Dichlorodihydrofluorescein Fluorescence Research Articles

Eriocitrin alleviates sevoflurane-induced cytotoxicity in HT22 cells via Nrf2 pathway

Purpose: To investigate the effect of eriocitrin on sevoflurane-induced neurotoxicity in mice.Methods: Mouse hippocampal neurons (HT22) were exposed to different concentrations of sevoflurane for 6 h and then incubated with different concentrations of eriocitrin for another 24 h. Cell viability was determined by CCK8 assay, while fluorescence intensity of dichlorodihydrofluorescein was used to evaluate reactive oxygen species. Enzyme linked immunosorbent assay (ELISA) was used to determine oxidative stress, and cellular apoptosis was determined by flow cytometry.Results: Sevoflurane exposure decreased HT22 cell viability, whereas incubation with eriocitrin increased viability of sevoflurane-treated HT22 cells (p < 0.05). Sevoflurane-induced increase in dichlorodihydrofluorescein fluorescence intensity was reduced by eriocitrin, but eriocitrin attenuated sevoflurane-induced increase in malondialdehyde, superoxide dismutase, and glutathione peroxidase in HT22 cells. Cell apoptosis increased after sevoflurane exposure, and eriocitrin suppressed apoptosis in sevoflurane-treated HT22 cells through downregulation of cleaved caspase-3 and cleaved caspase-9 (p< 0.05). Eriocitrin incubation enhanced protein expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1) in sevoflurane-treated HT22 cells (p < 0.05).Conclusion: Eriocitrin ameliorates sevoflurane-induced oxidative stress and inflammatory response in HT22 cells via activation of Nrf2/HO-1/NQO1 signaling. Thus, agent may be useful in the treatment of sevoflurane-induced toxicity, but in vivo studies are required to buttress this.

Protective effects of curcumin/cyclodextrin polymer inclusion complex against hydrogen peroxide-induced LO2 cells damage.

The objective of the present study was to explore the protective effects of the curcumin/cyclodextrin polymer (CUR/CDP) inclusion complex on hydrogen peroxide (H2O2)‐induced LO2 cells damage. In this study, a H2O2‐induced cells oxidative injury model was established to test the protective effects of the CUR/CDP inclusion complex. The cell viability of cells was detected by the thiazolyl blue tetrazolium bromide (MTT) assay. The extracellular lactate dehydrogenase (LDH) activity, catalase (CAT) activity, and malondialdehyde (MDA) level were detected by assay kits. The cellular reactive oxygen species (ROS) level was detected using the dichlorodihydrofluorescein (DCF) fluorescence assay. Western blotting analysis was conducted to assess the changes of phosphorylated‐p53 and caspase‐3. The results showed that 700 μM H2O2‐treated LO2 cells for 3 h resulted in a significant decrease of cell viability to 53.00 ± 1.68%, which established the cell oxidative injury model. Cells treated with H2O2 led to a significant increase of extracellular LDH activity, MDA content, and ROS level, and decreased CAT activity. Treatment with CUR/CDP significantly reversed the changes of the above indicators. Moreover, CUR/CDP treatment at 20 and 40 μg/ml inhibited H2O2‐induced increase in phosphorylated‐p53 and caspase‐3 expression, indicating that CUR/CDP suppressed cell apoptosis to alleviate liver injury. The results of those studies demonstrated that CUR/CDP had a protective effect on the oxidative damage of LO2 cells, and it could be developed as a new type of natural liver protection product to apply in the prevention of liver injury.

Exposures to Heat‐Inactivated SARS‐CoV‐2 Causes Reactive Oxygen Species Generation And Inflammatory Response in RAW 264.7 Cells

Introduction and ObjectivesNovel SARS‐CoV‐2 virus has been implicated in prompting a bold immune response that leads to severe Coronavirus disease 2019 (COVID‐19). Recent studies have shown that SARS‐CoV‐2‐infected monocytes and macrophages are stimulated to produce an overabundance of pro‐inflammatory cytokines and chemokines to generate a cytokine storm. Cytokines in excess can contribute to local tissue inflammation and the pathogenesis of COVID‐19. However, the mechanism by which SARS‐CoV‐2 signal macrophage‐derived inflammatory response remains unclear. In the present study, we used RAW 264.7 cells, a well‐characterized macrophage model, to study the in vitro effects of SARS‐CoV‐2 on reactive oxygen species (ROS) production and its potential role in the signal transduction of cytokine production.MethodsThe effect of SARS‐CoV‐2 on ROS and cytokine generation in macrophages was assessed by treating RAW 264.7 cells with SARS‐CoV‐2 heat inactivated virus (0‐20 million viral particles) or recombinant proteins for 24 hours. 2′,7′‐Dichlorodihydrofluorescein (2′,7′‐DCF) fluorescence analysis was utilized to quantify ROS generation within the RAW 264.7 macrophage cell line. Cell culture medium was sampled to quantify the levels of tumor necrosis factor (TNF) using enzyme‐linked immunosorbent assay (ELISA). To assess the effects of SARS‐CoV‐2 on mitochondrial function, cells were treated with SARS‐CoV‐2 heat inactivated virus (0‐20 million viral particles) for 24 hrs. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator.ResultsTreatment of RAW 264.7 cells with inactivated SARS‐CoV‐2 viral particles or recombinant proteins stimulated ROS production. Mitochondria‐derived superoxide and hydrogen peroxide production were increased in response to inactivated SARS‐CoV‐2 viral particles and recombinant protein exposure. The increased ROS generation is linked to macrophage activation induced by SARS‐CoV‐2 exposures. Along with the ROS generation, increased TNF production was observed.ConclusionsThe results of this study suggest that both SARS‐CoV‐2 viral proteins and heat‐inactivated viral particle exposures cause significant generation of ROS and cytokines by RAW 264.7 cells. ROS generation and the subsequent cytokine release apparently play a significant role in the pathogenesis associated with the SARS‐CoV‐2 viral infection. The imbalanced cellular defense system against oxidative stress commonly associated with aging could explain the increased occurrence of more severe SARS‐CoV‐2 illness in seniors and in patients with underlying health conditions. Based on the results from this study, we propose that antioxidants such as N‐acetyl‐L‐cysteine, resveratrol, or Vitamin E in combination with anti‐inflammatory drug could be used to control excess ROS and cytokines in patients with severe COVID‐19.

Senescence Is Associated With Elevated Intracellular Resting [Ca2 +] in Mice Skeletal Muscle Fibers. An in vivo Study.

Aging causes skeletal muscles to become atrophied, weak, and easily fatigued. Here, we have tested the hypothesis that normal aging in skeletal muscle cells is associated with Ca2+ intracellular dyshomeostasis and oxidative stress. Intracellular Ca2+ concentration ([Ca2+]i), resting intracellular Na+ concentration ([Na+]i) and reactive oxygen species (ROS) production were measured in vivo (superficial gastrocnemius fibers) using double-barreled ion-selective microelectrodes, and in vitro [isolated single flexor digitorum brevis fibers] using fluorescent ROS sensor CM-H2DCFDA in young (3 months of age), middle-aged (12 months of age), and aged (24 months of age) mice. We found an age-related increase in [Ca2+]i from 121 ± 4 nM in young muscle cells which rose to 255 ± 36 nM in middle-aged and to 409 ± 25 nM in aged cells. [Na+]i also showed an age-dependent elevation, increasing from 8 ± 0.5 mM in young muscle fibers, to 12 ± 1 mM in middle-aged and to 17 ± 1 mM in old muscle fibers. Using the fluorescent ROS sensor CM-H2DCFDA we found that these increases in intracellular cation concentrations were associated with significantly increased basal ROS production as demonstrated by age related increases in the rate of dichlorodihydrofluorescein fluorescence. To determine is this could be modified by reducing ROS and/or blocking sarcolemmal Ca2+ influx we administered flufenamic acid (FFA), a non-steroidal anti-inflammatory drug which is also a non-selective blocker of the transient receptor potential canonical channels (TRPCs), for 4 weeks to determine if this would have a beneficial effect. FFA treatment reduced both basal ROS production and muscle [Ca2+]i and [Na+]i in middle-aged and aged muscle fibers compared to fibers and muscles of untreated 12 and 24-months old mice. [Ca2+]i was reduced to 134 ± 8 nM in middle-aged muscle and to 246 ± 40 nM in muscle from aged mice. Likewise [Na+]i was reduced to 9 ± 0.7 mM in middle-aged muscles and to 13 ± 1 mM in muscle from aged mice. FFA treatment also reduced age associated increases in plasma interleukin 6 and tumor necrosis factor-alpha (TNF-α) concentrations which were elevated in 12 and 24-months old mice compared to young mice and decreased age-related muscle damage as indicated by a reduction in serum creatine kinase (CK) activity. Our data provides a direct demonstration that normal aging is associated with a significant elevation [Ca2+]i, [Na+]i, and intracellular ROS production in skeletal muscle fibers. Furthermore, the fact that FFA reduced the intracellular [Ca2+], [Na+], and ROS production as well as the elevated IL6, TNF-α, and CK levels, led us to suggest that its pharmacological effect may be related to its action both as a TRPC channel blocker and as an anti-inflammatory.

Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer’s disease and Parkinson’s disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Ascorbate exacerbates iron toxicity on intestinal barrier function against Salmonella infection

Ascorbic acid is often used to enhance iron absorption in nutritional interventions, but it produces pro-oxidant effects in the presence of iron. This study aimed to evaluate ascorbate’s role in iron toxicity on intestinal resistance against foodborne pathogens during iron supplementation/fortification. In polarized Caco-2 cell monolayers, compared to the iron-alone treatment, the iron-ascorbate co-treatment caused more than 2-fold increase in adhesion, invasion and translocation of Salmonella enterica serovar Typhimurium. According to 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase release and transepithelial electrical resistance, the iron-ascorbate co-treatment resulted in reduced cell viability and increased impairment of cell membrane and paracellular permeability compared to the iron-alone treatment. Butylated hydroxytoluene protected cells against these prooxidant toxicities of ascorbate. Ascorbate completely restored iron-induced intracellular oxidant burst and depletion of cytosolic antioxidant reserve, according to dichlorodihydrofluorescein fluorescence and intracellular reduced glutathione levels. In Salmonella-infected C57BL/6 mice, iron-ascorbate co-supplementation resulted in greater loss of body weight and appetite, lower survival rate, shorter colon length, heavier intestinal microvilli damage, and more intestinal pathogen colonization and translocation than the iron-alone supplementation. Overall, ascorbate would exacerbate iron toxicity on intestinal resistance against Salmonella infection through pro-oxidant impairment of intestinal epithelial barrier from extracellular side and/or by facilitating intestinal pathogen colonization.

Dioscin attenuates oxLDL uptake and the inflammatory reaction of dendritic cells under high glucose conditions by blocking p38 MAPK.

Dioscin has been shown to affect the regulation of metabolic diseases, including diabetes; however, the mechanism of action is still unclear. Under high glucose (HG) conditions, the expression of scavenger receptors and the uptake of oxidized low-density lipoprotein (oxLDL) are upregulated in dendritic cells (DCs), which are critical steps in atherogenesis and inflammation. In this study, the focus was on the impact of dioscin on the function of DCs. Immature DCs were cultured with: 5.5 mM glucose medium (control group); 30 mM glucose medium (HG group); HG + 10 mM dioscin; HG + 20 mM dioscin; HG + 30 mM dioscin; and HG + 40 mM dioscin. For subsequent experiments, 30 mM dioscin was used as the experimental concentration. Dichlorodihydrofluorescein fluorescence was used to measure the intracellular production of reactive oxygen species (ROS) in DCs. The expression levels of the scavenger receptors, including class A scavenger receptors (SR-A), CD36 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were determined via quantitative PCR. The protein expression of p38 mitogen-activated protein kinase (MAPK) was determined by western blotting. Furthermore, ELISA was used to detect the levels of interleukin (IL)-6, IL-10 and IL-12. Finally, DCs were incubated with diOlistic (Dil)-labeled oxLDL, and flow cytometry analysis was used to investigate the Dil-oxLDL-incorporated fraction. The incubation of DCs with dioscin inhibited the induction of ROS production, in a dose-dependent manner, under HG conditions. The upregulation of SR-A, CD36 and LOX-1 genes was partially abolished by dioscin, which also partially reversed p38 MAPK protein upregulation. Furthermore, increased secretion of IL-6 and IL-12, and decreased secretion of IL-10 in DCs, induced by HG, was also reversed by dioscin. To conclude, dioscin could attenuate the production of ROS, inflammatory cytokine secretion and oxLDL uptake by DCs in HG conditions by preventing the expression of scavenger receptors and p38 MAPK, thus playing a positive role in preventing atherogenesis.

A novel role of glutathione S-transferase A3 in inhibiting hepatic stellate cell activation and rat hepatic fibrosis

Background and aimsGlutathione S-transferase A3 (GSTA3) is known as an antioxidative protease, however, the crucial role of GSTA3 in liver fibrosis remains unclear. As a recently we developed water-soluble pyridone agent with antifibrotic features, fluorofenidone (AKF-PD) can attenuate liver fibrosis, present studies were designed to explore the role of GSTA3 in liver fibrosis and its modulation by AKF-PD in vivo and in vitro.MethodsRats liver fibrosis models were induced by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4). The two activated hepatic stellate cells (HSCs) lines, rat CFSC-2G and human LX2 were treated with AKF-PD respectively. The lipid peroxidation byproduct malondialdehyde (MDA) in rat serum was determined by ELISA. The accumulation of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescein fluorescence analysis. The expression of α-smooth muscle actin (α-SMA), fibronectin (FN), and phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 beta (GSK-3β) were detected by western blotting (WB).ResultsGSTA3 was substantially reduced in the experimental fibrotic livers and transdifferentiated HSCs. AKF-PD alleviated rat hepatic fibrosis and potently inhibited HSCs activation correlated with restoring GSTA3. Moreover, GSTA3 overexpression prevented HSCs activation and fibrogenesis, while GSTA3 knockdown enhanced HSCs activation and fibrogenesis resulted from increasing accumulation of ROS and subsequent amplified MAPK signaling and GSK-3β phosphorylation.ConclusionsWe demonstrated firstly that GSTA3 inhibited HSCs activation and liver fibrosis through suppression of the MAPK and GSK-3β signaling pathways. GSTA3 may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H⁺ ions, which is in part accomplished by Na⁺/H⁺ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na⁺/H⁺ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pH_i utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na⁺/H⁺ exchanger activity from Na⁺ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na⁺/H⁺ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na⁺/H⁺ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth inhibition of tumor cells.

붉은싸리버섯 추출물의 항산화 및 Human Neutrophil Elastase 저해활성

천연자원으로부터 항노화 화장품 신소재를 탐색하던 중, 국내 자생버섯의 일종인 붉은싸리버섯 자실체 추출물이 항산화 활성과 인체 호중구 엘라스타제 저해활성이 우수함을 확인하고 일련의 연구를 수행하였다. 붉은싸리버섯 추출물의 DPPH 라디칼 소거활성은 붉은싸리버섯 추출물 <TEX>$500{\mu}g/mL$</TEX> 처리시 <TEX>$117.0{\mu}g/mL$</TEX> (ascorbic acid 환산값)의 매우 우수한 소거활성을 나타냈다. Peroxy 라디칼 소거활성을 oxygen radical absorbance capacity (ORAC) assay 를 통하여 측정한 결과 붉은싸리버섯 추출물 1, 10, <TEX>$20{\mu}g/mL$</TEX> 처리 시, 각각 0.8, 5.2, 7.8 <TEX>$ORAC_{Roo}$</TEX> (trolox equivalents, <TEX>$1{\mu}M$</TEX>)로 농도 의존적으로 높은 소거활성을 나타냈다. 뿐만아니라 cellular antioxidant capacity를 DCF fluorescence intensity (% of control)로 조사한 결과에서도 붉은싸리버섯 추출물 <TEX>$20{\mu}g/mL$</TEX> 처리시 약 30% 이상 높은 항산화 활성을 나타냈다. Human neutrophil elastase 저해활성은 농도 의존적으로 저해활성을 나타냈으며 특히 에탄올 추출분획에서 <TEX>$ED_{50}$</TEX> 값은 <TEX>$42.9{\mu}g/mL$</TEX>이었다. 붉은싸리버섯 추출물은 Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Candida albicans (C. albicans), Aspergillus oryzae (A. oryzae) 균주 모두에서 항균활성은 나타나지 않았다. 또한 염증성 cytokine인 interleukin-10 및 interferon-<TEX>${\gamma}$</TEX> (IFN-<TEX>${\gamma}$</TEX>)의 생산 또는 분비 조절에는 영향을 미치지 않았다. 이상의 결과로 붉은싸리버섯 추출물은 항산화활성과 elastase 저해활성을 우수하여 피부에 자극이 없는 항노화 화장품 조성물로 유용하게 사용될 수 있음을 확인하였다. In searching for novel agents for skin anti-aging from natural resources, we found that the extract of the fruiting bodies of Ramaria formosa (R. formosa) had significant antioxidant and human neutrophil elastase (HNE) inhibitory activities. R. formosa extract exhibited a considerable DPPH radical scavenging activity with an antioxidant content of 117.0mg/mL (ascorbic acid equivalents) at the concentration of <TEX>$500{\mu}g/mL$</TEX>. The capacity of R. formosa extract to scavenge peroxy radicals measured by ORAC assay also showed dose-dependent antioxidant effect with <TEX>$ORAC_{Roo}$</TEX> (trolox equivalents, <TEX>$1{\mu}M$</TEX>) values of 0.8, 5.2, and 7.8 at the concentrations of 1, 10, and <TEX>$20{\mu}g/mL$</TEX>. The cellular antioxidant capacity of R. formosa extract was investigated by assaying the cellular fluorescence intensity using dichlorodihydrofluorescein (DCF). The cellular oxidative stress induced by AAPH, <TEX>$Cu^{2+}$</TEX> or <TEX>$H_2O_2$</TEX> in HepG2 cells was significantly attenuated by more than 30% at <TEX>$20{\mu}g/mL$</TEX> of R. formosa extract. HNE activity was reduced by treatment with R. formosa extract in a dose-dependent manner, and the <TEX>$ED_{50}$</TEX> value for the ethanol extract of R. formosa was <TEX>$42.9{\mu}g/mL$</TEX>. R. formosa extract did not exhibited antimicrobial activity against four microorganisms including Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Candida albicans (C. albicans), Aspergillus oryzae (A. oryzae). Furthermore, the extract did not affect the inflammatory cytokine production of interleukin-10 and interferon-<TEX>${\gamma}$</TEX> in NK92 cells. From the above results, we found that R. formosa extract has considerable antioxidant and elastase inhibitory effects, and does not stimulate immune cells. These findings suggest that R. formosa extract may be used as a bioactive component in cosmetic composition.

Nimodipine, an L-type calcium channel blocker attenuates mitochondrial dysfunctions to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

Parkinson’s disease (PD), the most common progressive neurodegenerative movement disorder, results from loss of dopaminergic neurons of substantia nigra pars compacta. These neurons exhibit Cav1.3 channel-dependent pacemaking activity. Epidemiological studies suggest reduced risk for PD in population under long-term antihypertensive therapy with L-type calcium channel antagonists. These prompted us to investigate nimodipine, an L-type calcium channel blocker for neuroprotective effect in cellular and animal models of PD. Nimodipine (0.1–10 μM) significantly attenuated 1-methyl-4-phenyl pyridinium ion-induced loss in mitochondrial morphology, mitochondrial membrane potential and increases in intracellular calcium levels in SH-SY5Y neuroblastoma cell line as measured respectively employing Mitotracker green staining, TMRM, and Fura-2 fluorescence, but only a feeble neuroprotective effect was observed in MTT assay. Nimodipine dose-dependently reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian syndromes (akinesia and catalepsy) and loss in swimming ability in Balb/c mice. It attenuated MPTP-induced loss of dopaminergic tyrosine hydroxylase positive neurons in substantia nigra, improved mitochondrial oxygen consumption and inhibited reactive oxygen species production in the striatal mitochondria measured using dichlorodihydrofluorescein fluorescence, but failed to block striatal dopamine depletion. These results point to an involvement of L-type calcium channels in MPTP-induced dopaminergic neuronal death in experimental parkinsonism and more importantly provide evidences for nimodipine to improve mitochondrial integrity and function.

Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling

Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺. Exposure of human erythrocytes to Ca²⁺ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca²⁺ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca²⁺.

Nocodazole Induced Suicidal Death of Human Erythrocytes

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca²⁺ entry, oxidative stress and ceramide.

Glutathione Depletion Induces Spermatogonial Cell Autophagy.

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.

Effects of TiO2 nanoparticles on ROS production and growth inhibition using freshwater green algae pre-exposed to UV irradiation

This study investigated the possibility that titanium dioxide nanoparticles (nano-TiO2) toxicity in Pseudokirchneriella subcapitata involves reactive oxygen species (ROS) production, using the dichlorodihydrofluorescein (DCF) assay. Algae were exposed to nano-TiO2 under laboratory fluorescent lamps supplemented with UV irradiation for 3h, with or without a UV filter. Results showed that nano-TiO2 increased ROS production in UV-exposed cells, with or without a UV filter (LOEC values were 250 and 10mg/L, respectively). Sublethal effects of nano-TiO2 on UV pre-exposed algae were also examined. Toxicity studies indicated that exposure to nano-TiO2 agglomerates decreased algal growth following 3h pre-exposure to UV, with or without a UV filter (EC50s were 8.7 and 6.3mg/L, respectively). The present study suggests that the growth inhibitory effects of nano-TiO2 in algae occurred at concentrations lower than those that can elevate DCF fluorescence, and that ROS generation is not directly involved with the sublethal effects of nano-TiO2 in algae.

Enhanced Suicidal Erythrocyte Death Contributing to Anemia in the Elderly

Background/Aims: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca²⁺-concentration ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. Methods: Blood was drawn from healthy young (n=11, age 31.3±1.7 years) and elderly (n=16, age 88.6±0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. Results: Elderly individuals suffered from severe anemia (hemoglobin 10.5±0.3 g/100 ml) despite enhanced number of reticulocytes (2.3±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5±0.2%) than in the young volunteers (1.3±0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca²⁺]_i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. Conclusions: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Suicidal erythrocyte death in end-stage renal disease.

Anemia in end-stage renal disease (ESRD) results mainly from erythropoietin and iron deficiency. Anemia could be confounded, however, by accelerated clearance of circulating erythrocytes because of premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+) concentration ([Ca(2+)]i), oxidative stress, and ceramide. The present study explored whether and how ESRD influences eryptosis. Blood was drawn from healthy volunteers (n = 20) as well as ESRD patients (n = 20) prior to and after hemodialysis. Phosphatidylserine exposure was estimated from annexin V binding, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, and ceramide from fluorescein-isothiocyanate-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes from healthy volunteers exposed in vitro for 24 h to plasma from healthy volunteers or ESRD patients prior to and following dialysis. The patients suffered from anemia (hemoglobin 10.1 ± 0.5 g/100 ml) despite 1.96 ± 0.34 % reticulocytes. The percentage of phosphatidylserine-exposing erythrocytes was significantly higher in ESRD patients (0.84 ± 0.09 %) than in healthy volunteers (0.43 ± 0.04 %) and was significantly increased immediately after dialysis (1.35 ± 0.13 %). The increase in phosphatidylserine exposure was paralleled by increase in [Ca(2+)]i, oxidative stress, and ceramide abundance. As compared to addition of plasma from healthy individuals, addition of predialytic but not of postdialytic plasma from ESRD patients increased phosphatidylserine exposure, [Ca(2+)]i, ROS, and ceramide abundance. In conclusion, both, dialyzable components of uremic plasma and dialysis procedure, trigger eryptosis at least in part by increasing erythrocyte [Ca(2+)]i, ROS, and ceramide formation. Anemia in uremia results in part from eryptosis, the suicidal erythrocyte death. Eryptosis in uremia is triggered in part by a dialyzable plasma component. Eryptosis in uremia is further triggered by dialysis procedure. Eryptosis in uremia is in part due to increased cytosolic Ca(2+) concentration. Eryptosis in uremia is further due to oxidative stress and ceramide formation.

High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells.

BackgroundBoth hyperglycaemia and dendritic cells (DCs) play causative roles in atherosclerosis. However, whether they interact in atherosclerosis remains uncertain. Therefore, we examined whether high glucose could regulate the expression of scavenger receptors responsible for oxidised low-density lipoprotein (oxLDL) uptake in DCs, a critical step in atherogenesis. In addition, we investigated the impact of glucose on DC maturation regarding changes in phenotype and cytokine secretion.MethodsImmature DCs were cultured with different concentrations of glucose (5.5 mmol/L, 15 mmol/L, 30 mmol/L) in the absence or presence of N-acetylcysteine (NAC), SB203580 or Bay11-7082 for 24 hours. We used 30 mmol/L mannitol as a high-osmolarity control treatment. The expression of the scavenger receptors SR-A, CD36 and LOX-1 was determined by real-time PCR and western blot analysis. Furthermore, DCs were incubated with DiI-labelled oxLDL. The DiI-oxLDL-incorporated fraction was investigated by flow cytometry analysis. The intracellular production of ROS in DCs was measured by dichlorodihydrofluorescein (DCF) fluorescence using confocal microscopy. Finally, flow cytometry analysis was used to investigate immunophenotypic protein expression (CD83 and CD86). Supernatant cytokine measurements were used for immune function assays.ResultsThe incubation of DCs with glucose enhanced, in a dose-dependent manner, the gene and protein expression of SR-A, CD36 and LOX-1. This effect was partially abolished by NAC, SB203580 and Bay11-7082. Incubation of DCs with mannitol (30 mmol/L) did not enhance these scavenger receptors’ expression. High glucose upregulated the production of ROS and expression of p38 MAPK in DCs. NAC partially reversed p38 MAPK upregulation. High glucose increased the oxLDL-uptake capacity of DCs. Blockage of the scavenger receptors SR-A and CD36 reduced oxLDL uptake, but blockage of LOX-1 did not. Furthermore, high-glucose (15 mmol/L or 30 mmol/L) treatment increased CD86 and CD83 in DCs. High glucose also increased IL-6 and IL-12 secretion and decreased IL-10 secretion.ConclusionHigh glucose can increase the expression of the scavenger receptors SR-A, CD36 and LOX-1, which can increase the oxLDL-uptake capacity of DCs. High glucose induces a proinflammatory cytokine profile in human DCs, leading to DC maturation. These results support the hypothesis that atherosclerosis is aggravated by hyperglycaemia-induced DC activation and oxLDL uptake.

Iron contributes to hepatocyte insulin resistance in a cell model of nonalcoholic fatty liver disease

Increased body iron stores are associated with increased severity of nonalcoholic fatty liver disease (NAFLD). To clarify the role(s) of iron in NAFLD, this study investigated its effects on surrogate disease markers in AML12 hepatocytes: reactive oxygen species (ROS), stress kinase activation, and insulin resistance. Control AML12 cells defined baseline ROS levels (dichlorodihydro‐fluorescein fluorescence) and maximum insulin response (phospho‐activation of protein kinase B: EC50 near 1 nM insulin, maximum at 100 nM). Treatment with 50 μM iron (given with the lipophilic chelator 8‐hydroxyquinoline) increased ROS levels 8‐fold (p&lt;0.01) and elevated phospho‐jnk and phospho‐p38. This was accompanied by a 33% decrease in response to 10 nM insulin (p&lt;0.01). Under conditions in which fat accumulation alone had little or no effect on ROS levels or insulin response, cells pre‐loaded with stearic acid appeared sensitized to iron (ROS increased 18‐fold (p&lt;0.01); insulin response decreased 51% vs. control), while cells treated with oleic acid appeared partially protected from iron (ROS levels increased 5‐fold (p&lt;0.01); insulin response decreased 24%). We conclude NAFLD progression in this in vitro model is best recapitulated by the combined effects of saturated fat and iron. Studies to further define iron‐sensitive signaling pathways and evaluate novel therapies are justified. Supported by NIH grant AT03448.

Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30min and a late increase at 6h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3μM) caused an increase in system xc− mediated 14C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system xc−. Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.