Mauro Velho de Castro Faria

Avaliação integrada do impacto do uso de agrotóxicos sobre a saúde humana em uma comunidade agrícola de Nova Friburgo, RJ

O impacto do uso de agrotoxicos sobre a saude humana e um problema que tem merecido atencao da comunidade cientifica em todo o mundo, sobretudo nos paises em desenvolvimento. O consumo de agrotoxicos na regiao sudeste do Brasil esta estimado em 12kg de agrotoxico/trabalhador/ano. Em algumas areas agricolas do Estado do Rio de Janeiro, como na regiao da Microbacia do Corrego de Sao Lourenco, Nova Friburgo, o consumo de agrotoxico foi estimado em 56kg de agrotoxico/trabalhador/ano. Elevados niveis de contaminacao humana e ambiental foram encontrados nesta regiao, como decorrencia do uso extensivo destes agentes quimicos. A avaliacao do impacto sobre a saude humana implica o conhecimento e a visualizacao da importância/magnitude relativa de cada uma das vias de contaminacao. Inumeros fatores, que, em geral, encontram-se inter-relacionados, contribuem para a situacao encontrada na Microbacia do Corrego de Sao Lourenco e a forma mais adequada de se avaliar toda a dimensao deste problema e o uso de uma abordagem integrada.

Análise da contaminação dos sistemas hídricos por agrotóxicos numa pequena comunidade rural do Sudeste do Brasil

Recent advances in analytical techniques allow identifying pesticide pollution in water systems. In small rural communities, the negative effects of pesticide pollution can be aggravated by the lack of infrastructure and adverse socioeconomic conditions. This study investigated pesticide pollution in potential water supply sources in a tomato growing area in Paty do Alferes, Rio de Janeiro State, Brazil. The study selected 27 points where five monthly samples were collected. Pesticide pollution was determined by analyzing acetyl-cholinesterase inhibition. In 19 of the 27 sample points, some pesticide pollution was detected, and in two points the pesticide pollution was above the permitted limits. The results thus proved the incidence of pesticide pollution in water sources in Paty do Alferes that could jeopardize the local populations health.

The anticancer drug perillyl alcohol is a Na/K-ATPase inhibitor

The monoterpene perillyl alcohol (POH) is a drug used in the treatment of several malignant tumors, including gliomas. The present study defines a POH inhibitory effect on Na/K-ATPase activity from kidney and brain guinea pig extracts and from a human glioblastoma cell line. This inhibition showed a high degree of selectivity toward the kidney enzyme expressing, as do glioblastoma cells, the α1 subunit. Kinetic studies with purified enzymes showed a noncompetitive POH inhibition profile to Na+ and K+ and an uncompetitive inhibition towards ATP. Furthermore, potassium activated p-nitrophenylphosfatase activity of these purified preparations was not inhibited by POH, suggesting that this drug, differently from the classical inhibitor ouabain, acted in the initial phase of the enzyme’s catalytic cycle. We suggest that POH antitumor action could be linked to its Na/K-ATPase binding properties.

Methyl parathion activation by a partially purified rat brain fraction

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway. The results from HPLC analysis did not show a peak corresponding to methyl paraoxon (O,O-dimethyl O-p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.

Interaction of stannous chloride leads to alteration in DNA, triphosphate nucleotides and isolated bases

Stannous chloride (SnCl2) is a reducing chemical agent used in several man-made products. SnCl2 can generate reactive oxygen species (ROS); therefore, studies have been carried out in order to better understand its damaging action in biological systems. In this work, calf thymus DNA, triphosphate nucleotides and isolated bases were incubated with SnCl2 and the results were analyzed through UV spectrophotometry. The presence of stannous ions altered the absorption spectra of all three isolates. The amount of stannous ions associated to DNA was measured by atomic absorption spectrophotometry. Data showed that more than 40% of the initial SnCl2 concentration was present in the samples. Our results are in accordance with the damaging potential of this salt and present evidence that stannous ions can complex with DNA, inducing ROS in its vicinity, which may be responsible for the observed lesions. (Mol Cell Biochem xxx: 173–179, 2005)

Reduced plasma nonesterified fatty acid levels and the advent of an acute lung injury in mice after intravenous or enteral oleic acid administration.

Although exerting valuable functions in living organisms, nonesterified fatty acids (NEFAs) can be toxic to cells. Increased blood concentration of oleic acid (OLA) and other fatty acids is detected in many pathological conditions. In sepsis and leptospirosis, high plasma levels of NEFA and low albumin concentrations are correlated to the disease severity. Surprisingly, 24 h after intravenous or intragastric administration of OLA, main NEFA levels (OLA inclusive) were dose dependently decreased. However, lung injury was detected in intravenously treated mice, and highest dose killed all mice. When administered by the enteral route, OLA was not toxic in any tested conditions. Results indicate that OLA has important regulatory properties on fatty acid metabolism, possibly lowering circulating fatty acid through activation of peroxisome proliferator-activated receptors. The significant reduction in blood NEFA levels detected after OLA enteral administration can contribute to the already known health benefits brought about by unsaturated-fatty-acid-enriched diets.

Murine lung injury caused by Leptospira interrogans glycolipoprotein, a specific Na/K-ATPase inhibitor

BackgroundLeptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation.MethodsWe injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE2 and LTB4). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells.ResultsLeptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor.ConclusionsGLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.

Development of a simple and low-cost enzymatic methodology for quantitative analysis of carbamates in meat samples of forensic interest.

Abstract:  Foods contaminated with a granulated material similar to Temik (a commercial pesticide formulation containing the carbamate insecticide aldicarb) are often involved in accidental ingestion, suicides, and homicides in Brazil. We developed a simple technique to detect aldicarb. This technique is based on the inhibition of a stable preparation of the enzyme acetylcholinesterase, and it is specially adapted for forensic purposes. It comprises an initial extraction step with the solvent methylene chloride followed by a colorimetric acetylcholinesterase assay. We propose that results of testing contaminated forensic samples be expressed in aldicarb equivalents because, even though all other carbamates are also potent enzyme inhibitors, aldicarb is the contaminant most frequently found in forensic samples. This method is rapid (several samples can be run in a period of 2 h) and low cost. This method also proved to be precise and accurate, detecting concentrations as low as 40 μg/kg of aldicarb in meat samples.

4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) inhibits Na+/K(+)-ATPase and Ras oncogene activity in cancer cells.

The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including glioma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed ~10 times greater in vitro growth inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the inhibition of both alpha-1 Na(+)/K(+)-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic activities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b displayed relatively similar inhibitory activities on purified guinea pig brain preparations that mainly express NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in gliomas, melanomas and non-small-cell lung cancers NSCLCs.

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion.

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hanks solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb+ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb+ in the absence and in the presence of ouabain. The optimal conditions for Rb+ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 degrees C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC(50) was 100 microM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.