Oral Azathioprine Leads to Higher Incorporation of 6-Thioguanine in DNA of Skin than Liver: The Protective Role of the Keap1/Nrf2/ARE Pathway

Abstract

Azathioprine is a widely used anti-inflammatory, immunosuppressive, and anticancer agent. However, chronic treatment with this drug is associated with a profoundly increased risk (in certain cases by more than 100-fold) of developing squamous cell carcinoma of the skin. Incorporation of its ultimate metabolite, thio-dGTP, in DNA results in partial substitution of guanine with 6-thioguanine which, combined with exposure to UVA radiation, creates a source of synergistic mutagenic damage to DNA. We now report that oral treatment with azathioprine leads to a much greater incorporation of 6-thioguanine in DNA of mouse skin than liver. These higher levels of 6-thioguanine, together with the fact that the skin is constantly exposed to UV radiation from the sun, may be responsible, at least in part, for the increased susceptibility of this organ to tumor development. Genetic upregulation of the Keap1/Nrf2/ ARE pathway, a major cellular regulator of the expression of a network of cytoprotective genes, reduces the incorporation of 6-thioguanine in DNA of both skin and liver following treatment with azathioprine. Similarly, pharmacologic activation of the pathway by the potent inducer sulforaphane results in lower 6-thioguanine incorporation in DNA and protects 6-thioguanine-treated cells against oxidative stress following exposure to UVA radiation. Protection is accompanied by increased levels of glutathione and induction of multidrug resistance-associated protein 4, an organic anion efflux pump that also exports nucleoside monophosphate analogues. Our findings suggest that activation of the Keap1/Nrf2/ARE pathway could reduce the risk for skin cancer in patients receiving long-term azathioprine therapy. Cancer Prev Res; 4(10); 1665-74. Ó2011 AACR.

Highly Potent Activation of Nrf2 by Topical Tricyclic Bis(Cyano Enone): Implications for Protection against UV Radiation during Thiopurine Therapy

“…As expected, the 6-TG levels in skin DNA were much higher than the corresponding levels in liver (Fig. 3), consistent with our previous observations (36). Compared with control animals, the levels of 6-TG in DNA isolated from ventral skin of TBE-31-treated animals were reduced by 30% (P ¼ 0.023).…”

“…28,34,35). We have observed a very similar level of 6-TG incorporation in skin DNA isolated from SKH-1 hairless mice that had been treated orally with azathioprine (36). In the same study, we also found that genetic upregulation of the Keap1/Nrf2/ARE pathway (by Keap1-knockdown) reduces the 6-TG DNA levels in murine skin and liver by approximately 30%.…”

“…Compared with their wild-type counterparts, Keap1-knockdown mice, in which the Keap1/Nrf2/ARE pathway is constitutively upregulated, have 30% lower levels of 6-TG in their DNA (36). As TBE-31 is a very potent inducer of the Keap1/Nrf2/ARE pathway both in vitro and in vivo (16, 17), we hypothesized that, similar to genetic upregulation of the pathway, pharmacologic induction by TBE-31 may affect the levels of 6-TG in DNA of wild-type mice that are receiving azathioprine.…”

“…MRP4 participates in the export of nucleoside monophosphate analogs (41)(42)(43), and its inducible gene expression is known to be regulated by Nrf2 (40,44,45). Furthermore, silencing of mrp4 increases the 6-TG incorporation in DNA of Hepa1c1c7 cells treated with this thiopurine (36). As TBE-31 caused an upregulation of the MRP4 mRNA levels, we next evaluated the effect of topical treatment with TBE-31 on the incorporation of 6-TG in DNA isolated from skin and liver of azathioprine-treated mice.…”

“…Due to limited quantities of blood, DNA that was extracted from 2 to 3 blood samples was pulled together before the oxidation step, such that the final number of independently analyzed samples was 3 for each group. The levels of 6-TG in DNA were determined as described (28,36): extracted DNA was oxidized with magnesium bis(monoperoxyphthalate) MMPP) in the dark for 30 minutes at room temperature and ethanol precipitated. Double-stranded DNA (120 mg) was denatured by heating at 90 C for 5 minutes, cooled on ice, and digested with 24U nuclease P1 (1 U/mL) for 1 hour at 50 C. After adjusting the pH to 8.0 with 20 mL of 1 mol/L Tris-Cl buffer (pH 8.0), the sample was incubated with alkaline phosphatase (2U) for 1 hour at 37 C. The resulting deoxynucleosides were separated by reverse phase high-performance liquid chromatography (HPLC) on Ascentis C18 column (Supelco, 250 mm x 4.6 mm, 5 mm) using Agilent 1100 system equipped with Agilent G1314A variable wavelength detector and Agilent G1321A fluorescence detector.…”