Similar Mutagenicity of Photoactivated Porphyrins and Ultraviolet A Radiation in Mouse Embryonic Fibroblasts: Involvement of Oxidative DNA Lesions in Mutagenesis

Abstract

Ultraviolet A (UVA) radiation is implicated in the etiology of human skin cancer. However, the underlying mechanism of carcinogenicity for UVA is not fully delineated. A mutagenic role for UVA has been suggested, which involves activation of endogenous photosensitizers generating oxidative DNA damage. We investigated the mutagenicity of UVA alone and in combination with δ-aminolevulinic acid (δ-ALA), a precursor of the intracellular photosensitizers porphyrins, in transgenic Big Blue mouse embryonic fibroblasts. A significant generation of 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxo-dG), a typical promutagenic oxidative DNA lesion, was observed in cells treated with a combination of δ-ALA (1 mM) and UVA (0.06 J/cm²) as quantified by high-pressure liquid chromatography−tandem mass spectrometry (p < 0.001; relative to the control). The steady-state level of 8-oxo-dG, however, remained unchanged in cells irradiated with UVA or treated with δ-ALA alone. Other photolesions including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were not detectable in cells treated with δ-ALA and/or irradiated with UVA as determined by terminal transferase-dependent polymerase chain reaction assay. Mutation analyses of the cII transgene in cells treated with a combination of δ-ALA and UVA showed an approximately 3-fold increase in mutant frequency relative to the control (p < 0.008), as well as a unique induced mutation spectrum as established by DNA sequence analysis (p < 0.005; 95% CI, 0.002−0.009). No mutagenic effects were observed in cells irradiated with UVA or treated with δ-ALA alone. The spectrum of mutations produced by δ-ALA plus UVA was characterized by a significantly increased frequency of G → T transversions (p < 0.0003; relative to the control), which are the hallmark mutations induced by 8-oxo-dG. Notably, the 8-oxo-dG-mediated mutagenicity of UVA plus δ-ALA is similar to that established previously for UVA alone at a mutagenic dose of 18 J/cm². We conclude that, in the presence of exogenous photosensitizers, UVA at a nonmutagenic dose induces mutations through the same mechanism as does a mutagenic dose of UVA per se.