Rat adult hepatocytes in primary pure and mixed monolayer culture. Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism.
The stabilities of several drug oxidation and conjugation pathways in adult rat hepatocytes were investigated in two systems: a primary pure culture lasting 3 days and a primary mixed culture (hepatocytes co-cultured with epithelial cells) lasting 10 days. The cytochrome P450 content in hepatocytes drastically declined within 48 hr in both culture systems. Cytochrome P450-dependent mixed function oxidase was measured by the O-dealkylation of ethoxyresorufin (EROD) and of pentoxyresorufin (PROD). UPD-glucuronosyl transferase (UDP-GT) activity was measured using 1-naphthol and morphine as substrates. In both culture systems, the activities of enzymes belonging to the 3-methylcholanthrene-inducible family, namely EROD and 1-naphthol UDP-GT, were much better maintained than those of PROD and morphine UDP-GT, which belong to the phenobarbitone-inducible family: in pure cultures, EROD and 1-naphthol UDP-GT activities declined to 60% of initial values within 3 days; in mixed cultures, EROD activity was stable throughout the 10 day culture period, whereas that of 1-naphthol UDP-GT was stable until day 4 but had declined to 70% of the initial value by day 8. In contrast, PROD and morphine UDP-GT activities declined to approx. 30% of the initial values within 2 days in both culture systems, and had dropped to approx. 10% of the initial value within 8 days in mixed culture. Reduced glutathione (GSH) levels fluctuated, but remained high throughout culture. GSH conjugation declined to 40% of initial values within 3 days in pure culture, whereas it remained relatively constant in mixed culture. Comparison of these two culture systems therefore showed that although the inclusion of epithelial cells did prolong hepatocyte viability, there was a change in relative enzyme activities in both systems, suggesting a shift towards a more de-differentiated drug metabolism pattern.
Niemann
C
C
Institut de Recherche sur la sécurité de Médicament, Centre de Recherches de Vitry-Alfortville, Vitry sur Seine, France.
Gauthier
J C
JC
Richert
L
L
Ivanov
M A
MA
Melcion
C
C
Cordier
A
A
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured / Q000201:enzymology; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008658:Inactivation, Metabolic; D008099:Liver / Q000201:enzymology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009020:Morphine / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D010078:Oxazines; C007649:ethoxyresorufin; D009020:Morphine; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D005978:Glutathione