Holst C Martina, Oredsson Stina M
Department of Cell and Organism Biology, Lund University, Animal Physiology Building, Helgonavägen 3B, SE-223 62 Lund, Sweden.
Toxicol In Vitro. 2005 Apr;19(3):379-87. doi: 10.1016/j.tiv.2004.10.005. Epub 2004 Dec 24.
Using three cytotoxicity assays, we have investigated the effect of the spermine analogue N1,N11-diethylnorspermine (DENSPM) on four human breast cancer cell lines with different known genetic lesions. Cells were seeded in 96 well plates and DENSPM was added 24 h later to give final concentrations from 0.1 to 100 microM. At 24, 48 and 72 h of treatment, the protein content was determined with a modified Lowry assay. Mitochondrial activity was determined with the AlamarBlue and MTT assays. These two assays differ with respect to where in the electron transport chain the reduction of the substrate takes place. Treatment with increasing concentrations of DENSPM resulted in differential responses in the four cell lines. There was a good of agreement between the protein content and the MTT assay showing increased negative effect with increased dose of DENSPM. The AlamarBlue assay on the other hand showed a stimulation of substrate reduction compared to control at DENSPM concentrations that were inhibitory according to the protein content and MTT assay. Thus, the data clearly show that the MTT and AlamarBlue assays are not equivalent. Importantly, the AlamarBlue assay presumably also reflects cytoplasmic reduction of the substrate through DENSPM-induced mechanisms.
我们使用三种细胞毒性测定方法，研究了精胺类似物N1,N11-二乙基亚精胺（DENSPM）对四种具有不同已知基因损伤的人乳腺癌细胞系的影响。将细胞接种于96孔板中，24小时后加入DENSPM，使其终浓度为0.1至100微摩尔。在处理24、48和72小时后，用改良的洛瑞法测定蛋白质含量。用阿拉玛蓝和MTT测定法测定线粒体活性。这两种测定方法在电子传递链中底物还原发生的位置方面存在差异。用浓度递增的DENSPM处理导致四种细胞系出现不同的反应。蛋白质含量与MTT测定结果之间有良好的一致性，表明随着DENSPM剂量的增加，负面影响增大。另一方面，根据蛋白质含量和MTT测定法，阿拉玛蓝测定法显示在DENSPM浓度具有抑制作用时，与对照相比底物还原受到刺激。因此，数据清楚地表明MTT和阿拉玛蓝测定法并不等效。重要的是，阿拉玛蓝测定法可能还通过DENSPM诱导的机制反映了底物的细胞质还原。
