EFFECT OF PHENOBARBITAL AND OTHER MODEL INDUCERS ON CYTOCHROME-P450 ISOENZYMES IN PRIMARY CULTURE OF DOG HEPATOCYTES

1. The effects of phenobarbital (PB), beta-naphthoflavone (beta-NF), o meprazole (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 6beta-hydroxylas e (6beta-OH-P), progesterone 16alpha-hydroxylase (16alpha-OH-P), proge sterone 21-hydroxylase (21-OH-P), 7-ethoxyresorufin O-deethylase (EROD ) activities and total cytochrome P450 content were approx. 50%. 3. Wh en the hepatocytes were cultured with PB, the enzyme activities increa sed time-and dose-dependently. MCOD, ECOD and PCOD activities increase d 5-8 fold with 2 mm PB in 96 h. Similar results were obtained for 6be ta-OH-P, 16alpha-OH-P and 21-OH-P activities, and total cytochrome P45 0. The effect of PB was abolished when 2.5 muM cycloheximide or 0.1 mu M actinomycin D was included in the culture. 4. Treatment of hepatocyt es with 40 muM 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 muM) incr eased EROD activity nearly 10-fold, and 25 muM Rif increased 6beta-OH- P activity approx. 8-fold, but ECOD only slightly. 6. Western blot ana lysis of microsomes from cultured dog hepatocytes with anti-rat CYP 2B 1 antibodies indicated that PB increased an immunochemically-reactive protein. The protein showed the same mobility as the major dog P450 is ozyme (cytochrome P450 PBD-2 or CYP 2B11) purified from liver microsom es of PB-treated male beagle dog. In a similar manner, induction of cy tochrome P450 PBD-1 (CYP 3A12) by PB was confirmed.