The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC(50) value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC(50) value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC(50) values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens.

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This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (±)- and (-)-methamidophos were administered at 50mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1mg/kg, i.m.) and one dose of calcium gluconate (5mg/kg, i.v.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs.

The aim of this study was to determine the no-observable-adverse-effect concentration (NOAEC) for trichlorfon, an antiparasitic agent used in aquaculture, in Piractus mesopotamicus (pacu) using acetylcholinesterase (AChE) activity as an end point. Fish were exposed 24 h/d for 15 d to different concentrations of trichlorfon in tanks of water for which a curve of dissipation was previously determined. Analysis of trichlorfon in water and fish plasma using gas chromatography with electron capture detection (GC-ECD) enabled measurement of limit of detection (LOD) and limit of quantification (LOQ), respectively, to be 3 and 10 ppb. Thirty-six hours after trichlorfon dilution in water, the concentration was below the LOD, and data showed that plasma concentrations did not exceed the LOQ. Apart from the 6.25 μg/L, all concentrations of trichlorfon significantly inhibited plasma and brain AChE activity compared to controls. The AChE activity levels returned to control values in 7 d. These data may be useful to determine the concentration of trichlorfon that destroys parasites without producing adverse effects in fish.

Many chiral pesticides are introduced into the environment as racemates, although their pesticidal activity is usually the result of preferential reactivity of only one enantiomer, while the other enantiomer may have toxic effects against nontarget organisms. Methamidophos (O,S-dimethyl phosphoramidothioate), a chiral compound, is an insecticide widely used in agriculture in both developed and developing countries. However, this pesticide has a high toxicity not only to targeted insects but also to human and animals. In the present study, the enantiomers of methamidophos were enantiomerically separated by a semipreparative chiral liquid chromatography at the multimilligram scale on a polysaccharide-based chiral stationary phase and a preliminary evaluation of their in vitro inhibition of plasma butyrylcholinesterase (BChE) of hens was performed. In the present study, our first effort was to resolve the racemic mixture of methamidophos and to that end reversed-phase, normal-phase, and polar organic elution conditions were investigated in four different polysaccharide-based chiral phases. The best performance was achieved on a cellulose tris(3,5-dimethylphenylcarbamate) phase under normal phase. This chromatographic condition allowed the separation of 225 mg of methamidophos enantiomers with a high degree of chiral purity (>98%) in a short analysis time. Significant differences were found between the concentration that causes 50% of enzyme inhibition (IC50) of the three isoforms of methamidophos. (-)-Methamidophos showed an IC50 approximately three times larger than the (+)-enantiomer for plasma BChE of hens.