João Bosco de Salles

Pseudomonas aeruginosa -induced production of free radicals by IFNγ plus TNFα-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?

We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNγ plus TNFα to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed. In this study we investigated the fate of IFNγ plus TNFα‐activated HUVEC that have harboured IC bacteria for up to 24 h. At 10 h, the endothelial cell killing of P. aeruginosa isolates exceeded 90%. IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC. P. aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH). Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability. Treatment of cells with the antioxidant α‐lipoic acid significantly increased the survival of infected cells. Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells. Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.

Cholinesterase activity of muscle tissue from freshwater fishes: Characterization and sensitivity analysis to the organophosphate methyl‐paraoxon

The biochemical characterization of cholinesterases (ChE) from different teleost species has been a critical step in ensuring the proper use of ChE activity levels as biomarkers in environmental monitoring programs. In the present study, ChE from Oreochromis niloticus, Piaractus mesopotamicus, Leporinus macrocephalus, and Prochilodus lineatus was biochemically characterized by specific substrates and inhibitors. Moreover, muscle tissue ChE sensitivity to the organophosphate pesticide methyl-paraoxon was evaluated by determining the inhibition kinetic constants for its progressive irreversible inhibition by methyl-paraoxon as well as the 50% inhibitory concentration (IC50) for 30 min for each species. The present results indicate that acetylcholinesterase (AChE) must be present in the muscle from P. mesopotamicus, L. macrocephalus, and P. lineatus and that O. niloticus possesses an atypical cholinesterase or AChE and butyrylcholinesterase (BChE). Furthermore, there is a large difference regarding the sensitivity of these enzymes to methyl-paraoxon. The determined IC50 values for 30 min were 70 nM (O. niloticus), 258 nM (P. lineatus), 319 nM (L. macrocephalus), and 1578 nM (P. mesopotamicus). The results of the present study also indicate that the use of efficient methods for extracting these enzymes, their kinetic characterization, and determination of sensitivity differences between AChE and BChE to organophosphate compounds are essential for the determination of accurate ChE activity levels for environmental monitoring programs.

Bioconcentration and Acute Intoxication of Brazilian Freshwater Fishes by the Methyl Parathion Organophosphate Pesticide

Three species of freshwater Brazilian fishes (pacu, Piaractus mesopotamicus; piavussu, Leporinus macrocephalus, and curimbatá, Prochilodus lineatus) were exposed to an acute dose of 5 ppm methyl parathion organophosphate pesticide. Three to five individuals per species were exposed, one at a time, to 40 liters tap water spiked with Folidol 600. Pesticide concentrations and cholinesterase (ChE) activities were evaluated in serum, liver, brain, heart, and muscle. The bioconcentration of methyl parathion was similar for all studied fishes. Brain tissue showed the highest pesticide concentration, reaching 80 ppm after exposure for 30 min to methyl parathion. Three to 5 hours of 5 ppm methyl parathion exposure provoked the death of all P. lineatus at 92% brain AChE inhibition, whereas fish from the other two species survived for up to 78 hours with less than 80% brain AChE inhibition. Our results indicate that acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon.

The importance of an efficient extraction protocol for the use of fish muscle cholinesterases as biomarkers

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL-1 NaCl or 1molL-1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed.

Efeitos in vivo de extratos de Abacateiro (Persea gratissima) e Mulungu (Erythrina mulungu) sobre atividades de enzimas citocromo P450 hepáticas de rato.

O uso de plantas com finalidades terapeuticas e uma das mais antigas praticas medicinais da humanidade, sendo baseado em evidencias historicas ou pessoais. Verifica-se uma tendencia a generalizacao do consumo de plantas medicinais e de seus derivados, devida a crenca de que tudo que e natural nao faz mal a saude. As plantas medicinais sao muito usadas como alternativa terapeutica, entretanto, sabe-se que muitos de seus componentes podem alterar as concentracoes plasmaticas de farmacos alopaticos atraves da inibicao ou inducao de enzimas do citocromo P450. Considerando o descrito acima, o objetivo deste trabalho foi avaliar os efeitos in vivo de extratos brutos de plantas medicinais sobre as atividades das enzimas 7-etoxicumarina O-desetilase (ECOD), 7-etoxirresorufina O-desetilase (EROD), 7-metoxirresorufina O -desmetilase (MROD) e 7-pentoxirresorufina O -despentilase (PROD) hepaticas de ratos Wistar. Assim, ratos machos foram tratados com extratos de folha de abacateiro ( Persea gratissima) e de casca de mulungu ( Erythrina mulungu ). Apos receberem os extratos por meio de gavagem por quatro dias consecutivos, os animais foram submetidos a eutanasia, sendo as atividades das enzimas determinadas por espectrofluorimetria em microssoma hepatico. N ossos resultados mostram que a administracao de extrato de abacateiro promoveu um aumento significativo das atividades da ECOD, MROD e PROD, enquanto a atividade de EROD sofreu uma reducao significativa. A administracao de extrato de casca de mulungu promoveu reducao significativa das atividades da ECOD, MROD e PROD, enquanto a atividade da EROD nao sofreu alteracao significativa. Diante do exposto, indicamos a necessidade de maior conhecimento dos efeitos de extratos de plantas sobre as atividades destas enzimas, de maneira a promover o uso racional e seguro de medicamentos.