Mechanistic characterization of the thioredoxin system in the removal of hydrogen peroxide

The thioredoxin system, which consists of a family of proteins, including thioredoxin (Trx), peroxiredoxin (Prx), and thioredoxin reductase (TrxR), plays a critical role in the defense against oxidative stress by removing harmful hydrogen peroxide (H₂O₂). Specifically, Trx donates electrons to Prx to remove H₂O₂ and then TrxR maintains the reduced Trx concentration with NADPH as the cofactor. Despite a great deal of kinetic information gathered on the removal of H₂O₂ by the Trx system from various sources/species, a mechanistic understanding of the associated enzymes is still not available. We address this issue by developing a thermodynamically consistent mathematical model of the Trx system which entails mechanistic details and provides quantitative insights into the kinetics of the TrxR and Prx enzymes. Consistent with experimental studies, the model analyses of the available data show that both enzymes operate by a ping-pong mechanism. The proposed mechanism for TrxR, which incorporates substrate inhibition by NADPH and intermediate protonation states, well describes the available data and accurately predicts the bell-shaped behavior of the effect of pH on the TrxR activity. Most importantly, the model also predicts the inhibitory effects of the reaction products (NADP⁺ and Trx(SH)₂) on the TrxR activity for which suitable experimental data are not available. The model analyses of the available data on the kinetics of Prx from mammalian sources reveal that Prx operates at very low H₂O₂ concentrations compared to their human parasite counterparts. Furthermore, the model is able to predict the dynamic overoxidation of Prx at high H₂O₂ concentrations, consistent with the available data. The integrated Prx–TrxR model simulations well describe the NADPH and H₂O₂ degradation dynamics and also show that the coupling of TrxR- and Prx-dependent reduction of H₂O₂ allowed ultrasensitive changes in the Trx concentration in response to changes in the TrxR concentration at high Prx concentrations. Thus, the model of this sort is very useful for integration into computational H₂O₂ degradation models to identify its role in physiological and pathophysiological functions.     

A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism

Cells are endowed with several overlapping peroxide-degrading systems whose relative importance is a matter of debate. In this study, three different sources of neural cells (rat hippocampal slices, rat C6 glioma cells, and mouse N2a neuroblastoma cells) were used as models to understand the relative contributions of individual peroxide-degrading systems. After a pretreatment (30 min) with specific inhibitors, each system was challenged with either H₂O₂ or cumene hydroperoxide (CuOOH), both at 100 μM. Hippocampal slices, C6 cells, and N2a cells showed a decrease in the H₂O₂ decomposition rate (23-28%) by a pretreatment with the catalase inhibitor aminotriazole. The inhibition of glutathione reductase (GR) by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) significantly decreased H₂O₂ and CuOOH decomposition rates (31-77%). Inhibition of catalase was not as effective as BCNU at decreasing cell viability (MTT assay) and cell permeability or at increasing DNA damage (comet test). Impairing the thioredoxin (Trx)-dependent peroxiredoxin (Prx) recycling by thioredoxin reductase (TrxR) inhibition with auranofin neither potentiated peroxide toxicity nor decreased the peroxide-decomposition rate. The results indicate that neural peroxidatic systems depending on Trx/TrxR for recycling are not as important as those depending on GSH/GR. Dimer formation, which leads to Prx2 inactivation, was observed in hippocampal slices and N2a cells treated with H₂O₂, but not in C6 cells. However, Prx-SO₃ formation, another form of Prx inactivation, was observed in all neural cell types tested, indicating that redox-mediated signaling pathways can be modulated in neural cells. These differences in Prx2 dimerization suggest specific redox regulation mechanisms in glia-derived (C6) compared to neuron-derived (N2a) cells and hippocampal slices.     

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.     

Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)₃, As(GS)₃, As(Penicillamine)₃, As(Mercaptoethanesulfonate)₃, As(Mercaptopurine)₃, As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)₃ which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ showed stronger cytotoxicity than the others. As(2-mercaptopyridine)₃ which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)₃ oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.     

Nicotinamide Nucleotide Transhydrogenase (Nnt) Links the Substrate Requirement in Brain Mitochondria for Hydrogen Peroxide Removal to the Thioredoxin/Peroxiredoxin (Trx/Prx) System

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H₂O₂) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H₂O₂ catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP⁺, provides the link between mitochondrial respiration and H₂O₂ detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H₂O₂ in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H₂O₂, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H₂O₂ catabolism, (b) decreased NADPH and increased NADP⁺ levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H₂O₂ and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H₂O₂ antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Thioredoxin reductase-2 is essential for keeping low levels of H(2)O(2) emission from isolated heart mitochondria

Respiring mitochondria produce H(2)O(2) continuously. When production exceeds scavenging, H(2)O(2) emission occurs, endangering cell functions. The mitochondrial peroxidase peroxiredoxin-3 reduces H(2)O(2) to water using reducing equivalents from NADPH supplied by thioredoxin-2 (Trx2) and, ultimately, thioredoxin reductase-2 (TrxR2). Here, the contribution of this mitochondrial thioredoxin system to the control of H(2)O(2) emission was studied in isolated mitochondria and cardiomyocytes from mouse or guinea pig heart. Energization of mitochondria by the addition of glutamate/malate resulted in a 10-fold decrease in the ratio of oxidized to reduced Trx2. This shift in redox state was accompanied by an increase in NAD(P)H and was dependent on TrxR2 activity. Inhibition of TrxR2 in isolated mitochondria by auranofin resulted in increased H(2)O(2) emission, an effect that was seen under both forward and reverse electron transport. This effect was independent of changes in NAD(P)H or membrane potential. The effects of auranofin were reproduced in cardiomyocytes; superoxide and H(2)O(2) levels increased, but similarly, there was no effect on NAD(P)H or membrane potential. These data show that energization of mitochondria increases the antioxidant potential of the TrxR2/Trx2 system and that inhibition of TrxR2 results in increased H(2)O(2) emission through a mechanism that is independent of changes in other redox couples.     

Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress

A dominant-negative, active-site mutant (C93S-Trx2) of mitochondrial thioredoxin-2 (Trx2) was expressed in cells to study the function of the thioredoxin system in protection against mitochondrial oxidative stress. C93S-Trx2 was detected as a disulfide with mitochondrial peroxiredoxin-3 (Prx3) but not peroxiredoxin-5 (Prx5). C93S-Trx2 enhanced sensitivity to cell death induced by tert-butylhydroperoxide or by tumor necrosis factor-alpha (TNF-alpha). In cells treated with buthionine sulfoximine (BSO) to deplete glutathione (GSH), endogenous Trx2 was oxidized, C93S-Trx2 potentiated toxicity, and overexpression of Trx2 protected against toxicity. Thus, the results show that Trx2 interacts with Prx3 in vivo and that the Trx2/Prx3 system functions in parallel with the GSH system to protect mitochondria from oxidative stress. The additive protection by Trx2 and GSH shows that Trx2 and GSH systems are both functionally important at low oxidative stress conditions.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k_cat and K_m, selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H₂O₂ production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.     

Mitochondrial 1-Cys-peroxiredoxin/thioredoxin system protects manganese-containing superoxide dismutase (Mn-SOD) against inactivation by peroxynitrite in Saccharomyces cerevisiae

Peroxynitrite is a reactive nitrogen species that can mediate protein tyrosine nitration, inactivating many proteins. We show that yeast mitochondrial peroxiredoxin (Prx1p), which belongs to the group 1-Cys-Prx, has thioredoxin-dependent peroxynitrite reductase activity. This activity was characterised in vitro with the recombinant mitochondrial Prx1p, the thioredoxin reductase Trr2p and the thioredoxin Trx3p, using a generator of peroxynitrite (SIN-1). Purified mitochondria from wild-type and null Prx1p or Trx3p yeast strains, exposed to SIN-1, showed a differential inactivation of manganese-containing superoxide dismutase activity. The above yeast strains were exposed to SIN-1 and examined under confocal microscopy. Prx1p or Trx3p-null cells showed a greater accumulation of peroxynitrite than wild-type ones. Our results indicate that this 1-Cys-Prx is a peroxynitrite reductase activity that uses reducing equivalents from NADPH through the mitochondrial thioredoxin system. Therefore, mitochondrial 1-Cys-peroxiredoxin/thioredoxin system constitutes an essential antioxidant defence against oxidative and nitrosative stress in yeast mitochondria.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Thioredoxin and glutaredoxin system proteins—immunolocalization in the rat central nervous system

Background

The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic–ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia–ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available.

Methods

The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia–ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina.

Results and conclusions

Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family.

General significance

We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia–ischemia insults and other disorders of the CNS.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Cytotoxicity induced by inhibition of thioredoxin reductases via multiple signaling pathways: Role of cytosolic phospholipase A2α‐dependent and ‐independent release of arachidonic acid

The thioredoxin (Trx) system, comprising Trx, the selenoprotein thioredoxin reductase (TrxR), and NADPH, functions as an antioxidant system. Trx has various biological activities including growth control and anti‐apoptotic properties, and the Trx system offers a target for the development of drugs to treat and/or prevent cancer. We evaluated the role of TrxR inhibition in the release of arachidonic acid (AA), cell toxicity, and intracellular signaling pathways in L929 mouse fibrosarcoma cells. Treatment with 1‐chloro‐2,4‐dinitrobenzene (DNCB, an inhibitor of TrxR) under conditions involving limited inhibition of TrxR activity in cells, released AA before causing cytotoxicity. Treatment with an inhibitor of p38 kinase, a downstream enzyme of the apoptosis signal‐regulating kinase 1 pathway, and pyrrophenone (an inhibitor of α‐type cytosolic phospholipase A₂, cPLA₂α) partially but significantly decreased the DNCB‐induced release of AA and cell death. The responses were much weaker in cPLA₂α knockdown L929 cells. Exogenously added AA showed cytotoxicity. DNCB increased intracellular reactive oxygen species (ROS) levels, and butylated hydroxyanisole (an antioxidant) reduced DNCB‐induced ROS formation and cell toxicity but not the phosphorylation of p38 kinase and release of AA. Auranofin, another inhibitor of TrxR having a different formula, released AA resulting in toxicity in L929 cells. DNCB caused the release of AA and cytotoxicity in A549 human lung carcinoma cells, and caused p38 kinase‐dependent toxicity in PC12 rat pheochromocytoma cells. Our data suggest that a dysfunctional Trx system triggers multiple signaling pathways, and that the AA released by cPLA₂α‐dependent and ‐independent pathways is important to cytotoxicity. J. Cell. Physiol. 219: 606–616, 2009. © 2009 Wiley‐Liss, Inc.     

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.     

Treatment of human cancer cells with selenite or tellurite in combination with auranofin enhances cell death due to redox shift

Selenium is an essential trace element incorporated as selenocysteine in 25 human selenoproteins. Among them are thioredoxin reductases (TrxR) and glutathione peroxidases, all central proteins in the regulation of the cellular thiol redox state. In this paper the effects of selenite and tellurite treatment in human cancer cells are reported and compared. Our results show that both selenite and tellurite, at relatively low concentrations, are able to increase the expression of mitochondrial and cytosolic TrxR in cisplatin-sensitive (2008) and -resistant (C13) phenotypes. We further investigated the cellular effects induced by selenite or tellurite in combination with the specific TrxR inhibitor auranofin. Selenite pretreatment induced a dramatic increase in auranofin cytotoxicity in both resistant and sensitive cells. Investigation of TrxR activity and expression levels as well as the cellular redox state demonstrated the involvement of TrxR inhibition and redox changes in selenite and auranofin combined action.     

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx’s keep many intracellular proteins reduced, including the peroxiredoxins (Prx’s). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx’s. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR’s) keep the Trx’s largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx’s and Prx’s. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx’s, Prx’s, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.     

Preferential elevation of Prx I and Trx expression in lung cancer cells following hypoxia and in human lung cancer tissues

Transient/chronic microenvironmental hypoxia that exists within a majority of solid tumors has been suggested to have a profound influence on tumor growth and therapeutic outcome. Since the functions of novel antioxidant proteins, peroxiredoxin I (Prx I) and II, have been implicated in regulating cell proliferation, differentiation, and apoptosis, it was of our special interest to probe a possible role of Prx I and II in the context of hypoxic tumor microenvironment. Since both Prx I and II use thioredoxin (Trx) as an electron donor and Trx is a substrate for thioredoxin reductase (TrxR), we investigated the regulation of Trx and TrxR as well as Prx expression following hypoxia. Here we show a dynamic change of glutathione homeostasis in lung cancer A549 cells and an up-regulation of Prx I and Trx following hypoxia. Western blot analysis of 10 human lung cancer and paired normal lung tissues also revealed an elevated expression of Prx I and Trx proteins in lung cancer tissues. Immunohistochemical analysis of the lung cancer tissues confirmed an augmented Prx I and Trx expression in cancer cells with respect to the parenchymal cells in adjacent normal lung tissue. Based on these results, we suggest that the redox changes in lung tumor microenvironment could have acted as a trigger for the up-regulation of Prx I and Trx in lung cancer cells. Although the clinical significance of our finding awaits more rigorous future study, preferential augmentation of the Prx I and Trx in lung cancer cells may well represent an attempt of cancer cells to manipulate a dynamic redox change in tumor microenvironment in a manner that is beneficial for their proliferation and malignant progression.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.