930 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Effect of Retinoic Acid (Vitamin A Acid) on Growth of Human Skin Epithelial Cells mm of Outgrowth from Explant (Days) Addition 3 6 9 12 Control 0.1 0.7 1.3 1.7 Retinoic acid (10 t•g) 0.1 1.2 1.8 2.6 20 - 15 - 0.1 0.5 1,0 5,0 I0.0 Retinoic Acid (,ug/ml) Figure 3. Effect of rctinoic acid on the keratinization of a 14-day culture of adult human skin In Fig. 3, the inhibition of keratin formation as a function of retinoic acid concentration is shown. In control cultures, approximately 20% of the cells stained orange-red at a concentration of 0.1-10 vg/ml a 90% inhibition of keratinization as determined by the orange G stain was observed. DISCUSSION The present series of experiments on epidermal cells in cell culture demonstrates a direct action of retinoic acid on the epidermal cell. At low concentrations of retinoic acid (0.1 t•g/ml), the growth of epider- mal cells is increased at concentrations greater than 10 t•g/ml, growth is inhibited. Since retinoic acid also inhibits the process of keratiniza- tion at the same concentrations that it increases epidermal growth, it is likely that the effect on growth at lower concentrations is a consequence

RETINOIC ACID AND EPITHELIAL CELLS 931 of a decreased rate of keratinization. Since epidermal cells cannot kera- tinize and divide simultaneously, an inhibition in either cellular process would be expected to increase the rate of the noninhibited process. These effects on epidermal cell growth in vitro correspond to observa- tions made in vivo by Sherman (1). In these experiments, vitamin A-- deficient animals displayed a reduced growth rate of epidermal tissues in addition to a progressive keratinization. Sherman concluded that mod- erate doses of the vitamin enhance growth, and toxic doses depress mitotic activity. Other studies in vitro have also shown a marked inhibition by vitamin A of vaginal and esophageal cornification (13, 14). It would appear clear that to be of topical use in the control of epidermal mitosis, high intracellular concentrations of retinoic acid must be achieved in order to inhibit epidermal growth. Although the antikeratinizing action of both vitamin A and retinoic acid has been well documented, both in vivo (1, 2) and in vitro (3, 4, 13, 14), the mechanism of action of vitamin A and its derivatives is still ob- scure. An interesting theory is that the vitamin may release cathepsins which inhibit keratinization (15). With the inhibition of keratiniza- tion, increased growth would be expected. The opportunity to study epidermal cells in vitro by histochemical and chemical methods in a partially defined medium should provide another system with which to obtain information on the role of retinoic acid in epidermal keratiniza- tion. ACKNOWLEDGMENT The technical assistance of Miss Mary Ellen Charlton is gratefully acknowledged. (Received June 18, 1970) (1) Sherman, B., The effect of vitamin A on epithelial mitosis in vitro and in vivo, J. Invest. Derrnatol., 37, 469 (1961). (2) Pinkus, H., and Hunter, R., Biometric analysis of the effect of oral vitamin A on human epidermis, Ibid., 42, 131 (1964). (3) Fell, H. B., and Mellanby, E. J., Metaplasia produced in cultures of chick ectoderm by high vitamin A, J. Physiol. (London), 119, 470 (1953). (4) Rothberg, S., The cultivation of embryonic chicken skin in a chemically defined medium, and the response of the epidermis to excess of vitamin A, J. Invest. Dermatol., 49, 35 (1967). (5) Studen, A., and Frey, J., Wirkung von Cortison auf die ruhende und die mit vitamin A oder testosteronproprionat zur proliferation gebrachte epidermis der Ratte, Derma- tologica, 104, 1 (1952).