Glutathione Homeostasis Research Articles

Exogenous nitric oxide requires endogenous hydrogen sulfide to induce the resilience through sulfur assimilation in tomato seedlings under hexavalent chromium toxicity

Nitric oxide (NO) and hydrogen sulfide (H2S), versatile signaling molecules, play multiple roles in plant growth, physiological and biochemical processes under heavy metal stress. However, the mechanisms through which NO in association with endogenous H2S mediated hexavalent chromium Cr(VI) toxicity mitigation are still not fully understood. Therefore, we investigated the role of NO and H2S in sulfur (S)-assimilation and the effect of NO on endogenous H2S, and cysteine (Cys) biosynthesis and maintenance of cellular glutathione (GSH) pool in tomato seedlings under Cr(VI) stress. Cr(VI) toxicity caused an increase in reactive oxygen species (ROS; O2•– and H2O2) formation and activity of chlorophyll (Chl) degrading enzyme [Chlorophyllase (Chlase)] and decrease in seedlings growth attributes, Chl a and b content, and activity of Chl synthesizing enzyme [δ-aminolevulinic acid dehydratase (δ-ALAD)], gas exchange parameters, S-assimilation, and Cys and H2S metabolism. An increase in the content of glycinebetaine (GB), total soluble carbohydrates (TSCs) and total phenols (TPls), and decrease in DNA damage and ROS in NO treated seedlings conferred Cr(VI) toxicity tolerance. Under Cr(VI) toxicity conditions, the inclusion of H2S scavenger hypotaurine (HT) in growth medium containing NO validated the role of endogenous H2S in S-assimilation, H2S and Cys and GSH metabolism by withdrawing activity of enzymes involved in S-assimilation [adenosine 5-phosphosulfatereductase (APS-R), ATP-sulfurylase (ATP-S)], in the biosynthesis of H2S [L-cysteine desulfhydrase (L-CD) and D-cysteine desulfhydrase (D-CD)], Cys [O-acetylserin (thiol) lyase (OAST-L)], and GSH [glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS)], and in antioxidant system. On the other hand, application of cPTIO [2-(4–32 carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide], a NO scavenger and HT diminished the effect of NO on internal H2S levels, Cys and glutathione homeostasis, and S-assimilation, which resulted in poor immunity against oxidative stress induced by Cr(VI) toxicity. The obtained results postulate that NO-induced internal H2S conferred tolerance of tomato seedlings to Cr(VI) toxicity and maintained better photosynthesis process and plant growth.

Metabolomics research on the hepatoprotective effect of cultured bear bile powder in α-naphthylisothiocyanate-induced cholestatic mice

Natural bear bile powder (NBBP) is a famous traditional medicine and has been widely used in clinic. However, access to the sources of bear bile is restricted; hence, it is essential to discover new substitutes for NBBP. Cultured bear bile powder (CBBP) is transformed from chicken bile and contains main ingredients as to NBBP. In the present study, the effect and potential mechanism of action of CBBP on cholestatic liver injury in-naphthylisothiocyanate (ANIT)-induced mouse model was explored using metabolomics. CBBP treatment ameliorated impaired hepatic dysfunction and tissue damage that induced by ANIT. Metabolomics showed there were 28 different metabolites induced by ANIT as compared with control mice, and 18 of which was reversed by CBBP. Pathway analysis revealed that those 18 metabolites are mainly involved in bile acid (BA) biosynthesis and D-glutamine and D-glutamate metabolism. Further LC-MS/MS analysis showed that CBBP and NBBP both reduced serum and liver levels of BAs, but increased their biliary levels. Additionally, CBBP and NBBP upregulated expression of BA efflux transporters, Mrp2, Mrp3, and Mrp4, and metabolic enzymes, Cyp2b10 and Ugt1a1 of liver tissue of cholestatic mice, increased the BA excretion and metabolism. Moreover, CBBP and NBBP treatment upregulated GCLc/GCLm expression, and restored glutathione metabolism. In conclusion, the protective effects of CBBP against cholestatic liver injury were similar to those of NBBP. Mechanistically, both CBBP and NBBP reversed the disruption in homeostasis of BAs and glutathione, alleviating damage to hepatocytes.

MS-222 induces biochemical and transcriptional changes related to oxidative stress, cell proliferation and apoptosis in zebrafish embryos

MS-222, the most widely used anaesthetic in fish, has been shown to induce embryotoxic effects in zebrafish. However, the underlying molecular effects are still elusive. This study aimed to investigate the effects of MS-222 exposure during early developmental stages by evaluating biochemical and molecular changes. Embryos were exposed to 50, 100 or 150 mg L−1 MS-222 for 20 min at one of three developmental stages (256-cell, 50% epiboly, or 1–4 somite stage) and oxidative-stress, cell proliferation and apoptosis-related parameters were determined at two time-points (8 and 26 hpf). Following exposure during the 256-cell stage, the biochemical redox balance was not affected. The genes associated with glutathione homeostasis (gstpi and gclc) were affected at 8 hpf, while genes associated with apoptosis (casp3a and casp6) and cellular proliferation (pcna) were found affected at 26 hpf. An inverted U-shaped response was observed at 8 hpf for catalase activity. After exposure at the 50% epiboly stage, the gclc gene associated with oxidative stress was found upregulated at 8 hpf, while gstpi was downregulated and casp6 was upregulated later on, coinciding with a decrease in glutathione peroxidase (GPx) activity and a non-monotonic elevation of protein carbonyls and casp3a. Additionally, MS-222 treated embryos showed a decrease in DCF-staining at 26 hpf. When exposure was performed at the 1–4 somite stage, a similar DCF-staining pattern was observed. The activity of GPx was also affected whereas RT-qPCR showed that caspase transcripts were dose-dependently increased (casp3a, casp6 and casp9). The pcna mRNA levels were also found to be upregulated while gclc was changed by MS-222. These results highlight the impact of MS-222 on zebrafish embryo development and its interference with the antioxidant, cell proliferation and cellular death systems by mechanisms still to be explained; however, the outcomes point to the Erk/Nrf2 signalling pathway as a target candidate.

Serum untargeted metabolomic changes in response to diet intervention in dogs with preclinical myxomatous mitral valve disease.

Myocardial energy deprivation plays a causal role in the development of heart failure. A cardiac protection blend (CPB) of nutrients including medium chain triglycerides, fish oil and other key nutrients was developed to slow the progression of canine myxomatous mitral valve disease (MMVD). A six-month dietary intervention demonstrated efficacy of CPB in slowing MMVD progression. Untargeted metabolomic analysis of serum from these dogs identified 102 differential metabolites (adjusted P < 0.05). The ratios of omega-6 to omega-3 fatty acid (FA) changed from 2.41 and 1.46 in control and CPB groups at baseline to 4.30 and 0.46 at 6 months respectively. A 2.7-fold increase of α-aminobutyrate, a myocardial modulator of glutathione homeostasis, was found in CPB dogs compared to 1.3-fold increase in control dogs. Arginine and citrulline, precursors of nitric oxide biosynthesis, were both increased 2-fold; caprate, a medium chain FA, was increased 3-fold; and deoxycarnitine, precursor of carnitine biosynthesis, was increased 2.5-fold in CPB dogs. Margarate and methylpalmitate decreased in response to CPB, a potential benefit in MMVD dogs as positive correlations were found between changes in both these FAs and left atrial diameter (r = 0.69, r = 0.87 respectively, adjusted P < 0.05). Sphingomyelins with very long chain saturated FAs associated with decreased risk of heart failure in humans were increased in MMVD dogs fed the CPB diet. Our data supports the hypothesis that CPB improves FA utilization and energetics, reduces oxidative stress and inflammation in MMVD dogs. More studies are needed to understand the roles of specific metabolites in MMVD.

Effect of di(2-ethylhexyl) phthalate on Nrf2-regulated glutathione homeostasis in mouse kidney.

Environmental toxicants such as phthalate have been involved in multiple health disorders including renal diseases. Oxidative damage is implicated in many alterations caused by phthalate especially the di(2-ethylhexyl) phthalate (DEHP), which is the most useful phthalate. However, information regarding its mechanism of renal damage is lacking. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates gene expression implicated in free radical scavenging and cytoprotection including the antioxidant glutathione (GSH) pathway. The aim of this study was to assess whether DEHP affects the Nrf2 pathway and the GSH concentration. Mice were divided into four groups: a control group and three groups treated with DEHP at different concentrations (5, 50, and 200 mg/kg body weight) for 30 days. Our results showed that DEHP altered the normal levels of serum biochemical parameters creatinine (CREA), urea, and lactate dehydrogenase (LDH). This phthalate caused oxidative damage through the induction of lipid peroxidation and protein oxidation as marked by increase of protein carbonyl (PC) and loss of protein-bound sulfhydryls (PSH). Simultaneously, DEHP treatment decreased the protein level of Nrf-2, HO-1, and GCLC (responsible of GSH synthesis) and decreased the GSH level. Inhibition of the Nrf2 pathway is related to the activation of the mitochondrial pathway of apoptosis. This apoptotic process is evidenced by an upregulation of p53 and Bax protein levels in addition to a downregulation of Bcl-2. Collectively, our data demonstrated that depletion of Nrf2 and GSH was associated with the elevation of oxidative stress and the activation of intrinsic apoptosis in mouse kidney treated with DEHP.

Protective Effects of Rice Peptide Oryza Peptide-P60 against Oxidative Injury through Activation of Nrf2 Signaling Pathway In Vitro and In Vivo.

We previously showed that commercially available rice peptide Oryza Peptide-P60 (OP60) increased the intracellular glutathione levels. This study aimed to evaluate the antioxidant potential of this peptide and assess its mechanism of action. Pretreatment of HepG2 cells with OP60 reduced the cytotoxicity caused by H2O2 or acetaminophen (APAP) (47.7 ± 1.3% or 12.2 ± 1.3% of the cytotoxicity for 5 mg/mL OP60 pretreatment compared to that in H2O2- or APAP-treated groups, respectively; p < 0.01) through the restoration of glutathione homeostasis. Moreover, OP60 elevated the mRNA level of genes encoding heavy and light subunits of γ-glutamylcysteine synthetase (γ-GCS) by 2.9 ± 0.1-fold and 2.7 ± 0.2-fold (p < 0.001), respectively, at 8 h and also increased the level of mRNA encoding other antioxidant enzymes. Besides, OP60 promoted Nrf2 nuclear translocation by 2.2 ± 0.3-fold (p < 0.05) after 8 h. Conversely, knockdown of Nrf2 inhibited the increase of the intracellular glutathione levels and suppressed the induction of antioxidant enzyme expression by OP60. In animal studies, OP60 prevented APAP-induced liver injury by suppressing glutathione depletion (from 0.19 ± 0.02 mmol/mg protein to 0.90 ± 0.02 mmol/mg protein; p < 0.01, by pretreatment with 500 mg/kg OP60) and increasing heavy subunit of γ-GCS and heme oxygenase-1 expression in the liver. Our results indicated that OP60 exhibits a cytoprotective effect via the Nrf2 signaling pathway and is one of the few peptides with excellent antioxidant properties.

Resemblance and Difference of Seedling Metabolic and Transporter Gene Expression in High Tolerance Wheat and Barley Cultivars in Response to Salinity Stress.

To elucidate inter-specific similarity and difference of tolerance mechanism against salinity stress between wheat and barley, high tolerant wheat cv. Suntop and sensitive cv. Sunmate and tolerant barley cv. CM72 were hydroponically grown in a greenhouse with 100 mM NaCl. Glutathione, secondary metabolites, and genes associated with Na+ transport, defense, and detoxification were examined to discriminate the species/cultivar difference in response to salinity stress. Suntop and CM72 displayed damage to a lesser extent than in Sunmate. Compared to Sunmate, both Suntop and CM72 recorded lower electrolyte leakage and reactive oxygen species (ROS) production, higher leaf relative water content, and higher activity of PAL (phenylalanine ammonia-lyase), CAD (cinnamyl alcohol dehydrogenase), PPO (polyphenol oxidase), SKDH (shikimate dehydrogenase), and more abundance of their mRNA under salinity stress. The expression of HKT1, HKT2, salt overly sensitive (SOS)1, AKT1, and NHX1 was upregulated in CM72 and Suntop, while downregulated in Sunmate. The transcription factor WRKY 10 was significantly induced in Suntop but suppressed in CM72 and Sunmate. Higher oxidized glutathione (GSSG) content was accumulated in cv. CM72 and Sunmate, but increased glutathione (GSH) content and the ratio of GSH/GSSG were observed in leaves and roots of Suntop under salinity stress. In conclusion, glutathione homeostasis and upregulation of the TaWRKY10 transcription factor played a more important role in wheat salt-tolerant cv. Suntop, which was different from barley cv. CM72 tolerance to salinity stress. This new finding could help in developing salinity tolerance in wheat and barley cultivars.

MiRNA Regulation of Glutathione Homeostasis in Cancer Initiation, Progression and Therapy Resistance.

Glutathione (GSH) is the most abundant antioxidant that contributes to regulating the cellular production of Reactive Oxygen Species (ROS) which, maintained at physiological levels, can exert a function of second messengers in living organisms. In fact, it has been demonstrated that moderate amounts of ROS can activate the signaling pathways involved in cell growth and proliferation, while high levels of ROS induce DNA damage leading to cancer development. Therefore, GSH is a crucial player in the maintenance of redox homeostasis and its metabolism has a role in tumor initiation, progression, and therapy resistance. Our recent studies demonstrated that neuroblastoma cells resistant to etoposide, a common chemotherapeutic drug, show a partial monoallelic deletion of the locus coding for miRNA 15a and 16-1 leading to a loss of these miRNAs and the activation of GSH-dependent responses. Therefore, the aim of this review is to highlight the role of specific miRNAs in the modulation of intracellular GSH levels in order to take into consideration the use of modulators of miRNA expression as a useful strategy to better sensitize tumors to current therapies.

Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery.

Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient. Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42. Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO2 ) levels. Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions.

New Insights into the Role of Glutathione in the Mechanism of Fever.

Glutathione is one of the most important and potent antioxidants. The development of pharmacological compounds that can either increase or decrease glutathione concentrations has allowed investigation into the role of glutathione in various biological processes, including immune responses. Recent findings have shown that glutathione not only affects certain factors involved in immunological processes but also modifies complex immune reactions such as fever. Until recently, it was not known why some patients do not develop fever during infection. Data suggest that fever induction is associated with oxidative stress; therefore, antioxidants such as glutathione can reduce pyrexia. Surprisingly, new studies have shown that low glutathione levels can also inhibit fever. In this review, we focus on recent advances in this area, with an emphasis on the role of glutathione in immune responses accompanied by fever. We describe evidence showing that disturbed glutathione homeostasis may be responsible for the lack of fever during infections. We also discuss the biological significance of the antipyretic effects produced by pharmacological glutathione modulators.

Untargeted metabolomics links glutathione to bacterial cell cycle progression.

Cell cycle progression requires the coordination of cell growth, chromosome replication, and division. Consequently, a functional cell cycle must be coupled with metabolism. However, direct measurements of metabolome dynamics remained scarce, in particular in bacteria. Here, we describe an untargeted metabolomics approach with synchronized Caulobacter crescentus cells to monitor the relative abundance changes of ~400 putative metabolites as a function of the cell cycle. While the majority of metabolite pools remains homeostatic, ~14% respond to cell cycle progression. In particular, sulfur metabolism is redirected during the G1-S transition, and glutathione levels periodically change over the cell cycle with a peak in late S phase. A lack of glutathione perturbs cell size by uncoupling cell growth and division through dysregulation of KefB, a K+/H+ antiporter. Overall, we here describe the impact of the C. crescentus cell cycle progression on metabolism, and in turn relate glutathione and potassium homeostasis to timely cell division.

N-acetylcysteine mitigates acute opioid withdrawal behaviors and CNS oxidative stress in neonatal rats

BackgroundNeonatal abstinence syndrome (NAS) is a significant problem. Opioid withdrawal induces oxidative stress and disrupts glutamate and glutathione homeostasis. We hypothesized that N-acetylcysteine (NAC) administered during acute opioid withdrawal in neonatal rats would decrease withdrawal behaviors and normalize CNS glutathione and glutamate.MethodsOsmotic minipumps with methadone (opioid dependent, OD) and saline (Sham) were implanted into Sprague Dawley dams 7 days prior to delivery. Pups were randomized to receive either naloxone plus saline or NAC (50–100 mg/kg), administered on postnatal day (PND) 7. We performed MR spectroscopy on PND6–7 before, 30 min, and 120 min after withdrawal. On PND7, we assessed withdrawal behaviors for 90 min after naloxone administration and summed scores during peak withdrawal period.ResultsMean summed behavioral scores were significantly different between groups (χ2 (2) = 10.49, p = 0.005) but not different between NAC/NAL/OD and Sham (p = 0.14): SAL/NAL/OD = 17.2 ± 4.2 (n = 10); NAC/NAL/OD = 11.3 ± 5.6 (n = 9); Sham = 6.5 ± 0.6 (n = 4). SAL/NAL/OD pups had decreased glutathione at 120 min (p = 0.01), while NAC/NAL/OD pups maintained pre-withdrawal glutathione (p = 0.26).ConclusionIn antenatal OD, NAC maintains CNS glutathione and mitigates acute opioid withdrawal in neonatal rats. This is the first study to demonstrate acute opioid withdrawal neurochemical changes in vivo in neonatal OD. NAC is a potential novel treatment for NAS.

Correlation Between Gamma-Glutamyl Transferase Activity and Glutathione Levels in Molecular Subgroups of Breast Cancer.

The gamma-glutamyl cycle catalyzed by gamma-glutamyl transferase (GGT) plays an important role in glutathione (GSH) homeostasis in the cell. In cells continuously exposed to the drug, the main phase of the enzymatic detoxification is the conjugation of the drug with GSH catalyzed by glutathione-S-transferase (GST). Conjugation of drugs with GSH is the first step in the development of chemotherapeutic drug resistance. In this study, we aimed to investigate the relationship between GGT and GSH in molecular subgroups of breast cancer patients. Serum GGT activity and GSH levels for patients diagnosed with breast cancer (n=58) and healthy controls (n=8) were measured by a spectrophotometric method and a colorimetric kit, respectively. GGT activity was significantly higher in the total patient group and in the molecular subgroups than those in the control groups (p<0.05). Serum GSH levels were higher in the patient groups compared to controls without reaching statistical significance (p>0.05). GGT activity was positively correlated with GSH levels in the total patients and healthy controls (p<0.001 and p<0.05, respectively). There was also a positive correlation between GGT activity and GSH levels in Luminal A, HER2-positive (Human epidermal growth factor receptor 2), and Triple-negative groups (p<0.05). This is the first study showing the relationship between GGT and GSH in molecular subgroups of breast cancer. An increase in GGT activity may affect intracellular GSH synthesis. Therefore, having a correlation between GGT and GSH in some molecular subgroups may affect the course of treatment in these patients.

Serum GGT and serum ferritin as early markers for metabolic syndrome.

Background:In India, the prevalence of lifestyle diseases like diabetes, hypertension, and metabolic syndrome (MetS) is showing an upward trend. Gamma glutamate transferase (GGT) and ferritin increase oxidant stress in the body through their role in glutathione homeostasis and iron metabolism, respectively. The increase in oxidant stress increases the inflammatory load, a risk factor for metabolic syndrome. These parameters are cheap, patient-friendly, and available in routine diagnostic labs compatible for follow-up, relieving the already overburdened healthcare system.Methodology:In a case-control study, samples of 77 cases of metabolic syndrome and 77 age and sex-matched controls were analyzed for serum GGT (by modified IFCC) and serum ferritin (by CLIA). Statistical analysis was done by SPSS 20.0 version.Results:The mean ± SD for ferritin and GGT were 101.58 ± 84.20 ng/dL and 36.67 ± 26.40 IU/L, respectively in cases, whereas in control group these values were 38.38 ± 29.26 ng/dL and 16.5 3 ± 6.79 IU/L (P < 0.001). Positive and significant correlation was seen between GGT with TG (r-value- 0.376/P-value-0.001) and GGT with waist circumference (r-value- 0.298/P-value- 0.022). A positive and significant correlation was seen between GGT and ferritin in cases with an r-value of 0.307 (P-value - 0.01).Conclusion:The increased values of GGT and ferritin in cases suggest an inflammatory load. The positive and significant correlation between GGT and triglyceride indicates its role in increasing oxidants’ stress leading to inflammation and the development of MetS. The association of ferritin with MetS though insignificant may be considered as a biomarker.

MiRNA Regulation of Glutathione Homeostasis in Cancer Initiation, Progression and Therapy Resistance

miRNA Regulation of Glutathione Homeostasis in Cancer Initiation, Progression and Therapy Resistance

Inhibition of multidrug resistance related protein by acetyl salicylic acid in cultured human lung cells

BACKGROUND: Multidrug resistance related proteins (MRP-proteins), members of the ATP binding cassette (ABC-) superfamily, are known to play an important role in regulation of glutathione homeostasis within cells, and hence in GSH dependent transport processes of xenobiotics. Many airborne compounds are hazardous inducing inflammatory reactions in lung and are suspected to play a substantial role in initiation and/or promotion of tumor formation. It is interesting to clarify whether and how anti-prostaglandins modify MRP function in normal human lung cells. Aspirin, an acetylated salicylate, is classified among the non-steroidal anti-inflammatory drugs (NSAIDs), reduce inflammatory signs and symptoms exhibiting a broad range of pharmacological activities (analgesic, antipyretic and antiplatelet properties). Cyclooxygenase (COX)-system catalyses the rate limiting step in prostanoids (prostaglandin (PG)) synthesis and is also involved in drug resistance and poor prognosis of many neoplastic diseases. Aspirin's ability to suppress prostaglandins and thromboxanes production is due to its irreversible inactivation of the cyclo-oxygenase (COX) enzyme. Furthermore, transport activity study of MRP1 in NHBEC, PLC and A549 under the effect of exogenously supplied PGE2 showed an increased activity. METHODOLOGY and RESULTS: In this study, both the localisation and description of the cellular distribution of MRP-1 under standard culture conditions in normal human bronchial epithelial cells (NHBEC), peripheral lung cells (PLC) and tumor lung cells (A549) was proved using indirect immunofluorescence microscopy. All substrates and incubation periods have been pre-checked for cellular toxicity in the MTT assay in order to guarantee that non toxic conditions are applied. CONCLUSION: COX inhibitor (acetylsalicylic acid 1,2mM) could significantly decrease the transport activity of MRP1 in NHBEC, PLC and A549 in 1 and 3 days trials.

Endurance training improves plasma superoxide dismutase activity in healthy elderly

We aimed to characterize the plasma redox homeostasis as underlying physiological mechanisms of specific training on healthy elderly. 51 healthy volunteers were trained to endurance, resistance, Neuro-Muscular Electrical Stimulation for 12 weeks, 3 sessions/w, all applied to lower limbs. We assessed ex-post quadriceps’ maximal voluntary contraction, handgrip strength, five-times-sit to stand and timed up-and-go tests, oxidative damage (lipid peroxidation, protein carbonyl groups), antioxidant enzyme activities (superoxide dismutase, Catalase, Glutathione peroxidase, Glutathione homeostasis), free thiols and total antioxidant status. We found significant difference in ex-post × protocol and in post-hoc analysis specifically for plasma superoxide dismutase activity in endurance training.

Linking Stored Red Blood Cell Metabolism to Transfusion Recipient Iron Homeostasis Pathophysiology in Critically-Ill Children

Background: Transfusion of packed red blood cells (pRBCs) is a common, life-saving therapy for many critically-ill children. However, routine blood bank storage results in the progressive accumulation of a series of RBC modifications, termed the "storage lesion," that reduce RBC recovery in vivo, may affect transfusion recipient physiology, and may be implicated in transfusion-related adverse events. In particular, circulating non-transferrin-bound iron (NTBI) in recipients can be increased after pRBC transfusion. NTBI promotes bacterial growth, induces oxidative injury, and inversely correlates with post-transfusion recovery, all of which may increase the risk of sepsis, multiorgan failure, and death. We aimed to determine whether the metabolic state of donor pRBCs at the time of transfusion correlates with recipient NTBI levels, which serves as a marker of hemolysis and iron homeostasis in critically-ill pediatric patients. Study Design/Methods: Critically-ill pediatric patients, aged &lt; 21-years-old with a minimum weight of 5kg in the pediatric intensive care unit at a single-site tertiary care hospital, who required pRBC transfusion, were prospectively enrolled after parental consent (n = 49 patients). Patient blood samples were obtained pre-transfusion and 4 hours following the patient's first qualifying pRBC transfusion, and analyzed for markers of iron and heme homeostasis, hemolysis, liver function, and inflammation utilizing standard laboratory methods. pRBC transfusate (1 mL) was frozen at -80°C immediately at the end of transfusion for each transfused patient. Hydrophilic and lipophilic compounds were extracted separately using methanol:acetonitrile:water or methanol, respectively, before analysis using high-throughput LC-MS based metabolomics and lipidomics platforms to determine the relative abundance of small molecule compounds. Metabolite levels in the pRBC transfusates were correlated with the differences in pre- and post-transfusion clinical laboratory values and selected patient parameters (e.g. blood type, patient age, and storage age of the pRBC unit at the time of transfusion), using Pearson correlation coefficients and associated p-values. Results: The relative levels of 221 unique metabolites and lipids in the pRBC transfusates were correlated with the corresponding transfusion recipient's clinical laboratory values and selected patient parameters. Analysis of Pearson's correlations between the levels of these compounds in the donor pRBC transfusates and the pre- and post-transfusion differences in the recipients' NTBI levels (ΔNTBI) demonstrated statistically significant correlations for 24 metabolites (R = 0.28-0.43, p = 0.0022-0.0476, see Table). Analysis of recipient clinical laboratory biomarkers associated with recipient ΔNTBI revealed a positive correlation with increases in serum iron (R = 0.41, p = 0.0034) but not with other changes in recipient clinical laboratory biomarkers. The age of the stored pRBCs at the time of transfusion did not correlate with ΔNTBI levels (R = 0.24, p = 0.0912). Conclusions: Statistically significant correlations of donor pRBC transfusate metabolites with transfusion recipient ΔNTBI are primarily seen with hypoxanthine, lactate, metabolites of glutathione homeostasis, amino acids, unsaturated free fatty acids, acyl-carnitines, and metabolites of indole and tryptophan metabolism. These data suggest that higher levels of oxidative stress, proteolysis, membrane remodeling in stored donor pRBCs, and, possibly, microbiome metabolism in the pRBC donor, are associated with significant increases in recipient plasma NTBI and serum iron levels, likely from hemolysis of storage-damaged RBCs. The levels of these metabolic biomarkers of the RBC storage lesion correlate better with ΔNTBI than the storage age of the donor unit, supporting the notion that pRBC metabolic age is not equivalent to pRBC chronologic age, and that the former may be more relevant to transfusion recipient pathophysiology in critically-ill children. Table. Disclosures Nemkov: Omix Technologies, Inc.: Equity Ownership, Other: Founder. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder.

Dual Targeting of ALDH1 and ALDH3: A Promising Therapeutic Approach in Acute Myeloid Leukemia

Background. The aldehyde dehydrogenasesfamily(ALDHs) play a major cytoprotective role in cells via detoxification of a wide range of aldehydes. Their functional activity also extends to modulation of cell proliferation, differentiation and survival of cancer cells. Nevertheless, the identity of the specific isoforms contributing to this in acute myeloid leukemia (AML), the underlying mechanisms and the therapeutic value of ALDH inhibitors remain elusive. Methods. We conducted a meta-analysis to investigate the impact of ALDHs on clinicopathological parameters and survival in AML patients. The expression of ALDHs and their corresponding enzymatic activities were monitored in AML patient-derived cells (n=83) and pharmacologically inhibited with DIMATE, an irreversible suicide inhibitor of ALDH1 and ALDH3. Targeting of ALDH was evaluated both, in vitro and in orthotopic xenograft tumor models and the molecular mechanisms associated were studied in loss-of-function experiments, luciferase reporter assays and combination-index for DIMATE-drug interactions. Results. We detected gene amplification or mRNA upregulation of ALDH1 and ALDH3 subfamilies in about 45% of AML (n = 200). The expression of these isoenzymes revealed shorter survival times in patients and strong impact on chemotherapy resistance. Lower enzymatic activity of ALDH was noted in AML patients in the favorable cytogenetic risk group. Consistently, the ALDH activity was significantly higher in patients with refractory AML (&gt;5% blasts after first line treatment) compared to non-refractory patients (p&lt;0,005). Simultaneous inhibition of ALDH1 and 3 activates JNK signaling, compromises glutathione homeostasis and induces a redox state imbalance that is amplified by the accumulation of malondialdehyde and 4-hydroxynonenal-protein adducts. This condition ultimately results in severe oxidative damage and cell death. In AML xenografts with high to moderate resistance to cytarabine, combination with DIMATE promoted strong synergistic responses. Conclusion. These results provide a proof of concept that AML with increased expression of ALDH1 and 3 may benefit from strategies including inhibitors of these isoenzymes, as single agents or in combination with chemotherapy to overcome patient-specific drug resistance. For these patients, who often have poor responses and no alternative therapeutic plan, DIMATE could be a promising option. Disclosures Perez-Alea: Advanced Biodesign: Employment, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties: MPA reports patent WO/2017/064247. Guillaume:Advanced Biodesign: Patents &amp; Royalties: GM reports patent WO/2017/064247. Ceylan:Advanced Biodesign: Patents &amp; Royalties: IC reports patent WO/2017/064247.

CBMT-02. UP-REGULATION OF Γ-GLUTAMYL-TRANSFERASE CAN BE USED TO IMAGE GLIOBLASTOMA USING HYPERPOLARIZED Γ-GLUTAMYL-[1-13C]GLYCINE MRS

Abstract γ-glutamyl-transferase (GGT) is a key enzyme in the γ-glutamyl cycle, which regulates glutathione homeostasis. The enzyme is localized on the outer cell membrane and cleaves glutathione to glutamate and cysteinylglycine. As such, it facilitates uptake of the amino acids essential for intracellular synthesis of glutathione (GSH), which is the major thiol anti-oxidant. GGT is upregulated in glioblastoma, but remains low in normal brain. It is therefore an attractive molecular imaging target for specific detection of glioblastoma. The goal of our study was therefore to assess for the first time the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) for imaging glioblastoma in orthotopic tumor-bearing rats. Athymic nude rats with U87 glioblastoma tumors or tumor-free controls were investigated. First, we confirmed that GGT expression was significantly higher in U87 tumors compared to normal rat brain tissue. GSH levels were also higher. Imaging studies were then performed by injecting HP γ-Glu-[1-13C]Gly and acquiring dynamic 13C MR spectra using a flyback spectral-spatial slab sequence on a preclinical Bruker 3T MR system. The dynamic data were analyzed by measuring the signal-to-noise (SNR) ratios of the substrate (γ-Glu-[1-13C]Gly) and the product ([1-13C]Glycine). Comparison of control and tumor-bearing rats showed no statistically significant difference in the SNR of the substrate. In contrast, the SNR of the product demonstrated a significantly higher level of HP [1-13C]Glycine in the tumor-bearing rats compared to controls. Consistent with the higher levels of HP [1-13C]Glycine, the [1-13C]Gly-to-γ-Glu-[1-13C]Glycine ratio was also significantly higher in tumor-bearing animals relative to controls (0.046±0.004 vs 0.021±0.008 respectively; p=0.03). Further studies are needed to assess the generality of our findings. Nonetheless, this study demonstrates for the first time the feasibility of using γ-Glu-[1-13C]Gly to monitor GGT activity and thus could serve as a new approach for monitoring the presence of tumor.