Phase 1 and Phase 2 drug metabolism in isolated epidermal cells from adult hairless mice and in whole human hair follicles.
A sensitive fluorimetric assay to determine both Phase 1 (oxidation) and Phase 2 (conjugation) drug metabolism in epidermal cells isolated from hairless mice, using ethoxycoumarin as a model substrate, is described. Ethoxycoumarin was metabolized by isolated epidermal cells via dealkylation to 7-hydroxycoumarin (7-OHC) and subsequent conjugation. Phase 1 metabolites were extracted in ether from the aqueous incubation media, back extracted into sodium hydroxide and determined fluorimetrically. Conjugated metabolites remaining in the aqueous phase were hydrolysed by the action of beta-glucuronidase and extracted and determined in a similar manner. The production of free 7-OHC by isolated epidermal cells was biphasic at all substrate concentrations tested, exhibiting an initial linear increase followed by a plateau phase. The plateau phase was attributable to the conjugation of 7-OHC produced in situ. Metabolism was inhibited by SKF 525A, carbon monoxide, and alpha-naphthoflavone. Endogenous supplies of reducing equivalents in the form of NADPH were adequate to attain maximal rates of metabolism. With human hair follicles both Phase 1 and Phase 2 activity was detectable in 7 out of 11 subjects. The assay has the advantages of being sensitive, producing single defined metabolites from both Phase 1 and Phase 2 metabolism; is readily adaptable to human skin samples.
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002248:Carbon Monoxide / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006197:Hair / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008812:Mice, Hairless; D011335:Proadifen / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014468:Umbelliferones / Q000378:metabolism
D001571:Benzoflavones; D003374:Coumarins; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C011512:alpha-naphthoflavone; C031477:7-hydroxycoumarin; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D011335:Proadifen