The UV components of sunlight (UVA and UVB) are implicated in the etiology of human skin cancer. The underlying mechanism of action for UVB carcinogenicity is well defined; however, the mechanistic involvement of UVA in carcinogenesis is not fully delineated. We investigated the genotoxicity of UVA1 versus UVB in the overall genome and in the p53 tumor suppressor gene in normal human skin fibroblasts. Immuno-dot blot analysis identified the cis-syn cyclobutane pyrimidine-dimer (CPD) as a distinctive UVBinduced lesion and confirmed its formation in the genomic DNA of UVA1-irradiated cells dependent on radiation dose. HPLC͞tandem MS analysis showed an induction of 8-oxo-7,8-dihydro-2-deoxyguanosine in the genomic DNA of UVA1-irradiated cells only. Mapping of DNA damages by terminal transferase-dependent PCR revealed preferential, but not identical, formation of polymeraseblocking lesions and͞or strand breaks along exons 5-8 of the p53 gene in UVB-and UVA1-irradiated cells. The UVB-induced lesions detected by terminal transferase-PCR were almost exclusively mapped to pyrimidine-rich sequences; however, the UVA1-induced lesions were mapped to purine-and pyrimidine-containing sequences along the p53 gene. Cleavage assays with lesion-specific DNA repair enzymes coupled to ligation-mediated PCR showed preferential, but not identical, formation of CPDs along the p53 gene in UVB-and UVA1-irradiated cells. Additionally, dosedependent formation of oxidized and ring-opened purines and abasic sites was established in the p53 gene in only UVA1-irradiated cells. We conclude that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts. U ltraviolet (UV) irradiation from the sun is linked to basal and squamous cell carcinomas of the skin and cutaneous malignant melanoma in humans (1-3). The etiologically relevant UV wavelengths for development of these diseases are UVA (320-400 nm) and UVB (280-320 nm) (4, 5). UVA constitutes the major proportion of the solar UV reaching the surface of the Earth (Ϸ95%). The remaining fraction is the residual UVB, which is not absorbed by passage through the stratospheric ozone layer (6, 7). Biologically, however, UVB is a few orders of magnitude more potent than UVA in inducing photocarcinogenesis (8).The carcinogenic effect of UVB is irrefutably ascribed to its ability to produce promutagenic DNA lesions including cis-syn cyclobutane pyrimidine-dimers (CPDs) and to a lesser extent pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs]. The carcinogenicity of UVA is partly attributed to its mutagenicity; however, the mechanistic involvement of UVA in mutagenesis remains controversial. The latter is caused by the ambiguous DNA damaging effects of this wavelength (4, 5).Generally, the poor absorption of UVA by DNA supports the notion that UVA indirectly triggers mutagenesis via photosensitization reactions. Presumably, UVA sensitizes intracellular chromophores, thereby generating reactive oxygen species, which in tu...

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm(2) produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage.

Methylglyoxal (MG) and related alpha-oxoaldehydes react with proteins, lipids and DNA to give rise to covalent adducts known as advanced glycation end-products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimers disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. In order to address these issues we have developed a sensitive and quantitative LC-ESI-MS/MS assay using the stable isotope dilution method with an 15N5-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3–12 adducts/107 dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant