Antioxidants & Redox Signaling

Several hypotheses have been proposed to explain the mechanisms of thalidomide teratogenesis, although none adequately accounts for the observed malformations and explains the basis for species specificity. Recent observations that thalidomide increases the production of free radicals and elicits oxidative stress, coupled with new insights into the redox regulation of nuclear transcription factors, lead to the suggestion that thalidomide may act through redox misregulation of the limb outgrowth pathways. Oxidative stress, as marked by glutathione depletion/oxidation and a shift in intracellular redox potential toward the positive, occurs preferentially in limbs of thalidomide-sensitive rabbits, but not in resistant rats. DNA binding of nuclear factor κ-B (NF-κB), a redox-sensitive transcription factor and key regulator of limb outgrowth, was shown to be significantly attenuated in rabbit limb cells and could be restored following the addition of a free radical spin-trapping agent, phenyl N-tert-butyl nitrone. The inability of NF-κB to bind to its DNA promoter results in the failure of limb cells to express fibroblast growth factor (FGF)-10 and twist in the limb progress zone (PZ) mesenchyme, which in turn attenuates expression of FGF-8 in the apical ectodermal ridge (AER). Failure to establish an FGF-10/FGF-8 feedback loop between the PZ and AER results in the truncation of limb outgrowth. We hypothesize that species-selective alterations in redox microenvironment caused by free radical production from thalidomide results in attenuation of the NF-κB-mediated gene expression that is responsible for limb outgrowth.

Oxidative stress (OS) plays a major role in chronic hepatitis C. Various OS markers have been found to be elevated in hepatitis C virus (HCV)-related liver disease. This study detected the presence of OS in serum and liver biopsy specimens of HCV patients. Reactive oxygen molecules (ROM) in sera of 54 HCV patients were compared with 23 controls. OS markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal, malondialdehyde, and thioredoxin were measured in liver biopsy specimens of 18 HCV patients with fibrosis staging F1 (six); F2 (two), F3 (four), and F4 (six). The interferon (IFN) response and hepatocellular carcinoma (HCC) occurrence in the presence of OS markers were also evaluated. The level of ROM in HCV patients was 318 ± 56.7 Carr compared with 248 ± 40.8 Carr in controls (p = 0.032). Multivariate analysis found age (p = 0.0236) to be the only independent variable associated with increase in ROM in sera. In liver biopsy specimens, OS markers were found mainly around the area of piecemeal necrosis or the periportal area. The presence of OS markers seemed to increase with fibrosis staging, although not significantly. The OS DNA damage marker 8-OHdG was detected in the nucleus of hepatocytes. Thirteen patients received IFN therapy. During the 4-year follow-up period, HCC developed in four nonresponders to IFN and in one untreated patient. OS markers were stained in both HCC cells and non-HCC cells in HCC patients. OS markers were found in serum and liver specimens of HCV-associated liver disease and in HCC tissue. Detection of OS markers may be important for monitoring disease progression in HCV patients. Antioxidant therapy in combination with antiviral therapy may minimize liver damage and aid in the prevention and subsequent development of HCC.

Redox control of cell physiology is one of the most important regulatory mechanisms in all living organisms. The thioredoxin system, composed of thioredoxin and thioredoxin reductase, has emerged as a key player in cellular redox-mediated reactions. For many years, only one thioredoxin system had been described in higher organisms, ubiquitously expressed in the cytoplasm of eukaryotic cells. However, during the last decade, we and others have identified and characterized novel thioredoxin systems with unique properties, such as organelle-specific localization in mitochondria or endoplasmic reticulum, tissue-specific distribution mostly in the testis, and features novel for thioredoxins, such as microtubule-binding properties. In this review, we will focus on the mammalian testis-specific thioredoxin system that comprises three thioredoxins exclusively expressed in spermatids (named Sptrx-1, Sptrx-2, and Sptrx-3) and an additional thioredoxin highly expressed in testis, but also present in lung and other ciliated tissues(Txl-2). The implications of these findings in the context of male fertility and testicular cancer, as well as evolutionary aspects, will be discussed.

Reactive oxygen species (ROS) are generated as toxic by-products of aerobic metabolism, but are also essential biomolecules in cell signaling. The thioredoxin (Trx) system is a major enzymatic system modulating ROS levels and is important for redox regulation of cellular function. It consists of Trx and thioredoxin reductase (TrxR), which reduces Trx using NADPH. Most, if not all, of the functions of Trx depend on the activity of TrxR. Mammalian TrxR enzymes are selenoproteins with broad substrate specificities, and alteration of cytosolic TrxR1 expression and activity is likely to be an important determinant for the control of cellular redox regulation. TrxR1 activity in cells seems to be modulated by an intricate interplay, involving a housekeeping type promoter in combination with alternative splice variants and transcriptional start sites, posttranscriptional regulation through AU-rich elements, inactivation by electrophilic agents and by itself modulating the effects of several key signaling molecules. TrxR1 activity is also intimately linked with several aspects of selenium metabolism, and hence selenoprotein function in general. Here, we summarize the current knowledge of these different levels of TrxR1 regulation in diverse cell types and in response to growth and signaling events.

Human cytosolic thioredoxin (Trx), which is the 12-kDa protein disulfide reductase with the Cys-Gly-Pro-Cys active site and a key component of cellular redox biochemistry and regulation, acts as cocytokine upon leaderless secretion. A 10-kDa C-terminally truncated thioredoxin (Trx80) comprising the 80 or 84 N-terminal amino acids is also secreted and present in plasma, where it originally was purified and identified as eosinophilic cytotoxicity enhancing factor. Recombinant Trx80 was discovered to be a potent mitogenic cytokine that stimulates growth of resting human peripheral blood mononuclear cells (PBMC) in a synthetic medium, an effect that Trx lacks. Trx80 is very different from Trx because it is a dimer lacking reductase activity and the cytokine activity is not dependent on the Cys residues of the Trx active-site motif. The primary targets of Trx80 in PBMC are monocytes that are activated to proliferate and increase expression of CD14, CD40, CD54, and CD86. Trx80 induces secretion of interleukin (IL)-12 in CD40+ monocytes from PBMC. Trx80 and IL-2 together were strongly synergistic to induce secretion of interferon-γ in PBMC. Trx80 is a potent cytokine for monocytes directing the immune system to a Th1 response via IL-12 production.

Most cells contain high levels of glutathione and multiple glutaredoxins, which utilize the reducing power of glutathione to catalyze disulfide reductions in the presence of NADPH and glutathione reductase(the glutaredoxin system). Glutaredoxins, like thioredoxins, may operate as dithiol reductants and are involved as alternative pathways in cellular functions such as formation of deoxyribonucleotides for DNA synthesis (by reducing the essential enzyme ribonucleotide reductase), the generation of reduced sulfur (via 3′-phosphoadenylylsulfate reductase), signal transduction, and the defense against oxidative stress. The three dithiol glutaredoxins of E. coli with the active-site sequence CPYC and a glutathione binding site in a thioredoxin/glutaredoxin fold display surprisingly different properties. These include the inducible OxyR-regulated 10-kDa Grx1 or the highly abundant 24-kDa glutathione S-transferase-like Grx2 (with Grx3 it accounts for 1% of total protein). Glutaredoxins uniquely reduce mixed disulfides with glutathione via a monothiol mechansim where only an N-terminal low pK _a Cys residue is required, by using their glutathione binding site. Glutaredoxins also catalyze formation of mixed disulfides (glutathionylation), which is an important redox regulatory mechanism, particularly in mammalian cells under oxidative stress conditions, to sense cellular redox potential.

To determine whether plasma levels of thioredoxin are associated with coronary spasm, we measured the plasma levels of thioredoxin in 170 patients who had <25% organic stenosis in coronary arteriography. According to the results of cardiac catheterization, we divided the patients into two groups: a coronary spastic angina group (n = 84) and a chest pain syndrome group (n = 86). The plasma levels of thioredoxin were significantly higher in the coronary spastic angina group than in the chest pain syndrome group (40.7 ± 4.1 versus 18.2 ± 1.1 ng/ml, p < 0.0001). Furthermore, the increased plasma levels of thioredoxin were associated with high disease activity indicated by the frequency of angina attacks (p = 0.0004). In multiple logistic regression analysis, the higher levels of thioredoxin [relative risk 14.8, 95% confidence interval (5.13-42.9), p < 0.0001] and current smoking [relative risk 3.39, 95% confidence interval (1.31-8.75), p = 0 .012] were significant and independent variables associated with coronary spasm. We demonstrated that the plasma levels of thioredoxin were increased in the coronary spastic angina group, and increased levels of thioredoxin were associated with high disease activity. The plasma levels of thioredoxin and current smoking were risk factors for coronary spastic angina, and they were independent from other traditional risk factors.

Thioredoxin (TRX) is induced by a variety of oxidative stimuli and shows cytoprotective roles against oxidative stress. To clarify the possibility of clinical application, we examined the effects of intravenously administered TRX in a model of transient focal cerebral ischemia in this study. Mature male C57BL/6j mice received either continuous intravenous infusion of recombinant human TRX (rhTRX) over a range of 1-10 mg/kg, bovine serum albumin, or vehicle alone for 2 h after 90-min transient middle cerebral artery occlusion (MCAO). Twenty-four hours after the transient MCAO, the animals were evaluated neurologically and the infarct volumes were assessed. Infarct volume, neurological deficit, and protein carbonyl contents, a marker of protein oxidation, in the brain were significantly ameliorated in rhTRX-treated mice at the dose of 3 and 10 mg/kg versus these parameters in control animals. Moreover, activation of p38 mitogen-activated protein kinase, whose pathway is involved in ischemic neuronal death, was suppressed in the rhTRX-treated mice. Further, rhTRX was detected in the ischemic hemisphere by western blot analysis, suggesting that rhTRX was able to permeate the blood-brain barrier in the ischemic hemisphere. These data indicate that exogenous TRX exerts distinct cytoprotective effects on cerebral ischemia/reperfusion injury in mice by means of its redox-regulating activity.

As oxidative stress plays a crucial role in the development and pathogenesis of hypertension, we analyzed the redox (reduction/oxidation) status in tissues from Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone SHR (SHRSP). Expressions of 8-hydroxy-2′-deoxyguanosine, a marker for oxidative stress-induced DNA damage, and protein carbonylation, a marker for oxidation status of proteins, were enhanced in aorta, heart, and kidney from SHR and SHRSP compared with WKY. The expression of redox regulating protein, thioredoxin (TRX), estimated by immunohistochemistry and western blot, and expression of TRX gene estimated by real-time RT-PCR were markedly suppressed in those tissues from SHR and SHRSP compared with WKY. Induction of TRX was impaired after angiotension II treatment in peripheral blood mononuclear cells isolated from SHR and SHRSP compared with those isolated from WKY. Although previous reports have shown that TRX is induced by a variety of oxidative stress in tissues, the present study shows the impaired induction of TRX in tissues from genetically hypertensive rats despite the relative increment of oxidative stress. Redox imbalance in essential organs may play a crucial role in the development and pathogenesis of hypertension.

Ebselen is a selanazal drug recently revealed as a highly efficient peroxiredoxin mimic catalyzing the hydroperoxide reduction by the mammalian thioredoxin system [thioredoxin (Trx), thioredoxin reductase(TrxR), and NADPH]. The mammalian Trx system is a dehydroascorbic acid reductase recycling ascorbic acid essential for cell functions. Here we report that ebselen strongly facilitated the recycling of ascorbic acid by the TrxR both with and without Trx present. Reduction of dehydroascorbic acid by TrxR has a pH optimum of 6.4, and only ∼55% of this activity at a physiological pH of 7.4. Ebselen at 6 µM enhances this reaction three-fold and with the same pH optimum of 6.4. The mechanism of the ebselen effect is suggested to involve reduction of dehydroascorbic acid by the ebselen selenol, a highly efficient two-electron reductant. Thus, ebselen acts as an antioxidant to lower the peroxide tone inside cells and to facilitate the recycling of dehydroascorbic acid to ascorbic acid, so as to increase the radical scavenging capacity of ascorbic acid directly or indirectly via vitamin E. The high ascorbic acid recycling efficiency of ebselen at pH 6.4 may play a major role in oxidatively stressed cells, where cytosol acidosis may trigger various responses, including apoptosis.

Bronchopulmonary dysplasia (BPD) is a major complication of premature infants who receive prolonged ventilatory support. The pathophysiology of BPD involves oxidant injury, baro/volutrauma, and disordered lung repair. Exposure of premature lung that is poorly adapted for air breathing (>3% oxygen in fetal lung) to a higher concentration of oxygen can cause significant oxidant injury. Cell growth and differentiation of the developing lung require selective and ordered cell division. As hyperoxia can increase the expression of cell-cycle checkpoints that can cause growth arrest of lung cells, in this report we examined the expression of checkpoint proteins p53 and p21 in a premature infant the baboon model of BPD. Additionally, we also determined whether enhanced apoptosis occurs in baboon BPD model. We have shown that p53 and p21 expression are increased in 125-day as well as 140-day premature baboons with BPD. We also demonstrate increased apoptosis in lung tissue of premature baboons with BPD. These results demonstrate that cell growth inhibition is a likely factor in the evolution of BPD. Additionally, lung cells may undergo increased apoptosis that can impair the repair process in the postventilatory recovery period.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants, which results in substantial morbidity. The pathophysiology of BPD includes oxidant injury, baro/volutrauma, and disordered lung repair. As lung development, differentiation, and repair require cell division, we hypothesized dysregulation of the cell cycle in oxygen exposure of premature infants that may contribute to the evolution of BPD. In this investigation, we studied the expression of cyclins and cyclin-dependent kinases (cdks) that regulate transition from G1 and G2 phases of the cell cycle. We report here that expression of cyclin D1, cyclin E, and cyclin A is modulated in premature baboons in respiratory distress. In addition, the expression of cdk1 or cdk4 was also modulated in these premature animals. The phosphorylation of retinoblastoma protein was progressively decreased in 125-day animals and in 140-day animals exposed to 6 or 14 days of PRN oxygen. These results indicate that due to altered cyclin and cdk expression, the repair of injured epithelium may proceed in a disordered manner that is characteristic of BPD. Thus, altered cell cycle regulation may be an important factor in the evolution of BPD.

The effect of hyperoxia on levels of DNA damage and global DNA methylation was examined in lung epithelial-like A549 cells. DNA damage was assessed by the single-cell gel electrophoresis (comet assay) and DNA methylation status by the cytosine extension assays. Cells exposed to ionizing radiation (0, 1, 2, 4, or 8 Gy) showed increasing rates of percentage of DNA in the tail and tail length with increasing radiation dose. When cells were exposed to room air (normoxia) for 1 day and 95% O₂ (hyperoxia) for 1, 2, 3, 4, and 5 days, data indicated that hyperoxia caused time-dependent increases in levels of (a) single strand breaks, (b) double strand breaks, and (c) 8-oxoguanine. Decreased DNA methylation also was observed at day 5 of hyperoxic exposure, suggesting that hyperoxia-induced DNA damage can influence patterns of DNA methylation in a lung-derived cell line.

Impaired neovascularization is associated with the pathologic presentation of bronchopulmonary dysplasia (BPD). To determine if neovascularization and factors that negatively influence blood vessel formation play a role in the evolution of BPD, we examined the temporospatial distribution of a protein known to inhibit fetal lung neovascularization with associated dysplastic lung formation, endothelial-monocyte activating polypeptide (EMAP) II. Immunohistochemical analysis of EMAP II in lung tissues of human infants with BPD indicated an elevation in EMAP II abundance as compared with control. Utilizing a baboon model, western analysis indicated that EMAP II was increased twofold in those baboons with pathologic signs of BPD as compared with gestational controls. Consistent with our findings in human tissues, immunohistochemistry and in situ hybridization demonstrate that EMAP II is highly expressed in the perivascular stroma and dysplastic lung periphery in neonatal baboons with BPD as compared with controls. Lastly, there is a premature acceleration in EMAP II's perivascular distribution in term newborn baboon as compared with gestational control. The marked increase in EMAP II's temporal expression, its distribution in the perivascular and dysplastic alveolar regions of the lungs, and the interruption in vasculogenesis in BPD suggest that neovascularization and factors that negatively influence blood vessel formation may play a role in BPD evolution.

In the cell, reducing and oxidizing molecules modulate the redox state. In embryonic and fetal growth, increased oxidative stress may be detrimental, but an oxidized state can also be beneficial. This is because redox may also affect key transcription factors that can alter gene expression during development. In addition, redox may impact on placentation and amniotic membrane integrity during pregnancy. Lastly, diseases of prematurity, such as necrotizing enterocolitis, retinopathy of prematurity, and chronic lung disease, may be modulated by redox in the premature. Because antioxidant therapies have not necessarily modified the outcome of these diseases, some debate exists as to this. Nonetheless, sufficient evidence suggests a role for redox throughout embryonic, fetal, and postnatal development. This evidence will be explored here.

Preterm neonates with respiratory distress are exposed not only to the relative hyperoxia ex utero, but also to life-saving mechanical ventilation with high inspired oxygen (O₂) concentrations, which is considered a major risk factor for the development of bronchopulmonary dysplasia, also referred to as chronic lung disease of infancy. O₂ toxicity is mediated through reactive oxygen species (ROS). ROS are constantly generated as byproducts of normal cellular metabolism, but their production is increased in various pathological states, and also upon exposure to exogenous oxidants, such as hyperoxia. Antioxidants, either enzymatic or nonenzymatic, protect the lung against the deleterious effects of ROS. Expression of various pulmonary antioxidants is developmentally regulated in many species so that the expression is increased toward term gestation, as if in anticipation of birth into an O₂-rich extrauterine environment. Therefore, the lungs of prematurely born infants may be ill-adapted for protection against ROS. While premature birth interrupts normal lung development, the clinical condition necessitating the administration of high inhaled O₂ concentrations may lead to permanent impairment of alveolar development. An understanding of the processes involved in lung growth, especially in alveolarization and vascularization, as well as in repair of injured lung tissue, may facilitate development of strategies to enhance these processes.

Free radicals have been implicated in the pathogenesis of a wide spectrum of human diseases. Premature infants are probably developmentally unprepared for extrauterine life in an oxygen-rich environment and exhibit a unique sensitivity to oxidant injury. Diseases associated with premature infants, including bronchopulmonary dysplasia, periventricular leukomalacia, intraventricular hemorrhage, retinopathy of prematurity, and necrotizing enterocolitis, have been linked to free radical-mediated cell and tissue injury. With the advent of therapies designed to combat the injurious effects of free radicals, the role of these highly reactive chemical molecules in the pathogenesis of neonatal diseases needs to be fully determined.

Thioredoxin is an important redox protein that is ubiquitously distributed. Thioredoxin exists in dynamic equilibrium between the oxidized and reduced forms, making it an ideal redox-regulatory protein. Thioredoxin, together with thioredoxin reductase and peroxiredoxins, forms a complete redox system that is similar to the glutathione system, but with distinct and divergent functions. This review provides a brief general summary of the thioredoxin system with particular emphasis on its role in premature birth and newborn physiology and disease states. Although extensive studies have examined the role of the thioredoxin system in antioxidant defense, cell proliferation, and signal transduction, further studies are needed to understand its role in embryogenesis and development. Such studies will facilitate our understanding of how thioredoxin may modulate newborn diseases via redox regulation.

Pulmonary surfactant, a complex of lipids and proteins, maintains alveolar integrity and participates in the control of host defense and inflammation in the lung. Surfactant proteins A, B, C, and D are important components of surfactant that play diverse roles in the surface tension reducing as well as host defense and inflammation control functions of surfactant. Hyperoxia or exposure of cells/tissues to elevated levels of oxygen occurs when high levels of oxygen are used to treat a variety of pulmonary disorders that include respiratory distress syndrome of premature infants, emphysema, sarcoidosis, end-stage lung diseases, and others. The lung serves as a primary target organ in hyperoxia, and hyperoxic lung injury is characterized by pulmonary edema, inflammation, and respiratory failure. Hyperoxic lung injury is associated with significant changes in the expression of surfactant proteins that likely serves as an adaptive response to elevated oxygen levels. In most animal species studied, hyperoxia increases the tissue expression of surfactant protein mRNAs. A limited number of studies have indicated that the increased tissue expression of surfactant protein mRNAs is associated with increased levels of surfactant proteins in the bronchoalveolar lavage.

Reactive oxygen species (ROS) are known to play an important role in the proliferation and viability of vascular smooth muscle cells. We have shown previously that treatment of fetal pulmonary arterial smooth muscle cells (FPASMC) with concentrations of 25 µM and higher of EUK-134, a superoxide dismutase/catalase mimetic, decreased cell viability via the induction of apoptosis. Here we demonstrate a dose-dependent decrease in serum-induced FPASMC growth at lower doses of EUK-134. This was due to the attenuation of FPASMC proliferation rather than the induction of apoptosis. Moreover, we found that the inhibition of FPASMC proliferation was observed using EUK-134 at concentrations as low as 5 µM. This inhibition of proliferation correlated with a 31% decrease in superoxide levels, as estimated using the oxidation of dihydroethidium. Flow cytometry revealed an increase in FPASMC in G₂ after 24 h of exposure to 10 µM EUK-134. This was associated with a twofold increase in levels of the cell-cycle regulatory protein p21. This, together with our previous data, suggests that ROS levels determine the rate of FPASMC proliferation and, when below a threshold level, trigger apoptosis. Titration of ROS with antioxidants may help to prevent, or reverse, the vascular remodeling manifest in many cardiovascular disease states.