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.     

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.     

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.     

INHIBITION OF CARBOXYLESTERASES IN SH-SY5Y HUMAN AND NB41A3 MOUSE NEUROBLASTOMA CELLS BY ORGANOPHOSPHORUS ESTERS

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SHSY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.     

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.     

Comparative non-cholinergic neurotoxic effects of paraoxon and diisopropyl fluorophosphate (DFP) on human neuroblastoma and astrocytoma cell lines

The objective of this study was to evaluate the comparative non-cholinergic neurotoxic effects of paraoxon, which is acutely neurotoxic, and diisopropyl fluorophosphate (DFP), which induces OPIDN, in the human neuroblastoma SY5Y and the human astrocytoma cell line CCF-STTG1. SY5Y cells have been studied extensively as a model for OP-induced neurotoxicity, but CCF cells have not previously been studied. We conducted a preliminary human gene array assay of OP-treated SY5Y cells in order to assess at the gene level whether these cells can distinguish between OP compounds that do and do not cause OPIDN. Paraoxon and DFP induced dramatically different profiles of gene expression. Two genes were upregulated and 13 downregulated by at least 2-fold in paraoxon-treated cells. In contrast, one gene was upregulated by DFP and none was downregulated at the 2-fold threshold. This finding is consistent with current and previous observations that SY5Y cells can distinguish between OPs that do or do not induce OPIDN. We also examined gene array results for possible novel target proteins or metabolic pathways for OP neurotoxicity. Protein levels of glucose regulated protein 78 (GRP78) revealed that paraoxon exposure at 3 microM for 24 h significantly reduced GRP78 levels by 30% in neuroblastoma cells, whereas DFP treatment had no effect. In comparison with SY5Y neuroblastoma cells, paraoxon and DFP (3 microM for 24 h) each significantly increased GRP78 levels by 23-24% in CCF astrocytoma cells. As we have previously evaluated intracellular changes in Ca(2+) levels in SY5Y cells, we investigated the effects of paraoxon and DFP on cellular Ca(2+) homeostasis in CCF by studying cytosolic and mitochondrial basal calcium levels. A significant decrease in the ratio of mitochondrial to cytosolic Ca(2+) fluorescence was detected in CCF cultures treated for either 1 or 3 days with 1, 3, 10, or 30 microM paraoxon. In contrast, treatment with DFP for 1 day had no significant effect on the ratio of mitochondrial to cytosolic Ca(2+) fluorescence; after 3 days treatment, only 30 microM decreased the ratio. These results are consistent with the finding that paraoxon induced a greater decrease than did DFP of intracellular esterase activity in CCF cells. The changes seen in the ratio of mitochondrial to cytosolic Ca(2+) represent a good indicator of the degree of injury induced by each chemical tested. This work further develops in vitro models that distinguish between compounds that cause OPIDN and those that induce acute neurotoxicity only. The study also exposes additional OP-induced toxicities that may be obscured in vivo.     

Types of adrenocorticoids and their effect on organophosphorus-induced delayed neuropathy in chickens

The present study examined the effects of a glucocorticoid and a mineralocorticoid on organophosphorus-induced delayed neuropathy (OPIDN) as previous investigations have indicated that an endogenous steroid with both properties could alter this syndrome in chickens. The glucocorticoid triamcinolone and the mineralocorticoid deoxycorticosterone were provided in the diet beginning 1 day before and continuing 10 days after triortho-tolyl phosphate (TOTP, 360 mg/kg po), phenyl saligenin phosphate (PSP, 2.5 mg/kg im), and diisopropyl phosphorofluoridate (DFP, 1 mg/kg sc). In a manner similar to that seen with corticosterone, a low concentration (0.1 ppm) of triamcinolone reduced and a high concentration (10 ppm) exacerbated clinical signs. Concentrations of deoxycorticosterone under 80 ppm also partially delayed or ameliorated ataxia induced by TOTP, PSP, and DFP, but a combination of 0.1 ppm triamcinolone and 80 ppm deoxycorticosterone was not more effective than triamcinolone alone. Peripheral nerve damage was noted in all chickens given organophosphorus compounds, whether or not they had been given corticoids. Both steroids induced hydroxylase activity, but effects on most other enzyme systems examined were unremarkable. High concentrations of triamcinolone (10 ppm) could, however, also reduce liver cytochrome P450 levels and liver cholinesterase activity. Exacerbation of OPIDN was most notable in chickens under highest stress, as indicated by elevated heterophil-to-lymphocyte ratios. The clinical, pathological, biochemical, and hematological indices of exposure to adrenocorticoids and agents inducing OPIDN in chickens were, therefore, similar for both a synthetic glucocorticoid and the endogenous steroid corticosterone.     

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.     

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.     

Use of the Biventer Cervicis Nerve-Muscle Preparation to Detect Early Changes following Exposure to Organophosphates Inducing Delayed Neuropathy

Use of the Biventer Cervicis Nerve-Muscle Preparation to DetectEarly Changes following Exposure to Organophosphates InducingDelayed Neuropathy. EL-FAWAL, H. A. N., JORTNER, B. S., ANDEHRICH, M.(l990). Fundam. Appl. Toxicol. 15, 108–120.Indices of organophosphorus (OP)-induced delayed neuropathy(OPIDN) in the hen model have traditionally been restrictedto the early inhibition of neuropathy target esterase (NTE)and ataxia with associated pathological changes in hind limbperipheral nerve which occur more than 7 days after OP exposure.The biventer cervicis nerve-muscle preparation was used to evaluateOPIDN in adult hens at various time periods after treatmentwith either the protoxicant tri-o-tolyl phosphate (TOTP), 360mg/kg po, or the active congener phenyl saligenin phosphate(PSP), 2.5 mg/kg im. NTE activity was 21 and 48% of controlfor TOTP and PSP, respectively, 4 days after administration.Clinical signs were notable by 10 days and progressed in severityto paralysis by 21 days. Partial clinical recovery was evidentat 37 days. Denervation hypersensitivity of biventer cervicismuscle to acetylcholine (ACh) was evident as early as 4 daysfollowing TOTP or PSP treatment. The sensitivity to ACh wasgreatest 21 days after OP administration, with partial recoveryat 37 days. Strength-duration curves (SDC) of preparations fromOP-treated hens showed an increase in excitability thresholdsand elevated rheobase with shorter chronaxie than did preparationsfrom controls as early as 4 days following treatment with eithercompound. SDC at 37 days indicated partial reinnervation. Peripheralnerve myelinated fiber degeneration and regeneration consistentwith these physiological changes was seen on histopathologicalexamination. This study suggests that the biventer cervicisnerve-muscle preparation may prove useful for detection of functionaland morphological changes that occur during the interval betweenNTE inhibition and appearance of clinical deficits.     

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.     

Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks

To examine the phenomenon of apparent age resistance of young chicks to organophosphate-induced delayed neuropathy (OPIDN), groups of either 2- or 10-week-old chicks were exposed subcutaneously daily for 4 days to the neuropathic organophosphate (OP), di-isopropylfluorophosphate (DFP, 1 mg/kg), the non-neuropathic OP, paraoxon (PO, 0.25 mg/kg) or atropine (20 mg/kg). Subsequently, all birds were examined at post-exposure intervals (calculated from the last day of exposure) for up to 56 days for neurological deficits and morphological lesions in the central and peripheral nervous systems (CNS, PNS). Clinically, none of the birds in the 2-week-old groups, or in the 10-week-old PO or atropine exposed groups had neurological deficits. However, all birds in the 10-week-old DFP exposed group developed ataxia by 7 days post-exposure (DPE) and then progressive paralysis. Therefore, all birds in the 10-week-old groups were killed at 14 DPE. Pathologically, the 2-week-old DFP exposed chicks had increasingly severe lesions of Wallerian-like degeneration predominantly in the spinal cord from 7 DPE and subsequently. In the 10-week-old DFP exposed chicks, the degenerative lesions of OPIDN were first detected in the CNS at 3 DPE and then with equally increasing severity in the CNS and PNS up to 14 DPE. A higher incidence of neuronal necrosis and chromatolysis in ventral motor horn neurons of spinal cord grey matter and in dorsal root ganglia occurred in both the DFP exposed age groups compared with those lesions in other groups. These results demonstrate that after neuropathic DFP exposure, 2-week-old chicks develop pathological lesions in the spinal cord without neurological deficits. In both age groups, onset of degenerative lesions in the spinal cord preceeded those in the PNS. The claim of apparent age resistance of chicks to OPIDN needs to be re-evaluated.     

Neurotoxicity of acute and repeated treatments of tabun, paraoxon, diisopropyl fluorophosphate and isofenphos to the hen.

The neuropathic potential of acute and repeated exposures of the phosphoramidates tabun (GA) and isofenphos (IFP), of diisopropyl fluorophosphate (DFP) and paraoxon (PO) were examined in the hen with treatments for up to 90 days via intramuscular injections of the highest tolerated doses with atropine protection. Plasma acetylcholinesterase (AChE), non-specific butyrylcholinesterase (BChE) and creatine kinase (CK) activities were measured in order to monitor whether the compounds were present at biologically active concentrations. Locomotor behavior was observed and tissues from the peripheral and central nervous systems were examined for signs of organophosphate-induced delayed neuropathy (OPIDN). No behavioral or histological evidence of OPIDN was observed after treatments with GA, IFP, PO, saline or atropine sulfate. DFP-treated birds displayed locomotor and neuropathological signs of OPIDN with a no effect level (NOEL) between 25 and 50 micrograms/kg.     

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

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

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.     

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.     

Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy

Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OP-induced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-o-cresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1 mg/kg i.m.) and calcium gluconate (Ca-glu 5 mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24 h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12 h after TOCP was given and, in the sciatic nerve, Ca²⁺ fell 56.4% 24 h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP.     

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.     

Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride

Certain sulfonates, like phenylmethanesulfonyl fluoride (PMSF), carbamates, and phosphinates, when given prior to neuropathic doses of organophosphates such as diisopropyl phosphorofluoridate (DFP), protect hens from organophosphate-induced delayed polyneuropathy (OPIDP). Protection was related to inhibition of the putative target of OPIDP, which is called Neuropathy Target Esterase (NTE). NTE inhibition above 70-80% in the nervous system of hens followed by a molecular rearrangement called aging initiates OPIDP. PMSF and other protective chemicals inhibit NTE but OPIDP does not develop because aging cannot occur. DFP (1 mg/kg sc) inhibited NTE above 70-80% in peripheral nerve and caused OPIDP in hens. Lower doses (0.3 and 0.5 mg/kg sc) caused about 40-60% NTE inhibition and no or marginal OPIDP. Chlorpyrifos (90 mg/kg po) also caused OPIDP. When repeated (30 mg/kg sc daily for 9 days) or single (5-120 mg/kg sc) doses of PMSF were given after either DFP or chlorpyrifos, OPIDP developed in birds treated with nonneuropathic doses of DFP and was more severe in birds treated with chlorpyrifos or higher doses of DFP. PMSF increased NTE inhibition to greater than 90%. Promotion of OPIDP with a single dose of PMSF (120 mg/kg sc) was obtained in birds up to 11 days after a marginally neuropathic dose of DFP (0.5 mg/kg sc). Promotion was also obtained with phenyl N-methyl N-benzyl carbamate (40 mg/kg iv) but not with non-NTE inhibitors in vivo such as paraoxon or benzenesulfonyl fluoride when given at maximum tolerated doses. These results indicate that protection from OPIDP is only one effect of PMSF because promotion of OPIDP is also observed depending upon the sequence of dosing. Either effect is always related to the doses of PMSF, which inhibit NTE.     

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.