Cholinesterase activity in the cup oyster Saccostrea sp. exposed to chlorpyrifos, imidacloprid, cadmium and copper.

In the present study, the sensitivity and concentration dependence of three functionally-defined components of cholinesterase activity (total: T-ChE; eserine-sensitive: Es-ChE; and eserine-resistant: Er-ChE) were quantified in the gill, digestive gland and adductor muscle of the tropical cup oyster Saccostrea sp., following acute (96h) aqueous exposure to commercial formulations of the organophosphate (OP) insecticide chlorpyrifos and the neonicotinoid (NN) imidacloprid (concentration range: 0.1-100mg/L), as well as to dissolved cadmium and copper (concentration range: 1-1000µg/L). Oysters (1.5-5.0cm shell length), field-collected from a boating marina in Santa Marta, Colombia (Caribbean Sea) were exposed in the laboratory to each substance at five concentrations. T-ChE, Es-ChE, and Er-ChE activity were quantified in the three tissues in pools of 5 individuals (3 replicates per concentration), before and after inhibition with the total cholinesterase inhibitor eserine (physostigmine, 100µM). Oysters exposed to chlorpyrifos, imidacloprid and Cd showed reduced T-ChE and Es-ChE activity in gills at highest exposure concentrations, with Es-ChE activity being inhibited proportionally more so than T-ChE, whereas Er-ChE activity showed no significant concentration-response. Digestive gland also showed diminished T-ChE, Es-ChE and Er-ChE activity for highest chlorpyrifos and Cd concentrations relative to controls, but an increase of T-ChE and Er-ChE activity at the highest imidacloprid concentration (100mg/L). For Cu, T-ChE, Es-ChE and Er-ChE activities in gills and digestive gland were elevated relative to controls in oysters exposed to Cu concentrations > 100µg/L. In adductor muscle, T-ChE, Es-ChE and Er-ChE activity showed no apparent pattern for any of the four xenobiotics and concentration levels tested. Although this study confirms acute (96h) concentration-dependent reduction of tissue T-ChE and Es-ChE activity in gills and digestive glands of Saccostrea sp. exposed to high concentrations of chlorpyrifos (100mg/L), significant changes in T-ChE, Es-ChE and Er-ChE were also caused by exposure to Cd and Cu at concentrations > 100µg/L and by exposure to imidacloprid (100mg/L), indicating that cholinesterase activity is not a specific biomarker of organophosphate exposure in this species, but, rather, a biomarker of diverse xenobiotic exposure.

Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys.

In this study, we investigated the possibility to improve a new behavioural bioassay (Swimming Speed Alteration test-SSA test) using larvae of marine cyst-forming organisms: e.g. the brine shrimp Artemia sp. and the rotifer Brachionus plicatilis. Swimming speed was investigated as a behavioural end-point for application in ecotoxicology studies. A first experiment to analyse the linear swimming speed of the two organisms was performed to verify the applicability of the video-camera tracking system, here referred to as Swimming Behavioural Recorder (SBR). A second experiment was performed, exposing organisms to different toxic compounds (zinc pyrithione, Macrotrol MT-200, and Eserine). Swimming speed alteration was analyzed together with mortality. The results of the first experiment indicate that SBR is a suitable tool to detect linear swimming speed of the two organisms, since the values have been obtained in accordance with other studies using the same organisms (3.05 mm s(-1) for Artemia sp. and 0.62 mm s(-1) for B. plicatilis). Toxicity test results clearly indicate that swimming speed of Artemia sp. and B. plicatilis is a valid behavioural end-point to detect stress at sub-lethal toxic substance concentrations. Indeed, alterations in swimming speed have been detected at toxic compound concentrations as low as less then 0.1-5% of their LC(50) values. In conclusion, the SSA test with B. plicatilis and Artemia sp. can be a good behavioural integrated output for application in marine ecotoxicology and environmental monitoring programs.

Ciguatoxin: more than an anticholinesterase.

Anticholinesterase action of ciguatoxin has been reported. A study of the pharmacology of the action of the toxin on the respiratory system suggests that additional properties may be involved in the respiratory failure-the usual cause of death in ciguatera poisoning.

Differential mRNA expression of acetylcholinesterase in the central nervous system of rats with acute and chronic exposure of sarin & physostigmine.

A time-course study was carried out to measure the acetylcholinesterase (AChE) gene expression in the brain of female rats exposed to different doses of sarin and physostigmine. Short-term effects were studied with an acute single subcutaneous dose (s.c.) of 80 microg kg(-1) (0.5 x LD(50)) sarin. Cortex and cerebellum showed a significant decline in AChE mRNA expression at 2.5, 24 and 72 h. Biochemical studies showed that plasma butrylcholinesterase (BChE) and brain AChE activities were significantly decreased at 2.5 h, which came back to near control values by 24 h in both cases. For long-term chronic studies, three groups of female rats received daily doses of physostigmine (0.1 mg kg(-1) day(-1)) intramuscularly (i.m.), sarin (15 microg kg(-1) day(-1)) s.c. independently and a combined dose of physostigmine (i.m.) (0.1 mg kg(-1) day(-1)) followed by sarin (s.c.) (15 microg kg(-1) day(-1)) continuously for 30 days. Differential AChE mRNA levels in cortex and cerebellum of rat brain were observed after 30 days and after a lag period of another 30 days with no further administration. Plasma (BChE) and brain (AChE) showed irregular inhibition profile in biochemical studies at 30 days and returned to control levels after 60 days. The acute single subcutaneous administration of sarin for short-term as well as chronic long-term studies showed that AChE inhibition alone does not lead to observed changes in mRNA expression of AChE gene. These observations further suggest that route of administration as well as dose exposure regimen also contributes to the regulation of AChE mRNA expression.

Toxicity and characterization of cholinesterase-inhibition induced by diisopropyl fluorophosphate in Artemia salina larvae.

The acute toxicity of diisopropyl fluorophosphate (DFP) on three age classes of Artemia salina was evaluated. An increase in toxicity of this organophosphorous (OP) compound was found following longer development of A. salina larvae. The effects of pretreatment with the non-selective muscarinic antagonist atropine, the two reversible acetylcholinesterase inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methoiodide (2-PAM), as individual and combined pretreatments, on DFP-induced lethality in 24h Artemia were also investigated. The lethal action of DFP was not prevented by pretreatment of 24h Artemia with atropine, physostigmine, and pyridostigmine, while 2-PAM proved effective against intoxication with this OP compound. The inhibitory effects of combinations of atropine (10(-5)M) plus 2-PAM or physostigmine were greater than those elicited by either drug alone, with the maximum protection afforded being 100%. Pretreatment with 2-PAM (10(-6)M) plus physostigmine or pyridostigmine was ineffective. These results suggest that the combinations of atropine plus 2-PAM or physostigmine are effective in the prevention of the lethal effects induced by DFP in A. salina larvae.

Respiratory failure triggered by cholinesterase inhibitors may involve activation of a reflex sensory pathway by acetylcholine spillover.

Respiration failure during exposure by cholinesterase inhibitors has been widely assumed to be due to inhibition of cholinesterase in the brain. Using a double chamber plethysmograph to measure various respiratory parameters, we observed long "end inspiratory pauses" (EIP) during most exposure that depressed breathing. Surprisingly, Colq KO mice that have a normal level of acetylcholinesterase (AChE) in the brain but a severe deficit in muscles and other peripheral tissues do not pause the breathing by long EIP. In mice, long EIP can be triggered by a nasal irritant. Eucalyptol, an agonist of cold receptor (TRPM8) acting on afferent sensory neurons and known to reduce the EIP triggered by such irritants, strongly reduced the EIP induced by cholinesterase inhibitor. These results suggest that acetylcholine (ACh) spillover from the neuromuscular junction, which is unchanged in Colq KO mice, may activate afferent sensory systems and trigger sensory reflexes, as reversed by eucalyptol. Indeed, the role of AChE at the cholinergic synapses is not only to accurately control the synaptic transmission but also to prevent the spillover of ACh. In the peripheral tissues, the ACh flood induced by cholinesterase inhibition may be very toxic due to interaction with non-neuronal cells that use ACh at low levels to communicate with afferent sensory neurons.

Screening for developmental neurotoxicity using PC12 cells: comparisons of organophosphates with a carbamate, an organochlorine, and divalent nickel.

BACKGROUND: In light of the large number of chemicals that are potential developmental neurotoxicants, there is a need to develop rapid screening techniques. OBJECTIVES: We exposed undifferentiated and differentiating neuronotypic PC12 cells to different organophosphates (chlorpyrifos, diazinon, parathion), a carbamate (physostigmine), an organochlorine (dieldrin), and a metal (divalent nickel; Ni2+) and examined indices of cell replication and differentiation for both short- and long-term exposures. RESULTS: In undifferentiated cells, all the agents inhibited DNA synthesis, with the greatest effect for diazinon, but physostigmine eventually produced the largest deficits in the total number of cells after prolonged exposure. The onset of differentiation intensified the adverse effects on DNA synthesis and changed the rank order in keeping with a shift away from noncholinergic mechanisms and toward cholinergic mechanisms. Differentiation also worsened the effects of each agent on cell number after prolonged exposure, whereas cell growth was not suppressed, nor were there any effects on viability as assessed with trypan blue. Nevertheless, differentiating cells displayed signs of oxidative stress from all of the test compounds except Ni2+, as evidenced by measurements of lipid peroxidation. Finally, all of the toxicants shifted the transmitter fate of the cells away from the cholinergic phenotype and toward the catecholaminergic phenotype. CONCLUSIONS: These studies point out the feasibility of developing cell-based screening methods that enable the detection of multiple end points that may relate to mechanisms associated with developmental neurotoxicity, revealing some common targets for disparate agents.

Pharmacological validation of a semi-automated in vitro hippocampal brain slice assay for assessment of seizure liability.

INTRODUCTION: Drug-induced seizures are a serious, life-threatening adverse drug reaction (ADR) that can result in the failure of drugs to be licensed for clinical use or withdrawn from the market. Seizure liability of potential drugs is traditionally assessed using animal models run during the later phases of the drug discovery process. Given the low throughput, high animal usage and high compound requirement associated with these assays, it would be advantageous to identify higher throughput, in vitro models that could be used to give an earlier assessment of seizure liability. The hippocampal brain slice is one possibility but conventionally allows recording from only one slice at a time. The aim of this study was to validate a semi-automated system (Slicemaster, Scientifica UK Ltd) which allows concurrent electrophysiological recording from multiple brain slices. METHODS: Conventional electrophysiological recording techniques were used to record electrically evoked synaptic activity from rat hippocampal brain slices. Population spikes (PS) were evoked at 30 s intervals by electrical stimulation of the Schaffer collateral pathway and were recorded using extracellular electrodes positioned in the CA1 cell body layer. Responses were quantified as PS areas (the area above and below the 0 mV line). The effects of eight validation compounds known to cause seizures in vivo and/or in the clinic were assessed. RESULTS: Seven out of eight compounds evoked a concentration-dependent increase in population spike (PS) area that was statistically significant at higher concentrations (P<0.05; ANOVA). At the highest test concentration the percentage effects (mean+/-s.e.m.), relative to vehicle, were: picrotoxin 212.9+/-28.8, pentylenetrazole (PTZ) 181.4+/-24.7, 4-AP 328.9+/-48.6, aminophylline 124.5+/-5.9, chlorpromazine 122.1+/-9.8, SNC-80 132.1+/-12.6 and penicillin 174.7+/-14.1. Physostigmine had no significant effect on PS area although a concentration-dependent change in the morphology of the response was evident. DISCUSSION: All validation compounds evoked a statistically significant effect on synaptic activity in the rat hippocampal slice. Although similar effects have been described previously, this is the first time that the effects of a pharmacologically diverse set of compounds have been assessed using a standardised brain slice assay. Given the low compound usage and relatively high throughput associated with this assay, the hippocampal brain slice assay may facilitate earlier testing of convulsant liability than is currently possible using in vivo models.

Cell proliferation and tumor formation induced by eserine, an acetylcholinesterase inhibitor, in rat mammary gland.

Environmental chemicals may be involved in the etiology of breast cancer. There is substantial evidence that breast cancer risk is associated with prolonged exposure to female hormones. Among these hormonal influences a leading role is attributed to the ovarian hormone estradiol, since breast cancer does not develop in the absence of ovaries. The rat mammary gland has special characteristics that make it an ideal organ for studying development, cell proliferation and transformation. In vivo and in vitro model systems for cell proliferation and mammary carcinogenesis have allowed morphological and biochemical analysis under different experimental conditions. The aim of this study was to examine the effect of eserine, an acetylcholinesterase inhibitor, as are the organophosphorous compounds malathion and parathion, and 17beta estradiol on cell proliferation and tumor formation that takes place in the rat mammary gland after in vivo and in vitro treatment. These studies showed that eserine and 17beta estradiol were capable of inducing carcinogenesis in the epithelium of rat mammary glands. It was found that there was a significant increase in the number of cells per duct of the 44-day-old rat mammary gland after the 10-day eserine treatment, compared to the control. A higher increase was observed in the animals treated for 10 days with eserine followed by 30-daily injections of estrogen in comparison to control animals. In 12 animals, two mammary tumors were directly developed in response to 17beta estradiol injected at 39 days of age with a latency period of 180 and 245 days, respectively. Such tumors were metastatic to the lung. These results suggest that terminal end buds are major targets related to rat mammary carcinogenesis and 17beta estradiol can be an initiator and promoter in this process.

Tissue distribution and characterization of cholinesterase activity in six earthworm species.

To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086+/-0.015 U mg(-1) and 0.235+/-0.036 U mg(-1) for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.

The early toxicology of physostigmine: a tale of beans, great men and egos.

Mid-19th century European visitors to Old Calabar, an eastern province of Nigeria, could not avoid becoming aware of native belief in the power of the seeds of a local plant to determine whether individuals were innocent or guilty of some serious misdemeanour. The seeds were those of a previously unknown legume and soon referred to as the ordeal bean of Old Calabar. Their administration was known locally as 'chop nut'. Missionaries who arrived in Calabar in 1846 estimated that chop nut caused some 120 deaths annually and documented the course of poisoning. The latter information and samples of the beans rapidly found their way to Scotland, the home of the missionaries' parent church, explaining why the early toxicology of physostigmine, quantitatively the most important of three active alkaloids in the beans, has such strong Scottish, predominantly Edinburgh, associations. However, it was 1855 before the first of many medical scientists, Robert Christison, a toxicologist of repute, investigated the effects of the beans to the extent of eating part of one himself and documenting the moderate, if not severe, consequences. A further 6 years were to pass before Balfour's comprehensive botanical description of the bean plant appeared. It was he who named it Physostigma venenosum. It was not so long until the next event, one that sparked more intensive and international interest in the beans. In 1863 a young Edinburgh ophthalmologist, Argyll Robertson, published a paper announcing the arrival of the first agent that constricted the pupil of the eye. The drug was an extract of Calabar beans and Argyll Robertson openly admitted that he had been alerted to its unusual property by his physician friend, Thomas Fraser. A minor flood of contributions on the ophthalmic uses of bean extracts followed in the medical press in the next few months; those on their systemic toxicity were fewer. Fraser's MD thesis, submitted to the University of Edinburgh in 1862 and clearly pre-dating Argyll Robertson's involvement with the beans, became generally available a few weeks after the appearance of Argyll Robertson's paper and was the first to address in detail the features of systemic administration of extracts of the beans. A major problem facing all early researchers of the beans was that of deciding how best to extract their active principle, a task made all the more difficult because bioassays were the only means of determining if the toxin was being tracked. The stability of extracts was an inevitable issue and the active principle finally became known as physostigma or physostigmine, after the botanical name of the parent plant. The features of physostigmine toxicity were soon exhaustively documented, both in animals and humans. How they were mediated was another matter altogether. Fraser maintained that muscular paralysis, the cardinal feature, was the result of depression of the spinal cord and was generally, but far from unanimously, supported. Of those who had reservations, Harley was the most prominent. He concluded that paralysis was secondary to effects on the motor nerve endings and, in so doing, came nearest to present-day knowledge at a time when acetylcholine, cholinesterases and cholinesterase inhibitors were not even imagined. Differences of opinion on the mode of action of the beans were to be expected and it is hardly surprising that they were not resolved. No standard formulation of physostigmine was available so the potency of those used would have varied from one investigator to another, the range of animals experimented upon was large while the number used by any researcher was commonly in single figures, more readily available cold-blooded creatures seemed less sensitive to physostigmine toxicity than warm-blooded ones and only Fraser determinedly pursued an answer; in general, the others made one foray into bean research then turned their attentions elsewhere. The same problems would beset other aspects of bean research. While Fraser did not get as close to the mode of action of physostigmine as Harley, he reigns supreme when it comes to antagonism between physostigmine and atropine. By this time, the 1870s had dawned and although the concept of antagonism between therapeutic agents was not new, it had little, if any, reliable scientific foundation. This was about to change; antagonism was becoming exciting and rational. Fraser's firm belief that physostigmine and atropine were mutually antagonistic at a physiological level was contrary to the conventional wisdom of his contemporaries. This alone would earn him a place in history but his contribution goes much, much further. Unlike any other at the time, he investigated it with scientific rigour, experimenting on only one species, ensuring as best he could the animals were the same weight, adjusting the doses of drugs he gave them for bodyweight, determining the minimum lethal dose of each drug before assessing their antagonistic effects, adopting a single, incontrovertible endpoint for efficacy and carrying out sufficient numbers of experiments to appear convincing in a later era where the statistical power of studies is all-important. To crown it all, he presented his results graphically. Fraser never claimed to have discovered the antagonism between physostigmine and atropine. Bartholow in 1873 did, based on work done in 1869. But his data hardly justify it. If anyone can reasonably claim this particular scientific crown it is an ophthalmologist, Niemetschek, working in Prague in 1864. His colleague in the same discipline, Kleinwächter, was faced with treating a young man with atropine intoxication. Knowing of the contrary actions of the two drugs on the pupil, Niemetschek suggested that Calabar bean extract might be useful. Kleinwächter had the courage to take the advice and his patient improved dramatically. Clearly, this evidence is nothing more than anecdotal, but the ophthalmologists were correct and, to the present day, physostigmine has had an intermittent role in the management of anticholinergic poisoning. The converse, giving atropine to treat poisoning with cholinesterase inhibitors, of which physostigmine was the first, has endured more consistently and remains standard practice today. It is salutary to realise that the doses and dosage frequency of atropine together with the endpoints that define they are adequate were formulated by Fraser and others a century and a half ago.

Carbamate nerve agent prophylatics exhibit distinct toxicological effects in the zebrafish embryo model.

Pyridostigmine bromide (PB) is an FDA-approved drug for the treatment of myasthenia gravis and a prophylactic pre-treatment for organophosphate nerve agent poisoning. Current methods for evaluating nerve agent treatments include enzymatic studies and mammalian models. Rapid whole animal screening tools for assessing the effects of nerve agent pre-treatment and post-exposure drugs represent an underdeveloped area of research. We used zebrafish as a model for acute and chronic developmental exposure to PB and two related carbamate acetylcholinesterase (AChE) inhibitors, neostigmine bromide (NB) and physostigmine (PS). Lethal doses and gross morphological phenotypes resulting from exposure to sub-lethal doses of these compounds were determined. Quantitative analyses of motility impairment and AChE enzyme inhibition were used to determine optimal dosing conditions for evaluation of the effects of carbamate exposures on neuronal development; ~50% impairment of response to startle stimuli and >50% inhibition of AChE activity were observed at 80 mMPB, 20 mM NB and 0.1 mM PS. PB induced stunted somite length, but no other phenotypic effects were observed. In contrast, NB and PS induced more severe phenotypic morphological defects than PB as well as neurite outgrowth mislocalization. Additionally, NB induced mislocalization of nicotinic acetylcholine receptors, resulting in impaired synapse formation. Taken together, these data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of carbamates and demonstrate the utility of the zebrafish model for distinguishing subtle structure-based differential effects of AChE inhibitors, which include nerve agents, pesticides and drugs.

A model for carbamate and organophosphate-induced emesis in humans.

In human volunteers, studies to assess the adverse effects of the carbamate anticholinesterase physostigmine showed that the intramuscular dose observed to induce emesis in 50% of subjects tested (ED50) was 28.1 (23.5-120.7) micrograms/kg. This dose reduced whole blood cholinesterase (ChE) activity to 60% of control values. Studies in marmosets to assess the behavioural toxicology of physostigmine showed that the corresponding ED50 and ChE activity values were 34.3 (21.5-55.8) micrograms/kg and 66% respectively. Sarin was also shown to induce emesis in marmosets, but only at doses that reduced erythrocyte ChE activity to 12% of control values. These data seem also to correspond with reports of organophosphate poisoning in humans. It is concluded that the marmoset may be a very good model of both carbamate and organophosphate-induced emesis in humans.

Syntheses, resolution, and structure-activity relationships of potent acetylcholinesterase inhibitors: 8-carbaphysostigmine analogues.

The synthesis of a series of 1,2,3,3a,8,8a-hexahydroindeno[2,1-b]pyrrole 5-alkylcarbamates and their resolution are reported. These compounds are structurally related to physostigmine with substitution of a methylene group in place of the NMe group at position 8 of physostigmine. Many of these 8-carbaphysostigmine analogues are more potent acetylcholinesterase inhibitors in vitro and less toxic in vivo than physostigmine. The (-)-enantiomer (e.g., 1d and 1g) possessing the same absolute configuration at C3a and C8a as that of physostigmine, is about 6 to 12-fold more potent at inhibiting acetylcholinesterase than the corresponding (+)-enantiomer (e.g., 1e and 1h).

New dibenzothiadiazepine derivatives with antidepressant activities.

A new series of 11-[(aminoalkyl)carbonyl] derivatives of 6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine 5,5-dioxide (10-39) were synthesized and evaluated for potential antidepressant activity in the apomorphine-induced hypothermia (Apo 16) test. Effects on reserpine-induced hypothermia and toxicity for the most potent antagonists of Apo 16 hypothermia were also studied. Structure-activity relationships are reported. Anticholinergic effects were evaluated for compound 12, identified as the most potent and least toxic in this series, by assessing physostigmine lethality. Compound 12 was also subjected to X-ray analysis.

Anti-tremor action of C10Dichol, a peripheral acetylcholine synthesis inhibitor.

1 Anti-tremor action of decamethylene bis-(hydroxyethyl)-dimethylammonium bromide (C10Dichol), a peripheral acetylcholine synthesis inhibitor, was investigated. 2 C10Dichol inhibited tremor induced by oxotremorine, nicotine and physostigmine and afforded partial protection from physostigmine-induced mortality in mice. 3 In non-paralysing doses, C10Dichol antagonized the neuromuscular effects of oxotremorine, nicotine and physostigmine. 4 Prior administration of C10Dichol failed to prevent tremor and neuromuscular paralysis induced by harmine and arecoline. 5 In the absence of any antimuscarinic property of C10Dichol, its neuromuscular effects appeared to be casually related to its anti-tremor action. 6 This study reveals a possibility for the development of peripherally acting anti-Parkinson drugs.

Prevention of physostigmine-, DFP-, and diazinon-induced acute toxicity by monoethylcholine and N-aminodeanol.

1. Choline, and the choline analogues monoethylcholine (MEC) and N-aminodeanol (NAD) were examined for prophylactic activity in acute acetylcholinesterase inhibitor toxicity in mice. The rank order of potency of the compounds was MEC greater than NAD greater than choline. 2. Simultaneous administration of MEC (60 mg kg-1) or NAD (200 mg kg-1) with physostigmine reduced lethality to 17 and 13% respectively. MEC (60 mg kg-1) completely protected against disopropylfluorophosphate (DFP) and diazinon toxicity, and NAD reduced lethality to 17% for both agents. Choline (200 mg kg-1) exhibited only negligible antidotal activity against the inhibitors. 3. In vitro concentrations of choline, MEC, and NAD, similar to the estimated concentration obtained in vivo in the acute toxicity study, produced mixed inhibition of mouse brain acetylcholinesterase. The inhibition was dose-related and was additive to the inhibition produced by the cholinesterase inhibitors. 4. All three analogues reduced ligand binding at the nicotinic, M1, and M2 receptors. The rank order of potencies for the analogues at each receptor was nicotinic: (choline greater than MEC greater than NAD), M1: (MEC greater than choline greater than NAD), and M2: (MEC greater than choline greater than NAD). 5. It is proposed that the analogues prevent acetylcholinesterase inhibitor toxicity peripherally by interacting with acetylcholinesterase, and/or by competing with acetylcholine for binding to cholinoceptors.

A rat mammary tumor model induced by the organophosphorous pesticides parathion and malathion, possibly through acetylcholinesterase inhibition.

Environmental chemicals may be involved in the etiology of breast cancers. Many studies have addressed the association between cancer in humans and agricultural pesticide exposure. Organophosphorous pesticides have been used extensively to control mosquito plagues. Parathion and malathion are organophosphorous pesticides extensively used to control a wide range of sucking and chewing pests of field crops, fruits, and vegetables. They have many structural similarities with naturally occurring compounds, and their primary target of action in insects is the nervous system; they inhibit the release of the enzyme acetylcholinesterase at the synaptic junction. Eserine, parathion, and malathion are cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. Atropine, a parasympatholytic alkaloid, is used as an antidote to acetylcholinesterase inhibitors. The aim of this study was to examine whether pesticides were able to induce malignant transformation of the rat mammary gland and to determine whether alterations induced by these substances increase the cholinergic activation influencing such transformation. These results showed that eserine, parathion, and malathion increased cell proliferation of terminal end buds of the 44-day-old mammary gland of rats, followed by formation of 8.6, 14.3, and 24.3% of mammary carcinomas, respectively, after about 28 months. At the same time, acetylcholinesterase activity decreased in the serum of these animals from 9.78 +/- 0.78 U/mL in the control animals to 3.05 +/- 0.06 U/mL; 2.57 +/- 0.15 U/mL; and 3.88 +/- 0.44 U/mL in the eserine-, parathion-, and malathion-treated groups, respectively. However, atropine alone induced a significant (p < 0.05) decrease in the acetylcholinesterase activity from the control value of 9.78 +/- 0.78 to 4.38 +/- 0.10 for atropine alone, to 1.32 +/- 0.06 for atropine in combination with eserine, and 2.39 +/- 0.29 for atropine with malathion, and there was no mammary tumor formation. These results indicate that organophosphorous pesticides induce changes in the epithelium of mammary gland influencing the process of carcinogenesis, and such alterations occur at the level of nervous system by increasing the cholinergic stimulation.

Lethal effects of physostigmine plus lithium in rats.

The interaction between the reversible cholinesterase inhibitor, physostigmine, and lithium chloride was studied in adult male rats. A combination of lithium plus physostigmine increased lethality more than that caused by either physostigmine or lithium alone. Scopolamine completely reversed this effect. These drug interactions may have clinical significance, since lithium plus cholinesterase inhibitors may be used together in the practice of medicine.

Neuropharmacological and behavioral evaluation of prostaglandin E2 and 11-thiol-11-desoxy prostaglandin E2 in the mouse and rat.

Prostaglandin E2 (PGE2) and 11-thiol-11-desoxy Prostaglandin E2 (SHPGE2) were evaluated in a variety of behavioral and neuropharmacological procedures that are sensitive to neuroleptics. Clozapine (C), thioridazine (T), haloperidol (H), and fluphenazine (F) were also tested for comparison. All agents except T suppressed avoidance responses in trained rats at one or more doses without concurrently disrupting escape behavior. T, H, and F dose-responsively antagonized lesioned rat rotational behavior at nontoxic doses. T, H, and F induced catalepsy at doses considerably higher than those effective on rotational behavior. SHPGE2, PGE2, and C did not cause catalepsy and did not show statistically significant dose-response antagonism of rotational behavior at less than toxic doses. All agents tested blocked d-amphetamine-induced lethality and caused motor incoordination dose-responsively. SHPGE2, PGE2, C, and T caused statistically significant blockade of physostigmine-induced lethality. H and F were ineffective against physostigmine lethality. It was concluded that SHPGE2 and PGE2 demonstrated, qualitatively, a spectrum of neuroleptic like properties remarkably similar to C.