Intracerebroventricular administration of N-acetylaspartic acid impairs antioxidant defenses and promotes protein oxidation in cerebral cortex of rats

N-acetylaspartic acid (NAA) is the biochemical hallmark of Canavan Disease, an inherited metabolic disease caused by deficiency of aspartoacylase activity. NAA is an immediate precursor for the enzyme-mediated biosynthesis of N-acetylaspartylglutamic acid (NAAG), whose concentration is also increased in urine and cerebrospinal fluid of patients affected by CD. This neurodegenerative disorder is clinically characterized by severe mental retardation, hypotonia and macrocephaly, and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain not fully understood, in the present study we investigated whether intracerebroventricular administration of NAA or NAAG elicits oxidative stress in cerebral cortex of 30-day-old rats. NAA significantly reduced total radical-trapping antioxidant potential, catalase and glucose 6-phosphate dehydrogenase activities, whereas protein carbonyl content and superoxide dismutase activity were significantly enhanced. Lipid peroxidation indices and glutathione peroxidase activity were not affected by NAA. In contrast, NAAG did not alter any of the oxidative stress parameters tested. Our results indicate that intracerebroventricular administration of NAA impairs antioxidant defenses and induces oxidative damage to proteins, which could be involved in the neurotoxicity of NAA accumulation in CD patients.     

Effect of aging on enzymatic antioxidant defenses in rat liver mitochondria.

Antioxidant defenses within liver mitochondria are pivotal in preventing liver damage from oxidative toxicants. In this study we determined the activities of glutathione peroxidase (GPO), superoxide dismutase (SOD) and glutathione reductase (GRD) in mitochondria from livers of variously aged Fischer 344 rats. A mixed pattern of age-associated alterations in mitochondrial antioxidant activities was observed. In male rats, GRD activity decreased in old age, whereas GPO and SOD activities increased. In female rats, GPO activity decreased with age, but SOD activity increased and GRD activity was unchanged. Age-associated decreases in antioxidant protection from mitochondrial enzymes appeared to be counterbalanced by increases in protection from other enzymes.     

Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide-challenged myocardium

PURPOSE: Pyruvate, a natural energy-yielding fuel in myocardium, neutralizes peroxides by a direct decarboxylation reaction, and indirectly augments the glutathione (GSH) antioxidant system by generating NADPH reducing power via citrate formation. The possibility that pyruvate's antioxidant actions could mediate its enhancement of contractile performance in prooxidant-challenged myocardium was investigated in isolated working guinea-pig hearts reversibly injured by hydrogen peroxide. METHODS: Hearts were challenged by 10 min perfusion with 100 microM H(2)O(2), followed by 90 min H(2)O(2)-free perfusion. Metabolic and antioxidant treatments (each 5m M) were administered at 30-90 min post-H(2)O(2). Phosphocreatine phosphorylation state, GSH/glutathione disulfide redox potential (GSH/GSSG) and key enzyme activities were measured in snap-frozen myocardium. RESULTS: H(2)O(2) exposure depleted myocardial energy and antioxidant reserves and produced marked contractile impairment that persisted throughout the H(2)O(2) washout period. Relative to untreated post-H(2)O(2) myocardium, pyruvate restored contractile performance, increased GSH/GSSG 52% and maintained phosphocreatine phosphorylation state; in contrast, lactate lowered cardiac performance and phosphorylation state. Neither the pharmacological antioxidant N -acetylcysteine (NAC) nor the pyruvate analog alpha-ketobutyrate increased cardiac function; both treatments increased GSH/GSSG but lowered phosphocreatine potential. H(2)O(2) partially inactivated aconitase, creatine kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), but all three enzymes spontaneously recovered during H(2)O(2) washout. Pyruvate did not further activate these enzymes and unexpectedly inhibited GAPDH by 60-70%. CONCLUSION: Pyruvate promoted robust contractile recovery of H(2)O(2)-challenged myocardium by the combination of citrate-mediated antioxidant mechanisms and maintenance of myocardial energy reserves.     

Propylthiouracil-induced congenital hypothyroidism upregulates vimentin phosphorylation and depletes antioxidant defenses in immature rat testis

Congenital hypothyroidism was induced in rats by adding 0.05% 6-propyl-2-thiouracil in the drinking water from day 9 of gestation, and continually up to postnatal day 15. Structural alterations observed by light microscopy of seminiferous tubules and by transmission electron microscopy of Sertoli cells of treated animals were consistent with hypothyroid condition. Hypothyroidism was also associated with high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 levels. Furthermore, the phosphorylation and the immunoreactivity of cytoskeletal-associated vimentin were increased without altering vimentin expression, suggesting an accumulation of insoluble and phosphorylated vimentin. These alterations in intermediate filament dynamics could result in loss of Sertoli cell cytoskeletal integrity and be somewhat related to the deleterious effects of hypothyroidism in testis. In addition, the mitochondrial alterations observed could also be related to defective cytoskeletal dynamics implying in cell damage. Moreover, we observed decreased oxygen consumption and unaltered lipid peroxidation in hypothyroid testis. However, we demonstrated decreased enzymatic and non-enzymatic antioxidant defenses, supporting an increased mitochondrial reactive oxygen species (ROS) generation, contributing to biochemical changes in hypothyroid testis. In addition, the changes in the testis histoarchitecture could be ascribed to cytoskeletal alterations, decreased antioxidant defenses, and increased ROS generation, leading to oxidative stress in the organ.     

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.     

Review of aerosolized hydrogen peroxide,vaporized hydrogen peroxide,and hydrogen peroxide gas plasma in the decontamination of filtering facepiece respirators

BackgroundCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to over 170?million cases worldwide with over 33.2?million cases and 594,000 deaths in the US alone as of May 31st, 2021. The pandemic has also created severe shortages of personal protective equipment, particularly of filtering facepiece respirators (FFRs). The Centers for Disease Control and Prevention (CDC) has issued recommendations to help conserve FFRs, as well as crisis standards, including four criteria required for decontamination of the traditionally single use respirators. This review is designed to provide an overview of the current literature on vaporized hydrogen peroxide (vHP), hydrogen peroxide gas plasma (HPGP), and aerosolized hydrogen peroxide (aHP) with respect to each of the four CDC decontamination criteria.MethodsPubMed and Medrxiv were queried for relevant articles. All articles underwent a title and abstract screen as well as subsequent full text screen by two blinded reviewers if indicated.ResultsSearches yielded 195 papers, of which, 79 were found to be relevant. Of those, 23 papers presented unique findings and 8 additional articles and technical papers were added to provide a comprehensive review. Overall, while there are potential concerns for all 3 decontamination methods, we found that vHP has the most evidence supporting its use in FFR decontamination consistent with CDC recommendation.ConclusionsFuture research is recommended to evaluate biological inactivation and real world fit failures after FFR reuse.     

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats

5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.     

The concentration of hydrogen peroxide in exhaled air depends on expiratory flow rate.

Hydrogen peroxide (H2O2) is known to be detectable in exhaled air. The present study aimed to determine whether the concentration of exhaled H2O2 depends on expiratory flow rate in order to make inferences on the site of its production within the lung. Breath condensate was collected in cooled Teflon tubes, at three different expiratorv flow rates, in 15 healthy or mild asthmatic subjects. Tests were repeated 2-5 times to assess reproducibility. Mean+/-SEM concentrations of H2O2 at flow rates of 140, 69 and 48 mL.s(-1) were 0.12+/-0.02, 0.19+/-0.02 and 0.32+/-0.03 microM, respectively. These values differed significantly from each other (p<0.001). For comparison, average coefficients of variability within repeated measurements at each of the three flow rates were 68, 62 and 82%, respectively. These data demonstrate that the concentration of exhaled hydrogen peroxide depends on expiratory flow rate. Since flow dependence is an indicator of production within the airways, this result suggests that, to a large extent, the exhaled hydrogen peroxide originates within the airways. However, even under strictly controlled conditions, a high degree of variability persists, which may limit the usefulness of exhaled hydrogen peroxide as a marker of airway inflammation.     

Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue

Studies of age-related changes based on the thiobarbituric acid (TBA) test appear to be inconsistent and contradictory. In our work, real basal (hypothetical, corrected to the zero concentration of atmospheric oxygen), basal (atmospheric oxygen-stimulated) and stimulated (L-glutamic and hydrochloric acids) levels of TBA-reactive products (TBARP) were estimated in the brain (hippocampus—HPC, cortex—COR, cerebellum—CRB) from young (3 months) and old (28 months) male and female Wistar rats. The values of basal levels of TBARP were different in young (HPC > COR > CRB) as well as in old animals (COR > CRB > HPC). Thus, the process of aging caused a significant decrease in the HPC (to 72%) and no change in the COR and CRB. Levels stimulated by L-glutamic acid were significantly decreased (COR,CRB) and unchanged (HPC) in old compared to young animals. The real basal levels estimated by regression analysis seem to be higher in the brain tissue damaged by aging. We discuss the usefulness of the TBA test applied to aged tissue, the effect of acidosis, the effectivity of L-glutamic acid to generate free radicals and the differences between individual brain areas.     

In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis

The hypothesis that hydrogen peroxide generation occurs in the corpora lutea of superovulated rats during luteolysis was tested using a peroxide-dependent inhibitor of catalase, 3-amino-1,2,4-triazole (AT). Luteal regression was induced during midpseudopregnancy by injection of 500 micrograms prostaglandin F2 alpha (PGF2 alpha) 1 h before administration of AT (0.1 g/kg, ip) and was confirmed by progesterone analysis of peripheral blood serum. Within groups of both PGF2 alpha-treated and untreated control rats, other rats also received ethanol (0.2 g/kg, ip), which prevents hydrogen peroxide-mediated inhibition of catalase by AT. Diluted homogenates of ovaries removed 1 h after AT administration were assayed for catalase activity by measuring the decrease in absorbance at 240 nm for 30 sec after the addition of hydrogen peroxide (10 mM). Ethanol-sensitive catalase inhibition by AT was significantly higher (47.9 +/- 3.38%) in samples from PGF2 alpha-treated groups than in controls (23.1 +/- 4.82%; P less than 0.01; n = 9). Similar increases in catalase inhibition by AT were found in luteal tissue of rats treated with PGF2 alpha 24 h earlier and in rats in which luteolysis was allowed to occur spontaneously in late pseudopregnancy. Hemoglobin an AT assays revealed that the changes in catalase activity were not the result of altered blood contamination or AT concentration in the luteal homogenates. Since catalase inhibition by AT is only seen in the presence of hydrogen peroxide, these results support the conclusion that an early and sustained component of corpus luteum regression is the generation of hydrogen peroxide in luteal tissue.     

Hydrogen peroxide inhibits iodide influx and enhances iodide efflux in cultured FRTL-5 rat thyroid cells

This study describes the effects of hydrogen peroxide on the two iodide transport systems, I influx and I efflux, in the cultured FRTL-5 rat thyroid cells. I influx was measured by the amount of I taken up by the cells during incubation with Na125I and NaI for 7 min, and I efflux was measured by calculating the rate of 125I release from the 125I-loaded cells in the presence and absence of 5 mmol/l H2O2. Exposure to greater than 100 mumol/l H2O2 for 40 min caused a significant inhibition of I influx; the inhibition was reversible and non-competitive with iodide. Thyroid Na+K+ ATPase activity, a major mechanism to drive I influx, decreased by 40% after the cells were exposed to 5 mmol/l H2O2 for 10 min. H2O2 enhanced I efflux only when Ca2+ was present in the medium. The mechanism of an enhanced I efflux by H2O2 appears to be mediated through the elevation of free cytosolic Ca2+ concentration. Our data indicate that H2O2 can affect I transport by inhibiting I influx and enhancing I efflux.     

Effect of exogenous hydrogen peroxide on myocardial function and structure in isolated rat heart.

A time- and dose-dependent effect of exogenous hydrogen peroxide was determined on myocardial function, structure, high energy phosphate and lipid peroxidation in the isolated perfused rat heart. Hydrogen peroxide induced a dose-dependent decrease in cardiac function whereas 200 microM hydrogen peroxide reduced +dP/dt to 50% of control value after 10 mins. The effect of 300 microM hydrogen peroxide was more severe after 15 mins; changes observed with this dose were reversible within 10 mins of perfusion, becoming irreversible after 15 mins. Lipid peroxidation and severe morphological damage were observed after 10 mins of perfusion with 300 microM hydrogen peroxide. When 16 mEq potassium ions were added in the perfusion buffer during hydrogen peroxide perfusion, the degree of tissue damage and loss of ATP were attenuated. However, lipid peroxidation was not inhibited by high potassium ions. When 0.25 microM N,N'-diphenyl-1,4-phenylenediamine, a potent antioxidant, was added to the perfusate, lipid peroxidation was totally inhibited and the degree of tissue damage was decreased. However, depletion of tissue ATP and functional deterioration were not influenced. These results suggest that hydrogen peroxide-mediated ATP loss was independent of lipid peroxidation.     

Increased hydrogen peroxide concentration in the exhaled breath condensate of stable COPD patients after nebulized N-acetylcysteine

BACKGROUND: The oxidative burden in the airways is a hallmark of chronic obstructive pulmonary disease (COPD). AIMS: This prospective, cross-over, placebo (PL)-controlled study was designed to investigate the effect of N-acetyl-l-cysteine (NAC) on hydrogen peroxide (H(2)O(2)), nitrites and nitrates (NO(2)(-)+NO(3)(-)), and thiol (RSH) concentrations in exhaled breath condensate (EBC) in stable COPD patients (n=19, aged 52.6+/-15.6 years, 10 females, mean FEV(1) 95.2+/-23.8%, FEV(1)/FVC 69.1+/-11.4%). METHODS: H(2)O(2), NO(2)(-)+NO(3)(-) and RSH concentrations in EBC were determined with homovanillic acid, NADPH-nitrite reductase assays and Ellman's reaction, respectively. RESULTS: Thirty minutes after nebulization, H(2)O(2) concentration increased if levels after NAC (0.45+/-0.25microM) and PL (0.17+/-0.17microM) were compared in COPD patients (p=0.002). This increased H(2)O(2) level in EBC was no longer observed either after 90min: 0.16+/-0.09microM (PL 0.17+/-0.15microM) or 3h: 0.12+/-0.07microM (PL 0.21+/-0.23microM) (p=0.5 and 0.2, respectively). The levels of NO(2)(-) and NO(3)(-) did not differ between NAC and PL. There was no significant difference in RSH levels between nebulized NAC and PL. After nebulized NAC, however, exhaled RSH increased from 1.42+/-1.69microM (0min) to 2.49+/-2.00microM (30min), and 1.71+/-1.83microM (180min) (p=0.009 and 0.03, respectively, compared with 0min). CONCLUSIONS: These data demonstrate that nebulized NAC transiently increases exhaled H(2)O(2) level, whereas it has no effect on other oxidative parameters.     

Histochemical demonstration of endothelial superoxide and hydrogen peroxide generation in ischaemic and reoxygenated rat tissues.

OBJECTIVE: The aims were to test and evaluate two novel and independent histochemical methods for detecting the initial postischaemic burst of superoxide and hydrogen peroxide in buffer perfused rat tissues during reflow after 60 min warm ischaemia. METHODS: The first is a high manganese/diaminobenzidine technique, in which superoxide oxidises Mn2+ to Mn3+, which in turn oxidises diaminobenzidine to form amber coloured polymers, observable by light microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidises diethylenetriaminepenta-acetate chelated Fe2+ to form intermediate species, which in turn oxidise diaminobenzidine similarly to Mn3+. Various isolated organs of the rat were rendered ischaemic for 60 min, and reperfused with oxygen or air equilibrated buffers containing diaminobenzidine and either Mn2+ or Fe2+. Tissues were fixed by perfusion with Trump's solution and processed for light microscopy. RESULTS: Both manganese and iron methods consistently showed the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells in lung, heart, and intestine of the rat during the first 2 to 3 min of reoxygenation after ischaemia. The histochemical reactions were nearly absent in non-manganese-treated and non-iron-treated controls. Superoxide dismutase strongly inhibited Mn2+/diaminobenzidine reaction product formation and catalase strongly inhibited Fe2+/diaminobenzidine reaction product formation, when tested in specially perfused lung preparations in which these specific antioxidant enzymes were concentrated. CONCLUSIONS: These histochemical techniques provide direct, visual evidence that a burst of reactive oxygen species is generated in postischaemic rat tissues. The Mn2+/diaminobenzidine and Fe2+/diaminobenzidine techniques permit investigation of the endothelium derived reactive oxygen by simple laboratory procedures available to almost any investigator at low marginal cost. The endothelial oxidants so revealed may be of pathophysiological significance in a variety of cardiovascular disorders.     

Effects of hydrogen peroxide on mitochondrial enzyme function studied in situ in rat heart myocytes

Summary Our previous work indicated that energy transduction, as measured by myocyte respiration, was inhibited by hydrogen peroxide, but the mitochondrial membrane potential was relatively unaffected. Therefore, we determined in the present study the critical steps in mitochondrial energy transduction by measuring the sensitivity to hydrogen peroxide of NADH-CoQ reductase, ATP synthase, and adenine nucleotide translocasein situ in myocytes. Adult rat heart cells were isolated using collagenase and incubated in the presence of 0.1–10 mM hydrogen peroxide for 30 min. Activities of NADH-CoQ reductase and oligomycin-sensitive ATP synthase were assayed enzymatically with sonicated myocytes, and adenine nucleotide translocase activities were determined by atractylosideinhibitable [¹⁴C]ADP uptake of myocytes, permeabilized by saponin. The NADH-CoQ reductase and ATP synthase activities were inhibited to 77% and 67% of control, respectively, following an exposure to 10 mM hydrogen peroxide for 30 min. The adenine nucleotide translocase activities were inhibited in a concentration- and time-dependent manner and by 10 mM hydrogen peroxide to 44% of control. The dose-response relationship indicated that the translocase was the most susceptible to hydrogen peroxide among the three enzymes studied. Combined treatment of myocytes with 3-amino-1,2,4-triazole, 1,3-bis(2-chloroethyl)-1-nitrosourea and diethyl maleate (to inactivate catalase, to inhibit glutathione reductase activity, and to deplete glutathione, respectively) enhanced the sensitivity of translocase to hydrogen peroxide, supporting the view that the cellular defense mechanism is a significant factor in determining the toxicity of hydrogen peroxide. The results indicate that hydrogen peroxide can cause dysfunction in mitochondrial energy transduction, principally as the result of inhibition of adenine nucleotide translocase.     

Elevated exhalation of hydrogen peroxide and thiobarbituric acid reactive substances in patients with community acquired pneumonia

BACKGROUND: Bacterial pneumonia involves influx of activated phagocytes into distal airways. These cells release oxidants including H2O2, that may be exhaled or induce peroxidative damage to lung tissues with formation of thiobarbituric reactive substances (TBARs). STUDY OBJECTIVES: To determine whether concentrations of H2O2 and TBARs in exhaled breath condensate (EBC) is elevated and correlate with systemic response to pneumonia during 10 days of hospital treatment. DESIGN: The concentration of H2O2 and TBARs was measured in EBC of 43 inpatients with community acquired pneumonia (CAP) and 20 healthy never smoked subjects over 10 days and were accompanied by monitoring of WBC count, serum concentration of C-reactive protein (CRP) and peroxyl radical-trapping capacity. RESULTS: Patients with CAP exhaled 4.6-, 3.7-, 3.9-, 3.3-times more H2O2 than healthy controls at 1st, 3rd, 5th and 10th day of treatment (P<0.05), respectively. EBC concentrations of TBARs were elevated at 1st and 3rd day. H2O2 and TBARs levels decreased along with treatment course. Correlation (P<0.05) was found between H2O2 levels and CRP and WBC count (r = 0.31) at 1st day and between TBARs and CRP at 5th (r = 0.34) and 10th day (r = 0.46). The mean H2O2 exhalation estimated over ten days of treatment correlated with pneumonic chest X-ray score (r = 0.42), CRP levels (r = 0.46) and WBC count (r = 0.33) at admission (P<0.05). CONCLUSIONS: Pneumonia is accompanied by oxidative stress in airways that moderately correlates with intensity of systemic inflammatory response. Determination of H2O2 in EBC may be helpful for non-invasive monitoring of oxidants production during lower respiratory tract infection.