CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver.
CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6 beta-hydroxy testosterone as a metabolite was formed. Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptor alpha(RXR alpha), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin. Similarly, the assay with a probe of HNF-4 alpha-binding motif indicated the formation of a large protein complex in the RFB culture. Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4 alpha, the large complexes binding to response elements of PXR or HNF-4 alpha in the RFB culture may contribute to up-regulation of CYP3A4 mRNA. In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4.
Iwahori
Tohru
T
Department of Biochemistry, The Jikei University School of Medicine, Tokyo, Japan.
Matsuura
Tomokazu
T
Maehashi
Haruka
H
Sugo
Ken
K
Saito
Masaya
M
Hosokawa
Masakiyo
M
Chiba
Kan
K
Masaki
Takahiro
T
Aizaki
Hideki
H
Ohkawa
Kiyoshi
K
Suzuki
Tetsuro
T
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019149:Bioreactors; D006528:Carcinoma, Hepatocellular; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010750:Phosphoproteins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000032:analysis; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D013739:Testosterone / Q000378:metabolism; D014157:Transcription Factors / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D004364:Pharmaceutical Preparations; D010750:Phosphoproteins; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin