Obesogenic polystyrene microplastic exposures disrupt the gut-liver-adipose axis.

Microplastics (MP) derived from the weathering of polymers, or synthesized in this size range, have become widespread environmental contaminants and have found their way into water supplies and the food chain. Despite this awareness, little is known about the health consequences of MP ingestion. We have previously shown that the consumption of polystyrene (PS) beads was associated with intestinal dysbiosis and diabetes and obesity in mice. To further evaluate the systemic metabolic effects of PS on the gut-liver-adipose tissue axis, we supplied C57BL/6J mice with normal water or that containing 2 sizes of PS beads (0.5 and 5 µm) at a concentration of 1 µg/ml. After 13 weeks, we evaluated indices of metabolism and liver function. As observed previously, mice drinking the PS-containing water had a potentiated weight gain and adipose expansion. Here we found that this was associated with an increased abundance of adipose F4/80+ macrophages. These exposures did not cause nonalcoholic fatty liver disease but were associated with decreased liver:body weight ratios and an enrichment in hepatic farnesoid X receptor and liver X receptor signaling. PS also increased hepatic cholesterol and altered both hepatic and cecal bile acids. Mice consuming PS beads and treated with the berry anthocyanin, delphinidin, demonstrated an attenuated weight gain compared with those mice receiving a control intervention and also exhibited a downregulation of cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor (PPAR) signaling pathways. This study highlights the obesogenic role of PS in perturbing the gut-liver-adipose axis and altering nuclear receptor signaling and intermediary metabolism. Dietary interventions may limit the adverse metabolic effects of PS consumption.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

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 ß-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.

Na/H Exchange Regulatory Factor 1 Deficient Mice Show Evidence of Oxidative Stress and Altered Cisplatin Pharmacokinetics.

(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.

Effects of cadmium and high-fat diet on essential metal concentration in the mouse 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.

Impact of sex, age and diet on the cysteine/cystine and glutathione/glutathione disulfide plasma redox couples in mice.

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.

Redox States of Protein Cysteines in Pathways of Protein Turnover and Cytoskeleton Dynamics Are Changed with Aging and Reversed by Slc7a11 Restoration in Mouse Lung Fibroblasts.

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.

Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing.

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.

Publisher Correction: Cadmium and High-Fat Diet Disrupt Renal, Cardiac and Hepatic Essential Metals.

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

Cadmium and High-Fat Diet Disrupt Renal, Cardiac and Hepatic Essential Metals.

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.

Plasma cysteine/cystine and glutathione/glutathione disulfide redox potentials in HIV and COPD patients.

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-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression.

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 (Eh(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 Eh(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 Eh(Cys/CySS) was calculated from the Nernst equation. The results showed that both Eh(Cys/CySS) and Eh(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 Eh(Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing Eh(Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore Eh(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 Eh(Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis.

Development, Prevention, and Treatment of Alcohol-Induced Organ Injury: The Role of Nutrition.

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.

Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice.

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as EhCySS. Cultured cells condition their media to reproduce physiological EhCySS, 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 media EhCySS before 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 extracellular EhCySS than 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.

Lung extracellular matrix and redox regulation.

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.

Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer.

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20mM, 24-48h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300µM, 24-48h) 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, 1mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1µM), was combined with 2DG+DHEA or DHEA-alone for 24h, 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, 20mM). 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.

Metabolomic analysis of the effects of chronic arsenic exposure in a mouse model of diet-induced Fatty liver disease.

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 point to potentially novel mechanisms by which these metabolic pathways could be altered by arsenic in the context of diet-induced fatty liver disease.

S-adenosylhomocysteine inhibits NF-κB-mediated gene expression in hepatocytes and confers sensitivity to TNF cytotoxicity.

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.

MetPP: a computational platform for comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry-based metabolomics.

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.

GSH or palmitate preserves mitochondrial energetic/redox balance, preventing mechanical dysfunction in metabolically challenged myocytes/hearts from type 2 diabetic mice.

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 ß-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 ß-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.

Simultaneous inhibition of glutathione- and thioredoxin-dependent metabolism is necessary to potentiate 17AAG-induced cancer cell killing via oxidative stress.

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 (20mM, 24h), BSO (1mM, 24h), and auranofin (500nM, 3h) 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 (15mM, 24h) 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.