The use of macroporous microcarriers for the large-scale growth of V79 cells genetically designed to express single human cytochrome P450 isoenzymes and for the characterization of the expressed cytochrome P450.
In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 x 10(5) cells/ml, approximately 1 x 10(7) cells/ml could easily be harvested after 6 days in the case of the V79h1A1 cells, resulting in a total of 3.6 x 10(10) cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 +/- 7.9 vs 14.5 +/- 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6 beta-hydroxylation activities. EROD, ECOD, and testosterone-6 beta-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6 beta-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.
Onderwater
R C
RC
Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit, The Netherlands.
Goeptar
A R
AR
Levering
P R
PR
Vos
R M
RM
Konings
P N
PN
Doehmer
J
J
Commandeur
J N
JN
Vermeulen
N P
NP
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D015786:Cytochromes b5 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008861:Microsomes / Q000378:metabolism
D007527:Isoenzymes; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1