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Supplementary Table 1 from RNAi-Mediated Silencing of Nuclear Factor Erythroid-2–Related Factor 2 Gene Expression in Non–Small Cell Lung Cancer Inhibits Tumor Growth and Increases Efficacy of Chemotherapy

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and β-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Chronic heavy alcohol consumption is a major risk factor for the development of liver steatosis, fibrosis, and cirrhosis, but the mechanisms by which alcohol causes liver damage remain incompletely elucidated. This group has reported that α4 nicotinic acetylcholine receptors (α4 nAChRs) act as sensors for alcohol in lung cells. This study tested the hypothesis that α4 nAChRs mediate the effects of alcohol in the liver.Expression of acetylcholine receptor subunits in mouse liver was determined by RNA sequencing (RNA-seq). α4 nAChR knockout (α4 KO) mice were generated in C57BL/6J mice by introducing a mutation encoding an early stop codon in exon 4 of Chrna4, the gene encoding the α4 subunit of the nAChR. The presence of the inactivating mutation was established by polymerase chain reaction and genomic sequencing, and the lack of α4 nAChR function was confirmed in primary fibroblasts isolated from the α4 KO mice. Wild-type (WT) and α4 KO mice were fed the Lieber-DeCarli diet (with 36% of calories from alcohol) or pair fed an isocaloric maltose-dextrin control diet for a 6-week period that included a ramping up phase of increasing dietary alcohol.Chrna4 was the most abundantly expressed nAChR subunit gene in mouse livers. After 6 weeks of alcohol exposure, WT mice had elevated serum transaminases and their livers showed increased fat accumulation, decreased Sirt1 protein levels, and accumulation of markers of oxidative stress and inflammation including Cyp2E1, Nos2, Sod1, Slc7a11, TNFα, and PAI1. All these responses to alcohol were either absent or significantly attenuated in α4 KO animals.Together, these observations support the conclusion that activation of α4 nAChRs by alcohol or one of its metabolites is one of the initial events promoting the accumulation of excess fat and expression of inflammatory mediators. Thus, α4 nAChRs may represent viable targets for intervention in chronic alcohol-related liver disease.

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.

Vinyl chloride is an organochlorine mainly used to manufacture its polymer polyvinyl chloride, which is extensively used in the manufacturing of consumer products. Recent studies suggest that chronic low dose vinyl chloride exposure affects glucose homeostasis in high fat diet-fed mice. Our data suggest that even in the absence of high fat diet, exposure to vinyl chloride (0.8 ppm, 6h/day, 5day/week, for 12 weeks) induces glucose intolerance (1.0 g/kg, i.p) in male C57BL/6 mice. This was accompanied with the depletion of hepatic glutathione and a modest increase in lung interstitial macrophages. Vinyl chloride exposure did not affect the levels of circulating immune cells, endothelial progenitor cells, platelet-immune cell aggregates, and cytokines and chemokines. The acute challenge of vinyl chloride-exposed mice with LPS did not affect lung immune cell composition or plasma IL-6. To examine the effect of vinyl chloride exposure on vascular inflammation and atherosclerosis, LDL receptor-KO mice on C57BL/6 background maintained on western diet were exposed to vinyl chloride for 12 weeks (0.8 ppm, 6h/day, 5day/week). Unlike the WT C57BL/6 mice, vinyl chloride exposure did not affect glucose tolerance in the LDL receptor-KO mice. Plasma cytokines, lesion area in the aortic valve, and markers of lesional inflammation in vinyl chloride-exposed LDL receptor-KO mice were comparable with the air-exposed controls. Collectively, despite impaired glucose tolerance and modest pulmonary inflammation, chronic low dose vinyl chloride exposure does not affect surrogate markers of cardiovascular injury, LPS-induced acute inflammation in C57BL/6 mice, and chronic inflammation and atherosclerosis in the LDL receptor-KO mice.

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

Graphical abstract, Highlights • Whole life exposure to low doses of cadmium results in a significant accumulation of metals in the testis. • Co-exposure to cadmium with a high fat diet increases cadmium levels. • Cadmium and high fat diet disrupt essential metal homeostasis in the testis., The effects of exposure to the environmental toxicant cadmium, in combination with obesity, on the metal content in mouse testis were evaluated. Starting in utero and continuing through to 10 or 24 weeks post-weaning, male mice were exposed to cadmium (0, 0.5 or 5 ppm), and fed either a low (LFD) or high fat diet (HFD) post-weaning. Testicular levels of cadmium and essential metals were determined 10 and 24 weeks post-weaning by ICP-MS. Similar to what has been previously observed in the liver, kidney, heart and brain, significant levels of cadmium accumulated in the testis under all exposure conditions. Additionally, HFD-fed animals accumulated more cadmium than did their LFD-treated counterparts. Both treatments affected essential metal homeostasis in the testis. These findings suggest that cadmium and obesity may compromise the reproductive potential in the male mouse by disrupting essential metal levels.

Slc7a11 is the key component of system Xc-, an antiporter that imports cystine (CySS) and exports glutamate. It plays an important role in cellular defense against oxidative stress because cysteine (Cys), reduced from CySS, is used for and limits the synthesis of glutathione (GSH). We have shown that downregulation of Slc7a11 is responsible for oxidation of extracellular Cys/CySS redox potential in lung fibroblasts from old mice. However, how age-related change of Slc7a11 expression affects the intracellular redox environment of mouse lung fibroblasts remains unexplored. The purpose of this study is to evaluate the effects of aging on the redox states of intracellular proteins and to examine whether Slc7a11 contributes to the age-dependent effects. Iodoacetyl Tandem Mass Tags were used to differentially label reduced and oxidized forms of Cys residues in primary lung fibroblasts from young and old mice, as well as old fibroblasts transfected with Slc7a11. The ratio of oxidized/reduced forms (i.e., redox state) of a Cys residue was determined via multiplexed tandem mass spectrometry. Redox states of 151 proteins were different in old fibroblasts compared to young fibroblasts. Slc7a11 overexpression restored redox states of 104 (69%) of these proteins. Ingenuity Pathway Analysis (IPA) showed that age-dependent Slc7a11-responsive proteins were involved in pathways of protein translation initiation, ubiquitin-proteasome-mediated degradation, and integrin-cytoskeleton-associated signaling. Gene ontology analysis showed cell adhesion, protein translation, and organization of actin cytoskeleton were among the top enriched terms for biological process. Protein-protein interaction network demonstrated the interactions between components of the three enriched pathways predicted by IPA. Follow-up experiments confirmed that proteasome activity was lower in old cells than in young cells and that upregulation of Slc7a11 expression by sulforaphane restored this activity. This study finds that aging results in changes of redox states of proteins involved in protein turnover and cytoskeleton dynamics, and that upregulating Slc7a11 can partially restore the redox states of these proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain a more oxidizing extracellular redox potential (E(h)(Cys/CySS)) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular E(h)(Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and E(h)(Cys/CySS) was calculated from the Nernst equation. The results showed that both E(h)(Cys/CySS) and E(h)(GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular E(h)(Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing E(h)(Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore E(h)(GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study reveals Slc7a11 is the key regulator of age-dependent changes in extracellular E(h)(Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis.

Exposure to the environmental toxicant cadmium (Cd) contributes to the development of obesity-associated diseases. Obesity is a risk factor for a spectrum of unhealthy conditions including systemic metabolic dyshomeostasis. In the present study, the effects of whole-life exposure to environmentally-relevant concentrations of Cd on systemic essential metal distribution in adult mice fed a high-fat diet (HFD) were examined. For these studies, male and female mice were exposed to Cd-containing drinking water for >2 weeks before breeding. Pregnant mice and dams with offspring were exposed to Cd-containing drinking water. After weaning, offspring were continuously exposed to the same Cd concentration as their parents, and divided into HFD and normal (low) fat diet (LFD) groups. At 10 and 24 weeks, mice were sacrificed and blood, liver, kidney and heart harvested for metal analyses. There were significant concentration dependent increases in Cd levels in offspring with kidney > liver > heart. Sex significantly affected Cd levels in kidney and liver, with female animals accumulating more metal than males. Mice fed the HFD showed > 2-fold increase in Cd levels in the three organs compared to similarly treated LFD mice. Cadmium significantly affected essential metals levels in blood, kidney and liver. Additionally, HFD affected essential metal levels in these three organs. These findings suggest that Cd interacts with HFD to affect essential metal homeostasis, a phenomenon that may contribute to the underlying mechanism responsible for the development of obesity-associated pathologies.

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.

Arsenic is a widely-distributed environmental component that is associated with a variety of cancer and non-cancer adverse health effects. Additional lifestyle factors, such as diet, contribute to the manifestation of disease. Recently, arsenic was found to increase inflammation and liver injury in a dietary model of fatty liver disease. The purpose of the present study was to investigate potential mechanisms of this diet-environment interaction via a high throughput metabolomics approach. GC×GC-TOF MS was used to identify metabolites that were significantly increased or decreased in the livers of mice fed a Western diet (a diet high in fat and cholesterol) and co-exposed to arsenic-contaminated drinking water. The results showed that there are distinct hepatic metabolomic profiles associated with eating a high fat diet, drinking arsenic-contaminated water, and the combination of the two. Among the metabolites that were decreased when arsenic exposure was combined with a high fat diet were short-chain and medium-chain fatty acid metabolites and the anti-inflammatory amino acid, glycine. These results are consistent with the observed increase in inflammation and cell death in the livers of these mice, and they point to potentially novel mechanisms by which these metabolic pathways could be altered by arsenic in the context of diet-induced fatty liver disease.

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.

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed asEhCySS. Cultured cells condition their media to reproduce physiologicalEhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the mediaEhCySSbefore and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellularEhCySSthan young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

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.

Arsenic is a ubiquitous contaminant in drinking water. Whereas arsenic can be directly hepatotoxic, the concentrations/doses required are generally higher than present in the US water supply. However, physiological/biochemical changes that are alone pathologically inert can enhance the hepatotoxic response to a subsequent stimulus. Such a '2-hit' paradigm is best exemplified in chronic fatty liver diseases. Here, the hypothesis that low arsenic exposure sensitizes liver to hepatotoxicity in a mouse model of non-alcoholic fatty liver disease was tested. Accordingly, male C57Bl/6J mice were exposed to low fat diet (LFD; 13% calories as fat) or high fat diet (HFD; 42% calories as fat) and tap water or arsenic (4.9 ppm as sodium arsenite) for ten weeks. Biochemical and histologic indices of liver damage were determined. High fat diet (± arsenic) significantly increased body weight gain in mice compared with low-fat controls. HFD significantly increased liver to body weight ratios; this variable was unaffected by arsenic exposure. HFD caused steatohepatitis, as indicated by histological assessment and by increases in plasma ALT and AST. Although arsenic exposure had no effect on indices of liver damage in LFD-fed animals, it significantly increased the liver damage caused by HFD. This effect of arsenic correlated with enhanced inflammation and fibrin extracellular matrix (ECM) deposition. These data indicate that subhepatotoxic arsenic exposure enhances the toxicity of HFD. These results also suggest that arsenic exposure might be a risk factor for the development of fatty liver disease in human populations.

Homeostasis of selenium (Se), a critical antioxidant incorporated into amino acids and enzymes, is disrupted by exposure to aryl hydrocarbon receptor (AhR) agonists. Here we examined the importance of dietary Se in preventing the toxicity of the most toxic polychlorinated biphenyl congener, 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), a potent AhR agonist. Male Sprague-Dawley rats were fed a modified AIN-93 diet with differing dietary Se levels (0.02, 0.2, and 2 ppm). Following 3 weeks of acclimatization, rats from each dietary group were given a single ip injection of corn oil (vehicle), 0.2, 1, or 5 μmol/kg body weight PCB 126, followed 2 weeks later by euthanasia. PCB exposure caused dose-dependent increases in liver weight and at the highest PCB 126 dose decreases in whole body weight gains. Hepatic cytochrome P-450 (CYP1A1) activity was significantly increased even at the lowest dose of PCB 126, indicating potent AhR activation. PCB exposure diminished hepatic Se levels in a dose-dependent manner, and this was accompanied by diminished Se-dependent glutathione peroxidase activity. Both these effects were partially mitigated by Se supplementation. Conversely, thioredoxin (Trx) reductase activity and Trx oxidation state, although significantly diminished in the lowest dietary Se groups, were not affected by PCB exposure. In addition, PCB 126–induced changes in hepatic copper, iron, manganese, and zinc were observed. These results demonstrate that supplemental dietary Se was not able to completely prevent the toxicity caused by PCB 126 but was able to increase moderately the levels of several key antioxidants, thereby maintaining them roughly at normal levels.

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.

Environmental arsenic exposure is a world-wide human health problem. Arsenic is associated with a wide range of diseases, including skin lesions, cardiovascular disease, liver disease, and cancers of the skin, lung, liver, and bladder. Metabolism of arsenic yields a variety of molecular species, each of which can have different biological effects. This review will discuss the reactivity of different chemical forms of arsenic with distinct molecular targets of toxicological relevance. A particular emphasis will be given to studies investigating the roles of arsenic–sulfur interactions in the etiology of disease.

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.

Intracellular deficiency of S-adenosylmethionine (AdoMet) and elevated serum concentrations of tumour necrosis factor α (TNF) are hallmarks of toxin-induced liver injury. In these models, the administration of either exogenous AdoMet or antibody/soluble receptor for TNF attenuates the injury. We have demonstrated previously that the administration of exogenous AdoMet to AdoMet-deficient rats attenuated lipopolysaccharide (LPS)-induced liver injury and serum TNF concentrations. Here we report that AdoMet lowered the amount of TNF secreted by LPS-stimulated murine macrophage cells (RAW 264.7) in a dose-dependent manner. The inhibition of TNF release was correlated with changes in the steady-state TNF mRNA concentrations. Changes in TNF mRNA were not due to its altered stability and might have been due to an attenuation of the transcription rate of the TNF gene. The inhibition of TNF release in RAW cells was not mediated by GSH because treatment with AdoMet did not increase intracellular GSH. In addition, N-acetylcysteine, whereas it did increase GSH concentration, had no effect on LPS-stimulated TNF release in these cells. Exogenous AdoMet also attenuated LPS-induced serum TNF levels in normal rats sensitized with lead. Thus AdoMet administration might exert its hepatoprotective effects at least in part by its inhibitory effect on expression of the gene for TNF.

Motivation: Due to the high complexity of metabolome, the comprehensive 2D gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) is considered as a powerful analytical platform for metabolomics study. However, the applications of GC×GC-TOF MS in metabolomics are not popular owing to the lack of bioinformatics system for data analysis. Results: We developed a computational platform entitled metabolomics profiling pipeline (MetPP) for analysis of metabolomics data acquired on a GC×GC-TOF MS system. MetPP can process peak filtering and merging, retention index matching, peak list alignment, normalization, statistical significance tests and pattern recognition, using the peak lists deconvoluted from the instrument data as its input. The performance of MetPP software was tested with two sets of experimental data acquired in a spike-in experiment and a biomarker discovery experiment, respectively. MetPP not only correctly aligned the spiked-in metabolite standards from the experimental data, but also correctly recognized their concentration difference between sample groups. For analysis of the biomarker discovery data, 15 metabolites were recognized with significant concentration difference between the sample groups and these results agree with the literature results of histological analysis, demonstrating the effectiveness of applying MetPP software for disease biomarker discovery. Availability: The source code of MetPP is available at http://metaopen.sourceforge.net Contact: xiang.zhang@louisville.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Young rats were maintained in a 10% oxygen atmosphere for 2, 6, and 10 days and administered normal rat chow and water ad libitum. Thereafter, their hepatic S-adenosyl-L-methionine (AdoMet) and activity and mRNA levels of AdoMet synthetase were assayed. AdoMet levels decreased by 45% after 10 days; hepatic AdoMet synthetase also declined by approximately 40%. In rats with low hepatic AdoMet, the mRNA level of AdoMet synthetase also declined by up to 80%. No significant change in AdoMet or AdoMet synthetase was noted in pair-fed normoxic rats. DNA hypomethylation was determined in terms of incorporation of [3H]methyl of AdoMet incorporated at unmethylated sites in DNA in reactions mediated by methylases HpaII and SssI. As compared to the normal hepatic DNA, [3H]methyl group incorporation in the 10-day hypoxic DNA was almost double in the HpaII-mediated reaction and approximately 10-fold in the SssI-mediated reaction. Hepatic tRNA methyltransferase activity doubled after 10 days of hypoxia. However, hypoxic rats showed no detectable mRNA transcripts for c-myc and c-fos oncogenes on Northern blot analysis. These observations show that because of subnormal activity of AdoMet synthetase, hypoxic liver is depleted of AdoMet, even when the animals are administered a complete diet. However, unlike rats on chronic lipotrope-deficient diets, hypoxic rats on a complete diet show no aberrant expression of oncogenes.

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle’s functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the β-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and β-stimulated function in db/db myocyte/heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient’s inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction.

International audience; Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high performance liquid chromatography. The increase in the NADH/NAD ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-methylpyrazole, an inhibitor the NAD-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD redox couple and the effects of ethanol on methionine metabolism in the liver.

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.

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.

The effect of dietary alcohol and protein intake on hepatic levels of S-adenosyl-L-methionine (SAMe), S-adenosylhomocysteine, SAMe synthetase, and phosphatidylethanolamine-N-methyltransferase (PEMT) was investigated in rats receiving the following diets for 28 days: 20% protein (group I); 20% protein plus 5% ethanol (group II); 8% protein (group III); and 8% protein plus 5% ethanol (group IV). The daily food intake of rats did not differ significantly between groups, but the gain in bodyweight in groups III and IV was significantly less than in group I — the control group. The mean hepatic SAMe level in rats in group IV was significantly lower (46% below normal; p < 0.02) than in the control group, although SAMe synthetase activity was essentially unchanged in all groups. PEMT activity was less than the mean for the control group in 5 of 8 group IV animals. These data indicate that the hepatic content of SAMe is sensitive to even a low intake of ethanol, with potential adverse effects on vital methyltransferase reactions utilising SAMe as a methyl donor.

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.