Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro.
Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha.
Duell
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EA
Department of Dermatology, University of Michigan, Ann Arbor 48109-0528.
Aström
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A
Griffiths
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CE
Chambon
P
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Voorhees
J J
JJ
D000287:Administration, Topical; D000328:Adult; D002352:Carrier Proteins / Q000502:physiology*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D018168:Receptors, Retinoic Acid; D012867:Skin / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014212:Tretinoin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D002352:Carrier Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; C019429:4-hydroxyretinoic acid; D003577:Cytochrome P-450 Enzyme System; D007654:Ketoconazole