Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion

Mouse embryonic fibroblasts derived from Nrf2-/- mice (NO) 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 NO 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 NO 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, NO 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 NO cells after 30-min of DQ exposure. However, increased levels of ROS were detected in NO cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not NO 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 NO cells. Thus the enhanced sensitivity of NO cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress. (c) 2006 Elsevier Inc. All rights reserved.