Dichlorodihydrofluorescein Diacetate Staining Research Articles

Deficiency in adiponectin exaggerates cigarette smoking exposure-induced cardiac contractile dysfunction: Role of autophagy

Second hand smoke is an independent risk factor for cardiovascular disease. Adiponectin (APN), an adipose-derived adipokine, has been shown to offer cardioprotective effect through an AMPK-dependent manner. This study was designed to evaluate the impact of adiponectin deficiency on second hand smoke-induced cardiac pathology and underlying mechanisms using a mouse model of side-stream smoke exposure. Adult wild-type (WT) and adiponectin knockout (APNKO) mice were placed in a chamber exposed to cigarette smoke for 1 hour daily for 40 days. Echocardiographic, cardiomyocyte function, and intracellular Ca2+ handling were evaluated. Autophagy and apoptosis were examined using western blot. 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining was used to evaluate reactive oxygen species (ROS) generation. Masson trichrome staining was employed to measure interstitial fibrosis. Our data revealed that adiponectin deficiency provoked smoke exposure-induced cardiomyopathy (compromised fractional shortening, disrupted cardiomyocyte function and intracellular Ca2+ homeostasis, apoptosis and ROS generation). In addition, these detrimental effects of side-stream smoke were accompanied by defective autophagolysosome formation, the effect of which was exacerbated by adiponectin deficiency. Blocking autophagolysosome formation using bafilomycin A1 (BafA1) negated the cardioprotective effect of rapamycin against smoke extract. Induction of autophagy using rapamycin and AMPKα activation using AICAR rescued against smoke extract-induced myopathic anomalies in APNKO mice. Our data suggest that adiponectin serves as an indispensable cardioprotective factor against side-stream smoke exposure-induced myopathic changes possibly through facilitating autophagolysosome formation.

Rifampicin attenuates rotenone-induced inflammation via suppressing NLRP3 inflammasome activation in microglia

A growing body of evidence has supported that environmental factors, such as exposure to heavy metal and pesticides, play an important role in the pathogenesis of Parkinson׳s disease (PD). Rotenone, the active ingredient in various pesticides, has been identified as an inducer of PD. It has been revealed that rotenone induces activation of microglia and generation of pro-inflammatory factors in PD. Our previous studies demonstrated that rifampicin possessed neural protective effect in PD. In this study, we aimed to study the effect of rifampicin on the inflammation induced by rotenone in microglia and the underlying mechanisms. Results demonstrated that rifampicin pretreatment significantly reduced rotenone-induced cytotoxicity and gene expression of IL-1β in BV2 microglia. Moreover, western blot analysis verified that rifampicin pretreatment suppressed NLRP3 inflammasome activation via inhibiting caspase-1 cleavage and protein expression of NLRP3. As it is indicated that reactive oxidative stress (ROS) is one of the activators for NLRP3 inflammasome, we further employed 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining and Rhodamine123 staining to detect intracellular ROS and mitochondrial membrane potential (MMP), respectively. Results confirmed that rifampicin obviously reduced intracellular ROS and reversed loss of MMP in BV2 cells treated by rotenone. Taken together, our data indicate that rifampicin pretreatment inhibits maturation of IL-1β and neuroinflammation induced by rotenone via attenuating NLRP3 inflammasome activation. Rifampicin might emerge as a promising candidate for modulating neuroinflammation in PD.

DNA hypomethylation predisposes sperm to DNA damage in individuals with varicocele

This study aimed to assess and compare global DNA methylation (GDM) between fertile men and infertile men with varicocele. In addition, we evaluated the correlations between DNA methylation with reactive oxygen species (ROS), protamine deficiency, and DNA integrity. Semen samples were collected from 44 men with grades II and III varicocele, and 15 fertile men for assessment of semen parameters, DNA methylation, DNA fragmentation, oxidative stress, and protamine deficiency. Samples were evaluated by the World Health Organization (WHO) criteria, immunostaining, the TUNEL assay, 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and chromomycin A3 (CMA3) staining. Semen parameters were significantly lower in individuals with varicocele compared to fertile men. The percentage of GDM and intensity of DCFH were reduced and the percentages of DCFH, TUNEL, and CMA3 positive sperm significantly increased in individuals with varicocele compared to fertile men. Correlation analysis revealed a negative significant relation between DNA methylation and DNA fragmentation, but not with the degree of protamine deficiency and ROS production. The results have shown that individuals with varicocele show increased DNA susceptibility to damage when DNA is hypomethylated. This phenomenon appears to be independent of ROS production.

Adiponectin induces apoptosis in hepatocellular carcinoma through differential modulation of thioredoxin proteins

Adiponectin blocks hepatocellular carcinoma (HCC) progression by inducing cell apoptosis through the modulation of C-Jun N-terminal kinase and mammalian target of rapamycin. However, the precise upstream signaling pathways or molecules remain elusive. In the present study, we analyzed the role of antioxidant protein thioredoxin (Trx) in adiponectin-induced apoptosis in HCC. Adiponectin treatment decreased the viabilities of both HepG2 and Huh7 HCC cells accompanied by increased accumulation of intracellular reactive oxygen species, as evidenced by 2′,7′-dichlorodihydrofluorescein diacetate staining. Pretreatment of these cells with the deoxidant N-acetylcysteine blocked the inhibitory effect of adiponectin. Levels of Trx2 protein in both HCC cells were significantly decreased, and the level of Trx1 was significantly inhibited in Huh7 cells while unchanged in HepG2 cells. However, the redox state of Trx1 was altered from reduced to the oxidized form following adiponectin treatment in HepG2 cells. Overexpression of both Trx proteins rescued adiponectin-induced cell apoptosis, whereas mutated Trx proteins were less effective. Further analysis suggested that both ASK1 and JNK signaling are involved in this process. Trx1 and Trx2 proteins also manifested protective effects on HCC cells in response to adiponectin treatment in a xenograft tumor model. Furthermore, high levels of Trx proteins and low adiponectin expression levels were found in primary human HCC samples compared with paracancerous tissues. These results suggest that Trx proteins play important roles in mediating adiponectin-induced HCC cell apoptosis, thus providing new insights into the pathogenesis of HCC and identifying adiponectin and Trx proteins as potential combinational therapeutic targets for the treatment of HCC.

1,25-dyhydroxyvitamin D3 attenuates L-DOPA-induced neurotoxicity in neural stem cells.

The neurotoxicity of levodopa (L-DOPA) on neural stem cells (NSCs) and treatment strategies to protect NSCs from this neurotoxicity remain to be elucidated. Recently, an active form of vitamin D3 has been reported to display neuroprotective properties. Therefore, we investigated the protective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) on L-DOPA-induced NSC injury. We measured cell viability via the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays and Annexin V/PI staining followed by flow cytometry, cell proliferation using the BrdU and colony-forming unit (CFU) assays, cell differentiation via immunocytochemistry, the levels of free radicals via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, apoptosis via DAPI and TUNEL staining, and intracellular signaling protein expression via Western blot. Antibody microarrays were also employed to detect changes in the expression of prosurvival- and death-related proteins. Treatment of NSCs with L-DOPA reduced their viability and proliferation. This treatment also increased the levels of free radicals and decreased the expression levels of intracellular signaling proteins that are associated with cell survival. However, simultaneous exposure to calcitriol significantly reduced these effects. The calcitriol-mediated protection against L-DOPA toxicity was blocked by the phosphoinositide 3-kinase (PI3K) inhibitor LY294004. L-DOPA also inhibited the expression of Nestin and Ki-67, and co-treatment with calcitriol alleviated these effects. The expression levels of GFAP, DCX, and Tuj1 were not significantly affected by treatment with L-DOPA or calcitriol. Calcitriol protects against L-DOPA-induced NSC injury by promoting prosurvival signaling, including activation of the PI3K pathway, and reducing oxidative stress.

Effect of carvedilol on atrial remodeling in canine model of atrial fibrillation.

We evaluated the effect of carvedilol, a beta-blocker with anti-oxidative action, against the atrial fibrillation (AF) inducibility, the development of atrial remodeling and the oxidative stress markers in a canine AF model. AF model was produced by performing 6-week rapid atrial stimulation in 15 dogs. The animals were divided into the following three groups: (I) pacing + carvedilol group (n=5); (II) pacing control group (n=5); and (III) non-pacing group (n=5). AF inducibility was gradually increased along the time course in the pacing control group. In the pacing + carvedilol group, the AF inducibility was suppressed especially in the latter phase of protocol in comparison with the pacing control group. Although carvedilol has beta-blocking effect, pacing control and pacing + carvedilol groups did not exhibit difference in the heart rate (177±13 vs. 155±13 bpm, P=0.08). On 8-hydroxy-2'-deoxyguanosine (8-OHdG), dihydroethidium and dichlorodihydrofluorescein diacetate staining, enhanced oxidative stress was observed in the atrial tissue in the pacing control, but not in the pacing + carvedilol group. Carvedilol suppressed AF inducibility and oxidative stress in the canine AF model.

Quality of bull spermatozoa after preparation by single-layer centrifugation

The present study aimed to evaluate the effect of single-layer centrifugation (SLC) through a species-specific colloid (Androcoll-B; patent pending, J. M. Morrell) on bull sperm quality. Computer-assisted sperm analysis of motility and flow cytometric analysis of sperm viability (SYBR-14/propidium iodide staining), chromatin integrity (acridine orange staining), reactive oxygen species production [Hoechst 33258-hydroethidine-2′,7′-dichlorodihydrofluorescein diacetate (HO-HE-DCFDA) staining], mitochondrial membrane potential (staining with JC-1 probe), and protein tyrosine phosphorylation (specific antibody staining) were performed on unselected and SLC-selected sperm samples. Single-layer centrifugation of bull spermatozoa resulted in the selection of a sperm population that had high mitochondrial membrane potential, a higher content of phosphorylated protein, and more reactive oxygen species than control samples. Sperm chromatin damage was lower in the SLC samples although sperm viability and motility did not differ between SLC samples and controls. These observations suggest that SLC of bull semen in a soybean-containing extender improved some, but not all, parameters of sperm quality.

The study of inducing apoptosis effect of fructose 1,6-bisphosphate on the papillary thyroid carcinoma cell and its related mechanism

This study aims to investigate the apoptosis-inducing effect of fructose 1,6-bisphosphate (F1,6BP) on the related mechanism of papillary thyroid carcinoma W3 and T cells. W3 cells were treated with F1,6BP alone or in combination with antioxidant catalase (CAT) or N-acetyl-L-cysteine (NAC). The changes of cell viability and cell nucleus morphology were examined by cell proliferation assay and Hoechst staining, and apoptosis levels of these cells were measured with flow cytometry. The changes of reactive oxygen species (ROS) level and the percentage of oxidized glutathione in total glutathione in W3 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining or colorimetry assay. At the same time, real-time fluorescence quantitative PCR was adopted to evaluate the expression levels of CAT and glutathione peroxidase (GSH-Px) mRNAs in W3 cells. F1,6BP inhibited the growth of W3 cells significantly, coupling with an increase in intracellular ROS level and the percentage of oxidized glutathione in total glutathione. Typical apoptotic morphological changes of the cell nucleus happened. The apoptosis rate and GSH-Px and CAT mRNAs expression levels were upregulated after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after W3 cells were pretreated with NAC and CAT. F1,6BP can induce the apoptosis of W3 cells through upregulating the generation of ROS, especially the production of H2O2.

Cytochrome P450 2E1 (CYP2E1) regulates the response to oxidative stress and migration of breast cancer cells

IntroductionThe cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis.MethodsCellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2′,7′-dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation.ResultsEctopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor.ConclusionsThese results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis, of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity.

Antitumor effect of fructose 1,6-bisphosphate and its mechanism in hepatocellular carcinoma cells

We aimed to investigate the antitumor effect and mechanism of fructose 1,6-bisphosphate (F1,6BP) in a hepatocellular carcinoma cell line. HepG2 cells were treated with different concentrations of F1,6BP alone or in combination with antioxidant N-acetyl-L-cysteine (NAC) or catalase (CAT), and cell proliferation assays were performed. Nuclear morphology was observed by fluorescence microscopy after Hoechst staining, and apoptosis was measured with flow cytometry. Changes in reactive oxygen species (ROS) levels in HepG2 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. A colorimetric assay was adopted to determine the percentage of oxidized glutathione in these cells. CAT and glutathione peroxidase (GSH-Px) mRNA expression levels in HepG2 cells were measured by real-time fluorescence quantitative PCR. HepG2 cell proliferation was significantly inhibited by F1,6BP, accompanied by an increase in intracellular ROS levels and oxidized glutathione. Upregulated apoptosis and characteristic nuclear morphological changes were observed, and the expression of CAT and GSH-Px mRNA was increased after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after pretreatment with NAC and CAT in HepG2 cells. In conclusion, F1,6BP can induce the apoptosis of HepG2 cells. The mechanism involved may be associated with the generation of ROS, especially the production of H2O2.

Phosphodiesterase-5 inhibitor tadalafil attenuates oxidative stress and protects against myocardial ischemia/reperfusion injury in type 2 diabetic mice

Diabetic patients exhibit increased risk for the development of cardiovascular diseases primarily because of impaired nitric oxide (NO) bioavailability. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil restores NO signaling and protects against ischemia/reperfusion (I/R) injury. In this study, we determined the effect of the long-acting PDE-5 inhibitor tadalafil on diabetes-associated complications and its role in attenuating oxidative stress after I/R injury in type 2 diabetic db/db mice. Adult male db/db mice (n=40/group) were randomized to receive dimethyl sulfoxide (10% DMSO, 0.2ml, ip) or tadalafil (1mg/kg in 10% DMSO, ip) for 28 days. After 28 days treatment, the hearts were isolated and subjected to 30min global ischemia followed by 60min reperfusion in the Langendorff mode. Infarct size was measured using computer morphometry of tetrazolium-stained sections. Cardiomyocytes were isolated from a subset of hearts and subjected to 40min simulated ischemia followed by 1h of reoxygenation (SI/RO). Dichlorodihydrofluorescein diacetate and JC-1 staining was used to measure reactive oxygen species (ROS) generation and mitochondrial membrane potential (Δψm), respectively. Another subset of hearts was used for the estimation of lipid peroxidation, glutathione, and the expression of myocardial pRac1, Rac1, gp91phox, p47phox, and p67phox by Western blot. Tadalafil treatment improved the metabolic status and reduced infarct size compared to the untreated db/db mice (21.2±1.8% vs 45.8±2.8%; p<0.01). The db/db mice showed enhanced oxidative stress in cardiomyocytes as indicated by a significant increase in ROS production. Cardiac NAD(P)H oxidase activity, lipid peroxidation, and oxidized glutathione were also increased in db/db mice compared to nondiabetic control animals. Tadalafil treatment in db/db mice suppressed oxidative stress, attenuated myocardial expression of pRac1 and gp91phox, and also preserved the loss of Δψm in cardiomyocytes after SI/RO. In conclusion, these results demonstrate that chronic treatment with tadalafil attenuates oxidative stress and improves mitochondrial integrity while providing powerful cardioprotective effects in type 2 diabetes.

Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after heterologous expression in Aspergillus nidulans

Neosartorya fischeri antifungal protein (NFAP) is a β-defensin-like peptide produced by the N. fischeri NRRL 181 isolate. In this study, we investigated the manifestation of the antimicrobial effect of NFAP via heterologous expression of the nfap gene in an NFAP-sensitive fungus, Aspergillus nidulans. Heterologous expression of the nfap gene was carried out in A. nidulans CS2902 using a pAMA1-based autonomous replicative vector construct. The effect of the produced NFAP on the germination of A. nidulans conidia was investigated by scanning electron microscopy (SEM), and by DAPI and Calcofluor white (CFW) staining. 2',7'-Dichlorodihydrofluorescein diacetate staining and an Annexin V-FITC Apoptosis Detection kit were used to reveal the accumulation of reactive oxygen species (ROS) and the possible apoptotic, necrotic effect. The impact of mono- and divalent cations on the antimicrobial activity of NFAP was also examined. Transformants expressing the nfap gene showed reduced hyphal growth compared with the untransformed strain. This effect was absent in the presence of mono- and divalent cations (50 and 100 mM KCl, Mg(2)SO(4), Na(2)SO(4)). Delayed and abnormal germination was observed in the case of transformants. Conidia developed short branching germination tubes with swollen tips. The great majority of germinating conidia were destroyed after 8 h of cultivation, although a few survived and developed into abnormal hyphae. Damage in the organization of the cell wall, the destruction of chitin filaments and the accumulation of nuclei at the broken hyphal tips were detected by SEM, DAPI and CFW staining. The accumulation of ROS and more frequent apoptotic, necrotic events were also observed in the case of the NFAP-producing A. nidulans strain.

Influence of NADPH Oxidase Activity On the Reactive Oxygen Species Level in Human Leukemic Cells

AbstractAbstract 4801Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic and leukemic cells. Reactive oxygen species (ROS) level is highly regulated. This regulation involves antioxydative enzymes and it has been recently described that leukemic stem cells (LSC) overexpress glutathione peroxydase 3 (Herault O et al, J. Exp. Med, 2012). This overexpression is associated with a decrease in ROS level and p38MAPK inactivation. ROS level in leukemic cells could be also regulated by the activity of ROS producers, such as NADPH oxidase, known to catalyze an electron transfer from NADPH to oxygen producing superoxides which could generate other downstream ROS. The expression of this enzymatic complex (NOX family, 6 isoforms) has been established in the plasma cell membrane of normal CD34+ hematopoietic progenitors (Piccoli C et al, Biochem. Biophys. Res. Commun., 2007). The aim of this study was to decipher the expression of NADPH oxydase components in various human acute myeloid leukemia (AML)Different leukemic cell lines were used according FAB classification: KG1a (MO/M1), KG1 (M1), HL60 (M2), Kasumi 1 (M2), NB4 (M3), ML2 (M4), THP1 (M5), U937 (M5), MV4–11 (M5), K562 (M6). The cells were cultured (2.105 cells/mL, 37°C in 95% humidified air and 5% CO2) in RPMI 1640 with 20mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100mg/mL streptomycin. The expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, P22phox and P40phox, P47phox, P67phox, NOXO1, NOXA1 was quantified by RT-qPCR (Universal Probe Library, Roche). NOX2 and its regulatory subunits expression was quantified by SDS-PAGE and western-blot experiments. The effects of diphenylene iodonium (DPI), a specific NOX inhibitor, were evaluated by ROS quantification using dichlorodihydrofluorescein diacetate (DCF-DA) staining followed by fluorimetry and flow cytometry analyses. The cells were washed twice in the physiological saline buffer (PBS) without calcium and magnesium, then incubated in PBS complemented with 0.5M MgCl2, 0.9M CaCl2, 20mM glucose (Picciocchi A et al, J. Biol. Chem., 2011) with or without 20μM DPI for 1 hour. The cells were distributed at 106cells per 200μL well in 96 wells plates. DCF-DA (10μM) was added to quantify the ROS level (flow cytometry) and to monitor ROS production kinetic (fluorimetry).NOX family genes expression showed that phagocyte oxidase NOX2 is expressed in all leukemic cell lines. Conversely the NOX2 isoforms were not expressed, or very weakly expressed in leukemic cell lines (NOX3 in KG1a; NOX4 in K562; DUOX1 in KG1a, KG1; DUOX2 in KG1a, KG1, HL60). P22phox, the second cytochrome b558 component was also expressed in all cell lines, this expression being higher than NOX2. The cytochrome b558 components were more expressed in differentiated leukemic cells (granulocytic and monocytic) than in undifferentiated cells (KG1a, KG1). NOX2 regulatory subunits were expressed in all leukemic cell lines, the lower level (especially P40phox, P47phox) being observed in KG1a. Proteins quantification confirmed RNA results. Cytochrome b558 components and regulatory subunits were expressed in all cell lines with a higher level in differentiated leukemias. Interestingly, the regulatory subunits were not observed in KG1a cells. Functional flow cytometry and fluorimetry studies revealed a decrease in ROS production in DPI exposed leukemic cell lines. This effect was higher in monocytic cell lines than in granulocytic and undifferentiated leukemias.In conclusion, NADPH oxidases are present in the AML cell membrane, and NOX contribution to the ROS level is higher in differentiated cells than in immature leukemias. Altogether these results suggest that NADPH oxidase is constitutively active in leukemic cells and influences the ROS level, suggesting a role in the pathophysiology of AML. Disclosures:No relevant conflicts of interest to declare.

Effect of droplet vitrification on mitochondrial membrane potential and developmental competence in two-cell mouse embryos

The accelerated cooling rate associated with vitrification reduces injuries attributed to cryopreservation and improves the post-freezing developmental competence of vitrified embryos. In this study, embryos were vitrified and warmed and morphologically evaluated for their development to blastocysts. Survival rates between the fresh (96.7%±3.8%) and vitrified embryos (90.7%±5.1%) did not differ significantly (P >0.05). The mitochondrial membrane potential of fresh control cells measured by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanide iodide staining was similar to that of cryoprotected and vitrified embryos. Mitochondrial staining with rhodamine 123 did not differ among the fresh, cryoprotected, and vitrified embryos. Moreover, the distribution of H2O2, assessed by 2′,7′-dichlorodihydrofluorescein diacetate staining, did not differ among the groups. The results showed that the developmental rate did not differ significantly among the fresh (87.8%±11.3%), cryoprotected (83.2%±7.6%), and vitrified 2-cell embryos (75.8%±14.2%). The mean number of the inner cell mass (ICM), trophectoderm (TE), and apoptotic cells was counted and statistically compared, and although the number of ICM and TE was decreased in the cryoprotected and vitrified embryos, there were no significant differences among the groups (P >0.05). During the cultivation period, randomly selected blastocysts from each group were stained using either 4′,6-diamidino-2-phenylindole and bisbenzimide or the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling technique. The incidence of apoptosis appeared to be almost identical in all the groups. Droplet vitrification could subsequently lead to high survival and developmental rates of cryopreserved mouse embryos.

Abstract 17038: Long-Acting Phosphodiesterase-5 Inhibitor Tadalafil Attenuates Oxidative Stress and Protects Against Myocardial Ischemia/Reperfusion Injury in Type 2 Diabetic Mice

Background: Diabetic patients exhibit increased risk for the development of cardiovascular diseases. Recent findings from our laboratory suggest that phosphodiesterase-5 (PDE-5) inhibitor, sildenafil, restores NO signaling and protects against ischemia/reperfusion injury. In this study, we determined the effect of long-acting PDE-5 inhibitor, tadalafil (TAD), on type 2 diabetes associated complications in db/db mice. Methods and Results: Adult male db/db mice (n=24/group) were randomized to receive: DMSO (10%, 0.2 ml, i.p.) or TAD (1 mg/kg, i.p) for 28 days. The mice were sacrificed and blood was collected for assessment of biochemical parameters. Cardiomyocytes were isolated from the hearts and subjected to 40 min simulated ischemia followed by 1 h of reoxygenation. Dichlorodihydrofluorescein diacetate and JC-1 staining were used to measure ROS generation and mitochondrial membrane potential (Δψm), respectively. A second subset of mice hearts were subjected to 30 min global ischemia followed by 60 min reperfusion in Langendorff mode. Infarct size was measured using computer morphometry of tetrazolium stained sections. A third subset of mice hearts were used for the estimation of lipid peroxidation, GSSH/GSH and the expression of myocardial pAKT and pAMPK by Western blot. TAD treatment reduced the body weight (48.5±1.5 g vs 51.9±1.3 g), fasting plasma glucose levels (466.1±41.4 mg/dl vs 567.7±29.7 mg/dl), insulin (2.97±0.67 ng/ml vs 14.42±3.15 ng/ml; p&lt;0.02) and triglycerides (91.2±12.3 mg/dl vs 190.5±38.5 mg/dl; p&lt;0.05) as compared to control db/db mice. TAD-treated mice also showed reduced infarct size compared to the control db/db mice (21.2±1.8% vs 45.8±2.8%; p&lt;0.01). Control db/db mice demonstrated enhanced oxidative stress as indicated by significantly increased ROS production, lipid peroxidation and GSSH/GSH ratio which were attenuated in TAD-treated mice. Furthermore, TAD treatment preserved the loss of Δψm and enhanced the expression of myocardial pAKT and pAMPK compared to the control (n=4/group; p&lt;0.05). Conclusion: Chronic treatment with TAD attenuates oxidative stress and improves mitochondrial integrity while providing powerful cardioprotective effects through Akt-AMPK signaling in type 2 diabetes.

Assessment of the involvement of oxidative stress and Mitogen-Activated Protein Kinase signaling pathways in the cytotoxic effects of arsenic trioxide and its combination with sulindac or its metabolites: sulindac sulfide and sulindac sulfone on human leukemic cell lines

The purpose of the study was to characterize the involvement of reactive oxygen species (ROS) in mediating the cytotoxic effects of arsenic trioxide (ATO) in combination with sulindac or its metabolites: sulfide (SS) and sulfone (SF) on human leukemic cell lines. Jurkat, HL-60, K562, and HPB-ALL cells were exposed to the drugs alone or in combinations. Cell viability was measured using WST-1 or XTT reduction tests and ROS production by dichlorodihydrofluorescein diacetate staining (flow cytometry). Modulation of (a) intracellular glutathione (GSH) level was done by using L: -buthionine sulfoximine (BSO) or diethylmaleate (DEM), (b) NADPH oxidase by using diphenyleneiodonium (DPI), and (c) MAP kinases by using SB202190 (p38), SP600125 (JNK), and U0126 (ERK) inhibitors. ATO cytotoxicity (0.5 or 1 μM) was enhanced by sulindacs, with higher activity showed by the metabolites. Strong cytotoxic effects appeared at SS and SF concentrations starting from 50 μM. The induction of ROS production seemed not to be the major mechanism responsible for the cytotoxicity of the combinations. A strong potentiating effect of BSO on ATO cytotoxicity was demonstrated; DEM (10-300 μM) and DPI (0.0025-0.1 μM; 72 h) did not influence the effects of ATO. Some significant decreases in the viability of the cells exposed to ATO in the presence of MAPK inhibitors comparing with the cells exposed to ATO alone were observed; however, the effects likely resulted from a simple additive cytotoxicity of the drugs. The combinations of ATO with sulindacs offer potential therapeutic usefulness.

Bone Marrow Mesenchymal Stromal Cells Regulate the Metabolism of H2O2 In Human Leukemic Cells.

AbstractAbstract 1058Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic cells and it has been recently established by the Guy Sauvageau's group (Institute for Research in Immunology and Cancer, MontreÌal, QC) that glutathione peroxydase 3 (GPx-3) promotes competitiveness of Hoxa9-Meis1 induced leukemic stem cells in which Gpx3 overexpression is concomitant of a decrease in H2O2 level and inactivation of p38 MAPK (Herault O et al, ASH annual meeting, 2010 - submitted). Leukemic cells located in the bone marrow (BM) are interacting with a microenvironment which plays a crucial role in the development and progression of leukemia. Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to the different hematopoietic microenvironmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells). The aim of our study was to evaluate the effects of MSC-contact on the GPx-3/H2O2/p38 MAPK axis in human leukemic cells and to assess the cell cycle changes associated with the modification of H2O2 metabolism.BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery, following a procedure approved by the local ethical committee. Nucleated cells harvested from the iliac crest were seeded (5.104 cells per cm2) in α-MEM supplemented with 10% fetal calf serum (FCS), 20 mmol/l L-glutamine, and 100 units/mL penicillin G. Cells were incubated at 37°C in 95% humidified air and 5% CO2. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA), and the cells were resuspended in culture medium and seeded at 103 cells per cm2 (passage 1 - P1). BM MSCs were used at P2 in all experiments. The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described (Delorme et al, Blood 2008, 111:2631). The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. KG1a cells were seeded at 1.5 105 cells/cm2 and cocultured with MSCs for 72 h. We have defined two distinct localizations of leukemic cells relative to MSC layer: those in supernatant (non-adherent cells) and cells adhering on the surface of MSCs. The expression of antioxydative enzymes, H2O2 level, p38 MAPK activation (T180/Y182), cell cycle, proliferation and immunophenotype of these two cell fractions were evaluated at day 0 and day 3. The expression of SOD1, SOD2, SOD3, CAT, TXN, TXN2, GLRX, GLRX2, GLRX3, GLRX5, PRDX, PRDX2, PDRX3, PRDX4, PRDX5, PRDX6, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GSR antioxydative genes and CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal ProbeLibrary, Roche). SDS-PAGE and western-blot experiments were realized to quantify GPx-3 expression and p38 MAPK activation. Flow cytometry studies were performed: (a) to quantify H2O2 level by dichlorodihydrofluorescein diacetate (DCF-DA) staining; (b) to analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated anti-phospho-histone H3 (ser10) antibodies; (c) to track the cell divisions with carboxyfluorescein diacetate N-succinimidyl ester (CFSE).Supernatant of MSCs did not modify the GPx-3/H2O2/p38 MAPK axis in KG1a cells. Conversely, when compared with cells in the supernatant of MSCs, adhering KG1a cells were characterized by the exclusive overexpression of GPX3 antioxydative gene, the induction of GPx-3 production, a major decrease in H2O2 concentration and the inactivation of p38 MAPK. These effects were concomitant of cell cycle inhibition: increase in G0 phase, decrease in S and M phase, overexpression of CDKN1A and reduced mitotic activity (CFSE).Altogether these findings suggest that the bone marrow microenvironment plays a key role in the regulation of the oxidative metabolism of leukemic cells by promoting the inhibition of the H2O2/p38 MAPK axis via the induction of GPx-3. Modulation of the GPx-3/H2O2/p38 MAPK pathway by targeting of microenvironmental interactions in leukemia may have clinical relevance and it will be important to verify if these results can be extended in vivo to other models of human leukemias. Disclosures:No relevant conflicts of interest to declare.

Radioprotective effect of geraniin via the inhibition of apoptosis triggered by γ-radiation-induced oxidative stress

The radioprotective effect of geraniin, a tannin compound isolated from Nymphaea tetragona Georgi var. (Nymphaeaceae), against γ-radiation-induced damage was investigated in Chinese hamster lung fibroblast (V79-4) cells. Geraniin recovered cell viability detected by MTT test and colony formation assay, which was compromised by γ-radiation, and reduced the γ-radiation-induced apoptosis by the inhibition of loss of the mitochondrial membrane potential. Geraniin protected cellular components (lipid membrane, cellular protein, and DNA) damaged by γ-radiation, which was detected by lipid peroxidation, protein carbonyl formation, and comet assay. Geraniin significantly reduced the level of intracellular reactive oxygen species generated by γ-radiation, which was detected using spectrofluorometer, flow cytometer, and confocal microscope after 2',7'-dichlorodihydrofluorescein diacetate staining. Geraniin normalized the superoxide dismutase and catalase activities, which were decreased by γ-radiation. These results suggest that geraniin protects cells against radiation-induced oxidative stress via enhancing of antioxidant enzyme activities and attenuating of cellular damage.

Celastrol Attenuates Hypertension-Induced Inflammation and Oxidative Stress in Vascular Smooth Muscle Cells via Induction of Heme Oxygenase-1

The aim of this study was to investigate the potential beneficial effects of celastrol, a compound with anti-inflammatory and antioxidant properties, on vascular smooth muscle cells (VSMCs) under hypertensive conditions. Hypertension was induced in rats by fructose feeding. Hypertensive rats were injected with celastrol, and systolic blood pressure (SBP) and diastolic BP (DBP) were monitored by the tail-cuff method. Insulin sensitivity in animals was measured by glucose tolerance test (GTT). Serum levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay. Real-time reverse transcription-PCR and western blot were applied to quantify mRNA and protein levels in tissues and primary cultured VSMCs. Generation of reactive oxygen species (ROS) was measured using lucigenin chemiluminescence for tissue homogenates and dichlorodihydrofluorescein diacetate staining for VSMC cells. Celastrol decreased both SBP and DBP while improving insulin sensitivity in fructose-induced hypertensive rats. Celastrol also inhibited vascular and cardiac hypertrophy. Hypertension augmented circulating and mRNA levels of inflammatory cytokines, and celastrol treatment suppressed their induction. Celastrol also blocked activation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and Akt signaling both in vivo and in vitro. More importantly, celastrol increased heme oxygenase-1 (HO-1) expression and activity, whereas zinc protoporphyrin 9 (ZnPP9), a HO-1 inhibitor, partially abolished the beneficial effects of celastrol on hypertensive rats and VSMCs. Finally, ROS generation in tissue homogenates and in VSMCs was reduced by celastrol. These findings suggest that celastrol attenuates hypertension-induced inflammation and oxidative stress in VSMCs via HO-1 induction, and this compound may therefore serve as a novel drug to treat hypertension.

Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial apoptosis

The mechanisms mediating hyperglycemia-mediated myocardial cell death are poorly defined. Since elevated flux through the hexosamine biosynthetic pathway (HBP) is closely linked with the diabetic phenotype, we hypothesized that hyperglycemia-mediated oxidative stress results in greater O-GlcNAcylation (HBP end product) of the proapoptotic peptide BAD, thereby increasing myocardial apoptosis. H9c2 cardiomyoblasts were exposed to high glucose (33 mM) +/- HBP modulators +/- antioxidant treatment for 5 days vs. matched controls (5.5 mM), and we subsequently evaluated apoptosis by immunoblotting, immunofluorescence staining, and caspase activity measurements. In vitro reactive oxygen species (ROS) levels were quantified by 2',7'-dichlorodihydrofluorescein diacetate staining (fluorescence microscopy and flow cytometry). We determined total and BAD O-GlcNAcylation, respectively, by immunoblotting and immunofluorescence microscopy. The current study shows that high glucose treatment of cells significantly increased the degree of apoptosis. In parallel, overall O-GlcNAcylation, BAD O-GlcNAcylation, and ROS levels were increased. HBP inhibition and antioxidant treatment attenuated these effects, while increased end product levels exacerbated it. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and colocalization and found increased dimerization in cells exposed to hyperglycemia. Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress and increased HBP activation and BAD O-GlcNAcylation in H9c2 cardiomyoblasts. Since greater BAD-Bcl-2 dimerization increases myocardial apoptosis, this pathway may play a crucial role in diabetes-related onset of heart diseases.