Cotovio, J., J. LeClaire, and R. Roguet. Cytochrome P450-Dependent Enzyme Activities in Normal Adult Human Keratinocytes and Transformed Human Keratinocytes. In Vitro Toxicol. 1997. 10(2): 207-216.

Human keratinocytes, which are the most abundant epidermal cell type, are increasingly used to study the cytotoxicity of topically applied compounds and preparations. The cytotoxicity of some compounds may be due to their metabolism in the skin, notably by keratinocytes known to express xenobiotic metabolizing enzymes (phase I and II). The use of normal adult human keratinoctyes (NHK) can be restricted by the small number of cells isolated and by the donor variability. Both disadvantages can be overcome by amplifying the cells or by using cell lines. For pharmacological and/or toxicologic studies, the metabolic capacities of the cell model used may first be determined comparatively to NHK. NHK isolated from breast skin, human keratinocytes cell lines immortalized either spontaneously (NCTC 2544, HaCaT) or by SV-40 transfection (SVK14) were studied for the presence of certain cytochrome P-450-dependent phase I enzyme activities. 7-ethoxy-coumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin-O-dealkylase (PROD) activities were measured after various culture conditions (subculture and cryopreservation). Induction by 3-methylcholanthrene (3-MC) as well as the effect of a mono-oxygenase activity inhibitor (proadifen), were also evaluated. Our results show that after subculture (up to the second passage), NHK retain CYP-dependent ECOD (1.2 to 3.6 pmol of product/h/mg protein) and EROD (1.6 to 5.3 pmol of product/h/mg protein) enzyme activities. These enzyme activities remain inducible by 3-MC (1 uM) in the same proportions as in primary culture (450 to 760 pmol of product/h/mg protein for ECOD and 220 to 365 pmol of product/h/mg protein for EROD). Similar studies of human keratinocyte cell lines also showed the presence of ECOD and EROD activities. These activities were inducible by 3-MC, but less so than in primary culture. PROD activity was not detected. These results are discussed with respect to the use of subcultured NHK or transformed kerationcyte cell lines, for toxicity screening studies of compounds that could be metabolized by the skin.