Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects

Abstract

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 μM), while α-tocopherol and the iron chelator o-phenanthroline had no effect and β-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

Introduction

There is good evidence that the carcinogenic potential of natural sunlight is mostly a consequence of DNA modifications that are generated by direct excitation of DNA in the UVB range of the spectrum, in particular cyclobutane pyrimidine dimers and (6–4) photoproducts 1, 2. However, some DNA damage is also generated indirectly via excitation of yet unidentified cellular chromophores at wavelengths at which DNA does not absorb. According to analysis by means of repair endonucleases, this indirect (photosensitizer-mediated) DNA damage is most efficient at wavelengths around 420 nm [3]. The spectrum of DNA modifications (damage profile) generated in cells irradiated at these wavelengths is quite different from that caused by direct excitation of DNA on the one hand and by hydroxyl radicals on the other, but similar to that induced in DNA by photosensitizers such as methylene blue, porphyrins and acridins, both under cell-free conditions and in cells 3, 4, 5, 6. The DNA damage consists to a large extent of oxidative base modifications sensitive to formamidopyrimidine-DNA glycosylase (Fpg protein), a repair endonuclease which is known to recognize 8-hydroxyguanine (7,8-dihydro-8-oxoguanine; 8-oxoG) residues and formamidopyrimidines (imidazole-ring opened purines) (Table 1), while single-strand breaks (SSB), sites of base loss (AP sites) and pyrimidine modifications sensitive to endonuclease III (5,6-dihydropyrimidine derivatives) are relatively rare. The relative yields of other modifications such as DNA-protein cross-links [13]or oxazolones [14]remain to be established.

That relatively high yields of 8-oxoG in cellular DNA are generated by photosensitizers such as riboflavin and hematoporphyrin has been demonstrated by HPLC analysis with electrochemical detection 15, 16. 8-oxoG has also been detected in cells exposed to radiation from a broad-spectrum sun-lamp [17]and to UVA 18, 19.

The generation of 8-oxoG by endogenous photosensitizers in cells and the well-established mutagenic activity of this lesion 20, 21, 22predict that visible light may have mutagenic and carcinogenic potential. Indications that the indirectly generated oxidative DNA modifications indeed contribute to the mutagenicity and carcinogenicity of solar radiation have been obtained by comparison of the mutation spectra observed after UVA or solar radiation on the one hand and UVB or UVC irradiation on the other. Thus, the percentage of G→T transversions, which is the prominent type of mutation caused by 8-oxoG, was higher in human skin cancers than in UVB-induced tumors in mice [23]. The action spectrum of photocarcinogenesis in hairless mice is at least compatible with a role of non-dimer modifications in skin cancer induction [24]. In bacteria, there is evidence from experiments with strains deficient in Fpg protein that UVA-induced Fpg-sensitive modifications indeed give rise to G→T transversions [25].

Here, we report on the extent of the oxidative DNA damage induced by visible light in several types of cells under various conditions and on experiments to estimate the mutagenic risk arising from this type of damage.