Heat shock-induced attenuation of hydroxyl radical generation and mitochondrial aconitase activity in cardiac H9c2 cells

A mild heat shock (hyperthermia) protects cells from apoptotic and necrotic deaths by inducing overexpression of various heat shock proteins (Hsps). These proteins, in combination with the activation of the nitric oxide synthase (NOS) enzyme, play important roles in the protection of the myocardium against a variety of diseases. In the present work we report that the generation of potent reactive oxygen species (ROS), namely ·OH in cardiac H9c2 cells, is attenuated by heat shock treatment (2 h at 42°C). Western blot analyses showed that heat shock treatment induced overexpression of Hsp70, Hsp60, and Hsp25. The observed ·OH was found to be derived from the superoxide (O₂^–·) generated by the mitochondria. Whereas the manganese superoxide dismutase (MnSOD) activity was increased in the heat-shocked cells, the mitochondrial aconitase activity was reduced. The mechanism of O₂^–· conversion into ·OH in mitochondria is proposed as follows. The O₂^–· leaked from the electron transport chain, oxidatively damages the mitochondrial aconitase, releasing a free Fe²⁺. The aconitase-released Fe²⁺ combines with H₂O₂ to generate ·OH via a Fenton reaction and the oxidized Fe³⁺ recombines with the inactivated enzyme after being reduced to Fe²⁺ by other cellular reductants, turning it over to be active. However, in heat-shocked cells, because of higher MnSOD activity, the excess H₂O₂ causes irreversible damage to the mitochondrial aconitase enzyme, thus inhibiting its activity. In conclusion, we propose that attenuation of ·OH generation after heat shock treatment might play an important role in reducing the myocardial ischemic injury, observed in heat shock-treated animals. proteins; free radicals; spin trapping; reactive oxygen species     

Discovery of adamantane based highly potent HDAC inhibitors

Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI₅₀ of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, in vitro. Compound 32 displays efficacy in human tumour animal xenograft model.     

In-Vitro Cytotoxicity and Cell Cycle Analysis of Two Novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC₅₀ of 3–5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G₁ phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model

Background

Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).

Methods and Findings

Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.

Conclusion

Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.     

In vivo bioassays of acute asbestosis and its correlation with ESR spectroscopy and imaging in redox status

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.     

Lipid peroxidation and antioxidant status in patients with periodontitis

Our aim was to assess the degree of oxidative stress in patients with periodontitis by measuring their levels of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and non-enzymatic antioxidants (vitamins E and C, reduced glutathione (GSH)). This study was conducted on 25 adult chronic periodontitis sufferers who were patients in Rajah Muthiah Dental College and Hospital, Annamalai University. The levels of TBARS and non-enzymatic antioxidants, and the activities of enzymatic antioxidants in the patients' plasma, erythrocytes and gingival tissues were assayed using specific colorimetric methods. The periodontitis sufferers had a significantly higher TBARS level than the healthy subjects. In the plasma, erythrocytes, erythrocyte membranes and gingival tissues of the periodontitis sufferers, enzymatic antioxidant activities were found to be significantly higher, whereas the levels of non-enzymatic antioxidants were significantly lower (except for reduced glutathione in the gingival tissues) relative to the parameters found for healthy subjects. The disturbance in the endogenous antioxidant defense system due to over-production of lipid peroxidation products at inflammatory sites can be related to a higher level of oxidative stress in patients with periodontitis.     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity

Alveolar macrophages are important host defense cells in the human lung that continuously phagocytose environmental and infectious particles that invade the alveolar space. Alveolar macrophages are prototypical alternatively activated macrophages, with up-regulated innate immune receptor expression, down-regulated costimulatory molecule expression, and limited production of reactive oxygen intermediates (ROI) in response to stimuli. Surfactant protein A (SP-A) is an abundant protein in pulmonary surfactant that has been shown to alter several macrophage (Mphi) immune functions. Data regarding SP-A effects on ROI production are contradictory, and lacking with regard to human Mphi. In this study, we examined the effects of SP-A on the oxidative response of human Mphi to particulate and soluble stimuli using fluorescent and biochemical assays, as well as electron paramagnetic resonance spectroscopy. SP-A significantly reduced Mphi superoxide production in response to the phorbol ester PMA and to serum-opsonized zymosan (OpZy), independent of any effect by SP-A on zymosan phagocytosis. SP-A was not found to scavenge superoxide. We measured Mphi oxygen consumption in response to stimuli using a new oxygen-sensitive electron paramagnetic resonance probe to determine the effects of SP-A on NADPH oxidase activity. SP-A significantly decreased Mphi oxygen consumption in response to PMA and OpZy. Additionally, SP-A reduced the association of NADPH oxidase component p47(phox) with OpZy phagosomes as determined by confocal microscopy, suggesting that SP-A inhibits NADPH oxidase activity by altering oxidase assembly on phagosomal membranes. These data support an anti-inflammatory role for SP-A in pulmonary homeostasis by inhibiting Mphi production of ROI through a reduction in NADPH oxidase activity.     

Modulatory Effect of Taurine on 7,12‐Dimethylbenz(a)Anthracene‐Induced Alterations in Detoxification Enzyme System,Membrane Bound Enzymes,Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue

The modulatory effect of taurine on 7,12‐dimethylbenz(a)anthracene (DMBA)‐induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na⁺/K⁺ATPase, Ca²⁺ATPase, and Mg²⁺ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione‐S‐transferase and UDP‐glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA‐induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA‐induced breast cancer.