Increased Reactive Oxygen Species Formation Research Articles

MSCs enhances the protective effects of valsartan on attenuating the doxorubicin-induced myocardial injury via AngII/NOX/ROS/MAPK signaling pathway.

Objective: To verify if AngII/NOX/ROS/MAPK signaling pathway is involved in Doxorubicin (DOX)-induced myocardial injury and if mesenchymal stem cells (MSCs) could enhance the protective effects of valsartan (Val) on attenuating DOX-induced injury in vitro.Methods: Reactive oxygen species (ROS) formation and the protein expression of AT1R, NOX2, NOX4, caspase-3, caspase-9 and MAPK signaling were assessed in H9c2 cardiomyocytes exposed to DOX for 24 h in the absence or presence of Val, NADPH oxidase inhibitor DPI or knockdown and overexpression of NADPH oxidase subunit: NOX2 and NOX4, co-culture with MSCs, respectively. Finally, MTT assay was used to determine the cell viability of H9c2 cells, MDA-MB-231 breast cancer cells and A549 pulmonary cancer cells under Val, DOX and Val+ DOX treatments.Results: DOX increased ROS formation and upregulated proteins expression of AT1R, NOX2, NOX4, caspase-3, caspase-9 and MAPK signaling including p-p38, p-JNK, p-ERK in H9c2 cells. These effects could be attenuated by Val, DPI, NOX2 siRNA and NOX4 siRNA. Meanwhile, overexpression of NOX2 and NOX4 could significantly increase DOX-induced ROS formation and further upregulate apoptotic protein expressions and protein expressions of MAPK signaling. MSCs on top of Val further enhanced the protective effects of Val on reducing the DOX-induced ROS formation and downregulating the expression of apoptotic proteins and MAPK signaling as compared with Val alone in DOX-treated H9c2 cells. Simultaneous Val and DOX treatment did not affect cell viability of DOX-treated MDA-MB-231 breast cancer cells or A549 pulmonary cancer cells but significantly improved cell viability of DOX-treated H9c2 cardiomyocytes.Conclusions: AT1R/NOX/ROS/MAPK signaling pathway is involved in DOX-induced cardiotoxicity. Val treatment significantly attenuated DOX-induced cardiotoxicity, without affecting the anti-tumor effect of DOX. MSCs enhance the protective effects of Val on reducing the DOX-induced toxicity in H9c2 cells.

Platinum nanoparticles Protect Against Lipopolysaccharide-Induced Inflammation in Microglial BV-2 Cells via Decreased Oxidative Damage and Increased Phagocytosis.

Neuroinflammation and oxidative stress cooperate to compromise the function of the central nervous system (CNS). Colloidal platinum nanoparticles (Pt NPs) are ideal candidates for reducing the deleterious effects of neuroinflammation since they act as free radical scavengers. Here we evaluated the effects of Pt NPs on several markers of lipopolysaccharide (LPS)-induced inflammation in cultured BV-2 microglial cells. BV-2 cells were treated with increased dilutions (1-100ppm) of Colloidal Pt and/or LPS (1-10µg/mL) at different exposure times. Three different protocols of exposure were used combining Pt NPs and LPS: (a) conditioning-protective effect (pre-post-treat), (b) therapeutic effect (co-treat) and (c) conditioning-therapeutic effect (pre-co-treat). After exposure to LPS for 24h, cells were used for assessment of cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, apoptosis and caspase-3 levels, cell proliferation, mitochondrial membrane potential, inducible nitric oxide (iNOS) activity, pro-inflammatory cytokine (IL-1β, TNF-α and IL-6) levels, and phagocytic activity. Low concentrations (below or equal to 10ppm) of Colloidal Pt prevented or ameliorated the LPS-induced increase in ROS formation, loss of mitochondrial membrane potential, induction of apoptosis, increase in LDH release, increase in pro-inflammatory cytokines and iNOS, inhibition of phagocytosis linked to microglial persistence in the M1 phase phenotype, loss of cell adhesion, differentiation and/or proliferation, as well as loss of cell viability. These protective effects were evident when cells were preconditioned with Pt NPs prior to LPS treatment. Collectively, the findings demonstrate that at low concentrations, Pt NPs can regulate the function and phenotype of BV-2 cells, activating protective mechanisms to maintain the microglial homeostasis and reduce inflammatory events triggered by the inflammatory insults induced by LPS. These preventive/protective effects on the LPS pro-inflammatory model are linked to the antioxidant properties and phagocytic activity of these NPs.

Mitochondrial Superoxide Dismutase in Cisplatin-Induced Kidney Injury.

Cisplatin is a chemotherapy agent commonly used to treat a wide variety of cancers. Despite the potential for both severe acute and chronic side effects, it remains a preferred therapeutic option for many malignancies due to its potent anti-tumor activity. Common cisplatin-associated side-effects include acute kidney injury (AKI) and chronic kidney disease (CKD). These renal injuries may cause delays and potentially cessation of cisplatin therapy and have long-term effects on renal function reserve. Thus, developing mechanism-based interventional strategies that minimize cisplatin-associated kidney injury without reducing efficacy would be of great benefit. In addition to its action of cross-linking DNA, cisplatin has been shown to affect mitochondrial metabolism, resulting in mitochondrially derived reactive oxygen species (ROS). Increased ROS formation in renal proximal convoluted tubule cells is associated with cisplatin-induced AKI and CKD. We review the mechanisms by which cisplatin may induce AKI and CKD and discuss the potential of mitochondrial superoxide dismutase mimetics to prevent platinum-associated nephrotoxicity.

The Abnormal Presence of Mitochondria in Circulating Red Blood Cells Cause an Increased Oxygen Consumption Rate, ROS Generation and Hemolysis in Patients with Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited blood disorder that affects millions of people worldwide, and it is caused by a mutation of the β-globin gene which results in the polymerization of HbS when deoxygenated. Patients with sickle cell disease experience severe pain, multi organ damage, and shortened life span. Changes arising from sickle hemoglobin (HbS) inside the red blood cell (RBC) leads to an imbalance of the oxidative reactions which increases reactive oxygen species (ROS) generation. ROS causes hemolysis and damages blood vessels. Previously, our lab demonstrated that SCD patients have a significant fraction of mitochondria retaining-RBCs which were associated with excessive levels of ROS. In addition immature mitochondria containing reticulocytes, R1 reticulocytes, are increased in the peripheral blood of SCD patients compared to control subjects. Here we report that the percentage of mitochondria retaining-RBCs directly correlated with the absolute number of reticulocytes (n=18,r=0.69, P< 0.001). As would be expected we were able to link mitochondria-retention with hemolysis. In addition, when we investigated energy metabolism we made the novel finding that SCD RBCs have an increased oxygen consumption rate (OCR). Mitochondrial-RBC retention and markers of hemolysis (serum bilirubin, and LDH) were compared by using Pearson's correlation coefficient analysis. The total serum bilirubin was directly associated with the percentage of mitochondria-containing RBCs in SCD patients' blood samples (r=0.31, n=41, p<0.04). A subgroup analysis showed a strong association between bilirubin and mitochondria-retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.55, n=17, P<0.02). Lactate dehydrogenase (LDH) is a known biomarker for hemolysis-and associated disease complications and early mortality in patients with SCD. There was a less significant association of percentage of mitochondria- retaining RBCs in SCD with serum LDH (r=0.02, n=40, P<0.2) when all patients were analyzed, however, a strong association was seen with serum LDH and mitochondria retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.67, n=17, P<0.002). We further investigated the mitochondrial RBCs energy metabolism in SCD as compared to controls in both human and mice. RBCs obtained from Townes SCD or control mice were seeded in an Agilent Seahorse XF 24-well plate using the Seahorse XF Base Medium with supplements, and we monitored the OCR and extracellular acidification rate (ECAR), in real time by Seahorse XFe-extracellular flux analyzer. OCR(pmol/min) standardized for protein was significantly higher in sickle cell RBCs of both human and mice origin compared to control. [((SCD patients(Hb SS): 6.92± 1.23, n=2 and control subjects (Hb AA)2.55±0.17, n=2, p<0.006)). ((Townes SCD (HbSS): 169.3± 30.8, n=3 and Transgenic control (HbAA):5.0±0.8, n=2, p<0.001))]. OCR was reduced to normal levels with oligomycin which is a known blocker of mitochondrial respiration. ECAR, a measure of glycolysis, was significantly higher levels in SCD patient and mouse samples compared to control. These findings support our premise that mitochondria retaining RBCs play a role in intravascular hemolysis. In addition, they indicate that RBC from SCD patients have increased oxygen consumption. Mitochondrial retention in RBCs have two possible mechanism of damage to the RBC, increased ROS formation and increased deoxygenated HbS polymer formation due to mitochondrial oxygen consumption. These results support further investigation into the role of mitochondrial energy metabolism in the pathogenesis of sickle cell disease. DisclosuresNo relevant conflicts of interest to declare.

Nrf2 activation is involved in osteogenic differentiation of periodontal ligament stem cells under cyclic mechanical stretch

During orthodontic treatment, mechanical stretch serves a crucial function in osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Up-regulated reactive oxygen species (ROS) level is a result of cyclic mechanical stretch in many cell types. Nuclear factor erythroid-2-related factor-2 (Nrf2) is a master regulator in various antioxidants expression. However, it is not known whether cyclic mechanical stretch could induce the ROS generation in PDLSCs and whether Nrf2 participated in this process. The present study was aimed to investigate the role of Nrf2 in PDLSCs under cyclic mechanical stretch. Our results showed that cyclic mechanical stretch increased ROS level and the nuclear accumulation of Nrf2 during osteoblast differentiation. Knocking down Nrf2 by siRNA transfection increased ROS formation and suppressed osteogenic differentiation in PDLSCs. T-BHQ, a Nrf2 activator, promoted the osteogenic differentiation in PDLSCs under cyclic mechanical stretch, and improved the microstructure of alveolar bone during orthodontic tooth movement in rats by employing micro-CT system. Taken together, Nrf2 activation was involved in osteogenic differentiation under cyclic mechanical stretch in PDLSCs. T-BHQ could promote the osteogenic differentiation in vitro and in vivo, suggesting a promising option for the remodeling of the alveolar bone during orthodontic tooth movement.

Modifications of proteins of membrane-cytoskeleton complex and production of reactive oxygen species in erythrocytes cryopreserved with polyethylene glycol

Protein modifications in the membrane-cytoskeleton complex (MCC) of human erythrocytes, as well as changes in the intensity of reactive oxygen species (ROS) production upon cell cryopreservation with polyethylene glycol (PEG) were investigated. The protein profile of ghosts of erythrocytes frozen with PEG has common features with both the control and cells frozen without cryoprotectant. PEG makes it possible to restrict the structural rearrangements of the main MCC proteins under the effect of extreme factors and to restrain the amount of high molecular weight polypeptide complexes induced by the protein-cross-linking reagent diamide at the control level, in contrast to cells frozen without a cryoprotectant. However, changes related to the protein peroxiredoxin 2 in ghosts of erythrocytes cryopreserved with PEG are also attributed to cells frozen without a cryoprotectant that may be associated with the activation of oxidative processes. This is evidenced by a 10-fold increase in ROS formation in erythrocytes frozen under PEG protection. Thus, upon cryopreservation of erythrocytes with PEG, certain disorders in MCC proteins may be associated with increased formation of ROS, which may contribute to the disorganization of the structural components of MCC and disrupt the stability of cryopreserved cells under physiological conditions.

The selective toxicity of superparamagnetic iron oxide nanoparticles (SPIONs) on oral squamous cell carcinoma (OSCC) by targeting their mitochondria.

In recent years, many researchers have madetremendous efforts into using nanotechnology in biomedical applications and science, such as magnetic resonance imaging, drug delivery, and in particular, oncological therapeutic via superparamagnetic iron oxide nanoparticles (SPIONs). Head and neck squamous cell carcinoma (HNSCC) and especially oral squamous cell carcinoma (OSCC) have been a serious and ongoing concern. There are manystrong emphases on the importance of toxic mechanisms due to oxidative stress and specifically, the changed cellular response. Therefore, our study was designed to evaluate the effects of SPIONs on OSCC mitochondriabecause of the usefulness of the application of these nanoparticlesin cancer treatment and diagnosis. An increased level of reactive oxygen species (ROS) is one of the substantial mechanisms found for SPIONs in this study,andinitially originated from disruption of the electron transfer chain shown by a decrease in mitochondrial succinate dehydrogenaseactivity. Increased ROS formation subsequently followed a decline of mitochondrial membrane potential, the release of mitochondrial cytochrome complex, and mitochondrial swelling in the OSCC mitochondria compared with almost no effect in normal mitochondria. In addition, the SPIONs decreased cell viability and increased lipid peroxidationlevel and caspase-3 activity in OSCC cells. The results represented that the exposure to the SPIONs induced selective toxicity only on the OSCC but not normal mitochondria. Based on our findings, we finally concluded that the SPIONs may be considered as a potential therapeutic candidate for the treatment of OSCC.

Redox-active phytoconstituents ameliorate cell damage and inflammation in rat hippocampal neurons exposed to hyperglycemia+Aβ1-42 peptide

Alzheimer's disease (AD) is the most common dementia causing progressive loss of memory and compromised cognitive functions. Although the neurotoxic mechanisms underlying AD have yet to be fully elucidated, hyperglycemia seems to trigger oxidative and inflammatory responses in the brain of afflicted patients. Removal of free radicals reduces the neurotoxic effects of hyperglycemia in AD models. In this study we investigated the neuroprotective effects of the antioxidant phytoconstituents oleuropein (OLE), rutin (RUT), luteolin (LUT) and S-allylcysteine (SAC) in an experimental model combining the exposure to high glucose (HG, mimicking chronic hyperglycemia) plus amyloid-β peptide 1–42 (Aβ1-42, mimicking AD) in primary hippocampal neurons. Cells were pre-treated with OLE, RUT, LUT or SAC (10–1000 nM), and then co-treated with high glucose (GLU, 150 mM) for 24 h plus 500 nM oligomeric Aβ1-42 for 24 h more. Cell viability and reactive oxygen species (ROS) formation were assessed as indices of survival/toxicity and oxidative stress, respectively. Activity/expression of antioxidant enzymes, toxic adducts, inflammatory molecules, mitochondrial membrane potential (ΔΨm) and the pattern of amyloid aggregation were also assessed. The GLU + Aβ1-42 treatment significantly decreased cell viability, increased ROS formation, reduced superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, augmented Advanced Glycation End Products- and 4-hydroxynonenal-adducts generation, increased 3-nitrotyrosine and inflammatory outcomes such as inducible nitric oxide synthase, interleukin 1β and Tumor Necrosis Factor α, decreased MMP and augmented amyloid aggregation. All phytoconstituents reduced in a differential manner all toxic endpoints, with SAC showing the highest efficacy in preventing loss of cell viability and oxidative damage, whereas RUT was most efficacious in mitigating inflammatory endpoints. Combined, the results of this study suggest that protection afforded by these compounds against GLU + Aβ1-42-induced cell damage in hippocampal neurons is attributable to their properties as redox modulators, which might act through a concerted mechanism oriented to reduce oxidative stress and neuroinflammation.

Borassus flabellifer L. crude male flower extracts alleviate cisplatin-induced oxidative stress in rat kidney cells

Objective: To investigate the effects of Borassus flabellifer L. extracts on antioxidant activity, maintenance of cellular redox, and mitochondrial function in cisplatin-induced kidney injury. Methods: The extracts of Borassus flabellifer were obtained from crude male flowers using ethyl acetate and methanol. The antioxidant potential was evaluated by 2,2-azino-bis-(3-ethylbenzothiazoline- 6-sulfonic acid), 2,2-diphenyl-1-picrylhydrazyl, and ferric reducing antioxidant power, and total phenolic content was also determined. Cytoprotective activity of ethyl acetate and methanolic extracts was assessed after kidney cells were treated with cisplatin. Oxidative stress was determined by glutathione (GSH) assay, and formation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential (ΔΨm) using 2’,7’-dichlorofluorescin diacetate and JC-10 assays, respectively. Results: Borassus flabellifer methanolic extract exhibited greater antioxidant activity than the ethyl acetate extract. Cytoprotective effect was demonstrated in both extracts, particularly in the ethyl acetate extract. The extracts showed protection against the cytotoxic effect of cisplatin by prevention of the increased GSSG and declined GSH/GSSG ratio. Both extracts also prevented the increase in ROS formation, and loss of ΔΨm. Conclusions: Both Borassus flabellifer extracts show antioxidant activity and cytoprotective effect against cisplatin-induced cytotoxicity of NRK-52E cells by preventing oxidative stress and maintenance of GSH redox status. Borassus flabellifer extracts may possess beneficial effects on the prevention of oxidative stress- induced cell injury.

Cardiomyocytes-specific deletion of monoamine oxidase B reduces irreversible myocardial ischemia/reperfusion injury

Monoamine oxidase B (MAO-B), a protein localized at the outer mitochondrial membrane, catalyzes the oxidative deamination of biogenic amines thereby producing reactive oxygen species (ROS). Increased ROS formation contributes to myocardial ischemia/reperfusion (I/R); however, the importance of different ROS producing enzymes for increased I/R-induced ROS formation and the subsequent I/R injury is still a matter of debate. Here we describe the first cardiomyocytes-specific MAO-B knockout mouse and test the hypothesis that lack of cardiomyocyte MAO-B protects the heart from I/R injury.A cardiac-specific and tamoxifen-inducible MAO-B knockout mouse (MAO-B KO) was generated using the Cre/lox system; Cre-negative MAO-Bfl/fl littermates served as controls (WT). Lack of MAO-B was verified by Western blot and immunohistochemistry. Cardiac function of MAO-B KO and WT was analyzed by echocardiography, quantification of mitochondrial ROS production, and measurement of myocardial infarct size (in % of ventricle) in hearts exposed to global I/R using the Langendorff technique. MAO-B protein expression was significantly down-regulated in MAO-B KO mice after two weeks of tamoxifen feeding followed by ten weeks of feeding with normal chow. ROS formation stimulated by the MAO-B-specific substrate β-phenylethylamin (PEA; 250 μM) was significantly lower in mitochondria isolated from MAO-B KO compared to WT hearts (WT 4.5 ± 0.8 a. u.; MAO-B KO 1.2 ± 0.3 a. u.). Echocardiography revealed no significant differences in LV dimensions as well as ejection fraction (EF) between WT and MAO-B KO mice (EF: WT 67.3 ± 8.8%; MAO-B KO 67.7 ± 6.5%). After I/R, infarct size was significantly lower in MAO-B KO hearts (WT 69.3 ± 15.1%; MAO-B KO 46.8 ± 12.0%). ConclusionLack of cardiomyocytes-specific MAO-B reduces infarct size suggesting that MAO-B activity contributes to acute reperfusion injury.

Acetoacetate enhancement of glucose mediated DNA glycation

Acetoacetate (AA) is a ketone body, which generates reactive oxygen species (ROS). ROS production is impacted by the formation of covalent bonds between amino groups of biomacromolecules and reducing sugars (glycation). Glycation can damage DNA by causing strand breaks, mutations, and changes in gene expression. DNA damage could contribute to the pathogenesis of various diseases, including neurological disorders, complications of diabetes, and aging. Here we studied the enhancement of glucose-mediated DNA glycation by AA for the first time. The effect of AA on the structural changes, Amadori and advanced glycation end products (AGEs) formation of DNA incubated with glucose for 4 weeks were investigated using various techniques. These included UV–Vis, circular dichroism (CD) and fluorescence spectroscopy, and agarose gel electrophoresis. The results of UV–Vis and fluorescence spectroscopy confirmed that AA increased the DNA-AGE formation. The NBT test showed that AA also increased Amadori product formation of glycated DNA. Based on the CD and agarose gel electrophoresis results, the structural changes of glycated DNA was increased in the presence of AA. The chemiluminescence results indicated that AA increased ROS formation. Thus AA has an activator role in DNA glycation, which could enhance the adverse effects of glycation under high glucose conditions.

SIRT3 Deficiency Sensitizes Angiotensin-II-Induced Renal Fibrosis.

Background: Sirtuin 3 (SIRT3) has a crucial role in the cardiovascular diseases. Our previous study revealed that SIRT3 knockout (SIRT3KO) promoted cardiac pericyte–fibroblast transition. In this study, we investigated the involvement of pericyte and iron in angiotensin II (Ang-II)-mediated renal fibrosis in the SIRT3KO mice. Methods and Results: NG2-DsRed mice and NG2-DsRed-SIRT3 knockout (SIRT3KO) mice were infused with saline or Ang-II (1000 ng/kg/min) for 4 weeks. Renal fibrosis, iron content and reactive oxygen species (ROS) were measured. Masson’s trichrome staining showed that SIRT3KO enhanced Ang-II-induced renal fibrosis. Immunostaining showed that Ang-II treatment increased the number of NG2-DsRed+ cells in the kidney, and SIRT3KO further enhanced NG2-DsRed+ cells. Moreover, SIRT3KO promoted pericyte differentiation into fibroblasts as evidenced by co-staining NG2-DsRed/FSP-1. Furthermore, DsRed/FSP-1+ and DsRed/transforming growth factor-β1 (TGF-β1)+ fibroblasts were elevated by SIRT3KO after Ang-II infusion. Ang-II-induced collagen I and TGF-β1 expression was also enhanced in the SIRT3KO mice. SIRT3KO significantly exacerbated Ang-II-induced iron accumulation. This was accompanied by an increase in acetyl-p53, HO-1 and FPN expression. Further, SIRT3KO sensitized Ang-II-induced upregulation of p47phox and gp91phox together with increased ROS formation in the kidney. Conclusion: Our study suggests that SIRT3 deficiency sensitized Ang-II-induced renal fibrosis by the mechanisms involved in promoting differentiation of pericytes into fibroblasts, exacerbating iron overload and accelerating NADPH oxidase-derived ROS formation.

Airborne particulate matter (PM2.5) triggers cornea inflammation and pyroptosis via NLRP3 activation

Although studies have demonstrated that fine particulate matter (PM2.5) induces ocular surface damage, PM2.5 exposure causes cornea toxicity is not entirely clear. The aim of this study is to investigate the role of the nod-like receptor family pyrin domain containing three (NLRP3) inflammasome-mediated pyroptosis in PM2.5-related corneal toxicity. Human corneal epithelial cells (HCECs) were exposed to different concentrations of PM2.5, and the cell viability, expressions of NLRP3 inflammasome mediated pyroptosis axis molecules and intracellular reactive oxygen species (ROS) formation were measured in HCECs. Animal experiments were undertaken to topically apply PM2.5 suspension to mouse eyes for three months and the pyroptosis related molecules in the mouse corneas were measured. ResultsOur results showed a dose-dependent decrease of HCEC viability in the PM2.5-treated cells. NLRP3 inflammasome-mediated pyroptosis axis (NLRP3, ASC, GSDMD, caspase-1, IL-1β, and IL-18) were activated in the PM2.5-treated HCECs, accompanied by increased ROS formation. Further in vivo study confirmed the activation of this pathway in the mouse corneas exposed to PM2.5. In conclusion, this study provids novel evidence that PM2.5 induces corneal toxicity by triggering cell pyroptosis.

Evaluation of Cytotoxic Activity of Betanin Against U87MG Human Glioma Cells and Normal Human Lymphocytes and Its Anticancer Potential Through Mitochondrial Pathway

Recent studies revealed an antioxidant activity and anticancer efficiency of betanin. In this study, we investigated the cytotoxic effects and the possible mechanisms of betanin-induced apoptosis against U87MG human glioma cells and compared the results to those of human normal lymphocytes. MTT assay, caspase-3 activation assays in cells and succinate dehydrogenases (SDH), mitochondrial swelling, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and cytochrome C release assays in isolated mitochondria were obtained from U87MG human glioma cells and noncancerous human lymphocytes The results illustrated the significant cytotoxic effect of betanin on U87MG human glioma cells, with a concentration value that inhibits 50% of the cell growth of 7 µg/ml after 12 h of treatment. MTT assay demonstrated that the betanin is selectively toxic to U87MG human glioma cells, and betanin induced cell apoptosis via activation of caspase-3 along with modulation of apoptosis-related mitochondria. Meanwhile, betanin selectively increased ROS formation, mitochondria swelling, MMP decrease, and cytochrome c release in cancerous mitochondria but in normal mitochondria. Based on the evidence obtained from this study, it is concluded that the betanin is a promising natural compound to fight U87MG human glioma cells via induction of apoptosis through activation of intrinsic pathways.

Investigating the mechanism by which Naproxen and Prometryn causes Cardiovascular dysfunction

Background and ObjectiveCardiovascular disease (CVD) is the leading cause of death and a major cause of disability worldwide. Previous studies have shown that the long‐term and chronic use of nonsteroidal anti‐inflammatory drugs (NSAIDs) increases the risk of developing stroke and cardiovascular diseases. Naproxen is an FDA‐approved NSAID that is currently used to treat several medical conditions such as pain, osteoarthritis, rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, tendonitis, etc. Acetaminophen, the active ingredient in Tylenol, is not an NSAID but it has been associated with increased risk of liver toxicity. Similarly, the long‐term exposures to pesticides such as Prometryn amongst others have been established to be dangerous to human lives and capable to disrupt the physiology of different organs in the body, including the cardiovascular system. Prometryn, a diamino‐1,3,5‐triazine, is used as a selective pre‐ and post‐emergence herbicide. It is used to control annual grasses and broadleaf weeds in cotton and celery. This study aimed to investigate the effects of Naproxen, Acetaminophen, and Prometryn in cardiomyocytes and the underlying mechanisms by which it causes cardiovascular dysfunction.MethodsThe embryonic cardiomyocyte cell line H9c2 is commonly used in numerous in vitro studies because morphological parameters of their cells resemble immature embryonic cardiomyocytes. It has been previously shown that H9c2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. H9c2 cardiomyocytes were plated in a 96 well plate at a concentration of 4000 cells/well. The cells were then treated with different concentrations of Naproxen, Acetaminophen, and Prometryn for 24h at 37°C and 5% CO2. The cell viability, reactive oxygen species (ROS) formation, and mitochondrial membrane potential (ΔΨm) were examined.ResultsH9c2 cells treated with 100μM Acetaminophen and 20μM Prometryn showed significant decreases (&gt; 30%) in cell viability. Hydrogen peroxide (H2O2), which was used as a positive control, also resulted in significant decreases in cell viability at 100μM. ROS formation was significantly increased in the presence of 200–400μM Naproxen. Acetaminophen showed significant amount of ROS formation at a higher concentration of 200μM while Prometryn also showed a significant increase in ROS formation at a higher concentration of 30μM. The effect of varying concentrations of Naproxen showed that ΔΨm was decreased by ~30% at 100μM. Prometryn decreased ΔΨm by ~20% at 10–20μM while Acetaminophen also decreased ΔΨm by ~20% at 100–200μM.ConclusionThese results suggest that both concentration and exposure time are important for NSAID‐induced cardiotoxicity. Acetaminophen, which is not an NSAID, increased ROS generation and decreased ΔΨm at higher concentration. This experimental data suggests that Naproxen, Acetaminophen, and Prometryn causes cardiomyocyte dysfunction at varying concentrations.

Electrochemical oxidation of paraben compounds and the effects of byproducts on neuronal activity

Some organic recalcitrant compounds are not degraded by conventional water treatment systems, making necessary the use of advanced technologies to eliminate these substances. Advanced Oxidation Processes (AOPs) have been extensively proposed to remove emerging contaminants aiming potable water reuse, but literature barely addresses neurotoxic effects of AOPs residual byproducts. These processes involve high costs associated with the electricity, maintenance and oxidizing agent used. However, electrochemical AOPs are techniques based on electron transfer, thus being a clean form of energy and very efficient in the degradation of organic pollutants. Parabens are naturally found in plant sources but most are chemically synthesized, requiring careful treatment to not disturb the environment. In this study, a mixture of parabens (10 mg L−1 each) was degraded by an electrochemical oxidation (EO) system with a Ti/Pt anode. Some parameters, such as the current density (25, 75 and 125 A m−2) and the electrolyte type and concentration (1.5, 3.0 and 5.0 g NaCl L−1 and 3.0 g Na2SO4 L−1) were changed. The best results were obtained with 125 A m−2 and 3.0 g NaCl L−1, which led to the complete degradation of the parabens present in the mixture, after 10 min. In addition to these studies neurotoxicity tests were also performed using the solutions of interest, before and after the EO treatment. It was observed, using the reactive oxygen species (ROS) fluorescent indicator H2DCFDA, that the non-treated solution caused an increase in ROS formation with a signal amplitude of 0.84 ± 0.20 above the baseline. After the EO process the parabens mixture did not lead to a significant ROS change.The solution to bridge the problem of high electricity costs may be replacing it with solar energy, low cost catalysts and other treatment processes involving renewable and eco-friendly energy.

Increased formation of reactive oxygen species during tumor growth: Ex vivo low-temperature EPR and in vivo bioluminescence analyses

Previous studies have shown that reactive oxygen species (ROS) such as superoxide or hydrogen peroxide generated at low levels can exert a tumor-promoting role via a redox-signaling mechanism. Reports also suggest that both tumorigenesis and tumor growth are associated with enhanced ROS formation. However, whether ROS levels or ROS-derived oxidative marker levels increase during tumor growth remains unknown. In this study, in vivo bioluminescence imaging with a boronate-based pro-luciferin probe was used to assess ROS formation. Additionally, probe-free cryogenic electron paramagnetic resonance was used to quantify a characteristic aconitase [3Fe4S]+ center that arises in the tumor tissue of mouse xenografts from the reaction of the native [4Fe4S]2+ cluster with superoxide. Results indicated that tumor growth is accompanied by increased ROS formation, and revealed differences in oxidant formation in the inner and outer sections of tumor tissue, respectively, demonstrating redox heterogeneity. Studies using luciferin and pro-luciferin probes enabled the assessment of tumor size, ROS formation, and bioenergetic status (e.g., ATP) in luciferase-transfected mice tumor xenografts. Probe-free ex vivo low-temperature electron paramagnetic resonance can also be translated to clinical studies.

Ginsenosides Rb1 and Rg1 Protect Primary Cultured Astrocytes against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function.

This study aimed to evaluate whether ginsenosides Rb1 (20-S-protopanaxadiol aglycon) and Rg1 (20-S-protopanaxatriol aglycon) have mitochondrial protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in primary mouse astrocytes and to explore the mechanisms involved. The OGD/R model was used to mimic the pathological process of cerebral ischemia-reperfusion in vitro. Astrocytes were treated with normal conditions, OGD/R, OGD/R plus Rb1, or OGD/R plus Rg1. Cell viability was measured to evaluate the cytotoxicity of Rb1 and Rg1. Intracellular reactive oxygen species (ROS) and catalase (CAT) were detected to evaluate oxidative stress. The mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were measured to evaluate mitochondrial function. The activities of the mitochondrial respiratory chain (MRC) complexes I–V and the level of cellular adenosine triphosphate (ATP) were measured to evaluate oxidative phosphorylation (OXPHOS) levels. Cell viability was significantly decreased in the OGD/R group compared to the control group. Rb1 or Rg1 administration significantly increased cell viability. Moreover, OGD/R caused a significant increase in ROS formation and, subsequently, it decreased the activity of CAT and the mtDNA copy number. At the same time, treatment with OGD/R depolarized the MMP in the astrocytes. Rb1 or Rg1 administration reduced ROS production, increased CAT activity, elevated the mtDNA content, and attenuated the MMP depolarization. In addition, Rb1 or Rg1 administration increased the activities of complexes I, II, III, and V and elevated the level of ATP, compared to those in the OGD/R groups. Rb1 and Rg1 have different chemical structures, but exert similar protective effects against astrocyte damage induced by OGD/R. The mechanism may be related to improved efficiency of mitochondrial oxidative phosphorylation and the reduction in ROS production in cultured astrocytes.

Severe but not moderate hyperoxia of newborn mice causes an emphysematous lung phenotype in adulthood without persisting oxidative stress and inflammation

BackgroundPreterm newborns typically require supplemental oxygen but hyperoxic conditions also damage the premature lung. Oxygen-induced lung damages are mainly studied in newborn mouse models using oxygen concentrations above 75% and looking at short-term effects. Therefore, we aimed at the investigation of long-term effects and their dependency on different oxygen concentrations.MethodsNewborn mice were exposed to moderate vs. severe hyperoxic air conditions (50 vs. 75% O2) for 14 days followed by a longer period of normoxic conditions. Lung-related parameters were collected at an age of 60 or 120 days.ResultsSevere hyperoxia caused lower alveolar density, enlargement of parenchymal air spaces and fragmented elastic fibers as well as higher lung compliance with peak airflow limitations and higher sensitivity to ventilation-mediated damages in later life. However, these long-term lung structural and functional changes did not restrict the voluntary physical activity. Also, they were not accompanied by ongoing inflammatory processes, increased formation of reactive oxygen species (ROS) or altered expressions of antioxidant enzymes (superoxide dismutases, catalase) and lung elasticity-relevant proteins (elastin, pro-surfactant proteins) in adulthood. In contrast to severe hyperoxia, moderate hyperoxia was less lung damaging but also not free of long-term effects (higher lung compliance without peak airflow limitations, increased ROS formation).ConclusionsSevere but not moderate neonatal hyperoxia causes emphysematous lungs without persisting oxidative stress and inflammation in adulthood. As the existing fragmentation of the elastic fibers seems to play a pivotal role, it indicates the usefulness of elastin-protecting compounds in the reduction of long-term oxygen-related lung damages.

Oxidative status of prostate and its dynamics against etiotropic antimicrobial chemotherapy in patients with chronic bacterial prostatitis

The study objective is to investigate some initial characteristics of prostate oxidative status and their dynamics during the course of etiotropic antimicrobial monochemotherapy in patients with chronic bacterial prostatitis (CBP).Materials and methods. The work is based on treatment of 90 men aged 24–46 years (mean age 38.2 ± 1.4 years) with CBP (the treatment group), and 30 clinically healthy men aged 20–45 years (mean age 35.5 ± 1.5 years) (control group). All men underwent the same type of comprehensive examination during the study, and the results of the examination of the men of the control group were taken as reference values of the conditional norm. Complaints and anamnesis were collected. General physical, special urological, microbiological, laboratory, sonographic studies were performed. The etiotropic antimicrobial monochemotherapy was selected according to the results of microbiological studies of the secretion of the prostate gland, and levofloxacin (500 mg within 28 days) was chosen as a base drug for the subgroup of treatment (n = 15) isolated from the treatment group in the 2nd stage of the study. To assess the characteristics of the prostate oxidative status and its dynamics during etiotropic antimicrobial monochemotherapy, various modifications of biochemical studies of the secretion of the prostate were performed (reactive oxygen species (ROS), lipid peroxidation products (diol conjugates, malonic dialdehyde), activity of superoxide dismutase).Results. In patients with CBP in the secretion of the prostate due to increased ROS formation, increased functional activity (free radical aggression), increased reactions of lipid peroxidation and increased functional load on prostatic superoxide dismutase. Antimicrobial monochemotherapy of CBP was characterized by a microbiological efficacy of 86.7 % and was accompanied by a positive dynamics of a number of clinical and laboratory parameters of chronic bacterial prostatitis, however, this did not lead to the complete elimination of pain syndrome and oxidative disorders in the secretion of the prostate, and also did not significantly improve the quality life of patients and prostate secretory function. In patients with CBP a significant positive relationship was found between the amount of ROS and superoxide dismutase activity in the secretion of the prostate (n = 90; r = 0.413; p = 0.001); and the positive relationship between the amount of ROS in the secretion of the prostate and the clinical pain index (n = 90, r = 0.304, p = 0.001), which reflected the essential role of free radical prostatic aggression as an non-infectious component of the multifactorial pathogenesis of the pain syndrome in CBP.Conclusion. The persistence of residual oxidative disorders and functional deficits in the prostate after a standard course of microbial monochemotherapy justifies the advisability of the additional administration of antioxidants and antihypoxic agents in CBP.