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). Startin g from 2 X 10(5) cells/ml, approximately 1 X 10(7) cells/ml could easi ly be harvested after 6 days in the case of the parental V79 cells, or after 11 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 diff erence spectra with a Soret maximum around 450 nm. CYP levels in micro somes derived from the V79h1A1 cells of 14 pmol/mg protein were achiev ed. Importantly, no CYP was detected in microsomal fractions of the pa rental V79 cells. Cytochrome b5 levels could also be measured by diffe rence spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growt h 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 (CYP h1A1) in microsomal fractions derived from the large-scale growth of V 79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin- O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testost erone-6 beta-hydroxylation activities. EROD, ECOD, and testosterone-6 beta-hydroxylation activities of the V79h1A1 microsomes were 40 pmol r esorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6 beta-hydroxytestosterone/min/pmol CYPh1A1, respectivel y, indicating the presence of a highly active human CYP1A1 enzyme syst em. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporou s microcarriers is suitable for large-scale growth of V79 cells expres sing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes co ntaining human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes contain ing human CYP isoenzymes may substantially facilitate the production o f sufficient quantities of human metabolites to allow isolation and id entification in an early stage of development of pharmacologically int eresting drugs. (C) 1996 Academic Press, Inc.