Neurotoxic potential of six organophosphorus compounds in adult hens.

The neurotoxic potential of trichlorfon, diazinon, phosmet, dichlorvos, phosphamidon and coumaphos was evaluated for their ability to inhibit brain neurotoxic esterase (NTE) activity in adult hens. Leptophos was used as a reference neurotoxic agent. All compounds were administered at high single oral doses and the NTE and acetylcholinesterase (AchE) activities were measured at 24 h and 6 w later. With the exception of leptophos, all compounds produced severe cholinergic signs associated with > 80% inhibition of brain AchE at 24 h. On the other hand, brain NTE activity was 86% inhibited by leptophos and to lesser extents by trichlorfon (76%), phosphamidon (74%), coumaphos (70%) and dichlorvos (70%). However, none of the latter compounds produced clinical delayed neurotoxicity as was observed with leptophos. It was concluded that trichlorfon, phosphamidon, coumaphos and dichlorvos are potentially neurotoxic because of their ability to inhibit brain NTE activity, but the extent of inhibition required for development of clinical delayed neurotoxicity (> 80%) is not likely to occur with any of these compounds due to their severe cholinergic activity.     

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.     

Neurotoxic esterase in peripheral nerve: Assay,inhibition, and rate of resynthesis

The potential neurotoxicity of organophosphate esters is usually evaluated by measuring neurotoxic esterase (NTE) inhibition in brains taken from dosed birds. An improved method to measure NTE in peripheral nerve has been developed and used to compare brain, spinal cord, and peripheral nerve NTE inhibition both in vitro and in vivo after a single dose of several organophosphates. Brain and spinal cord NTE activities are a good mirror of NTE activity of the sciatic nerve. The rate of resynthesis of NTE in peripheral nerves after inhibition with an effective dose of a neurotoxic organophosphate was similar to that in other nervous tissues.     

Delayed neurotoxicity of leptophos and related compounds: differential effects of subchronic oral administration of pure, technical grade and degradation products on the hen

Pure and technical grade leptophos (O-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonothioate) and some of its degradation products were screened for delayed neurotoxicity following daily oral administration to hens. Minor modification of the leptophos molecule did not abolish its neurotoxic effect. In contrast, the hydrolytic products, 4-bromo-2,5-dichlorophenol and phenylphosphonic acid, had no neurotoxic action. The effectiveness of esters to induce delayed neurotoxicity was, in descending order: O-2,5-dichlorophenyl O-methyl phenylphosphonothioate (desbromoleptophos) >; pure leptophos > technical grade leptophos > O-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonate (leptophos oxon). Leptophos oxon was only weakly effective as a neurotoxic agent probably because it was unstable in the body and by the time it had reached the target for neurotoxic action it had been hydrolyzed to nonneurotoxic products. Histopathologic examination of hens that died during the study or were killed 30 days after completion of treatment showed marked axon and myelin degeneration in the sciatic, peroneal, and tibial nerves and spinal cord of most hens. The lesions in peripheral nerves were generally seen earlier and in fewer hens than those in spinal cord. The most consistent histopathological changes were degeneration of axons and myelin in the spinal cord. Leptophos oxon was the most potent inhibitor of brain and plasma cholinesterases. Also, the activity of plasma butyryl cholinesterase was more inhibited than brain acetylcholinesterase. Controls consisted of four groups of hens given daily oral doses of 10 mg/kg tri-o-cresyl phosphate (TOCP), 1.0 mg/kg O,O-diethyl-O-4-nitrophenyl phosphorothioate (parathion), and an empty gelatin capsule. TOCP-treated hens developed delayed neurotoxicity while those given parathion showed initial leg weakness but subsequently recovered without developing delayed neurotoxicity. Control hens receiving gelatin capsules remained normal. This study suggests that changes in structure of leptophos as it is absorbed, metabolized, accumulated, and eliminated in vivo, significantly affect the development and severity of delayed neurotoxicity. The chicken continues to be an ideal model for screening organophosphorus pesticides for their capacity to produce delayed neurotoxicity.     

Delayed neurotoxicity of leptophos: toxic effects on the nervous system of hens.

Delayed neurotoxicity in hens fed a single oral dose of leptophos, O-(4-bromo-2,5-dichlorophenyl) O-methyl phenylphosphonothioate, has been studied. Three doses of the insecticide were given: 200, 400, and 800 mg/kg. Leptophos produced neurotoxicity in hens similar to that reported for other neurotoxic organophosphorus compounds. Thus leptophos caused ataxia which progressed to paralysis and death, caused loss of appetite and weight, and reduced the number and weight of eggs laid. Acetylcholinesterase in red blood cells showed an initial inhibition, then a substantial recovery. On the other hand, plasma cholinesterase, after initial inhibition and recovery, was severely inhibited as the signs of neurotoxicity progressed. Histological examination revealed marked axon and myelin degeneration of the sciatic nerve and spinal cord. Less severe changes were seen in the medulla, and nonspecific degeneration of muscle fibers was present.     

Triphenyl phosphite neurotoxicity in the hen: inhibition of neurotoxic esterase and of prophylaxis by phenylmethylsulfonyl fluoride

The neuropathic syndrome resulting in the cat and the rat from single or multiple doses of the phosphorous acid ester tiphenyl phosphite (TPP) has been reported to differ from the syndrome caused by numerous phosphoric acid esters, which is known as organophosphorous compound-induced delayed neurotoxicity (OPIDN). Since the hen is used to test compounds for OPIDN, we chose to study the neurotoxicity of single subcutaneous doses of TPP using this animal model. TPP (1000 mg/kg) produced progressive ataxia and paralysis which began to develop 5–10 days after dosing. Similar signs were observed when subcutaneous doses of the OPIDN-causing agents tri-o-cresyl phosphate (TOCP) or diisopropyl phosphorofluoridate (DFP) were administered. The minimum neurotoxic dose of TPP was 500 mg/kg. Prior administration of phenylmethylsulfonyl fluoride (PMSF) prevented the development of a neuropathy induced by DFP, but did not fully protect the hens from TPP or TOCP. PMSF slowed, but did not prevent, the neuropathy caused by TOCP. PMSF reduced the neurotoxicity of 500 mg/kg TPP, but increased the neurotoxicity of 1000 mg/kg TPP. TPP was found to be a very potent inhibitor of neurotoxic esterase (NTE), the putative target site for OPIDN, in vitro, with a k_i of about 2.1×10⁵ M^–1min^–1. Equimolar doses of either TPP (1000 mg/kg) and TOCP (1187 mg/kg) caused over 80% inhibition of neurotoxic esterase (NTE) in brain and sciatic nerve. This high level of NTE inhibition persisted for several weeks. This prolonged inhibition probably accounts for the inability of PMSF to block the neurotoxicity of TOCP. The dose-response curve for NTE inhibition 48 h after dosing indicated that a level of 70% inhibition correlated with the neurotoxicity of TPP.Subneurotoxic doses of TPP and DFP were found to have an additive effect which could be blocked by PMSF. These results indicate that TPP can cause OPIDN in the hen. The synergism between PMSF and the higher dose of TPP suggests the presence of a second neurotoxic effect as well.     

Six-month daily treatment of sheep with neurotoxic organophosphorus compounds

The delayed neurotoxic effects of tri-o-cresyl-phosphate (TOCP), O-methyl-O-(4-bromo-2,5-dichlorophenyl) phenylphosphonothioate (leptophos), and O-ethyl O-(4-nitrophenyl) phenylphosphonothioate (EPN) at 5, 5, and 1 mg/kg/day, respectively, on male sheep were studied during 6 months of daily oral treatment under field conditions. A vehicle-control group of sheep given corn oil (0.1 ml/kg/day) only was used for comparison. All sheep were killed 24 h after the 180th daily treatment. Blood, brain, spinal cord, and sciatic nerve tissues were taken for histological and/or biochemical examinations. The results indicated that leptophos induced severe ataxia and paralysis in sheep following about 4 months of treatment. TOCP produced either mild ataxia or lameness in two of four sheep during the last week of experiment. On the other hand, none of the EPN-treated sheep showed clinical signs of neurotoxicity during the course of the experiment at the dosage tested. These clinical results were supported by histological findings and also by biochemical results with neurotoxic esterase (NTE) measurements. In the case of leptophos-treated sheep, numerous prominent degenerative lesions of axons were observed in spinal cords and brains. Similar but somewhat less numerous lesions were noted in sheep treated with TOCP. No histological changes were observed in similar tissues taken from EPN-treated sheep. The results also indicated that, for chronic exposure to these neurotoxic organophosphorus compounds in sheep, a threshold in excess of 60-70% prolonged inhibition of brain NTE, or 50-60% inhibition of spinal cord NTE must be exceeded to initiate clinical and/or histological neurotoxic effects.     

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.     

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.     

Evidence for the existence of neurotoxic esterase in neural and lymphatic tissue of the adult hen

Hen brain and spinal cord contain a number of esterases that hydrolyze phenyl valerate (PV). Most of this activity is sensitive to inhibition by micromolar concentrations of paraoxon. Included among the paraoxon-resistant esterases is neurotoxic esterase (NTE), which is inhibited in vivo and in vitro by certain organophosphorus compounds, such as mipafox, which cause delayed neurotoxicity. Since published information on the NTE content of non-neural tissues was heretofore lacking, a comprehensive study was undertaken of the occurrence of this enzyme in tissues of the adult hen (Gallus gallus domesticus), the species of choice in the study of organophosphorus-induced delayed neurotoxicity. Complete differential titration curves of PV esterase activity were obtained by preincubation of each tissue homogenate with a wide range of concentrations of paraoxon, a non-neurotoxic compound, plus or minus mipafox, a neurotoxic compound, followed by PV esterase assay. Brain NTE activity was determined to be 2426 ± 104 nmoles · min^?1 · (g wet weight)^?1 (mean ± S.E.M.). Titration of other tissues resulted in the following NTE activities, expressed as percentages of brain NTE activity: spinal cord (21%), peripheral nerve (1.7%), gastrocnemius muscle (0%), pectoralis muscle (0%), heart (14%), liver (0%), kidney (0%), spleen (70%), spleen lymphocytes (26%), and blood lymphocytes (24%). Using an abbreviated procedure, erythrocytes and plasma showed no NTE activity. These results indicate that NTE has limited distribution among the tissues of the adult hen and is present in lymphatic as well as neural tissue.     

Comparative effectiveness of organophosphorus protoxicant activating systems in neuroblastoma cells and brain homogenates.

The ability of bromine and rat liver microsomes (RLM) to convert organophosphorus (OP) protoxicants to esterase inhibitors was determined by measuring acetylcholinesterase (AChE) and neuropathy target esterase (NTE) inhibition. Species specific differences in susceptibility to esterase inhibition were determined by comparing the extent of esterase inhibition observed in human neuroblastoma cells and hen, bovine, and rodent brain homogenates. OP protoxicants examined included tri-o-tolyl phosphate (TOTP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), leptophos, fenitrothion, fenthion, and malathion. Bromine activation resulted in greater AChE inhibition than that produced by RLM activation for equivalent concentrations of fenitrothion, malathion, and EPN. For EPN and leptophos, bromine activation resulted in greater inhibition of NTE than RLM. Only preincubation with RLM activated TOTP; resultant inhibition of AChE was less in hen brain (13 +/- 3%) than in neuroblastoma cells (73 +/- 1%) at 10(-6) M. In contrast, 10(-6) M RLM-activated TOTP produced more inhibition of hen brain NTE (89 +/- 6%) than NTE of human neuroblastoma cells (72 +/- 7%). Human neuroblastoma cells and brain homogenates from hens, the accepted animal model for study of OP-induced neurotoxicity, were relatively similar in sensitivity to esterase inhibition. Homogenates from hens were more sensitive to NTE inhibition induced by phenyl saligenin phosphate (PSP), an active congener of TOTP, than were homogenates from less susceptible species (mouse, rat, bovine). AChE of hen brain homogenates was also more sensitive than homogenates from other species to malaoxon, the active form of malathion.     

Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications

Fatty acid amide hydrolase (FAAH) plays an important role in nerve function by regulating the action of endocannabinoids (e.g., anandamide) and hydrolyzing a sleep-inducing factor (oleamide). Several organophosphorus pesticides and related compounds are shown in this study to be more potent in vivo inhibitors of mouse brain FAAH than neuropathy target esterase (NTE), raising the question of the potential toxicological relevance of FAAH inhibition. These FAAH-selective compounds include tribufos and (R)-octylbenzodioxaphosphorin oxide with delayed neurotoxic effects in mice and hens plus several organophosphorus pesticides (e.g., fenthion) implicated as delayed neurotoxicants in humans. The search for a highly potent and selective inhibitor for FAAH relative to NTE for use as a toxicological probe culminated in the discovery that octylsulfonyl fluoride inhibits FAAH by 50% at 2 nM in vitro and 0.2 mg/kg in vivo and NTE is at least 100-fold less sensitive in each case. More generally, the studies revealed 12 selective in vitro inhibitors for FAAH (mostly octylsulfonyl and octylphosphonyl derivatives) and 9 for NTE (mostly benzodioxaphosphorin oxides and organophosphorus fluoridates). The overall in vivo findings with 16 compounds indicate the expected association of AChE inhibition with acute or cholinergic syndrome and >70% brain NTE inhibition with delayed neurotoxic action. Surprisingly, 75-99% brain FAAH inhibition does not lead to any overt neurotoxicity or change in behavior (other than potentiation of exogenous anandamide action). Thus, FAAH inhibition in mouse brain does not appear to be a primary target for organophosphorus pesticide-induced neurotoxic action (cholinergic or intermediate syndrome or delayed neurotoxicity).     

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.     

Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides

Organophosphorus (OP) compound-induced inhibition of acetylcholinesterase (AChE) and neuropathy target esterase explains the rapid onset and delayed neurotoxic effects, respectively, for OP insecticides and related compounds but apparently not a third or intermediate syndrome with delayed onset and reduced limb mobility. This investigation tests the hypothesis that fatty acid amide hydrolase (FAAH), a modulator of endogenous signaling compounds affecting sleep (oleamide) and analgesia (anandamide), is a sensitive target for OP pesticides with possible secondary neurotoxicity. Chlorpyrifos oxon inhibits 50% of the FAAH activity (IC50 at 15 min, 25 degrees C, pH 9.0) in vitro at 40--56 nM for mouse brain and liver, whereas methyl arachidonyl phosphonofluoridate, ethyl octylphosphonofluoridate (EOPF), oleyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (oleyl-BDPO), and dodecyl-BDPO give IC50s of 0.08--1.1 nM. These BDPOs and EOPF inhibit mouse brain FAAH in vitro with > or =200-fold higher potency than for AChE. Five OP pesticides inhibit 50% of the brain FAAH activity (ED50) at <30 mg/kg 4 h after ip administration to mice; while inhibition by chlorpyrifos, diazinon, and methamidophos occurs near acutely toxic levels, profenofos and tribufos are effective at asymptomatic doses. Two BDPOs (dodecyl and phenyl) and EOPF are potent inhibitors of FAAH in vivo (ED50 0.5--6 mg/kg). FAAH inhibition of > or =76% in brain depresses movement of mice administered anandamide at 30 mg/kg ip, often leading to limb recumbency. Thus, OP pesticides and related inhibitors of FAAH potentiate the cannabinoid activity of anandamide in mice. More generally, OP compound-induced FAAH inhibition and the associated anandamide accumulation may lead to reduced limb mobility as a secondary neurotoxic effect.     

Delayed neurotoxicity from long-term low-level topical administration of leptophos to the comb of hens.

The production of delayed neurotoxicity in hens following percutaneous administration of leptophos [O-(4-bromo-2,5-dichlorophenyl) O-methyl phenylphosphonothioate] has been investigated. By applying a solution of the insecticide in acetone to the comb, seven groups of three laying hens were given daily a single percutaneous dose of 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, or 20.0 mg/kg of leptophos for 183–323 days. All hens given 0.5–20 mg/kg developed ataxia. The group of hens given a daily dose of 0.1 mg/kg of leptophos showed no abnormalities in gait or behavior. The severity of the clinical condition depended on the size of the daily applied dose. The “latent period” and “total administered dose” before onset of ataxia depended on the daily topically applied dose. The most consistent histopathological changes were degeneration of myelin and axons in spinal cords of intoxicated birds. The severity of change was greatest in hens receiving the highest doses. While plasma cholinesterase was inhibited in all treated birds, plasma acid phosphatase activity was significantly increased. The present investigation shows that long-term low-dose applicaton of leptophos to the comb produced delayed neurotoxicity in hens similar to that reported for the oral administration of this compound.     

Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase

Single doses of organophosphorus compounds (OP) which covalently inhibit neuropathy target esterase (NTE) can induce lower-limb paralysis and distal damage in long nerve axons. Clinical signs of neuropathy are evident 3 weeks post-OP dose in humans, cats and chickens. By contrast, clinical neuropathy in mice following acute dosing with OPs or any other toxic compound has never been reported. Moreover, dosing mice with ethyloctylphosphonofluoridate (EOPF) - an extremely potent NTE inhibitor - causes a different (subacute) neurotoxicity with brain oedema. These observations have raised the possibility that mice are intrinsically resistant to neuropathies induced by acute toxic insult, but may incur brain oedema, rather than distal axonal damage, when NTE is inactivated. Here we provide the first report that hind-limb dysfunction and extensive axonal damage can occur in mice 3 weeks after acute dosing with a toxic compound, bromophenylacetylurea. Three weeks after acutely dosing mice with neuropathic OPs no clinical signs were observed, but distal lesions were present in the longest spinal sensory axons. Similar lesions were evident in undosed nestin-cre:NTEfl/fl mice in which NTE had been genetically-deleted from neural tissue. The extent of OP-induced axonal damage in mice was related to the duration of NTE inactivation and, as reported in chickens, was promoted by post-dosing with phenylmethanesulfonylfluoride. However, phenyldipentylphosphinate, another promoting compound in chickens, itself induced in mice lesions different from the neuropathic OP type. Finally, EOPF induced subacute neurotoxicity with brain oedema in both wild-type and nestin-cre:NTEfl/fl mice indicating that the molecular target for this effect is not neural NTE.     

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 Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens

Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitorof neurotoxk esterase (NTE), is a known potentiator of organophosphorus-induceddelayed neurotoxicity (OPIDN)- The ability of PMSF posttreatment(90 mg/kg, sc, 4 hr after the last PSP injection) to modifydevelopment of delayed neurotoxicity was examined in 2-, 5-,and 8-week-old White Leghorn chickens treated either one, two,or three times (doses separated by 24 hr) with the neuropathicOP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTEactivity was measured in the cervical spinal cord 4 hr afterthe last PSP treatment. Development of delayed neurotoxicitywas measured over a 16-day postexposure period. All PSP-treatedgroups exhibited >97% NTE inhibition regardless of age ornumber of OP treatments. Two-week-old birds did not developclinical signs of neurotoxicity in response to either singleor repeated OP treatment regimens nor following subsequent treatmentwith PMSF. Five-week-old birds were resistant to the clinicaleffects of a single PSP exposure and were minimally affectedby repeated doses. PMSF posttreatment, however, significantlyamplified the clinical effects of one, two, or three doses ofPSP. A single exposure to PSP induced slight to moderate signsof delayed neurotoxicity in 8-week-old birds with more extensiveneurotoxicity being noted following repeated dosing. As with5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicitywhen given after one, two, or three doses of PSP in 8-week-oldbirds. Axonal degeneration studies supported the clinical findings:PMSF posttreatment did not influence the degree of degenerationin 2-week-old chickens but resulted in more severe degeneration(relative to PSP only exposure) in cervical cords from both5- and 8-week-old birds. The results indicate that PMSF doesnot alter the progression of delayed neurotoxicity in very young(2 weeks of age) chickens but potentiates PSP-induced delayedneurotoxicity in the presence of 0–3% residual NTE activityin older animals. We conclude that posttreatment with neuropathicor nonneuropathic NTE inhibitors, following virtually completeNTE inhibition by either single or repeated doses of a neuropathicagent in sensitive age groups, can modify both the clinicaland morphological indices of delayed neurotoxicity. This studyfurther supports the hypothesis that potentiation of OPIDN occursthrough a mechanism unrelated to NTE.     

Delayed neuropathy in adult Peking ducks induced by some organophosphorus esters

Trio-o-cresyl phosphate (TOCP), leptophos [O-methyl O-(4-bromo-2,5,-dichlorophenyl) phenylphosphonothioate] and cyanofenphos [O-ethyl O-(4-cyanophenyl) phenyl-phosphonothioate] were used to determine whether adult peking ducks would exhibit neurotoxicity after exposure to such chemicals. Clinical, histopathological, and specific biochemical tests were used to detect the neurologic dysfunctions that were induced by these neurotoxic agents. Ducks were orally treated with TOCP or leptophos at 100 or 10 mg/kg X d for 30 d, respectively. Another group of ducks received cyanofenphos at 4 mg/kg X d for 10 d. All the TOCP- and leptophos-treated ducks developed clinical signs of delayed neuropathy, as manifested by ataxia and paralysis. Two of the cyanofenphos-treated ducks died from cholinergic effect during the course of dosing. Surviving ducks of this group completely recovered from the cholinergic effect 2 or 3 d after finishing the dosing regimen. However, they developed signs of delayed neurotoxicity 10-17 d later. Surviving ducks of all groups were sacrificed for biochemical and/or histopathologic tests 1 d after the last treatment or when they became paralyzed. Histopathologic examinations indicated that degenerative lesions of axons consistent with the type occurring in delayed neurotoxicity were seen in all TOCP-, leptophos-, or cyanofenphos-treated ducks and were specially evident in sections of spinal cord. Biochemically, it was found that duck brain neurotoxic esterase (NTE) activity was inhibited in vivo to less than 15% of control levels as measured 24 h after the last treatment with TOCP, leptophos, or cyanofenphos. These results indicate that adult peking ducks could be used to screen organophosphorus compounds for delayed toxic neuropathy.     

Differential sensitivity to the delayed neurotoxin tri-o-tolyl phosphate in several avian species

Adult white leghorn chickens, ring-necked pheasants, mallards, bobwhites, and Japanese quail were administered single oral doses of tri-o-tolyl phosphate (TOTP) at levels of 125, 250, 500, and 1000 mg/kg body weight. Corn oil served as the vehicle control. At 24 h after dosing, half the birds from each group were killed for determination of whole-brain neurotoxic esterase (NTE) activity. The remaining birds were maintained for 21 d. Daily observations for the development of clinical signs typical of delayed neurotoxicity were begun 7 d after dosing and continued for the subsequent 14 d. In both the Japanese quail and bobwhite, all doses of TOTP resulted in NTE inhibition in excess of 70%, yet no birds of either species developed ataxia or paralysis. However, in the mallard none of the doses of TOTP caused inhibition of NTE activity greater than 61% nor resulted in the development of clinical signs. In the pheasant, all doses of TOTP caused at least a 70% inhibition of whole-brain NTE activity, yet only birds receiving 500 and 1000 mg/kg developed clinical signs. In the chicken, all TOTP doses caused inhibition of NTE in excess of 80%, and all doses resulted in clinical signs typical of delayed neurotoxicity.