Human keratinocyte cultures as models of cutaneous esterase activity.
A reproducible, quantifiable assay has been developed for the measurement of esterase activity in human keratinocyte cultures, using the model substrate 4-methyl umbelliferyl heptanoate (MUH) which is hydrolysed to the fluorescent metabolite 4-methyl umbelliferone (MU). Activity was assessed in two human keratinocyte cell lines, NCTC 2544 and SVK-14, and in freshly isolated human breast keratinocytes from primary culture to passage 3. V(max) values for MUH hydrolysing activity in the two cell lines showed that the less differentiated cell line NCTC 2544 (V(max) = 23.00 +/- 2.84) expresses a much higher activity than SVK-14s (V(max) = 13.28 +/- 1.42) which are more differentiated and able to form a cornified envelope. Activity in the freshly isolated human breast keratinocytes decreased with time in culture in all three donors tested, which is also likely to relate to the extent of cell differentiation. In human skin, xenobiotic esters penetrating the stratum corneum may be exposed to changing levels of hydrolysing esterases as they are absorbed across the epidermal cell layers. The assay for MUH hydrolysis will be a useful tool for the study of esterase activity in populations of human keratinocytes in vitro.
Barker
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CL
FRAME Alternatives Laboratory, Department of Human Morphology, The Medical School, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.
Clothier
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RH