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Highlights • Integrative GEMs can reveal the metabolic responses of rice to salinity stress. • Photosynthesis and hexose utilization were downregulated by salinity stress. • Photorespiration was enhanced and could contribute to redox homeostasis under salinity stress. • Flux predictions, metabolite levels and photosynthesis parameters were in good agreement., Rice is one of the most economically important commodities globally. However, rice plants are salt susceptible species in which high salinity can significantly constrain its productivity. Several physiological parameters in adaptation to salt stress have been observed, though changes in metabolic aspects remain to be elucidated. In this study, rice metabolic activities of salt-stressed flag leaf were systematically characterized. Transcriptomics and metabolomics data were combined to identify disturbed pathways, altered metabolites and metabolic hotspots within the rice metabolic network under salt stress condition. Besides, the feasible flux solutions in different context-specific metabolic networks were estimated and compared. Our findings highlighted metabolic reprogramming in primary metabolic pathways, cellular respiration, antioxidant biosynthetic pathways, and phytohormone biosynthetic pathways. Photosynthesis and hexose utilization were among the major disturbed pathways in the stressed flag leaf. Notably, the increased flux distribution of the photorespiratory pathway could contribute to cellular redox control. Predicted flux statuses in several pathways were consistent with the results from transcriptomics, end-point metabolomics, and physiological studies. Our study illustrated that the contextualized genome-scale model together with multi-omics analysis is a powerful approach to unravel the metabolic responses of rice to salinity stress.

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens., Highlights • Oxidative damage to the lens epithelium may lead to cataract. • Cells treated with N-acetylcysteine amide exhibited the highest viability. • Drugs that both increase GSH and scavenge ROS may protect lens epithelium.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions., Graphical abstract This review discusses common pitfalls in studies of acetaminophen hepatotoxicity.Image 1

Background Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli. Methods Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay. Results Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA. Conclusion Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence., Graphical abstract Image 1, Highlights • MIF augments phagosomal HOCl production. • This results in increased oxidation of bacterial glutathione. • MIF increases superoxide production in response to soluble stimuli. • This results in increased NET formation.

Objectives: The deficit of Glyoxalase I (Glo1) and the subsequent increase in methylglyoxal (MG) has been reported to be one the five mechanisms by which hyperglycemia causes diabetic late complications. Aldo-keto reductases (AKR) have been shown to metabolize MG; however, the relative contribution of this superfamily to the detoxification of MG in vivo, particularly within the diabetic state, remains unknown. Methods: CRISPR/Cas9-mediated genome editing was used to generate a Glo1 knock-out (Glo1−/−) mouse line. Streptozotocin was then applied to investigate metabolic changes under hyperglycemic conditions. Results: Glo1−/− mice were viable and showed no elevated MG or MG-H1 levels under hyperglycemic conditions. It was subsequently found that the enzymatic efficiency of various oxidoreductases in the liver and kidney towards MG were increased in the Glo1−/− mice. The functional relevance of this was supported by the altered distribution of alternative detoxification products. Furthermore, it was shown that MG-dependent AKR activity is a potentially clinical relevant pathway in human patients suffering from diabetes. Conclusions: These data suggest that in the absence of GLO1, AKR can effectively compensate to prevent the accumulation of MG. The combination of metabolic, enzymatic, and genetic factors, therefore, may provide a better means of identifying patients who are at risk for the development of late complications caused by elevated levels of MG. Keywords: Advanced glycation end products, Glyoxalase 1, Reactive metabolites, Methylglyoxal, Diabetic complications, Aldo-keto reductases

Monocytes/macrophages are thought to be recruited to the renal interstitium during calcium oxalate (CaOx) kidney stone disease for crystal clearance. Mitochondria play an important role in monocyte function during the immune response. We recently determined that monocytes in patients with CaOx kidney stones have decreased mitochondrial function compared to healthy subjects. The objective of this study was to determine whether oxalate, a major constituent found in CaOx kidney stones, alters cell viability, mitochondrial function, and redox homeostasis in THP-1 cells, a human derived monocyte cell line. THP-1 cells were treated with varying concentrations of CaOx crystals (insoluble form) or sodium oxalate (NaOx; soluble form) for 24 h. In addition, the effect of calcium phosphate (CaP) and cystine crystals was tested. CaOx crystals decreased cell viability and induced mitochondrial dysfunction and redox imbalance in THP-1 cells compared to control cells. However, NaOx only caused mitochondrial damage and redox imbalance in THP-1 cells. In contrast, both CaP and cystine crystals did not affect THP-1 cells. Separate experiments showed that elevated oxalate also induced mitochondrial dysfunction in primary monocytes from healthy subjects. These findings suggest that oxalate may play an important role in monocyte mitochondrial dysfunction in CaOx kidney stone disease., Graphical abstract fx1, Highlights • Oxalate is a major constituent of calcium oxalate (CaOx) kidney stones and can be found in either soluble or insoluble forms. • CaOx crystals are required for CaOx kidney stone formation. • Monocytes/macrophages play an important role in crystal clearance. • Oxalate causes mitochondrial dysfunction and disrupts redox homeostasis in monocytes.

Objective We previously demonstrated that diabetes exacerbates stroke-induced brain injury, and that this correlates with brain methylglyoxal (MG)-to-glutathione (GSH) status. Cerebral injury was reversed by N-acetylcysteine (NAC). Here we tested if the pro-thrombotic phenotype seen in the systemic circulation and brain during diabetes was associated with increased MG-glycation of proteins, and if NAC could reverse this. Methods The streptozotocin (STZ)-induced mouse model of type 1 diabetes was used. Thrombus formation in venules and arterioles (pial circulation) was determined by intravital videomicroscopy using the light-dye method. Circulating blood platelet-leukocyte aggregates (PLAs) were analyzed by flow cytometry 1 wk before other measurements. GSH and MG levels in platelets were measured by HPLC. MG-modified proteins, glutathione peroxidase-1 (GPx-1), and superoxide dismutase-1 (SOD1) levels were detected in platelets by western blot at 20 weeks. Proteins involved in coagulation were quantified by ELISA. NAC (2 mM) was given in drinking water for 3 weeks before the terminal experiment. Results Thrombus development was accelerated by diabetes in a time-dependent manner. % PLAs were significantly elevated by diabetes. Plasma activated plasminogen activator inhibitor type 1 levels were progressively increased with diabetes duration, with tail bleeding time reduced by 20 wks diabetes. Diabetes lowered platelet GSH levels, GPx-1 and SOD-1 expression. This was associated with higher MG levels, and increased MG-adduct formation in platelets. NAC treatment partly or completely reversed the effects of diabetes. Conclusion Collectively, these results show that the diabetic blood and brain become progressively more susceptible to platelet activation and thrombosis. NAC, given after the establishment of diabetes, may offer protection against the risk for stroke by altering both systemic and vascular prothrombotic responses via enhancing platelet GSH, and GSH-dependent MG elimination, as well as correcting levels of antioxidants such as SOD1 and GPx-1., Graphical abstract fx1, Highlights • Diabetes elevates dicarbonyl stress leading to enhanced thrombosis in the brain. • Glutathione levels decrease leading to impaired elimination of methylglyoxal in platelets during diabetes. • Platelet proteins are glycated and platelets form aggregates with leukocytes in diabetes. • Diabetes increases circulating levels of plasminogen activator inhibitor-1. • NAC, via GSH synthesis, reverses the platelet activation, protein glycation and pro-coagulation responses & protects against thrombosis in the diabetic brain.

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1 mM. In contrast, low and moderate doses of DEM (10–100 µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100 µM DEM, but not 1 mM DEM, whereas only 1 mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms., ISSN:2213-2317, Redox Biology, 11

Neurodegenerative diseases are linked to the accumulation of specific protein aggregates, suggesting an intimate connection between injured brain and loss of proteostasis. Proteostasis refers to all the processes by which cells control the abundance and folding of the proteome thanks to a wide network that integrates the regulation of signaling pathways, gene expression and protein degradation systems. This review attempts to summarize the most relevant findings about the transcriptional modulation of proteostasis exerted by the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2). NRF2 has been classically considered as the master regulator of the antioxidant cell response, although it is currently emerging as a key component of the transduction machinery to maintain proteostasis. As we will discuss, NRF2 could be envisioned as a hub that compiles emergency signals derived from misfolded protein accumulation in order to build a coordinated and perdurable transcriptional response. This is achieved by functions of NRF2 related to the control of genes involved in the maintenance of the endoplasmic reticulum physiology, the proteasome and autophagy., This paper was funded by grant SAF2016-76520-R of the Spanish Ministry of Economy and Competitiveness. MP is recipient of a FPU fellowship of Autonomous University of Madrid.

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset., Graphical abstract fx1

RAS proteins are critical regulators of signaling networks controlling diverse cellular functions such as cell proliferation and survival and its mutation are among the most powerful oncogenic drivers in human cancers. Despite intense efforts, direct RAS-targeting strategies remain elusive due to its “undruggable” nature. To that end, bulk of the research efforts has been directed towards targeting upstream and/or downstream of RAS signaling. However, the therapeutic efficacies of these treatments are limited in the long run due to the acquired drug resistance in RAS-driven cancers. Interestingly, recent studies have uncovered a potential role of RAS in redox-regulation as well as the interplay between ROS and RAS-associated signaling networks during process of cancer initiation and progression. More specifically, these studies provide ample evidence to implicate RAS as a redox-rheostat, manipulating ROS levels to provide a redox-milieu conducive for carcinogenesis. Importantly, the understanding of RAS-ROS interplay could provide us with novel targetable vulnerabilities for designing therapeutic strategies. In this review, we provide a brief summary of the advances in the field to illustrate the dual role of RAS in redox-regulation and its implications in RAS signaling outcomes and also emerging redox-based strategies to target RAS-driven cancers. Keywords: Cancer, RAS, Reactive Oxygen Species

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses., Graphical abstract Image 1, Highlights • A newly-validated plasma PDI assay shows high inter- but low intra-individual variability. • PDI-poor plasma carries protein signatures related to coagulation/immunoinflammation • PDI-rich plasma protein signatures relate to cell adhesion/housekeeping functions. • Endothelial exposure to such plasmas triggers corresponding gene and secretome patterns. • PDI-poor plasma impairs endothelial adhesion and migration vs. PDI-rich plasma.

Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H2O2, O2•-, ONOO-, thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O2•-. This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based., Graphical abstract In step 1 of the catalytic cycle, cationic Mn(III) substituted pyridylporphyrin (MnP) becomes oxidized from Mn(II) to a high-valent, highly oxidizing O=Mn(IV) species with an oxidant such as H2O2. This species closes the catalytic cycle by oxidizing protein cysteines and/or GSH in step 2 and gets ready for another cycle. In step 3 oxidation of the protein and/or glutathione results in protein S-glutathionylation.fx1, Highlights • Mn porphyrins were synthesized as SOD mimics. • The redox potential of the Mn porphyrins makes them equally able to act as oxidants and reductants which in turn allows them to act as catalytic redox cycling agents in vivo. • Mn porphyrins react equally well with H2O2, O2•-, ONOO-; cellular redox environment determines which species react with Mn porphyrins in vivo. • Mn porphyrins catalyze protein S-glutathionylation; global protein S-glutathionylation modulates cellular redox regulatory signaling networks. • Mn porphyrins act as chemo/radiosensitizers of tumor cells, but chemo/radioprotectors of normal tissue in pre-clinical disease models. • Clinical trials are underway to test Mn porphyrin efficacy in patients.

Endothelial dysfunction due to vascular inflammation and oxidative stress critically contributes to the etiology of atherosclerosis. The intracellular redox environment plays a key role in regulating endothelial cell function and is intimately linked to cellular thiol status, including and foremost glutathione (GSH). In the present study we investigated whether and how the dietary flavonoid, quercetin, affects GSH status of human aortic endothelial cells (HAEC) and their response to oxidative stress. We found that treating cells with buthionine sulfoximine to deplete cellular GSH levels significantly reduced the capacity of quercetin to inhibit lipopolysaccharide (LPS)-induced oxidant production. Furthermore, incubation of HAEC with quercetin caused a transient decrease and then full recovery of cellular GSH concentrations. The initial decline in GSH was not accompanied by a corresponding increase in glutathione disulfide (GSSG). To the contrary, GSSG levels, which were less than 0.5% of GSH levels at baseline (0.26±0.01 vs. 64.7±1.9 nmol/mg protein, respectively), decreased by about 25% during incubation with quercetin. As a result, the GSH: GSSG ratio increased by about 70%, from 253±7 to 372±23. These quercetin-induced changes in GSH and GSSG levels were not affected by treating HAEC with 500 µM ascorbic acid phosphate for 24 h to increase intracellular ascorbate levels. Incubation of HAEC with quercetin also led to the appearance of extracellular quercetin-glutathione conjugates, which was paralleled by upregulation of the multidrug resistance protein 1 (MRP1). Furthermore, quercetin slightly but significantly increased mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic and modifier subunits. Taken together, our results suggest that quercetin causes loss of GSH in HAEC, not because of oxidation but due to formation and cellular export of quercetin-glutathione conjugates. Induction by quercetin of GCL subsequently restores GSH levels, thereby suppressing LPS-induced oxidant production., Graphical abstract fx1, Highlights • Glutathione mediates the antioxidant effects of quercetin in human aortic endothelial cells. • Quercetin affects cellular levels of GSH and GSSG, resulting in an increased redox ratio. • Quercetin forms conjugates with GSH, which are rapidly excreted from the cells. • Quercetin induces glutamate-cysteine ligase and multidrug resistance protein 1 via Nrf2 activation.

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at −80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions., Highlights • Glutathione disulfide (GSSG) liposomes were developed to deliver GSSG into cells. • The GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds. • The delivered GSSG is confirmed to be functionally effective through protein S-glutathionylation.

Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit., Graphical abstract fx1

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress., Highlights • Construction of stably integrated Mrx1-roGFP2 in C. glutamicum. • Highly reducing basal EMSH of ~− 296 mV along the growth curve. • Mtr and SigH contribute to basal EMSH in C. glutamicum. • KatA and OxyR required for EMSH regeneration under oxidative stress. • Live imaging revealed stable biosensor expression and fluorescence.

Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological ‘gasotransmitter’, in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media – such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) – protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 μM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 μM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs., Graphical abstract fx1, Highlights • Carbon monoxide-releasing molecules (CORMs) are used for experimental CO delivery. • CORM-3 is a potent antimicrobial, but is reportedly beneficial to mammalian cells. • We demonstrate CORM-3 releases little CO in buffers and cell culture media. • Redox-active Ru2+ is the biological agent, binding tightly to metabolites e.g. thiol. • These results necessitate a major reappraisal of the biological effects of CORMs.

Objective Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6−/− compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. Methods We used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6−/− cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. Results We find that Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6−/− mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6−/− mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6-deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6−/− mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. Conclusion The protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6−/− mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit., Highlights • Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression and damage to the neurovascular unit. • The level of methylglyoxal (MG), which plays a crucial role in the development of DR is reduced in Trpc1/4/5/6−/− mice. • The protection against DR in Trpc1/4/5/6−/− mice may be mediated by an increase in GLO1 activity and protein expression.

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity., Graphical abstract fx1, Highlights • Uric acid decreased microbicide activity and release of cytokines by dHL-60 cells. • Uric acid decreased HOCl in cells and in the myeloperoxidase/Cl-/H2O2 system. • Uric acid induces a pro-oxidant redox imbalance. • HOCl is crucial for Pseudomonas aeruginosa killing by dHL-60.

Association of oxidative stress with carcinogenesis is well known, but not understood well, as is pathophysiology of oxidative stress generated during different types of anti-cancer treatments. Moreover, recent findings indicate that cancer associated lipid peroxidation might eventually help defending adjacent nonmalignant cells from cancer invasion. Therefore, untargeted metabolomics studies designed for advanced translational and clinical studies are needed to understand the existing paradoxes in oncology, including those related to controversial usage of antioxidants aiming to prevent or treat cancer. In this short review we have tried to put emphasis on the importance of pathophysiology of oxidative stress and lipid peroxidation in cancer development in relation to metabolic adaptation of particular types of cancer allowing us to conclude that adaptation to oxidative stress is one of the main driving forces of cancer pathophysiology. With the help of metabolomics many novel findings are being achieved thus encouraging further scientific breakthroughs. Combined with targeted qualitative and quantitative methods, especially immunochemistry, further research might reveal bio-signatures of individual patients and respective malignant diseases, leading to individualized treatment approach, according to the concepts of modern integrative medicine. Keywords: Carcinogenesis, Cancer, Oxidative stress, Lipid peroxidation, 4-hydroxynonenal, Glutathione, Metabolomics, Immunochemistry, Biomarkers, Omics science

Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses., Graphical abstract fx1, Highlights • Cytoprotection against tBHP-evoked oxidative stress by morin is Nrf2-regulated. • Morin prevents Nrf2-destabilization via PHLPP2-Akt-GSK3β-Fyn kinase pathway. • In silico docking studies confirmed PHLPP2 activity inhibition by morin. • Morin mitigates APAP induced cytotoxicity by suppressing PHLPP2 pathway.

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20 mM, 24–48 h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300 µM, 24–48 h) increased clonogenic cell killing in both human prostate (PC-3 and DU145) and human breast (MDA-MB231) cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH) synthesis (l-buthionine sulfoximine; BSO, 1 mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48 h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1 µM), was combined with 2DG+DHEA or DHEA-alone for 24 h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20 mM). Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1) oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC) were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231). Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing., Graphical abstract, Highlights • Inhibition of both glycolysis and pentose cycle causes oxidative stress in human breast and prostate cancer cells. • Combining inhibition of glycolysis and pentose cycle with inhibition of thioredoxin reductase enhances cell killing of these human cancer cells. • The toxicity and oxidative stress is selective for cancer vs. normal cells.

Ascorbic acid is synthesised by eukaryotes, the known exceptions being primates and some other animal groups which have lost functional gulonolactone oxidase. Prokaryotes do not synthesise ascorbate and do not need an ascorbate supply, so the functions that are essential for mammals and plants are not required or are substituted by other compounds. The ability of ascorbate to donate electrons enables it to act as a free radical scavenger and to reduce higher oxidation states of iron to Fe2+. These reactions are the basis of its biological activity along with the relative stability of the resulting resonance stabilised monodehydroascorbate radical. The importance of these properties is emphasised by the evolution of at least three biosynthetic pathways and production of an ascorbate analogue, erythroascorbate, by fungi. The iron reducing activity of ascorbate maintains the reactive centre Fe2+ of 2-oxoglutarate-dependent dioxygenases (2-ODDs) thus preventing inactivation. These enzymes have diverse functions and, recently, the possibility that ascorbate status in mammals could influence 2-ODDs involved in histone and DNA demethylation thereby influencing stem cell differentiation and cancer has been uncovered. Ascorbate is involved in iron uptake and transport in plants and animals. While the above biochemical functions are shared between mammals and plants, ascorbate peroxidase (APX) is an enzyme family limited to plants and photosynthetic protists. It provides these organisms with increased capacity to remove H2O2 produced by photosynthetic electron transport and photorespiration. The Fe reducing activity of ascorbate enables hydroxyl radical production (pro-oxidant effect) and the reactivity of dehydroascorbate (DHA) and reaction of its degradation products with proteins (dehydroascorbylation and glycation) is potentially damaging. Ascorbate status influences gene expression in plants and mammals but at present there is little evidence that it acts as a specific signalling molecule. It most likely acts indirectly by influencing the redox state of thiols and 2-ODD activity. However, the possibility that dehydroascorbylation is a regulatory post-translational protein modification could be explored., Graphical abstract fx1, Highlights • The function and metabolism of ascorbate is compared between plants and mammals. • Ascorbate readily reduces radicals and Fe/Cu. • Ascorbate is synthesised by plants, protists and animals via different pathways. • Fungi synthesise the analogue D-erythroascorbate. • Plants and photosynthetic protists use ascorbate peroxidase to remove H2O2.

Highlights • α-Cypermethrin induces toxicity in rats when administered at single high doses. • In humans, exposure is primarily via contaminated food. • Continuous dietary exposure does not cause oxidative stress or hepatotoxicity in rats. • Single-dose gastric-intubation studies may have overestimated its toxicity in rats., α-Cypermethrin is a widely used insecticide and, at high doses, induces oxidative stress in mammals. Curcumin is an antioxidant phytochemical commonly used for food coloring and flavoring. We aimed to investigate the effects of continuous dietary exposure to low doses of α-cypermethrin, as is the case in exposed humans, on oxidative stress and its potential prevention by dietary curcumin. Four groups of ten male Wistar rats were ad libitum-fed a control diet or identical diets fortified with α-cypermethrin (350 mg/kg diet), curcumin (1000 mg/kg diet), or α-cypermethrin and curcumin (350 and 1000 mg/kg diet, respectively) for 7 weeks. α-Cypermethrin accumulated in adipose tissues and was detectable in kidney, liver, and brains. Dietary α-cypermethrin did not alter concentrations of malondialdehyde, ascorbic and uric acid, retinol, liver damage markers, or the activities of CAT and SOD, but reduced vitamin E in blood. α-Cypermethrin did not affect malondialdehyde or reduced glutathione concentrations in any of the tissues, but significantly increased glutathione disulfide in kidney and subcutaneous adipose tissue. In conclusion, dietary exposure to small doses of α-cypermethrin did not induce oxidative stress in rats and may be less toxic than exposure to comparable quantities administered as single high doses by gastric intubation.

Oxidative stress-induced decrease in tissue or systemic glutathione (GSH) and damage to the vascular endothelium of the blood-brain barrier such as occurs in diabetes or stroke will have important implications for brain homeostasis. Endothelial proliferation or repair is crucial to preserving barrier function. Cell proliferation has been associated with increased intracellular GSH, but the kinetic and distribution of GSH during cell cycle is poorly understood. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) GSH distribution (6-h interval) during proliferation in a human brain microvascular endothelial cell line (IHEC). Control IHECs exhibited two peak S-phases of the cell cycle at 48 and 60 h post seeding that temporally corresponded to peak nuclear GSH levels and expression of cdk1, the S-to-G2-to-M checkpoint controller, suggesting a link between cell cycle progression and nuclear GSH. Sustained inhibition of GSH synthesis delayed S-to-G2/M cell transition; cell arrest in the S-phase was correlated with decreased total nuclear GSH and increased nuclear expressions of chk2/phospho-chk2 and GADPH. The temporal correspondence of nuclear chk2 activation and GAPDH expression with S-phase prolongation is consistent with enhanced DNA damage response and extended time for DNA repair. Strikingly, when GSH synthesis was restored, cell transit time through S-phase remained delayed. Significantly, total nuclear GSH remained depressed, indicating a time lag between restored cellular GSH synthetic capacity and recovery of the nuclear GSH status. Interestingly, despite a delay in cell cycle recovery, nuclear expressions of chk2/phospho-chk2 and GAPDH resembled those of control cells. This means that restoration of nuclear DNA integrity preceded normalization of the cell cycle. The current results provide important insights into GSH control of endothelial proliferation with implications for cell repair or wound healing in recovery post-oxidative damage., Graphical abstract Highlights ► Increased nuclear-to-cytosol GSH was associated with brain endothelial cell (IHEC) cell cycle progression. ► Inhibition of GSH synthesis decreased nuclear GSH and delayed S-to-G2 transition. ► S-phase prolongation was linked to elevated DNA damage response. ► Recovery of nuclear GSH and normal cell cycle lagged behind the restoration of GSH synthesis.

Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)–glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH]2:[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space., Graphical abstract, Highlights • Glutathionylation status of a 1-Cys glutaredoxin is a readout for [GSH]:[GSSG]. • Compartment-specific [GStot] is given by [GSH]:[GSSG] and [GSH]2:[GSSG]. • [GStot] is higher in the ER than in the cytosol of human cells.

Background The oxidative modifications of bioactive macromolecules have important roles in carcinogenesis. Of particular interest are lipid peroxidation products, which are involved in the activation of Nrf2 and endocannabinoids that affect cancer progression. Methods In lung cancer tissues (squamous cell lung carcinoma - SCC and adenocarcinoma - AC), the glutathione peroxidase and catalase activity and glutathione level, together with the expression of Nrf2 and its activators/inhibitors were estimated. The oxidative modifications of DNA (8-hydroxy-2′-deoxyguanosine and N7-methylguanine), endocannabinoids (anandamide and 2- arachidonylglyceriol), their receptors (CB1/2, TRV1, GPR55), phospholipid fatty acids (arachidonic, linoleic and docosahexaenoic), and reactive aldehydes (4-hydroxynonenal, 4-oxononenal and malondialdehyde) were determined. Results Tumour tissues showed lower antioxidant capacity than healthy tissues, which was accompanied by lower levels of fatty acids and higher levels of reactive aldehydes. Disturbances in antioxidant capacity and enhanced DNA oxidative modifications were observed in 88% of AC patients and 81% of SCC patients. The 4-hydroxynonenal-Histidine adducts were detected in the necrotic and stromal cells in all tumours. These findings were associated with the enhanced Nrf2 activity, especially in AC. The strong difference between the cancer subtypes was evident in the levels of endocannabinoids, with an increase in 89% of SCC and a decrease in 85% of AC patients being observed. Additionally, the increase in the expression of CB1/2 receptors was observed only in 82% of AC, while the expression of VR1 and GPR55 was enhanced in 79% of SCC and 82% of AC patients. Conclusions This study shows significant differences in the redox status, Nrf2 pathway and endocannabinoid system between SCC and AC tissues. Understanding the relation between the various lipid mediators and antioxidants in different lung cancer subtypes may be beginning for further research on the effective anticancer therapy., Graphical abstract fx1, Highlights • AC tissues show lower antioxidant capacity than SCC. • Nrf2/ARE pathway is activated stronger in AC than in SCC. • AC exhibits more lipid oxidative modifications than SCC. • HNE-protein adducts are detected in the necrotic and stromal cells in SCC and AC. • SCC exhibits increase in endocannabinoids level while in AC reverse effect is observed.

Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50–60% in the IMS, but Grx2 is increased by 1.4–2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron–sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron–sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ., Graphical abstract ., Highlights • Glutaredoxins play key roles in cellular redox regulation, which is sensitive to aging-dependent dysregulation. • Grx1 is diminished in the intermembrane space of mitochondria from aged heart; matrix Grx2 is increased but mostly in an inactive form. • The inactive Grx2 is selectively activated by superoxide. • Mitochondrial glutaredoxin changes may contribute to dysregulation of redox homeostasis during aging. • Changes in in situ activities of heart mitochondrial Grx1 and Grx2 with aging provide mechanistic insights for future studies.

Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper–zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity., Graphical abstract, Highlights • Multiple myeloma (MM) displays intrinsic/adaptive resistance to bortezomib (BTZ). • An up-regulation of antioxidant levels is observed in BTZ-resistant MM cell lines. • Inhibition of CuZnSOD increases BTZ cytotoxicity in BTZ naïve/resistant cells. • We propose disulfiram as a combination chemotherapy drug to inhibit relapse in MM.

Highlights • PDIL1;1 efficiently catalyzed both disulfide bond formation and disulfide bond reduction. • Two redox-active sites of PDIL1;1 were involved in disulfide reduction. • Disulfide reduction activity of PDIL1;1 increased with increasing GSH concentration., Protein disulfide isomerases (PDIs), a family of thiol-disulfide oxidoreductases that are ubiquitous in all eukaryotes, are the principal catalysts for disulfide bond formation. Here, we investigated three rice (Oryza sativa) PDI family members (PDIL1;1, PDIL1;4, and PDIL2;3) and found that PDIL1;1 exhibited the highest catalytic activity for both disulfide bond formation and disulfide bond reduction. The activity of PDIL1;1-catalyzed disulfide bond reduction, in which two redox-active sites were involved, was enhanced by increasing the glutathione concentration. These results suggest that PDIL1;1 plays primary roles in both disulfide bond formation and disulfide bond reduction, which allow for redox control of protein quality and packaging.

Anthocyanins are a class of phytochemicals that have generated considerable interest due to their reported health benefits. It has been proposed that commonly consumed anthocyanins, such as cyandin-3-O-β-glucoside (C3G), confer cellular protection by stimulating biosynthesis of glutathione (GSH), an endogenous antioxidant. Currently, it is unknown whether the health effects of dietary anthocyanins are genetically determined. We therefore tested the hypothesis that anthocyanin-induced alterations in GSH homeostasis vary by genetic background. Mice representing five genetically diverse inbred strains (A/J, 129S1/SvImJ, CAST/EiJ, C57BL/6J, and NOD/ShiLtJ) were assigned to a control or 100 mg/kg C3G diet (n=5/diet/strain) for six weeks. GSH and GSSG levels were quantified in liver, kidney, heart, pancreas, and brain samples using HPLC. The C3G diet promoted an increase in renal GSH concentrations, hepatic GSH/GSSG, and cardiac GSH/GSSG in CAST/EiJ mice. C3G treatment also induced an increase in pancreatic GSH/GSSG in C57BL/6J mice. In contrast, C3G did not affect GSH homeostasis in NOD/ShiLtJ mice. Surprisingly, the C3G-diet caused a decrease in hepatic GSH/GSSG in A/J and 129S1/SvImJ mice compared to controls; C3G-treated 129S1/SvImJ mice also exhibited lower total glutathione in the heart. Overall, we discovered that C3G modulates the GSH system in a strain- and tissue-specific manner. To our knowledge, this study is the first to show that the redox effects of anthocyanins are determined by genetic background., Graphical abstract fx1, Highlights • Effects of the anthocyanin C3G on the GSH system are genetically determined. • C3G causes oxidizing shifts in GSH redox potential in A/J and 129S1/SvImJ mice. • Effects of C3G on GSH were most significant in liver and least potent in brain.