HYDROXY ACIDS IN COSMETIC FORMULATIONS 279 DISCUSSION In this study, according to the methodology employed, we had the opportunity to evaluate and compare the effects of several HAs and HA esters. The HA esters have pH close to skin pH, a pH of 4.5-5.0, and thus they present a low irritant potential and, like the HAs, their application on the skin can cause both epidermal and dermal effects (6). Among the studied substances, only the salicylic esters mixture did not increase the epithelium thickness significantly, probably because its pH and molecular mass are higher than other studied substances. The other actives had a significant advantage over the vehicle in improving the epidermal thickness, probably due to cutaneous hydration. This result is consistent with data reported by other authors (6), where the treatment with glycolic acid, lactic acid, and citric acid caused an increase in skin thickness, with an increase in epidermal thickness. Glycolic acid, lactic acid, the fruit acids mixture, and malic acid provoked the hydration effect by intra- and extracellular edema. This effect is very interesting because it can improve cutaneous hydration, and it is not limited to the upper cell layers but is also present in the deeper ones. The application of the cream and the cream plus glycolic acid, lactic acid, and fruit mixture formulations caused an increase in the cellular volume of the basal layer and of the spinous layer. In the presence of the malic acid ester an increase in cellular volume was observed only in the spinous layer. The increase in cellular volume was at the expense of nuclear volume and/or an increase in cytoplasmatic volume (intracellular hydration). No inflammation was evident. The increased nuclear volume is probably related to the increased cellular synthesis activity and intense mitotic activity (13). Thus the active substances provoked an increased nuclear volume that may suggest that the nuclei are in intensive activity and that as a result a cellular function stimulus is occurring. The presence of glycolic acid and lactic acid in the cream formulation caused an increase in the nuclear volume of the basal layer, statistically different from the other groups. This result coincided with that of Smith (14), who studied the effects of alpha hydroxy acids (AHAs: glycolic acid, lactic acid, malic acid), and demonstrated that the glycolic acid and lactic acid were more effective in cellular renewal stimulation. Among the active substances studied, only the glycolic acid and the lactic acid caused a significant increase in the thickness of the stratum corneum, which indicates that this layer is in the process of exfoliation. Van Scott and Yu (4) demonstrated that AHAs in topical application reduce the thick- ness of hyperkeratotic stratum corneum, decreasing the corneocyte cohesion. After treat- ment the stratum corneum is less strongly linked to the interior part of the epidermis. Multiple mechanisms may be involved. Low concentrations of AHAs diminish corneo- cyte cohesion (2). The effect occurs at the lower levels of the stratum corneum, and it may involve a dynamic process operative at a particular step of keratinization, like the modification of the ionic bonding. Another mechanism involved is enzymatic inhibition, induced by AHAs, of the reactions of sulphate transferase, phosphotransferase, and kinases, leading to fewer electronegative sulphate and phosphate groups on the outer wall of corneocytes and resulting in the diminishing of the cohesion forces (1).

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: