Altered Glutathione Metabolism Research Articles

Combinatorial lipidomics and proteomics underscore erythrocyte lipid membrane aberrations in the development of adverse cardio-cerebrovascular complications in maintenance hemodialysis patients

Patients on maintenance hemodialysis exhibit a notably higher risk of cardio-cerebrovascular complications that constitute the major cause of death. Preceding studies have reported conflicting associations between traditional lipid measures and clinical outcome in dialysis patients. In this prospective longitudinal study, we utilized quantitative lipidomics to elucidate, at molecular resolution, changes in lipidome profiles of erythrocyte and plasma samples collected from maintenance hemodialysis patients followed up for 86 months (≈7 years). Primary outcome was defined as cardiovascular-related deaths or new-onset cardio-cerebrovascular events. Cox regression model uncovered plasma/erythrocyte lipids associated with incident cardio-cerebrovascular events in the erythrocyte cohort (n = 117 patients, 37 events) and plasma cohort (n = 45 patients, 11 events), respectively. Both the erythrocyte lipid panel [PA 40:5, PI 34:2, PC 42:6, AUC = 0.83] and plasma lipid panel [PC O-34:1, GM3 18:1; O2/25:0, TG 44:1(16:1_28:0), AUC = 0.94] significantly improved the prediction of cardio-cerebrovascular-related outcome compared to the base model comprising age, sex and dialysis vintage alone. Our findings underscore the pathophysiological significance of anionic phospholipid accretion in erythrocytes in the development of cardio-cerebrovascular complications in dialysis patients. In particular, distorted membrane lipid asymmetry leads to compromised membrane deformability, aberrant cell-cell interactions and altered glutathione metabolism in the erythrocytes of high-risk individuals even at relatively early stage of hemodialysis. Our findings thus underscore the importance of maintaining the RBC pool to lower the risk of cardio-cerebrovascular complications in dialysis patients.

The Second Highest Prevalence of Celiac Disease Worldwide: Genetic and Metabolic Insights in Southern Brazilian Mennonites.

Celiac disease (CD), despite its high morbidity, is an often-underdiagnosed autoimmune enteropathy. Using a modified version of the Brazilian questionnaire of the 2013 National Health Survey, we interviewed 604 Mennonites of Frisian/Flemish origin that have been isolated for 25 generations. A subgroup of 576 participants were screened for IgA autoantibodies in serum, and 391 participants were screened for HLA-DQ2.5/DQ8 subtypes. CD seroprevalence was 1:29 (3.48%, 95% CI = 2.16-5.27%) and biopsy-confirmed CD was 1:75 (1.32%, 95% CI = 0.57-2.59%), which is superior to the highest reported global prevalence (1:100). Half (10/21) of the patients did not suspect the disease. HLA-DQ2.5/DQ8 increased CD susceptibility (OR = 12.13 [95% CI = 1.56-94.20], p = 0.003). The HLA-DQ2.5 carrier frequency was higher in Mennonites than in Brazilians (p = 7 × 10-6). HLA-DQ8 but not HLA-DQ2.5 carrier frequency differed among settlements (p = 0.007) and was higher than in Belgians, a Mennonite ancestral population (p = 1.8 × 10-6), and higher than in Euro-Brazilians (p = 6.5 × 10-6). The glutathione pathway, which prevents reactive oxygen species-causing bowel damage, was altered within the metabolic profiles of untreated CD patients. Those with lower serological positivity clustered with controls presenting close relatives with CD or rheumatoid arthritis. In conclusion, Mennonites have a high CD prevalence with a strong genetic component and altered glutathione metabolism that calls for urgent action to alleviate the burden of comorbidities due to late diagnosis.

PD17-02 UNIVERSAL LOSS OF COMPLEX I IN ONCOCYTOMA IS ASSOCIATED WITH DEFECTIVE MITOPHAGY VIA ALTERED GLUTATHIONE METABOLISM

You have accessJournal of UrologyCME1 Apr 2023PD17-02 UNIVERSAL LOSS OF COMPLEX I IN ONCOCYTOMA IS ASSOCIATED WITH DEFECTIVE MITOPHAGY VIA ALTERED GLUTATHIONE METABOLISM Lin Lin, Neal Patel, Blake Wilde, Lucia Fernandez-del-Rio, Eirini Christodoulou, Shinji Ohtake, Randy Caliliw, Orian Shirihai, Xiaowu Gai, Heather Christofk, David Shackelford, and Brian Shuch Lin LinLin Lin More articles by this author , Neal PatelNeal Patel More articles by this author , Blake WildeBlake Wilde More articles by this author , Lucia Fernandez-del-RioLucia Fernandez-del-Rio More articles by this author , Eirini ChristodoulouEirini Christodoulou More articles by this author , Shinji OhtakeShinji Ohtake More articles by this author , Randy CaliliwRandy Caliliw More articles by this author , Orian ShirihaiOrian Shirihai More articles by this author , Xiaowu GaiXiaowu Gai More articles by this author , Heather ChristofkHeather Christofk More articles by this author , David ShackelfordDavid Shackelford More articles by this author , and Brian ShuchBrian Shuch More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal oncocytoma (RO) is the most commonly resected benign renal neoplasm. ROs are characterized by a dense accumulation of dysfunctional mitochondria possibly resulting by increased mitochondrial biogenesis and defective mitophagy. Although it is well known that ROs harbor recurrent inactivating mutations in mitochondrial genes encoding Complex I (NADH dehydrogenase) in the electron transport chain, the mechanism of defective mitophagy remains unclear. Here, we hypothesize that mitophagy inhibition in RO is regulated by Complex I loss, leading to increased glutathione that oxidizes key players in the mitophagy pathway. METHODS: RO and normal kidney (NK) tissues were obtained from patients undergoing surgical resection. DNA was isolated from frozen RO samples (n=18) and matched whole blood. Long-range PCR was performed to sequence mitochondrial DNA (mtDNA). Protein expression was analyzed using Western Blot. Complex I-IV activity, as measured by oxygen consumption rate, was analyzed by the Seahorse XF96 analyzer. Gene expression changes were evaluated using the Nanostring nCounter human metabolic pathway panel and analyzed by Rosalind. Metabolic profiles were measured using liquid chromatography-tandem mass spectrometry (LC-MS). Statistical calculations were performed using Graphpad Prism. RESULTS: A total of 13 ROs (72.2%) had mtDNA Complex I loss-of-function (LoF) mutations with 8 (44.4%) having high allele frequency (>50%). Compared to NK, nearly all ROs had reduced Complex I activity with a compensatory increase of Complex IV activity, irrespective of identified mtDNA mutation. On the protein level, there was a universal loss of Complex I with resultant compensatory increase of other complexes regardless of mtDNA status. Gene expression profiling revealed increased activation of AMPK pathway in ROs as well as altered glutathione metabolism, including increased biosynthesis and decreased breakdown. Metabolic profiling confirmed that both reduced and oxidized forms of glutathione were significantly enriched in ROs compared to NKs. The mechanism of glutathione inhibition of mitophagy is currently being investigated with possible candidates having dysregulated oxidation. CONCLUSIONS: The universal loss of Complex I activity in ROs is independent of mtDNA mutations. Complex I loss in RO is associated with increased glutathione, which in turn may result in impaired mitophagy. We continue to develop models to interrogate key drivers in this process. Source of Funding: AUA Urology Care Foundation Residency Research Award AUA Research Scholar Award © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e495 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lin Lin More articles by this author Neal Patel More articles by this author Blake Wilde More articles by this author Lucia Fernandez-del-Rio More articles by this author Eirini Christodoulou More articles by this author Shinji Ohtake More articles by this author Randy Caliliw More articles by this author Orian Shirihai More articles by this author Xiaowu Gai More articles by this author Heather Christofk More articles by this author David Shackelford More articles by this author Brian Shuch More articles by this author Expand All Advertisement PDF downloadLoading ...

Rejuvenating muscle stem cells with the glutathione system

Aging results from the combination of complex processes still largely undefined. In this issue, Benjamin etal. use multiomic analysis to reveal a causative role of altered glutathione (GSH) synthesis and metabolism in age-dependent muscle stem cell (MuSC) dysfunction, casting light on novel mechanisms regulating stem cell function and on therapeutic approaches to improve defective regeneration in the aged muscle.

Glutathione: pharmacological aspects and implications for clinical use

Glutathione is a tripeptide found in many tissues which plays a pivotal role in critical physiological processes such as maintenance of redox balance, reduction of oxidative stress by enhancement of metabolic detoxification of both xenobiotic and endogenous compounds, and regulation of immune system function. Glutathione depletion is associated with many chronic degenerative diseases and loss of function with aging and altered glutathione metabolism has been implicated in central nervous system diseases, frailty and sarcopenia, infected state, chronic liver diseases, metabolic diseases, pulmonary and cardiovascular diseases. Therefore, the glutathione status may be an important biomarker and treatment target in various chronic, age-related diseases. Here we describe the main pharmacological aspects of glutathione, focusing on its synthesis and role in several vital functions including antioxidant defense, detoxification of xenobiotics and modulation of immune function and fibrogenesis and the clinical implications of its depletion and we discuss the different strategies for increasing glutathione cellular levels either by providing specific precursors and cofactors or directly administering the tripeptide.

Contrasting Metabolic Profiles of TGFβ2 and TNFα in the Induction of Retinal Epithelial‐Mesenchymal Transition (EMT)

Epithelial‐mesenchymal‐transition (EMT) is a process whereby epithelial cells transdifferentiate into matrix‐producing mesenchymal cells. EMT of retinal pigment epithelial cells (RPE) plays a crucial role in severe ocular pathologies including retinal fibrosis. Emerging evidence implicates metabolic and mitochondrial dysfunction as key drivers of EMT. Both transforming growth factor‐beta 2 (TGFβ2) and tumor necrosis factor‐alpha (TNFα) have the capacity to induce EMT in RPE; however, little is known about their impact on mitochondrial metabolism. We hypothesized that TGFβ2 and TNFα should share similar metabolic effects, i.e. repressed mitochondrial function and enhanced glycolysis, to support EMT of RPE. Matured human primary RPE (H‐RPE) were treated with TGFβ2 or TNFα (both at 10 ng/ml) for up to 5 days for Seahorse metabolic assays (Mito and Glycolytic Stress Tests), qPCR and untargeted metabolomics analysis (West Coast Metabolomics Center) analyzed using MetaboAnalyst. Both TGFβ2 and TNFα induced the upregulation of mesenchymal genes (fibronectin and matrix metalloproteinase‐2) in H‐RPE. While TNFα significantly upregulated inflammatory genes (IL‐6, IL‐8, IL‐1β, MCP‐1), no changes were evident in TGFβ2‐treated cells. Metabolically, TGFβ2 induced the “Warburg effect” in H‐RPE with enhanced glycolysis and a suppression of oxidative phosphorylation capacity. Surprisingly, TNFα showed the opposite effect with enhanced oxidative phosphorylation (higher basal levels of oxygen consumption and maximal respiration) and reduced glycolysis and glycolytic capacity. Metabolomics profiling showed distinct metabolomics signatures associated with TGFβ2‐ and TNFα‐treated primary RPE cells. Of the 188 metabolites tested, 7 metabolites were significantly changed in TGFβ2‐treated cells and 26 in TNFα‐treated cells. Enrichment analysis depicted altered glutathione metabolism, proline metabolism, aminoacyl‐tRNA synthesis, nicotinamide metabolism, pentose phosphate pathway and arachidonic acid metabolism in TNFα‐treated cells whereas TGFβ2‐treated cells showed perturbations in the biosynthesis of fatty acids, tryptophan metabolism, linoleic acid metabolism and arachidonic acid metabolism. The divergent bioenergetic rewiring governing TGFβ2 and TNFα in their induction of EMT may be linked to their differential effect on inflammation. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences will enable the development of more effective drug targets for combating retinal fibrosis.

Frizzled-7 Identifies Platinum-Tolerant Ovarian Cancer Cells Susceptible to Ferroptosis.

Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7- cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7-β-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential "persister cancer cell" phenotype. SIGNIFICANCE: Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

Integrated Metagenomic and Metabolomic Analyses of the Effect of Astragalus Polysaccharides on Alleviating High-Fat Diet-Induced Metabolic Disorders.

Most herbal polysaccharides possess multiple benefits against metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD) and obesity. However, the underlying mechanisms are largely unknown. Here, male C57BL/6J mice were fed with chow or high-fat diet (HFD) with or without Astragalus polysaccharides (APS) supplementation, and gut microbial profile and metabolite profile were studied by metagenomic sequencing and untargeted metabolomics, respectively. APS was effective in alleviating HFD-induced metabolic disorders, with the alteration of gut microbiota composition and function. A total of 188 species, which mainly from Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria phyla, and 36 metabolites were markedly changed by HFD and revered by APS. Additionally, the altered glutathione metabolism and purine metabolism pathways were identified by both metagenomic function analysis and metabolite pathway enrichment analysis. Furthermore, the gut microbial alteration was associated with the changes of key intestinal metabolites. We found 31 and 20 species were correlated with purine metabolism and glutathione metabolism, respectively. Together, our results showed significant metagenomic and metabolomic changes after HFD feeding and APS intervention, revealed the potential correlation between gut microbial species and metabolites, and highlighted mechanisms of herb-derived polysaccharides by modulating gut microbiome and host metabolism underlying their benefits on metabolic disorders.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

The brominated flame retardant BDE 47 upregulates purine metabolism and mitochondrial respiration to promote adipocyte differentiation

Adipocyte differentiation is closely associated with obesity and obesity-induced metabolic disorders. Epidemiological studies have demonstrated the association of obesity with environmental pollutants, such as polybrominated diphenyl ethers (PBDEs), common flame retardants in various consumer products. However, their obesogenic effects and mechanism are underexplored. We employed non-targeted metabolomics studies based on liquid chromatography-high resolution mass spectrometry to determine how 2,2′,4,4′-tetra-brominated biphenyl ether (BDE 47), one of the main congeners of PBDEs detected in human tissue, promotes adipocyte differentiation of mouse preadipocyte 3 T3-L1 cells. The promoting effects of BDE 47 exposure (5 or 10 μM) on adipocyte differentiation were confirmed by enhancing lipid accumulation and expression levels of biomarkers of adipogenesis. For the first time, we demonstrated that BDE 47 upregulated purine metabolism and altered glutathione metabolism to promote oxidative stress and uric acid production in adipocytes. BDE 47 also elevated mitochondrial respiration and glycolysis in adipocytes to induce more ATP to combat oxidative stress. Antioxidant treatments, including the suppression of xanthine oxidase, inhibited the effects of BDE 47 on inducing oxidative stress and lipid accumulation. BDE 47 may be a potential environmental obesogen by providing a permissive oxidative environment to induce adipocyte differentiation.

Distinct Metabolic Properties of MDS Stem Cells Provide Novel Opportunities for Therapeutic Intervention

This project is focused on characterizing the malignant stem cells that drive the pathogenesis of MDS, with the goal of developing improved therapeutic strategies. To this end, we have established methods by which MDS stem cells can be identified, isolated and characterized in xenograft models. These approaches have been employed to permit global metabolomic and transcriptomic analyses, which have subsequently led to modeling novel therapeutic regimens.Initial studies exploited previous work in acute myelogenous leukemia to identify candidate phenotypic markers of stem cell malignancy. These efforts demonstrated that up-regulation of CD123 in the hematopoietic stem cell compartment identifies MDS stem cells as they transition from low risk to high risk stages of pathogenesis. Thus, using cell sorting, we are able to isolate early (CD123-) vs. late (CD123+) stage stem cells from MDS patient specimens and subject them to the experimental analyses outlined below. We first performed a transcriptomic study that demonstrated a massive increase in cellular protein translation machinery through significant increases in ribosomal proteins as stem cells progress to advanced stages of MDS. To functionally validate these findings, MDS stem cell populations were cultured with the fluorescent protein substrate OP-puro, which detects newly synthesized polypeptide chains. CD123+ stem cells strongly increase protein synthesis levels (~13-fold increase). Given the established role of protein translation homeostasis in hematopoietic stem cells [Signer et al., 2014], this finding indicates a major change in cellular metabolism as well as a potential therapeutic entry point.To further characterize cellular changes occurring as MDS stem cells evolve, we performed global mass spectrometry-based metabolomic analyses which further demonstrates increased protein biosynthesis as well as altered glutathione metabolism (elevated oxidized glutathione). Taken together, these findings indicate major metabolic changes in MDS stem cells as they acquire increasing malignant phenotypes.Next, we investigated signaling related to increased ribosomal protein production. Specifically, we examined the hypothesis that ribosomal subunit binding of MDM2, may play a role in pathogenesis. Intriguingly, our data show that the specific subunits known to bind MDM2 are increased in CD123+ MDS stem cells. Consistent with this observation, MDM2 is elevated as well. This finding indicates that increased ribosomal protein levels may function to inhibit p53 activity, thereby enhancing pathogenic outgrowth of MDS stem cells.Using the mechanistic insights outlined above, we explored novel therapeutic strategies. First, we examined drugs known to selectively target metabolism through altering protein synthesis and targeting ribosomal proteins (e.g. homoharringtonine, HHT)). In addition, given the prevalent role of hypomethylating agents in current MDS treatment regimens, we also examined how these inhibitors interact with 5-azacytidine (5-aza). In vitro studies indicate that multiple protein synthesis inhibitors selectively eradicate MDS stem cells (CD123+). In addition, combination with 5-aza yielded additive to synergistic eradication of MDS stem cells. We have demonstrated that high risk MDS specimens effectively engraft the NSG-S strain of immune deficient mice, when T cell depletion and a busulfan conditioning regimen are employed. Using this model, we transplanted primary MDS specimens and challenged using the regimens above. Treatment with HHT demonstrated selective eradication of MDS stem cells, with a significant differential toxicity observed in multiple samples (50-60% selective ablation). Finally, analysis of protein synthesis inhibitors in combination with the hypomethylating agent 5-aza demonstrated potent efficacy in targeting the MDS stem cell population with greater then additive toxicity when compared to single agent treatment(70-80% selective ablation).Taken together, these data show that changes in metabolic properties represent a critical inflection point in the pathogenesis and progression of the MDS. Focusing on such changes, we have identified novel pharmacological approaches that may effectively target the MDS stem cell population. Importantly, these approaches function well in conjunction with commonly used agents used in the treatment of MDS. DisclosuresPollyea:Alexion: Other: advisory board; Celgene: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding.

Biomolecules damage and redox status abnormalities in Fabry patients before and during enzyme replacement therapy

Fabry disease (FD) is caused by deficient activity of the lysosomal enzyme α-galactosidase A. Its substrates, mainly globotriaosylceramide (Gb3), accumulate and seem to induce other pathophysiological findings of FD. Once enzyme replacement therapy (ERT) is not completely efficient on preventing disease progress in FD patients, elucidating the underlying mechanisms in FD pathophysiology is essential to the development of additional therapeutic strategies. We investigated 58 Fabry patients (23 male and 35 female) subdivided into two groups (at diagnosis and during long-term ERT) and compared them to healthy individuals. Fabry patients at diagnosis presented altered glutathione (GSH) metabolism (higher GSH levels, lower glutathione peroxidase – GPx – and normal glutathione reductase – GR - activities), higher lipid peroxidation levels (thiobarbituric acid reactive species - TBARS - and malondialdehyde - MDA), nitric oxide (NO.) equivalents and urinary Gb3. Fabry patients on ERT presented GSH metabolism similar to controls, although lipid peroxidation and urinary levels of NO. equivalents remained higher whereas Gb3 levels were lower than at diagnosis but still higher than controls. These data demonstrated that redox impairment occurs in Fabry patients before and after ERT, probably as a consequence of Gb3 accumulation, providing targets to future therapy approaches using antioxidants in combination with ERT in FD.

Changes to Intermediary Metabolites in Sporadic and LRRK2 Parkinson's Disease Demonstrated by Proton Magnetic Resonance Spectroscopy.

Background. Parkinson's disease (PD) remains a clinical diagnosis and biomarkers are needed to detect the disease as early as possible. Genetically determined PD provides an opportunity for studying metabolic differences in connection with disease development. Objectives. To study the levels of intermediary metabolites in cerebrospinal fluid (CSF) from patients with PD, either of sporadic type or in carriers of the LRRK2 p.G2019S mutation. Methods. Results from patients with sporadic PD or LRRK2-PD were compared with asymptomatic LRRK2 mutation carriers and healthy control individuals. CSF was analysed by proton MR spectroscopy (1H-MRS) giving reliable results for 16 intermediary metabolites. Partial least squares discriminant analysis (PLS-DA) was applied to study group differences. Results. PLS-DA distinguished PD patients from healthy individuals based on the metabolites identified in CSF, with 2-hydroxybutyrate, glutamine, and dimethyl sulphone largely contributing to the separations. Conclusion. Speculatively, all three metabolites could alter concentration in response to metabolic changes connected with neurodegeneration; glutamine as a means of removing excess nitrogen from brain, dimethyl sulphone as an anti-inflammatory agent, and 2-hydroxybutyrate in connection with altered glutathione metabolism. Potentially, 1H-MRS is a promising tool for identifying novel biomarkers for PD.

Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines

There is an increasing recognition that mitochondrial dysfunction is associated with autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction and how mitochondrial abnormalities might interact with other physiological disturbances such as oxidative stress. Reserve capacity is a measure of the ability of the mitochondria to respond to physiological stress. In this study, we demonstrate, for the first time, that lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) have an abnormal mitochondrial reserve capacity before and after exposure to reactive oxygen species (ROS). Ten (44%) of 22 AD LCLs exhibited abnormally high reserve capacity at baseline and a sharp depletion of reserve capacity when challenged with ROS. This depletion of reserve capacity was found to be directly related to an atypical simultaneous increase in both proton-leak respiration and adenosine triphosphate-linked respiration in response to increased ROS in this AD LCL subgroup. In this AD LCL subgroup, 48-hour pretreatment with N-acetylcysteine, a glutathione precursor, prevented these abnormalities and improved glutathione metabolism, suggesting a role for altered glutathione metabolism associated with this type of mitochondrial dysfunction. The results of this study suggest that a significant subgroup of AD children may have alterations in mitochondrial function, which could render them more vulnerable to a pro-oxidant microenvironment as well as intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxins. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors.

Abstract P3-01-05: Metabolomic analysis of breast cancer cell lines: Clues to the metabolic fingerprint of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis and lower survival rate. IBC is characterized by peculiar clinical and biological features and resistance to standard treatments. IBC has shown the capacity to spread early through lymphatic channels and blood vessels causing the specific inflammatory signs. Currently, there are no specific therapeutic options for IBC patients because of very few molecular alterations associated with IBC. Thus, there is an unmet need to identify distinguished features of IBC that differentiate the disease from non-inflammatory breast cancer (nIBC). Metabolomic analysis of cancer cells holds the promise to identify unique chemical fingerprints of important cellular processes. In this study we investigated the global biochemical profiles of IBC and nIBC cells. METHODS: Three IBC cell lines, SUM190, SUM149, FC-IBC-02, and three nIBC cell lines, MCF-7, MDA-MB-231, MDA-MB-468, with five replicates for each cell line, were analyzed in this study. Metabolomic profiling analysis was performed by Metabolon, Inc (Durham, NC) on the gas chromatography/mass spectroscopy and ultrahigh performance liquid chromatography/mass spectroscopy platforms as previously described (Proc. Natl. Acad. Sci. U. S. A. 108, 3270-3275, 2011). A total of 347 metabolites were detected. Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (p≤0.001), as well as those approaching significance (0.001<p<0.05). RESULTS: Comparison of global biochemical profiles for IBC and nIBC cell lines revealed numerous statistically changed metabolites at a p-value threshold of p<0.001. Statistical comparisons also revealed a clear metabolic separation of samples due to cell line differences. Several biochemical pathways emerged as being commonly altered across all IBC cell lines compared to all nIBC cell lines. Notable observations include alterations in inflammatory signaling, energy metabolism, and cell proliferation. Signatures of inflammation were elevated and inflammatory signaling pathways showed activation in IBC cell lines. In energy metabolism, the cellular energy for IBC cells appeared to be derived from amino acid catabolism to a greater extent than in nIBC cells. Furthermore, fatty acids was significantly elevated in IBC cell lines, that may indicate a reduced cellular proliferation and increased glycogen storage in IBC cells. Moreover, an altered methylation and glutathione metabolism were shown in IBC cell lines. CONCLUSIONS: Our study is the first to demonstrate difference of metabolic profiles between IBC and nIBC cells. Future studies comparing global biochemical profiles in tissue biopsy and blood from IBC and nIBC patients may help to confirm the relevance of these findings in cell lines. Metabolic targets may provide more effective and specific therapeutic strategy for the treatment of IBC patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-05.

Targeted disruption of the nucleosomal binding protein HMGN5 results in altered glutathione metabolism and mild hepatic dysfunction

High mobility group nucleosome‐binding protein 5 (HMGN5) is a chromatin architectural protein that regulates gene expression by decompacting higher‐order chromatin structure; however, the physiological role of this protein is unknown. To examine the function of HMGN5 in vivo, we generated mice lacking the chromatin binding domain of the protein. Compared to wild type littermates, male Hmgn5−/Y mice had elevated serum albumin (p=0.03), fasting non‐HDL cholesterol (p=0.03), fasting triglycierides (p=0.05), and alanine transaminase (p=0.08), suggesting a mild hepatic dysfunction. An UPLC‐QTOF‐MS‐based metabolomic study in Hmgn5−/Y mice identified a 41% increase in hepatic glutathione (p=0.02) and significantly (p<0.05) decreased urinary concentrations of compounds related to the glutathione precursor glycine, such as betaine (−62%), phenylacetylglycine (−50%), and creatine (−42%). Microarray analysis and validation by real‐time PCR revealed that the expression of two genes affecting glutathione function, Gpx6 and Hk1, was significantly (p<0.05) decreased in Hmgn5−/Y mouse liver tissue. In sum, these data suggest that functional loss of the chromatin remodeling protein HMGN5 leads to transcriptional changes that alter glutathione metabolism resulting in mild hepatic dysfunction.Grant Funding Source: NIH/NCI/CCR Intramural Program

Implications of Altered Glutathione Metabolism in Aspirin-Induced Oxidative Stress and Mitochondrial Dysfunction in HepG2 Cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.

The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis

BackgroundGlutathione has a wide range of functions; it is an endogenous anti-oxidant and plays a key role in the maintenance of intracellular redox balance and detoxification of xenobiotics. Several studies have indicated that children with autism spectrum disorders may have altered glutathione metabolism which could play a key role in the condition.MethodsA systematic literature review and meta-analysis was conducted of studies examining metabolites, interventions and/or genes of the glutathione metabolism pathways i.e. the γ-glutamyl cycle and trans-sulphuration pathway in autism spectrum disorders.ResultsThirty nine studies were included in the review comprising an in vitro study, thirty two metabolite and/or co-factor studies, six intervention studies and six studies with genetic data as well as eight studies examining enzyme activity.ConclusionsThe review found evidence for the involvement of the γ-glutamyl cycle and trans-sulphuration pathway in autistic disorder is sufficiently consistent, particularly with respect to the glutathione redox ratio, to warrant further investigation to determine the significance in relation to clinical outcomes. Large, well designed intervention studies that link metabolites, cofactors and genes of the γ-glutamyl cycle and trans-sulphuration pathway with objective behavioural outcomes in children with autism spectrum disorders are required. Future risk factor analysis should include consideration of multiple nutritional status and metabolite biomarkers of pathways linked with the γ-glutamyl cycle and the interaction of genotype in relation to these factors.

Metabolic Profiling of 3-Nitropropionic Acid Early-Stage Huntington’s Disease Rat Model Using Gas Chromatography Time-of-Flight Mass Spectrometry

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH3HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 microg Hg/g and 0.4 microg Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 microg Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks.