Paradoxical Roles of Antioxidant Enzymes: Basic Mechanisms and Health Implications.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate "paradoxical" outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of "antioxidant" nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that "paradoxical" roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways.

Catalase-deficient mice induce aging faster through lysosomal dysfunction.

BACKGROUND: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice. METHODS: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group. RESULTS: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice. CONCLUSIONS: This study unveils the new role of catalase and its role in lysosomal function during aging. Video abstract.

Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils.

The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice.

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Glutaredoxin 2 (Grx2) gene deletion induces early onset of age-dependent cataracts in mice.

Glutaredoxin 2 (Grx2) is an isozyme of glutaredoxin1 (thioltransferase) present in the mitochondria and nucleus with disulfide reductase and peroxidase activities, and it controls thiol/disulfide balance in cells. In this study, we investigated whether Grx2 gene deletion could induce faster age-related cataract formation and elucidated the biochemical changes effected by Grx2 gene deletion that may contribute to lens opacity. Slit lamp was used to examine the lenses in Grx2 knock-out (KO) mice and age-matched wild-type (WT) mice ages 1 to 16 months. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice, and the progression of cataracts was also much faster than the age-matched controls. Lenses of KO mice contained lower levels of protein thiols and GSH with a significant accumulation of S-glutathionylated proteins. Actin, αA-crystallin, and ßB2-crystallin were identified by Western blot and mass spectroscopy as the major S-glutathionylated proteins in the lenses of 16-month-old Grx2 KO mice. Compared with the WT control, the lens of Grx2 KO mice had only 50% of the activity in complex I and complex IV and less than 10% of the ATP pool. It was concluded that Grx2 gene deletion altered the function of lens structural proteins through S-glutathionylation and also caused severe disturbance in mitochondrial function. These combined alterations affected lens transparency.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization.

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes, and Glrx overexpression inhibits VEGF-induced EC migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx-overexpressing EC from Glrx transgenic (TG) mice showed impaired migration and network formation and secreted higher levels of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared with wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Noncanonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC and enhanced NF-κB activity, which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-κB-dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt explains part of the mechanism of redox-regulated VEGF signaling.

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease.

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.

Thioredoxin 1 enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction: a study using thioredoxin 1 transgenic mice.

Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin 1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild-type (W) and Trx1 transgenic (Trx1(Tg/+)) mice were randomized into W sham (WS), Trx1(Tg/+) sham (TS), WMI, and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS, p-GSK-3ß, HIF-1α, ß-catenin, VEGF, Bcl-2, and survivin expression in TMI compared to WMI. Echocardiography performed 30days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12, and subsequent activation of Akt/GSK-3ß/ß-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.

Ablation of glutaredoxin-1 attenuates lipopolysaccharide-induced lung inflammation and alveolar macrophage activation.

Protein S-glutathionylation (PSSG), a reversible posttranslational modification of reactive cysteines, recently emerged as a regulatory mechanism that affects diverse cell-signaling cascades. The extent of cellular PSSG is controlled by the oxidoreductase glutaredoxin-1 (Grx1), a cytosolic enzyme that specifically de-glutathionylates proteins. Here, we sought to evaluate the impact of the genetic ablation of Grx1 on PSSG and on LPS-induced lung inflammation. In response to LPS, Grx1 activity increased in lung tissue and bronchoalveolar lavage (BAL) fluid in WT (WT) mice compared with PBS control mice. Glrx1(-/-) mice consistently showed slight but statistically insignificant decreases in total numbers of inflammatory cells recovered by BAL. However, LPS-induced concentrations of IL-1ß, TNF-α, IL-6, and Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) in BAL were significantly decreased in Glrx1(-/-) mice compared with WT mice. An in situ assessment of PSSG reactivity and a biochemical evaluation of PSSG content demonstrated increases in the lung tissue of Glrx1(-/-) animals in response to LPS, compared with WT mice or PBS control mice. We also demonstrated that PSSG reactivity was prominent in alveolar macrophages (AMs). Comparative BAL analyses from WT and Glrx1(-/-) mice revealed fewer and smaller AMs in Glrx1(-/-) mice, which showed a significantly decreased expression of NF-κB family members, impaired nuclear translocation of RelA, and lower levels of NF-κB-dependent cytokines after exposure to LPS, compared with WT cells. Taken together, these results indicate that Grx1 regulates the production of inflammatory mediators through control of S-glutathionylation-sensitive signaling pathways such as NF-κB, and that Grx1 expression is critical to the activation of AMs.

Postischemic deactivation of cardiac aldose reductase: role of glutathione S-transferase P and glutaredoxin in regeneration of reduced thiols from sulfenic acids.

Aldose reductase (AR) is a multifunctional enzyme that catalyzes the reduction of glucose and lipid peroxidation-derived aldehydes. During myocardial ischemia, the activity of AR is increased due to the oxidation of its cysteine residues to sulfenic acids. It is not known, however, whether the activated, sulfenic form of the protein (AR-SOH) is converted back to its reduced, unactivated state (AR-SH). We report here that in perfused mouse hearts activation of AR during 15 min of global ischemia is completely reversed by 30 min of reperfusion. During reperfusion, AR-SOH was converted to a mixed disulfide (AR-SSG). Deactivation of AR and the appearance of AR-SSG during reperfusion were delayed in hearts of mice lacking glutathione S-transferase P (GSTP). In vitro, GSTP accelerated glutathiolation and inactivation of AR-SOH. Reduction of AR-SSG to AR-SH was facilitated by glutaredoxin (GRX). Ischemic activation of AR was increased in GRX-null hearts but was attenuated in the hearts of cardiospecific GRX transgenic mice. Incubation of AR-SSG with GRX led to the regeneration of the reduced form of the enzyme. In ischemic cardiospecific AR transgenic hearts, AR was co-immunoprecipitated with GSTP, whereas in reperfused hearts, the association of AR with GRX was increased. These findings suggest that upon reperfusion of the ischemic heart AR-SOH is converted to AR-SSG via GSTP-assisted glutathiolation. AR-SSG is then reduced by GRX to AR-SH. Sequential catalysis by GSTP and GRX may be a general redox switching mechanism that regulates the reduction of protein sulfenic acids to cysteines.

Catalase deficiency induces reactive oxygen species mediated pexophagy and cell death in the liver during prolonged fasting.

Peroxisomes are dynamic organelles that participate in a diverse array of cellular processes, including ß-oxidation, which produces a considerable amount of reactive oxygen species (ROS). Although we showed that catalase depletion induces ROS-mediated pexophagy in cells, the effect of catalase deficiency during conditions that favor ROS generation remains elusive in mice. In this study, we reported that prolonged fasting in catalase-knockout (KO) mice drastically increased ROS production, which induced liver-specific pexophagy, an autophagic degradation of peroxisomes. In addition, increased ROS generation induced the production of pro-inflammatory cytokines in the liver tissues of catalase-KO mice. Furthermore, there was a significant increase in the levels of aspartate transaminase and alanine transaminase as well as apparent cell death in the liver of catalase-KO mice during prolonged fasting. However, an intra-peritoneal injection of the antioxidant N-acetyl-l-cysteine (NAC) and autophagy inhibitor chloroquine inhibited the inflammatory response, liver damage, and pexophagy in the liver of catalase-KO mice during prolonged fasting. Consistently, genetic ablation of autophagy, Atg5 led to suppression of pexophagy during catalase inhibition by 3-aminotriazole (3AT). Moreover, treatment with chloroquine also ameliorated the inflammatory response and cell death in embryonic fibroblast cells from catalase-KO mice. Taken together, our data suggest that ROS-mediated liver-specific pexophagy observed during prolonged fasting in catalase-KO mice may be responsible for the process associated with hepatic cell death.

Catalase deficiency facilitates the shuttling of free fatty acid to brown adipose tissue through lipolysis mediated by ROS during sustained fasting.

BACKGROUND: Fatty acids (FA) derived from adipose tissue and liver serve as the main fuel in thermogenesis of brown adipose tissue (BAT). Catalase, a peroxisomal enzyme, plays an important role in maintaining intracellular redox homeostasis by decomposing hydrogen peroxide to either water or oxygen that oxidize and provide fuel for cellular metabolism. Although the antioxidant enzymatic activity of catalase is well known, its role in the metabolism and maintenance of energy homeostasis has not yet been revealed. The present study investigated the role of catalase in lipid metabolism and thermogenesis during nutrient deprivation in catalase-knockout (KO) mice. RESULTS: We found that hepatic triglyceride accumulation in KO mice decreased during sustained fasting due to lipolysis through reactive oxygen species (ROS) generation in adipocytes. Furthermore, the free FA released from lipolysis were shuttled to BAT through the activation of CD36 and catabolized by lipoprotein lipase in KO mice during sustained fasting. Although the exact mechanism for the activation of the FA receptor enzyme, CD36 in BAT is still unclear, we found that ROS generation in adipocytes mediated the shuttling of FA to BAT. CONCLUSIONS: Taken together, our findings uncover the novel role of catalase in lipid metabolism and thermogenesis in BAT, which may be useful in understanding metabolic dysfunction.

Attenuation of doxorubicin-induced cardiac injury by mitochondrial glutaredoxin 2.

While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein-glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury.

Glutaredoxin 1 regulates cigarette smoke-mediated lung inflammation through differential modulation of I{kappa}B kinases in mice: impact on histone acetylation.

Glutaredoxin 1 (Glrx1) is a small dithiol protein that regulates the cellular redox state and redox-dependent signaling pathways via modulation of protein glutathionylation. IkappaB kinase (IKK), an essential enzyme for NF-kappaB activation, can be subjected to S-glutathionylation leading to alteration of its activity. However, the role of Glrx1 in cigarette smoke (CS)-induced lung inflammation and chromatin modifications are not known. We hypothesized that Glrx1 regulates the CS-induced lung inflammation and chromatin modifications via differential regulation of IKKs by S-glutathionylation in mouse lung. Glrx1 knockout (KO) and wild-type (WT) mice were exposed to CS for 3 days and determined the role of Glrx1 in regulation of proinflammatory response in the lung. Neutrophil influx in bronchoalveolar lavage fluid and proinflammatory cytokine release in lung were increased in Glrx1 KO mice compared with WT mice exposed to CS, which was associated with augmented nuclear translocation of RelA/p65 and its phospho-acetylation. Interestingly, phosphorylated and total levels of IKKalpha, but not total and phosphorylated IKKbeta levels, were increased in lungs of Glrx1 KO mice compared with WT mice exposed to CS. Ablation of Glrx1 leads to increased CS-induced IKKbeta glutathionylation rendering it inactive, whereas IKKalpha was activated resulting in increased phospho-acetylation of histone H3 in mouse lung. Thus, targeted disruption of Glrx1 regulates the lung proinflammatory response via histone acetylation specifically by activation of IKKalpha in response to CS exposure. Overall, our study suggests that S-glutathionylation and phosphorylation of IKKalpha plays an important role in histone acetylation on proinflammatory gene promoters and NF-kappaB-mediated abnormal and sustained lung inflammation in pathogenesis of chronic inflammatory lung diseases.

Glutathione peroxidase 1-deficient mice are more susceptible to doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H(2)O(2), which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H(2)O(2). In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H(2)O(2) generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

Absence of glutaredoxin1 increases lens susceptibility to oxidative stress induced by UVR-B.

We investigated if the absence of glutaredoxin1, a critical protein thiol repair enzyme, increases lens susceptibility to oxidative stress caused by in vivo exposure to ultraviolet radiation type B (UVR-B). Glrx(-/-) mice and Glrx(+/+) mice were unilaterally exposed in vivo to UVR-B for 15 min. Groups of 12 animals each received 4.3, 8.7, and 14.5 kJ/m(2) respectively. 48 h post UVR-B exposure, the induced cataract was quantified as forward lens light scattering. Cataract morphology was documented with darkfield illumination photography. Glutathione (GSH/GSSG) content was analyzed in Glrx(-/-) and Glrx(+/+) lenses. UVR-B exposure induced anterior sub-capsular cataract (ASC) in Glrx(-/-) and Glrx(+/+) mice. In Glrx(-/-) lenses the opacities extended further towards the lens equator than in wild type animals (Glrx(+/+)). Lens light scattering in Glrx(-/-) mice was increased in all dose groups compared to lenses with normal glutaredoxin1 function. The difference was more pronounced with increasing exposure dose. Lens sensitivity for UVR-B induced damage was significantly higher in Glrx(-/-) lenses compared to Glrx(+/+) lenses. The Glrx gene provides a 44% increase of protection against close to threshold UVR-B induced oxidative stress compared to the absence of the Glrx gene. In conclusion, the absence of glutaredoxin1 increases lens susceptibility to UVR-B induced oxidative stress in the mouse.

Prevention of ischemia/reperfusion-induced cardiac apoptosis and injury by melatonin is independent of glutathione peroxdiase 1.

Free-radical generation is one of the primary causes of myocardial ischemia/reperfusion (I/R) injury. Melatonin is an efficient free-radical scavenger and induces the expression of antioxidant enzymes. We have previously shown that melatonin can prevent free-radical-induced myocardial injury. To date, the mechanism underlying melatonin's cardioprotective effect is not clear. In this study, we assessed the ability of melatonin to protect against I/R injury in mice deficient in glutathione peroxidase 1 (Gpx1). Mice hearts were subjected to 40 min of global ischemia in vitro followed by 45 min of reperfusion. Myocardial I/R injury (expressed as % of recovery of left ventricular developed pressure x heart rate) was exacerbated in mice deficient in Gpx1 (51 +/- 3% for Gpx1+/+ mice versus 31 +/- 6% for Gpx1(-/-) mice, P < 0.05). Administration of melatonin for 30 min protected against I/R injury in both Gpx1+/+ mice (72 +/- 4.8%) and Gpx1(-/-) mice (63 +/- 4.7%). This protection was accompanied by a significant improvement in left ventricular end-diastolic pressure and a twofold decrease in lactate dehydrogenase (LDH) level released from melatonin-treated hearts. In another set of experiments, mice were subjected to 50 min of ligation of the left descending anterior coronary artery in vivo followed by 4 hr of reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly larger in Gpx1(-/-) mice than in Gpx1+/+ mice (75 +/- 9% versus 54 +/- 6%, P < 0.05) and were reduced significantly in melatonin-treated mice (31 +/- 3.7% Gpx1(-/-) mice and 33 +/- 6.0% Gpx1+/+ mice). In hearts subjected to 30 min of coronary artery occlusion followed by 3 hr of reperfusion, melatonin-treated hearts had significantly fewer in situ oligo ligation-positive myocytes and less protein nitration. Our results demonstrate that the cardioprotective function of melatonin is independent of Gpx1.

Control of Her-2 tumor immunity and thyroid autoimmunity by MHC and regulatory T cells.

Immune reactivity to self-antigens in both cancer and autoimmune diseases can be enhanced by systemic immune modulation, posing a challenge in cancer immunotherapy. To distinguish the genetic and immune regulation of tumor immunity versus autoimmunity, immune responses to human ErbB-2 (Her-2) and mouse thyroglobulin (mTg) were tested in transgenic mice expressing Her-2 that is overexpressed in several cancers, and HLA-DRB1*0301 (DR3) that is associated with susceptibility to several human autoimmune diseases, as well as experimental autoimmune thyroiditis (EAT). To induce Her-2 response, mice were electrovaccinated with pE2TM and pGM-CSF encoding the extracellular and transmembrane domains of Her-2 and the murine granulocyte macrophage colony-stimulating factor, respectively. To induce EAT, mice received mTg i.v. with or without lipopolysaccharide. Depletion of regulatory T cells (Treg) with anti-CD25 monoclonal antibody enhanced immune reactivity to Her-2 as well as mTg, showing control of both Her-2 and mTg responses by Treg. When immunized with, Her-2xDR3 and B6xDR3 mice expressing H2(b)xDR3 haplotype developed more profound mTg response and thyroid pathology than Her-2 or B6 mice that expressed the EAT-resistant H2(b) haplotype. In Her-2xDR3 mice, the response to mTg was further amplified when mice were also immunized with pE2TM and pGM-CSF. On the contrary, Her-2 reactivity was comparable whether mice expressed DR3 or not. Therefore, induction of Her-2 immunity was independent of DR3 but development of EAT was dictated by this allele, whereas Tregs control the responses to both self-antigens. These results warrant close monitoring of autoimmunity during cancer immunotherapy, particularly in patients with susceptible MHC class II alleles.

Overexpression of glutaredoxin-2 reduces myocardial cell death by preventing both apoptosis and necrosis.

Mitochondrial glutaredoxin-2 (Glrx2) has been recognized as an important redox regulator in mammalian organs including heart. To date no investigations have addressed the potential role of Glrx2 in cardiac disorders. The present study examined if myocardial overexpression of Glrx2 in the heart could rescue the cardiac cells from apoptosis and necrosis induced by ischemia and reperfusion. The human Glrx2 transgene was created by placing a full-length cDNA fragment encoding human mitochondrial Glrx2 downstream to the 5' flanking sequence and promoter of the mouse alpha-myosin heavy chain gene. The isolated hearts from Glrx2 transgenic mice and non-transgenic (wild type) littermates [on c57BL/6xC3H hybrid background] were subjected to 30 min of global ischemia followed by 2 h of reperfusion via working mode. The hearts from Glrx2 transgenic mice displayed significantly improved contractile performance and reduced myocardial infarct size and cardiomyocyte apoptosis. There was a reduction in cytochrome c release and activation of caspase 3 and caspase 9. Glrx2 overexpression also reduced the ischemia/reperfusion-mediated loss of mitochondrial cardiolipin, decreased the activities of reactive oxygen species (ROS) and preserved GSH/GSSG ratio. Glrx2 mediated survival signal appeared to be stemmed from PI-3-kinase-Akt survival signaling pathway and involved the activation of redox sensitive transcription factor NFkappaB and antiapoptotic protein Bcl-2. The results indicated a crucial role of mitochondrial Glrx2 in cardioprotection.

Role of glutaredoxin-1 in cardioprotection: an insight with Glrx1 transgenic and knockout animals.

This study examined if glutaredoxin-1 (Glrx1), a redox-regulator of thioredoxin superfamily, plays any role in the redox signaling of ischemic myocardium. The hearts were subjected to 30 min of coronary occlusion followed by 24 h of reperfusion. Another group of hearts was rendered tolerant to ischemia (preconditioned, PC) by four cyclic episodes of 5 min ischemia each followed by another 10 min of reperfusion, which was then subjected to 30 min ischemia and 24 h of coronary occlusion. While ischemia/reperfusion had no effect on Glrx1 expression, adaptation to ischemia resulted in the up-regulation of Glrx1 expression, which was inhibited by cadmium, a known inhibitor of Glrx1. CdCl(2) also abolished cardioprotection afforded by PC as evidenced by its ability to partially increase myocardial infarct size without affecting cardiomyocyte apoptosis. The amount of ROS was significantly decreased in the PC heart, which was abolished by CdCl(2). The cardioprotective role of Glrx1was further confirmed with Glrx1 transgenic and knockout mice. The mouse hearts overexpressing Glrx1 exhibited significantly improved post-ischemic ventricular recovery and reduced myocardial infarct size while hearts deficient in Glrx1 exhibited depressed functional recovery and increased infarct size as compared to the wild-type hearts. Furthermore, Glrx1-overexpressing hearts exhibited reduced and Glrx1-deficient hearts exhibited increased ROS production during ischemia and reperfusion. Adapted hearts showed increased Akt phosphorylation that was inhibited by CdCl(2). The amount of Bcl-2 protein expression was not affected by the inhibition of Glrx1. Taken together, the results of this study implicate a role of Glrx1 in cardioprotection and redox signaling of the ischemic myocardium.