Análise fitoquímica e avaliação das atividades anti-inflamatória, antipeçonhenta e citotóxica de extratos aquosos de Aspidosperma pyrifolium e Ipomoea asarifolia

Envenomation caused by venomous animals, mainly scorpions and snakes, are a serious matter of public health. Tityus serrulatus is considered the most venomous scorpion in South America because of the high level of toxicity of its venom. It is responsible for causing serious accidents, mainly with kids. The species Bothrops jararaca is a serpent that has in its venom a complex mixture of enzyme, peptides and other molecules. The toxins of the venom of B. jararaca induce local and systemic inflammatory responses. The treatment chosen to serious cases of envenomation is the intravenous administration of the specific antivenom. However, the treatment is not always accessible to those residents in rural areas, so that they use medicinal plant extracts as the treatment. In this context, aqueous extracts, fractions and isolated compounds of Aspidosperma pyrifolium (pereiro) and Ipomoea asarifolia (salsa, salsa-brava), used in popular medicine, were studied in this research to evaluate the anti-inflammatory activity in the peritonitis models induced by carrageenan and peritonitis induced by the venom of the T. serrulatus (VTs), and in the local oedema model and inflammatory infiltrate induced by the venom of the B. jararaca, administrated intravenously. The results of the assays of cytotoxicity, using the MTT, showed that the aqueous extracts from the plant species presented low toxicity to the cells that came from the fibroblast of the mouse embryo (3T3).The chemical analysis of the extracts by High Performance Liquid Chromatography revealed the presence of the rutin flavonoid, in A. pyrifoliu, and rutin, clorogenic acid and caffeic acid, in I. asarifolia. Concerning the pharmacological evaluation, the results showed that the pre-treatment using aqueous extracts and fractions reduced the total leukocyte migration to the abdominal cavity in the peritonitis model caused by the carrageenan and in the peritonitis model induced by the T. serulatus venom. Yet, these groups presented anti-oedematous activity, in the local oedema model caused by the venom of the B. jararaca, and reduced the inflammatory infiltrate to the muscle. The serum (anti-arachnid and anti-bothropic) specific to each venom acted inhibiting the inflammatory action of the venoms and were used as control. The compounds identified in the extracts were also tested and, similar to the plant extracts, showed meaningful anti-inflammatory effects, in the tested doses. Thus, these results are indicating the potential anti-inflammatory activity of the plants studied. This is the first research that evaluated the possible biological effects of the A. pyrifolium and I. asarifolia, showing the biological potential that these species have.

Extração, caracterização e prospecção de atividades farmacológicas de polissacarídeos sulfatados da macroalga verde Caulerpa prolifera

This study aimed to extract, characterize and conduct a prospective analysis of pharmacological activities of sulfated polysaccharides from green seaweed Caulerpa prolifera. Seven fractions (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) were obtained from C. prolifera by alkaline proteolysis followed by sequential precipitation in acetone. The physicochemical analyzes indicated that C. prolifera synthesizes a homogalactan (CP-0.9) and different populations of sulfated heteropolysaccharides. In the analysis of anticoagulant activity, all fractions except CP-0.3, influenced the intrinsic coagulation pathway. All fractions showed antioxidant activity in six different assays being more pronounced in hydrogen peroxide scavenging assay, especially CP-0.3, CP-0.7 and CP-0.9 (which obtained 61% of hydrogen peroxide scavenging), in ferric chelation assay (especially CP-0.9 with 56% chelation) and cupric chelation assay (especially CP-2.0 with 78% chelation). With respect to immunomodulatory activity, the presence of CP-0.3, CP-0.7 and CP-0.9 showed an immunogenic potential, increasing the production of nitric oxide (NO) by 48, 142 and 163 times, respectively. Conversely, the NO synthesis fell 73% after the activation of macrophages by LPS, incubated concurrently with CP-2.0. The anti-adipogenic activity of the fractions was also evaluated and CP-1.5 was able to reduce the differentiation of pre-adipocytes (3T3-L1) into adipocytes by 60%, without affecting the cell viability. The fractions CP-0.3, CP-0.5 and CP-0.9 reduced the viability of the HeLa cells (human cervical adenocarcinoma) by 55% and CP-1.5 reduced the viability of the 786-0 cells (human renal adenocarcinoma) by 75%. Leishmanicidal activity and microbicide effect against Carbapenem-resistant Klebsiella pneumoniae (KPC) have not been identified. However, the viability of Staphylococcus epidermidis was reduced by 23.8% in the presence of CP -1.5. All fractions were able to change the formation of calcium oxalate crystals. CP-0.3, CP-0.5 and CP-1.1 only promoted the formation of COD type crystals with a very small size (1 μm). Confocal microscopy and zeta potential data of crystals formed in the presence of the samples showed that the polysaccharides present in the fractions must interact with calcium ions present throughout the crystal lattice, affecting the growth and morphology of crystals The results described herein indicate that the fractions rich in polysaccharides obtained from the green seaweed C. prolifera present a multi therapeutic potential, and subsequent purification steps, as well as research on the mechanisms of action by which these polymers act should be investigated.

Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.

Alterações hepáticas na expressão gênica e atividade da catalase e superóxido dimutase em ratos diabéticos induzidos por estreptozootocina

The correlation between the type 1 diabetes mellitus and oxidative stress have been described in several studies, however its underlying mechanisms are not fully elucidated. The present work aimed to evaluate the effects of four weeks of streptozootocin-induced (STZ) diabetes in the redox homeostasis of rat hepatocytes. Thus, the liver of male Wistar rats from control and diabetic groups were collected and the activity and expression of antioxidant enzymes, as well the main markers of oxidative stress and content of H2O2 in these tissues were measured. The diabetes induced the activity of superoxide dismutase (SOD) and the gene expression of its mitochondrial isoform, SOD2. However, the expression of SOD1, the cytoplasmic isoform, was reduced by this disease. The activity and expression of catalase (CAT), as well the expression of glutathione peroxidase 1 (GPX1) and peroxiredoxin 4 (PRX4) were drastically reduced in the hepatocytes of diabetics rats. Even with this debility in the peroxidases mRNA expression, the content of H2O2 was reduced in the liver of diabetics rats when compared to the control group. The diabetes caused an increase of lipid peroxidation and a decrease of protein thiol content, showing that this disease causes distinct oxidative effects in different cell biomolecules. Our results indicate that four week of diabetes induced by STZ is already enough to compromise the enzymatic antioxidant systems of the hepatocytes.

Dextranas e seus derivados fosforilados: obtenção e avaliação do potencial antioxidante, imunomodulatório e anticoagulante

Glucans are polysaccharides with different pharmacological and biological activities described. However, there are some reports about the activities of the glucan type α (alpha). In this context, a group of α-D-glucans called dextrans extracted from Leuconostoc mesenteroides bacteria, with molecular weights of 10 (D10), 40 (D40) and 147 (D147) kDa and their phosphorylated derivatives P10, P40 and P147, were evaluated as for their antioxidant, anticoagulant and immunomodulatory potential for the first time, in order to elucidate compounds with potent activities and low toxicity. Infrared spectroscopy analysis, monosaccharide composition and chemical dosages showed that these dextrans are the same polysaccharide, but with different molecular weights, besides confirming the success of phosphorylation. None presented with anticoagulant features. The reducing power test showed that D147 was twice as potent as other dextrans. On the other hand, all six samples showed similar activity (50%) when it came to scavenging the OH radical. To the superoxide ion scavenging, only D10 had a pronounced activity (50%). D40 was the single native dextran that presented with immunomodulatory features since it double stimulated the proliferation of murine macrophages (RAW 264.7) and double the release of nitric oxide by the cells, both in the absence and presence of lipopolysaccharides (LPS). In addition, D40 showed a greater scavenging activity (50%) for the hydrogen peroxide, which caused it to also be the more potent dextran when it came to inhibiting lipid peroxidation (70%). On other hand, P147 showed the highest iron and copper ion chelation activity (~85%). P10 proved be the most effective compound to macrophage proliferation. The results point toward dextrans with a 40 kDa weight as being ideal for antioxidant and immunomodulatory use, could be supplemented with phosphorylated derivatives. However, future studies with the D40 and other similarly dextrans are to confirm this hypothesis.

Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização estrutural e atividades farmacológicas do alginato obtido da alga Dictyopteris delicatula (J.V. Lamouroux., 1809) e seu derivado sulfatado

Marine algae are rich sources of various structural compounds which recently has been increasingly studied as a new source of bioactive substances. The alginate, as come as fucans, are considered the main acidic polysaccharides found in brown seaweed. This molecule consists a linear natural polysaccharide, non-sulfated, and presents monosaccharides: acid β-D-mannuronic (M) and α-L-guluronic acid (G); in a vast amount compositions and threads. Alginate has been widely applied in food and pharmaceutical industries because of its ability to retain water, forming films and gels as well as thickening, stabilizing and form emulsions. In this work we aimed to extract, structurally characterize, compare and analyze the possible pharmacological activities of native alginate molecule obtained from brown seaweed Dyctiopteris delicatula (DYN), and its chemically sulfated derivative (DYS). The alginate structure and composition molecule can be proven through chemical dosing, that showed low protein contamination and high sugar level, existence and separation of M and G blocks in the descending paper chromatography, infrared spectroscopy and nuclear magnetic resonance. Molecule sulfation was proven with sulphate dosage, resulting in 28.56% sulphate in molecule; electrophoresis, verify metachromasia with toluidine blue; and infrared spectroscopy, that showed a characteristic band at 1221cm-1 corresponding a sulfate group vibration. For the pharmacological activities the tests was: antioxidant activity, changes in cell function (MTT test) and anticoagulant test. In the antioxidant activity we observed that DYN showed better results in the kidnapping of hydroxyl radicals and ferric chelation compared to DYS, this had the best result in the total antioxidant capacity. Both showed similar activity in reducing power and the kidnapping radicals DPPH. In MTT test DYN and DYS had not proliferative and cytotoxic activity in fibroblast cells (3T3) and showed antiproliferative and cytotoxic activity in cancer cell lines HeLa and B16 melanoma. In anticoagulant assay DYN showed good activity in the intrinsic pathway of blood coagulation, and a small activity in the extrinsic pathway, in the other hand DYS showed only a very small activity in the extrinsic pathway, but cannot come to be regarded as an anticoagulant agent. From these results it can be concluded that the alginate was extracted and sulfated, revealing a potential compound to be used in the pharmaceutical industry as an anticoagulant agent, antioxidant and antitumor and the sulfation has not been conclusively important to performance in the tested pharmacological activities

Receptores de citocinas proinflamatórias na pré-eclâmpsia

Preeclampsia is a disease specific of human pregnancy that affects 3-8% of pregnant women, and it is one of the three leading causes of maternal mortality and morbidity. The disease is characterized by hypertension and proteinuria after the 20th week of gestation. The risk factors for this disease are not completely understood but appear to include dysregulation of the immune response arising from defects in placentation, environmental and genetic factors. This study aimed to determine whether the variation in the amount of proinflammatory cytokine receptors IL-1R2, IL-6R and TNF-αR1 would be involved in preeclampsia. They were recruited women with preeclampsia (n=24) and women who evolved during pregnancy without changes in blood pressure (n=12) were recruited. Clinical and laboratory data were collected. The cytokine receptors (IL-1R2, TNF-αR1 and IL-6R) were assessed in mononuclear cells isolated from peripheral blood using flow cytometry (Control = 8; PE = 24). C-reactive protein (CRP) was determined by CRP ultrasensitive method (Control = 7; PE = 18) was performed using sera pregnant women. Women with preeclampsia had higher weight at the beginning of the pregnancy (p=0.0171) and lower gestational age at delivery (0.0008). Classical monocytes were decreased in preeclampsia but not intermediate or non-classical monocytes. The frequency of IL-1R2 pro inflammatory cytokine receptors is decreased in women with PE only in the subpopulation of non-classical monocytes (p = 0.0011). TNF-αR1 receptor and IL-6R, had a decreased frequency in the three subpopulations of monocyte (classic, intermediate and non-classical) when compared to women with normal pregnancy. An increase in IL-1R2 receptor in TCD4+ lymphocytes, but a decrease in TNF-receptor and IL-6R in women with preeclampsia were found. No differences in the frequency of those receptors in CD3+/CD8+ in preeclampsia. There was no difference in C-reactive protein in preeclampsia. The reduction in the amount of IL-1R2, TNF- αR1 and IL-6R monocytes and lymphocytes can be involved in the regulation of inflammation observed in preeclampsia, contributing to disease.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

Avaliação das atividades farmacológicas de fucoidans obtidos do extrato bruto de fucoidan comercial de Fucus vesiculosus

Fucoidan is a term used to define heteropolysaccharides that are composed of less than 90% L-fucose. The exception to this rule is the homofucoidan obtained from the seaweed Fucus vesiculosus. This fucoidan can be purchased from SIGMA Co. and have been used in various research for evaluation of their pharmacological activities. However, it is not a pure molecule. In fact, it is a mix of several fucoidan molecules. In this work, were obtained, from acetone precipitation, and biochemically characterized, four fucoidan molecules from SIGMA-ALDRICH Co. fucoidan to evaluate their anticoagulant, antioxidant, antiadipogenic, immunomodulatory and antiurolithiatic activities. In anticoagulant activity, evaluated by aPTT assay, fucoidans F0.9, F1.1 and F2.0 increased eightfold the coagulation time, compared to the control, when a mass of 10 μg was used. To PT test, only fucoidan F0.9 was capable of increase the coagulation time, compared to control. In the total antioxidant capacity assay (TAC), the fucoidan F2.0 showed 400 ascorbic acid equivalents, while fucoidan F0.5, the lest effective, 38 equivalents. In respect to the effect on pre-adipocyte cell lines (3T3-L1) adipogenesis, was observed that fucoidan F1.1 and F2.0 reduced the adipogenesis and this effect was associated to the reduction in the expression of regulatoy proteins C/EBPα, C/EBPβ and PPARγ. On the other hand, fucoidans F0.5 and F0.9 induced increased expression of these regulatory proteins. Furthermore, fucoidan F2.0 induced hydrolysis of triglycerides present in the interior of adipocytes. The immunomodulatory effect was evaluated and observed that the presence of fucoidans F0.5 , F1.1 and F2.0 significantly reduced the production of nitric oxide by activated macrophages with LPS specially fucoidan F2.0 that in 100 μg/mL, reduced about 55% the effect caused by LPS. Relative to the effect upon the formation of calcium oxalate crystals, fucoidan F0.5 was more effective in reduce the aggregation of the crystals and this effect it was not significantly different regarding the effect caused by the crude. Besides, fucoidan F0.5 only promoted the formation of COD type crystals, while fucoidans F1.1 and F2.0 did not influence the formation of crystals compared with the control. The results described in this study indicate that the commercial crude fucoidan of Fucus vesiculosus it’s a mix of several fucoidan which, in turn, have different chemical compositions besides having different pharmacological activities. The use of these fucoidans it´s indicated according the pharmacological activity to be evaluated.

Avaliação das atividades farmacológicas de fucoidans obtidos do extrato bruto de fucoidan comercial de Fucus vesiculosus

Fucoidan is a term used to define heteropolysaccharides that are composed of less than 90% L-fucose. The exception to this rule is the homofucoidan obtained from the seaweed Fucus vesiculosus. This fucoidan can be purchased from SIGMA Co. and have been used in various research for evaluation of their pharmacological activities. However, it is not a pure molecule. In fact, it is a mix of several fucoidan molecules. In this work, were obtained, from acetone precipitation, and biochemically characterized, four fucoidan molecules from SIGMA-ALDRICH Co. fucoidan to evaluate their anticoagulant, antioxidant, antiadipogenic, immunomodulatory and antiurolithiatic activities. In anticoagulant activity, evaluated by aPTT assay, fucoidans F0.9, F1.1 and F2.0 increased eightfold the coagulation time, compared to the control, when a mass of 10 μg was used. To PT test, only fucoidan F0.9 was capable of increase the coagulation time, compared to control. In the total antioxidant capacity assay (TAC), the fucoidan F2.0 showed 400 ascorbic acid equivalents, while fucoidan F0.5, the lest effective, 38 equivalents. In respect to the effect on pre-adipocyte cell lines (3T3-L1) adipogenesis, was observed that fucoidan F1.1 and F2.0 reduced the adipogenesis and this effect was associated to the reduction in the expression of regulatoy proteins C/EBPα, C/EBPβ and PPARγ. On the other hand, fucoidans F0.5 and F0.9 induced increased expression of these regulatory proteins. Furthermore, fucoidan F2.0 induced hydrolysis of triglycerides present in the interior of adipocytes. The immunomodulatory effect was evaluated and observed that the presence of fucoidans F0.5 , F1.1 and F2.0 significantly reduced the production of nitric oxide by activated macrophages with LPS specially fucoidan F2.0 that in 100 μg/mL, reduced about 55% the effect caused by LPS. Relative to the effect upon the formation of calcium oxalate crystals, fucoidan F0.5 was more effective in reduce the aggregation of the crystals and this effect it was not significantly different regarding the effect caused by the crude. Besides, fucoidan F0.5 only promoted the formation of COD type crystals, while fucoidans F1.1 and F2.0 did not influence the formation of crystals compared with the control. The results described in this study indicate that the commercial crude fucoidan of Fucus vesiculosus it’s a mix of several fucoidan which, in turn, have different chemical compositions besides having different pharmacological activities. The use of these fucoidans it´s indicated according the pharmacological activity to be evaluated.