EFFECT OF ZINC PYRITHIONE ON SKIN CELLS 5 Table I Effect of ZPT, DS and SPT on 3H-TdR Incorporation intoJTC-17 Cells and Guinea Pig Keratinocytes and Its Reversibility 3H-TdR Guinea Pig Keratinocytes (cpm/dish) % Inhibition ZPT DS DP-300 JTC-17 cells Control 11854 _+ 263 a 1.0/xg/ml (3 hr b) 4963 + 174 58.1 1.0/xg/ml wash c 8423 + 168 28.9 0.5/xg/ml (7 hr) 1380 + 273 88.4 1.0/xg/ml (3 hr) 5966 _+ 30 49.7 1.0/xg/ml wash 11028 + 132 7,0 1,0/xg/ml (3 hr) 11245 + 414 5.1 3H-TdR (cpm/104 cell) Sodium pyrithione Control 1936 + 286 - (S P T) 0.25/xg/ml 1739 -+ 345 10.2 (3 hr b) 0.50/xg/ml 1073 _+ 241 44.8 1.00/xg/ml 256 + 67 86.8 Control 2306 + 234 1.00/xg/ml 2178 + 240 5.5 2.00/xg/ml 2196 + 98 4.8 DP-300 5.00/xg/ml 1814 _+ 146 21.3 (3 hr) 5.00/xg/ml wash 2353 + 716 -2.0 10.00/xg/ml 1302 _+ 354 43.5 10.00/xg/ml wash 2513 + 147 -8.9 a) Mean and standard deviation n = 9, b) Incubation hour. 3H-TdR was incubated for the last 1 hr. c) 5 hr after replacement with fresh medium. EXAMINATION USING SYNCHRONIZED CELLS In order to confirm if ZPT primarily influences cellular DNA synthesis, JTC-17 cells synchronized by excess TdR (2mM), followed by the addition of amethopterin (10 -6 M) and adenosine (10-' M), were assayed for S phase initiation after removal of amethopterin and adenosine. Figure 4 shows that JTC-17 cells synchronized at the beginning of S phase are released into an original cell cycle following removal of amethopterin, thereby inducing DNA synthesis within about an 8 hr period, as shown by the pulse incorporation of 3H-TdR for 30 min. In the case of addition of ZPT at 1.0 •tg/ml concentration, the DNA synthesis starting in early S phase was almost completely suppressed. Further, differences in inhibitory effects depending on the time of addition were examined to determine the active phase in the cell cycle for the ZPT's action. Thus, synchronized cells of JTC-17 were incubated with 1 •tCi/ml of 3H-TdR immediately after or 2.5 hr prior to the addition of ZPT, and the accumulated incorporation of 3H-TdR was compared in both. Results (Figure 5) show the same inhibitory pattern in both cases, indicating that ZPT can act during all periods of DNA synthesis to suppress it. COMPARISON WITH OTHER CELLULAR SYNTHESES Comparison of inhibitory effects using TdR, UR, and leucine incorporations (Table II) revealed that ZPT and DS have fewer inhibitory effects on 3H-UR and 3H-Leucine incorporations compared to 3H-TdR incorporation. Despite about a 90% inhibition of

6 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS x5x10 3 3 1 Control 3Omi Z PT 1.0,•g/•l 3H TdR 1/tCi pulse-label ing 3 4 5 6 7 8 hours after removal of Amethopterin Figure 4. Effect of ZPT on synchronizedJTC-17 cells at early S phase as shown by pulse 3H-TdR uptake for 30 min. ZPT was added at 1.0/•g/ml immediately after removal of amethopterin. No lethal effect on the treated cells was observed with phase contrast microscopy during 8 hr. 3H-TdR incorporation, only 40% and 55% inhibitions of UR and leucine incorporations respectively were seen at the concentration of 1.0 !ag/ml of ZPT and DS. INHIBITORY EFFECT ON CELL GROWTH Addition of ZPT to normal medium in the range of 0.04-0.4/xg/ml caused JTC-17 cells to have a growth inhibition in 5 to 14 days after initiation of culture in comparison with growth in normal medium (Figure 6). This growth inhibition varied depending on the initial concentrations of the seeded cells. Thus, at a low concentration of 3 x 104 cells/dish, about 27.9% growth inhibition was observed with 0.2/xg/ml of ZPT (Figure 6A) while a much lower inhibitory effect of 12.7% was demonstrated at a higher seed concentration of 3.2 x 105 cells/dish (Figure 6B). In the latter case, even 0.4/xg/ml of ZPT exhibits less growth inhibition than is seen at the 3 x 104 cells/dish concentra- tion. DISCUSSION The present study using cultured human skin cells has demonstrated that ZPT has a capacity to inhibit reversibly mammalian DNA synthesis by influencing a cellular