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 beta-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.

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.