Cytochrome P450 expression-induction profile and chemically mediated alterations of the WIF-B9 cell line.
BACKGROUND
WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF-B9 cell line as a model for analysing drug-induced hepatic effects. The drug metabolism potential of WIF-B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized.


RESULTS
Competitive reverse transcriptase-PCR revealed that four rat (1A1, 2B1/2, 2E1 and 4A1) and four human (1A1, 2Cs, 2D6 and 2E1) CYP mRNA isoforms were constitutively expressed in WIF-B9 cells. The rat CYP forms were expressed at levels 2-4 orders of magnitude higher than the human forms. Exposure for 20-72 h to increasing concentrations of CYP reference inducers (beta-naphthoflavone, 3-methyl cholanthrene, dexamethasone, phenobarbital, clofibrate and pregnenolone 16alpha-carbonitrile) revealed that the rat CYP 1A1, 1A2, 3A1, 3A2 and 4A1 and human CYP 1A1 and 2Cs mRNAs were inducible. Rat CYP 1A1 and 1A2 were the most inducible isoforms since they were overexpressed up to 100-fold after 20-48 h of treatment with beta-naphthoflavone. Human CYP 1A1 and 2Cs mRNAs were induced 3-fold after 48 h of treatment with phenobarbital. Other mechanisms involved in hepatotoxicity were explored using microscopy and immunofluorescence. The WIF-B9 cell line exhibited fragmentation and dilatation of bile canaliculi upon exposure to erythromycin, and to isoniazid and cytochalasins, respectively. Monensin promoted cell depolarization and cytoplasmic granulation. Ethionine promoted cytoplasmic vacuolation and dilatation of the Golgi structures.


CONCLUSIONS
These results indicate that the CYP expression and induction profiles and the morphological features of WIF-B9 cells allow prediction in vitro of the induction and hepatotoxicity profiles of chemical entities.
Biagini
Christine
C
Pfizer Global R&D, Safety Sciences Europe, ZI Pocé sur Cisse, Amboise, France. Christine.biagini@pfizer.com
Bender
Virginie
V
Borde
Françoise
F
Boissel
Elodie
E
Bonnet
Marie-Claire
MC
Masson
Marie-Thérèse
MT
Cassio
Doris
D
Chevalier
Stephan
S
D000818:Animals; D001648:Bile Canaliculi / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects; D004790:Enzyme Induction; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D006822:Hybrid Cells; D007527:Isoenzymes / Q000378:metabolism; D008854:Microscopy, Electron; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System