Protective role of Nrf2 in zinc oxide nanoparticles-induced lung inflammation in female mice and sexual dimorphism in susceptibility.

BACKGROUND: Zinc oxide nanoparticles (ZnO-NPs) are currently employed in various products such as rubber, paint, and cosmetics. Our group reported recently that Nrf2 protein provides protection against pulmonary inflammation induced by ZnO-NPs in male mice. The current study investigated the effect of Nrf2 deletion on the lung inflammatory response in female mice exposed to ZnO-NPs. METHODS: An equal number of female Nrf2-/- mice and female Nrf2+/+ mice (24 each) were allocated into three equal groups, and each was exposed to ZnO-NPs at either 0, 10 or 30 µg ZnO-NPs/mouse through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were examined 14 days later to determine the number of inflammatory cells, the protein level, and for scoring inflammation histopathologically. The mRNA levels of Nrf2-dependent antioxidant enzymes and proinflammatory cytokine in lung tissue were also measured. RESULTS: Exposure to ZnO-NPs increased all types of BALF cells and lung inflammation scores in both of female Nrf2-null (Nrf2-/-) and wild-type (Nrf2+/+) mice, and Nrf2 deletion enhanced ZnO-NPs-induced increase in the number of eosinophils in BALF. Exposure to ZnO-NPs dose-dependently increased the level of oxidized glutathione (GSSG), and mRNA levels of proinflammatory cytokines/chemokines; KC, MIP-2, IL-6, IL-1ß and MCP-1 only in wild-type mice. Nrf2 deletion decreased total glutathione levels and basal mRNA levels of SOD1 and NQO1, and increased the basal mRNA level of above proinflammatory cytokines/chemokines. Nrf2 deletion enhanced ZnO-NPs-induced downregulation of GcLc, GR and TGF-ß and upregulation of HO-1 and TNF-α. Taken together with our previous results in male mice, our results showed a lower susceptibility of females to lung tissue inflammation, relative to males, irrespective of Nrf2 deletion, and that enhancement of ZnO-NPs-induced upregulation of HO-1 and TNF-α and downregulation of GcLc, GR and TGF-ß by deletion of Nrf2 is specific to female mice. CONCLUSION: We conclude that Nrf2 provides protection in female mice against increase in BALF eosinophils, probably through down-regulation of proinflammatory cytokines/chemokines and upregulation of oxidative stress-related genes. The study also suggests lower susceptibility to lung tissue inflammation in female mice relative to their male counterparts and the synergistic effects of Nrf2 and exposure to ZnO-NPs on mRNA expression of GcLc, GR, HO-1, TGF-ß or TNF-α in female mice.

Enhanced epithelial sodium channel activity in neonatal Scnn1b mouse lung attenuates high oxygen-induced lung injury.

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen ([Formula: see text])-induced lung injury. Exposure to high [Formula: see text] from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG (400 µM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 µM) or amiloride (1 µM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10 µM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high [Formula: see text] exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.

Effect of protein glutathionylation on neuronal cytoskeleton: a potential link to neurodegeneration.

Neurons are highly susceptible to oxidative stress and oxidation of cytoskeletal proteins is considered one of the first steps of neurodegeneration. Protein glutathionylation is a key event in the redox regulation of protein function and constitutes a sensor of tissue oxidative stress in patho-physiological conditions. In this study, we analyzed for the first time tubulin glutathionylation and its relation to neurites degeneration. For this purpose, we exposed motoneuronal cells to the physiological oxidant glutathione disulfide (GSSG) and we analyzed the extent and morphology of axonal changes caused by protein glutathionylation in these cells. Then we studied the effect of glutathionylation on the distribution of stable and dynamic microtubules in the same cells. Our results indicate that oxidative stress conditions determined by an increased intracellular level of oxidized glutathione may cause an alteration of the cytoskeleton organization and function leading to axon degeneration. These findings might contribute to understand the sequence of pathogenic events involved in the axonal degeneration that characterizes many diseases of the nervous system associated with oxidative stress.

Protective effects of a glutathione disulfide mimetic (NOV-002) against cisplatin induced kidney toxicity.

NOV-002 is a glutathione disulfide (GSSG) mimetic with chemoprotective activity. Previous and ongoing clinical studies demonstrate a significantly improved 1-year survival and decreased tumor progression rates in non-small cell lung (NSCLC) and ovarian cancer patients when NOV-002 was included in cisplatin containing regimens. In order to understand this chemoprotective property, we employed as an animal model of kidney toxicity, 8-week-old Bl6 mice that were treated with a single nephrotoxic dose of cisplatin (15 mg/kg, ip) and sacrificed on Day 5. One group of animals was treated with NOV-002 (15 mg/kg, im) daily. NOV-002-treated mice had significantly lower levels of plasma creatinine compared to mice treated with cisplatin alone (4.7 vs 2.9 mg/dL, respectively). Moreover, NOV-002 protected the kidneys from cisplatin mediated proximal tubule damage, including dilation of tubules and the presence of protein casts. Since cisplatin-induced nephrotoxicity can be mediated by a glutathione-platinum conjugate catalyzed by gamma-glutamyl-transpeptidase (GGT) and glutathione is an endogenous substrate of GGT, the protective effect of NOV-002 in the kidney may be attributed to its ability to act as a competitive substrate for the enzyme.

Glutathione disulfide induces neural cell death via a 12-lipoxygenase pathway.

Oxidized glutathione (GSSG) is commonly viewed as a byproduct of GSH metabolism. The pathophysiological significance of GSSG per se remains poorly understood. Adopting a microinjection approach to isolate GSSG elevation within the cell, this work identifies that GSSG can trigger neural HT4 cell death via a 12-lipoxygenase (12-Lox)-dependent mechanism. In vivo, stereotaxic injection of GSSG into the brain caused lesion in wild-type mice but less so in 12-Lox knockout mice. Microinjection of graded amounts identified 0.5 mM as the lethal [GSSG]i in resting cells. Interestingly, this threshold was shifted to the left by 20-fold (0.025 mM) in GSH-deficient cells. This is important because tissue GSH lowering is commonly noted in the context of several diseases as well as in aging. Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner. GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells. Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death. Strategies directed at improving or arresting cellular GSSG clearance may be effective in minimizing oxidative stress-related tissue injury or potentiating the killing of tumor cells, respectively.