Brain cholinergic involvement in the rapid development of tolerance to the hypothermic action of ethanol

The enzymes of the cholinergic system have been investigated in discrete brain regions in mice treated with repeated injection of ethanol. Male mice kept under controlled environmental conditions were treated with ethanol (3 g/kg/day) for 3 days. Animals were sacrificed 1 hr after ethanol injections. Brain regions studied were cerebral cortex, cerebellum, midbrain, hypothalamus, medulla oblongata, amygdala, and hippocampus. The administration of a single dose of ethanol resulted in significant increase (P less than 0.05) in choline acetyltransferase (ChAT) activity in all different brain regions. Repeated injections of ethanol at the 2nd and 3rd day did not result in any further rise in ChAT activity of the brain regions studied except for the midbrain. The results also show that acetylcholinesterase (AChE) activities increased significantly (P less than 0.05) in the pons and hippocampus in acutely ethanol intoxicated animals. The repeated injection of ethanol resulted in significant increase in AChE activities of the cortex and the amygdala. Meanwhile, animals developed tolerance to the hypothermic action of ethanol after ethanol third injection. The results of the present investigation indicate that the rapid development of the hypothermic tolerance to ethanol might be mediated by the brain cholinergic system.     

Acrylonitrile has Distinct Hormetic Effects on Acetyl-Cholinesterase Activity in Mouse Brain and Blood that are Modulated by Ethanol

Acrylonitrile(AN) is a neurotoxin both in animals and humans, but its effects on acetylcholinesterase (AChE) activity remain controversial. This study aimed to determine the dose-response effects of AN on AChE activity and the modulatory role of ethanol pre-treatment. A total of 144 Kunming mice were randomly divided into 18 groups: nine groups received 5% ethanol in their drinking water, and the remaining nine groups received regular tap water. One week later, both the ethanol and tap water only groups were given an intraperitoneal injection of AN at the following doses: 0 (control), 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 mg AN/kg body weight. AChE activity was determined on whole blood and brain 24 h later. Blood AChE activity was higher in AN-injected mice than in controls at all doses. AChE activity in blood increased in a dose-dependent manner, peaking at 0.156 mg/kg, after which a gradual decrease ensued, displaying a β-typed dose-response relationship. In contrast, brain AChE activity, following a single AN injection, was consistently lower than in control mice, and continued to fall up to a dose of 0.313 mg/kg, and thereafter increased gradually with higher doses. Mice receiving a 20 mg/kg dose of AN exhibited AChE brain activity indistinguishable from that of control mice, demonstrating a typical U-typed dose-response relationship. The activity of AChE in the blood and brain of the AN + ethanol-treated groups displayed a shift to the right, and the magnitude of the decrease in AChE activity induced by AN was attenuated relative to the AN-only group. These results suggest that AN affects AChE activity in both mouse blood and brain in a hormetic manner. Pretreatment with ethanol modifies the effect of AN on AChE, indicating that parent AN has a more prominent role than its metabolites in modulating enzyme activity.     

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.     

Effect of intrauterine exposure to cocaine on acetylcholinesterase in primary cultures of fetal mouse brain cells.

The effect of in utero exposure to cocaine on the developmental pattern of acetylcholinesterase (AChE), a major regulator of the central nervous system neurotransmitter acetylcholine, was studied in fetal brain cell cultures collected from mice on gestational day 15 after maternal exposure to cocaine from gestational days 6-14. A significant decrease in total and specific activity (expressed per mg of protein) of acetylcholinesterase was seen throughout the culture period in the cells grown from the cocaine-exposed animals as compared to controls (p less than .002). Similarly, the total protein content of the brain cells grown from the cocaine-exposed animals was significantly decreased as compared to controls (p less than .03). Utilizing AChE as a biochemical marker, these studies have shown that in utero exposure to cocaine has an adverse effect on the normal developmental pattern of the production of acetylcholinesterase in cholinergic neurons in the fetal mouse brain.     

Effect of ethanol on the rat gastrointestinal cholinergic enzymes

Male Sprague-Dawley rats weighing between 180 and 220 g and maintained under controlled lighting and temperature conditions were used in this experiment. Animals were given ethanol (3 g/kg, p.o.) 24 h after fasting. One group was given ethanol at 10.00 h (light phase) and the other at 22.00 h (dark phase). One hour later, the treated animals with their proper controls were sacrificed and the mucosa of the stomach, duodenum, ileum and colon were separated and assayed for choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities. Data obtained indicate that the administration of ethanol resulted in significant decline (p less than 0.01) in ChAT activity in the stomach and the colon during the light phase. A significant increase (p less than 0.01) in ChAT activity was also noted in the ileum during the dark phase. There was a significant decrease (p less than 0.01) in AChE activity in the stomach during the dark phase. The administration of ethanol also resulted in a significant decline in AChE activity (p less than 0.05) in the duodenum and the colon (p less than 0.01). The results obtained indicate that the gastrointestinal changes caused by ethanol administration may be related to changes in the cholinergic enzymes of the mucosa of the gastrointestinal tract.     

The hematotoxic effects of inhaled benzene on peripheral blood,bone marrow,and spleen cells are increased by ingested ethanol

For 13 weeks, groups of $\text{C57Bl}\text{6J}$ male mice were exposed to 300 ppm benzene by inhalation, 6 hr/day, 5 days/week, and to 5 or 15% ethanol in the drinking water, 4 days/week. The number and type of blood cells in the peripheral blood, marrow, and spleen were determined at regular intervals. Anemia and lymphocytopenia were observed in the peripheral blood of all benzene and benzene/ethanol-treated groups. These cytopenias, however, were more severe in the benzene/ethanol-treated mice. In addition, there was a transient increase of normoblasts in the peripheral blood of benzene/ethanol-treated mice which was not observed in mice treated with benzene alone or in control mice. Groups exposed to benzene or benzene/ethanol displayed reduced marrow and splenic cellularities but the reductions were more severe in mice exposed to benzene/ethanol. Specifically, these reduced cellularities were characterized by decreased numbers of lymphocytes in both marrow and spleen and decreased numbers of granulocytes and normoblasts in the marrow. In benzene/ethanol-treated mice a transient increase in the numbers of splenic normoblasts was observed which coincided with the transient appearance of normoblasts in the peripheral blood of these animals. This condition was not observed in mice treated with only benzene or only ethanol. Mice exposed to benzene/ethanol presented a greater degree of atypical cellular morphology than mice treated with benzene alone. These data suggest that the ingestion of ethanol increases the hematotoxicity of inhaled benzene.     

Cyclophosphamide effects on fetal mouse cephalic acetylcholinesterase

Pregnant C3H mice were exposed to a teratogenic dose of cyclophosphamide (CP, 20 mg/kg body weight) on the 10th day after copulation. Embryos or fetuses were examined 2–9 days later for gross abnormalities, weighed, and assayed for cephalic acetylcholinesterase (EC 3.1.1.7, AChE) activity, and haemoglobin content. In embryos examined 2 days after CP administration, fetal weight and brain weight were less than controls, but cephalic AChE was higher (p<0.001). The higher cephalic AChE was associated with a higher absorbance for haemoglobin, although at 14 days there was no apparent difference in the cephalic AChE and absorbance for haemoglobin, compared with controls. At 19 days, fetal weight, brain weight and brain AChE activity were less than controls (p<0.001 in each case). The raised cephalic AChE activity in 12-day embryos is explained by the presence of blood, which appears to have resorbed by day 14, whereas the reduced brain AChE activity in 19-day fetuses is accounted for by the growth inhibitory effects of the drug.     

Effects of arsenic on cholinergic parameters in brain in vitro

The effects of sodium arsenite (arsenite) on the cholinergic system in the brain of the mouse were investigated in vitro and compared with those of N-ethylmaleimide (NEM) and iodoacetate, both of which are alkylating sulfhydryl reagents. Arsenite, at concentrations greater than 10⁻⁴M, inhibited depolarized and nondepolarized release of acetylcholine (ACh) from cerebral slices, the synthesis of ACh in the slices, high-affinity uptake of choline into synaptosomes and activity of acetylcholinesterase (AChE). On the other hand, arsenite potentiated dose-dependently the activity of choline acetyltransferase (ChAT). N-Ethylmaleimide and iodoacetate showed inhibitory effects similar to those of arsenite. However, some exceptions were that N-ethylmaleimide did not have any effect on the nondepolarized release of ACh while iodoacetate had no effect on high affinity uptake of choline and activity of AChE. In contrast to arsenite, N-ethylmaleimide and iodoacetate inhibited the activity of ChAT. Neither of arsenite, N-ethylmaleimide nor iodoacetate showed any effect on the binding of [³H]quinuclidinyl benzilate to muscarinic ACh receptors. Although arsenite is thought to inhibit the cholinergic system in brain in vivo, its potentiating effect on ChAT and inhibition of AChE may reduce this harmful effect.     

Production of a lesion on liver rough endoplasmic reticulum after prolonged ethanol ingestion and its reversal through abstinence

Prolonged ethanol ingestion by adult rats resulted in an inhibition of amino acid incorporation in vitro by liver membrane-bound ribosomes. The same treatment, however, produced a stimulation in the activity of free ribosomes. The addition of endoplasmic reticulum membranes, extracted from the microsomal fraction of control or ethanol-treated rats, had an inhibitory effect on the protein synthetic activities of free ribosomes. However, the membrane preparations from the livers of ethanol-treated animals had relatively greater inhibitory activity if compared with the similar preparation from the control livers. The observed effect of ethanol intake on liver protein synthesis in vitro could be almost completely reversed if the animals abstained from ethanol for a period of 4 weeks.     

Brain growth during ethanol-induced hypoplasia

The inhibition of fetal brain growth resulting from in utero ethanol exposure may impair central nervous system (CNS) development and thereby result in mental retardation. Studies of ethanol-induced brain hypoplasia using chick embryos have shown that the early development of the chick is significantly growth inhibited by a single dose of ethanol (1.0 g/kg) given at the start of incubation (day 0). However, this level of ethanol exposure has been reported to have no effect on chick weight measured at hatching, suggesting that the weights of ethanol-treated chicks were regained during their development. The present experiments were undertaken to determine the biochemical changes associated with the varying growth rates believed to occur in the alcohol-treated embryos. The results indicated that between days 5 and 8 of development, the rates of DNA and protein synthesis (measured as radioactive thymidine and leucine incorporation, respectively) were inhibited by ethanol. The growth inhibition was highly correlated with blood alcohol content and there were associated increases in brain prostaglandin E (PGE) levels relative to vehicle-treated embryos. Further, there was a significant, inverse correlation between brain cyclic AMP content and individual brain weight. By day 10, the ethanol-treated embryos remained smaller than controls but their rates of DNA and protein synthesis were comparable to those of control animals. The normal rates of synthesis observed on day 10 appeared to correlate with clearance of the ethanol dose and with restoration of normal brain levels of PGE relative to 10-day vehicle-dosed embryos.     

Ethanol-induced delayed male puberty in mice is not due to impaired Leydig cell function

The present study was performed to evaluate the involvement of reduced testosterone in ethanol-induced impairment of male reproductive tract development. In vivo and in vitro gonadotropin (hCG)-stimulated steroidogenesis were examined in CFW mice as a function of chronic ethanol treatment during pubertal development. Chronic ethanol treatment from ages 20 to 49 days impaired testicular growth from ages 35 days (29 +/- 2 mg vs 37 +/- 2 mg for pair-fed controls) to 50 days (42 +/- 2 mg vs 63 +/- 2 mg for pair-fed controls). Consistent with a reduction in testicular weight, testicular content of androstenedione, testosterone, and dihydrotestosterone (DHT) was depressed in ethanol-treated mice. At age 50 days, the content (expressed as pg/testis) of androstenedione, testosterone, and DHT was reduced in ethanol-treated animals by 49%, 31%, and 38%, respectively, as compared to that of their respective controls. However, no difference in plasma (ng/mL) or testicular (pg/mg protein) concentrations of steroids was observed. Except for the DHT response at ages 35 to 40 days, neither in vivo nor in vitro steroidogenesis was impaired by chronic ethanol treatment at ages 26 to 50 days; similarly, the acute ethanol effect on steroidogenesis was unaffected. However, an adaptive increase (54%-173%) in the in vivo testosterone response to hCG was seen at ages 26 to 40 days. The data indicate that 1) chronic ethanol treatment does not impair gonadotropin-stimulated steroidogenesis or result in tolerance to acute ethanol effects on steroidogenesis in older animals; and 2) ethanol-induced reduction in testosterone is not a likely mechanism for delayed sexual maturation.     

Sex differences in the recovery of brain acetylcholinesterase activity following a single exposure to DFP

Male and female C57BL, DBA, and C3H mice were injected intraperitoneally with a single 6.33 mg/kg dose of diisopropylphosphofluoridate (DFP). The time course of recovery of acetylcholinesterase (AChE) activity as well as effects on choline acetyltransferase (ChAT) activity and brain muscarinic and nicotinic receptors were measured. DFP treatment did not affect ChAT activity or the muscarinic and nicotinic receptors. Near control levels of AChE activity were regained in female mice within the first 20 days. However, levels of whole brain AChE activity remained depressed for as long as 40 days following a single dose of DFP in male mice. An analysis of the recovery of AChE activity in several brain regions indicated that control activity was regained in striatum, hindbrain, and hippocampus, but not in cortex, midbrain, and hypothalamus. These data are discussed in terms of potential neurotoxicity induced by a single dose of DFP.     

Assessment of acetylcholinesterase activity in <Emphasis Type="Italic">Clarias gariepinus</Emphasis> as a biomarker of organophosphate and carbamate exposure

The objective of this study was to investigate the response of acetylcholinesterase (AChE) activities in Clarias gariepinus in response to Organophosphates (Ops) and carbamate exposure. The AChE activities were determined in plasma, and eye and brain homogenates of unexposed and exposed fish using Ellman’s method and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) chromophore. The baseline AChE activities in plasma, eyes and brain tissues in unexposed fish were comparable between males and females (P > 0.05). Concentrations of pesticides that inhibited 50% (IC₅₀) of AChE activities in brain homogenates following in vitro exposures were 0.003, 0.03, 0.15, 190, 0.2, 0.003 and 0.002 μM for carbaryl, chlorfenvinphos, diazinon, dimethoate, fenitrothion, pirimiphosmethyl and profenofos, respectively. The in vivo dose–effect relationships were assessed using chlorfenvinphos and carbaryl at different concentrations that ranged from 0.0003 to 0.06 μM and 0.0005 to 0.05 μM, respectively. Acetylcholinesterase activities were comparable in plasma, and eye and brain homogenates from control and carbaryl-exposed fish. Following exposure of fish to chlorfenvinphos at concentrations above 0.03 μM, a significant inhibition of AChE activities in plasma (84%) and eye homogenate (50%) was observed. The AChE activities in brain homogenate were comparable between chlorfenvinphos-exposed fish and controls. Because carbaryl cause reversible inhibition of AChE activities was found to be more potent than chlorfenvinphos that cause irreversible inhibition following in vitro exposure. Contrary, carbaryl was less potent than chlorfenvinphos after in vivo exposure possibly due to more rapid biotransformation of carbaryl than chlorfenvinphos. Findings from this study have demonstrated that inhibition of AChE activity in C. gariepinus is a useful biomarker in assessing aquatic environment contaminated by anticholinesterases.     

Effect of cholinesterase inhibitor and exercise on choline acetyltransferase and acetylcholinesterase activities in rat brain regions

This study sought to determine whether the choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes in the brain were affected in a regionally selective manner by chemical and physical stressors: 1) subacute administration of physostigmine (Phy); 2) exercise; and 3) the combination of these two stressors. ChAT and AChE activities in corpus striatum were significantly decreased due to Phy as well as Phy + exercise. This suggests that corpus striatum is affected by chemical stressors but more so by the combination of chemical and physical stressors. The brainstem is the only region which showed inhibition of ChAT activity due to exercise. Subacute Phy also inhibited brainstem ChAT activity. The hippocampus showed significant decrease in ChAT activity due to Phy + exercise but not due to Phy alone. These results suggest that the brain regions involved with control of motor, autonomic and cognitive functions were affected by subacute Phy and exercise. These data are consistent with the hypothesis that the responsiveness of these brain regions to different stressors is a function of the level of ongoing cholinergic activity and that elevations in ACh levels due to AChE inhibition may have long-term effects on the regulation of ChAT and AChE activities through a negative feedback mechanism.     

Region selective increase in activities of CNS cholinergic marker enzymes during learning of memory tasks in aged rats

The effects of learning memory tasks on activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex (FC), hippocampus (HC) and cerebellum of aged rat brains were studied in comparison with those of young adult rats. Aged rats were significantly inferior than young adult rats in both active avoidance (two-way shuttle box) and water-filled multiple T-maze learning. ChAT activity in the FC of aged rats was significantly increased after 5 days of training in an active-avoidance learning task. ChAT activity in the HC of aged rats was also significantly increased after 6 days of training in a water-filled multiple T-maze. These changes did not occur in young adult rats after either 2 or 5 days of active avoidance training, or in aged rats after 10 days of training, both of which were after the maximum level of learning of active avoidance task had been attained. AChE activity was significantly lower in the FC and HC of nontrained aged rats when compared with that of nontrained young adult rats. The reduced activity of AChE in both brain regions of nontrained aged rats rose to almost the same level as that in young adult rats in nontrained and trained states in an active avoidance task. From these findings, it is hypothesized that the task-dependent elevation in the activities of the central nervous system (CNS) cholinergic marker enzymes in trained aged rats may be compensatory changes to keep a relevant level of neurotransmission in the face of specific motor and/or cognitive insults.     

The influence of 2-/o-cresyl/-4 H-1 : 3 : 2-benzodioxa-phosphorin-2-oxide (CBDP) on organophosphate poisoning and its therapy.

The aim of the experiments was to obtain more information on the toxicity of organophosphates and protection against them. Pretreatment of mice with CBDP increased the s.c. toxicity of soman 19.1-fold, and its i.p. toxicity 17.8-fold. The protective effect of atropine and the oximes HS-3, HS-6 and HI-6 in soman poisoning was much greater in CBDP pretreated than in control animals. Atropine + HI-6 raised the s.c. LD50 of soman in the CBDP pretreated animals from 6.8 micrograms/kg to 166 micrograms/kg (PI = 24.3), but in control animals the i.p. LD50 was only raised from 370 micrograms/kg to 608 micrograms/kg (PI = 0.6). CBDP inhibited blood and brain AChE activity, but had no effect on aliesterase (AE) activity in plasma, liver and brain of mice in vivo. CBDP increased s.d. toxicities of sarin 11-fold, of tabun 5-fold and of VX 0.24-fold. The protective index of atropine + HS-3 in sarin poisoning, as in the case of soman poisoning, was much higher in CBDP pretreated than in control animals (20.1 : 13.6), only slightly higher in tabun poisoning (4.3 : 3.4) and in the case of VX poisoning lower in CBDP pretreated than in control animals (32 : 47). The results indicate that CBDP potentiates soman, sarin and tabun toxicities mainly by blocking their binding to non-specific sites in the body.     

Acute and chronic studies with the anticholinesterase Huperzine A: effect on central nervous system cholinergic parameters

High affinity choline transport, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were assessed in rats after acute and chronic administration of the AChE inhibitor Huperzine A. Acute treatment: Forty-five min after a single injection of Huperzine A (0.5 mg/kg i.p.) the activity of AChE was significantly decreased by 15-30% in hippocampus, striatum and septum. The activity of ChAT was not altered. In the hippocampus high affinity choline transport was attenuated by 25%, whereas no effect in the striatum was observed. After 90 min, both inhibition of AChE and attenuation of high affinity choline transport had returned to control values. A dose of 0.1 mg/kg (i.p.) did not produce significant effects. Similar results were obtained with physostigmine (0.25 mg/kg), although the duration of inhibition of AChE was shorter than that with Huperzine A. Chronic treatment: After 5 days (twice a day), at 0.5 mg/kg, the activity of AChE was significantly reduced by 20-30% in every region of the brain studied. High affinity choline transport in the hippocampus was reduced by 28%, 45 min after the last injection, but in the striatum there was no effect. The activity of ChAT was not affected in any region of the brain studied. Thus, acute or chronic treatment with Huperzine A: did not alter ChAT; reduced high affinity choline transport in the hippocampus in a transient manner; and had a longer duration of action as an AChE inhibitor than physostigmine. Moreover, tolerance to low-toxicity doses of Huperzine A was minimal, contrary to what has been observed with other inhibitors of AChE.     

Effects of copper,zinc and paraquat on acetylcholinesterase activity in carp (Cyprinus carpio L.)

The effects of pH and substrate concentration on acetylcholinesterase (AChE) activity were studied in serum, brain, heart and muscle of common carp (Cyprinus carpio L.). Effects of zinc chloride, paraquat (1,1′-dimethyl-4,4′-dipyridinium dichloride) and copper sulphate from in vivo and in vitro exposure were studied on these AChE activities. Zinc chloride did not decrease AChE activity in any of the organs studied in vivo or in vitro. In contrast, paraquat, competitively, and copper sulphate, in a mixed way, inhibited acetylcholinesterase activity. Inhibition of AChE in fish exposed to these two pesticides may serve as an indicator of hazard due to application of these chemicals in the natural environment.     

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

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.