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Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures

In Vitro Technologies Inc., Wingertstrasse 25, D64390 Erzhausen, Germany; 1450 South Rolling Road, Baltimore, MD 21227, USA; hewittn{at}invitrotech.com

Dietmar Utesch

Institute of Toxicology, Merck KGaA, Frankfurter Strasse 250, D-64271 Darmstadt, Germany

Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)).

CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional. Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions. In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis. However, this effect was substrate and species dependent and was not always the same in fresh and CP cells. CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation. CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells. Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture. Speciesspecific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures.

In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.

Key Words: cryopreserved • dog • hepatocytes • metabolism • monkey • rat

Human & Experimental Toxicology, Vol. 23, No. 6, 307-316 (2004)
DOI: 10.1191/0960327104ht453oa


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