The Influence of Structural Patterns on Acute Aquatic Toxicity of Organic Compounds.

Investigation of the influence of molecular structure of different organic compounds on acute toxicity towards Fathead minnow, Daphnia magna, and Tetrahymena pyriformis has been carried out using 2D simplex representation of molecular structure and two modelling methods: Random Forest (RF) and Gradient Boosting Machine (GBM). Suitable QSAR (Quantitative Structure - Activity Relationships) models were obtained. The study was focused on QSAR models interpretation. The aim of the study was to develop a set of structural fragments that simultaneously consistently increase toxicity toward Fathead minnow, Daphnia magna, Tetrahymena pyriformis. The interpretation allowed to gain more details about known toxicophores and to propose new fragments. The results obtained made it possible to rank the contributions of molecular fragments to various types of toxicity to aquatic organisms. This information can be used for molecular optimization of chemicals. According to the results of structural interpretation, the most significant common mechanisms of the toxic effect of organic compounds on Fathead minnow, Daphnia magna and Tetrahymena pyriformis are reactions of nucleophilic substitution and inhibition of oxidative phosphorylation in mitochondria. In addition acetylcholinesterase and voltage-gated ion channel of Fathead minnow and Daphnia magna are important targets for toxicants. The on-line version of the OCHEM expert system (https://ochem.eu) were used for a comparative QSAR investigation. The proposed QSAR models comply with the OECD principles and can be used to reliably predict acute toxicity of organic compounds towards Fathead minnow, Daphnia magna and Tetrahymena pyriformis with allowance for applicability domain estimation.

Asociación de la exposición ocupacional a plaguicidas organofosforados con el daño oxidativo y actividad de acetilcolinesterasa / Association of occupational exposure to pesticides organophosphate oxidative damage and activity acetylcholinesterase

Los plaguicidas organofosforados (POF) poseen una acción anticolinesterásica utilizada como indicador de intoxicación crónica del trabajador agrícola por POF. En esta población, los POF pueden actuar como pro-oxidantes, afectando la actividad de las enzimas antioxidantes y probablemente generar daños crónicos. Objetivo. Determinar la influencia de la exposición ocupacional a POF sobre el daño oxidativo y actividad de acetilcolinesterasa en trabajadores agrícolas. Metodología. Se realizó un estudio observacional, analítico, retrospectivo y comparativo en 45 trabajadores del Comité de Sanidad Vegetal de Durango A.C. (CESAVEDAC). Se evaluó la actividad de acetilcolinesterasa lipoperoxidación y capacidad antioxidante total en plasma, además de evaluar las concentraciones de colesterol, triglicéridos, glucosa. Resultados. El daño oxidativo se asocia a la exposición a POF (p=0.003) siendo 3.21 veces mayor el riesgo de desarrollar daño oxidativo en el grupo expuesto a POF, además existe una asociación entre la inhibición de acetilcolinesterasa y la exposición a POF (p=0.01) siendo 2.92 veces mayor el riesgo de presentar dicha inhibición con respecto al grupo no expuesto. Conclusiones. Existe una influencia negativa de la exposición ocupacional a POF sobre el daño oxidativo y la actividad de acetilcolinesterasa en la población de trabajadores agrícolas estudiada (AU)

Organophosphate pesticides (OPs) are considered as negative risk factors for the health POF farm workers. The anticholinesterase action these compounds have been used as an indicator POF chronic OP poisoning. Some authors have suggested that agricultural workers in the Ops can act as pro-oxidants, affecting the activity POF antioxidant enzymes and generating chronic damage likely. Objective. This study was conducted to determine the influence POF occupational exposure to OPs in oxidative damage and acetylcholinesterase activity in farmworkers Plant Health Committee POF Durango A.C (CESAVEDAC). Methodology. An observational, analytical, retrospective comparative study was conducted on 45 workers. The activity POF acetylcholinesterase as a biomarker POF toxicity assessed, indicator POF oxidative damage to lipid peroxidation antioxidant capability. Results. Was found that there is an association between oxidative damage and exposure to OPs (p = 0.003) being 3.21 times greater risk POF developing oxidative damage when it is exposed to OPs, moreover, there is an association between inhibition POF acetylcholinesterase and exposure to OPs (p = 0.01), 2.92 times higher risk POF show inhibition activity acetylcholinesterase when it is exposed to OPs Conclusions. There is a negative influence POF occupational exposure to OPs on oxidative damage and acetylcholinesterase activity in farmworkers (AU)

Kinetic and physicochemical properties of brain acetylcholinesterase from the peacock bass (Cichla ocellaris) and in vitro effect of pesticides and metal ions.

Brain acetylcholinesterase (AChE; EC 3.1.1.7) from peacock bass (Cichla ocellaris) was characterized and the effect of organophosphorus and carbamate pesticides as well as ions and heavy metals was evaluated. The kinetic parameters K(m) and V(max) were determined as 0.769 mM and 0.189 U/mg of protein respectively. Optimal pH and temperature were found to be 8.0 and 45°C. The enzyme retained approximately half of the activity after incubation at 50°C for 30 min. Total cholinesterase activity on brain of this species can be ascribed to AChE according to selective inhibitors analysis (neostigmine, eserine and BW284c5 reduced its activity whereas no effect was noticed for Iso-OMPA). Seven pesticides (five organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate - TEPP and two carbamates: carbaryl and carbofuran) showed inhibitory effects on C. ocellaris AChE. However, the strongest effect was observed with carbofuran (IC(50)=0.21 µM and K(i)=2.57 × 10(-3) µM). The following ions (1 mM) showed to inhibit its activity (decrescent order): Hg(2+)>As(3+)>Cu(2+)>Zn(2+). EDTA(2-) did not affect enzyme activity. The present study provides assay conditions and data to suggest this enzyme as in vitro biomarker of organophosphorus and carbamate pesticides in routine environmental screening programs.

Acetilcolinesterase, butirilcolinesterase, carboxilesterase e a resistência de peixes neotropicais aos pesticidas organofosforados / Acetylcholinesterase, butyrylcholinesterase, carboxylesterase and the resistance of neotropical fishes to organophosphorus pesticides

Todos os agrupamentos humanos que se organizam para o trabalho usam rios, lagos ou lagoas como depósitos para a decomposição de matéria indesejável. A contaminação do meio aquático por herbicidas e agrotóxicos derivados de práticas agrícolas se tornou, faz tempo, um problema de importância mundial. Precisamos de informações detalhadas sobre a bioquímica da intoxicação de peixes nativos para avaliarmos quais os efeitos de agrotóxicos sobre os processos bioquímicos que mantêm o ciclo de vida dos peixes em águas do Brasil. Organofosfatos, que são agrotóxicos de uso disseminado, podem interargir com as B-esterases butirilcolinesterase (EC 3.1.1.8) e carboxilesterase (EC 3.1.1.1) presentes no fígado e no plasma. Tanto a butirilcolinesterase (BChE) como a carboxilesterase (CarbE), se presentes em concentrações relativamente elevadas, agem como limpadores estequiométricos ("scavengers" moleculares) por ligarem o átomo de fósforo do grupo P=O com a hidroxila de uma serina presente nos seus sítios ativos. Em nossos resultados observamos que curimbatá possui a CarbE plasmática (IC50 74 nM) mais sensível ao organofosfato metilparaoxon quando comparado ao pacu (IC50 691 nM). Isolamos CarbE dos plasmas de curimbatá e pacu. Piavassu não possui uma atividade expressiva de CarbE no sangue, por isso não a isolamos. O tipo e a distribuição das esterases nos tecidos são particulares da espécie. Curimbatá tem alta atividade de CarbE no fígado (237,8 U.g-1) e no sangue (29,85 U.mL-1), pacu é dotado de alta atividade de BChE (134,0 U.g-1) e CarbE (149,6 U.g-1) no fígado, mas o piavussu conta apenas com a BChE do sangue (17,87 U.g-1). Este arsenal enzimático foi suficiente para proteger as AChE de cérebro, músculo e coração das três espécies e evitar a sua intoxicação leve por 0,2 mg metilparation/L. A abordagem cinético-bioquímica para conhecer a inibição das esterases presentes nos tecidos de diferentes espécies de peixes por agrotóxicos é uma ferramenta útil...

Every human group who organizes to work together uses rivers, lakes or ponds as places in which undesirable substances are deposited for decomposition. Contamination of the aquatic environment with herbicides and pesticides derived from agrucultural practices has become a problem of global importance since a long ago. We need detailed information on the biochemistry of the poisoning of native fish to assess the effects of pesticides on the biochemical processes that maintain fishes' life cycle in waters of Brazil. Organophosphates, which are widely used pesticides, can interact with the B-esterases butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) in plasma and liver. Butyrylcholinesterase (BChE) and carboxylesterase (CarbE) in relatively high concentrations act as stoichiometric scavengers by linking the phosphorus atom of the P=O group with the serine's hydroxyl they have in their active site. Our results show that the CarbE of curimbata plasma (IC50 74 nm) is more sensitive to the organophosphate metilparaoxon than CarbE of pacu plasma (IC50 691 nm). We isolated CarbE from curimbata and pacu's plasma. Piavussu does not have an expressive activity of CarbE in plasma, so we did not isolate it. The type and distribution of esterases in tissues are peculiar to a species. Curimbata has high CarbE activity in the liver (237.8 U.g-1) and blood (29.85 U.mL-1), pacu is equipped with high activities of BChE (134.0 U.g-1) and CarbE (149.6 U.g-1) in the liver and piavussu relies only on BChE of blood (17.87 U.g-1). This enzymatic arsenal was sufficient to protect AChE from brain, muscle and heart of the three species and protect them against mild intoxication by methilparathion (0.2 mg/L). The biochemical kinetic approach that allows understanding of the inhibition of the esterases in tissues of different fish species is a good tool capable of anticipating the harmful consequences of these drugs.

Pro-apoptotic protein-protein interactions of the extended N-AChE terminus.

The N-terminally extended "synaptic" acetylcholinesterase variant N-AChE-S operates to promote apoptosis; however, the protein partners involved in this function remain unknown. Here, we report that when microinjected to fertilized mouse oocytes, N-AChE-S caused embryonic death as early as the zygotic stage. To identify the putative protein partners involved, we first tried yeast two hybrid screening, but this approach failed, probably because of the N-AChE-S-induced lethality. In contrast, sequence analysis and a corresponding peptide array revealed possible partners, which were validated by co-immunoprecipitation. These include the kinases GSK3, Aurora and GAK, the membrane integrin receptors, and the death receptor FAS. Each of these could potentially modulate N-AChE-S-induced apoptosis with possible therapeutic value for the treatment of Alzheimer's disease.

Edema pulmonar no cardiogénico asociado con la ingestión de carbamato / Non-cardiogenic pulmonary edema associated with the ingestion of carbamate

Se presenta el caso de una joven mujer que ingirió un raticida, cuyo componente principal fue el carbamato aldicarb, en un intento de suicidio. Este causó las manifestaciones características de los inhibidores de la acetilcolinesterasa, destacándose un cuadro reversible de edema pulmonar no cardiogénico.

This was the case of a young woman that attempted to commit suicide ingesting a rat killer, which main component was aldicarb, a carbamate. This casued the typical manifestations of the inhibitors of acetylcholinesterase, standing out a reversible clinical piciture of non-cardiogenic pulmonary edema.

A peptide derived from acetylcholinesterase is a pivotal signalling molecule in neurodegeneration.

It is now widely accepted that acetylcholinesterase (AChE) also displays non-cholinergic functions, completely independent of cholinergic transmission. Indeed, AChE has been implicated in a variety of trophic and toxic actions in a range of different systems. However, it is still uncertain what part of the AChE molecule may be responsible for these actions, and indeed via what receptor. Recent work has identified a peptide towards the C-terminus of the AChE molecule that appears to have very similar effects to non-cholinergic AChE itself. This action is to enhance calcium entry, in acute and chronic preparations across a trophic-toxic spectrum, depending on concentration applied and/or duration of exposure.

Acetylcholinesterase-Abeta complexes are more toxic than Abeta fibrils in rat hippocampus: effect on rat beta-amyloid aggregation, laminin expression, reactive astrocytosis, and neuronal cell loss.

Neuropathological changes generated by human amyloid-beta peptide (Abeta) fibrils and Abeta-acetylcholinesterase (Abeta-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Abeta-AChE complexes trigger a more dramatic response in situ than Abeta fibrils alone as characterized by the following features observed 8 weeks after treatment: 1). amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Abeta aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Abeta-AChE deposits observed in vivo probably recruited endogenous Abeta peptide; 2). the appearance of laminin expressing neurons surrounding Abeta-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3). an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4). a stronger neuronal cell loss in comparison with Abeta-injected animals. We conclude that the hippocampal injection of Abeta-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Abeta-AChE complexes are more toxic than Abeta fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimer's disease brains.

A 45-kDa acetylcholinesterase protoxin of Aeromonas hydrophila: purification and immunogenicity in fish.

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.

PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid25-35 fragment, glutamate, and hydrogen peroxide.

This work addresses the differential effects of several oxidative insults on two neuronal cell lines, PC12 and Neuro 2a cells, extensively used as neuronal models in vitro. We measured cellular damage using the cytotoxic assays for MTT reduction and LDH release and found that acetylcholinesterase (AChE)-amyloid-beta-peptide (Abeta) complexes, Abeta25-35 fragment, glutamate and H2O2 were over 200-fold more toxic to PC12 than to Neuro 2a cells. 17alpha and 17beta estradiol were able to protect both cell types from damage caused by H2O2 or glutamate. By contrast, other insults not related to oxidative stress, such as those caused by the nonionic detergent Triton X-100 and serum deprivation, induced a similar level of damage in both PC12 and Neuro 2a cells. Considering that the Abeta peptide, H2O2 and glutamate are cellular insults that cause an increase in reactive oxygen species (ROS), the intracellular levels of the antioxidant compound, glutathione were verified. Neuro 2a cells were found to have 4- to 5-fold more glutathione than PC12 cells. Our results suggest that Neuro 2a cells are less susceptible to exposure to AChE-Abeta complexes, Abeta25-35 fragment, glutamate and H2O2 than PC12 cells, due to higher intracellular levels of antioxidant defense factors.

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.

Toxic effects of acetylcholinesterase on neuronal and glial-like cells in vitro.

Acetylcholinesterase (AChE), the enzyme involved in the hydrolysis of the neurotransmitter acetylcholine, has been implicated in non-cholinergic actions which may play a role in neurodegenerative diseases such as Alzheimer's disease. To study the potential cytotoxicity of brain AChE, the effects of affinity purified AChE were analyzed on neuronal (Neuro 2a) and glial-like (B12) cells. LDH release and MTT reduction assays showed that AChE was toxic; the toxicity was dependent on the enzyme concentration, time of incubation and cellular density. The toxic effect of AChE was not related to its catalytic activity, since the anti-cholinesterase drug BW284C51 and heat inactivation were unable to block the effects of the enzyme. When cells were incubated at 4 degrees C, toxicity was completely blocked, in contrast to cells incubated at 37 degrees C. The presence of serum in the culture medium inhibited the toxic effects of AChE. Cytoplasmic shrinkage, condensation and fragmentation of nucleus as well as DNA strand breaks detected with the TUNEL technique indicated that apoptotic cell death is involved in the effect of AChE. Considering that we have previously shown that AChE promotes the assembly of beta-amyloid peptide into neurotoxic amyloid fibrils, it is conceivable that the neurotoxicity of AChE shown here may play a role in the neuronal degeneration observed in Alzheimer's disease.

Estrogen protects neuronal cells from the cytotoxicity induced by acetylcholinesterase-amyloid complexes.

The senile plaques present in Alzheimer's disease (AD) are composed of a core of amyloid beta-peptide (Abeta) plus several proteins including acetylcholinesterase (AChE). Recently we found that AChE forms complexes with the Abeta peptide in vitro and that these are more cytotoxic than Abeta fibrils alone. Considering that estrogen has been reported to act as a protective agent against Abeta-induced cytotoxicity, the effect of 17beta-estradiol was studied in rat pheochromocytoma (PC12) and mouse neuroblastoma (Neuro 2a) cells exposed to either Abeta alone or AChE-Abeta complexes. Estrogen showed a powerful protective effect in response to the challenge of AChE-Abeta complexes as well as with Abeta fibrils. This was also the case for other cytotoxic agents such as glutamate and H2O2. Our results suggest a common mechanism for cellular protection by estrogen against the toxicity of both Abeta fibrils and AChE-Abeta complexes, likely avoiding the free radical apoptotic pathway.

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non-amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N-glycans of the 'complex' and 'hybrid' types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non-denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

Effects of the acetylcholinesterase toxin of Aeromonas hydrophila on the central nervous system of fish.

The purified acetylcholinesterase (AcChE) toxin, crude extracellular products (ECP) or viable virulent Aeromonas hydrophila were injected intraperitoneally into rainbow trout in different sublethal and lethal doses. When fish showed signs of morbidity, brain tissue was excised and assayed for acetylcholinesterase activity. In all cases there was a large increase in AcChE activity (about 40-fold for purified AcChE-toxin). This was shown to be due to an accumulation of the fish's own AcChE and not the bacterial toxin. Nevertheless, the latter was detected in brain homogenates from fish in all treatment groups using a rabbit antiserum to the purified toxin to probe Western blots of brain homogenates, demonstrating that the toxin does gain access to brain tissue and is produced during in vivo infection. The results strongly suggest that this toxin plays a central role in the pathogenesis of A. hydrophila infection.

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish.

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 micrograms g-1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.