Dihydrodichlorofluorescein Diacetate Research Articles

Anti-cancer Effect and Active Phytochemicals of Houttuynia cordata Thunb. against Human Breast Cancer Cells.

Houttuynia cordata Thunb (HCT) is a medicinal herb used in Southeast Asia. Aim of this work: This study aimed at investigating the cytotoxicity of this plant extract and fractions towards human breast cancer MDA-MB-231 and MCF-7 cells. HCT's phytoactive compounds are determined. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of cell death was measured by staining with annexin V-FITC and propidium iodide (PI) employing flow cytometry technique. The oxidative stress was measured by using 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE+) fluorescent probes and using a fluorescence microplate reader. HCT phytochemicals were characterized by high performance liquid chromatography (HPLC). The proliferation of MDA-MB-231 and MCF-7 cells was dramatically decreased by the crude extract and individual fraction of HCT. Ethyl acetate was the solvent fraction with the highest toxicity against MCF-7 cells, followed by dichloromethane, crude, and hexane fractions, respectively, whereas in MDA-MB231 cells, dichloromethane, crude, hexane, and ethyl acetate fractions each had the strongest impact, respectively. The methanol fraction had no effect on either cell line up to 200 μg/ml. The extract and fractions were less harmful to the NIH3T3 normal murine fibroblast cell line. The mode of both cell death was apoptosis evidenced by the increase of cell population stained with annexin V-FITC and PI. The fluorescence probes of both DCFH-DA and DHE in MDA-MB-231 cell line were enhanced. Phenolic acids included chlorogenic acid (CA), gallic acid (GA), transcoumaric acid (TCA), vanillic acid (VA), and syringic acid (SA), as well as flavonoids like quercetin and rutin, were identified as the active phytochemicals in the crude and fractions by using HPLC method. MDA-MB-231cells underwent apoptosis via oxidative stress when induced with HCT hexane fraction. Phenolic acids and flavonoids were identified in HCT's extract and fractions.

Metformin promotes apoptosis of A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio and ROS levels

Purpose: To investigate the influence of metformin on apoptosis of pulmonary carcinoma cells (A549), and the associated mode of action.Methods: Pulmonary carcinoma cells in logarithmic growth phase were treated with graded concentrations of metformin, and the anti-proliferative and apoptotic effects of the drug were measured using MTT assay and flow cytometry, respectively. The levels of reactive oxygen species (ROS) in A549 cell suspension were determined with 2, 7- dihydrodichlorofluorescein diacetate (DCFH-DA) assay. The expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), mammalian target of rapamycin (mTOR), and bax/bcl-2 ratio were measured using Western blotting and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).Results: Metformin significantly promoted A549 cell apoptosis, but suppressed its proliferative potential in a dose- and time-based fashion. The levels of ROS, superoxide anion and MDA in A549 cells were significantly and dose-dependently increased by metformin (p < 0.05). Moreover, metformin markedly upregulated the mRNA and protein expressions of p-AMPK as well as bax/bcl-2 ratio, but had no impact on the expression level of mTOR (p < 0.05).Conclusion: Metformin promotes apoptosis in A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio, and ROS levels, and hence may play a role in lung cancer therapy.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2′,7′-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Effect of Hyperbaric Oxygen Therapy on Acute Liver Injury and Survival in a Rat Cecal Slurry Peritonitis Model.

Background: The effects of hyperbaric oxygen therapy (HBOT) in sepsis remain unclear. This study evaluated its effects on acute liver injury and survival in a rat model. Methods: Cecal slurry peritonitis was induced in male rats, which were then randomly allocated into the HBOT and control groups. In the survival experiment, six 90 min HBOT sessions (2.6 atmospheres absolute 100% oxygen) were performed over 48 h; the survival rate was determined 14 days after sepsis induction. In the acute liver injury experiment, three HBOT sessions were performed, followed by liver and plasma harvesting, 24 h after sepsis induction. Serum levels of alanine aminotransferase (ALT), interleukin (IL)-6, and IL-10 were measured, and the hepatic injury scores were determined. Reactive oxygen species (ROS) generation was detected by 2′,7′-dihydrodichlorofluorescein diacetate (H2DCF-DA) assay. Western blot assays assessed protein kinase B (Akt), phosphorylated-Akt (p-Akt), glycogen synthase kinase (GSK)-3β, phosphorylated-GSK-3β, and cleaved caspase-3 levels. Results: Survival in the HBOT group (57.1%) was significantly higher than that in the controls (12.5%, p = 0.029), whereas IL-6, IL-10, and ALT levels were significantly lower in the HBOT group. The ROS generation was significantly inhibited to a greater extent in the HBOT group than in the control group. Additionally, in the HBOT group, the p-Akt and p-GSK-3β increased significantly and cleaved caspase-3 levels decreased significantly. Conclusions: HBOT showed a beneficial effect on acute liver injury and rat survival by enhancing the Akt signaling pathway and decreasing apoptosis.

Glutamate-Cysteine Ligase Catalytic Subunit Attenuated Hepatitis C Virus-Related Liver Fibrosis and Suppressed Endoplasmic Reticulum Stress

The study aimed to clarify the role and molecular mechanism of glutamate-cysteine ligase catalytic subunit (GCLC) in modulating Hepatitis C virus (HCV)-related liver fibrosis. Twenty patients with HCV-related liver fibrosis and 15 healthy controls were enrolled. Differentially expressed plasma mRNAs were detected by digital gene expression profile analysis and validated by qRT-PCR. Hepatic histopathology was observed by H&E and Masson stained liver sections. The mRNA and protein expression of GCLC, endoplasmic reticulum (ER) stress markers, and inflammatory and fibrogenic factors were detected in liver tissues from patients with HCV-related hepatic fibrosis and HCV core protein-expressing LX-2. The GCLC-overexpressing LX-2 were established by transfecting puc19-GCLC plasmid. Then, glutathione and reactive oxygen species (ROS) levels were measured respectively by spectrophotometric diagnostic kit and dihydrodichlorofluorescein diacetate kit. GCLC were dramatically down-regulated in HCV-related fibrotic livers and activated HSCs, which companied with up-regulation of ER stress-related genes, including inositol-requiring 1 (IRE1) and glucose-regulated protein 78 (GRP78). Also, the proinflammatory and profibrogenic gene, including nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNFα), and transforming growth factor 1(TGFβ1), was highly upregulated. Overexpression of GCLC in hepatic stellate cells could suppress α-SMA and collagen I expression, produce hepatic GSH and reduce ROS, and down-regulate IRE1, GRP78, NF-κB, TNF-α, and TGFβ1 expression. GCLC was a negative regulatory factor in the development of HCV-related liver fibrosis and might be a potential therapeutic target for liver fibrosis.

Extracts of Moringa oleifera lack cytotoxicity and attenuate oleic acid-induced steatosis in an in vitro HepG2 model

Moringa oleifera Lam. has received much praise over its potential benefits to health and nutrition, however, not much literature is available to support claims of hepatic safety. The liver, as primary xenobiotic metaboliser, is subject to high levels of cytotoxicity. As such, hepatotoxicity has been a consistent hurdle during the drug discovery and development process. The aim of this study was to assess the in vitro hepatotoxic properties of both hot water and methanol extracts of M. oleifera leaves by assessing common mechanisms of cytotoxicity in the HepG2 cell line. Also, the ability of these extracts to protect against in vitro oleic acid-induced steatosis was investigated.Tentative phytochemical profiling was done by ultra-performance liquid chromatography coupled to a photodiode array detector and high-definition mass spectrometer. In vitro cytotoxicity was assessed by sulforhodamine B staining (cell density), dihydro-dichlorofluorescein diacetate cleavage (reactive oxygen species), monochlorobimane adduct formation (reduced glutathione), JC-1 ratiometry (mitochondrial membrane potential), nile red staining (fatty acids), Ac-DEVD-AMC cleavage (caspase-3/7 activity), thiobarbituric acid reactive species production (lipid peroxidation), chemiluminescence (adenosine triphosphate; ATP), propidium iodide distribution (cell cycle) and Annexin V-FITC dual staining (mode of cell death). The protective action of the extracts was determined using an oleic acid-induced model of steatosis.Phytochemical profiling tentatively identified several chemicals within the extracts. Viability assessment indicated slight cytotoxicity at 100 μg/mL after both the hot water and methanol extract exposure (approximately 8–14%), however, this was not reflected by cell density results. The cell cycle remained unperturbed, apart from a slight increase in G0/G1-phase. Although mitochondrial depolarisation was evident, especially after exposure to the methanol extract, it did not translate to cytotoxicity in the form of oxidative stress. Reactive oxygen species generation was only observed at 100 μg/mL hot water extract exposure (1.99-fold), with the rest of the values being below baseline. Slight lipid peroxidation (1.48-fold at 100 μg/mL) was observed after methanol extract exposure. The GSH was unaltered. Fatty acid levels reduced, especially after methanol extract exposure (0.43-fold at 100 μg/mL), which may be indicative of increased mitochondrial respiration. Caspase-3/7 activity and ATP were decreased slightly. Methanol extracts were particularly active in protecting cells from oleic acid-induced cytotoxicity and fatty acid accumulation, with a decrease of up to 27% and 4.1-fold at 3.2 μg/mL, respectively.Although the in vitro results suggest hepatic safety, further investigation is needed to ascertain the underlying mechanisms of cytotoxicity at concentrations above 100 μg/mL, particularly the effects on mitochondrial membrane stability. Although mitochondrial membranes were depolarised, this may assist with the reduction of fatty acid levels and offer protection against oleic acid-induced steatosis.

Phytochemical-induced reactive oxygen species and endoplasmic reticulum stress-mediated apoptosis and differentiation in malignant melanoma cells

BackgroundPhytochemicals are derived from plants, vegetables and daily products and exert chemopreventive effects. Malignant melanoma is highly metastatic, and melanoma patients can develop chemotherapeutic resistance against conventional melanoma therapies. MethodsIn the present study, we investigated the anti-cancer effect of the phytochemicals kaempferol (Kaem), genistein (Gen), and 3′3-diindolylmethane (DIM) on melanoma cell viability. We also evaluated the altered expression of cell cycle-related genes. We verified the production of intracellular reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress at both the protein and cellular level using a western blot, TUNEL assay, and Dihydrodichlorofluorescein diacetate (DCF-DA) assay. ResultsTreatment of A375SM melanoma cells with phytochemicals resulted in inhibition of cell growth. Treatment with phytochemicals increased the gene expression of p21 and decreased the gene expression of cyclin E and/or cyclin B. The three phytochemicals activated the ROS-p38-p53 apoptotic pathway by increasing the level of phosphorylated p38 MAPK and p53, and they activated the ER stress-mediated apoptotic pathway by increasing the level of phosphorylated eIF2α and C/EBP homologous protein (CHOP). Both the ROS-p38-p53 and ER stress-mediated pathway induced the mitochondrial apoptotic pathway by attenuating Bcl-2 expression and upregulating BAX. Detection of morphological changes demonstrated that Kaem and Gen can induce differentiation in A375SM cell line. ConclusionThese results indicate that phytochemicals are potentially useful in treatments for melanoma due to their ability to inhibit melanoma cell growth and division via the ROS and ER stress pathway.

Antifungal mode of action of macrocarpal C extracted from Eucalyptus globulus Labill (Lan An) towards the dermatophyte Trichophyton mentagrophytes.

BackgroundThe fresh leaves of Eucalyptus globulus Labill. (Lan An) have been used in Chinese medicine for many years to treat dermatomycosis. Macrocarpal C was isolated from this herb and identified as its major antifungal component by bioassay-guided purification. This study aims to investigate the antifungal activity of macrocarpal C against Trichophyton mentagrophytes, which can cause tinea pedis.MethodsFresh leaves of E. globulus were extracted with 95 % ethanol, and the resulting ethanolic extracts were dried before being partitioned with n-hexane. The n-hexane layer was then subjected to chromatographic purification to give macrocarpal C. The antifungal minimum inhibitory concentration (MIC) of macrocarpal C was determined using the standard M38-A2 method described by the Clinical Laboratory Standards Institute (CLSI). The mode of action of macrocarpal C was elucidated using three in vitro assays, including (1) a fungal membrane permeability test using SYTOX® Green; (2) a reactive oxygen species (ROS) production test using 5-(and-6)-carboxy-2′,7′-dihydrodichlorofluorescein diacetate as a cell-permeable fluorogenic probe; and (3) a DNA fragmentation test based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) detection. Terbinafine hydrochloride and nystatin were used as positive controls.ResultsThe suppression in the growth of T. mentagrophytes following its treatment with macrocarpal C was associated with an increase in the permeability of the fungal membrane (P = 0.0043 when compared to control); an increase in the production of intracellular ROS (P = 0.0063); and the induction of apoptosis as a consequence of DNA fragmentation (P = 0.0007).ConclusionThis study demonstrated that the antifungal action of macrocarpal C was associated with increases of membrane permeability, intracellular ROS and DNA fragmentation.

Abstract 915: Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells

Abstract BRB powder, extracts and metabolites have been shown to inhibit tumorigenesis in oral and other sites in animal models. Here we examined the effects of an anthocyanin-enriched BRB extract, and protocatechuic acid (PA), a major BRB anthocyanin metabolite, on mutagenesis and DNA adduct formation by DBP-dihydrodiol, and DBP diolepoxide (DBPDE) (the proximate and ultimate carcinogenic metabolites, resp. of DBP) in lacI rat oral fibroblasts. DBP was poorly metabolized to genotoxic metabolites by the oral fibroblasts, but DBP-dihydrodiol was mutagenic and produced DNA adducts. DBP-dihydrodiol (0.5 uM) induced a mutant fraction of 14.1 +/- 3.9 mutants/10E5 pfu in the target cII gene. This was reduced in a concentration-dependent fashion to 9.1 +/- 2.4 by 0.1-1uM PA– a 35% reduction. Mutagenesis by DBP-dihydrodiol was also reduced in a concentration-dependent fashion by BRB extract and at the highest concentration (20ug/ml) mutagenesis was reduced by 68%. The major DNA adduct produced by 0.5 uM DBP-dihydrodiol was anti-DBPDE-dA, as assessed by L.C.-M.S.-M.S. Its levels were reduced from 13.7 +/- 1.2 to 7.8 +/- 1.9 adducts/10E6 dA by 3uM protocatechuic acid, and from 6.8 +/- 0.7 to 3.2 +/- 0.4 by 66 ug/ml BRB extract. The inhibitions were statistically significant. In contrast, no significant effect PA on mutagenesis by 140 nM DBPDE was observed, with mutant fractions similar to that of DBPDE (3.2 +/- 0.45 mutants/10E5 pfu) being obtained over a range of 0.1 - 1 uM PA. Similarly 10-50 ug/ml BRB extract had no effect on DBPDE-induced mutagenesis. Consistent with these results, levels of total DBPDE adducts were unchanged over a range of PA concentrations, with the high dose of 1uM PA yielding, 4.6 +/- 0.7 adducts/10E5 nucleotides and 4.5 +/- 0.9 without PA. Presumably the short halflife of DBPDE precludes detoxification, or scavenging of DBPDE. The effects of PA and BRB extract on oxidative stress in a human oral cancer cell line (SCC 1483, retromolar trigone) were also examined. The redox sensitive fluorescent probe, dihydrodichlorofluorescein diacetate (DHDCF) was added to the cells at 5uM and BRB (0.5-5 ug/ml) or PA (0.02-0.2 uM) was added shortly afterwards. 2 hr later all doses of BRB extract and PA inhibited the oxidation of DHDCF to its fluorescent product, in a dose-dependent manner. At 5ug/ml BRB extract the increase in fluorescence was reduced from 304 +/- 3.3 to 177 +/- 7.1 relative fluorescence units (RFU) and at 0.2 uM PA the increase was reduced to 224 +/- 25 RFU. In summary, BRB extract and PA inhibited mutagenesis and DNA adduct formation by DBP-dihydrodiol, but not DBPDE. BRB extract and PA were also effective inhibitors of oxidative stress. These mechanisms may a play a role in the protective effects of BRB on oral and other cancers in experimental animals. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Seungjin Kim, Ying Zhou, Wieslawa Kosinska, Ana-Vera Golgotiu, Shantu Amin, Gary D. Stoner, Yuan-Wan Sun, Kun-Ming Chen, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2015-915

Abstract 3872: Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions

Abstract Zinc oxide nanoparticles (ZnO-NP) are used in a variety of applications including cosmetics and pharmaceuticals. The common utilization of ZnO-NPs results in an increasing environmental release with unknown health effects. Recent studies have shown that ZnO-NP exposure cause cellular toxicity including DNA-damages. Discussed mechanisms are interactions of the ZnO-NP with intracellular structures and molecules after internalization, extracellular or intracellular dissociation of the particles, and the generation of ROS. In the present study we evaluated the genotoxic properties of ZnO-NP as well as the cellular DNA damage response in dependence of oxidative distress and ZnO-NP dissociation into Zn2+. For this purpose, we exposed the human epithelial cell line A549 to defined ZnO-NP (100μg/ml). Genotoxic effects of the particles were evaluated by Comet assay and γH2AX-foci, measuring DNA double-strand breaks (DNA-DSBs). Dissociation of ZnO-NP and the intracellular increase of Zn2+ were performed with spectrofluorimetric and cLSM measurements by the use of FluoZin3. Oxidative distress was determined by the compound dihydrodichlorofluorescein diacetate (H2DCF-DA). The Activation of the DNA damage response pathway was proven by analyses of H2AX-, ATM-, Chk1- and Chk2 phosphorylation, and p21, p53 up-regulation after ZnO-NP exposure by western blot or ELISA. To figure out, if the genotoxic potential of ZnO-NP is dependent of released Zn2+ or the generation of ROS we inhibited oxidative distress by the antioxidant N-acetyl-L-cysteine (NAC) and Zn2+ were extracellular chelated by diethylene triamine pentaacetic acid (DTPA). We found a fast extracellular dissociation of ZnO-NP and a quick intracellular increase of Zn2+. Furthermore, we ascertained an activation of H2AX induced by ZnO-NP; this phosphorylation is totally inhibited by DTPA. ZnO-NP activated the DSB-damage response indicated by ATM-, Chk2-phosphorylation as well as an up-regulation of p53 and p21. Pretreatment with NAC scavenging ROS leads to a reduced and Zn2+ complexation to no activation of ATM-Chk2-DNA damage pathway. The ATR/Chk1 pathway was not activated by ZnO-NP. We demonstrated ZnO-NP induced DNA-damages and the activation of the ATM/Chk2 DNA damage response pathway but not the ATR/Chk1 pathway, indicating that ZnO-NP exposure induces DNA-DSBs. Scavanging of ROS leads to a slight reduction of DNA-DSBs but extracellular Zn2+ compelxation to a complete impairment suggesting that the mechanism of ZnO-NP genotoxicity is not mediated via ROS-generation but rather a direct interaction of dissociated Zn-ions with the DNA. Citation Format: Julia Heim, Nawaz Muhammad Tahir, Anke Kaltbeitzel, Volker Mailänder, Hartmut Lüddens, Wolfgang Tremel, Jürgen Brieger. Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2015-3872

Involvement of HDL receptor SR-BI-mediated vitamin e uptake in murine neural tube closure

Involvement of HDL receptor SR-BI-mediated vitamin e uptake in murine neural tube closure

Salinity-induced noise in membrane potential of Characeae Chara australis: effect of exogenous melatonin.

Salt sensitive Characeae Chara australis responds to 50 mM NaCl by a prompt appearance of noise in the trans-membrane potential difference (PD). The noise diminishes with time in saline and PD depolarization, leading to altered current-voltage characteristics that could be modeled with H(+)/OH(-) channels. Beilby and Al Khazaaly (JMB 230:21-34, 2009) suggested that the noise might arise from cooperative transient opening of H(+)/OH(-) channels. Presoaking cells in 10 μM melatonin over 24 h abolished the noise in some cells, postponed its appearance in others or changed its characteristics. As melatonin is a very effective antioxidant, we postulated opening of H(+)/OH(-) channels by reactive oxygen species (ROS). Measurement of ROS using dihydrodichlorofluorescein diacetate confirmed substantial reduction in ROS production in melatonin-treated cells in saline and sorbitol media. However, ROS concentration decreased as a function of time in saline medium. Possible schemes for activation of H(+)/OH(-) channels under salinity stress are considered.

Leishmania donovani Prevents Oxidative Burst-mediated Apoptosis of Host Macrophages through Selective Induction of Suppressors of Cytokine Signaling (SOCS) Proteins

One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.

Cyclosis-mediated transfer of H2O2 elicited by localized illumination of Chara cells and its relevance to the formation of pH bands

Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H2O2 produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H2O2, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H2O2 first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H2O2 released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H(+)/OH(-)-permeable channels in the plasma membrane.

Overexpression of glutathione peroxidase 4 prevents β-cell dysfunction induced by prolonged elevation of lipids in vivo

We have shown that oxidative stress is a mechanism of free fatty acid (FFA)-induced β-cell dysfunction. Unsaturated fatty acids in membranes, including plasma and mitochondrial membranes, are substrates for lipid peroxidation, and lipid peroxidation products are known to cause impaired insulin secretion. Therefore, we hypothesized that mice overexpressing glutathione peroxidase-4 (GPx4), an enzyme that specifically reduces lipid peroxides, are protected from fat-induced β-cell dysfunction. GPx4-overexpressing mice and their wild-type littermate controls were infused intravenously with saline or oleate for 48 h, after which reactive oxygen species (ROS) were imaged, using dihydrodichlorofluorescein diacetate in isolated islets, and β-cell function was assessed ex vivo in isolated islets and in vivo during hyperglycemic clamps. Forty-eight-hour FFA elevation in wild-type mice increased ROS and the lipid peroxidation product malondialdehyde and impaired β-cell function ex vivo in isolated islets and in vivo, as assessed by decreased disposition index. Also, islets of wild-type mice exposed to oleate for 48 h had increased ROS and lipid peroxides and decreased β-cell function. In contrast, GPx4-overexpressing mice showed no FFA-induced increase in ROS and lipid peroxidation and were protected from the FFA-induced impairment of β-cell function assessed in vitro, ex vivo and in vivo. These results implicate lipid peroxidation in FFA-induced β-cell dysfunction.

Abstract 2918: Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer.

Abstract MYC is a potent oncogene that transcriptionally regulates 15 percent of the genome and is found to be deregulated in approximately 70 percent of cancers (c-MYC), including breast cancer. Activation of c-MYC induces association between its required partners, MYC-associated factor X (MAX) or MAX dimerization protein (MAD), regulating pro-survival and pro-apoptotic pathways, respectively. c-MYC has proved to be a difficult molecular target of inhibition. Only recently have there been strategies providing a way to inhibit c-MYC-dependent tumors and its downstream targets. However, dietary factors like, long-chain fatty acids (LCFA), have been shown to change activity and expression levels of c-MYC in cell culture and animal models. LCFAs have also been reported to induce apoptosis through c-MYC. The present study evaluated whether changes in c-MYC expression or activity were responsible for induction of oxidative stress and apoptosis in response to docosahexaenoic acid (DHA, C22:6) supplementation in breast cancer. c-MYC mRNA and protein levels were determined in vitro in the breast cancer BT-474, MDA-MB-231, and the non-transformed MCF-10A cell lines. qPCR results showed increases in c-MYC mRNA expression upon PUFA treatment in both BT-474 and MDA-MB-231 cancer lines, but not in the MCF-10A non-transformed line. Protein analysis revealed no changes in total c-MYC expression, however total phosphorylation at Thr58 and Ser62 are significantly upregulated in a dose-response manner upon DHA supplementation in the BT-474 cell line. Remarkably, transcriptional activity of c-MYC is highly upregulated as well, suggesting that c-MYC activity increases in response to DHA treatment. Using dihydrodichlorofluorescein diacetate (H2-DCF-DA) assay, which measures intracellular reactive oxygen species (ROS) and oxidative stress, we found increases in response to DHA treatment in all cell lines. Finally, the increase in oxidative stress also resulted in an increase in apoptosis, which we suggest is through c-MYC activation. Further analysis of the c-MYC isoform status, association between MYC-MAX and MYC-MAD complexes, as well as inducers and inhibitors of c-MYC are currently underway to define specific targets where DHA is applying some of its anti-cancer effects. Ultimately, with a goal that this research will reveal dietary targets of DHA that could be used to enhance efficacy to current therapies. Citation Format: Michael Mouradian, Haviva Kobany, Christopher R. Douglas, Nam D. Hoang, Keith D. Kikawa, Ronald S. Pardini. Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2013-2918

The abilities of selenium dioxide and selenite ion to coordinate DNA-bound metal ions and decrease oxidative DNA damage

Several transition metals react with H2O2 and produce reactive oxygen species (ROS) responsible for oxidative damage linked to many diseases and disorders, and species that form coordination complexes with these metal ions show promise as antioxidants. The present study demonstrates that metal-mediated radical and non-radical oxidative DNA damage decreases when selenium dioxide (SeO2) and sodium selenite (Na2SeO3) are present. Radical-induced damage is associated with production of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), which arises from ROS generated at or near the guanine base, and the selenium compounds reduce Fe(II)-, Cr(III)- and Cu(II)-mediated radical damage to differing degrees based on the identity of the metal ion and the order in which the metals, selenium compounds and DNA are combined. Radical damage arising from Fe(II) and Cr(III) decreases substantially when they are pre-incubated with the selenium compounds prior to adding DNA. Non-radical damage is associated with oxidation of the adenine base in the presence of high H2O2 concentrations through an ionic mechanism, and this type of damage also decreases significantly when the selenium compounds are allowed to interact with the metal ions before adding DNA. Fluorescence studies using dihydrodichlorofluorescein diacetate (DCF-DA) to probe ROS formation indicate that the majority of the SeO2- and SeO32−-metal systems in combination with H2O2 (no DNA present) produce ROS to the same degree as the metal/H2O2 systems in the absence of the selenium compounds, suggesting that selenium–metal complexes react with H2O2 in a sacrificial manner that protects DNA from oxidative damage.

13 REACTIVE OXYGEN SPECIES IN STORED STALLION SEMEN

The quality of cooled semen doses for AI varies considerably between stallions. One of the factors affecting sperm quality may be the content of reactive oxygen species (ROS), which are known to affect fertility in some species. The objective of this study was to measure the ROS content of cooled stored stallion semen doses for AI as part of an evaluation of sperm quality and to correlate it with pregnancy rates. Ejaculates (3 per stallion) were collected from 14 stallions at a commercial stud farm and were extended in INRA 96 (IMV Technologies) as standard cooled semen doses for AI. After transporting the semen doses overnight to the laboratory at the Swedish University of Agricultural Sciences (Uppsala, Sweden) in an insulated container with a cold pack, aliquots were evaluated for sperm quality and ROS content by staining with hydroethidine and dihydrodichlorofluorescein diacetate and measuring fluorescence by flow cytometry (Guthrie and Welch 2006 J. Anim. Sci. 84, 2089–2100). Seven sperm sub-populations were quantified: superoxide positive or negative, living (S+L and S–L, respectively); superoxide positive, dead (S+D); and hydrogen peroxide positive or negative, living or dead (P+L, P–L, P+D, and P–D, respectively). Sperm motility was measured by computer-assisted sperm motility analysis (Sperm Vision, Minitube), membrane integrity by flow cytometry, and chromatin structure using the sperm chromatin structure assay (all assays described in Morrell et al. 2011 Theriogenology 76, 1424–1432). Pregnancy rates following AI with cooled semen doses from the same stallions were available later in the year. The effect of stallion was tested by ANOVA. Correlations were calculated between the proportions of sperm stained for S or P and other sperm quality parameters and also with pregnancy rates (SAS version 9.1, SAS Institute Inc., Cary, NC, USA). The effect of stallion was significant on all variables measured (P < 0.01). There were no significant correlations between percentages of S+L or P+L spermatozoa and progressive motility, membrane integrity, or DNA fragmentation index (%DFI). There was a trend for the proportion of P-L spermatozoa to be correlated with progressive motility (r = 0.51; P < 0.07) whereas the proportions of S+D and P–D were negatively correlated with progressive motility (r = –0.66 and –0.61; P < 0.02). The proportion of S+D and chromatin damage were negatively correlated (r = –0.54; P < 0.05). There was a trend for the proportions of S+D and P+D to be negatively related to the overall pregnancy rate (r = –0.52; P < 0.07, and r = –0.58; P < 0.05, respectively). The proportions of superoxide and hydrogen peroxide-containing live spermatozoa were not correlated to stallion fertility or other parameters of semen quality in stored semen samples. In contrast, a significant negative relationship was found between the proportion of dead spermatozoa producing superoxide radicals and progressive motility, and between dead spermatozoa producing superoxide radicals and chromatin damage. However, many factors contribute to sperm quality and fertility, with many interactions between them. More work is needed to unravel these different effects and determine which factors can be used to predict stallion fertility.

High-Content Imaging Technology for the Evaluation of Drug-Induced Steatosis Using a Multiparametric Cell-Based Assay

In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2′,7′-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.

Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling

We have shown before that constitutive DNA damage signaling represented by H2AX-Ser139 phosphorylation and ATM activation in untreated normal and tumor cells is a reporter of the persistent DNA replication stress induced by endogenous oxidants, the by-products of aerobic respiration. In the present study we observed that exposure of normal mitogenically stimulated lymphocytes or tumor cell lines A549, TK6 and A431 to metformin, the specific activator of 5'AMP-activated protein kinase (AMPK) and an inhibitor of mTOR signaling, resulted in attenuation of constitutive H2AX phosphorylation and ATM activation. The effects were metformin-concentration dependent and seen even at the pharmacologically pertinent 0.1 mM drug concentration. The data also show that intracellular levels of endogenous reactive oxidants able to oxidize 2',7'-dihydro-dichlorofluorescein diacetate was reduced in metformin-treated cells. Since persistent constitutive DNA replication stress, particularly when paralleled by mTOR signaling, is considered to be the major cause of aging, the present findings are consistent with the notion that metformin, by reducing both DNA replication stress and mTOR-signaling, slows down aging and/or cell senescence processes.