Alterations In Antioxidant Capacity Research Articles

Exposure to polystyrene nanoplastics and PCB77 induced oxidative stress, histopathological damage and intestinal microbiota disruption in white hard clam Meretrix lyrata

The toxic effects of organic pollutants and nanoplastics on fish have been extensively studied, but there is limited research available on their combined toxicity to bivalves. This research aimed to investigate the accumulation and ecotoxicological impacts such as antioxidant capacity, histopathology and intestinal microbiota in white hard clam Meretrix lyrata, resulting from 7 days of single and mixture exposure to 3,3′,4,4′-tetrachlorobiphenyl (PCB77, 0.1 mg/L) and polystyrene nanoplastics (PS-NPs, 80 nm, 1 mg/L). Our findings revealed that PS-NPs accumulated in various tissues such as the intestine, gill, mantle, foot, and siphon. And when compared to the PCB-PSNPs (PP) co-exposure group, the intestinal fluorescence intensity mediated by plastic particles in the PS-NPs (PS group) was significantly higher. The gill, digestive gland, and intestine were all damaged to varying extent by single exposure to PS-NPs or PCB77, according to histopathological analysis, which was aggravated by PP group. Moreover, the co-exposure induced a higher level of oxidative stress, which reflected by increase of activities of superoxide dismutase, catalase, glutamate oxaloacetate transaminase and glutamic-pyruvic transaminase and malondialdehyde content. In addition, the intestine microbial composition was dramatically altered by the combined exposure, reducing the abundance of probiotics such as Firmicutes, thereby posing a great threat to the health and metabolism of M. lyrata. In conclusion, our findings showed that PS-NPs and PCB77 co-exposure induced a higher toxicity to M. lyrata, including histopathological changes, altered antioxidant capacity and intestinal microbiota disruption. This study provides novel insights into PCB77 and PS-NPs' combined toxicity to marine organisms and its underlying molecular mechanisms of ecotoxicological effects.

Mental Health Disorders Due to Oxidant and Antioxidants Imbalance: A Literature Review Study

Health is a significant thing in human life because it is one of the supports for human activity. By living a healthy life, everyone can be productive socially and economically to achieve their desired life goals. Oxidative stress is an imbalance between the cellular production of Reactive Oxygen Species (ROS) and antioxidant countermeasures. The high oxygen consumption and lipid-rich environment in the brain are thought to be highly susceptible to oxidative stress or redox imbalance. Various kinds of literature show accumulating evidence of implications for free radical-mediated pathology, altered antioxidant capacity, neurotoxicity, and inflammation in neuropsychiatric disorders. Oxidative stress contributes to the specific clinical symptomatology of complex and debilitating psychiatric illnesses. This complex and heterogeneous oxidative stress disorder negatively impacts the quality of life, morbidity or mortality, and cognitive function, known as depression. Oxidative stress has received much attention concerning psychiatric illness and has also been suggested as a contributing factor to depression.

Study on the change of flavonoid glycosides to aglycones during the process of steamed bread containing tartary buckwheat flour and antioxidant, α-glucosidase inhibitory activities evaluation in vitro

Tartary buckwheat dough (120 g) was used to produce steamed tartary buckwheat bread (STB). Alterations in antioxidant capacity and α-glucosidase inhibitory activity levels, and the concentrations of various flavonoid compounds, were investigated after each processing stage. It was identified that aglycone (quercetin and kaempferol) levels were increased after dough formation and fermentation stages. These stages were also associated with higher antioxidant capacity and α-glucosidase inhibitory activity. After steaming, flavonoid compound and antioxidant activity levels were slightly decreased in comparison to those observed after dough formation and fermentation steps, although they were higher than in raw flour.To clarify the anti-diabetic potential of individual flavonoid compounds, the antioxidant and α-glucosidase inhibitory activities of four distinct flavonoids were investigated. The results of the present study revealed that aglycones have more potent anti-diabetic effects than their corresponding glycosides, which may explain the change in antioxidant and α-glucosidase inhibitory activities of samples taken after the different processing stages. Correlation analysis also indicated that flavonoids’ being in their aglycone form was positively associated with higher antioxidant and α-glucosidase inhibitory activities. Overall, these data suggest that deglycosylation of flavonoids, which occurs during the preparation of STB, contributes to higher hypoglycemic potential in tartary buckwheat bread products.

Use of Parabens (Methyl and Butyl) during the Gestation Period: Mitochondrial Bioenergetics of the Testes and Antioxidant Capacity Alterations in Testes and Other Vital Organs of the F1 Generation.

Since the mid-1920s, parabens have been widely used as antimicrobial preservatives in processed foods and beverages, pharmaceuticals, and cosmetic products. Paraben use continues to generate considerable controversy, both in the general population and in the scientific community itself. The primary purpose of our study was to determine whether parabens (methyl and butyl at concentrations of 100 and 200 mg/kg body weight by subcutaneous injection) during pregnancy of adult female Wistar rats can have an impact on the F1 generation. As far as we know, we are the first to demonstrate that using parabens during pregnancy has negative repercussions on the mitochondrial bioenergetics and antioxidant activity of testicular germ cells in the F1 generation. Our study showed that there was a 48.7 and 59.8% decrease in the respiratory control index with 100 and 200 mg/kg of butylparaben, respectively. Cytochrome c oxidase activity was significantly inhibited (45 and 51%) in both groups. In addition, 200 mg/kg butylparaben promoted a marked decrease in citrate synthase activity, indicating that mitochondrial content decreased in the germ cells, especially spermatocytes and spermatids. Mitochondrial ROS production increased in groups exposed to parabens in a concentration-dependent manner, especially the butyl one (102 and 130%). The groups exposed to butylparaben showed an increase in superoxide dismutase (SOD) and catalase (CAT) activities, while glutathione reductase (GR) and glutathione S-transferase (GST) decreased. With methylparaben, only differences in SOD and GR were observed; for the latter, this only occurred with the highest concentration. The glutathione (GSH)/glutathione disulfide (GSSG) ratio did not undergo any significant change. However, there was a considerable increase in hydroperoxide content in animals exposed to butylparaben, with 100 and 200 mg/kg resulting in 98.6 and 188% increase, respectively. Furthermore, several other organs also showed alterations in antioxidant capacity due to paraben use. In summary, our study demonstrates that paraben use during pregnancy will cause severe changes in the mitochondrial bioenergetics and antioxidant capacity of testicular germ cells and the antioxidant capacity of several other F1 generation organs.

Circadian zinc feeding regime in laying hens related to laying performance, oxidation status, and interaction of zinc and calcium

This study investigated that circadian zinc (Zn) feeding regime affected laying performance, Zn and calcium (Ca) status, antioxidant capacity and gene expression of circadian clock, and Ca and Zn transporter in laying hens. In total, 162 of 21-wk Hyline Sophie laying hens were assigned randomly into 3 groups including CON group (Control Zn, basal diets supplemented 60 mg/kg Zn), HL group (high-low Zn, basal diets supplemented 120 mg/kg Zn—basal diets), and LH group (low-high Zn, basal diets—basal diets supplemented 120 mg/kg Zn), which were fed at 0,530 h and 1,530 h, respectively. Blood, tibia, duodenum, and eggshell gland samples were collected at 8 h intervals with starting at 0,000 h in 1 d after 10 wk of experiment. Compared with CON group: 1) Feed conversion ratio (FCR) of LH and HL group decreased significantly (P < 0.05); 2) in serum, total antioxidant capacity and CuZn-superoxide dismutase (SOD) at 0,000 h increased significantly, as well as Ca and Zn concentration of tibia at 0,800 h in LH group (P < 0.05); 3) in duodenum, mRNA expression of calbindin-d28k (CaBP) and NCX1 at 1,600 h in HL group upregulated significantly, as well as Per2 and Per3 at 0,000 h, CLOCK, Cry2, Per2, and Per3 at 1,600 h (P < 0.05). But, Zn5 at 0,800 h in HL group downregulated significantly (P < 0.05). 4) In eggshell gland, the mRNA expression of CaBP at 0,000 h and Zn5 at 1,600 h in HL group downregulated significantly (P < 0.05). However, SOD at 1,600 h in HL group upregulated significantly, as well as Cry1 and Per3 at 0,800 h in HL group upregulated significantly (P < 0.05). In conclusion, circadian Zn feeding diet regime was beneficial to improvement of FCR. The regulation of laying hens' circadian rhythms affected Zn and Ca transporter and interrelationship between Ca and Zn metabolism, also altered antioxidant capacity in present study. Therefore, circadian Zn feeding regime can be considered as a new method to improve laying performance in laying hens.

Investigating the Role of Antioxidants in Protecting Saccharomyces cerevisiae from Oxidative Damage

The free radical theory of aging suggests that cellular aging is due to the buildup of oxygen‐derived free radicals which damage biological molecules. These free radicals, known as reactive oxygen species (ROS), are produced as a secondary product of oxidative phosphorylation in the mitochondria. Cells maintain existing mechanisms to neutralize ROS, via both enzymatic and non‐enzymatic means. The thiol antioxidants, such as glutathione, form disulfide bonds to reduce the oxidative potential of ROS. Multiple studies have implicated alterations of the glutathione‐glutaredoxin system in tissue degeneration and cellular aging, and genetic mutations in antioxidant enzymes have been correlated with altered antioxidant capacity and lifespan in Drosophila, PC‐12 neuronal cells, and yeast. In this study, GSH2, glutathione synthetase, was deleted from S. cerevisiae to investigate the effect of altering the endogenous antioxidant balance. We hoped to mimic the changes that are believed to occur in the earliest stages of neurodegenerative diseases, such as Alzheimer's and Parkinson's. ΔGSH2 deletion mutants were pre‐treated with the commercially available thiol antioxidant cysteamine to determine its ability to protect the cells from oxidative damage caused by hydrogen peroxide. We hypothesized that, due to the structural similarities between cysteamine and glutathione, cysteamine would act as a substitute for glutathione when GSH2 was lost. Here we show alterations in gene and protein expression of relevant glutathione‐glutaredoxin system proteins as a result of the loss of GSH2 function.This work is supported by the Biology Department at Franklin College and by a grant from the Indiana Academy of Science.

Future climate alleviates stress impact on grassland productivity through altered antioxidant capacity

Predicting future ecosystem functioning requires a mechanistic understanding of how plants cope with different stressors under future climate conditions with elevated CO2 concentrations and warmer temperatures. Nonetheless, studies of stress responses under combined elevated CO2 and warming remain scarce.We assembled grassland communities in sunlit, climate-controlled greenhouses and subjected these to three stressors (drought, zinc toxicity, nitrogen limitation) and their combinations. Half of the communities were exposed to ambient climate conditions (current climate) and the other half were continuously kept at 3°C above ambient temperatures and at 620ppm CO2 (future climate).Across all stressors and their combinations, future climate-grown plants coped better with stress, i.e. above-ground biomass production was reduced less in future than in current climate. Among several tested potential biochemical and ecophysiological stress-relief mechanisms, we found three mutually non-exclusive mechanisms underpinning an improved stress protection under future climate conditions: (i) altered sugar metabolism; (ii) up-regulated levels of total antioxidant capacity and polyphenols; and (iii) more efficient use of ascorbate and glutathione as antioxidants.

Evaluation of antioxidant capacity and inflammatory cytokine gene expression in horses fed silibinin complexed with phospholipid

To evaluate antioxidant capacity and inflammatory cytokine gene expression in horses fed silibinin complexed with phospholipid. 5 healthy horses. Horses consumed increasing orally administered doses of silibinin phospholipid during 4 nonconsecutive weeks (0 mg/kg, 6.5 mg/kg, 13 mg/kg, and 26 mg/kg of body weight, twice daily for 7 days each week). Dose-related changes in plasma antioxidant capacity, peripheral blood cell glutathione concentration and antioxidant enzyme activities, and blood cytokine gene expression were evaluated. Plasma antioxidant capacity increased throughout the study period with increasing dose. Red blood cell nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase I activity decreased significantly with increasing doses of silibinin phospholipid. No significant differences were identified in glutathione peroxidase activity, reduced glutathione or oxidized glutathione concentrations, or expression of tumor necrosis factor α, interleukin-1, or interleukin-2. Minor alterations in antioxidant capacity of healthy horses that consumed silibinin phospholipid occurred and suggest that further study in horses with liver disease is indicated.

Functional Diversity of the Glutathione Peroxidase Gene Family Among Human Populations: Implications for Genetic Predisposition to Disease and Drug Response

To analyze the human genetic variation of glutathione peroxidases (GPX), estimating the functional differences among human populations and suggesting interethnic differences in predisposition to disease and drug response. Using 1000 Genomes Project data, we analyzed 723 GPX variants in 1092 individuals belonging to 14 populations. Combining functional prediction analyses of coding and noncoding variants, we developed a method to estimate haplotype functionality. GPX rare variants have a higher functional impact than common variants. The frequency among Asian patients of haplotypes associated with normal functionality is significantly higher for GPX1 and lower for GPX3 than for non-Asian patients; no adaptation signals in GPX1 and GPX3 were found in Asian patients. GPX1 and GPX3 differences may be associated with alterations in antioxidant capacity and redox regulation, which suggests diverse susceptibility to complex disease and diverse response to relevant drugs in Asians compared with individuals with other ethnic origins.

Increased maternal cytokine production and congenital heart defects

Congenital heart defects (CHDs) are a major cause of infant mortality. Most CHDs are thought to result from genetic, lifestyle, and environmental factors that include maternal obesity, diabetes, toxicant exposure, and alterations in anti-oxidant capacity. Since these well-documented risk factors are also associated with immune dysregulation, we sought to compare the maternal immune response in mothers carrying a fetus with a CHD with those mothers whose pregnancies were not affected by any birth defect. We conducted a case-control study to examine the maternal cytokine profile using multiplex technology in pregnant mothers (subject mean=26 weeks’ gestation). This investigation revealed that whole blood cultures derived from case mothers produced higher levels of certain cytokines and chemokines compared with cultures from control subjects when activated with mitogen. Cultures from case subjects produced higher levels of IL-10, IL-13, IL-4, IL-5, IL-17, and IL-6, when stimulated with mitogen compared with control subjects. Plasma levels of chemokine MIP-1α were higher in cases compared with controls. In contrast, C-reactive protein levels were not statistically different. These results demonstrate the need to further examine the maternal cytokine signature in CHD-affected pregnancies. This information could pave the way toward maternal immunotherapeutic intervention to prevent CHDs, and novel biomarker discovery to improve pre-natal diagnosis.

Evaluation of Oxidative Stress and Antioxidant Status in Diabetic and Hypertensive Women during Labor

Pregnancy in insulin-dependent diabetes mellitus is associated with a greater incidence of fetal abnormality. Animal studies suggested that increased free-radical production and antioxidant depletion may contribute to this risk. The objective of this work was to evaluate oxidative stress and antioxidant capacity in hypertensive, diabetics, and healthy control women during labor. Simultaneous determination of antioxidant enzymes activities, namely glutathione peroxidase (GSH-Px), glutathione reductase (GSH-red), superoxide dismutase (SOD), total antioxidant, and lipid peroxides measured as thiobarbituric acid-reactive substances (TBARS) levels, were carried out in maternal plasma during labor. Plasma GSH-Px activity was found to be significantly increased as it doubled in hypertensive, and diabetic women when compared with healthy control women (P < 0.05). In contrast, plasma SOD activity was significantly decreased in both groups when compared to the control group (P < 0.05). No significant differences were detected in GSH-Red activity between diabetic, hypertensive and control groups. Alterations in antioxidant enzyme activities were accompanied by a significant increase in the levels of plasma lipid peroxides in hypertensive and diabetic women during labor. Plasma levels of total antioxidants were significantly increased in diabetic women as compared with the control group. Based on our results, it may be concluded that enhanced generation of oxidative stress causes alteration of antioxidant capacity in diabetic and hypertensive women during labor. Alterations in antioxidant and prooxidant components may result in various complications including peroxidation of vital body molecules which may be regarded as an increased risk factor for pregnant women as well as the fetus.

P-1274 - Possible pathophysiological role of the relationship between levels of nitric oxide and bilirubin in patients with schizophrenia

Introduction Today we have a large number of articles that investigate oxidative stress and potential role of nitric oxide (NO) in the pathophysiology of schizophrenia and a lot of evidence of altered antioxidant capacity. Interesting scientifically facts is that bilirubin acts as an endogenous scavenger of NO, giving him the role of antioxidant that is reduced in schizophrenia. Objectives Antioxidant properties of bilirubin through its interaction with the nitric propose that bilirubin-nitric oxide as a new biomarker of oxidative / nitrosative stress. Aims The aim of our study was to investigate possible correlation between serum levels of nitric oxide and bilirubin in patient's whit schizophrenia. Methods The study was consisted of 50 patients with schizophrenia and 50 healthy controls. We investigated the levels of nitric oxide, which is determined by conversion of nitrate to nitrite and then measuring with Greiss reagent, but only in group patients we measured the mean levels of bilirubin, within of course of illness. Results Mean of NO between group patients and controls are statistically significant (CI = 13.31–27.29, t = 5.863, p = 0.0001). The average value of total bilirubin in patients suffering from schizophrenia was presented with SD = 11.77; 6.06 ± 0.86, and is statistically significant differences between the flow of diseases with the level of bilirubin, where the highest values detected markedly with the first hospitalization. Conclusion Antioxidant capacity of schizophrenia decreases with the progress of the disease. We can deduce that imbalanced between NO and bilirubin participates in the pathogenesis basis of schizophrenia.

Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase

BackgroundCellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network.ResultsStable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins.ConclusionsOur results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.

Rapid electrochemical screening of antioxidant capacity (RESAC) of selected tea samples

Antioxidants found in the daily diets behave as chemopreventive agents to inactivate potentially harmful reactive oxygen species present in our organisms. Consequently, it will be quite important to have easy-to-use diagnostic tools to dose antioxidant food ingredients. A method for a robust and rapid electrochemical screening of antioxidant capacity (RESAC) has been developed. To assess the suitability of the developed testing method, different tea varieties were evaluated for their antioxidant capacity. Results obtained with the RESAC method were highly correlated with conventional photometric antioxidant assay like FRAP (0.9457) and DPPH (0.9511). The newly developed RESAC method is easy to use, fast and at a low price. This method is well suited for quality control and may facilitate future studies regarding alteration of antioxidant capacity during growth, storage and processing of food or medicinal plants.

DNA damage and plasma antioxidant indices in Bangladeshi type 2 diabetic patients

Background and aim Diabetes mellitus is a complex metabolic disorder characterized by a disturbance in glucose metabolism. Recent evidence suggests that increased oxidative stress as well as alteration of antioxidant capacity may be related to the complications seen in patients with type 2 diabetes. The aim of this study was to measure serum antioxidant status in type 2 diabetic patients and to assess its relationship with oxidative DNA damage. Methods A total of 57 subjects were included in this study. Of these, 32 were type 2 diabetic patients and 25 were non-diabetic subjects. Comet assay was used to quantify the level of DNA damage in lymphocytes. Spectrophotometric methods were used to assess serum levels of malondialdehyde (MDA) and protein carbonyl, and serum activity of superoxide dismutase (SOD) and the protein thiol (P-SH) group. Results A significant increase in mean comet tail DNA, indicating DNA damage, was observed in diabetic patients compared with controls. Diabetic patients had significantly higher levels of MDA and protein carbonyl in parallel with significant decreases in levels of SOD and the P-SH group compared with controls. Serum SOD was also inversely correlated with the increase in comet tail DNA. Conclusion These results indicate the presence of significant lipid peroxidation, protein oxidation and oxidative DNA damage in patients with diabetes. Perturbation of glucose homoeostasis was associated with an increase in oxidants and a concomitant decrease of antioxidant enzymes in the type 2 diabetic patients’ blood. The present study suggests that the status of oxidant–antioxidant imbalance may be one of the mechanisms leading to the DNA damage detected in the lymphocytes of type 2 diabetic patients.

Altered antioxidant capacity in human renal cell carcinoma: Role of glutathione associated enzymes

Purpose We aimed to discern the role of glutathione (GSH) associated enzymes in maintaining high GSH levels in renal cell carcinoma (RCC) of the clear cell type and analyze RCC enzyme antioxidant capacity. Since changes in cellular redox balance in RCC might also be related to alterations of glutathione S-transferase (GST) phenotype, GST class α and π expression was also explored. Methods and materials Human kidney specimens of tumor and distant nontumor regions were obtained from 15 patients with RCC at the time of surgery. The activities of GSH-replenishing enzymes, γ-glutamylcysteine synthetase (γ-GCS), γ-glutamyl transferase (γ-GT), and glutathione reductase (GR), as well as the activities of antioxidant enzymes glutathione peroxidase (GPX) and catalase (CAT) were determined spectrophotometrically. GST α and π class expression was determined by immunoblot. Results In the course of renal cancerization, significant changes appear in the activities of GSH-replenishing and antioxidant enzymes. The activity of the key enzyme of GSH synthesis, γ-GCS, is up-regulated ( P < 0.001), while the activities of γ-GT and GR are down-regulated in renal tumors compared to nontumor tissue ( P < 0.001 and P < 0.05, respectively). Activities of GPX and CAT were also down-regulated ( P < 0.001 and P < 0.05, respectively) in RCC. Changes in enzyme antioxidant capacity in RCC were associated with decreased GST class α ( P < 0.001) and unchanged GST π expression at the protein level. Conclusions Changes in redox status in RCC as a consequence of decreased enzyme antioxidant capacity, together with altered GST α expression, may be important factors in development and tumor growth. The up-regulation of γ-GCS and high levels of GSH in RCC may be an attempt to limit injury caused by oxidative stress.

Rapid electrochemical method to screen antioxidative capacity of biological matrices (RESAC)

One aspect involved in initiation and development of cardiovascular diseases and cancers are oxidative processes, leading to the generation of hydroxyl radicals and peroxyl compounds [1]. There is growing body of evidence that the chemopreventive effects of antioxidants contribute to inactivate „reactive oxygen species“ [2–4]. In all cases, assessment of antioxidant capacity of our food or other biological matrices is of utmost importance. The majority of free radicals are good oxidants. Therefore, a good antioxidant maybe at first a reductant. The wide mixture of antioxidants with varying redox potentials in plant foods may interact in a synergistic manner, enabling effective protection against oxidation at low concentration levels [5]. Measurement of electrochemical oxidizability should be an appropriate method to assess antioxidant capacity of a biological sample. The aim of the present study was to apply a recently developed electrochemical method for a rapid screening of antioxidant capacity (RESAC) of biological matrices. The electrochemical detector used was equipped with a glassy carbon electrode, operating at a potential of +600 mV. Calibration of the electrochemical method with common photometric standard methods DPPH [6] and FRAP [7] has been done. Results obtained indicate a good correlation between RESAC and these conventional photometric methods (DPPH r=0,935, n=12; FRAP r=0,992, n=12). After calibration, applicability of the method has been shown with selected tea and green coffee samples. The RESAC-method is a rapid and convenient assay based on electrochemical oxidation for the assessment of antioxidant capacity. The method is well suited for quality and specification control and may facilitate future studies regarding alteration of antioxidant capacity during growth, storage and processing of food or medicinal plants.

Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase

Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2).

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.

Dietary supplementation with (R)-α-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24–28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-α-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.