The use of human hepatocyte cultures to study the induction of cytochrome P-450.
We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively.
Kostrubsky
V E
VE
University of Pittsburgh Medical Center, Department of Pathology, Pittsburgh, Pennsylvania 15261, USA.
Ramachandran
V
V
Venkataramanan
R
R
Dorko
K
K
Esplen
J E
JE
Zhang
S
S
Sinclair
J F
JF
Wrighton
S A
SA
Strom
S C
SC
D000904:Antibiotics, Antitubercular / Q000494:pharmacology / Q000633:toxicity; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D017239:Paclitaxel / Q000494:pharmacology / Q000633:toxicity; D012333:RNA, Messenger / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology / Q000633:toxicity; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D000904:Antibiotics, Antitubercular; D000972:Antineoplastic Agents, Phytogenic; D065607:Cytochrome P-450 Enzyme Inhibitors; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D017239:Paclitaxel; D012293:Rifampin