2-pyridine Aldoxime Methiodide Research Articles

Antidotal action of atropine sulfate against insecticide benfuracarb poisoning in rats.

The antidotal action of atropine sulfate and 2-pyridine aldoxime methiodide (2-PAM) against poisoning attributable to the new procarbamate insecticide benfuracarb [(ethyl N-[2,3-dihydro-2,2-dimethylbenzofuran-7-yloxycarbonyl (methyl) aminothio]-N-isopropyl-beta-alaninate] was compared utilizing rats as our experimental model. Both the intraperitoneal and oral administrations of these antidotes were examined after five, ten, fifteen and thirty minutes exposure periods, following treatment with benfuracarb at dose levels approximating LD50 and LD100. The results obtained demonstrate that both the intraperitoneal and oral administrations of atropine sulfate blocked or significantly reduced the toxic effects of benfuracarb and protected the animals from death. The intraperitoneal administration route appears to be more effective than was the oral route. In addition, the administration of atropine sulfate after the shorter period (up to 15 minutes), following exposure to benfuracarb, improved antidotal action, particularly with the LD100 dose of benfuracarb. It is suggested that atropine sulfate antagonizes benfuracarb poisoning by blocking acetylcholine (ACh) receptors, as many other carbamate insecticides, since benfuracarb was an in vivo cholinesterase (ChE) inhibitor and the toxic effect of benfuracarb was reduced by atropine sulfate. 2-PAM, however, did not significantly block or reduce the toxic effects of benfuracarb.

Acetylcholinesterase in organic solvents for the detection of pesticides: Biosensor application

Acetylcholinesterase (AChE) from electric eel, in a free or immobilized state, can be used for the detection of insecticides. This system is convenient because of the selectivity and specificity of the inhibition of AChE by organophosphorus and carbamate insecticides. However, these pesticides are highly soluble only in organic solvents. This article deals with the determination of the activity of AChE in organic solvents. Firstly, the effect of different organic solvents on activity was determined using the free enzyme. The AChE activity was maintained using the free enzyme. The AChE activity was maintained depending on the nature of the solvent. The results were applied to the biosensing system and a new method for the detection of organophosphorus and carbamate insecticides was developed. A correlation between the AChE activity and a physico-chemical parameter, was found in order to predict the effect of the solvent on enzyme activity. Upon comparison of the correlation curve obtained with free and immobilized enzyme, it appeared that immobilization enhanced the stability of the enzyme and increased the number of usable organic solvents. The inhibition of AChE by organophosphorus and carbamate insecticides was tested in organic solvents and the limit of detection determined. The inhibitory capacity of AChE was maintained in most organic solvents. The reactivation of immobilized enzyme with 2-pyridine aldoxime methiodide (2-PAM) allowed the repeated use of the same enzyme electrode. The application of the biosensor for the detection of organophosphorus and carbamate insecticides in organic solvents using chemical knowledge will be useful for the detection of pesticide residues present in water and food at very low levels.

Field results using cholinesterase reactivation techniques to diagnose acute anticholinesterase poisoning in birds and fish

The inhibition of brain cholinesterase (ChE) activity in organophosphate (OP)- or carbamate (CB)-poisoned animals is usually determined by comparison with the normal activity in control specimens. Alternatively, the activity of pesticide-inhibited ChE can be restored, ideally up to normal levels, using simple in vitro procedures, thereby providing a reference value against which ChE inhibition can be estimated. The activity of phosphorylated ChE can be increased by the addition of the nucleophilic reagent pyridine 2-aldoxime methiodide (2-PAM), while the spontaneous reactivation of carbamylated ChE can be enhanced by dilution. These procedures can also be used to differentially diagnose OP and CB poisoning. Brain ChE inhibition in birds and fish from field poisoning incidents was determined using reactivation techniques and normal activity data from separate control specimens. In OP-poisoned birds, the mean inhibition relative to 2-PAM-treated samples and controls ranged from 77–82% and 84–87%, respectively. The corresponding values for OP-poisoned fish were 48–82% and 66–86%, respectively. In CB-poisoned birds, the mean inhibition relative to diluted samples and controls ranged from 22–56% and 33–76%, respectively. The reactivation of carbamylated ChE was further enhanced by increasing the dilution ratio and lengthening the incubation period. Although ChE activity was not fully restored to normal levels in many individuals, diagnostically significant inhibition was demonstrated in most cases. In OP-poisoned fish held 24 h postmortem at 20°C, 2-PAM restored brain ChE activity to that of similarly handled, unexposed controls. After postmortem storage for 48 h, ChE activities in reactivated and inhibited samples were statistically indistinguishable due to the aging of the phosphorylated ChE. Despite the limitations imposed by aging, ChE reactivation is a potentially useful diagnostic technique, particularly when control data are unavailable or difficult to obtain.

A study on acute organophosphorus poisoning--changes in the activity and isoenzyme patterns of serum cholinesterase in human poisoning

Various organophosphorus compounds with low acute toxicity levels are widely used as insecticides. Human acute poisoning by organophosphates has often occurred accidentally. We determined the activity and isoenzyme patterns of serum cholinesterase (ChE) obtained from 13 human patients who attempted suicide with various organophosphates, i.e. Fenitrothion, Malathion, Isoxathion, Pyridaphenthion and Trichlorfon, and studied on the changes in the activity and isoenzyme patterns of serum ChE after ingestion. The following results were obtained. 1) Twenty ChE isoenzyme bands from normal human serum were detected by electrophoretic separation on polyacrylamide gradient gel. The main bands in the ChE isoenzyme pattern in normal serum were bands 4 and 5 which had the highest activity of acetylcholinesterase (AChE) with a molecular weight of 600,000-800,000, and bands 7, 12, 14, 17 and 18. 2) Inhibition of serum ChE activity was more severe as the amount ingested increased in patients who took Fenitrothion and Malathion. Reactivation of serum ChE activity was very slow in patients treated with PAM (2-pyridine aldoxime methiodide) in the late stage of ingestion or whose symptoms reappeared. 3) There were no differences in the patterns of serum ChE isoenzyme by organophosphorus compound. Band 7 disappeared in the serum ChE isoenzyme of almost every patient, and bands 12, 18, 14 and 17 of the serum ChE isoenzyme disappeared successively with the decline of serum ChE activity. Only band 5 of the isoenzyme remained in cases who had serum ChE activity lower than 5% of normal. 4) All 13 patients were treated with PAM and atropine immediately after being admitted to hospitals. We could not clearly determine the efficacy of PAM on reactivation of serum ChE activity and isoenzyme, because it was impossible in human poisoning to compare PAM efficacy with no treatment and with pre- and post-PAM treatment. 5) The activity and isoenzyme patterns of serum ChE recovered rapidly after combined hemoperfusion and hemodialysis treatment (HP-HD treatment) of the patients poisoned with Malathion. But HP-HD treatment had no effect on poisoning by Fenitrothion and Isoxathion. These findings demonstrated the changes in the activity and isoenzyme pattern of serum ChE in patients poisoned with several organophosphates after PAM and HP-HD treatment.

電界効果型トランジスタを用いたバイオ化学センサによる農薬類の計測

Acetylcholine sensor based on ISFET as a pH sensor, using the immobilized acetylcholine esterase (AChE) in the BSA membrane are reported for the characteristics and for measuring chemicals. We attempted the application of an enzyme sensor based on ISFET as an effective and simple potentiometric sensor for the detection of chemicals, as irreversible and reversible inhibitors. New data on the analysis of chemicals are reported using the present acetylcholine sensor based on the inhibition of the immobilized AChE. Reactivation of immobilized AChE after inhibition with reversible and irreversible inhibitors, was carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection for PHC and Nicotine as reversible inhibitors, and Malathion and Dimethoate as irreversible inhibitors was found to be in the ranges of 5×10-5-5×10-4 M, 10-5-10-4 M, 10-6-10-4 and 2.5×10-5-10-3 M, respectively.The present study was also developed for an electrochemical sensor for toxic substances which works by measuring the inhibition of the enzyme activity. We suggested that these inhibition rates (e.g., I50) by the biosensor may be important as a biological index to biological systems with regard to toxic matters.

Studies on the therapeutic effect of 2-pyridine aldoxime methiodide (2-PAM) in mammals following organophosphorus compound (OP)-poisoning (report II): aging of OP-inhibited mammalian cholinesterase.

We studied the ability of 2-PAM to reactivate cholinesterase (ChE) inhibited by organophosphorus compounds (OPs) and aging. We estimated the reactivation rate with 2-PAM following inhibition of ChE by fenitrothion, methylparathion or ethylparathion using erythrocytes of rat and rabbit and rat brain. The period of time during which inhibited ChE could be reactivated was shorter in the case of inhibition by fenitrothion or methylparathion than in the case of inhibition by ethylparathion. This results suggest that aging is related to the presence of the alkyl group in OPs, and occurs faster in the case of inhibition by OPs with an O,O-dimethyl moiety than in the case of inhibition by OPs with an O,O-diethyl moiety.

Studies on the therapeutic effect of 2-pyridine aldoxime methiodide (2-PAM) in mammals following organophosphorus compound-poisoning (report III): distribution and antidotal effect of 2-PAM in rats.

The metabolic fate of 2-PAM and its antidotal effect on organophosphorus compound poisoning in rats were studied. When 14C-2-PAM was administered intravenously, the amount of 14C reaching the brain was small. Following administration by intramedullary injection, 14C was present in high concentrations in the brain, and 72-90% of the 14C present in the brain corresponded to the unchanged form of 2-PAM. 2-PAM was rapidly excreted into the urine and feces following either intramedullary or intravenous administration. The half-life of 2-PAM in the brain following intramedullary administration was 1.52 hr. Intramedullary administration of 2-PAM to rats poisoned with fenitrothion or malathion enabled their survival and induced reactivation of brain cholinesterase.

Inhibition of avian esterases by organophosphorus insecticides: Problems of reactivation and storage

Brain and serum esterase activities were measured in starlings (Sturnus vulgaris) dosed with corn oil, demeton-S-methyl (S-[2-(ethyl thio)ethyl]O,O-dimethyl phosphorothioate), chlorpyrifos (O,O-diethylO-(3,5,6-trichloro-2-pyridinyl) phosphorothioate) or triazophos (O,O-diethylO-(1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate). Activity was assayed before and after (1) storage at 20°C and (2) chemical reactivation by pyridine-2-aldoxime methiodide (P2AM). During storage, significant spontaneous reactivation occurred in samples from birds dosed with chlorpyrifos or triazophos but not in the birds dosed with demeton-S-methyl or corn oil. The degree of chemical reactivation of samples was dependent on the compound administered, the type of esterase and the time after dosing. Brain acetylcholinesterase appeared to be more readily reactivated than serum esterases by P2AM. Following dosing with the diethyl organophosphorus compounds, spontaneous or chemical reactivation sometimes resulted in levels of esterase activity above those measured before dosing. This indicates that release and/orde novo synthesis had occurred. It is important to take into account spontaneous reactivation during storage when assessing the inhibition of esterase activity.

Aspartate-70 to glycine substitution confers resistance to naturally occurring and synthetic anionic-site ligands on inovo produced human butyrylcholinesterase

The "atypical" allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nucleotide substitution conferring a change of aspartate-70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the "atypical" BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine-425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of the resultant recombinant BuChE variants. The presence of the Gly-70 mutation alone was found to render the enzyme resistant to 100 microM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the "normal," Asp-70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly-70 BuChE failed to be reactivated by 10 mM of the cholinesterase-specific oxime pyridine 2-aldoxime methiodide (2-PAM); a concentration restoring about 50% of activity in the "normal" Asp-70 recombinant enzyme. The Pro-425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly-70 mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dichlorvos on blood cholinesterase activities of cattle

SUMMARY Inhibitory effects of dichlorvos (2,2-dichlorovinyl dimethyl phosphate, ddvp) inhibitory effects on erythrocyte acetylcholinesterase (AChE) and plasma cholinesterase (ChE) activities of steers were characterized after treatments in vitro and in vivo (cutaneous application). The rates of in vitro inhibition were markedly influenced by ddvp concentration and incubation time. The activities of inhibited enzymes failed to reactivate spontaneously and had little response to treatment with 2-pyridine aldoxime methiodide (2-pam). After gel-filtration chromatography, however, the inhibited enzymes had remarkable spontaneous reactivation and reactivation by 2-PAM treatment, indicating interference of excess unreacted ddvp in the reactivation process. Repeated cutaneous applications of a ddvp-containing livestock spray caused marked and characteristic decreases of AChE and ChE activities in blood of treated steers; however, the effects were transient because activities of both enzymes regenerated gradually. The nature of these in vivo trends suggests that spontaneous and de novo synthetic mechanisms could be responsible for complete recovery of both enzyme activities.

Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase

Acetylcholine esterase electrodes, based on glass, Pd/PdO and Ir/IrO 2 electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some organophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine esterase. Reactivation of immobilized AChE after inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working buffer and acetylcholine, whereas reactivation after inhibition with irreversible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be 10 −5M whereas for paraoxon, methyl parathion and malathion are found to be 10 −9 M and 10 −10 M.

Disposition kinetics and urinary excretion of 2-pyridine aldoxime methiodide in sheep.

Disposition kinetics and urinary excretion of 2-pyridine aldoxime methiodide in sheep.

Effect of fixatives on rat brain acetylcholinesterase activity

Biochemical measurement of acetylcholinesterase (AChE) activity on sonicates of samples of rat cortex, striatum and cerebellum which had been treated with either 4% formaldehyde (HCHO) or 4% HCHO/0.5% glutaraldehyde suggest that fixation of tissue causes a rather large loss (50–80%) in AChE activity which may vary considerably from region to region of brain. Attempts to protect the activity by treatment with diisopropyl fluorophosphate (DFP) before fixation and reactivation after fixation with pyridine-2-aldoxime methiodide (PAM) were unsuccessful. The results suggest care must be exercised in attempting to use AChE histochemistry on fixed tissue for quantitative measurements.

Effects of organophosphorus compounds and PAM on cholinesterase activity in rat tissues

Various organophosphorus compounds are widely used in the world. Each organophosphorus compound has some differences in toxic symptoms, inhibition of ChE (cholinesterase) and the efficacy of PAM (2-pyridine aldoxime methiodide). This paper reports on the effects of three organophosphorus compounds (1/2 LD50, p. o.) and PAM on ChE activity in rat tissues including the relation to toxic signs. The results were as follows:1. Toxic signs appeared in the respiratory function of rats immediately after administration of Dichlorvos, and these signs disappeared rapidly. But in the case of rats administered Fenitrothion or Pyridaphenthion, serious toxic signs such as salivation, dyspnoea, lacrimation and tremors caused decreased body weight and rise of hematcrit value, and these signs continued for several days.2. ChE activity in rat tissues administered organophosphorus compounds spontaneously recovered further after the toxic signs disappeared. But there was a clear relation between the inhibition levels of tissue ChE and the appearance of toxic signs in rats administered three organophosphorus compounds. With special reference to the correlation between salivation and the inhibition level of ChE activity in salivary glands, serious salivary secretion was observed in rats where ChE activity in the glands was inhibited severely by administration of Fenitrothion or Pyridaphenthion. It was clear that salivary secretion was induced by organophosphorus compounds when the activity of ChE in salivary glands was inhibited by more than 50% of normal activity.3. The half-time (r1/2) for spontaneous recovery of tissues' ChE activity in rats following poisoning with organophosphorus compounds was different by tissues. But the r1/2 for recovery of serum and liver ChE activity was the same as the approximate 20h for three organophosphorus compounds, and those were shorter than other tissues' ChE. The r1/2 for recovery of erythrocytes ChE was longest in tissues, particularly those of rats treated with Fenitrothion was as long as 410h.4. As expected, atropine was unable entirely to reactivate tissue ChE activity inhibited by three organophosphorus compounds. The antidote PAM was capable of reactivating ChE activity in only serum and erythrocytes of rats administered Pyridaphenthion, but it couldn't reactivate ChE activity in brain, liver, kidneys, lung and salivary glands for pyridaphenthion, nor ChE activity in any tissues for Dichlorvos and Fenitrothion.Therefore, it was clear that each ChE in rat tissues has many differences in the speed of spontaneous recovery and the reactivating effect of PAM following poisoning with organophosphorus compounds. The qualitative differences of ChE by each tissue will be made clear in the future by a study on ChE isoenzyme of rat tissues.

Acute malathion toxicosis and related enzymatic alterations in Bubalus bubalis: antidotal treatment with atropine, 2-PAM, and diazepam.

Oral administration of malathion (MTH) in sublethal (100 mg/kg) or minimal lethal (125 mg/kg) doses in buffalo calves produced toxicity with an onset within 15-20 min and peak effects including severe tremors and convulsions within 40-60 min. Various antidotal drugs were administered alone or in combination at the time of peak malathion toxicity (within 1 h) and were assessed for their ability to alleviate signs of cholinergic toxicity. Blood cholinesterase and aminotransferases activities were monitored at various times. A combination of atropine sulfate (0.5 mg/kg, 1/4 iv and 3/4 im) and pyridine 2-aldoxime methiodide (2-PAM, 20 mg/kg, iv) reversed the clinical evidence of malathion toxicity within 15 min. The combination of atropine sulfate and diazepam (0.75 mg/kg, iv) prevented death and cholinergic signs of toxicity except for weak muscular fasciculations, which persisted for 30-60 min. Atropine sulfate alone was less effective and also did not reverse malathion-induced biochemical changes. In contrast, administration of either 2-PAM (10-30 mg/kg, iv) or diazepam (0.5-1.0 mg/kg, iv) alone accentuated malathion toxicity. Thus, the combination of atropine sulfate and 2-PAM was the most effective antidotal treatment in acute malathion toxicity.

Toxicological properties of O,O,S-trialkyl phosphorothioates.

The effect of atropine, 2-pyridine aldoxime methiodide (2-PAM), and several O,O,O-trialkylphosphorothioates on poisoning of rats by a series of O,O-dimethyl and O,O-diethyl S-alkyl phosphorothioates was investigated. Atropine and 2-PAM successfully protected rats treated with O,O-diethyl S-n-propyl and S-i-propyl phosphorothioates, while the O,O,O-trialkyl phosphorothioates were effective in protecting rats treated with O,O-dimethyl S-methyl and S-ethyl phosphorothioates. O,O-Dimethyl and O,O-diethyl S-i-propyl phosphorothioates also were examined for in vitro and in vivo inhibition of rat plasma, red blood cell, and brain cholinesterase. Overall, the results indicated that two different mechanisms, cholinergic and noncholinergic, are involved in intoxication by the O,O,S-trialkyl phosphorothioates.

Possible antidotes in severe intoxication with fenitrothion in rats.

We investigated antidotal effects of nine drugs against acute fenitrothion poisoning in rats. The antidotal effects were evaluated by a relief from toxic signs, an increased survival ratio and a prolonged surviving time. In the poisoning with approximate LD50 fenitrothion (500 mg/kg), oximes such as 2-PAM (2-pyridine aldoxime methiodide) and TPMM (1-(methyl morphorinium)-3-(4-hydroxyiminomethylpyridinium) propane dibromide), diphenhydramine and parasympatholytics such as atropine, scopolamine and biperiden significantly increased survival ratio and/or prolonged surviving time. These effective drugs did not sufficiently depress the toxic signs, except that the parasympatholytics markedly blocked salivation, miosis and motor ataxia. In contrast, reduced glutathione and central depressants such as diazepam and phenobarbital made the poisoning serious. In the poisoning with 100% lethal dose of fenitrothion (800 mg/kg), the parasympatholytics were more effective than 2-PAM and diphenhydramine, and they elevated the survival ratio up to 20-40%. The best therapy against the severe poisoning with 100% lethal dose of fenitrothion was confirmed to be the repeated and combined treatment with atropine and 2-PAM as established in parathion poisoning, resulting in 90% survival ratio and considerable alleviation from the toxic signs.

Effect of organophosphate pesticides on the activities of lecithin-cholesterol acyltransferase and cholinesterase in rat serum.

The lecithin-cholesterol acyltransferase activity in rat serum as well as that in human plasma was examined by using sonicated dispersions of lecithin and cholesterol mixtures as substrates. When dispersions with molar ratio of 1.2 and 5.9 were used as substrates, the acyltransferase activity in rat serum, in contrast to that in human plasma, was greater with the substrate dispersion having a molar ratio of 1.2 than with that of 5.9. In addition, the acyltransferase activity in rat serum with the dispersion having a molar ratio of 0.9 was inhibited upon addition of increasing amounts of lecithin, but that in human plasma for the same dispersion was stimulated and the maximum acyltransferase activity was obtained at a molar ratio of approximately 7. On the basis of these results, the inhibitory actions of organophosphate pesticides on the acyltransferase and cholinesterase in rat serum were investigated. The acyltransferase activity in rat serum was inhibited by the addition of 1×10-3M dimethyldichlorovinylphosphate (DDVP) and methylparathion. The decreased acyltransferase activity in rat serum was not restored by the addition of 1×10-3M 2-pyridine aldoxime methiodide (PAM). Similarly, cholinesterase activity in rat serum was almost completely inhibited by the addition of 1×10-5M DDVP. However, the decreased cholinesterase activity was restored by the addition of 1×10-3M PAM. In addition, the acyltransferase and cholinesterase activities in serum obtained from rats administered DDVP (35 mg/kg body) in vivo were inhibited to approximately 83-87% and 35-37% of those in control rat serum, respectively.

Conjugation of triaziquinone to immunoglobulin G by a thiolation procedure catalyzed by 2-pyridinealdoxime methiodide

Thiolation of immunoglobulin G (IgG) with DL-N-acetylhomocycteinethiolactone, catalyzed by 2-pyridinealdoxime methiodide, incorporated new sulfur groups into IgG. Triaziquinone was subsequently conjugated to the sulfur groups. Triaziquinone-IgG complex retained the alkylating activity of the drug and the immunological activity of the antibody. The conjugation procedure was inhibited by the thiol-blocking agent methyl methanethiolsulfonate.

Calorimetric Determination of Phosphorylated Serum Cholinesterase and Its Reactivation by an Antidote

Abstract Serum cholinesterase, inhibited in vitro up to 92% by 0, 0 dimethyl-2, 2-dichlorovinyl phosphate (4.52 × 10−9 mole/ml of 40% serum), was reactivated up to 35% with 2-pyridine aldoxime methiodide (3.5 × 10−6 mole/ml of 40% serum). Evaluation was based on the reaction rate between cholinesterase and acetylcholine chloride (2.2 × 10−6 mole) measured with a microcalorimeter. It was thus possible to approximate the “normal” or pre-exposure level of serum cholinesterase, while simultaneously determining the extent of poisoning.