GSSG Ratio Research Articles

Effect of luteolin on the gene level expression of apoptosis-associated speck-like protein containing a caspase recruitment domain of NLRP3 inflammasome and NF-κB in rats subjected to experimental pancreatitis- influence of HSP70.

Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis. Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5weeks and cerulein (20μg/kg body weight i.p.,thrice weekly for the last 3weeks of the experimental period). Additionally, group 2 and 4 rats received 2mg/kg body weight of luteolin orally from third week. Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman's rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio. The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

The dynamics of nigrostriatal system damage and neurobehavioral changes in the rotenone rat model of Parkinson’s disease

Subcutaneous administration of rotenone to rats is currently a widely used method of reproducing Parkinson's disease (PD) symptoms, due to its convenience and effectiveness. Despite this, its influence on the temporal dynamics of parkinsonism development has yet to be investigated. The present study characterizes behavioral and neurochemical disruptancies underlying the dynamics of parkinsonism development in rats, induced by chronic subcutaneous administration of 2 mg/kg rotenone over the course of 18 days. In this article, the presence of two stages of pathology development in the model in question – the premotor and motor disability stages - are illustrated through a complex assessment of animal behavior, the development of an original neurological symptoms scale, and the establishment of the dynamics of histological and neurochemical changes in the brain. The premotor stage was observed up to 3 days of rotenone administration, and was characterized by a decrease in the motivational component of behavior, shown both in the food-getting task and in the “sucrose preference” test. A 30 % decrease in the number of cells in the substantia nigra pars compacta by the 3rd day of rotenone administration was also shown during the premotor stage. No changes in the metabolism of dopamine and other monoamine mediators were observed at this time. At the same time, acute administration of rotenone caused an increase in the GSH / GSSG ratio by 69 %. The motor stage developed after a decrease in the number of cells in the SNpc by more than 30 %, and was characterized by changes in the dopaminergic system, leading up to a 71 % reduction in dopamine levels in the striatum. It was shown that starting from 4 to 6 days of rotenone injection, experimental group animals begin to develop motor symptoms of Parkinson's disease, including bradykinesia, rigidity and postural instability. The development of motor impairment in all rats of this group was accompanied by significantly reduced activity of the antioxidant system in brain frontal lobe tissue homogenates, as compared to intact rats. Thus, in the used model of rotenone-induced parkinsonism, the dynamics of neuropathology development are described and the premotor stage of the disease is highlighted, which allows future using of this model in developing new approaches for treatment of parkinsonism at an early stage.

Metabolic and oxidative impairments in human salivary gland cells line exposed to MeHg

Background/aimThe ingestion of contaminated seafood by MeHg is considered the main route of human exposure, turning the salivary gland one important target organ. The salivary glands play critical roles in maintaining oral health homeostasis, producing saliva that maintains the oral microbiota, initiation of the digestion of macromolecules, and being essential in maintaining the integrity of the adjacent soft tissues and teeth. Thus, this study aimed to investigate the effects of MeHg exposure on human salivary gland cells line. MethodsCells were exposed to 1–6 μM of MeHg for 24 h, and analysis of toxicity was performed. Based on these results, the LC50 was calculated and two concentrations were chosen (0.25 and 2.5 μM MeHg) to evaluate intracellular mercury (Hg) accumulation (THg), metabolic viability and oxidative stress parameters (GSH:GSSG ratio, lipid peroxidation, protein oxidation and DNA damage). ResultsThe results demonstrated accumulation of THg as we increased the MeHg concentrations in the exposure and, the higher the dose, the lower is the cell metabolic response. In addition, the 2.5 μM MeHg concentration also triggered oxidative stress in human salivary gland cells by depleting the antioxidant competence of GSH:GSSG ratio and increasing lipid peroxidation and proteins carbonyl levels, but no damages to DNA integrity. ConclusionIn conclusion, although these two elected doses did not show lethal effects, the highest dose triggered oxidative stress and new questionings about long-term exposure models are raised to investigate furthers cellular damages to human salivary gland cells caused by MeHg exposure to extrapolate in a translational perspective.

Alterations in glutathione redox homeostasis among adolescents with obesity and anemia

The reduced (GSH)-to-oxidized (GSSG) glutathione ratio represents a dynamic balance between oxidants and antioxidants. However, redox status in adolescents with obesity and anemia has not been investigated. This study investigated the association of erythrocyte GSH redox status (GSH, GSH:GSSG ratio, and glutathione peroxidase [GPx] activity) with anemia and adiposity in adolescents. This case–control study nested in a cross-sectional study enrolled 524 adolescents (268 boys; 256 girls). The prevalence of anemia in overweight and obesity (OWOB) was 5.2% in boys and 11.7% in girls. The GSH:GSSG ratio and GPx activity were significantly higher in girls than in boys (p < 0.001), in anemic than in non-anemic subjects (p < 0.001), and in OWOB than in normal-weight subjects (p < 0.001). Similarly, significantly higher GSH: GSSG level (p < 0.001) and GPx activity (p < 0.001) were found in subjects with 90th percentile waist circumference than in those with < 90th percentile. GPx and GSH:GSSG were positively associated with anemia after adjusting for age, sex, and body mass index (adjusted odds ratio, adjOR [95% confidence interval, CI] 2.18 [1.44–3.29]) or tertiles (adjOR [95% CI], T3 = 2.49 [1.03–6.01]). A similar association was noted for GSH and GPx. A compensatory increased redox defense mechanism exists in anemia and obesity among adolescents without metabolic disturbances.

Redox forms of glutathione in malignant lesions of the stomach with varying aggressiveness degrees

Aim. To study the levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as the thiol status in gastric cancer (GC) tumors of various histological types and grades. Materials and methods. The indicators were determined by ELISA methods in tumor, peritumoral and visually intact tissues obtained during surgery from 52 patients with GC: 18 patients had a G1-2 adenocarcinoma (AC), 8 with G3 AC, 6 with signet ring cell cancer (SRCC), 14 with combined gastric lesions (CGL) – AC with signet ring cell fragments, 6 with patients with a component of undifferentiated cancer, G4. Results. In the groups of patients with low-differentiated and undifferentiated tumors, the GSH content in the tumor tissue and the peritumoral zone was higher than in the group of patients with well- and moderately-differentiated tumors. Tumor GSH levels in G3 AC and SRCC exceeded the values in visually intact tissues. Moreover, in the visually intact tissue of patients with SRCC, GSH level was reduced relative to G1-2 AC and CGL. GSH in all tissues of patients with CGL was higher than in patients with G1-2 AC. The lowest level of GSSG in the tumor tissue was registered in SRCC: 27.5% lower than in G1-2 AC and 30.3% lower than in G3 AC. Patients with undifferentiated tumors (G4 AC) had the highest GSH content in all studied tissues: by 29.9% in tumor; by 40.7% in peritumoral zone; and in visually intact tissue not only GSH, but also GSSG was increased by 22.5–25.5% in comparison with AC G1-2. G4 AC was also characterized by a sharp increase in the thiol status in tumor tissues by 80.2 and 89.9% higher than in visually intact and peritumoral tissues, and it was statistically higher than in AC G1-2, AC G3, SRCC and CGL. The ratio of GSH and GSSG was the most informative. Conclusion . Poor AC differentiation (in the row G1-2, G3, G4) and a change of histological tumor type (AC, SPL and SRCC), i.e. an increase in tumor aggressiveness, were accompanied by the enhancement of reductive processes in tumor tissue, as evidenced by the statistically significant increase in the GSH/GSSG coefficient and a sharp increase in the thiol status in G4 AC.

Сравнительный анализ влияния эпибрассинолида и 6-бензиламинопурина на ключевые компоненты глутатионового комплекса в корнях проростков пшеницы в условиях засоления

The study carried out a comparative analysis of the effect of 24 epibrassinolide (EB) and 6-benzylaminopurine (BAP) on the growth and state of the main components of the glutathione system in the roots of wheat seedlings under the action of 2% NaCl, which for the first time revealed the ability of these phytohormones to similarly stabilize stress-induced decrease GSH / GSSG ratio, positively regulate the activity of glutathione reductase and glutathione-S-transferase. A comparable level of protective effect of BAP and EB on root length was revealed. The data obtained indicate that endogenous cytokinins may play the role of hormonal intermediates in the implementation of the protective.

Bmal1 Regulates the Redox Rhythm of HSPB1, and Homooxidized HSPB1 Attenuates the Oxidative Stress Injury of Cardiomyocytes.

Oxidative stress is the main cause of acute myocardial infarction (AMI), which is related to the disorder of the regulation of Bmal1 on the redox state. HSPB1 form homologous-oxidized HSPB1 (homooxidized HSPB1) to resist oxidative damage via S-thiolated modification. However, it is still unclarified whether there is an interaction between the circadian clock and HSPB1 in myocardial injury. A total of 118 AMI patients admitted and treated in our hospital from Sep. 2019 to Sep. 2020 were selected to detect the plasma HSPB1 expression and the redox state. We divided the AMI patients into three subgroups: morning-onset AMI (5 : 00 am to 8 : 00 am; Am-subgroup, n = 38), noon-onset AMI (12 : 00 pm to 15 : 00; Pm-subgroup, n = 45), and night-onset AMI (20 : 00 pm to 23 : 00 pm; Eve-subgroup, n = 35) according to the circadian rhythm of onset. The Am-subgroup had remarkably higher cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and B-type natriuretic peptide (BNP) but lower left ventricular ejection fraction (LVEF) than the Pm-subgroup and Eve-subgroup. Patients complicated with cardiogenic shock were significantly higher in the Am-subgroup than in the other two groups. The homooxidized HSPB1 in plasma markedly decreased in the Am-subgroup. The HSPB1C141S mutant accelerated H9c2 cell apoptosis, increased reactive oxygen species (ROS), and decreased reduced-glutathione (GSH) and the ratio of reduced-GSH and GSSG during oxidative stress. Importantly, we found that the redox state of HSPB1 was consistent with the oscillatory rhythm of Bmal1 expression in normal C57B/L mice. The circadian rhythm disorder contributed to decrease Bmal1 and homooxidized HSPB1 in cardiomyocytes of C57BL/6 mice. In addition, Bmal1 and homooxidized HSPB1 decreased in neonatal rat cardiomyocytes exposed to H2O2. Knockdown of Bmal1 led to significant attenuation in homooxidized HSPB1 expression, whereas overexpression of Bmal1 increased homooxidized HSPB1 expression in response to H2O2. Our findings indicated that the homooxidized HSPB1 reduced probably the AMI patients' risk of shock and target organ damage, which was associated with Bmal1 regulating the redox state of HSPB1.

Effects of Feeding Dried Fruit Pomaces as Additional Fibre-Phenolic Compound on Meat Quality, Blood Chemistry and Redox Status of Broilers.

Simple SummaryFruit juice production resulted in a considerable amount of by-products that are rich in phenolic compounds. Several studies have already reported that polyphenols seemed to have antioxidant, anti-inflammatory and hypolipidemic properties. For this reason, fruit extracts have been widely used as a human food supplement for health promotion and disease prevention. However, little information about their application in animal feeds is available. The aim of this study was to investigate whether 3% or 6% apple, blackcurrant and strawberry dietary inclusion could have a positive effect on meat quality, blood chemistry and redox status of broiler chickens. Overall, the obtained results seem encouraging as both 3% and 6% fruit pomaces diets did not impair carcass traits and meat quality. Moreover, fruit pomaces groups showed lower blood triglycerides and improved renal function with lower creatinine level. Regarding antioxidant activity, all fruit pomaces improved the redox status in liver, breast and blood. No differences have been recorded between 3% and 6% diets. From a productive and biological point of view, the use of fruit pomaces in broiler chicken nutrition seems to be promising, in particular, 3% dietary inclusion seems to be preferable as higher fibre level can impair nutrient digestibility in poultry.The present study investigated the effects of apple (A), blackcurrant (B) and strawberry (S) dried pomaces on meat quality, blood chemistry and redox status of broiler chickens. A total of 480 Ross-308 male broilers were divided into 8 dietary treatments containing 3% and 6% of cellulose preparation (C), A, B or S. Six birds/group were slaughtered at 35 days of age and blood samples were collected. Carcass traits and meat quality were determined on the Pectoralis major muscles, recording nonsignificant differences. Antioxidant activity was evaluated in serum, liver and breast muscle. In serum, fruit pomaces lowered triglycerides, creatinine and atherogenic index (p < 0.05). Regarding redox status, in serum, ACW (antioxidant capacity of water-soluble substances) and ACL (antioxidant capacity of lipid-soluble substances) were greater in A (p < 0.001). In breast, ACW and ACL were higher in B and S compared to C (p < 0.05). In liver, ACL was greater in B and S compared to C (p < 0.001) and in higher dosage compared to low (p = 0.036). GSSG (oxidized glutathione) concentration was lower in A, whereas A, B and S presented a higher GSH (reduced glutathione)/GSSG ratio. The results showed that fruit pomaces could represent promising feed ingredients for broilers, improving serum, meat and tissue antioxidant parameters.

Antioxidant activity as a response to cadmium pollution in three durum wheat genotypes differing in salt-tolerance

AbstractDurum wheat is commonly used in various food industry industries and cultivated worldwide. A serious problem with the species cultivation is its capability to accumulate cadmium (Cd) in the grains. The aim of this study is to investigate whether antioxidant activity may be used as a marker of Cd tolerance in durum wheat. The experiment involved three durum wheat genotypes/lines differing in salt tolerance. The plant response to Cd was appraised based on the activity of ascorbate–glutathione (AsA–GSH) cycle enzymes, ascorbate-to-dehydroascorbate ratio, reduced-to-oxidized glutathione ratio (GSH:GSSG), as well as Cd content in the seeds. The highest activity of dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase was noted in control plants of salt-sensitive cultivar “Tamaroi.” In the presence of Cd, activity of these enzymes was considerably reduced. “Tamaroi” plants demonstrated also the highest Cd content in the grain. In conclusion, we identified the cultivar “Tamaroi” as most susceptible to cadmium, and the level of durum wheat sensitivity to the element can be evaluated based on a significant decrease in the activity of AsA–GSH cycle enzymes and GSH:GSSG ratio.

Investigating the mechanism of vanadium toxicity in freshwater organisms

Vanadium (V) could present a risk for aquatic organisms from the Alberta oil sands region, if present in high concentrations. An industry pilot project has used petroleum coke (PC) as a sorbent to remove organic toxicants from oil sands process-affected water (OSPW), but it also caused V to leach from PC into the OSPW, reaching concentrations of up to 7 mg V/L (a level known to be toxic to aquatic organisms). Vanadium is a transition metal with several oxidation states, which could potentially elicit its toxicity through either ion imbalance or oxidative stress. This study investigated the effect of V on Daphnia magna and Oncorhynchus mykiss. Daphinds and O. mykiss were exposed to concentrations of V up to their respective calculated median lethal concentration (LC50): 3 mg V/L for D. magna and 7 mg V/L for O. mykiss. For both organisms, the influence of V on sodium flux and whole body sodium was evaluated. Its effect on whole body calcium and the oxidative stress responses in O. mykiss at the gill and liver levels was also studied. Results suggested that 3.1 mg V/L for D. magna and 6.8 mg V/L for O. mykiss caused an overall increase in sodium influx in both the daphnids and rainbow trout. However, concentrations of V ranging between 0.2 and 4 mg V/L for D. magna and 1.8 and 6 mg V/L for O. mykiss reduced whole body sodium in both organisms and whole body calcium in O. mykiss. Concentrations above 3.6 mg V/L caused significant lipid peroxidation in the gills and liver of rainbow trout, while 1.9 mg V/L produced a substantial decrease in the fish gill GSH:GSSG ratio, but no change in the ratio between these thiols in the liver. Concentrations of 6.62 mg V/L sharply increased catalase activity in the liver but not in the gills. Neither liver nor gill superoxide dismutase was altered by V. Overall, results suggest that both ion imbalance and oxidative stress are part of the mechanism of toxicity of V in D. magna and O. mykiss and that further research is warranted to fully elucidate the mechanism(s) of V toxicity in aquatic organisms.

Exposure of Rats to Multiple Oral Doses of Dichloroacetate Results in Upregulation of Hepatic Glutathione Transferases and NAD(P)H Dehydrogenase [Quinone] 1.

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

\u03b2-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.

24-Epibrassinolide protects against ethanol-induced behavioural teratogenesis in zebrafish embryo

Embryonic studies have demonstrated the neurotoxic, teratogenic, and neurobehavioral toxicity of ethanol (EtOH). Although multiple mechanisms may contribute to these effects, oxidative stress has been described as the major damage pathway. In this regard, natural antioxidants have the potential to counteract oxidative stress-induced cellular damage. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proved antioxidant properties, in EtOH-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 1 % EtOH, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 24 h. Following exposure, biochemical evaluations were made at 26 hpf, developmental analysis was made throughout the embryo-larval period, and behavioural responses were evaluated at 120 hpf. Exposure to 1 % EtOH caused an increase in the number of malformations, which were diminished by 24-EPI. In addition, EtOH induced an accumulation of GSSG and consequent reduction of GSH:GSSG ratio, indicating the involvement of oxidative mechanisms in the EtOH-induced effects. These were reverted by 24-EPI as proved by the GSSG levels and GSH:GSSG ratio that returned to control values. Furthermore, exposure to EtOH resulted in behavioural deficits at 120 hpf as observed by the disrupted response to an aversive stimulus, suggesting the involvement of neurotoxic mechanisms. 24-EPI restored the behavioural deficits observed in a dose-dependent manner. The absence of effects in the embryos exposed solely to 24-EPI showed its safety during the exposure period. In conclusion, EtOH caused developmental teratogenicity and behavioural toxicity by inducing glutathione changes, which were prevented by 24-EPI.

SO049HYPOXIA INDUCIBLE FACTOR-PROLYL HYDROXYLASE (HIF-PH) INHIBITION COUNTERACTS THE RENAL ENERGY METABOLISM ALTERATIONS IN THE EARLY STAGES OF DIABETIC KIDNEY DISEASE

Abstract Background and Aims Hypoxia inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors (also known as HIF stabilizers) increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. Considering that HIF induces the expression of various genes, HIF stabilizers might have pleiotropic effects on the progression of kidney diseases as well as improvement in anemia. Interestingly, HIF induces the metabolic reprogramming from tricarboxylic acid (TCA) cycle to glycolysis as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies have shown accumulation of glucose and TCA cycle metabolites in diabetic renal tissue, which might be related to the progression of diabetic kidney disease (DKD). We hypothesized that HIF stabilization might reverse these metabolism alterations and conducted a proof-of-concept study using enarodustat (JTZ-951), an oral HIF-PH inhibitor. Method We utilized the streptozotocin-induced diabetic rat and alloxan-induced diabetic mouse models. Animals were divided into three groups: Control (normal animals eating normal food), DKD (diabetic animals eating normal food) and DKD+enarodustat (diabetic animals eating food mixed with enarodustat). Blood, urine and kidney samples were collected two weeks after grouping. Metabolism status in renal tissue were compared between groups from transcriptome and metabolome perspectives. Results Although plasma creatinine levels were not different between groups, enarodustat tended to reverse diabetic renal changes such as urinary albumin excretion, glomerulomegaly and glomerular basement membrane thickening (Figure 1). Transcriptome analysis has revealed that enarodustat counteracts the diabetic renal metabolism alterations; fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, while glucose metabolism was upregulated by enarodustat. These symmetric metabolism alterations were confirmed by metabolome analysis (Figure 2); glycolysis and TCA cycle metabolites were accumulated, and amino acids were reduced in diabetic renal tissue, while these metabolism alterations were mitigated by enarodustat. Moreover, enarodustat alleviated the accumulation of glutathione disulfide (GSSG) in diabetic renal tissue and thus showed higher glutathione (GSH)/GSSG ratio, which suggested that enarodustat relieved oxidative stress in DKD. Conclusion HIF stabilization counteracts the renal energy metabolism alterations in the early stages of DKD, in association with the improvement in urinary albumin excretion and renal pathological abnormalities. Our study suggests that HIF stabilization may serve as a potential intervention targeting dysregulated energy metabolism of diabetic kidneys.

PSAT1 Regulated Oxidation-Reduction Balance Affects the Growth and Prognosis of Epithelial Ovarian Cancer.

IntroductionA growing number of studies have found that the serine-glycine biosynthesis pathway is highly activated for biosynthesis in cancer progression and metastasis. Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step of the serine-glycine biosynthesis pathway; the effects and mechanism of PSAT1 in epithelial ovarian cancer (EOC) remains unclear.Materials and MethodsThe expression of PSAT1 in clinical EOC samples and normal ovarian tissues was conducted by RT-PCR, Western blot, and immunohistochemical staining. Survival analysis of PSAT1 in ovarian cancer was performed by using the public database. Following the downregulation of PSAT1, the cell growth, cell apoptosis, and cell cycle in ovarian cancer cells were respectively examined by the soft agar colony formation assay and flow cytometry analysis. Then the glutathione (GSH) levels, the GSH/GSSG ratio, the NADPH/NADP ratio, and the cellular reactive oxygen species (ROS) levels were tested to analyze the oxidation-reduction balance in PSAT1-depleted ovarian cancer cells.ResultsPSAT1 is markedly over-expressed in clinical EOC samples (n = 90) compared to that in normal ovarian tissues (n = 10), and the expression of PSAT1 is correlated with histological subtype, FIGO stage, histological grade, lymph node metastasis, distant metastasis and the presence of ascites. Public database analysis shows that higher PSAT1 indicates poor survival in EOC patients. Downregulation of PSAT1 in EOC cells inhibits growth, induces apoptosis and cell cycle arrest in vitro. EOC cells with high PSAT1 levels have increased a higher GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio and lower reactive oxygen species (ROS) content. The cancer-killing effects of PSAT1 knockdown are reversed by exogenous glutathione. PSAT1 participates in cancer growth by regulating oxidation-reduction balance.ConclusionTherefore, these results highlight the potential of PSAT1 inhibitors or metabolic substrate deprivation as therapeutic strategies for treating patients with EOC, especially those with advanced stages of cancer.

Induction of Glutathione Transferases and NAD(P)H Quinone Dehydrogenase 1 in Rats Dosed with Dichloroacetate

Dichloroacetate (DCA) is an investigational drug for treatment of children and adults with genetic or acquired mitochondrial diseases and cancer. The pharmacokinetics and dynamics of DCA vary by subject age. For example, the chronic use of DCA in adults is associated with the development of reversible peripheral neuropathy, a side effect that is seldom observed in children, who metabolize DCA more quickly. The dechlorination of DCA to its inactive metabolite, glyoxylate, is catalyzed by maleylacetoacetate isomerase/glutathione transferase zeta 1 (MAAI/GSTZ1). GSTZ1 is also critical in tyrosine catabolism through trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). However, DCA causes mechanism‐based inactivation of GSTZ1, resulting in inhibition of both DCA metabolism and isomerization of MAA and MA. The resulting tissue accumulation of DCA, MAA, and MA may be a mechanism of toxicity. Studies in GSTZ1(−/−) mice showed induction of antioxidant enzymes and GSTs A1/A2, M1 and M5, as well as an increase in the GSSG:GSH ratio, suggesting that GSTZ1 deficiency causes oxidative stress. In this study, we determined if loss of the expression and activity of GSTZ1 in rat tissues following multiple DCA dosages that mimicked therapy resulted in similar effects. Adult (52 wk) female Sprague Dawley rats (8 per group) and young (4 wk) male and female Sprague Dawley rats (4 per group) were dosed with sodium DCA (100 mg/kg) or sodium acetate (100 mg/kg) by oral gavage daily for 8 consecutive days. On the 9th day, rats were euthanized, livers were removed and cytosolic and mitochondrial subcellular fractions were prepared. Protein expression was assessed by Western blot using antibodies to A, M, P, T and O‐class GSTs and to NAD(P)H quinone dehydrogenase 1 (NQO1), gamma‐glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by LC/MS/MS. Enzyme activity was measured in hepatic cytosol using 1‐chloro‐2,4‐dinitrobenzene (CDNB), 1,2‐dichloro‐4‐nitrobenzene (DCNB), and 2,6‐dichloroindophenol (DCPIP) as substrates. In comparison to acetate‐treated controls, GST activities with CDNB and DCNB were higher in DCA‐treated rats irrespective of age (p &lt; 0.05), as was the relative expression of GSTA1/A2 (p &lt; 0.01), whereas DCA‐treated adults displayed higher levels of GSTM1 (p &lt; 0.05) and GSTO1 (p &lt; 0.05). Changes in the expression of P and T‐class GSTs following multiple doses of DCA were not observed for either group. NQO1 expression and activity were higher in juveniles after DCA dosing (p &lt; 0.05). GSH concentrations were increased by DCA in adults but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA‐mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. These findings will be important when interpreting outcomes of ongoing clinical trials of DCA in children and adults.Support or Funding InformationSupported in part by NIH RO1‐GM099871.

Nicotinamide N-methyltransferase expression in SH-SY5Y human neuroblastoma cells decreases oxidative stress.

Nicotinamide N-methyltransferase (NNMT) plays a central role in cellular metabolism, regulating pathways including epigenetic regulation, cell signalling, and energy production. Our previous studies have shown that the expression of NNMT in the human neuroblastoma cell line SH-SY5Y increased complex I activity and subsequent ATP synthesis. This increase in ATP synthesis was lower than the increase in complex I activity, suggesting uncoupling of the mitochondrial respiratory chain. We, therefore, hypothesised that pathways that reduce oxidative stress are also increased in NNMT-expressing SH-Y5Y cells. The expression of uncoupling protein-2 messenger RNA and protein were significantly increased in NNMT-expressing cells (57% ± 5.2% and 20.1% ± 1.5%, respectively; P = .001 for both). Total GSH (22 ± 0.3 vs 35.6 ± 1.1 nmol/mg protein), free GSH (21.9 ± 0.2 vs 33.5 ± 1 nmol/mg protein), and GSSG (0.6 ± 0.02 vs 1 ± 0.05 nmol/mg protein; P = .001 for all) concentrations were significantly increased in NNMT-expressing cells, whereas the GSH:GSSG ratio was decreased (39.4 ± 1.8 vs 32.3 ± 2.5; P = .02). Finally, reactive oxygen species (ROS) content was decreased in NNMT-expressing cells (0.3 ± 0.08 vs 0.12 ± 0.03; P = .039), as was the concentration of 8-isoprostane F2α (200 ± 11.5 vs 45 ± 2.6 pg/mg protein; P = .0012). Taken together, these results suggest that NNMT expression reduced ROS generation and subsequent lipid peroxidation by uncoupling the mitochondrial membrane potential and increasing GSH buffering capacity, most likely to compensate for increased complex I activity and ATP production.

Donor brain stem death and cardiac transplantation causes mitochondrial dyscoupling and oxidative stress

Introduction: Mitochondria are essential for adequate cardiovascular function and are important in the generation of reactive oxygen species (ROS). However, mitochondrial function and ROS post donor brain stem death (BD) and heart transplantation HTx is poorly understood and may contribute to donor heart dysfunction (DHD) and post HTx graft failure (GF). Objectives∖Aims: To examine mitochondrial function and ROS production in hearts both after 24hrs of BSD and post HTx. Methods: Donor sheep underwent BD (BD n=13) or sham instrumentation (SH, n=8) and monitored for 24hrs followed by heart procurement or cold static storage. Orthotopic HTx followed, in 7∖13 BD (BD-Tx) and 4/8 SH donors (SH-Tx). Mitochondrial respirometry was assessed using the Oroboros Oxygraph using carbohydrate (CHO) or fatty acid (FAO) substrates for both ventricles. O2 flux control ratios (FCR) were calculated in LEAK (no ADP) and Complex- I (CI) & II (CII) OXPHOS (with ADP) respiration states. Tissue levels of 3-nitrotyrosine and glutathione:reduced glutathione (GSH:GSSG) ratio were assessed using plate based assays. Results: BD caused a significant increase in LEAK RV proton slip. Both ventricles showed a trend toward higher O2 consumption at CI (known driver of ROS) and a significant decrease in CII respiration for both substrates. Post HTx showed mitochondrial dyscoupling evidenced by a trend toward higher CI O2 consumption, significantly higher RV proton slip, and depressed LV and RV CII respiration. For the LV, GSH:GSSG ratios were significantly lower for BD, BD-Tx and SH-Tx. For the RV, GSH:GSSG ratios were non-significantly depressed post BD both pre and post Tx. Conclusions: We have shown that donor BD and Tx caused significant mitochondrial dyscoupling and higher oxidative stress. Mitochondrial function, particularly CI ROS production may then contribute to DHD and consequently post-HTx GF. Therapeutically targeting mitochondrial ROS could improve patient prognosis.

Centaurium erythraea extract reduces redox imbalance and improves insulin expression and secretion in pancreatic β-cells exposed to oxidative and nitrosative stress

Oxidative stress is one of the major mechanisms that underlies the damage of pancreatic ?-cells and defects in insulin secretion in diabetes. As herbal preparations can alleviate oxidative stress through their redox-active secondary metabolites, in this study we investigated the cytoprotective effects of Centaurium erythraea extract (CEe) against H2O2- and SNP-induced oxidative/nitrosative stress in Rin-5F ?-cells. The antioxidant activity of CEe and its effect on cell survival and insulin expression/secretion were evaluated. The CEe increased cell viability and ameliorated the disturbance of redox homeostasis in H2O2- and SNP-treated cells by decreasing DNA damage, lipid peroxidation and protein S-glutathionylation. The CEe restored GSH homeostasis in H2O2-treated ?-cells and attenuated the SNP-induced disturbance of the GSH/ GSSG ratio. The H2O2- and SNP-induced disruption of CAT, GPx, GR, MnSOD and CuZnSOD activities was adjusted by the CEe towards control values, as well as mRNA and protein levels of GPx, MnSOD and CAT. The CEe increased insulin expression/secretion particularly in H2O2-treated ?-cells, which was in accordance with the more pronounced antioxidant effect of the CEe observed in H2O2-treated ?-cells as compared to SNP-treated cells. These findings support the beneficial effect of the CEe in preventing or slowing down ?-cell damage and dysfunction caused by oxidative/nitrosative stress during diabetes development.

Arsenic-related oxidative stress in experimentally-dosed wild great tit nestlings

Arsenic (As) is broadly distributed due to natural and anthropogenic sources, and it may cause adverse effects in birds. However, research on other elements (Pb, Hg and Cd) has been prioritized, resulting in scarce data on As exposure and related effects in wild birds. One of the mechanisms responsible for As toxicity is oxidative stress. Therefore, the aim of this study was to investigate if environmentally relevant As levels affected oxidative stress biomarkers in great tits (Parus major). This is the first field experiment studying the effects of As on oxidative stress in wild passerines. Wild great tit nestlings were orally dosed with sodium arsenite (Control: water, Low dose: 0.2 μg g−1 d−1 and High dose: 1 μg g−1 d−1; from day 3 to day 13 post-hatching). We intended to reach As concentrations similar to those at which passerines are exposed to at actual polluted areas. We compared the responses to the experimental manipulations (High, Low and Control groups) with those in an As/metal-exposed population breeding close to a Cu–Ni smelter in Finland (Smelter group). A set of antioxidants (tGSH, GSH:GSSG ratio, CAT, SOD, GST and GPx), and oxidative damage biomarkers (lipid peroxidation, protein carbonylation, 8-hydroxy-2′-deoxyguanosine formation in DNA, and telomere length) were explored in blood. Arsenic administration had no significant effect on most of the biomarkers measured: only the CAT activity was lower in the High As group and the GPx activity was enhanced in the Smelter group compared to the Control. Our results suggest that the dose and duration of the As exposure was not enough to induce oxidative damage in red cells of great tit nestlings. In spite of this, nestlings dosed with 1 μg g−1 d−1 of sodium arsenite showed non-significantly higher oxidative stress biomarkers than controls, suggesting that we were close to an effect level for the redox-defense system. Oxidative effects at equivalent As levels combined with other stressors cannot be dismissed.