ETHYLMORPHINE N-DEMETHYLASE ACTIVITY AS A MARKER FOR CYTOCHROME-P450 CYP3A ACTIVITY IN RAT HEPATIC MICROSOMES

The purpose of this study was to evaluate the selectivity and sensitiv ity of ethylmorphine N-demethylase (EMD) as an indicator of chemically -induced cytochrome P450 CYP3A activity in liver microsomes of rats fo llowing treatment with selective enzyme inducers. Male and female Spra gue-Dawley (CD(R)) rats were dosed with either pregnenolone-16 alpha-c arbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (beta NF; 100 mg/kg pe r day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), iso niazid (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 subseq uently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P < 0.01) in EMD activity were obser ved in microsomes from PB-, DEX-and PCN-dosed animals compared with un treated controls. Microsomes from ISO-and beta NF-dosed males showed a reduction (P < 0.05) in EMD activity when compared with control micro somes, and CF was without effect. In females, EMD activities were sign ificantly increased in microsomes from PCN, DEX and PB-dosed but not b eta NF, ISO, or CF-dosed animals. As expected on the basis of sex-rela ted differences in gene expression, EMD activities in untreated animal s were considerably higher in males than females, attributable to cons titutive CYP3A and CYP2C11 activities. The selectivity of EMD for indu ced CYP3A was confirmed on the basis of inhibition studies with select ed steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclu sion, for both sexes, the greatest elevations (approximate to 3-13-fol d) in EMD activity were observed in microsomes from rats dosed with DE X, a potent archetypal inducer with lesser but significant increases n oted for PCN and PB, indicating that EMD is a reliable indicator of in duced rat hepatic cytochrome P450 CYP3A activity. (C) 1998 Elsevier Sc ience Ireland Ltd. All rights reserved.