Protein S-palmitoylation enhances profibrotic signaling in response to cadmium.

Cadmium (Cd) is a naturally occurring, toxic environmental metal found in foods. Humans do not have an efficient mechanism for Cd elimination; thus, Cd burden in humans increases with age. Cell and mouse studies show that Cd burden from low environmental levels of exposure impacts lung cell metabolism, proliferation signaling and cell growth as part of disease-promoting profibrotic responses in the lungs. Prior integrative analysis of metabolomics and transcriptomics identified the zDHHC11 transcript as a central functional hub in response to Cd dose. zDHHC11 encodes a protein S-palmitoyltransferase, but no evidence is available for effects of Cd on protein S-palmitoylation. In the present research, we studied palmitoylation changes in response to Cd and found increased protein S-palmitoylation in human lung fibroblasts that was inhibited by 2-bromopalmitate (2-BP), an irreversible palmitoyltransferase inhibitor. Mass spectrometry-based proteomics showed palmitoylation of proteins involved in divalent metal transport and in fibrotic signaling. Mechanistic studies showed that 2-BP inhibited palmitoylation of divalent metal ion transporter ZIP14 and also inhibited cellular Cd uptake. Transcription analyses showed that Cd stimulated transforming growth factor (TGF)-ß1 and ß3 expression within 8 h and lung fibrotic markers α-smooth muscle actin, matrix metalloproteinase-2, and collagen 1α1 gene expression and that these effects were blocked by 2-BP. Because 2-BP also blocked palmitoylation of proteins controlled by TGFß1, these results show that palmitoylation impacts Cd-dependent fibrotic signaling both by enhancing cellular Cd accumulation and by supporting post-translational processing of TGFß1-dependent proteins.

Low-dose vanadium pentoxide perturbed lung metabolism associated with inflammation and fibrosis signaling in male animal and in vitro models.

Vanadium is available as a dietary supplement and also is known to be toxic if inhaled, yet little information is available concerning the effects of vanadium on mammalian metabolism when concentrations found in food and water. Vanadium pentoxide (V+5) is representative of the most common dietary and environmental exposures, and prior research shows that low-dose V+5 exposure causes oxidative stress measured by glutathione oxidation and protein S-glutathionylation. We examined the metabolic impact of V+5 at relevant dietary and environmental doses (0.01, 0.1, and 1 ppm for 24 h) in human lung fibroblasts (HLFs) and male C57BL/6J mice (0.02, 0.2, and 2 ppm in drinking water for 7 mo). Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that V+5 induced significant metabolic perturbations in both HLF cells and mouse lungs. We noted 30% of the significantly altered pathways in HLF cells, including pyrimidines and aminosugars, fatty acids, mitochondrial and redox pathways, showed similar dose-dependent patterns in mouse lung tissues. Alterations in lipid metabolism included leukotrienes and prostaglandins involved in inflammatory signaling, which have been associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other disease processes. Elevated hydroxyproline levels and excessive collagen deposition were also present in lungs from V+5-treated mice. Taken together, these results show that oxidative stress from environmental V+5, ingested at low levels, could alter metabolism to contribute to common human lung diseases.NEW & NOTEWORTHY We used relevant dietary and environmental doses of Vanadium pentoxide (V+5) to examine its metabolic impact in vitro and in vivo. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we found significant metabolic perturbations, with similar dose-dependent patterns observed in human lung fibroblasts and male mouse lungs. Alterations in lipid metabolism included inflammatory signaling, elevated hydroxyproline levels, and excessive collagen deposition were present in V+5-treated lungs. Our findings suggest that low levels of V+5 could trigger pulmonary fibrotic signaling.

Metabolomics of V2O5 nanoparticles and V2O5 nanofibers in human airway epithelial BEAS-2B cells.

Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V2O5) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V2O5 nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V2O5 nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V2O5 nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V2O5 nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V2O5 nanoparticles, and cholesterol and sialic acid metabolism associations with V2O5 nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V2O5 nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V2O5 nanomaterials.

MTOR-initiated metabolic switch and degeneration in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a particularly vulnerable tissue to age-dependent degeneration. Over the life span, the RPE develops an expanded endo-lysosomal compartment to maintain the high efficiency of phagocytosis and degradation of photoreceptor outer segments (POS) necessary for photoreceptor survival. As the assembly and activation of the mechanistic target of rapamycin complex 1 (mTORC1) occur on the lysosome surface, increased lysosome mass with aging leads to higher mTORC1 activity. The functional consequences of hyperactive mTORC1 in the RPE are unclear. In the current study, we used integrated high-resolution metabolomic and genomic approaches to examine mice with RPE-specific deletion of the tuberous sclerosis 1 (Tsc1) gene which encodes an upstream suppressor of mTORC1. Our data show that RPE cells with constitutively high mTORC1 activity were reprogramed to be hyperactive in glucose and lipid metabolism. Lipolysis was suppressed, mitochondrial carnitine shuttle was inhibited, while genes involved in fatty acid (FA) biosynthesis were upregulated. The metabolic changes occurred prior to structural changes of RPE and retinal degeneration. These findings have revealed cellular events and intrinsic mechanisms that contribute to lipid accumulation in the RPE cells during aging and age-related degeneration.

Environmental Cadmium Enhances Lung Injury by Respiratory Syncytial Virus Infection.

Cadmium (Cd) is a naturally occurring environmental toxicant that disrupts mitochondrial function at occupational exposure levels. The impacts of Cd exposure at low levels through dietary intake remain largely uncharacterized. Human respiratory syncytial virus (RSV) causes severe morbidity, which can require hospitalization and result in death in young children and elderly populations. The impacts of environmental Cd exposure on the severity of RSV disease are unknown. Herein, we used a mouse model to examine whether Cd pre-exposure at a level of dietary intake potentiates pulmonary inflammation on subsequent infection with RSV. Mice were given Cd or saline in drinking water for 28 days. Subsets of these mice were infected with RSV at 5 days before the end of the study. Cd pre-exposure caused relatively subtle changes in lung; however, it elevated the IL-4 level and altered metabolites associated with fatty acid metabolism. After RSV infection, mice pre-exposed to Cd had elevated lung RSV titer and increased inflammation, as measured by histopathology, immune cell infiltration, cytokines, and chemokines. RSV infection after Cd pre-exposure also caused widespread perturbation in metabolism of glycerophospholipids and amino acids (Trp, Met, and Cys, branched-chain amino acids), as well as carnitine shuttle associated with mitochondrial energy metabolism. The results show that Cd burden by dietary intake potentiates RSV infection and severe disease with associated mitochondrial metabolic disruption.

Antiatherogenic Effect of Resveratrol Attributed to Decreased Expression of ICAM-1 (Intercellular Adhesion Molecule-1).

Objective- Increasing evidence shows that resveratrol has antiatherogenic effects, but its underlying mechanisms are unknown. Thus, we evaluated the molecular mechanisms underlying the antiatherogenic effect of resveratrol. Approach and Results- Using the previously established mouse atherosclerosis model of partial ligation of the left carotid artery, we evaluated the role of resveratrol in antiatherosclerosis. We attempted to determine the mechanisms associated with focal adhesions using vascular endothelial cells. The results showed that resveratrol stimulated focal adhesion kinase cleavage via resveratrol-increased expression of lactoferrin in endothelial cells. Furthermore, we found that an N-terminal focal adhesion kinase fragment cleaved by resveratrol contained the FERM (band 4.1, ezrin, radixin, and moesin)-kinase domain. Furthermore, resveratrol inhibited lipopolysaccharide-stimulated adhesion of THP-1 human monocytes by decreased expression of ICAM-1 (intercellular adhesion molecule-1). A decreased ICAM-1 level was also observed in the left carotid artery of mice treated with resveratrol. To understand the relationship between resveratrol-induced antiinflammation and focal adhesion disruption, endothelial cells were transfected with FERM-kinase. Ectopically expressed FERM-kinase, the resveratrol-cleaved focal adhesion kinase fragment, was found in the nuclear fraction and inhibited the transcription level of icam-1 via the Nrf2 (nuclear factor erythroid 2-related factor 2)-antioxidant response element complex. Finally, ectopically expressed FERM-kinase blocked tumor necrosis factor-α- or IL- (interleukin) stimulated monocytic binding to endothelial cells. Conclusions- Our results show that resveratrol inhibits the expression of ICAM-1 via transcriptional regulation of the FERM-kinase and Nrf2 interaction, thereby blocking monocyte adhesion. These suppressive effects on the inflammatory mechanism suggest that resveratrol delayed the onset of atherosclerosis.

xMWAS: a data-driven integration and differential network analysis tool.

Summary: Integrative omics is a central component of most systems biology studies. Computational methods are required for extracting meaningful relationships across different omics layers. Various tools have been developed to facilitate integration of paired heterogenous omics data; however most existing tools allow integration of only two omics datasets. Furthermore, existing data integration tools do not incorporate additional steps of identifying sub-networks or communities of highly connected entities and evaluating the topology of the integrative network under different conditions. Here we present xMWAS, a software for data integration, network visualization, clustering, and differential network analysis of data from biochemical and phenotypic assays, and two or more omics platforms. Availability and implementation: https://kuppal.shinyapps.io/xmwas (Online) and https://github.com/kuppal2/xMWAS/ (R). Contact: kuppal2@emory.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Redox Systems Biology of Nutrition and Oxidative Stress.

Diet and nutrition contribute to both beneficial and harmful aspects of oxidative processes. The harmful processes, termed oxidative stress, occur with many human diseases. Major advances in understanding oxidative stress and nutrition have occurred with broad characterization of dietary oxidants and antioxidants, and with mechanistic studies showing antioxidant efficacy. However, randomized controlled trials in humans with free-radical-scavenging antioxidants and the glutathione precursor N-acetylcysteine have provided limited or inconsistent evidence for health benefits. This, combined with emerging redox theory, indicates that holistic models are needed to understand the interplay of nutrition and oxidative stress. The purpose of this article is to highlight how recent advances in redox theory and the development of new omics tools and data-driven approaches provide a framework for future nutrition and oxidative stress research. Here we describe why a holistic approach is needed to understand the impact of nutrition on oxidative stress and how recent advances in omics and data analysis methods are viable tools for systems nutrition approaches. Based on the extensive research on glutathione and related thiol antioxidant systems, we summarize the advancing framework for diet and oxidative stress in which antioxidant systems are a component of a larger redox network that serves as a responsive interface between the environment and an individual. The feasibility for redox network analysis has been established by experimental models in which dietary factors are systematically varied and oxidative stress markers are linked through integrated omics (metabolome, transcriptome, proteome). With this framework, integrated redox network models will support optimization of diet to protect against oxidative stress and disease.

Myeloperoxidase oxidation of methionine associates with early cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease progressively worsens from infancy to adulthood. Disease-driven changes in early CF airway fluid metabolites may identify therapeutic targets to curb progression.CF patients aged 12-38 months (n=24; three out of 24 later denoted as CF screen positive, inconclusive diagnosis) received chest computed tomography scans, scored by the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) method to quantify total lung disease (PRAGMA-%Dis) and components such as bronchiectasis (PRAGMA-%Bx). Small molecules in bronchoalveolar lavage fluid (BALF) were measured with high-resolution accurate-mass metabolomics. Myeloperoxidase (MPO) was quantified by ELISA and activity assays.Increased PRAGMA-%Dis was driven by bronchiectasis and correlated with airway neutrophils. PRAGMA-%Dis correlated with 104 metabolomic features (p<0.05, q<0.25). The most significant annotated feature was methionine sulfoxide (MetO), a product of methionine oxidation by MPO-derived oxidants. We confirmed the identity of MetO in BALF and used reference calibration to confirm correlation with PRAGMA-%Dis (Spearman's ρ=0.582, p=0.0029), extending to bronchiectasis (PRAGMA-%Bx; ρ=0.698, p=1.5×10-4), airway neutrophils (ρ=0.569, p=0.0046) and BALF MPO (ρ=0.803, p=3.9×10-6).BALF MetO associates with structural lung damage, airway neutrophils and MPO in early CF. Further studies are needed to establish whether methionine oxidation directly contributes to early CF lung disease and explore potential therapeutic targets indicated by these findings.

Selenium Supplementation Alters Hepatic Energy and Fatty Acid Metabolism in Mice.

Background: Human and animal studies have raised concerns that supplemental selenium can increase the risk of metabolic disorders, but underlying mechanisms are unclear. Objective: We used an integrated transcriptome and metabolome analysis of liver to test for functional pathway and network responses to supplemental selenium in mice. Methods: Male mice (8-wk-old, C57BL/6J) fed a standard diet (0.41 ppm Se) were given selenium (Na2SeO4, 20 µmol/L) or vehicle (drinking water) for 16 wk. Livers were analyzed for selenium concentration, activity of selenoproteins, reduced glutathione (GSH) redox state, gene expression, and high-resolution metabolomics. Transcriptomic and nontargeted metabolomic data were analyzed with biostatistics, bioinformatics, pathway enrichment analysis, and combined transcriptome-metabolome-wide association study (TMWAS). Results: Mice supplemented with selenium had greater body mass gain from baseline to 16 wk (55% ± 5%) compared with controls (40% ± 3%) (P < 0.05); however, no difference was observed in liver selenium content, selenoenzyme transcripts, or enzyme activity. Selenium was higher in the heart, kidney, and urine of mice supplemented with selenium. Gene enrichment analysis showed that supplemental selenium altered pathways of lipid and energy metabolism. Integrated transcriptome and metabolome network analysis showed 2 major gene-metabolite clusters, 1 centered on the transcript for the bidirectional glucose transporter 2 (Glut2) and the other centered on the transcripts for carnitine-palmitoyl transferase 2 (Cpt2) and acetyl-CoA acyltransferase (Acaa1). Pathway analysis showed that highly associated metabolites (P < 0.05) were enriched in fatty acid metabolism and bile acid biosynthesis, including acylcarnitines, triglycerides and glycerophospholipids, long-chain acyl-coenzyme As, phosphatidylcholines, and sterols. TMWAS of body weight gain confirmed changes in the same pathways. Conclusions: Supplemental selenium in mice alters hepatic fatty acid and energy metabolism and causes increases in body mass. A lack of effect on hepatic selenium content suggests that signaling involves an extrahepatic mechanism.

Hypoxia inhibits expression and function of mitochondrial thioredoxin 2 to promote pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 h. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 wk. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.

Redox dynamics of manganese as a mitochondrial life-death switch.

Sten Orrenius, M.D., Ph.D., pioneered many areas of cellular and molecular toxicology and made seminal contributions to our knowledge of oxidative stress and glutathione (GSH) metabolism, organellar functions and Ca+2-dependent mechanisms of cell death, and mechanisms of apoptosis. On the occasion of his 80th birthday, we summarize current knowledge on redox biology of manganese (Mn) and its role in mechanisms of cell death. Mn is found in all organisms and has critical roles in cell survival and death mechanisms by regulating Mn-containing enzymes such as manganese superoxide dismutase (SOD2) or affecting expression and activity of caspases. Occupational exposures to Mn cause "manganism", a Parkinson's disease-like condition of neurotoxicity, and experimental studies show that Mn exposure leads to accumulation of Mn in the brain, especially in mitochondria, and neuronal cell death occurs with features of an apoptotic mechanism. Interesting questions are why a ubiquitous metal that is essential for mitochondrial function would accumulate to excessive levels, cause increased H2O2 production and lead to cell death. Is this due to the interactions of Mn with other essential metals, such as iron, or with toxic metals, such as cadmium? Why is the Mn loading in the human brain so variable, and why is there such a narrow window between dietary adequacy and toxicity? Are non-neuronal tissues similarly vulnerable to insufficiency and excess, yet not characterized? We conclude that Mn is an important component of the redox interface between an organism and its environment and warrants detailed studies to understand the role of Mn as a mitochondrial life-death switch.

Redox theory of aging: implications for health and disease.

Genetics ultimately defines an individual, yet the phenotype of an adult is extensively determined by the sequence of lifelong exposures, termed the exposome. The redox theory of aging recognizes that animals evolved within an oxygen-rich environment, which created a critical redox interface between an organism and its environment. Advances in redox biology show that redox elements are present throughout metabolic and structural systems and operate as functional networks to support the genome in adaptation to environmental resources and challenges during lifespan. These principles emphasize that physical and functional phenotypes of an adult are determined by gene-environment interactions from early life onward. The principles highlight the critical nature of cumulative exposure memories in defining changes in resilience progressively during life. Both plasma glutathione and cysteine systems become oxidized with aging, and the recent finding that cystine to glutathione ratio in human plasma predicts death in coronary artery disease (CAD) patients suggests this could provide a way to measure resilience of redox networks in aging and disease. The emerging concepts of cumulative gene-environment interactions warrant focused efforts to elucidate central mechanisms by which exposure memory governs health and etiology, onset and progression of disease.

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice.

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1ß, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation.

Computational Metabolomics: A Framework for the Million Metabolome.

"Sola dosis facit venenum." These words of Paracelsus, "the dose makes the poison", can lead to a cavalier attitude concerning potential toxicities of the vast array of low abundance environmental chemicals to which humans are exposed. Exposome research teaches that 80-85% of human disease is linked to environmental exposures. The human exposome is estimated to include >400,000 environmental chemicals, most of which are uncharacterized with regard to human health. In fact, mass spectrometry measures >200,000 m/z features (ions) in microliter volumes derived from human samples; most are unidentified. This crystallizes a grand challenge for chemical research in toxicology: to develop reliable and affordable analytical methods to understand health impacts of the extensive human chemical experience. To this end, there appears to be no choice but to abandon the limitations of measuring one chemical at a time. The present review looks at progress in computational metabolomics to provide probability-based annotation linking ions to known chemicals and serve as a foundation for unambiguous designation of unidentified ions for toxicologic study. We review methods to characterize ions in terms of accurate mass m/z, chromatographic retention time, correlation of adduct, isotopic and fragment forms, association with metabolic pathways and measurement of collision-induced dissociation products, collision cross section, and chirality. Such information can support a largely unambiguous system for documenting unidentified ions in environmental surveillance and human biomonitoring. Assembly of this data would provide a resource to characterize and understand health risks of the array of low-abundance chemicals to which humans are exposed.

Nuclear Thioredoxin-1 Overexpression Attenuates Alcohol-Mediated Nrf2 Signaling and Lung Fibrosis.

BACKGROUND: Alcohol abuse, which impairs antioxidant defenses and promotes acute lung injury, increases Nrf2 nuclear translocation but nevertheless inhibits its activation of the antioxidant response element (ARE). Thioredoxin-1 (Trx1) is required for optimal Nrf2 binding and activation of the ARE, and we hypothesized that its inhibition contributes to impaired Nrf2-ARE signaling in the alcoholic lung. METHODS: Lung tissue and primary lung fibroblasts (PLFs) were isolated from C57/BL6 wild-type (WT) and transgenic mice overexpressing the human Trx1 gene with a nuclear localizing sequence (NLS-Tg); some mice consumed alcohol in water prior to lung tissue and PLF isolation; in some mice, acute lung injury was induced with intratracheal bleomycin. In other experiments, PLFs were isolated from WT and NLS-Tg mice and then exposed to alcohol. Finally, PLF isolated from WT mice were transfected with Trx1 expression vector containing either a cytosolic localized sequence (NES) or a nuclear localized sequence (NLS) prior to alcohol exposure. RESULTS: Alcohol treatment in vivo or in vitro decreased Trx1 expression, and bleomycin-treated alcohol-fed mice had fibrotic disrepair in their lungs. In parallel, whereas alcohol exposure in vitro increased TGFß1 expression and decreased Nrf2-ARE activity in PLF from WT mice, these effects were not observed in PLF from NLS-Tg mice. Finally, selective overexpression of Trx1 in the nucleus but not in the cytosol preserved Nrf2-ARE activity during alcohol exposure. CONCLUSIONS: Although alcohol-induced redox stress actually promotes Nrf2 nuclear translocation, the coincident suppression of Trx1 impairs Nrf2-ARE activity within the nuclear compartment. Nuclear overexpression of Trx1 restored Nrf2-ARE activity and attenuated alcohol-induced TGFß1 expression and alcohol-induced exaggerate response to bleomycin-induced acute lung injury.

Thiol/disulfide redox states in signaling and sensing.

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.

Disturbed flow induces systemic changes in metabolites in mouse plasma: a metabolomics study using ApoE⁻/⁻ mice with partial carotid ligation.

Disturbed blood flow (d-flow) occurring in branched and curved arteries promotes endothelial dysfunction and atherosclerosis, in part, by altering gene expression and epigenomic profiles in endothelial cells. While a systemic metabolic change is known to play a role in atherosclerosis, it is unclear whether it can be regulated by local d-flow. Here, we tested this hypothesis by carrying out a metabolomics study using blood plasma samples obtained from ApoE(-/-) mice that underwent a partial carotid ligation surgery to induce d-flow. Mice receiving sham ligation were used as a control. To study early metabolic changes, samples collected from 1 wk after partial ligation when endothelial dysfunction occurs, but before atheroma develops, were analyzed by high-resolution mass spectrometry. A metabolome-wide association study showed that 128 metabolites were significantly altered in the ligated mice compared with the sham group. Of these, sphingomyelin (SM; m/z 703.5747), a common mammalian cell membrane sphingolipid, was most significantly increased in the ligated mice. Of the 128 discriminatory metabolites, 18 and 41 were positively and negatively correlated with SM, respectively. The amino acids methionine and phenylalanine were increased by d-flow, while phosphatidylcholine and phosphatidylethanolamine were decreased by d-flow, and these metabolites were correlated with SM. Other significantly affected metabolites included dietary and environmental agents. Pathway analysis showed that the metabolic changes of d-flow impacted broad functional networks. These results suggest that signaling from d-flow occurring in focal regions induces systemic metabolic changes associated with atherosclerosis.

Metabolome-wide association study of phenylalanine in plasma of common marmosets.

Little systematic knowledge exists concerning the impacts of cumulative lifelong exposure, termed the exposome, on requirements for nutrients. Phenylalanine (Phe) is an essential dietary amino acid with an aromatic ring structure similar to endogenous metabolites, dietary compounds and environmental agents. Excess plasma Phe in genetic disease or nutritional deficiency of Phe has adverse health consequences. In principle, structurally similar chemicals interfering with Phe utilization could alter Phe requirement at an individual level. As a strategy to identify components of the exposome that could interfere with Phe utilization, we tested for metabolites correlating with Phe concentration in plasma of a non-human primate species, common marmosets (Callithrix jacchus). The results of tests for more than 5,000 chemical features detected by high-resolution metabolomics showed 17 positive correlations with Phe metabolites and other amino acids. Positive and negative correlations were also observed for 33 other chemicals, which included matches to endogenous metabolites and dietary, microbial and environmental chemicals in database searches. Chemical similarity analysis showed many of the matches had high structural similarity to Phe. Together, the results show that chemicals in marmoset plasma could impact Phe utilization. Such chemicals could contribute to early lifecycle developmental disorders when neurological development is vulnerable to Phe levels.

Selective targeting of the cysteine proteome by thioredoxin and glutathione redox systems.

Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system.