Characterization of a primary hepatocyte culture system for toxicological studies.
An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.
Zurlo
J
J
Division of Toxicological Sciences, Johns Hopkins Center for Alternatives to Animal Testing, Baltimore, Maryland 21202-6709, USA.
Arterburn
L M
LM
D000418:Albumins / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D003975:Diazepam / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010969:Plastics; D051381:Rats; D011916:Rats, Inbred F344; D013739:Testosterone / Q000378:metabolism; D018675:Toxicity Tests
D000418:Albumins; D010969:Plastics; D013739:Testosterone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D005978:Glutathione; D003975:Diazepam; D004121:Dimethyl Sulfoxide