Time-dependent variations of drug-metabolising enzyme activities (DMEs) in primary cultures of rabbit hepatocytes.
In the present study, time-dependent variations of drug-metabolising enzyme activities (DMEs) in primary cultures of rabbit hepatocytes, a species of economic importance in Mediterranean countries, were investigated. Cross-bred rabbits were anesthetised and their livers perfused in situ by a two-step collagenase technique; cells suspensions were filtered, seeded in collagen-coated dishes and cultivated at 37 degrees C in a controlled atmosphere for 24 and 72 h. Cytochrome P450 and b(5) contents as well as the catalytic activity of some P450-dependent monooxygenases were measured in subcellular fractions obtained by differential ultracentrifugation; microsomal proteins were also subjected to immunoblotting, using antibodies to rat P4501A, 2B, 2E1 and 3A isoforms. The activity of some microsomal hydrolytic enzymes was also determined. As regards conjugative enzymes, glutathione content and activities of glutathione S-transferase, uridindiphosphoglucuronosyl-transferase, acetyl-transferase and 1,2-epoxibuthane glutathione transferase were assayed. An overall reduction of the catalytic activity was observed 72 h after plating, reaching in certain instances the level of statistical significance. On the whole, our data confirm those previously reported with hepatocytes obtained from other species; however, the evidence that DMEs were still measurable after 72 h supports the usefulness of this in vitro method for drug metabolism studies in the rabbit as well.
Institut Nationale de la Recherche Agronomique, Laboratoire de Pharmacologie et Toxicologie, UR66, 180 chemin de Tournefeuille, St. Martin du Touch, BP 3, 31931 Toulouse cedex, France.
C
C
Eeckhoutte
A Giuliano
AG
Albo
M
M
Carletti
A Rossetto
AR
Giaccherino
P
P
Galtier
C
C
Nebbia
M
M
Dacasto
D000818:Animals; D000906:Antibodies; D018929:Cell Culture Techniques; D003577:Cytochrome P-450 Enzyme System / Q000494:pharmacology*; D005978:Glutathione / Q000032:analysis / Q000378:metabolism; D005982:Glutathione Transferase / Q000494:pharmacology*; D022781:Hepatocytes / Q000473:pathology; D015151:Immunoblotting; D007700:Kinetics; D011817:Rabbits
D000906:Antibodies; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; D005978:Glutathione