In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices.
With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.
Glöckner
R
R
Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, 07740 Jena, Germany. rglo@mti-n.uni-jena.de
Steinmetzer
P
P
Lupp
A
A
Danz
M
M
Müller
D
D
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015925:Cryopreservation; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D008867:Microtomy; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D010634:Phenobarbital / Q000494:pharmacology; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar
D012333:RNA, Messenger; D011285:Pregnenolone Carbonitrile; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C465138:Cyp3a23-3a1 protein, rat; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D010634:Phenobarbital