hydantoin (Sp) and guanidinohydantoin (Gh).
Figure 2
Figure 2
Sequencing gel showing the concentration dependence and site-specificity of DNA oxidation by Cr(VI) and ascorbate. Lanes 1 and 2 are untreated DNA control with and without piperidine treatment, respectively. Lane 3 shows the Maxam/Gilbert G/A lane. Lanes 4–8 show the Cr(VI)/Asc-treated DNA following piperidine cleavage in order of decreasing concentrations of Cr(VI)/Asc of (50/500, 25/250, 12.5/125, 6.2/62, 3.1/31 μM).
Figure 3
Figure 3
SDS–PAGE gel showing lesion-specific reductive trapping of 8-oxoG and Sp in duplex DNA by the BER glyco-sylases NEIL2 and hOGG1. Lane 1, 8-oxoG + NaCNBH3 control; lane 2, Sp + NaCNBH3 control; lane 3, unmodified DNA + NaCNBH3 control; lane 4, 8-oxoG-containing DNA + NEIL2 + NaCNBH3; lane 5, Sp-containing DNA + NEIL2 + NaCNBH3; lane 6, unmodified DNA + NEIL2 + NaCNBH3; lane 7, 8-oxoG-containing DNA + hOGG1 + NaCNBH3; lane 8, Sp-containing DNA + hOGG1 + NaCNBH3; lane 9, unmodified DNA + hOGG1 + NaCNBH3.
Figure 4
Figure 4
Trapped BER glycosylases in Cr(VI)/Asc-oxidized duplex DNA. Lane 1, 50/500 μM Cr(VI)/Asc with NEIL2; lane 2, 50/500 μM Cr(VI)/Asc with hOGG1; lane 3, 25/250 μM Cr(VI)/Asc with NEIL2; lane 4, 25/250 μM Cr(VI)/Asc with hOGG1; lane 5, 15–150 μM Cr(VI)/Asc with NEIL2; lane 6, 15/150 μM Cr(VI)/Asc with hOGG1.
Figure 5
Figure 5
(A) SDS–PAGE gel showing the dose-dependent trapping of NEIL2 to Cr(VI)/Asc-treated duplex DNA. Lanes 1–6 are in order of decreasing Cr(VI)/Asc concentrations of 50/500, 25/250, 12.5/125, 6.25/62.5, 3.1/31, and 1.5/15 μM, respectively. Lane 7 is control DNA with 500 μM ascorbate only. (B) Densitometry of the NEIL2-trapped DNA bands as a function of Cr(VI)/Asc concentration. Ascorbate is at 10-fold molar excess to Cr(VI).
Figure 6
Figure 6
Dose-dependent formation of 8-oxoG in DNA with respect to Cr(VI) concentration.
Figure 7
Figure 7
Characteristic mass spectra of the 18O-Sp isotopic standard. B = free base, and M = the free nucleoside.
Figure 8
Figure 8
LC–ESI-MS (SIM) ion chromatogram profiles for a typical Cr(VI) oxidized DNA digest showing the m/z 324 of the spiked 18O-Sp internal standard (A) and the m/z 322 for 16O-Sp formed from the Cr(VI)/Asc reaction (B).
Figure 9
Figure 9
LC–ESI-MS calibration curve for Sp.
Figure 10
Figure 10
Formation of Sp and 8-oxoG from reaction of Cr(VI)/Asc with respect to the percentage of guanine oxidized in the DNA duplex oligonucleotide.
Figure 11
Figure 11
General reaction mechanism proposed for the formation of Sp from guanine.
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