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.     

Inhibition of Hen Brain Acetylcholinesterase and Neurotoxic Esterase by Chlorpyrifos in Vivo and Kinetics of Inhibition by Chlorpyrifos Oxon in Vitro: Application to Assessment of Neuropathic Risk

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate;Dursban) is a widely used broad-spectrum organophosphorus (OP)insecticide. Because some OP compounds can cause a sensory-motordistal axonopathy called OP compound-induced delayed neurotoxicity(OPIDN), CPS has been evaluated for this paralytic effect. Earlystudies of the neurotoxicity of CPS in young and adult hensreported reversible leg weakness but failed to detect OPIDN.More recently, a human case of mild OPIDN was reported to resultfrom ingestion of a massive dose (about 300 mg/kg) in a suicideattempt. Subsequent experiments in adult hens (the currentlyaccepted animal model of choice for studies of OPIDN) showedthat doses of CPS in excess of the LD₅₀ in atropine-treatedanimals inhibited brain neurotoxic esterase (NTE) and producedmild to moderate ataxia. Considering the extensive use of CPSand its demonstrated potential for causing OPIDN at supralethaldoses, additional data are needed to enable quantitative estimatesto be made of the neuropathic risk of this compound. Previouswork has shown that the ability of OP insecticides to causeacute cholinergic toxicity versus OPIDN can be predicted fromtheir relative tendency to inhibit the intended target, acetylcholinesterase(AChE), versus the putative neuropathic target, NTE, in braintissue. The present study was designed to clarify the magnitudeof neuropathic risk associated with CPS exposures by measuringhen brain AChE and NTE inhibition following dosing in vivo anddetermining the bimolecular rate constant of inhibition (k₁)for each enzyme by the active metabolite, CPS oxon (CPO), invitro. CPS administered to atropine-treated adult hens at 0,75, 150, and 300 mg/kg po in corn oil produced mean values forbrain AChE inhibition 4 days after dosing of 0, 58, 75, and86%, respectively, and mean values for brain NTE inhibitionof 0, 21, 40, and 77%, respectively. Only the high dose (sixtimes the unprotected LD₅₀ in hens) produced NTE inhibitionabove the presumed threshold of 70%, and these animals werein extremis from cholinergic toxicity at the time of euthanizationdespite continual treatment with atropine. When 150 mg/kg CPSpo in corn oil was given to atropine-treated hens on Day 0,inhibition on Days 1, 2,4, 8, and 16 for brain AChE was 86,82, 72, 44, and 29%, respectively, and for brain NTE was 30,28, 38, 29, and 6%, respectively. No signs of OPIDN were observedin any of the animals during the 16-day study period. Kineticstudies of the inhibition of hen brain AChE and NTE by CPO invitro demonstrated that CPO exhibits high potency and extraordinaryselectivity for its intended target, AChE. The k₁, values were15.5 µM^–1 min^–1 for AChE and 0.145 µM^–1min^–1 for NTE. The calculated fixed-time (20-min) I₅₀values were 2.24 nM for AChE and 239 nM for NTE, yielding anI₅₀ ratio for NTE/AChE of 107. These results may be comparedwith data compiled for other OP compounds with respect to NTE/AChEI₅₀ ratios and the corresponding doses required to produce OPIDNrelative to the LD₅₀. In general, NTE/AChE I₅₀ ratios greaterthan 1 indicate that the dose required to produce OPIDN is greaterthan the LD₅₀. Taken together, the results of this study indicatethat acute exposures to CPS would not be expected to cause OPIDNexcept under extreme conditions such as attempted suicides involvingmedically assisted survival of doses considerably in excessof the LD₅₀.     

Inhibition of extension outgrowth in differentiating rat C6 glioma cells by chlorpyrifos and chlorpyrifos oxon: effects on microtubule proteins.

The aim of this work was to assess the toxic effects of the phosphorothionate insecticide chlorpyrifos (CPF) and its major in vivo metabolite chlorpyrifos oxon (CPO) on differentiating rat C6 glioma cells. At sublethal concentrations (1-10 microM), both compounds were able to inhibit the development of extensions from C6 cells induced to differentiate by sodium butyrate. Western blot analysis of C6 cell lysates revealed that 4 h exposure to CPF was associated with decreased levels of the cytoskeletal protein MAP1B compared to controls, whereas the levels of the cytoskeletal proteins tubulin and MAP2c were not significantly affected. Western blot analysis of extracts of cells treated with CPO showed a significant, concentration-dependent decrease in the levels of tubulin after 24 h. MAP-1B levels were also significantly decreased. The above changes were not temporally related to acetylcholinesterase (AChE) inhibition. These results suggest that both CPF and CPO can exert toxic effects directly on glial cell differentiation and that the latter compound has a potent effect on the microtubule network.     

Mice treated with a nontoxic dose of chlorpyrifos oxon have diethoxyphosphotyrosine labeled proteins in blood up to 4 days post exposure, detected by mass spectrometry

Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is an established biomarker of exposure to organophosphorus poisons (OP). Inhibition of activity is due to covalent binding of the OP to the active site serine. Mass spectrometry has made it possible to monitor OP exposure by analyzing OP adducts on tyrosine in proteins that have no active site serine. Our goal was to test the hypothesis that OP-tyrosine may serve as a biomarker of OP exposure in mice. A MALDI-TOF mass spectrometry strategy to analyze diethoxyphosphate-tyrosine of m/z 318 was developed. The adduct was synthesized by incubating l-tyrosine with chlorpyrifos oxon at pH 8.1. The adduct eluted from a reverse phase HPLC column with 22-23% acetonitrile. The fragmentation spectrum of the m/z 318 precursor ion confirmed its identity as diethoxyphosphate-tyrosine. Diethoxyphosphate-tyrosine was isolated from chlorpyrifos oxon treated mouse albumin after digesting the protein with pronase. Mice (n=3 per group) were treated with a nontoxic dose of chlorpyrifos oxon (3 mg/kg) and a toxic dose (10 mg/kg transdermally). The pronase digested plasma yielded diethoxyphosphate-tyrosine up to 120 h after treatment with 3 mg/kg chlorpyrifos oxon and up to 144 h after 10 mg/kg. In contrast plasma AChE activity returned to normal after 24-72 h. In conclusion MALDI-TOF mass spectrometry can be used to diagnose exposure to chlorpyrifos oxon days after AChE inhibition assays are uninformative.     

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.     

Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase

For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphorylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have k(i)'s that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the K(d) (binding affinity) and k(2) (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose-response curve.     

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.     

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.     

Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates

Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (PhP; R = alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios > 1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.     

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.     

Diethylphosphorylation of rat cardiac M2 muscarinic receptor by chlorpyrifos oxon in vitro

The acute toxicity of chlorpyrifos oxon (CPO), the metabolically-activated form of the major organophosphorus insecticide chlorpyrifos, is attributable to diethylphosphorylation of acetylcholinesterase at its esteratic site. As a secondary effect, CPO is known to compete with agonist binding to the M2 muscarinic acetylcholine receptor (mAChR). This study tests the hypothesis that [ethyl-1,2-(3)H]CPO labels the M2 mAChR in rat cardiac membrane proteins. Of four labeled protein regions observed, only one had an apparent molecular mass (70-75 kDa) consistent with that of glycosylated M2 mAChR. It was identified as M2 muscarinic receptor by Western blotting and immunoprecipitation using a cardiac-specific M2 mAChR monoclonal antibody, providing the first direct evidence for diethylphosphorylation of a muscarinic receptor. This may be a functionally important M2 mAChR site, but the toxicological relevance and species and organ specificity of diethylphosphorylation are unknown.     

In vitro effects of organophosphorus anticholinesterases on muscarinic receptor-mediated inhibition of acetylcholine release in rat striatum

The aim of the present study was to evaluate the in vitro modulation of muscarinic autoreceptor function by the organophosphorus (OP) anticholinesterases chlorpyrifos oxon, paraoxon, and methyl paraoxon. Acetylcholine (ACh) release was studied by preloading slices from rat striatum with [3H]choline and depolarizing with potassium (20 mM) in perfusion buffer containing hemicholinium-3 (to prevent reuptake of radiolabeled choline). Under these conditions, chlorpyrifos oxon, paraoxon, and methyl paraoxon (0.1-10 microM) all reduced ACh release in a concentration-dependent manner. Addition of the carbamate acetylcholinesterase (AChE) inhibitor physostigmine (20 microM) to the perfusion buffer also decreased ACh release. When physostigmine was present, the three oxons had no additional effect on ACh release. Concentration-dependent inhibition of AChE activity in striatal slices perfused with chlorpyrifos oxon (0.1, 1, and 10 microM) suggested AChE inhibition was responsible for oxon-mediated alterations in ACh release. To differentiate between direct and indirect actions of the OP toxicants on muscarinic autoreceptors, we compared the effects of the oxons on ACh release under two conditions, i.e., tissues were perfused with buffer containing only hemicholinium-3 or with buffer containing hemicholinium-3, physostigmine, and the nonselective muscarinic receptor blocker atropine (100 nM). In the presence of only hemicholinium-3, concentration-dependent inhibition of ACh release was again noted for all oxons, similar to the effects of the muscarinic agonists carbachol and cis-dioxolane. In the presence of physostigmine and atropine, the relative potencies of all agents were markedly reduced. Interestingly, carbachol, cis-dioxolane, paraoxon, and methyl paraoxon all decreased ACh release as before, but chlorpyrifos oxon (100-300 microM) actually increased ACh release. Together, the results suggest that chlorpyrifos oxon, paraoxon, and methyl paraoxon can activate muscarinic autoreceptors indirectly through inhibition of AChE. Both paraoxon and methyl paraoxon also directly activate whereas chlorpyrifos oxon blocks muscarinic autoreceptor function. Qualitative differences in the direct actions of these oxons at this presynaptic regulatory site could contribute to differential toxicity with high-dose exposures.     

Chlorpyrifos: Assessment of Potential for Delayed Neurotoxicity by Repeated Dosing in Adult Hens with Monitoring of Brain Acetylcholinesterase, Brain and Lymphocyte Neurotoxic Esterase, and Plasma Butyrylcholinesterase Activities

Previous work has shown that acute exposures to chlorpyrifos(CPS; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) cannotproduce >70% inhibition of brain neurotoxic esterase (NTE)and cause organophosphorus compound-induced delayed neurotoxicity(OPIDN) unless the dose is well in excess of the LD50, necessitatingaggressive therapy for cholinergic toxicity. The present studywas carried out to determine if repeated doses of CPS at themaximum tolerated daily dose without prophylaxis against cholinergictoxicity could cause cumulative inhibition of NTE and OPIDN.Adult hens were dosed daily for 20 days with CPS (10 mg/kg/daypo in 2 ml/kg corn oil) or corn oil (vehicle control) (2 ml/kg/daypo) and observed for an additional 4 weeks. Brain acetylcholinesterase(AChE), brain and lymphocyte NTE, and plasma butyrylcholinesterase(BuChE) activities were assayed on Days 0 (control only), 4,10, 15, 20, and 48. During Days 4–20, brain AChE and plasmaBuChE activities from CPS-treated hens were inhibited 58–70%and 49–80% of contemporaneous controls, respectively.At 4 weeks after the end of dosing, brain AChE activity in treatedbirds had recovered to 86% of control and plasma BuChE activitywas 134% of control. Brain and lymphocyte NTE activities oftreated animals throughout the study were 82–99% and 85–128%of control, respectively. Neither brain nor lymphocyte NTE activitiesin treated hens exhibited cumulative inhibition. The 18% inhibitionof brain NTE seen on days 10 and 20 was significant, but substantiallybelow the putative threshold for OPIDN. Body weight of treatedhens decreased 10–25% during Days 4–20 and recoveredto 87% of control by the end of the study. Some treated hensdeveloped a slight staggering gait during the first week ofdosing, which disappeared by the second week. Throughout the4-week observation period, all hens appeared normal and wereable to perch on a horizontal rod. The results indicate thatdaily dosing with CPS at a level sufficient to cause significantloss of body weight as well as marked inhibition of brain AChEand plasma BuChE resulted in no significant change in lymphocyteNTE activity, a maximum inhibition of brain NTE of 18%, no cumulativeinhibition of lymphocyte or brain NTE, and no clinical signsof OPIDN.     

Paraoxonase protects against chlorpyrifos toxicity in mice

Paraoxonase can hydrolyze paraoxon (PO), chlorpyrifos-oxon (CPO) and other organophosphates. Previous studies have indicated that the levels of serum paraoxonase can influence the toxicity of PO and CPO. In the present study we have investigated whether exogenous paraoxonase administered to mice would offer protection toward the acute toxicity of a phosphorothioate, chlorpyrifos (CPS). Paraoxonase was purified from rabbit serum and injected i.v., or i.v. plus i.p., in mice. Inhibition of acetylcholinesterase (AChE) in brain, diaphragm, plasma and red blood cells was measured as an index of CPS (100 mg/kg) toxicity. Administration of paraoxonase 30 min before CPS increased plasma enzyme activity toward CPO by 35-fold, and protected against its toxicity; protection was still present at 24 h, when enzyme activity was still 20-fold over basal. When paraoxonase was given 30 min after CPS, a significant protection against CPS toxicity was still observed, while after 3 h the protective effect was decreased. To mimic conditions of severe acute poisoning, a higher dose of CPS (150 mg/kg) was also administered. Administration of paraoxonase 30 min after this exposure abolished cholinergic signs and significantly protected against AChE inhibition. These results indicate that exogenous paraoxonase offers significant protection against CPS toxicity when administered both before and after the organophosphate, suggesting that it may be considered as a potential additional treatment of organophosphate poisoning.     

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound‐induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20‐min IC₅₀ values as well as ED₅₀ values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC₅₀(AChE)/IC₅₀(NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose‐dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED₅₀(AChE)/ED₅₀(NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.     

Serine hydrolase KIAA1363: toxicological and structural features with emphasis on organophosphate interactions

Serine hydrolase KIAA1363 is highly expressed in invasive cancer cells and is the major protein in mouse brain diethylphosphorylated by and hydrolyzing low levels of chlorpyrifos oxon (CPO) (the activated metabolite of a major insecticide). It is also the primary CPO-hydrolyzing enzyme in spinal cord, kidney, heart, lung, testis, and muscle but not liver, a pattern of tissue expression confirmed by fluorophosphonate-rhodamine labeling. KIAA1363 gene deletion using homologous recombination reduces CPO binding, hydrolysis, and metabolism 3-29-fold on incubation with brain membranes and homogenates determined with 1 nM [(3)H-ethyl]CPO and the inhibitory potency for residual CPO with butyrylcholinesterase as a biomarker. Studies with knockout mice further show that KIAA1363 partially protects brain AChE and monoacylglycerol lipase from CPO-induced in vivo inhibition. Surprisingly, mouse brain KIAA1363 and AChE are similar in in vitro sensitivity to seven methyl, ethyl, and propyl but not higher alkyl OP insecticides and analogues, prompting structural comparisons of the active sites of KIAA1363 and AChE relative to OP potency and selectivity. Homology modeling based largely on the Archaeoglobus fulgidus esterase crystal structure indicates that KIAA1363 has a catalytic triad of S191, D348, and H378, a GDSAG motif, and an oxyanion hole of H113, G114, G115, and G116. Excellent selectivity for KIAA1363 is achieved on OP structure optimization with long alkyl chain substituents suggesting that KIAA1363 has larger acyl and leaving group pockets than those of AChE. KIAA1363 reactivates faster than AChE presumably due to differences in the uncoupling of the catalytic triad His upon phosphorylation. The structural modeling of KIAA1363 helps us understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.     

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

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

Effects of chlorpyrifos oxon on M2 muscarinic receptor internalization in different cell types

The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 microM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [(3)H]-N-methylscopolamine (NMS). CPO alone (100 microM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.     

Cannabinoid CB1 receptor as a target for chlorpyrifos oxon and other organophosphorus pesticides

Binding of the endocannabinoid anandamide or of Delta(9)-tetrahydrocannabinol to the agonist site of the cannabinoid receptor (CB1) is commonly assayed with [3H]CP 55,940. Potent long-chain alkylfluorophosphonate inhibitors of agonist binding suggest an additional, important and closely-coupled nucleophilic site, possibly undergoing phosphorylation. We find that the CB1 receptor is also sensitive to inhibition in vitro and in vivo by several organophosphorus pesticides and analogs. Binding of [3H]CP 55,940 to mouse brain CB1 receptor in vitro is inhibited 50% by chlorpyrifos oxon at 14 nM, chlorpyrifos methyl oxon at 64 nM and paraoxon, diazoxon and dichlorvos at 1200-4200 nM. Some 15 other organophosphorus pesticides and analogs are less active in vitro. The plant defoliant tribufos inhibits CB1 in vivo, without cholinergic poisoning signs, by 50% at 50 mg/kg intraperitoneally with a recovery half-time of 3-4 days, indicating covalent derivatization. [3H-ethyl]Chlorpyrifos oxon may be suitable for radiolabeling and characterization of this proposed nucleophilic site.     

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 +/- 8 nM amperometrically and 42 +/- 2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 +/- 3 and 70 +/- 14 nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highly correlated between brain and lymphocyte (r = .994) and between brain and blood (r = .997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.