Riboflavin activated by ultraviolet A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C

Abstract

An increasingly popular theory ascribes UVA (>320 -400 nm) carcinogenicity to the ability of this wavelength to trigger intracellular photosensitization reactions, thereby giving rise to promutagenic oxidative DNA damage. We have tested this theory both at the genomic and nucleotide resolution level in mouse embryonic fibroblasts carrying the lambda phage cII transgene. We have also tested the hypothesis that inclusion of a cellular photosensitizer (riboflavin) can intensify UVA-induced DNA damage and mutagenesis, whereas addition of an antioxidant (vitamin C) can counteract the induced effects. Cleavage assays with formamidopyrimidine DNA glycosylase (Fpg) coupled to alkaline gel electrophoresis and ligation-mediated PCR (LM-PCR) showed that riboflavin treatment (1 M) combined with UVA1 (340 -400 nm) irradiation (7.68 J/cm 2 ) or higher dose UVA1 irradiation alone induced Fpg-sensitive sites (indicative of oxidized and/or ring-opened purines) in the overall genome and in the cII transgene, respectively. Also, the combined treatment with riboflavin and UVA1 irradiation gave rise to single-strand DNA breaks in the genome and in the cII transgene determined by terminal transferasedependent PCR (TD-PCR). A cotreatment with vitamin C (1 mM) efficiently inhibited the formation of the induced lesions. Mutagenicity analysis showed that riboflavin treatment combined with UVA1 irradiation or high-dose UVA1 irradiation alone significantly increased the relative frequency of cII mutants, both mutation spectra exhibiting significant increases in the relative frequency of G:C 3 T:A transversions, the signature mutations of oxidative DNA damage. The induction of cII mutant frequency was effectively reduced consequent to a cotreatment with vitamin C. Our findings support the notion that UVA-induced photosensitization reactions are responsible for oxidative DNA damage leading to mutagenesis. ultraviolet A radiation ͉ photosensitizer ͉ antioxidant ͉ skin cancer A large body of evidence exists regarding the association between solar UV irradiation and human skin carcinogenesis (1, 2). Sunlight UV wavelengths that reach the surface of the earth are UVA (Ͼ320-400 nm) and UVB (280-320 nm), with shorter wavelengths (UVC) being completely absorbed by stratospheric oxygen (O 2 ) (1, 3). The biologically relevant UVA and UVB have been extensively studied as the etiologic factors for skin cancer (4). The mechanistic involvement of UVB in carcinogenesis rests upon the ability of this wavelength to induce promutagenic cis-syn cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs), and Dewar valence photoisomers (4). However, the underlying mechanism of action for UVA carcinogenicity is not fully delineated (4). Despite the weak absorbance of UVA by DNA (3), a genotoxic mode of action for UVA has been demonstrated (1). Yet, the exact process through which UVA exerts genotoxicity remains elusive (4).A widely recognized theory ascribes UVA genotoxicity to its ability to trigger intracellular photosensitizat...

Ultraviolet A within Sunlight Induces Mutations in the Epidermal Basal Layer of Engineered Human Skin

“…7 UVA mutagenesis has also been suggested to result from oxidative DNA base modifications, such as 7, 8-dihydro-8-oxoguanine. 55,56 While a high proportion of G:CϾT:A transversions, considered to be the mutagenic hallmark of 7, 8-dihydro-8-oxoguanine, was found in UVA-irradiated rodent cells 57,8 and healthy human skin exposed to 40 J/cm 2 UVA1 three times a week for 2 weeks, 58 none were identified in UVA-irradiated EHS in this study. It is generally accepted that G:CϾA:T transitions are UVB fingerprints.…”

“…G:CϾC:G transversions are likely to be formed by reactive oxygen species, and were frequently seen with UVB and UVA in the current study. These lesions were not found in the hrpt gene of UVA-and UVB-irradiated primary human fibroblasts, 38 nor in cII 57 and lacI transgenes 8,60 in UVA-irradiated Big Blue mouse embryonic fibroblasts. In contrast a similar frequency of A:TϾG:C transitions were induced by UVA and UVB in the hprt gene of human fibroblasts, 38 but only one was found in ssUV-irradiated EHS.…”

“…6 UVA is likely to contribute to skin cancer development. Indeed, UVA is mutagenic in mammalian cells, 7,8 human skin tissues, 9 and induces skin tumors in animal models. 10,11 UVA also causes immunosuppression in humans 2 and has been implicated in the development of malignant melanoma in humans.…”

3-Methyladenine DNA glycosylase is important for cellular resistance to psoralen interstrand cross-links

“…2, panels A and B; Supplemental Figure 3A). UVA induction of γ-H2AX foci formation was previously shown [44] and is probably the consequence of oxidized DNA lesions [45,46] creating sites of single strand DNA and/or stalled replication forks that could also be sites for γ-H2AX foci formation [47][48][49][50]. UVA treated wild-type and Aag−/− cell cultures had many cells with 11-30 foci per cell by 6 hours after treatment (data not shown) but very few cells with >50 foci per cell (12% for both wild-type and Aag−/−; Fig.…”

Antioxidant and Chemotherapeutic Potential of Curcuma Amada Rhizome Extract on Benzo(a)pyrene Induced Cervical Carcinoma in Sprague Dawley Rats

“…GSH constitutes the first line of defense against free radicals and plays an important role in Palanisamy and Samiappan the detoxification of electrophilic substances and prevention of cellular oxidative stress [36]. Several studies have shown that many radical scavengers, including naturally occurring compounds such as vitamin C, have been found to possess a strong antioxidant activity related to a strong scavenging capacity [37].…”