Osburn William O, Wakabayashi Nobunao, Misra Vikas, Nilles Tricia, Biswal Shyam, Trush Michael A, Kensler Thomas W
Johns Hopkins University Bloomberg School of Public Health, Department of Environmental Health Sciences, Baltimore, MD, USA.
Arch Biochem Biophys. 2006 Oct 1;454(1):7-15. doi: 10.1016/j.abb.2006.08.005. Epub 2006 Aug 23.
Mouse embryonic fibroblasts derived from Nrf2-/- mice (N0) and Nrf2+/+ mice (WT) have been used to characterize both basal and diquat (DQ)-induced oxidative stress levels and to examine Nrf2 activation during exposure to DQ-generated superoxide anion. Microarray analysis revealed that N0 cells have similar constitutive mRNA expression of genes responsible for the direct metabolism of reactive oxygen species but decreased expression of genes responsible for the production of reducing equivalents, repair of oxidized proteins and defense against lipid peroxidation, compared to WT cells. Nonetheless, the basal levels of ROS flux and oxidative damage biomarkers in WT and N0 cells were not different. Diquat dibromide (DQ), a non-electrophilic redox cycling bipyridylium herbicide, was used to generate intracellular superoxide anion. Isolated mitochondria from both cell lines exposed to DQ produced equivalent amounts of ROS, indicating a similar cellular capacity to generate ROS. However, N0 cells exposed to DQ for 24-h exhibited markedly decreased cell viability and aconitase activity as well as increased lipid peroxidation and glutathione oxidation, relative to WT cells. 2',7'-Dichlorofluorescein fluorescence was not increased in WT and N0 cells after 30-min of DQ exposure. However, increased levels of ROS were detected in N0 cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not N0 cells following a 24-h exposure to DQ. DQ exposure resulted in activation of an antioxidant response element-luciferase reporter gene, as well as induction of Nrf2-regulated genes in WT, but not N0 cells. Thus the enhanced sensitivity of N0 cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress.
源自Nrf2基因敲除小鼠（N0）和Nrf2基因野生型小鼠（WT）的小鼠胚胎成纤维细胞已被用于表征基础和百草枯（DQ）诱导的氧化应激水平，并研究暴露于DQ产生的超氧阴离子期间Nrf2的激活情况。微阵列分析显示，与WT细胞相比，N0细胞具有负责活性氧直接代谢的基因相似的组成型mRNA表达，但负责产生还原当量、修复氧化蛋白和抵御脂质过氧化的基因表达降低。尽管如此，WT和N0细胞中ROS通量和氧化损伤生物标志物的基础水平并无差异。二溴百草枯（DQ），一种非亲电氧化还原循环联吡啶除草剂，被用于产生细胞内超氧阴离子。暴露于DQ的两种细胞系分离出的线粒体产生等量的ROS，表明产生ROS的细胞能力相似。然而，相对于WT细胞，暴露于DQ 24小时的N0细胞表现出明显降低的细胞活力和乌头酸酶活性，以及增加的脂质过氧化和谷胱甘肽氧化。DQ暴露30分钟后，WT和N0细胞中的2',7'-二氯荧光素荧光没有增加。然而，DQ处理13小时后，在N0细胞中检测到ROS水平升高，但WT细胞中未检测到。此外，暴露于DQ 24小时后，WT细胞中的总谷胱甘肽浓度增加，但N0细胞中未增加。DQ暴露导致WT细胞中抗氧化反应元件荧光素酶报告基因的激活，以及Nrf2调控基因的诱导，但N0细胞中未出现。因此，N0细胞增强的敏感性并不反映抗氧化能力的基础差异，而是反映了对持续氧化应激产生适应性反应的能力受损。
