An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005–3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis–Menten kinetics, with a K_m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis–Menten kinetics, with a k_i of 3048 nM⁻¹ h⁻¹, and a K_D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k_i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k_is of 1.2 and 19.3 nM⁻¹ h⁻¹, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.     

Concentration-dependent binding of chlorpyrifos oxon to acetylcholinesterase.

The organophosphorus insecticides have been known for many years to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase. The interactions of the activated, toxic insecticide metabolites (termed oxons) with acetylcholinesterase have been studied extensively for decades. However, more recent studies have suggested that the interactions of certain anticholinesterase organophosphates with acetylcholinesterase are more complex than previously thought since their inhibitory capacity has been noted to change as a function of inhibitor concentration. In the present report, chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) was incubated with human recombinant acetylcholinesterase in the presence of p-nitrophenyl acetate in order to better characterize kinetically the interactions of this oxon with enzyme. Determination of the dissociation constant, Kd, and the phophorylation rate constant, k2, for chlorpyrifos oxon with a range of oxon and p-nitrophenyl acetate concentrations revealed that Kd, but not k2, changed as a function of oxon concentration. Changes in p-nitrophenyl acetate concentrations did not alter these same kinetic parameters. The inhibitory capacity of chlorpyrifos oxon, as measured by ki (k2/Kd), was also affected as a result of the concentration-dependent alterations in binding affinity. These results suggest that the concentration-dependent interactions of chlorpyrifos oxon with acetylcholinesterase resulted from a different mechanism than the concentration-dependent interactions of acetylthiocholine. In the latter case, substrate bound to the peripheral anionic site of acetylcholinesterase has been shown to reduce enzyme activity by blocking the release of the product thiocholine from the active site gorge. With chlorpyrifos oxon, the rate of release of 3,5,6-trichloro-2-pyridinol is irrelevant since the active site is not available to interact with other oxon molecules after phosphorylation of Ser-203 has occurred.     

Interactive toxicity of the organophosphorus insecticides chlorpyrifos and methyl parathion in adult rats

The acute interactive toxicity following exposure to two common organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), was investigated in adult male rats. Oral LD1 values were estimated by dose-response studies (CPF = 80 mg/kg; MPS = 4 mg/kg, in peanut oil, 1 ml/kg). Rats were treated with both toxicants (0.5 or 1 x LD1) either concurrently or sequentially, with 4-h intervals between dosing. Functional signs of toxicity (1-96 h) and cumulative lethality (96 h) were recorded. Rats treated with CPF (1 x LD1) did not show any signs of toxicity although MPS (1 x LD1) elicited slight to moderate signs (involuntary movements) within 1-2 h. Concurrent exposure (LD1 dosages of both CPF and MPS) caused slight signs of toxicity only apparent between 24 and 48 h after dosing. When rats were treated sequentially with MPS first followed by CPF 4 h later, slight signs of toxicity were noted between 6 and 24 h, whereas reversing the sequence resulted in 100% lethality within 1 h of the second dosage. Following exposure to lower dosages (0.5 x LD1), the CPF first group showed higher signs of cholinergic toxicity compared with MPS first or concurrent groups. Cholinesterase inhibition in plasma, diaphragm, and frontal cortex was generally higher in rats treated sequentially with CPF first than in those treated initially with MPS from 4 to 24 h after dosing. Plasma and liver carboxylesterase inhibition at 4 h was also significantly higher in the CPF first (62-90%) compared with MPS first (22-43%) group, while at 8 and 24 h, there was no significant difference between any of the treatment groups. ChE inhibition assays to evaluate in vitro hepatic detoxification of oxons indicated that carboxylesterase (CE)- and A-esterase-mediated pathways are markedly less important for methyl paraoxon (MPO) than chlorpyrifos oxon (CPO) detoxification. CPF pretreatment blocked hepatic detoxification of methyl paraoxon while MPS pretreatment had minimal effect on hepatic CPO detoxification ex vivo. These findings suggest that the sequence of exposure to two insecticides that elicit toxicity through a common mechanism can markedly influence the cumulative action at the target site (acetylcholinesterase, AChE) and consequent functional toxicity.     

Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B(max) and K(d) for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B(max) did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B(max), would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K(d) represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K(d), and dimethylphosphorylation of Ser203 decreasing K(d). These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.     

Life without acetylcholinesterase: the implications of cholinesterase inhibitor toxicity in AChE-knockout mice

The acetylcholinesterase (AChE)-knockout mouse is a new tool for identifying physiologically relevant targets of organophosphorus toxicants (OP). If AChE were the only important target for OP toxicity, then mice with zero AChE would have been expected to be resistant to OP. The opposite was found. AChE−/− mice were more sensitive to the lethality of DFP, chlorpyrifos oxon, iso-OMPA, and the nerve agent VX. A lethal dose of OP caused the same cholinergic signs of toxicity in mice with zero AChE as in mice with normal amounts of AChE. This implied that the mechanism of toxicity of a lethal dose of OP in AChE−/− mice was the same as in mice that had AChE, namely accumulation of excess acetylcholine followed by overstimulation of receptors. OP lethality in AChE−/− mice could be due to inhibition of BChE, or to inhibition of a set of proteins. A search for additional targets used biotinylated-OP as a marker. In vitro experiments found that biotinylated-OP appeared to label as many as 55 proteins in the 100,000 × g supernatant of mouse brain. Chlorpyrifos oxon bound a set of proteins (bands 12, 41, 45) that did not completely overlap with the set of proteins bound by diazoxon (bands 9, 12, 41, 47) or dichlorvos (bands 12, 23, 24, 32, 44, 45, 51) or malaoxon (band 9). These results support the idea that a variety of proteins could be interacting with a given OP to give the neurotoxic symptoms characteristic of a particular OP.     

Concentration-dependent kinetics of acetylcholinesterase inhibition by the organophosphate paraoxon.

For decades the interaction of the anticholinesterase organophosphorus compounds with acetylcholinesterase has been characterized as a straightforward phosphylation of the active site serine (Ser-203) which can be described kinetically by the inhibitory rate constant k(i). However, more recently certain kinetic complexities in the inhibition of acetylcholinesterase by organophosphates such as paraoxon (O,O-diethyl O-(p-nitrophenyl) phosphate) and chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) have raised questions regarding the adequacy of the kinetic scheme on which k(i) is based. The present article documents conditions in which the inhibitory capacity of paraoxon towards human recombinant acetylcholinesterase appears to change as a function of oxon concentration (as evidenced by a changing k(i)), with the inhibitory capacity of individual oxon molecules increasing at lower oxon concentrations. Optimization of a computer model based on an Ordered Uni Bi kinetic mechanism for phosphylation of acetylcholinesterse determined k(1) to be 0.5 nM(-1)h(-1), and k(-1) to be 169.5 h(-1). These values were used in a comparison of the Ordered Uni Bi model versus a k(i) model in order to assess the capacity of k(i) to describe accurately the inhibition of acetylcholinesterase by paraoxon. Interestingly, the k(i) model was accurate only at equilibrium (or near equilibrium), and when the inhibitor concentration was well below its K(d) (pseudo first order conditions). Comparisons of the Ordered Uni Bi and k(i) models demonstrate the changing k(i) as a function of inhibitor concentrations is not an artifact resulting from inappropriate inhibitor concentrations.     

Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells

Previous work in our laboratory has shown that sub-lethal concentrations (1-10 μM) of chlorpyrifos (CPF), diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon (CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions similar to our previous work, sub-lethal concentrations (1-10 μM) of CPO were found to inhibit N2a cell differentiation. However, unlike previous studies with DZ and DZO, there was a high level of sustained inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly, further work will help to determine whether the persistent inhibition of AChE by CPO can account for the different effects induced by CPO and DZO on the levels of total and phosphorylated NFH.     

Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC₅₀ values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k_inact/K_I) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC₅₀ values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.     

Toxicokinetics of methyl paraoxon in the dog

The toxicokinetics of methyl paraoxon, the active metabolite of the organophosphorus insecticide methyl parathion, were studied in non-anaesthetized dogs after intravenous (2.5 mg/kg) and oral (15 mg/kg) administration of methyl paraoxon. After intravenous administration, distribution and elimination occured very rapidly and using the data from 5 min post-injection, the plasma concentration versus time curves could be fitted to a one-compartment open model. The mean half-life of elimination was 9.7 min, the average volume of distribution 1.76 l/kg and the average plasma clearance 126 ml/kg/min. After oral administration, peak plasma concentrations were obtained within 3–16 min, and the bioavailability varied from 5 to 71%. The hepatic extraction of methyl paraoxon measured in anaesthetized dogs, was high (70–92%). Comparison of the urinary excretion after intravenous and oral administration in two dogs indicated a gastrointestinal absorption of more than 60%. The kinetics of methyl paraoxon were linear in the dose range tested.     

Comparison of chlorpyrifos-oxon and paraoxon acetylcholinesterase inhibition dynamics: potential role of a peripheral binding site.

The primary mechanism of action for organophosphorus (OP) insecticides, like chlorpyrifos and parathion, is to inhibit acetylcholinesterase (AChE) by their oxygenated metabolites (oxons), due to the phosphorylation of the serine hydroxyl group located in the active site of the molecule. The rate of phosphorylation is described by the bimolecular inhibitory rate constant (k(i)), which has been used for quantification of OP inhibitory capacity. It has been proposed that a peripheral binding site exists on the AChE molecule, which, when occupied, reduces the capacity of additional oxon molecules to phosphorylate the active site. The aim of this study was to evaluate the interaction of chlorpyrifos oxon (CPO) and paraoxon (PO) with rat brain AChE to assess the dynamics of AChE inhibition and the potential role of a peripheral binding site. The k(i) values for AChE inhibition determined at oxon concentrations of 1-100 nM were 0.206 +/- 0.018 and 0.0216 nM(-1)h(-1) for CPO and PO, respectively. The spontaneous reactivation rates of the inhibited AChE for CPO and PO were 0.084-0.087 (two determinations) and 0.091 +/- 0.023 h(-1), respectively. In contrast, the k(i) values estimated at a low oxon concentration (1 pM) were approximately 1,000- and 10,000-fold higher than those determined at high CPO and PO concentrations, respectively. At low concentrations, the k(i) estimates were approximately similar for both CPO and PO (150-180 [two determinations] and 300 +/- 180 nM(-1)h(-1), respectively). This implies that, at low concentrations, both oxons exhibited similar inhibitory potency in contrast to the marked difference exhibited at higher concentrations. These results support the potential importance of a secondary peripheral binding site associated with AChE kinetics, particularly at low, environmentally relevant concentrations.     

Relative inhibitory potencies of chlorpyrifos oxon,chlorpyrifos methyl oxon,and mipafox for acetylcholinesterase versus neuropathy target esterase

The relative inhibitory potency (RIP) of an organophosphorus (OP) inhibitor against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) may be defined as the ratio [k(i)(AChE)/k(i)(NTE)], where k(i) is the bimolecular rate constant of inhibition for a given inhibitor against each enzyme. RIPs greater than 1 correlate with the inability of ageable OP inhibitors or their parent compounds to produce OP compound-induced delayed neurotoxicity (OPIDN) at doses below the LD50. The RIP for chlorpyrifos oxon (CPO) is >1 for enzymes from hen brain homogenate, and the parent compound, chlorpyrifos (CPS), cannot produce OPIDN in hens at sublethal doses. This study was carried out to test the hypothesis that the RIP for the methyl homologue of CPO, chlorpyrifos methyl oxon (CPMO), is >1 and greater than the RIP for CPO. Mipafox (MIP), an OP compound known to produce OPIDN, was included for comparison. Hen brain microsomes were used as the enzyme source, and k(i) values (mean +/- SE, microM(-1) min(-1)) were determined for AChE and NTE (n = 3 and 4 separate experiments, respectively). The k(i) values for CPO, CPMO, and MIP against AChE were 17.8 +/- 0.3, 10.9 +/- 0.1, and 0.00429 +/- 0.00001, respectively, and for NTE were 0.0993 +/- 0.0049, 0.0582 +/- 0.0013, and 0.00498 +/- 0.00006, respectively. Corresponding RIPs for CPO, CPMO, and MIP were 179 +/- 9, 187 +/- 4, and 0.861 +/- 0.011, respectively. The results demonstrate that RIPs for CPO and CPMO are comparable, markedly different from that for MIP, and >1, indicating that CPS methyl, like CPS, could not cause OPIDN at sublethal doses.     

Comparison of the reactivation rates of acetylcholinesterase modified by structurally different organophosphates using novel pyridinium oximes

A novel panel of oximes were synthesized, which have displayed varying degree of reactivation ability towards different organophosphorus (OP) modified cholinesterases. In the present article, we report a comparative reactivation profile of a series of quaternary pyridinium-oximes for electric eel acetylcholinesterase (EEAChE) inhibited by the organophosphorus (OP) inhibitors methyl paraoxon (MePOX), ethyl paraoxon (POX; paraoxon) and diisopropyl fluorophosphate (DFP) that are distinguishable as dimethoxyphosphoryl, diethoxyphosphoryl and diisopropoxyphosphoryl AChE-OP-adducts. Most of the 59-oximes tested led to faster and more extensive reactivation of MePOX- and POX-inhibited EEAChE as compared to DFP-modified EEAChE. All were effective reactivators of three OP-modified EEAChE conjugates showing 18–21% reactivation for DFP-inhibited AChE and ≥45% reactivation for MePOX- and POX-inhibited EEAChE. Oximes 7 and 8 showed k_r values better than pralidoxime (1) for DFP-inhibited EEAChE. Reactivation rates determined at different inhibition times showed no significant change in k_r values during 0–90 min incubation with three OPs. However, a 34–72% decrease in k_r for MePOX and POX and > 95% decrease in k_r for DFP-inhibited EEAChE was observed after 24 h of OP-exposure (aging).     

Reactivating and protective effects of pyridinium compounds in human erythrocyte acetylcholinesterase inhibition by organophosphates in vitro

The reactivation of human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by O-ethyl-S-2-di-isopropylaminoethyl methylphosphonothioate (VX) and the protection against AChE inhibition by O-1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) was studied with sixteen quaternized pyridinium compounds. TMB-4 which is known as a good reactivator of AChE inhibited by organophosphates proved to be the most effective reactivator. Of the tested newly synthetised compounds 3 were fairly good reactivators of methylethoxyphosphonylated AChE. These compounds have 2 pyridinium rings connected by a dimethylether link and a hydroxiiminomethyl group in position 2 of one pyridinium ring, while the radicals of the other pyridinium ring are benzoylcarbonyl, cyclohexylcarbonyl or amidocarbonyl residue.The rate of reactivation with these compounds followed a two-phase pattern, being fast at the beginning and then slowing down to an equilibrium. Kinetic treatment of the first-phase reaction course yielded the second-order rate constants of reactivation. All 3 compounds had similar reactivating efficiency (k _r values range from 0.8×10³ to 3.6×10³ M^–1 min^–1) and in effective concentrations (1 to 100 M) they also inhibited AChE (K _i(app) values range from 0.11 to 0.19 mM). Their reactivating properties were not better than those revealed by TMB-4 (k _r= 19.4×10³ M^–1 min^–1) which was tested as a reference compound.HGG-12, HGG-42 and HI-6 were also found to exert a good protective effect against AChE inhibition by Soman; no protection was obtained with TMB-4.     

Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma

Chlorpyrifos oxon (CPO) is the active metabolite of the pesticide chlorpyrifos that inhibits cholinesterases at high reaction rates. Chlorpyrifos is of major concern because it causes some ten thousand fatalities each year, mostly due to suicidal attempts. Notwithstanding, toxicokinetic studies on chlorpyrifos in humans are scarce and CPO has not been detected hitherto in human blood. Knowledge of the concentration and the time course of CPO in poisonings would be helpful to better design antidotal strategies, particularly with oximes. Owing to the exceptionally fast covalent binding to butyrylcholinesterase we searched for an enzyme-based assay for CPO determination. We succeeded in a simple procedure where CPO is titrated with purified equine butyrylcholinesterase. The assay requires less than 0.2mL EDTA plasma and allows the quantification of CPO down to 0.5nM. CPO is first extracted from plasma with n-pentane, thereby largely excluding the majority of the more hydrophilic pesticide oxons from possible cross-reactions. When chlorpyrifos incorporation is ascertained the assay may be considered largely specific. The new procedure enabled the assessment of the extent of reversible binding of CPO to human albumin, amounting to 85% under physiological conditions. The assay allowed the quantification of CPO in the plasma of a poisoned patient, where the active metabolite was about two orders of magnitude lower than chlorpyrifos. Similar to the parent compound its oxon showed the same tendency to persist for longer periods, thus calling for a change of the usual oxime dosage regimen.     

Kinetic interactions of a homologous series of bispyridinium monooximes (HGG oximes) with native and phosphonylated human acetylcholinesterase

Inhibition of acetylcholinesterase (AChE) is the main toxic mechanism of organophosphorus compounds (OP) and reactivation of OP-inhibited AChE by oximes is a mainstay of antidotal treatment. The inadequate efficacy of clinically used oximes led to the synthesis of numerous new compounds in the past decades to identify more effective reactivators. Despite of extensive in vitro reactivation studies the structural features for the development of effective oximes are not well understood. In the present study we investigated the kinetic interactions of a homologous series of bispyridinium monoximes bearing C1 to C12 alkylketone groups on the second pyridinium ring with native and cyclosarin-inhibited human AChE. We observed a correlation of the length of the alkyl side chain with an up to 20-fold increased affinity towards native AChE. The effect of the alkyl side chain on the affinity and reactivity towards phosphonylated AChE was moderate, except of a markedly reduced reactivity of C10 and C12 oximes. In comparison to the reference oxime HI-6 all HGG oximes had a lower reactivating potency and these oximes are not considered as promising compounds for the reactivation of cyclosarin-inhibited AChE.     

Dichlorvos,chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase,detected by mass spectrometry in human plasma following deliberate overdose

The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide‐adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60–84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.     

Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase

A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE -/-) versus wild type (AChE +/+) mice indicated that while these OPs inhibited axonal growth in AChE+/+ DRG neurons, they had no effect on axonal growth in AChE -/- DRG neurons. However, transfection of AChE -/- DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.     

Sperm chromatin alteration and DNA damage by methyl-parathion, chlorpyrifos and diazinon and their oxon metabolites in human spermatozoa

Extensive use of organophosphorous pesticides (OP) by young men represents a public health problem. Toxicity of OP mainly results in neurotoxicity due to their oxygen analogues (oxons), formed during the OP oxidative activation. OP alter semen quality and sperm chromatin and DNA at different stages of spermatogenesis. Oxons are more toxic than the parent compounds; however, their toxicity to spermatogenic cells has not been reported. We evaluated sperm DNA damage by several OP compounds and their oxons in human spermatozoa from healthy volunteers incubated with 50-750 microM of methyl-parathion (MePA), methyl-paraoxon (MePO), chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), diazinon (DZN) or diazoxon (DZO). All concentrations were not cytotoxic (evaluated by eosin-Y exclusion), except 750 microM MePO. Oxons were 15% to 10 times more toxic to sperm DNA (evaluated by the SCSA parameter, %DFI) than their corresponding parent compounds, at the following order: MePO>CPO=MePA>CPF>DZO>DZN, suggesting that oxon metabolites participate in OP sperm genotoxicity.