2002 ANNUAL SCIENTIFIC SEMINAR 315 SUBSURFACE UV DAMAGE IN SKIN Yash K. Kamath, Ph.D., Sigrid B. Ruetsch and B. Yang TRI Princeton, Princeton, NJ 08540 INTRODUCTION Sunscreens are extensively used in skin care preparations. The efficacy of these compounds as skin protectors is often determined by the effect on the surface of the skin. Many of the studies do not explicitly deal with the changes which occur in the subsurface regions of the epidermis of the skin. In the past, at TRI, we have studied the photodegradation of hair using sunscreens and have shown the effectiYeness of some of these compounds in protecting hair [ 1]. We have studied the photo-protection of synthetic fibers in parallel, using UV stabilizers. In these studies we have observed that photodamage does not always occur by direct impact of a photon with the segment of a molecule, but can also take place by secondary free radical reactions in locations far removed from the regions of photon impact. This happens by the diffusion of free radicals within the material. The environmental conditions, which affect the diffusion of free radicals will have an impact on the rate and extent of damage. We have used UV microspectrophotometry and microfiuorometry to characterize the damage. UV microspectrophotometry can locate damage within the sample ff measurements are done on appropriately prepared cross-sections. Microfiuorometry is ideally suited to evaluate surface damage. In this work we have investigated the protective effect of a water soluble sunscreen benzophenone4 and zinc oxide and titanium dioxide. All this work was done "in vitro" on Yucatan pig skin obtained from Charles River Labs, Windham, ME. Thin sections of the epidermis were excised from the sample and were exposed to Xenon radiation from an Atlas Weather-O-meter. Benzophenone was applied as a solution (10%). Zinc oxide and titanium dioxides were applied in the form of dispersions, leaving a film on the surface. The radiation simulated the sun spectrum and the exposure conditions were similar to 45 ø Miami summer sunlight. The energy density in the different wavelength ranges of the spectrum were: 250- 300 nm - 0.012mW/cm2 300400nm - 5.06mW/cm 2, and 400-800nm - 36.SmW/cm 2 . The peak intensity at 340nm was 0.3W/m 2. The temperature was 50øC at 50% RH. The skin samples were microtomed and the UV spectra of the sections in different regions of the epidermis were obtained. Some specimens were also prepared for microfluorometric study by staining the surface with Rhodamme B. The specimen was excited with green light and the intensity of fluorescence emission was recorded. E•mple fluoromicrographs were also made to demonstrate the effect of sunscreens on the protection of the surface. RESULTS AND DISCUSSION UV Microspectrophotometric Studies: The [IV spectra of these samples were obtained from the spinous cell region of the epidermis, which is shown in Figure 1. Typical UV spectra of the untreated skin before and after exposure indicate that the absorbance in the 280 and 320nm region increases with UV exposure. The subsurface protective effect of benzophenone4 can be seen clearly from Figure 2, in which the spectra of the benzophenone treated skin are shown before and after UV exposure. The increases in absorbance in the 280 and 320nm region are suppressed by the sunscreen. Figure 3 shows similar UV spectra for hair treated with TiO2. High absorbances are observed in the 280nm region indicating that surface deposition of titanium dioxide does not protect the subsurface regions of the skin.

316 JOURNAL OF COSMETIC SCIENCE Microfluorometric Studies: Fluorescence intensities of surface of untreated and sunscreen treated skin are shown in Figure 4. The fluorescence intensities of benzophenone4 treated skin are similar to those observed for the untreated skin, suggesting that benzophenone4 does not protect the surface. On the other hand, both zinc and titanium oxides are effective in protecting the surface. In sunrotary, this study indicates that benzophenone-4 which is capable of penetrating into the skin protects the subsurface regions of the skin but does not protect the surface, probably because its concentration on the surface is very small, or whatever was present on the surface has been destroyed by the radiation. The oxides forming a film on the surface absorb and scatter the radiation so that the surface is protected. However, the free radicals generated in the process diffuse into the subsurface regions of the skin to damage the cells in this area. Therefore, for effective protection of the skin, it seems as though we need a combination of sunscreens which form a film on the surface and also diffuse into the subsurface regions. Figure I 240 •O • 3QQ 320 3dO 3•Q • 4 nn 4•) Wavd•gth (nn• Figure 2 24O 2•} 2•} 300 320 3dO 3•0 3eO 4 nn 4•) Wavele•,th " Unprotected " Benzophenone-4 ,. -, ß UV Exposure (h) UV Exposure (h) TiO 2 ZnO ß r' "-. ß ß • E•mm (h) • E•mu• •) Figure 3 Figure 4 REFERENCES [l] S.B. Ruetsch, Y. Kamath and H.-D. Weigmann (2001). Photodegradation of human hair: a microscopy study. In:Paolo U. Giacomoni (Ed), Sun Protection in Man. Elsevier, Amsterdam, The Netherlands, Chapter 9, pp. 175-205.