Increase In GSSG Research Articles

Effects of H9N2 avian influenza virus infection on metabolite content and gene expression in chick DF1 cells

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.

Erythrocytic Reduced/Oxidized Glutathione and Serum Thiol/Disulfide Homeostasis in Patients with Opioid Use Disorder.

This study aimed to evaluate oxidative damage by measuring erythrocytic reduced/oxidized glutathione as an intracellular thiol pool and serum thiol/disulfide homeostasis as an extracellular thiol pool in patients with opioid use disorder. In this prospective cross-sectional study, 33 male patients diagnosed with opioid use disorder and 30 healthy male controls were included. Sociodemographic characteristics and psychometric analyzes were performed and addiction characteristics (duration and amount of heroin use, usage methods) were recorded. For the evaluation of oxidative balance, intracellular reduced-oxidized glutathione (reduced glutathione and oxidized glutathione), and extracellular thiol-disulfide (native thiol and disulfide) levels were measured. There was a decrease in reduced glutathione and native thiol levels and an increase in GSSG and SS levels. Similarly, while oxidized/reduced glutathione, oxidized/total glutathione%, and disulfide/native thiol % ratios increased, the ratio of reduced glutathione/total glutathione% and native thiol/total thiol% decreased. Moreover, a positive correlation was found between the level of both intracellular and extracellular oxidant molecules and the duration and amount of opioid use. Impaired intracellular reduced glutathione/oxidized glutathione and extracellular disulfide/native thiol homeostasis were found in patients with opioid use disorder. The intracellular and extracellular oxidative stress may cause complications related to chronic opioid use.

The role of oxidative stress, tumor and inflammatory markers in colorectal cancer patients: A one-year follow-up study

Oxidative stress (OS) and inflammation are known to play an important role in colorectal cancer (CRC). This study analyzed tumor, inflammatory and OS markers in CRC patients and in a control group. In addition, the evolution of these markers was evaluated after one-year of follow-up treatment. This was a longitudinal and prospective, observational study in 80 CRC patients who were candidates for tumor resection surgery and/or chemo-radiotherapy treatment and a healthy control group (n = 60). Subsequently, catalase (CAT), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSSG/GSH ratio in serum and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and F2-IsoProstanes (F2-IsoPs) in urine at 1, 6 and 12 months after treatment was analyzed. Tumor markers (CEA and CA 19.9), as well as inflammatory markers—leukocytes, neutrophils, neutrophil/lymphocyte (N/L) index, platelets, fibrinogen, C-reactive protein (CRP), and interleukin 6 (IL6)— were also analyzed. As expected, levels of CEA and CA 19.9 and markers of inflammation, except CRP, were significantly higher in CRC compared to the control group. Regarding OS markers, a decrease in CAT and GSH and an increase in GSSG, GSSG/GSH ratio, 8-oxodG and F2-IsoPs were found in CRC patients compared to healthy controls at baseline. After treatment, an improvement of their inflammation profile was accompanied by a progressive recovery of antioxidant enzyme activities and the decline of oxidative byproducts both in serum and urine. Based on the results obtained, we propose the assay of urinary 8-oxodG and F2-IsoPs, as well as serum CAT, GSH, GSSG as a marker for the evaluation of OS and the clinical follow-up of CRC patients.

Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

While redox processes play a vital role in maintaining intracellular homeostasis by regulating critical signaling and metabolic pathways, supra-physiological or sustained oxidative stress can lead to adverse responses or cytotoxicity. Inhalation of ambient air pollutants such as particulate matter and secondary organic aerosols (SOA) induces oxidative stress in the respiratory tract through mechanisms that remain poorly understood. We investigated the effect of isoprene hydroxy hydroperoxide (ISOPOOH), an atmospheric oxidation product of vegetation-derived isoprene and a constituent of SOA, on intracellular redox homeostasis in cultured human airway epithelial cells (HAEC). We used high-resolution live cell imaging of HAEC expressing the genetically encoded ratiometric biosensors Grx1-roGFP2, iNAP1, or HyPer, to assess changes in the cytoplasmic ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), and the flux of NADPH and H2O2, respectively. Non-cytotoxic exposure to ISOPOOH resulted in a dose-dependent increase of GSSG:GSH in HAEC that was markedly potentiated by prior glucose deprivation. ISOPOOH-induced increase in glutathione oxidation were accompanied by concomitant decreases in intracellular NADPH. Following ISOPOOH exposure, the introduction of glucose resulted in a rapid restoration of GSH and NADPH, while the glucose analog 2-deoxyglucose resulted in inefficient restoration of baseline GSH and NADPH. To elucidate bioenergetic adaptations involved in combatting ISOPOOH-induced oxidative stress we investigated the regulatory role of glucose-6-phosphate dehydrogenase (G6PD). A knockout of G6PD markedly impaired glucose-mediated recovery of GSSG:GSH but not NADPH. These findings reveal rapid redox adaptations involved in the cellular response to ISOPOOH and provide a live view of the dynamic regulation of redox homeostasis in human airway cells as they are exposed to environmental oxidants.

Naphthalimide Fluorescent Skeleton for Facile and Accurate Quantification of Glutathione.

Glutathione (GSH), the most prevalent nonprotein thiol in biological systems, acts as both an antioxidant to manipulate intracellular redox homeostasis and a nucleophile to detoxify xenobiotics. The fluctuation of GSH is closely related to the pathogenesis of diverse diseases. This work reports the construction of a nucleophilic aromatic substitution-type probe library based on the naphthalimide skeleton. After an initial evaluation, the compound R13 was identified as a highly efficient GSH fluorescent probe. Further studies demonstrate that R13 could readily quantify GSH in cells and tissues via a straightforward fluorometric assay with a comparable accuracy to the results from the HPLC. We then used R13 to quantify the content of GSH in mouse livers after X-ray irradiation, revealing that irradiation-induced oxidative stress leads to the increase of oxidized GSH (GSSG) and depletion of GSH. In addition, probe R13 was also applied to investigate the alteration of the GSH level in the Parkinson's mouse brains, showing a decrease of GSH and an increase of GSSG in Parkinson's mouse brains. The convenience of the probe in quantifying GSH in biological samples facilitates further understanding of the fluctuation of the GSH/GSSG ratio in diseases.

Effect of ginsenoside CK combined with cisplatin on the proliferation and migration of human cervical cancer HeLa cells via Ras/ERK/MAPK pathway

Ginsenoside (G-CK) has good chemo preventive and anticancer effects. However, its antimetastatic effects and underlying mechanisms on cervical cancer (CC) are still not clear. This study found that both G-CK and DDP inhibited the proliferation of HeLa cells in a concentration-dependent manner, and the combination blocked cell growth and colony-forming ability, and inhibited cell migration and EMT processes. The signature molecule of EMT, E-cadherin, was up-regulated, and both N-cadherin and vimentin were down-regulated and cell migration was affected through inhibition of RAS/MEK/ERK and PI3K/AKT signaling pathways. DDP could induce oxidative stress damage in HEK293 cells, as evidenced by the accumulation of ROS, decrease in GSH levels and oxidative stress antioxidant enzymes (CAT, SOD and GSH-Px) and increase in GSSG and MDA levels, which proved that G-CK exerted inhibitory on HeLa cell proliferation and EMT in combination with traditional anti-cancer drug and protected normal cells from DDP-induced oxidative stress damage.

Quantification of reduced and oxidized glutathione in wine by MALDI-TOF MS

Reduced glutathione (GSH) is one of the important antioxidants in the winemaking and aging process and it also regulates the color and flavor of wines. Accurate and rapid quantification of GSH is crucial in wine quality control. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to quantify GSH and oxidized glutathione (GSSG) of wine in a high-throughput way. Quinoline-5,8-dione was used as a label to react with GSH, and the derivative of GSH, glutamate-cysteine-alanine, acted as the internal standard (IS). The quantitative decrease of GSH and the increase in GSSG were detected with a relative standard deviation of ≤ 5%, after the wines were exposed to air from one to seven days, and the concentration changes of GSH and GSSG were measured by simple dilution. All the results suggested the feasibility and potential of this method for wider applications in the quantification of GSH and GSSG.

L-Carnitine prevents memory impairment induced by post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a genuine obstructing mental disorder. As indicated by the name, it is related to the patients' stress augmented by life-threatening conditions or accidents. The PTSD has linked to oxidative stress that can result in neurodegeneration. L-carnitine (L-CAR) is known for its antioxidant properties, which can protect against neuronal damage. In the current study, we investigated the beneficial effects of L-CAR on the memory impairment induced by PTSD using a rat model. A model of single-prolonged stress (a cycle of restraining, forced swimming, rest, and finally diethyl ether exposure for 2 h, 20 min, 15 min, and 1-2 min, respectively) was used to induce PTSD-like behavior. Intraperitoneal L-CAR treatment (300 mg/kg/day) was introduced for four weeks. Both memory and special learning were evaluated utilizing the radial arm water maze (RAWM). Moreover, the levels of glutathione peroxidase (GPx), glutathione reduced (GSH), and glutathione oxidized (GSSG) were assessed as biomarkers oxidative stress in the hippocampus. The results demonstrated that both the short and long-term memories were impaired by PTSD/SPS model (P < 0.05), while L-CAR treatment prevented this memory impairment in PTSD rats. Besides, L-CAR prevented the reduction in GPx activity and increase in GSSG, which were altered in the hippocampus of the PTSD/SPS rats (P < 0.05). Levels of GSH were not changed in PTSD and/or L-CAR rats. L-CAR administration prevented short- and long-term memories' impairments induced in the PTSD/SPS rat model. This is probably related to its antioxidant effects in the hippocampus.

Organophosphorus pesticides exhibit compound specific effects in rat precision-cut lung slices (PCLS): mechanisms involved in airway response, cytotoxicity, inflammatory activation and antioxidative defense

Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is the predominant toxic mechanism, OP may induce pneumonia and formation of lung edema after poisoning and during clinical treatment as life-threatening complication. To investigate the underlying mechanisms, rat precision-cut lung slices (PCLS) were exposed to the OP parathion, malathion and their biotransformation products paraoxon and malaoxon (100–2000 µmol/L). Airway response, metabolic activity, release of LDH, cytokine expression and oxidative stress response were analyzed. A concentration-dependent inhibition of airway relaxation was observed after exposure with the oxon but not with the thion-OP. In contrast, cytotoxic effects were observed for both forms in higher concentrations. Increased cytokine expression was observed after exposure to parathion and paraoxon (IL-6, GM-CSF, MIP-1α) and IL-6 expression was dependent on NFκB activation. Intracellular GSH levels were significantly reduced by all four tested OP but an increase in GSSG and HO-1 expression was predominantly observed after malaoxon exposure. Pretreatment with the antioxidant N-acetylcysteine reduced malaoxon but not paraoxon-induced cytotoxicity. PCLS as a 3D lung model system revealed OP-induced effects depending on the particular OP. The experimental data of this study contribute to a better understanding of OP toxicity on cellular targets and may be a possible explanation for the variety of clinical outcomes induced by different OP.

Asthma-like symptoms and oxidative stress in adults from the GEIRD Cohort

BACKGROUND AND AIM: Oxidative Stress (OS) is an imbalance between reactive oxygen species production and scavenging. This pre-pathological condition could be involved in the perpetuation and amplification of airway inflammation and other chronic diseases. The present study aimed at investigating OS in both healthy and asthma-like-diseased subjects. METHODS: Overall, 208 subjects were randomly selected from the GEIRD Cohort (Gene Environment Interaction in Respiratory Diseases). Based on the positive response to the Methacholine Challenge Testing or the obstructive impairment tested by spirometry, 124 volunteers were classified as “healthy”, while 84 as suffering from Asthma-Like Symptoms (A-LSs). Subjects participated after providing their informed consent, underwent anthropometric measurements, and provided a urinary sample. Urinary 15-F2t-IsoP and GSSG were quantified (ELISA technique) as biomarkers of OS. Urinary cotinine was measured as biomarker of tobacco smoking exposure. All biomarkers were normalised by creatinine. The association between OS and A-LSs was tested using two Multivariable Linear Regression Models and log(e)-transformed variables. RESULTS:Subjects were aged from 18 to 64 years and 41.3% were males. Sex, age, height and weight were not significantly affecting parameter estimates of any models, thus they have been excluded. We observed a significant increase of 15-F2t-IsoP in subjects suffering from A-LSs compared to healthy subjects (+1.58 folds, p 0.001), even adjusting for cotinine and GSSG. Similarly, when considering GSSG as dependent variable, we found a GSSG increase of 1.79 folds in symptomatic subjects compared to healthy ones, adjusting for cotinine and 15-F2t-IsoP. Finally, 15-F2t-IsoP levels were redoubled for each unit of increase of urinary cotinine (p 0.001). CONCLUSIONS:In conclusion, our findings suggest that 15-F2t-IsoP and GSSG are reliable and sensitive monitors of OS in various respiratory diseases, including A-LSs. Additionally, urinary 15-F2t-IsoP is effective to quantify some of the in vivo effects of tobacco smoking exposure. KEYWORDS: Molecular epidemiology, Respiratory outcomes, Asthma

Grx2 Regulates Skeletal Muscle Mitochondrial Structure and Autophagy.

Glutathione is an important antioxidant that regulates cellular redox status and is disordered in many disease states. Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that plays a pivotal role in redox control by catalyzing reversible protein deglutathionylation. As oxidized glutathione (GSSG) can stimulate mitochondrial fusion, we hypothesized that Grx2 may contribute to the maintenance of mitochondrial dynamics and ultrastructure. Here, we demonstrate that Grx2 deletion results in decreased GSH:GSSG, with a marked increase of GSSG in primary muscle cells isolated from C57BL/6 Grx2−/− mice. The altered glutathione redox was accompanied by increased mitochondrial length, consistent with a more fused mitochondrial reticulum. Electron microscopy of Grx2−/− skeletal muscle fibers revealed decreased mitochondrial surface area, profoundly disordered ultrastructure, and the appearance of multi-lamellar structures. Immunoblot analysis revealed that autophagic flux was augmented in Grx2−/− muscle as demonstrated by an increase in the ratio of LC3II/I expression. These molecular changes resulted in impaired complex I respiration and complex IV activity, a smaller diameter of tibialis anterior muscle, and decreased body weight in Grx2 deficient mice. Together, these are the first results to show that Grx2 regulates skeletal muscle mitochondrial structure, and autophagy.

Nuclear Factor Erythroid 2-Related Factor 2 Activation Might Mitigate Clinical Symptoms in Friedreich's Ataxia: Clues of an "Out-Brain Origin" of the Disease From a Family Study.

Friedreich’s ataxia (FRDA) is the most frequent autosomal recessive ataxia in western countries, with a mean age of onset at 10–15 years. Patients manifest progressive cerebellar and sensory ataxia, dysarthria, lower limb pyramidal weakness, and other systemic manifestations. Previously, we described a family displaying two expanded GAA alleles not only in the proband affected by late-onset FRDA but also in the two asymptomatic family members: the mother and the younger sister. Both of them showed a significant reduction of frataxin levels, without any disease manifestation. Here, we analyzed if a protective mechanism might contribute to modulate the phenotype in this family. We particularly focused on the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the first line of antioxidant defense in cells, and on the glutathione (GSH) system, an index of reactive oxygen species (ROS) detoxification ability. Our findings show a great reactivity of the GSH system to the frataxin deficiency, particularly in the asymptomatic mother, where the genes of GSH synthesis [glutamate–cysteine ligase (GCL)] and GSSG detoxification [GSH S-reductase (GSR)] were highly responsive. The GSR was activated even in the asymptomatic sister and in the proband, reflecting the need of buffering the GSSG increase. Furthermore, and contrasting the NRF2 expression documented in FRDA tissues, NRF2 was highly activated in the mother and in the younger sister, while it was constitutively low in the proband. This suggests that, also under frataxin depletion, the endogenous stimulation of NRF2 in asymptomatic FRDA subjects may contribute to protect against the progressive oxidative damage, helping to prevent the onset of neurological symptoms and highlighting an “out-brain origin” of the disease.

Investigation of some biochemical parameters of wild and cultured Myrtus communis L. fruits subjected to different conservation methods

In this study, wild and cultivated white Myrtus communis L. (“myrtle”) fruits were investigated for their vitamin levels (A, B, C and E), carotenes (lycopene, β-carotene), functional peptides (glutathione, ghrelin), oxidative stress markers (GSSG and MDA), total phenolic and flavonoid substances, antioxidant capacity (DPPH, TEAC) and essential elements (Se, Zn, Fe, Cu and Mn). The results showed that both myrtle fruits can be considered as the good source of vitamins, antioxidants and elements. The preservation (sun or microwave-drying) methods for this seasonal fruit caused a significant (p < 0.05) decrease in their biochemical and bio-pharmacological content compared to fresh or frozen fruits. On the contrary, preservation resulted a significant increase in GSSG and MDA levels. The amounts of Se, Zn, Fe, Cu and Mn in wild myrtle fruit were found as 0.58, 205, 228.0, 37.22 and 24.3 µg/g dw, respectively.

Effect Of Graded Exercise On Ror And Blood Oxidative-stress In Trained And Untrained Subjects

Submaximal graded exercise testing is a commonly used method to assess cardiovascular stress. Reserve of repolarization of the heart (RoR) is an ECG-based, non-invasive method to monitor the heart’s stress response, and assesses cardiac cells ability to reestablish their membrane potential. RoR has been shown to be a useful indicator of cardiovascular disease risk in cardiac patients. This study examined RoR difference between trained (T) and untrained (U) individuals and correlate this outcome with blood stress markers. PURPOSE: To determine if RoR and blood stress markers in response to a graded exercise in T and U cohorts differ. METHODS: Thirty-nine (male and female) subjects (23.6 ±5.6 yrs) were recruited. Subjects arrived after overnight fast between 7-9 am and rested for 20 minutes. Subjects completed a fitness questionnaire to determine training status (T or U). ECGs (12 lead) were monitored before, during and after exercise to obtain RoR. A graded walking test (GXT) on a treadmill until 85% of estimated maximum heart rate was performed. Blood obtained at rest and immediately after exercise were analyzed using HPLC for glutathione (oxidized [GSSG], reduced [GSH], total [TGSH]). Repeated measures ANOVAs were utilized to analyze the results using SPSS v24 with significance set at α = 0.05. RESULTS: There were significantly lower resting HRs (p = .023) and higher workloads achieved during testing (p = .002) between T vs U groups. Resting RoR (independent of group) was significantly reduced from 75±5% pre-test to 26±10% RoR at the end of exercise (p < .001). Final stage RoR was significantly lower for T compared to U group (T: 20±9.4%; U: 31±9.4%, p = .041), but T group performed significantly greater stages (p=0.002). The GXT induced a reduction in blood GSH (Pre 366 ±161 μM, Post 273±180 μM, p < .001) and an increase of GSSG (Pre 135 ±62 μM, Post 157±83 μM, p = .038) with no difference between groups. GSSG/TGSH decreased after GXT (p=0.028) independent of group. CONCLUSIONS: These data suggest that a GXT induces a different stress response in T and U individuals. The oxidative stress at end GXT was similar but needed more workloads to get to this same internal stress level in the T group. Further studies are needed to ascertain stress responses with RoR and relative workloads.

Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

Antioxidant status of rats' blood and liver affected by sodium selenite and selenium nanoparticles.

BackgroundSelenium is an essential element; however, at higher doses, it can be toxic. Therefore, alternative nanotechnological solutions are required to overcome toxicological issues, rather than conventional alternatives. Nanoparticles show new and promising properties that may be able to suppress toxicity while maintaining the positive effects of selenium on an organism. The aim of the experiment was to determine the influence of sodium selenite and selenium nanoparticles (SeNPs) on the antioxidant status of rats.MethodsThe males of the outbreed rat strain Wistar albino were selected as a model organism. Animals were fed different forms of selenium. The control group was given a mixture without selenium addition, whereas other groups were fed a mixture containing sodium selenite, Se-49, and Se-100 SeNPs respectively. The duration of the trial was 30 days.ResultsAnalysis of blood and liver was performed where the concentration of reduced (GSH) and oxidised (GSSG) glutathione, and total selenium content were measured. In the liver, a significant reduction in GSSG was found for all experiment groups. Blood samples showed a significant reduction in GSH and an increase in GSSG.DiscussionThese results show that SeNPs may be an alternative to dietary selenium for animal organisms.

Leptin Maintained Zinc Homeostasis Against Glutamate-Induced Excitotoxicity by Preventing Mitophagy-Mediated Mitochondrial Activation in HT22 Hippocampal Neuronal Cells.

Developmental seizure-induced long-term neuronal hyperexcitation is partially mediated by regenerative mossy fiber sprouting in hippocampus. Yet, there are no effective drugs available to block this pathological process. Recently, leptin has been shown to prevent the sprouting of hippocampal mossy fibers and abnormalities in the neurobehavioral parameters. However, their underlying molecular mechanisms are largely unknown. The purpose of this study was to determine the effect of glutamate on the parameters of zinc homeostasis, mitochondrial functions, and mitophagy regulating factors, as well as to investigate the protective effects of leptin against cytotoxicity of glutamate in murine HT22 hippocampal neuronal cells. Cells were assigned to one of the four groups as follows: control group, leptin alone group, glutamate injury group, and leptin pretreatment group. Our results demonstrated that glutamate induced a decrease in superoxide dismutase, GSH (glutathione), and mitochondrial membrane potential and an increase in GSSG (oxidized glutathione), mitochondrial reactive oxygen species, and supplementation of leptin blocked the toxic effect of glutamate on cell survival. The glutamate-induced cytotoxicity was associated with an increase in mitophagy and intracellular zinc ion levels. Furthermore, glutamate activated the mitophagy markers PINK1, Parkin, and the ratio of LC3-II/LC3-I, as well as increased the expression of zinc transporter 3 (ZnT3). Leptin corrected these glutamate-caused alterations. Finally, the mitophagy inhibitor, CsA, significantly reduced intracellular zinc ion content and ZnT3 expression. These results suggest that mitophagy-mediated zinc dyshomeostasis and mitochondrial activation contributed to glutamate-induced HT22 neuronal cell injury and that leptin treatment could counteract these detrimental effects, thus highlighting mitophagy-mediated zinc homeostasis via mitochondrial activation as a potential strategy to counteract neuroexcitotoxicity.

Melatonin prevents memory impairment induced by high-fat diet: Role of oxidative stress

Consumption of high-fat diet (HFD) induces oxidative stress in the hippocampus that leads to memory impairment. Melatonin has antioxidant and neuroprotective effects. In this study, we hypothesized that chronic administration of melatonin can prevent memory impairment induced by consumption of HFD. Melatonin was administered to rats via oral gavage (100mg/kg/day) for 4 weeks. HFD was also instituted for the same duration. Behavioral studies were conducted to test spatial memory using the radial arm water maze. Additionally, oxidative stress biomarkers were assessed in the hippocampus. Results showed that HFD impaired both short- and long- term memory (P<0.05), while melatonin treatment prevented such effects. Furthermore, melatonin prevented HFD-induced reduction in levels of GSH, and ratio of GSH/GSSG, and increase in GSSG in the hippocampus. Melatonin also prevented reduction in the catalase activity in hippocampus of animals on HFD. In conclusion, HFD induced memory impairment and melatonin prevented this impairment probably by preventing alteration of oxidative stress in the hippocampus.

Vulnerability of marsh frog Pelophylax ridibundus to the typical wastewater effluents ibuprofen, triclosan and estrone, detected by multi-biomarker approach

Pharmaceutical and personal care products (PPCPs) are the environmental pollutants of growing concern. The aim of this study was to indicate the effects of typical PPCPs on the marsh frog Pelophylax ridibundus. We treated male frogs with waterborne ibuprofen (IBU, 250ng·L−1), triclosan (TCS, 500ng·L−1), or estrone (E1, 100ng·L−1) for 14days. Common vulnerability of the frogs was detected from dramatic decrease of Zn, total and metalated metallothionein (MT) concentrations, Zn/Cu ratio, the elevation of activity of glutathione-S-transferase, cathepsin D and DNA instability in the liver, the depletion of cholinesterase in the brain and cortisol in the blood plasma in all exposures. Nevertheless, lipofuscin concentration in the liver was always decreased. The groups were best distinguished by cytochrome P450 (CYP450) activity determined by ELISA. The exposure to IBU caused lesser damage, but elevated the levels of oxyradicals and glutathione (GSH and GSSG) and lysosomal membrane instability. Exposures to TCS and E1 provoked the endocrine disturbance (increased levels of vitellogenin and thyrotropin in blood plasma), decreased lactate dehydrogenase activity and increased level of pyruvate in the liver. TCS caused the increase of GSSG by 7.3 times and lactate levels. Only E1 lead to decrease of deiodinase activity in the liver, activation of CYP450 and caspase-3 and efflux of cathepsin D from lysosomes. Spectrophotometric and ELISA assays of MTs and CYP450 gave distinct results in E1-group. Broad disruption of the hormonal pathways caused by E1 could be of concern for the health status of frogs in their habitats.

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

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 1mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4h 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 4h. 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.