NADPH Levels Research Articles

Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium simulating blood plasma led to a twofold decrease in the ATP level into them. Besides, the cell sizes as well as intracellular sodium concentration were increased. These findings could be the result of a disruption in ion transporters` functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH), decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The reason was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and probably, by decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.

Inhibiting G6PD by quercetin promotes degradation of EGFR T790M mutation

EGFRT790M mutation causes resistance to the first-generation tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, the therapeutic options for sensitizing first TKIs and delaying the emergence of EGFRT790M mutant are limited. In this study, we show that quercetin directly binds with glucose-6-phosphate dehydrogenase (G6PD) and inhibits its enzymatic activity through competitively abrogating NADP+ binding in the catalytic domain. This inhibition subsequently reduces intracellular NADPH levels, resulting in insufficient substrate for methionine reductase A (MsrA) to reduce M790 oxidization of EGFRT790M and inducing the degradation of EGFRT790M. Quercetin synergistically enhances the therapeutic effect of gefitinib on EGFRT790M-harboring NSCLCs and delays the acquisition of the EGFRT790M mutation. Notably, high levels of G6PD expression are correlated with poor prognosis and the emerging time of EGFRT790M mutation in patients with NSCLC. These findings highlight the potential implication of quercetin in overcoming EGFRT790M-driven TKI resistance by directly targeting G6PD.

G6PD Deficiency: Exploring the Relationship with Different Medical Disorders

G6PD deficiency (G6PDD) is associated with oxidative stress resulting from an imbalance between reactive oxygen species (ROS) production and the body's ability to counteract. In this review, we explore the adverse effects of G6PDD on diverse physiological processes and disease outcomes. Past studies have demonstrated the association between G6PDD and various other diseases, indicating a link between G6PDD to heightened oxidative stress by accelerating virus replication, worsening infection severity, and weakening the body's defense mechanisms. Such stress is critical in the destruction of red blood cells (RBCs) during infections and has a detrimental impact on redox signaling, ultimately impacting cell health and promoting cancer development. Furthermore, it impairs endothelial function by lowering the nitric oxide (NO) level and increasing stress, resulting in detrimental cardiac consequences and reduced myocardial antioxidant capacity. Because ROS contributes to inflammation, this imbalance causes conditions such as early atherosclerosis. It also compromises the functionality of NO-regulated bronchodilators and conditions such as G6PDD exacerbate the risks of kidney damage. Elevated ROS levels can also induce harm in retinal tissues, blood vessels, brain cells, and Beta-cells, hence quickening the progression of diseases like Diabetic Retinopathy. Furthermore, oxidative stress plays a significant role in cerebral ischemic pathogenesis, contributing to neurodegenerative disorders. Additionally, decreased NADPH level is vital for NO synthesis as it can impact blood vessel relaxation and can potentially lead to ischaemic priapism. Investigating the association between G6PDD and other medical conditions is crucial as it helps to identify possible approaches to mitigate oxidative stress, thereby preventing associated complications and diseases, particularly in situations where current treatment options are insufficient.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis MethodsIn this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. ResultsAfter PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. ConclusionIn ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

Quercetin ameliorates ferroptosis of rat cardiomyocytes viaactivation of the SIRT1/p53/SLC7A11 signalingpathway to alleviate sepsis‑induced cardiomyopathy.

Sepsis‑induced cardiomyopathy (SIC) is a manifestation of multiple organ failure as a result of sepsis and is a serious threat to life. Here, the effect and mechanisms of quercetin (QUE) in SIC were assessed. It was found that patients with SIC expressed lower serum levels of glutathione peroxidase 4 (GPX4) and SIRT1 but higher levels of CK‑MB, cTnI, TNF‑α, and IL‑6 compared with healthy individuals. A dose of 80µM QUE increased the viability and reduced the ferroptosis of H9C2 cells treated with 1.0µg/ml LPS invitro. The administration of QUE decreased the levels of MDA, NADPH, lipid peroxidation and cytoplasmic cytochrome C and upregulated the levels of GSH and TOM 20, thus exerting an anti‑oxidative effect via mediating SIRT1 expression. It also activated the SIRT1/p53/SLC7A11 signaling pathway to reduce cellular Fe2+ and PTGS2 levels, decreased cell apoptosis rate, and upregulated the levels of GPX4 and ferritin to inhibit ferroptosis of H9C2 cells invitro. Injection of QUE into rats activated the SIRT1/p53/SLC7A11 signaling pathway, reduced the levels of CK‑MB, cTnI, inflammatory cell infiltration, MDA, NADPH, cytoplasmic cytochrome C, cellular Fe2+, and PTGS2 but upregulated the levels of GSH, TOM 20, GPX4, and ferritin to alleviate SIC in a dose‑dependent manner invivo. To conclude, QUE exerted an anti‑ferroptotic effect via activation of the SIRT1/p53/SLC7A11 signaling pathway to dampen SIC both invivo and invitro. These findings highlighted a potential therapeutic strategy for the management of SIC.

Protective mechanism of fruit vinegar polyphenols against AGEs-induced Caco-2 cell damage.

Accumulation of advanced glycation end products (AGEs) is linked with development or aggravation of many degenerative processes or disorders. Fruit vinegars are rich in polyphenols that can be a good dietary source of AGEs inhibitors. In this study, eight kinds of vinegars were prepared. Among them, the highest polyphenol and flavonoid content were orange vinegar and kiwi fruit vinegar, respectively. Ferulic acid, vanillic acid, chlorogenic acid, p-coumaric acid, caffeic acid, catechin, and epicatechin were main polyphenols in eight fruit vinegars. Then, we measured the inhibitory effect of eight fruit vinegars on fluorescent AGEs, and found that orange vinegar had the highest inhibitory rate. Data here suggested that orange vinegar and its main components catechin, epicatechin, and p-coumaric acid could effectively reduce the level of ROS, RAGE, NADPH and inflammatory factors in Caco-2 cells. Our research provided theoretical basis for the application of orange vinegar as AGEs inhibitor.

DMT1 differentially regulates mitochondrial complex activities to reduce glutathione loss and mitigate ferroptosis.

Mitochondria are vital for energy production and redox homeostasis, yet knowledge of relevant mechanisms remains limited. Here, through a genome-wide CRISPR-Cas9 knockout screening, we have identified DMT1 as a major regulator of mitochondria membrane potential. Our findings demonstrate that DMT1 deficiency increases the activity of mitochondrial complex I and reduces that of complex III. Enhanced complex I activity leads to increased NAD+ production, which activates IDH2 by promoting its deacetylation via SIRT3. This results in higher levels of NADPH and GSH, which improve antioxidant capacity during Erastin-induced ferroptosis. Meanwhile, loss of complex III activity impairs mitochondrial biogenesis and promotes mitophagy, contributing to suppression of ferroptosis. Thus, DMT1 differentially regulates activities of mitochondrial complex I and III to cooperatly suppress Erastin-induced ferroptosis. Furthermore, NMN, an alternative method of increasing mitochondrial NAD+, exhibits similar protective effects against ferroptosis by boosting GSH in a manner similar to DMT1 deficiency, shedding a light on potential therapeutic strategy for ferroptosis-related pathologies.

Fuels for ROS signaling in plant immunity.

Reactive oxygen species (ROS) signaling has an important role in plant innate immune responses and is primarily mediated by NADPH oxidase, also known as respiratory burst oxidase homologs (RBOHs) in plants. NADPH serves as a fuel for RBOHs and limits the rate or amount of ROS production. Molecular regulation of RBOHs has been extensively studied; however, the source of NADPH for RBOHs has received little attention. Here, we review ROS signaling and the regulation of RBOHs in the plant immune system with a focus on NADPH regulation to achieve ROS homeostasis. We propose an idea to regulate the levels of NADPH as part of a new strategy to control ROS signaling and the corresponding downstream defense responses.

Propyl gallate postharvest treatment improves the storability of longans by regulating the metabolisms of respiratory and disease-resistance substances

The influences of 0.5 mM propyl gallate, a ROS scavenger, on the metabolisms of respiratory and disease-resistance substances in ‘Fuyan’ longan fruit during storage at 15 °C were explored. Compared to the control longan, propyl gallate-treated longan displayed a higher rate of commercially acceptable fruit, the lower indices of pulp breakdown and fruit disease, and a lower rate of fruit weight loss. Additionally, propyl gallate-treated longan exhibited a lower rate of fruit respiration, the lower activities of SDH, PGI, CCO, AAO, PPO and AOX, and the lower levels of NAD and NADH, but the higher activities of NADK and G-6-PDH + 6-PGDH, and the higher levels of NADP and NADPH. Furthermore, propyl gallate-treated longan manifested the higher activities of CHI, GLU, PAL, POD, CAD, C4H and 4-CL, and the higher contents of lignin. These data indicated that, propyl gallate-improved the storability of longan was related to the fact that propyl gallate reduced respiration and increased disease resistance of longan through decreasing the respiratory pathways of EMP and TCA cycle, and raising PPP respiratory pathway, as well as through increasing the metabolism of disease-resistance substances. Above findings suggest that 0.5 mM propyl gallate has potential application in prolonging the shelf-life and restraining the fruit spoilage of postharvest fresh longan.

Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1/PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage

Increasing evidence shows that metabolic factors are involved in the pathological process of osteoarthritis (OA). Lactate has been shown to contribute to the onset and progression of diseases. While whether lactate is involved in the pathogenesis of OA through impaired chondrocyte function and its mechanism remains unclear. This study confirmed that serum lactate levels were elevated in OA patients compared to healthy controls and were positively correlated with synovial fluid lactate levels, which were also correlated with fasting blood glucose, high-density lipoprotein, triglyceride. Lactate treatment could up-regulate expressions of the lactate receptor hydroxy-carboxylic acid receptor 1 (HCAR1) and lactate transporters in human chondrocytes. We demonstrated the dual role of lactate, which as a metabolite increased NADPH levels by shunting glucose metabolism to the pentose phosphate pathway, and as a signaling molecule up-regulated NADPH oxidase 4 (NOX4) via activating PI3K/Akt signaling pathway through receptor HCAR1. Particularly, lactate could promote reactive oxygen species (ROS) generation and chondrocyte damage, which was attenuated by pre-treatment with the NOX4 inhibitor GLX351322. We also confirmed that lactate could increase expression of catabolic enzymes (MMP-3/13, ADAMTS-4), reduce the synthesis of type II collagen, promote expression of inflammatory cytokines (IL-6, CCL-3/4), and induce cellular hypertrophy and aging in chondrocytes. Subsequently, we showed that chondrocyte damage mediated by lactate could be reversed by pre-treatment with N-Acetyl-l-cysteine (NAC, ROS scavenger). Finally, we further verified in vivo that intra-articular injection of lactate in Sprague Dawley (SD) rat models could damage cartilage and exacerbate the progression of OA models that could be countered by the NOX4 inhibitor GLX351322. Our study highlights the involvement of lactate as a metabolic factor in the OA process, providing a theoretical basis for potential metabolic therapies of OA in the future.

Wheat dihydroflavonol-4-reductase like gene confers disease resistance by modulating NADPH pool and lignin biosynthesis

Hm1 is the first cloned disease resistance gene (R) from maize which encodes an enzyme that detoxifies Helminthosporium carbonum (HC) toxin from the fungal pathogen Cochliobolus carbonum. The homologous genes for Hm1 have been mined in various crop plants and their sequences are showed similar to dihydroflavonol-4-reductase (DFR), a key enzyme in flavonoid biosynthesis, therefore they are collectively named dihydroflavonol-4-reductase like (DFRL). The biological functions of DFRL are largely elusive. In the present study, DFRL gene was cloned from wheat, namely TaDFRL. TaDFRL was barely expressed in leaf, stem and root tissues; however, its expression level was rapidly increased following rust infection. Biochemical analysis showed that TaDFRL had the broad spectrum of substrate preference, including dihydroflavonol, flavonol and flavone, and could use both NAD and NADP as co-enzyme, which was quite distinct from DFR. Overexpressing TaDFRL in tobacco altered NAD(H) and NADP(H) pools towards to high NADPH levels. Subsequently, the gene expression of cinnamyl alcohol dehydrogenase (CAD) was up-regulated and lignin accumulation was increased. These brought about to enhance resistance to wildfire disease in tobacco plants. This research provides novel insights into DFRL mechanism, which boost host defense responses by elevating NADPH level and lignin biosynthesis.

Hsp27 over expression protect against cadmium induced nephrotoxicity in Drosophila melanogaster

Cadmium (Cd) exposure to the animals including humans is reported as nephrotoxic compounds i.e., disturbing redox status (increase oxidative stress), mitochondrial dysfunction, renal cell death and altered transporters in the renal system. Hsp27 (a small heat shock protein) has been shown as one of the modulators in the renal dysfunction and increased against the Cd induced toxicity. However, no studies are reported on the genetic modulation of stress protein against the Cd-induced nephrotoxicity. The current study aimed to examine the protective role of hsp27 overexpression against the Cd-induced nephrotoxicity using Drosophila melanogaster as an animal model. D. melanogaster renal system includes nephrocytes and Malpighian tubules (MTs) that show the functional similarity with mammalian kidney nephron. Overexpression of the hsp27 was found to reduce the Cd induced oxidative stress, rescue cell death in MTs of Cd exposed D. melanogaster larvae. The rescued GSH level, NADPH level and glucose 6 phosphate dehydrogenase (G6PD) activity were also observed in the MTs of the Cd exposed organism. Function (efflux activity and fluid secretion rate) of the MTs was restored in Cd exposed hsp27 overexpressed larvae. Further, results were confirmed by restored brush border microvilli density and reduced uric acid level. Tissue specific knockdown of hsp27 developed Cd like phenotypes in MTs and the phenotypes enhanced in Cd exposed condition. The present study clearly shows the role of hsp27 overexpression in restoration of the MTs function and protection against the Cd induced renal toxicity.

ETS1 Ameliorates Hyperoxia-Induced Bronchopulmonary Dysplasia in Mice by Activating Nrf2/HO-1 Mediated Ferroptosis.

Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD. Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP+/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1. ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe2+) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression. ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.

Exploring the Potential of a Genome-Reduced Escherichia coli Strain for Plasmid DNA Production.

The global demand for nucleic acid-based vaccines, including plasmid DNA (pDNA) and mRNA vaccines, needs efficient production platforms. However, conventional hosts for plasmid production have encountered challenges related to sequence integrity due to the presence of insertion sequences (ISs). In this study, we explored the potential of a genome-reduced Escherichia coli as a host for pDNA production. This strain had been constructed by removing approximately 23% of the genome which were unessential genes, including the genomic unstable elements. Moreover, the strain exhibits an elevated level of NADPH, a coenzyme known to increase plasmid production according to a mathematical model. We hypothesized that the combination of genome reduction and the abundance of NADPH would significantly enhance pDNA production capabilities. Remarkably, our results confirmed a three-fold increase in pDNA production compared to the widely employed DH5α strain. Furthermore, the genome-reduced strain exhibited heightened sensitivity to various antibiotics, bolstering its potential for large scale industrial pDNA production. These findings suggest the genome-reduced E. coli as an exciting candidate for revolutionizing the pDNA industry, offering unprecedented efficiency and productivity.

S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells.

Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein's cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO's bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.

Hemolysis and Metabolic Lesion of G6PD Deficient RBCs in Response to Dapsone Hydroxylamine in a Humanized Mouse Model.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans (~5% of all individuals). G6PD deficiency (G6PDd) is caused by an unstable enzyme and manifests most strongly in red blood cells (RBCs) that cannot synthesize new protein. G6PDd RBCs have decreased ability to mitigate oxidative stress due to lower levels of NADPH, as a result of a defective pentose phosphate pathway. Accordingly, oxidative drugs can result in hemolysis and potentially life-threatening anemia in G6PDd patients. Dapsone is a highly useful drug for treating a variety of pathologies but oral dapsone is contraindicated in patients with G6PDd due to oxidative stress-induced anemia. Dapsone must be metabolized to become hemolytic. Dapsone hydroxylamine (DDS-NOH) has been implicated as the major hemolytic dapsone metabolite, but this has never been tested on G6PDd RBCs with in vivo circulation as a metric. Moreover, the metabolic lesion caused by DDS-NOH is unknown. We report that RBCs from a novel humanized mouse expressing the human Mediterranean G6PD-deficient variant have increased sensitivity to DDS-NOH. In addition, we show that DDS-NOH damaged RBCs can either undergo sequestration (with subsequent return to circulation) or permanent removal in a dose-dependent manner, with G6PD-sufficient RBCs mostly being sequestered, and G6PDd RBCs mostly being permanently removed. Finally, we characterize the metabolic lesion caused by DDS-NOH in G6PDd RBCs and report a blockage in terminal glycolysis resulting in a cellular accumulation of pyruvate. These findings confirm DDS-NOH as a hemolytic metabolite and elucidate metabolic effects of DDS-NOH on G6PDd RBCs. Significance Statement These findings confirm that dapsone hydroxylamine, an active metabolite of dapsone, causes in vivo clearance of murine RBCs expressing a human variant of deficient G6PD, an enzymopathy that affects half a billion individuals (G6PD deficiency). Both cellular mechanisms of clearance (sequestration vs. destruction) and specific metabolic disturbances caused by dapsone hydroxylamine are elucidated, providing novel mechanistic understanding.

CISPLATIN - AN OVERVIEW OF ITS EFFICIENCY AND TOXICITY

Cisplatin is the first heavy metal compound that has been found to possess antineoplastic activity. It is effective in treating testicular, ovarian, head and neck, bladder, cervical, esophageal tumors, and small cell lung carcinoma. Approximately 1% of cisplatin that enters the cell interacts with DNA, forming DNA-cisplatin bonds. Both apoptosis and necrosis can be found in the same population of cells exposed to cisplatin, and the mode of cell death depends on the cisplatin concentration and metabolic state of the target cell. In the bloodstream, the platinum component of cisplatin binds to the blood's proteins (hemoglobin, albumin and transferrin), and other significant portion binds to the glutathione and other cysteine-rich biomolecules. Cisplatin impairs the mitochondrial and cell antioxidant defense system (decreases GSH, NADPH levels, GCH/GSSG ratio, and increases GSSG levels) leading to oxidative stress. There are three main mechanisms of cell resistance to cisplatin: (1) enhanced repair of cisplatin-induced DNA lesions, (2) decrease in uptake and/or increase in efflux and (3) inactivation of cisplatin intracellularly. The usage of cisplatin is limited due to its toxicity and side effects, which include neurotoxicity (numbness and tingling, paresthesia, reduced deep tendon reflexes), nephrotoxicity (renal insufficiency, hypomagnesemia), ototoxicity (tinnitus and bilateral high-frequency hearing loss), cardiotoxicity (changes in electric heart activity, congestive heart failure), gastrotoxicity (nausea, vomiting, and dyspepsia), etc. So far, there has been no effective, clinically administered, therapy for cisplatin-induced toxicity.

Effect of postharvest nicotinamide treatment on NAD+ metabolism and redox status in strawberry fruit during storage

The increased activity of PARP enzymes is associated with a deficiency of NAD+, as well as with a loss of NADPH and ATP, and consequent deterioration of the redox state in fruits. In this study, we checked whether treatment with nicotinamide (NAM) would affect PARP-1 expression and NAD+ metabolism in strawberry fruit during storage. For this purpose, strawberry fruits were treated with 10 mM NAM and co-treated with NAM and UV-C, and then stored for 5 days at 4 °C. Research showed that nicotinamide contributes to reducing oxidative stress level by reducing PARP-1 mRNA gene expression and the protein level resulting in higher NAD+ availability, as well as improving energy metabolism and NADPH levels in fruits, regardless of whether they are exposed to UV-C. The above effects cause fruits treated with nicotinamide to be characterised by higher anti-radical activity, and a lower level of reactive oxygen species in the tissue.

Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells.

Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.