Hengstler J G, Ringel M, Biefang K, Hammel S, Milbert U, Gerl M, Klebach M, Diener B, Platt K L, Böttger T, Steinberg P, Oesch F
Institute of Toxicology, Mainz, Germany.
Chem Biol Interact. 2000 Feb 15;125(1):51-73. doi: 10.1016/s0009-2797(99)00141-6.
The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenzyme-dependent regio and stereoselective testosterone hydroxylations were 1.6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, as questionable these induction factors were similar to those of cultures with freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3-methylcholanthrene (5 microM; 72 h) induced exclusively the formation of 7alpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocytes. This specificity also correlates to that obtained in rats. Although these induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products were reduced when compared to fresh cultures. For instance, 6beta-hydroxytestosterone, the main metabolite in solvent controls was reduced to 79%, 7alpha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16beta-hydroxytestosterone, the main metabolite after induction with PB, was reduced to 52%. Similarly, EROD activity after induction with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and validation is required, the data show that cytochrome P450 activities can clearly be induced in co-cultures of cryopreserved hepatocytes, in a fashion which for the investigated inducers, is similar to that in cultures from freshly isolated hepatocytes and similar to the in vivo situation.
肝细胞培养在药物相互作用研究中已得到广泛应用。然而，使用人肝细胞培养的主要障碍是人类肝细胞仅偶尔可得。这个问题可以通过冷冻保存来克服。尽管冷冻保存的肝细胞已被推荐用于短期悬浮培养，但关于酶活性诱导的研究，需要在培养中对冷冻保存的肝细胞进行更长时间的维持，这代表了一个新的研究领域。在本研究中，我们建立了一种技术，该技术允许使用冷冻保存的肝细胞制备大鼠肝细胞共培养物。在用苯巴比妥（0.75 mM；72小时）孵育后，对于2α-、2β-、6α-、6β-、7α-、15β-、16α-和16β-羟基睾酮以及4-雄烯-3,17-二酮，同工酶依赖性区域和立体选择性睾酮羟基化的诱导因子分别为1.6、2.2、1.0、2.1、5.6、2.4、3.6、4.5和0.9。对于诱导因子小于2倍的情况，由于这些诱导因子与新鲜分离肝细胞培养物的诱导因子相似，且各个羟基化产物的诱导模式与体内情况相似，因此这些诱导因子值得怀疑。此外，在冷冻保存肝细胞的培养物中，3-甲基胆蒽（5 microM；72小时）仅诱导7α-羟基睾酮的形成（6.6倍）。这种特异性也与在大鼠中获得的特异性相关。尽管这些诱导因子在冷冻保存的培养物中显然令人满意，但与新鲜培养物相比，主要睾酮羟基化产物的绝对活性降低。例如，溶剂对照中的主要代谢产物6β-羟基睾酮降低到79%，用3-MC诱导后的主要代谢产物7α-羟基睾酮降低到66%，用PB诱导后的主要代谢产物16β-羟基睾酮降低到52%。同样，与新鲜培养物相比，冷冻保存培养物中用3-甲基胆蒽诱导后的EROD活性降低到62%。尽管需要进一步优化和验证，但数据表明，在冷冻保存肝细胞的共培养物中，细胞色素P450活性可以明显被诱导，对于所研究的诱导剂，其诱导方式与新鲜分离肝细胞培养物中的诱导方式相似，且与体内情况相似。
