ROS Synthesis Research Articles

Abstract LB-212: Antioxidant therapy reprograms macrophages from protumor M2 to antitumor M1

Abstract Macrophages (Mϕ) have high plasticity and can polarize to two extremes, pro-inflammatory M1 Mϕ and immuno-suppressive M2 Mϕ. In triple negative breast cancer, tumor-associated Mϕ (TAM) have a pro-tumor M2-like phenotype and are associated with decreased survival. Thus, it is vital to develop treatments to reprogram TAM away from protumor M2 Mϕ toward antitumor M1 Mϕ. A recent study reported a lipid peroxide scavenger selectively inhibits M2 Mϕ markers and cytokines. However, little is known about the differences in ROS metabolism between M1 Mϕ and M2 Mϕ. Our hypothesis is pro-tumorigenic M2 Mϕ possess a distinct profile of pro- and anti-oxidant enzymes, as well as a different sensitivity to ROS modulation versus the anti-tumor M1 Mϕ. Our results show M2 Mϕ, derived from primary human monocytes, have a significant 20-30% reduction in intracellular ROS levels versus M1 Mϕ by DCFH and DHE staining. Extracellular ROS synthesis was dramatically reduced by ~70% in M2 Mϕ, in part due to reduced mRNA expression of Nox2, Nox5, and their accessory protein CYBA in M2 Mϕ. Another contributing factor to lower ROS levels is that M2 Mϕ have significantly increased Catalase and Gpx4 mRNA expression, as well as increased Cu/ZnSOD and Gpx1 protein and activity. These data suggest M2 Mϕ have increased ROS metabolism versus M1 Mϕ, which may provide an advantage in the oxidative tumor microenvironment. Furthermore, EcSOD re-expression in MB231, a triple negative breast cancer cell line, inhibited the MB231-mediated increase in M2 Mϕ markers and decrease in M1 Mϕ markers. We moved forward utilizing Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE), a small molecule SOD mimetic, shown to have anti-tumor effects. MnTE treatment increased M1 Mϕ markers, IL-12b and CD86, while concurrently decreasing M2 Mϕ markers, IL-10, CD163, and CD206. MnTE also inhibited the ability of M2 Mϕ to inhibit T cell activation suggesting ROS is required for M2 Mϕ polarization and function. Increasing ROS levels by addition of exogenous H2O2, lead to increased M2 markers and decreased M1 markers, which implicates the oxidative tumor microenvironment in promoting a M2-like TAM phenotype. These studies provide the rationale for antioxidant treatment of breast cancer to reprogram TAM from pro-tumorigenic M2-like TAM phenotype to a more tumoricidal M1-like phenotype. Citation Format: Brandon Griess, Kaustubh Datta, Melissa Teoh-Fitzgerald. Antioxidant therapy reprograms macrophages from protumor M2 to antitumor M1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2017-LB-212

Miltirone protects human EA.hy926 endothelial cells from oxidized low-density lipoprotein-derived oxidative stress via a heme oxygenase-1 and MAPK/Nrf2 dependent pathway

BackgroundOxidized low-density lipoprotein (ox-LDL) is an underlying cause of endothelial dysfunction, which is an early event in the pathogenesis of atherosclerosis. In our previous study, we established an ARE-driven luciferase reporter system and screened out several potential Nrf2 activators from Salvia miltiorrhiza Bunge. PurposeSince miltirone showed the most potent ARE-driven luciferase activity, the aim of this study was to test the protective role of miltirone against oxidative stress in endothelial cell and to investigate the underlying mechanistic signaling pathways. Study Design/MethodIn the present study, miltirone increased the expression of nuclear translocation and transcriptional activities of NF-E2-related factor 2 (Nrf2), which led to augmented expression of antioxidant-response element (ARE)-dependent heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1). Inhibition of Nrf2/HO-1 by RNA interference abolished miltirone-induced cytoprotective effects against ox-LDL, which suggested that Nrf2 and the downstream expression of HO-1 are required for the functional effects of miltirone. Ox-LDL-stimulated mitogen-activated protein kinase activation, ROS production, and miltirone dramatically inhibited synthesis of ROS, as well as decreased SOD and glutathione S-transferase (GST) in human EA.hy926 endothelial cells. ResultsMiltirone-induced Nrf2 and HO-1 expression was related to mitogen-activated protein kinase (MAPK) pathways. The activation of MAPK was partially dependent on the phosphorylation of the c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, but not P38 MAPK signaling. However, miltirone-induced Nrf2/HO-1 expression can only be effectively blocked by JNK inhibitor SP600125. ConclusionOur findings reveal that miltirone exerts protective functions on endothelial cells in response to ox-LDL-induced oxidative stress, and does so via Nrf2/HO-1, which provides novel insights into the antioxidant capacity of miltirone.

Circulating immune complexes of calves with bronchopneumonia modulate the function of peripheral blood leukocytes: In vitro evaluation

In this work we studied if circulating immune complexes (CIC) of calves with bronchopneumonia have the capacity to modulate function of peripheral blood leukocytes of healthy cattle.CIC of three month old calves (6 healthy and 6 diseased) were isolated by PEG precipitation. Peripheral blood mononuclear cells (MNCs) and granulocytes from healthy calves and cows were the CIC responder cells in in vitro tests.The most remarkable increase of adhesiveness to polystyrene and ROS synthesis (assessed by NBT test) was detected in cows' granulocytes stimulated with CIC of diseased calves. Results of MTT test showed that CIC of both healthy and diseased calves reduced granulocytes' viability. The strongest effect of inhibition of cows' granulocytes resulted from CIC of diseased calves. CIC only moderately reduced spontaneous viability of calves' MNCs. Again, the strongest effect of CIC isolated from diseased calves was observed. In contrast to the low impact of CIC on non-stimulated cells, their inhibitory effect on viability of mitogen stimulated MNCs was very strong. With CFSE assay we showed that both types of CIC stimulated spontaneous, but inhibited mitogen induced proliferation of calves' MNCs. Propidium iodide staining reviled that CIC increased apoptosis/necrosis of both non-stimulated and mitogen stimulated MNCs.CIC of both healthy and diseased calves modulated the function of peripheral blood MNCs and granulocytes, but a stronger effect of CIC of diseased calves was shown. The age of the donors (calves or cows) of the responder cells, and the activation state of these cells, were also of influence.

Cytotoxic effect of proanthocyanidins from a Mexican endemic blackberry (Rubus liebmannii) on the synthesis of ROS on two murine glioma cell lines

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion, and it indicates possible cellular damage. In tumor cells the accelerated metabolism produces a high number of ROS. Brain tumors represent the third cause of death in cancer patient's woldwide. Gliomas are the most frequents tumors of the neuroepitelial tissue, and they are graded on a scale from I to IV based on their malignancy. The alternative medicine offers some options of natural origin as possible therapeutic agents against neoplastic cells, such as proanthocyanidins extracted from berries and other fruits and grains. Here, the objective was to determine the cytotoxic effect of proanthocyanidins from a wild blackberry (R. liebmanii) from Mexico on two murine glioma cell lines (C6 and RG2), and determinate the formation of ROS. After 22 and 46 hours of incubation (1 × 105 cells/mL) at 37°C, the viability was determined with two concentrations of proanthocyanidins (0.5 y 100 μg/mL) extracts by 24 and 48 h, respectively, in 96 well plates. ROS concentration was determined with spectrophometric method using 2′,7′‐DCFH‐diacetate (DCFH‐DA). Our results show that the neoplastic cells has a dose dependent cytotoxic response and the major effect was at 48 hours of exposition at 100 μg/mL in RG2 (>80%) vs the control. The results obtained will allow us to suggest suitable compounds for the development of alternative treatment against tumors of glial origin.Support or Funding InformationPROFAPI2014, Universidad Autonoma de Sinaloa, Mexico

SIRT3 prevents bleomycin induced lung fibrosis in mice by blocking mitochondrial DNA damage and ROS synthesis

Background and objectiveTissue fibrosis is one of the most critical mechanisms in the pathogenesis of wide range of diseases. Fibrosis is also hallmark of organ dysfunction associated with aging. The process of fibrosis begins with transformation of fibroblast into myofibroblast, a cell which deposits extracellular matrix proteins and contributes to scar formation. Recently a group of NAD‐dependent deacetylases, called sirtuins, have been identified which are capable of regulating variety of cellular processes including extending lifespan of the organism. One of them is SIRT3, which is considered guardian of mitochondria. SIRT3 activation has been shown to ameliorate many aging‐associated diseases including development of metabolic syndrome, heart failure, hearing loss and cancer. SIRT3 activation has been also linked to longevity of the man. This study was undertaken to examine the role of SIRT3 in protecting the lungs from development of fibrosis.Methods and ResultsOne of the most important contributors of tissue fibrosis is activation of transforming growth factor‐β (TGFβ1) signaling. To determine the role of SIRT3 in the induction of fibrosis, we treated human fetal lung fibroblasts (IMR‐90) with TGFβ1 (10ng/ml) for 48 hours. The results showed that there was a significant increase in the expression of fibrotic markers, including smooth muscle alpha‐actin (α‐SMA), type I collagen and fibronectin in TGFβ1 treated cells. TGFβ1 treatment also caused depletion of endogenous SIRT3 levels, which correlated with increased ROS production, DNA damage and subsequent reduction in levels of OGG1, a major DNA glycosylase which hydrolyzes oxidized‐guanine (8‐Oxo‐dG) and protects the DNA from oxidative damage. We also found increased expression of inflammosome markers such as caspase‐1, and IL‐1β in TGFβ1 treated IMR‐90 cells. These changes were blocked when cells were infected with adenovirus expressing wild‐type SIRT3, but not the mutant, thus suggesting the ability of SIRT3 to block transformation of lung fibroblasts to myofibroblasts. To determine the anti‐fibrotic effect of SIRT3 in vivo, we induced lung fibrosis in mice by intra‐tracheal instillation of bleomycin for three weeks. As compared to wild‐type controls, SIRT3‐KO mice showed exacerbated fibrosis after bleomycin treatment, as measured by increased expression of fibrotic marker collagen‐1 and inflammasome marker caspase 1 and subsequent decreased levels of OGG1. However, the transgenic mice having whole body SIRT3 over expression were protected from bleomycin‐induced lung fibrosis.ConclusionThese data demonstrate that loss of mitochondrial SIRT3 promotes transformation of lung fibroblasts to myofibroblasts and development of lung fibrosis. Therefore, SIRT3 activation may be considered a medical therapy for the management of lung fibrosis.Support or Funding InformationThis study was supported by the NIH‐RO1 grants HL117041 and HL111455.

A simple, fast and inexpensive method to assess salt stress tolerance of aerial plant part: Investigations in the mandarin group

For grafted plants, salt stress tolerance of the aerial plant part is poorly documented. Thus, we developed a simple, fast and inexpensive method to identify tolerant genotypes. Twigs of 14 mandarin accessions that we previously analyzed as seedlings were cut in solution to prevent embolism and were then evaluated in salt stress condition for a week. Physiological parameters such as gas exchanges, leaf Cl− and Na+, as well as the presence of H2O2 and the activity of enzymes involved in ROS synthesis and detoxification processes were analyzed. One accession known to be tolerant as rootstock was shown to be sensitive with limited Cl− translocation from the solution to the shoot while sensitive accessions when grown as seedlings presented limited wilting symptoms and accumulated large leaf Cl− content. A model is proposed to explain the different strategies of the plant to cope with high toxic ion content. This method allows separation of the root compartment, where ion exclusion mechanisms may exist and have an impact on the salt stress tolerance of the whole plant.

The effect of cryptogein with changed abilities to transfer sterols and altered charge distribution on extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene transcription in Nicotiana tabacum

Cryptogein, a protein from oomycete Phytophthora cryptogea, induces a hypersensitive cell death in Nicotiana tabacum. We prepared a new series of cryptogein mutant proteins with altered abilities to bind sterols and with altered charge distribution in the proteins. The effect of the mutations on the cryptogein ability to induce plant defence mechanisms associated with hypersensitive cell death were examined. Our results with new mutants support the previous findings that the sterol binding does not influence synthesis of ROS, cytosol acidification and development of leaf necrosis as these events seem to be more likely affected by the charge distribution and the overall protein structure. This hypothesis was also applicable on other mechanisms involved in the execution of plant cell death such as the NO generation, the stimulation of lipid peroxidation (determination of malondialdehyde and hydroxy fatty acids levels) and LOX gene transcription. In addition, the ability to bind sterols was found to serve not only for pathogen utilisation in its own metabolism but also to have an important function for the destabilization of plant membrane facilitating the pathogen spread inside the plant tissue as well as intensively contributing to the development of plant cell death. Considering the insertion of charged amino acid residues in the protein structure, the change localized in the protein surface affected its biological activity more effectively than that change inside the protein cavity. Moreover, the insertion of negative charged amino acids influenced mainly the events involved in the early phase of defence reaction, while the positive residues affected especially the necrotic activity of cryptogein.

Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3.

Honokiol (HKL) is a natural biphenolic compound derived from the bark of magnolia trees with anti-inflammatory, anti-oxidative, anti-tumor and neuroprotective properties. Here we show that HKL blocks agonist-induced and pressure overload-mediated, cardiac hypertrophic responses, and ameliorates pre-existing cardiac hypertrophy, in mice. Our data suggest that the anti-hypertrophic effects of HKL depend on activation of the deacetylase SIRT3. We demonstrate that HKL is present in mitochondria, enhances SIRT3 expression nearly two-fold and suggest that HKL may bind to SIRT3 to further increase its activity. Increased SIRT3 activity is associated with reduced acetylation of mitochondrial SIRT3 substrates, MnSOD and OSCP. HKL-treatment increases mitochondrial rate of oxygen consumption and reduces ROS synthesis in wild-type, but not in SIRT3-KO cells. Moreover, HKL-treatment blocks cardiac fibroblast proliferation and differentiation to myofibroblasts in SIRT3-dependent manner. These results suggest that HKL is a pharmacological activator of SIRT3 capable of blocking, and even reversing, the cardiac hypertrophic response.

SIRT3 Blocks Doxorubicin Induced Cardiac Hypertrophic Response by Suppressing ROS Synthesis from Mitochondria

Background and ObjectiveDoxorubicin is widely used to treat different types of cancers. Most serious adverse effect of doxorubicin is cardiomyopathy. Here we investigated whether the mitochondrial deacetylase SIRT3 can attenuate doxorubicin induced cardiac hypertrophy in mice.Methods and ResultsDoxorubicin treatment induced increased ROS levels in cardiomyocytes, which was significantly reduced by the overexpression of SIRT3. Further, SIRT3 overexpression significantly decreased doxorubicin induced cardiomyocyte death. Doxorubicin also induced exacerbated mitochondrial DNA damage in SIRT3 knockout cardiac fibroblasts (CF) as well as in mouse embryonic fibroblasts (MEFs). These results suggest that SIRT3 is capable of reducing doxorubicin induced ROS synthesis from mitochondria, thereby blocking its deleterious effects. To examine the cardioprotective effects of SIRT3 from doxorubicin induced damage in vivo, we treated wild type and cardiac‐specific SIRT3 over‐expressing transgenic mice with a cumulative dose of 15mg/kg of doxorubicin. Mice expressing cardiac‐specific SIRT3 (Sirt3‐Tg) showed 33% reduced HW/BW ratio compared to control mice. Echocardiographic evaluation of hearts showed significantly reduced fractional shortening in control mice compared to Sirt3‐Tg mice (24.6 vs 34.7 %, P<0.05)). Further, Masson's trichrome staining showed significantly reduced fibrosis in doxorubicin treated Sirt3‐Tg mice, compared to controls, thus suggesting that SIRT3 is capable of blocking doxorubicin‐induced cardiac hypertrophic response.ConclusionOur study suggests that SIRT3 is an endogenous negative regulator of doxorubicin induced cardiac hypertrophic response.

Abstract 15445: The Sirtuin SIRT3 Blocks Doxorubicin-induced Cardiac Hypertrophic Response of Mice

Background and objective: Doxorubicin is a chemotherapeutic drug widely used to treat variety of cancers. One of the serious side effects of doxorubicin is its toxicity to the heart. Previously, we have shown that overexpression of SIRT3 blocks the hypertrophic response of the heart to agonist treatments. This study was undertaken to investigate whether SIRT3 can also attenuate the doxorubicin-induced cardiac hypertrophic response in mice. Methods and results: Neonatal rat cardiomyocytes overexpressed with SIRT3 and treated with doxorubicin (10μM) showed 28% reduced mean fluorescent intensity for CM-H 2 DCFDA dye, compared to mock infected control cells treated with doxorubicin, thus suggesting that SIRT3 was capable of blocking doxorubicin-induced ROS synthesis in cardiomyocytes. To examine the cardioprotective effects of SIRT3 in doxorubicin-induced cardiotoxicity in vivo ; we used a cumulative dose of 15mg/kg of doxorubicin for two different time points. One group of mice was treated intraperitoneally with 5mg/kg doxorubicin or an equal volume of saline every two weeks for a total of three doses. Transgenic mice having cardiac specific expression of SIRT3 (SIRT3-Tg) showed 33% reduced HW/BW ratio compared to control mice. Echocardiographic evaluation of hearts showed significantly reduced fractional shortening in control mice, compared to SIRT3-Tg mice (24.6 vs 34.7 %, P<0.05). SIRT3-Tg mice also showed significantly reduced fetal gene expression for ANF, βMHC and collagen-1 as determined by RT-PCR. Masson’s trichrome staining showed significantly reduced fibrosis in doxorubicin treated SIRT3-Tg mice compared to its control. Furthermore, electron microscopic analysis showed preserved mitochondrial and sarcomeres structures in doxorubicin treated SIRT3-Tg hearts, whereas in wild-type hearts these structures were highly disorganized. Second group of mice that received 15mg/kg dose for two weeks also showed similar results. Contrary to this, whole body SIRT3 knockout mice showed exacerbated cardiac hypertrophic response compared to wild-type mice in response to doxorubicin treatment. Conclusion: These results demonstrated that SIRT3 is an endogenous negative regulator of doxorubicin-induced cardiac hypertrophic response.

Reactive oxygen species regulates expression of iron–sulfur cluster assembly protein IscS of Leishmania donovani

The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron–sulfur cluster (ISC) system that serves as a sulfur donor for Fe–S clusters biogenesis. Fe–S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe–S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe–S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe–S clusters/proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H2O2, menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe–S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani.

The alternative Medicago truncatula defense proteome of ROS-defective transgenic roots during early microbial infection.

ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation.

The SIRT3 deacetylase blocks cardiac fibrosis by targeting TGFβ1‐Smad3 signaling (1152.6)

Cardiac fibrosis after myocardial injury is a major challenge in the medicine today to preserve heart function. At the cellular level, activation of TGFβ1 signaling is considered a major contributor of cardiac fibrosis. However, the underlying mechanism of TGFβ activation is not yet completely understood. SIRT3 is a mitochondrial deacetylase which is activated by exercise and calorie restriction. SIRT3 regulates variety of mitochondrial functions, including metabolism, ROS synthesis and apoptosis. SIRT3KO mice spontaneously develop cardiac fibrosis with age. This study was undertaken to test the hypothesis whether SIRT3 deficiency leads to transformation of cardiac fibroblasts (CF) into myofibroblasts (myoFB) and excessive synthesis of extracellular matrix (ECM). CF obtained from adult rat hearts were subjected to SIRT3 knockdown (KD) by using siRNA. Differentiation of CF to myoFB was determined by increased expression of myoFB markers, like smooth muscle α‐actin, fibronectin and collagen1. The results showed robust expression of myoFB markers in SIRT3KD cells. To test whether SIRT3 deficiency contributed to this phenotype, we re‐expressed SIRT3 and found that this intervention changed back myoFB phenotype of SIRT3‐deficient cells to normal CF. Similar results were obtained when TGFβ1‐induced myoFB were over expressed with SIRT3, thus demonstrating the ability of SIRT3 to block transformation of CF into myoFB and synthesis of excessive ECM. Mechanistic studies showed increased levels of TGFβ1 and Smad3 in SIRT3KD cells, which were resistant to use of the anti‐oxidant, N‐acetyl cysteine. These data thus demonstrated an anti‐fibrotic role of SIRT3, which is independent of its ability to regulate cellular oxidative stress.Grant Funding Source: NIH‐NHLBI

Immune effector activities in challenged rohu, Labeo rohita after vaccinating with Aeromonas bacterin

The present study assessed the efficacy of formalin inactivated Aeromonas vaccines by determining the cellular immune-effector activities such as production of oxidative radical (ROS) and nitrite and lymphocyte proliferation in challenged rohu, Labeo rohita upon vaccination with Aeromonas hydrophila (AH), Aeromonas caviae (AC) and a cocktail of both (CKTL). The relative percentage survival was much higher in AH group (68.89) than in the AC (28.89) and CKTL (20.00) groups. The vaccinated L. rohita showed high immune-effector activities in the first 15days post-vaccination, which subsequently lowered at different post-challenge periods. The vaccination activated the synthesis of ROS in leucocytic cells of all vaccinated groups up on challenge with A. hydrophila-C36 strain as immediate protection against the bacteria and the activities of blood phagocytes progressively waned as the disease process progressed. The production of nitrite was higher in AH group when stimulated with OMP antigen than that of somatic antigen and LPS; while in AC group, both OMP and somatic antigen stimulation resulted in higher nitrite production. On the other hand, the cocktail group showed higher nitrite production upon somatic antigen stimulation followed by OMP and LPS. A high level of non-specific lymphocyte proliferation was observed on the 10th day post-vaccination and the 5th day post-challenge periods and an antigen specific proliferation when stimulated with somatic and OMP antigens on the 15th day post-challenge. The stimulation by specific somatic and OMP antigens resulted in higher HK leucocyte proliferation in A. hydrophila vaccinated group. The experiment revealed that the highest protection was generated in the AH group compared to the other two groups and this might be explained by the specific cellular immune response generated in this vaccinated group against the specific antigen as assessed at 15days post-challenge.

27-hydroxycholesterol and the expression of three estrogen-sensitive proteins in MCF7 cells

The principal aim of this study was to analyze in estrogen receptor-positive MCF7 cells the response of three estrogen-dependent proteins to 27-hydroxycholesterol (27OHC), a major circulating cholesterol metabolite. Immunofluorescence, immunoblotting and immunogold labelling analyses of MCF7 cells exposed for up to 72 h to 2 nM estradiol (E2) or to 2 µM 27OHC demonstrated similar responses in the expression of MnSOD and ERβ compared to the non-stimulated cells. Thus, the results confirm 27OHC's function as a novel selective estrogen receptor modulator (SERM). The epithelial to mesenchymal transition (EMT), observed in MCF7 cells stimulated for longer than 48 h with 2 µM 27OHC, was accompanied by lower immunoreactive levels of nuclear FOXM1 in comparison to E2-treated cells. The results presented in this study are discussed taking into consideration the relationship of hypercholesterolemia, 27OHC production, ROS synthesis and macrophage infiltration, potentially occurring in obese patients with ERα-positive, infiltrated mammary tumors.

The activity of G-ROS and the predominant role of Type II reaction in the photodynamic therapy using 9-hydroxypheophorbide-α for HeLa cell lines

Photodynamic therapy (PDT) is a treatment modality that destroys the tumor. It activates the photosensitizer with the light of a specific wavelength, where the light is well absorbed by the photosensitizer, thus causing a fatal injury and thereby leading to a tumor necrosis. To date, a hematoporphyrin-derived photosensitizer has been widely used. It is disadvantageous, however, in that it causes a long-term photo-toxicity and has a poor selectivity for the tumor. This had led to the development of a chlorophyll-derived photosensitizer. We conducted this study to elucidate the mechanisms by which the activity of ROS is involved in the PDT using a novel type of chlorophyll-derived photosensitizer, 9-hydroxypheophorbide-α (9-HpbD-α), for the HeLa cell lines. Besides, we also attempted to determine which reaction plays a predominant role in the synthesis of ROS, either Type I reaction or Type II one, when both reactions are involved in the synthesis of ROS during the PDT using 9-HpbD-α. Our results showed not only that the activity of ROS is involved in the PDT using 9-HpbD-α in human uterine cervical cancer cell lines but also that the mechanisms of PDT are based on Type II reaction where the singlet oxygen is involved.

Fluoride as a pro-inflammatory factor and inhibitor of ATP bioavailability in differentiated human THP1 monocytic cells

Chronic exposure of humans to fluorine compounds in the air, water and food may be atherogenic via the activation of oxidative stress and increased ROS production. The most important factor that promotes the formation of ROS seems to be the oxidoreduction of electron carriers in the critical points of the respiratory chain, which depends, among other things, on the cellular demand for ATP. This paper examines the effect of fluorides in concentrations determined in human serum on the intracellular synthesis of ROS, the activity of the respiratory chain enzymes and the synthesis of ATP via oxidative and substrate-level phosphorylation. The incubation of macrophages in fluoride solutions significantly decreased the amount of synthesized cellular ATP and increased formation of ROS and apoptosis in a dose-dependent pattern. The addition of respiratory chain inhibitors resulted in a significant decrease in the synthesized ROS. Sodium fluoride probably promotes oxidative stress in macrophages, which is manifested by a strong increase in ROS synthesis and a decrease in ATP. We suppose that fluoride may destabilize the action of respiratory chain. Our results indicate that the respiratory chain is the main site of ROS synthesis. One cannot exclude the stimulating role of fluorine compounds on the formation of ROS that is independent of the respiratory chain.

μ‐Opioid receptor‐stimulated synthesis of reactive oxygen species is mediated via phospholipase D2

We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

CsA and FK506 up-regulate eNOS expression: Role of reactive oxygen species and AP-1

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.