1. An enzyme-linked immunosorbent assay (ELISA) using specific antisera has been developed to quantify individual cytochrome P450 (P450) enzymes (1A1/2, 2B11, 2C21 and 3A12) in dog liver microsomes. 2. The specific contents of CYP1A1/2, 2B11, 2C21 and 3A12 in untreated male dog liver microsomes determined by the ELISA were 17, 48, 160 and 69 pmol/mg protein respectively, corresponding to 4, 10, 34 and 15% of total optically determined P450 respectively. These P450 enzymes in untreated female dog liver microsomes showed almost similar amounts and relative proportions to those observed in male dog liver microsomes. 3. The oral treatment of male dogs with phenobarbital (PB), rifampicin (Rif) or beta-naphthoflavone (beta-NF) induced significant increases in the contents of CYP1A1/2 (12-fold by beta-NF), 2B11 (16-fold by PB), 2C21 (2-fold by PB) and 3A12 (5-fold by PB and Rif), resulting in marked proportional alterations of the P450 enzymes in dog liver microsomes. 4. This ELISA method will be a useful tool for investigating possible influences (induct on/suppression) of xenobiotics on the expression of P450 enzymes in dog liver.

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1. The effects of phenobarbital (PB), beta-naphthoflavone (beta-NF), omeprazole (Omep) and rifampicin (Rif) on drug-metabolizing activities in dog hepatocytes, cultured with William's medium E, were examined. 2. The drug metabolizing activities of the hepatocytes decreased during culture; 7-ethoxycoumarin O-deethylase (ECOD) activity was nearly 70% of initial value at 72 h, but 7-methoxycoumarin O-demethylase (MCOD), 7-propoxycoumarin O-depropylase (PCOD), progesterone 6 beta-hydroxylase (6 beta-OH-P), progesterone 16 alpha-hydroxylase (16 alpha-OH-P), progesterone 21-hydroxylase (21-OH-P), 7-ethoxyresorufin O-deethylase (EROD) activities and total cytochrome P450 content were approx. 50%. 3. When the hepatocytes were cultured with PB, the enzyme activities increased time- and dose-dependently. MCOD, ECOD and PCOD activities increased 5-8 fold with 2 mM PB in 96 h. Similar results were obtained for 6 beta-OH-P, 16 alpha-OH-P and 21-OH-P activities, and total cytochrome P450. The effect of PB was abolished when 2.5 microM cycloheximide or 0.1 microM actinomycin D was included in the culture. 4. Treatment of hepatocytes with 40 microM beta-NF for 72 h resulted in 25-fold elevation of EROD activity. beta-NF enhanced PCOD activity approx. six-fold, while ECOD increased only slightly, and 7-MCOD negligibly. 5. Omep (100 microM) increased EROD activity nearly 10-fold, and 25 microM Rif increased 6 beta-OH-P activity approx. 8-fold, but ECOD only slightly. 6. Western blot analysis of microsomes from cultured dog hepatocytes with anti-rat CYP 2B1 antibodies indicated that PB increased an immunochemically-reactive protein. The protein showed the same mobility as the major dog P450 isozyme (cytochrome P450 PBD-2 or CYP 2B11) purified from liver microsomes of PB-treated male beagle dog. In a similar manner, induction of cytochrome P450 PBD-1 (CYP 3A12) by PB was confirmed.

1. Effects of rifampicin (Rif) on the contents of cytochrome P450 (P450) enzymes (CYP1A1/2, 2B11, 2C21 and 3A12) assessed by enzyme-linked immunosorbent assay and catalytic activities (ethoxyresorufin O-deethylase, and testosterone 6 beta-, 16 alpha- and 16 beta-hydroxylase; 6 beta-, 16 alpha- and 16 beta-OHT) in dog liver microsomes were compared between liver lobes of both the male and female dogs. 2. In the control dogs, the contents of individual P450 enzymes and their activities showed no significant differences between individual liver lobes and between the sexes. 3. Rif treatment (10 mg/kg/day, p.o. for 7 days) induced substantial increases in the content of CYP3A12 and 6 beta- and 16 beta-OHT activities, and slight increases in the content of CYP2B11 and 16 alpha-OHT activity, and their elevated levels were virtually the same between liver lobes. The magnitudes of the elevation of the CYP3A12 level and 6 beta- and 16 beta-OHT activities compared with control levels appeared to be greater in the female dogs. However, the ratios of their magnitudes (CYP3A content/6 beta-OHT activity and CYP3A content/16 beta-OHT activity) showed no differences between the sexes. 4. In both the control and Rif-treated dogs, the activities of 6 beta- and 16 beta-OHT were specifically inhibited by anti-CYP3A12 antiserum, and 16 alpha-OHT activity was specifically inhibited by anti-CYP2B11 and anti-CYP2C21 antiserum. 5. These results indicate that Rif treatment induces the expression of CYP3A12 protein, and correlates well with the elevation of its catalytic activity (6 beta- and 16 beta-OHT), and that the female dog is more responsive to Rif treatment as compared with the male.