Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate

Abstract

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may arise from Fenton reaction, which also induces the formation of *OH; the structures of the proposed intrastrand cross-link lesions, however, have not been determined. Here, we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of an intrastrand cross-link lesion, i.e., G wedge T, where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G wedge T. In addition, the yield of the intrastrand cross-link was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-formyl-2'-deoxyuridine. The induction of intrastrand cross-link lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Figure 1

Selected-ion chromatograms (SICs) for the monitoring of the m/z 570 → 472 [a, for unlabeled d(G^T)] and 573 → 475 [b, for labeled d(G^T)] transitions in the Cu/H₂O₂/ascorbate-treated calf thymus DNA after enzymatic digestion. Shown in the insets are the product-ion spectra of the [M + H]⁺ ions of the unlabeled and labeled d(G^T).

Figure 2

Cu(II)/ H₂O₂/ascorbate-induced formation of 8-oxodG (•), 5-FodU (○) and 5-HmdU (θ) in the calf thymus DNA. The values represent the mean ± SD from three independent oxidation and quantification experiments. The inset shows that the yields of the three single-base lesions were proportional to the concentrations of Fenton-type reagents at low dose range.

Figure 3

Cu(II)/ H₂O₂/ascorbate-induced formation of d(G^T) in the calf thymus DNA. The values represent the mean ± SD from three independent oxidation and quantification experiments. The inset shows that the formation of d(G^T) was proportional to the concentration of Fenton-type reagents at low dose range.

Figure 4

(a) Formation of 5-FodU, 5-HmdU, and 8-oxodG under different oxidation conditions. (b) Formation of d(G^T) under different oxidation conditions. The data represent mean ± SD from three independent experiments. No Cu2+: 400 μM H₂O₂ and 4 mM ascorbate; No ascorbate: 400 μM H₂O₂ and 50 μM Cu²⁺; with DMSO: 400 μM H₂O₂, 50 μM Cu²⁺, 4 mM ascorbate and 8 mM DMSO; without DMSO: 400 μM H₂O₂, 50 μM Cu²⁺ and 4 mM ascorbate.

Figure 4

(a) Formation of 5-FodU, 5-HmdU, and 8-oxodG under different oxidation conditions. (b) Formation of d(G^T) under different oxidation conditions. The data represent mean ± SD from three independent experiments. No Cu2+: 400 μM H₂O₂ and 4 mM ascorbate; No ascorbate: 400 μM H₂O₂ and 50 μM Cu²⁺; with DMSO: 400 μM H₂O₂, 50 μM Cu²⁺, 4 mM ascorbate and 8 mM DMSO; without DMSO: 400 μM H₂O₂, 50 μM Cu²⁺ and 4 mM ascorbate.

Scheme 1

Scheme 2