Triazole antifungal agents promote UV-induced DNA damage by increasing oxidative stress

Abstract

Purpose : Immunosuppression often necessitates long-term use of antimicrobial agents to prevent life-threatening infections like aspergillosis. Voriconazole (Vcz), a second-generation triazole antifungal agent, is commonly used as first-line prophylactic therapy. Vcz is associated with an increased incidence of photodistributed squamous cell carcinoma (SCC). Eyelid cancers account for 5-10% of all cutaneous malignancies with basal cell and SCCs being most common. The strong photo-distribution of Vcz-induced SCCs suggests UV-related tumorigenesis. To understand the tumorigenic effect of Vcz, we performed in vitro studies to investigate the mechanism underlying Vcz-induced cSCC development.

Methods : Primary human keratinocytes (PHKs) were cultured with 25uM Vcz, fluconazole (Fcz), or vehicle (DMSO) for 24 hours; and exposed to UV irradiation at 25mJ/cm². Single cell gel electrophoresis (Comet Assay) was used to assess DNA strand breaks pre- and post-UV exposure. Protein cell extracts were collected, and pp53BP1 and pp38MAPK levels were measured by western blotting using capillary electrophoresis. Levels of nuclear 8-oxo-dG in PHKs exposed to UV and/or Vcz were assayed by immunofluorescence. Cyclobutane pyrimidine dimer (CPD) quantification was performed by in-situ western blot analysis using a Licor Odyssey infrared scanner. Amplex Red Catalase assay kit was used to measure catalase activity.

Results : Vcz-treated, UV-irradiated PHKs exhibited significantly increased DNA damage (p < 0.001) by comet assay compared to controls. Levels of pp53BP1 and pp38MAPK, biomarkers for DNA damage, were increased in both Vcz- and Fcz-treated cells compared to control cells. Treatment with either Vcz or Fcz led to significant (P<0.01) increases in 8-oxoG formation, an oxidative stress marker. Interestingly, no changes in formation or repair of CPD were detected. Instead, in vitro catalase assays showed that Vcz directly inhibits the enzyme independent of UV exposure.

Conclusions : Collectively, our data supports the conclusion that Vcz and its first generation counterpart Fcz promotes UV-induced DNA damage by increasing oxidative stress. Although there are no reports of increased SCC associated with Fcz, our data suggests prolonged use of Fcz could promote UV-induced DNA damage and increase the risk of SCC.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.