RESUMO
The present work aimed to compare the small, neutral and monoaromatic oxime, isatin-3-oxime (isatin-O), to the commercial ones, pralidoxime (2-PAM) and obidoxime, in a search for a new potential reactivator for acetylcholinesterase (AChE) inhibited by the pesticide paraoxon (AChE/POX) as well as a novel potential scaffold for further synthetic modifications. The multicriteria decision methods (MCDM) allowed the identification of the best docking poses of those molecules inside AChE/POX for further molecular dynamic (MD) studies, while Ellman's modified method enabled in vitro inhibition and reactivation assays. In corroboration with the theoretical studies, our experimental results showed that isatin-O have a reactivation potential capable of overcoming 2-PAM at the initial moments of the assay. Despite not achieving better results than obidoxime, this molecule is promising for being an active neutral oxime with capacity of crossing the bloodâ»brain barrier (BBB), to reactivate AChE/POX inside the central and peripheral nervous systems. Moreover, the fact that isatin-O can also act as anticonvulsant makes this molecule a possible multipotent reactivator. Besides, the MCDM method showed to be an accurate method for the selection of the best docking poses generated in the docking studies.
Assuntos
Inibidores da Colinesterase/farmacologia , Reativadores da Colinesterase/química , Reativadores da Colinesterase/farmacologia , Modelos Moleculares , Oximas/química , Oximas/farmacologia , Paraoxon/química , Paraoxon/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left â¼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.
Assuntos
Acetilcolina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Traqueia/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Atropina/farmacologia , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Inibidores da Colinesterase/farmacologia , Estimulação Elétrica , Cobaias , Hemicolínio 3/farmacologia , Hexametônio/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organofosfatos/farmacologia , Paraoxon/farmacologia , Piperidinas/farmacologia , Tetrodotoxina/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/inervação , ômega-Conotoxina GVIA/farmacologiaRESUMO
An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.
Assuntos
Lipase/isolamento & purificação , Lipase/metabolismo , Penaeidae/anatomia & histologia , Penaeidae/enzimologia , Animais , Ativação Enzimática/efeitos dos fármacos , Hemócitos/enzimologia , Cinética , Lactonas/farmacologia , Lipase/antagonistas & inibidores , Orlistate , Paraoxon/farmacologia , Especificidade da Espécie , Relação Estrutura-Atividade , TemperaturaRESUMO
We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.
Assuntos
Butirilcolinesterase/sangue , Peixes/sangue , Sequência de Aminoácidos , Animais , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/metabolismo , Butiriltiocolina/metabolismo , Clorpirifos/análogos & derivados , Clorpirifos/farmacologia , Inibidores da Colinesterase/farmacologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Intoxicação por Organofosfatos , Paraoxon/análogos & derivados , Paraoxon/farmacologia , Intoxicação/prevenção & controle , Alinhamento de Sequência , Tetraisopropilpirofosfamida/farmacologiaRESUMO
Acetylcholinesterase (AChE) sensitivity to the organophosphorus (OP) pesticide methyl-paraoxon was measured in fourteen species of Neotropical marine and freshwater fish found in the waters of Brazil. The rate constant for phosphorylation, kp, the dissociation constant, kd, the second order rate constant, ki, and the IC50 value were measured at 28 degrees C in pH 7.5 buffer for AChE extracted from brain. In addition, the substrate affinity constant, km, was measured with acetylthiocholine. The IC50 for 30 min of inhibition ranged from 123 nM (Prochilodus lineatus) to 3340 nM (Percophis brasiliensis), which corresponded to ki values of 187-6.9 mM(-1) min(-1). A 10-fold range in kp values from 0.21 min(-1) (Paralonchurus brasiliensis) to 2.1 min(-1) (Dules auriga) was associated with a 37-fold range in kd values from 4 to 150 microM. These large differences in reactivity with methyl-paraoxon were not reflected in the binding affinity for acetylthiocholine; km values were approximately 0.1-0.3 mM for all species. These results predict that the amino acid sequence involved in AChE sensitivity differs in these fishes, and that consequently some fish species may be resistant to the toxicity of methyl-paraoxon.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/enzimologia , Inibidores da Colinesterase/farmacologia , Paraoxon/análogos & derivados , Paraoxon/farmacologia , Animais , Peixes , Cinética , FosforilaçãoRESUMO
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.
Assuntos
Inibidores da Colinesterase/análise , Inibidores da Colinesterase/farmacologia , Análise de Injeção de Fluxo/métodos , Paraoxon/análise , Paraoxon/farmacologia , Ácido Acético/análise , Acetilcolina , Acetilcolinesterase/metabolismo , Animais , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Espectrofotometria/métodosRESUMO
1. Studies were carried out on rural workers in Brazil to determine the decrease in the activity of plasma butyrylcholinesterase (BChE), erythrocyte cholinesterase (AChE) associated with exposure to organophosphorus pesticides (OP). The goal of this work is to help prevent injury to these workers. 2. In developing countries the distance between area of pesticide use and reference laboratories is a drawback for analytical techniques, since cholinesterase activity determinations require fresh blood samples. Field methodologies can be a useful alternative to laboratory tests, however they are not as sensitive as those found in laboratories. 3. The modification of Ellman's Method presented in this paper allows blood samples to be frozen and maintain enzymatic stability: 7 days for AChE and 3 days for BChE. The proposed method is also more sensitive than Ellman's Method Modified by Magnotti (EMMM). 4. The results suggest that the Ellman Method Modified by Oliveira-Silva (EMMOS) is valid for monitoring procedures. This method represents an important contribution to the process of monitoring OP exposures, since the evaluations no longer have to be conducted near the site of OP use.
Assuntos
Acetilcolinesterase/sangue , Butirilcolinesterase/sangue , Criopreservação , Monitoramento Ambiental/métodos , Eritrócitos/enzimologia , Exposição Ocupacional/análise , Paraoxon/análogos & derivados , Estabilidade Enzimática , Eritrócitos/efeitos dos fármacos , Congelamento , Humanos , Paraoxon/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE.
Assuntos
Acetilcolinesterase/fisiologia , Inibidores da Colinesterase/farmacologia , Venenos Elapídicos/farmacologia , Proteínas do Olho/fisiologia , Retina/embriologia , Animais , Benzenamina, 4,4'-(3-oxo-1,5-pentanodi-il)bis(N,N-dimetil-N-2-propenil-), Dibrometo/farmacologia , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Agregação Celular , Técnicas de Cultura de Células/métodos , Células Cultivadas/efeitos dos fármacos , Embrião de Galinha , Proteínas do Olho/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Paraoxon/farmacologia , Fisostigmina/farmacologia , Retina/citologia , Retina/enzimologiaRESUMO
The organophosphorate pesticides are highly toxic for insects and mammals, but their effects in the male reproductive tract are scarcely known. Many alterations induced by organophosphorate pesticides have been described, such as: cytogenetic alterations in germinal cells, oligozoospermia and teratozoospermia in the mouse. Parathion, the pesticide mostly utilized in Chilean agriculture, is rapidly metabolized to paraoxon, the active metabolite, in mammalian organisms. The purpose of this study is to evaluate the effect of Parathion and paraoxon on different morphological and functional parameters of the sperm. Human spermatozoa were incubated with Parathion and paraoxon at different concentrations (0.05, 0.1, 0.2, 0.4 and 0.8 mM). Vitality (tripan blue and eosin tests), acrosome reaction (triple stain test), plasma membrane integrity (HOS-test), and chromatin stability (sodium thioglycolate test) were determined. The observations were done by optical microscopy at 1000x of magnification and three hundred sperms were evaluated for each treatment. The results indicated that Parathion and paraoxon increase the percent of sperm with acrosome reaction and also increase the percentage of sperm with chromatin decondensation in a dose-dependent manner. The vitality and plasma membrane integrity decrease significantly in a dose-dependent manner. The results suggest a direct action of Parathion and paraoxon on the different parameters studied. The morphofunctionality of sperm is altered significatively, suggesting that Parathion and paraoxon, thanks to their alkylating and electrophylic properties, could act on DNA and proteins respectively, to elicit these changes
Assuntos
Humanos , Masculino , Técnicas In Vitro , Inseticidas Organofosforados/farmacologia , Paration/farmacologia , Espermatozoides/citologia , Inseticidas Organofosforados/farmacologia , Paraoxon/farmacologiaRESUMO
The organophosphorate pesticides are highly toxic for insects and mammals, but their effects in the male reproductive tract are scarcely known. Many alterations induced by organophosphorate pesticides have been described, such as: cytogenetic alterations in germinal cells, oligozoospermia and teratozoospermia in the mouse. Parathion, the pesticide mostly utilized in Chilean agriculture, is rapidly metabolized to paraoxon, the active metabolite, in mammalian organisms. The purpose of this study is to evaluate the effect of Parathion and paraoxon on different morphological and functional parameters of the sperm. Human spermatozoa were incubated with Parathion and paraoxon at different concentrations (0.05, 0.1, 0.2, 0.4 and 0.8 mM). Vitality (tripan blue and eosin tests), acrosome reaction (triple stain test), plasma membrane integrity (HOS-test), and chromatin stability (sodium thioglycolate test) were determined. The observations were done by optical microscopy at 1000x of magnification and three hundred sperms were evaluated for each treatment. The results indicated that Parathion and paraoxon increase the percent of sperm with acrosome reaction and also increase the percentage of sperm with chromatin decondensation in a dose-dependent manner. The vitality and plasma membrane integrity decrease significantly in a dose-dependent manner. The results suggest a direct action of Parathion and paraoxon on the different parameters studied. The morphofunctionality of sperm is altered significatively, suggesting that Parathion and paraoxon, thanks to their alkylating and electrophylic properties, could act on DNA and proteins respectively, to elicit these changes.
Assuntos
Inseticidas/farmacologia , Paration/farmacologia , Espermatozoides/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Paraoxon/farmacologia , Espermatozoides/citologiaAssuntos
Inibidores da Colinesterase/farmacologia , Monitoramento Ambiental/métodos , Esterases/análise , Inseticidas/farmacologia , Moluscos/efeitos dos fármacos , Poluentes Químicos da Água/farmacologia , Animais , Argentina , Esterases/antagonistas & inibidores , Fenitrotion/farmacologia , Moluscos/enzimologia , Paraoxon/farmacologia , Paration/farmacologiaRESUMO
Organophosphates comprise a group of chemical compounds extensively used in farming as insecticides, which cause accidental poisoning in animals and men and are also used in suicide attempts. The toxicity of these compounds is due especially to the cardiac and respiratory impairment in consequence of autonomic nervous system disorders. However, it is known that some of these products induce a myopathy in experimental animals and humans. This myopathy is characterized by muscle cell degeneration, involving above all the respiratory muscles. Based on the fact that this involvement certainly enhances the respiratory impairment, this study offers an experimental method for routine evaluation of organophosphate myotoxicity, using a minimal and sufficient battery of stains and histochemical reactions, for muscle necrosis quantification. For this purpose, albino rats (Wistar) treated with the organophosphate paraoxon, were used both with and without antidotes (atropine or pralidoxime). Muscle fiber necrosis in the diaphragm of the rats treated with paraoxon or paraoxon and atropine, that affected about 15% of the fibers in some areas, was detected. In the group treated with paraoxon and pralidoxime, a minimal necrosis was seen, revealing a protective role of this later antidote during the development of myopathy.