In cardiac muscle cells, both adrenergic agonists and antagonists induce reactive oxygen species from NOX2 but mutually attenuate each other's effects.

In cardiac muscle cells adrenergic agonists stimulate the generation of reactive oxygen species, followed by redox signaling. We postulated that the antagonists would attenuate such reactive oxygen species generation by the agonists. H9c2 cardiac myoblasts, neonatal rat cardiac myocytes, and HEK293 cells expressing ß1/ß2 adrenoceptors were stimulated with several agonists and antagonists. All the agonists and antagonists independently generated reactive oxygen species; but its generation was minimum whenever an agonists was added together with an antagonist. We monitored the Ca++ signaling in the treated cells and obtained similar results. In all treatment sets, superoxide and H2O2 were generated in the mitochondria and the cytosol respectively. NOX2 inhibitor gp91ds-tat blocked reactive oxygen species generation by both the agonists and the antagonists. The level of p47phox subunit of NOX2 rapidly increased upon treatment, and it translocated to the plasma membrane, confirming NOX2 activation. Inhibitor studies showed that the activation of NOX2 involves ERK, PI3K, and tyrosine kinases. Recombinant promoter-reporter assays showed that reactive oxygen species generated by both the agonists and antagonists modulated downstream gene expression. Mice injected with the ß-adrenergic agonist isoproterenol and fed with the antagonist metoprolol showed a robust induction of p47phox in the heart. We conclude that both the agonism and antagonism of adrenoceptors initiate redox signaling but when added together, they mutually counteract each other's effects. Our study thus highlights the importance of reactive oxygen species in adrenoceptor agonism and antagonism with relevance to the therapeutic use of the ß blockers.

Mitoapocynin, a mitochondria targeted derivative of apocynin induces mitochondrial ROS generation and apoptosis in multiple cell types including cardiac myoblasts: a potential constraint to its therapeutic use.

Mitoapocynin is a triphenylphosphonium conjugated derivative of apocynin that specifically locates to the mitochondria. It has been developed as a mitochondrially targeted therapeutic antioxidant. We attempted to attenuate the mitochondrial ROS induced in H9c2 cardiac myoblast cells treated with norepinephrine. Mitoapocynin was a poor quencher of total ROS as detected by the fluoroprobe DCFH-DA. Using mitochondrial superoxide specific probe MitoSoxRed, we found that 5-10 µM mitoapocynin itself induces superoxide over and above that is generated by the norepinephrine treatment. A supposedly control molecule to mitoapocynin, the synthetic compound PhC11TPP, having the triphenylphosphonium group and a benzene moiety with C11 aliphatic chain spacer was also found to be a robust inducer of mitochondrial ROS. Subsequent assays with several cell lines viz., NIH3T3, HEK293, Neuro2A, MCF-7 and H9c2, showed that prolonged exposure to mitoapocynin induces cell death by apoptosis that can be partially prevented by the general antioxidant N-acetyl cysteine. Analyses of mitochondrial electron transport complexes by Blue Native Polyacrylamide gel electrophoresis showed that both mitoapocynin and PhC11TPP disrupt the mitochondrial Complex I and V, and in addition, PhC11TPP also damages the Complex IV. Our data thus highlights the limitations of the therapeutic use of mitoapocynin as an antioxidant.

Computational prediction and experimental validation of the activator function of C2-ß-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone on pancreatic and hepatic hexokinase.

This study identifies and validates hexokinase type 4 (HK4), an isozyme of hexokinase in the liver and pancreas, as an important target of C2-ß-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (ßdGT), a xanthone glucoside suggested to have antidiabetic property. In the study, we applied the computational pipeline of molecular docking followed by the molecular dynamics simulations to shortlist potential ßdGT protein targets. The analysis of protein dynamics and the binding free energy (ΔG) led us to the identification of HK4 as a key ßdGT target, whereby the binding mode and domain dynamics suggested the activator function of ßdGT. ßdGT bound to the allosteric site of the isozyme ∼13 Å away from the substrate (glucose)-binding site. The binding free energy of the ligand-protein complex was energetically feasible (ΔG, -41.61 kcal/mol) and the cleft angle deviation between the two (small and large) domains of HK4 revealed differential HK4 dynamics in response to ßdGT binding. 3D structure analysis of the isozyme-ligand complex highlighted the role of Arg63, Glu67 and Lys458 in ligand stabilization and hydrophobic interactions mediated by Tyr214 and Met235. Experimental validation of the results of computational analysis confirmed the activator function of ßdGT on HK4. The study has implication in diabetes as ßdGT may be used to lower the blood glucose level by activating hepatic and pancreatic hexokinase without the risk of hypoglycemia.Communicated by Ramaswamy H. Sarma.

Gestational Exposure to Pesticides Induces Oxidative Stress and Lipid Peroxidation in Offspring that Persist at Adult Age in an Animal Model.

Pesticide exposure may induce biochemical alterations including oxidative stress and lipid peroxidation. However, in the context of developmental origin of health and disease, putative trans-generational effect of exposure to pesticides are insufficiently studied. We therefore aimed to evaluate the biochemical effect of gestational exposure to four pesticides on female Wistar rats and their offspring at adult age. We studied 30 female nulliparous Wistar rats divided into 5 equal groups. Group 1 served as the control group and received distilled water while group 2, 3, 4 and 5 received orally pesticide 1 (imidacloprid), pesticide 2 (chlorpyrifos), pesticide 3 (imidacloprid + lambda cyhalothrin) and pesticide 4 (oxamyl) respectively once daily throughout gestation at a dose equivalent to 1/10 lethal dose 50. The mothers were followed up until one month post gestation. The offspring were followed up from birth until adult age (12 weeks). In all animals at each time point we evaluated malondialdehyde (MDA), oxidative stress and liver function enzymes. There was similar variation of total body weight in all the groups during and after gestation. However, Female Wistar rats of the exposed groups had significant alterations in liver SOD (-30.8% to +64.1%), catalase (-38.8% to -85.7%) and GSH (-29.2% to -86.5%) and; kidney catalase (> 100%), GSH (> 100%). Moreover, MDA, alanine transaminase (ALT) and aspartate transaminase (AST) levels were significantly higher in pesticide exposed rats compared to the control group. Similar alterations in antioxidant enzymes, MDA and liver function enzymes were observed in offspring of treated rats evidenced at weaning and persisting until adult age. Exposure to pesticides causes oxidative stress and lipid peroxidation in exposed female Wistar rats and their offspring. The persistence in offspring at adult age suggests transgenerational adverse effects.

Eugenol-rich Fraction of Syzygium aromaticum (Clove) Reverses Biochemical and Histopathological Changes in Liver Cirrhosis and Inhibits Hepatic Cell Proliferation.

BACKGROUND: Dried flower bud of Syzygium aromaticum (clove) is rich in eugenol, an antioxidant and antiinflammatory compound that can protect liver against injury. Clove, besides eugenol, also contains other pharmacologically active phytochemicals such as ß-sitosterol and ascorbic acid. This study reports the effect of eugenol-rich fraction (ERF) of clove on liver cirrhosis induced by thioacetamide. METHODS: Cirrhosis of the liver, which predisposes to hepatocellular carcinoma, was induced by administering thioacetamide (0.03%) in drinking water for 16 weeks. Cirrhotic animals were divided into two groups; the treated group was administered ERF for 9 weeks, one week after discontinuation of thioacetamide, while the other group received normal saline for a similar duration of time. RESULTS: The treatment with ERF, as determined by histopathology and through a battery of biochemical markers of hepatic injury, oxidative stress and drug metabolizing enzymes, significantly ameliorated the signs of liver cirrhosis. It lowered the elevated levels of alkaline phosphatase, γ-glutamyl transferase and other biochemical changes in liver cirrhosis. Histopathology of the liver corroborated the effect of ERF with biochemical findings. ERF treatment further inhibited cell proliferation, as demonstrated by reduced [(3)H]-thymidine uptake. CONCLUSIONS: Data provide evidence supporting the protective action of ERF on liver cirrhosis. The study assumes significance because cirrhosis predisposes the liver to cancer, which is characterized by abnormal cell proliferation. ERF in this study is reported to inhibit hepatic cell proliferation and at the same time decrease oxidative stress, which might be the mechanism of protection against liver cirrhosis.

Mineral pitch stimulates humoral, cellular and innate immune responses in mice.

CONTEXT: Mineral pitch (MP), a traditional medicine, is proposed to boost immunity in conditions that suppress Th1 cytokines such as AIDS/HIV, tuberculosis, leishmaniasis and cancer. OBJECTIVE: This study investigates the immunoregulatory mechanisms of MP in innate, humoral and cell-mediated immunity. MATERIALS AND METHODS: Mice given MP (100, 200, 300 or 400 mg/kg, orally) for 10 consecutive days were immunized intravenously with goat RBC or ovalbumin, and investigated for plaque-forming cells (PFC), hemagglutination titer, hypersensitivity response, lymphocyte proliferation and macrophage function. RESULTS: MP increased PFC (330.2 versus 182.2/106 splenocytes) in mice immunized with goat RBC and elicited ovalbumin-specific IgG titer at 400 mg/kg. Increase in Th1 immunity was correlated with the increased level of IFN-γ (724 versus 470 pg/ml) and decreased IL-4 (96 versus 178 pg/ml). CD4⁺/CD3⁺ ratio and delayed-type hypersensitivity response also increased to, respectively, 20.62 ± 0.59 (versus 16.47 ± 0.72) and 1.59 ± 0.12 (versus 0.87 ± 0.10 mm) in MP-treated mice. MP increased lymphocyte proliferation (11.14 ± 0.60 versus 5.81 ± 0.40 SI) and macrophage phagocyte response (0.24 ± 0.02 versus 0.15 ± 0.009), expressed as absorbance at 570 nm, but decreased nitrite production (17.4 ± 1.10 versus 24.3 ± 1.30 µM/106 cells). We also observed an increased bone marrow cellularity (24.5 ± 1.10 versus 17.10 ± 0.70 cells/femur) and WBC count (12 667 ± 377 versus 9178 ± 213 cells/mm³) following MP treatment. There was no sign of toxicity at 400 mg/kg, 1/12th of reported LD50. CONCLUSION: MP elicits a dose-dependent Th1 immune response.

Aqueous Extract of Dried Flower Buds of Syzygium aromaticum Inhibits Inflammation and Oxidative Stress.

This study reports the beneficial effect of aqueous extract of dried flower buds of Syzygium aromaticum (clove) in acute and chronic inflammation. Inflammation was induced in rats by injecting carrageenan in hind paw or implanting cotton pellet in the axilla. Administration of the extract (1 g/kg body weight) inhibited the formation of oedema induced by carrageenan and decreased granuloma in cotton pellet granuloma model. The extract, when compared with the disease control, is reported to decrease the elevated levels of succinate dehydrogenase (p<0.001), xanthine oxidase (p<0.05) and lipid peroxidation, and increase the activity of catalase (p<0.001) and glutathione peroxidase (p<0.01) in the two animal models. Potential role of xanthine oxidase in inflammation and the ability of the extract to alleviate oxidative stress and inflammation is discussed. The study advocates the use of aqueous extract, rather than the isolated bioactive principle for various reasons.