U.v.-induced DNA damage and its repair in human skin in vivo studied by sensitive immunohistochemical methods.
Antibodies specific for u.v.-induced DNA damage were raised in rabbits, and used to study damage and repair of nuclear DNA in nude mouse and human skin in vivo by immuno-fluorescence and immunoperoxidase techniques. Purification of the antibodies by affinity chromatography strongly reduced unspecific background staining. In situ denaturation of nuclear DNA with 70 mM NaOH in 70% ethanol increased the sensitivity of the assay approximately 10-fold. Absorption experiments indicated that the specificity of the antibodies was primarily directed against pyrimidine dimers in single stranded DNA. Immunofluorescence and immunoperoxidase staining were essentially equally sensitive and positive responses using these techniques were already apparent in epidermal cell nuclei after 0.5 minimal erythemal dose (MED) of u.v. light. At higher doses, such as 2 MED, the staining was strong in all the epidermal layers and could also be observed in dermis. Even so, removal of antibody binding sites was well under way at 4-5 h post-irradiation and essentially complete after 24 h. Visible light increased the rate of repair, indicating the involvement of a photoreactivation enzyme in human skin in vivo.