Cellular Oxidation Research Articles

Malignant hyperthermia‐associated mutation of RyR1 induces mitochondrial Ca2+ overload in the cardiomyocytes

Although human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations mainly show skeletal muscle disorders including malignant hyperthermia (MH), sudden cardiac death (SCD) in these MH families in the conscious condition without anesthesia are also frequently found in the clinical case studies. However, the molecular mechanism underlying cardiac phenotypes in human MH family is completely unknown. Our group previously reported that a low level of RyR1 is expressed in the mitochondria, but not in the sarcoplasmic reticulum (SR) in the rat and mouse hearts, which serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also showed, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state. Moreover, ex‐vivo YS heart developed significantly higher number of multiple ventricular extrasystoles by β‐adrenergic stimulation compared those observed in the wild‐type (WT) hearts. Therefore, our main hypothesis is that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handing in cardiomyocyte. Using isolated mitochondria loaded with Ca2+ or membrane‐potential sensitive dyes under the confocal microscope, we found that YS mitochondria have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential. In addition, YS cardiomyocytes exhibit higher basal cytosolic Ca2+ concentration as well as slower cytosolic Ca2+ clearance compared to WT. Finally, pretreatment of RyR1 blocker dantrolene cancelled these changes in YS mitochondria and cardiomyocyes and normalized their Ca2+ handing profiles similar to those in WT. In summary, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, which may alter cytosolic Ca2+ handling, induce cellular oxidation, and increase the arrhythmogenic events in MH.Support or Funding Information16SDG27260248 AHA Scientist Development Grant

Obesity, bariatric surgery and oxidative stress.

Obesity refers to the accumulation of fatty tissues and it favors the occurrence of oxidative stress. Alternatives that can contribute to body weight reduction have been investigated in order to reduce the production of reactive oxygen species responsible for tissue damage. The aim of the current study was to assess whether the oxidant and antioxidant markers of obese women before and after bariatric surgery were able to reduce oxidative damage. We have assessed 16 morbidly obese women five days before and 180 days after the surgery. The control group comprised 16 non-obese women. Levels of thiobarbituric acid-reactive substances, carbonylated proteins, reduced glutathione and ascorbic acid were assessed in the patients' plasma. Levels of lipid peroxidation and protein carbonylation in the pre-surgical obese women were higher than those of the controls and post-surgical obese women. Levels of reduced glutathione in the pre-surgical obese women were high compared to the controls, and declined after surgery. Levels of ascorbic acid fell in the pre--surgical obese women compared to the control and post-surgical obese women. Body weight influences the production of reactive oxygen species. Bariatric surgery, combined with weight loss and vitamin supplementation, reduces cellular oxidation, thus reducing tissue damage.

Oxidized and degraded mitochondrial polynucleotides (DeMPs), especially RNA, are potent immunogenic regulators in primary mouse macrophages

Certain mitochondrial components can act as damage-associated molecular patterns (DAMPs) or danger signals, triggering a proinflammatory response in target (usually immune) cells. We previously reported the selective degradation of mitochondrial DNA and RNA in response to cellular oxidative stress, and the immunogenic effect of this DNA in primary mouse astrocytes. Here, we extend these studies to assess the immunogenic role of both mitochondrial DNA and RNA isolated from hydrogen peroxide (HP) treated HA1 cells (designated “DeMPs” for degraded mitochondrial polynucleotides) using mouse bone marrow derived macrophages (BMDMs), a conventional immune cell type. DeMPs and control mitochondrial DNA (cont mtDNA) and RNA (cont mtRNA) were transfected into BMDMs and cell-free media analyzed for the presence of proinflammatory cytokines (IL-6, MCP-1, and TNFα) and Type I interferon (IFN-α and IFN-β). Cont mtDNA induced IL-6 and MCP-1 production, and this effect was even greater with DeMP DNA. A similar response was observed for Type I interferons. An even stronger induction of proinflammatory cytokine and type 1 interferons was observed for cont mtRNA. However, contrary to DeMP DNA, DeMP RNA attenuated rather than potentiated the cont mtRNA cytokine inductions. This attenuation effect was not accompanied by an IL-10 or TGFβ anti-inflammatory response. All DeMP effects were observed at multiple oxidant concentrations. Finally, DeMP production and immunogenicity overlaps with cellular adaptive response and so may contribute to cellular oxidant protection. These results provide new insight into the immunogenicity of mitochondrial polynucleotides, and identify new roles and selective consequences of cellular oxidation.

Effects of Combinational Procyanidins in Grape Seed Extract and Exercise on the Levels of Glucose and Blood Lipid Profile

The oxygenation process is essential for life and cellular activity, and therefore for physical exercise. During physical exercise an oxidative stress situation is produced, which can result in cellular oxidation and alterations in the immune system, causing cells to age prematurely. To prevent this, it is recommended to use high concentrations of antioxidants to minimize this process. The phenolic compounds of the flavonoid family, among which are the procyanidins, have shown neuroprotective effects, and cardioprotective effects. The cardiovascular protection and antiatherogenic effects of procyanidins have been attributed to the antioxidant properties of these phenolic compounds derived from its chemical structure. Different studies have shown the cardioprotective effects of chronic ingestion of procyanidins on models of hyperglycemia and dyslipidemia. This study aims to present the properties of these compounds and their protective effects on the cardiovascular system, setting markers of the primary action of procyanidins from grape seed extract (obtained by discarding the pulp and preserving the seeds) in plasma. A secondary aim of this study was to provide information on the molecular mechanisms by which these substances exert their cardioprotective effect and implement these into a recommended dietary regime. This would be used in appropriate doses and complement the protective and regulatory effect of exercise.

FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2.

Obesity-linked metabolic disease is mechanistically associated with the accumulation of proinflammatory macrophages in adipose tissue, leading to increased reactive oxygen species (ROS) production and chronic low-grade inflammation. Previous work has demonstrated that deletion of the adipocyte fatty acid-binding protein (FABP4/aP2) uncouples obesity from inflammation via upregulation of the uncoupling protein 2 (UCP2). Here, we demonstrate that ablation of FABP4/aP2 regulates systemic redox capacity and reduces cellular protein sulfhydryl oxidation and, in particular, oxidation of mitochondrial protein cysteine residues. Coincident with the loss of FABP4/aP2 is the upregulation of the antioxidants superoxide dismutase (SOD2), catalase, methionine sulfoxide reductase A, and the 20S proteasome subunits PSMB5 and αβ. Reduced mitochondrial protein oxidation in FABP4/aP2-/- macrophages attenuates the mitochondrial unfolded-protein response (mtUPR) as measured by expression of heat shock protein 60, Clp protease, and Lon peptidase 1. Consistent with a diminished mtUPR, FABP4/aP2-/- macrophages exhibit reduced expression of cleaved caspase-1 and NLRP3. Secretion of interleukin 1β (IL-1β), in response to inflammasome activation, is ablated in FABP4/aP2-/- macrophages, as well as in FABP4/aP2 inhibitor-treated cells, but partially rescued in FABP4/aP2-null macrophages when UCP2 is silenced. Collectively, these data offer a novel pathway whereby FABP4/aP2 regulates macrophage redox signaling and inflammasome activation via control of UCP2 expression.

A reversible carnitine palmitoyltransferase I (CPT1) inhibitor offsets chronic lymphocytic leukemia cell proliferation

Crucial for CLL development and progression are iterative cycles of cell re-activation and proliferation in lymphoid tissues. Since cellular fatty acid (FA) import and oxidation (FAO) were recently reported to be upregulated in CLL, we explored in-vitro effects of ST1326, a reversible inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), on leukemic cells subject to activating microenvironment-mimicking stimuli. ST1326 induced dose-dependent mitochondrial dysfunction and cell death, which were remarkably higher in activated/proliferating than quiescent CLL cells. Drug sensitivity was observed irrespective of the presence of TP53 alterations or chromosomal abnormalities. ST1326 cytotoxicity was associated with decreased levels of intracellular acetylCoA and phosphorylated STAT3, known to favor CLL cell proliferation and upregulate antiapoptotic Bcl-2 family members. Indeed, rising of Mcl-1 and Bcl-xl expression in response to microenvironment stimulation was impaired by ST1326. Drug combination experiments with the BH3-mimetic ABT-199/Venetoclax, whose effects are counteracted by Mcl-1/Bcl-xl and cell proliferation, showed strong ST1326-mediated potentiation of ABT-199 cytotoxicity in activated/proliferating CLL cells. The data indicate that CLL cells turning to an activated/proliferating state become more dependent on FAO and more sensitive to FAO-antagonists, and pave the way for ST1326 as an adjuvant tool in anti-CLL drug-combination regimens with drugs that loose efficacy on proliferating leukemic cells.

Atherogenicity of Monosaccharides, Disaccharides and Artificial Sweeteners in the Lipid-Laden Macrophage Model System: Cell Culture and Mice Studies

Background: Glucose is known to enhance macrophage foam cell formation and atherosclerosis development. However, the role of other monosaccharides, disaccharides or artificial sweeteners in macrophage atherogenicity remains unclear.Objective: We thus compared their effects on oxidative status, cholesterol, and triglycerides accumulation which regulate foam cell formation.Results: Supplementation of C57/BL6 mice for four weeks with sweeteners revealed that glucose, fructose, mannose, lactose or sucrose significantly increased hepatic lipid peroxidation and cholesterol accumulation, as well as mouse peritoneal macrophages (MPM) generation of ROS and lipid content. Supplementation with artificial sweeteners showed no significant pro-oxidative/atherogenic effects in the mice liver or aorta. Yet, cyclamate and sucralose significantly increased MPM ROS generation, and all artificial sweeteners increased MPM cholesterol content. In cultured J774A.1 macrophage cell line, glucose demonstrated the most pro-oxidative/atherogenic effects and significantly increased reactive oxygen species (ROS) generation (by 80%), cellular protein oxidation (by 119%), the accumulation of cholesterol and triglycerides (by 65% and 51%, respectively), and the macrophage phagocytosis capacity (by 177%). Mechanistically, glucose attenuated HDL-mediated cholesterol efflux from macrophages (by 17%) and enhanced their triglyceride biosynthesis rate (by 51%). Although to a lesser extent, mannose or cyclamate demonstrated pro-oxidative/ atherogenic effects and significantly increased cellular ROS generation, cholesterol content, triglyceride content and macrophage phagocytosis capacity.Conclusions: Taking together, the above results indicate the key pro-oxidative/atherogenic role for glucose as compared to other monosaccharides, as well as disaccharides or artificial sweeteners. Finally, the detrimental pro-atherogenic effects on macrophage foam cell formation of mannose or cyclamate, and to a lesser extent fructose, aspartame and saccharin are now clearly shown.

Foliar applications of alpha-tocopherol improves the composition of fresh pods of Vigna radiata subjected to water deficiency

Alpha-tocopherol (α-Toc), so-called vitamin E, is a low molecular weight lipophilic antioxidant that generally protects plants from stress-induced cellular oxidation. It is well known that exogenously applied α-Toc is effective in improving plant growth and developmental processes under adverse environmental conditions. The current study was performed to determine the best concentration of α-Toc [0 (no spray), 100, 200, and 300 mg L-1] that could improve the biological yield and chemical constituents of fresh green pods of mung bean (Vigna radiata) and hence their quality under varying water regimes. Foliar spray of α-Toc significantly improved total soluble proteins, chlorophyll a and b, total soluble sugars, proline, phenolics, total free amino acids, nonreducing sugars, and activities of SOD, POD, and CAT under water stress conditions. However, no prominent change was observed in reducing sugars and biological yield due to externally applied α-Toc in either mung bean cultivar subjected to either water regime. Both mung bean cultivars (Cyclone 7008 and Cyclone 8009) showed similar behavior in chlorophyll a and b, while cv. Cyclone 7008 was superior with respect to the concentrations of total soluble sugars and total free amino acids, whereas Cyclone 8009 was better in activities of CAT, SOD, and POD. Overall, externally applied 200 and 300 mg L-1 α-Toc considerably enhanced the activities of antioxidant enzymes (SOD, POD and CAT), chlorophyll a and b, proline, and total phenolic contents in both mung bean cultivars.

Cellular Hydration and Oxidation As Phenotype Modifiers in Sickle Cell Anemia

Although Sickle Cell Anemia (SCA) is caused by a single nucleotide mutation in the beta globin gene, there is broad phenotypic variability in affected individuals. It would be highly advantageous to be able to predict which SCA patients are most likely to suffer severe complications and which are likely to benefit from specific treatments. Since two of the major pathologic mechanisms in SCA are erythrocyte dehydration and chronic inflammation, alterations in the expression and/or function of proteins affecting these processes may be responsible for heterogeneity in SCA phenotype. We have developed a 23-gene Next-Generation sequencing panel to identify variants in genes involved in erythrocyte hydration and in reactive oxygen species (ROS) generation and reduction in patients with SCA. We have collected blood samples from an initial cohort of 18 SCA patients with severe phenotype, defined by history of stroke or abnormal transcranial Doppler velocities, and 13 SCA patients with mild phenotype for DNA preparation and for specialized testing to evaluate erythrocyte hydration status and ROS generation.Advia Automated Cell Counter results provided the clearest indications of erythrocyte dehydration in patient blood samples. The ability to examine reticulocyte parameters made it possible to evaluate hydration regardless of the frequency of transfusion. Reticulocyte CHCM, MCV, and percent hyperdense cells were significantly different between the two groups and indicative of a greater degree of dehydration in the severe phenotype group.Osmoscans (using a LoRRca ektacytometer) demonstrated a highly significant left shift indicative of erythrocyte dehydration in both mild and severe groups relative to controls (O min and O hyper values significantly decreased). Both phenotypic groups also demonstrated a significantly lower EI max in relation to controls, indicating decreased deformability of sickle erythrocytes. Deformability scans revealed significant increases in SS ½ and decreases in EI max in both groups in relation to the controls. There were significant differences between mild and severe phenotype groups in osmoscan O min and O hyper values and in deformability assay SS ½ and EI max values. The severe group had values closer to the normal values, most likely due to the contribution of transfused blood in the severe phenotype group. There were no significant differences between the mild and severe SCA phenotype groups in intracellular cation levels (K+ and Na+) measured by flame emission spectroscopy, although both groups had significantly higher intracellular Na+ compared to normal controls.Erythrocyte ROS detection was performed using a flow cytometry DCFDA assay. Both mild and severe phenotype groups demonstrated a highly significant increase in ROS compared to controls. However there was not a significant difference in erythrocyte ROS between the mild and severe phenotype groups.DNA from patient blood samples has been sequenced using the Haloplex target enrichment system and Illumina high-throughput sequencing. We analyzed 23 genes that are likely candidates to be involved in erythrocyte hydration (ABCB6, ABCG5, ABCG8, AQP1, AQP3, ATP2B4, KCNN4, PIEZO1, RHAG, SLC9A1, SLC12A4, SLC12A6, SLC12A7, SLC2A1, SLC4A1, STOM, TRPC6, XK) or in ROS production or reduction (G6PD, NOX1, CYBB, NOX4, NOX5). At least 19 variants classified as possibly damaging have been detected in 10 of the genes. Future study will be directed toward exploring the phenotypic implications of identified variants.Results of this study should further our understanding of the pathogenesis of SCD and help identify biomarkers of disease severity which may ultimately help guide clinical management for individual patients. They may also suggest novel targets for development of new therapies for SCD based on the modulation of erythrocyte hydration and inflammation. DisclosuresQuinn:Amgen: Research Funding; Eli Lilly: Research Funding; Silver Lake Research Corporation: Consultancy. Joiner:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Lenalidomide Inhibits Thioredoxin Reductase (TrxR) in Multiple Myeloma (MM) Cells but Direct Inhibition of Trxr and Thioredoxin (Trx) Can Bypass Requirement of Cereblon (CRBN)

IntroductionA unique biological characteristic of MM plasma cells (PCs) is the substantial production of immunoglobulin proteins. This process requires disulfide bond formations that in turn generate large amounts of H2O2. The Trx-TrxR reductase system exists to combat this oxidative stress; however, cytotoxicity can occur when the cells' antioxidative capacity is overwhelmed. Recently, we demonstrated that IMiDs inhibit peroxidase-mediated decomposition of H2O2, and that lenalidomide sensitivity in MM correlates with the degree of antioxidative capacity (Sebastian et al. 2015 ASH). In this study we sought to better understand this inhibitory process.MethodsWe used various human MM cell lines with different sensitivity to lenalidomide. Cell viability was assessed by the MTT assay. Antioxidative capacity was measured via the total cellular oxidation of reduced flavin adenine dinucleotide (FADH2) and reduced nicotinamide adenine dinucleotide (NAD(P)H) after H2O2treatment. A CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and lentivirus system was used to over-express CRBN in MM cells.ResultsWe found that IMiDs inhibit TrxR mediated intracellular H2O2 decomposition in a CRBN dependent fashion. Unlike lenalidomide, the direct TrxR inhibitor auranofin was very effective in inducing cell death in MM cell lines, in a CRBN independent fashion. By using MM.1R isogeneic cells with and without CRBN, and the CRBN-overexpressing OCIMY-5 cell line (transfected with wt-CRBN) we showed that auranofin treatment caused cytotoxicity irrespective of CRBN level of expression, while lenalidomide was only effective in CRBN expressing cells. Clinically achievable concentrations of auranofin treatment induced cell signals similar to lenalidomide, including degradation of IKZF1, IKZF3. As with lenalidomide, the sensitivity of CRBN expressing cell lines to auranofin correlated with their basal antioxidative capacity (MM.1S cells are sensitive and have with lower anti-oxidative capacity while RPMI-8226 is resistant due to its high anti-oxidative capacity).We also found that targeting Trx (downstream effector of TrxR) with the direct inhibitor PX12 overcame this cellular anti-oxidative capacity and inhibited cell proliferation in both RPMI-8226 and MM.1S in a CRBN independent fashion. Combination of auranofin with bortezomib showed a synergistic effect, but only in cells with lower anti-oxidative capacity. In contrast, PX-12, showed a synergistic effect with bortezomib in all analyzed MM cells lines, irrespective of their anti-oxidative capacity and CRBN expression.ConclusionsIMiDs inhibit TrxR-mediated intracellular H2O2decomposition in CRBN expressing MM. While the direct TrxR inhibitor, auranofin, bypassed the CRBN requirement for inducing cytotoxicity in MM, its cytotoxic effect could be overcome by the cells antioxidative capacity. Direct Trx inhibition (such as PX-12) seems like a potent strategy for MM therapy to overcome cellular CRBN requirements and higher anti-oxidative capacity cells. DisclosuresBergsagel:Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Stewart:celgene: Consultancy. Fonseca:Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Janssen: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Janssen: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; BMS: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Ecotoxicological assessment of dewatered drinking water treatment residue for environmental recycling

ABSTRACTThe beneficial recycle of drinking water treatment residue (DWTR) in environmental remediation has been demonstrated in many reports. However, the lack of information concerning the potential toxicity of dewatered DWTR hinders its widespread use. The present study examined the ecotoxicity of dewatered aluminum (Al) and iron (Fe) DWTR leachates to a green alga, Chlorella vulgaris. Data from the variations of cell density and chlorophyll a content suggested that algal growth in DWTR leachates was inhibited. The algal cellular oxidation stress was initially induced but completely eliminated within 72 h by antioxidant enzymes. The expression of three photosynthesis-related algae genes (psaB, psbC, and rbcL) also temporarily decreased (within 72 h). Moreover, the algal cells showed intact cytomembranes after exposure to DWTR leachates. Further investigation confirmed that inhibition of algal growth was due to DWTR-induced phosphorus (P) deficiency in growth medium, rather than potentially toxic contaminants (e.g. copper and Al) contained in DWTR. Interestingly, the leachates could potentially promote algal growth via increasing the supply of new components (e.g. calcium, kalium, magnesium, and ammonia nitrogen) from DWTR. In summary, based on the algae toxicity test, the dewatered Fe/Al DWTR was nontoxic and its environment recycling does not represent an ecotoxicological risk to algae.

378 - Overexpression of Glutaredoxin Averts F-Actin Loss in Spines of Primary Cortical Neurons Derived from Mouse Model of Alzheimer's Disease

Oxidative stress is known to be one of the major contributors towards the pathogenesis of Alzheimer’s disease (AD) potentially leading to amyloid plaques, neurofibrillary tangles and loss of dendritic spine morphology. As a marker for ongoing oxidative stress, we observed increased levels of reactive oxygen species (ROS) in primary cortical neurons from APPSwe/PS1ΔE9 (APP/PS1) transgenic mouse model of AD, compared to wild type littermates. Thiol oxidation and protein glutathionylation are important targets during oxidative stress, resulting in perturbation of cellular thiol homeostasis. Glutaredoxins and other thiol disuflide oxidoreductases are important enzymes involved in reversing protein thiol oxidation in the brain. We hypothesized that overexpressing Grx1 (glutaredoxin) may partially reverse the adverse effects of oxidative stress in AD neurons. We overexpressed human glutaredoxin using AAV6 virus and confirmed overexpression of glutaredoxin using biochemical and immunostaining methods. Actin, an important neuronal cytoskeletal filament, is important for maintenance of spine morphology and function. It is also a target of cellular oxidation undergoing gluathionylation, leading to its conversion from the functional fibrillar form (F-actin) to the globular (G-actin) form. Other studies from our laboratory have shown increased actin oxidation in early stages of AD-related pathology leading to synaptic dysfunction using a combination of cellular, in vivo and post-mortem human brain samples. We also observed loss of F-actin in APP/PS1 neurons compared to their wild-type littermates, as indicated by reduced phalloidin staining. Importantly, overexpression of Grx1 reversed this F-actin loss, and preserved the F-actin microarchitecture in spines. Our results suggest that thiol oxidation events, such as protein glutathionylation including that of actin, play an important role in the synaptic structural and functional deficits seen in AD.

Panax ginseng Fraction F3 Extracted by Supercritical Carbon Dioxide Protects against Oxidative Stress in ARPE-19 Cells.

In our previous work, the ethanolic extract of Panax ginseng C. A. Meyer was successively partitioned using supercritical carbon dioxide at pressures in series to yield residue (R), F1, F2, and F3 fractions. Among them, F3 contained the highest deglycosylated ginsenosides and exerted the strongest antioxidant and anti-inflammatory activities. The aim of this study was to investigate the protective effects of P. ginseng fractions against cellular oxidative stress induced by hydrogen peroxide (H2O2). Viability of adult retinal pigment epithelium-19 (ARPE-19) cells was examined after treatments of different concentrations of fractions followed by exposure to H2O2. Oxidative levels (malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), and reactive oxygen species (ROS)) and levels of activity of antioxidant enzymes were assessed. Results showed that F3 could dose-dependently protected ARPE-19 cells against oxidative injury induced by H2O2. F3 at a level of 1 mg/mL could restore the cell death induced by H2O2 of up to 60% and could alleviate the increase in cellular oxidation (MDA, 8-OHdG, and ROS) induced by H2O2. Moreover, F3 could restore the activities of antioxidant enzymes suppressed by H2O2. In conclusion, F3 obtained using supercritical carbon dioxide fractionation could significantly increase the antioxidant capacity of P. ginseng extract. The antioxidant capacity was highly correlated with the concentration of F3.

Insights into the fluoride-resistant regulation mechanism of Acidithiobacillus ferrooxidans ATCC 23270 based on whole genome microarrays.

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S(0) substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity.

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.

Linking genotoxicity and cytotoxicity with membrane fluidity: A comparative study in ovarian cancer cell lines following exposure to auranofin

Auranofin, an organogold compound classified as an anti-rheumatic agent is under phase 2 clinical trials for re-purposing to treat recurrent epithelial ovarian cancer. We have reported earlier that Breast cancer 1, early onset (BRCA1) mutant ovarian cancer cells exhibit increased sensitivity to auranofin. BRCA1 is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from DNA repair capability of cancer cells, membrane fluidity is also implicated in modulating resistance to chemotherapeutics. We report here that membrane fluidity influences the sensitivity of ovarian cancer cell lines, OVCAR5 and IGROV1, to auranofin. Electron spin resonance (ESR) analysis revealed a more fluidized membrane in IGROV1 compared to OVCAR5. Interestingly, IGROV1 cells were more sensitive to auranofin induced cytotoxicity than OVCAR5. In comparison to OVCAR5, IGROV1 cells also exhibited an increased number of DNA double strand breaks (DSBs) upon auranofin treatment as assessed by 53BP1 immunostaining. Furthermore, correlation analysis demonstrated a strong positive correlation (r=0.856) between membrane fluidity and auranofin sensitivity in these cell lines. Auranofin-treated IGROV1 cells also exhibited increased cellular oxidation and apoptosis. Anti-oxidant, N-acetyl cysteine (NAC) inhibited the cellular oxidation and apoptosis in auranofin-treated ovarian cancer cells suggesting reactive oxygen species (ROS) mediate the anti-cancer properties of auranofin. Overall, our study suggests that auranofin mediates its cytotoxicity via ROS production in ovarian cancer cells which correlates positively with membrane fluidity.

포도주 부산물의 총 플라보노이드와 총 페놀 함량 및 항산화 효과

포도주 가공 중에 발생하는 포도주 부산물의 항산화 활성 검토를 위해 포도주 부산물을 유기용매로 추출 및 분획하여 총 플라보노이드 및 총 페놀 함량을 측정하고 포도주 부산물의 항산화 효과를 분석하였다. 포도주 부산물의 분획 및 추출물의 총 플라보노이드의 양을 측정한 결과 MeOH 추출물은 A+M 추출물보다 높은 함량의 총 플라보노이드를 함유하는 것으로 나타났고 분획물들 중에서는 85% aq. MeOH 분획물이 105.1±0.93 mg/g으로 가장 높았으며 총 페놀 함량은 MeOH 추출물과 n-BuOH 분획물에서 높은 함량을 나타내었다. DPPH를 통한 라디칼 소거활성능을 측정한 결과, 0.5 mg/ml 첨가농도에서 MeOH 추출물은 74%의 소거율을 나타내었으며 분획물들 중에서는 n-BuOH 분획물이 72%의 소거활성을 나타내었다. 또한 ABTS+를 통한 라디칼 소거활성능을 측정한 결과, 0.5 mg/ml 첨가농도에서 85% aq. MeOH 및 n-BuOH 분획물들은 각각 90% 및 92% 소거활성을 나타내어 동일한 BHT 농도에서 92%인 것과 비교해 보았을 경우 합성항산화제만큼 높은 항산화능을 나타내었다. 세포내 활성 산소종 소거 효과를 나타내는 ROS 실험을 통해 0.1 mg/ml 첨가농도에서 MeOH 추출물과 n-BuOH 분획물은 각각 64%와 60%의 소거 활성을 나타었다. 이상의 결과를 종합해 보면 포도 및 포도씨와 비교했을 때 포도주 부산물의 경우 총 플라보노이드 및 총 페놀 함유량은 다소 낮았지만 항산화 효과도 지속되는 것으로 여겨지므로 향후 포도주 부산물의 응용이 기대되어진다. We investigated the flavonoid and phenol contents and antioxidant effect of wine by-product extract. Antioxidant effects were measured with 1,1-diphenyl-2-picryhydrazyl (DPPH) and 2.2'-azino-bis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS+) assays. Cellular reactive oxygen species (ROS) were measured with the dichlorofluorescein-diacetate (DCFH-DA) assay. The flavonoid and phenol contents of the methanol (MeOH) extract were greater than those of the acetone+methylene chloride (A+M) extract. Among fractions, the 85% aqueous methanol (85% aq. MeOH) fraction contained the highest flavonoid contents, while the n-BuOH fraction had more phenol contents. In the DPPH and ABTS assays, the MeOH extract showed a scavenging effect greater than that of the A+M extract (p<0.05). The n-BuOH fraction (0.5 mg/ml concentrations) showed scavenging effects of 72% and 92%, respectively, in the DPPH and ABTS assays (p<0.05). However, the 85% aq. MeOH fraction showed a 90% scavenging effect in the DPPH assay only. In 120 min ROS production assay, all tested fractions dose-dependently decreased cellular ROS production induced by H₂O₂ in comparison with that produced by exposure to the extract-free control. The MeOH extract showed a higher sinhibitory effect on cellular ROS producing than that of the A+M extract at all concentrations tested. Treatment with the n-BuOH fraction (0.1 mg/ml concentrations) inhibited cellular ROS production by 60%. These results indicate that the n-BuOH fraction of wine by-product extract inhibited cellular oxidation and may contain valuable bioactive compounds such as flavonoids and phenols.

C2C12 골격근 세포에서 FAT/CD36 발현 조절에 있어 Insulin-like growth factor-I이 미치는 영향

본 연구에서는 C2C12 근육 세포의 분화 과정에 있어 IGF-I이 지방산의 수송을 담당하는 지방산 수송체인 FAT/CD36의 mRNA 및 단백질 발현에 미치는 영향에 대해 알아보았다. 그 결과 근육세포의 분화 과정에 있어 FAT/CD36의 단백질과 mRNA 발현이 분화 시간 의존적으로 유의하게 증가하였으며, IGF-I의 처리에 의해서도 유의하게 조절되었음을 알 수 있었다. 이는 IGF-I이 골격근 세포의 성장 및 분화를 촉진하여 근육 관련 유전자들의 발현을 조절하는 기능뿐만 아니라, 골격근의 주요 에너지원으로 사용되는 지방산의 수송을 담당하는 FAT/CD36의 발현에도 영향을 미친다는 것으로 해석할 수 있겠다. 향후 IGF-I이 골격근 세포에서 FAT/CD36의 발현에 영향을 미침으로써 골격근의 지방산 흡수와 산화율을 조절하는지, 그에 따라 지방대사에 어떠한 영향을 미치는지에 대한 연구가 필요할 것이며, 이와 관련된 신호전달 체계 및 기전에 대한 연구도 진행 되어야 할 것이다. Fatty acid transporters are key mediators of skeletal muscle lipid metabolism. Several protein groups have been implicated in cellular long-chain fatty acid uptake or oxidation, including fatty acid transporter proteins (FATPs), the plasma membrane fatty acid-binding protein (FABPpm), and the fatty acid translocase (FAT/CD36). FAT/CD36 is highly expressed in skeletal muscle and known to be regulated by various factors such as exercise and hormones. Insulin-like growth factor-I (IGF-I) is a well-known regulator of skeletal muscle cells. However, it has not been studied whether there is any interaction between IGF-I and FAT/CD36 in skeletal muscle cells. In this study, the effects of IGF-I treatment on FAT/CD36 induction were examined. Differentiated C2C12 cells were treated with 20 ng/ml of IGF-I at different time points. Treatment of C2C12 cells with IGF-I resulted in increased FAT/CD36 mRNA and protein expression. After 24 and 48 hr of IGF-I treatment, FAT/CD36 mRNA increased 89% and 24% respectively. The increase of both proteins returned to the control level after 72 hr of IGF-I treatment, suggesting that the FAT/CD36 gene is regulated pretranslationally by IGF-I in skeletal muscle cells. These results suggest that IGF-I can regulate the expression of FAT/CD36 in skeletal muscle cells. In conclusion, IGF-I induces a rapid transcriptional modification of the FAT/CD36 gene in C2C12 skeletal muscle cells and has modulating effects on fatty acid uptake proteins as well as oxidative proteins.

Redox Sensitivities of Global Cellular Cysteine Residues under Reductive and Oxidative Stress.

The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD), and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but instead had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,β-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.