THE EFFECT OF U. V. IRRADIATION ON ENZYME SYSTEMS IN THE EPIDERMIS By W. COFFEY, P. FINKELSTEIN, and K. LADEN* Presented Seplember 19, 1962, Seminar, New York City THE PHYSIOLOGICAL effect of ultraviolet light irradiation upon the epidermis and dermis has been extensively studied from many points of view. Thus the effect of U. V. light on erythema production, melanin formation, long-term changes in dermal collagen, induction of cancer and histological changes in the epidermis have been, and continue to be, important areas of research (1-6). It is interesting to note, however, that attempts to identify the initial biochemical alterations induced by U. V. light have been in general un- successful. Indeed most of the changes brought about by exposure to U. V. light do not manifest themselves until at least several hours after exposure. Thus, for example, erythema begins to develop two to three hours after irradiation (1) and histopathological changes, twenty-four hours later (7). Recently, Daniels et al. (8) using histochemica! procedures have examined the histochemical, enzymatic and cellular changes occurring in the epidermis after U. V. exposure. While histological and some histo- chemical changes were observed as soon as four hours after irradiation, no histologically demonstrable enzymatic changes were noted unti! extensive cellular damage was observed. ':' It was the purpose of this investigation to see if any signs of damage re- sulting from U. V. irradiation could be detected at fairly short periods of time after exposure. Since one might expect enzymatic changes to precede any marked histological changes, our investigation has centered around the effect of U. V. irradiation on some of the enzyme systems present in the skin. The enzymes we chose to investigate were those involved in glucose metabolism and two transaminase enzyme systems. EXPERIMENTAL Preparation of Tissue Homogenates--Male rats (200 q- 25 gm.) of the Sprague-Dawley strain were used in all experiments. Four days prior to * The•Toni Company, Div. of Gillette, Chicago 54, Ill.

56 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS the day of the experiment, the animals were anesthetized with ether and the hair plucked from the dorsal area. The plucking was done four days before the experiment to allow the skin flare reaction caused by the hair removal to subside. Both control and treated animals were sacrificed by cervical fracture and their pelts quickly removed. After scraping away the sub- cutaneous fat, the pelt was stretched over a chilled drum. The epidermis was then scraped from the dermis with a scalpel, and after weighing on a Roller-Smith precision balance, placed into a Potter-Elvehjem homogeniz- ing tube. A small pair of scissors was next used to mince the tissue, and then a calculated amount of KC1-KHCO• homogenizing fluid (sufFicient fluid to prepare a 5 per cent homogenate) was added and the contents homogenized for three to five minutes in the cold. For spectrophotometric studies, the homogenate was centrifuged at 5000 r.p.m. in a Precision Vari-Hi Speed Centricone centrifuge for fifteen minutes and the resultant supernatant liquid was used. Injury Inducing ProceduresFour days after plucking, the animals were exposed to U. V. light (Westinghouse R. S. 275W Sun Lamp) for twenty- four minutes at a distance of 101/2 in. This exposure was sufficient to pro- duce a reaction comparable to a first-degree burn. The animals were then sacrificed at varying time periods after exposure. Control animals were plucked four days prior to the experiment, but were not exposed to U. V. light. Glucose Oxidation--Glucose oxidation was determined mariometrically in a Warburg apparatus by measuring oxygen uptake. The complete system contained homogenate, buffer (phosphate pH 7.4), Mg ++, ATP, TPN and glucose with KOH in the center well. TPN Reduction--Glucose oxidation via the hexose monophosphate shunt was determined spectrophotometrically by following the rate of TPN-H formation at 340 m•. The system contained epidermal extract, buffer (phosphate pH 8.0), Mg ++, ATP, TPN and glucose. Transaminase .4ctivity--Transaminase activity was followed spectro- photometrically via the oxidation of DPN.H. Both glutamic-oxalacetic trans•minase and glutamic-pyruvic transaminase were studied. The complete system contained: epidermal extract, buffer (phosphate pH 7.4), aspattic acid or alanine, DPN.H, lactic dehydrogenase and alpha keto glutaric acid. RESULTS Initial experiments in this investigation were aimed at studying the effect of U. V. light irradiation on glucose metabolism by rat skin. Rats were irradiated in the manner described and sacrificed at one-half hour, one hour, one and one-half hours and two hours after exposure. Epidermal homogenates were prepared from the exposed sites, and glucose metabolism