Determination Of Reactive Oxygen Species Research Articles

Synthesis and Biological Evaluation of Novel 2-imino-4-thiazolidinones as Potential Antitumor Agents for Glioblastoma.

The purpose of our study was to explore the molecular hybridization between 2- imino-4-thizolidione and piridinic scaffolds and its potential antitumor activity. Glioblastoma is the most aggressive glioma tumor clinically diagnosed malignant and highly recurrent primary brain tumor type. The standard of treatment for a glioblastoma is surgery, followed by radiation and chemotherapy using temozolomide. However, the chemoresistance has become the main barrier to treatment success. 2-imino-4-thiazolidinones are an important class of heterocyclic compounds that feature anticancer activity; however the antiglioblastoma activity is yet to be explored. To synthesize and characterize a series of novel 2-imino-4-thiazolidinones and evaluate their antiglioblastoma activity. The 2-imino-4-thiazolidinone (5a-p) was synthesized according to the literature with modifications. Compounds were identified and characterized using spectroscopic analysis and X-ray diffraction. The antitumor activity was analyzed by 3-(4,5- dimethyl)-2,5-diphenyltetrazolium bromide (MTT) assay both in primary astrocyte and glioma (C6). Apoptosis and cell cycle phase were determined by flow cytometry analysis. The expression of caspase-3/7 was measured by luminescence assay. Oxidative stress parameters as: Determination of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD) activity, Catalase (CAT) activity and total sulfhydryl content quantification were analyzed by colorimetric assays according to literature. Among sixteen synthesized compounds, three displayed potent antitumor activities against tested glioblastoma cell line showed IC50 values well below the standard drug temozolomide. Therefore, compounds 5a, 5l and 5p were evaluated using cell cycle and death analysis, due to potent toxicity (2.17±1.17, 6.24±0.59, 2.93±1.12μM, respectively) in C6 cell line. The mechanism of action studies demonstrated that 5a and 5l induced apoptosis significantly increase the percentage of cells in Sub-G1 phase in the absence of necrosis. Consistent with these results, caspase-3/7 assay revealed that 5l presents pro-apoptotic activity due to the significant stimulation of caspases-3/7. Moreover, 5a, 5l and 5p increased antioxidant defense and decreased reactive oxygen species (ROS) production. The compounds were synthesized with good yield and three of these presented (5a, 5l and 5p) good cytotoxicity against C6 cell line. Both affected cell cycle distribution via arresting more C6 cell line at Sub-G1 phase promoting apoptosis. Furthermore, 5a, 5l and 5p modulated redox status. These findings suggest that these compounds can be considered as promising lead molecules for further development of potential antitumor agents.

Novel tungsten disulfide (WS2) nanosheets for photocatalytic degradation and electrochemical detection of pharmaceutical pollutants

In the present work, a new strategy of “Freeing two birds with one key” was implemented for the photocatalytic degradation and electrochemical sensing of antibiotic nitrofurantoin (NFT) using tungsten disulfide (WS2) nanosheets. Novel WS2 nanosheets were synthesized through a facile hydrothermal technique. The successful formation of WS2 nanosheets was confirmed by several characterization techniques such as HRTEM, FESEM, XRD, FTIR, UV–Vis spectroscopy, and BET isotherms. WS2 nanosheets modified paper electrodes demonstrated excellent electrocatalytic activity for the reduction of NFT with a wide linear range of 500 μM to 0.01 μM, a lower detection limit of 0.06 μM, and good selectivity. Furthermore, photocatalysis study revealed that WS2 nanosheets exhibited superior photocatalytic activity, successfully degrading 90% of NFT in 120 min. The scavenger study was also investigated for the determination of reactive oxygen species (ROS).

THE IMPACT OF SUCCINATE DERIVATIVE PHENSUCCINAL ON MITOCHONDRIAL FUNCTION AND REDOX STATUS IN THE HEART OF RATS WITH TYPE 2 DIABETES

Dysfunction of cardiac mitochondria appears to play a substantial role in cardiomyopathy and is a promising therapeutic target for many cardiovascular diseases. Persistent hyperglycaemia and hyperlipidemia are believed to be the main causes of increased oxidative stress, mitochondrial dysfunctions, fibrosis and apoptosis of cardiomyocytes in diabetes. We have previously shown that the low-toxic succinate derivative – Phensuccinal (beta-phenylethylamide-2 hydroxy-succinanylic acid, Phe) possesses antioxidant and anti-inflammatory properties. The aim of this study was to assess the effects of Phe on the mitochondrial function and oxidative status in the heart of rats with T2DM. Materials and Methods. T2DM was induced in Wistar rats by a high-caloric diet during 14 weeks combined with intraperitoneal injections of 25 mg/kg streptozotocin twice per week. All diabetic animals were divided into two groups: treated with vehicle or with Phe (in dose 50 mg/kg/day) for four weeks after diabetes induction. Redox status of rats’ heart mitochondria was estimated by determination of reactive oxygen species (ROS) production, GSH level and activity of antioxidant enzymes (Mn-superoxide dismutase, glutathione peroxidase and glutathione reductase). Mitochondrial function was determined by activity of aconitase, succinate dehydrogenase and cytochrome C oxidase in rats’ cardiomyocytes. Results. It was established that Phe inhibited oxidative stress in isolated heart mitochondria of rats with T2DM, which was confirmed by decreasing ROS production and increasing GSH level compared to diabetic rats. The use of Phe led to a normalization of antioxidant enzymes (Mn-superoxide dismutase and glutathione peroxidase) activity in the heart of diabetic rats. In addition, Phe improved metabolic activity of the heart mitochondria activating aconitase and succinate dehydrogenase in cardiomyocytes. We can suggest that Phe ameliorate mitochondrial dysfunction decreasing oxidative stress and preventing deficiency in complex II of ETC in the heart of rats with type 2 diabetes. Conclusion. The data of the present study confirmed the positive effect of Phe on redox homeostasis and functional state of the heart mitochondria in diabetic rats. We suggest that the use of Phe may contribute to the amelioration of cardiovascular risk in type 2 diabetes

Nanostructured systems increase the in vitro cytotoxic effect of bullfrog oil in human melanoma cells (A2058).

The aim of this work was to investigate the in vitro cytotoxic effect of previously developed nanocapsules, nanoemulsion, and microemulsion based on bullfrog oil (BFO) against human melanoma cells (A2058). The nanosystems were produced as described in previous studies and characterized according to droplet/particle distribution and zeta potential. The biocompatibility was evaluated by the determination of the hemolytic potential against human erythrocytes. The cytotoxicity assessment was based on MTT and cell death assays, determination of Reactive Oxygen Species (ROS) levels, and cell uptake. The nanosystems were successfully reproduced and showed hemolytic potential smaller than 10% at all oil concentrations (50 and 100µg.mL-1) (p<0.05). The MTT assay revealed that the nanosystems decreased the mitochondrial activity up to 92±2% (p<0.05). The study showed that the free BFO induced cell apoptosis, while all the nanostructured systems caused cell death by necrosis associated with a ROS overproduction. This can be related to the increased ability of the nanostructured systems to deliver the BFO across all cellular compartments (membrane, cytoplasm, and nucleus). Finally, these results elucidate the in vitro BFO nanosystems cytotoxic effect against human melanoma cells (A2058), revealing the emulsified ones as the most cytotoxic systems. Overall, the findings suggest that the safety and antineoplastic activity of these systems can be further investigated by in vivo studies.

Role of NADPH oxidase-mediated hydrogen peroxide in 5-aminolevulinic acid induced photooxidative stress tolerance in pear leaves

The beneficial effects of 5-aminolevulinic acid (ALA) on abiotic stress have been demonstrated in several plant species. However, little is known about the signal transduction pathways triggered by ALA in the photooxidative stress response. In this study, we investigated the effects of ALA on methyl viologen (MV)-induced photooxidative stress tolerance in detached pear leaves (Pyrus ussuriensis). Furthermore, we also examined the involvement of NADPH oxidase (RBOH)-mediated H2O2 as a signaling molecule in ALA-induced photooxidative stress tolerance. Our results showed that exogenous ALA alleviated the observed decreases in chlorophyll content and maximum potential photosystem II efficiency under MV-induced stress. Exogenous ALA also effectively reduced MV-induced oxidative damage and enhanced the activities of antioxidative enzymes (SOD and CAT). In situ determination of reactive oxygen species revealed that ALA induced H2O2 production in the pear leaves. Importantly, ALA-induced tolerance was abolished by an RBOH inhibitor. Further investigation revealed that the transient overexpression of RBOHF in tobacco leaves alleviated MV-induced damage by enhancing the activities of SOD and CAT. This study provides new insights into the mechanisms by which ALA improves photooxidative tolerance in pear leaves.

Hsa_circ_0003204 Knockdown Weakens Ox-LDL-Induced Cell Injury by Regulating miR-188-3p/TRPC6 Axis in Human Carotid Artery Endothelial Cells and THP-1 Cells.

Background: Circular RNAs (circRNAs) are involved in atherosclerosis (AS) development. However, the function and mechanism of circRNA hsa_circ_0003204 (circ_0003204) in carotid artery AS remain unclear.Methods: Oxidized low-density lipoprotein (ox-LDL)-treated human carotid artery endothelial cells (HCtAECs) and THP-1 cells were used as cell models of carotid artery AS. Relative levels of circ_0003204, microRNA-188-3p (miR-188-3p), and transient receptor potential canonical channel 6 (TRPC6) were detected by quantitative reverse transcription–polymerase chain reaction or Western blotting. The targeting relationship between circ_0003204 or TRPC6 and miR-188-3p was assessed via dual-luciferase reporter analysis and RNA immunoprecipitation. Cell proliferation was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was analyzed via assessing cell caspase-3 activity, apoptosis, and apoptosis-related protein. Inflammatory response was analyzed via analysis of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Oxidative stress was assessed via determination of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD).Results: Circ_0003204 and TRPC6 levels were elevated, and miR-188-3p expression declined in ox-LDL-treated HCtAECs and THP-1 cells. Circ_0003204 could regulate TRPC6 expression via mediating miR-188-3p. Circ_0003204 silencing weakened ox-LDL-induced viability inhibition and apoptosis in HCtAECs, and inflammatory response and oxidative stress in THP-1 cells via regulating miR-188-3p. MiR-188-3p overexpression attenuated ox-LDL-induced injury in HCtAECs and THP-1 cells by targeting TRPC6.Conclusion: Circ_0003204 knockdown mitigated ox-LDL-induced injury in HCtAECs and THP-1 cells via regulating the miR-188-3p/TRPC6 axis, indicating that circ_0003204 might play an important role in carotid artery AS.

The effects of agomelatine on endoplasmic reticulum stress related to mitochondrial dysfunction in hippocampus of aging rat model

BackgroundAgomelatine, a novel antidepressant, is a melatonin MT receptor agonist and serotonin 5HT2C receptor antagonist. In this study, agomelatine was used to investigate the molecular mechanisms of hippocampal aging associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptosis, all of which led to short-term memory impairment. MethodHippocampal aging was induced in male Wistar rats by d-galactose (D-gal) intraperitoneal injection (100 mg/kg) for 14 weeks. During the last 4 weeks of D-gal treatment, rats were treated with agomelatine (40 mg/kg) or melatonin (10 mg/kg). At the end of the experiment, all rats were assessed for short-term memory by using the Morris water maze test. Subsequently, rats were sacrified and the hippocampus was removed from each rat for determination of reactive oxygen species (ROS), malondialdehyde (MDA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays; and immunohistochemistry related to ER stress, mitochondrial dysfunction, and apoptosis. ResultsAgomelatine suppressed the expression of the aging-related proteins P16 and receptor for advanced glycation endproducts (RAGE), the expression of NADPH oxidase (NOX) 2 and 4, and ROS production. This treatment also shifted the morphology of astrocytes and microglia toward homeostasis. Furthermore, agomelatine decreased inositol-requiring enzyme 1 (pIRE1), protein kinase R-like endoplasmic reticulum kinase (pPERK), and chaperone binding immunoglobulin protein (BiP), leading to suppression of ER stress markers C/EBP homologous protein (CHOP) and caspase-12. Agomelatine reduced Ca2+ from the ER and stabilized the mitochondrial membrane stability, which was denoted by the BCL2 Associated X (Bax)/B-cell lymphoma 2 (Bcl2) balance. Agomelatine decreased cleaved caspase-3 production and the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive area, and glutamate excitotoxicity was prevented via suppression of N-methyl-d-aspartate (NMDA) receptor subunit expression. Agomelatine exhibited effects that were similar to melatonin. ConclusionAgomelatine improved neurodegeneration in a rat model of hippocampal aging by attenuating ROS production, ER stress, mitochondrial dysfunction, excitotoxicity, and apoptosis.

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.

SNAT2/SLC38A2 Confers the Stemness of Gastric Cancer Cells via Regulating Glutamine Level.

Glutamine (Gln) is essential for cancer progression, however, few studies have been conducted to investigate the roles of Gln transporters in gastric cancer stem cells (CSCs). This work aims to explore the roles of Gln transporters in gastric cancer cell stemness. We collected spheres formed by gastric cancer (GC) cells through a 3-dimensional (3D) semisolid culture system which has been shown to hold CSC-like traits. Lentivirus package was used to construct GC cells with SNAT2 overexpression. Analysis of sphere-formation, stemness marker expression, ALDH activity were used to detect the effects of Gln transporters on GC cell stemness. Determination of reactive oxygen species (ROS) and Gln consumption combined with the methods analyzing cell stemness were performed to explore the underlying mechanisms. Gln consumption was upregulated in GC spheres compared to the parental GC cells. The Gln transporter SNAT2 was highly expressed in GC spheres compared to that in the parental GC cells. SNAT2 overexpression significantly increased the Gln consumption in GC cells and increased the expression of stemness markers, sphere-formation ability and ALDH activity. Notably, SNAT2-mediated promoting effects on GC cell stemness were rescued by Gln deprivation. What's more, high expression of SNAT2 was associated with a poor GC patient survival through different online datasets. SNAT2 can promote the stemness of GC cells in a Gln-dependent manner.

Estrogen Decreases Cytoskeletal Organization by Forming an ERα/SHP2/c-Src Complex in Osteoclasts to Protect against Ovariectomy-Induced Bone Loss in Mice.

Loss of ovarian function is closely related to estrogen (E2) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E2 to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E2 injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E2 decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E2/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E2 exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E2 exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p–Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E2/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E2-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.

Photo induced antibacterial activity of CeO2/GO against wound pathogens

Infections are the main cause for delayed wound healing. Graphene oxide (GO) and cerium oxide nanoparticles are used for many biomedical applications because of their biocompatibility and therapeutic property. Cerium Oxide (CeO2)/Graphene Oxide (GO) nanocomposite was synthesized in this work. It shows good antimicrobial property against wound pathogens. The aggregation of CeO2 particles on GO results in the generation of reactive oxygen species (ROS) and inhibits microbial growth. The synthesized nanocomposites were analysed by X-ray diffraction, Transmission electron microscopy, Raman Spectroscopy, UV–vis diffuse reflectance spectroscopy and Fourier transformation-IR spectroscopy. The antimicrobial activity of CeO2/GO nanocomposite was tested against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) Staphylococcus aureus (S. aureus) and Salmonella typhi (S. typhi). Well diffusion method, growth curve studies, bio film inhibition studies and colony count studies were done in both presence and absence of visible light. The mechanistic investigation was done by the determination of reactive oxygen species (ROS), Lactate dehydrogenase leakage (LDH) assay and DNA fragmentation assay.

Noble metal alloy nanoparticles coated flexible MoS2 paper for the determination of reactive oxygen species

The monitoring of reactive oxygen species (ROS) in biological system has been occupying research hotspots recently, since the biochemical alterations caused by the overproduction of ROS are the main incentives of diseases and accelerated aging process. In this work, we proposed an effective and simple strategy for the construction of high-performance ROS electrochemical sensor. Noble metal alloy nanoparticles (AuPt nanoparticles) with high catalytic activity were spontaneously coated on the freestanding metallic molybdenum disulfide (MoS2) paper independent of any auxiliary conditions. Results have found that the abundant defects and electrons distributed on the metallic MoS2 paper could provide active sites for the nucleation and growth of noble metal nanoparticles. Besides, the excellent mechanical properties of the MoS2 paper promote the formation of flexible sensors. The fabricated MoS2 paper-based sensor was demonstrated to detect ROS with the advantages of wide linear range, prominent selectivity and flexibility, satisfactory detection stability, as well as simple and convenient preparation process. Furthermore, the desirable results obtained in the real sample experiments operated in plant extract pave the way for further real-time monitoring of plant physiological status to provide valuable information for guidance during plant growth.

Insights into the Photocatalytic Bacterial Inactivation by Flower-Like Bi2WO6 under Solar or Visible Light, Through in Situ Monitoring and Determination of Reactive Oxygen Species (ROS)

This study addresses the visible light-induced bacterial inactivation kinetics over a Bi2WO6 synthesized catalyst. The systematic investigation was undertaken with Bi2WO6 prepared by the complexation of Bi with acetic acid (carboxylate) leading to a flower-like morphology. The characterization of the as-prepared Bi2WO6 was carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), specific surface area (SSA), and photoluminescence (PL). Under low intensity solar light (&lt;48 mW/cm2), complete bacterial inactivation was achieved within two hours in the presence of the flower-like Bi2WO6, while under visible light, the synthesized catalyst performed better than commercial TiO2. The in situ interfacial charge transfer and local pH changes between Bi2WO6 and bacteria were monitored during the bacterial inactivation. Furthermore, the reactive oxygen species (ROS) were identified during Escherichia coli inactivation mediated by appropriate scavengers. The ROS tests alongside the morphological characteristics allowed the proposition of the mechanism for bacterial inactivation. Finally, recycling of the catalyst confirmed the stable nature of the catalyst presented in this study.

Analysis of the synergistic antifungal mechanism of eugenol and citral

The synergistic effects of different essential oils can not only reduce their dosage and cost, but also improve their biological activity. The aim of this study was to evaluate the synergistic antifungal effect of eugenol and citral (SEC) on Penicillium roqueforti. Determination of reactive oxygen species (ROS) and malondialdehyde content indicated that SEC could induce membrane lipid peroxidation, and the contribution of citral was greater than that of eugenol. In addition, downregulation of the gene encoding an NADPH oxidase subunit indicated that the ROS burst induced by SEC was mediated by NADPH oxidase. Scanning electron microscope (SEM) and transmission electron microscope (TEM), propidium iodide treatment, DNA gel electrophoresis and cell leakage experiments showed that SEC destroyed the integrity of the cell membrane and internal structures, and degraded the cell contents, and the contribution of eugenol was greater than that of citral. These results showed that SEC inhibited the activity of P. roqueforti more effectively than eugenol or citral alone, by inducing membrane lipid peroxidation and promoting the ability of eugenol to destroy the cell membrane. The combined agents eventually caused leakage of the cell contents, and ultimately cell death.

Zinc-Phosphate Nanoparticles as a Novel Anticancer Agent: An In Vitro Evaluation of Their Ability to Induce Apoptosis.

In the current study, zinc-phosphate nanoparticles (ZnPNPs) were investigated for the first time due to their anticancer activity against breast cancer Michigan Cancer Foundation-7 (MCF-7) cell line. The modification of such nanoparticles (NPs) was further examined for physicochemical characterization using various techniques such as powder X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential calculation, field emission scanning electron microscopy (FESEM), energy-dispersed spectroscopy (EDS), and Fourier transform infrared (FTIR) spectroscopy. Then, the newly fabricated ZnPNPs were tested for their in vitro cell cytotoxicity against breast cancer MCF-7 cells and noncancerous human embryonic kidney HEK293 cells, using MTT assay as a colorimetric one to assess cell metabolic activity for 24h. The apoptotic efficacy of the NPs was subsequently confirmed through data obtained from Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit and cell cycle analysis. Determination of reactive oxygen species (ROS) generation was further performed via flow cytometry. Additionally, the expression of tumor suppressor genes p53 was analyzed using real-time polymerase chain reaction (PCR). Also, the prepared NPs showed a mean particle size of 38nm. The measurements correspondingly showed that the cytotoxicity of MCF-7 cells depends on the concentration of NPs (IC50 = 80.112μg/mL). MCF-7 cells were associated with initiation of apoptotic pathway in cells. Additionally, flow cytometry revealed cell cycle arrest in sub-G1 phase. ROS production was also obtained after treatment with IC50 concentration. According to annexin V-FITC/PI staining kit data, the percentage of early and late apoptotic cells was 78.2% in those treated with ZnPNPs. Moreover, the real-time PCR results demonstrated the ability of NPs in upregulating p53 gene expression. In summary, the data demonstrated that fabricated ZnPNPs had prominence to act as antitumor agents in breast cancer therapy.

Hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration: promoting mitochondrial autophagy

Objective To evaluate the relationship between the hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration (OGD/R) and mitochondrial autophagy. Methods Hippocampal neurons isolated from healthy Sprague-Dawley rats (24 h after birth) were cultured in vitro, seeded in polylysine-coated 6-well plates at a density of 7×105 cells/well and then divided into 5 groups (n=30 each) using a random number table method: control group (C group), OGD/R group, OGD/R+ H2 group, OGD/R plus 3-methyladenine (3-MA) group (OGD/R+ 3-MA group), and OGD/R plus H2 plus 3-MA group (OGD/R+ H2+ 3-MA group). The cells were cultured for 24 h in normal culture atmosphere (75%N2-20%O2-5%CO2) in group C, and cells were subjected to oxygen-glucose deprivation for 2 h followed by O2-glucose supply for 24 h to establish the model of OGD/R injury in OGD/R, OGD/R+ H2, OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups.The cells were cultured for 24 h in a hydrogen-rich incubator (60% H2-10% O2-5% CO2-25% N2) after establishing the model in group OGD/R+ H2.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in normal culture atmosphere after establishing the model in group OGD/R+ 3-MA.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in hydrogen-rich incubator after establishing the model in group OGD/R+ H2+ 3-MA.The cell survival rate was measured using MTT assay.DCFH-DA fluorescent probe was applied for determination of reactive oxygen species (ROS) activity.The mitochondrial membrane potential was measured using a JC-10 assay kit.The neuronal apoptosis was detected by flow cytometry, and apoptosis rate was calculated.The expression of mitophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), PINK1 and Parkin was determined by Western blot, and LC3Ⅱ/LC3Ⅰ ratio was calculated. Results Compared with group C, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in OGD/R and OGD/R+ H2 groups (P<0.05). Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the apoptosis rate and ROS activity were decreased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in group OGD/R+ H2(P<0.05), and the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in group OGD/R+ 3-MA (P<0.05). Compared with group OGD/R+ H2, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups (P<0.05). Conclusion Hippocampal neuron-protective mechanism of hydrogen against OGDR injury is related to promoting mitochondrial autophagy in rats. Key words: Hydrogen; Hypoxia-ischemia, brain; Reperfusion injury; Hippocampus; Neurons; Mitochondria; Autophagy

Cerium and erbium effects on Daphnia magna generations: A multiple endpoints approach

Cerium (Ce, CeCl3) and Erbium (Er, ErCl3) are increasingly used in many electronic devices facilitating the alteration of their biogeochemical cycles (e.g. e-waste). Previous surveys stated that their environmental concentrations due to natural or anthropogenic events can reach up to 161 μg/L in ore mine effluent for Ce with a mean water concentration of 0.79 μg/L, and 11.9 μg/L for Er in ore mine effluents with a mean water concentration of 0.004 μg/L. Their potential effects onto aquatic organisms are still relatively unexplored. In this study, long-term multigenerational effects on Daphnia magna were assessed using various exposure times (3, 7, 14, and 21 days) in three generations (F0, F1 and F2). Each generation was exposed to environmental concentrations of Ce and Er (0.54 and 0.43 μg/L, respectively – mean values) and effects included organisms' size, parental reproduction, and survival, determination of reactive oxygen species (ROS), enzymatic activity (superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST)), gene expression of ATP-binding cassette (ABC) transporter, and uptake.Results evidenced that chronic multi-generational exposure of daphnids to Ce and Er reduced survival, growth and reproduction, decreasing ROS, SOD and CAT from F0 to F2. Ce reduced the number of generated offsprings after each generation, while Er delayed the time of offsprings emergence, but not their number. ROS, SOD, CAT and GST evidenced that Er is slightly more toxic than Ce. Up- and downregulation of genes was limited, but Ce and Er activated the ABC transporters. Uptake of Ce and Er decreased through exposure time and generations.

The accumulation of reactive oxygen species in root tips caused by autotoxic allelochemicals – A significant factor for replant problem of Angelica sinensis (Oliv.) Diels

Continuous cropping obstacles are widespread problems in agriculture but the reasons for their occurrence are generally considered complicated. Autotoxic allelopathy has been thought to be a critical factor in replant problems. To investigate the autotoxic allelochemicals of Angelica sinensis (Oliv.) Diels, whose cultivation has been frequently hampered by replant failure, five compounds were isolated from the rhizosphere soil and identified as imperatorin (1), α-spinasterol (2), vanillin (3), dibutyl phthalate (4) and ferulic acid (5). Bioassays showed that compounds 1, 3 and 5 significantly inhibited the growth of A. sinensis and L. sativa seedlings. The existence of the allelochemicals in the soil environment of A. sinensis was further confirmed and quantified by high-performance liquid chromatography (HPLC). The total concentration of these allelochemicals in the rhizosphere soil reached 17.67 μg/g. Determination of reactive oxygen species (ROS) in the root tips of the treated seedlings indicated that the generation of ROS-induced oxidative damage could be an essential factor in the autotoxicity of the allelochemicals. These findings provide new insights for understanding the mechanism of autotoxicity and replant failure in the cultivation of A. sinensis. This could be helpful for the development of a strategy to eliminate some replant problems in agriculture.11Abbreviations: ANOVA, analysis of variance; CC, column chromatography; DCFH-DA, 2’,7’-Dicholrofluorecein diacetate; GC–MS, gas chromatography−mass spectrometry; LSD, least significant difference; PTLC, preparative thin layer chromatography; ROS, reactive oxygen species; TLC, Thin layer chromatography.

Chronic toxicity of treated and untreated aqueous solutions containing imidazole-based ionic liquids and their oxydized by-products

In the present work, an experimental study is presented aimed at assessing the chronic toxicity of three imidazole-based ionic liquids, i.e. imidazole (IM), 1-methylimidazole (1MIM), 1-ethyl-3-methyl-imidazolium chloride (1E3MIM), and 1-butyl-3-methyl-imidazolium chloride (1B3MIM), generally considered as environmentally friendly surrogates of traditional industrial solvents. In this study Daphnia magna was used as test organism due to its wide application in the ecotoxicological literature of ionic liquids, monitoring both the cumulative survival of exposed organisms, and their reproductive parameters. The intracellular oxidative stress of daphnids was also assessed through the determination of Reactive Oxygen Species (ROS) and Catalase activity (CAT). The chronic toxicity of their oxidized by-products (BPs), generated by advanced oxidation treatment with UV254/H2O2, was finally evaluated. Four generations of BPs were considered, each formed at reaction times higher than those required for the complete removal of the parent compounds. Results indicate that IM and 1MIM have a moderate chronic toxicity, which mainly affects reproductive parameters. On the contrary, 1E3MIM and 1B3MIM showed significantly higher chronic toxicity effects resulting in a significant increase in the mortality of exposed organisms compared to the controls. UV/H2O2 treatment of the compounds did not always reduce the observed effects, since the generated BPs have, in some cases, higher chronic toxicity than their corresponding parent compounds. Chronic toxic effects remained significant up to the fourth generation of BPs in the cases of 1E3MIM and 1B3MIM, whereas they were found to be negligible from the second generation of BPs in the case of IM and 1MIM. The results of oxidative stress measurements confirmed the previous findings, suggesting a potential risk for the aquatic ecosystem induced by the mentioned compounds and their BPs.

Age-related changes in vascular responses to angiotensin-(1-7) in female mice.

The vasodilatory effect of angiotensin-(1-7) seems to vary between sexes, and estradiol (E2) can modulate the magnitude of the Ang-(1-7) vasodilatory response in female rats. However, there are few studies addressing the influence of sex on the age-related vasodilatory effect of Ang-(1-7). Here, we evaluated the vasodilatory response to Ang-(1-7) on vascular ageing. Ang-(1-7) dose–response curves were determined in mice aortic rings from males (old and young) and females (E2 treated/non-treated old and young) mounted in an isolated organ chamber. Abdominal aortic rings were used for protein expression analysis and determination of reactive oxygen species (ROS) and nitric oxide (NO) production. Our results showed that the Ang-(1-7) vasodilatory effect was absent in aorta from old females, contrasting with a full response in vessels from young females. The Ang-(1-7) vasodilatory effect was restored by E2 replacement in old females. A robust increase in Mas receptor, SOD2, NRF-2 and NOX2 expression was observed in aorta from old females, which was normalized by E2. This effect of E2 was also associated with lower production of ROS and normal levels of NO. In conclusion, our data demonstrated that pathways involved in the Ang-(1-7) vasodilatory response in female mice is affected by hormonal changes in ageing and rescued by E2.