Major Intracellular Antioxidant Research Articles

Evaluation of a novel pyridinium cation-linked styryl-based boronate probe for the detection of selected inflammation-related oxidants

Reactive oxygen and nitrogen species (RONS) are a range of chemical individuals produced by living cells that contribute to the proper functioning of organisms. Cells under oxidative and nitrative stress show excessive production of RONS (including hydrogen peroxide, H2O2, hypochlorous acid, HOCl, and peroxynitrite, ONOO−) which may result in a damage proteins, lipids, and genetic material. Thus, the development of probes for in vivo detection of such oxidants is an active area of research, focusing on molecular redox sensors, including boronate-caged fluorophores. Here, we report a boronate-based styryl probe with a cationic pyridinium moiety (BANEP+) for the fluorescent detection of selected biological oxidants in vitro and in vivo. We compare the chemical reactivity of the BANEP+ probe toward H2O2, HOCl, and ONOO− and examine the influence of the major intracellular non-enzymatic antioxidant molecule, glutathione (GSH). We demonstrate that, at the physiologically relevant GSH concentration, the BANEP+ probe is efficiently oxidized by peroxynitrite, forming its phenolic derivative HNEP+. GSH does not affect the fluorescence properties of the BANEP+ and HNEP+ dyes. Finally, we report the identification of a novel type of molecular marker, with the boronate moiety replaced by the iodine atom, formed from the probe in the presence of HOCl and iodide anion. We conclude that the reported chemical reactivity and structural features of the BANEP+ probe may be a basis for the development of new red fluorescent probes for in vitro and in vivo detection of ONOO−.

PSI-14 Weaning Stress Increases Bile Cysteine and Cysteinylglycine Concentrations in Pigs

Abstract Weaning is a critical time for pigs that is associated with nutritional, social, and environmental stressors that lead to reduced feed intake and poor nutrient utilization. Cysteine is a sulfur amino acid and a key constituent of glutathione (GSH), a major intracellular antioxidant and potential regulator of intestinal redox status. Glutathione is present in biliary secretions and may be an important source of cysteine to intestine in newly weaned pigs. The objective of this study was to determine amino thiol and amino acid concentrations in bile. Piglets (initial BW = 7,233 ± 683 g) were either weaned on day 21 of age (W; n = 9) and euthanized after 2 d or remained on the sow until day 23 of age (NW; n = 9) and were euthanized. Weaned pigs were fasted and allowed free access to water. After euthanasia, bile (directly from the gallbladder), jejunum, and ileum samples were collected. Bile amino acids and bile amino thiols were analyzed by reversed phase HPLC following precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and 4-fluoro-7-aminosulfonylbenzofurazan, respectively. Reverse transcription quantitative real-time PCR was performed to measure cysteine transporter expression in jejunum and ileum. Cysteine concentration was greater in W than NW pigs (1,203 versus 324 ± 99 µmol/L; P < 0.0001). Both γ-glutamylcysteine (7.1 versus 2.2 ± 2.1 µmol/L), the direct precursor for GSH synthesis, and cysteinylglycine (203 versus 110 ± 30 µmol/L, the direct product of GSH degradation, were greater in W pigs than in NW pigs (P < 0.05). Bile GSH concentration tended to be greater in W than NW pigs (9.1 versus 5.3 ± 2.8 µmol/L; P = 0.07). Taurine, derived from cysteine oxidation, was greater in W than NW pigs (180 versus 91 ± 23 µmol/L; P < 0.05), whereas glutamate (11.4 versus 8.8 ± 1.9 µmol/L) and glycine (463 versus 403 ± 62 µmol/L), additional amino acid constituents of GSH, were not different between groups (P > 0.10). Expression of cysteine transporters SLC1A4, SLC1A5, SLC6A14, SLC6A19, SLC7A9, and SLC7A11 in jejunum and ileum were not different between groups (P > 0.10). Despite no feed intake after weaning, cysteine concentrations increase in bile. While this is likely due to bile accumulation in the gallbladder after weaning, high bile cysteine, cysteinylglycine, and glycine concentrations could potentially be a source of these amino acids for enterocytes during weaning stress.

Antioxidant Effect of a Dietary Supplement Containing Fermentative S-Acetyl-Glutathione and Silybin in Dogs with Liver Disease

Oxidative stress is often involved in liver disease progression. Liver is the primary site for the synthesis of glutathione (GSH), the major intracellular antioxidant. GSH erythrocyte concentration can decrease in case of liver damage. So, the use of food supplements with antioxidant capacity has been reported in the veterinary literature. In this case-control study, we tested a new supplement containing S-acetyl-glutathione (SAG), silybin, and other antioxidant ingredients in dogs affected by liver disease. After two weeks of supplement administration, we were able to report a significant increase in the level of erythrocyte GSH in the treated (TRT) group, nearly reaching the physiological limit at the end of the study. In addition, most of the key liver parameters are significantly reduced in the TRT group by the end of the trial. The results of this study support the effectiveness of the tested complementary feed, which may be helpful in managing dogs with liver conditions.

Serum levels of reduced glutathione, oxidized glutathione, and glutathione reductase activity in minor recurrent aphthous stomatitis patients

Background/purposeRecurrent aphthous stomatitis (RAS) is one of the most prevalent oral mucosa diseases with unknown etiology. Reduced glutathione (GSH) is a major intracellular non-protein physiological antioxidant, and it has been demonstrated that GSH deficiency may be related to cardiovascular, immune, and diabetes. The purpose of this investigation was to evaluate the potential roles of GSH, oxidized glutathione (GSSG), and glutathione reductase (GR) in the etiopathogenesis of minor recurrent aphthous stomatitis (MiRAS). Materials and methodsThe study comprised 87 patients with idiopathic MiRAS and 90 race-, age-, and gender-matched healthy individuals. The spectrophotometric method was used to determine serum GSH and GSSG concentrations as well as GR activity. The GSSG/GSH ratios were subsequently computed. For statistical evaluation, the independent sample t test, Pearson's chi-square test, Mann–Whitney U test, Kruskal–Wallis H test, and Binary logistic regression analysis were used. ResultsThe serum GSSG level, GR activity and GSSG/GSH ratio were statistically higher in MiRAS patients, whereas the concentration of serum GSH was significantly decreased. With the exception of GR, serum GSSG, GSH, and GSSG/GSH were all significantly associated with MiRAS. Serum GSSG can be regarded as a risk factor, whereas serum GSH and GSSG/GSH maybe considered as protective factors against the occurrence of MiRAS. ConclusionGSSG may be a potential danger factor to MiRAS and GSH may be a protective factor, while GR may not play an important role in the aetiopathogenesis of MiRAS.

Plasma glutathione as a risk marker for the severity and functional outcome of acute atherothrombotic and cardioembolic stroke

ObjectiveGlutathione (GSH) is a major intracellular thiol-containing antioxidant. We tried to determine whether blood plasma GSH level is a marker for the severity of the two subtypes of acute stroke (large-artery atherosclerosis, LA and cardioembolic, CE). Forty-three patients with LA and 36 patients with CE aged 65 (47–82) years were included in the study. Thirty-one patients with cerebral microangiopathy were included for comparison. Total (t) and reduced (r) GSH levels were determined at admission. Neurological deficit was assessed by the National Institutes of Health Stroke Scale (NIHSS) on the first day, functional outcome and independence were assessed by the modified Rankin scale (mRs) and Bartel index (BI), respectively, after 21 days.ResultsThe tGSH and rGSH levels in acute stroke were significantly lower than cerebral microangiopathy patients. Low tGSH (≤ 1.45 μM) and rGSH (≤ 30 nM) levels were risk markers for stroke severity at admission (NIHSS > 10) in patients with LA: age and gender adjusted odds ratio (AOR) was 4.95, 95% coincidence interval (CI) 1.31–18.7, AOR = 9.141, CI 1.84–45.3 for t- and rGSH, respectively. A low level of rGSH (≤ 30 nM) was found as risk marker for functional independence (BI ≤ 60: AOR = 15.9, CI 2.22–114.2) in patients with LA. Low tGSH level (≤ 1.1 μM) was associated with the reduction of poor outcome risk (mRs > 2: AOR = 0.154, CI 0.029–0.809) in CE group.ConclusionsLow t- and rGSH levels may be considered potential risk markers for severity and insufficient functional independence in LA. Conversely, low tGSH level reduce the risk of poor stroke outcome only for CE.

Protective Role of Glutathione in the Hippocampus after Brain Ischemia.

Stroke is a major cause of death worldwide, leading to serious disability. Post-ischemic injury, especially in the cerebral ischemia-prone hippocampus, is a serious problem, as it contributes to vascular dementia. Many studies have shown that in the hippocampus, ischemia/reperfusion induces neuronal death through oxidative stress and neuronal zinc (Zn2+) dyshomeostasis. Glutathione (GSH) plays an important role in protecting neurons against oxidative stress as a major intracellular antioxidant. In addition, the thiol group of GSH can function as a principal Zn2+ chelator for the maintenance of Zn2+ homeostasis in neurons. These lines of evidence suggest that neuronal GSH levels could be a key factor in post-stroke neuronal survival. In neurons, excitatory amino acid carrier 1 (EAAC1) is involved in the influx of cysteine, and intracellular cysteine is the rate-limiting substrate for the synthesis of GSH. Recently, several studies have indicated that cysteine uptake through EAAC1 suppresses ischemia-induced neuronal death via the promotion of hippocampal GSH synthesis in ischemic animal models. In this article, we aimed to review and describe the role of GSH in hippocampal neuroprotection after ischemia/reperfusion, focusing on EAAC1.

Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders

Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Human Platelets Stored at Room Temperature Demonstrate Diminished Resistance to Oxidative Stress Compared to Those Stored at 4°C

Background: Current blood banking practices dictate that platelets can only be stored at room temperature (RT) for up to seven days because of functional decline and risk of bacterial overgrowth. A lower storage temperature has the potential to slow platelet metabolism and reduce oxidative damage during storage. In the current study, we quantified by mass spectrometry small molecule thiols and disulfides as markers of oxidative stress in platelets stored in plasma at RT or 4°C. Because glutathione (GSH), γGlu-Cys-Gly, is the major thiol in platelets, we also analyzed metabolites (amino acids and dipeptides) involved in GSH synthesis and degradation cycles. Method: Platelets were acquired from five healthy donors and adjusted to 3 × 1011/L in plasma. The platelets from each donor were split into two bags and stored in plasma at RT or 4°C (with or without agitation, respectively) for up to 14 days. Aliquots from each group were taken at days 0, 3, 7, and 14, and N-ethylamaleimide (NEM) was added to block free thiols before storing the samples at -80°C until analysis. Total and reduced forms of GSH and cysteine including protein-bound disulfides were quantified by mass spectrometry as described previously [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]. Paired t-tests were used to evaluate the differences between the groups. Results: GSH is the major intracellular antioxidant that maintains a reducing environment within cells. After 7 days of storage at RT, reduced GSH decreased drastically from 13.0±1.2 pre-storage to 1.7±0.6 µM (p <0.005), an 85% decrease. In contrast, GSH in platelets stored at 4°C decreased at a much slower rate, with over 60% and 50% GSH remaining after 7 and 14 days of storage, respectively, indicating that platelets stored at 4°C maintain higher reducing capacities than those stored at RT. The decrease in reduced GSH can be caused by oxidation and/or degradation. If it is primarily due to oxidation, total GSH ― the sum of reduced and oxidized (disulfides) forms ― should remain relatively constant. The total GSH in the stored platelets decreased in a manner similar to the reduced GSH at both RT and 4°C, suggesting that degradation, not oxidation, was responsible for the decrease in reduced GSH. We then determined the quantities of metabolites related to GSH synthesis and degradation and found significant increases in the total amounts of the amino acids Glu, Cys, and Gly, suggesting that more GSH was degraded than synthesized in platelets during storage. We also examined the changes of Cys during storage at RT and 4°C because Cys is the main thiol in plasma and the platelets were stored in 100% plasma. Protein-bound Cys was significantly elevated in the samples stored at RT compared to those stored at 4°C, the differences being observed as early as day 3 and continuing to day 14 day (p<0.01). We previously identified protein-bound Cys as a marker for oxidative stress in plasma [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]; its elevation is therefore indicative of increased oxidation of the platelet sample during RT storage compared to 4°C storage. Summary: In this study, we found that the ability of platelets to withstand oxidative stress during storage is severely compromised in platelets stored at RT as opposed to 4°C. This storage lesion affected both intracellular (GSH) and extracellular (Cys) antioxidant molecules and involved 2 mechanisms: GSH degradation and Cys oxidation. We are currently attempting to determine whether the differences observed result from increased metabolic rate in the RT-stored platelets or from greater exposure to ambient air caused by the need for agitation. These studies will identify targets for improving the quality and shelf-life of platelets stored for transfusion. Disclosures No relevant conflicts of interest to declare.

A Ketogenic Diet Is Protective Against Atherosclerosis in Apolipoprotein E Knockout Mice

Abstract Objectives A ketogenic diet (KD) positively impacts cardiovascular disease (CVD) risk factors yet its effect on atherosclerosis, the main cause of CVD, is elusive. We hypothesize that, when compared to a high-fat diet (HF), KD protects from atherosclerosis. Methods Seven-week-old male Apoe−/− mice, a model for human atherosclerosis, were fed ad libitum (%kcal) KD (81-fat, 1-carbohydrate, 18-protein; n = 4) or HF (40-fat, 42-carbohydrate, 18-protein; n = 5). After 4, 8 and 12 weeks, plasma was collected and used to (1) quantify beta-hydroxybutyrate levels (OH-But) by a colorimetric assay; or (2) assess systemic inflammation, a key feature associated with atherosclerosis, using a panel of inflammatory cytokines; or (3) explore diet-driven changes in levels of atherosclerosis-relevant metabolites using a targeted metabolomic approach by triple quadrupole mass spectrometry. At the endpoint, mice were euthanized and their perfusion-fixed aortas were subjected to 3-D analysis by magnetic resonance imaging to quantify the extent of atherosclerosis. Data were reconstructed using Matlab and segmented to obtain atherosclerotic plaque volumes using Avizo 9.0. Results The inflammatory cytokines were significantly (P < 0.05, Student's t-test) elevated in HF-mice compared to KD-mice after 12 weeks; MIP-1α, (25 ± 13 vs 13 ± 4 pg/mol), MCP-1 (165 ± 67 vs 69 ± 23 pg/mol), TNF-α(51 ± 17 vs 35 ± 5 pg/mol), and IL-1β (2 ± 2 vs 0.6 ± 0.1 pg/mol). OH-But levels were always significantly higher in KD-mice than in HF-mice, thus confirming the presence of ketosis in KD-mice. Significant changes in the plasma metabolome of KD-mice, when compared to HF-mice, were present for dimethylated arginines, gut-derived metabolites, certain bile acids, some acylcarnitines and ceramides, certain sphingolipids and cholesterol esters, and constituents of the major intracellular antioxidant glutathione. Lastly, aortic plaque burden was significantly decreased in KD mice (2.77 ± 1.02%) than in HF mice (13.94 ± 3.72%). Conclusions KD was associated with decreased inflammation, changes in several metabolic intermediates and an atheroprotective effect based on MRI analysis. Funding Sources Huck Institutes of the Life Sciences; Graduate Program in Nutritional Sciences.

Association of GPx1 P198L and CAT C-262T Genetic Variations With Polycystic Ovary Syndrome in Chinese Women.

Background: Oxidative stress plays an important role in the pathogenesis of polycystic ovary syndrome (PCOS). Glutathione peroxidase 1 (GPx1) and catalase (CAT) are the major intracellular antioxidant enzymes that can detoxify hydrogen peroxide into water, preventing cellular injury from reactive oxygen species. The aim of the present study was to investigate the association of GPx1 P198L (Pro198Leu, C559T, rs1050450) and CAT C-262T (rs1001179) genetic polymorphisms with the risk of PCOS and evaluate the effects of the genotypes on clinical, hormonal, metabolic and oxidative stress parameters in Chinese women.Methods: This is a case-control study of 654 patients with PCOS and 535 controls. The GPx1 P198L, CAT C-262T, and superoxide dismutase 2 (SOD2) A16V genotypes were determined by polymerase chain reaction amplification and restriction analysis. Clinical, hormonal, metabolic and oxidative stress parameters were also analyzed.Results: The frequencies of the PL + LL genotype (14.1 vs. 8.4%) and L allele (7.3 vs. 4.4%) of GPx1 P198L polymorphism were significantly higher in patients with PCOS than in control subjects. Genotype (PL + LL) remained a significant predictor for PCOS in prognostic models including age, body mass index (BMI), insulin resistance index, total cholesterol, triglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol as covariates (OR = 2.105, 95%CI: 1.330–3.331, P = 0.001). Patients carrying the L allele had relatively high average ovarian volume, waist circumference, and malondialdehyde levels (P < 0.07) compared with patients with the PP genotype. We also demonstrated that the subjects with both GPx1 L and SOD2 A alleles further increase the risk of PCOS compared with the individuals carrying the PP/VV genotype after adjusting for age and BMI (OR = 5.774, 95%CI: 2.243–14.863, P < 0.001). However, no significant differences were observed in the frequencies of the CAT C-262T genotypes and alleles between PCOS and control groups.Conclusions: The GPx1 P198L, but not CAT C-262T, genetic polymorphism is associated with the risk of PCOS in Chinese women.

Drug repurposing studies of PARP inhibitors as a new therapy for inherited retinal degeneration.

The enzyme poly-ADP-ribose-polymerase (PARP) has important roles for many forms of DNA repair and it also participates in transcription, chromatin remodeling and cell death signaling. Currently, some PARP inhibitors are approved for cancer therapy, by means of canceling DNA repair processes and cell division. Drug repurposing is a new and attractive aspect of therapy development that could offer low-cost and accelerated establishment of new treatment options. Excessive PARP activity is also involved in neurodegenerative diseases including the currently untreatable and blinding retinitis pigmentosa group of inherited retinal photoreceptor degenerations. Hence, repurposing of known PARP inhibitors for patients with non-oncological diseases might provide a facilitated route for a novel retinitis pigmentosa therapy. Here, we demonstrate and compare the efficacy of two different PARP inhibitors, BMN-673 and 3-aminobenzamide, by using a well-established retinitis pigmentosa model, the rd1 mouse. Moreover, the mechanistic aspects of the PARP inhibitor-induced protection were also investigated in the present study. Our results showed that rd1 rod photoreceptor cell death was decreased by about 25-40% together with the application of these two PARP inhibitors. The wealth of human clinical data available for BMN-673 highlights a strong potential for a rapid clinical translation into novel retinitis pigmentosa treatments. Remarkably, we have found that the efficacy of 3 aminobenzamide was able to decrease PARylation at the nanomolar level. Our data also provide a link between PARP activity with the Wnt/β-catenin pathway and the major intracellular antioxidant concentrations behind the PARP-dependent retinal degeneration. In addition, molecular modeling studies were integrated with experimental studies for better understanding of the role of PARP1 inhibitors in retinal degeneration.

Folate/Vitamin B12 Supplementation Combats Oxidative Stress-Associated Carcinogenesis in a Rat Model of Colon Cancer

Folate and vitamin B12 deficiency is associated with depletion of the major intracellular antioxidant glutathione, and oxidative stress is emerging as an etiological mechanism for colon cancer. Azoxymethane (AOM), a potent carcinogen, induces colon cancer in rats by causing pathophysiological changes and oxidative stress. We investigated the synergistic effect of folate and vitamin B12 supplementation against AOM-induced carcinogenesis and oxidative stress in rat colon. Adult male rats were distributed into four groups: 1) Basal diet only; 2) AOM injection (15 mg/kg once per week in weeks 5 and 6); 3) Folate and vitamin B12 supplemented diet; 4) Folate and B12 diet with AOM injection. After 16 weeks, rats were sacrificed, colon tissue dissected, indicators of oxidative stress were measured, and immunohistochemical and ultrastructural changes were evaluated. AOM-injected rats showed oxidative stress, evident by glutathione depletion, oxidation of cellular proteins, and DNA oxidative damage. AOM increased mucosal levels of antiapoptotic and proapoptotic proteins Bcl2 and Bax and caused ultrastructure changes in colonic cell organelles. Folate and vitamin B12 supplementation decreased the level of oxidative stress and ameliorated the cytotoxic effects of AOM. In this in vivo experimental model of colon cancer, folate and vitamin B12 supplementation combats carcinogen-induced oxidative stress.

F8. SEARCHING FOR A STRATIFICATION MARKER FOR ANTIOXIDANT USE IN SCHIZOPHRENIA AND BIPOLAR DISORDER: A META-ANALYSIS OF MRS STUDIES OF ANTERIOR CINGULATE GLUTATHIONE

BackgroundGlutathione [GSH] is a major intracellular antioxidant that disposes peroxides and protects neurons and glial cells from oxidative stress. In both schizophrenia and bipolar disorder, atypical levels of GSH has been demonstrated, particularly in the anterior cingulate cortex (ACC), though no consistent results have emerged due to limitations in sample size. Examining the state of GSH deficit in schizophrenia is a critical step when attempting to correct putative redox imbalance in this illness using agents such as N-Acetyl Cysteine (NAC). We conducted a meta-analysis to investigate the aberrations in GSH levels in the ACC of patients with schizophrenia and bipolar disorder measured using magnetic resonance spectroscopy (MRS).MethodsMedline, Google Scholar, Ovid Online and EMBASE databases were searched for studies published until September 2017. Search terms included magnetic resonance spectroscopy, MRS, schizophrenia, psychosis, psychotic, bipolar disorder, glutathione, GSH. We included all 1H-MRS studies reporting GSH values for patients satisfying DSM or ICD based criteria for a primary psychotic disorder (SCZ) or bipolar disorder (BPAD) in comparison to a healthy controls (HC) group. We screened all identified abstracts, filtered studies that did not satisfy inclusion criteria, hand-searched references and contacted experts to locate further studies. We excluded studies that reported only on comorbid illnesses, did not compare patients and HCs, or failed to report data required to construct effect size metrics. After initial screening, a total of 261 patients and 185 controls were considered for the meta-analysis from the SCZ group; 464 patients and 245 controls were considered for the meta-analysis from the BPAD group. A random-effects, inverse-weighted variance model was used to calculate the pooled effect size. Mean values were extracted and verified independently. Effect sizes were computed based on excel macro, produced by Major Depressive Disorder Neuroimaging Database investigators.ResultsContrary to our expectations, in SCZ, there were no significant differences in ACC GSH in patients compared to HC (RFX p= 0.74; 95% CI, -0.24 to 0.17; FFX p= 0.71; heterogeneity p = 0.58). In BPAD, there were highly significant differences in the ACC GSH, with patients having higher GSH concentrations than HC (RFX: p= 0.0003; 95% CI, 0.14 to 0.5; heterogeneity p=0.70). In the BPAD group, the mean effect size (SMD) was d= 0.32, indicating a small to medium sized difference. A network meta-analysis revealed significantly higher GSH levels in BPAD compared to SCZ (RFX p= 0.01; 95% CI, 0.08 to 0.63; SMD=0.36; heterogeneity p = 0.71). There were several methodological issues in the reported studies. Notably, most acquisitions were not optimized to collect GSH spectra; polymorphisms in the glutamate-cysteine ligase catalytic gene (GCLC) were not quantified in most studies; voxel placements were not standardized across the published studies; majority of patients were medicated, in various stages of illness.DiscussionThere are no major differences in concentration of ACC glutathione in the anterior cingulate cortex in patients with schizophrenia, though in bipolar disorder, GSH levels appear elevated. Given that GSH is the most readily accessible cortical redox marker in vivo, current status of MRS literature is insufficient to prepare for stratified therapeutics with antioxidants among patients with schizophrenia. Nevertheless, abnormalities in the redox system may be more pronounced in bipolar disorder compared to schizophrenia, and could serve to guide stratification in samples lacking diagnostic clarity (e.g. in First Episode Psychosis clinics).

Rapamycin Protects Skin Fibroblasts from Ultraviolet B-Induced Photoaging by Suppressing the Production of Reactive Oxygen Species

Background/Aims: Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin photoaging. Murine dermal fibroblasts (MDFs) were subjected to a series of 4 sub-cytotoxic UVB doses (120 mJ/cm²), resulting in changes in cell shape, DNA damage, cell cycle arrest, extracellular matrix variations, reactive oxygen species (ROS) generation, and alterations in major intracellular antioxidant and cellular autophagy levels. Rapamycin (RAPA) is a new macrolide immunosuppressive agent that is primarily used in oncology, cardiology, and transplantation medicine and has been found to extend the lifespan of genetically heterogeneous mice. Several studies have shown that RAPA may have anti-aging effects in cells and organisms. Thus, in this study, we explored the effects and mechanisms of RAPA against the photoaging process using a well-established cellular photoaging model. Methods: We developed a stress-induced premature senescence (SIPS) model through repeated exposure of MDFs to ultraviolet B (UVB) irradiation. The cells were cultured in the absence or presence of RAPA for 48 h. Senescent phenotypes were assessed by examining cell viability, cell morphology, senescence-associated β-galactosidase (SA-β-gal) expression, cell cycle progression, intracellular ROS production, matrix metalloproteinase (MMP) synthesis and degradation, extracellular matrix (ECM) component protein expression, alterations in major intracellular antioxidant levels, and the cellular autophagy level. Results: Compared with the UVB group, pretreatment with RAPA (5 µM) significantly decreased the staining intensity and percentage of SA-β-gal-positive cells and preserved the elongated cell shape. Moreover, cells pretreated with RAPA showed inhibition of the reduction in the type I collagen content by blocking the UVB-induced upregulation of MMP expression. RAPA also decreased photoaging cell cycle arrest and downregulated p53 and p21 expression. RAPA application significantly attenuated irradiation-induced ROS release by modulating intracellular antioxidants and increasing the autophagy level. Conclusions: Our study demonstrated that RAPA elicited oxidative damage in vitro by reducing ROS accumulation in photoaged fibroblasts. The anti-aging effect can be attributed to the maintenance of normal antioxidant and cellular autophagy levels. However, determination of the definitive mechanism requires further study.

Abstract 105: Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate at 7%. The oncogene KRAS that is mutationally activated in over 90% of PDACs, has been shown to be involved in regulating redox homeostasis. Hence, perturbation of oxidative balance might provide a therapeutic window to effectively treat pancreatic cancer. One strategy to achieve this is to target the tripeptide glutathione (GSH) – a major intracellular antioxidant. Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported, predominantly as cystine (CSSC) that is then reduced intracellularly to Cys. In cancer, intracellular Cys synthesis has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation, which includes maintenance of oxidative balance through GSH synthesis. We hypothesize that this increased requirement for Cys/CSSC import in tumor cells will make them selectively sensitive to prolonged depletion of these amino acids in the serum by a genetically engineered human enzyme called Cyst(e)inase. In addition, we believe that combining Cyst(e)inase with other redox balance perturbing agents will produce a synergistic therapeutic effect. In our study, Cyst(e)inase treatment of cultured pancreatic cancer cell lines (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Sensitivity to Cyst(e)inase was correlated with ROS accumulation (Panc1&amp;gt;MIA-PaCa2&amp;gt;BxPC3). Panc1 cells exhibited G2-arrest and apoptotic cell death following 24 hours of treatment whereas BxPC3 cells underwent only a G1-arrest and no cell death even after 72 hours of treatment. Further mechanistic investigation showed activation of AMP kinase and other stress related kinases (p38, ERK and JNK), and DNA damage signaling (ATM) only in the more sensitive cell lines. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway was observed in all 3 cell lines. Cyst(e)inase displayed synergistic cytotoxicity when combined with buthionine sulfoximine (GSH synthesis inhibitor), auranofin (thioredoxin reductase inhibitor), sulfasalazine (inhibitor of cystine import) and the natural compound curcumin known to increase intracellular ROS, indicating that concurrently inhibiting alternative cellular antioxidant pathways or directly increasing intracellular ROS might improve the anti-tumor efficacy of Cyst(e)inase. The effect of biweekly intraperitoneal Cyst(e)inase treatment on growth of pancreatic cancer cell xenografts in nude mice will also be reported. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for pancreatic cancer. Citation Format: Sabin Kshattry, Achinto Saha, Shira Cramer, John DiGiovanni, Stefano Tiziani, Nathalie Munoz, George Georgiou, Everett Stone. Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2017-105

Abstract 2132: Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth

Abstract Prostate cancer (PCa) is the most common non-skin neoplasm and second leading cause of cancer death in men in the USA. The major drawbacks of PCa treatment is the development of resistance to androgen ablation therapy. Due to abnormal growth and genetic alterations, cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than the normal cells. The L-cysteine (Cys) containing tripeptide, glutathione (GSH) is the major intracellular antioxidant and is essential for the survival and proliferation of cancer cells. Under conditions of elevated ROS, endogenous Cys production is insufficient for GSH synthesis. This necessitates uptake of Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. Cys is a non-essential amino acid in animals; therefore, eliminating Cys and CSSC uptake should selectively impact tumors that display increased ROS production, without causing an adverse effect on normal physiology. However, inhibition of xCT(-) alone is insufficient because free Cys is still imported via other transporters. A superior approach is the elimination of Cys and CSSC through the action of an enzyme that converts these amino acids into non-toxic products. Based on the idea that enzyme mediated systemic depletion of the serum Cys/CSSC pool would constitute a powerful and completely novel therapeutic approach, we developed a genetically engineered and pharmacologically optimized human enzyme called cyst(e)inase. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular Cys and CSSC pool in mice. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts and reduced tumor growth in PCa xenografts. Mechanistically, cyst(e)inase treatment increased AMPK phosphorylation, reduced mTORC1 activity, formation of LC3 II as well as modulation of several cell cycle proteins including p27, c-Myc, CDK2, CDK4, pRB, E2F4 and cyclins A, D1 and E1. Further studies showed cyst(e)inase produced synergistic effects with a GSH synthesis inhibitor, buthionine sulfoximine and the natural compound, curcumin for cell growth inhibition and ROS production in vitro. Cyst(e)inase also showed synergistic tumor growth inhibition with curcumin in a xenograft model of human castrate resistant PCa cells. Collectively, enzyme-mediated depletion of serum Cys and CSSC pool suppresses the growth of prostate tumors, and is very well tolerated. These results suggest that cyst(e)inase represents a potentially safe and effective therapeutic modality as a single agent or in combination for the treatment of prostate and possibly other cancers. Citation Format: Achinto Saha, Shira L. Cramer, Sabin Kshattry, Stefano Tiziani, Everett Stone, George Georgiou, John DiGiovanni. Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2132. doi:10.1158/1538-7445.AM2017-2132

Glutathione system in Wolfram syndrome 1‑deficient mice.

Wolfram syndrome 1 (WS) is a rare neurodegenerative disease that is caused by mutations in the Wolfram syndrome 1 (WFS1) gene, which encodes the endoplasmic reticulum (ER) glycoprotein wolframin. The pathophysiology of WS is ER stress, which is generally considered to induce oxidative stress. As WS has a well‑defined monogenetic origin and a model for chronic ER stress, the present study aimed to characterize how glutathione (GSH), a major intracellular antioxidant, was related to the disease and its progression. The concentration of GSH and the activities of reduction/oxidation system enzymes GSH peroxidase and GSH reductase were measured in Wfs1‑deficient mice. The GSH content was lower in most of the studied tissues, and the activities of antioxidative enzymes varied between the heart, kidneys and liver tissues. The results indicated that GSH may be needed for ER stress control; however, chronic ER stress from the genetic syndrome eventually depletes the cellular GSH pool and leads to increased oxidative stress.

Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l-methionine (met) and l-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759-770, 2017.

GSH: A Double-Edged Sword

As the most abundant thiol compound, the reduced form of glutathione (GSH) has won a great title of being a major intracellular antioxidant. Indeed, GSH has been well known to play a major role in the detoxification of reactive oxygen species (ROS) and electrophilic compounds either via direct reaction with the ROS or electrophiles or serving as an electron donor for enzymatic antioxidants, including glutathione peroxidase, glutathione S-transferase, and glutaredoxin. On the other hand, under certain conditions, GSH may become a detrimental factor for the host cells. In this context, several recent studies suggest that GSH may augment bacterial virulence, promotes tumorigenesis, and even contributes to the development of anxiety. This Research Highlights article briefly surveys the classical and atypical biological activities of GSH to better define this famous intracellular tripeptide as a double-edged sword.

Abstract 367: Assessing the therapeutic efficacy of Cyst(e)inase to induce oxidative stress mediated cytotoxicity in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma is the only cancer in the US with a dismal one-digit 5-year survival rate (7%). Its oncogenic program relies on upregulated ROS antioxidant mechanisms to maintain oxidative balance. Hence, perturbation of this balance might be an effective therapeutic strategy for pancreatic cancer. A major intracellular antioxidant is the tripeptide glutathione (GSH). Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported [predominantly as cystine (CSSC) that is reduced intracellularly to Cys]. In cancer, de novo synthesis of Cys has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation. This intricate balance between ROS and antioxidants, and the excessive requirement for Cys/CSSC import in tumor cells, in part to maintain oxidative balance through GSH synthesis, potentially provides a therapeutic window. We believe that prolonged depletion of the serum Cys and CSSC pool by a genetically engineered human enzyme called Cyst(e)inase may provide a pancreatic cancer therapeutic avenue either as a monotherapy or in combination with other agents with low or minimal toxicity. In this study, Cyst(e)inase treatment (3-500 nM) of cultured pancreatic cancer cell lines (MIA-PaCa2, BxPC3, Panc1) caused a dose-dependent decrease in survival and intracellular GSH content. Further mechanistic investigation showed an increase in intracellular ROS, activation of AMP kinase and autophagy signaling, as well as inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway. There was also a concentration dependent reduction in cyclin D1 after treatment with the enzyme. Cyst(e)inase displayed synergistic cytotoxicity when combined with the natural compound curcumin. Notably, a single intraperitoneal 100 mg/kg dose of PEGylated Cysteinase in mice was able to deplete serum CSSC below 5 μM (normal is ∼ 70 μM) for over 3 days with a half-life of ∼4 days. Longer treatment with Cyst(e)inase at this dose given 4 times/week for 6 weeks did not produce significant signs of toxicity. This in vivo dose also inhibits the growth of prostate cancer cells in xenograft tumor models. Ongoing experiments to evaluate the in vivo efficacy of Cyst(e)inase for inhibition of pancreatic tumor growth as well as further mechanistic studies will be presented. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for several cancers including pancreatic cancer. Citation Format: Sabin Kshattry, Achinto Saha, Shira Cramer, Everett M. Stone, George Georgiou, John DiGiovanni. Assessing the therapeutic efficacy of Cyst(e)inase to induce oxidative stress mediated cytotoxicity in pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 367.