Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes

In the present study, we evaluated the inducibility of cytochrome P-450 (CY P) CYP1A, CYP2B, CYP3A, and CYP4A by beta-naphthoflavone, phenobarbital, de xamethasone, and clofibric acid, respectively, in primary hepatocyte cultur es prepared from both fresh and cryopreserved rat hepatocytes. Rat hepatocy tes were successfully thawed and cultured after cryopreservation in liquid nitrogen for up to 1 month. Percentage of total recovery, viable cell recov ery, and final viability of the cells were 68%, 72%, and 85%, respectively. Regardless of whether they were cryopreserved or not, cultured hepatocytes exhibited near-normal morphology. Treatment of cryopreserved hepatocytes w ith beta-naphthoflavone caused an 8-fold increase in 7-ethoxyresorufin O-de alkylase (CYP1A1/2) activity, with an EC50 of 1.5 mu M; treatment with phen obarbital caused a 26-fold increase in 7-pentoxyresorufin O-dealkylase (CYP 2B1/2) activity, with an EC50 of 10 mu M; treatment with dexamethasone caus ed a 10-fold increase in testosterone 6 beta-hydroxylase (CYP3A1/2) activit y, with an EC50 of 1.3 mu M, whereas treatment with clofibric acid caused a 3-fold increase in lauric acid 12-hydroxylase (CYP4A1-3) activity, with an EC50 of 170 mu M. The induction of CYP1A, CYP2B, CYP3A, and CYP4A enzymes by these inducers was confirmed by Western immunoblotting. The patterns of P-450 induction in cryopreserved rat hepatocytes, in terms of concentration response, reproducibility, magnitude, and specificity of response, were si milar to those observed in freshly isolated hepatocytes. Additionally, the magnitude and specificity of induction was similar to that observed in vivo in rats. In conclusion, under the conditions examined, cryopreserved rat h epatocytes appear to be a suitable in vitro system for evaluating xenobioti cs as inducers of P-450 enzymes.