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Drug Metabolism and Disposition Fast Forward
First published on July 25, 2006; DOI: 10.1124/dmd.105.008342

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Received for publication November 30, 2005.
Revised July 18, 2006.
Accepted for publication July 19, 2006.

Cytochrome P450 Expression of Cultured Rat Small Hepatocytes After Long-Term Cryopreservation

Hidekazu Ooe 1, Junko Kon 1, Shigeki Miyamoto 2, Yoshiyasu Ozone 3, Shin-ichi Ninomiya 3, Toshihiro Mitaka 1*

1 Dept. Pathophysiol., Cancer Research Institute, Sapporo Medical University School of Medicine 2 First Department of Surgery, Sapporo Medical University School of Medicine 3 Daiichi Pure Chemical Co. Ltd.

* Address correspondence to: E-mail: tmitaka{at}sapmed.ac.jp

Abstract

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study we investigated whether cryopreserved SHs could express cytochrome P450s (CYP), whether CYP expression was induced by appropriate inducers, and whether CYP activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16{alpha}-carbonitrile (PCN), and ethanol (EtOH) were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3MC and PCN, respectively. Although Matrigel did not upregulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs though the activities in both were about 30% of that of MHs. Profiles of 14C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by HPLC. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of CYPs after long-term cryopreservation.


Key words: CYP expression, CYP gene regulation, CYP induction, CYP1A, CYP2B, CYP2E, CYP3A, hepatocytes




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