Cytochrome P450 3A Expression and Activity in the Rabbit Lacrimal Gland: Glucocorticoid Modulation and the Impact on Androgen Metabolism

• Published in: Investigative ophthalmology & visual science Vol. 46; no. 12; pp. 4697 - 4706
• Main Authors: Attar, Mayssa, Ling, Kah-Hiing John, Tang-Liu, Diane D.-S, Neamati, Nouri, Lee, Vincent H. L
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01.12.2005; Association for Research in Vision and Ophtalmology
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Biological and medical sciences; Blotting, Western; Chromatography, High Pressure Liquid; Dexamethasone - pharmacology; Enzyme Inhibitors - pharmacology; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic - physiology; Glucocorticoids - pharmacology; Hydroxylation; Lacrimal Apparatus - drug effects; Lacrimal Apparatus - enzymology; Male; Microsomes - enzymology; Quinolines - metabolism; Rabbits; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Receptors, Steroid - genetics; Receptors, Steroid - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Testosterone - metabolism; Vertebrates: nervous system and sense organs; Androgen; Enzyme; Cytochrome P450; Rabbit; Activity; Lagomorpha; Metabolism; Glucocorticoid; Vertebrata; Mammalia; Lacrimal gland; Animal; Modulation; Ophthalmology
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.05-0139

Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly prescribed glucocorticoid and can be inhibited by known CYP3A inhibitors.