Monocrotaline induces acutely cerebrovascular lesions, astrogliosis and neuronal degeneration associated with behavior changes in rats: A model of vascular damage in perspective.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites playing an important role as phytotoxins in the plant defense mechanisms and can be present as contaminant in the food of humans and animals. The PA monocrotaline (MCT), one of the major plant derived toxin that affect humans and animals, is present in a high concentration in Crotalaria spp. (Leguminosae) seeds and can induce toxicity after consumption, characterized mainly by hepatotoxicity and pneumotoxicity. However, the effects of the ingestion of MCT in the central nervous system (CNS) are still poorly elucidated. Here we investigated the effects of MCT oral acute administration on the behavior and CNS toxicity in rats. Male adult Wistar were treated with MCT (109 mg/Kg, oral gavage) and three days later the Elevated Pluz Maze test demonstrated that MCT induced an anxiolytic-like effect, without changes in novelty habituation and in operational and spatial memory profiles. Histopathology revealed that the brain of MCT-intoxicated animals presented hyperemic vascular structures in the hippocampus, parahippocampal cortex and neocortex, mild perivascular edema in the neocortex, hemorrhagic focal area in the brain stem, hemorrhage and edema in the thalamus. MCT also induced neurotoxicity in the cortex and hippocampus, as revealed by Fluoro Jade-B and Cresyl Violet staining, as well astrocyte reactivity, revealed by immunocytochemistry for glial fibrillary acidic protein. Additionally, it was demonstrated by RT-qPCR that MCT induced up-regulation on mRNA expression of neuroinflammatory mediator, especially IL1ß and CCL2 in the hippocampus and cortex, and down-regulation on mRNA expression of neurotrophins HGDF and BDNF in the cortex. Together, these results demonstrate that the ingestion of MCT induces cerebrovascular lesions and toxicity to neurons that are associated to astroglial cell response and neuroinflammation in the cortex and hippocampus of rats, highlighting CNS damages after acute intoxication, also putting in perspective it uses as a model for cerebrovascular damage.

Saponin-rich fraction from Agave sisalana: effect against malignant astrocytic cells and its chemical characterisation by ESI-MS/MS.

Astrocytic tumour cells derived from human (GL-15) and rat (C6) gliomas, as well as non-tumoural astrocytic cells, were exposed to the saponin-rich fraction (SF) from Agave sisalana waste and the cytotoxic effects were evaluated. Cytotoxicity assays revealed a reduction of cell viability that was more intensive in glioma than in non-tumoural cells. The SF induced morphological changes in C6 cells. They were characterised by cytoplasmic vacuole formation associated with increase in the formation of acidic lysosomes. The SF was subjected to purification on Sephadex LH-20, which characterised three probable steroidal saponins (sisalins) by electrospray ionisation mass spectrometry multistage (ESI-MSn). Sisalins from sisal may be responsible for the cytotoxicity, which involves cytoplasmatic vacuole formation and selective action for glioma cells.

Involvement of astrocytic CYP1A1 isoform in the metabolism and toxicity of the alkaloid pyrrolizidine monocrotaline.

Monocrotaline (MCT) and its pyrrole derivative, dehydromonocrotaline (DHMC), interact with molecular targets in cells of the central nervous system. DHMC presents higher toxicity than MCT indicating that its metabolism of MCT is a critical step of this alkaloid toxicity. This study sought to elucidate the metabolism and the toxicity of MCT in C6 astrocyte cell line and primary cultures of rat astrocytes by investigating metabolic enzymatic mechanisms of the cytochrome P450 (CYP) system and conjugation with glutathione. Treatment with omeprazole (OMP) (20 µM), a non-specific inducer of CYP450 induced approximately 10-fold increase in CYP1A1 activity after 2 h of treatment. Similarly, the 7-Ethoxyresorufin-O-deethylase (EROD) activity was induced by treatment with MCT (100-500 µM), indicating that the P450 CYP1A1 isoform was active and involved in the metabolism of MCT. Analysis of conjugation with glutathione showed a significant depletion of GSH after MCT (500 µM) treatment, and this was partially reversed by pretreatment with a P450 inhibitor (cimetidine 100 µM). These results suggest that not only the alkaloid MCT but, also its metabolite may deplete GSH. Rosenfeld staining showed intense vacuolization after MCT treatment, which was partially inhibited in the presence of a P450 activator. MTT test showed that association of MCT with OMP induced a reduction in cell viability in C6 and primary astrocytic cells. These results demonstrate that MCT is metabolized by astrocytic CYP1A1 to generate metabolites that can deplete GSH. Moreover, changes in the activity of the P450 enzymes interfere with the cytotoxic effects induced by the alkaloid.

In vitro ovicidal and larvicidal activity of Agave sisalana Perr. (sisal) on gastrointestinal nematodes of goats.

This study describes the in vitro anthelmintic activity of aqueous extracts (AE), ethyl acetate extracts (EE), flavonoid fractions (FF) and saponin fractions (SF) obtained from sisal waste (Agave sisalana) against gastrointestinal nematodes of goats. The activity of these extracts was evaluated by performing inhibition of egg hatch (EHA) and larval migration (LMI) assays. The EC(50) results of the EHA corresponded to 4.7, 0.1 and 0.05 mg/mL for EE, EA and FF, respectively. The SF fraction showed no ovicidal activity. The percent efficacies that were observed for the LMI were 50.3, 33.2 and 64.1% for the AE, EE and SF, respectively. The FF fraction did not show activity against the larvae. The analysis of the FF fraction indicates the presence of a homoisoflavonoid. This report suggests that the A. sisalana has activity in vitro against gastrointestinal nematodes of goats. This effect is likely related to the presence of homoisoflavonoid and saponin compounds, which have different actions for specific stages of nematode development.

[Effects of aqueous extracts of Mentha piperita L. and Chenopodium ambrosioides L. leaves in infective larvae cultures of gastrointestinal nematodes of goats]. / Efeitos dos extratos aquosos de folhas de Mentha piperita L. e de Chenopodium ambrosioides L. sobre cultivos de larvas infectantes de nematóides gastrintestinais de caprinos.

Phitotherapy has been frequently utilized in parasitism control for numerous animal species. The aim of this experiment was to evaluate the in vitro effects of aqueous extracts of Mentha piperita L. and Chenopodium ambrosioides L. leaves in larvae cultures of gastrointestinal nematodes of goats. Six different concentrations of M. piperita extracts (196; 150.7; 115.9; 89.1; 68.5 e 52.7 mg/mL) and C. ambrosioides extracts (110,6; 85; 65,3; 50,2; 38,6 e 29,6 mg/mL) were used for the treatment of larvae cultures, in triple assays. Distilled water and doramectin were used in larvae cultures as negative and positive controls, respectively. The results revealed a reduction of more than 95% of the infective larvae when M. piperita extracts were used in the concentrations of 115.9 and 196 mg/mL, and C. ambrosioides extract in the concentration of 110.6 mg/mL, supporting the effect of these extracts in the in vitro treatment of gastrointestinal nematodes of goats.

Otimização de metodologia por CCD para determinação de aflatoxina M1 em leite de cabra e investigação de sua ocorrência no Estado da Bahia / Optimization of methodology by TLC for determining aflatoxin M1 in goat’s milk and investigation of its occurrence in the State of Bahia

A aflatoxina M1 (AFM1) é um produto da biotransformação da aflatoxina B1, a qual é excretada no leite de animais que ingerem alimentos contaminados. Os objetivos deste trabalho foram otimizar o método por cromatografia em camada delgada (CCD) para análise de AFM1 em leite de cabra e avaliar sua ocorrência no Estado da Bahia. Foram coletadas 100 amostras de leite em cinco propriedades, localizadas na região do Recôncavo Baiano, durante o período de novembro de 2000 a agosto de 2002. A AFM1 foi determinada no leite por meio de método de CCD conforme Sabino et al. (1989) modificado com a utilização de acetato de chumbo na fase de purificação para promover a precipitação de proteínas do substrato, possibilitando a visualização da AFM1 na placa cromatográfica. Os limites de detecção e quantificação obtidos pela técnica modificada foram de 0,2 e 0,5μg/L, respectivamente, com percentual de recuperação de 89,6% e coeficiente de variação igual a zero. Estes resultados revelaram que esta metodologia mostrou-se eficiente para a determinação de AFM1 em leite caprino. Em todas as amostras analisadas não foi detectada a presença de AFM1, demonstrando a boa qualidade deste produto quanto à contaminação por esta toxina

Ocorrência de aflatoxinas em amendoim e seus produtos comercializados no Estado da Bahia durante o ano de 2002 / Occurrence of Aflatoxins in peanut and peanut products commercialized in the state of Bahia during the year 2002

Este estudo teve como objetivo avaliar a contaminação por aflatoxinas (B1,B2,G1 e G2) em amostras de amendoim comercializadas em alguns municípios do estado da Bahia. Foram analisadas 100 amostras, sendo 33 de amendoim e 67 de produtos a base de amendoim. A coleta das amostras foi realizada aleatoriamente em diferentes estabelecimentos comerciais da cidade de Salvador (75 amostras) e em municípios do estado da Bahia (25 amostras) durante o período de janeiro a outubro de 2002. A extração foi realizada com acetonitrila:água (84:16, v/v), e a quantificação das aflatoxinas procedida pelo método de Cromatografia Líquida de Alta Eficiência (CLAE), empregando detecção por fluorescência. Das 31 amostras de amendoim analisadas, 29 (93,55 por cento) foram positivas, sendo que 17 (54,84 por cento), apresentaram teor de contaminação por B1 + B2 + G1+G2 superior a 20 ug/kg. Para as 69 amostras de derivados de amendoim, apenas 6 (8,7 por cento) apresentaram contaminação por aflatoxinas B1+B2+G1+G2 superior ao limite. Os resultados ainda revelaram que 20(80 por cento) do total de amostras coletadas nos municípios do Estado (25) e 38 (50,7 por cento) das 75 amostras procedentes de Salvador foram positivas. Os resultados ressaltam a necessidade de um controle eficaz dos níveis de aflatoxinas destes produtos na região