Detection of UVR-induced DNA damage in mouse epidermis in vivo using alkaline elution.
Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320-400 nm) or indirectly by UVB (280-320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kJ/m2 (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine were administered (intraperitoneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kJ/m2 (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kJ/m2 (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.
Kinley
J S
JS
Department of Biophysics, Norwegian Radium Hospital, Oslo.
Brunborg
G
G
Moan
J
J
Young
A R
AR
D000818:Animals; D003561:Cytarabine / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000528:radiation effects*; D004249:DNA Damage; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D005260:Female; D006918:Hydroxyurea / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D014466:Ultraviolet Rays
D003561:Cytarabine; D004247:DNA; D006918:Hydroxyurea