Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats
Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats
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DOI：
10.1016/s0006-2952(00)00233-1
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发表时间：
2000-05-01
影响因子：
5.8
通讯作者：
Cascales, M
中科院分区：
文献类型：
--
作者：
Andrés, D;Sanz, N;Cascales, M
环孢霉素A（CsA）的体外作用进行了研究，在参考生产的活性氧（过氧化物和超氧阴离子）和细胞酶介导的抗氧化防御肝细胞分离的大鼠2个月和12个月。将肝细胞的原代培养物在0至50 μ M浓度的环孢菌素存在下孵育24小时，并将乳酸脱氢酶释放到培养基中作为细胞毒性和膜溶解的参数进行评价。通过使用2 '，7'-二氯二氢荧光素二乙酸酯定量过氧化物，并通过二氢乙锭的荧光评价超氧阴离子水平。测定了过氧化氢酶、锰超氧化物歧化酶和铜锌超氧化物歧化酶的酶活性和基因表达。CsA的细胞毒性显着较高的肝细胞从大鼠12个月相比，年龄2个月。细胞内过氧化物含量呈剂量依赖性增加，而超氧阴离子细胞内水平略有下降，CsA从0-50 μ M增加。在细胞培养物中的细胞内过氧化物在0-50 μ M CsA的范围内的逐步增加与细胞活力的损失，并伴随着显着较高水平的Mn-和Cu，Zn-超氧化物歧化酶的酶活性和mRNA，过氧化氢酶活性和mRNA略有增加。我们的结论是，在原代肝细胞培养物中，CsA的细胞毒性是剂量依赖性的，在两个年龄组和显着较高的文化从12个月大的大鼠相比，从2个月大的动物。抗氧化酶系统的基因表达的非协调调节，即过氧化氢酶和Mn-和Cu，Zn-超氧化物歧化酶，证明在更大程度上在肝细胞从老年组的大鼠，可能是参与CsA毒性的机制之一。(C)2000 Elsevier Science Inc.
The in vitro effect of cyclosporine A (CsA) was studied in reference to the production of reactive oxygen species (peroxides and superoxide anion) and to cell enzyme-mediated antioxidant defence in hepatocytes isolated from rats aged 2 and 12 months. Primary cultures of hepatocytes were incubated in the presence of concentrations of cyclosporine in the range of 0 to 50 mu M for 24 hr, and the release of lactate dehydrogenase into the culture medium was evaluated as a parameter of cytotoxicity and membrane lysis. Peroxides were quantified by using 2',7'-dichlorodihydrofluorescein diacetate, and superoxide anion levels were evaluated by the fluorescence of dihydroethidium. Enzyme activity and gene expression of catalase and Mn- and Cu,Zn-superoxide dismutase were also assayed. CsA cytotoxicity was significantly higher in hepatocytes from rats aged 12 months when compared to those aged 2 months. Intracellular peroxide content resulted in a dose-dependent increase, while the anion superoxide intracellular level slightly decreased as CsA increased from 0-50 mu M. The progressive increase in intracellular peroxides in cell cultures in the range from 0-50 mu M CsA was associated with the loss of cell viability and accompanied by significantly higher levels of Mn- and Cu,Zn-superoxide dismutase enzyme activities and mRNAs, and slight increases in catalase activity and mRNA. We conclude that, in primary hepatocyte cultures, the cytotoxicity of CsA was dose-dependent in both age groups and significantly higher in cultures from 12-month-old rats when compared to those from 2-month-old animals. The non-coordinated regulation of the gene expression of antioxidant enzyme systems, i.e. catalase and Mn- and Cu,Zn-superoxide dismutases, evidenced to a greater extent in hepatocytes from the older group of rats, could be one of the mechanisms involved in CsA toxicity. (C) 2000 Elsevier Science Inc.