EFFECT OF ZINC PYRITHIONE ON SKIN CELLS 3 cold trichloroacetic acid (TCA), followed by successive treatments with 70%, 90% and 100% EtOH for dehydration. The TCA insoluble fraction was dissolved in 0. Sml of Soluene©-350 (Packard) and mixed with 10 ml of toluene based scintillation fluid. The radioactivity was counted in a liquid scintillation spectrometer. Cell counts were carried out using a hemocytometer after trypsinization of cells adhering to the glass coverslips. KERATINOCYTE CULTURE Primary cultures of keratinocytes were initiated from the ears of guinea pigs (Hartley strain) according to the method of Christophers (6). The ear skin was floated overnight on 0.25% trypsin in Hank's buffer solution (pH 7.2) at 4øC. The epidermis was separated from the skin, and the epidermal cells were removed by very gentle scraping with a scalpel and filtered through cotton into a tube containing the medium. At 72 hr after seeding at 105 cell/cm 2, incorporation experiments were carried out in the same manner as described above. SYNCHRONOUS CULTURE To obtain synchronized cells in S-phase, a double treatment with excess TdR and amethopterin was used according to the method of Ebina et al. (7). Briefly, JTC-17 cells were first synchronized with excess TdR (2raM) for 24 hr. After replacement with fresh medium for 10 hr, medium containing 10-6M amethopterin and 5 x 10-5M adenosine was added for 16 hr. Then the cultures were washed rapidly with Hank's solution 3 times and received fresh medium for starting DNA synthesis in early S phase. RESULTS INHIBITORY EFFECT OF ZPT ON DNA SYNTHESIS Figure 2A shows the effect of ZPT on the incorporation of 3H-TdR intoJTC-17 cells. Results indicated that ZPT added at the level of 0.25-1.0 •g/ml is effective in suppressing the DNA synthesis of the cell by approximately 20-90%. This inhibitory effect at 1.0 •g/ml was maximal after a 2 hr period (Figure 3), and was found to be diminished within 3 to 6 hr after removal of ZPT from the culture medium (Figure 2B). A similarly active antidandruff agent, DS also exhibits a similar dose dependent inhibition of DNA synthesis with a reversible effect (Figure 2). However, an active antimicrobial agent, DP-300, which has been found to have no antidandruff effect in vivo (1), shows no significant inhibition even at the level of 2.0/xg/ml, although it demonstrates reversible inhibition at high doses of 5/xg/ml and 10/xg/ml (Table I). Table I also shows that ZPT and DS but not DP-300 have a similar reversible anti-DNA synthesizing effect on keratinocytes from the guinea pig ear. Sodium pyrithione, a Zn lacking derivative of ZPT, was also highly effective in suppressing the DNA synthesis of the JTC-17 cells with a similar dose dependence (Table I). Observation of cultures with phase contrast microscopy demonstrated that there was no lethal or cytotoxic appearance occurring at least within 8 hr of our experiments.

4 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (A) [ (B) I•] ZPT [] DS Control 0.25 0.5 1.0/tg/ml hour after removal 1.0•og ml Control 0 3 6 Figure 2. Effects of ZPT and DS on 3H-TdR incorporation into JTC-17 cells (A) and their reversibility (B). Cells were seeded at approximately 2 x 10 • cells/15 mm diameter glass coverslip. After 3 hr incubation with ZPT and DS (13tg/ml), cells were washed three times with Hank's buffer and placed in fresh medium. 3H-thymidine (! 3tCi/ml) was added for the last ! hr of the incubation period. 3100 - 050 - 1.0/tg/ml z P T J TC-17 Cell 2 3 4 5 6 Incubation hour Figure 3. Inhibition of 3H-TdR uptake by ZPT plotted against the incubation time. Experiments were in triplicate.