Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells.
We have earlier reported on determining UV-induced DNA damage in murine epidermal cell suspensions by flow cytometric analysis of the fluorescence from a fluorescein isothiocyanate-labeled antibody (H3) directed against thymine dimers (T < > T). Here we present an optimization of the technique for analysis of epidermal cell suspensions from 4 mm biopsies from human skin. Cells with different DNA contents can easily be distinguished in flow cytometry by the intensity of DNA-specific 7-amino-actinomycin D fluorescence. Genuine G2-M-phase cells can further be distinguished from cell doublets by pulse-shape discrimination. Thus, T < > T levels in individual cells with different DNA contents (i.e. G0-G1, S or G2-M phases) can be determined after in vivo exposure of human skin to environmentally relevant UVB (280-315 nm) doses. The method was applied to measure the decrease of T < > T in nonreplicating cells (G0-G1 phase) and replicating cells (S phase or G2-M phase) from seven volunteers exposed to twice their minimal erythema dose. The reduction in the average T < > T-specific fluorescence at 24 h after exposure was 46% (ranging between 16% and 66%) for the G0-G1 cells and 70% (ranging between 37% and 100%) for the S + G2-M cells. The difference was statistically highly significant. Determination of individual DNA repair capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to identify individuals with low repair proficiencies and increased risk of developing skin cancers.
Berg
R J
RJ
Department of Dermatology, University Hospital Utrecht, The Netherlands.
de Bueger
S C
SC
Guikers
K
K
van Weelden
H
H
van Vloten
W A
WA
van der Leun
J C
JC
de Gruijl
F R
FR
D002455:Cell Division / Q000502:physiology / Q000528:radiation effects; D004249:DNA Damage; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D011740:Pyrimidine Dimers / Q000378:metabolism* / Q000528:radiation effects*; D012867:Skin / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D014466:Ultraviolet Rays / Q000009:adverse effects*
D011740:Pyrimidine Dimers