Rate Of Superoxide Generation Research Articles

Self-Reporting Conjugated Polymer Nanoparticles for Superoxide Generation and Detection.

Conjugated polymer nanoparticles (CPNs or Pdots) have become increasingly popular fluorophores for multimodal applications that combine imaging with phototherapeutic effects. Reports of CPNs in photodynamic therapy applications typically focus on their ability to generate singlet oxygen. Alternatively, CPN excited states can interact with oxygen to form superoxide radical anion and a CPN-based hole polaron, both of which can have deleterious effects on fluorescence properties. Here, we demonstrate that CPNs prepared from the common conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT, also known as F8BT) generate superoxide upon irradiation. We use the same CPNs to detect superoxide by doping them with a superoxide-responsive hydrocyanine dye developed by Murthy and co-workers. Superoxide induces off-to-on fluorescence switching by converting quenching hydrocyanine dyes to fluorescent cyanine dyes that act as fluorescence resonance energy transfer (FRET) acceptors for PFBT chromophores. Amplified FRET from the multichromophoric CPNs yields fluorescence signal intensities that are nearly 50 times greater than when the dye is excited directly or over 100 times greater when signal readout is from the CPN channel. The dye loading level governs the maximum amount of superoxide that induces a change in fluorescence properties and also influences the rate of superoxide generation by furnishing competitive excited state deactivation pathways. These results suggest that CPNs can be used to deliver superoxide in applications in which it is desirable and provide a caution for fluorescence-based CPN applications in which superoxide can damage fluorophores.

The Effects of Combined 1-Methylcyclopropene and Melatonin Treatment on the Quality Characteristics and Active Oxygen Metabolism of Mango Fruit during Storage.

In this study, mango fruit (Tainong No. 1) was treated with either 0.1 mg/L 1-methylcyclopropene (1-MCP) alone or with a combination of 0.1 mg/L 1-MCP and 0.2 mM melatonin (MT). The mango fruit was then stored for 10 days at 25 °C and 85-90% relative humidity. Quality characteristics and the active oxygen metabolism of postharvest mangoes were evaluated every 2 days. Compared to untreated mango fruit, those with the treatments of 1-MCP alone or 1-MCP + MT had a better appearance and higher levels of soluble sugar, ascorbic acid, and titratable acidity. Moreover, these treatments prevented the loss of fruit firmness, successfully delayed the escalation of a* and b* values, and reduced malondialdehyde content and superoxide anion generation rate. After 10 days of storage, mango fruit treated by 1-MCP alone or 1-MCP + MT exhibited increased activities of antioxidant enzymes such as ascorbate peroxidase, catalase, superoxide dismutase, and other peroxidases; nevertheless, the two treatment protocols maintained higher mango total phenolic content only at the later stage of storage. These findings suggest that mango fruit treated with 1-MCP alone or with 1-MCP + MT improves the quality characteristics and antioxidant activities. Moreover, compared to 1-MCP treatment alone, 1-MCP + MT-treated mangoes exhibited higher quality and a stronger regulation of active metabolism during storage.

Glutathione Upregulates the Expression of KATP Channels and Vasorelaxation Responses and Inhibits mPTP Opening and Oxidative Stress in the Heart Mitochondria of Old Rats.

In the present work, we investigated the effect of exogenous glutathione in old rats on the expression of ATP-sensitive potassium (KATP) channels, the mitochondrial permeability transition pore (mPTP) opening in the heart, and the vasorelaxation responses of isolated aortic rings to activation of KATP channels. Experiments were performed on adult (6 months) and old (24 months) male Wistar rats, which were divided into three groups: adult, old, and glutathione-treated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg 1 hour before the studies. The mRNA expression of KATP channels was determined using reverse transcription and real-time polymerase chain reaction analysis. The effect of glutathione administration on mPTP opening, relaxation responses of isolated aortic rings, and oxidative stress markers was studied. It was shown that the expression levels of Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and levels of reduced glutathione were significantly increased in glutathione-treated old rats (by 8.3, 2.8, 13.1, and 1.5-fold, respectively), whereas the levels of oxidative stress markers (hydrogen peroxide, diene conjugates, malondialdehyde, and rate of superoxide generation) in heart mitochondria and mPTP opening were significantly reduced. Relaxation of aortic rings was significantly increased in response to the actions of KATP channel openers flocalin and pinacidil in glutathione-treated animals, which was prevented by glibenclamide. Thus, the administration of exogenous glutathione to old rats resulted in a significant increase in the expression levels of the Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and a decrease in oxidative stress. This was accompanied by inhibition of mPTP opening and enhancement of vasorelaxation responses to activation of KATP channels.

Superoxide Generation by Nicotinamide Coenzymes

Nicotinamide coenzymes can generate superoxide radicalsin alkaline environment. Their formation was registered by reduction of nitroblue tetrazolium (NBT) present in the buffer with formation of diformasan. Inhibition of diformazan formation occurs when superoxide dimutase (SOD) is added to the system, thus confirming generation of O─●. The highest superoxide generating activity was observed with NADPH. In the case of NADPH and NADH, the rate of superoxide generation was significantly lower (by approximately 50%). No O─● was detected when NAD was used under the same conditions and in the same time; however, 4 h later, diformasan was detected in the same sample. The superoxide generating activity decreased in the following order: NADPH > NADH ? NADP > NAD. Other compounds tested (adenosine, ADP and ATP) did not generate superoxide radicals even after prolonged incubation. In a cell, where a local changes in the pH of the environment are possible, nicotinamide coenzymes can be potential sources of O─● and thus participate in cell signaling. A change in pH can initiate this process.

Storage Quality Variation of Mushrooms (Flammulina velutipes) after Cold Plasma Treatment.

Flammulina velutipes is susceptible to mechanical damage, water loss, microbial growth, and other factors that lead to postharvest deterioration, thereby shortening the storage period. The purpose of this study was to analyze the effects of cold plasma treatment on the physicochemical properties and antioxidant capacity of F. velutipes during storage at 4 °C for 21 days. Compared to the control group, cold plasma cold sterilization (CPCS) treatment (150 Hz, 95 kV for 150 s) effectively inhibited the growth and multiplication of microorganisms on the surface of F. velutipes, with no significant effect on the fresh weight change and the superoxide anion generation rate, but with a higher postharvest 1,1-dephenyl-2-picrylhydrzyl (DPPH) clearance rate. Moreover, CPCS increased antioxidant enzyme activities, delayed both malondialdehyde (MDA) accumulation and vitamin C loss, inhibited the browning reaction and polyphenol oxidases (PPO) activity and protected F. velutipes cell membrane from disruption. In general, CPCS not only achieved bacteriostatic effects on F. velutipes during storage, but also reduced cell damage from free radical oxidation, resulting in better postharvest quality and longer shelf life.

The accelerated evolution of human cytochrome c oxidase – Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.

Acidic electrolyzed water improves the postharvest quality of jujube fruit by regulating antioxidant activity and cell wall metabolism

Disease development of ‘Lingwu Long’ jujube fruit treated with acidic electrolyzed water (AEW) at 4 ± 1℃ for storing 30 d was evaluated by the reactive oxygen species (ROS) metabolism, phenylpropanoid metabolism, and cell wall metabolism. Compared to the control, jujube fruit treated with AEW had a lower disease index. At the end of storage, AEW treatment maintained lower malondialdehyde (2.1 μmol kg−1) and superoxide anion generation rate (41.5 nmol g−1 min−1) compared to the control, while enhancing catalase (127.2 U g−1) and superoxide dismutase activities (244.2 U g−1). AEW treatment increased 1,1-diphenyl-2-picrylhydrazyl scavenging ability and hydrogen peroxide. The higher contents of ascorbic acid and glutathione were related to higher activities of ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase in AEW-treated jujube fruit. Moreover, the highest activities of phenylalanine ammonia lyase (21.1 U g−1), cinnamate-4-hydroxylase (304.2 U g−1), and 4-coumarate-CoA ligase (6.4 U g−1) and contents of total phenolics (5.3 g GAE kg−1) and total flavonoids (0.3 mg RE g−1) were observed in AEW-treated fruit compared to the control. Higher firmness, Na2CO3-soluble pectin, cellulose, and hemicellulose and lower water-soluble pectin in AEW-treated fruit were due to lower activities of polygalacturonase, pectinate lyases, β-galactosidase, and cellulase. As a result, AEW could be used for decreasing decay occurrence and maintaining storage quality of jujube fruit.

Metabolomics and Transcriptomics Analysis of Pollen Germination Response to Low-Temperature in Pitaya (Hylocereus polyrhizus).

Cross-pollination can improve the percentage of fruit set and fruit weight for most red flesh varieties in pitaya. The technology of pollen storage was very important for successful cross-pollination. However, till present, the technology of pollen storage is unsatisfactory in pitaya production. In this study, pitaya pollen stored at low temperature was taken as the research object, and its physicochemical indexes, metabolomics, and transcriptomics were studied. The results showed that in vitro pollen germination rate decreased significantly with the increase in storage time. Soluble sugar and soluble protein content of pollen peaked on the first day of storage, whereas its relative conductivity, and manlondialdehyde (MDA) and proline contents increased gradually during storage. At the same time, the antioxidant enzyme system of pollen was also affected. Superoxide dismutase (SOD) activity decreased, while the activities of catalase (CAT) and peroxidase (POD) increased and superoxide anion generation rate increased gradually during storage. According to the metabolomics results, amino acid, peptide, nucleotide, plant hormone, terpene, alcohol, phenol, flavonoid, sterol, vitamin, ester, sphingolipid, and ketone contents increased significantly during storage, whereas flavonoid and pigment contents declined gradually. During pollen storage, the gene expressions related to carbohydrate metabolism, protein metabolism, acid and lipid metabolism, sterol metabolism, plant hormone metabolism, and signal transductions were significantly downregulated. With KEGG pathway analysis, isoquinoline alkaloid biosynthesis, tyrosine metabolism, alanine, aspartate, and glutamate metabolism of pollen were affected significantly during low-temperature storage. Correlation analysis showed that the gene expression patterns of HuRP2, HuUPL1, and HuAAT2 had significant effects on pollen germination. D-arabinose 5-phosphate and myricetin were positively correlated with pollen germination rate, which was valuable for studying preservation agents. In this study, the changes in pollen during low-temperature storage were described from the level of metabolites and genes, which could provide theoretical support for the research and development of pollen long-term storage technology in pitaya.

Metastatic Colorectal Cancer: Redox Metabolism and Malignancy

Investigating the tumor microenvironment has a direct impact on the understanding of the mechanisms which regulate cancer progression and treatment. Tumor cells undergoing profound changes in their own intrinsic metabolism affect the microenvironment. Colorectal cancer (CRC) is the third most common cancer with the predominant metastases (Mts) into the liver. Mts in liver tissues (LT), adjacent to Mts (AMT) and remote tissues (RMT, taken at the distance of 5 cm from Mts) from 25 patients with metachronous liver Mts after the liver resection as well as blood and urine were studied by electron paramagnetic resonance (EPR) including the spin-trapping EPR for the detection and quantification of the activity of N2 iron-sulfur proteins, levels of NO-N2 complexes, labile iron pool (LIP), lactoferrin (Lf), superoxide and NO radicals. Activity of metalloproteinase MMP-2 and MMP-9 were determined. In adjacent and remote LT low activity of N2 in mitochondrial electron transport chain (EPR signal with g=1.94), loss of functions of detoxification system (cytochrome P450, g=2.25), appearance and growth of NO-N2 complexes (g=2.007) are obtained. Intensive EPR signals from LIP (g=2.2-2.4) and Lf (g=4.3) are registered. Superoxide generation rates were of up to 6 times higher than in the reference LT tissues and blood (p<0.001). NO levels are of 1.7 times higher for the AMT compared to RMT (p<0.05) while being 15 times higher for blood comparing to the reference species (p<0.001). Activity of MMP-2 and MMP-9 was registered both in AMT and RMT and is in 1.7 times higher in ALT (p<0.05). The obtained results can be used to estimate the functional state of organs and tissues with distant metastases, the risk of recurrence, to correct the antitumor therapeutic procedures.

The Molecular and Functional Characterization of the Durum Wheat Lipoxygenase TdLOX2 Suggests Its Role in Hyperosmotic Stress Response.

In plants, lipoxygenases (LOXs) are involved in various processes, such as growth, development, and response to stress cues. In the present study, the expression pattern of six durum wheat LOX-encoding genes (TdLpx-B1.1, TdLpx-B1.2, TdLpx-A2, TdLpx-B2, TdLpx-A3 and TdLpx-B3) under hyperosmotic stress was investigated. With osmotic (0.42 M mannitol) and salt (0.21 M NaCl) stress imposed at the early stages of seedling growth, a strong induction of the TdLpx-A2 gene expression in the shoots paralleled an equally strong increase in the LOX activity. Enhanced levels of malondialdehyde (MDA) and increased rates of superoxide anion generation were also observed as a result of the stress imposition. Sequence analysis of the TdLOX2 encoded by the TdLpx-A2 gene revealed that it belonged to the type-1 9-LOX group. When overexpressed in E. coli, TdLOX2 exhibited normal enzyme activity, high sensitivity to specific LOX inhibitors, with 76% and 99% inhibition by salicylhydroxamic and propyl gallate, respectively, and a preference for linoleic acid as substrate, which was converted exclusively to its corresponding 13-hydroperoxide. This unexpected positional specificity could be related to the unusual TV/K motif that in TdLOX2 replaces the canonical TV/R motif of 9-LOXs. Treatment of seedlings with propyl gallate strongly suppressed the increase in LOX activity induced by the hyperosmotic stress; the MDA accumulation was also reduced but less markedly, whereas the rate of superoxide anion generation was even more increased. Overall, our findings suggest that the up-regulation of the TdLpx-A2 gene is a component of the durum wheat response to hyperosmotic stress and that TdLOX2 may act by counteracting the excessive generation of harmful reactive oxygen species responsible for the oxidative damages that occur in plants under stress.

Fractal and Topological Analyses and Antioxidant Defense Systems of Alfalfa (Medicago sativa L.) Root System under Drought and Rehydration Regimes

Alfalfa (Medicago sativa L.) is a nutritious forage plant in dryland regions with strong drought tolerance and broad adaptability. To understand the architectural characters and physiological functions of the root systems in response to different drought stress and rehydration, two experiments were conducted in a glasshouse. Experiment 1 used three alfalfa cultivars and four soil moisture treatments in a split-plot design with four replicates to investigate the response of root systems of different alfalfa cultivars to drought stress and subsequent rehydration. Experiment 2 used one alfalfa cultivar and four soil moisture treatments in a randomized complete block (RCB) design with three replicates to measure reactive oxidant species and antioxidant defense systems of alfalfa root. The soil moisture treatments included (1) 75–80% of the water-holding capacity (CK); (2) 60–65% of the water-holding capacity and subsequent rehydration to 75–80% of the water-holding capacity; (3) 45–50% of the water-holding capacity and subsequent rehydration to 60–65% and 75–80% of the water-holding capacity; and (4) 30–35% of the water-holding capacity and subsequent rehydration to 45–50%, 60–65% and 75–80% of the water-holding capacity. Our results demonstrated that plants had higher root biomasses (mean = 0.77 g), fractal dimensions (1.47), fractal abundances (3.84), and branching ratios (0.39) under severe water deficit and rehydration than under less stressful soil moisture conditions. Plant root systems tended to exhibit herringbone branching (Topological Index = 0.63) when soil moisture was at 60–65% water-holding capacity with the soil being then rehydrated, whereas they tended to have a smaller lateral root length, root surface area, root volume, and branching ratio and larger root diameter under more favorable soil moisture conditions for less stressful soil moisture treatments. Severe water stress and subsequent considerably improved superoxide (O2−) generation rate, the content of hydrogen peroxide (H2O2) and Malondialdehyde (MDA), and non-enzyme antioxidant glutathione (GSH), and reduced the content of ascorbate (AsA) and activity of catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX). It is concluded that Medicago sativa L. cv Zhaodong (ZD) and cv Aohan (AH) exhibited higher drought resistances than cv Golden empress (GE) using topological and fractal analyses. This could be a key contributor to altering optimum soil moisture conditions and screening alfalfa cultivars based on root system architecture and antioxidant defense systems. The molecular functions warrant further investigation which could help explain the observed root phenotypes after drought conditions and rehydration.

Possible role of seasonality and harmful algal blooms (HAB) on the oxidative and nitrosative metabolisms in hemocytes

Bivalves survive to biotoxin consumption but their metabolism could be affected. The objective of this work was to study the oxidative and nitrosative changes in the hemocytes of the mussel Mytilus edulis platensis in different seasons, including spring, characterized by the appearance of harmful algal blooms (HAB). Reactive species generation rate (measured as the 2′,7′dichlorofluorescein diacetate oxidation rate) was increased by 2.5- and 8.3-fold in hemocytes from spring and summer, respectively, as compared to winter samples. Neither total Fe nor labile Fe pool content was changed in the three seasons. Superoxide anion generation rate was 3-fold higher in spring as compared to winter and summer samples. Catalase content in spring cells were significantly higher as compared to winter (60%) and summer (3-fold increase) but glutathione-S-transferase activity only increased compared to summer season (125% increase). Lipid radical content in spring samples was 140 and 50% higher as compared to cells from winter and summer, respectively. Nitric oxide and nitro-tyrosine content were significantly higher in samples from spring as compared to values obtained either in winter or summer cells. Considering the aspects that influence metabolism, changes in temperature seem to mainly affect the oxidative over the nitrosative condition of the hemocytes. Nevertheless, HAB biotoxins seem as a contributing factor to affect not only reactive oxygen species generation, antioxidant activity and protein/lipid damage, but also the nitrosative metabolism. In this regard, the changes in the nitric oxide content are new and critical evidence that HAB-related toxins could affect reactive nitrogen species metabolism.

Redox state of adipose tissue for patients with gastric cancer and its connection with the body mass index and distance from the tumor

Excess body weight has been causally linked to an increased risk of different cancer types, including gastric cancer but the mechanisms underlying this relationship are not well understood. Superoxide generation rate, activity of complex I in electron transport chain of mitochondria, activity of matrix metalloproteinase (MMP-2 and 9) of adipose tissues (AT) of patients with gastric cancer in AT located adjacent to tumor (ATAT) and at a distance of 3 cm (ATD) are measured to follow the connection of the redox state with some of the microenvironment indicators (HIF-1α, CD68, Plin5), body mass index (BMI) and cancer metastasis.Superoxide generation rate in ATAT positively correlates with BMI (r = 0.59, p < 0.05) being 4 times higher than in control (p < 0.05). MMP-2, 9 activities in ATAT positively correlate with BMI (r = 0.67, p < 0.05) being 3.3-4.0 higher than in control (p < 0.05). In ATD a statistically significant increase of MMP-2 activity is found. In ATAT for the group of patients with distant metastasis (M1) the superoxide generation rate, MMP-2, 9 activities are about 2 times higher (p < 0.05) than in the subgroup without distant metastases (M0). M1 is also characterized by the increased values of HIF-1α+ (factor 1.25), CD68+ (factor 1.4) and Plin5+ (factor 2.1) compared to M0 category in tumor tissues (p < 0.05). The results can be used for better understanding the mechanism(s) of symbiosis of tumor and adipose tissues as well as serve as a basis for new therapeutic approaches.

Evaluation of salt tolerant mungbean (Vigna radiata L.) Genotypes on growth through bio-molecular approaches

This study was conducted to obtain saline tolerant mungbean genotypes through evaluating growth, biochemical and molecular parameters, and possible salt tolerant mechanisms were studied in different salt sensitive genotypes. Thirteen prescreened mungbean genotypes were grown on 0, 40, 80 and 120 mMNaCl induced salinity and evaluated by germination percentage, shoot and root length, superoxide (O2•-) generation rate, concentration of H2O2, lipid peroxidation (as malondialdehyde, MDA), methylglyoxal (MG), K+/Na+ and proline content in leaves. Based on these parameters, genotypes BD-10588, BD-6894 and IR-01 were selected as tolerant genotypes. For studying oxidative stress tolerance mechanism, BD-10588 and IR-01 were used as tolerant and BD-6887 and BD-10741 as susceptible genotypes, and comparative ROS (O2•- and H2O2), and MDA as well as LOX activity between the two groups were determined. Analysis of activities of ROS metabolizing antioxidant enzymes strongly suggested that superoxide dismutase in tolerant genotypes provided first line protection from salt induced O2•-. Higher catalase (CAT) and ascorbate peroxidase (APX) played major role in H2O2 metabolism in tolerant genotypes. Both specific and in-gel activities of glutathione peroxidase (GPX) strongly proved the H2O2 metabolism for reducing oxidative damage in both tolerant and susceptible genotypes. However, higher peroxidase activity was important for mitigating salt stress in susceptible mungbean genotypes. Therefore, SOD, APX and GPX are very important for protecting salt mediated oxidative damage in mungbean genotype.&#x0D; Bangladesh J. Agril. Res. 44(3): 469-492, September 2019

Hydroxyl radical and non-hydroxyl radical pathways for trichloroethylene and perchloroethylene degradation in catalyzed H2O2 propagation systems

Catalyzed H2O2 propagations (CHP) is characterized by the most robust reactivity of any of the in situ chemical oxidation (ISCO) technologies because it generates the strong oxidant hydroxyl radical along with nucleophiles + reductants, such as superoxide radical. The most common groundwater contaminants, trichloroethylene (TCE) and perchloroethylene (PCE), were used as model contaminants in evaluating the effect of hydrogen peroxide (H2O2) dosage on contaminant destruction kinetics. Both TCE and PCE degradation rates increased with H2O2 dosages up to 0.1 M, and then decreased with higher H2O2 dosages. Parallel reactions conducted with the addition of the hydroxyl radical scavenger 2-propanol and the hydroxyl radical-specific probe nitrobenzene confirmed that hydroxyl radical is primarily responsible for TCE and PCE degradation; however, 5–20% of their degradation was attributed to a non-hydroxyl radical mechanism. Reactions conducted with the superoxide probe tetranitromethane showed that superoxide generation rates increased with increasing H2O2 doses. These results were confirmed by electron spin resonance spectroscopy. Therefore, the non-hydroxyl radical pathway for TCE and PCE degradation at H2O2 ≥0.5 M was likely via nucleophilic attack by superoxide. The results of this research demonstrate that contaminants present in the aqueous phase that are reactive with hydroxyl radical require only low doses of H2O2 (≤0.1 M), but subsurface systems contaminated with species not reactive with hydroxyl radical (e.g., carbon tetrachloride) require H2O2 concentrations ≥0.5 M.

OXIDATIVE AND MUTAGENIC EFFECTS OF LOW INTENSITY GSM 1800 MHz MICROWAVE RADIATION

Despite a significant number of epidemiological studies on potential carcinogenicity of microwave radiation (MWR) from wireless devices and a bulk of experimental studies on oxidative and mutagenic effects of low intensity MWR, the discussion on potential carcinogenicity of low intensity MWR is going on. This study aims to assess oxidative and mutagenic effects of low intensity MWR from a typical commercial model of a modern smartphone. The model of developing quail embryos has been used for the assessment of oxidative and mutagenic effects of Global System for Mobile communication (GSM) 1800MHz MWR from a commercial model of smartphone. The embryos were exposed in ovo to 0.32µW/cm2, discontinuously- 48s- On, 12s- Off, during 5days before and 14days through the incubation period. The exposure of quail embryos before and during the incubation period to low intensity GSM 1800MHz has resulted in expressive statistically significant oxidative effects in embryonic cells, including a 2-fold increase in superoxide generation rate and 85% increase in nitrogen oxide generation rate, damages of DNA integrity and oxidative damages of DNA (up to twice increased levels of 8-oxo-dG in cells of 1-day old chicks from the exposed embryos). Finally, the exposure resulted in a significant, almost twice, increase of embryo mortality. The exposure of model biological system to low intensity GSM 1800MHz MWR resulted in significant oxidative and mutagenic effects in exposed cells, and thus should be recognized as a significant risk factor for living cells.

Rectal Cancer: Redox State of Venous Blood and Tissues of Blood Vessels from Electron Paramagnetic Resonance and Its Correlation with the Five-Year Survival.

A role of pro- and antioxidants for reducing rectal cancer (RC) incidence in operative, preoperative, and postoperative treatments is still disputable and controversial. The redox state of venous blood and tissues of blood vessels of 60 patients with RC (T2-4N0-2M0G2) and 20 donors is studied by means of the conventional and spin-trapping electron paramagnetic resonance (EPR). The intensity of the signals from ceruloplasmin (CP), transferrin (TF), and labile iron pool (LIP) at temperature T = 77 K as well as superoxide generation rate and nitric oxide (NO) levels at T = 300 K is measured. The reduced CP and TF activity and decreased NO levels increased LIP levels and superoxide-generating rates are detected in blood species. Correlation analysis for the five-year survival rate as a function of the extracted values is done. The results show that the intensities of the corresponding EPR signals from the “native” and “trapped” paramagnetic centers can be potentially used for the understanding of the molecular mechanisms underlying the RC progression and treatment.

Oxidative damage and antioxidative indicators in 48h germinated rice embryos during the vitrification-cryopreservation procedure.

Cu/Zn SOD and other genes may be critical indicators of a stress response to reactive oxygen species (ROS) accumulation in 48 h germinated rice embryos subjected to vitrification cryopreservation. In the current study, reactive oxygen species (ROS) accumulation was investigated in 48h germinated rice embryos during the vitrification-cryopreservation process. We found that vitrification-cryopreservation significantly affected ROS levels, especially superoxide anion levels, in 48h germinated rice embryos. Malonaldehyde content in the apical meristems of germinated embryos was significantly positively correlated with the rate of superoxide anion generation and the highest levels of malonaldehyde content were reached after vitrification treatment. Cell viability in 48h germinated embryos was significantly negatively correlated with the rate of superoxide anion generation, malonaldehyde content, and electrolyte leakage. Spatial and temporal patterns in ROS accumulation in these embryos existed during the vitrification procedure. Among the vitrification-cryopreservation treatments we assessed, the preculture treatment was found to stimulate superoxide anion generation and to activate the response system in the apical meristems of germinated embryos. Loading treatments motivated the catalase and ascorbate peroxidase activities. During the vitrification-dehydration treatment, oxidative stress reached the highest levels causing an antioxidative response. This response involved antioxidant enzymes promoting detoxification of ROS. Based on a comprehensive correlation analysis involving ROS accumulation, cell viability, the activities of antioxidant enzymes, and gene expression profiles, Cu/Zn SOD, CAT1, APX7, GR2, GR3, MDHAR1, and DHAR1 may be critical indicators of oxidative stress affected by the vitrification-cryopreservation treatments. The investigation of these antioxidative responses in 48h germinated rice embryos may, therefore, provide useful information with respect to plant vitrification-cryopreservation.

Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125–750 μM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria.

Muscle specific MnSOD deficiency leads to complex II‐specific inactivity of ETC and contractile dysfunction, but increases muscle mass

Multiple pathological conditions exhibit excess mitochondrial ROS and muscle weakness in parallel, including aging, denervation and cancer cachexia. To directly test the role of mitochondrial ROS in skeletal muscle weakness and atrophy we generated a skeletal muscle specific knockout mouse model in which MnSOD (Sod2), the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre‐Lox approach driven by a constitutively active human skeletal actin promoter (HSA‐Cre). Female wild type and MnSOD deficient (mSod2 KO) mice were studied at 6–8 months of age. In mSod2 KO mice, the observed rate of superoxide generation from isolated skeletal muscle mitochondria was significantly elevated, and hydrogen peroxide generation using permeabilized fibers increased by ~5 fold. ATP generation rate and electron transport chain (ETC) activity were severely reduced in response to Complex II linked substrate, suggesting superoxide‐induced complex II inactivity. Reduced ATP generation rate and ETC activity were consistent with severe exercise intolerance in mSod2 KO mice. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we first tested in situ contractile function using gastrocnemius. Maximum isometric specific force was decreased by ~30 % in mSod2 KO mice with direct stimulation on muscle, but the reduction in force was further diminished (~50%) after stimulation of the sciatic nerve suggesting neuromuscular disruption. Morphological assessment exhibited increases in NMJ size and fragmentation in mSod2 KO mice supporting that mitochondrial dysfunction can feed back to initiate NMJ disruption. Surprisingly, gastrocnemius muscle mass was increased in mSod2 KO mice, which was associated with increased numbers of central nuclei and hyperplasia via fiber branching. In summary, our data suggest that excess mitochondrial superoxide caused a selective loss of complex II activity and mitochondrial defects, leading to severe exercise intolerance. Increased cytoplasmic hydrogen peroxide induced necrosis‐mediated regeneration, which increased muscle mass by fiber branching.Support or Funding InformationNIH/NIA to Dr. Van Remmen (R01 AG050676) and a VA Merit grant to HVRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.