Guanine sulphinate is a major stable product of photochemical oxidation of DNA 6-thioguanine by UVA irradiation

Abstract

The DNA of patients taking the immunosuppressant and anticancer drugs azathioprine or 6-mercaptopurine contains 6-thioguanine (6-TG). The skin of these patients is selectively sensitive to ultraviolet A radiation (UVA) and they suffer an extremely high incidence of sunlight-induced skin cancer with long-term treatment. DNA 6-TG interacts with UVA to generate reactive oxygen species, which oxidize 6-TG to guanine sulphonate (G(SO3)). We suggested that G(SO3) is formed via the reactive electrophilic intermediates, guanine sulphenate (G(SO)) and guanine sulphinate (G(SO2)). Here, G(SO2) is identified as a significant and stable UVA photoproduct of free 6-TG, its 2'-deoxyribonucleoside, and DNA 6-TG. Mild chemical oxidation converts 6-TG into G(SO2), which can be further oxidized to G(SO3)-a stable product that resists further reaction. In contrast, G(SO2) is converted back to 6-TG under mild conditions. This suggests that cellular antioxidant defences might counteract the UVA-mediated photooxidation of DNA 6-TG at this intermediate step and ameliorate its biological effects. In agreement with this possibility, the antioxidant ascorbate protected DNA 6-TG against UVA oxidation and prevented the formation of G(SO3).

Figure 1.

6-TG/UVA photoproducts. (A) Identification of G^SO2 as a major photoproduct. 6-TG (0.1 mM in aqueous solution) was irradiated with 21 kJ/m² UVA. Products were separated by HPLC System 1 as described in ‘Experimental Procedures’ section and the eluate monitored at 320 nm. (Top panel) Unirradiated 6-TG. (Second panel) 6-TG after 21 kJ/m² UVA. (Third panel) As second panel, with authentic G^SO2 added before HPLC. (Bottom panel) Authentic G^SO2 (prepared by mild I₂ oxidation of 6-TG) alone. (B) Quantitation of photoproducts. 6-TG [as in (A)] was irradiated with UVA at a dose rate of 0.07 kJ/m²/s. Samples were removed and analysed on HPLC. The four peaks of 320 mn absorbance with retention times of 2.8 min (filled square), 3.5 min (open circle), 6.5 min (filled triangle) and 14.8 min (open triangle) were identified as G^SO3, G^SO2, G and G-S-G as described in the text. Unaltered 6-TG (filled circle) eluted at 9 min. Quantitation was at the absorbance maximum for each product and by comparison to authentic standard compounds. G^SO3 (A₃₂₅), G^SO2 (A₃₂₀), G (A₂₇₃), 6-TG (A₃₄₀) and G-S-G (A₃₃₁). Products are expressed mole % of unirradiated 6-TG. A representative of three independent experiments is shown. (C) 6-TG oxidation by MMPP. 6-TG (0.1 mM) was treated for 10 min at 20°C with MMPP at the final molar ratio indicated. Products were separated by HPLC System 2 and eluates monitored at 320 nm. Unchanged 6-TG elutes at 9 min, G^SO2 at 3.5 min and G^SO3 at 3.0 min. (D) G^SO2 oxidation by MMPP. G^SO2 (0.1 mM) was treated for 10 min at 20° with MMPP at the molar ratios shown. Products were separated by HPLC System 3.

Figure 2.

UVA oxidation of 6-TG 2′-deoxyribonucleoside. 6-TGdR (20 µM aqueous solution) was irradiated with UVA in neutral conditions (10 mM Tris–HCl pH 7.5). Products were immediately separated by HPLC System 4. Quantitation of unaltered 6-TGdR (filled circle) and dG^SO2 (open circle) by their A₃₄₂, dG^SO3 (filled square) by its fluorescence at 410 nm, dG (filled triangle) by its A₂₆₀ were by comparison to authentic standard compounds. Products are expressed mole % of unirradiated 6-TG. A representative of three independent experiments is shown.

Figure 3.

UVA oxidation of 6-TG in oligonucleotide. (A) Single-stranded 8-mer oligo A₇TG₁ (AAAAXAAA where X = 6-TG; 50-µM aqueous solution) was irradiated with UVA, then digested to 2′-deoxyribonucleosides by nuclease P1 (1 h, 50°, pH 4.7) followed by alkaline phosphatase as described in ‘Experimental Procedures’ section. Products were separated and quantitated as described in legend to Figure 2. Products are expressed mole % of unirradiated 6-TG. A representative of three independent experiments is shown. (B) 6-TGdR (20 µM aqueous solution) was irradiated with 15 kJ/m² UVA, half the sample was immediately analysed by HPLC. The other half was subjected to the same conditions of temperature and pH as for nuclease P1 (pH 4.7, 1 h at 50°C) and alkaline phosphatase digestion before HPLC analysis. The means and SD of three independent experiments are shown. (C) Corrected photoproduct yield. The amount of dG^SO2 destroyed under acidic conditions was calculated based on yield of dG from the irradiated A₇TG₁ and the known dG formation after UVA irradiation of 6-TGdR at pH 7. The total yield of dG^SO2 = dG^SO2 measured + dG^SO2 calculated from (dG measured in A₇TG₁ oligo digested at pH 4.7—dG measured from irradiated 6-TGdR at neutral pH from Figure 2).

Figure 4.

Chemical oxidation of 6-TG, G^SO2 and G^SO3. (A) Rose Bengal and light treatment of 6-TG. Aqueous 6-TG (0.1 mM) was irradiated with visible light (200 W) at a dose of 360 kJ in the presence of 0.5 mM Rose Bengal. Products were separated by HPLC System 1. The relevant part of the absorbance (300 nm) trace is shown. (B) Rose Bengal and light treatment of G^SO2 and G^SO3. Aqueous G^SO2 (upper panels) or G^SO3 (lowest panel) (both 0.1 mM) were irradiated with visible light for the indicated times in the presence of 0.5 mM Rose Bengal. Products were separated by HPLC System 3. (C) Oxidation of G^SO2. Aqueous G^SO2 (0.1 mM) was incubated with the indicated concentrations of Fe(NH₄)₂SO₄, and products analysed immediately by HPLC System 3.

Figure 5.

Ascorbate protects against oxidation. (A) Protection of 6-TG against photochemical destruction. The 6-TG (0.1 mM in aqueous solution) was irradiated with the doses shown in the presence of different concentrations of ascorbate. Photochemical destruction of 6-TG was monitored by the reduction in A₃₄₂ spectrophotometrically. (open circle) no ascorbate (filled circle) ascorbate:6-TG 3: 1 (filled square) 12: 1 (open square) 25: 1 (filled triangle) 100: 1. (B) Protection of DNA 6-TG. A 11-mer oligonucleotide (1 µM) (CAGXAATTCGC where X = 6-TG) was UVA irradiated in the presence of ascorbate as indicated; 0 µM ascorbate (filled square), 10 µM (open circle) or 20 µM (filled circle). Conversion of 6-TG to G^SO3 in the intact oligonucleotide was monitored fluorimetrically (λ_ex 320 nm; λ_em410 nm).

Figure 6.

Reversion of G^SO2 by Na₂S. Aqueous G^SO2 (0.1 mM) was mixed with Na₂S at RT to the final concentration indicated and the sample was immediately analysed by HPLC System 3. Products were detected by A_{320 nm}. The known position of elution of 6-TG is shown arrowed.

Figure 7.

The 6-TG Oxidation: products and reactions. (A) Structures of 6-TG and oxidation products. (B) Reaction scheme for 6-TG. (O) represents oxidizing treatment and, in particular the ¹O₂ that is generated by the interaction of 6-TG with UVA. The more favourable reactions are shown with bold arrows.