Maria Rita Rodrigues

Superoxide-dependent oxidation of melatonin by myeloperoxidase.

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.

Interferon-gamma independent oxidation of melatonin by macrophages

Abstract: Mononuclear phagocytes appear to synthesize kynurenine‐like products from the oxidation of biologically active indole compounds including melatonin, catalyzed by interferon (IFN)‐γ‐inducible enzyme indoleamine 2,3‐dioxygenase (IDO). Concanavalin A (Con A) is a plant lectin that induces interferon‐gamma (IFN‐γ) production by T cells. In this study we investigated whether Con A‐primed peritoneal macrophages are able to oxidize melatonin to N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK). The AFMK production was accompanied by chemiluminescence. It was found that Con A‐primed but not resident macrophages produce AFMK. Surprisingly, Con A‐primed macrophages from IFN‐γ‐deficient mice were as effective as macrophages from IFN‐γ‐sufficient mice in oxidizing melatonin. Moreover, addition of an inhibitor of IDO (1‐methyltryptophan) did not affect melatonin oxidation. Con A‐primed but not resident macrophages have a significant content of myeloperoxidase (MPO) and inhibition of MPO by azide completely blocked chemiluminescence and AFMK production. Thus, our findings provide evidence that melatonin oxidation by macrophages may occur through a mechanism dependent of MPO and independent of IFN‐γ and IDO activity.

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the May–Grunwald–Giemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils. Copyright

Intracellular localization of myeloperoxidase in murine peritoneal B-lymphocytes and macrophages.

Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.

PREVALENCE OF PARACOCCIDIOIDOMYCOSIS INFECTION BY INTRADERMAL REACTION IN RURAL AREAS IN ALFENAS, MINAS GERAIS, BRAZIL

This study aimed to estimate the prevalence of paracoccidioidal infection by intradermal reaction (Delayed-Type Hypersensitivity, DTH) to Paracoccidioides brasiliensis in rural areas in Alfenas, Southern Minas Gerais (MG) State, Brazil, and to assess risk factors (gender, occupation, age, alcohol intake and smoking) associated with infection. We conducted a population-based cross-sectional study using intradermal tests with gp 43 paracoccidioidin in 542 participants, who were previously contacted by local health agents and so spontaneously attended the test. Participants underwent an interview by filling out a registration form with epidemiological data and were tested with an intradermal administration of 0.1 mL of paracoccidioidin in the left forearm. The test was read 48 hours after injection and was considered positive if induration was greater than or equal to 5 mm. Out of 542 participants, 46.67% were positive to the skin test. Prevalence increased in accordance with an increase of age. There was statistical significance only for males. Occupation, alcohol intake and smoking habits were not significantly associated with the risk of paracoccidioidomycosis infection. There is relevance of paracoccidioidomycosis infection in such rural areas, which suggests that further epidemiological and clinical studies on this mycosis should be done in the southern part of Minas Gerais State.

Effects of the Consumption of Caffeinated and Decaffeinated Instant Coffee Beverages on Oxidative Stress Induced by Strenuous Exercise in Rats

Many authors attribute the antioxidant activity of brewed coffee to its caffeine content. In addition, caffeine intake has been associated with increased performance during physical exercise. This study analyzed the in vivo effects of drinking caffeinated and decaffeinated instant coffee (8%, w/v) on oxidative stress and antioxidant enzyme activity in the anterior tibialis muscles of rats subjected to intense exercise. It was observed that exercise induced lipid peroxidation (estimated using malondialdehyde) and protein oxidation (evaluated by determining the formation of carbonyl groups) in the muscle (P < 0.05). Decaffeinated instant coffee and caffeine solution did not exhibit antioxidant activity in vivo. Caffeinated instant coffee beverage intake did not induce changes in superoxide dismutase and glutathione peroxidase activities but was able to diminish lipid and protein oxidation in the anterior tibialis muscles of rats after exercise (P < 0.05), contributing to a reduction in the oxidative stress triggered by exercise.

Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.

Aminoguanidine treatment increased NOX2 response in diabetic rats: Improved phagocytosis and killing of Candida albicans by neutrophils.

In this study, we show that aminoguanidine (AMG), an inhibitor of protein glycation, increases the NOX2 (phagocyte NADPH oxidase) response and microbicidal activity by neutrophils, regardless of diabetic status. The non-enzymatic glycation of proteins, yielding irreversible advanced glycation end products (AGEs), is involved in the development of diabetes complications, including alterations of signaling pathways and the generation of reactive oxygen species by phagocytes. The phagocytes produce ROS (reactive oxygen species) through activation of the NOX2 complex, which generates superoxide. The purpose of this study was to evaluate the effect of hyperglycemia and the glycation of proteins on the NOX2 activity of neutrophils and its implications for cellular physiology, with a focus on the microbicidal activity of these cells. We treated diabetic rats with AMG and evaluated neutrophil ROS generation and Candida albicans killing ability. We observed a large increase in the microbicidal activity of peritoneal neutrophils from AMG-treated rats. The increase was independent of diabetic status and myeloperoxidase activity. Collectively, our results suggest that AMG has an immunomodulator role that triggers an increase in the microbicidal response of neutrophils mainly related to reactive oxygen species production by NOX2.

Hepatoprotective and Antioxidant Activities of Oil from Baru Almonds (Dipteryx alata Vog.) in a Preclinical Model of Lipotoxicity and Dyslipidemia

The oil obtained from baru (Dipteryx alata Vog.) almonds exhibits high energy value and is reported in popular medicine for the treatment of rheumatic diseases and reproductive disturbances. Although baru oil is used in domestic cuisine, the chemical characterization of this oil and its effects on lipid metabolism are still poorly understood. Therefore, this study evaluated the fatty acid (FA) profile and the effects of baru oil on liver and aorta in a murine model of dyslipidemia. The chromatographic profile of baru oil showed high levels of unsaturated FAs, especially oleic acid. Saturated FAs, such as palmitic and lignoceric acids, were found in lower amounts. Hypercholesterolemia was induced in male Wistar rats by daily administration of a lipid emulsion by gavage for 15 weeks. Biochemical and histopathological analysis were performed on serum, aorta, and liver. The results demonstrated that animals developed marked hypercholesterolemia, liver steatosis, and increased lipid peroxidation in the aorta. Treatment with baru oil attenuated lipid peroxidation and drastically reduced liver damage, especially ballooning degeneration and steatosis. By restricting vascular and hepatic injury, this oil showed potential applicability as a functional food, reinforcing its use in popular medicine and domestic cuisine.

Ethanolic extract of Passiflora edulis Sims leaves inhibits protein glycation and restores the oxidative burst in diabetic rat macrophages after Candida albicans exposure

This study was conducted to evaluate the effects of the ethanolic extract of Passiflora edulis leaves on blood glucose, protein glycation, NADPH oxidase activity and macrophage phagocytic capacity after Candida albicans exposure in diabetic rats. The Passiflora edulis Sims leaves were dried to 40°C, powdered, extracted by maceration in 70% ethanol, evaporated under reduced pressure and lyophilised. The biochemical tests performed were total phenolic content (TP) as determined by the Folin-Ciocalteu assay, trapping potential DPPH assay and total iron-reducing potential. Diabetes was induced by alloxan injection. Protein glycation was determined by AGE and fructosamine serum concentrations. Extract-treated diabetic animals demonstrated lower fructosamine concentrations compared with the diabetic group. Our results suggest that ethanolic Passiflora edulis Sims leaf extraction may have beneficial effects on diabetes and may improve glycaemic control in diabetic rats.