Publications by authors named "James R Roede"

45 Publications

Trisomy 21 results in modest impacts on mitochondrial function and central carbon metabolism.

Free Radic Biol Med 2021 Jun 12;172:201-212. Epub 2021 Jun 12.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, USA; Linda Crnic Institute for Down Syndrome, School of Medicine, University of Colorado, Aurora, CO, USA. Electronic address:

Down syndrome (DS) is the most common genetic cause of intellectual disability. Mechanistically, oxidative stress and mitochondrial dysfunction are reported to be etiological factors for many of the DS-related comorbidities and have previously been reported in a number of in vitro and in vivo models of DS. The purpose of this study was to test for the presence of mitochondrial dysfunction in fibroblast cells obtained via skin biopsy from individuals with DS, and to assess the impact of trisomy 21 on central carbon metabolism. Using extracellular flux assays in matched dermal fibroblasts from euploid and DS individuals, we found that basal mitochondrial dysfunction is quite mild. Stressing the cells with a cocktail of mitochondrial stressors revealed a significant mitochondrial deficit in DS cells compared to euploid controls. Evaluation of extracellular acidification rate did not reveal a baseline abnormality in glycolysis; however, metabolomic assessments utilizing isotopically labeled glucose and glutamine revealed altered central carbon metabolism in DS cells. Specifically, we observed greater glucose dependency, uptake and flux into the oxidative phase of the pentose phosphate pathway in DS fibroblasts. Furthermore, using induced pluripotent stem cells (iPSC) we found that mitochondrial function in DS iPSCs was similar to the previously published studies employing fetal cells. Together, these data indicate that aberrant central carbon metabolism is a candidate mechanism for stress-related mitochondrial dysfunction in DS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2021.06.003DOI Listing
June 2021

Cystathionine γ-lyase promotes estrogen-stimulated uterine artery blood flow via glutathione homeostasis.

Redox Biol 2021 04 8;40:101827. Epub 2020 Dec 8.

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 12700 E. 19th Avenue, Aurora, CO, 80045, USA; Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 12700 E. 19th Avenue, Aurora, CO, 80045, USA. Electronic address:

During pregnancy, estrogen (E) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (HS) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E stimulation. We quantified serum and uterine artery NO metabolites (NO), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E stimulation of UBF. Renal blood flow was E-insensitive for both genotypes. While serum and uterine artery NO were similar between genotypes at baseline, E decreased NO in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823052PMC
April 2021

Derangement of hepatic polyamine, folate, and methionine cycle metabolism in cystathionine beta-synthase-deficient homocystinuria in the presence and absence of treatment: Possible implications for pathogenesis.

Mol Genet Metab 2021 02 11;132(2):128-138. Epub 2021 Jan 11.

Medicine and University of Colorado School of Medicine, Aurora, CO 80045, USA.

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5'-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5'-methylthioadenosine. HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one‑carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one‑carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgme.2021.01.003DOI Listing
February 2021

Trisomy 21 impairs PGE2 production in dermal fibroblasts.

Prostaglandins Other Lipid Mediat 2021 Apr 5;153:106524. Epub 2021 Jan 5.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, USA; Linda Crnic Institute for Down Syndrome, Aurora, Colorado, USA. Electronic address:

The triplication of human chromosome 21 results in Down syndrome (DS), the most common genetic form of intellectual disability. This aneuploid condition also results in an enhanced risk of a spectrum of comorbid conditions, such as leukemia, early onset Alzheimer's disease, and diabetes. Individuals with DS also display an increased incidence of wound healing complications and resistance to solid tumor development. Due to this unique phenotype and the involvement of eicosanoids in key comorbidities like poor healing and tumor development, we hypothesized that cells from DS individuals would display altered eicosanoid production. Using age- and sex-matched dermal fibroblasts we interrogated this hypothesis. Briefly, assessment of over 90 metabolites derived from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 systems revealed a possible deficiency in the COX system. Basal gene expression and Western blotting experiments showed significantly decreased gene expression of COX1 and 2, and COX2 protein abundance in DS fibroblasts compared to euploid controls. Further, using two different stressors, scratch wound or LPS, we found that DS fibroblasts could not upregulate COX2 abundance and prostaglandin E2 production. Together, these findings show that dermal fibroblasts from DS individuals have a deficient COX2 response, which may contribute to wound healing complications and tumor resistance in DS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.prostaglandins.2020.106524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7965340PMC
April 2021

Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease.

Free Radic Biol Med 2021 01 3;162:65-76. Epub 2020 Dec 3.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, 80045, USA. Electronic address:

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2020.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889696PMC
January 2021

Time-dependent simvastatin administration enhances doxorubicin toxicity in neuroblastoma.

Toxicol Rep 2020 22;7:520-528. Epub 2020 Apr 22.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO 80045, United States.

Statins have a primary indication for the reduction and management of hypercholesterolemia; however, evidence shows that statins have the ability to increase the toxicity of chemotherapeutics within cancer cells by inducing anti-proliferative, anti-metastatic, and anti-angiogenic effects. More recently, lipophilic statins have shown complex interaction with energy metabolism, specifically acute mitochondrial dysfunction and delayed inhibition of glycolysis. With the goal to demonstrate that statin-mediated enhancement of chemotherapeutics is time-dependent, we hypothesized that the lipophilic statin simvastatin, in conjunction with variable co-exposure of doxorubicin or cisplatin, will enhance the toxicity of these drugs in neuroblastoma. Utilizing human SK-N-AS neuroblastoma cells, we assessed cell proliferation, necrosis, caspase activation, and overall apoptosis of these cells. After determining the toxicity of simvastatin at 48 h post-treatment, 10μM was chosen as the intervention concentration. We found that significant cell death resulted from 1.0μM dose of doxorubicin with 24 h pre-treatment of simvastatin. On the other hand, simvastatin enhancement of cisplatin toxicity was only observed in the co-exposure model. As doxorubicin has strict dosage limits due to its primary off-target toxicity in cardiac muscle, we further compared the effects of this drug combination on rat H9C2 cardiomyoblasts. We found that simvastatin did not enhance doxorubicin toxicity in this cell line. We conclude that simvastatin provides time-dependent sensitization of neuroblastoma cells to doxorubicin toxicity, and our results provide strong argument for the consideration of simvastatin as an adjuvant in doxorubicin-based chemotherapy programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxrep.2020.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184108PMC
April 2020

Simvastatin Induces Delayed Apoptosis Through Disruption of Glycolysis and Mitochondrial Impairment in Neuroblastoma Cells.

Clin Transl Sci 2020 05 6;13(3):563-572. Epub 2020 Feb 6.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, Colorado, USA.

Simvastatin, a commonly used cholesterol-lowering drug, inhibits the mevalonate pathway involved in the synthesis of the mitochondrial electron carrier coenzyme Q10 (CoQ10), as well as other bioenergetics substrates. The purpose of this study was to investigate simvastatin exposure on mitochondrial respiration, metabolic fuel preferences, and glucose utilization. We hypothesized that simvastatin at a noncytotoxic dose will impair energy metabolism in human neuroblastoma cells. SK-N-AS cells were exposed at acute and chronic time points and evaluated in a Seahorse XF analyzer, revealing decreased mitochondrial and glycolytic parameters. Flow cytometry showed a significant induction of apoptosis in simvastatin-treated cells at 48 hours. Finally, multiple techniques were used to show that simvastatin-mediated impairment of bioenergetics is more complex than CoQ10 depletion or hampered glucose uptake. Therefore, the data reported here represent a biphasic hit to mitochondria followed by reduction in glucose and glutamine metabolism in neuroblastoma; adding mechanism to potential pleotropic effects of statins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cts.12740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214657PMC
May 2020

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes.

Cell Chem Biol 2020 02 22;27(2):206-213.e6. Epub 2019 Nov 22.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA. Electronic address:

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395678PMC
February 2020

SNARE proteins rescue impaired autophagic flux in Down syndrome.

PLoS One 2019 12;14(11):e0223254. Epub 2019 Nov 12.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO, United States of America.

Down syndrome (DS) is a chromosomal disorder caused by trisomy of chromosome 21 (Ts21). Unbalanced karyotypes can lead to dysfunction of the proteostasis network (PN) and disrupted proteostasis is mechanistically associated with multiple DS comorbidities. Autophagy is a critical component of the PN that has not previously been investigated in DS. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts and other DS cell models. Following induction of autophagy by serum starvation, DS fibroblasts displayed impaired autophagic flux indicated by autophagolysosome accumulation and elevated p62, NBR1, and LC3-II abundance, compared to age- and sex-matched, euploid (CTL) fibroblasts. While lysosomal physiology was unaffected in both groups after serum starvation, we observed decreased basal abundance of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) family members syntaxin 17 (STX17) and Vesicle Associated Membrane Protein 8 (VAMP8) indicating that decreased autophagic flux in DS is due at least in part to a possible impairment of autophagosome-lysosome fusion. This conclusion was further supported by the observation that over-expression of either STX17 or VAMP8 in DS fibroblasts restored autophagic degradation and reversed p62 accumulation. Collectively, our results indicate that impaired autophagic clearance is a characteristic of DS cells that can be reversed by enhancement of SNARE protein expression and provides further evidence that PN disruption represents a candidate mechanism for multiple aspects of pathogenesis in DS and a possible future target for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223254PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850524PMC
March 2020

Toxicant-mediated redox control of proteostasis in neurodegeneration.

Curr Opin Toxicol 2019 Feb 28;13:22-34. Epub 2018 Dec 28.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045.

Disruption in redox signaling and control of cellular processes has emerged as a key player in many pathologies including neurodegeneration. As protein aggregations are a common hallmark of several neuronal pathologies, a firm understanding of the interplay between redox signaling, oxidative and free radical stress, and proteinopathies is required to sort out the complex mechanisms in these diseases. Fortunately, models of toxicant-induced neurodegeneration can be utilized to evaluate and report mechanistic alterations in the proteostasis network (PN). The epidemiological links between environmental toxicants and neurological disease gives further credence into characterizing the toxicant-mediated PN disruptions observed in these conditions. Reviewed here are examples of mechanistic interaction between oxidative or free radical stress and PN alterations. Additionally, investigations into toxicant-mediated PN disruptions, specifically focusing on environmental metals and pesticides, are discussed. Finally, we emphasize the need to distinguish whether the presence of protein aggregations are contributory to phenotypes related to neurodegeneration, or if they are a byproduct of PN deficiencies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cotox.2018.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785977PMC
February 2019

Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles.

Nanotoxicology 2020 03 25;14(2):145-161. Epub 2019 Sep 25.

Colorado Center for Nanomedicine and Nanosafety, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Zinc oxide nanoparticles (ZnO NPs) are used in numerous applications, including sunscreens, cosmetics, textiles, and electrical devices. Increased consumer and occupational exposure to ZnO NPs potentially poses a risk for toxicity. While many studies have examined the toxicity of ZnO NPs, little is known regarding the toxicological impact of inherent defects arising from batch-to-batch variations. It was hypothesized that the presence of varying chemical defects in ZnO NPs will contribute to cellular toxicity in rat aortic endothelial cells (RAECs). Pristine and defected ZnO NPs (oxidized, reduced, and annealed) were prepared and assessed three major cellular outcomes; cytotoxicity/apoptosis, reactive oxygen species production and oxidative stress, and endoplasmic reticulum (ER) stress. ZnO NPs chemical defects were confirmed by X-ray photoelectron spectroscopy and photoluminescence. Increased toxicity was observed in defected ZnO NPs compared to the pristine NPs as measured by cell viability, ER stress, and glutathione redox potential. It was determined that ZnO NPs induced ER stress through the PERK pathway. Taken together, these results demonstrate a previously unrecognized contribution of chemical defects to the toxicity of ZnO NPs, which should be considered in the risk assessment of engineered nanomaterials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/17435390.2019.1668067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036006PMC
March 2020

A biplot correlation range for group-wise metabolite selection in mass spectrometry.

BioData Min 2019 4;12. Epub 2019 Feb 4.

6Department of Industrial Engineering, Hanyang University, Seoul, 04763 South Korea.

Background: Analytic methods are available to acquire extensive metabolic information in a cost-effective manner for personalized medicine, yet disease risk and diagnosis mostly rely upon individual biomarkers based on statistical principles of false discovery rate and correlation. Due to functional redundancies and multiple layers of regulation in complex biologic systems, individual biomarkers, while useful, are inherently limited in disease characterization. Data reduction and discriminant analysis tools such as principal component analysis (PCA), partial least squares (PLS), or orthogonal PLS (O-PLS) provide approaches to separate the metabolic phenotypes, but do not offer a statistical basis for selection of group-wise metabolites as contributors to metabolic phenotypes.

Methods: We present a dimensionality-reduction based approach termed 'biplot correlation range (BCR)' that uses biplot correlation analysis with direct orthogonal signal correction and PLS to provide the group-wise selection of metabolic markers contributing to metabolic phenotypes.

Results: Using a simulated multiple-layer system that often arises in complex biologic systems, we show the feasibility and superiority of the proposed approach in comparison of existing approaches based on false discovery rate and correlation. To demonstrate the proposed method in a real-life dataset, we used LC-MS based metabolomics to determine spectrum of metabolites present in liver mitochondria from wild-type (WT) mice and thioredoxin-2 transgenic (TG) mice. We select discriminatory variables in terms of increased score in the direction of class identity using BCR. The results show that BCR provides means to identify metabolites contributing to class separation in a manner that a statistical method by false discovery rate or statistical total correlation spectroscopy can hardly find in complex data analysis for predictive health and personalized medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13040-019-0191-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360680PMC
February 2019

Aberrant expression of redox regulatory proteins in patients with concomitant primary Sclerosing cholangitis/inflammatory bowel disease.

Exp Mol Pathol 2018 08 29;105(1):32-36. Epub 2018 May 29.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, United States. Electronic address:

Objective: Primary Sclerosing Cholangitis (PSC) is a severe cholestatic liver disease characterized by progressive peri-biliary tract inflammation, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%-80% of PSC patients (PSC/IBD). We previously reported dysregulation of key anti-oxidant pathways in PSC/IBD. The objective of this study was to expand previous data by examining the abundance of thioredoxins (Trx) in PSC/IBD.

Methods: Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, the protein abundance of thioredoxin, thioredoxin reductase (TrxR1), and their downstream substrates peroxiredoxins was assessed.

Results: Western blot analyses of thioredoxin and peroxiredoxin abundance revealed significant increases in abundance of Trx1 and TrxR1 whereas expression of thioredoxin-interacting protein was significantly decreased in PSC/IBD. Concurrently, abundance of cytosolic peroxiredoxins was not significantly impacted. The abundance of mitochondrial Trx2, along with peroxiredoxins 3, 5 and 6 were significantly decreased by concurrent PSC/IBD. Histological staining of Trx1/TrxR1 revealed elevated nuclear Trx1 and TrxR1 staining within cholangiocytes as well as an overall periportal increase in expression in PSC/IBD. An examination of additional anti-oxidant responses reveal suppression of gamma-glutamylcysteine synthetase and heme oxygenase (HO-1) whereas expression of the protein chaperone glucose regulated protein 78 increased suggesting elevated cellular stress in PSC/IBD.

Conclusions: Results herein suggest that in addition to severe dysregulation of anti-oxidant responses, cholestasis impacts both cytosolic/nuclear (Trx1) as well as mitochondrial (Trx2) redox signaling and control pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexmp.2018.05.012DOI Listing
August 2018

Redox Equivalents and Mitochondrial Bioenergetics.

Methods Mol Biol 2018 ;1782:197-227

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University, Atlanta, GA, USA.

Mitochondrial energy metabolism depends upon high-flux and low-flux electron transfer pathways. The former provide the energy to support chemiosmotic coupling for oxidative phosphorylation. The latter provide mechanisms for signaling and control of mitochondrial functions. Few practical methods are available to measure rates of individual mitochondrial electron transfer reactions; however, a number of approaches are available to measure steady-state redox potentials (E ) of donor/acceptor couples, and these can be used to gain insight into rate controlling reactions as well as mitochondrial bioenergetics. Redox changes within the respiratory electron transfer pathway are quantified by optical spectroscopy and measurement of changes in autofluorescence. Low-flux pathways involving thiol/disulfide redox couples are measured by redox Western blot and mass spectrometry-based redox proteomics. Together, the approaches provide the opportunity to develop integrated systems biology descriptions of mitochondrial redox signaling and control mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7831-1_12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986360PMC
February 2019

Acute Maneb Exposure Significantly Alters Both Glycolysis and Mitochondrial Function in Neuroblastoma Cells.

Toxicol Sci 2018 09;165(1):61-73

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, Colorado 80045.

The pesticides paraquat (PQ) and maneb (MB) have been described as environmental risk factors for Parkinson's disease (PD), with mechanisms associated with mitochondrial dysfunction and reactive oxygen species generation. A combined exposure of PQ and MB in murine models and neuroblastoma cells has been utilized to further advance understanding of the PD phenotype. MB acts as a redox modulator through alkylation of protein thiols and has been previously characterized to inhibit complex III of the electron transport chain and uncouple the mitochondrial proton gradient. The purpose of this study was to analyze ATP-linked respiration and glycolysis in human neuroblastoma cells utilizing the Seahorse extracellular flux platform. Employing an acute, subtoxic exposure of MB, this investigation revealed a MB-mediated decrease in mitochondrial oxygen consumption at baseline and maximal respiration, with inhibition of ATP synthesis and coupling efficiency. Additionally, MB-treated cells showed an increase in nonmitochondrial respiration and proton leak. Further investigation into mitochondrial fuel flex revealed an elimination of fuel flexibility across all 3 major substrates, with a decrease in pyruvate capacity as well as glutamine dependency. Analyses of glycolytic function showed a substantial decrease in glycolytic acidification caused by lactic acid export. This inhibition of glycolytic parameters was also observed after titrating the MB dose as low as 6 μM, and appears to be dependent on the dithiocarbamate functional group, with manganese possibly potentiating the effect. Further studies into cellular ATP and NAD levels revealed a drastic decrease in cells treated with MB. In summary, MB significantly impacted both aerobic and anaerobic energy production; therefore, further characterization of MB's effect on cellular energetics may provide insight into the specificity of PD to dopaminergic neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfy116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135636PMC
September 2018

Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

FASEB J 2018 03 3;32(3):1265-1280. Epub 2018 Jan 3.

Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Aurora, Colorado, USA.

Cystathionine β-synthase-deficient homocystinuria (HCU) is a poorly understood, life-threatening inborn error of sulfur metabolism. Analysis of hepatic glutathione (GSH) metabolism in a mouse model of HCU demonstrated significant depletion of cysteine, GSH, and GSH disulfide independent of the block in trans-sulfuration compared with wild-type controls. HCU induced the expression of the catalytic and regulatory subunits of γ-glutamyl ligase, GSH synthase (GS), γ-glutamyl transpeptidase 1, 5-oxoprolinase (OPLAH), and the GSH-dependent methylglyoxal detoxification enzyme, glyoxalase-1. Multiple components of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant-response regulatory axis were induced without any detectable activation of Nrf2. Metabolomic analysis revealed the accumulation of multiple γ-glutamyl amino acids and that plasma ophthalmate levels could serve as a noninvasive marker for hepatic redox stress. Neither cysteine, nor betaine treatment was able to reverse the observed enzyme inductions. Taurine treatment normalized the expression levels of γ-glutamyl ligase C/M, GS, OPLAH, and glyoxalase-1, and reversed HCU-induced deficits in protein glutathionylation by acting to double GSH levels relative to controls. Collectively, our data indicate that the perturbation of the γ-glutamyl cycle could contribute to multiple sequelae in HCU and that taurine has significant therapeutic potential for both HCU and other diseases for which GSH depletion is a critical pathogenic factor.-Maclean, K. N., Jiang, H., Aivazidis, S., Kim, E., Shearn, C. T., Harris, P. S., Petersen, D. R., Allen, R. H., Stabler, S. P., Roede, J. R. Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201700586RDOI Listing
March 2018

Comment on: Extra-virgin olive oil ameliorates cognition and neuropathology of the 3xTg mice: role of autophagy.

Authors:
James R Roede

Ann Clin Transl Neurol 2017 10 5;4(10):762. Epub 2017 Sep 5.

Department of Pharmaceutical Sciences Skaggs School of Pharmacy and Pharmaceutical Sciences University of Colorado Aurora Colorado.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634343PMC
October 2017

Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH.

Free Radic Biol Med 2017 12 6;113:280-290. Epub 2017 Oct 6.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH).

Methods: Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics.

Results: Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively.

Conclusions: These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704928PMC
December 2017

Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation.

Alcohol Clin Exp Res 2017 Oct 14;41(10):1705-1714. Epub 2017 Sep 14.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

Background: Chronic ethanol (EtOH) consumption is a major cause of liver disease worldwide. Oxidative stress is a known consequence of EtOH metabolism and is thought to contribute significantly to alcoholic liver disease (ALD). Therefore, elucidating pathways leading to sustained oxidative stress and downstream redox imbalances may reveal how EtOH consumption leads to ALD. Recent studies suggest that EtOH metabolism impacts mitochondrial antioxidant processes through a number of proteomic alterations, including hyperacetylation of key antioxidant proteins.

Methods: To elucidate mechanisms of EtOH-induced hepatic oxidative stress, we investigate a role for protein hyperacetylation in modulating mitochondrial superoxide dismutase (SOD2) structure and function in a 6-week Lieber-DeCarli murine model of EtOH consumption. Our experimental approach includes immunoblotting immunohistochemistry (IHC), activity assays, mass spectrometry, and in silico modeling.

Results: We found that EtOH metabolism significantly increased the acetylation of SOD2 at 2 functionally relevant lysine sites, K68 and K122, resulting in a 40% decrease in enzyme activity while overall SOD2 abundance was unchanged. In vitro studies also reveal which lysine residues are more susceptible to acetylation. IHC analysis demonstrates that SOD2 hyperacetylation occurs near zone 3 within the liver, which is the main EtOH-metabolizing region of the liver.

Conclusions: Overall, the findings presented in this study support a role for EtOH-induced lysine acetylation as an adverse posttranslational modification within the mitochondria that directly impacts SOD2 charge state and activity. Last, the data presented here indicate that protein hyperacetylation may be a major factor contributing to an imbalance in hepatic redox homeostasis due to chronic EtOH metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acer.13473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626652PMC
October 2017

The burden of trisomy 21 disrupts the proteostasis network in Down syndrome.

PLoS One 2017 21;12(4):e0176307. Epub 2017 Apr 21.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO, United States of America.

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176307PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400264PMC
September 2017

Comparative Proteomic Analysis of Carbonylated Proteins from the Striatum and Cortex of Pesticide-Treated Mice.

Parkinsons Dis 2015 9;2015:812532. Epub 2015 Aug 9.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO 80045, USA.

Epidemiological studies indicate exposures to the herbicide paraquat (PQ) and fungicide maneb (MB) are associated with increased risk of Parkinson's disease (PD). Oxidative stress appears to be a premier mechanism that underlies damage to the nigrostriatal dopamine system in PD and pesticide exposure. Enhanced oxidative stress leads to lipid peroxidation and production of reactive aldehydes; therefore, we conducted proteomic analyses to identify carbonylated proteins in the striatum and cortex of pesticide-treated mice in order to elucidate possible mechanisms of toxicity. Male C57BL/6J mice were treated biweekly for 6 weeks with saline, PQ (10 mg/kg), MB (30 mg/kg), or the combination of PQ and MB (PQMB). Treatments resulted in significant behavioral alterations in all treated mice and depleted striatal dopamine in PQMB mice. Distinct differences in 4-hydroxynonenal-modified proteins were observed in the striatum and cortex. Proteomic analyses identified carbonylated proteins and peptides from the cortex and striatum, and pathway analyses revealed significant enrichment in a variety of KEGG pathways. Further analysis showed enrichment in proteins of the actin cytoskeleton in treated samples, but not in saline controls. These data indicate that treatment-related effects on cytoskeletal proteins could alter proper synaptic function, thereby resulting in impaired neuronal function and even neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2015/812532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546751PMC
September 2015

Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney.

Redox Biol 2015 Dec 6;6:33-40. Epub 2015 Jul 6.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA. Electronic address:

In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2015.06.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511634PMC
December 2015

Short-term oral atrazine exposure alters the plasma metabolome of male C57BL/6 mice and disrupts α-linolenate, tryptophan, tyrosine and other major metabolic pathways.

Toxicology 2014 Dec 4;326:130-41. Epub 2014 Nov 4.

Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; Interdisciplinary Toxicology Program, University of Georgia, Athens, GA 30602, USA. Electronic address:

Overexposure to the commonly used herbicide atrazine (ATR) affects several organ systems, including the brain. Previously, we demonstrated that short-term oral ATR exposure causes behavioral deficits and dopaminergic and serotonergic dysfunction in the brains of mice. Using adult male C57BL/6 mice, the present study aimed to investigate effects of a 10-day oral ATR exposure (0, 5, 25, 125, or 250mg/kg) on the mouse plasma metabolome and to determine metabolic pathways affected by ATR that may be reflective of ATR's effects on the brain and useful to identify peripheral biomarkers of neurotoxicity. Four hours after the last dosing on day 10, plasma was collected and analyzed with high-performance, dual chromatography-Fourier-transform mass spectrometry that was followed by biostatistical and bioinformatic analyses. ATR exposure (≥5mg/kg) significantly altered plasma metabolite profile and resulted in a dose-dependent increase in the number of metabolites with ion intensities significantly different from the control group. Pathway analyses revealed that ATR exposure strongly correlated with and disrupted multiple metabolic pathways. Tyrosine, tryptophan, linoleic acid and α-linolenic acid metabolic pathways were among the affected pathways, with α-linolenic acid metabolism being affected to the greatest extent. Observed effects of ATR on plasma tyrosine and tryptophan metabolism may be reflective of the previously reported perturbations of brain dopamine and serotonin homeostasis, respectively. ATR-caused alterations in the plasma profile of α-linolenic acid metabolism are a potential novel and sensitive plasma biomarker of ATR effect and plasma metabolomics could be used to better assess the risks, including to the brain, associated with ATR overexposure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tox.2014.11.001DOI Listing
December 2014

Mitochondrial metabolomics using high-resolution Fourier-transform mass spectrometry.

Methods Mol Biol 2014 ;1198:43-73

Clinical Biomarkers Laboratory, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University, 615 Michael Street, Atlanta, GA, 30322, USA.

High-resolution Fourier-transform mass spectrometry (FTMS) provides important advantages in studies of metabolism because more than half of common intermediary metabolites can be measured in 10 min with minimal pre-detector separation and without ion dissociation. This capability allows unprecedented opportunity to study complex metabolic systems, such as mitochondria. Analysis of mouse liver mitochondria using FTMS with liquid chromatography shows that sex and genotypic differences in mitochondrial metabolism can be readily distinguished. Additionally, differences in mitochondrial function are readily measured, and many of the mitochondria-related metabolites are also measurable in plasma. Thus, application of high-resolution mass spectrometry provides an approach for integrated studies of complex metabolic processes of mitochondrial function and dysfunction in disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1258-2_4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318503PMC
June 2015

Thiol-reactivity of the fungicide maneb.

Redox Biol 2014 18;2:651-5. Epub 2014 Apr 18.

Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University, 615 Michael Street, Atlanta, GA 30322, USA.

Maneb (MB) is a manganese-containing ethylene bis-dithiocarbamate fungicide that is implicated as an environmental risk factor for Parkinson's disease, especially in combination with paraquat (PQ). Dithiocarbamates inhibit aldehyde dehydrogenases, but the relationship of this to the combined toxicity of MB + PQ is unclear because PQ is an oxidant and MB activates Nrf2 and increases cellular GSH without apparent oxidative stress. The present research investigated the direct reactivity of MB with protein thiols using recombinant thioredoxin-1 (Trx1) as a model protein. The results show that MB causes stoichiometric loss of protein thiols, reversibly dimerizes the protein and inhibits its enzymatic activity. MB reacted at similar rates with low-molecular weight, thiol-containing chemicals. Together, the data suggest that MB can potentiate neurotoxicity of multiple agents by disrupting protein thiol functions in a manner analogous to that caused by oxidative stress, but without GSH depletion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2014.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052523PMC
January 2015

Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity.

Toxicol Sci 2014 May 4;139(1):59-73. Epub 2014 Feb 4.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University, Atlanta, Georgia 30322.

Cadmium (Cd) exposure contributes to human diseases affecting liver, kidney, lung, and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Our previous mouse and cell studies showed that environmental low-dose Cd exposure altered protein redox states resulting in stimulation of inflammatory signaling and disruption of the actin cytoskeleton system, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated the effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria to gain insight into associated toxicological mechanisms and functions. We analyzed redox states of liver mitochondrial proteins by redox proteomics using isotope coded affinity tag (ICAT) combined mass spectrometry. Redox ICAT identified 2687 cysteine-containing peptides (peptidyl Cys) of which 1667 peptidyl Cys (657 proteins) were detected in both control and Cd-exposed samples. Of these, 46% (1247 peptidyl Cys, 547 proteins) were oxidized by Cd more than 1.5-fold relative to controls. Bioinformatics analysis using MetaCore software showed that Cd affected 86 pathways, including 24 Cys in proteins functioning in branched chain amino acid (BCAA) and 14 Cys in proteins functioning in fatty acid (acylcarnitine/carnitine) metabolism. Consistently, high-resolution metabolomics data showed that Cd treatment altered levels of BCAA and carnitine metabolites. Together, these results show that mitochondrial protein redox and metabolites are targets in Cd-induced hepatotoxicity. The results further indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfu018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007108PMC
May 2014

Transcriptome-metabolome wide association study (TMWAS) of maneb and paraquat neurotoxicity reveals network level interactions in toxicologic mechanism.

Toxicol Rep 2014 24;1:435-444. Epub 2014 Jul 24.

Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University, Atlanta, GA 30322, United States; Clinical Biomarkers Laboratory, Emory University, Atlanta, GA 30322, United States.

A combination of the herbicide paraquat (PQ) and fungicide maneb (MB) has been linked to Parkinson's disease. Previous studies show that this involves an additive toxicity with at least two different mechanisms. However, detailed understanding of mixtures is often difficult to elucidate because of the multiple ways by which toxic agents can interact. In the present study, we used a combination of transcriptomics and metabolomics to investigate mechanisms of toxicity of PQ and MB in a neuroblastoma cell line. Conditions were studied with concentrations of PQ and MB that each individually caused 20% cell death and together caused 50% cell death. Transcriptomic and metabolomic samples were collected at time points prior to significant cell death. Statistical and bioinformatic methods were applied to the resulting 30,869 transcripts and 1358 metabolites. Results showed that MB significantly changed more transcripts and metabolites than PQ, and combined PQ + MB impacted more than MB alone. Transcriptome-metabolome-wide association study (TMWAS) showed that significantly changed transcripts and metabolites mapped to two network substructures, one associating with significant effects of MB and the other included features significantly associated with PQ + MB. The latter contained 4 clusters of genes and associated metabolites, with one containing genes for two cation transporters and a cation transporter regulatory protein also recognized as a pro-apoptotic protein. Other clusters included stress response genes and transporters linked to cytoprotective mechanisms. MB also had a significant network structure linked to cell proliferation. Together, the results show that the toxicologic mechanism of the combined neurotoxicity of PQ and MB involves network level interactions and that TMWAS provides an effective approach to investigate such complex mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053767PMC
http://dx.doi.org/10.1016/j.toxrep.2014.07.006DOI Listing
July 2014

Serum metabolomics of slow vs. rapid motor progression Parkinson's disease: a pilot study.

PLoS One 2013 22;8(10):e77629. Epub 2013 Oct 22.

Division of Pulmonary, Allergy and Critical Care Medicine, Emory University, Atlanta, Georgia, United States of America.

Progression of Parkinson's disease (PD) is highly variable, indicating that differences between slow and rapid progression forms could provide valuable information for improved early detection and management. Unfortunately, this represents a complex problem due to the heterogeneous nature of humans in regards to demographic characteristics, genetics, diet, environmental exposures and health behaviors. In this pilot study, we employed high resolution mass spectrometry-based metabolic profiling to investigate the metabolic signatures of slow versus rapidly progressing PD present in human serum. Archival serum samples from PD patients obtained within 3 years of disease onset were analyzed via dual chromatography-high resolution mass spectrometry, with data extraction by xMSanalyzer and used to predict rapid or slow motor progression of these patients during follow-up. Statistical analyses, such as false discovery rate analysis and partial least squares discriminant analysis, yielded a list of statistically significant metabolic features and further investigation revealed potential biomarkers. In particular, N8-acetyl spermidine was found to be significantly elevated in the rapid progressors compared to both control subjects and slow progressors. Our exploratory data indicate that a fast motor progression disease phenotype can be distinguished early in disease using high resolution mass spectrometry-based metabolic profiling and that altered polyamine metabolism may be a predictive marker of rapidly progressing PD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077629PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805572PMC
May 2014

Characterization of plasma thiol redox potential in a common marmoset model of aging.

Redox Biol 2013 18;1:387-93. Epub 2013 Jul 18.

Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University, Atlanta, GA, USA.

Due to its short lifespan, ease of use and age-related pathologies that mirror those observed in humans, the common marmoset (Callithrix jacchus) is poised to become a standard nonhuman primate model of aging. Blood and extracellular fluid possess two major thiol-dependent redox nodes involving cysteine (Cys), cystine (CySS), glutathione (GSH) and glutathione disulfide (GSSG). Alteration in these plasma redox nodes significantly affects cellular physiology, and oxidation of the plasma Cys/CySS redox potential (E hCySS) is associated with aging and disease risk in humans. The purpose of this study was to determine age-related changes in plasma redox metabolites and corresponding redox potentials (E h) to further validate the marmoset as a nonhuman primate model of aging. We measured plasma thiol redox states in marmosets and used existing human data with multivariate adaptive regression splines (MARS) to model the relationships between age and redox metabolites. A classification accuracy of 70.2% and an AUC of 0.703 were achieved using the MARS model built from the marmoset redox data to classify the human samples as young or old. These results show that common marmosets provide a useful model for thiol redox biology of aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2013.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757708PMC
May 2015

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

Mol Cell Proteomics 2013 Nov 14;12(11):3285-96. Epub 2013 Aug 14.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.M113.030437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820939PMC
November 2013
-->