Intracellular Superoxide Dismutase Activity Research Articles

The Bioflavonoid Fustin, Isolated from Cotinus coggygria Heartwood, Restores Intracellular Activity of Superoxide Dismutase in a Rat Model of Trinitrobenzensulfonic Acid-induced Colitis

The Bioflavonoid Fustin, Isolated from Cotinus coggygria Heartwood, Restores Intracellular Activity of Superoxide Dismutase in a Rat Model of Trinitrobenzensulfonic Acid-induced Colitis

Novel antioxidant peptides from sheep plasma protein hydrolysates: Purification, identification and cytoprotective effects against H2O2-induced oxidative stress.

This study sought to purify and identify antioxidant peptides from sheep (Ovis aries) plasma protein hydrolysates and assess their protective impacts on H2O2-induced Caco-2 cells. The purification process involved reversed high-performance liquid chromatography, anion-exchange chromatography, and Sephadex G-25. Three peptides, namely Trp-Glu-Glu-Pro-Ala-Met (WEEPAM), Ser-Leu-His-Phe-Met-Glu (SLHFME), and His-Cys-Thr-Thr-Phe-Met-Ile, with molecular weights of 761.84, 762.87, and 852.03Da, respectively, were identified by liquid chromatography with tandem mass spectrometry. Among the three antioxidant peptides, superoxide radical (O2 -) radical scavenging capacity of WEEPAM and SLHFME was not significantly different from glutathione (GSH) (p>0.05), while their 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity was greater than GSH (p<0.05). WEEPAM revealed increased antioxidant activity after pepsin and trypsin hydrolysis under an in vitro digestion model. In addition, WEEPAM inhibited oxidative damage in Caco-2 cells by significantly reducing reactive oxygen species accumulation, early apoptosis, malondialdehyde formation, and increasing intracellular superoxide dismutase, glutathione peroxidase, and catalase activities.

Neuroprotective Potential of Thunbergia laurifolia Lindl Leaf Extracts Against Beta Amyloid-induced Neurotoxicity: An in vitroModel of Alzheimer’s Disease

Background: Beta-amyloid peptide (Aβ) induces oxidative stress, contributing to Alzheimer’s disease (AD) initiation and progression. This study aims to explore Thunbergia laurifolia ( T. laurifolia) leaf extract’s protective effects on Aβ25–35-induced oxidative stress and cell injury in SH-SY5Y cells and investigate underlying mechanisms. Materials and Methods: SH-SY5Y cells were treated with T. laurifolia leaf extract in the presence or absence of Aβ25–35. After 24 h, neuroprotective effects were assessed using cell viability and lactate dehydrogenase (LDH) assays. Caspase-3/7 activity, intracellular reactive oxygen species (ROS) levels, catalase (CAT), and superoxide dismutase (SOD) activities were measured to examine mechanisms. Total flavonoid and phenolic content assays were performed. Results: The findings showed that exposure to Aβ25–35 led to a notable rise in oxidative stress in SH-SY5Y cells, as evidenced by increased levels of ROS. Additionally, Aβ25–35 treatment increased caspase-3/7 activity and LDH release and decreased cell viability. However, T. laurifolia extract effectively suppressed ROS production, attenuated caspase-3/7 activity, and concentration-dependently reduced Aβ25–35-induced neurotoxicity. LDH release decreased, and cell viability increased. SOD and CAT activities also increased after T. laurifolia treatment. The extract had total phenolic and flavonoid contents of 178.5 ± 6.86 and 32.51 ± 1.26 mg/g, respectively. Conclusion: T. laurifolia extract demonstrated neuroprotective effects against Aβ25–35-induced injury in SH-SY5Y cells. These effects were attributed to reduced oxidative stress, elevated SOD and CAT activity, and suppressed caspase-3/7 activity. T. laurifolia extract shows potential as an alternative or therapeutic approach to AD mediated by Aβ. Nevertheless, further research is needed to elucidate the mechanism by which T. laurifolia ameliorates neuronal cell death induced by Aβ25–35.

Empagliflozin ameliorates diabetic cardiomyopathy probably via activating AMPK/PGC-1α and inhibiting the RhoA/ROCK pathway.

Diabetic cardiomyopathy (DCM) increases the risk of hospitalization for heart failure (HF) and mortality in patients with diabetes mellitus. However, no specific therapy to delay the progression of DCM has been identified. Mitochondrial dysfunction, oxidative stress, inflammation, and calcium handling imbalance play a crucial role in the pathological processes of DCM, ultimately leading to cardiomyocyte apoptosis and cardiac dysfunctions. Empagliflozin, a novel glucose-lowering agent, has been confirmed to reduce the risk of hospitalization for HF in diabetic patients. Nevertheless, the molecular mechanisms by which this agent provides cardioprotection remain unclear. To investigate the effects of empagliflozin on high glucose (HG)-induced oxidative stress and cardiomyocyte apoptosis and the underlying molecular mechanism. Twelve-week-old db/db mice and primary cardiomyocytes from neonatal rats stimulated with HG (30 mmol/L) were separately employed as in vivo and in vitro models. Echocardiography was used to evaluate cardiac function. Flow cytometry and TdT-mediated dUTP-biotin nick end labeling staining were used to assess apoptosis in myocardial cells. Mitochondrial function was assessed by cellular ATP levels and changes in mitochondrial membrane potential. Furthermore, intracellular reactive oxygen species production and superoxide dismutase activity were analyzed. Real-time quantitative PCR was used to analyze Bax and Bcl-2 mRNA expression. Western blot analysis was used to measure the phosphorylation of AMP-activated protein kinase (AMPK) and myosin phosphatase target subunit 1 (MYPT1), as well as the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and active caspase-3 protein levels. In the in vivo experiment, db/db mice developed DCM. However, the treatment of db/db mice with empagliflozin (10 mg/kg/d) for 8 wk substantially enhanced cardiac function and significantly reduced myocardial apoptosis, accompanied by an increase in the phosphorylation of AMPK and PGC-1α protein levels, as well as a decrease in the phosphorylation of MYPT1 in the heart. In the in vitro experiment, the findings indicate that treatment of cardiomyocytes with empagliflozin (10 μM) or fasudil (FA) (a ROCK inhibitor, 100 μM) or overexpression of PGC-1α significantly attenuated HG-induced mitochondrial injury, oxidative stress, and cardiomyocyte apoptosis. However, the above effects were partly reversed by the addition of compound C (CC). In cells exposed to HG, empagliflozin treatment increased the protein levels of p-AMPK and PGC-1α protein while decreasing phosphorylated MYPT1 levels, and these changes were mitigated by the addition of CC. Adding FA and overexpressing PGC-1α in cells exposed to HG substantially increased PGC-1α protein levels. In addition, no sodium-glucose cotransporter (SGLT)2 protein expression was detected in cardiomyocytes. Empagliflozin partially achieves anti-oxidative stress and anti-apoptotic effects on cardiomyocytes under HG conditions by activating AMPK/PGC-1α and suppressing of the RhoA/ROCK pathway independent of SGLT2.

MiR-218-5p promotes trophoblast infiltration and inhibits endoplasmic reticulum/oxidative stress by reducing UBE3A-mediated degradation of SATB1.

This research evaluated the effects of miR-218-5p on trophoblast infiltration and endoplasmic reticulum/oxidative stress during preeclampsia (PE). The expression of miR-218-5p and special AT-rich sequence binding protein 1 (SATB1) in placental tissues from 25 patients with PE and 25 normal pregnant subjects was determined using qRT-PCR and western blotting. Cell invasion and cell migration were detected by performing Transwell assays and scratch assays, respectively. MMP-2/9, TIMP1/2, HIF-1α, p-eIF2α, and ATF4 expression in cells was assessed through western blotting. Intracellular reactive oxygen species were detected using 2,7-dichlorodihydrofluorescein diacetate, and intracellular malondialdehyde and superoxide dismutase activities were determined with kits. Dual-luciferase and RNA pull-down assays were performed to verify the interaction between miR-218-5p and UBE3A. Co-immunoprecipitation and western blotting were used to detect the ubiquitination levels of SATB1. A rat model of PE was established, and an miR-218-5p agomir was injected into rat placental tissues. The pathological characteristics of placental tissues were detected via HE staining, and MMP-2/9, TIMP1/2, p-eIF2α, and ATF4 expression in rat placental tissues was determined through western blotting. MiR-218-5p and SATB1 were expressed at low levels, while UBE3A was highly expressed in the placental tissues of patients with PE. The transfection of an miR-218-5p mimic, UBE3A shRNA, or an SATB1 overexpression vector into HTR-8/SVneo cells promoted trophoblast infiltration and inhibited endoplasmic reticulum/oxidative stress. It was determined that UBE3A is a target of miR-218-5p; UBE3A induces ubiquitin-mediated degradation of SATB1. In PE model rats, miR-218-5p alleviated pathological features, promoted trophoblast infiltration, and inhibited endoplasmic reticulum/oxidative stress. MiR-218-5p targeted and negatively regulated UBE3A expression to inhibit ubiquitin-mediated SATB1 degradation, promote trophoblast infiltration, and inhibit endoplasmic reticulum/oxidative stress.

A Se-enriched Grifola frondosa polysaccharide induces macrophage activation by TLR4-mediated MAPK signaling pathway

Se-polysaccharide (Se-GFP-22) from Se-enriched Grifola frondosa has double and cooperative activities of polysaccharide and Se. To delineate the underlying mechanism and signaling cascade involved in immune-stimulatory property of Se-GFP-22, the production of cellular mediators and key proteins in signaling pathway was examined. Results showed that Se-GFP-22 exhibited no cytotoxic and had a high capacity to promote macrophage phagocytosis, up-regulate interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide (NO) productions, as well as the relative messenger RNA (mRNA) expressions. In Se-GFP-22-induced macrophages, intracellular superoxide dismutase (SOD) activity was significantly increased to protect cells from oxidative injury. However, Se-GFP-22 induced macrophage activation was suppressed when the toll-like receptor 4 (TLR4) signaling pathway was blocked by a specific TLR4 inhibitor. According to the western blot analysis and the use of specific inhibitors against the mitogen-activated protein kinases (MAPK) signaling pathway, we speculated that Se-GFP-22 activated RAW264.7 macrophages through the TLR4-mediated MAPK signaling pathway. This study provides a molecular basis for the potential of Se-GFP-22 as a novel immune-stimulatory agent.

Reactive oxygen metabolism in the proliferation of Korean pine embryogenic callus cells promoted by exogenous GSH

Exogenous glutathione (GSH) promotes the proliferation of embryogenic callus (EC) cells in Korean pine in the course of somatic embryogenesis, and reactive oxygen species (ROS) may play an important role in regulating the proliferation of EC cells by exogenous GSH. However, the concrete metabolic response of ROS is unclear. In this study, two cell lines of Korean pine with high proliferative potential 001#-001 (F, Fast proliferative potential cell line is abbreviated as F) and low proliferative potential 001#-010 (S, Slow proliferative potential cell line is abbreviated as S) were used as test materials. The responses of ROS-related enzymes and substances to exogenous GSH and L-Buthionine-sulfoximine (BSO) were investigated in EC cells. The results showed that the exogenous addition of GSH increased the number of early somatic embryogenesis (SEs) in EC cells of both F and S cell lines, decreased the amount of cell death in both cell lines. Exogenous addition of GSH promoted cell division in both cell lines, increased intracellular superoxide dismutase (SOD) and catalase (CAT) activities, inhibited intracellular hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) production, and increased NO/ROS ratio. In conclusion, the exogenous GSH promoting the proliferation of Korean pine EC cells, the activity of intracellular antioxidant enzymes was enhanced, the ROS level was reduced, and the resistance of cells to stress was enhanced.

Bioactive polyphenols separated from hemp seed shells ameliorate H2 O2 -induced oxidative stress injury in human umbilical vein endothelial cells.

In the present study, we investigated the protection of hemp seed polyphenols on human umbilical vein endothelial cells (HUVEC) from H2 O2 -mediated oxidative stress injury. Fractions with different polarities were obtained by separating the hemp seed extract using HPD300 macroporous resin-packed column. The fraction, desorbed by 50% ethanol, is rich in polyphenol (789.51±21.92mg GAE/g) and has the highest antioxidant activity in vitro. HPLC-QTOF-MS/MS identified the main polyphenol components in hemp seed shells: 4 hydroxycinnamic acid amides and 15 lignanamides. The protective effects of hemp seed polyphenol against oxidative-stress injury in HUVEC cells were evaluated by cell viability, intracellular antioxidant parameters, and cell apoptosis assay. After HUVEC cells were precultured with 50µg/ml hemp seed polyphenols, the cell viability increased significantly from 53.07±2.46% (model group) to 80.65±1.32% (p<0.01). In addition, the pretreatment of HUVEC cells with polyphenol could substantially increase their intracellular superoxide dismutase activity and reduce their intracellular reactive oxygen species level, malondialdehyde content, and lactate dehydrogenase leakage index. These findings demonstrate the defensive potential of hemp seed polyphenol in reducing the incidence of cardiovascular disease. PRACTICAL APPLICATION: Hemp seed shell waste is produced while producing hemp seed kernel and has abundant phenolic compounds. This research showed that hemp seed polyphenol has potent antioxidant activity in vitro and protects HUVEC cells against H2 O2 -induced oxidative stress injury, suggesting that hemp seed polyphenol has the defensive potential to reduce the incidence of cardiovascular disease. These results indicated that polyphenol separated from hemp seed shells is valuable for further research and development, which will improve the utilization rate of hemp seed.

A Carotenoid- and Nuclease-Producing Bacterium Can Mitigate Enterococcus faecalis Transformation by Antibiotic Resistance Genes.

Dissemination of antibiotic resistance genes (ARGs) through natural transformation is facilitated by factors that stabilize extracellular DNA (eDNA) and that induce reactive oxygen species (ROS) that permeabilize receptor cells and upregulate transformation competence genes. In this study, we demonstrate that Deinococcus radiodurans can mitigate this ARG dissemination pathway by removing both eDNA and ROS that make recipient cells more vulnerable to transformation. We used plasmid RP4 as source of extracellular ARGs (tetA, aphA, and blaTEM-2) and the opportunistic pathogen Enterococcus faecalis as receptor. The presence of D. radiodurans significantly reduced the transformation frequency from 2.5 ± 0.7 × 10-6 to 7.4 ± 1.4 × 10-7 (p < 0.05). Based on quantification of intracellular ROS accumulation and superoxide dismutase (SOD) activity, and quantitative polymerase chain reaction (qPCR) and transcriptomic analyses, we propose two mechanisms by which D. radiodurans mitigates E. faecalis transformation by ARGs: (a) residual antibiotics induce D. radiodurans to synthesize liposoluble carotenoids that scavenge ROS and thus mitigate the susceptibility of E. faecalis for eDNA uptake, and (b) eDNA induces D. radiodurans to synthesize extracellular nucleases that degrade eARGs. This mechanistic insight informs biological strategies (including bioaugmentation) to curtail the spread of ARGs through transformation.

Responses of Issatchenkia terricola WJL-G4 upon Citric Acid Stress.

This study aimed to elucidate the responses of a novel characterized Issatchenkia terricola WJL-G4 against citric acid stress by performing physiological analysis, morphology observation, and structural and membrane fatty acid composition analysis. The results showed that under citric acid stress, the cell vitality of I. terricola WJL-G4 was reduced. The cell morphology changed with the unclear, uncompleted and thinner cell wall, and degraded the cell structure. When the citric acid concentration was 20 g/L, I. terricola WJL-G4 could tolerate citric acid and maintain the cell structure by increasing the intracellular pH, superoxide dismutase activity, and contents of unsaturated fatty acids. As the citric acid concentration was ≥80 g/L, the stress has exceeded the cellular anti-stress ability, causing substantial cell damage. The cell membrane permeability, the content of membrane lipids, malondialdehyde and superoxide anion increased, but the intracellular pH and superoxide dismutase activities decreased, accompanying the increase of citric acid concentrations. The findings of this work provided a theoretical basis for the responsive mechanism of I. terricola WJL-G4 under high concentrations of citric acid, and can serve as a reference for biological acid reduction in fruit processing.

A Novel Virus-Like Agent Originated From Genome Rearrangement of Porcine Circovirus Type 2 (PCV2) Enhances PCV2 Replication and Regulates Intracellular Redox Status In Vitro.

Genome rearrangement occurs to porcine circovirus type 2 (PCV2) during in vitro and in vivo infections, and a number of rearranged PCV2 genomes have been isolated and characterized. This study was conducted to investigate the role of the rearranged PCV2 (rPCV2) in PCV2 replication and the biological effect of rPCV2 in host cells. Two whole rPCV2 genome sequences (358 nt and 1125 nt in length) were synthesized and recombinant plasmids pBSK(+)-rPCV2 (pBSK(+)-1125 and pBSK(+)-358) were constructed. A novel virus-like agent (rPCV2-1125) was rescued by in vitro transfection of porcine kidney cell line (PK-15) and porcine alveolar macrophage 3D4/21 cells. The data indicate that rPCV2-1125 significantly enhanced PCV2 replication in vitro. Furthermore, rPCV2-1125 led to oxidative stress in host cells, as indicated by decreased intracellular glutathione (GSH) and total superoxide dismutase (SOD) activities, as well as increased malondialdehyde (MDA) levels. These results provide new insights into genome rearrangement of PCV2 and will contribute to future studies of PCV2 replication and associated mechanisms.

Cytotoxic Effects of Garcinia mangostana Pericarp Extract on Cancer Cell Lines.

Garcinia mangostana, commonly also called mangosteen, is an evergreen tropical tree, and its pericarps have been used in traditional herbal medicine for different diseases. The anticancer efficacy of the ethanolic extract from the pericarps of Garcinia mangostana was investigated in human prostate cancer cells (PC3), melanoma cells (B16F10), breast cancer cells (MCF7), and glioblastoma (U87) cell lines. 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability. Propidium iodide (PI) staining and analysis on a flow cytometer were used to identify apoptosis. Action on cell migration was evaluated by scratch assay and gelatin zymography. Furthermore, the level of intracellular reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activity was measured. Moreover, we investigated the synergistic efficacy with several combinations of Garcinia mangostana extract (GME) with doxorubicin. GME reduced cell viability in malignant cell dose time-dependently. GME-induced sub- G1 peak in flow cytometry histogram of treated cells control representing apoptotic cell death is involved in GME toxicity. Furthermore, GME exhibited inhibitory effects on the migration ability of U87 cells, which was accompanied by inhibition in the activity and expression of MMP2 (matrix metalloproteinase-2). Besides, GSH level and SOD activity were significantly reduced while there was an increase in ROS and MDA concentration following 24 hr of GME treatment. Moreover, a combination of GME (1.5-25 μg/mL) with Dox (6 μg/mL) displayed synergistic efficacy and cell growth inhibition. In conclusion, GME could cause cell death in PC3, MCF7, U87, and B16F10 cell lines, in which apoptosis plays an imperative role. Plant extract decreased the migration ability of the cells by inhibiting the activity and expression of Matrix metalloproteinases (MMPs). G. mangostana could be a promising therapeutic strategy to treat cancer in the future.

Testosterone and dihydrotestosterone modulate the redox homeostasis of endothelium.

The predominance of cardiovascular diseases among men compared to premenopausal women has been attributed to testosterone, which is implicated in vascular remodeling. Molecular mechanisms underlying its role have not been clarified but oxidative stress-induced inflammation may be important. We therefore investigated in vitro the effects of testosterone and dihydrotestosterone, (a nonaromatized androgen), on redox homeostasis in absence (basal conditions) and after corticotropin-releasing hormone-induced pro-oxidant action in macroendothelial cells. More specifically, we explored their role on well-established antioxidant enzymes activity, namely endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that both androgens significantly increased the intracellular reactive oxygen species levels, endothelial nitric oxide synthase activity, nitric oxide concentration as well as superoxide dismutase activity and decreased catalase activity. These effects of Testosterone and DHT were reversed in the presence of the androgen receptor antagonist, flutamide. Moreover, testosterone and dihydrotestosterone similarly enhanced the stimulatory effect of corticotropin-releasing hormone on intracellular reactive oxygen species levels and superoxide dismutase activity but did not influence the inhibitory effect on endothelial nitric oxide synthase activity, nitric oxide release and catalase activity. Finally, androgens did not have a detectable effect on glutathione levels or the glutathione/glutathione plus glutathione disulfide ratio. Our results reveal that testosterone and DHT rise the intracellular redox threshold of the endothelial cell and increases NO synthesis. These findings suggest that the action of testosterone is affected by the redox status of the endothelium and help to explain its controversial effects on the cardiovascular system.

ITRAQ-based quantitative proteomic reveals proteomic changes in Serratia sp. CM01 and mechanism of Cr(Ⅵ) resistance

ObjectiveSerratia sp. CM01 is a wild strain with the resistance and reduction ability of chromium(Ⅵ). The aim of this study it to investigate the underlying mechanisms of the Cr(Ⅵ) tolerance and reduction of strain CM01, and to explore its response to environmental pollution pressure at the molecular level. MethodsThe iTRAQ technique was utilized to investigate the differentially expressed protein patterns related to the Cr(Ⅵ)-resistance in wild-type strain CM01 and domesticated CM01. RT-qPCR was used to verify the expression levels of several functional genes. The cell surface hydrophobicity and autoaggregation, the intracellular glucose content, and the total superoxide dismutase (SOD) activity were determined. ResultsIn total, 2750 proteins were detected and identified in WT CM01 and domesticated CM01. Compared with WT CM01, the iTRAQ results of 646 proteins were found to be significantly differentially expressed in domesticated CM01. There were 343 up-regulated and 303 down-regulated proteins, which mainly related to carbohydrate metabolism, stress responses, amino acid metabolism and some other systems. RT-qPCR results showed that the expression level of seven genes in domesticated CM01 were consistent with the iTRAQ proteomic profiles. The cell surface hydrophobicity, self-aggregation, intracellular glucose content and total SOD activity of domesticated CM01 with Cr(Ⅵ) treatment were significantly higher than without Cr(Ⅵ) treatment. ConclusionDomesticated CM01 displayed a complex biological network to exhibit the tolerance of Cr(Ⅵ), which may be attributed to the following aspects: (a) CM01 reduced the consumption of glucose by inhibiting the metabolism of carbohydrates, which was an energy-saving survival mode. (b) The inositol phosphate metabolism pathway played an important role. (c) Oxidative stress proteins enhanced the adaptability. (d) CM01 enhanced biosynthesis of hydrophobic amino acids to resistance to Cr(Ⅵ). (e) Several key systems and proteins, such as UvrABC system, Lon protease, porin OmpC, also may play an important role.

Evaluation of the oxidation stability and anti-cancer cell activity of Paeonia ostii seed oil and its linolenic acid fractions delivered as microemulsions

In this study, oil-in-water microemulsions (MEs) were developed and used as a stable carrier for delivery of Paeonia ostii seed oil (PSO), a fatty acid mixture (FAM), and α-linolenic acid (ALA). Dynamic light scattering (DLS) was used to measure the particle size and polydispersity index (PDI) of the ME. The effects of temperature, exposure to light, and the addition of antioxidants on the peroxide values of the loads were investigated. The MEs were composed of droplets with diameters between 15 and 35 nm and PDIs lower than 0.5, and the addition of antioxidants had no significant effect on the particle size and dispersibility. Temperature, more than light, was the factor that most significantly promoted doxidation, and the influence of the addition of different antioxidants was higher by butylated hydroxytoluene (BHT), followed by vitamin C and vitamin E. Viability and morphology of A549 human lung adenocarcinoma cells treated with MEs were studied. The three evaluated MEs caused damage to cell membrane and inhibited the growth of cancer cells in a dose-dependent manner. Data on intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in cancer cells indicated that the treatment with MEs stimulated oxidative stress response and induced oxidative damage. These findings showed the latent capacity of MEs for protection of nutritional factors and for drug delivery.

Superoxide Dismutase (SOD) Activity in Cryopreserved Semen of Itik Pinas-Khaki (Anas platyrhynchos L.)

Cryopreservation induces oxidative stress on sperm due to an increase in the number of reactive oxygen species (ROS), thereby resulting in decreased sperm quality. ROS's destructive potential is normally counteracted in sperm by their innate antioxidant system consisting of enzymes, which include superoxide dismutase (SOD). This study aimed to assess the quality of semen from Itik Pinas-Khaki (IP-Khaki) drakes that were cryopreserved with either 4.5% DMSO or 7.0% glycerol as cryoprotectant through evaluation of total sperm motility (%) and determination of SOD activity (U/mL). Here, semen samples were collected from 12 sexually mature IP-Khaki drakes, an improved egg-type breed of Philippine mallard duck, and processed using modified reported cryopreservation procedure for ducks. Results showed that post-thawing total sperm motility averages of 12.04±5.61% using 4.5% DMSO and 13.99±5.28% using 7.0% glycerol were comparable. Moreover, similar SOD activity levels of 0.39±0.18 U/mL with 4.5% DMSO and 0.33±0.21 U/mL with 7.0% glycerol in 2.00 x 108 IP-Khaki sperm cells were also observed. The observed very low intracellular SOD activity indicates severe damage to sperm cells due to cryopreservation, which resulted in a comparably low total sperm motility with either of the cryoprotectants. Thus, the cryopreservation protocol used is not the optimum for IP-Khaki semen based on the observed considerable decline in sperm motility and very low SOD activity after cryopreservation.

FC 103PROTEOME WIDE OXIDATIVE STRESS PROFILING IN MESOTHELIAL CELLS INDUCED BY PERITONEAL DIALYSIS FLUID

Abstract Background and Aims Reactive oxygen species (ROS) in the peritoneal cavity may result both from CKD and the specific composition of peritoneal dialysis fluids (PDF). Elevated cellular oxidative stress is defined as a cellular oxidant/antioxidant imbalance which impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. So far only single targets or mediators of oxidative stress were investigated in mesothelial cells exposed to PD fluids. Here, we aim to analyze the broad impact and also identify individual targets of ROS during PD. Using the developed technique the anti-oxidative effect of alanyl-glutamine (AlaGln) supplementation of PDF was characterized on the proteome level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (100 µm hydrogen peroxide (H2O2)) and PD-fluid induced stress. Levels of oxidative stress were first evaluated by intracellular ROS levels and superoxide dismutase activity. Oxidative stress levels induced by PDF were titrated to comparable levels of H2O2 treatment to be able to characterize redox modifications and the effect of addition of 8 mM AlaGln. To detect alterations of the redox proteome we adapted and refined an approach combining redox-sensitive isobaric mass tags and high-performance liquid chromatography coupled to mass spectrometry (LC/MS). We used a sequential combination of direct and indirect labeling of redox-sensitive cysteine residues. Results Exposure to PDF increased intracellular ROS production and accumulation as well as cell damage assessed by LDH-release compared to control cells. Cells exposed to AlaGln supplemented PDF showed less cell damage compared to PDF alone. Addition of AlaGln not only reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but also led to different proteins being affected by redox modifications. The carefully optimized highly sensitive LC/MS-based redox proteomics workflow allowed identification of 5537 proteins of which 2614 contained a labeled cysteine. H2O2 treatment resulted in a shift of median oxidation from 11% under control conditions to 36%. While PDF alone increased the oxidation level to 31%, AlaGln supplemented PDF only led to 15% oxidation. Pathway analysis of proteins that changed their oxidation level &amp;gt;50% following the treatment were subjected to molecular pathway analysis revealing distinct differences. PDF exposure leads to regulation of general cell processes like regulation of glucokinase, RNA-binding and SUMOylation, addition of AlaGln regulated more specific signaling pathways for example fibrosis related pathways like TGF-ß and SMAD signaling. Conclusion Redox proteomics of peritoneal cells could represent a novel tool for the identification of mediators of uraemia and PD-induced pathomechanisms, and also to evaluate anti-oxidant pharmacological interventions to improve PD outcomes.

The Effects of Triclosan on Physiological and Photosynthetic Characteristics of Chlorella vulgaris

Triclosan has been widely used as addition ingredient in personal care and medical antibacterial products, and the increasing amounts of triclosan discharged in aquatic environments pose a potential risk to aquatic ecological systems. In this study, we investigated the effects of exposure to varying triclosan concentrations on the growth, chlorophyll fluorescence and antioxidant enzyme activity of Chlorella vulgaris. The results showed that low-concentration triclosan (&lt;0.75 mg/L) can stimulate the growth of Chlorella vulgaris, whereas 1.05 mg/L triclosan exhibited significant inhibition. Low-concentration triclosan (&lt;0.75 mg/L) could improve the tolerance and utilization ability of Chlorella vulgaris in relation to strong light. We observed a significant increase in the malondialdehyde content of Chlorella vulgaris exposed to 1.05 mg/L triclosan. The intracellular superoxide dismutase and catalase (CAT) activities of Chlorella vulgaris exposed to triclosan were higher than the control groups, and the increase in this activity was positively correlated with the concentration of triclosan. The results also showed that excessive H2O2 may in turn damage the CAT structure and eventually inactivate CAT activity when Chlorella vulgaris is exposed to 1.05 mg/L triclosan. This study provided a theoretical basis which can be used to evaluate the ecological risk of triclosan in the aquatic environment.

Arsenate toxicity to the marine microalga Chlorella vulgaris increases under phosphorus-limited condition.

To understand the arsenic (As) toxicity to aquatic organisms in the phosphors-polluted aquatic ecosystem, the growth, the physiological response of Chlorella vulgaris exposed to As (V), and the underlying mechanism were investigated under different phosphorus (P) levels (0, 6, 13, 32 μM). Results showed that As toxicity to the marine microalga C. vulgaris was enhanced under P-limited condition. P supply distinctly altered the effect of As on the light-harvesting efficiency of photosystem. Insufficient P supply also resulted in an enhanced level of membrane integrity loss, which probably facilitated As entering cells and led to stronger toxicity to C. vulgaris under low P supply. At high concentrations of As, the relative superoxide dismutase (SOD) activity was significantly enhanced. When phosphorus was limited, the activation of peroxidase (POD) was significantly enhanced after adding As (V). When intracellular SOD activity was at its highest level, the level of membrane peroxidation (MDA) was also at the highest level, and membrane peroxidation level was positively related to the level of membrane integrity loss (Pearson R2=0.8977). These results suggested that alternation of light-harvesting efficiency of photosystem and As-induced oxidative damage, resulting in membrane peroxidation and integrity loss, were the possible mechanism of As toxicity to C. vulgaris. This study provided insight into the understanding of As toxicity to algae in the eutrophication aquatic system and the potential application of algae in As remediation.

Supernatants of Bifidobacterium longum and Lactobacillus plantarum Strains Exhibited Antioxidative Effects on A7R5 Cells.

Vascular reactive oxygen species (ROS) play an essential role in cardiovascular diseases and the antioxidative effects of probiotics have been widely reported. To screen the probiotic strains that may prevent cardiovascular diseases, we tested the antioxidative effects of supernatants of different Bifidobacterium and Lactobacillus strains on A7R5 cells. Preincubation with supernatants of B. longum CCFM752, L. plantarum CCFM1149, or L. plantarum CCFM10 significantly suppressed the angiotensin II-induced increases in ROS levels and increased catalase (CAT) activity in A7R5, whereas CCFM752 inhibited NADPH oxidase activation and CCFM1149 enhanced the intracellular superoxide dismutase (SOD) activity simultaneously. Treatment with CCFM752, CCFM1149, or CCFM10 supernatants had no significant impact on transcriptional levels of Cat, Sod1, Sod2, Nox1, p22phox, or p47phox, but altered the overall transcriptomic profile and the expression of genes relevant to protein biosynthesis, and up-regulated the 60S ribosomal protein L7a (Rpl7a). A positive correlation between Rpl7a expression and intracellular CAT activity implied that Rpl7a may participate in CAT synthesis in A7R5. Supernatant of CCFM752 could also down-regulate the expression of NADPH oxidase activator 1 (Noxa1) and angiotensinogen in A7R5. Collectively, the probiotic strains CCFM752, CCFM1149, and CCFM10 exhibited antioxidative attributes on A7R5 cells and might help to reduce the risk of cardiovascular diseases.