Inhibitor Report for: Paraoxon

Acetylcholinesterase (EC 3.1.1.7; AChE), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission, be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human AChE (hAChE) in solution occurs through a C-terminal 4-helix bundle (4HB) at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R(P) enantiomer of sarin promotes a ten-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6 or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of a S(P)-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS (TR-SAXS) occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wild-type hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket towards the 4HB dimerization interface 25 A away.

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.

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.

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of =360 (to a k(cat)/K(M) of =7 x 10(5) M(-1) s(-1)) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.

The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.

Acetylcholinesterase (EC 3.1.1.7; AChE), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission, be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human AChE (hAChE) in solution occurs through a C-terminal 4-helix bundle (4HB) at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R(P) enantiomer of sarin promotes a ten-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6 or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of a S(P)-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS (TR-SAXS) occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wild-type hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket towards the 4HB dimerization interface 25 A away.

Lipases (E.C. 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates such as triacylglycerols and phospholipids. Candida antarctica Lipase B (CALB) acts in aqueous as well as in low-water media, thus being of considerable biochemical significance with high interest also for its industrial applications. The hydrolysis reaction follows a two-step mechanism, or 'interfacial activation', with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Once positioned within the catalytic triad, substrates are then hydrolysed, and products released. However, the intermediate steps of substrate transfer from the lipidic-aqueous phase to the enzyme surface and then down to the catalytic site are still unclear. By inhibiting CALB with ethyl phosphonate and incubating with glyceryl tributyrate (2,3-di (butanoyloxy) propyl butanoate), the crystal structure of the lipid-enzyme complex, at 1.55A resolution, shows the tributyrin in the limbus region of active site. The substrate is found 10A above the catalytic Ser, with the glycerol backbone pre-aligned for further processing by key interactions via an extended water network with alpha-helix10 and alpha-helix5. The findings offer new elements to elucidate the mechanism of substrate recognition, transfer and catalysis of Candida antarctica Lipase B (CALB) and lipases in general.

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100degC and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl--D-xylopyranoside monoacetates as substrates in a -xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 resolution, respectively, revealing a classicalpha/beta-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 , respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.

Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicated in roles that include plant defense, plant development, and secondary metabolism. We have cloned, overexpressed, purified, and crystallized a carboxylesterase from the kiwifruit species Actinidia eriantha (AeCXE1). The structure of AeCXE1 was determined by X-ray crystallography at 1.4 A resolution. The crystal structure revealed that AeCXE1 is a member of the alpha/beta-hydrolase fold superfamily, most closely related structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an 11 A deep hydrophobic gorge, contains the conserved catalytic triad residues Ser169, Asp276, and His306. Kinetic analysis using artificial ester substrates showed that the enzyme can hydrolyze a range of carboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties. This preference was supported by the discovery of a three-carbon acyl adduct bound to the active site Ser169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon (IC50 = 1.1 muM) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 muM). The structure of AeCXE1 with paraoxon bound was determined at 2.3 A resolution and revealed that the inhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of the enzyme. The structural information for AeCXE1 provides a basis for addressing the wider functional roles of carboxylesterases in plants.

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.

A series of achiral, chiral, and racemic mixtures of paraoxon analogues containing various combinations of methyl, ethyl, isopropyl, or phenyl substituents were synthesized as probes of the stereochemical constraints within the active site of phosphotriesterase. The kinetic constants for these paraoxon analogues with the enzyme varied significantly with the size of substituents surrounding the phosphorus center. These results indicate that binding and catalysis depend significantly on the relative size and orientation of the two subsites that must accommodate the coordination of the alkyl or aryl substituents within the enzyme active site. Individual enantiomers of paraoxon analogues were also synthesized and the stereochemical specificity for phosphotriesterase determined. In general, the kinetic constants, kcat and kcat/Km, for the (-)-enantiomers of these phosphotriesters were 1-2 orders of magnitude greater than the (+)-enantiomers. In every case, the preferred isomer is of the SP-configuration. For example, the kcat/Km for SP-(-)-ethyl phenyl p-nitrophenyl phosphate is 1.8 x 10(8) M-1 s-1 but is only 1.8 x 10(6) M-1 s-1 for the RP-(+)-isomer. These results suggest that one enantiomer is positioned for hydrolysis more favorably than the other enantiomer. The inactivation of acetylcholinesterase with the same series of organophosphate nerve agents was also measured. The stereoisomer that more rapidly inactivates human acetylcholinesterase is hydrolyzed more slowly than its enantiomer by the phosphotriesterase.

The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate nerve agents such as paraoxon (diethyl p-nitrophenyl phosphate) with a turnover number of approximately 10(4) s(-1). The active site of the enzyme has been shown to be composed of a binuclear Zn2+ complex with a bridging hydroxide. The utilization of chiral phosphotriesters has demonstrated that the overall hydrolytic reaction occurs with net inversion of stereochemistry at the phosphorus center. The stereochemical constraints of the active site have been probed by the synthesis and characterization of paraoxon analogs. One or both of the two ethoxy substituents of paraoxon have been replaced with various combinations of methyl, isopropyl, or phenyl groups. Racemic mixtures and individual enantiomers were tested as substrates for the phosphotriesterase. In general, the kinetic constants (k(cat) and k(cat)/Km) for the (-)-enantiomers were one to two orders of magnitude greater than the (+)-enantiomer. Conversely, acetylcholinesterase was more rapidly inactivated by the (+)-enantiomers than the (-)-enantiomers. These results were examined in the context of the three-dimensional structure of the bacterial phosphotriesterase.

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.

1. Paraoxon concentration was estimated by means of inhibition kinetics observed with electric eel acetylcholinesterase (AChE) which was determined by a modified Ellman procedure. In human plasma, paraoxon was stabilized by inactivation of paraoxonase with EDTA and aluminon and by inhibition of butyrylcholinesterase with ethopropazine. Paraoxon (1-50 ng) was recovered at 86+/-1.7% (mean+/-s.e.m.) in ether extracts from 0.5 ml samples of spiked stabilized plasma. It could be stored without loss at - 20 degrees C for at least 1 month. 2. The enzyme-based assay was applied to follow the paraoxon plasma concentrations in three suicidal patients with severe parathion poisoning. In poisoning with excessive doses and initial paraoxon concentrations above 500 nM, therapeutic obidoxime concentrations of approximately 10 microM failed to essentially reactivate erythrocyte AChE in vivo, while reactivatability ex vivo was nearly complete. With the plasma concentrations of paraoxon dropping below 100 nM, however, reactivation by obidoxime became significant. Unexpectedly, paraoxon levels occasionally reincreased during treatment and resulted in re-inhibition of AChE, bearing some resemblance to the Intermediate Syndrome. 3. The paraoxon concentrations measured fitted satisfactorily the values calculated from the kinetic constants previously obtained for AChE inhibition and obidoxime-induced reactivation in vitro. This indicates that diethylphosphoryloxime formation during obidoxime-induced reactivation does not markedly contribute to the re-inhibition of AChE as observed in vitro.

1. The effects of two model inducers of the cytochrome P450 system, phenobarbital (PB) and beta-naphthoflavone (NF), on the toxicity of paraoxon were studied in rats. 2. Paraoxon toxicity was measured by inhibition of brain acetylcholinesterase (AChE) activity. 3. PB treatment did not affect the toxicity of paraoxon, whereas NF increased the inhibition of brain AChE. PB administration slightly increased the activities of some peripheral cholinesterases and carboxylesterases, as well as liver microsomal paraoxonase (Pxase). 4. NF administration, in contrast, decreased the activities of peripheral esterases. Serum Pxase activity was reduced by both inducers. 5. Hepatic CYP2B and CYP1A were markedly induced by PB and NF, respectively. 6. Cytochrome P450 isoenzymes induced by PB or NF seemed not to be critical in the detoxification of paraoxon in vivo. NF caused a general reduction of peripheral esterases, which led to an increase in paraoxon toxicity. 7. The results indicated the great importance of peripheral cholinesterases and carboxylesterases as a detoxifying mechanism of paraoxon. The role of serum paraoxonase was not critical.

Trout were exposed to an aqueous solution of 75 ng/ml paraoxon for 5 days at 12 degrees C. The relationships among paraoxon concentration in water and target organs, AChE inhibition, and carboxylesterase (CaE) detoxification of paraoxon were characterized quantitatively by development of a PBPK-PD model. The PKPD model structure consisted of brain, heart, liver, kidney, and remainder of the body, which were interconnected by blood circulation. The paraoxon tissue/blood partition coefficients were: plasma/water, 1.46; liver/plasma, 5.89; brain/plasma, 3.90; heart/plasma, 2.91; kidney/plasma, 0.45; and blood/plasma, 0.91. Turnover of AChE was characterized from a dose-response study, in which its zero-order synthesis rate and first-order degradation rate constant were determined in several tissues; for brain they were 7.67 pmol/min and 7.31 x 10(-5) hr(-1). The uptake and depuration clearances of paraoxon (Cl(u) = 0.651 and Cl(d) = 0.468 ml min(-1) g body wt(-1)) were determined using a compartmental model. During continuous water exposure to paraoxon, AChE activity in the tissues declined to new steady state values that were maintained by the synthesis of new AChE. CaE was shown by simulation to be an important pathway for detoxification of paraoxon.

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.

To establish the dose dependency of phospholipase A2 (PLA2) inhibition by the organophosphorus compound (OPC) paraoxon (POX), human platelet membranes were incubated after Ca2+ removal (to inactivate the PLA2) with 0.3, 1 and 3 microg ml(-1) POX for 5, 30 and 60 min each. The PLA2 activity (pmol mg[-1] protein min[-1]) was measured after subsequent enzyme reactivation. The PLA2 activity in native platelets was considered to be 100%; all other measured values are expressed as a percentage thereof. Data were analysed with the Mann-Whitney Wilcoxon rank order test and ANOVA. Statistical significance was assumed for P < or = 0.01. Paraoxon inhibited in a dose-dependent manner the PLA2 activity. Different incubation times of the inactive PLA2 with POX did not have any additional effect on the activity reduction after activation. At the tested POX concentrations the PLA2 activity was 42 +/- 5.4%, 29 +/- 3.4% and 15 +/- 6.6%, respectively. The corresponding butyrylcholine esterase (BChE) activities were <<1% of the baseline activity. Phospholipase A2 is less sensitive to POX inhibition than BChE and, at clinically achievable POX concentrations, shows a clear dose dependency. Further work is needed to elucidate the exact mechanism and time dependency of the phenomenon.

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.

In rats, the phosphorothionate insecticide parathion exhibits greater toxicity than chlorpyrifos, while in catfish the toxicities are reversed. The in vitro inhibition of brain acetylcholinesterase (AChE) by the active metabolites of the insecticides and the rates at which these inhibitor-enzyme complexes undergo reactivation/ aging were investigated in both species. Rat AChE was more sensitive to inhibition than catfish AChE as demonstrated by greater bimolecular rate constants (ki) in rats than in catfish. In both species, chlorpyrifos-oxon yielded higher ki's than paraoxon. The higher association constant (KA) of chlorpyrifos-oxon than paraoxon in both species and the lack of significant differences in the phosphorylation constants (kp) suggest that association of the inhibitor with AChE is the principal factor in the different potencies between these two inhibitors. In catfish, the ki of chlorpyrifos-oxon was 22-fold greater than that of paraoxon, while in rats it was 9-fold greater, suggesting that target site sensitivity is an important factor in the higher toxicity of chlorpyrifos to catfish but not in the higher toxicity of parathion to rats. No spontaneous reactivation of phosphorylated catfish AChE occurred and there were no differences in the first oder aging constants (ka) between compounds. For phosphorylated rat AChE, there were no differences in the first order reactivation constants (kr) but the ka for chlorpyrifos-oxon was significantly greater than that for paraoxon. This difference suggests that the steric positioning of the diethyl phosphate in the esteratic site is not the same between the two compounds, leading to differences in aging.

Orthologous E3 and EST23 carboxylesterases have been enriched over 200-fold from organophosphate (OP) susceptible strains of Lucilia cuprina and Drosophila melanogaster, respectively. Mutants of E3 are associated with OP resistance but no resistance mutations of EST23 are known. The behaviours of the two enzymes were very similar during purification which involved differential centrifugation followed by three or four ion exchange and gel filtration chromatographic steps. Nondenaturing polyacrylamide gel electrophoresis and histochemical staining for esterase activity revealed no other esterases in the enriched material. Two-dimensional polyacrylamide gel electrophoresis (native followed by denaturing) showed that a major 70-kDa component of each preparation comigrates with E3 and EST23 activities, respectively. Kinetic properties of the enzymes are also very similar. Estimates of Km, Kcat, and Kcat/Km for alpha-naphthyl acetate are 42 +/- 18 μM, 19 sec-1, and 4.6 x 10(5) M-1 sec-1, respectively, for E3, and 62 +/- 25 μM, 23 sec-1, and 3.7 x 10(5) M-1 sec-1, for EST23. Both enzymes are potently inhibited by dibrom and less potently by another OP, diisopropylflurophosphate. E3 is also potently inhibited by paraoxon, whereas EST23 is at least 8-fold less susceptible to inhibition by paraoxon. This supports previous analyses of crude homogenates which showed that E3 is more susceptible to inhibition by paraoxon and fenitrooxon than is EST23 or the target site for OP action, acetylcholinesterase. It is proposed that the unusual affinity of E3 for such OPs is a necessary precondition for mutations that enable it to confer OP resistance on L. cuprina.

The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via A-esterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAE Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 degrees) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAE gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (Ph 7.7 at 5 degrees). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 4.5 to 5.6. Following native-PAGE analysis, activity stains with beta-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10-5 M) verified that A-esterase activity was associated with both proteins. Spectropho-tometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.

Paraoxon (O,O-diethyl O-p-nitrophenyl phosphate) is the neurotoxic metabolite of the insecticide parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate). The effects of organophosphorus compounds on peripheral synapses have been attributed to inhibition of cholinesterase and to direct actions on muscarinic and nicotinic receptors, but less is known about the actions of organophosphorus compounds, including paraoxon, in the central nervous system. We investigated initially the effects of paraoxon on spontaneous transmitter release by recording miniature postsynaptic currents (MPSCs) from cultured rat hippocampal neurons using the whole-cell mode of the patch-clamp technique. Paraoxon (0.3 microM) in the presence of tetrodotoxin (0.3 microM) and atropine (1 microM) caused a significant increase in the frequency of gamma-aminobutyric acid- and glutamate-mediated MPSCs, but did not change the peak amplitudes or decay-time constants of these MPSCs. In contrast, application of nicotinic agonists or antagonists did not change the MPSC frequency. The presynaptic effect of paraoxon shown here was not mediated by actions on muscarinic or nicotinic receptors, or by elevated acetylcholine levels secondary to inhibition of cholinesterase. In addition, agonists were applied to assess the postsynaptic effects of paraoxon on excitatory and inhibitory amino acid receptors. Paraoxon (30 microM-1 mM) blocked the ion channels of glycine, gamma-aminobutyric acidA, N-methyl-D-aspartic acid and nicotinic acetylcholine receptors, but not the ion channels of kalnate- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. The combined effects of paraoxon on spontaneous transmitter release and on the functions of several ligand-gated receptors may constitute mechanisms relevant to the neurotoxicity 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

The inhibition and aging of acetylcholinesterase (AChE) in fingerling channel catfish (lctalurus punctatus) brain tissue was studied after single in vivo exposures to high levels of chlorpyrifos (0.25 mg/L), chlorpyrifos-oxon (7 micrograms/L), parathion (2.5 mg/L), or paraoxon (30 micrograms/L). Exposure to both parent compounds produced identical initial inhibition (95%), but in the later sampling times there was significantly more inhibited AChE in the chlorpyrifos-treated fish than in the parathion-treated fish (47% and 28%, respectively, on d 16). There were higher levels of aged AChE following chlorpyrifos exposure than following parathion exposure, but differences were not significant. Exposure to both oxons produced initial inhibition greater than 90%, and patterns of recovery and aging were statistically similar between both compounds; no significant inhibition was observed after d 11. The similar patterns of inhibition, recovery, and aging between the two oxon treatments, which have similar lipophilicities, suggest that the greater amount of AChE inhibition and aging observed in the chlorpyrifos-treated fish compared with the parathion-treated fish probably results from the higher lipophilicity of chlorpyrifos than of parathion. Overall, the prolonged brain AChE inhibition exhibited in catfish exposed to phosphorothionates is not the result of aging of the inhibited enzyme but is the result of either a slow rate or a lack of spontaneous reactivation.

Annealed murine erythrocytes were employed as a carrier model to antagonize the toxic effects of organophosphorus agents. These resealed cells containing a recombinant phosphotriesterase provided striking protection against the lethal effect of paraoxon, an active metabolite of an agricultural pesticide, parathion. Phosphotriesterase hydrolyzes paraoxon to the less-toxic 4-nitrophenol and diethylphosphate. This enzyme was encapsulated into carrier erythrocytes by hypotonic dialysis with subsequent resealing and annealing. These carrier cells were administered to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recipient animals were subsequently challenged with paraoxon and a marked protection was noted. Protection of free enzyme and encapsulated enzyme was compared and the encapsulated enzyme was found to persist longer and possess much greater efficacy. Less serum cholinesterase inhibition also was observed with this enhanced protection. These results indicate that the erythrocyte carrier alone is quite effective in the antagonism of organophosphorus intoxication. Moreover, when these carrier cells were administered in combination with 2-PAM and/or atropine, a marked synergism was observed.

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 microM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of beta-oxidation might be correlated with the lower level of acetylcholine synthesis.

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.

OBJECTIVE (1) Retrospective evaluation of the clinical course of carbamate poisoning and the effect of oxime therapy in children. (2) In vitro study of the effect of oximes on the reactivation of carbamylated cholinesterase. DESIGN: (1) Clinical survey: The records of 26 children intoxicated with carbamates were examined retrospectively. The poisoning agents in all cases were positively identified as methomyl or aldicarb by gas chromatography-mass spectrometry. (2) Laboratory study: The direct effect of obidoxime and of pralidoxime on acetylcholinesterase activity in vitro was investigated in normal human packed red blood cells pretreated with an organophosphate (paraoxon) or a carbamate (aldicarb or methomyl). CLINICAL SETTING: Pediatric intensive care unit of a teaching hospital. PATIENTS: Twenty-six infants and young children (aged 1 to 8 years) admitted to the pediatric intensive care unit with severe carbamate intoxication. INTERVENTIONS: All cases had been treated with repeated doses of atropine sulfate (0.05 mg/kg) administered every 5 to 10 minutes until muscarinic symptoms disappeared. Obidoxime chloride (Toxogonin, 6 mg/kg) was administered on admission, and again after 4 to 5 hours. RESULTS: Predominant symptoms were related to central nervous system and nicotinic effects. All the patients showed marked improvement within several hours and recovered completely within 24 hours. None of the children deteriorated and none showed exacerbation of cholinergic symptoms after obidoxime treatment. In vitro, oximes reactivated acetylcholinesterase inhibited with paraoxon, whereas no significant effect of oximes on carbamylated enzyme activity was observed. CONCLUSIONS: Based on the recovery of all cases, as compared with other reports of carbamate poisoning treated with atropine alone, it is concluded that, in the case of aldicarb or methomyl poisoning, oxime therapy apparently does not contribute to the recovery of poisoned patients. In cases of poisoning by an unknown pesticide or of mixed poisoning, oxime therapy can prove beneficial because no negative effects of the therapy can be discerned.

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.

Rats were administered high sublethal intraperitoneal dosages of the phosphorothionate insecticides parathion, methyl parathion, and chlorpyrifos, and their oxons. Acetylcholinesterase activities in cerebral cortex and medulla oblongata and aliesterase activities in liver and plasma were monitored at 2 hr and 1, 2, and 4 days after exposure. The maximal inhibition of brain acetylcholinesterase activity was not immediate with parathion and chlorpyrifos, reflecting the time required for bioactivation of the phosphorothionates as well as the effectiveness of the aliesterases to inactivate much of the hepatically generated oxons. In contrast, brain acetylcholinesterase activities were more quickly inhibited following administration of paraoxon and chlorpyrifos-oxon, which do not require bioactivation. Brain acetylcholinesterase was also rapidly inhibited following administration of methyl parathion and methyl paraoxon, reflecting the low sensitivity of the aliesterases to methyl paraoxon. Aliesterases were inhibited to a greater extent than acetylcholinesterase at each sampling time with parathion and chlorpyrifos and their oxons, whereas the reverse was true with methyl parathion and methyl paraoxon. All of the above patterns correlate with the in vitro sensitivities of acetylcholinesterase and aliesterases to the oxons. The very prolonged inhibition of esterase activities following chlorpyrifos treatment probably results from its substantially greater lipophilicity compared to the other compounds, which would allow it to be stored and released for gradual bioactivation. The data reported indicate that the disposition and effects of different phosphorothionate insecticides will be influenced by the sensitivities of target and nontarget esterases for their oxons and by their lipophilicity, and that predictions of in vivo responses can be made from in vitro data.

A number of lines of evidence suggest that serum paraoxonase is protective against poisoning by organophosphorus substrates of this enzyme. Birds that have very low levels of paraoxon hydrolyzing activity in their sera are very susceptible to parathion poisoning. Rabbits, which have a sevenfold higher enzyme level compared with rats, have a fourfold higher resistance to paraoxon poisoning than rats. Rabbit paraoxonase hydrolyzes chlorpyrifos-oxon with a much higher turnover number than does rat paraoxonase, resulting in a very high resistance of rabbits to chlorpyrifos toxicity. Direct tests of paraoxonase protection have been carried out by injecting purified rabbit enzyme into rats. The protection achieved was higher for chlorpyrifos-oxon than for paraoxon, probably due to the high hydrolytic activity of the rabbit enzyme for chlorpyrifos-oxon. In humans, a substrate-dependent polymorphism of serum paraoxonase is observed, where one isoform of paraoxonase has a high turnover number for paraoxon and the other a low turnover number. Both isoforms appear to hydrolyze chlorpyrifos-oxon and phenylacetate at the same rate. Cloning and sequencing of the human paraoxonase cDNAs has elucidated the molecular basis of the polymorphism. Arginine at position 192 determines high paraoxonase activity, and glutamine at this position, low paraoxonase activity. In addition to the polymorphism, a 13-fold variation in serum enzyme levels within a given genetic class is seen. The experiments reported here demonstrate that rabbit paraoxonase injected into mice provides protection against the parent insecticide chlorpyrifos as well as the toxic oxon. These results suggest that serum paraoxonase status may serve as a biomarker for insecticide susceptibility in humans.

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 neuropathic potential of acute and repeated exposures of the phosphoramidates tabun (GA) and isofenphos (IFP), of diisopropyl fluorophosphate (DFP) and paraoxon (PO) were examined in the hen with treatments for up to 90 days via intramuscular injections of the highest tolerated doses with atropine protection. Plasma acetylcholinesterase (AChE), non-specific butyrylcholinesterase (BChE) and creatine kinase (CK) activities were measured in order to monitor whether the compounds were present at biologically active concentrations. Locomotor behavior was observed and tissues from the peripheral and central nervous systems were examined for signs of organophosphate-induced delayed neuropathy (OPIDN). No behavioral or histological evidence of OPIDN was observed after treatments with GA, IFP, PO, saline or atropine sulfate. DFP-treated birds displayed locomotor and neuropathological signs of OPIDN with a no effect level (NOEL) between 25 and 50 micrograms/kg.

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.

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.

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.

The effects of lethal (2.0 mg/kg) and high sublethal (1.3 mg/kg) dosages of the organophosphate acetylcholinesterase (AChE) inhibitor paraoxon on FR10 performance rate was determined 1 and 2 days after intoxication. The lethal doses were antidoted with either centrally acting atropine sulfate (AS), or atropine methyl bromide (AMB) or atropine methyl nitrate (AMN), both quaternary salts and not expected to act centrally. AChE inhibition in the brain was about 35-60% on the second day after treatment. AS yielded a small transient depression in performance, while AMB and AMN yielded severe deficits, with incomplete recovery. Performance was depressed by 1.3 mg/kg paraoxon by 52% and 34% on days 1 and 2, respectively, while performance was more greatly depressed by the lethal dose, especially with the noncentrally acting antidotes: AS, 67 and 48%; AMB, 81 and 55%; AMN, 91 and 78%. However, a low dose of AS with 2 mg/kg paraoxon resulted in very severe, nonrecovering deficits. A lethal dose of the nonpersistent anti-AChE eserine sulfate, antidoted with a low dose of AS, yielded no deficits. Thus, a high level, acute intoxication with paraoxon yields behavioral deficits which are attenuated by high levels of a centrally acting muscarinic receptor antagonist. The paraoxon-induced performance deficits or their recovery do not correlate directly with AChE inhibition.

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.

Hydrolytic "A"-esterase activities of various tissues of rat (plasma, liver, kidney, brain and intestinal mucosa) against selected OP esters of diverse structure as potential substrates (paraoxon, di-n-propyl paraoxon, di-n-butyl paraoxon, chlorpyrifos oxon, di-(4-phenyl butyl) phosphorofluoridate and the chiral isomers of ethyl 4-nitrophenyl phenylphosphonate) were studied. We have developed a sensitive and widely applicable assay depending on measuring decline in residual inhibitory power of any chosen OP against horse serum cholinesterase: for seven compounds examined so far I50s against BCHE ranged from 0.07 to 70 nM, and it is easy to monitor loss of OP starting from an initial 25 microM concentration. Progressive destruction rates were always highest in liver and plasma with activity sometimes detectable in kidney, brain but not in intestinal mucosa, but the ratios of activity between tissues differed for different substrates. At 25 microM/37 degrees/pH 7.2 hydrolysis rates ranged from 8500 nmol/min/g liver for di-(4-phenylbutyl) phosphorofluoridate down to 0.8 nmol/min for the butyl analogue of paraoxon; the rate for L(-) isomer of EPN oxon (23 nmol/min/g liver) was greater than 2x that for the D(+) isomer and for paraoxon. From our data we conclude that several OP hydrolases exist whose identity may be further characterised by use of selective substrates

Two organophosphorus compounds, paraoxon and phenyl saligenin cyclic phosphate, as well as p-nitrophenol and phenol which are structurally related to paraoxon, were tested for their effects on interleukin 2 (IL2) production and responsiveness by rat splenocytes in vitro. Three of the four compounds inhibited mitogen-induced lymphocyte proliferation as well as IL2 production and responsiveness. However, phenyl saligenin cyclic phosphate produced maximal inhibition at a much lower concentration (0.5 microM) than p-nitrophenol (200 microM) or paraoxon (200 microM). Phenol was not inhibitory at any concentration tested (up to 250 microM). Since the production of and response to IL2 are key events in immune responses, compounds which suppress these events can be identified as potential suppressors of host resistance to disease.

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.

Esterases which can hydrolyse organophosphates without being inhibited by them are termed "A" esterases. Using paraoxon and pirimiphos-methyl oxon as substrates, high "A" esterase activity is found in the liver and plasma or serum of a range of mammalian species. In a study of serum "A" esterases of sheep and humans, over 80% of the activity separated into the high density lipoprotein (HDL) fraction following ultracentrifugation. When HDL fractions from sheep serum were run on Sepharose gel columns, most of the paraoxonase activity separated as a single peak of estimated molecular weight 360,000, which corresponds to that of HDL2 of humans. During the course of purification of "A" esterases by three different column procedures, contrasting esterase elution profiles were obtained with organophosphate and pyrethroid substrates. This was strong evidence for the existence of multiple forms of HDL "A" esterases. Levels of "A" esterase activity in plasma and liver of birds were much lower than those of mammals. This appears to be the main reason why birds are much more susceptible than mammals to organophosphates such as pirimiphos-methyl and diazinon which form active oxons that are good substrates for mammalian "A" esterases. No "A" esterase was detected in strains of rust red flour beetle (Tribolium castaneum) which were resistant to organophosphates. Similar observations have been made with strains of other insects resistant to organophosphates, raising the question to what extent esterases of this type are present in insects.

The toxicity of acephate to four species of aquatic insects, as well as the metabolism and cholinesterase-inhibiting properties of the chemical in the rat were studied. The results indicated that mayfly larvae were very sensitive to the toxic effects of acephate, whereas larvae of the stonefly, damselfly and mosquito were much less sensitive. In the rat, orally-administered acephate was rapidly absorbed from the intestines and severely inhibited the cholinesterases in the blood and brain. The enzymes began to recover after 24 hours, while the chemical was completely eliminated within three days. The amount of methamidophos observed in the liver was extremely low. The cholinesterase-inhibiting properties of acephate and methamidophos were compared in vitro to that of paraoxon, a known strong anticholinesterase. Enzymes from four vertebrates were used. In all cases, except one, acephate was found to be six orders of magnitude weaker than paraoxon, whereas methamidophos was three orders weaker. Trout brain cholinesterase was the exception; it was as sensitive to paraoxon as it was to methamidophos. Finally, four cholinesterases were inhibited with methamidophos, and their ability to reactivate spontaneously or to recover by induction with pyridine aldoxime methiodide (PAM) in vitro were determined. The results suggested that methamidophos-inhibited cholinesterases did not reactivate spontaneously; instead the enzymes remained inhibited either in a phosphorylated or an aged state. The significance of these results are discussed in relation to the use of acephate for forest insect pests.

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.

Using paraoxon and pirimiphos-methyl as substrates, much of the "A'-esterase activity of sheep serum was found to be in the high-density lipoprotein (HDL) fraction. A method was developed for the partial purification of "A'-esterases by the preparation of a lipoprotein fraction, followed by preparative polyacrylamide gel electrophoresis. The properties of the partially purified preparations of "A'-esterase were studied. Although four different preparations all contained a major protein unit, which resembled the core protein of HDL, there was evidence of differences between preparations with regard to substrate specificity, suggesting the existence of multiple enzyme forms. Gel filtration of serum samples indicated that paraoxonase activity is expressed by proteins with mol. wts greater than 200,000, strongly suggesting that the "A'-esterase activity of the lipoprotein fraction is present in one or more forms of HDL2. The dependence of "A'-esterase activity upon Ca2+ and the problem of esterase classification are discussed.

The position in porcine pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) of the serine reacting specifically with emulsified or micellar diethyl p-nitrophenyl phosphate has been investigated. This serine which appears to be involved in lipase adsorption to insoluble triglyceride interfaces, is at position 152 in the enzyme chain. The sequence around this amino acid is: His-Val-Ile-Gly-His-Ser-Leu-Gly.

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.

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.