title: Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes.
abstract: The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague-Dawley (CD) rats were dosed with either pregnenolone-16alpha-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (betaNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P < 0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and betaNF-dosed males showed a reduction (P < 0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not betaNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (approximately 3-13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.
authors.affiliation: Drug Safety Evaluation Department, Pfizer Central Research, Groton, CT 06340, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Amacher
authors.forename: S J
authors.initials: SJ
authors.lastname: Schomaker
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001189:Aryl Hydrocarbon Hydroxylases; D015415:Biomarkers / Q000032:analysis; D002994:Clofibrate / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000276:immunology; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005037:Ethylmorphine-N-Demethylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D005260:Female; D007538:Isoniazid / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology; D010634:Phenobarbital / Q000494:pharmacology; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012737:Sex Factors; D019324:beta-Naphthoflavone / Q000494:pharmacology
chemical_list: D000906:Antibodies; D015415:Biomarkers; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011285:Pregnenolone Carbonitrile; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005037:Ethylmorphine-N-Demethylase; D002994:Clofibrate; D007538:Isoniazid; D010634:Phenobarbital