Total Reactive Oxygen Species Levels Research Articles

Successful vitrification of early-stage porcine cloned embryos

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.

Guarana: Stability-Indicating RP-HPLC method and safety profile using microglial cells

Paullinia cupana Kunth is a compound with therapeutic potential due to the presence of methylxanthines and tannins. In this sense, the precise and safe quantification of these compounds is of fundamental importance. From this, the objective of this work was to develop and validate a stability-indicating RP-HPLC method for the simultaneous determination of theobromine, theophylline, caffeine, catechin and epicatechin in powdered guarana. In addition, the safety profile of guarana exposure in microglia cells was evaluated through different colorimetric and fluorimetric experiments. Chromatographic conditions suitable were obtained using RP-18 column (250 mm × 4.5 mm, 5 μm) at 280 nm. Mobile phase constituted of A: water (0.1 % trifluoroacetic acid pH 4.2) and B: methanol:acetonitrile (25:75 v/v) 90:10 v/v (A:B) at 1 mL.min−1 flow rate. The in vitro tests of cell viability, nitric oxide and formation of total reactive oxygen species levels demonstrated that when in contact with microglia cells, guarana presents safety concentrations of 3.91–15.63 mg.mL−1. The results demonstrated, specificity, linearity, precision, accuracy, and robustness of the proposed method. Based on in vitro tests, it is believed that, in these concentrations, guarana may exert a neuroprotective activity, preventing the activation of microglia, possibly due to the presence of bioactive molecules.

Dihydroartemisinin initiates ferroptosis in glioblastoma through GPX4 inhibition.

It has been demonstrated from previous studies about the killing effect of dihydroartemisinin (DHA) on glioblastoma, which involves multiple aspects: cytotoxicity, cell cycle arrest and invasion inhibition. DHA has the advantages of low cytotoxicity to normal cells, selective killing effect and low drug resistance, making it one of the popular anti-tumor research directions. Ferroptosis is a newly discovered form of cell death characterized by iron dependence and lipid reactive oxygen species (ROS) accumulation. In the present study, we found differences in the expression of transferrin receptors in normal human astrocytes (NHA) and glioblastoma cells (U87 and A172), which may be one of the mechanisms of DHA selective killing effect. Through the determination of ferroptosis-related protein expression, we found that the significant decrease of GPX4, accompanied by the constant expression of xCT and ACSL4, suggesting GPX4 was a pivotal target for DHA-activated ferroptosis in glioblastoma. Total and lipid ROS levels were increased and all these results could be reversed by the ferroptosis inhibitor, ferrostatin-1. These findings demonstrated ferroptosis would be a critical component of cell death caused by DHA and GPX4 was the main target. All these results provide a novel treatment direction to glioblastoma. The association between ferroptosis and polyamines is also discussed, which will provide new research directions for ferroptosis caused by DHA in glioblastoma.

Nrf2 activation protects auditory hair cells from cisplatin-induced ototoxicity independent on mitochondrial ROS production

Cisplatin is a well-known and commonly used chemotherapeutic agent. However, cisplatin-induced ototoxicity limits its clinical use. Previous studies have shown an important role of reactive oxygen species (ROS) accumulation in the pathogenesis of cisplatin-induced ototoxicity. In many cell types, the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE) protect against oxidative stress by suppressing ROS. Here our results showed that cisplatin injury reduced Nrf2 expression and inhibited Nrf2 translocation in HEI−OC1 cells and Nrf2 activator tert-butylhydroquinone (TBHQ) rescued hair cells from cisplatin induced apoptosis by suppressing the total cellular ROS accumulation. Moreover, we found that decreased ROS accumulation induced by TBHQ didn’t depend on mitochondrial derived ROS production, indicating that Nrf2 activation alleviated cisplatin induced oxidative stress and apoptosis through mitochondrial-independent ROS production. Therefore, we provide a potential strategy of prevention and treatment for cisplatin-induced ototoxicity by Nrf2 activation. In conclusion, Nrf2 activation protects auditory hair cells from cisplatin-induced ototoxicity through suppressing the total cellular ROS levels which arise from sources other than mitochondria.

Effect Of Iron And Diazoxide On Muscle Strength Development, Oxidative Stress And Lipid Profile In Diabetic Rats

Diabetes is a global health problem. Oxidative stress plays an essential role in the development of complications, among the most frequent symptoms is muscle fatigue. Diabetes also contributes to a high cardiovascular risk attributed to dyslipidemia. Diazoxide (dzx) is a drug that acts on mitoKATP. Studies indicate that this drug improves the development of muscle strength; on the other hand, it is known that iron overload (IO) increases oxidative stress, while a reduction has a protective role. The objective was to evaluate the effect of iron and dzx on the development of muscle strength, oxidative stress, and lipid profile in diabetic rats. Male Wistar rats (250g) were used in eight groups: control, diabetics, IO, diabetic + IO, low iron, diabetic + low iron, dzx, diabetic + dzx. Soleus muscle and extensor digitorum longus (EDL) of an extremity were extracted to perform the muscle tension tests, and the other limb was used to obtain homogenates to measure lipid oxidation, glutathione levels and total reactive oxygen species (ROS). Finally, the serum was collected to measure lipid profile. Rats fed with a low iron diet and dzx administration improved muscle strength and fatigue took longer to appear. The groups fed with IO had reduced muscle strength development. Regarding the lipid peroxidation, the groups of diabetic rats fed with a low iron diet (0.34 nM/mg of protein) and dzx administration (0.033 nM/mg of protein) had the lower levels of lipid peroxidation. As for the glutathione levels, diabetic rats treated with dzx obtained the highest levels of reduced glutathione (1.65 μmol of GSH/mg of protein) and oxidized glutathione (GSSG) levels in diabetic rats with a low iron diet obtained the lowest values (0.82 μmol of GSH/mg of protein). Concerning total ROS levels, diabetic rats treated with dzx got the lowest levels (2.80 DCF*mg of protein). As for the lipid profile, diabetic rats treated with dzx obtained lower levels (ẋ =105 mg/dL). Very low‐density lipoprotein (VLDL) levels were high in the group of diabetic rats without treatment (ẋ=58.06 mg/dL), and the group of diabetics + dzx obtained similar levels to the control group (ẋ=21.1 mg/dL). Dzx acts as a mitoKATP opener, previous studies reported that the opening of these channels provokes a decrease in oxidative stress during fatigue and also activates the electron transport chain, which promotes a constant production of ATP, which is necessary for energy production and in this way the generation of muscular strength. On the other hand, iron plays an essential role in the formation of the hydroxyl radical, which is the most dangerous radical, this agrees with the results we obtained in the groups that had an IO. The higher levels of oxidative stress and muscular strength were lower in IO groups; however, the iron restriction had a protective effect on oxidative stress levels. Regarding the lipid profile, both dzx and low iron diets were shown to have a positive impact on the lipid profile by mechanisms that are not yet known.

A Comorbidity Model of Myocardial Ischemia/Reperfusion Injury and Hypercholesterolemia in Rat Cardiac Myocyte Cultures

IntroductionThe use of comorbidity models is crucial in cardioprotective drug development. Hypercholesterolemia causes endothelial and myocardial dysfunction, as well as aggravates ischemia/reperfusion (I/R)-induced myocardial injury. Endogenous cardioprotective mechanisms against I/R are impaired in hyperlipidemic and hyperglycemic in vivo animal models. Therefore, our aim was to develop a medium throughput comorbidity cell-based test system of myocardial I/R injury, hypercholesterolemia and hyperglycemia that mimics comorbidity conditions.MethodsCardiac myocytes isolated from neonatal or adult rat hearts were cultured in control or in three different hypercholesterolemic media with increasing cholesterol content (hiChol) or hiChol + hyperglycemic medium, respectively. Each group was then subjected to simulated ischemia/reperfusion (SI/R) or corresponding normoxic condition, respectively. Cholesterol uptake was tested by Filipin staining in neonatal cardiac myocytes. Cell viability, total cell count and oxidative stress, i.e., total reactive oxygen species (ROS) and superoxide level were measured by fluorescent assays.ResultsNeonatal cardiac myocytes took up cholesterol from the different hiChol media at a concentration-dependent manner. In normoxia, viability of hiChol neonatal cardiac myocytes was not significantly changed, however, superoxide levels were increased as compared to vehicle. After SI/R, the viability of hiChol neonatal cardiac myocytes was decreased and total ROS level was increased as compared to vehicle. HiChol combined with hyperglycemia further aggravated cell death and oxidative stress in normoxic as well as in SI/R conditions. Viability of hiChol adult cardiac myocytes was significantly decreased and superoxide level was increased in normoxia and these changes were further aggravated by SI/R. HiChol combined with hyperglycemia further aggravated cell death, however level of oxidative stress increased only in normoxic condition.ConclusionHiChol rat cardiac myocytes showed reduction of cell viability and increased oxidative stress, which were further aggravated by SI/R and with additional hyperglycemia. This is the first demonstration that the combination of the current hypercholesterolemic medium and SI/R in cardiac myocytes mimics the cardiac pathology of the comorbid heart with I/R and hypercholesterolemia.

Does myoinositol supplement improve sperm parameters and DNA integrity in patients with oligoasthenoteratozoospermia after the freezing-thawing process?

Sperm cryopreservation is a routine method in andrology and IVF laboratory. However, the sperm quality and its fertilizing capacity have been decreased during this process. The purpose of this experiment was to determine the role of myoinositol as a supplement in amelioration of total and progressive sperm motility, DNA fragmentation, total antioxidant capacity (TAC), reactive oxygen species (ROS), and lipid peroxidation after the freezing-thawing process on patients with oligoasthenoteratozoospermia (OAT) syndrome. Semen samples obtained from 40 patients were divided into two aliquots and freezed with simple and 2mg/mL myoinositol (MYO) supplemented freezing media. All samples were thawed and assessed after one month. Semen parameters were analyzed in terms of the motility by CASA, the level of total ROS by fluorimetry, TAC and MDA by colorimetric assay and finally DNA fragmentation by TUNEL assay. Our results clearly showed that MYO could improve total (37.46 vs. 12.91, p < 0.001) and progressive motility (21.92 vs. 6.49, p < 0.001) in experimental group compared to control group. A higher TAC level was observed in the MYO treated group in comparison to control group (1.11 vs. 0.91, p = 0.05). While MYO supplementation could not be effective on ROS level, it reduced DNA fragmentation of sperm after freeze-thaw process (p = 0.01). Therefore, MYO could be a good supplement for sperm freezing to reduce the detrimental effects of freezing process especially on DNA integrity, which is an important factor in the success of ART, in OAT suffered patients.

Genetic Activation of NRF2 By KEAP1 Inhibition Induces Fetal Hemoglobin Expression and Triggers Anti-Oxidant Stress Response in Erythroid Cells

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017). In the present work, we employed CRISPR Cas9 mediated knockout (KO) of KEAP1 in HUDEP2 cells and CD34+ derived erythroid cells to study its impact on downstream gene and protein expression relevant to sickle cell disease such as fetal hemoglobin (HbF) induction and anti-oxidant stress responses. Genetic KO of KEAP1 by six different gRNAs resulted in populations with indel levels from 30 to 95%, as determined by a mutation specific digital droplet PCR (ddPCR) and next-generation sequencing (NGS). LC/MS confirmed KEAP1 knockdown at the protein level. Consequently, NRF2 protein level in the nuclear extract was elevated in KEAP1 KO cells as demonstrated by nuclear NRF2 bound to antioxidant response element (ARE) in a DNA-binding assay. qPCR analysis revealed a robust and significant induction of the NRF2 dependent enzyme, NAD(P)H quinone dehydrogenase 1 (NQO1) and γ-globin. Gene expression levels of NQO1 and γ-globin inductions correlated significantly with the indel percentages; cell populations with ~80% indels showed about 20-fold induction in NQO1, and about 10-fold induction in γ-globin gene expression levels. Upon differentiation for 7 days in culture, KEAP1 KO cells showed up to 4-fold induction in γ-globin protein levels measured by flow cytometry and HPLC. Up-regulation of HbF and NQO1 protein were confirmed using LC/MS analysis. Additionally, KEAP1 KO clonal populations continued to show robust induction of NQO1 and HbF at gene and protein expression levels. To elucidate the mechanism of γ-globin induction, chromatin immunoprecipitation (ChIP) analysis in the KEAP1 KO cells demonstrated enrichment of NRF2 recruitment in the ARE sequences of the promoter regions of NQO1 and γ-globin genes. Next, KEAP1 KO was carried out in bone marrow derived CD34+ cells using a CRISPR Cas9 RNP system. In agreement with our observations in HUDEP2 cells, knockdown of KEAP1 in CD34+ cells resulted in induction of the expression of γ-globin, NQO1 and HO1. Additionally, treatment with CDDO-Me, an NRF2 activator compound, resulted in a 4-fold induction in γ-globin mRNA levels in primary SCD patient derived CD34+ cells (by ddPCR), a 2.5-fold induction in HbF (by flow cytometry), and a 2.5-fold up-regulation in both Aγ and Gγ globin levels (by ultra-performance liquid chromatography). When CDDO-Me treated cells were subjected to ex-vivo hypoxia challenge, we observed a dose dependent inhibition in sickling with a maximal decrease of 55% at 250 nM, as evaluated by IDEAS imaging flow cytometry. We further evaluated the antioxidant response with NRF2 activation in these cell systems. KEAP1 KO in HUDEP2 cells evaluated using RNA-sequencing showed enrichment of genes that participate in various protective mechanisms including NRF2 mediated oxidative stress response and glutathione redox reaction as analyzed by ingenuity pathway analysis (IPA). We subjected KEAP1 KO CD34+ cells to 4% hypoxia for 2 hours and observed a reduction in total reactive oxygen species levels as measured by flow cytometry compared to the control cells. These findings suggest that Nrf2 activation in erythroid cells offer a protective phenotype by improving the oxidative stress defense mechanisms, a well established pathophysiological manifestation of SCD. In conclusion, KEAP1 inhibition in erythroid cell lineage and subsequent NRF2 activation leads to HbF induction and improvement in the antioxidant stress enzymes. Overall these data demonstrate a multi-faceted potential benefit of NRF2 activation in sickle cell disease. Disclosures Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

The efficacy of oxidation reduction potential(ORP) in male infertility and its relationship with seminal leukocyte concentration

Reactive oxygen species (ROS) in semen has been reported to have negative effect to male fertile capacity, and recent studies reported the efficacy of oxidation-reduction potential (ORP) which reflects the balance of oxidants and antioxidants in semen. The source of ROS in semen is considered as immature spermatozoa and seminal leukocytes, but the detail is still unknown. The aim of this study is to evaluate the relationship between the concentration of seminal leukocytes and oxidative stress level using ROS, ORP. Retrospective study. Between April 2018 and March 2019, 29 infertile males who visited Reproduction Centre of Yokohama City University Medical Center were enrolled. All patients underwent semen analysis and measurement of ROS and ORP levels. The ROS level in semen was measured using Monolight 3010TM Luminometer and the ORP level was measured using MiOXSYS SystemTM. The concentration of peroxidase-positive leukocytes were evaluated using myeloperoxidase staining (Endtz test). The relationship between ROS levels, ORP levels, the concentration of leukocytes and semen parameters were evaluated using correlation analysis. The sperm concentration and motility were 39.6±36.6×106/ml, 30.0±18.9 %, respectively. The total ROS level was 9328.9 (±18851) Relative Light Units, and the ORP level was 46.1(±38.7) mV. The ROS level was significantly correlated with ORP level (r=0.79, p<0.01). The concentration of leukocytes measured by Endtz test was positively correlated with both ORP level (r=0.46, p=0.023) and ROS level (r=0.82, p<0.01). ORP level was negatively correlated with sperm concentration (r= -0.41, p=0.026), while ROS level didn’t show significant correlation with every semen parameters. To our knowledge, this is the first study that showed the significant correlation between ORP levels and seminal leukocytes concentration. Our study suggested that peroxidase-positive leukocyte is one of the main source of ROS in semen. Although ROS was strongly correlated with leukocyte concentration, ORP level is considered to have better potential to reflect the sperm quality.

Lactobacillus frumenti improves antioxidant capacity via nitric oxide synthase 1 in intestinal epithelial cells.

Oxidative damages have adverse effects on mammals. Growing studies have focused on exploring new antioxidants. Here, we report that Lactobacillus frumenti increases the total antioxidation capacity activities and decreases the total reactive oxygen species levels in porcine intestinal epithelial cells. Comparative proteomics revealed that expressions of peroxiredoxin 2, isocitrate dehydrogenase 1, NAD(P)H dehydrogenase quinone 1, antioxidant protein 1, and metallothionein-2A, which are associated with antioxidant defense system, were significantly increased with L. frumenti treatment. In germ-free mice, L. frumenti treatment also remarkably improves the intestinal antioxidant capacity. We further illustrated that nitric oxide production-mediated by nitric oxide synthase 1 activation is essential for L. frumenti-induced improvements in intestinal epithelial antioxidant capacity and barrier function. This study suggested that L. frumenti may be a potential probiotic used to prevent oxidative stress-induced aging and diseases in mammals.-Nie, Y., Hu, J., Hou, Q., Zheng, W., Zhang, X., Yang, T., Ma, L., Yan, X. Lactobacillus frumenti improves antioxidant capacity via nitric oxide synthase 1 in intestinal epithelial cells.

PS1007 NORMAL AND LEUKEMIC BONE MARROW STROMAL CELLS HAVE DIFFERENT CAPACITIES FOR MITOCHONDRIA TRANSFER TO ACUTE MYELOID LEUKEMIA CELLS

Background:Recent data pointed to the association of disease progression and relapse in acute myeloid leukemia (AML) with the evidence of functional mitochondria (mt), and reactive oxygen species (ROS) transference from bone marrow stromal cells (BMSCs) to the blasts cells. Nevertheless, differential performances in this transference process among normal and leukemic‐BMSCs in co‐culture systems were not well established.Aims:To evaluate the presence of a favoring microenvironment for the AML's cells survival provided by leukemic‐BMSCs through the reduction of the cellular damage associated with mt transference process in co‐culture arrangements.Methods:BMSCs were isolated and expanded from bone marrow aspirates of 7 patients diagnosed with de novo AML (Based on European Leukemia Net risk stratification system: 1 favorable, 2 intermediate I and 4 adverse; median age: 51y; 57% male) and 4 age‐and sex‐matched hematologically healthy donors (median age: 30y; 66% males). By adherence, BMSCs were expanded until the fourth passage and immunophenotypically characterized for presence of CD73/CDw90/CD105 expression with CD34/CD45 negative staining. For co‐culture experiments, 3 AML cell lines (Kasumi‐1, OCI/AML3, and THP1) were used after synchronization by thymidine block and the collection of fractions following the protocol of Jing et al. for normal hematopoietic stem cells (Jing et al, Hematologica 2010). Cells were evaluated for total (H2DCFDA) and mitochondrial (MitoSOX) ROS production, mitochondrial content (Mitotracker) and proliferation index (Ki‐67) by flow cytometry after 3 and 5 days of co‐culture. For phase‐bright/dim cells, the analyses were conducted discerning CD45+/CD73+ cells, being bright cells those in loosely adhered to the surface and dim those tightly attached to BMSCs. Gene expression was evaluated for genes related with BMSCs metabolism, such as TNFA, SLIT2 and PRGC1A. Results:BMSCs isolated from AML patients presented reduced CFU‐F capacity in comparison to healthy‐BMSCs (P &lt; .05). In co‐culture, OCI/AML3 cells exhibited increased proliferation and reduced mtROS in non‐adherent cell phase after co‐culture with leukemic‐BMSCs (P &lt; .05). No differences regarding proliferation, ROS production and mt levels were observed for the other cell lines in this population. Regarding phase‐bright, THP1 cells had increased proliferation rate at day 5 when co‐cultured with leukemic‐BMSCs (110%) than those normal‐BMSCs co‐culture (81%; P = .04). Analyzing all AML cells combined, the mtROS levels are reduced in the phase‐bright CD45+ fraction when co‐cultured with leukemic‐BMSCs compared to healthy‐BMSCs (P = .005). In agreement, the AML CD73+ cells exhibited a reduction of 40% in the mt levels (P = .014). THP1 cells in closely contact with leukemic‐BMSCs (phase‐dim) showed decreased total ROS levels in 5 days of co‐culture (P = .005) associated with reduction in the mt content (P &lt; .05). OCI/AML3 cells (CD45+) exhibited increased proliferation in phase‐dim compartment (P &lt; .05). Take together, the AML cell lines showed reduced total/mtROS levels with increased mt number (P &lt; .05). Equal to phase‐bright, leukemic phase‐dim cells (CD73+) had a reduction in mt levels (P &lt; .05). Finally, the relative expression of SLIT2 and PRGC1A present a significantly decrease in AML‐BMSCs cells in comparison to healthy BMSCs (P &lt; .05).Summary/Conclusion:Our data suggest in mt donation capacity from BMSCs to AML blasts in healthy and leukemic BMCS. Therefore providing a further understanding of the complex nature of tumor metabolism, not only in the blasts cells, but also within the microenvironment which supports it.

Cyclic stretch induced oxidative stress by mitochondrial and NADPH oxidase in retinal pigment epithelial cells

BackgroundVitreomacular adhesion (VMA) has been reported to associated with age-related macular degeneration (AMD). Understanding the mechanisms underlying cyclic stretch induced in retinal pigment epithelial cells (RPE) may be important for the treatment of VMA-related AMD.MethodCyclic stretch (1HZ, 20% elongation) was applied to cultured ARPE-19 cells for 15 min, 2 h, 6 h, 12 h, 24 h by flexcell FX-5000 Tension system. Total reactive oxygen species (ROS) were detected using DCFH-DA. Mitochondrial superoxide were detected using MitoSOX Red mitochondrial superoxide indicator. NADPH oxidases (NOX) and signaling pathways, such as p38 and PKC, were detected using western blot. Apocycin (Apo) were used as NOX inhibitors.ResultHigh levels of total ROS were detected from 15 min to 24 h, whereas mitochondrial superoxide were higher only in early time. NOX2 were significantly increased at 24 h. NOX4 were significantly increased at 2 h and reach its peak at 24 h. P-p38 was significantly increased at 12 h and 24 h. P-PKC was significantly increased at 15 min and kept a persistent high level. The upregulated expression of NOX4 by cyclic stretch can be significantly decreased under p-PKC inhibitor other than p-p38 inhibitor.ConclusionCyclic stretch induce oxidative stress from both mitochodrial and NADPH oxidase in RPE cells, which may prompt oxidative damage in VMA-related AMD.

Adverse effects, expression of defense-related genes, and oxidative stress-induced MAPK pathway in the benzo[α]pyrene-exposed rotifer Brachionus rotundiformis

To examine the adverse effects of the benzo[α]pyrene (B[α]P), the monogonont rotifer Brachionus rotundiformis was exposed to various concentration of B[α]P (0 [control], 1, 10, and 100 μg/L) and measured life cycle parameters (e.g., mortality, fecundity [cumulated number of offspring], and lifespan), reactive oxygen species (ROS), antioxidant enzymatic activity of glutathione S-transferase (GST). In addition, defense-related transcripts (e.g., glutathione S-transferases [GSTs], ATP binding cassette [ABCs] transporters) and Western blot analysis of mitogen-activated protein kinase (MAPK) signaling pathway were investigated in B[α]P-exposed rotifer. In this study, the total intracellular ROS level and GST activity were significantly increased (P < 0.05), while fecundity and lifespan were also significantly (P < 0.05) reduced in a concentration dependent manner in B[α]P-exposed B. rotundiformis. In addition, transcriptional regulation of GSTs and ABC transporters were significantly upregulated and downregulated (P < 0.05), respectively, suggesting that B[α]P can induce oxidative stress leading to induction of antioxidant system and detoxification mechanism. In addition to detoxification-related genes, B[α]P-exposed B. rotundiformis showed the increased levels of the p-JNK and p-p38, suggesting that B[α]P can activate MAPK signaling pathway in B. rotundiformis.

Synergistic Interaction Between Zinc and Reactive Oxygen Species Amplifies Ischemic Brain Injury in Rats.

Background and Purpose- Although intracellular zinc accumulation has been shown to contribute to neuronal death after cerebral ischemia, the mechanism by which zinc keeps on accumulating to cause severe brain damage remains unclear. Herein the dynamic cause-effect relationships between zinc accumulation and reactive oxygen species (ROS) production during cerebral ischemia/reperfusion are investigated. Methods- Rats were treated with zinc chelator, ROS scavenger, mitochondria-targeted ROS inhibitor, or NADPH oxidase inhibitor during a 90-minute middle cerebral artery occlusion. Cytosolic labile zinc, ROS level, cerebral infarct volume, and neurological functions were assessed after ischemia/reperfusion. Results- Zinc and ROS were colocalized in neurons, leading to neuronal apoptotic death. Chelating zinc reduced ROS production at 6 and 24 hours after reperfusion, whereas eliminating ROS reduced zinc accumulation only at 24 hours. Furthermore, suppression of mitochondrial ROS production reduced the total ROS level and brain damage at 6 hours after reperfusion but did not change zinc accumulation, indicating that ROS is produced mainly from mitochondria during early reperfusion and the initial zinc release is upstream of ROS generation after ischemia. Suppression of NADPH oxidase decreased ROS generation, zinc accumulation, and brain damage only at 24 hours after reperfusion, indicating that the majority of ROS is produced by NADPH oxidase at later reperfusion time. Conclusions- This study provides the direct evidence that there exists a positive feedback loop between zinc accumulation and NADPH oxidase-induced ROS production, which greatly amplifies the damaging effects of both. These findings reveal that different ROS-generating source contributes to ischemia-generated ROS at different time, underscoring the critical importance of spatial and temporal factors in the interaction between ROS and zinc accumulation, and the consequent brain injury, after cerebral ischemia/reperfusion.

The effects of crocetin supplementation on the blastocyst outcome, transcriptomic and metabolic profile of in vitro produced bovine embryos

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1 μM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Lupeol against high-glucose-induced apoptosis via enhancing the anti-oxidative stress in rabbit nucleus pulposus cells.

This study aimed to investigate the potential mechanism and value of lupeol in inhibiting high-glucose-induced apoptosis in rabbit nucleus pulposus cells (NPCs). NPCs were divided into four groups: control (CON), high glucose (HG), LUP, and HG + LUP. Viability, reactive oxygen species (ROS) levels, and apoptosis were examined in NPCs. The protein expression levels of Bax, Bcl-2, cytochrome C, and caspase 9/3 were measured using reverse transcription-polymerase chain reaction and Western blot assay. The apoptotic rate and total ROS level of the HG group significantly increased compared with the CON group (P < 0.01). The total ROS level in the HG + LUP group significantly decreased compared with the HG group(P < 0.05). The mRNA expression of Bcl-2 was significantly upregulated, whereas the expression of Bax, cytochrome C, and caspase 9/3 was downregulated in the HG + LUP group compared with those in the HG group(P < 0.05).The Western blot assay showed that the expression of Bcl-2 was upregulated, but the expression of Bax, cytochrome C, and caspase 9/3 was significantly downregulated in the HG + LUP group compared with the HG group (P < 0.05). Lupeol inhibited high-glucose-induced apoptosis in NPCs by enhancing the anti-oxidative stress in the mitochondria. This study suggested lupeol as a potential therapeutic drug for treating intervertebral disc degeneration under hyperglycaemic conditions. These slides can be retrieved under Electronic Supplementary Material.

Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni.

Campylobacter is a leading foodborne pathogen worldwide. Biofilm formation is an important survival mechanism that sustains the viability of Campylobacter under harsh stress conditions. Iron affects biofilm formation in some other bacteria; however, the effect of iron on biofilm formation has not been investigated in Campylobacter. In this study, we discovered that ferrous (Fe2+) and ferric (Fe3+) iron stimulated biofilm formation in Campylobacter jejuni. The sequestration of iron with an iron chelator prevented the iron-mediated biofilm stimulation. The level of total reactive oxygen species (ROS) in biofilms was increased by iron. However, the supplementation with an antioxidant prevented the total ROS level from being increased in biofilms by iron and also inhibited iron-mediated biofilm stimulation in C. jejuni. This suggests that iron promotes biofilm formation through oxidative stress. Based on the results of fluorescence microscopic analysis, Fe2+ and Fe3+ enhanced both microcolony formation and biofilm maturation. The levels of extracellular DNA and polysaccharides in biofilms were increased by iron supplementation. The effect of iron on biofilm formation was also investigated with 70 C. jejuni isolates from raw chicken. Regardless of the inherent levels of biofilm formation, iron stimulated biofilm formation in all tested strains; however, there were strain variations in iron concentrations affecting biofilm formation. The biofilm formation of 92.9% (65 of 70) strains was enhanced by either 40 μM Fe2+ or 20 μM Fe3+ or both (the iron concentrations that enhanced biofilm formation in C. jejuni NCTC 11168), whereas different iron concentrations were required to promote biofilms in the rest of the strains. The findings in this study showed that Fe2+ and Fe3+ contributed to the stimulation of biofilm formation in C. jejuni through oxidative stress.

Kinin B1 Receptor Knockdown Prevents DOCA‐Salt Hypertension by Modulating Mitochondrial Oxidative Stress in the Brain

The imbalance between reactive oxygen species (ROS) production and anti‐oxidant capacity in the central nervous system results in oxidative stress and is implicated in the pathogenesis of hypertension. Deoxycorticosterone acetate (DOCA)‐salt hypertension is associated with increased expression of the pro‐inflammatory kinin B1 receptor (B1R) in the brain. We previously reported that DOCA‐salt hypertension is attenuated in global B1R knockout (B1RKO) mice. However, the functional role of B1R in inducing oxidative stress within the brain during neurogenic hypertension has not been fully studied. In this study, we tested the hypothesis that the beneficial effects of B1R knockdown are mediated by reduction in mitochondrial oxidative stress in the brain. DOCA‐salt treatment (1mg/g body wt. DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower blood pressure (BP; telemetry) level in B1RKO mice (121 ±2 mmHg, n=12) compared to wild‐type controls (WT) (138 ±3 mmHg, n=12). DOCA‐salt treatment resulted in decreased catalase (79.1 ±5.6 vs. 150.3 ±10.4 mmol/min/ml, p&lt;0.01), total superoxide dismutase (SOD, 4.19 ±0.3 vs. 5.97 ±0.3 U/ml, p&lt;0.01), and the mitochondrial antioxidant manganese SOD (0.67 ±0.09 vs. 1.58 ±0.05 U/ml, p&lt;0.01) activities in hypothalamic homogenates of WT mice, which were prevented in B1RKO mice (134.8 ±4.6 mmol/min/ml, 5.89 ±0.1 U/ml, and 1.68 ±0.22 U/ml, respectively, p&lt;0.01). WT mice treated with DOCA‐salt showed increased oxidative stress as indicated by increased total ROS levels in the hypothalamus as measured using electron paramagnetic resonance spectrometer. This increase in ROS production was not observed in B1RKO mice treated with DOCA‐salt. Furthermore, DOCA‐salt hypertension resulted in decreased phosphorylation of eNOS‐ser1177 in the PVN (35 ±5 % decrease, n=9, p&lt;0.05 vs WT), which was prevented in B1RKO mice. In addition, the gene expression of PPAR Gamma coactivator (PGC) 1‐Alpha and Uncoupling protein‐2 (UCP‐2), two important genes involved in mitochondrial function, were decreased in the PVN of DOCA‐salt treated WT mice but not in B1RKO mice. Together, these data provide evidence that kinin B1R knockdown reduces mitochondrial oxidative stress, improves antioxidant and nitric oxide signaling, and there by attenuates the development of DOCA‐salt hypertension.Support or Funding InformationThe American Heart Association (15SDG25720021)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension.

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced pulmonary hypertension (PH) and right ventricular (RV) remodelling.We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-targeted antioxidant MitoQ under these conditions.The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly, MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV remodelling after chronic hypoxia as well as after PAB.Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

Superoxide Anion Production by the Spermatozoa of Men with Varicocele: Relationship with Varicocele Grade and Semen Parameters.

PurposeTo investigate the pathophysiological role of superoxide anion and total reactive oxygen species (ROS) production by the spermatozoa of men with varicocele and its relationship with varicocele grade and semen parameters.Materials and MethodsThis prospective study included 34 men with grade II–III varicocele, regardless of their fertility status. The control group consisted of 13 healthy men. Semen characteristics were examined according to the 2010 World Health Organization criteria. The swim-up method was used for sperm preparation. Total ROS and superoxide anion production was assayed by luminol- and lucigenin-dependent chemiluminescence (CL), respectively.ResultsThe men with varicocele had significantly higher total ROS and superoxide anion levels than the healthy control subjects (2.9±0.4 relative light unit (RLU) vs. 2.4±0.1 RLU, p=0.001 for luminol-dependent CL and 2.8±0.4 RLU vs. 2.3±0.2 RLU, p=0.002 for lucigenin-dependent CL). Cases of grade III varicocele had significantly higher superoxide anion and total ROS levels than grade II cases and control subjects (p<0.001). Superoxide anion and total ROS levels were negatively correlated with all semen parameters.ConclusionsThe superoxide anion levels produced by spermatozoa were significantly higher in varicocele patients than in control subjects. ROS production was related to increased varicocele grade, impaired semen concentration, and abnormal morphology in men with varicocele. Our findings suggest that superoxide anion overproduction may be an important step in the cascade of ROS-related damage to spermatozoa, resulting in impaired semen parameters in patients with varicocele.