The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers.
Changes in the isoenzyme pattern of cytochrome P450 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline 4-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P450. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7 alpha, 2 alpha, 6 beta, 16 alpha and 17 sites (androstenedione). During culture, these microsomal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 24 hr and to 15% after 96 hr. The overall decline of cytochrome P450-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P450-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7 alpha site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P450 isoenzymes in vitro.
Wortelboer
H M
HM
UTOX, Research Institute of Toxicology (RITOX), University of Utrecht, The Netherlands.
de Kruif
C A
CA
van Iersel
A A
AA
Falke
H E
HE
Noordhoek
J
J
Blaauboer
B J
BJ
D000818:Animals; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D006900:Hydroxylation; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1