Inhibitor Report for: Disulfoton

A 75-year-old woman attempted suicide by ingesting a large quantity of granular Di-Syston which is an organophosphate insecticide containing 5% disulfoton (ethylthiometon). On admission, the total plasma phosphorodithioate sulfone concentration (disulfoton and its metabolites, phosphorodithioate sulfoxide and its sulfone) determined by gas chromatography was 1095 ng/mL. After gastric lavage, the concentration gradually decreased to 505 ng/mL. However, it began to increase again 20 h after admission and reached the peak concentration (1322 ng/mL) at 56 h. It was concluded that the secondary elevation of the plasma concentration was due to the prolonged absorption of the organophosphate from the residual granules in the stomach, despite the early gastric lavage. Pralidoxime iodide administration temporarily restored erythrocyte cholinesterase activity to almost normal and inhibited the excessive, delayed reduction of cholinesterase activity. It is recommended that poisoning with the granular form of disulfoton should be treated with repetitive or prolonged gastric and intestinal lavage, charcoal, and a continuous intravenous infusion of pralidoxime iodide in addition to atropine sulfate.

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [3H]QNB binding and only at concentrations greater than 100 microM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [3H]CD, which binds with high affinity (approximate KD = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.

Brain cholinesterase (ChE) activities of songbirds collected in pecan groves 6 to 7 hr after separate applications of the organophosphorus pesticides, phosalone and disulfoton, were compared to mean ChE activities of controls (normals) as a measure of insecticide exposure. In general, reduction of brain ChE activity greater than or equal to 2 standard deviations below the control mean indicates exposure to an anticholinesterase compound. Phosalone had little effect on brain ChE activity of birds from treated groves; only slight to moderate (21 to 38%) ChE inhibition was detected in blue jays (Cyanocitta cristata) and red-bellied woodpeckers (Melanerpes carolinus). However, 11 of 15 blue jays from disulfoton-treated groves had moderate to severe ChE depression, ranging from 32 to 72%. Inhibition greater than or equal to 50% of normal may be diagnostic for cause of death. Direct mortality was not observed, but studies have shown that bird carcasses disappear rapidly from agricultural areas, many within 24 hr. We recommend additional field studies of the effects of disulfoton to wildlife, since large wheat-growing areas in the western United States are being considered for disulfoton treatment to control the Russian wheat aphid (Diuraphis noxia).

Cereal leaf beetle, Oulema melanopus (L.), invaded northern Alabama and Georgia more than a decade ago and since has become an economic pest of winter wheat and other cereal crops in the southeastern United States. A series of trials was conducted beginning in 1995 to determine optimal rate and timing of applications of selected foliar insecticides for managing cereal leaf beetle in soft red winter wheat. These trials, cage studies with larvae, and a manual defoliation experiment were used to provide information on cereal leafbeetle yield loss relationships and to develop economic decision rules for cereal leaf beetle in soft red winter wheat. Malathion, methomyl, carbaryl, and spinosad effectively controlled larval infestations when treatments were applied after most eggs had hatched. Encapsulated endotoxin of Bacillus thuringiensis, methyl parathion, and disulfoton applied at the lowest labeled rates were not effective treatments. Organophosphate insecticides generally were not effective when applied before most eggs had hatched. The most effective treatments were the low rates of lambda cyhalothrin when applied early while adults were still laying eggs and before or near 50% egg hatch. These early applications applied at or before spike emergence virtually eliminated cereal leaf beetle injury. The manual defoliation study demonstrated that defoliation before spike emergence has greater impact on grain yield and yield components than defoliation after spike emergence. Furthermore, flag leaf defoliation causes more damage than injury to lower leaves. Grain test weight and kernel weight were not affected by larval injury in most trials. Regression of larval numbers and yield losses calculated a yield loss of 12.65% or 459 kg/ha per larva per stem, which at current application costs suggested an economic threshold of 0.4 larvae per stem during the spike emergence to anthesis stages.

Phorate and disulfoton are organophosphate insecticides containing three oxidizable sulfurs, including a thioether. Previous studies have shown that only the thioether is oxygenated by flavin-containing monooxygenase (FMO) and the sole product is the sulfoxide with no oxygenation to the sulfone. The major FMO in lung of most mammals, including non-human primates, is FMO2. The FMO2*2 allele, found in all Caucasians and Asians genotyped to date, codes for a truncated, non-functional, protein (FMO2.2A). Twenty-six percent of individuals of African descent and 5% of Hispanics have the FMO2*1 allele, coding for full-length, functional protein (FMO2.1). We have here demonstrated that the thioether-containing organophosphate insecticides, phorate and disulfoton, are substrates for expressed human FMO2.1 with Km of 57 and 32 microM, respectively. LC/MS confirmed the addition of oxygen and formation of a single polar metabolite for each chemical. MS/MS analysis confirmed the metabolites to be the respective sulfoxides. Co-incubations with glutathione did not reduce yield, suggesting they are not highly electrophilic. As the sulfoxide of phorate is a markedly less effective acetylcholinesterase inhibitor than the cytochrome P450 metabolites (oxon, oxon sulfoxide or oxon sulfone), humans possessing the FMO2*1 allele may be more resistant to organophosphate-mediated toxicity when pulmonary metabolism is an important route of exposure or disposition.

Water samples from 30 lakes in Canada and the northeastern United States were analyzed for the occurrence of 27 current-use pesticides (CUPs). Eleven CUPs were frequently detected in lakes receiving agricultural inputs as well as in remote lakes hundreds of kilometers from known application areas. These included the triazine herbicide atrazine and its desethylated degradation product; the herbicides alachlor, metolachlor, and dacthal; the organophosphate insecticides chlorpyrifos, diazinon, and disulfoton; the organochlorine insecticides alpha-endosulfan and lindane; and the fungicides chlorothalonil and flutriafol. For six of the pesticides, empirical half-distances on the order of 560 to 1,820 km were estimated from the water-concentration gradient with latitude. For most of the pesticides, a suite of assessment models failed to predict such atmospheric long-range transport behavior, unless the effect of periods of lower hydroxyl radical concentrations and dry weather were taken into account. Observations and model results suggest that under the conditions prevailing in south-central Canada (relatively high latitude, low precipitation rates), many CUPs will be able to undergo regional-scale atmospheric transport and reach lakes outside areas of agricultural application. When assessing the potential of fairly reactive and water-soluble substances to undergo long-range transport, it is imperative to account for periods of no precipitation, to assure that degradation rate constants are correct, and to apply oxidant concentrations that are valid for the region and time period of interest.

Chronic and acute exposure to organophosphate (OP) pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. It is suggested that other brain proteins are involved. Effects of commonly used organophosphate pesticides on rat neuronal alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. Several OP pesticides, e.g., parathion-ethyl, chlorpyrifos and disulfoton, inhibited the ACh-induced ion current with potencies in the micromolar range. The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of nAChR inhibition with the potency of AChE inhibition demonstrated that some OPs inhibit nAChRs more potently than AChE. Binding experiments on alpha4beta2 nAChRs showed that the OPs noncompetitively interact with nAChRs. The inhibitory effects on nAChRs are adequately described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It is concluded that OPs interact directly with neuronal alpha4beta2 nAChRs to inhibit the agonist-induced response. This implicates that neuronal alpha4beta2 nAChRs are additional targets for some OP pesticides.

Increasing rates of insecticide use against the coffee leaf minerLeucoptera coffeella(Guerin-Meneville) and field reports on insecticide resistance led to an investigation of the possible occurrence of resistance of this species to some of the oldest insecticides used against it in Brazil: chlorpyrifos, disulfoton, ethion and methyl parathion. Insect populations were collected from ten sites in the state of Minas Gerais, Brazil and these populations were subjected to discriminating concentrations established from insecticide LC99s estimated for a susceptible standard population. Eight of the field-collected populations showed resistance to disulfoton, five showed resistance to ethion, four showed resistance to methyl parathion, and one showed resistance to chlorpyrifos. The frequency of resistant individuals in each population ranged from 10 to 93% for disulfoton, 53 to 75% for ethion, 23 to 76% for methyl parathion, and the frequency of resistant individuals in the chlorpyrifos resistant population was 35%. A higher frequency of individuals resistant to chlorpyrifos, disulfoton and ethion was associated with greater use of insecticides, especially other organophosphates. This finding suggests that cross-selection, mainly between organophosphates, played a major role in the evolution of insecticide resistance in Brazilian populations of L. coffeella. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide and triphenyl phosphate) suggested that cytochrome P450-dependent monooxygenases may play a major role in resistance with minor involvement of esterases and glutathione S-transferases.

The goal of this study was to develop a sensitive in vitro bioassay for quantification of the total esterase inhibiting potency of low concentrations of organophosphate and carbamate insecticides in relatively small rainwater samples. Purified acetylcholinesterase (AChE) from electric eel (Electrophorus electricus) and carboxylesterases from a homogenate of honeybee heads (Apis mellifera) were used as esterases, each having different affinities for the substrates S-acetylthiocholine-iodide (ATC) and N-methylindoxylacetate (MIA). MIA hydrolysis by honeybee homogenate was more sensitive to inhibition by organophosphate insecticides than ATC hydrolysis by purified AChE, although the latter parameter is often used for in vitro monitoring of esterase inhibitors. The higher sensitivity of carboxylesterases is attributed to the instant formation of a reversible Michaelis-Menten complex with the inhibitor, which competes with MIA for the active sites of the free enzymes. This dose-dependent instant inhibition can be quantified with kinetics for competitive inhibition at dichlorvos concentrations < 16 nM. At similar concentrations, purified AChE was not instantly inhibited, whereas both AChE and carboxylesterases were irreversibly and progressively inhibited at higher dichlorvos concentrations (IC50(10min) >/= 0.1 microM). Honeybee homogenate mediated MIA hydrolysis was applied as the most sensitive enzyme-substrate combination for experiments with fractionated extracts of 4 rainwater samples collected in a natural conservation area. Most esterase inhibiting potency was found in the polar methanol fraction, with recalculated concentrations equivalent to 12-125 ng dichlorvos per liter rainwater.

We examined the effects of organophosphate exposure on mRNA expression levels of synaptic- and target tissue-specific proteins in rats. We treated rats with a single dose of Disulfoton (O,O-diethyl S-2-ethylthioethyl phosphorodithioate) and used quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure the time course of changes in the levels of mRNAs encoding acetylcholinesterase (AChE), nicotinic acetylcholine receptor (nAChR), beta-enolase (MSE), and gamma-enolase (NSE) in soleus muscles and sciatic nerves. The expression levels of synaptic genes encoding AChE in both tissues were significantly decreased, with a nadir at 12h after the administration, and this down-regulation lasted for up to 30 days after administration. Similarly, the level of nAChR mRNA in soleus muscle also decreased, with a nadir at 48 h after administration and a return to 95% of that of the control levels by 30 days after administration. These results indicate that administration of organophosphate can decrease AChE and nAChR expression in the neuromuscular junction, and are suggestive of multiple mechanisms of down-regulation of both AChE and nAChR, some of which might involve alterations at the transcriptional level. The transcript level of the target tissue-specific gene encoding MSE in soleus muscle was slightly decreased, with a nadir at 48 h after administration, and was still lower than that of the control level after 30 days. In contrast, the level of the NSE transcript in sciatic nerve significantly increased within 2 h, and this up-regulation was sustained until 30 days after administration. Although the functions of either of these enolases are not completely established, up-regulation of NSE mRNA may be a marker for the nervous system abnormality following organophosphate exposure. All of these phenomena may contribute to the long-lasting neurotoxic effects observed after developmental exposure to organophosphates.

An organophosphate (OP)-resistant clone (OR-0) of the greenbug, Schizaphis graminum, showed 27.2-, 19.9-, 65.8-, 44.5-, 18.2-, 4.8-, and 3.1-fold greater resistance to dimethoate, omethoate, disulfoton, demeton-S-methyl, parathion, methyl parathion, and chlorpyrifos, respectively, than an OP-susceptible clone (OSS). General esterase and glutathione S-transferase activities were similar in the OSS and OR-0 clones, whereas cytochrome P450 O-demethylase activities were below the detection limit in both greenbug clones. In contrast, acetylcholinesterase (AChE) activity in the OR-0) clone was 2.3-fold higher than that in the OSS clone. Kinetic studies also revealed that AChE from the OR-0 clone was 2.1-, 2.1-, 1.6-, 4.4-, and 1.3-fold less sensitive to inhibition by methyl paraoxon, paraoxon, demeton-S-methyl, omethoate, and demeton S, respectively, than AChE from the OSS clone (P < 0.05). However, Northern blot analysis of mRNA, using a 296-bp AChE cDNA fragment generated by seminested PCR as a homologous probe, indicated that the increased activity of AChE in the OR-0 clone was not due to the overexpression of the AChE gene. Our overall results suggest that the increased AChE activity in the OR-0 clone most likely is due to structural modifications of AChE, leading to both an increased catalytic activity with the model substrate acetylthiocholine and a reduced sensitivity to inhibition by OP compounds.

Technical grade disulfoton (DiSyston) was fed to Beagle dogs (four animals per sex and treatment level) at nominal concentrations of 0, 0.5, 4 and 12 ppm for 1 year. The purpose of this study was to characterize the potential general and neurovisual toxicity according to routine Environmental Protection Agency (EPA) guideline requirements, and by use of ancillary ocular and neurologic tests established in this Laboratory. Ophthalmological tests included: ocular tissue cholinesterase and histopathology, electroretinography (ERG), tracking, refractivity, intraocular pressure and pachymetry (corneal thickness) measurements. Neurological examinations included; peripheral and cranial reflex tests, task performance tests, gait and behavioral observations, and rectal temperature measurements. Plasma, erythrocyte and corneal cholinesterase were significantly depressed at 4 and 12 ppm in both sexes. Brain cholinesterase was depressed at 4 and 12 ppm in females. Retinal cholinesterase was depressed at 4 ppm in females and at 12 ppm in males. Ciliary body cholinesterase was depressed at 12 ppm in both sexes. Despite these cholinergic effects, there were no ophthalmologic findings in measurements of ERG, tracking, refractivity, intraocular pressure or pachymetry. There were no clinical neurology findings related to compound administration. We conclude that 0.5 ppm was a no-observable effect level (NOEL), and effects were limited to cholinesterase changes that had no detectable physiologic impact. This study demonstrates that special mechanistic investigations incorporated within guideline studies, enhances scientific integrity and can minimize the need for dedicated organ system studies.

Sulprofos, disulfoton, azinphos-methyl, methamidophos, trichlorfon, and tebupirimphos were screened for neurotoxic potential, in accordance with U.S. EPA (FIFRA) requirements. Each organophosphate was administered through the diet for 13 weeks to separate groups of Fischer 344 rats at four dose levels, including a vehicle control. For each study, 12 rats/sex/dietary level were tested using a functional observational battery (FOB), automated measures of activity (figure-8 maze), and detailed clinical observations, with half of the animals perfused at term for microscopic examination of neural and muscle tissues. Separate groups of satellite animals (6/sex/dietary level) were used to measure the effect of each treatment on plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity. The results show that measures of ChE activity were consistently the most sensitive indices of exposure and assisted in the interpretation of findings. All treatment-related neurobehavioral findings were ascribed to cholinergic toxicity, occurring only at dietary levels that produced more than 20% inhibition of plasma, RBC, and brain ChE activity. Neurobehavioral tests provided no evidence of additional cumulative toxicity after 8 weeks of treatment. The FOB and motor activity findings did not alter the conclusions and generally did not reduce the neurobehavioral no-observed-effect level (NOEL) for any of the six compounds, relative to the results from detailed clinical observations as conducted in these studies. The one exception occurred with tebupirimphos, where the NOEL for motor activity was one dose level lower than the NOEL for the FOB and clinical observations. These results support the value of detailed clinical observations to screen for the neurotoxic potential of organophosphates and a general standard of more than 20% inhibition of brain ChE activity for cholinergic neurotoxicity.

Milligram quantities of sulfoxides, sulfones, and oxygen analogue (oxon) sulfones of the insecticides disulfoton, phorate, and terbufos were prepared by selective oxidation. Pure insecticides or acetone extracts of granular formulations served as reactants. Structures of 9 compounds were confirmed by comparing their electron impact mass spectra (EI-MS), obtained by the direct inlet system with published data. Thin-layer chromatography (TLC) on silica gel was used to screen oxidation products and to purify products. Products were detected by spraying plates with PdCl2 reagent and exposing to iodine vapors. An esterase inhibition technique gave low detection limits, which are promising for residue analysis.

Mature male rainbow trout (Oncorhynchus mykiss) were exposed for 28 days to 0, 1, 5, and 20 micrograms/liter disulfoton, i.e., to concentrations well below any macroscopically visible effect due to the primary acute toxic mechanism of acetylcholine esterase inhibition. In an attempt to reveal sublethal injury of disulfoton in rainbow trout, ultrastructural and stereological parameters were recorded in the liver as the central organ of xenobiotic metabolism in fish. Quantitative methods were definitely not able to replace qualitative techniques because, except for mitochondria, peroxisomes, and hepatocellular lipid inclusions, stereological analysis revealed only insignificant variations of hepatocellular components, whereas hepatocytes displayed a complex pattern of numerous delicate qualitative alterations. Effects were most evident within cisternae of the rough endoplasmic reticulum (RER), thus suggesting modifications of protein metabolism. Structural alterations included degenerative effects such as dilation and vesiculation of RER cisternae, formation of concentric RER arrays and augmentation of smooth endoplasmic reticulum, dilation of Golgi cisternae, and the development of cytoplasmic myelinated bodies as well as stacks of membranous material within mitochondria. Structural integrity and augmentation of peroxisomes and mitochondria as well as increased activity of the Golgi system were indicative of adaptive/compensative reactions following disulfoton treatment. In fact, adaptive effects seemed more pronounced than degenerative phenomena resulting in only minor disturbances in hepatocyte structure following disulfoton exposure. Because most effects had to be classified as unspecific responses to environmental or xenobiotic stressors, no distinct mode of sublethal action can be suggested for disulfoton.

Repeated exposure to organophosphorus (OP) insecticides results in a decrease of muscarinic acetylcholine receptors (MRs) in the central nervous system. OP-induced MR down-regulation in vivo is modeled by chronic in vitro exposure to muscarinic agonists. Many studies, both in vivo and in vitro, indicate that the treatment-induced decrease in MR number is accompanied by a decrease in the mRNA levels of specific MR subtypes. In this study, the in vivo effects of OP exposure on the mRNA levels of three MR subtypes (m1, m2, and m3) were examined in brain tissue and in peripheral mononuclear cells, which express the m3 subtype. Adult male Sprague-Dawley rats were orally administered disulfoton (2 mg/kg/day) for 14 days, and a subset of exposed animals was allowed to recover for 28 days. This treatment caused a 28% and 81% decrease, respectively, in [3H]-QNB binding and acetylcholinesterase activity in the cortex, similar to that observed in previous studies; after recovery, these levels had returned to 99% and 90%, respectively, of controls. There was a significant decrease in m1 mRNA levels in hippocampus (23%) after disulfoton treatment, while no change was observed in the cortex, corpus striatum, medulla, or cerebellum. The m2 subtype mRNA was significantly decreased in both hippocampus (24%) and medulla (19%), but not in cortex, striatum, or cerebellum. m3 mRNA levels were significantly decreased in cortex (10%), but no change was observed in hippocampus, medulla, cerebellum, or lymphocytes. After recovery, no differences in m1 or m3 mRNA levels were observed in any tissue examined, whereas the decrease in m2 mRNA in the hippocampus remained significant (29%). These results indicate that OP exposure can differentially regulate mRNA levels for MR subtypes in different brain areas, and suggest that m2 muscarinic receptors in the hippocampus are most affected by this treatment.

A 75-year-old woman attempted suicide by ingesting a large quantity of granular Di-Syston which is an organophosphate insecticide containing 5% disulfoton (ethylthiometon). On admission, the total plasma phosphorodithioate sulfone concentration (disulfoton and its metabolites, phosphorodithioate sulfoxide and its sulfone) determined by gas chromatography was 1095 ng/mL. After gastric lavage, the concentration gradually decreased to 505 ng/mL. However, it began to increase again 20 h after admission and reached the peak concentration (1322 ng/mL) at 56 h. It was concluded that the secondary elevation of the plasma concentration was due to the prolonged absorption of the organophosphate from the residual granules in the stomach, despite the early gastric lavage. Pralidoxime iodide administration temporarily restored erythrocyte cholinesterase activity to almost normal and inhibited the excessive, delayed reduction of cholinesterase activity. It is recommended that poisoning with the granular form of disulfoton should be treated with repetitive or prolonged gastric and intestinal lavage, charcoal, and a continuous intravenous infusion of pralidoxime iodide in addition to atropine sulfate.

Repeated exposures to organophosphorus (OP) insecticides has been shown to cause a decrease of cholinergic muscarinic receptors (mAChR) in brain and in peripheral tissues. These changes are believed to be involved in the development of tolerance to OP toxicity and may play a role in cognitive dysfunctions observed following repeated OP exposure. Recently, mAChRs identified in circulating lymphocytes have been shown to be modulated similarly to brain mAChRs following repeated OP exposure, suggesting that these peripheral cells may be useful as indicators of mAChR changes in the central nervous system. This study was designed to further investigate whether mAChRs on lymphocytes could serve as a biomarker for changes in brain mAChRs during prolonged OP exposure and during recovery from such exposure. Using the mAChR antagonist [3H]quinuclidinyl benzilate (QNB) to label mAChRs, we found that exposure to disulfoton for 14 days (2 mg/kg/day by gavage) caused a significant decrease (25-35%) in muscarinic receptors density in the cerebral cortex, hippocampus, and striatum, as well as in circulating lymphocytes. The decline of mAChR density in lymphocytes paralleled those observed in brain, particularly in cortex and hippocampus, during exposure to disulfoton; however, while brain mAChR levels recovered slowly after termination of exposure and remained significantly reduced 4 weeks after the last treatment, [3H]QNB binding in lymphocytes recovered rapidly within 1 week. Similarly, lymphocyte acetylcholinesterase (AChE) activity was significantly inhibited and correlated well with brain AChE activity during exposure, but the recovery was rapid relative to AChE activity in brain.

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [3H]QNB binding and only at concentrations greater than 100 microM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [3H]CD, which binds with high affinity (approximate KD = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.

Brain cholinesterase (ChE) activities of songbirds collected in pecan groves 6 to 7 hr after separate applications of the organophosphorus pesticides, phosalone and disulfoton, were compared to mean ChE activities of controls (normals) as a measure of insecticide exposure. In general, reduction of brain ChE activity greater than or equal to 2 standard deviations below the control mean indicates exposure to an anticholinesterase compound. Phosalone had little effect on brain ChE activity of birds from treated groves; only slight to moderate (21 to 38%) ChE inhibition was detected in blue jays (Cyanocitta cristata) and red-bellied woodpeckers (Melanerpes carolinus). However, 11 of 15 blue jays from disulfoton-treated groves had moderate to severe ChE depression, ranging from 32 to 72%. Inhibition greater than or equal to 50% of normal may be diagnostic for cause of death. Direct mortality was not observed, but studies have shown that bird carcasses disappear rapidly from agricultural areas, many within 24 hr. We recommend additional field studies of the effects of disulfoton to wildlife, since large wheat-growing areas in the western United States are being considered for disulfoton treatment to control the Russian wheat aphid (Diuraphis noxia).

Memory impairment is one of the recurrent complaints of agricultural workers repeatedly exposed to organophosphorus insecticides. In an effort to establish an animal model for such behavioral effects, which would allow studying its underlying biochemical mechanism(s), in this study we evaluated spatial memory in animals following repeated organophosphate exposure. Male Long-Evans rats were given daily i.p. injections of either diisopropylfluorophosphate (DFP; 1 mg/kg/day) or disulfoton (O,O-diethyl S-[2-(ethylthio)ethyl] phosphorodithioate; 2 mg/kg/day) for 14 days. Acetylcholinesterase activity was inhibited 71-77% in the cortex, hippocampus, and striatum of rats treated with DFP, and 73-74% in those treated with disulfoton. Binding of [3H]quinuclidinyl benzilate ([3H]QNB) to cholinergic muscarinic receptors in the same brain areas was reduced 16-28% in organophosphate-treated rats. This decrease was due to a reduction in muscarinic receptor density (Bmax) with no changes in receptor affinity. At the end of the treatment rats were tested for spatial memory using the spontaneous alternation task in a T-maze. Rates of true spontaneous alternation were 64.4, 45.0, and 44.8% in animals which received corn oil, DFP, or disulfoton, respectively (P less than 0.05). These results indicate that prolonged inhibition of acetylcholinesterase caused by repeated organophosphate exposure alters spatial memory functions in rats, as well as causing a loss of muscarinic receptors. Considering the role of the cholinergic system in cognitive processes, these biochemical alterations could be related to the observed behavioral changes and may offer a potential explanation of the memory impairment reported by workers chronically exposed to organophosphates.

A simple and efficient cleanup method for gas chromatographic determination of 23 organophosphorus pesticides in crops including onion is described. The sample was extracted with acetone. The extract was purified with coagulating solution, which contained ammonium chloride and phosphoric acid, and then filtered by suction. The filtrate was diluted with NaCl solution and reextracted with benzene. The organic layer was evaporated and injected into a gas chromatograph equipped with a flame photometric detector (FPD) and fused silica capillary columns (0.53 mm id) coated with silicone equivalent to OV-1701, OV-1, and SE-52. Onion extract, which contained FPD interferences, was cleaned up on a disposable silica cartridge. Recoveries of most organophosphorus pesticides from spiked crops: mandarin orange, tomato, spinach, sweet pepper, broccoli, lettuce, and onion at levels of 0.02-0.28 ppm, exceeded 80%, but the water-soluble pesticides dichlorvos and dimethoate gave poor recoveries in all crops; the nonpolar pesticides disulfoton, chlorpyrifos, fenthion, prothiophos, and leptophos were not recovered quantitatively in spinach, sweet pepper, broccoli, and lettuce. IBP, edifenphos, phosmet, and pyridaphenthion were not recovered from onion because of adsorption to the silica cartridge. The detection limits ranged from 1.25 to 17.5 ppb on a crop basis.