Recovery Of Acetylcholinesterase Activity Research Articles

Relation between Serum Cholinesterase and Mortality among Patients with OP Poisoning

Introduction: Serum cholinesterase levels can indicate the prior presence of cholinesterase inhibition even after recovery of acetyl cholinesterase activity by pralidoxime in organophosphorus poisoning. In acute poisoning, manifestations generally occur only after more than 50% of cholinesterase is inhibited. Methodology: 36 patients presenting with history of Organo-phosphorus poisoning and feature of respiratory failure (requiring ventilatory support) were included in the present study. Results: In the present study 27% were died. Conclusion: Higher mortality was seen patients having mild suppression of SCE activity. Key words: Acetylcholinesterase, OP poisoning, Mortality

The recovery of acetylcholinesterase activity and the progression of neuropathological and pathophysiological alterations in the rat basolateral amygdala after soman-induced status epilepticus: Relation to anxiety-like behavior

Organophosphorus nerve agents are powerful neurotoxins that irreversibly inhibit acetylcholinesterase (AChE) activity. One of the consequences of AChE inhibition is the generation of seizures and status epilepticus (SE), which cause brain damage, resulting in long-term neurological and behavioral deficits. Increased anxiety is the most common behavioral abnormality after nerve agent exposure. This is not surprising considering that the amygdala, and the basolateral nucleus of the amygdala (BLA) in particular, plays a central role in anxiety, and this structure suffers severe damage by nerve agent-induced seizures. In the present study, we exposed male rats to the nerve agent soman, at a dose that induce SE, and determined the time course of recovery of AChE activity, along with the progression of neuropathological and pathophysiological alterations in the BLA, during a 30-day period after exposure. Measurements were taken at 24 h, 7 days, 14 days, and 30 days after exposure, and at 14 and 30 days, anxiety-like behavior was also evaluated. We found that more than 90% of AChE is inhibited at the onset of SE, and AChE inhibition remains at this level 24 h later, in the BLA, as well as in the hippocampus, piriform cortex, and prelimbic cortex, which we analyzed for comparison. AChE activity recovered by day 7 in the BLA and day 14 in the other three regions. Significant neuronal loss and neurodegeneration were present in the BLA at 24 h and throughout the 30-day period. There was no significant loss of GABAergic interneurons in the BLA at 24 h post-exposure. However, by day 7, the number of GABAergic interneurons in the BLA was reduced, and at 14 and 30 days after soman, the ratio of GABAergic interneurons to the total number of neurons was lower compared to controls. Anxiety-like behavior in the open-field and the acoustic startle response tests was increased at 14 and 30 days post-exposure. Accompanying pathophysiological alterations in the BLA – studied in in vitro brain slices – included a reduction in the amplitude of field potentials evoked by stimulation of the external capsule, along with prolongation of their time course and an increase in the paired-pulse ratio. Long-term potentiation was impaired at 24 h, 7 days, and 14 days post-exposure. The loss of GABAergic interneurons in the BLA and the decreased interneuron to total number of neurons ratio may be the primary cause of the development of anxiety after nerve agent exposure.

Toxicokinetic and toxicodynamic model for diazinon toxicity—mechanistic explanation of differences in the sensitivity of Daphnia magna and Gammarus pulex

A mechanistic toxicokinetic and toxicodynamic model for acute toxic effects (immobilization, mortality) of the organothiophosphate insecticide diazinon in Daphnia magna is presented. The model was parameterized using measured external and internal (whole-body) concentrations of diazinon, its toxic metabolite diazoxon, and the inactive metabolite 2-isopropyl-6-methyl-4-pyrimidinol, plus acetylcholinesterase (AChE) activity measured during exposure to diazinon in vivo. The toxicokinetic and toxicodynamic model provides a coherent picture from exposure to the resulting toxic effect on an organism level through internally formed metabolites and the effect on a molecular scale. A very fast reaction of diazoxon with AChE (pseudo first-order inhibition rate constant k(i) = 3.3 h(-1)) compared with a slow formation of diazoxon (activation rate constant k(act) = 0.014 h(-1)) was responsible for the high sensitivity of D. magna toward diazinon. Recovery of AChE activity from inhibition was slow and rate-determining (99% recovery within 16 d), compared with a fast elimination of diazinon (99% elimination within 17 h). The obtained model parameters were compared with toxicokinetic and toxicodynamic parameters of Gammarus pulex exposed to diazinon from previous work. This comparison revealed that G. pulex is less sensitive because of a six times faster detoxification of diazinon and diazoxon and an approximately 400 times lower rate for damage accrual. These differences overcompensate the two times faster activation of diazinon to diazoxon in G. pulex compared to D. magna. The present study substantiates theoretical considerations that mechanistically based effect models are helpful to explain sensitivity differences among different aquatic invertebrates.

Acetylcholinesterase based assay of eleven organophosphorus pesticides: finding of assay limitations

The study includes findings about limitations of acetylcholinesterase (AChE) based assay. Eleven organophosphorus pesticides: chlorpyrifos ethyl, chlorpyrifos methyl, DFP, dichlorvos, dimethoate, fenthion, paraoxon ethyl, paraoxon methyl, phosalone, pirimiphos methyl and pirimiphos ethyl were photometrically assayed using AChE as a recognition element. The study was carried out in order to find approachability of AChE based assay. In the first round, common organic solvents were tested for interfering in assay, since samples collection and extraction is a necessary part in samples processing. Isopropanol was found as the most convenient due to minimal inhibition not exceeding 5%. Though all analysed pesticides inhibit AChE in vivo, some of them are toxic after metabolisation. We found AChE based assay approachable for assay of DFP, paraoxons, and dichlorvos. These are oxoforms of organophosphorus pesticides. From thioforms of assayed pesticides, only fenthion was able significantly inhibit AChE in vitro. Electrochemical biosensor with AChE attached on platinum electrode was used for confirmation of interaction pesticide – AChE and complex stability estimation. DFP, paraoxons and dichlorvos were allowed to interact with AChE in biosensor. These pesticides were settled firmly in AChE active site as no spontaneous recovery of AChE activity was observed.

Response and recovery of acetylcholinesterase activity in freshwater shrimp, Paratya australiensis (Decapoda: Atyidae) exposed to selected anti-cholinesterase insecticides

The toxicity of carbaryl, chlorpyrifos, dimethoate and profenofos to the freshwater shrimp, Paratya australiensis was assessed by measuring acetylcholinesterase (AChE) inhibition after 96 h exposures. Shrimp exposed to these pesticides exhibited significant AChE inhibition, with mortality in shrimp corresponding to 70–90% AChE inhibition. The sensitivity of P. australiensis to the four pesticides based on AChE inhibition can be given as chlorpyrifos>profenofos>carbaryl>dimethoate. Recovery of AChE activity was followed in shrimp after 96 h exposures to carbaryl, chlorpyrifos and dimethoate. Recovery after exposure to the carbamate pesticide carbaryl was more rapid than for the two organophosphorus pesticides, chlorpyrifos and dimethoate. The slow recovery of depressed AChE activity may mean that affected organisms in the natural system are unable to sustain physical activities such as searching for food or eluding predators. To investigate the ecological significance of AChE inhibition, chemotaxis behaviour was assessed in shrimp exposed to profenofos for 24 h. Abnormal chemotaxis behaviour in the exposed shrimp was observed at concentrations representing 30–50% AChE inhibition. A clear relationship existed between the depression of AChE activity and observed chemotaxis responses, such as approaching and grasping the chemoattractant source. These results suggest that in vivo toxicity tests based on this specific biomarker are sensitive and present advantages over conventional acute tests based on mortality. Behavioural studies of test organisms conducted in conjunction with measurement of AChE inhibition will provide data to clarify the toxic effects caused by sublethal chemical concentrations of anti-cholinesterase compounds.

(44) An ex vivo approach for the evaluation of reversible inhibitors as potential pretreatments against organophosphate toxicity

Several studies demonstrated that pretreatment with reversible acetylcholinesterase (AChE) inhibitor, such as (pyridostigmine) PYR, improved the survival of animals intoxicated by organophosphate nerve agents (OP). These compounds temporarily inhibited a fraction of the enzyme and protected it from inactivation by nerve agents. An important criterion for effective pretreatment is that it must ensure the recovery of the protected fraction of the enzyme. We thus designed a simple ex vivo method to investigate the recovery of AChE activity, that was protected by PYR, prior to irreversible inhibition by an OP, using a modified Ellman assay. Results show that our approach is suitable for routine use and can successfully predict the potential use of various AChE inhibitors as pretreatment drugs against OP nerve agent intoxication.

The effect of oral pyridostigmine bromide on the recovery of whole blood cholinesterase activity following ex vivo exposure to soman

Background RBC acetylcholinesterase (AChE) and serum butyrylcholinesterase (BChE) inhibition are considered good biomarkers for protection against exposure to nerve agents. These enzymes may be sequestered (reversibly bound) by pyridostigmine bromide (PB) to protect against the lethal, irreversible binding to organophosphates such as soman (GD). Methods Twenty-four human subjects were given either 30 mg PB orally (n=19) or nothing (n=5). Blood samples were obtained pre- and 2.5, 5, 8, and 24 hours post-dosing. The samples were analyzed for AChE and BChE activity and for PB concentration, using validated spectrophotometric and HPLC/MS methods, respectively. Additionally, the recovery of AChE activity over time following ex vivo exposure of the samples to GD was quantified. Results Maximal inhibition of AChE (29.9%) and concentration of PB (17.1 ng/mL) was noted at 2.5h. AChE activity returned to 95% of pre-dose values at 24h. A linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following GD exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Conclusion Preliminary data analysis confirms previous findings that PB is an effective pre-treatment drug against soman nerve agent exposure, and that RBC-AChEI is a marker of protection. Complete data and analysis will be presented. Clinical Pharmacology & Therapeutics (2004) 75, P87–P87; doi: 10.1016/j.clpt.2003.11.332

Acetylcholinesterase activity in copepods ( Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants

From April 1997 to June 1998, 14 measurements of acetylcholinesterase (AChE) enzymatic activity were performed with the copepod, Tigriopus brevicornis, collected at five stations in the Vilaine River estuary (South Brittany, France). Simultaneously, four chemical analyses of triazines and one analysis of total pesticides in water were undertaken. AChE activity levels in T. brevicornis were compared to the levels measured at a reference site not exposed to effluents from Vilaine River. Results reveal significant differences between AChE activity levels depending on location of stations in the plume of the river with an increasing gradient of activity from the upstream to the downstream stations, thus indicating that neurotoxic contaminants are mainly brought by the river. The average degree of AChE inhibition between the reference site and the most upstream site is 70–80% during spring in 1997 and 1998. In May 1997, live copepods from the different sites were brought back and transferred to clean seawater. After 14 days, recovery of AChE activity was almost total when compared to the control. Moreover, using a linear regression model and the atrazine concentration as marker of the presence of pesticides, low levels of AChE activity were significantly explained by atrazine concentration in water.

Recovery of acetylcholinesterase activity in the common carp (Cyprinus carpio L.) after inhibition by organophosphate and carbamate compounds.

Recovery of acetylcholinesterase activity in the common carp (Cyprinus carpio L.) after inhibition by organophosphate and carbamate compounds.

Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature

Chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) is a broad-spectrum organophosphorus (OP) insecticide. Anticipated increases in the already extensive use of this compound have prompted this reassessment of its neurotoxicity. Because chlorpyrifos and other OP insecticides are designed to produce acute cholinergic effects through inhibition of acetylcholinesterase (AChE) and some OP compounds can cause OP compound-induced delayed neurotoxicity (OPIDN) via chemical modification of neurotoxic esterase (neuropathy target esterase, NTE), this review focuses on the capacity of chlorpyrifos to precipitate these and other adverse neurological consequences. Chlorpyrifos exhibits only moderate acute toxicity in many mammalian species, due largely to detoxification of the active metabolite, chlorpyrifos oxon, by A-esterases. Rats given large doses of chlorpyrifos (sc in oil) have prolonged inhibition of brain AChE, possibly due to slow release of the parent compound from a depot. Associated cognitive and motor deficits return to normal well before recovery of AChE activity and muscarinic receptor down-regulation, as expected from classic tolerance. Controlled studies of OP compound exposures in humans also indicate that cognitive dysfunction requires substantial AChE inhibition. Information is relatively sparse on neurological dysfunction that is secondary to theoretical reproductive, developmental, or immunological effects, but the best available data indicate that such effects are unlikely to result from exposures to chlorpyrifos. In accord with the much greater inhibitory potency of chlorpyrifos oxon for AChE than for NTE, clinical reports and experimental studies indicate that OPIDN from acute exposures to chlorpyrifos requires doses well in excess of the LD50, even when followed by repeated doses of the OPIDN potentiator phenylmethanesulfonyl fluoride (PMSF). Likewise, studies in hens show that subchronic exposures at the maximum tolerated daily dose do not result in OPIDN. Although exposure to chlorpyrifos as a result of normal use is unlikely to produce classical OPIDN, a recent report stated that mild reversible sensory neuropathy had occurred in eight patients who had been exposed subchronically to unknown amounts of chlorpyrifos. It is not clear whether these cases represent an incorrect linkage of cause and effect, a newly disclosed reversible sensory component of OPIDN, or an entirely new phenomenon. The question of the potential for chlorpyrifos to cause this mild sensory neuropathy could be resolved by the use of quantitative tests of sensory function in animal experiments and/or prospective studies of humans with known exposures to chlorpyrifos.

Recovery of acetylcholinesterase activity after irreversible inhibition by organophosphorous compounds in embryonic development

1. Recovery of acetylcholinesterase (AChE) activity was studied using the embryos of sea urchins Strongylocentrotus intermedius and S. nudus, embryos of axolotl Ambystoma mexicanum and in the chick embryo muscle culture treated by “irreversible” organophosphorous inhibitors (OPI). 2. ChE activity was assayed by a modified Ellman's procedure. 3. It follows from the data obtained that, unlike the plutei of sea urchins and the monolayer culture of chick embryo muscle cells, the embryos of axolotl show a compensatory increase in AChE biosynthesis after inhibition by OPI. 4. This mechanism is assumed to be related to the presence of a well developed neuromuscular system in the A. mexicanum embryos. 5. It is possible that acetylcholine accumulated as a result of partial AChE inhibition is responsible for the compensatory increase in AChE biosynthesis.0

Comparative toxicity, cholinergic effects, and tissue levels of S, S, S,-tri- n-butyl phosphorotrithioate (DEF) to channel catfish (ictalurus punctatus) and blue crabs (callinectes sapidus)

The acute neurotoxic effects of S, S, S-tri- n-butyl phosphorotrithioate (DEF) on juvenile channel catfish and adult blue crabs was examined using short-term exposures in aerated, static aquaria. The effects on acetylcholinesterase (AChE) activity in catfish brain and skeletal muscle, and in crab ganglia, was studied. In addition, recovery of AChE activity was followed after transferring the animals to DEF-free water. The results indicate that both species exhibit pronounced anticholinesterase effects due to DEF. Recovery of AChE activity is quite slow in nervous tissues of both animals and catfish skeletal muscle, with effects persisting for several weeks following a single acute exposure. Tissue levels of DEF, as well as its rate of disappearance, were followed in several tissues of each species. The highest levels were detected in fish liver, but high levels were also detected in fish brain. Levels in crab ganglia were an order of magnitude lower than those in fish brain, but the anticholinergic effects were similar in both species. Disappearance was biphasic from tissues in both animals, with relatively long half-lives in target tissues.

Recovery of acetylcholinesterase activity after acute organophosphate treatment of CNS reaggregate cultures

The effect of a single dose of diisopropylfluorophosphate on acetylcholinesterase (AChE) activity was examined in mouse CNS reaggregate cultures. At 17–24 days in culture, reaggregates were treated with 0.6 mg/liter DFP for 15 min. The recovery of AChE activity was examined in culture. After treatment, 5.4 ± 1.19% of control AChE activity remained. By 24 hr, 31.6 ± 6.4% of control activity had returned and the recovery of activity was essentially complete by 7 days after treatment. Recovery of AChE activity after DFP treatment required protein synthesis, since there was no recovery in the presence of cycloheximide. After treatment with the reversible inhibitor, physostigmine at 0.5 mg/ml, recovery of AChE activity was complete within 24 hr after treatment. These results indicate that CNS reaggregate cultures not only express differentiated functions of the CNS, but also have the capacity to turn over proteins, and thus may provide a good model system in which to examine mechanisms of toxicity.

Recovery of Acetyicholinesterase Activity after Acute Organophosphate Treatment of CNS Reaggregate Cultures

The effect of a single dose of diisopropylfluorophosphate on acetylcholinesterase (AChE) activity was examined in mouse CNS reaggregate cultures. At 17–24 days in culture, reaggregates were treated with 0.6 mg/liter DFP for 15 min. The recovery of AChE activity was examined in culture. After treatment, 5.4 ± 1.19% of control AChE activity remained. By 24 hr, 31.6 ± 6.4% of control activity had returned and the recovery of activity was essentially complete by 7 days after treatment. Recovery of AChE activity after DFP treatment required protein synthesis, since there was no recovery in the presence of cycloheximide. After treatment with the reversible inhibitor, physostigmine at 0.5 mg/ml, recovery of AChE activity was complete within 24 hr after treatment. These results indicate that CNS reaggregate cultures not only express differentiated functions of the CNS, but also have the capacity to turn over proteins, and thus may provide a good model system in which to examine mechanisms of toxicity.

Degeneration and regeneration of neuromuscular junctions in chicken iris muscle after crush of the ciliary nerves: A study of ultrastructural changes and of cholinergic enzymes

Degeneration of neuromuscular junctions in the iris muscle was observed to have occurred 4 days after ciliary nerve crush. By day 10 reinnervation had commenced and by day 30 the maturation of neuromuscular junctions was nearly complete. The loss and recovery of acetylcholinesterase activity in the iris muscle paralleled the denervation reinnervation process, with recovery being completed by day 30, whereas the loss in the activity of choline acctyltransferase had not yet completely recovered at this time. The acetylcholinesterase activity localized cytochcmically at synaptic sites followed the same trend as the total activity in the iris muscle, whereas acetylcholinesterase localizcd at myo-muscular junctions showed only slight changes. The acetylcholinesterase molecular forms displayed changes in their relative proportions, which could be related to the time course of the denervation-reinnervation process and to the cytochemical localization of the activity.

Recovery of acetylcholinesterase activity from fenitrothion-induced inhibition in the freshwater field crab (Oziotelphusa senex senex).

Recovery of acetylcholinesterase activity from fenitrothion-induced inhibition in the freshwater field crab (Oziotelphusa senex senex).

Analysis of acetylcholinesterase synthesis and transport in the rat hippocampus: recovery of acetylcholinesterase activity in the septum and hippocampus after administration of diisopropylfluorophosphate

The site and rate of synthesis, as well as the transport dynamics, of newly synthesized acetylcholinesterase (AChE) has been studied in the septum and hippocampus of the rat. Histochemical and biochemical techniques were employed to study time-dependent changes in AChE activity of the medial septal nucleus and hippocampus following systemic administration of the anticholinesterase diisopropyl-fluorophosphate. In both septum and hippocampus, an initially rapid phase of recovery was followed by a slower recovery with similar rate constants for the two regions. Analysis of AChE synthesis in the septo-hippocampal system revealed that recovery of activity in the septum(t 1/2for recovery= 140h) preceded that in the hippocampus(t 1/2for recovery= 400h). The data suggested that the bulk of septal AChE in the hippocampus is transported via the slow component of axoplasmic flow (about 2 mm/day). A fast component may exist, but the experimental arrangement did not permit the clear demonstration of any rapid axoplasmic flow.

A specific antidote against lethal alkyl phosphate intoxication. IV. Effects in brain

The recovery of acetylcholinesterase activity in brain following injection of alkyl phosphates (DFP, paraoxon, and OMPA) has been studied without and in combination with PAM. Whole brains of mice were used throughout the experiments. 1. For the evaluation of the enzyme activity in the intact cell during lifetime, a procedure has been used in which the tissue has been extracted with chloroform. The values obtained with this method were much higher during the first 24-hr. period than in homogenized tissue without chloroform extraction. This result indicates that considerable amounts of alkyl phosphate must be present, during the 24-hr. period following injection, in the extracellular fluid and in lipide, unable to reach the intracellular esterase. After that period of time no measurable difference was found between the two techniques; apparently all the extracellular alkyl phosphate had been removed. The new method proved to be greatly superior compared to those previously used for evaluating intracellular enzyme activity during life. 2. The esterase activity after LD 50 of paraoxon is about 25–30% of the initial, whether the animal dies or survives. The activity after injection of DFP is much lower than that after injection of paraoxon, about 5% of the initial. The rate of recovery is slow and not markedly different whether DFP or paraoxon is used. 3. After injection of 35 mg./kg. of OMPA, which is close to a sure lethal dose, inhibition of only 10% was found. 4. In contrast to the marked rise of enzyme activity in diaphragm following the administration of PAM, no effect of this compound is detectable on the enzyme activity in brain with amounts which protect the animal against death. Only by repeated injections of PAM, a 25 % increase of enzyme activity was found following injection of paraoxon; when DFP was used no clear-cut reactivation by PAM was observed, even after repeated injections. These results indicate that PAM in concentrations adequate to preserve life does not readily penetrate into the brain. Since, however, whole brains were used for the determinations, the experiments do not exclude that in some parts of the brain PAM restored enzyme activity important for survival. 5. The availability of PAM makes it possible to apply much higher amounts of DFP or paraoxon compatible with life than ever used before. Therefore, the minimum enzyme activity in brain compatible with life has been determined with extreme doses. Repeated injections of LD 50 of either DFP or paraoxon did not seem to influence markedly the concentration as compared with the first injection. However, after a single very high dose of DFP (50 mg./kg., i.e., about 12-fold LD 50 ) against which PAM in combination with atropine is still able to protect, the hydrolytic activity was found to be 2.4 mg./g./hr. or about 2% of the initial. This value was found at the peak of inhibition. Two hours later, the value was about doubled. The significance of these findings is discussed.