Inhibitor Report for: DFP

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BCHE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BCHE, and foreshadow the design of recombinant BCHEs with tailored scavenging properties.

Rats treated daily with diisopropylfluorophosphate (DFP) (0.5 mg/kg, sc), an inhibitor of acetylcholinesterase (AChE) activity, exhibited the symptoms of cholinergic hyperactivity between Days 3 and 5 similar to those observed 15 min after a single acute dosage (1.5 mg/kg, sc). A significant (p less than 0.05) decrease in the activities of both AChE and cholinesterase (BuChE) (greater than 80%) occurred in muscles and in brain regions and of aliesterases in liver (greater than 92%) at this time. Further administration of DFP (0.5 mg/kg, for 7-14 days) led to behavioral tolerance, where symptoms of toxicity disappeared such as muscle fasciculations, tremors, and muscle necrosis. The activity of aliesterases in liver and AChE in muscles significantly (p less than 0.01) recovered, while no such recovery was seen in brain AChE. DFP toxicity was potentiated in rats that were pretreated with BuChE inhibitors, such as iso-OMPA (3 mg/kg, sc) or mipafox (0.05 mg/kg, sc), 30 min prior to DFP (0.5 mg/kg, sc). The severity of cholinergic hyperactivity and inhibition of aliesterase in liver, AChE and BuChE activity in brain and muscles was greater when compared to the effects of DFP alone. Both iso-OMPA and mipafox completely abolished the tolerance development to DFP, since no animal survived more than 5 days of combined treatment. The observed adaptation to DFP toxicity appears to be due to recovery of aliesterase, BuChE, and AChE activity as well as decreased nicotinic binding sites at the neuromuscular junction, as previously reported.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue SCPEP1 as potential targets of organophosphates (OP). CatA could be stably inhibited by low microM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 microM followed by VR (KI = 2.8 microM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 x 10(3) M(-1)sec(-1) and 1.2 x 10(3) M(-1)sec(-1), respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KI(DFP) = 13 microM and 11 microM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BCHE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BCHE activity.

We describe the responses of single units in the awake (24 cells) or urethane-anesthetized (37 cells) rat somatosensory cortex during repeated iontophoretic pulses (1.0 s, 85 nA) of acetylcholine, both before and after systemic treatment with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (i.p., 0.3-0.5 LD50). The time-course of the response to acetylcholine pulses differed among cortical neurons but was characteristic for a given cell. Different time-courses included monophasic excitatory or inhibitory responses, biphasic (excitatory-inhibitory, inhibitory-excitatory, excitatory-excitatory, and inhibitory-inhibitory), and triphasic (excitatory-excitatory-inhibitory, inhibitory-inhibitory-excitatory, and inhibitory-excitatory-inhibitory) responses. Although the sign and time-course of the individual responses remained consistent, their magnitude fluctuated across time; most cells exhibited either an initial increase or decrease in response magnitude followed by oscillations in magnitude that diminished with time, gradually approaching the original size. The time-course of the characteristic response to an acetylcholine pulse appeared to determine direction and rate of change in response magnitude with successive pulses of acetylcholine. Diisopropylfluorophosphate treatment, given 1 h after beginning repeated acetylcholine pulses, often resulted in a gradual increase in spontaneous activity to a slightly higher but stable level. Superimposed on this change in background activity, the oscillations in the response amplitude reappeared and then subsided in a pattern similar to the decay seen prior to diisopropylfluorophosphate treatment. Our results suggest that dynamic, homeostatic mechanisms control neuronal excitability by adjusting the balance between excitatory and inhibitory influences within the cortical circuitry and that these mechanisms are engaged by prolonged increases in extracellular acetylcholine levels caused by repeated pulses of acetylcholine and by acetylcholinesterase inhibition. However, this ability of neurons in the cortical neuronal network to rapidly adjust to changes in extracellular levels of acetylcholine questions the potential efficacy of therapeutic treatments designed to increase ambient levels of acetylcholine as a treatment for Alzheimer's disease or to enhance mechanisms of learning and memory.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

This study was aimed to investigate the possibility of modifying the rate of aging of diisopropylfluorophosphate-inhibited neuropathy target esterase (NTE) of hen brain. This reaction on NTE occurs with a half-time of 7.4 min. Atropine was effective in decreasing the rate of aging on DFP-inhibited NTE and this effect was time- and concentration-dependent. Atropine was also a weak but progressive inhibitor of NTE activity (I50 = 80 mM) and this reaction appears to be reversible at lower atropine concentrations. Among compounds containing oxime functional groups only OPAB, having longer methylene chain and being more lipophylic than other oximes usually used in acetylcholinesterase (AChE) reactivation studies, was effective in decreasing the rate of aging on DFP-inhibited NTE. However, when atropine and oximes were used together we have obtained a potentiating and/or synergistic effect which was most significant with combination of atropine and TMB-4(Trimedoxime) giving up to a 15-fold decrease in the rate of aging reaction. The efficacy of this particular combination was concentration-dependent. We have also discussed similarities and differences in aging reaction occurring on NTE and AChE.

Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BCHE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BCHE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BCHE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BCHE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BCHE, allowing some regeneration by spontaneous hydrolysis.

The feasability of PET determination of myocardial muscarinic acetylcholine receptor (mAChR) density has been demonstrated in dogs and humans. The results of the PET method, however, were not validated by a direct comparison with the in vitro determination of mAChR density. METHODS: Left ventricular mAChR concentrations were studied in beagle dogs at baseline and after a 5- or a 11-day treatment with the irreversible acetylcholinesterase inhibitor di-isopropylfluorophosphate (DFP). The determination of mAChR densities were performed in vivo using PET, 11C-MQNB, the three-injection protocol and the compartmental model previously described. In a parallel group of dogs, determination of mAChR density was performed in vitro using 3H-(-)-MQNB. RESULTS: In control dogs (n = 4), PET left ventricular density of mAChR was 61.1 +/- 8.1 pmol/ml tissue. In the 5-day DFP-treated animals (n = 3), Bmax decreased to 38.2 +/- 8.3 pmol/ml tissue (-38%; p = 0.005 versus control). In the 11-day DFP-treated animals (n = 3), Bmax was 34.7 +/- 5.5 pmol/ml tissue (-43%; p = 0.003). There was no change in the affinity constant either at 5 or 11 days. In control dogs, Bmax, measured in vitro, was 9.53 +/- 0.93 pmol/g tissue. In the 5-day DFP-treated animals, Bmax decreased to 6.2 +/- 0.9 pmol/g tissue (-35%; p = 0.003). In the 11-day DFP-treated animals, Bmax was 5.1 +/- 0.6 pmol/g tissue (-47%; p = 0.003 versus control). At that time, there was no change in affinity constant. On the fifth and 11th days, myocardial acetylcholinesterase activity was reduced by 88% and 90%, respectively. CONCLUSION: The in vivo and in vitro methods showed a similar decrease in mAChR density while for both methods affinity constant remained unchanged. This study validates the ability of PET and of the compartmental model to in vivo quantify changes in mAChR density.

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.

Raman spectra of human butyrylcholinesterase (BCHE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BCHE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% alpha-helices, 26% beta-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BCHE, but 39% helical structure, 31% beta-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BCHE conjugates and 36% helical structure, 34% beta-sheets, 20% turns and 10% undefined structure for 'aged' soman-BCHE conjugates. The approximately 10% decrease of alpha-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BCHE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BCHE and soman-BCHE

Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.

Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AChE) activities was compared in brain and spinal cords of adult While Leghorn hens and adult male Long Evan rats 4-48 hr after administration of triortho-tolyl phosphate (TOTP po, 50-500 mg/kg to hens; 300-1000 mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1-2.5 mg/kg to hens; 5-24 mg/kg to rats), mipafox (3-30 mg/kg ip to hens and rats), diisopropyl phosphorofluoridate (DFP sc, 0.25-1.0 mg/kg to hens; 1-3 mg/kg to rats), dichlorvos (5-60 mg/kg ip to hens; 600-2000 mg/kg to rats), and carbaryl (300-560 mg/kg ip to hens; 30-170 mg/kg to rats). Inhibitions of NTE and AChE were dose-related after administration of all compounds to both species. Hens and rats given TOTP, PSP, mipafox, and DFP demonstrated delayed neuropathy 3 weeks later, with spinal cord lesions and clinical signs more notable in hens. Ratios of NTE/AChE inhibition in hen spinal cord, averaged over the doses used, were 2.6 after TOTP, 5.2 after PSP, 1.3 after mipafox, and 0.9 after DFP, which contrast with 0.53 after dichlorvos, 1.0 after malathion, and 0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9 after TOTP, 2.6 after PSP, 1.0 after mipafox, 0.62 after DFP, 1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl. The lower NTE/AChE ratios in rats given dosages of the four organophosphorus compounds that caused delayed neuropathy interferred with survival, an effect that was not a problem in hens.

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.

Intraspecies variation has been found to affect the physiological, behavioral, and biochemical responses to a variety of neurotoxicants, including the organophosphate diisopropyl fluorophosphate (DFP). However, there is little information on long-term physiological responses to neurotoxicant exposure using strain as a dependent variable. In the present study, radiotelemetry methodology was used to continuously monitor core temperature, heart rate, and motor activity for 4 d following administration of 1.5 mg/kg DFP (sc) in four common strains of rat: Sprague-Dawley (SD), Long-Evans (LE), Fischer 344 (F344), and Wistar (WST). The F344 rat was least susceptible to DFP in terms of both a minimal hypothermic response and recovery of the day-night difference in core temperature. The SD strain was unusual in that its heart rate was elevated relative to the other strains after DFP, in spite of a marked decrease in core temperature and motor activity. The LE strain exhibited the largest reduction in core temperature and heart rate following DFP. Serum and brain cholinesterase activity (ChE) measured 3 h after administration of 1.0 mg/kg DFP also indicated strain effects. The F344 showed less inhibition in these variables compared to the other strains, a response that may explain its attenuated thermoregulatory response to DFP. Overall, the inbred F344 rat demonstrated better resistance to DFP compared to the outbred strains. Therefore, the impact of genetic differences on sensitivity to neurotoxicants such as DFP could be an important tool in understanding the mechanism of action of these agents.

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 diisopropylfluorophosphate (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.

Organophosphate (OP) exposure can be lethal at high doses while lower doses may impair performance of critical tasks. The ability to predict such effects for realistic exposure scenarios would greatly improve OP risk assessment. To this end, a physiologically based model for diisopropylfluorophosphate (DFP) pharmacokinetics and acetylcholinesterase (AChE) inhibition was developed. DFP tissue/blood partition coefficients, rates of DFP hydrolysis by esterases, and DFP-esterase bimolecular inhibition rate constants were determined in rat tissue homogenates. Other model parameters were scaled for rats and mice using standard allometric relationships. These DFP-specific parameter values were used with the model to simulate pharmacokinetic data from mice and rats. Literature data were used for model validation. DFP concentrations in mouse plasma and brain, as well as AChE inhibition and AChE resynthesis data, were successfully simulated for a single iv injection. Effects of repeated, subcutaneous DFP dosing on AChE activity in rat plasma and brain were also well simulated except for an apparent decrease in basal AChE activity in the brain which persisted 35 days after the last dose. The psychologically based pharmacokinetic (PBPK) model parameter values specific for DFP in humans, for example, tissue/blood partition coefficients, enzymatic and nonenzymatic DFP hydrolysis rates, and bimolecular inhibition rate constants for target enzymes were scaled from rodent data or obtained from the literature. Good agreement was obtained between model predictions and human exposure data on the inhibition of red blood cell AChE and plasma butyrylcholinesterase after an intramuscular injection of 33 micrograms/kg DFP and at 24 hr after acute doses of DFP (10-54 micrograms/kg), as well as for repeated DFP exposures.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BCHE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BCHE, and foreshadow the design of recombinant BCHEs with tailored scavenging properties.

Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187-199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low-salt and high-salt detergent-free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X-100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low-salt, high-salt and detergent) resolved almost exclusively as a single peak between 6.9-7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic subunits and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X-100, Brij 96) in the gradient was a component of the detergent-soluble extract, indicating the existence of a minor amphiphilic form. In low-salt-soluble and high-salt-soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate-labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66-70 kDa with the active site being located in a 30-kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this 'non-cholinergenic' nematode secretion.

The effect of phosphotriesterase (PTE) on cholinesterase (ChE) activities was studied with exposures to different organophosphates in mice. Paraoxon (PO) (1.0 mg/kg, ip) almost totally inhibited serum ChE activity. This activity, however, recovered to the normal level within 24 hr. The PTE pretreatment (16.8 U/animal, 2.5 micrograms/10 g body wt, iv 10 min before the organophosphate) accelerated this reactivation. The same phenomenon was also seen in vitro. In vitro with human serum, there was only minimal reactivation of the inhibited ChE. PTE, however, reactivated it significantly. The PTE-pretreated mice (168 U/animal, 30 micrograms/10 g body wt, iv) tolerated even 50 mg/kg of PO without showing any remarkable signs of intoxication. In PTE-untreated animals, however, PO doses as low as 1.0 and 1.5 mg/kg caused severe signs of poisoning. PTE (16.8 U/animal, 4 micrograms/10 g body wt, iv) reduced the inhibition of brain and serum ChE activities after PO and diisopropyl fluorophosphate exposure. In sarin and soman intoxications, PTE decreased only slightly the inhibition of ChE activities. The results indicate that PTE pretreatment given iv prevents the inhibition of ChE activities after certain organophosphates and it also hastens the recovery of activities after PO poisoning.

Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the "tail" that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 microM (-)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (-)-vesamicol.

In reaction of hydrolysis of choline and thiocholine esters of carbonic acids at 25 degrees C, cholinesterase activity of the blood serum from the fish A. ballerus has been studied by modified Ellman's method and potentiometric titration method. The activity is maximal in pH region 7.5-9.0 and is not inhibited by high concentration of substrates. Michaelis constants and maximal rates for the enzyme reactions were determined. Butyrylcholine and butyrylthiocholine were hydrolyzed with the highest rates by the serum. Some of the organophosphorus inhibitors (diisopropylfluorphosphate and DDVF) inhibit cholinesterase activity of the blood serum significantly faster, whereas some of the carbamates (aminostygmin, eserine, etc.) inhibit it significantly slower than typical butyrylcholinesterase from horse blood serum and typical acetylcholinesterase of human erythrocytes. Besides, with respect to the sensitivity to inhibitors and some other properties, fish blood serum cholinesterase differs from other known cholinesterases.

Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [3H]QNB binding and only at concentrations greater than 100 microM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [3H]CD, which binds with high affinity (approximate KD = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.

Cholinesterases of porcine left ventricular heart muscle were characterized with respect to substrate specificity and inhibition kinetics with organophosphorus inhibitors N,N'-di-isopropyl-phosphorodiamidic fluoride (Mipafox), di-isopropylphosphorofluoridate (DFP), and diethyl p-nitro-phenyl phosphate (Paraoxon). Total myocardial choline ester hydrolysing activity (234 nmol/min/g wet wt with 1.5 mM acetylthiocholine, ASCh; 216 nmol/min/g with 30 mM butyrylthiocholine, BSCh) was irreversibly and covalently inhibited by a wide range of inhibitor concentrations and, using weighted least-squares non-linear curve fitting, residual activities as determined with four different substrates in each case were fitted to a sum of up to four exponential functions. Quality of curve fitting as assessed by the sum of squares reached its optimum on the basis of a three component model, thus, indicating the presence of three different enzymes taking part in choline ester hydrolysis. Final classification of heart muscle cholinesterases was obtained according to both substrate hydrolysis patterns with ASCh, BSCh, acetyl-beta-methylthiocholine and propionylthiocholine, and second-order rate constants for the reaction with organophosphorus inhibitors Mipafox, DFP, and Paraoxon. One choline ester-hydrolysing enzyme was identified as acetylcholinesterase (EC 3.1.1.7), and one as butyrylcholinesterase (EC 3.1.1.8). The third enzyme with relative resistance to organophosphorus inhibition was classified as atypical cholinesterase.

The protective action of 1,2,3,4-tetrahydro-9-aminoacridine (THA) against the long-lasting inactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE) brought about by diisopropylfluorophosphate (DFP) and physostigmine, as well as by neostigmine in the case of AChE only, was evaluated by a dilution technique using Electrophorus electricus AChE and horse serum BCHE as target enzymes. In parallel experiments, the ability of physostigmine itself to protect these enzymes from DFP was evaluated and compared with that of THA. THA pretreatment was seen to prevent in a dose-dependent manner the inhibition of both AChE and BCHE. However, it was appreciably more potent towards AChE than towards BCHE. THA mean EC50 values for protecting AChE against 10, 40 and 100 microM DFP were 0.04, 0.16 and 0.45 microM, respectively; against 1 microM physostigmine the value was 1.8 microM and against 1.2 microM neostigmine it was 3.0 microM. The THA mean EC50 value for protecting BCHE against 3 microM physostigmine was 0.55 microM and the values for protecting against 3, 10 and 40 microM DFP were 1.5, 3 and greater than 10 microM, respectively. The protective action of THA was time independent: recovery of the maximal enzymic activity was immediate upon dilution. Unlike THA, the protective action of physostigmine developed progressively after dilution and was maximal within 3-4 (AChE) or 6-8 hr (BCHE). Under our experimental conditions, 0.3 microM physostigmine protected approximately 70% of AChE from 40 microM DFP and 5 microM physostigmine protected 9 and 47% of BCHE from 40 and 3 microM DFP, respectively. The results of this work suggest that THA exerts its protective action by shielding the active site of AChE and BCHE from the attack of the inactivating agents on account of its higher enzymic affinity, whereas the protective action of physostigmine against DFP takes advantage also of the carbamylation of the enzyme. These results are in line with the hypothesis that protection of AChE is the primary mechanism responsible for the antidotal action of THA against organophosphorus poisoning.

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.

The effect of repeated administration of the organophosphate anticholinesterases, soman (pinacolyl methylphosphonofluoridate) and DFP (diisopropylfluorophosphate) on core temperature was investigated in mice. Mice were implanted with telemetry transmitters for the monitoring of core temperature. Following repeated administration of soman (3-10 injections), tolerance (as defined by a decrease in the organophosphate-induced hypothermia upon subsequent administration) to the organophosphate-induced hypothermia was evident after the 5th injection; however, there was cross-tolerance to oxotremorine hypothermia as early as after the 3rd injection of soman. Following repeated administration of DFP, there was no tolerance to the DFP-induced hypothermia following 5 injections, whereas cross-tolerance to oxotremorine was evident following the 5th injection. The organophosphate-induced hypothermia may have another component which contributes to the response. It is proposed that the cross-tolerance to oxotremorine hypothermia after subchronic administration of an anticholinesterase is representative of the functionality of muscarinic cholinergic receptor coupling.

Acetylcholine esterase inhibitors block cholinergic neurotransmission. This blockade can be reversed by oximes. However, a universally effective esterase reactivator does not exist. A new H-oxime, HL 7, was tested on rat diaphragm strips. Electrically evoked contractions were blocked by di-2-propyl fluorophosphate (DFP), tabun, sarin and soman. Whereas pralidoxime, obidoxime and HI 6 reversed the blockade induced by three of these organophosphorus compounds, HL 7 restored the contractions after short blockade induced by all four organophosphorus compounds tested.

1. The inhibition of cholinesterase and carboxylesterase activities in the diisopropyl fluorophosphate (DFP) intoxication, and the inducibility of organophosphate (OP) detoxicating enzymes was studied in rats. 2. In phenobarbital (PB)-, but not in beta-naphthoflavone (NF)-pretreated rats, the activities of DFP-inhibited cholinesterases were 70-120% higher than in non-pretreated rats. Also the inhibition of the microsomal and cytosolic carboxylesterase activity in liver was efficiently antagonized by BP, but not by NF. 3. In vitro the microsomes from PB-treated rats detoxicated DFP probably by O-dealkylation, since no fluoride was released from DFP. Glutathione S-transferase did not detoxicate DFP. 4. 7-Pentoxyresorufin O-dealkylase, a specific enzyme of cytochrome P450IIB subfamily, was induced by PB, flumecinol, isosafrole and NF by 167- 61-, 26- and 1.6-fold, respectively. 7-Ethoxyresorufin O-deethylase, a marker enzyme of cytochrome P450IA subfamily, was induced by those agents 5-, 4-, 31- and 94-fold, given in the same order. Glutathione S-transferase, paraoxonase and DFPase activities were increased 0-72% by the tested inducers. 5. The results suggest that the cytochrome P450IIB subfamily, inducible by PB, participates in DFP detoxication by O-dealkylation. Its induction probably causes the protection against the cholinesterase inhibition by OPs.

Catalytic properties of human blood erythrocyte acetylcholinesterase and horse blood serum butyrylcholinesterase immobilized and nonimmobilized in the gelatin membrane have been comparatively studied. Cholinesterase immobilization induces an increase in the Michaelis constant value and a decrease in the maximum rate value in reactions of enzymic hydrolysis of thiocholine ethers, but exerts no effect on these kinetic parameters in case of enzymic hydrolysis of indophenylacetate. The effect of reversible inhibitors: galanthamine, N-methyl-4-piperidinyl benzylate and 1,2,3,4-tetrahydro-9-aminoacridine (tacrine), as well as of irreversible inhibitors: O-ethyl-O-(4-nitrophenyl)ethyl phosphonate (armin), diisopropyl fluorophosphate (DFP), O,O-diethyl-O-(4-nitrophenyl) phosphate (paraoxon) and O,O-dimethyl-O-(2,2-dichlorovinyl) phosphate (DDVP) on immobilized cholinesterases is weaker as compared with the effect on nonimmobilized enzymes. The results obtained are discussed for the effect of immobilization on the catalytically active enzyme surface.

This experiment examined the effects of repeated exposure to diisopropylfluorophosphate (DFP), an organophosphate anticholinesterase, on the retention and reversal of a visual discrimination and on the number of muscarinic receptors in the brain. Rats were trained in a serial reversal procedure. After achieving stable performance, the rats were divided into two groups. One group received repeated injections of DFP, the other group received injections. To determine whether DFP-treated rats would be more sensitive than normal rats to stresses on the cholinergic system, each rat was injected with saline or one of three doses of scopolamine, a muscarinic receptor blocker, prior to testing on every 6th day. DFP alone caused no impairment in performance. Scopolamine produced a greater impairment in DFP-treated rats than in control rats. Similar results were obtained in a second behavioral task, match-to-sample in a water maze, using the same DFP treatment protocol and only one dose of scopolamine. The number of muscarinic receptors and acetylcholinesterase activity levels were reduced on the 2nd and 15th day after the end of DFP treatment. These results demonstrate that although repeated exposure to organophosphate anticholinesterases may not alter discrimination behavior directly, it may compromise the central nervous system so that it cannot react normally when challenged.

Subacute (daily) administration of diisopropylfluorophosphate (DFP) to male swine (Yorkshire white) resulted in a 97% inhibition of cholinesterase and a decrease of [3H]quinuclidinyl benzilate [( 3H]QNB) binding sites in homogenates of striata by approximately 50% after 14 days. The maximal density of receptors (Bmax) decreased from 2.1 +/- 0.3 to 1.0 +/- 0.2 pmole/mg protein. There was no significant change in the dissociation constant (Kd) for [3H]QNB binding (control: 52.6 +/- 10.7 pM; 7-day: 57 +/- 2.8 pM). Carbachol displacement of [3H]QNB binding yielded data best fit by a two-binding site model. The dissociation constants were KiL = 115 +/- 62 microM (55 +/- 3%) and KiH = 1.8 +/- 0.7 microM (45 +/- 3%), respectively, for the low- and high-affinity states. Seven-Day treatment with DFP reduced the percentage of high-affinity receptors to 22 +/- 8.6%, but affected neither the low- nor the high-affinity Kd (100 +/- 20 and 2 +/- 0.6 microM). With the addition of Mg2+, striatal homogenates had low- and high-affinity receptors in the proportion of approximately 1 to 1. In the presence of Gpp(NH)p + Mg2+ the ratio of high- to low-affinity receptors was 3:1 in homogenates of control tissue (to 26 +/- 5%). This treatment had no effect on this ratio in homogenates of tissue from 7-day DFP-treated swine (3:1) since it was already 3:1. Pirenzepine displacement of [3H]QNB binding was best described by a two-binding site model, with Ki values of 38 +/- 14 and 201 +/- 78 nM, which represent 74 and 26% of the binding sites, respectively. The high affinity Kd value was unchanged following 7 days of DFP treatment (24 +/- 5 nM). There appears to be little change in the displacement curves for pirenzepine inhibition of [3H]QNB binding. This suggests that about 75% of the receptors are of the M1 subtype. Thus, subacute administration of DFP causes not only a decrease in the number of receptors, but also a change in the proportion of agonist affinity states which is related to the interaction of the guanine nucleotide binding protein and the muscarinic receptor.

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.

1. The effect of cold environment on the acute toxicity of organophosphates (OP), without and with atropine-oxime treatment, was studied in rats and mice by exposing them to +5 and -5 degrees C temperature. The tested OPs and oximes (given intraperitoneally) were diisopropylfluorophosphate (DFP), isopropyl methylphosphonofluoridate (sarin) and dichlorovinyl phosphate (DDVP), pralidoxime (PAM) and obidoxime. 2. An exposure to low environmental temperature decreased the effectiveness of atropine-oxime therapy in OP poisoned rats and mice, evaluated by means of acute LD50 values. 3. The lowering of environmental temperature did not influence the ability of PAM to reactivate tissue cholinesterase in rats intoxicated by 0.5 x LD50 doses of DFP. 4. The acute toxicity of atropine and oximes was not affected by cold environment in rats, but in mice it was increased by 1.1-2.1 times. 5. The decrease in the effectiveness of atropine-oxime therapy at cold environment may be explained by the observation that the cold temperature sensitizes the animals to the inhibition of brain acetylcholinesterase by OP.

The LD50s and ED50s for inhibition of acetylcholinesterase (AChE) in whole mouse brain by DFP (diisopropylfluorophosphate), sarin (methylphosphonofluoridic acid 1-methyl ethyl ester), soman (methylphosphonofluoridic acid 1,2,2-trimethyl propyl ester), and tabun (dimethylphosphoramidocyanidic acid ethyl ester) were compared after iv administration. The LD50s of DFP, sarin, soman, and tabun in ICR (Institute for Cancer Research) mice were 3.40, 0.109, 0.042, and 0.287 mg/kg, respectively. The recovery of AChE activity in whole mouse brain after sub-LD50 doses of these agents was slow and did not reach control values by 14 d after iv administration. AChE activity was inhibited in a dose-dependent manner in whole mouse brain, as well as in six brain regions (cortex, hippocampus, striatum, midbrain, medulla-pons, and cerebellum). None of these brain areas appeared to be particularly sensitive to AChE inhibition. The ED50s for DFP, sarin, soman, and tabun for inhibition of AChE in whole mouse brain were approximately 19, 38, 69, and 66% of their respective LD50s. Because of the differential potencies between lethality and inhibition of AChE, it is concluded that the lethality of these agents is due to more factors than simply the inhibition of AChE within the brain.

1. Rats were used for studies on organophosphate (OP) toxicity both in acute and chronic cold exposure. Furthermore the effects of OPs on tissue acetyl- and butyrylcholinesterase activities were studied in the cold environment. 2. No change in the toxicity of dichlorovinyl phosphate (DDVP) was observed whereas that of diisopropylphosphofluoridate (DFP) increased 1.5-fold at +5 degrees C. 3. Chronic exposure to cold produced no change in DFP toxicity. 4. The survival time in acute cold exposure (1.1 x LD50 DFP) was longer than in chronic exposure or at +20 degrees C. 5. In control rats, chronic cold exposure increased blood BCHE and decreased BCHE in lungs. 6. A dose-dependent inhibition of cholinesterases was observed. 7. AcChE in the liver of chronically cold exposed rats was more sensitive to DFP inhibition compared to acute exposure. 8. Blood AcChE activity correlated only to AcChE in brain and lungs in rats.

The neurotoxicities of single doses of a chemical warfare agent VX [phosphonothioic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl) O-ethyl ester], a metabolite of the agricultural chemical parathion, paraoxon, PO (phosphonothioic acid, diethyl paranitrophenyl ester), and the known neuropathic agents DFP] phosphorofluoridic acid, bis(1-methylethyl) ester] and TOCP (phosphoric acid, tri-o-tolyl ester) were compared in the chicken. Single injections (subcutaneous, sc) of VX as high as 150 micrograms/kg (5 times the LD50, intramuscular, im) were tolerated by laying tens if atropine and 2-pralidoxime were used as antidotes before and immediately after injection. The 150 of VX for inhibition of chicken brain acetylcholinesterase was approximately 5 X 10(-10). Plasma acetylcholinesterase, but not butyrylcholinesterase, was depressed 2 h after injections of 2-20 micrograms VX/kg im without antidotes. Levels of plasma enzymes such as creatine kinase, indicative of tissue damage, were increased after exposure to both VX and PO. Injections of up to 150 micrograms/kg of VX with antidotes did not cause locomotor or histological signs of organophosphorus-induced delayed neuropathy, but single injections of 400 mg TOCP/kg did.

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.

Acetylcholinesterases, butyrylcholinesterases, and carboxylesterases appear to form kinetically a homologous enzyme series with respect to many substrates and inhibitors. The present paper evaluates the interaction of aprophen with acetylcholinesterases, butyrylcholinesterases, and carboxylesterases with respect to protecting the enzyme from organophosphate and carbamate inhibition, accelerating pralidoxime iodide (2-PAM) regeneration of the diisopropylphospho-enzyme, and comparing the inhibition and regeneration kinetics of a soluble mammalian acetylcholinesterase with that of bovine erythrocyte acetylcholinesterase. The irreversible inhibition kinetics of diisopropyl fluorophosphate (DFP) and eserine inhibition of fetal bovine serum acetylcholinesterase were typical of other acetylcholinesterases as indicated by the bimolecular inhibition rate constants, ki, of 7.7 +/- 1.3 X 10(4) M-1 min-1 and 2.9 +/- 1.7 X 10(6) M-1 min-1, respectively. Similarly, the bimolecular regeneration rate constant, kr, for 2-PAM regeneration of the diisopropylphospho-acetylcholinesterase was 14.7 M-1 min-1. The bimolecular rate constants, ki and kr, were not statistically perturbed when the reaction was monitored in the presence of aprophen with the fetal bovine serum acetylcholinesterase. Human serum butyrylcholinesterase was partially protected from DFP inhibition by aprophen with no detectable change in the bimolecular inhibition rate constant, ki. The regeneration of the diisopropylphospho-butyrylcholinesterase by 2-PAM was accelerated in the presence of aprophen by a factor of 2.7 over that of 2-PAM alone (8.4 +/- 2.2 M-1 min-1 to 23.1 +/- 2.6 M-1 min-1 respectively). Neither the inhibition (DFP) nor the regeneration (2-PAM) kinetics observed for the carboxylesterase was perturbed by the presence of aprophen.

The protective action of eseroline--(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3 a, 8-trimethyl-pyrrolo[2,3-b]indol-5-ol--salicylate against (DFP) diisopropyl fluorophosphate and carbamate poisoning of cholinesterases (ChEs) has been examined in-vitro with human erythrocytes and purified preparations of electric eel acetylcholinesterase (AChE) and of horse serum butyrylcholinesterase (BCHE), and in-vivo using mice. Eseroline afforded 50% protection (ED 50) of erythrocyte AChE against inactivation by 1 microM DFP, physostigmine or neostigmine, at concentrations of 4.3, 22 and 23.5 microM, respectively, while for eel AChE protection against 10 and 30 microM DFP, 0.3 and 1 microM physostigmine and 1 microM neostigmine the eseroline ED 50 values were 0.3, 0.4, 0.7, 1.9 and 5.6 microM, respectively. On the other hand, up to 0.3 mM eseroline did not appreciably affect the inhibitory action of the same drugs on horse serum BCHE. Eseroline concentrations in the range 0.1-1 mM were able to reactivate 20-42% of erythrocyte AChE previously inhibited by 100 microM physostigmine, but failed to reactivate the DFP (10 microM)-pretreated enzyme to any extent. Finally, eseroline salicylate injected into mice (10 mg kg-1 s.c.) protected an average of 82 and 26% of the animals against lethal doses of DFP (7 mg kg-1 s.c.) and physostigmine sulphate (1 mg kg-1 i.p.) respectively, which were administered 15 min later. These results indicate that the protective activity of eseroline correlates well with its own anti-ChE profile, and that the effectiveness of the protection depends largely on the rate of AChE inhibition by the agents used to inactivate the enzyme.

Rats treated daily with diisopropylfluorophosphate (DFP) (0.5 mg/kg, sc), an inhibitor of acetylcholinesterase (AChE) activity, exhibited the symptoms of cholinergic hyperactivity between Days 3 and 5 similar to those observed 15 min after a single acute dosage (1.5 mg/kg, sc). A significant (p less than 0.05) decrease in the activities of both AChE and cholinesterase (BuChE) (greater than 80%) occurred in muscles and in brain regions and of aliesterases in liver (greater than 92%) at this time. Further administration of DFP (0.5 mg/kg, for 7-14 days) led to behavioral tolerance, where symptoms of toxicity disappeared such as muscle fasciculations, tremors, and muscle necrosis. The activity of aliesterases in liver and AChE in muscles significantly (p less than 0.01) recovered, while no such recovery was seen in brain AChE. DFP toxicity was potentiated in rats that were pretreated with BuChE inhibitors, such as iso-OMPA (3 mg/kg, sc) or mipafox (0.05 mg/kg, sc), 30 min prior to DFP (0.5 mg/kg, sc). The severity of cholinergic hyperactivity and inhibition of aliesterase in liver, AChE and BuChE activity in brain and muscles was greater when compared to the effects of DFP alone. Both iso-OMPA and mipafox completely abolished the tolerance development to DFP, since no animal survived more than 5 days of combined treatment. The observed adaptation to DFP toxicity appears to be due to recovery of aliesterase, BuChE, and AChE activity as well as decreased nicotinic binding sites at the neuromuscular junction, as previously reported.

We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10(-5) M eserine) that differs from acetylcholinesterase (AChE, EC 3.1.1.7) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10(-5) M BW 284C51, but rapidly inactivated by 10(-8) M diisopropylfluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 X 10(-3) M AcSCh. (d) It does not cross-react with anti-AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the "nonspecific" or pseudocholinesterase (pseudo-ChE, EC 3.1.1.8) that exists in other species, although it is closer to "true" AChE than classic pseudo-ChE in several respects. Thus, it shows the highest Vmax with acetyl-, and not propionyl- or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo-ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo-ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo-ChE exists in several molecular forms, including collagen-tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo-ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X-100 within days.

Cholinesterases in hen brain were characterized with respect to inhibition kinetics and substrate specificity. Three organophosphorus inhibitors were used: diethyl p-nitrophenyl phosphate (Paraoxon, E 600), di-isopropylphosphorofluoridate (DFP), and N,N'-di-isopropylphosphorodiamidic fluoride (Mipafox). The kinetics of irreversible cholinesterase inhibition were studied using two substrates, acetylthiocholine and butyrylthiocholine. The inhibition curves were analysed by the method of iterative elimination of exponential functions. Final classification of the different enzymes was done by combining two inhibitors in sequential inhibition expts. Six cholinesterases were shown to hydrolyse choline esters in hen brain, one was identified as acetylcholinesterase (EC 3.1.1.7) and one as cholinesterase (EC 3.1.1.8). Four enzymes can be classified as intermediate type cholinesterases according to their substrate specificity and to their inhibition constants. The possible role of different brain cholinesterases for the development of atypical symptoms following organophosphate intoxication is discussed.

Compounds which enhance cholinergic activity have been reported to interact with opioid drugs. We have shown, using the hot-plate test in mice that di-isopropylfluorophosphate potentiates the antinociceptive activity of alfentanil but has no effect on the activity of morphine or fentanyl. Administration of atropine and pralidoxime as a treatment for DFP poisoning does not reverse this effect, and itself potentiates morphine antinociception. The results suggest that a cholinergic/opioid interaction is dependent on the opioid studied, and may have clinical importance when opioid drugs are required in patients poisoned by irreversible anticholinesterases.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.