280 JOURNAL OF COSMETIC SCIENCE The histopathologic results observed in our paper show hyperkeratosis and parakeratosis. The nucleus is gone in normal stratum corneum cells but it persists in incompletely keratinized cells. It is believed that such cells undergo differentiation too rapidly for destruction to be completed (13). When the AHAs stimulate the cellular renewal very quickly, there is not enough time for cellular maturation and nucleus cells may be present in the stratum corneum (3). The histopathologic analysis also allowed us visualize an apparently increased dermis in all groups studied, when compared with the control, and it was more evident in the presence of glycolic acid and lactic acid. Treatment with cream plus lactic acid resulted in a thicker, hypercellular, and hydrated dermis when compared with application of glycolic acid. No inflammation was evident. In this paper as in others (15), we have speculated that AHAs may promote collagen synthesis in mouse skin, and so other studies will be conducted by our research group to assess if the increased cell proliferation in the dermis can represent the presence of new collagen. The effect of glycolic acid in collagen synthesis was evaluated by Kim and Won (16), who found increased cell proliferation and collagen synthesis with glycolic acid treat- ment when compared with a control and with malic acid treatment. The results of Moy et al. (17) also showed that glycolic acid causes an increase in collagen synthesis i, vitro. Ditre et al. (6) verified that treatment with AHAs causes an approximate 25 % increase in skin thickness. The epidermis was thicker, and the dermal changes included increased thickness, increased acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen. Dinardo (10) has also demonstrated that increased glycosaminoglycan along with in- creased dermal hydration occurs with the presence of glycolic acid. Collagen deposition is increased. Our results suggest a correlation between the active substances studied and the pH, i.e., the free acid concentration in the formulation. The HAs stimulated the cell renewal of the skin when in acid form. The HA ester formulations with a pH of 4.5-5.0 were less effective. Smith (3) related the cellular renewal induced by AHAs with the pH formu- lation. Various acids were examined for their ability to increase cell renewal at various pHs, as they were neutralized to salt form. Similar results were observed for all acids tested. As pH increased, the ability to stimulate cell renewal diminished. Our results have demonstrated that, in the experimental conditions of the present study, glycolic acid lactic and acid stimulate cellular renewal (increase of mitosis) as a result of the exfoliation process. The differences between hairless mouse and human skin mean that caution is needed in interpreting the results. Nevertheless, the results obtained in this research contribute toward orienting and elucidating the possible effects of these hydroxy acids on the epidermis. CONCLUSIONS Under the present experimental conditions we conclude that:

HYDROXY ACIDS IN COSMETIC FORMULATIONS 281 1. All the formulations studied, including the cream without hydroxy acids, present an epithelium hydradon effect and a more detached horny layer. 2. Hydroxy acids and their derivatives, except salicylic esters of lipophilic acid, in the cream formulation cause a significant total epidermal thickness increase in hairless mice in a fifteen-day application period. The alterations observed were due to an increase in extracellular hydradon and increases in cellular volume (intracellular hydradon) and nuclear volume (intensive cellular activity), because there was no increase in the number of cells. 3. Glycolic acid and lactic acid are more effective in the increase of cellular volume in the basal and spinous layers (with increase in the nuclear and cytoplasmatic volume) when compared with other study groups. 4. Hydroxy acids are more effective when compared with the control group and with their derivatives, mainly in the cellular renovation and in the process of exfoliation. 5. The results show that the cosmetic benefits from the use of hydroxy acids are caused by the modification of the epidermis. ACKNOWLEDGMENTS This work is part of a Master's thesis submitted by Ldcia Helena Terenciani Rodrigues to the Faculty of Pharmaceutical Sciences of Ribeir•o Preto, University of S•o Paulo. Ldcia Helena Terenciani Rodrigues received a graduate (Master's) fellowship from CAPES. The authors thank Dr. Ana Maria Roselino, Dr. Geraldo Maia Campos, and Dr. Ruberval A. Lopes for valuable discussion and suggestions. REFERENCES (1) E. Berardesca and H. Maibach, AHA mechanisms of action, Cosmet. Toiletr., 110, 30-31 (1995). (2) E.J. Van Scott and R.J. Yu, Hyperkeratinization, corneocyte cohesion, and alpha hydroxy acids, J. Am. Acad. Dermatol., 11, 867-879 (1984). (3) W. P. Smith, Hydroxy acids and skin aging, Cosmet. Toiletr., 109, 4108 (1994). (4) E.J. Van Scott and R.J. Yu, Alpha-hydroxy-acids: Therapeutic potentials. Can. J. Dermatol., 1, 108-112 (1989). (5) E.J. Van Scott and R.J. Yu, Alpha-hydroxy-acids: Produceres for use in clinical practice, Cutis, 93, 222-228 (1998). (6) C.M. Ditre, T. D. Griffin, G. F. Murphy, H. Sueki, B. Telegan, W. C. Johnson, R.J. Yu, and E.J. Van Scott, Effects of o•-hydroxy acids on photoaged skin: A pilot clinical, histologic, and ultrastruc- tural study, J. Am. Acad. DermatoL, 34, 187-195 (1996). (7) R. W. Siegfried, Formulating with alpha hydroxyacids, D.C.I., 5, 30-34 (1995). (8) M. G. Rubin, PH is an important element in determining the safety and efficacy of AHAs, Suppl. Cosmet. Dermatol., 5, 14-15 (1996). (9) W. P. Smith, Comparative effectiveness of o•-hydroxy acids on skin properties. Int. J. Cosmet. Sd., 18, 75-83 (1996). (10) J. C. Dinardo, Studies show cumulative irritation potential based on pH, Suppl. Cosmet. Dermatol., 5, 12-13 (1996). (11) S. Bennett, D. Scholz, G.J. Brooks, D. F. Parish, and F. Burmeister, Extratos mistos de frutas: Novo enfoque para renovaq2o da pele, Cosmet. Toiletr., 7, 59 (1995). (12) L. Celleno, A Study to Assess the Antiacne Activity of ot-Hydroxy Acid Esters, Universit• Cattolica del Sacro Cuore, Facolth di Medicina e Chirugia "Agostino Gemelli," Scula Diretta a Fini Speciali per Tecnici Cosmetici (1994).