Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray.
Hepatocyte multicellular aggregates (spheroids), which maintain high expression of liver functions, have been advocated as a useful culture technique for various cell-based assays. In this study, we investigated the drug metabolic function of a hepatocyte spheroid microarray (HSM) chip, which contained an array of 672 spheroids of primary rat hepatocytes within a 100-mm(2) region in the center of a poly(methylmethacrylate) plate (24 x 24 mm) and used an alkoxyresorufin (ethoxy-, methoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase assay system. Ethoxyresorufin O-dealkylase (EROD) activity of the HSM chip initiated by 3-methylcholanthrene (3-MC), an inducer of cytochrome P450 enzymes, was 5- to 10-fold higher than that of monolayer hepatocytes, with activity being maintained for at least 2 weeks. We also demonstrated that 3-MC induced EROD, methoxyresorufin O-dealkylase (MROD) and benzyloxyresorufin O-dealkylase (BROD) activities in the HSM chip, while sodium phenobarbital (P450 inducer) induced pentoxyresorufin O-dealkylase (PROD), BROD, EROD and MROD activities. Induction of these activities was confirmed by increased gene expression of the related P450 enzymes. These results showed that the HSM chip had a good response to P450 inducers and that function was maintained for long periods of time. The HSM chip therefore may be a promising cellular platform for drug metabolic assays using hepatocytes.
Sakai
Yusuke
Y
Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
Tanaka
Tomoko
T
Fukuda
Junji
J
Nakazawa
Kohji
K
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D057005:Bioengineering; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018874:Spheroids, Cellular / Q000201:enzymology / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; C076307:methoxyresorufin-O-demethylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1