Standardization of clinical cholinesterase measurements

Previous studies showed that commonly used kits for measuring cholinesterases were not optimal for determining acetylcholinesterase (AChE) activity. Clinical use of different kits and methodologies resulted in AChE levels being reported in different units and activities that were not reproducible among laboratories. Findings such as these led to a revision in California regulations (covering AChE measurements for pesticide worker safety) calling for clinical laboratories to standardize their findings. The laboratories were contacted and invited to participate in a split-sample study of human blood AChE and nonspecific cholinesterase (BChE) assays. Participating laboratories measured erythrocyte (RBC) AChE and/or plasma BChE from undiluted and 50% diluted blood, according to their practices. Aliquots of blood samples were shipped to University of California Davis for measurement, using an optimized semiautomated plate reader version of the method of Ellman. Nine of 25 laboratories sent samples for comparison. Two others performed their own comparisons and submitted data to the state. Best correlations were obtained with BChE activity. Correlations (r(2)) were .88 or above for four of five laboratories for BChE, and above .9 for two of seven laboratories for AChE. Reasons for poor correlations may include difficulties in pipetting RBCs, storage, and processing. A bovine AChE RBC ghost "standard" was devised and tested. Activity of the preparation was maintained at -70 degrees C for approximately 11 months. A test with an East coast laboratory resulted in a high correlation, demonstrating the reliability of the RBC ghost standard and that one laboratory can replicate the AChE findings of another. The overall poor correlation of interlaboratory cholinesterase results points to the need to further standardize sample handling and assay methods.     

Potential of two new oximes in reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by organophosphate compounds: An in vitro study

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzyme. Oximes are compounds generally used to reverse the ChE inhibition caused by OP agents. In this study, we compared the in vitro reactivation potency of two new oximes (oxime 1: butane-2,3-dionethiosemicarbazone; oxime 2: 3-(phenylhydrazono) butan-2-one) against the inhibition on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities induced by chlorpyrifos, diazinon and malathion. Oximes used clinically (obidoxime and pralidoxime) were used as positive control. For this study, human blood (erythrocytes for AChE determination and plasma for BChE determination) was used and different concentrations of oximes (1–100 μM) were tested. The concentrations of OP used were based on the IC₅₀ for AChE and BChE. Results demonstrated that obidoxime was more effective in reactivate the AChE inhibition induced by OP compounds. However, both newly developed oximes achieved similar reactivations rates that pralidoxime for chlorpyrifos and diazinon-inhibited AChE. For BChE reactivation, none of evaluated oximes achieved positives rates of reactivation, been obidoxime able to reactivate malathion-inhibited BChE only in 24% at the highest concentration. We conclude that both newly developed oximes seem to be promising reactivators of OP-inhibited AChE.     

Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals

Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)‐selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (?)‐huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (?)‐huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (?)‐huperzine A is not required for measuring BChE activity in normal or ‘exposed’ blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high‐throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide manufacturing workers

Oxidative stress status and acetylcholinesterase (AChE) activity were studied in blood samples obtained from 45 organophosphorous (OP)-formulating pesticide workers with a minimum work history of 1 year in the age range of 23-55. Controls were age-matched workers of a food-making factory. They were evaluated for oxidative stress markers, including thiobarbituric acid-reactive substances (TBARS) indicator of lipid peroxidation (LPO), ferric-reducing ability of plasma (FRAP) indicator of total anti-oxidant capacity, total thiol (SH) groups and gamma glutamyl transpeptidase (GGT) levels in blood and AChE activity in erythrocytes. The results show marked inhibition of AChE activity, increased TBARS, decreased FRAP and decreased thiol group levels in workers. The reduction in activity of AChE correlated well with increased TBARS and decreased FRAP in OP formulators. It is concluded that OP-formulating workers are exposed to more oxidative stress. The measurement of erythrocyte AChE activity in pesticide workers who formulate OPs can be a good monitoring factor and is recommended to be performed in a regular manner.     

Design, synthesis and biological evaluation of new 2-benzoxazolinone derivatives as potential cholinesterase inhibitors for therapy of alzheimer's disease

Currently acetylcholinesterase inhibitor (AChEI) therapy is one of the most frequently used methods in the treatment of Alzheimer's disease; tacrine, donepezil, rivastygmine and galantamine are applied in different stages of AD. In the present study, we propose a new series of 2-benzoxazolinone derivatives as potential cholinesterase inhibitors. These compounds were synthesized by condensation of 6-chloro acetyl-2-benzoxa zolinone with the corresponding amine and evaluated as acetylcholinesterase inhibitors using the colorimetric Ellman's method. Selectivity and the IC50 values were determined for the received derivatives. All tested compounds exhibited the inhibitory activity towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Compound 3e showed stronger activity than the standard tacrine, and compound 3a showed activity similar to that of tacrine for AChE. Compounds 3a, 3b, 3c, and 3e showed stronger activity than the standard donepezil towards the inhibition of BChE, and the compound 3e showed stronger activity than donepezil towards AChE.     

Diagnostic aspects of organophosphate poisoning

Organophosphate (OP)-type chemical warfare agents (nerve agents) present a constant threat to the population. Sensitive and specific methods for the detection and verification of exposure to nerve agents are required for diagnosis, therapeutic monitoring, health surveillance and forensic purposes. Determination of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood remains a mainstay for the fast initial screening but lacks sensitivity and specificity. Quantitative analysis of nerve agents and their degradation products in plasma and urine by mass spectrometric methods may prove exposure but is limited to hours or days after the incident due to the short residence time of the analytes. Investigation of protein adducts extends the time interval between exposure and sampling and may be suitable to detect low-level exposure. Definitive prove of exposure requires a spectrum of different methods, expensive and sophisticated equipment and will be limited to specialized laboratories.     

Comparative effects of oral chlorpyrifos exposure on cholinesterase activity and muscarinic receptor binding in neonatal and adult rat heart

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M(2) muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M(2) receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M(2) receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age-related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac cholinesterase (ChE) activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1x LD(10): neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1x LD(10), relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (approximately 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC(50) values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4h after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity.     

Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: A kinetic analysis

The widespread use of organophosphorus compounds (OPs) as pesticides and the frequent misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the high clinical relevance of OP poisoning. The toxic symptomatology is caused by inhibition of acetylcholinesterase (AChE). A mainstay of standard antidotal treatment is atropine for antagonizing effects mediated by over stimulation of muscarinic ACh-receptors and oxime to reactivate OP-inhibited AChE. For therapeutic monitoring of oxime treatment in OP poisoning, measurement of erythrocyte AChE is suitable because erythrocyte AChE is an easily accessible surrogate for synaptic AChE. However, measurement of erythrocyte AChE is not standard practice. In contrast, determination of plasma butyrylcholinesterase (BChE) activity is in routine use for monitoring the benefit of oxime therapy. As oxime efficacy is limited with certain OPs (e.g. dimethoate, tabun, soman) alternative therapeutic approaches, e.g. the application of scavengers (BChE) which may sequester OPs before they reach their physiological target, are under investigation.     

Effects of the organophosphate fenthion for control of the red-billed quelea Quelea quelea on cholinesterase and haemoglobin concentrations in the blood of target and non-target birds

The red-billed quelea bird Quelea quelea is one of sub-Saharan Africa's most damaging pests, attacking small-grain crops throughout semi-arid zones. It is routinely controlled by spraying its breeding colonies and roosts with organophosphate pesticides, actions often associated with detrimental effects on non-target organisms. Attributions of mortality and morbidity of non-targets to the sprays are difficult to confirm unequivocally but can be achieved by assessing depressions in cholinesterase activities since these are reduced by exposure to organophosphates. Here we report on surveys of birds caught before and after sprays that were examined for their blood cholinesterase activities to assess the extent to which these became depressed. Blood samples from birds were taken before and after sprays with fenthion against red-billed quelea in colonies or roosts, and at other unsprayed sites, in Botswana and Tanzania and analysed for levels of haemoglobin (Hb) and activities of whole blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Background activities of AChE, BChE and Hb concentrations varied with bird species, subspecies, mass, age and gender. Contrary to expectation, since avian erythrocytes are often reported to lack cholinesterases, acetylcholinesterase activities in pre-spray samples of adult birds were positively correlated with Hb concentrations. When these factors were taken into account there were highly significant declines (P?相似文献   

Butyrylcholinesterase in guinea pig lung lavage: a novel biomarker to assess lung injury following inhalation exposure to nerve agent VX

Respiratory disturbances play a central role in chemical warfare nerve agent (CWNA) induced toxicity; they are the starting point of mass casualty and the major cause of death. We developed a microinstillation technique of inhalation exposure to nerve agent VX and assessed lung injury by biochemical analysis of the bronchoalveolar lavage fluid (BALF). Here we demonstrate that normal guinea pig BALF has a significant amount of cholinesterase activity. Treatment with Huperzine A, a specific inhibitor of acetylcholinesterase (AChE), showed that a minor fraction of BALF cholinesterase is AChE. Furthermore, treatment with tetraisopropyl pyrophosphoramide (iso-OMPA), a specific inhibitor of butyrylcholinesterase (BChE), inhibited more than 90% of BChE activity, indicating the predominance of BChE in BALF. A predominance of BChE expression in the lung lavage was seen in both genders. Substrate specific inhibition indicated that nearly 30% of the cholinesterase in lung tissue homogenate is AChE. BALF and lung tissue AChE and BChE activities were strongly inhibited in guinea pigs exposed for 5 min to 70.4 and 90.4 microg/m3 VX and allowed to recover for 15 min. In contrast, BALF AChE activity was increased 63% and 128% and BChE activity was increased 77% and 88% after 24 h of recovery following 5 min inhalation exposure to 70.4 microg/m3 and 90.4 mg/m3 VX, respectively. The increase in BALF AChE and BChE activity was dose dependent. Since BChE is synthesized in the liver and present in the plasma, an increase in BALF indicates endothelial barrier injury and leakage of plasma into lung interstitium. Therefore, a measure of increased levels of AChE and BChE in the lung lavage can be used to determine the chronology of barrier damage as well as the extent of lung injury following exposure to chemical warfare nerve agents.     

Reaction kinetics of biotinylated organophosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase

A biotinylated organophosphate could be useful for identifying proteins that react with organophosphorus toxicants (OP). FP-biotin, 10-(fluoroethoxyphosphinyl)-N-(biotinamidopentyl)decanamide, was synthesized and found to be stable in methanol and chloroform but less stable in water. Because acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are known to be sensitive targets of OP, their reactivity with FP-biotin was tested. The rate constant for reaction with human AChE was 1.8 x 10(7) M(-1) min(-1), and for human BChE, it was 1.6 x 10(8) M(-1) min(-1). A phosphorus stereoisomer, constituting about 50% of the FP-biotin preparation, appeared to be the reactive species. The binding affinity was estimated to be >85 nM for AChE and >5.8 nM for BChE. It was concluded that FP-biotin is a potent OP, well-suited for searching for new biomarkers of OP exposure.     

B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts

The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated.JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities.Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.     

Benefits of butyrylcholinesterase reactivability testing in organophosphate poisoning

Organophosphate (OP) poisoning continues to represent an important medical issue through its high prevalence among toxic pathologies and through its severity. In diagnosing this toxicological disorder, the most frequently utilized and available laboratory test remains the assessment of plasma cholinesterase - butyrylcholinesterase (BChE) - activity. Despite the reluctance of many researchers on the usefulness of serum BChE for kinetic analysis in OP intoxications, we have tested a recently proposed protocol, which is safe, non-expensive, easy to perform, appropriate to distinguish between an aged cholinesterase and a still reactivable one. Our aim was to validate the usefulness of this protocol, studying a series of 23 consecutive patients acutely intoxicated with OP, admitted in a regional Emergency Hospital, over a 1-year period. Introducing the proposed test in the routine of monitoring OP-intoxicated patients has resulted in the identification of a pattern with a funnel aspect, consequence of the initial possibility to increment the degree of BChE activity. This funnel shape defines the presence of reactivability, while its absence demonstrates the lack of obidoximes effect, due to cholinesterase's ageing process. This method consisted in an advantage for the diagnosis, having the potential of improving prognostic evaluation and therapeutic orientation in OP intoxications.     

Weak inhibitors protect cholinesterases from stronger inhibitors (dichlorvos): in vitro effect of tiapride

Metoclopramide is a benzamide dopamine receptor antagonist and serotonine receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition, metoclopramide is a weak and reversible inhibitor of cholinesterases. The authors have previously shown that metoclopramide has a cholinesterase protective effect against inhibition by organophosphates (OPs). The putative mode of protective action of metoclopramide is, when administered in excess, competion for the active site of the enzyme with the more potent OP. In the present paper the authors present their results using another benzamide with weak cholinesterase inhibitory properties, tiapride (TIA). The purpose of the study was to quantify in vitro the extent of TIA-conferred protection, using dichlorvos (dichlorovinyl dimethyl phosphate; DDVP) as an inhibitor. DDVP is a moderately toxic (LD50 in rats in the milligram range), non-neuropathic OP. The substance is responsible for a large number of accidental or suicidal exposures. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different DDVP and TIA concentrations and IC50 was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC50 of DDVP increases with the TIA concentration in a linear manner. The protective effect of TIA on cholinesterase could be of practical relevance in the treatment of OP poisoning. The authors conclude that in vivo testing of TIA as an OP protective agent is warranted.     

Butyrylcholinesterase in Guinea Pig Lung Lavage: A Novel Biomarker to Assess Lung Injury Following Inhalation Exposure to Nerve Agent VX

Respiratory disturbances play a central role in chemical warfare nerve agent (CWNA) induced toxicity; they are the starting point of mass casualty and the major cause of death. We developed a microinstillation technique of inhalation exposure to nerve agent VX and assessed lung injury by biochemical analysis of the bronchoalveolar lavage fluid (BALF). Here we demonstrate that normal guinea pig BALF has a significant amount of cholinesterase activity. Treatment with Huperzine A, a specific inhibitor of acetylcholinesterase (AChE), showed that a minor fraction of BALF cholinesterase is AChE. Furthermore, treatment with tetraisopropyl pyrophosphoramide (iso-OMPA), a specific inhibitor of butyrylcholinesterase (BChE), inhibited more than 90% of BChE activity, indicating the predominance of BChE in BALF. A predominance of BChE expression in the lung lavage was seen in both genders. Substrate specific inhibition indicated that nearly 30% of the cholinesterase in lung tissue homogenate is AChE. BALF and lung tissue AChE and BChE activities were strongly inhibited in guinea pigs exposed for 5 min to 70.4 and 90.4 μ g/m³ VX and allowed to recover for 15 min. In contrast, BALF AChE activity was increased 63% and 128% and BChE activity was increased 77% and 88% after 24 h of recovery following 5 min inhalation exposure to 70.4 μ g/m³ and 90.4 μg/m³, respectively. The increase in BALF AChE and BChE activity was dose dependent. Since BChE is synthesized in the liver and present in the plasma, an increase in BALF indicates endothelial barrier injury and leakage of plasma into lung interstitium. Therefore, a measure of increased levels of AChE and BChE in the lung lavage can be used to determine the chronology of barrier damage as well as the extent of lung injury following exposure to chemical warfare nerve agents.     

Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted tissue samples

Background and purpose:

Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N-[¹⁴C]methylpiperidin-4-yl acetate ([¹⁴C]MP4A) and (R)-N-[¹⁴C]methylpiperidin-3-yl butyrate ([¹⁴C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively.

Experimental approach:

We investigated the kinetics of hydrolysis of [¹⁴C]-MP4A and [¹⁴C]-MP3B_R by cholinesterases, and evaluated the selectivity of [¹⁴C]MP4A and [¹⁴C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC₅₀ values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [¹⁴C]MP4A and [¹⁴C]MP3B_R.

Key results:

AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [¹⁴C]-MP4A and [¹⁴C]MP3B_R respectively. The AChE selectivity of [¹⁴C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC₅₀ values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2-fold diluted human whole blood were much higher than those in 120-fold diluted blood. In addition, the IC₅₀ values of donepezil in monkey brain were dramatically decreased as the tissue was diluted.

Conclusions and implications:

This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents.     

Synthesis of novel 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(substituted/-nonsubstituted benzal)hydrazone derivatives and acetylcholinesterase and butyrylcholinesterase inhibitory activities in vitro

In this study thirteen 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(substituted/nonsubstituted benzal)hydrazone V derivatives were synthesized as acetylcholinesterase and butyrylcholinesterase inhibitors. Ten of the synthesized compounds were synthesized for the first time in this study and the rest of them had been synthesized in a previous study. The structures of compounds V were elucidated by IR, 1H-NMR and MASS spectra. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChe) inhibitory activities of V derivatives were measured using Ellman's method. While some of the 6-substituted-3(2H)-pyridazinone-2-propyl-3-(substituted/-nonsubstituted benzal)hydrazone V derivatives exhibited significant AChE inhibitory activity, none of the compoundsshowed BChE inhibitory activity. Theseresults indicate that V derivatives were AChE inhibitors with AChE/ BChE selectivity.     

The effect of pralidoxime chloride in the assay of acetylcholinesterase using 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent).

Pralidoxime chloride (PAM) hydrolyzes acetylthiocholine, the substrate used in the assay of red cell cholinesterase. The thiocholine that is produced forms a yellow complex when Ellman's reagent is used in the assay. This was tested in blood samples of patients who were treated with PAM after organophosphorus (OP) poisoning and after the observation of an immediate increase in absorption of light at 412 nm.     

Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase

Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 microg/ml of human G117H BChE in plasma as well as 2 microg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 microg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal.     

Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry.

The classical laboratory tests for exposure to organophosphorus toxicants (OP) are inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood. In a search for new biomarkers of OP exposure, we treated mice with a biotinylated organophosphorus agent, FP-biotin. The biotinylated proteins in muscle were purified by binding to avidin-Sepharose, separated by gel electrophoresis, digested with trypsin, and identified from their fragmentation patterns on a quadrupole time-of-flight mass spectrometer. Albumin and ES1 carboxylesterase (EC 3.1.1.1) were found to be major targets of FP-biotin. These FP-biotinylated proteins were also identified in mouse plasma by comparing band patterns on nondenaturing gels stained for albumin and carboxylesterase activity, with band patterns on blots hybridized with Streptavidin Alexa-680. Two additional FP-biotin targets, AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), were identified in mouse plasma by finding that enzyme activity was inhibited 50-80%. Mouse plasma contained eight additional FP-biotinylated bands whose identity has not yet been determined. In vitro experiments with human plasma showed that chlorpyrifos oxon, echothiophate, malaoxon, paraoxon, methyl paraoxon, diazoxon, diisopropylfluorophosphate, and dichlorvos competed with FP-biotin for binding to human albumin. Though experiments with purified albumin have previously shown that albumin covalently binds OP, this is the first report of OP binding to albumin in a living animal. Carboxylesterase is not a biomarker in man because humans have no carboxylesterase in blood. It is concluded that OP bound to albumin could serve as a new biomarker of OP exposure in man.