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...

Background & Aims-Dietary exposure to aflatoxin B 1 (AFB 1 ), in addition to other known factors, increases risk for human hepatocellular carcinoma (HCC). HCCs from AFB 1 -exposed individuals frequently have distinct TP53 mutations, such as G to T transversions in the 2 nd guanine of codon 249 (AGG to AGT), and a characteristic mutational spectrum predominated by G:C to T:A mutations.

Despite the predominance of ultraviolet A (UVA) relative to UVB in terrestrial sunlight, solar mutagenesis in humans and rodents is characterized by mutations specific for UVB. We have investigated the kinetics of repair of UVA- and UVB-induced DNA lesions in relation to mutagenicity in transgenic mouse fibroblasts irradiated with equilethal doses of UVA and UVB in comparison to simulated-sunlight UV (SSL). We have also analyzed mutagenesis-derived carcinogenesis in sunlight-associated human skin cancers by compiling the published data on mutation types found in crucial genes in nonmelanoma and melanoma skin cancers. Here, we demonstrate a resistance to repair of UVB-induced cis-syn cyclobutane pyrimidine-dimers (CPDs) together with rapid removal of UVA-induced oxidized purines in the genome overall and in the cII transgene of SSL-irradiated cells. The spectra of mutation induced by both UVB and SSL irradiation in this experimental system are characterized by significant increases in relative frequency of C-->T transitions at dipyrimidines, which are the established signature mutation of CPDs. This type of mutation is also the predominant mutation found in human nonmelanoma and melanoma tumor samples in the TP53, CDKN2, PTCH, and protein kinase genes. The prevailing role of UVB over UVA in solar mutagenesis in our test system can be ascribed to different kinetics of repair for lesions induced by the respective UV irradiation.

Acrolein is an endogenous metabolite and a ubiquitous environmental pollutant. Recently, it has been suggested that acrolein is a major etiologic agent for tobacco smokingrelated lung cancer. Despite the known DNA-damaging effects of acrolein, its mutagenicity to mammalian cells remains uncertain. We have investigated acrolein-induced DNA damage in relation to mutagenesis, with special focus on DNA repair, in mouse and human cells. We mapped the formation of acrolein-induced DNA adducts and the kinetics of repair of the induced lesions in the cII transgene, the mutational target, in acrolein-treated transgenic mouse fibroblasts. Acrolein-DNA adducts were formed preferentially at specific nucleotide positions, mainly at G:C base pairs, along the cII transgene. The induced acrolein-DNA adducts were moderately resistant to DNA repair. Quantification of cII mutant frequency in acrolein-treated cells, however, revealed that acrolein was not mutagenic to these cells at doses sufficient to produce DNA adducts. Determination of supF mutant frequency in DNA repair-proficient and DNA repair-deficient human fibroblasts transfected with acrolein-treated plasmids confirmed a lack of acrolein mutagenicity. Because CpG methylation may intensify acrolein-DNA adduction, we examined whether the extent of CpG methylation in the supF gene can determine acroleininduced mutagenesis in human cells. Enhancement of acrolein-DNA adduction by methylating CpGs in the supF sequence did not elicit a mutagenic response in human fibroblasts, however. We conclude that acrolein is not mutagenic to mouse and human fibroblasts, regardless of DNA repair capacity or methylation status of CpGs, possibly because of a highly accurate replication bypass of the induced lesions. [Cancer Res 2007;67(24):11640-7]

Much of the cancer-causing effects of ultraviolet radiation from the sun have been linked to the formation of dimerized DNA bases. These dimeric DNA photoproducts include the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine(6–4)pyrimidone photoproducts [(6–4)PPs). CPDs are highly mutagenic and are produced in substantial quantities by UVB radiation. These dimers can form between any two adjacent pyrimidines and can involve thymine, cytosine, or 5-methylcytosine. Very recently, a sixth DNA base, 5-hydroxymethylcytosine (5hmC) has been identified and characterized as a normal component of mammalian DNA. Here, we investigated the formation of CPDs at different DNA sequences containing 5hmC following irradiation with UVA, UVB, or UVC light sources. We show that the formation of CPDs at dipyrimidines containing 5hmC occurs at different DNA sequences but is not enhanced relative to cytosine or 5-methylcytosines at the same sequence positions. In fact, in some sequence contexts, CPDs containing 5hmC are formed at very low levels. Nonetheless, CPD formation at 5hmC pyrimidines is expected to be biologically relevant since three types of human skin-derived cells, fibroblasts, keratinocytes and melanocytes, all contain detectable levels of this modified base.