Gebhardt R, Wegner H, Alber J
Physiologisch-chemisches Institut der Universität Tübingen, Germany.
Cell Biol Toxicol. 1996 Apr;12(2):57-68. doi: 10.1007/BF00143356.
The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 micromol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including induction of cytochrome P450-dependent enzymes and steady-state kinetics of biotransformation, as well as cytotoxic and protective effects of different drugs.
将肝细胞和上皮细胞系与新开发的灌流系统共同培养，用于药物代谢和细胞毒性的体外研究。这种方法提高了肝细胞的活力，并增强了其生物转化能力的诱导。如通过3-甲基胆蒽或苯并蒽诱导7-乙氧基试卤灵O-脱乙基酶活性所证明的，灌流系统中共同培养的肝细胞对这些诱导剂的反应比无灌流时更敏感，这很可能是由于在前一种条件下诱导剂浓度稳定（稳态），而在后一种条件下浓度迅速下降。灌流方法使得在单次或连续孵育期间确定药物代谢动力学成为可能。在用3-甲基胆蒽和苯巴比妥诱导后，灌流共同培养物中洛那唑酸向单羟基化产物的I相代谢与体内产生的报道值密切接近（87%），而在固定共同培养物中仅能达到52%。同样，在灌流共同培养物中可以更精确地检测到细胞毒性作用。如果细胞用0.2 mmol/L半乳糖胺预处理3小时，用递增浓度的甲萘醌进行灌流时，分别在低浓度和高浓度下差异性地杀死上皮RL-ET-14细胞和肝细胞，而在固定共同培养物中未观察到差异效应，只有较高浓度对两种细胞具有细胞毒性。仅在灌流共同培养物中观察到用S-腺苷甲硫氨酸孵育可预防高达250 μmol/L甲萘醌浓度的细胞毒性，而在固定培养物中仅注意到使50%细胞损伤的细胞毒性浓度有轻微向更高值的偏移。这些结果证明了灌流共同培养物在药物代谢研究中的广泛应用，包括细胞色素P450依赖性酶的诱导和生物转化的稳态动力学，以及不同药物的细胞毒性和保护作用。
