Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes.
There is growing evidence to suggest that solar radiation-induced, oxidative DNA damage may play an important role in skin carcinogenesis. Numerous methods have been developed to sensitively quantitate 8-oxo-2'deoxyguanosine (8-oxodG), a recognised biomarker of oxidative DNA damage. Immunoassays may represent a means by which the limitations of many techniques, principally derived from DNA extraction and sample workup, may be overcome. We report the evaluation of probes to thymine dimers and oxidative damage in UV-irradiated cells and the DNA derived therefrom. Thymine dimers were most readily recognised, irrespective of whether in situ in cells or in extracted DNA. However, using antibody-based detection the more subtle oxidative modifications required extraction and, in the case of 8-oxodG, denaturation of the DNA prior to successful recognition. In contrast, a recently described novel probe for 8-oxodG detection showed strong recognition in cells, although appearing unsuitable for use with extracted DNA. The probes were subsequently applied to examine the relative induction of lesions in cells following UV irradiation. Guanine-glyoxal lesions predominated over thymine dimers subsequent to UVB irradiation, whereas whilst oxidative lesions increased significantly following UVA irradiation, no induction of thymine dimers was seen. These data support the emerging importance of oxidative DNA damage in UV-induced carcinogenesis.
Cooke
M S
MS
Oxidative Stress Group, University of Leicester, Leicester Royal Infirmary, UK. msc5@le.ac.uk
Mistry
N
N
Ladapo
A
A
Herbert
K E
KE
Lunec
J
J
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002461:Cell Line, Transformed; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000737:chemistry* / Q000528:radiation effects; D009691:Nucleic Acid Denaturation; D010084:Oxidation-Reduction; D011740:Pyrimidine Dimers / Q000032:analysis; D013539:Simian virus 40; D012878:Skin Neoplasms / Q000209:etiology; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; D000080242:8-Hydroxy-2'-Deoxyguanosine; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine