Lii C K, Wang S T, Chen H W, Sheen L Y
Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan, Republic of China.
Arch Toxicol. 1996;70(12):822-9. doi: 10.1007/s002040050345.
The effect of culture medium on glutathione (GSH) dependent detoxification defence system of primary cultured hepatocyte from either male or female rats was studied. Intracellular reduced (GSH) and oxidized glutathione (GSSG), and six GSH-related enzyme activities, including GSH peroxidase (GSH Px), GSH reductase (GSH Rd), cytosolic GSH S-transferase (cGST), microsomal GSH S-transferase (mGST), gamma-glutamyl transpeptidase (GTP), and gamma-glutamylcysteine synthetase (GCS), were investigated during a 6-day culture. Media free of fetal bovine serum (FBS) and with 2.5 or 10% FBS were used. Whatever the medium, there was an initial decrease of intracellular GSH and GSSG, a threefold increase of GSH at day 3 and fourfold increase of GSSG at day 4, later decreasing to their original level at day 6. The activities of all six GSH-related enzymes of male and female hepatocytes remained relatively stable during the first 72h, then gradually decreased to 50-80% of initial activities. With the exception of cGST, time-course profiles of other enzyme activities were not significantly different among various media. In both sexes, higher cGST activity was maintained for cells cultured in the presence of FBS. Results of immunoblotting analysis of cytosolic GST isozymes indicate that the placental form of GST (Yp) was markedly increased after plating and the extent of increase of Yp was higher in the presence of FBS. Despite the culture medium, the level of GST isoform Ya was maintained steadily for 6 days, however, Yb was maintained during the first 3 days and then decreased. In terms of the gender difference, GSH Px and GTP activities of hepatocytes from females were significantly greater than of males over the entire culture period. Results indicate that FBS seems not to be absolutely essential in maintaining GSH level and most of the GSH-related enzyme activities in rat hepatocytes. Furthermore, GSH levels and GSH-related enzyme activities of hepatocytes from female rats were similar to those from male rats.
研究了培养基对雄性或雌性大鼠原代培养肝细胞中谷胱甘肽（GSH）依赖性解毒防御系统的影响。在为期6天的培养过程中，检测了细胞内还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG），以及六种与GSH相关的酶活性，包括谷胱甘肽过氧化物酶（GSH Px）、谷胱甘肽还原酶（GSH Rd）、胞质谷胱甘肽S-转移酶（cGST）、微粒体谷胱甘肽S-转移酶（mGST）、γ-谷氨酰转肽酶（GTP）和γ-谷氨酰半胱氨酸合成酶（GCS）。使用了不含胎牛血清（FBS）以及含有2.5%或10%FBS的培养基。无论使用何种培养基，细胞内GSH和GSSG最初都会下降，第3天GSH增加三倍，第4天GSSG增加四倍，随后在第6天降至原始水平。雄性和雌性肝细胞的所有六种与GSH相关的酶活性在最初的72小时内保持相对稳定，然后逐渐降至初始活性的50 - 80%。除cGST外，其他酶活性在不同培养基中的时间进程曲线没有显著差异。在两性中，在FBS存在下培养的细胞保持较高的cGST活性。胞质GST同工酶的免疫印迹分析结果表明，接种后胎盘型GST（Yp）显著增加，且在FBS存在下Yp的增加程度更高。无论培养基如何，GST同工型Ya的水平在6天内保持稳定，然而，Yb在最初3天保持不变，然后下降。就性别差异而言，在整个培养期间，雌性肝细胞的GSH Px和GTP活性显著高于雄性。结果表明，FBS似乎并非维持大鼠肝细胞中GSH水平和大多数与GSH相关的酶活性所绝对必需的。此外，雌性大鼠肝细胞的GSH水平和与GSH相关的酶活性与雄性大鼠相似。
