Cytochrome P450 enzyme activity and protein expression in primary porcine enterocyte and hepatocyte cultures

1. A method for the isolation and cultivation of porcine hepatoytes and por cine duodenal enterocytes for the investigation of drug oxidation reactions has been established. 2. Hepatocytes as well as enterocytes metabolized ethoxyresorufin (EROD) an d ethoxycoumarin (ECOD) effectively, the rate being 31 +/- 17 pmol/h dish ( EROD) and 9530 +/- 4062 pmol/h dish (ECOD) in the case of hepatocytes, and 9 +/- 4 pmol/h dish (EROD) and 510 +/- 467 pmol/h.dish (ECOD) in the case o f enterocytes. Diazepam, another CYP monooxygenase substrate, was also meta bolized by porcine hepatocytes bur not with porcine enterocytes, thus indic ating differences in the metabolic competence of the liver and the gut. 3. The ability to induce enzymes responsible for the metabolism of ethoxyre sorufin and ethoxycoumarin was investigated in vitro on treatment of the ce ll cultures with either 50 mu M 3-methylcholanthrene (3-MC) or 50 mu M beta -naphthoflavone (beta-NF). With enterocyte cultures, ECOD activity was indu cible up to 20-fold, whereas EROD remained unchanged following treatment wi th either 3-MC or beta-NF. 4. Western blotting provided additional evidence for the expression of CYP1 A1 and CYP3A4 ar the protein level and treatment of cultured enterocytes tt with 30 mu M Aroclor 1254 or 50 mu M beta-NF resulted in enhanced expressi on of the CYP1A protein, and CYP3A4 protein expression was induced followin g treatment with 50 mu M DEX, 2 mM PB, 30 mu M Aroclor 1254 or 50 mu M beta -NF. 5. The metabolism of diazepam was also investigated with bilculovirus-expre ssed human CYP enzymes (2C8, 2C9-ARG, 2C9-CYS, 2C19, 3A4, 3A4+cytochrome b( 5) and 3A5) and evidence was obtained to suggest the formation of temazepam and oxazepam by enzymes of the CYP3A subfamily. Small amounts (32+/-12 ng/ ml) of desmethyidiazepam were additionally recovered in microsomal preparat ions of all CYP-transfected cell lines. 6. In conclusion, porcine duodenal enterocytes can successfully be cultured for a short period and may be used as a tool for studying intestinal metab olism whereas porcine hepatocytes can be cultured for prolonged periods (> 10 days) reliably to investigate hepatic drug oxidation reactions.