Vitamin E inhibits the UVAI induction of "light" and "dark" cyclobutane pyrimidine dimers, and oxidatively generated DNA damage, in keratinocytes

Abstract

Solar ultraviolet radiation (UVR)-induced DNA damage has acute, and long-term adverse effects in the skin. This damage arises directly by absorption of UVR, and indirectly via photosensitization reactions. The aim of the present study was to assess the effects of vitamin E on UVAI-induced DNA damage in keratinocytes in vitro. Incubation with vitamin E before UVAI exposure decreased the formation of oxidized purines (with a decrease in intracellular oxidizing species), and cyclobutane pyrimidine dimers (CPD). A possible sunscreening effect was excluded when similar results were obtained following vitamin E addition after UVAI exposure. Our data showed that DNA damage by UVA-induced photosensitization reactions can be inhibited by the introduction of vitamin E either pre- or post-irradiation, for both oxidized purines and CPD (including so-called "dark" CPDs). These data validate the evidence that some CPD are induced by UVAI initially via photosensitization, and some via chemoexcitation, and support the evidence that vitamin E can intervene in this pathway to prevent CPD formation in keratinocytes. We propose the inclusion of similar agents into topical sunscreens and aftersun preparations which, for the latter in particular, represents a means to mitigate on-going DNA damage formation, even after sun exposure has ended.

Figure 1

(A) UVAI-induced oxidizing species dose-response in: control (EtOH) or vitamin E (+Vit E) pre-treated groups; cells were treated with vitamin E for 24 h followed by UVAI irradiation. The production of oxidising species was determined by H₂DCFDA fluorescence, coupled with flow cytometry. Results represent the mean of three independent experiments ±SEM. The UVAI dose-responses were determined by linear regression analyses. R² was >0.98 and slopes were very significantly different from zero (p < 0.0001). (B) Effect of vitamin E supplementation on intracellular HaCaT GSH levels. HaCaTs were supplemented with vitamin E for 24 h prior to UVAI irradiation. Data are expressed as the means ± SEM of three independent experiments. **p < 0.01.

Figure 2

Effect of pre-UVAI incubation with vitamin E on the formation of oxidized purines and CPD. Mean percentage of tail DNA was determined following UVAI doses of 5 and 10 J/cm². Results are the mean ± SEM of three independent experiments; ***p < 0.001, **p < 0.01 for selected comparisons.

Figure 3

Induction and repair of UVAI-induced oxidized purines and CPDs, determined by the T4endoV- and hOGG1-modified comet assay. HaCaT keratinocytes were irradiated with 5 J/cm² UVA and were left to repair for different time periods. At 0 h, the dotted line mainly represents the formation of “light” CPD. The subsequent increase, with a peak at 1 h, represents the formation of “dark” CPD and their repair (1–2.5 h). The dashed line, which joins the dotted line, represents the proposed, differential repair of “light” CPD. The results are the mean ± SEM of three independent experiments.

Figure 4

UVAI-induced oxidizing species dose-response in: control (EtOH) or vitamin E (+Vit E) treated groups; cells were treated with vitamin E for 2.5 h, after UVA irradiation. Production of oxidising species was determined by H₂DCFDA fluorescence. Results represent the mean of three independent experiments ± SEM. The UVA dose-responses were determined by linear regression analyses. R² was >0.92 and slopes were very significantly different from zero (p < 0.0001).

Figure 5

Effects of post-UVAI incubation on oxidized purine and CPD formation. Mean percentage of tail DNA was determined following UVA doses of 5 and 10 J/cm² and treatment of cells with vitamin E for 2.5 h. Results are the mean ± SEM of three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05 for selected comparisons.

Figure 6

Effect of vitamin E pre-incubation on UVC-induced formation of CPD and oxidized purines, determined by T4endoV- and hOGG1-modified comet assays, respectively. The results represent mean (±SEM) percentage tail DNA in HaCaT cells exposed to UVC for 10 s, and have been corrected for baseline levels of damage and SB/ALS. Results are the mean ± SEM of three independent experiments; *p < 0.05 for selected comparisons.

Figure 7

Emission spectrum of the “UVA spot”. This was determined by a Bentham DM150 double monochromator spectroradiometer through the plastic lid of a petri dish in which cells were irradiated, at a distance of 39 cm. Also shown is the absorption spectrum of a vitamin E solution (0.1 mg/mL in ethanol).

Figure 8

Representative images of (A) hOGG1-modified comet assay analysis of untreated cells; (B) hOGG1-modified comet assay analysis of cells treated with 10 J/cm² UVA; (C) hOGG1-modified comet assay analysis of cells treated with 10 J/cm² UVA, with post-irradiation with vitamin E; (D) T4endoV-modified comet assay analysis of cells treated with UVC for 10 s; and (E) T4endoV-modified comet assay analysis of cells treated with UVC for 10 s, after pre-incubation with vitamin E.