Biotransformation of estradiol in the human keratinocyte cell line HaCaT: Metabolism kinetics and the inhibitory effect of ethanol

Purpose. The aim of our study was to investigate the kinetics of beta-estradiol (E-2) metabolism in the human keratinocyte cell line HaCaT and to estimate the effect of the potential inhibitor ethanol on the biotransformation reaction. Methods. The formation rates of estrone (E-1) in dependence on substrate concentrations were determined in HaCaT cells using tritium labelled E-2. Experiments were conducted with and without addition of dehydroepiandrosterone (DHEA) and ethanol. Possible toxic effects on the cells due to ethanol were investigated by cytotoxicity tests. Results. The metabolism of E-2 in HaCaT cells exhibited Michaelis-Menten kinetics with K-m and V-max values of 3.5 mu M and 216 pmol x mg(-1) protein x h(-1), respectively. The reaction was inhibited by DHEA and ethanol. The alcohol showed a reversible competitive inhibition mechanism for concentrations of 4 to 8% (v/v). Lower ethanol concentrations had no effect, whereas levels greater than or equal to 10% significantly decreased cell viability leading to a different inhibition mechanism. Conclusions. The HaCaT cell line seems to be a suitable model for studying enzyme kinetics equivalent to the human skin. The concentration dependent inhibitory effect of ethanol observed in this cell line may be relevant for the transdermal E-2 application in patients.