Cholinesterase activity in Japanese quail dusted with carbaryl.

Japanese quail (Coturnix coturnix japonica) were dusted with 5% carbaryl to determine if this topical treatment would alter plasma and brain cholinesterase activities. Within 6 hours after dusting, plasma cholinesterase activity was depressed compared with controls, the depression averaging 20% for females and 27% for males. By 24 hours the cholinesterase activity of females had returned to normal, but the cholinesterase activity of males remained depressed. Brain cholinesterase activity was not affected by the treatment, and there were no overt toxic signs.     

Plasma and erythrocyte cholinesterase values for the common long-nosed armadillo, Dasypus novemcinctus

Plasma and erythrocyte cholinesterase activities were determined for 40 free-living and 12 captive common long-nosed armadillos (Dasypus novemcinctus) in order to establish normal values for monitoring pesticide exposure. Plasma cholinesterase activity ranged from 105 to 549 U/liter with no sexual or seasonal differences. Plasma values from captive animals were significantly lower than those from wild armadillos. Erythrocyte cholinesterase activity ranged from 2,915 to 15,126 U/liter with no differences detected between captive and wild animals or between sexes. However, erythrocyte cholinesterase values varied seasonally. Erythrocyte and plasma cholinesterase activities were not significantly correlated. Packed cell volume ranged from 24 to 51% and did not vary significantly between captive and wild samples, between sexes or among seasons. However, both whole blood and erythrocyte cholinesterase activities showed significant negative correlations with packed cell volume. Controlled experiments are needed to find the factors responsible for the statistically significant difference between plasma cholinesterase activities of captive and wild armadillos. The seasonal variation in erythrocyte cholinesterase activity and the negative correlation between erythrocyte cholinesterase activity and packed cell volume can be explained by an hypothesis that relates the variation in erythrocyte cholinesterase activity to variation in erythrocyte turnover rate. Future work should involve experiments to test this hypothesis.     

Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.     

The effect of hypophysectomy upon cholinesterase activity in the forelimbs of regenerating adult Notophthalmus news

In order to study endocrine influence upon cholinesterase activity during regeneration, adult newts were hypophysectomized either prior to limb transection or during regeneration. Homogenates of limb tissues were assayed for cholinesterase activity during each stage of regeneration.In animals with pituitaries intact, cholinesterase activity in regenerating limb tissues decreases soon after amputation, and then it rises to the level of activity in intact limbs of normal animals, during the period of differentiation. In hypophysectomized newts there seems to be no alteration of this basic pattern of activity, but removal of the pituitary does result in more elevated levels of enzymatic activity. In the intact forelimbs of control newts undergoing regeneration, cholinesterase activity greatly increases as the other transected limb begins to regenerate but it returns to normal as regeneration progresses. If these animals are hypophysectomized, no such increase is observed during the early stages of regeneration. Rather, there is an initial decrease in cholinesterase activity that is followed by an increase in such activity.These data are compatible with the hypothesis that the pituitary modulates cholinesterase activity in the limb tissues of adult newts.     

Tissue distribution and characterization of cholinesterase activity in six earthworm species

To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086+/-0.015 U mg(-1) and 0.235+/-0.036 U mg(-1) for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.     

CHOLINESTERASE ACTIVITY OF THE MOTOR ENDPLATE IN ISOLATED MUSCLE MEMBRANE

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, K_m value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 10⁸ acetylcholine molecules in 1 msec. Since it is estimated that 10⁸ cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 10⁵. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.     

Cholinesterase assay by gas--solid chromatography.

The determination of serum cholinesterase activity was based on the hydrolysis of butyrylcholine chloride, solvent extraction, and the quantitative estimation of the hydrolytic product, butyric acid, by gas-solid chromatography. Using this procedure, eptimum conditions of pH and buffer strength for the cholinesterase activity were pH 8.0 and 0.05 m Tris-HCl, respectively. The cholinesterase activities in blood samples from healthy subjects, as determined by this method, were in the range of normal values reported in the literature. The effects of eserine and other drugs on the cholinesterase activity were also reported. The high accuracy (1.1%) and high sensitivity which permit the determination of butyric acid at levels down to 1 μg/ml make this procedure attractive as a serum cholinesterase assay.     

A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.     

Effect of food deprivation on histomorphology and cholinesterase activity of taste buds in ratst

The histomorphology and cholinesterase activity of the taste buds and the gustatory nerve fibre sin well-fed, in protein and protein-calorie deficient rats have been investigated. The nerve fibre arborisation in the taste buds is predominantly nonmyelinated and shows degenerative changes ranging from initial swelling to disintegration, fragmentation and finally complete disappearance with the increasing degree and duration of food deprivation. Coincident with these changes in the nerve fibre, the taste bud also shows various stages of degeneration. By contrast, the cholinesterase activity in the gustatory papillae shows an initial increase during the first week followed by a decline in the activity during the succeeding weeks; a second peak of cholinesterase activity appears during 4–6 weeks. The cholinesterase activity is barely detectable after the 8th week. In the more severely protein calorie restricted groups, the cholinesterase changes are more pronounced and abrupt in onset and show a total disappearance by 4–5 weeks.     

Secretory cholinesterase of Ancylostoma ceylanicum: effect of tubulin binding agents and benzimidazole anthelmintics.

Ancylostoma ceylanicum, the human hookworm parasite, exhibited significant secretion of cholinesterase when maintained in vitro in RPMI-1640 medium. Secretion of the enzyme was linear up-to 4 hours of incubation. About 40 percent of the total cholinesterase activity was localized in the soluble fraction, while remaining activity was associated with the particulate fraction of the nematode. Exposure of the hookworms to colchicine in vitro caused significant inhibition in secretion of the enzyme by the parasite with concomitant accumulation of cholinesterase within the adult worms. Vinblastine did not show noticeable effect on the enzyme secretion as well as activity within the parasite. Incubation of hookworms with some benzimidazole anthelmintics viz., mebendazole or albendazole significantly reduced the capacity of the worms to secrete cholinesterase and increase in enzyme activity within the parasite. Adult worms recovered from mebendazole treated hamsters exhibited about 3 fold greater activity of cholinesterase as well as significantly lower capacity to secrete cholinesterase in vitro as compared to the worms recovered from untreated animals. These observations indicate role of microtubules in the secretion of cholinesterase by hookworms and as a target for the action of benzimidazole anthelmintics.     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.     

Characterization of embryonic cholinesterase in chick limb bud by colorimetry and disk electrophoresis

Summary In the chondrogenic blastema of the chick limb bud an histochemical cholinesterase activity related to aggregation of the chondroblasts was described by Drews and Drews (1972). This cholinesterase activity was termed embryonic cholinesterase (Drews 1975). In the present study embryonic cholinesterase from the limb bud has been characterized by colorimetry and disk electrophoresis and compared to cholinesterase from the myotomes of the same embryos. Embryonic cholinesterase comprises both acetylcholinesterase and butyrylcholinesterase activity. The banding patterns of embryonic cholinesterase from limb bud and of cholinesterase from myotomes are identical. These findings support the hypothesis that the cholinergic system is involved in the regulation of embryonic development.This study is dedicated to Prof. Dr. W. Graumann on occasion of his 65th birthday     

Polymorphism of human blood serum cholinesterase in populations of Evenks and Yakuts in Krasnoyarsk Territory

Polymorphism for E2 locus of human serum cholinesterase was studied in populations of evenks and yakuts of Krasnoyarsky region by the method of starch gel electrophoresis. Thee frequencies of C5+ phenotype correspond to C5+ frequencies in other mongoloid populations of Siberia. The activity of cholinesterase was determined by semiquantitative technique. Three individuals with a sharply decreased cholinesterase activity have been revealed.     

Combined effect of short-term dehydration and sublethal acute oral dicrotophos exposure confounds the diagnosis of anticholinesterase exposure in common quail (Coturnix coturnix) using plasma cholinesterase activity

Common Quail (Coturnix coturnix) were subjected to controlled and replicated experiments in the summer of 2008 to investigate the effects of short-term dehydration on cholinesterase activity in brain and plasma and the interaction between dehydration and exposure to the organophosphorus pesticide dicrotophos in these same tissues. Our objective was to determine if dehydration could confound the diagnosis of anticholinesterase exposure using inhibition of cholinesterase activity in quail tissues. The effect of dehydration was quantified using measures of plasma osmolality and hematocrit. Dicrotophos exposure caused significant inhibition of cholinesterase activity in brain, while the effects of dehydration and interaction were not significant. Dehydration caused significant duration-dependent increases in plasma osmolality and hematocrit. Dehydration also caused a significant increase in plasma cholinesterase activity. Variation in the change in plasma cholinesterase activity in response to dehydration was significantly and positively correlated with dehydration-induced variation in both the change in plasma osmolality and the change in hematocrit. These correlations suggest that plasma cholinesterase activity in quail is not limited to plasma but occupies some larger pool of the extracellular fluid volume, and we suggest lymph is part of that pool. The effects of dehydration on plasma cholinesterase activity masked the inhibitory effects of dicrotophos. Here, the combination of dehydration and dicrotophos exposure produced plasma cholinesterase activity that was not significantly different from reference and pre-exposure values, confounding the diagnosis of anticholinesterase exposure in dehydrated, dicrotophos-exposed quail. A method to adjust plasma cholinesterase activities for the confounding effects of dehydration and enable the diagnosis of anticholinesterase exposure in dehydrated, dicrotophos-exposed quail was developed. Clinicians and practitioners responsible for the diagnosis of anticholinesterase exposure in birds are cautioned that dehydration, commonly observed in sick wildlife, may mask the effect of anticholinesterases on plasma cholinesterase activity.     

Nano-Intercalated Organophosphorus-Hydrolyzing Enzymes in Organophosphorus Antagonism

A dendritic poly(2-alkyloxazoline)-based polymer was studied as a new carrier system for the organophosphorus-hydrolyzing recombinant enzymes, organophosphorus acid anhydrolase and organophosphorus hydrolase. Paraoxon (PO) and diisopropylfluorophosphate (DFP) were used as model organophosphorus compounds. Changes in plasma cholinesterase activity were monitored. The cholinesterase activity was proportional to the concentrations of DFP or PO. Plasma cholinesterase activity was higher in animals receiving enzyme and oxime before the organophosphates than in the oxime-only pretreated groups. These studies suggest that cholinesterase activity can serve as an indicator for the in vivo protection by the nano-intercalated organophosphorus acid anhydrolase or organophosphorus hydrolase against organophosphorus intoxications. These studies represent a practical application of polymeric nano-delivery systems as enzyme carriers in drug antidotal therapy.     

Contribution to histochemical distribution of non-specific cholinesterase activity in sensory ganglia of some mammals

The activity of non-specific cholinesterase was demonstrated histochemically in satellite cells of the spinal ganglia from adult rat, cat, rabbit and baboon. The spinal ganglia of newborn rats displayed distinct intraneuronal reactivity for non-specific cholinesterase while a low reactivity was observed in satellite cells. The spinal and trigeminal ganglia of adult mice contained satellite cells with non-specific cholinesterase reactivity only sporadically. Most of reaction product for non-specific cholinesterase activity (from low to high intensity) was found in perikarya of the neurons. Spinal and trigeminal ganglia of the same mice embryo exhibited diffuse staining for non-specific cholinesterase activity remaining in the spinal ganglia of newborn mice. The trigeminal ganglia of newborn mice exhibited, however, more differentiated pattern of the positive reaction for non-specific cholinesterase like adult animals. The pattern of histochemical distribution of non-specific cholinesterase activity in trigeminal and spinal ganglia from mice of various ages corresponds with morphological differentiation and maturation undergoing in a rostrocaudal wave. Intraneuronal presence of non-specific cholinesterase activity in sensory ganglia during development and in adult animals gives a new possibilities for explanation of the functional involvement of this enzyme in the nervous system.     

Effects of single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats

The effects of a single or repeated dermal administration of methyl parathion on motor function, learning and memory were investigated in adult female rats and correlated with blood cholinesterase activity. Exposure to a single dose of 50 mg/kg methyl parathion (75% of the dermal LD(50)) resulted in an 88% inhibition of blood cholinesterase activity and was associated with severe acute toxicity. Spontaneous locomotor activity and neuromuscular coordination were also depressed. Rats treated with a lower dose of methyl parathion, i.e. 6.25 or 12.5 mg/kg, displayed minimal signs of acute toxicity. Blood cholinesterase activity and motor function, however, were depressed initially but recovered fully within 1-3 weeks. There were no delayed effects of a single dose of methyl parathion on learning acquisition or memory as assessed by a step-down inhibitory avoidance learning task. Repeated treatment with 1 mg/kg/day methyl parathion resulted in a 50% inhibition of blood cholinesterase activity. A decrease in locomotor activity and impairment of memory were also observed after 28 days of repeated treatment. Thus, a single dermal exposure of rats to doses of methyl parathion which are lower than those that elicit acute toxicity can cause decrements in both cholinesterase activity and motor function which are reversible. In contrast, repeated low-dose dermal treatment results in a sustained inhibition of cholinesterase activity and impairment of both motor function and memory.     

Cholinesterase inhibitory effects of Rhizophora lamarckii, Avicennia officinalis, Sesuvium portulacastrum and Suaeda monica: Mangroves inhabiting an Indian coastal area (Vellar Estuary)

Alzheimer's disease is a progressive neurodegenerative illness accounting for approximately 50% of all types of dementia in elderly people. The only symptomatic treatment proven effective to date is the use of cholinesterase inhibitors to augment surviving cholinergic activity. The purpose of this study is to investigate cholinesterase inhibitory activity of mangroves as an alternative medicine for the treatment of Alzheimer's disease. About nine mangrove plants, which were used as folk medicine in tropical countries, were collected from Parangipettai, Vellar estuary, Tamilnadu, India. Nile Tilapia muscle homogenate was used as source of enzyme. Inhibitory effect of methanolic leaf extract was assessed under in vitro condition by incubating various concentration of the extract with total cholinesterase and butyryl cholinesterase and assessing their residual activities by Ellman's colorimetric method. The results showed that of the nine plants screened Rhizophora lamarckii, Suaeda monica, Avicennia officinalis and Sesuvium portulacastrum showed 50% inhibitory activity to both TChE and BChE at concentrations less than 2 mg/mL when compared to other plant extracts, which was comparable to the standard drug Donepezil. Phytochemical analysis showed the presence of alkaloids in high concentration which might be correlated to its cholinesterase inhibitory activity.     

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay

Ellman’s assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman’s assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman’s method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman’s method into two successive steps that may be convenient for some applications.     

BRAIN ACETYLCHOLINE AND CHOLINESTERASE: EFFECT OF PHENOTHIAZINES AND PHYSOSTIGMINE INTERACTION IN RATS1

Abstract— The effect of phenothiazines either alone or in combination with physostigmine on whole brain acetylcholine concn and cholinesterase activity has been investigated in male rats. Phenothiazines (chlorpromazine, trifluperazine and thioridazine) when injected alone had no significant effect on brain acetylcholine concentration. Pretreatment with chlorpromazine and thioridazine significantly enhanced the physostigmine-induced increase in brain acetylcholine concn and inhibition of cholinesterase activity. However, trifluperazine had no significant effect on the physostigmine-induced increase in brain acetylcholine concentration and inhibition of cholinesterase activity. The potentiation of the physostigmine-induced increase in brain acetylcholine concn by phenothiazines may be due to (1) increased acetylcholine turnover secondary to the blockade of dopamine receptors by neuroleptic drugs and.