Further studies toward a mouse model for biochemical assessment of neuropathic potential of organophosphorus compounds

Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound‐induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct‐acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED₅₀ ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

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.     

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.     

The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds.

The ability of endogenous carboxylesterase (CaE) to protect against the lethal effects of a variety of organophosphorus (OP) compounds was examined in rats. The in vivo protection provided by endogenous CaE was measured by the difference in the LD50 values of OP compounds in control rats and rats whose CaE activity had been inhibited by sc injection with 2 mg/kg of 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide. Endogenous CaE provided significant protection against the in vivo toxicity of soman, sarin, tabun, and paraoxon, but not against dichlorvos, diisopropyl fluorophosphate, or ethoxymethyl-S-[2-(diisopropylamino)ethyl] thiophosphonate (VX). The relationship between the in vivo CaE protection against OP compounds and their relative reactivities with CaE and acetylcholinesterase (AChE) was evaluated by measuring the in vitro bimolecular rate constants (ki) for inhibition of plasma CaE and brain AChE. Except for VX, ki values for CaE inhibition varied less than 10-fold while ki values for AChE inhibition varied 10(5)-fold. The degree of in vivo inhibition of CaE by equitoxic doses of the OP compounds increased as the CaE/AChE ki ratio increased. However, the protective ratio of the LD50 values in control vs CaE-inhibited rats decreased as the CaE/AChE ki ratio increased. This inverse relationship between in vivo CaE protection and relative in vitro reactivity for CaE suggested that CaE detoxication is more important for highly toxic OP compounds (i.e., compounds with high AChE ki values and low LD50 values) than for less toxic compounds.     

Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 microL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area.     

The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.     

Comparative studies of two organophosphorus compounds in the mouse

A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 μmol/kg s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 μmol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE.     

Inhibition of calcium-stimulated ATPase in the hen brain P2 synaptosomal fraction by organophosphorus esters: relevance to delayed neuropathy

Organophosphorus (OP) compounds have been reported to inhibit Ca/Mg-ATPase, but the relevance of this inhibition to organophosphate-induced delayed neuropathy (OPIDN) has not been explored. To determine if inhibition of this enzyme was related to the development of OPIDN, neuropathic and nonneuropathic OP compounds were sted for their ability to inhibit Ca-stimulated ATPase activity in the P2 synaptosomal fraction from hen brain. Following in vitro exposure to 10(-3) to 10(-5) M OP compounds, Ca-stimulated ATPase activity was inhibited by chlorpyrifos, chlorpyrifos-oxon, phenyl saligenin phosphate (PSP), and tri-o-tolyl phosphate (TOTP), but not by parathion, paraoxon, or diisopropyl fluorophosphate (DFP). Further investigation of inhibition induced by chlorpyrifos determined that inhibition was noncompetitive with respect to calcium and ATP. OP compound hydrophobicity was well correlated with in vitro inhibition of Ca-stimulated ATPase, suggesting that OP compounds interact with membrane lipids, and this interaction may contribute to the noncompetitive inhibition of Ca-stimulated ATPase that was observed. Subsequent to in vivo exposure, DFP, but not PSP, produced inhibition of Ca-stimulated ATPase activity in the hen brain P2 synaptosomal fraction. These data indicate that inhibition of Ca-stimulated ATPase activity is not correlated to neuropathic potential and demonstrate that inhibition of Ca/Mg-ATPase is not responsible for OPIDN.     

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.     

Variation between three strains of rat: inhibition of neurotoxic esterase and acetylcholinesterase by tri-o-cresyl phosphate

The present study is concerned with the involvement of strain differences in rodent sensitivity to organophosphorous compound-induced delayed neurotoxicity (OPIDN). The inhibitory effect of three doses of tri-o-cresyl phosphate (TOCP) on neurotoxic esterase (NTE) and acetylcholinesterase (AChE) in brain was compared in three strains of rat: Long-Evans (LE) animals, which have been reported to be sensitive to the neurotoxic effects of TOCP, and Sprague-Dawley (SD) or Fischer 344 (F344) strains, with which negative results have been obtained. Differences in basal levels were found for NTE (LE greater than F344) greater than SD, with a range of 4.87-7.47 nmol phenylvalerate hydrolyzed/mg protein), but not AChE. Strain differences in inhibition by TOCP were found with both assays, with Sprague-Dawley animals being much less sensitive to esterase inhibition than either Long-Evans or Fischer 344 rats. The ED50 values for NTE inhibition were estimated to be 458, 209, and 288 mg/kg for SD, F344, and LE rats, respectively. The ED50 values for AChE inhibition were estimated to be 1007, 408, and 420 mg/kg for SD, F344, and LE rats, respectively. Liver microsomes from the Fischer animals had less cytochrome P-450 than those from the other two strains. Differences in the ability of the strains to either form or inactivate the active metabolite of TOCP may account for the variation observed. While metabolism may play a role in the differences in the level of NTE inhibition in SD rats compared to the LE strain, it cannot account for the lack of sensitivity of the F344 animals to OPIDN. These results may be important in selecting a strain for the study of the toxic effects of organophosphorous compounds in rats.     

Acute and delayed effects of fenthion in young chicks

The effects of desbromoleptophos, fenitrothion, and fenthion on brain acetylcholinesterase (AChE), brain neurotoxic esterase (NTE), and walking were investigated in immature chicks, below the age of organophosphorus ester-induced delayed neurotoxicity (OPIDN). Seventy-five milligrams per kilogram of the delayed neurotoxicant desbromoleptophos (DBL) and 100 mg/kg of the nonneurotoxicant fenithrothion (FTR) were given orally to 8-d-old chicks. Five milligrams per kilogram of the suspected neurotoxicant fenthion (FEN) was administered topically for 7 d, in 4 different age groups. Behavioral testing was performed for treated and control chicks on various days after treatment. Brain NTE and AChE assays were carried out for treated and control chicks on each day of behavioral testing. NTE and AChE inhibition were around 80 and 50%, respectively, 24 h after treatment, for the chicks treated with DBL. NTE returned to normal levels by 20 d and AChE by 6 d after treatment. FTR caused 56% AChE inhibition but not NTE inhibition 24 h after treatment. NTE inhibition for the FEN-treated chicks never exceeded 25% during the whole period of the experiment, whereas 65 and 54% inhibition of AChE was seen in two age groups. DBL and FEN significantly altered the gait of treated chicks, but the non-OPIDN-inducing FTR did not. FEN-treated chicks developed an atypical ataxia at the normal age for onset of sensitivity to OPIDN. Minimal NTE inhibition, long latency for the development of ataxia, and immaturity of the chicks at treatment distinguish FEN-induced functional deficits from classical OPIDN.     

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC(50) value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC(50) value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC(50) values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens.     

Preliminary characterisation of an in vitro paradigm for the study of the delayed effects of organophosphorus compounds: hen embryo brain spheroids

Organophosphate induced delayed neuropathy (OPIDN) has been studied extensively but the mechanisms of toxicity remain unclear. It is generally accepted that the inhibition and ageing (dealkylation) of the B-esterase neuropathy target esterase (NTE) is integral to axonal loss. At present, the only way of detecting compounds that induce OPIDN is the hen test, an animal model. In this study, we preliminary validated hen embryo brain spheroids (HEBS) for the study of organophosphate (OP) toxicity. Hen brain spheroids have been characterised previously, although they have never been fully optimised for OP testing. We optimised the levels of acetylcholine esterase (AChE) and neuropathy target esterase by adapting the culture technique and using chemically defined media. Spheroid cultures were maintained for 35 days and viability and enzyme levels were monitored over this time. Levels of AChE and NTE in this system remained stable over the 35 day period. Using transmission electron microscopy, we have shown synaptogenesis within HEBS earlier than previously suggested in spheroid culture. These studies indicate that HEBS may be useful for the study of OP-induced toxicity and that the long-term stability of the cultures makes it an ideal candidate for studying OPIDN.     

Effects of multiple dosing of fenthion, fenitrothion, and desbromoleptophos in young chicks

The effects of multiple doses of desbromoleptophos, fenitrothion, and pure fenthion on brain acetylcholinesterase (AChE), brain neurotoxic esterase (NTE), and walking were investigated in immature chicks, below the age of sensitivity to organophosphorus ester-induced delayed neurotoxicity (OPIDN). Ten milligrams per kilogram per day of delayed neurotoxicant desbromoleptophos (DBL), 15 mg/kg.d of the non-neurotoxicant fenitrothion (FTR), and 3 mg/kg.d of the suspected neurotoxicant fenthion (FEN) were given orally for 7 d to 3-d-old chicks. Behavioral testing was performed for treated and control chicks on various days after treatment. Brain NTE and AChE assays were carried out for treated and control chicks on each day of behavioral testing. DBL altered gait and inhibited both NTE and AChE; FEN altered gait and inhibited AChE but not NTE; and FTR did not affect gait, while inhibiting AChE but not NTE. NTE and AChE inhibition were 70% and 55%, respectively, 24 h after the last treatment, for the chicks treated with DBL. NTE returned to normal levels by around d 25 and AChE by 20 d after the last treatment. FTR caused more than 50% AChE inhibition but no NTE inhibition, 24 h after last treatment. NTE inhibition for the FEN-treated chicks never exceeded 11% during the whole period of the experiment, whereas 54% inhibition of AChE was seen 1 d after last treatment. DBL and FEN significantly altered the gait of treated chicks, but the non-OPIDN-inducing FTR did not. This study confirms that alterations in the gait of young chicks are not direct consequences of either NTE or AChE inhibition, and that fenthion-induced functional deficits can be distinguished from classical OPIDN.     

In vitro and in vivo assessment of the effect of impurities and chirality on methamidophos-induced neuropathy target esterase aging.

In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(-) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(-) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.     

Characterization of binding sites for diisopropyl phosphorofluoridate in spinal cord cytosol

Gel filtration chromatography was performed on cytosol preparation of hen spinal cord to find molecular target(s) for organophosphorus-induced delayed neurotoxicity (OPIDN). Three binding peaks of [(3)H]diisopropyl phosphorofluoridate (DFP), an organophosphate that induces OPIDN, were separated from the cytosol preparation. The activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) that has been proposed as a screening method for OPIDN eluted in the fractions within these two DFP binding peaks. However, the other peak had none of the activities of AChE and NTE. Therefore, this DFP binding proteins in cytosol may be peculiar to the pathogenesis of OPIDN.     

Isofenphos and an in vitro activation assay for delayed neuropathic potential

Organophosphorus compounds (OPs) that cause organophosphorus ester-induced delayed neuropathy (OPIDN) generally inhibit neurotoxic esterase (NTE). However, the assay itself, when conducted in vitro, misses OPs that are activated into OPIDN-causing agents in the body. A preparation of liver mixed-function oxidases and brain NTE was used to rapidly detect activations of OPs. The compounds (0.1 mM or less) to be tested were incubated with microsomes isolated from livers of phenobarbital-treated chick embryos (P-450 content averaged 1.81 +/- 0.27 nmol/mg protein, means +/- SD, N = 5) and NTE (average of 13.8 nmol/min/mg protein) from untreated chick embryo brains. The NTE was separated by calcium precipitation and its activity assayed as usual. The low inhibitions of NTE of compounds that were not neurotoxic (parathion, Diazinon) did not increase in the presence of NADPH; inhibitions of NTE of compounds that required activation (leptophos, S,S,S-tri-n-butyl phosphorotrithioate, and tri-o-cresyl phosphate) greatly increased with NADPH. Both the recently identified neuropathic OP isofenphos (IFP) and its oxon required activation to inhibit NTE (inhibitions of 20 and 80%, respectively). Evidence is presented that the possible neuropathic metabolite is des-N-isopropyl IFP oxon.