Judy B. de Haan

Mice with a Homozygous Null Mutation for the Most Abundant Glutathione Peroxidase, Gpx1, Show Increased Susceptibility to the Oxidative Stress-inducing Agents Paraquat and Hydrogen Peroxide*

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (−/−) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H2O2. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (−/−) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg·kg−1 (approximately 1 7 the LD50of wild-type controls). The effects of paraquat were dose-related. In the 30 mg·kg−1-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulatesGpx1 in normal cells, reinforcing a role forGpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (−/−) mice are more susceptible to H2O2; 30% of neurons fromGpx1 (−/−) mice were killed when exposed to 65 μm H2O2, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H2O2. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest thatGpx1 (−/−) mice may be valuable for studying the role of H2O2 in neurodegenerative disorders.

Lack of the Antioxidant Enzyme Glutathione Peroxidase-1 Accelerates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice

Background— Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. Methods and Results— ApoE-deficient (ApoE−/−) and ApoE/GPx1 double-knockout (ApoE−/−GPx1−/−) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE−/−GPx1−/− aortas compared with diabetic ApoE−/− aortas. This increase was accompanied by increased macrophages, &agr;-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE−/−GPx1−/− mouse aortas. These findings were observed despite upregulation of other antioxidants. Conclusions— Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE−/− mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

NADPH Oxidase 1 Plays a Key Role in Diabetes Mellitus–Accelerated Atherosclerosis

Background— In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Methods and Results— Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E–deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E–deficient mice with GKT137831 attenuated atherosclerosis development. Conclusions— These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Nox1 Plays a Key Role in Diabetes Accelerated Atherosclerosis

Background— In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Methods and Results— Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E–deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E–deficient mice with GKT137831 attenuated atherosclerosis development. Conclusions— These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three‐fold in the glutathione peroxidase‐1 (Gpx‐1) –/– mouse compared with the wild‐type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx‐1 –/– mouse compared with the wild‐type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase‐3 expression is present at an earlier time point in the Gpx‐1 –/– mice when compared with the wild‐type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx‐1 –/– mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx‐1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.

Cu/Zn superoxide dismutase mRNA and enzyme activity, and susceptibility to lipid peroxidation, increases with aging in murine brains

To protect against reactive oxygen species, prokaryotic and eukaryotic cells have developed an antioxidant defence mechanism where O2- is converted to H2O2 by superoxide dismutase (Sod), and in a second step, H2O2 is converted to H2O by catalase (Cat) and/or glutathione peroxidase (Gpx). If Sod levels are increased without a concomitant Gpx increase, then the intermediate H2O2 accumulates. This intermediate could undergo the Fentons reaction, generating hydroxyl radicals which may lead to lipid peroxidation in cells. In this study, we investigate the expression of Sod1, Gpx1 and susceptibility to lipid peroxidation during the aging process in mouse brains. We demonstrate that the mRNA levels and enzyme activity of Sod1 are higher in brains from adult mice compared to neonatal mice. Furthermore, we show that a linear increase in Sod1 mRNA and enzyme activity occurs with aging (1-100 weeks). On the contrary, we find that the mRNA and enzyme activity for Gpx1 does not increase with aging in mouse brains. In addition, our results demonstrate that the susceptibility of murine brains to lipid peroxidation increases with aging. The data in this study are consistent with the notion that reactive oxygen species may contribute to the aging process in mammalian brains. These results are discussed in relation to the normal aging process in mammals, and to the premature aging and mental retardation in Down syndrome.

Reactive oxygen species and their contribution to pathology in Down syndrome.

Publisher Summary In addition to mental retardation, individuals with Down syndrome suffer from congenital heart defects, in utero growth retardation, increased susceptibility to infection; and a higher incidence of leukemia. These individuals show abnormalities of the viscerocranium those result in the characteristic facial features of Down syndrome; and features of premature aging. All individuals develop Alzheimer-type neuronal pathology. The involvement of reactive oxygen species (ROS) in some of the Down syndrome pathologies was initiated after assignment of the gene coding for Cu/Zn-superoxide dismutase (also known as Sodl) to chromosome21 in humans. All individuals with Down syndrome are either trisomic for the entire chromosome or part thereof. Increased gene dosage for Sodl has been proposed to contribute to the premature aging and/or mental retardation that occur as part of the syndrome. In gaining an understanding of how elevated Sodl levels may contribute to various pathologies in Down syndrome, this chapter delineates the physiological role for Sodl within cells. Elevated Sodl activity in Down syndrome could result in the accumulation of H 2 0 2 , which through the Fenton reaction could lead to a loss of cellular function through damage to macromolecules. The chapter discusses in detail the Sodl and Gpx1 expression levels in Down syndrome, premature aging of Down syndrome individuals, and apoptosis related to Down syndrome—some of the neurodegenerative symptoms and thymic destruction in Down syndrome may be because of the oxidative stress caused by the imbalance in the Sod/Gpx1 ratio, which in turn triggers apoptosis directly or confers an increased susceptibility of cells to apoptotic stimuli. Down syndrome is a situation where the antioxidant balance is affected because of gene dosage. Damage to biologically important macromolecules resulted because of the inability to prevent oxidative interactions. This accumulated macromolecular damage may be responsible for the abnormalities that are seen as part of the syndrome.

Expression of Copper/Zinc Superoxide Dismutase and Glutathione Peroxidase in Organs of Developing Mouse Embryos, Fetuses, and Neonates

ABSTRACT: The rise in antioxidant enzyme activity in the lungs of late-gestation fetuses is thought to be caused by the preparation of the pulmonary antioxidant system for birth. However, recent data have shown that such a rise also occurs in the livers of late-gestation fetuses. Consequently, this surge cannot solely be ascribed to the preparation of the pulmonary antioxidant system for birth. In this study we examine the expression of copper/zinc superoxide dismutase (Sod1) and glutathione peroxidase (Gpx1) in various organs of late-gestational mouse fetuses. Furthermore, we compare the expression of these genes in organs of fetuses, neonates, and adult mice. These studies were carried out to investigate whether the change in mRNA levels for these two genes is related to a developmental change in oxidant stress. Our data demonstrate that an increase in both Sod1 and Gpx1 mRNA occurs in lungs and livers of late-gestational mouse fetuses. The brain demonstrates an increase in Sod1 expression at or around the time of birth, the kidney shows an elevation in Gpx1 mRNA levels, and the heart fails to demonstrate a surge in both Sod1 and Gpx1 mRNA levels. Our data show that the liver is the organ with the highest levels of Sod1 and Gpx1 mRNA in embryos and neonates (immediately after birth). In the adult, the liver has the highest levels of Sod1 mRNA and the spleen the highest level of Gpx1 mRNA. These data suggest that the levels of Sod1 and Gpx1 mRNA are unrelated to oxygen consumption and to oxygen tension exposure of individual organs and do not necessarily appear to occur in the lung solely in preparation for birth. The reasons for the increase in antioxidant enzyme(s) mRNA levels in late gestation are more complex and may involve other factors.

Nrf2 Activators as Attractive Therapeutics for Diabetic Nephropathy

Diabetes is the major cause of chronic kidney disease worldwide (1) with treatment options focused primarily on glucose control, blood pressure, lipid lowering, and the blockade of the renin-angiotensin system (2). However, despite intensive metabolic control and other interventions (3), the unrelenting decline in kidney function means that for many patients the condition progresses to overt kidney failure. This underpins the urgent need for novel approaches to manage the ever-increasing number of patients with diabetes and chronic kidney disease. One approach that is attracting attention is the use of compounds to bolster the natural cytoprotective responses of the body. The transcription factor NF-E2–related factor 2 (Nrf2), together with its negative regulator, Kelch-like ECH-associated protein 1 (Keap1), is considered one of the most important cellular defense mechanisms to combat oxidative stress (4) with a particular role in the regulation of phase II detoxifying enzymes (Fig. 1). In particular, NADPH quinone oxidoreductase, glutathione S-transferase, heme oxygenase-1, and g-glutamylcysteine synthetase are wellstudied targets of Nrf2 that are upregulated through the antioxidant response element found in the promoters of these genes (5). Therefore, the coordinated upregulation of genes coding for detoxification, antioxidant, and antiinflammatory regulators is seen as a potential therapeutic strategy to protect against insults such as inflammation and oxidative stress that are known to be enhanced by the diabetic milieu. It is therefore not surprising that attention has focused on identifying small molecule activators of the Nrf2/Keap1 pathway. Many chemically diverse activators have already been identified (6), including the glutathione peroxidase-1 mimetic ebselen (7), sulforaphane found in cruciferous vegetables (8), caffeic acid phenethylester from the bee product propolis (9), cinnamic aldehyde (found in cinnamon bark), and most recently, bardoxolone methyl (10,11). Many have shown promising actions relevant to diabetes complications. For example, activation of Nrf2 by sulforaphane is able to suppress hyperglycemia-induced oxidative stress and metabolic dysfunction in human microvascular endothelial cells (8). A preclinical study by Zheng et al. (12) published in this edition of Diabetes also demonstrates the potential utility of the Nrf2 agonists, sulforaphane and cinnamic aldehyde, for improving the metabolic profile and reducing renal injury in mice with streptozotocin-induced diabetes. Specifically, this treatment was associated with reduced oxidative stress and attenuated induction of the profibrotic mediator transforming growth factor-b, the growth inhibitory protein p21, and extracellular matrix proteins in the diabetic kidney. Importantly, sulforaphane and cinnamic aldehyde failed to protect against renal injury in diabetic Nrf2 knockout mice, suggesting that their renoprotective actions are specifically mediated via activation of Nrf2. Furthermore, specifically silencing Nrf2 also increased matrix synthesis. Rather than directly targeting Nrf2, many agonists appear to work by suppressing its endogenous inhibitor, Keap1. Indeed, the study by Zheng et al. (12) showed that the specific silencing of Keap1 using small interfering RNA was able to reduce the expression of transforming growth factor-b and matrix proteins in human renal mesangial cells under both normal and high glucose conditions. However, limited knowledge of the structural biology of Nrf2-Keap1 means that the precise way in which small molecule agents might interact with Keap1 is still to be fully elucidated. One common feature appears to be their reactivity with the sulfhydryl groups of the Keap1 protein. A recent study by Kobayashi et al. (13), using a zebrafish model of a Keap1 mutation, has shown that certain classes of Nrf2 activators display a greater propensity to modify certain sulfhydryl groups within Keap1. In their classification, sulforaphane and several other Nrf2 activators modified Cys151, while the prostaglandin activators required Cys273 for their activation. Hydrogen peroxide, on the other hand, modified multiple cysteine residues of Keap1 (14), suggesting that the specificity of these small molecule activators may reside in the specific modifications of Keap1 cysteine residues. It is likely that more detailed understanding of the molecular interactions between Nrf2-Keap1 and these small molecule activators will pave the way for additional therapeutic interventions. Beyond actions on renal fibrosis, small molecule activators of Nrf2 may also have direct actions on renal function. In a recently published double-blind, randomized, placebo-controlled trial using the Nrf2 activator, bardoxolone methyl (15), rapid improvements in the estimated glomerular filtration rate have been noted in patients with type 2 diabetes and impaired renal function (estimated glomerular filtration rate 20–45 ml/min/1.73 m). That these changes were observed within 4 weeks and were largely reversible when the drug was discontinued suggests a direct hemodynamic effect of this strategy. Given that current intervention appears to slow the decline in renal function by less than 1 ml/min/1.73 m per year at best (16), sustained improvements with bardoxolone methyl of between 5–10 ml/min/1.73 m potentially represent a major advance over standard therapies. Indeed, the result of this clinical trial is encouraging, and it is sure to lead to a flurry From the Diabetic Complications Division, Oxidative Stress Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. Corresponding author: Judy B. de Haan, judy.dehaan@bakeridi.edu.au. DOI: 10.2337/db11-1072 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. See accompanying original article, p. 3055.

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse

Abstract Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H2O2 accumulation), are presented. Although in most instances accumulation of H2O2 affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.