Marisa Freitas

Nickel induces apoptosis in human neutrophils

Nickel is an ubiquitous transition metal that is industrially applied in many forms, which inevitably leads to a high degree of occupational and environmental exposure. Over-exposure to nickel can produce a variety of adverse effects on human health, including allergy and lung and nasal cancers. In the present study, it is demonstrated, for the first time, that nickel [(Ni(II)] (as a nickel nitrate salt) at concentrations that may be attained in vivo, induces neutrophils’ apoptosis by the intrinsic pathway. The use of diphenyleneiodonium, a NADPH oxidase inhibitor, delayed Ni(II)-induced apoptosis, suggesting that NADPH oxidase-derived reactive oxygen species and subsequent signaling could contribute to this event. This is an important finding since increased apoptosis mediated by nickel may disrupt the physiological activities of neutrophils, with potential impact in its immunological and antimicrobial role.

Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

OBJECTIVES To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA). DESIGN AND METHODS Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca(2+)](i)) was measured using Fluo-3 AM, a calcium-sensitive dye. RESULTS EDTA provided the highest number of isolated neutrophils/mL of blood (1.7x10(6)+/-1.5x10(5)) when compared with citrate (0.46x10(6)+/-0.95x10(5)) and heparin (0.66x10(6)+/-0.15x10(5)). EDTA originated less degree of PMA-induced activation (370+/-30%) relatively to citrate (830+/-98%) and heparin (827+/-77%). [Ca(2+)](i) was lower with EDTA (122+/-11 nM) when compared with citrate and heparin (150+/-13 and 230+/-30 nM). CONCLUSION The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.

Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation.

Rosmarinic acid is a polyphenolic compound and main constituent of Rosmarinus officinalis and has been shown to possess antioxidant and anti‐inflammatory properties. We aimed to evaluate the anti‐inflammatory properties of rosmarinic acid and of an extract of R. officinalis in local inflammation (carrageenin‐induced paw oedema model in the rat), and further evaluate the protective effect of rosmarinic acid in rat models of systemic inflammation: liver ischaemia–reperfusion (I/R) and thermal injury models. In the local inflammation model, rosmarinic acid was administered at 10, 25 and 50 mg/kg (p.o.), and the extract was administered at 10 and 25 mg/kg (equivalent doses to rosmarinic acid groups) to male Wistar rats. Administration of rosmarinic acid and extract at the dose of 25 mg/kg reduced paw oedema at 6 hr by over 60%, exhibiting a dose–response effect, suggesting that rosmarinic was the main contributor to the anti‐inflammatory effect. In the liver I/R model, rosmarinic acid was administered at 25 mg/kg (i.v.) 30 min. prior to the induction of ischaemia and led to the significant reduction in the serum concentration of transaminases (AST and ALT) and LDH. In the thermal injury model, rosmarinic acid was administered at 25 mg/kg (i.v.) 5 min. prior to the induction of injury and significantly reduced multi‐organ dysfunction markers (liver, kidney, lung) by modulating NF‐κB and metalloproteinase‐9. For the first time, the anti‐inflammatory potential of rosmarinic acid has been identified, as it causes a substantial reduction in inflammation, and we speculate that it might be useful in the pharmacological modulation of injuries associated to inflammation.

Zinc, cadmium and nickel increase the activation of NF-κB and the release of cytokines from THP-1 monocytic cells.

The sustained activation of the transcription factor nuclear factor κB (NF-κB) by metal-activated signalling pathways can lead to chronic inflammatory processes and related diseases, including carcinogenesis. The aim of the present work was to clarify the effect of zinc, nickel and cadmium on NF-κB activation in the THP-1 human monocytic leukemia cell line. The production of the NF-κB downstream pro-inflammatory mediators tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-6 and IL-8 was also evaluated due to their important roles in the pathogenesis of chronic inflammatory and autoimmune diseases and, ultimately, in the development of cancer. The results obtained demonstrated that zinc, nickel and cadmium significantly activate NF-κB, and the release of the chemokine IL-8. Cadmium also induced the release of TNF-α and IL-6 in THP-1 monocytic cells, which may indicate some potential to induce deleterious effects through this pathway.

Arsenic, antimony, and other trace element contamination in a mine tailings affected area and uptake by tolerant plant species.

The study was conducted to characterize mineralogical and elemental composition of mine tailings in order to evaluate the environmental hazards, and identify the metal accumulation potential of native plant species from São Domingos mine, one of the long-term activity mines of the Iberian Pyrite Belt dating back to pre-Roman times. The mine tailings including soils and different plant species from São Domingos were analyzed for determination of tailings characteristics and chemical element contents in tailings and plants. The large amounts of mining wastes are causing significant adverse environment impacts due to acid mine drainage production and mobilization of potentially toxic metals and metalloids in residential areas, agricultural fields, downstreams, and rivers. The typical mineralogical composition is as follows: quartz, micas, K-feldspar, olivine-group minerals, magnetite, goethite, hematite, jarosite, and sulfides. The mine tailings were highly contaminated by As, Ag, Cr, Hg, Sn, Sb, Fe, and Zn; and among them, As and Sb, main contaminants, attained the highest concentrations except Fe. Arsenic has exhibited very good correlations with Au, Fe, Sb, Se, and W; and Sb with As, Au, Fe, Se, Sn, and W in tailings. Among the all plant species, the higher concentrations of all the metals were noted in Erica andevalensis, Erica australis, Echium plantagium, and Lavandula luisierra. Considering the tolerant behavior and abundant growth, the plant species Erica australis, Erica andevalensis,Lavandula luisierra,Daphne gnidium, Rumex induratus, Ulex eriocladus, Juncus, and Genista hirsutus are of major importance for the rehabilitation and recovery of degraded São Domingos mining area.

Lichens (Parmelia sulcata) time response model to environmental elemental availability

Parmelia sulcata transplants, collected in a non-polluted area, were exposed to new atmospheric conditions at six stations, of which five were located near power plants and one at an unpolluted area. Data were collected for a 1-year period, on rainfall, airborne particulates, elemental deposition and on lichen elemental composition. The study was carried out to quantify the relationships between elemental availability and elemental accumulation in lichens. A mathematical model was developed, involving a simple representation of the lichen, and a memory loss function which describes the time relations for the lichens progressive representation of its new ambient conditions. The collected data set was used to test the model. The results indicate that the environmental data on Na, Al and Pb could be reproduced and predicted by the model, including the variability patterns. Half-memory times were calculated for Na, Al and Pb as 200, 500 and 600 days, respectively, based on using all station data. However, further improvements are still needed, including the clarification of differences in model outcomes between stations. Copyright (C) 1998 Elsevier Science B.V.

Inhibition of LOX by flavonoids: a structure-activity relationship study.

The lipoxygenase (LOX) products have been identified as mediators of a series of inflammatory diseases, namely rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, atherosclerosis and certain types of cancer. Hence, LOX inhibitors are of interest for the modulation of these phenomena and resolution of the inflammatory processes. During LOX activity, peroxyl radical complexes are part of the reaction and may function as sources of free radicals. Thus antioxidants, such as flavonoids, capable of inhibiting lipid peroxidation and scavenging free radicals, may act as LOX inhibitors. The aim of this work was to assess the structure-activity relationship among a series of flavonoids concerning 5-LOX inhibition, through a systematic study of the inhibition of the formation of LTB4 in human neutrophils. The type of inhibition of the flavonoids was further studied using soybean LOX, type I, and Saturation Transfer Difference (1)H NMR (STD-(1)H NMR) was used to characterize the binding epitopes of the compounds to LOX-1. The obtained results reinforce flavonoids as effective inhibitors of LTB4 production in human neutrophils. It was also possible to establish a structure/activity relationship for the inhibitory activity and the type of inhibition.

Optimization of experimental settings for the analysis of human neutrophils oxidative burst in vitro

The evaluation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by neutrophils is currently a matter of extensive research, with scientific reports showing an enormous variability on the detection methods as well as the concentration of the detecting probes. Also the incubation media used to test neutrophils and the respective ionic concentration, as well as the glucose concentration, varies enormously from study to study. This variability often results in different sensibility and/or response of neutrophils to stimulating agents, which can be a focus of confounding and sometime contradictory results among reports. Thus, the main objective of the present study was to appraise and compare the effect of commonly described buffering media [phosphate buffer saline (PBS), Hanks balanced salt solution (HBSS) and Tris buffer], with or without glucose, on the activation of human neutrophils by phorbol myristate acetate (PMA), using different detection probes [luminol amplified chemiluminescence, dihydrorhodamine 123 fluorescence or cytochrome c reduction (UV/vis spectrometry)]. It was observed that the choice of incubation media as well as the methodology used to detect neutrophils oxidative burst has an enormous influence on posterior results. Independently of buffer, the presence of glucose is important, as a source of NADPH through pentose phosphate pathway (PPP). From the obtained results, we advise the use of HBSS, with the glucose concentration of 0.55mM. This incubation media provided the best performance of neutrophils allowing the use of lower concentrations of the tested probes as well as of the stimulating agent, PMA.

Antioxidant activity of unexplored indole derivatives: synthesis and screening.

The present study envisaged the development of novel antioxidant candidates using the indole scaffold. Several tryptophan and tryptamine derivatives were synthesized, in particular prenylated indole compounds, and their scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) was investigated. The library substitution pattern included several alkyl chains at positions N-1, C-2 of the indole nucleus, including prenyl and isopentyl chain, as well as different groups at the side chain (C-3) that allowed the investigation of a possible radical stabilization. The results obtained showed that tryptophan (8), tryptamine (9), N-phthaloyl tryptamine (5) and N-prenyl tryptophan (13) were the most active against peroxyl radical (ROO(•)) with activities higher than Trolox, which was used as control. The scavenging of hypochlorous acid (HOCl) was also evaluated and tryptophan (8) and tryptamine (9) showed IC(50) of 3.50 ± 0.4 and 6.00 ± 0.60 μM, respectively. Significant activity was also found for the N-prenyl tryptophan (13) with an IC(50) of 4.13 ± 0.17 μM and C-2 prenylated derivative (14), with 4.56 ± 0.48 μM. The studies were extended to RNS and best results were obtained against peroxynitrite anion (ONOO(-)) in the presence of NaHCO(3). N-alkylated tryptophan (18) showed a high activity with an IC(50) of 14.0 ± 6.8 μM. The results show that the tested compounds are effective scavengers of ROS and RNS, and suggest that the radical stabilization is strongly dependent on the type of substituents on the indolic moiety and on their relative positions. In addition, the radical dissipation inside the indolic system is mandatory for the observed antioxidant activity.

Synthesis and antioxidant properties of new chromone derivatives.

Nowadays, the recognition of the benefits of antioxidants is eliciting an increasingly interest in the search for new molecules with improved activity. The aim of the present work was to search for improved reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavengers by testing new structures of 2-styrylchromones (2-SC) and 3-substituted flavones, which were synthesised by the Baker-Venkataraman approach. The new compounds were also tested for their metal chelating capacity and reducing activity. The obtained results showed that the methylation of hydroxyl groups decreases the scavenging of ROS and RNS by 2-SC. The decrease in the scavenging activities was, generally, more evident when the methylation occurred in B-ring, except for O2*- and (1)O(2). On the other hand, the introduction of a substituent, either hydroxyl or methoxyl, in position 8 was sometimes favourable and others unfavourable to the scavenging activities, depending on the reactive species. In conclusion, the study of the antioxidant properties of the new 2-SC and flavones allowed establishing new structure-activity relationships and brought out, in some cases, pharmacophores with improved activity.