Azathioprine and UVA light generate mutagenic oxidative DNA damage

Abstract

Oxidative stress and mutagenic DNA lesions formed by reactive oxygen species (ROS) are linked to human malignancy. Clinical treatments inducing chronic oxidative stress may therefore carry a risk of therapy-related cancer. We suggest that immunosuppression by azathioprine (Aza) may be one such treatment. Aza causes the accumulation of 6-thioguanine (6-TG) in patients' DNA. Here we demonstrate that biologically relevant doses of ultraviolet A (UVA) generate ROS in cultured cells with 6-TG-substituted DNA and that 6-TG and UVA are synergistically mutagenic. A replication-blocking DNA 6-TG photoproduct, guanine sulfonate, was bypassed by error-prone, Y-family DNA polymerases in vitro. A preliminary analysis revealed that in five of five cases, Aza treatment was associated with a selective UVA photosensitivity. These findings may partly explain the prevalence of skin cancer in long-term survivors of organ transplantation.

Fig. 1

Biological consequences of UVA irradiation of DNA 6-TG. (A) UVA generates intracellular ROS in cells with DNA 6-TG. HCT116 cells were grown for 24 hours in medium containing 1 μM 6-TG in the presence or absence of 10 mM HU. In the absence of HU, 6-TG replaced approximately 0.2% of DNA guanine. After thorough washing with phosphate-buffered saline, 6-TG–treated cells were incubated with CM-H2DCFDA and irradiated with 3 kJ/m² of UVA as described (25). Green fluorescence generated by the reaction between CM-H2DCFDA and oxygen free radicals was analyzed by FACS or fluorescence microscopy. HCT116 cells grown without 6-TG and treated with H₂O₂ served as a control for ROS generation. Representative photomicrographs of fluorescent cells are shown. Scale bar, 20 μm. Note the absence of nuclear fluorescence in cells treated with HU. (B) 6-TG sensitizes cells to UVA. HCT116 (circles), A2780 (squares), or CHOD422 (triangles) cells were grown for 48 hours in medium containing 1, 0.1, or 0.1 μM 6-TG, respectively. Treated cells were irradiated with the UVA doses shown and replated in medium without 6-TG. Clonal survival was determined after 10 days. Open symbols: Survival after 30 kJ/m² of UVA administered to the same cells grown in the absence of 6-TG. (C) Mutagenesis by 6-TG/UVA. CHOD422 cells were grown for 48 hours in 0.1 μM 6-TG, washed, and irradiated with 1 kJ/m² of UVA. The aprt mutation frequency was calculated by determining the number of 8-azaadenine–resistant colonies (26). Results are the means of five independent determinations ± SD. The mean spontaneous mutation frequency was 4.1 × 10⁻⁶ (range: 2.1 × 10⁻⁶ to 6.0 × 10⁻⁶), and the mean for 6-TG+UVA was 12.6 × 10⁻⁶ (range: 7.1 × 10⁻⁶ to 19.4 × 10⁻⁶).

Fig. 2

Photochemical reactions of 6-TG. (A) Conversion of free 6-TG into a fluorescent product by UVA. An aqueous solution of 6-TG (0.1 mM) was irradiated with UVA at a dose rate of 0.1 kJ/m²/s. The progress of the reaction was monitored simultaneously by A₃₄₂ (solid circles) and by fluorescence (open circles). Excitation was at 324 nm and emission at 410 nm. The same reaction was carried out in the presence of the free-radical scavenger N-acetyl-L-cysteine (triangles) and monitored by fluorescence. (B) UVA irradiation converts 6-TG into a fluorescent product. 6-TG was irradiated in solution with 10 kJ/m² of UVA, and the products were analyzed by reverse-phase HPLC. Column eluates were monitored simultaneously by A₃₄₂ (left panel) and fluorescence (right panel). 6-TG is converted from a UVA-absorbing compound with minimal fluorescence into an earlier-eluting highly fluorescent product (arrowed) that does not absorb at 342 nm. (solid line, unirradiated; dashed line, irradiated). (C) The fluorescent 6-TG photoproduct is also formed in DNA. Unirradiated (left panel) or UVA-irradiated (50 kJ/m², right panel) 18-mer oligonucleotides that contained a single 6-TG were digested to deoxynucleosides with P1 nuclease and acid phosphatase. These were separated by HPLC. Eluates were monitored by A₃₄₂ and fluorescence at 410 nm. (Left) In digests of unirradiated oligonucleotides, 6-TGdR elutes at 21 min. It is detected by A₃₄₂ but has minimal fluorescence. (Right) After irradiation, no 342-nm–absorbing material is detectable, and a major fluorescent product elutes coincident with the fluorescent UVA photoproduct of authentic 6-TGdR (fig. S2) at 12 min (arrow). (solid line, unirradiated; dashed line, irradiated).

Fig. 3

Primer extension by KF and photoproduct bypass by a Y-family DNA polymerase. (A) 22-mer templates containing G (left) or a single 6-TG (right) were irradiated with the UVA doses indicated. They were annealed to a ³²P end-labeled 14-mer primer that terminated immediately 3′ to the site-specific 6-TG. The primer/templates were used to direct replication by KF. Polymerization was for 60 s at 37°C. Products were analyzed by denaturing gel electrophoresis. The arrow indicates the full-length 22-mer product. (B) Photoproduct bypass by a Y-family DNA polymerase. Primer and templates containing unirradiated template G (control), unirradiated template 6-TG (6-TG), or irradiated (100 kJ/m²) template 6-TG (6-TG + UVA) were supplemented with all four deoxynucleoside triphosphates (dNTPs) and increasing amounts of purified human DNA polymerase η. After 10 min of incubation at 37°C, products were separated by denaturing gel electrophoresis. (C) Nucleotide insertion opposite 6-TG and the 6-TG photoproduct by a Y-family DNA polymerase. Primer and templates containing irradiated template 6-TG were incubated with human DNA polymerase η in the presence of a single dNTP as indicated. After 10 min of incubation at 37°C, products were separated by denaturing polyacrylamide gel electrophoresis. The positions of unaltered primer (−1) and the product that is two nucleotides longer (+1) are indicated.

Fig. 4

Skin photosensitivity in patients treated with Aza. The MED for UVA was determined in five patients who were about to begin a course of Aza treatment for polymorphic light eruption, Crohn's disease, ulcerative colitis, pemphigus vulgaris, or recurrent erythema multiforme. MED measurements were repeated 3 months after beginning Aza treatment (1 to 2 mg/kg/day). Before treatment, black bars; during treatment, white bars.