Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes.
OBJECTIVE
Small hepatocytes (SH), which are hepatic progenitor cells, were isolated from an adult rat liver. SH in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The morphological changes of SH may be correlated with hepatic maturation. Cytochrome P450s (CYP) are drug-metabolizing enzymes and the expression is one of hepatic differentiated functions. However, it is well known that the re-expression and maintenance of CYP activity are very difficult in cultured hepatocytes. We investigated the expression of CYP and the enzymatic activities in long-term cultured SH.


METHODS
SH were isolated from adult rat livers and SH colonies were collected, replated on new dishes, and then cultured. CYP1A1/2, CYP2B1, CYP3A2, CYP4A1, and CYP2E1 were induced by the addition of 3-methylcholanthrene, phenobarbital, pregnenolone-16alpha-carbonitrile, clofibric acid, and ethanol, respectively. Immunocytochemistry, immunoblots, and enzyme activities were examined.


RESULTS
SH could differentiate into mature hepatocytes by the addition of Matrigel and re-express constitutive CYPs. The expression of CYP1A1/2, CYP2B1, CYP3A2, and CYP4A1 dose-dependently increased and the amounts gradually increased with time in culture, especially in the cells treated with Matrigel. Activities of CYP1A, CYP2B, CYP3A and CYP2E in SH treated with Matrigel induced by each of the inducers were approximately 120-fold, 2.8-fold, 6.4-fold and 0.8-fold higher than in the control.


CONCLUSIONS
The matured SH could re-express the constitutive CYP and recover inducibility, not only of protein expression but also of enzyme activities.
Miyamoto
Shigeki
S
Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, S-1 W-17 Chuo-ku, Sapporo 060-8556, Japan.
Hirata
Koichi
K
Sugimoto
Shinichi
S
Harada
Keisuke
K
Mitaka
Toshihiro
T
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D009940:Organoids / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D051381:Rats; D017207:Rats, Sprague-Dawley
D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System