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 murin e epidermal cell suspensions by flow cytometric analysis of the fluore scence from a fluorescein isothiocyanate-labeled antibody (H3) directe d against thymine dimers (T >$($) over bar T). Here we present an opti mization of the technique for analysis of epidermal cell suspensions f rom 4 mm biopsies from human skin. Cells with different DNA contents c an easily be distinguished in flow cytometry by the intensity of DNA-s pecific 7-amino-actinomycin D fluorescence. Genuine G(2)-M-phase cells can further be distinguished from cell doublets by pulse-shape discri mination. Thus, T >$($) over bar T levels in individual cells with dif ferent DNA contents (i.e. G(0)-G(1), S or G(2)-M phases) can be determ ined 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 >$($) over bar T in nonreplicating cells (G(0)-G(1) phase) and r eplicating cells (S phase or G(2)-M phase) from seven volunteers expos ed to twice their minimal erythema dose. The reduction in the average T >$($) over bar T-specific fluorescence at 24 h after exposure was 46 % (ranging between 16% and 66%) for the G(0)-G(1) cells and 70% (rangi ng between 37% and 100%) for the S + G(2)-M cells. The difference was statistically highly significant. Determination of individual DNA repa ir capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to ide ntify individuals with low repair proficiencies and increased risk of developing skin cancers.