Wilfried Siefken

Comparison of Activities Dependent on Glutathione S-Transferase and Cytochrome P-450 IA1 in Cultured Keratinocytes and Reconstructed Epidermal Models

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.

Effects of Glyceryl Glucoside on AQP3 Expression, Barrier Function and Hydration of Human Skin

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.

NAD(P)H:quinone reductase activity in human epidermal keratinocytes and reconstructed epidermal models

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.

Regulation of cholesterol synthesis by oleic and palmitic acid in keratinocytes

Abstract: Cholesterol synthesis is essential for homeostasis of the epidermis, being required for both cell division and differentiation, as well as maintenance of the epidermal permeability barrier. Cholesterol synthesis in keratinocytes has been demonstrated to be regulated by sterol levels and the barrier function of the stratum corneum. Cholesterol synthesis in the epidermis is correlated with changes in mRNA levels for key enzymes, such as HMG‐CoA synthase and HMG‐CoA reductase, which have been previously demonstrated to be coordinately regulated by the sterol regulatory element binding proteins (SREBPs). In this study we demonstrate that a functional sterol regulatory element is required for sterol regulation of HMG‐CoA synthase in keratinocytes. We also investigate the regulation of cholesterol synthesis by fatty acids, which are another important constituent of the stratum corneum lipids. Palmitic and oleic acid inhibit 14C‐labelled acetate incorporation into sterols in a similar manner to sterols. However, unlike sterols, 50 μM oleic acid increase the steady state mRNA levels of HMG‐CoA synthase and the activity of the HMG‐CoA synthase promoter. The addition of 50 μM oleic acid to 25‐hydroxycholesterol results in an enhancement of the inhibitory effect of the sterol on promoter activity. The inhibition of acetate incorporation into sterols in human keratinocytes by 50 μM palmitic and 50 μM oleic acid is not due to regulation of HMG‐CoA synthase at the level of transcription.