Inhibitor Report for: Methylparathion

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.

Methyl parathion is a widely used agricultural insecticide, and the recent unlicensed use of this compound in homes has led to the evacuation of approximately 1100 persons in Mississippi. Although the primary concern in such cases of acute exposure is neurotoxicity, a few organophosphorus compounds apparently have immunotoxic effects at dosages that do not produce neurotoxic symptoms. The purpose of the present study was to determine if this is the case for methyl parathion. Female B6C3F1 mice were exposed to methyl parathion by gavage, daily for 7, 14, 2 1, or 28 d (at 6 mg/kg/d). Exposure for 14-28 d produced significant, dose-responsive inhibition of acetylcholin-esterase (the target molecule for methyl parathion-induced neurotoxicity) in brain or plasma, indicating that the compound was active. The following immunological parameters were evaluated: white blood cell counts and differentials, spleen and thymus weight and cellularity, splenic natural killer cell activity, nitrite production by peritoneal macrophages following activation in vitro, antibody response to sheep erythrocytes in vitro and in vivo, the cytotoxic T lymphocyte response to allogeneic tumor cells, and resistance to Streptococcus agalactiae and B16F10 melanoma cells. Methylparathion at 1 or 3 mg/kg/d significantly increased splenic natural killer cell activity. Nitrite production by macrophages was increased in mice treated with 1, 3, or 6 mg/kg/d. The antibody response to sheep erythrocytes in vitro was significantly suppressed, but the humoral response to sheep erythrocytes in vivo was not affected. The cytotoxic T-lymphocyte response to allogeneic tumor cells was not significantly affected. Host resistance was not significantly decreased. Although it remains possible that immunological parameters not tested here may be affected by methyl parathion, the present results do not suggest substantial immunotoxic potential for this compound.

Oxidative damage was quantified in the liver of rats by measuring the levels of 8-OH-2-deoxyguanosine (8-OH-2DG) relative to 2-deoxyguanosine in DNA after treating rats for 10 days at a total dose of 1 mg/kg/day with a mixture of the 15 pesticides most commonly found in Italian foods (comprised of dithiocarbamate, benomyl, procymidone, methidathion, chlorpyrifos-ethyl, parathion-methyl, chlorpropham, parathion, vinclozolin, chlorfenvinphos, pirimiphos ethyl, thiabendazole, fenarimol, diphenylamine and chlorothalonil). We fractionated this pesticide mixture into subgroups in order to determine which molecules, if any, induced DNA oxidative damage. The administration of diphenylamine (0.09-1.4 mg/kg/day) and chlorothalonil (0.13-1 mg/kg/day) induced a dose-dependent increase in 8-OH-2DG levels in liver DNA. The other 13 pesticides of the mixture on the contrary, did not produce oxidative liver DNA damage. These results indicate that the toxicity of low doses of pesticide mixtures present in food might be further reduced by eliminating diphenylamine and chlorothalonil.

Approximately 200 citrus samples from markets of the Valencian Community (Spain) were analyzed to establish their residue levels in 12 organophosphorus pesticide residues during the 1994-1995 campaign. The organophosphorus pesticides carbophenothion, chlorpyriphos, chlorfenvinphos, diazinon, ethion, fenitrothion, malathion, methidation, methylparathion, phosmet, quinalphos, and tetradifon were simultaneously extracted by matrix solid-phase dispersion and determined by gas chromatography-mass spectrometry using selected ion monitoring mode. A total of 32.25% contained pesticide residues and 6.9% exceeded the European Union Maximum Residue Levels (MRLs). The pesticides found in the samples with residues above MRLs were carbophenothion, ethion, methidathion, and methyl parathion. Lower level residues of these and the other pesticides studied (except diazinon) were frequently found. The estimated daily intake of the 12 organophosphorus pesticide residues during the studied period was 4.87 x 10(-4) mg/kg body weight/day. This value is lower than the provisional tolerances dairy intakes proposed by the Food and Agriculture Organization and the World Health Organization.

Cholinesterase (ChE) in brain and muscle was quickly inhibited during a 48-hr in vivo exposure to chlorpyrifos (0.1 ppm), parathion (0.15 ppm), and methyl parathion (8 ppm) in mosquitofish (Gambusia affinis). ChE remained inhibited during a 96-hr nonexposure period. Brain ChE reached peak inhibition by 12 hr after exposure to parathion and chlorpyrifos and by 4 hr after exposure to methyl parathion. All insecticides caused greater than 70% ChE inhibition by 4 hr in muscle. There was no recovery of ChE after 4 days of nonexposure in either brain or muscle. Hepatic aliesterases (AliE) were quickly and greatly inhibited (> 70% by 4 hr) after exposure to parathion and chlorpyrifos but not after exposure to methyl parathion. Exposure to methyl parathion required 24-36 hr to inhibit hepatic AliE to the same level as that following parathion and chlorpyrifos exposures at 4 hr. Exposure to all insecticides eventually resulted in greater than 80% inhibition of AliE. None of the test groups treated with insecticides showed any signs of significant recovery of AliE during the 4 days of nonexposure. Nonprotein sulfhydryl (NPSH) concentrations were lower than controls after 24 hr of exposure and 96 hr after recovery for all compounds. Exposure to methyl parathion lowered NPSH concentrations greater than the other compounds. Hepatic AliE appear capable of affording some protection of ChE from inhibition following parathion or chlorpyrifos exposures, but considerably less protection against methyl parathion.

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway. The results from HPLC analysis did not show a peak corresponding to methyl paraoxon (O,O-dimethyl O-p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.

A rapid procedure has been developed that allows a single-step, selective extraction and cleanup of organophosphate (OP) pesticide residues from milk dispersed on solid-matrix diatomaceous material filled into disposable cartridges by means of light petroleum saturated with acetonitrile and ethanol. Recovery experiments were carried out on homogenized commercial milk (3.6% fat content) spiked with ethanolic solutions of 24 OP pesticides, viz., ethoprophos, diazinon, dimethoate, chlorpyrifos-methyl, parathion-methyl, chlorpyrifos-ethyl, malathion, isofenphos, quinalphos, ethion, pyrazophos, azinphosethyl, heptenophos, omethoate, fonofos, pirimiphos-methyl, fenitrothion, parathion, chlorfenvinphos, phenthoate, methidathion, triazophos, phosalone, azinphos-methyl, at levels ranging for the different OP pesticides from 0.02 mg/kg to 1.11 mg/kg. Average recoveries of four replicates were in the range 72-109% for the different OP pesticides, with relative standard deviations (R.S.D.) from ca. 1 to 19%, while dimethoate and omethoate were not recovered. Coextracted fatty material amounted to an average of about 4.0 mg/ml of milk. The extraction procedure requires about 30 min. The main advantages are that extraction and cleanup are carried out in a single step, emulsions do not occur, several samples can be run in parallel by a single operator, reusable glassware is not needed and simple operations are required.

Adult male whitethroated munias, Lonchura malabarica (Aves; Passeriformes), were orally administered with methyl parathion (O, O-dimethyl O-(4-nitrophenyl) phosphorothioate), an extensively used organophosphate pesticide, in graded sublethal dose (5 micro g-, or 10 micro g-, or 20 micro g/100 g body wt/day) for variable durations (1-, 5-, or 10 day/s) during their peak reproductive activities in an annual gonadal cycle. No subtle changes in the feeding behavior, mobility, and body weight were noted between the control and different groups of pesticide-fed birds. As a result of the treatment, the paired testicular weight became reduced significantly only after 10 days at 10 micro g and 20 micro g dose levels, but significant decrease in the number of tubules containing healthy germ cells occurred even after single administration of methyl parathion (MP) at the lowest dose (5 micro g/100 g). With the increase in dose and progress of treatment, the number of tubules with healthy germ cells became gradually decreased. The activity of acetylcholinesterase (AChE) in both the brain and testes of MP-treated birds was inhibited in a dose and duration dependent manner. A significant negative correlation was observed between the number of tubules containing degenerated germ cells in the testis and the AChE activity in both the brain and testes of MP-administered birds. However, no remarkable changes in the cytomorphological features, including the nuclear diameter of Leydig cells, were noted in any testis of the pesticide-treated munias. The results of the present investigation suggest that methyl parathion ingestion is harmful to male gametogenic functions in the studied passeriform bird, and the given pesticide may exert its antigonadal effect by impairing cholinergic functions of the brain and/or testes.

The acute toxicity of chlorpyrifos, methylchlorpyrifos, parathion and methylparathion to three age classes of Artemia salina was determined. In general, A. salina 24-h old was less sensitive to these organophosphorous insecticides (OPI) than A. salina 48-h old and A. salina 48-h old was significantly more tolerant than A. salina 72-h old, in contrast, chlorpyrifos was equally toxic to A. salina 48- and 72-h old. There were some differences among the three age classes of A. salina in the relative order of toxicity of OPI tested. The rank order of toxicity to A. salina 48-h old was methylparathion < parathion < methyl-chlorpyrifos < chlorpyrifos, while to A. salina 24- and 72-h old it was methylparathion = parathion < methyl-chlorpyrifos < chlorpyrifos. The protective effect of the cholinergic antagonists atropine, hexamethonium, pirenzepine and 11-(2-((diethyl-amino)methyl)-1-piperidinylacetyl)-5, 11-dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepine-6-one (AF-DX 116) and a cholinesterase-reactivating oxime 2-pyridine aldoxime methochloride (2-PAM) on the mortality due to four selected OPI in Artemia salina 24-h old was investigated. The lethal action of OPI tested was completely prevented by pretreatment of Artemia salina 24-h old with 2-PAM (10(-5) M) and atropine (10(-4 )M). However no concentration of hexamethonium, pirenzepine or AF-DX 116 protected 100% of the animals poisoned by LC84 of the OPI selected, maximum protection obtained was 71 to 88%. In contrast, the maximum inhibition of mortality obtained with AF-DX 116 pretreatment was about 55% because this compound was used at concentrations which were non toxic to control Artemia salina. Atropine, hexamethonium, pirenzepine, AF-DX 116 and 2-PAM afforded 50 % protection (IC50) of Artemia salina against mortality by LC84 of the OPI selected at concentrations in the range of 6.62x10(-7)-1.6x10(-6) M, 2. 38x10(-4)-2.05x10(-3)M, 8.91x10(-7)-1.24x10(-6) M, 9.66x10(-8)-1. 34x10(-7 )M, and 1.95x10(-8)-2.73x10(-8 )M, respectively. Pretreatment of atropine plus 2-PAM to determine whether this combination afforded greater inhibition of the lethality induced by four OPI tested than pretreatment with either atropine or 2-PAM alone was investigated. Atropine (10(-5) M) in combination with 2-PAM (10(-7 )M) inhibited completely the acute toxicity of all OPI tested, while the pretreatment with atropine (10(-6) M) plus 2-PAM at the same concentration gave a inhibition of mortality (about 62%) significantly greater than each antagonist alone (about 14 and 46%, respectively).

A multiresidue extraction method based on matrix solid-phase dispersion (MSPD) is optimized for the extraction and gas chromatographic screening of eighteen insecticides (aldrin, carbophenothion, captafol, chlorpyriphos, chlorfenvinphos, diazinon, dicofol, alpha-endosulfan, beta-endosulfan, ethion, fenitrothion, folpet, methidathion, malathion, methyl-azinphos, methyl-parathion, phosmet, and tetradifon) from oranges. After optimization of different parameters, such as type of solid phase used and the amount of solid phase or eluent, recoveries ranged from 67 to 102% with relative standard deviations ranging from 2 to 10%. The limits of detection, calculated as 3 times the baseline noise ranged from 2 to 171 micrograms/kg. These limits of detection were about 10 times lower than the maximum residue levels established by the European Community. Compared with classical methods, the described procedure is simple, less labour intensive and does not require preparation and maintenance of equipment. Troublesome emulsions, such as those frequently observed in liquid-liquid partitioning did not occur.

The impact of Methyl parathion and Carbaryl was evaluated on an ecologically important soil Collembola, Cyphoderus sp. Enzyme characteristics demonstrate substrate optimum at 1 10(-2) mol/L temperature optimum at 30 degrees C with a pH requirement of 8.0. In vivo inhibition of whole body AChE reveals higher degree of inhibition by LD50 dose of Methyl parathion as compared to that of Carbaryl where maximum inhibition was noticed at the agricultural dose.

The study examined the interactions between a commercial formulation of methyl parathion (CF-MP) and a commercially formulated product of permethrin (CF-PMT) in male rats. The acute toxicity (LD50 values) and brain cholinesterase activity were investigated as toxicological endpoints. Results indicated that CF-MP modified the acute toxicity of CF-PMT. When animals were treated with a mixture, the addition of 380 mg/kg of CF-MP reduced the LD50 of CF-PMT by only 9.0%; however, when rats received CF-MP at 464 mg/kg, the LD50 of CF-PMT was reduced by 37% (P < 0.001). Also, CF-PMT decreased the CF-MP-induced inhibition of cholinesterase activity by 50% (P < 0.05). It was interesting to observe that xylene, which is the most abundant component in the vehicle of both formulations, had no effect on the CF-MP-induced inhibition of the cholinesterase activity. There was no relation between lethality and the inhibition of the brain cholinesterase activity in rats treated with mixtures containing CF-MP+CF-PMT or with either commercially formulated product alone. Considering the increased toxicity observed in rats treated with CF-PMT+CF-MP, it would be advisable to investigate further the interactions between both pesticides.

Lymphocytes obtained from 5 healthy donors were incubated with a mixture of 15 pesticides commonly found in foods of central Italy (dithiocarbamates (20.7%), benomyl (19.6%), thiabendazole (14.9%), diphenylamine (14.4%), chlorthalonil (13.1%), procymidone (8.0%), methidathion (2.3%), chlorpyrifos-ethyl (2%), fenarimol (1.9%), parathion-methyl (1%), chlorpropham, parathion, vinchlozolin, chlorfenvinphos and pirimiphos-ethyl (< 1%)). The percent of each pesticide in the mixture was proportional to its average concentration in foods. Incubated with the lymphocytes at a concentration of 1-20 micrograms/ml the pesticide mixture did not induce significant variations in the number of hypodiploid, hyperdiploid and polyploid cells or in the number of chromosome and chromatid aberrations. On the contrary, we observed a dose-dependent increase in the number of nonsynchronous centromeric separations which reached the level of 37.9% at 20 micrograms/ml of pesticide mixture in the incubation medium. This effect was not observed when benomyl was excluded from the mixture. These data show that the removal of benomyl could decrease the toxicity of pesticide residues present in human food.

Study was conducted to find out the correlation between red blood cholinesterase (RBC ChE) and plasma butyryl cholinesterase (BCHE) activities and toxic signs of oral methylparathion (MPT) and their recovery pattern with or without atropine treatment in female rats. Enzyme activity was estimated before and after an oral dose of MPT (7.5 mg/kg-1) at various time intervals upto 120 hr. Antidote groups received atropine (10 mg/kg-1, i.p.), either alone or with diazepam (2.5 mg/kg-1, i.p.), at the onset of toxic signs. Inhibition of enzyme activity served as definite index of acute toxicity of MPT. RBC ChE activity correlated with the intensity of toxic signs in no-antidote rats, while in atropine treated groups, there was no correlation. BCHE levels did not correlate with toxic signs in any of the groups except in the fatal group. The resynthesis of both the enzymes was complete in 120 hr study and did not synchronize with the recovery pattern of animals from toxic signs. Compared to BCHE, RBC ChE activity was found to be a more sensitive indicator for the diagnosis of severity of MPT toxicity.

Rats were administered high sublethal intraperitoneal dosages of the phosphorothionate insecticides parathion, methyl parathion, and chlorpyrifos, and their oxons. Acetylcholinesterase activities in cerebral cortex and medulla oblongata and aliesterase activities in liver and plasma were monitored at 2 hr and 1, 2, and 4 days after exposure. The maximal inhibition of brain acetylcholinesterase activity was not immediate with parathion and chlorpyrifos, reflecting the time required for bioactivation of the phosphorothionates as well as the effectiveness of the aliesterases to inactivate much of the hepatically generated oxons. In contrast, brain acetylcholinesterase activities were more quickly inhibited following administration of paraoxon and chlorpyrifos-oxon, which do not require bioactivation. Brain acetylcholinesterase was also rapidly inhibited following administration of methyl parathion and methyl paraoxon, reflecting the low sensitivity of the aliesterases to methyl paraoxon. Aliesterases were inhibited to a greater extent than acetylcholinesterase at each sampling time with parathion and chlorpyrifos and their oxons, whereas the reverse was true with methyl parathion and methyl paraoxon. All of the above patterns correlate with the in vitro sensitivities of acetylcholinesterase and aliesterases to the oxons. The very prolonged inhibition of esterase activities following chlorpyrifos treatment probably results from its substantially greater lipophilicity compared to the other compounds, which would allow it to be stored and released for gradual bioactivation. The data reported indicate that the disposition and effects of different phosphorothionate insecticides will be influenced by the sensitivities of target and nontarget esterases for their oxons and by their lipophilicity, and that predictions of in vivo responses can be made from in vitro data.

OBJECTIVES To estimate the frequency of the intermediate syndrome in organophosphorus-poisoned patients, and examine its relationship to cholinesterase inhibition and electromyographic findings. Muscle biopsies were available in some patients. DESIGN: A 3-yr prospective study. SETTING: University teaching hospital intensive care unit. PATIENTS: Consecutive patients with acute organophosphorus poisoning (n = 19). MEASUREMENTS AND MAIN RESULTS: We determined the frequency of the intermediate syndrome in poisonings with various organophosphates, duration of (acetyl) cholinesterase inhibition and metabolite excretion, evolution of alterations on repetitive nerve stimulation, type and frequency of muscle lesions. A total of eight of 19 patients developed an intermediate syndrome. In some patients, short relapses of muscarinic symptoms superimposed on the intermediate syndrome. Agents such as methylparathion, fenthion, and dimethoate carry a high risk, but we also noted a prolonged intermediate syndrome in an ethyl-parathion-poisoned patient. Prolonged and severe cholinesterase inhibition occurred during the intermediate syndrome in all patients, and metabolite excretion was prolonged. As the intermediate syndrome evolved, repetitive nerve stimulation initially demonstrated decrement, then increment, and finally, normal responses. Necrotic fibers were noted in muscle biopsies, but these fibers were too sparse to explain severe muscle weakness and were similar in patients with and without the intermediate syndrome. No patients developed delayed neuropathy. CONCLUSIONS: The intermediate syndrome is not rare. Although it is more likely to occur with some organophosphates, it is not confined to a few distinct compounds. This syndrome coincides with prolonged cholinesterase inhibition, and is not due to muscle fiber necrosis. When viewed together, the clinical and electromyographic features are best explained by combined pre- and postsynaptic dysfunction of neuromuscular transmission. The intermediate syndrome is not related to an incipient delayed neuropathy.

To determine the toxicological effects of complex mixtures of pesticides, we obtained data on 100 pesticide residues in common foods of central Italy. Fifteen pesticides were more regularly detected at higher levels (dithiocarbamates, benomyl/carbendazim, thiabendazole, diphenylamine, chlorthalonil, procymidone, fenarimol, chlorpropham, vinchlozolin, methidathion, chlorpyriphos-ethyl, parathion-methyl, parathion, chlorfenviphos, pirimiphos-ethyl). Using itemized data on daily food consumption in Italy, we calculated that the average exposure for an adult subject was 716 micrograms/day, ranging from 148 micrograms of dithiocarbamates to 1 microgram of pirimiphos-ethyl. We made a mixture of these 15 pesticides at concentrations proportional to the ratio determined in foods and tested it with the Salmonella-microsome assay, with and without metabolic activation with PCB-induced rat liver S9. No mutagenic activity was observed at concentrations up to 500 micrograms/plate. We also tested the same mixture at concentrations ranging from 0.1 to 20 micrograms/ml on human lymphocytes in vitro, and observed a slight but statistically significant increase in sister-chromatid exchanges at 1 microgram/ml. We also administered the mixture in corn oil by gavage to Wistar rats at doses of 1, 10, and 100 micrograms/kg. After 24 hr the ratio between bone marrow polychromatic and normochromatic lymphocytes (a sign of cellular toxicity) was decreased by the exposure, but we did not observe a significant increase in the frequency of micronuclei. We conclude that the pesticide mixture did not have appreciable genotoxic activity in the assays used.

Sublethal doses of methyl parathion (o, o-dimethyl-o-nitrophenyl thiophosphate) injected intraperitoneally to 7th day old developing albino rat pups induced alterations in the inhibitory GABAergic system of CNS. A substantial simulation of the inhibitory system was noticed. A profound increase was found in the level of the inhibitory transmitter, GABA on methyl parathion injection. An increase in the activity levels of the enzymes glutamic acid decarboxylase and 4-aminobutyrate-2-oxoglutarate-amino transferase in the cortex, brain stem and spinal cord of the CNS was found. It is observed that methyl parathion causes potentiation of the inhibitory transmission (GABAergic system) in the wake of inducing suppression of cholinergic system in CNS of developing rat pups.

We measured in nine patients, poisoned by organophosphorus agents (ethyl parathion, ethyl and methyl parathion, dimethoate, or bromophos), erythrocyte and serum cholinesterase activities, and plasma concentrations of the organophosphorus agent. These patients were treated with pralidoxime methylsulphate (Contrathion), administered as a bolus injection of 4.42 mg.kg-1 followed by a continuous infusion of 2.14 mg.kg-1/h, a dose regimen calculated to obtain the presumed "therapeutic" plasma level of 4 mg.l-1, or by a multiple of this infusion rate. Oxime plasma concentrations were also measured. The organophosphorus agent was still detectable in some patients after several days or weeks. In the patients with ethyl and methyl several days or weeks. In the patients with ethyl and methyl parathion poisoning, enzyme reactivation could be obtained in some at oxime concentrations as low as 2.88 mg.l-1; in others, however, oxime concentrations as high as 14.6 mg.l-1 remained without effect. The therapeutic effect of the oxime seemed to depend on the plasma concentrations of ethyl and methyl parathion, enzyme reactivation being absent as long as these concentrations remained above 30 micrograms.l-1. The bromophos poisoning was rather mild, cholinesterases were moderately inhibited and increased under oxime therapy. The omethoate inhibited enzyme could not be reactivated.

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.

To study the molecular origin of the altered regulation of butyrylcholinesterase (BCHE) in nervous system tumors, BCHE complementary DNA (cDNA) sequences from human glioblastoma and neuroblastoma cDNA libraries were compared with BCHE cDNAs from normal fetal and adult tissues. A single 2.6-kilobase BCHE cDNA sequence was found in all normal tissues, whereas an additional alternatively terminated BCHE cDNA clone was found in both tumor libraries. The tumor-specific cDNA contained a 3',0.7-kilobase nontranslatable extension, as well as several nucleotide alterations in the normal polyadenylation site. Single-base mutations in the coding region of this unusual BCHE cDNA infer two amino acid substitutions: Asp70----Gly and Ser425----Pro. The Asp70----Gly change has recently been implicated with "atypical" BCHE, which is deficient in its capacity to hydrolyze succinylcholine. The 3.6-kilobase mRNA was less abundant in RNA blot hybridization than the 2.6-kilobase mRNA, which is in agreement with the low ratios between the 3.6- and 2.6-kilobase BCHE cDNA clones in glioblastoma and neuroblastoma libraries. Furthermore, size fractionation and microinjection of glioblastoma polyadenylated RNA, followed by enzyme activity and selective inhibition measurements, demonstrated two peaks of functional BCHE mRNA, the heavier one probably reflecting the longer transcripts. Chromosomal mapping of the 0.7-kilobase 3' fragment by in situ hybridization localized it to a unique 3q26-ter position, where we recently found an inheritably amplified "silent" defective CHE gene in a family exposed to the cholinesterase inhibitor methyl parathion. Our findings confirm previous genetic linkage mapping of the functional CHE gene to the 3q26-ter position and demonstrate that extended functional mRNA transcripts encoding a BCHE form with two modified amino acids are produced from this gene in glioblastoma and neuroblastoma cells.

Cholinesterases are ubiquitous carboxylesterase type B enzymes capable of hydrolyzing the neurotransmitter acetylcholine which are transiently expressed in multiple germline, embryonic, and tumor cells. The acute poisoning effects of various organophosphorous compounds are generally attributed to their irreversible covalent interaction with cholinesterases and block of their catalytic activities. We have recently found a de novo inheritable amplification of a CHE gene encoding defective butyrylcholinesterase (acylcholine acyl hydrolase; EC 3.1.1.8) in a family under prolonged exposure to the agricultural organophosphorous insecticide methyl parathion. Further analysis revealed that both the CHE and the ACHE genes, encoding acetylcholinesterase (acetylcholine acetyl hydrolase; EC 3.1.1.7), are amplified in leukemias and platelet disorders and that the tumorigenic expression of these genes in ovarian carcinomas is associated with their frequent coamplification in these tumors. The amplification of CHE and ACHE genes in normal and tumor tissues might be analogous to the well-known amplification of other genes encoding target proteins to toxic compounds. As such, it could provide cells a selection advantage when exposed to organophosphorous poisons. Further, since cholinesterases appear to play developmentally important roles in multiple cell types, the amplification and overexpression of their corresponding genes might affect fertility, be related to the progression of various tumor types, and bear upon the ecological and clinical risks involved with the common use of organophosphorous poisons.

The purpose of this study was to determine the effects of subchronic administration of the organophosphate methylparathion (MPTH) during gestation on behavior and development of brain cholinergic neurons in the offspring. Pregnant rats received daily po doses of MPTH from Day 6 through Day 20 of gestation at doses causing no (1.0 mg/kg) or minimal (1.5 mg/kg) visible signs of maternal toxicity. Acetylcholinesterase (AChE) and choline acetyltransferase (CAT) activities, and [3H]quinuclidinyl benzilate (QNB) binding to muscarinic receptors, were determined in several brain regions at 1, 7, 14, 21, and 28 days postnatal age and in maternal brain at Day 19 of gestation. Prenatal exposure to 1.5 mg MPTH/kg reduced AChE and increased CAT activity in all brain regions at each developmental period and in maternal brain. Similar exposure to 1.0 mg MPTH/kg caused a significant but smaller and less persistent reduction in AChE activity but no change in brain CAT activity of the offspring. Both doses of MPTH decreased the Bmax of 3H-QNB binding in maternal frontal cortex but did not alter the postnatal pattern of 3H-QNB binding. In parallel studies, prenatal exposure to MPTH did not affect a variety of behaviors. However, cage emergence, accommodated locomotor activity, and operant behavior in a mixed paradigm were impaired in rats exposed to 1.0 but not to 1.5 mg/kg MPTH. No morphological changes were observed in hippocampal or cerebellar tissue. Thus, subchronic prenatal exposure to MPTH altered postnatal development of cholinergic neurons and caused subtle alterations in selected behaviors of the offspring.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.