Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance

Liquid and gas chromatography are commonly used to measure organophosphorus pesticides. However, these methods are relatively time consuming and require a tedious sample pretreatment. Here, we applied the localized surface plasmon resonance (LSPR) of gold nanoparticles covalently coupled with acetylcholinesterase (AChE) to create a biosensor for detecting an example of serial signals responding to paraoxon in the range of 1-100 ppb by an AChE modified LSPR sensor immersing in a 0.05 mM ACh solution. The underlying mechanism is that paraoxon prevents acetylcholine chloride (ACh) reacting with AChE by destroying the OH bond of serine in AChE. We found that the AChE modified LSPR sensors prepared by incubation with 12.5 mU/mL of AChE in phosphate buffer solution at pH 8.5 room temperature for 14 h have the best linear inhibition response with a 0.234 ppb limit of paraoxon detection. A 14% of inhibition on the sensor corresponds to the change of paraoxon concentration from 1 to 100 ppb. The sensor remained 94% of its original activity after six cycles of inhibition with 500 ppb paraoxon followed with reactivation of AChE by 0.5 mM 2-pyriding-aldoxime methoiodide (2-PAM). In addition, the sensor retains activity and gives reproducible results after storage in dry state at 4 degrees C for 60 days. In conclusion, we demonstrated that the AChE modified LSPR sensors can be used to determine the concentration of paraoxon biosensor with high sensitive and stable characteristics.     

The effect of high and low dosages of paraoxon in β-naphthoflavone-treated rats

Aliesterases (carboxylesterases) are serine esterases that can serve a protective role for the target acetylcholinesterase (AChE) during organophosphorus insecticide intoxication because the former esterases are alternate phosphorylation sites. The levels of aliesterase activity in liver and plasma and AChE activity in brain regions were investigated after the intravenous administration of paraoxon (P = O) into female rats. The rats were pretreated intraperitoneally with β-naphthoflavone (BNF), which decreases hepatic aliesterase activity following a 3 day in vivo treatment, and/or tri-o-totyl phosphate (TOTP) to inhibit aliesterases. The liver aliesterases were inhibited less by P = O in BNF-treated rats than in control rats, which suggests that either BNF exposure may have resulted in aliesterases that are less sensitive to P = O inhibition or BNF may have altered P = O's availability. The BNF treatment did not seem to alter the degree of inhibition of the brain AChE activity following the low dosage of paraoxon (0.04 mg/kg). However, the brain AChE activity in the P = O/TOTP/BNF-treated rats was lower than that in the P = O/TOTP-treated rats, suggesting that BNF also caused changes in systems affecting the disposition of P = O in addition to the changes in the hepatic aliesterases. At the high dosage of paraoxon (0.12 mg/kg), the AChE and aliesterase activities showed a pattern similar to that of the low dosage. This suggests that the aliesterases, as altered by BNF exposure, even when nearly completely inhibited, did not alter the response of the target enzyme, AChE, and, therefore, the magnitude of the toxic response. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 263–268, 1997.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.     

Evidence for a noncholinergic function of acetylcholinesterase during development of chicken retina as shown by fasciculin

Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE.     

Effect of preceding physostigmine administration on neuromuscular transmission disturbances in rats caused by dichlorphos and phospholine

The reported experiments were carried out on male Wistar rats. Under general anaesthesia with chloral hydrate in situ physiological preparations were made consisting of sciatic nerve and anterior tibialis muscle. Physostigmine was injected subcutaneously and was followed after from 15 to 60 minutes by intravenous injection of DDVP or phospholine. Physostigmine effect on the blockade of tetanic muscular contractions was studied after administration of these organophosphate inhibitors of acetylcholinesterase. At the same time, the effect of these substances on acetylcholinesterase was determined in the skeletal muscle. It was found that physostigmine in a dose of 125 microgram/kg protected the tibialis muscle against the development of blockade of tetanic response. The protective effect was greatest when the time between physostogmine injection and the subsequent administration of these organophosphate inhibitors was 30--40 minutes. In the same observation period the activity of AChE in the tibialis muscle of rats was found to be highest after physostigmine injection before administration of DDVP or phospholine.     

Comparison Between the Effects of Botulinum Toxin-Induced Paralysis and Denervation on Molecular Forms of Acetylcholinesterase in Muscles

Abstract: Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms in the fast extensor digitorum longus muscle and in the slow soleus muscle of the rat was carried out on days 4, 8, and 14 after induction of muscle paralysis by botulinum toxin type A (BoTx). The results were compared with those observed after muscle denervation. In addition, the ability of BoTx-paralyzed muscles to resynthesize AChE was studied after irreversible inhibition of the preexistent enzyme by diisopropyl phosphorofluoridate. Major differences were observed between the effects of BoTx treatment and nerve section on AChE in the junctional region of the muscles. A precipitous drop in content of the asymmetric A12 AChE form was observed after denervation, whereas its decrease was much slower and less extensive in the BoTx-paralyzed muscles. Recovery of junctional AChE and of its A12 form after irreversible inhibition of the preexistent AChE in BoTx-paralyzed muscles was nevertheless very slow. It seems that a greater part of the junctional A12 AChE form pertains to a fraction with a very slow turnover that is rapidly degraded after denervation but not after BoTx-produced muscle paralysis. The postdenervation decrease in content of junctional A12 AChE is therefore not primarily due to muscle inactivity. The extrajunctional molecular forms of AChE seem to be regulated mostly by muscle activity because they undergo virtually identical changes both after denervation and BoTx paralysis. The differences observed in this respect between the fast and slow muscles after their inactivation must be intrinsic to muscles.     

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions

When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.     

Histopathology, histochemistry and acetylcholinesterase activity after repetitive administration of fluostigmine to albino rat

Histopathological, histochemical and biochemical investigations were performed on the brain, sciatic nerve, skeletal muscle, heart, liver and kidney of rats which were given 5% of LD50 dose of DFP for 10 days. A decrease in AChE activity, degeneration of neurons and necrotic changes in the nuclei of hypothalamus, degeneration of myelin sheaths in sciatic nerve, a decrease in succinic dehydrogenase activity in the myocardium, and a minimal decrease of acid phosphatase activity (AcPh) in the liver were found. The biochemical determination of AChE level indicated about 30% AChE activity in erythrocytes and tibialis muscle, and 40% in the brain 1 hr after the last dose of the inhibitor and 80% and 50% respectively on the 7th day after poisoning in relation to normal values.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

Recovery of the acetylcholinesterase activity in a monolayer culture of chick myoblasts after irreversible inhibition by an organophosphorus inhibitor

The recovery of the acetylcholine esterase (AChE) activity after the irreversible inhibition with an organophosphorus inhibitor B-156 was studied in a developing monolayer culture of chick myoblasts. The culture was obtained from muscles of posterior limbs of the 11 day old chick embryos. The AChE activity was estimated by the modified Ellman method from the moment of inoculation to the stage of spontaneous contractions of muscle fibres. After the B-156 treatment the AChE activity of muscle cells decreased, then started to increase and the maximum recovery of activity, below the initial level, was attained within roughly 2 days after the treatment. The AChE activity in the treated culture somewhat decreased thereafter. The lower the inhibitor concentration, i.e. the lower the value of the initial AChE inhibition, the higher the starting rate and degree of recovery of the AChE activity. The results obtained suggest that, unlike the multilayer culture of muscle tissue at later stages of differentiation no compensatory enhancement of AChE biosynthesis after irreversible inhibition of this enzyme by an organophosphorus inhibitor is observed in the monolayer culture of chick myoblasts at the early stages of myogenesis.     

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.     

Intracellular marking of skeletal muscle cells with horseradish peroxidase in combination with a stain for cholinesterase

A procedure is described by which muscle fibers can be electrophysiologically studied, intracellulary marked with horseradish peroxidase (HRP), and stained for acetylcholinesterase (AChE) activity to identify nerve endings. Following electrophysiological characterization, 4% HRP is injected through the recording microelectrode with small depolarizing pulses. Muscles are fixed, washed, and stained with a Koelle procedure for AChE modified by extending the incubation period to 15 hours to identify small nerve endings. The HRP is visualized with 3,3'-diaminobenzidine tetrahydrochloride or benzidine dihydrochloride. Stained muscles are embedded in Epon and sectioned. Lucifer yellow dye is also demonstrated to be an excellent intracellular marker of muscle fibers but it cannot be combined with AChE staining.