RESUMO
INTRODUCTION AND AIMS: The Chilean Altiplano ecosystem is conserved free from contaminants and pollutants because of the absence of major local human activities such as agriculture or other industries. We studied the effects of paraoxon on proliferation and apoptosis of testicular cells during active spermatogenesis in Dugesia gonocephala collected from a pristine river (Guacollo) in the Altiplano region nearby Visviri town, Chile. MATERIALS AND METHODS: Adult planarians were incubated in varying concentrations of paraoxon (0.8, 0.4, 0.04, 0.004, and 0.0004 mM) for 4 h. After 3 h of incubation, bromodeoxyuridine (BrdU) was added. Effects on cell proliferation (BrdU) and apoptosis (Apaf-1) were determined by immunohistochemistry. RESULTS: Paraoxon concentrations of 0.4 and 0.8 mM caused 100% mortality in the respective treatment groups. The lowest tested concentration (0.0004 mM) caused a significant increase on cell proliferation in the seminiferous tubules, as well as an increase in the number of apoptotic cells. All other tested concentrations significantly inhibited cell proliferation and induced apoptosis. CONCLUSIONS: Paraoxon inhibits DNA synthesis and induces apoptosis during spermatogenesis in adult planarians from a high-altitude, pollution-free environment. This could suggest its use as a biosensor or biomarker for contamination with agro pesticides.
Assuntos
Inseticidas/toxicidade , Paraoxon/toxicidade , Planárias/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Chile , Água Doce/química , Inseticidas/análise , Paraoxon/análise , Planárias/metabolismo , Poluentes Químicos da Água/análiseRESUMO
An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.
Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais/instrumentação , Diclorvós/análise , Eletrônica/instrumentação , Análise de Injeção de Fluxo/instrumentação , Paraoxon/análogos & derivados , Praguicidas/análise , Biomimética/instrumentação , Biomimética/métodos , Técnicas Biossensoriais/métodos , Misturas Complexas/análise , Diclorvós/química , Eletroquímica/instrumentação , Eletroquímica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Paraoxon/análise , Paraoxon/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , LínguaRESUMO
Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes-AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 x 10(-11) and 2.7 x 10(-9) mol L(-1), respectively), the B1 enzyme for carbofuran (4.5 x 10(-9) mol L(-1)), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 x 10(-7) mol L(-1)). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.
Assuntos
Acetonitrilas , Acetilcolinesterase/metabolismo , Técnicas Biossensoriais/métodos , Carbaril/análise , Carbofurano/análise , Diclorvós/análise , Paraoxon/análogos & derivados , Animais , Carbaril/metabolismo , Carbofurano/metabolismo , Diclorvós/metabolismo , Drosophila melanogaster/enzimologia , Electrophorus/metabolismo , Malus/química , Estrutura Molecular , Paraoxon/análise , Paraoxon/metabolismo , Praguicidas/análise , Praguicidas/metabolismoRESUMO
A spectrophotometric enzymatic flow injection (FI) system for the determination of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method was based on the determination of the acetic acid formed by the enzymatic reaction of the acetylcholinesterase, immobilized on glass beads, with the substrate acetylcholine. The acetic acid formed permeates through a PTFE membrane and is received by a solution (pH 7.0) containing the acid-base indicator Bromocresol Purple (B.C.P.), leading to a pH change and therefore to a color change. The variation of the absorbance of the solution is detected spectrophotometrically at 400 nm. The determination of paraoxon is related to its inhibitory action on the enzyme. Therefore the analytical signal is the difference between the signal that corresponds to the free and the one that corresponds to the inhibited enzyme, considering a fixed acetylcholine concentration. The correlation between the peak height and paraoxon concentration at a given acetylcholine concentration is linear in the range from 5.0 x 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 (r = 0.998) of paraoxon, with a relative estimated standard deviation (R.S.D.) of +/- 1.7% (n = 10) considering a solution containing 5.0 x 10(-6) mol L-1 of paraoxon and a solution containing 5.0 x 10(-3) mol L-1 of acetylcholine. Therefore, the quantitative limit detection is about 2.5 x 10(-7) of paraoxon (3 sigma). A 1,1'-trimethylene-bis(4-formylpyridinium bromide)dioxime (TMB-4) solution was used to reactivate the enzyme.