Induced Reactive Oxygen Species Formation Research Articles

The complexation of atmospheric Brown carbon surrogates on the generation of hydroxyl radical from transition metals in simulated lung fluid

Atmospheric particulate matter (PM) poses great adverse effects through the production of reactive oxygen species (ROS). Various components in PM are acknowledged to induce ROS formation, while the interactions among chemicals remain to be elucidated. Here, we systematically investigate the influence of Brown carbon (BrC) surrogates (e.g., imidazoles, nitrocatechols and humic acid) on hydroxyl radical (OH) generation from transition metals (TMs) in simulated lung fluid. Present results show that BrC has an antagonism (interaction factor: 20–90 %) with Cu2+ in OH generation upon the interaction with glutathione, in which the concentrations of BrC and TMs influence the extent of antagonism. Rapid OH generation in glutathione is observed for Fe2+, while OH formation is very little for Fe3+. The compositions of antioxidants (e.g., glutathione, ascorbate, urate), resembling the upper and lower respiratory tract, respond differently to BrC and TMs (Cu2+, Fe2+ and Fe3+) in OH generation and the degree of antagonism. The complexation equilibrium constants and site numbers between Cu2+ and humic acid were further analyzed using fluorescence quenching experiments. Possible complexation products among TMs, 4-nitrocatechol and glutathione were also identified using quadropule-time-of-flight mass spectrometry. The results suggest atmospheric BrC widely participate in complexation with TMs which influence OH formation in the human lung fluid, and complexation should be considered in evaluating ROS formation mediated by ambient PM.

Photoenhanced Radical Formation in Aqueous Mixtures of Levoglucosan and Benzoquinone: Implications to Photochemical Aging of Biomass-Burning Organic Aerosols.

The photochemical aging of biomass-burning organic aerosols (BBOAs) by exposure to sunlight changes the chemical composition over its atmospheric lifetime, affecting the toxicological and climate-relevant properties of BBOA particles. This study used electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping agent, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), high-resolution mass spectrometry, and kinetic modeling to study the photosensitized formation of reactive oxygen species (ROS) and free radicals in mixtures of benzoquinone and levoglucosan, known BBOA tracer molecules. EPR analysis of irradiated benzoquinone solutions showed dominant formation of hydroxyl radicals (•OH), which are known products of reaction of triplet-state benzoquinone with water, also yielding semiquinone radicals. In addition, hydrogen radicals (H•) were also observed, which were not detected in previous studies. They were most likely generated by photochemical decomposition of semiquinone radicals. The irradiation of mixtures of benzoquinone and levoglucosan led to substantial formation of carbon- and oxygen-centered organic radicals, which became prominent in mixtures with a higher fraction of levoglucosan. High-resolution mass spectrometry permitted direct observation of BMPO-radical adducts and demonstrated the formation of •OH, semiquinone radicals, and organic radicals derived from oxidation of benzoquinone and levoglucosan. Mass spectrometry also detected superoxide radical adducts (BMPO-OOH) that did not appear in the EPR spectra. Kinetic modeling of the processes in the irradiated mixtures successfully reproduced the time evolution of the observed formation of the BMPO adducts of •OH and H• observed with EPR. The model was then applied to describe photochemical processes that would occur in mixtures of benzoquinone and levoglucosan in the absence of BMPO, predicting the generation of HO2• due to the reaction of H• with dissolved oxygen. These results imply that photoirradiation of aerosols containing photosensitizers induces ROS formation and secondary radical chemistry to drive photochemical aging of BBOA in the atmosphere.

Vanillic acid alleviates methamphetamine-induced mitochondrial toxicity in cardiac mitochondria via antioxidant activity and inhibition of MPT Pore opening: an in-vitro study

BackgroundMethamphetamine is widely abused in all parts of the world. It has been reported that short-term and long-term methamphetamine exposure could damage the dopaminergic system and induce cardiomyopathy and cardiotoxicity via mitochondrial dysfunction and oxidative stress. Vanillic acid (VA), a phenolic acid compound derived from plants, is known for its antioxidant and mitochondrial protection properties.MethodsIn the current study we used VA for attenuating of Methamphetamine-induced mitochondrial toxicity in cardiac mitochondria. Isolated mitochondria obtained from rat heart were grouped as: control, methamphetamine (250 µM), VA (10, 50 and 100 µM) was cotreated with methamphetamine (250 µM) and VA (100 µM) alone. After 60 min, mitochondrial fraction including: succinate dehydrogenases (SDH) activity, mitochondrial membrane potential (MMP), mitochondrial swelling, mitochondrial glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation (LPO) were evaluated.ResultsMethamphetamine exposure significantly disrupted mitochondrial function and induced ROS formation, lipid peroxidation, GSH depletion, MMP collapse and mitochondrial swelling, while VA significantly increased SDH activity as indicator of mitochondrial toxicity and dysfunction. VA also significantly decreased ROS formation, lipid peroxidation, mitochondrial swelling, MMP collapse and depletion of GSH in cardiac mitochondria in the presence of methamphetamine.ConclusionThese findings suggested that VA is able to reduce methamphetamine-induced mitochondrial dysfunction and oxidative stress. Our results demonstrate that VA could potentially serve as a promising and accessible cardioprotective agent against methamphetamine-induced cardiotoxicity, via antioxidant and mitochondrial protection properties.

Characterization of the absorption properties of 5 nm spherical gold nanoparticles functionalized with dodecanothiol and without functionalization with potential therapeutic applications

Hybrid nanosystems have useful properties for preparing therapeutic systems. Among the most commonly used inorganic components in hybrid nanosystems are gold nanoparticles (AuNP). The design of these nanosystems may require AuNP of hydrophilic or hydrophobic nature. Upon irradiation of AuNP, reactive oxygen species (ROS) are formed, and the temperature of the surrounding medium rises, depending on the size, shape and structure of the nanoparticle. The aim of this work is to evaluate whether irradiating 5 nm spherical gold nanoparticles both ‘bare’ (AuNP) and functionalized with dodecanethiol (AuNPf) with a Nd:YAG pulsed laser (30 ps, and 10 Hz) at wavelengths of 532 nm (0.031 J cm−2) and 1064 nm (1.91 J cm−2) produces ROS and heat sufficiently to induce cytotoxicity, or to demonstrate whether functionalization significantly influences such processes. It was verified by UV–vis spectrophotometry with ABMA and DCPIP that AuNP and AuNPf in solution induced ROS formation. They also produce a moderate elevation of the temperature of the surrounding medium. Irradiation of T47D cells at 532 nm in the presence of AuNP significantly decreased cell viability (>60%) compared to irradiation in the absence of NP (<20%). This effect was less evident when irradiating at 1064 nm because the cytotoxicity in the absence of AuNPs was very high (80%). AuNPf showed in solution a similar behavior to AuNP but due to the solvent in the suspension it was not possible to evaluate the cell toxicity caused by AuNPf. The results obtained indicate that it is feasible to design hybrid nanosystems for therapy from these 5 nm NPs, which would have photodynamic and photothermal effect.

Biological Consequences of Vanadium Effects on Formation of Reactive Oxygen Species and Lipid Peroxidation

Lipid peroxidation (LPO), a process that affects human health, can be induced by exposure to vanadium salts and compounds. LPO is often exacerbated by oxidation stress, with some forms of vanadium providing protective effects. The LPO reaction involves the oxidation of the alkene bonds, primarily in polyunsaturated fatty acids, in a chain reaction to form radical and reactive oxygen species (ROS). LPO reactions typically affect cellular membranes through direct effects on membrane structure and function as well as impacting other cellular functions due to increases in ROS. Although LPO effects on mitochondrial function have been studied in detail, other cellular components and organelles are affected. Because vanadium salts and complexes can induce ROS formation both directly and indirectly, the study of LPO arising from increased ROS should include investigations of both processes. This is made more challenging by the range of vanadium species that exist under physiological conditions and the diverse effects of these species. Thus, complex vanadium chemistry requires speciation studies of vanadium to evaluate the direct and indirect effects of the various species that are present during vanadium exposure. Undoubtedly, speciation is important in assessing how vanadium exerts effects in biological systems and is likely the underlying cause for some of the beneficial effects reported in cancerous, diabetic, neurodegenerative conditions and other diseased tissues impacted by LPO processes. Speciation of vanadium, together with investigations of ROS and LPO, should be considered in future biological studies evaluating vanadium effects on the formation of ROS and on LPO in cells, tissues, and organisms as discussed in this review.

Attenuation of reactive oxygen species (ROS) generation in the cultured retinal cells under high glucose conditions

The excess reactive oxygen species (ROS) formation due to diabetes-induced hyperglycemia is believed to play a central role in the pathophysiology of diabetic complications including diabetic retinopathy. However, the mechanism of excess ROS generation due to hyperglycemia is still uncertain. In this study, we measured ROS levels under the influence of low and hyperglycaemic conditions in the cultured retinal Muller (TR-MUL) and bovine retinal endothelial cells (BRECs) to better elucidate the basis of oxidative stress. The most common fluorescent dye, CM-H2DCFDA was used to examine the ROS level in the cultured retinal cells after incubations with high (30 mM) and low glucose (5.5 mM), and treatments with metabolites (pyruvate, glutamate, and glutamine), oxidizing agents (diamide, CuSO4), and endotoxin, lipopolysaccharide (LPS). Our results showed high glucose did not increase ROS levels in both TR-MUL and BRECs cells; on the contrary, high glucose resulted in a significant decrease in ROS levels compared to low glucose (P < 0.05). Diamide and CuSO4 treatment induced a large increase in ROS levels in the TR-MUL cells at low glucose, while high glucose significantly reduced its levels (P < 0.05). Pyruvate and glutamate ameliorated, while glutamine augmented the ROS level in the TR-MUL cells. LPS induced ROS formation, while high glucose incubation significantly reduced its level (p < 0.05). The results of this study do not support the high glucose-induced increase in ROS levels in the cultured retinal cells; rather, high glucose appeared to have ameliorative effects on oxidative stress, induced due to low or euglycemic conditions.

Cerium oxide decorated 5-fluorouracil loaded chitosan nanoparticles for treatment of hepatocellular carcinoma

Reactive oxygen species (ROS) play a crucial role in the mammalian system in both normal and pathological conditions. Hence, this work prepared and characterized the ROS responsive cerium oxide nanoparticles (CeO2 NPs) decorated 5-fluorouracil (5FU) loaded chitosan (CS) nanoparticles (CS-5FU NPs) for enhanced anticancer activity in hepatocellular carcinoma (HepG2 cells). CeO2 NPs decorated CS-5FU NPs were found to be spherical in shape and black dense aggregated particles sized 200 nm. The functional properties and cubic crystalline structure of CeO2 NPs were studied by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis, respectively. Further, CS-5FU-CeO2 NPs attenuated the 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) induced ROS formation in mouse embryonic fibroblasts (NIH3T3 cells) while enhancing apoptosis in HepG2 cells by controlled delivery of 5FU. Furthermore, CS-5FU-CeO2 NPs have not exhibited toxicity to red blood cells (RBCs) and chick chorioallantoic membrane (CAM). Hence, this work concluded that CS-5FU-CeO2 NPs synergistically enhanced anticancer activity in HepG2 cells through the regulation of ROS.

Reactive Oxygen Species Formed by Metal and Metal Oxide Nanoparticles in Physiological Media-A Review of Reactions of Importance to Nanotoxicity and Proposal for Categorization.

Diffusely dispersed metal and metal oxide nanoparticles (NPs) can adversely affect living organisms through various mechanisms and exposure routes. One mechanism behind their toxic potency is their ability to generate reactive oxygen species (ROS) directly or indirectly to an extent that depends on the dose, metal speciation, and exposure route. This review provides an overview of the mechanisms of ROS formation associated with metal and metal oxide NPs and proposes a possible way forward for their future categorization. Metal and metal oxide NPs can form ROS via processes related to corrosion, photochemistry, and surface defects, as well as via Fenton, Fenton-like, and Haber–Weiss reactions. Regular ligands such as biomolecules can interact with metallic NP surfaces and influence their properties and thus their capabilities of generating ROS by changing characteristics such as surface charge, surface composition, dissolution behavior, and colloidal stability. Interactions between metallic NPs and cells and their organelles can indirectly induce ROS formation via different biological responses. H2O2 can also be generated by a cell due to inflammation, induced by interactions with metallic NPs or released metal species that can initiate Fenton(-like) and Haber–Weiss reactions forming various radicals. This review discusses these different pathways and, in addition, nano-specific aspects such as shifts in the band gaps of metal oxides and how these shifts at biologically relevant energies (similar to activation energies of biological reactions) can be linked to ROS production and indicate which radical species forms. The influences of kinetic aspects, interactions with biomolecules, solution chemistry (e.g., Cl− and pH), and NP characteristics (e.g., size and surface defects) on ROS mechanisms and formation are discussed. Categorization via four tiers is suggested as a way forward to group metal and metal oxide NPs based on the ROS reaction pathways that they may undergo, an approach that does not include kinetics or environmental variations. The criteria for the four tiers are based on the ability of the metallic NPs to induce Fenton(-like) and Haber–Weiss reactions, corrode, and interact with biomolecules and their surface catalytic properties. The importance of considering kinetic data to improve the proposed categorization is highlighted.

Cytotoxicity of three graphene-related materials in rainbow trout primary hepatocytes is not associated to cellular internalization

As a consequence of increasing production and use of graphene-related materials (GRM), their release into the aquatic environment is likely to be expected. Development of appropriate model systems to assess their potential toxicity toward aquatic organisms is undoubtedly needed. Of particular relevance are primary cultures of fish hepatocytes, since they maintain similar functionalities as those of the original tissue. Isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to ranges of concentrations of different forms of GRM, two graphene oxides (GO) of sheet-like structure and one tubular-shaped carbon nanofiber (CNF) in the presence or absence of fetal bovine serum (FBS) for 24 and 72 h. Metabolic activity, cell membrane integrity, lysosomal function, reactive oxygen species (ROS) formation and interaction with cytochrome P450 1 A enzyme were assessed by using AlamarBlue, 5-carboxyfluorescein diacetate-acetoxymethyl ester, neutral red uptake, dichlorofluorescein and 7-ethoxyresorufin-O-deethylase (EROD) assays, respectively. In the presence of FBS, GO affected metabolic activity and cell membrane integrity more than CNF, whilst absence of serum further reduced cell viability in GRM-exposed cells. GRM did not alter lysosomal function nor did it induce ROS formation or EROD activity. Intracellular uptake was observed only in the case of CNF when incubated without FBS. Primary hepatocytes from rainbow trout appear to be a suitable model to screen for cytotoxicity and to reveal any interaction with GRM. Results emphasize the role of serum proteins in the toxicological responses following exposure to GRM with important implications for the environmental risk assessment of these nanomaterials.

In vitro toxicological evaluation of mesoporous silica microparticles functionalised with carvacrol and thymol

The cytotoxicity of carvacrol- and thymol-functionalised mesoporous silica microparticles (MCM-41) was assessed in the human hepatocarcinoma cell line (HepG2). Cell viability, lactate dehydrogenase (LDH) activity, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), lipid peroxidation (LPO) and apoptosis/necrosis analyses were used as endpoints. The results showed that both materials induced cytotoxicity in a time- and concentration-dependent manner, and were more cytotoxic than free essential oil components and bare MCM-41. This effect was caused by cell-particle interactions and not by degradation products released to the culture media, as demonstrated in the extract dilution assays. LDH release was a less sensitive endpoint than the MTT (thiazolyl blue tetrazolium bromide) assay, which suggests the impairment of the mitochondrial function as the primary cytotoxic mechanism. In vitro tests on specialised cell functions showed that exposure to sublethal concentrations of these materials did not induce ROS formation during 2 h of exposure, but produced LPO and ΔΨm alterations in a concentration-dependent manner when cells were exposed for 24 h. The obtained results generally support the hypothesis that the carvacrol- and thymol-functionalised MCM-41 microparticles induced toxicity in HepG2 cells by an oxidative stress-related mechanism that resulted in apoptosis through the mitochondrial pathway.

Enhancement of Proton Therapy Efficiency by Noble Metal Nanoparticles Is Driven by the Number and Chemical Activity of Surface Atoms

Proton-based radiotherapy is a modern technique for the treatment of solid tumors with significantly reduced side effects to adjacent tissues. Biocompatible nanoparticles (NPs) with high atomic numbers are known to serve as sensitizers and to enhance treatment efficacy, which is commonly believed to be attributed to the generation of reactive oxygen species (ROS). However, little systematic knowledge is available on how either physical effects due to secondary electron generation or the particle surface chemistry affect ROS production. Thereto, ligand-free colloidal platinum (Pt) and gold (Au) NPs with well-controlled particle size distributions and defined total surface area are proton-irradiated. A fluorescence-based assay is developed to monitor the formation of ROS using terephthalic acid as a cross-effect-free dye. The findings indicate that proton irradiation (PI)-induced ROS formation sensitized by noble metal NPs is driven by the total available particle surface area rather than particle size or mass. Furthermore, a distinctive material effect with Pt being more active than Au is observed which clearly indicates that the chemical reactivity of the NP surface is a main contributor to ROS generation upon PI. These results pave the way towards an in-depth understanding of the NP-induced sensitizing effects upon PI and hence a well-controlled enhanced therapy.

Anticancer Activities of &lt;i&gt;Phlogacanthus pulcherrimus &lt;/i&gt;T. Anderson Leaves Extract on MCF-7 Breast Cancer Cells

Phlogacanthus pulcherrimus T. Anderson (PPT) is an edible plant found in the northern and northeastern regions of Thailand. There is limited information about the anti-breast cancer activity of the ethanolic leaf extract of PPT. This study aimed to evaluate the effects of an ethanolic leaf extract of PPT on MCF-7 breast cancer cell lines. The biological effects, including cytotoxicity, cell apoptosis, colony formation, reactive oxygen species (ROS) formation and cell migration, were determined by a means of sulforhodamine B (SRB), acridine orange/ethidium bromide (AO/EB) staining, a clonogenic assay, flow cytometry and a scratch wound healing assay, respectively. The results demonstrated that the PPT extract showed cytotoxic on MCF-7 cells in a dose-dependent manner with 50% inhibitory concentration (IC50) values of 119.9 ± 12.1 and 51.3 ± 4.7 μg/mL at 24 h and 48 h incubation, respectively. In addition, the extract exhibited cell apoptosis in a dose-dependent manner when used at a concentration of 50–100 μg/mL and inhibited colony formation with an IC50 value of 26.0 ± 2.0 μg/mL when compared with the control group. The extract induced ROS formation in a dose-dependent manner when used at a concentration of 50–100 μg/mL. The extract suppressed MCF-7 cell migration, with significant effect at 25 μg/mL. These results indicate that PPT ethanolic leaf extract has an anticancer activity against MCF-7 breast cancer cells and may be useful for prevention and treatment of breast cancer.

Polycyclic Aromatic Hydrocarbons from Fine Particulate Matter Induce Oxidative Stress and the Inflammatory Response in Human Vocal Fold Fibroblast Cells.

Polycyclic aromatic hydrocarbons (PAHs) are toxicants in particulate matter (PM). The vocal fold, part of the larynx and a key structure for voicing, is always in contact with air. In recent epidemic studies, PM was shown to cause laryngitis; however, the basic mechanism has not been evaluated. In the present study, intracellular reactive oxygen species (ROS) and proinflammatory cytokine levels were analyzed after exposing human vocal fold fibroblasts (hVFFs) to PM standard reference material (SRM 2786). Expression levels of the aryl hydrocarbon receptor (AhR) and Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) were also evaluated. PM induced ROS formation and proinflammatory cytokines via the AhR CYP1A1 pathway and caused lipid peroxidation and DNA damage. Blocking AhR or CYP1A1 production using siRNAs significantly decreased ROS production and IL-6 and IL-9 expression in PM-exposed hVFFs, thus protecting the cells against oxidative stress. These results confirm that PAHs in PM play an important role in cell damage and inflammation, confirming a basic pathophysiologic relationship between PM exposure and laryngitis.

Role of Novel Redox Regulator, MnTnBuOE‐2‐PyP 5+ in Rotenone Induced 4‐HNE Aggresosome Degradation through Autophagy in Cardiomyocytes

Oxidative stress has been linked to pathogenesis of cardiovascular disease. Mitochondria is the major site where reactive oxygen species (ROS) are produced and mainly generated from complex I and complex III of electron transport chain. ROS leads to generation of reactive aldehydes known as 4-hydroxynonenal (4-HNE) through lipid peroxidation. 4-HNE modifies protein through covalent adduction thereby causing their crosslinking, aggregation, and inactivation. It has been little known about degradation of 4-HNE adducted proteins. Autophagy is a dynamic process which maintains cellular homeostasis by removing damaged organelles and protein. There has been use of antioxidant in cardiovascular disease in order to detoxify ROS and ameliorate disease process. Superoxide dismutase (SOD) mimetic, MnTnBuOE-2-PyP5+ has shown to be promising treatment in oxidative stress induced disease pathology because of its specificity to superoxide (O −), high lipophilicity and less toxic profile. In this study, we are trying to explore the role of, MnTnBuOE-2-PyP5+on rotenone induced 4-HNE aggresosome formation and its degradation process in HL-1 cardiomyocyte. Rotenone, a complex I inhibitor treatment (500nM) for 24 hours increased ROS in HL-1 cardiomyocytes. Rotenone treatment led to increase accumulation 4-HNE aggresosome in HL-1 cardiomyocytes. Rotenone treatment induce increase in LC3-II shown by immunoblotting and immunofluorescence. Pre-treatment with MnTnBuOE-2-PyP5+(20 µM) for 24 hours attenuated rotenone induced ROS formation. In addition, pre-treatment with mimetic decreased 4-HNE aggresosome and LC3-II. Rotenone induced increase in autophagosome was attenuated by pre-treatment with MnTnBuOE-2-PyP5+ as shown by fluorescent-tagged LC3 (tfLC3). Rotenone induced tubulin hyperacetylation through ROS mediated pathway which was attenuated by MnTnBuOE-2-PyP5+. These results indicate that pre-treatment with MnTnBuOE-2-PyP5+ decrease rotenone induced 4-HNE aggresosome by enhancing their degradation process.

Physiological and Molecular Alterations of Phycobionts of Genus Trebouxia and Coccomyxa Exposed to Cadmium.

Several studies on aeroterrestrial microalgae are unravelling their resistance mechanisms to different abiotic stressors, including hazardous metals, pointing to their future role as bioremediation microorganisms. In the present study, physiological and molecular alterations of four phycobionts of genus Trebouxia (T. TR1 and T. TR9) and Coccomyxa (C. subellipsoidea and C. simplex) exposed to Cd were studied. Cd accumulation and subcellular distribution, cell wall structure, production of biothiols (GSH and phytochelatins), reactive oxygen species (ROS) formation, expression of key antioxidant genes and ROS-related enzymes were evaluated to determine the physiological differences among the four microalgae, with the aim to identify the most suitable microorganism for further biotechnological applications. After 7 days of Cd exposure, Coccomyxa algae showed higher capacity of Cd intake than Trebouxia species, with C. subellipsoidea being the highest Cd accumulator at both intracellular and, especially, cell wall level. Cd induced ROS formation in the four microalgae, but to a greater extent in both Coccomyxa algae. Trebouxia TR9 showed the lowest Cd-dependent oxidative stress probably due to glutathione reductase induction. All microalgae synthetized phytochelatins in response to Cd but in a species-specific and a dose-dependent manner. Results from this study agree with the notion that each microalga has evolved a distinct strategy to detoxify hazardous metals like Cd and to cope with oxidative stress associated with them. Coccomyxa subellipsoidea and Trebouxia TR9 appear as the most interesting candidates for further applications.

Tyramine and amyloid beta 42: A toxic synergy

AbstractBackgroundIn synaptic cleft, trace amines play a crucial role as neurotransmitter signaling through the trace amine associated receptors. Tyramine, an important trace amine formed by decarboxylation of tyrosine, has been reported to be implicated in various diseases including schizophrenia, Parkinson's disease, migraine and Huntington's disease. However, there are no investigations to find its connection with Alzheimer's disease (AD).MethodGenetically modified Saccharomyces cerevisiae BY4743 producing different forms of amyloid beta 42 (Aβ42) peptide including native Aβ42 and green fluorescent protein (GFP) fused to Aβ42 were treated with various concentrations of tyramine. Reactive oxygen species (ROS) produced in these strains were measured using 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCF‐DA) staining in a flow cytometric analysis. Growth inhibition of the yeast strains producing GFP and GFP‐Aβ42 were analysed in presence of tyramine. Petites generated due to tyramine were estimated in both these strains and petite frequency calculated.ResultTyramine was found to induce ROS formation in S. cerevisiae BY4743 as well as its genetically modified variants. ROS formation in yeast strains producing native Aβ42 treated with tyramine was significantly synergistically higher as compared to experimental controls. Similarly, yeast strains producing GFP‐Aβ42 was found to be inhibited more in media containing ethanol as sole carbon source compared to GFP producing controls in presence of 3 mM tyramine. Additionally, petite frequency in yeast strains producing GFP‐Aβ42 were found to increase significantly from around 20% to nearly 80% when grown in presence of tyramine.ConclusionThese findings suggest that tyramine in presence of Aβ42 is synergistically toxic to yeast cells by inducing ROS formation and causing petite mutation, implying possible cause of mitochondrial damage evident in AD patients. The study also demonstrates yeast as model to study compounds that have potential to exacerbate AD.

Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes

Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.

Seleninic Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability.

Organoselenium compounds are well-known glutathione peroxidase (GPx) mimetics that possess antioxidants/prooxidant properties and are able to modulate the concentration of reactive oxygen species (ROS), preventing oxidative stress in normal cells or inducing ROS formation in cancer cells leading to apoptosis. The purpose of this study was the synthesis of potent GPx mimics with antioxidant and anticancer activity along with improved bioavailability, as a result of good solubility in protic solvents. As a result of our research, glutathione peroxidase (GPx) mimetics in the form of water-soluble benzeneseleninic acid salts were obtained. The procedure was based on the synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids, through the oxidation of benzisoselenazol-3(2H)-ones or analogous arenediselenides with an amido group, which were further converted to corresponding potassium salts by the treatment with potassium tert-butanolate. All derivatives were tested as potential antioxidants and anticancer agents. The areneseleninic acid salts were significantly better peroxide scavengers than analogous acids and the well-known organoselenium antioxidant ebselen. The highest activity was observed for the 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt. The strongest cytotoxic effect against breast cancer (MCF-7) and human promyelocytic leukemia (HL-60) cell lines was found for 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt and the 2-(N-ethylcarboxyamido)benzeneselenenic acid, respectively. The structure–activity correlations, including the differences in reactivity of benzeneseleninic acids and corresponding salts were evaluated.

NAD hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages.

Mycobacterium tuberculosis (Mtb) kills infected macrophages through necroptosis, a programmed cell death that enhances mycobacterial replication and dissemination. The tuberculosis necrotizing toxin (TNT) is the major cytotoxicity factor of Mtb in macrophages and induces necroptosis by NAD+ hydrolysis. Here, we show that the catalytic activity of TNT triggers the production of reactive oxygen species (ROS) in Mtb-infected macrophages causing cell death and promoting mycobacterial replication. TNT induces ROS formation both by activating necroptosis and by a necroptosis-independent mechanism. Most of the detected ROS originate in mitochondria as a consequence of opening the mitochondrial permeability transition pore. However, a significant part of ROS is produced by mechanisms independent of TNT and necroptosis. Expressing only the tnt gene in Jurkat T-cells also induces lethal ROS formation indicating that these molecular mechanisms are not restricted to macrophages. Both the antioxidant N-acetyl-cysteine and replenishment of NAD+ by providing nicotinamide reduce ROS levels in Mtb-infected macrophages, protect them from cell death, and restrict mycobacterial replication. Our results indicate that a host-directed therapy combining replenishment of NAD+ with inhibition of necroptosis and/or antioxidants might improve the health status of TB patients and augment antibacterial TB chemotherapy.

Mitochondrial and lysosomal protective agents ameliorate cytotoxicity and oxidative stress induced by cyclophosphamide and methotrexate in human blood lymphocytes.

Cyclophosphamide (CYP) and methotrexate (MTX) have been evaluated for their ability to induce toxicity in human peripheral blood lymphocytes (PBLs) and the protective role of mitochondrial and lysosomal stabilizing agents. The potential toxicity effects of CYP and MTX were measured in vitro by cellular parameters assays such as cellular viability, reactive oxygen species (ROS) formation, mitochondrial membrane permeability transition (mitochondrial membrane potential (MMP)) collapse, lysosomal membrane damage, intracellular reduced glutathione (GSH), extracellular oxidized glutathione (GSSG), and lipid peroxidation. Separately, human lymphocytes were treated with concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 ng/mL for CYP and 1, 2, 5, and 10 µg/mL for MTX for 6 h. Statistical evaluations showed that CYP and MTX significantly decreased the cell viability at the three highest concentrations when compared with both the negative and solvent controls. In addition, CYP and MTX were significantly induced ROS formation, MMP collapse, lysosomal membrane damage, lipid peroxidation, and GSH depletion compared with the controls. Mitochondrial and lysosomal protective agents like cyclosporine A and chloroquine, respectively, decreased cytotoxicity and oxidative stress induced by CYP and MTX. The present results indicate that CYP and MTX are toxic to human PBLs and their toxicity could be ameliorated by mitochondrial and lysosomal protective agents.