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.     

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.     

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 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.     

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.     

In vitro sensitivity of cholinesterases and [3H]oxotremorine-M binding in heart and brain of adult and aging rats to organophosphorus anticholinesterases

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [³H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [³H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.     

The interaction of the phosphorothioate insecticides chlorpyrifos and parathion and their oxygen analogues with bovine serum albumin

The distribution and subsequent toxicity of hazardous chemicals can be influenced by their interactions with plasma proteins. In the present study reversible binding of the phosphorothioate insecticides chlorpyrifos and parathion to fatty acid-free bovine serum albumin (BSA) was examined using the technique of equilibrium dialysis. Computer analyses of the binding data revealed that chlorpyrifos and parathion each bound reversibly to a single class of binding sites on BSA, with apparent KD values of 3.4 +/- 0.1 and 11.1 +/- 0.3 microM, respectively. Additionally, the maximal number of binding sites for each insecticide per molecule of BSA was one. Displacement studies using both chlorpyrifos and parathion indicated that each was a competitive inhibitor of the other's binding, suggesting that they were bound to the same site. Incubation of chlorpyrifos oxon or paraoxon with a 1% solution of BSA resulted in limited, EDTA-insensitive formation of 3,5,6-trichloro-2-pyridinol or p-nitrophenol, respectively. Pretreatment of BSA with 5 mM paraoxon, chlorpyrifos oxon, or 1 mM diisopropylfluorophosphate did not alter this activity, suggesting that these reactions resulted from an esterase-like capacity of BSA, and not from phosphorylation of BSA by these oxons.     

In vitro effects of chlorpyrifos, parathion, methyl parathion and their oxons on cardiac muscarinic receptor binding in neonatal and adult rats.

Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular the m(2) subtype, in tissues from adult rats. As both the density and agonist affinity states of cardiac muscarinic receptors (primarily m(2)) have been reported to change in an age-related manner, we evaluated the relative in vitro sensitivity of cardiac muscarinic receptors in tissues from neonatal (7-11 days of age) and adult (90 days of age) rats to selected organophosphorus compounds (chlorpyrifos, parathion, methyl parathion and their oxygen analogs or oxons). The effects of the cholinergic agonist carbachol (100 pM-5 microM) or an organophosphorus toxicant (50 pM-10 microM) on muscarinic receptor binding were determined using the nonselective muscarinic ligand [3H]quinuclidinyl benzilate or the m(2)-preferential ligand [3H]oxotremorine-M acetate. Carbachol displaced [3H]oxotremorine labeling in adult and neonatal membranes in a relatively similar manner (IC(50)=7-20 nM). The oxons all displaced [3H]oxotremorine binding in a concentration-dependent manner, with chlorpyrifos oxon being the most potent (IC(50): neonates, 15 nM; adults, 7 nM) and efficacious (maximum displacement: neonates, 42%; adults, 56%). Interestingly, methyl parathion was an extremely potent displacer of [3H]oxotremorine binding in adult tissues (IC(50)=0.5 nM, maximum displacement=37%) but had no effect in neonatal tissues. The displacement of [3H]oxotremorine binding by chlorpyrifos oxon (10 microM) was still apparent after washing the tissues, suggesting the oxon irreversibly blocked agonist binding to the receptor while interaction with MePS appeared reversible. As effective concentrations of the oxons were relatively similar to their anticholinesterase potencies, these findings suggest that direct interaction with cardiac muscarinic receptors by some organophosphorus agents may occur at relevant exposure levels and contribute to cardiac 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.     

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.     

Interactions of rat brain acetylcholinesterase with the detergent Triton X-100 and the organophosphate paraoxon.

Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci.41, 8-20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the (app)V(max) for hydrolysis of acetylthiocholine, without affecting the (app)K(m) or the (app)K(ss). However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM-100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the (app)k(i). These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels.     

Each lipase has a unique sensitivity profile for organophosphorus inhibitors.

Lipases sensitive to organophosphorus (OP) inhibitors play critical roles in cell regulation, nutrition, and disease, but little is known on the toxicological aspects in mammals. To help fill this gap, six lipases or lipase-like proteins are assayed for OP sensitivity in vitro under standard conditions (25 degrees C, 15 min incubation). Postheparin serum lipase, lipoprotein lipase (LPL) (two sources), pancreatic lipase, monoacylglycerol (MAG) lipase, cholesterol esterase, and KIAA1363 are considered with 32 OP pesticides and related compounds. Postheparin lipolytic activity in rat serum is inhibited by 14 OPs, including chlorpyrifos oxon (IC50 50-97 nM). LPL (bovine milk and Pseudomonas) generally is less inhibited by the insecticides or activated oxons, but the milk enzyme is very sensitive to six fluorophosphonates and benzodioxaphosphorin oxides (IC50 7-20 nM). Porcine pancreatic lipase is very sensitive to dioctyl 4-nitrophenyl phosphate (IC50 8 nM), MAG lipase of mouse brain to O-4-nitrophenyl methyldodecylphosphinate (IC50 0.6 nM), and cholesterol esterase (bovine pancreas) to all of the classes of OPs tested (IC50 < 10 nM for 17 compounds). KIAA1363 is sensitive to numerous OPs, including two O-4-nitrophenyl compounds (IC50 3-4 nM). In an overview, inhibition of 28 serine hydrolases (including lipases) by eight OPs (chlorpyrifos oxon, diazoxon, paraoxon, dichlorvos, and four nonpesticides) showed that brain acetylcholinesterase is usually less sensitive than butyrylcholinesterase, liver esterase, cholesterol esterase, and KIAA1363. In general, each lipase (like each serine hydrolase) has a different spectrum of OP sensitivity, and individual OPs have unique ranking of potency for inhibition of serine hydrolases.     

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.     

The developmental neurotoxicity of organophosphorus insecticides: a direct role for the oxon metabolites

Several extensively used organophosphorus ester (OP) insecticides are phosphorothionates. The oxon metabolites of phosphorothionates have long been known to be responsible for the acute cholinergic neurotoxicity associated with OP poisoning. In addition, there is now sufficient evidence to suggest that the oxon metabolites may also be directly responsible for the particular neurotoxicity that phosphorothionate insecticides, and especially chlorpyrifos (CP) and diazinon (DZ), are known to inflict on the developing organism. In vitro data reveal that the oxons, which are present at increased levels in the developing brain, have the ability to directly disrupt, at toxicologically relevant doses, separately a number of neurodevelopmental processes, including those of neuronal proliferation, neuronal differentiation, gliogenesis and apoptosis. In most cases, the effects of the oxons are very potent. Inhibition of neuronal and glial cell differentiation by the oxons in particular is up to 1000-times stronger than that caused by their parent phosphorothionates. The neurodevelopmental toxicity of the oxons is not related to the inhibition of the enzymatic activity of acetylcholinesterase (AChE), but may be due to direct oxon interference with the morphogenic activity that AChE normally shows during neurodevelopment. Other possible direct targets of the oxons include neurodevelopmentally important cell signaling molecules and cytoskeletal proteins which have been found to be affected by the oxons and to which covalent binding of the oxons has been recently shown. Future studies should aim at confirming the developmental neurotoxic capacity of the oxons under in vivo conditions and they must also be extended to include OP parent insecticides with a PO moiety.     

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.     

The Relationship between PON1 phenotype and PON1-192 genotype in detoxification of three oxons by human liver.

Phosphorothioate pesticides (OP) such as diazinon, chlorpyrifos, and parathion are activated to highly toxic oxon metabolites by the cytochromes P450 (P450s), mainly in the liver. Simultaneously, the P450s catalyze detoxification of OP to nontoxic dearylated metabolites. The oxon is then detoxified to the dearylated metabolite by PON1, an A-esterase present in the liver and blood serum. The aims of this study were to define the influence of PON1-192 genotype and phenotype on the capacity of human liver microsomes (n = 27) to detoxify the oxons diazoxon, chlorpyrifos-oxon, and paraoxon. Near physiological assay conditions were used to reflect as closely as possible metabolism in vivo and because the hydrolytic activity of the allelic variants of PON1-192 are differentially affected by a number of conditions. The rates of hydrolysis of diazoxon, chlorpyrifos-oxon, and paraoxon varied 5.7-, 16-, and 56-fold, respectively, regardless of PON1-192 genotype. Individuals with the PON1-192RR genotype preferentially hydrolyzed paraoxon (p < 0.01), and the R allele was associated with higher hydrolytic activity toward chlorpyrifos-oxon, but not diazoxon. There were strongly significant relationships between phenylacetate and paraoxon hydrolysis (p < 0.001) and phenylacetate and chlorpyrifos-oxon hydrolysis (p < 0.001), but not between phenylacetate and diazoxon hydrolysis. These data highlight the importance of PON1 phenotype for efficient hydrolysis of paraoxon and chlorpyrifos-oxon, but environmental and yet unknown genetic factors are more important than PON1-192 genotype in determining capacity to hydrolyze diazoxon.     

Inhibition of trans-permethrin hydrolysis in human liver fractions by chloropyrifos oxon and carbaryl

Permethrin, a pyrethroid insecticide, is one of several deployment-related chemicals that have been suggested as causative agents for Gulf War related illnesses. Hydrolysis of trans-permethrin (tPMT) is a major route of detoxication and a potential locus for interactions with chemicals with similar use patterns. This study examined the potential inhibitory effects of chlorpyrifos, carbaryl, pyridostigmine bromide and the insect repellent N,N-diethyl-m-toluamide (DEET) on tPMT hydrolysis in human liver fractions. Although chlorpyrifos was not inhibitory, its toxic metabolite, chlorpyrifos oxon, strongly and irreversibly inhibited tPMT hydrolysis at low concentrations (cytosolic and microsomal Ki values of 3 and 16 nM, respectively). Carbaryl, a known anticholinesterase agent, showed non-competitive inhibition kinetics, with Ki values two orders of magnitude higher than those for chlorpyrifos oxon. Although DEET was much less effective than either chlorpyrifos oxon or carbaryl, equimolar concentrations inhibited up to 45% of tPMT hydrolysis. Pyridostigmine bromide showed no inhibitory effects. This study suggests that interaction potential between organophosphorus and pyrethroid insecticides should be considered in safety assessments when both insecticides are deployed simultaneously.     

Characterization of the in vitro kinetic interaction of chlorpyrifos-oxon with rat salivary cholinesterase: a potential biomonitoring matrix

The primary mechanism of action for organophosphorus (OP) insecticides such as chlorpyrifos (CPF) involves the inhibition of acetylcholinesterase (AChE) by their active oxon metabolites resulting in a wide range of neurotoxic effects. These oxons also inhibit other cholinesterases (ChE) such as butyrylcholinesterase (BuChE), which represents a detoxification mechanism and a potential biomarker for OP insecticide exposure/response. Salivary biomonitoring has recently been explored as a practical method for examination of chemical exposure, however, there are few studies exploring the use of saliva for OP insecticides. To evaluate the use of salivary ChE as a biological monitor for OP insecticide exposure, a modified Ellman assay in conjunction with a pharmacodynamic model was used to characterize salivary ChE in adult male Sprague-Dawley rats. Comparison of rat saliva, brain, and plasma ChE activity in the presence of selective inhibitors of AChE and BuChE (BW284C51 and iso-OMPA, respectively) with different ChE substrates indicated that rat salivary ChE activity is primarily associated with BuChE (>95%). Further characterization of rat salivary BuChE kinetics yielded an average total BuChE active site concentration of 1.20+/-0.13 fmol ml(-1) saliva, an average reactivation rate constant (Kr) of 0.070+/-0.008 h(-1), and an inhibitory rate constant (Ki) of approximately 9 nM(-1) h(-1). The pharmacodynamic model successfully described the in vitro BuChE activity profile as well as the kinetic parameters. These results support the potential utility of saliva as a biomonitoring matrix for evaluating occupational and environmental exposure to CPF and other OP insecticides.     

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.     

Degradation by rat tissues in vitro of organophosphorus esters which inhibit cholinesterase

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 BuChE 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.