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Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As3+)-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As3+ can displace zinc (Zn2+) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As3+ could displace Zn2+ from ZRANB2, altering its structure, expression, and splicing function. As3+/Zn2+ binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As3+ (0 or 5 μM, 0-72 h; or 0-5 μM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As3+ bound to, as well as displaced Zn2+ from, each zfm. Also, Zn2+ displaced As3+ from As3+-bound zfm's acutely, albeit transiently. As3+ exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As3+ exposures, starting at 100 nm. We conclude that As3+ exposure displaces Zn2+ from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.

Chronic obstructive pulmonary disease (COPD) is prevalent in patients infected with HIV. The purpose of this study was to test the hypothesis that systemic oxidation correlates with loss of lung function in subjects with COPD, and that HIV infection can contribute to creating such an environment. Subjects were recruited at the University of Louisville in the following groups: HIV-infected (n = 36), COPD (n = 32), HIV and COPD (n = 28), and uninfected controls with normal lung function (n = 34). HIV infection was assessed by viral load and CD4 cell counts. Pulmonary function was determined by spirometry, and plasma was collected for measurement of cysteine (Cys), cystine (CySS), glutathione (GSH) and GSH disulfide (GSSG) by HPLC followed by estimation of redox potentials (Eh) using the Nernst equation. Results showed that patients with COPD had more oxidized plasma Eh Cys/CySS than patients with normal lung function, but plasma Eh GSH/GSSG was unaltered. In addition, there was a correlation between the extent of plasma Eh Cys/CySS oxidation and loss of lung function, and this correlation remained even after correcting for age, sex, race and body mass index. HIV infection per se was not associated with increased oxidation of plasma Eh Cys/CySS, but plasma Eh Cys/CySS was more oxidized in patients with lower CD4-positve T cell counts. In patients with both HIV infection and COPD, there was a significant correlation between CD4 cell counts and lung function. Thus, systemic oxidation correlated with decreased lung function in subjects with COPD and decreased CD4 counts in subjects infected with HIV. Thus, factors contributing to plasma Eh Cys/CySS may represent novel mechanisms underlying the increased prevalence of COPD in people living with HIV.

Age, sex and diet are well-established risk factors for several diseases. In humans, each of these variables has been linked to differences in plasma redox potentials (Eh) of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples. Mice have been very useful for modeling human disease processes, but it is unknown if age, sex and diet affect redox couples in mice as they do in humans. The purpose of the present study was to examine the effects of these factors on plasma redox potentials in C57BL/6J mice. We found that age had no effect on either redox couple in either sex. Plasma Eh Cys/CySS and Eh GSH/GSSG were both more oxidized (more positive) in females than in males. A 24-hour fast negated the sex differences in both redox potentials by oxidizing both redox couples in male mice, while having no effect on Eh Cys/CySS and a smaller effect on Eh GSH/GSSG in female mice. A diet with excess sulfur amino acids reduced the plasma Eh Cys/CySS in females to a level comparable to that seen in male mice. Thus, sex-specific differences in plasma Eh Cys/CySS could be normalized by two different dietary interventions. Some of these findings are consistent with reported human studies, while others are not. Most strikingly, mice do not exhibit age-dependent oxidation of plasma redox potentials. Care must be taken when designing and interpreting mouse studies to investigate redox regulation in humans.

Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an ‘end-stage’ process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation–reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to provide a comprehensive review of this field, but rather to highlight what has been learned and to raise interest in this area in need of much attention., Graphical abstract fx1

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20 mM, 24–48 h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300 µM, 24–48 h) increased clonogenic cell killing in both human prostate (PC-3 and DU145) and human breast (MDA-MB231) cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH) synthesis (l-buthionine sulfoximine; BSO, 1 mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48 h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1 µM), was combined with 2DG+DHEA or DHEA-alone for 24 h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20 mM). Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1) oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC) were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231). Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing., Graphical abstract, Highlights • Inhibition of both glycolysis and pentose cycle causes oxidative stress in human breast and prostate cancer cells. • Combining inhibition of glycolysis and pentose cycle with inhibition of thioredoxin reductase enhances cell killing of these human cancer cells. • The toxicity and oxidative stress is selective for cancer vs. normal cells.

Alcohol and nutrition have the potential to interact at multiple levels. For example, heavy alcohol consumption can interfere with normal nutrition, resulting in overall malnutrition or in deficiencies of important micronutrients, such as zinc, by reducing their absorption or increasing their loss. Interactions between alcohol consumption and nutrition also can affect epigenetic regulation of gene expression by influencing multiple regulatory mechanisms, including methylation and acetylation of histone proteins and DNA. These effects may contribute to alcohol-related organ or tissue injury. The impact of alcohol–nutrition interactions has been assessed for several organs and tissues, including the intestine, where heavy alcohol use can increase intestinal permeability, and the liver, where the degree of malnutrition can be associated with the severity of liver injury and liver disease. Alcohol–nutrition interactions also play a role in alcohol-related lung injury, brain injury, and immune dysfunction. Therefore, treatment involving nutrient supplementation (e.g., with zinc or S-adenosylmethionine) may help prevent or attenuate some types of alcohol-induced organ damage.

Sexually transmitted infections affect hundreds of millions of people worldwide. Both human immunodeficiency virus (HIV-1 and -2) and herpes simplex virus-2 (HSV-2) remain incurable, urging the development of new prevention strategies. While current prophylactic technologies are dependent on strict user adherence to achieve efficacy, there is a dearth of delivery vehicles that provide discreet and convenient administration, combined with prolonged-delivery of active agents. To address these needs, we created electrospun fibers (EFs) comprised of FDA-approved polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(DL-lactide-co-ε-caprolactone) (PLCL), to provide sustained-release and in vitro protection against HIV-1 and HSV-2. PLGA and PLCL EFs, incorporating the antiretroviral, tenofovir disoproxil fumarate (TDF), exhibited sustained-release for up to 4 weeks, and provided complete in vitro protection against HSV-2 and HIV-1 for 24 hr and 1 wk, respectively, based on the doses tested. In vitro cell culture and EpiVaginal tissue tests confirmed the safety of fibers in vaginal and cervical cells, highlighting the potential of PLGA and PLCL EFs as multipurpose next-generation drug delivery vehicles.

Background Chronic alcohol exposure results in liver injury that is driven in part by inflammatory cytokines such as tumor necrosis factor-α (TNF). Hepatocytes are normally resistant to the cytotoxic effects of TNF, but they become sensitized to TNF by chronic alcohol exposure. Recently, we reported that the decrease in the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) that occurs with alcoholic liver injury renders hepatocytes sensitive to TNF cytotoxicity. The purpose of this study was to determine whether inhibition of the transcription factor nuclear factor-kappaB (NF-κB) contributed to TNF-induced cell death in hepatocytes with high levels of SAH. Methods Primary human hepatocytes or HepG2 cells were pre-incubated with a combination of adenosine plus homocysteine to increase SAH levels. Following exposure to TNF, viability was determined by the MTT assay, and activation of the NF-κB pathway was assessed by measuring degradation of cytosolic IκB-α, phosphorylation and translocation of NF-κB to the nucleus, and expression of NF-κB-dependent genes. TNF-induced apoptotic signaling pathways were assessed by monitoring levels of the anti-apoptotic protein, A20, and cleavage products of the caspase-8 substrate, RIP1. Results NF-κB-mediated gene expression was inhibited in cells with high SAH, despite the fact that TNF-induced degradation of the cytoplasmic inhibitor IκB-α and accumulation of NF-κB in the nucleus persisted for much longer. In contrast to control cells, the NF-κB that accumulated in the nucleus of cells with high SAH levels was not phosphorylated at serine 536, a modification associated with activation of the transactivation potential of this transcription factor. The inhibition of transactivation by NF-κB resulted in lower mRNA and protein levels of the anti-apoptotic protein A20 and increased cleavage of RIP1. Conclusions High SAH levels inhibited NF-κB-mediated gene expression and sensitized primary hepatocytes and HepG2 cells to the cytotoxic effects of TNF. It is likely that crosstalk with other transcription factors is perturbed under these conditions, resulting in still other changes in gene expression.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [l-buthionine-S,R-sulfoximine (BSO) and auranofin] were combined with the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20 mM, 24 h), BSO (1 mM, 24 h), and auranofin (500 nM, 3 h) were combined with 17AAG, cell killing was significantly enhanced in three human cancer cell lines (PC-3, SUM159, MDA-MB-231). Furthermore, the toxicity of this drug combination was significantly greater in SUM159 human breast cancer cells, relative to HMEC normal human breast epithelial cells. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant NAC (15 mM, 24 h) was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH- and Trx-dependent metabolism is necessary to sensitize human breast and prostate cancer cells to 2DG+17AAG-mediated killing via enhancement of thiol-dependent oxidative stress. These results suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemosensitization in human cancer cells.

Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non–small cell lung cancer. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth, and results in increased sensitivity to chemotherapeutic drug–induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance. [Cancer Res 2008;68(19):7975–84]

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.

Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.

Thioredoxin 1 (Trx1) is a key redox control system within the nucleus, yet little is known about the sensitivity of nuclear Trx1 to oxidative stress. The present study compared oxidant-induced changes in the redox states of nuclear Trx1, cytoplasmic Trx1, and cellular glutathione (GSH). Nuclear Trx1 was more reducing than cytoplasmic Trx1 and cellular GSH in proliferating cells. tert-Butylhydroperoxide caused an increase in the total amount of nuclear Trx1, but this was accompanied by a 60 mV oxidation. Thus, the increase in nuclear Trx1 levels did not correspond to an increase in the overall reducing capacity of Trx1 in the nucleus.

Glutathione (GSH) and thioredoxin (Trx), the two major biological thiol-disulfide redox systems, provide overlapping but complementary functions with respect to detoxification and maintenance of the intracellular redox state. GSH, in conjunction with GSH reductase, GSH-S-transferase, glutaredoxin, and NADPH, provides protection against a wide variety of oxidants and electrophiles. Likewise, Trx provides protection against oxidative stress through its interactions with Trx reductase, peroxiredoxins, and NADPH. GSH is present in millimolar concentrations within cells, and thus has a high capacity for detoxification. Trx, on the other hand, is present in micromolar concentrations, and, therefore, has a lower capacity than GSH for detoxification and repair. However, because Trx contains two cysteines in its active site, Trx is more suited than GSH for 2-electron reduction of proteins, as occurs in the reduction of protein disulfides. Thus, the high capacity of GSH and the efficiency of Trx complement one another in maintenance of thiol-disulfide redox environments. During recent years, considerable evidence has accumulated to show that redox signaling mechanisms function in cell regulation and growth control. Agents altering GSH concentration affect transcription of detoxification enzymes, cell proliferation and apoptosis [1]. In principle, either GSH, GSH S-conjugates, GSSG or the redox state of the GSH/GSSG couple could provide a mechanistic control or signal for functional changes. Both GSH loss and GSH oxidation have been associated with increased expression of the rate-limiting enzyme of GSH synthesis, glutamate:cysteine ligase (GLCL), and several other detoxification systems, including glutathione S-transferase (GST) and NAD(P)H: quinone reductase (N:QR). A loss or oxidation of GSH also occurs in association with differentiation both in vitro and in vivo and during apoptosis. In contrast, increases in GSH and/or a reduction of the GSH/GSSG pool are associated with growth stimulation by nutrients and growth factors. Thus, the balance of GSH and GSSG may not only reflect oxidative stress but also may reflect changes in redox signaling and control. Less is known about how the redox state of thioredoxin is maintained and the influence of Trx redox on cellular functions. In vitro assays have clearly shown that the oxidized and reduced forms of Trx

Glutathione (GSH) has a central role in the maintenance of the thiol-disulfide redox state in mammalian cells. GSH synthesis can be physiologically limited by the availability of cysteine (Cys), and Cys and its precursors are variable in the human diet. The purpose of this study was to determine the effect of severe Cys deficiency and readdition of Cys on the redox state of the GSH/glutathione disulfide (GSSG) pool in human colon carcinoma HT29 cells. Cells were cultured in Cys- (and cystine-)limiting medium for 48 h followed by culture in medium containing either Cys or cystine for 24 h. GSH and GSSG were measured by HPLC. Cys limitation decreased cellular GSH and GSSG concentrations with an associated >80 mV oxidation of the GSH/GSSG redox state. Upon addition of either Cys or its disulfide cystine (CySS), redox of GSH/GSSG recovered in 4 h, whereas GSH concentration continued to increase over 12 h. Maximal GSH concentrations attained were 200% of control cell values. These results show that severe Cys deficiency can have marked effects on cellular redox state but that redox recovers rapidly upon resupply. The magnitude of oxidation during Cys limitation in this cell model is sufficient to result in a >100-fold change in the reduced/oxidized ratio of redox-sensitive dithiol/disulfide motifs in proteins. If redox changes occur in vivo in association with variations in dietary Cys and its precursors, these changes could have important physiologic effects through altered redox signaling and control of cell proliferation and apoptosis.

Hepatic deficiency of S-adenosylmethionine (AdoMet) is a critical acquired metabolic abnormality in alcoholic liver disease (ALD) and in many experimental models of hepatotoxicity. Subnormal AdoMet, elevated serum tumor necrosis factor (TNF), and endotoxemia (LPS) are hallmarks of ALD and experimental liver injury. AdoMet deficiency is attributed to its subnormal synthesis, but mechanisms for increased TNF are not known. AdoMet deficiency may affect the critical balance of proinflammatory (e.g., TNF) and antiinflammatory [e.g., interleukin (IL)-10] cytokines. Rats maintained on a choline-deficient diet with limited amounts of methionine (MCD diet) developed AdoMet deficiency. When challenged with LPS, rats fed MCD diet had significantly increased serum TNF levels and worse liver injury compared with findings for controls. Exogenous AdoMet attenuated liver injury and serum TNF levels. Results of in vitro studies with the use of RAW 264.7 cells demonstrated that exogenous AdoMet supplementation lowered LPS-induced TNF formation in a dose-dependent manner, and AdoMet deficiency enhanced TNF secretion and TNF gene expression. AdoMet also dose-dependently decreased LPS-stimulated TNF production from monocytes obtained from patients with alcoholic hepatitis. Finally, AdoMet supplementation stimulated production of the antiinflammatory cytokine IL-10. Interleukin-10 plays a critical role in the modulation of TNF production, and IL-10 may inhibit hepatic fibrosis. This article will review (1) the role of AdoMet in ALD/liver injury, (2) the role of TNF/proinflammatory cytokines in ALD, (3) potential roles of AdoMet in TNF/proinflammatory cytokine regulation in ALD, and (4) conclusions and future directions.

The redox-sensitive transcription factor NFκB mediates the expression of genes involved in inflammation and cell survival. Thioredoxin reductase-1 (TR1) and its substrate thioredoxin-1 (Trx1) act together to reduce oxidized cysteine residues within the DNA binding domain of NFκB and promote maximal DNA binding activity in vitro. It is not clear, however, if NFκB is regulated via this mechanism within living cells. The purpose of the present study was to determine the mechanism of NFκB modulation by TR1 in cells stimulated with the inflammatory cytokine tumor necrosis factor-α (TNF). In both control cells and in cells depleted of TR1 activity through chemical inhibition or siRNA knock down, TNF stimulation resulted in degradation of the cytoplasmic NFκB inhibitor IκB-α and translocation of NFκB to the nucleus. Similarly, the DNA binding activity and redox state of NFκB were unaffected by TR1 depletion. In contrast, NFκB-mediated gene expression was markedly inhibited in cells lacking TR1 activity, suggesting that the transactivation potential of NFκB is sensitive to changes in TR1 activity. Consistent with this concept, phosphorylation of the transactivation domain of NFκB was inhibited in the presence of curcumin. Surprisingly, another TR1 inhibitor, 1-chloro-2,4-dinitrobenzene, had no effect, and siRNA knock down of TR1 actually increased phosphorylation at this site. These results demonstrate that TR1 activity controls the transactivation potential of NFκB, and that more than one mechanism may mediate this effect.

Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes the reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5'-promoter-flanking region of Srx1 identified multiple antioxidant-response elements (AREs) that are highly conserved. Reporter and chromatin-immunoprecipitation assays demonstrated that ARE1 at -228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H2O2), whereas overexpression of Srx1 protected against H2O2-mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to nonemphysematous lungs together with a decline in Nrf2 protein. Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung.

Reactive oxygen species (ROS) were once viewed only as mediators of toxicity, but it is now recognized that they also contribute to redox signaling through oxidation of specific cysteine thiols on regulatory proteins. Cells in sparse cultures have increased ROS relative to confluent cultures, but it is not known whether protein redox states are affected under these conditions. The purpose of the present study was to determine whether culture conditions affect the redox state of thioredoxin-1 (Trx1), the protein responsible for reducing most oxidized proteins in the cytoplasm and nucleus. The results showed that Trx1 was more oxidized in sparse HeLa cell cultures than in confluent cells. The glutathione pool was also more oxidized, demonstrating that both of the major cellular redox regulating systems were affected by culture density. In addition, the total amount of Trx1 protein was lower and the subcellular distribution of Trx1 was different in sparse cells. Trx1 in sparse cultures was predominantly nuclear whereas it was predominantly cytoplasmic in confluent cultures. This localization pattern was not unique to HeLa cells as it was also observed in A549, Cos-1 and HEK293 cells. These findings demonstrate that Trx1 is subject to changes in expression, redox state and subcellular localization with changing culture density, indicating that the redox environments of the cytoplasm and the nucleus are distinct and have different requirements under different culture conditions.

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.

Nrf-2 is a redox-sensitive transcription factor that is activated by an oxidative signal in the cytoplasm but has a critical cysteine that must be reduced to bind to DNA in the nucleus. The glutathione (GSH) and thioredoxin (TRX) systems have overlapping functions in thiol/disulfide redox control in both the cytoplasm and the nucleus, and it is unclear whether these are redundant or have unique functions in control of Nrf-2-dependent signaling. To test whether GSH and Trx-1 have distinct functions in Nrf-2 signaling, we selectively modified GSH by metabolic manipulation and selectively modified Trx-1 expression by transient transfection. Cytoplasmic activation of Nrf-2 was measured by its nuclear translocation and nuclear activity of Nrf-2 was measured by expression of a luciferase reporter construct containing an ARE4 from glutamate cysteine ligase. Results showed that tert-butylhydroquinone (TBHQ), a transcriptional activator that functions through Nrf-2/ARE, promoted Nrf-2 nuclear translocation by a type I (thiylation) redox switch which was regulated by GSH not by Trx-1. In contrast, the ARE reporter was principally controlled by nuclear-targeted Trx-1 and not by GSH. The data show that the GSH and TRX systems have unique, compartmented functions in the control of transcriptional regulation by Nrf-2/ARE.

Thioredoxins (Trx) are members of an evolutionarily conserved family of redox-active proteins containing a conserved active site dithiol motif. Trx supports diverse reduction reactions, including several of direct toxicologic interest, but relatively little information is available concerning the roles of Trx under specific toxicologic conditions. Accumulating evidence suggests that Trx serves a partially overlapping and highly complementary role to the glutathione (GSH) system in protecting against toxicity. GSH and Trx both function in the reduction of peroxides through the action of multiple GSH peroxidases and Trx peroxidases (peroxiredoxins), respectively. However, GSH is a small molecule that is present at millimolar concentrations, thereby providing a potential mechanism for elimination of alkylating electrophiles. In contrast, even though Trx is only present at micromolar or submicromolar concentrations, its dithiol motif makes it suited to reverse oxidative changes to proteins, including reduction of protein disulfides, methioninyl sulfoxides, and cysteinyl sulfenic acids. Moreover, Trx functions in redox-sensitive signal transduction, transcriptional activation of stress response genes, ribonucleotide reduction in synthesis of deoxyribonucleotides for DNA repair, and post-injury cell proliferation. Molecular studies show that the predominant cytoplasmic/nuclear form, Trx-1, and the mitochondrial form, Trx-2, both protect against oxidative stress, that both are essential for embryonic development, and that Trx-1 is inducible in response to oxidative stress. Because of the differences between GSH and Trx in distribution, catalytic activities and reactivities with electrophiles, particularly with the important role to be played by glutathione S-transferases, considerable research is needed to clarify their respective roles in protection against specific toxicologic conditions.

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.

Cellular redox, maintained by the glutathione (GSH)- and thioredoxin (Trx)-dependent systems, has been implicated in the regulation of a variety of biological processes. The redox state of the GSH system becomes oxidized when cells are induced to differentiate by chemical agents. The aim of this study was to determine the redox state of cellular GSH/glutathione disulfide (GSH/GSSG) and Trx as a consequence of progression from proliferation to contact inhibition and spontaneous differentiation in colon carcinoma (Caco-2) cells. Results showed a significant decrease in GSH concentration, accompanied by a 40-mV oxidation of the cellular GSH/GSSG redox state and a 28-mV oxidation of the extracellular cysteine/cystine redox state in association with confluency and increase in differentiation markers. The redox state of Trx did not change. Thus the two central cellular antioxidant and redox-regulating systems (GSH and Trx) were independently controlled. According to the Nernst equation, a 30-mV oxidation is associated with a 10-fold change in the reduced/oxidized ratio of a redox-sensitive dithiol motif. Therefore, the measured 40-mV oxidation of the cellular GSH/GSSG couple or the 28-mV oxidation of the extracellular cysteine/cystine couple should be sufficient to function in signaling or regulation of differentiation in Caco-2 cells.