Background/Aim: Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. Methods: In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. Results: AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. Conclusion: GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin.

There is an increasing need for in vitro testing of compounds for topical application. Reconstructed epidermal models may provide a suitable and relevant model for screening compounds that may affect the activities of phase I and II enzymes involved in epidermal detoxification. In this study, we measured the activity of a phase I enzyme, cytochrome P450 IA1, i.e. 7-ethoxyresorufin-O-deethylase (EROD) and 7-ethoxycoumarin-O-deethylase (ECOD) activities, and that of a phase II enzyme, glutathione S-transferase (GST). The enzyme activities were determined in cultured keratinocytes, reconstructed epidermal models and samples of human epidermis or hair follicle. EROD activity was detected in cultured keratinocytes and was induced by 3-methylcholanthrene (3-MC) and β-naphthoflavone. The level of induction increased with increasing confluence. Induced EROD activity could be inhibited by clotrimazole in a dose-dependent manner. However, EROD activity was not detected in either hair follicles or untreated epidermal models but could be induced by 3-MC. The ability to induce EROD activity in epidermal models was batch dependent, and clotrimazole was able to inhibit the induced EROD activity. ECOD activity was detected in untreated models and paralleled EROD activity. GST activity was detected in cultured keratinocytes and all epidermal models. GST activity in models was equal or higher than the activity in epidermal samples. Reconstructed skin models may be useful to study the effects of non-water-soluble topical formulations on xenobiotic metabolism.

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Synthesis of cholesterol, via the isoprenoid/mevalonate pathway, is required for keratinocyte growth and differentiation, and maintenance of the stratum corneum lipid lamellae. 3-hydroxy-3-methylglutaryl coenzyme A synthase catalyzes the first step in isoprenoid/mevalonate synthesis and under some conditions controls the flux into the pathway. We have investigated whether selected growth factors and hormones could increase 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA in keratinocytes. Northern blotting was used to demonstrate that 10 microg per ml insulin and 0.1 microg per ml epidermal growth factor both increased steady-state levels of 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA by 2.5 and 6-fold, respectively. Epidermal growth factor and insulin also increased 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme activity. 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter activity in a luciferase reporter construct was increased 2-fold by insulin and 2.9-fold by epidermal growth factor. When a mutation in the sterol regulatory element was introduced into the 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter, activity was not increased by insulin, but was increased by epidermal growth factor. Mutation of an AP-1 site in the 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter did not affect the increase in activity following treatment with insulin or epidermal growth factor. Therefore, 3-hydroxy-3-methylglutaryl coenzyme A synthase expression in keratinocytes is regulated by insulin and epidermal growth factor by different mechanisms. These results suggest a role for hormones and growth factors in the control of epidermal cholesterol synthesis.

Reconstructed epidermal models may provide a suitable and relevant model for screening compounds such as quinones, which affect the activities of phase I and II enzymes involved in epidermal detoxification. Reconstructed epidermis may also allow the study of the metabolism of topically applied compounds by the phase I and II enzymes. We demonstrate that NAD(P)H:quinone reductase (NQR) activity is present in three different types of reconstructed epidermal models and that levels vary depending on the type of model. We also determined the inter- and intrabatch variability and demonstrate that NQR activity can be significantly inhibited by dicumarol treatment. The NQR activity in reconstructed epidermis is similar to that in human epidermis and lower than in cultured keratinocytes. Therefore reconstructed epidermis is a more suitable model for testing the effects of topically applied compounds on NQR activity or the metabolism of the compound by NQR.