Caio Rodrigo Dias Assis

Characterization of acetylcholinesterase from the brain of the Amazonian tambaqui (Colossoma macropomum) and in vitro effect of organophosphorus and carbamate pesticides

In the present study, acetylcholinesterase (AChE) from the brain of the Amazonian fish tambaqui (Colossoma macropomum) was partially characterized and its activity was assayed in the presence of five organophosphates (dichlorvos, diazinon, chlorpyrifos, and tetraethyl pyrophosphate [TEPP]) and two carbamates (carbaryl and carbofuran) insecticides. Optimal pH and temperature were 7.0 to 8.0 and 45°C, respectively. The enzyme retained approximately 70% of activity after incubation at 50°C for 30 min. The insecticide concentration capable of inhibiting half of the enzyme activity (IC50) for dichlorvos, chlorpyrifos, and TEPP were calculated as 0.04 µmol/L, 7.6 µmol/L, and 3.7 µmol/L, respectively. Diazinon and temephos did not inhibit the enzyme. The IC50 values for carbaryl and carbofuran were estimated as 33.8 µmol/L and 0.92 µmol/L, respectively. These results suggest that AChE from the juvenile C. macropomum brain could be used as an alternative biocomponent of organophosphorus and carbamate biosensors in routine pesticide screening in the environment.

EFFECT OF Moringa oleifera FLOWER EXTRACT ON LARVAL TRYPSIN AND ACETHYLCHOLINESTERASE ACTIVITIES IN Aedes aegypti

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality.

Comparative effect of pesticides on brain acetylcholinesterase in tropical fish.

Monitoring of pesticides based on acetylcholinesterase (AChE; EC 3.1.1.7) inhibition in vitro avoids interference of detoxification defenses and bioactivation of some of those compounds in non-target tissues. Moreover, environmental temperature, age and stress are able to affect specific enzyme activities when performing in vivo studies. Few comparative studies have investigated the inter-specific differences in AChE activity in fish. Screening studies allow choosing the suitable species as source of AChE to detect pesticides in a given situation. Brain AChE from the tropical fish: pirarucu (Arapaima gigas), cobia (Rachycentron canadum) and Nile tilapia (Oreochromis niloticus) were characterized and their activities were assayed in the presence of pesticides (the organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate- TEPP and the carbamates: carbaryl and carbofuran). Inhibition parameters (IC₅₀ and Ki) for each species were found and compared with commercial AChE from electric eel (Electrophorus electricus). Optimal pH and temperature were found to be 8.0 and 35-45 °C, respectively. A. gigas AChE retained 81% of the activity after incubation at 50 °C for 30 min. The electric eel enzyme was more sensitive to the compounds (mainly carbofuran, IC₅₀ of 5 nM), excepting the one from A. gigas (IC₅₀ of 9 nM) under TEPP inhibition. These results show comparable sensitivity between purified and non-purified enzymes suggesting them as biomarkers for organophosphorus and carbamate detection in routine environmental and food monitoring programs for pesticides.

Kinetic and physicochemical properties of brain acetylcholinesterase from the peacock bass (Cichla ocellaris) and in vitro effect of pesticides and metal ions.

Brain acetylcholinesterase (AChE; EC 3.1.1.7) from peacock bass (Cichla ocellaris) was characterized and the effect of organophosphorus and carbamate pesticides as well as ions and heavy metals was evaluated. The kinetic parameters K(m) and V(max) were determined as 0.769 mM and 0.189 U/mg of protein respectively. Optimal pH and temperature were found to be 8.0 and 45°C. The enzyme retained approximately half of the activity after incubation at 50°C for 30 min. Total cholinesterase activity on brain of this species can be ascribed to AChE according to selective inhibitors analysis (neostigmine, eserine and BW284c5 reduced its activity whereas no effect was noticed for Iso-OMPA). Seven pesticides (five organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate - TEPP and two carbamates: carbaryl and carbofuran) showed inhibitory effects on C. ocellaris AChE. However, the strongest effect was observed with carbofuran (IC(50)=0.21 μM and K(i)=2.57 × 10(-3) μM). The following ions (1 mM) showed to inhibit its activity (decrescent order): Hg(2+)>As(3+)>Cu(2+)>Zn(2+). EDTA(2-) did not affect enzyme activity. The present study provides assay conditions and data to suggest this enzyme as in vitro biomarker of organophosphorus and carbamate pesticides in routine environmental screening programs.

Fish Cholinesterases as Biomarkers of Organophosphorus and Carbamate Pesticides

Due to reasons that last for decades, environmental monitoring of pesticides is an urgent need. Contamination by pesticides is an important public health problem, mainly in developing countries. It is estimated that only 0.1% of the applied pesticides in fact reach the target pests, while the rest spreads throughout the environment (Hart and Pimentel, 2002). In addition, among the 500,000 deaths a year related to pesticides in the developing world, approximately 200,000 occur due to the use of organophosphorus (OP) and carbamates (CB) pesticides (Eddleston et al., 2008). These are among the most important classes of insecticides/acaricides in usage and billing (Nauen and Bretschneider, 2002). The primary and most known target for the action of organophosphorus and carbamate compounds is a family of enzymes (Cholinesterases; ChEs) formed by: acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). The first is synthesized in hematopoiesis, occurs in the brain, endplate of skeletal muscle, erythrocyte membrane, and its main function is to regulate neuronal communication by hydrolyzing the ubiquitous neurotransmitter acetylcholine in synaptic cleft (Quinn, 1987; Silman and Sussman, 2005). The second is synthesized in liver and is present in plasma, smooth muscle, pancreas, adipocytes, skin, brain and heart (Cokugras, 2003). Although its physiological function is not well defined, BChE is pointed as one of the main detoxifying enzymes able to hydrolyze or scavenge a broad range of xenobiotic compounds like cocaine, heroine, anaesthetics, and pesticides (Soreq and Zakut, 1990; Taylor, 1991; Cokugras, 2003; Nicolet et al., 2003). Some studies hypothesize that one of the functions of BChE is to protect AChE against anticholinesterasic agents (Whitaker, 1980; Whitaker, 1986). Pezzementi and Chatonnet (2010) reported that ChEs emerged from a family of proteins with adhesion properties. Both play other roles in the neuronal tissue, particularly in neuronal differentiation and development, cell growth, adhesion and signalling. In addition, AChE participates even in hematopoietic differentiation (Chatonnet and Lockridge, 1989; Taylor, 1991; Johnson and Moore, 2000; Silman and Sussman, 2005). Moreover, AChE and BChE are different concerning several other aspects: while AChE has an in vivo half-life of 120 days, BChE lasts 7-12 days. AChE is inhibited by substrate excess and BChE is activated by substrate excess (Lopez-Carillo and Lopez-Cervantes, 1993; Cokugras, 2003). AChE is selectively inhibited by propidium, DDM, caffeine, Nu1250, 62c47

Aluminium sulfate exposure: A set of effects on hydrolases from brain, muscle and digestive tract of juvenile Nile tilapia (Oreochromis niloticus).

Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1μg·mL-1 (G1) and 3μg·mL-1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.

Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions

This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68μM (dichlorvos); 4.35μM (carbaryl) and 0.28μM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7μM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.

Levantamento epidemiológico da hanseníase no nordeste brasileiro durante o período de 2001-2010

E uma doenca infecciosa, cronica, de grande importância para a saude publica devido a sua magnitude e seu alto poder incapacitante, atingindo principalmente a faixa etaria economicamente ativa. Ela acomete principalmente a pele e os nervos perifericos, mas tambem se manifesta como uma doenca sistemica comprometendo articulacoes, olhos, testiculos, gânglios e outros orgaos. O alto potencial incapacitante da hanseniase esta diretamente relacionado a capacidade de penetracao do Mycobacterium leprae na celula nervosa e seu poder imunogenico. Com este trabalho, objetivou-se delinear a situacao epidemiologica dos Estados da Regiao Nordeste, durante o periodo de 2001 a 2010, baseado nos registros nos relatorios de situacao epidemiologica do Sistema Nacional de Vigilância em Saude, publicados no ano 2011. Segundo informacoes do Sinan/SVS-MS, em 2010, a situacao epidemiologica da hanseniase no Brasil, era de 34.894 casos novos, 2.461 (7,1%) em menores de 15 anos, coeficiente geral de deteccao 18,2/100 mil habitantes. O coeficiente de deteccao < 15 anos foi de 5,4/100 mil habitantes. Queda significante no coeficiente de deteccao em todas as regioes geograficas de 4,0% ao ano e 31,5% no periodo em media nos ultimos 10 anos. Especificamente, nos Estados da regiao Nordeste, apesar da importante reducao do coeficiente de prevalencia de hanseniase em Alagoas (atualmente: 1,1 casos/10 mil habitantes), Bahia (1,9 casos/10 mil habitantes), Ceara (1,1 casos/10 mil habitantes), Maranhao (5,7 casos/10 mil habitantes), Paraiba (1,5 casos/10 mil habitantes), Pernambuco (2,7 casos/10 mil habitantes), Piaui (3,5 casos/10 mil habitantes) e Sergipe (1,2 casos/10 mil habitantes), os Estados da regiao Nordeste demandam intensificar as acoes para eliminacao da doenca, justificadas por um padrao de media endemicidade segundo os parâmetros de prevalencia. O Rio Grande do Norte, embora tenha havido uma importante reducao do coeficiente de prevalencia de hanseniase, que atualmente e de 0,74 casos/10 mil habitantes, o Estado demanda intensificacao das acoes para eliminacao da doenca em alguns municipios que ainda nao alcancaram o nivel de eliminacao. Os dados revelam a importância das acoes da vigilância sanitaria de cada Estado para a diminuicao e posterior erradicacao desta enfermidade.

Doenças tropicais negligenciadas na região nordeste do Brasil

Doencas tropicais negligenciadas correspondem a um grupo de doencas infecciosas que afetam populacoes de baixa renda nos tropicos. A Organizacao Mundial de Saude lista entre elas a malaria, a leishmaniose, a esquistossomose, a oncocercose, a filariose linfatica, a doenca de chagas, tripanossomiase africana, a hanseniase, dengue, ulcera de Buruli (ou doenca de Buruli), a cisticercose, a equinococose, a bouba, a raiva, o tracoma e algumas helmintiases transmitidas pelo solo (Ascaris lumbricoides, Trichuris trichiura e os ancilostomideos). No Brasil, varias sao endemicas. Em seu relatorio anual pelo Ministerio da Saude, a partir do Sistema de Nacional de Vigilância e Saude, quatro dessas doencas sao mencionadas como negligenciadas (esquistossomose, tracoma, oncocercose e a filariose) para todos os Estados da Federacao, mais o Distrito Federal. Neste trabalho visualizaremos as principais caracteristicas das doencas tropicais negligenciadas citadas pelo Sistema de Nacional de Vigilância e Saude do Brasil para a Regiao Nordeste. Para o levantamento teorico foram utilizados dados e informacoes constantes no site oficial da Organizacao Mundial de Saude e no banco de bases Scientific Eletronic Library On-line (SciELO), tomando por base a lista de procura pelas doencas negligenciadas listadas nos relatorios anuais do Sistema de Nacional de Vigilância e Saude do Brasil, em seu site oficial, publicada no ano base de 2011, a saber: esquistossomose, tracoma, oncocercose e a filariose, para os seguintes Estados da Federacao: Alagoas, Bahia, Ceara, Maranhao, Paraiba, Pernambuco, Piaui, Rio Grande do Norte e Sergipe. Todos os dados de internacoes, prevalencia e de obitos, tanto a nivel nacional quando estadual, sao oriundos dos Inqueritos Epidemiologicos realizados pelo Ministerio da Saude (MS), Secretaria de Vigilância em Saude (SVS). Todas as imagens dos inqueritos de tracoma sao advindas dos relatorios por Estados, publicados pelo Ministerio da Saude (MS). Os dados demograficos e imagens foram provenientes do censo realizado no ano de 2010 pelo Instituto Brasileiro de Geografia e Estatistica. Nos relatorios pesquisados, foram encontrados dados sobre a esquistossomose e para o traco em todos os Estados. Para Filariose apenas para o Estado de Pernambuco. Para oncocercose nao foi encontrado dados epidemiologicos no relatorio. A maioria dos Estados apresentaram areas endemicas para a esquistossomose. A presenca de alta e media prevalencia em municipios do Estado reforcam a necessidade de implementacao e fortalecimento das acoes de vigilância epidemiologica e controle tanto da esquistossomose quanto do tracoma. Atraves desse aparato epidemiologico sobre as doencas tropicais negligenciadas e possivel mapear as principais areas e fomentar acoes para o controle das doencas estudadas neste trabalho.

High sucrolytic activity by invertase immobilized onto magnetic diatomaceous earth nanoparticles

Graphical abstract