Extracellular Reactive Oxygen Species Levels Research Articles

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

BackgroundGenerating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm.ResultsIn this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique.ConclusionOverall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals.Graphical

Combined ROS-related therapy improves the efficacy of multipleneoantigen vaccines against LUAD

Lung adenocarcinoma (LUAD) is classified as Non-Small-Cell Lung Cancer (NSCLC), one of the most malignantcancers due to its poor prognosis and fast progression. Its resistance to chemotherapy, immunosuppressive tumormicroenvironment (TME), and frequent immune evasion also impose enormous obstacles in treating LUAD. However,previous research found that these tumor cells resistant to chemotherapies commonly increase their production ofintracellular reactive oxygen species (ROS) due to high mitochondria activity. Antioxidants like glutathione (GSH)are also highly produced to maintain their redox homeostasis. Therefore, the depletion of antioxidants which can leadto intratumoral accumulation of ROS to cytotoxic levels, provides a new therapeutic target. Besides, the high levelof extracellular ROS also significantly contributes to the immunosuppressive characteristic of TME, which exhaustsactivated T cells. This work provides the framework for a novel combinational therapy based on a multiple-neoantigenvaccine to avoid immune evasion. Two types of nanoparticles further boost the vaccine’s efficacy against tumors, onedepleting intratumoral GSH and scavenging extracellular ROS. Anticipated results are shown from various aspects,hoping the innovative treatment can effectively improve the survival rate with few side effects.

Rosmarinic acid decreases viability, inhibits migration and modulates expression of apoptosis-related CASP8/CASP3/NLRP3 genes in human metastatic melanoma cells

Cutaneous melanoma is the most aggressive type of skin cancer; it is difficult to treat, and has been highlighted in recent years due to increasing numbers of cases worldwide. The use of antitumoral therapeutics for this neoplasm has been associated with severe side effects, low quality of life, and resistance. We aimed in this study to explore the effect of the phenolic compound rosmarinic acid (RA) on human metastatic melanoma cells. SK-MEL-28 melanoma cells were treated for 24 h with different concentrations of RA. In parallel, peripheral blood mononuclear cells (PBMCs) also were treated with RA under the same experimental conditions to verify the cytotoxic effect on non-tumoral cells. Then, we assessed cell viability and migration, levels of intracellular and extracellular reactive oxygen species (ROS), as well as nitric oxide (NOx), non-protein thiols (NPSH), and total thiol (PSH). Gene expression of the caspase 8, caspase 3 and NLRP3 inflammasome was evaluated by RT-qPCR. The enzymatic activity of the caspase 3 protein was assessed by a sensitive fluorescent assay. Fluorescence microscopy was employed to corroborate the effects of RA on melanoma cell viability, mitochondria transmembrane potential and apoptotic bodies formation. We found that RA potently reduces melanoma cell viability and migration after 24 h of treatment. On the other hand, it has no cytotoxic effect on non-tumoral cells. The fluorescence micrographics indicated that RA reduces transmembrane potential of mitochondria and induces apoptotic bodies formation. Moreover, RA significantly decreases intracellular and extracellular ROS levels, and increases the antioxidant defenders NPSH and PSH. A remarkable feature found in our study was that RA strongly upregulates the gene expression of the caspase 8 and caspase 3, and downregulates NLRP3 inflammasome expression. Similar to gene expression, RA greatly increases the enzymatic activity of caspase 3 protein. Taken together, we have shown for the first time that RA reduces cell viability and migration of human metastatic melanoma cells, in addition to modulates apoptosis-related gene expression. We suggest that RA may have the potential to be used in a therapeutic perspective, particularly for CM cell treatment.

A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen

AimsThe well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2’,7’-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methodsThe fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2’,7’-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findingsThe 2’,7’-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407–12769) vs. 1356 (range 470–2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. SignificanceBased on our pilot experimental data, the microplate-based 2’,7’-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.

The relationship between reactive oxygen species, DNA fragmentation, and sperm parameters in human sperm using simplified sucrose vitrification with or without triple antioxidant supplementation.

ObjectiveThis study examined whether the addition of triple antioxidants (3A)—10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid—in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa.MethodsWe analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated.ResultsThe cryopreserved spermatozoa had significantly reduced percentages of motility (p<0.05) and viability (p<0.05). Antioxidant supplementation non-significantly improved these parameters (p>0.05). No significant differences were found in sperm morphology between the fresh and frozen-thawed groups (p>0.05). After freezing, the extracellular ROS levels in the frozen-thawed groups were significantly higher (p<0.05) than in the fresh group. However, we did not find any differences in intracellular ROS parameters among these groups (p>0.05). The SDF was higher in the SuV and Vapor groups than in the fresh group, but without statistical significance (p=0.075 and p=0.077, respectively).ConclusionCryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.

Baicalein May Act as a Caloric Restriction Mimetic Candidate to Improve the Antioxidant Profile in a Natural Rodent Model of Aging.

Caloric restriction (CR) is the most effective intervention for extending the life span of vertebrate and invertebrate aging models. Calorie restriction mimetics (CRMs), which are synthetic or natural chemicals that mimic the biochemical, hormonal, and physiological consequences of calorie restriction, are being researched for antiaging benefits. Baicalein is a plant-derived polyphenol that has the potential of antioxidant, anti-inflammatory, and autophagy inducer. The objective of this study is to evaluate the antiaging, anti-inflammatory, and antioxidant role of Baicalein in erythrocyte membrane and plasma, and evaluate the efficacy of Baicalein to act as a CRM candidate. This study evaluates the effect of Baicalein on aging biomarkers in normal and aged rats. We study various pro- and antioxidant markers, erythrocyte membrane transporters, and eryptosis. Baicalein supplementation in male Wistar rats significantly alleviated pro-oxidant markers and improved antioxidant profile. Improvement was also observed in age-induced alterations in membrane transporters, and eryptosis. Based on the aforementioned observations we conclude that Baicalein has the potential to maintain extracellular reactive oxygen species levels and redox homeostasis during the aging process, an effect that is similar to CR. Thus, Baicalein may be a potent CRM candidate for antiaging interventions.

Antioxidant Power on Dermal Cells by Textiles Dyed with an Onion (Allium cepa L.) Skin Extract.

In this study, the phenol loading and antioxidant activity of wool yarn prepared with the aqueous extract of onion (Allium cepa L.) skin was enhanced by implementing the dyeing process with the use of alum as a mordant. Spectrophotometric and chromatographic methods were applied for the characterization of polyphenolic substances loaded on the wool yarn. The antioxidant/anti-inflammatory properties were evaluated by determining the level of intra- and extra-cellular reactive oxygen species (ROS) production in keratinocytes and dermal fibroblasts pre-treated with lipopolysaccharide put in contact with artificial sweat. An elevated dye uptake on wool was observed for the pre-mordanted sample, as demonstrated by high absorbance values in the UV-Visible spectral range. Chromatographic results showed that protocatechuic acid and its glucoside were the main phenolic acid released in artificial sweat by the wool yarns, while quercetin-4′-glucoside and its aglycone quercetin were more retained. The extract released from the textile immersed in artificial sweat showed a significant reducing effect on the intra-and extracellular ROS levels in the two cell lines considered. Cytofluorimetric analyses demonstrated that the selected mordant was safe at the concentration used in the dyeing procedure. Therefore, alum pre-mordanted textiles dyed with onion-skin extracts may represent an interesting tool against skin diseases.

Preliminary Data Monitoring In Vivo Sources of Reactive Oxygen Species Production in Human Obesity

Introduction Excessive reactive oxygen species (ROS) production is a prominent feature of obesity-related conditions including impairments in endothelial function. NADPH oxidases (Nox) and mitochondria are major sources of ROS production within the vasculature. However, studies involving direct measurement of in vivo ROS production are limited in humans. Thus, the relative contributions of Nox- and mitochondrial-derived ROS in human obesity remain to be fully characterized. The objective of this study was to determine the predominant sources of in vivo ROS production in the skeletal muscle microvasculature of individuals with obesity. We hypothesize that both Nox- and mitochondria-dependent ROS production are upregulated in human obesity. Methods Two sedentary men with Class II and above obesity (body mass index ≥ 35 kg/m2) and the Metabolic Syndrome were studied. Microdialysis was used for measurements of in vivo skeletal muscle ROS production. Microdialysis probes were perfused with saline containing Amplex Ultrared, horseradish peroxidase and superoxide dismutase to measure local hydrogen peroxide and superoxide concentrations. To assess ROS production from distinct sources, microdialysis probes were perfused with the following: apocynin (non-specific Nox inhibitor), MitoTEMPO (mitochondrial-ROS inhibitor), and GKT 137831 (specific Nox 4 inhibitor). Results The concentrations of hydrogen peroxide and superoxide decreased upon addition of apocynin to microdialysis probes (mean ± SEM; hydrogen peroxide: 1.18 ± 0.58 to 0.76 ± 0.01 μM; superoxide: 1.47 ± 0.46 to 1.10 ± 0.62 μM). MitoTEMPO perfusion elicited an increase in hydrogen peroxide (1.12 ± 0.76 to 1.51 ± 1.08 μM) and decrease in superoxide (1.47 ± 0.46 to 0.78 ± 0.10 μM). The combination of apocynin plus MitoTEMPO resulted in concentrations of hydrogen peroxide (0.22 ± 0.10 μM) and superoxide (0.23 ± 0.06 μM) that were 69.7% and 79.1% lower than apocynin alone. In addition, the hydrogen peroxide (0.56 ± 0.25 μM) and superoxide (0.43 ± 0.18 μM) concentrations with co-perfusion of apocynin and GKT were 26.3% and 60.9% lower than with apocynin alone. Conclusions Our preliminary data suggest that mitochondria and Nox together are significant sources of ROS production within the skeletal muscle microvasculature of obese individuals. The local inhibition of Nox- and mitochondrial-derived ROS resulted in marked decreases in extracellular ROS levels within the skeletal muscle microvasculature. Although the small sample size currently limits our interpretation of the results, this study is anticipated to become the first to report on distinct sources of in vivo ROS production in human obesity.

The extracellular reactive oxygen species levels in primary in vitro culture of human ovarian granulosa and cumulus cells

Abstract In the female reproductive tract, reactive oxygen species (ROS) may exert physiological and pathophysio-logical effects. Although ROS play an essential role as the signaling molecules, their excessive accumulation contributes to the pathogenesis of many reproductive processes. In the ovarian follicle, ROS affect multiple physiological processes, including oocyte maturation and fertilization. However, a lack of studies showing to which extend ovarian granulosa and cumulus cells can contribute to the development of oxidative stress within the ovarian follicle. In the presented research, the extracellular ROS accumulation level was investigated using GCs and CCs primary in vitro cultures. The obtained results demonstrated a steady decrease in extracellular ROS level during GCs primary culture. By contrast, ROS concentration in CCs conditioned medium increased gradually between the first and the seventh days of culture. The observed changes may reflect the proliferation status and metabolic activity of GCs and CCs during in vitro culture. Additionally, the elevated ROS level at respective points of time could occur as a consequence of culture in atmospheric oxygen. The distinct function and localization within the ovarian follicle may explain the differences between GCs and CCs oxygen metabolism. Running title: Reactive oxygen species in primary culture of human follicular cells

Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury.

Acidosis is among the least studied secondary injury mechanisms associated with neurotrauma. Acute decreases in brain pH correlate with poor long‐term outcome in patients with traumatic brain injury (TBI), however, the temporal dynamics and underlying mechanisms are unclear. As key drivers of neuroinflammation, we hypothesized that microglia directly regulate acidosis after TBI, and thereby, worsen neurological outcomes. Using a controlled cortical impact model in adult male mice we demonstrate that intracellular pH in microglia and extracellular pH surrounding the lesion site are significantly reduced for weeks after injury. Microglia proliferation and production of reactive oxygen species (ROS) were also increased during the first week, mirroring the increase in extracellular ROS levels seen around the lesion site. Microglia depletion by a colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622, markedly decreased extracellular acidosis, ROS production, and inflammation in the brain after injury. Mechanistically, we identified that the voltage‐gated proton channel Hv1 promotes oxidative burst activity and acid extrusion in microglia. Compared to wildtype controls, microglia lacking Hv1 showed reduced ability to generate ROS and extrude protons. Importantly, Hv1‐deficient mice exhibited reduced pathological acidosis and inflammation after TBI, leading to long‐term neuroprotection and functional recovery. Our data therefore establish the microglial Hv1 proton channel as an important link that integrates inflammation and acidosis within the injury microenvironment during head injury.

Staphylococcus aureus resists UVA at low irradiance but succumbs in the presence of TiO2 photocatalytic coatings

The aim of this study was to evaluate the bactericidal effect of reactive oxygen species (ROS) generated upon irradiation of photocatalytic TiO2 surface coatings using low levels of UVA and the consequent killing of Staphylococcus aureus. The role of intracellular enzymes catalase and superoxide dismutase in protecting the bacteria was investigated using mutant strains. Differences were observed in the intracellular oxidative stress response and viability of S. aureus upon exposure to UVA; these were found to be dependent on the level of irradiance and not the total UVA dose. The wild type bacteria were able to survive almost indefinitely in the absence of the coatings at low UVA irradiance (LI, 1 mW/cm2), whereas in the presence of TiO2 coatings, no viable bacteria were measurable after 24 h of exposure. At LI, the lethality of the photocatalytic effect due to the TiO2 surface coatings was correlated with high intracellular oxidative stress levels. The wild type strain was found to be more resistant to UVA at HI compared with an identical dose at LI in the presence of the TiO2 coatings. The UVA-irradiated titania operates by a “stealth” mechanism at low UVA irradiance, generating low levels of extracellular lethal ROS against which the bacteria are defenceless because the low light level fails to induce the oxidative stress defence mechanism of the bacteria. These results are encouraging for the deployment of antibacterial titania surface coatings wherever it is desirable to reduce the environmental bacterial burden under typical indoor lighting conditions.

Restoring Effects of Natural Anti-Oxidant Quercetin on Cellular Senescent Human Dermal Fibroblasts.

The oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the functional decline and disability that characterizes aging. The anti-oxidant flavonoid, quercetin, is a plant polyphenol that may be beneficial for retarding the aging process. We examined the restoring properties of quercetin on human dermal fibroblasts (HDFs). Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs. To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified. Silencing LPL increased the expression levels of senescence proteins such as p16INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs. Silencing of LPL and KCNE2 decreased the expression levels of anti-oxidant enzymes such as superoxide dismutase and catalase. Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment. Taken together, these results suggest that quercetin has restoring effect on the cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of anti-oxidant enzymes in aged HDFs.

Quantifying Extracellular ROS Levels in Individual Pancreatic Islets using an Optical Sensor and Microfluidic Device

The metabolism of pancreatic islet beta-cells is negatively regulated by reactive oxygen species (ROS), which is a toxic byproduct of the electron transport chain and excessive aerobic respiration. During oxidative stress it is evident that ROS levels increase and result in apoptotic signaling. Chronic oxidative stress is associated with beta-cell failure and type 2 diabetes. Oxidative stress increases expression of antioxidant enzymes as a protective mechanism that paradoxically reduces glucose stimulated insulin secretion. To remove ROS, the cell uses superoxide dismutase (SOD) an enzyme that catalyzes the conversion of superoxide into hydrogen peroxide and oxygen. The hydrogen peroxide is then converted into water and oxygen via glutathione peroxidase or catalase. Therefore, both hydrogen peroxide (H2O2) release and molecular oxygen (O2) consumption of islets can serve as proxies to measure conversion rates in the tissue. We have designed a microfluidic device to hold individual pancreatic islets separately in chambers optimized for live cell confocal imaging. This device puts islets very close to the planar slide and also collects islet effluent in a well-defined tail, thus offering us two positions to place a sensor: planar slide and solution. We will first modify the device by introducing a clear fluorescent sensor for oxygen directly beneath the islets. We are currently exploring RuII(bpy)3 [Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate as an optical sensor which has excitation and emission maxima at 450nm, and 600nm, respectively. The ruthenium compound will serve as a seed layer on the base of the device that will increase in fluorescence intensity when oxygen levels decrease. Furthermore, we are exploring putting the dye in solution and using other sensors such as palladium and platinum compounds that react to oxygen. This device will enable toxicity screening of living pancreatic islets and other types of cells.

Chasing stress signals – Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea

Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA.

Abstract 83: Modulation of Macrophage Function via Metabolism by Trypanosoma cruzi

Chagas heart disease is an inflammatory cardiomyopathy which presents with mononuclear infiltrates in the interstitium and myocardial fibrosis in the chronic phase. Incomplete clearance by macrophages of the etiologic agent, Trypanosoma cruzi , is a significant cause of chronic disease development in approximately 30% of those serologically positive for the blood-borne parasite. The differential metabolic status, anaerobic glycolysis and mitochondria-dependent oxidative phosphorylation, are respectively associated with pro-inflammatory (M1) and anti-inflammatory (M2) functional activation of macrophages. Reactive oxygen species (ROS) have been shown to be an intracellular signal for glycolysis while peroxisome proliferator-activated receptors (PPARs) that enhance fatty acid oxidation provide transcription control of macrophage functional state. In our studies using diverse T. cruzi isolates, we showed that SylvioX10 (virulent), but not TCC (non-virulent), isolates are able to differentially control extracellular and intracellular ROS levels in macrophages. We found in macrophages infected with SylvioX10, the nuclear expression of PPAR-α was increased by 18 hours post-infection, and mitochondrial metabolic activity was similar to that of not-infected and M2 controls; which indicates anti-inflammatory function of macrophages, and therefore prohibiting T. cruzi clearance. In our ongoing studies, we are examining the impact of PPAR-α inhibitors in modulating the metabolic gene expression profile, functional phenotype and parasite survival in macrophages. Our data will provide the first indication that host macrophages have deficient pro-inflammatory capacity due to sub-optimal glucose oxidation, and enhancing the metabolism that supports T. cruzi clearance will provide a valuable basis for a strategy to arrest Chagas disease progression.

Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin

ObjectiveThe first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin.Approach and ResultsTo address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).ConclusionsAortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure.

Background and AimsAcute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF.MethodsPrimary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H2O2 were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.ResultsFibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.ConclusionsReg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.

Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization

The toxicity of TiO2 engineered nanoparticles (NPs) to the marine microalga Nitzschia closterium was investigated by examining growth inhibition, oxidative stress and uptake. The results indicated that the toxicity of TiO2 particles to algal cells significantly increased with decreasing nominal particle size, which was evidenced by the 96 EC50 values of 88.78, 118.80 and 179.05mg/L for 21nm, 60nm and 400nm TiO2 particles, respectively. The growth rate was significantly inhibited when the alga was exposed to 5mg/L TiO2 NPs (21nm). Measurements of antioxidant enzyme activities showed that superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were first induced and subsequently inhibited following exposure to 5mg/L TiO2 NPs. The depletion of antioxidant enzymes with a concomitant increase in malondialdehyde (MDA) levels and reactive oxygen species (ROS) posed a hazard to membrane integrity. A combination of flow cytometry analysis, transmission electron microscopy and Ti content measurement indicated that TiO2 NPs were internalized in N. closterium cells. The level of extracellular ROS, which was induced by TiO2 NPs under visible light, was negligible when compared with the intracellular ROS level (accounting for less than 6.0% of the total ROS level). These findings suggest that elevated TiO2 nanotoxicity in marine environments is related to increased ROS levels caused by internalization of TiO2 NPs.

Oviductal Microsomal Epoxide Hydrolase (EPHX1) Reduces Reactive Oxygen Species (ROS) Level and Enhances Preimplantation Mouse Embryo Development1

Somatic cell-embryo coculture enhances embryo development in vitro by producing embryotrophic factor(s) and/or removing harmful substances from the culture environment. Yet, the underlying molecular mechanisms on how somatic cells remove the toxicants from the culture medium remain largely unknown. By using suppression subtractive hybridization, we identified a number of mouse oviductal genes that were up-regulated when developing preimplantation embryos were present in the oviduct. Epoxide hydrolase 1, microsomal (Ephx1 previously known as mEH) was one of these genes. EPHX1 detoxifies genotoxic compounds and participates in the removal of reactive oxygen species (ROS). The transcript of Ephx1 increases in the oviductal epithelium at the estrus stage and in Day 3 of pregnancy as well as in the uterus of ovariectomized mice injected with estrogen or progesterone. Human oviductal epithelial cells OE-E6/E7 express EPHX1 and improve mouse embryo development in vitro. Addition of an EPHX1 inhibitor, cyclohexene oxide (CHO) or 1,1,1-trichloropropene 2,3-oxide (TCPO), to the culture medium increased intracellular and extracellular ROS levels of OE-E6/E7 cells and suppressed the beneficial effect of the cells on embryo development; CHO and TCPO at these concentrations had no adverse effect on OE-E6/E7 growth and embryo development in vitro. Taken together, EPHX1 in oviductal cells may enhance the development of cocultured embryos by protecting them from oxidative stress. Our result supports the notion that somatic cell coculture may enhance embryo development via removal of deleterious substances in the culture medium.

Analysis of chemokines and reactive oxygen species formation by rat and human neutrophils induced by microcystin-LA, -YR and -LR

Microcystins (MC), a family of heptapeptide toxins produced by some genera of Cyanobacteria, have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The present study reports the effects of MC-LA, MC-YR and MC-LR (1 and 1000 nM) on human and rat neutrophils functions in vitro. Cell viability, DNA fragmentation, mitochondrial membrane depolarization and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MC increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2 αβ (CINC-2 αβ) and extracellular ROS levels in human and rat neutrophils. Apart from neutrophil presence during the inflammatory process of MC-induced injury, our results suggest that hepatic neutrophil accumulation is further increased by MC-induced neutrophil-derived chemokine.