Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus

The aim of this study was to determine the toxicity of the aqueous extract of neem leaves, a product extensively used in fish-farms as alternative for the control of fish parasites and fish fry predators, for the neotropical fish Prochilodus lineatus. The 24 It LC(50) of neem leaf extract for juveniles P lineatus was estimated as 4.8 g L(-1); the fish were then exposed for 24 h to 2.5, 5.0 and 7.5 g L(-1) or only clean water (control). Plasma glucose levels were higher in fish exposed to 2.5 g L(-1) and 5.0 g L(-1) neem extract, relative to control, indicating a typical stress response. Neem extract did not interfere with the osmoregulating capacity of the fish, as their plasma sodium, chloride, total protein and osmolarity did not change. The presence of the biopesticide interfered with the antioxidant defense system of P. lineatus, as there was a decrease in liver catalase activity at all neem concentrations and the detoxifying enzyme glutathione-S-transferase was activated in fish exposed to 5.0 g L(-1). Fish exposed to all neem extract concentrations exhibited damaged gill and kidney tissue. These results indicate that although neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming it does cause functional and morphological changes in this fish species. (c) 2006 Elsevier B.V. All rights reserved.

Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4μM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

Rational design of an organometallic glutathione transferase inhibitor.

Double trouble: A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor (see picture). The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

A peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related to sulfur amino acid metabolism

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine -lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

Location of a potential transport binding site in a sigma class glutathione transferase by x-ray crystallography.

The crystal structure of the sigma class glutathione transferase from squid digestive gland in complex with S-(3-iodobenzyl)glutathione reveals a third binding site for the glutathione conjugate besides the two in the active sites of the dimer. The additional binding site is near the crystallographic two-fold axis between the two alpha 4-turn-alpha 5 motifs. The principal binding interactions with the conjugate include specific electrostatic interactions between the peptide and the two subunits and a hydrophobic cavity found across the two-fold axis that accommodates the 3-iodobenzyl group. Thus, two identical, symmetry-related but mutually exclusive binding modes for the third conjugate are observed. The hydrophobic pocket is about 14 A from the hydroxyl group of Tyr-7 in the active site. This site is a potential transport binding site for hydrophobic molecules or their glutathione conjugates.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

In Vivo Models for Measuring Placental Glutatione-S-transferase (GST-P 7-7) Levels: A Suitable Biomarker for Understanding Cancer Pathogenesis

The Glutatione-S-transferases (GSTs) comprise a family of enzymes closely associated with the cell detoxification of xenobiotics. GSTs exist as homo- or heterodimers and have been grouped into at least seven distinct classes. The main function of GSTs is to catalyze the conjugation of reduced glutathione (GSH) to an electrophilic site of a broad range of potentially toxic and carcinogenic compounds, thereby making such compounds less dangerous and enabling their ready-excretion. Placental GST, known as GST-P 7-7, is the main isoform found in normal placental tissue and comprises 67% of the total GST concentration in this tissue. During development, GST-P 7-7 decreases in concentration and is absent in adult tissues. Interestingly, GST-P 7-7 expression has been detected in adult tissues after exposure to carcinogenic agents in several experimental test systems, being considered a reliable biomarker of exposure and susceptibility in early phases of carcinogenesis. In this article, we review a series of studies involving GST-P 7-7 expression as a suitable tool for understanding cancer pathogenesis, especially cancer risk.

Polimorfismos GSTM1, GSTT1 e GSTP1 da enzima Glutationa S-transferase como fatores moduladores do fenótipo na anemia falciforme

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Determinação de aflatoxina B1 em rações e aflatoxina M1 no leite de propriedades do Estado de São Paulo

A ocorrência de aflatoxina B1 (AFB1) em rações e aflatoxina M1 (AFM1) no leite cru foi avaliada em propriedades leiteiras situadas na região nordeste do Estado de São Paulo, Brasil, de outubro de 2005 a fevereiro de 2006. A análise de aflatoxinas foi efetuada utilizando-se colunas de imunoafinidade para purificação dos extratos, sendo a quantificação realizada através de cromatografia líquida de alta eficiência. A AFB1 foi detectada em 40% das rações em níveis de 1,0 a 19,5 μg.kg-1. A concentração de AFM1 em 36,7% de amostras de leite positivas variou de 0,010 a 0,645 μg.L-1. Somente uma amostra de leite estava acima do limite de tolerância adotado no Brasil (0,5 μg.L-1) para AFM1. Concluiu-se que as concentrações de aflatoxinas na ração e no leite foram relativamente baixas, embora a alta frequência das aflatoxinas nas amostras analisadas indique a necessidade de contínuo monitoramento a fim de prevenir a contaminação de ingredientes e rações destinadas ao gado leiteiro.

Influência do treinamento físico aeróbio no transporte mitocondrial de ácidos graxos de cadeia longa no músculo esquelético: papel do complexo carnitina palmitoil transferase

O ácido graxo (AG) é uma importante fonte de energia para o músculo esquelético. Durante o exercício sua mobilização é aumentada para suprir as necessidades da musculatura ativa. Acredita-se que diversos pontos de regulação atuem no controle da oxidação dos AG, sendo o principal a atividade do complexo carnitina palmitoil transferase (CPT), entre os quais três componentes estão envolvidos: a CPT I, a CPT II e carnitina acilcarnitina translocase. A função da CPT I durante o exercício físico é controlar a entrada de AG para o interior da mitocôndria, para posterior oxidação do AG e produção de energia. Em resposta ao treinamento físico há um aumento na atividade e expressão da CPT I no músculo esquelético. Devido sua grande importância no metabolismo de lipídios, os mecanismos que controlam sua atividade e sua expressão gênica são revisados no presente estudo. Reguladores da expressão gênica de proteínas envolvidas no metabolismo de lipídios no músculo esquelético, os receptores ativados por proliferadores de peroxissomas (PPAR) alfa e beta, são discutidos com um enfoque na resposta ao treinamento físico.

Interação entre consumo alimentar e polimorfismos da GSTM1 e GSTT1 no risco para o câncer de cabeça e pescoço: estudo caso-controle em São Paulo, Brasil

O objetivo foi investigar a interação entre fatores dietéticos e polimorfismos de enzimas de metabolização de xenobióticos (GSTM1 e GSTT1) associadas ao câncer de cabeça e pescoço em um estudo caso controle de base hospitalar, no Município de São Paulo, Brasil. Participaram 103 casos incidentes, histologicamente confirmados, e 101 controles. O consumo alimentar foi obtido por um questionário de frequência alimentar validado. Os polimorfismos GSTM1 e GSTT1 foram avaliados pelo método PCR. Observou-se aumento de risco no mais alto tercil de consumo de carne bovina na presença do alelo nulo da GSTM1 (OR = 10,79; IC95%: 2,17-53,64) e GSTT1 (OR = 3,41; IC95%: 0,43-27,21). Considerando-se a razão entre alimentos de origem animal e vegetal, verificou-se para o tercil intermediário a OR = 2,02 (IC95%: 0,24-16,0) e no tercil superior OR = 3,23 (IC95%: 0,40-25,92). Os resultados apontam para uma possível interação entre o consumo de carne e variantes polimórficas dos genes GSTM1 e GSTT1 na modulação do risco para o câncer de cabeça e pescoço, influenciados pelo consumo de alimentos de origem vegetal.

Predictive values of aspartate aminotransferase and gamma-glutamyl transferase for the hepatic accumulation of copper in cattle and buffalo

Ten cattle and 10 buffalo were divided into 2 groups (control [n = 8] and experimental [n = 12]) that received daily administration of copper. Three hepatic biopsies and blood samples were performed on days 0, 45, and 105. The concentration of hepatic copper was determined by spectrophotometric atomic absorption, and the activities of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) were analyzed. Regression analyses were done to verify the possible existing relationship between enzymatic activity and concentration of hepatic copper. Sensitivity, specificity, accuracy, and positive and negative predictive values were determined. The serum activities of AST and GGT had coefficients of determination that were excellent predictive indicators of hepatic copper accumulation in cattle, while only GGT serum activity was predictive of hepatic copper accumulation in buffalo. Elevated serum GGT activity may be indicative of increased concentrations of hepatic copper even in cattle and buffalo that appear to be clinically healthy. Thus, prophylactic measures can be implemented to prevent the onset of a hemolytic crisis that is characteristic of copper intoxication.