Coecke S, Segaert A, Vercruysse A, Rogiers V
Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
Toxicol In Vitro. 1993 Jul;7(4):487-91. doi: 10.1016/0887-2333(93)90052-7.
Until now hormonal regulation of flavin-containing monooxygenase (FMO) has been studied to a limited extent and all experiments reported so far have been carried out in vivo using invasive techniques. In order to examine whether in vitro models could be applied to study the regulation of FMO, pure cultures of rat hepatocytes and their co-cultures with primitive bile-duct epithelial cells have been used. Since the addition of foetal calf serum (FCS) to the medium, usually done to improve cell attachment, could interact with some hormones, the effect of its removal on FMO activity has been investigated. Furthermore, to find out if in vitro results comparable with the in vivo situation could be obtained, phenobarbital was added and its effect on the FMO system was measured. In pure hepatocyte cultures FMO activity declined continuously as a function of culture time, and after 6 days it was only 15% of that obtained for freshly isolated hepatocytes. In contrast, in co-cultures after an initial decrease a steady-state situation was maintained at a level of approximately 37% of the initial value. This observation was noted from day 7 onwards and for at least 1 wk. It was also observed that in both culture systems, the various media had no statistically significant effect on FMO activity expressed in nmol methimazole/min/mg microsomal protein. In pure hepatocyte cultures, however, addition of FCS and phenobarbital significantly increased the microsomal protein content per culture dish in comparison with the value obtained for serum-free medium. In the latter case deterioration and loss of hepatocytes in the absence of FCS occurred. In conclusion, the expression of FMO is better and longer maintained in co-cultures than in pure cultures. The removal of FCS from the medium in co-cultures does not affect FMO activity. Since addition of phenobarbital has no inducing effects on FMO activity, as is also the case in vivo, the results support the idea of using co-cultures of rat hepatocytes as an in vitro model for regulation studies of FMO.
迄今为止，对含黄素单加氧酶（FMO）的激素调节的研究还很有限，而且目前报道的所有实验都是在体内采用侵入性技术进行的。为了研究体外模型是否可用于研究FMO的调节，已使用大鼠肝细胞的纯培养物及其与原始胆管上皮细胞的共培养物。由于通常为改善细胞贴壁而向培养基中添加胎牛血清（FCS）可能会与某些激素相互作用，因此研究了去除FCS对FMO活性的影响。此外，为了确定是否能获得与体内情况相当的体外结果，添加了苯巴比妥并测量了其对FMO系统的影响。在纯肝细胞培养物中，FMO活性随培养时间持续下降，6天后仅为新鲜分离肝细胞的15%。相比之下，在共培养物中，最初下降后会维持在约初始值37%的稳态水平。从第7天起至少持续1周都观察到了这一现象。还观察到，在两种培养系统中，各种培养基对以nmol甲巯咪唑/分钟/毫克微粒体蛋白表示的FMO活性均无统计学上的显著影响。然而，在纯肝细胞培养物中，与无血清培养基相比，添加FCS和苯巴比妥显著增加了每个培养皿中的微粒体蛋白含量。在后一种情况下，无FCS时肝细胞会发生退化和损失。总之，与纯培养物相比，FMO在共培养物中的表达更好且维持时间更长。共培养物中从培养基中去除FCS不会影响FMO活性。由于添加苯巴比妥对FMO活性没有诱导作用，这与体内情况相同，因此这些结果支持将大鼠肝细胞共培养物用作FMO调节研究的体外模型这一观点。
