Organophosphate-induced Delayed Neurotoxicity Research Articles

Changes of Neuropathy Target Esterase Affect Phospholipid Homeostasis in SK-N-SH Cells

Neuropathy target esterase (NTE), is a membrane protein located in the endoplasmic reticulum (ER). NTE has the activity of phospholipase B and can catalyze the deacylation of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) to glycerylcholine (GPC). It is phosphorylated and aged by organophosphorus compounds (OPs), that induce delayed neuropathy in humans and sensitive animals. Our previous study has reported that the disruption of ER phospholipid homeostasis caused by the NTE inhibition may contribute to the initiation of the organophosphate-induced delayed neurotoxicity (OPIDN), while it is unknown how the disturbed phospholipid homeostasis initiates OPIDN. It is difficult to change phospholipids in in vivo experiments. Therefore, an in vitro model is urgently needed to explain the role of phospholipid homeostasis disorders in OPIDN. In this study, we altered the expression of NTE in SK-N-SH cells and determined its phospholipid component by using HPLC-MS. Our results showed that the changes of NTE affected the levels of PC, sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylserine (LPS), phosphatidyl-glycerol (PG), and phosphatidylinositol (PI). Our results were consistent with the in vivo results. Furthermore, our findings indicate that the SK-N-SH cell model is a significantly useful method for the further research on how the changes of phospholipid homeostasis initiate the OPIDN, which is easier than the in vivo experiments in practice.

Disturbed phospholipid homeostasis in endoplasmic reticulum initiates tri-o-cresyl phosphate-induced delayed neurotoxicity.

Tri-o-cresyl phosphate (TOCP) is a widely used organophosphorus compound, which can cause a neurodegenerative disorder, i.e., organophosphate-induced delayed neurotoxicity (OPIDN). The biochemical events in the initiation of OPIDN were not fully understood except for the essential inhibition of neuropathy target esterase (NTE). NTE, located in endoplasmic reticulum (ER), catalyzes the deacylation of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) to glycerophosphocholine (GPC). The present study aims to study the changes of ER phospholipids profile as well as levels of important intermediates of phospholipid synthesis such as diacylglycerol (DAG) and phosphatidic acid (PA) at the initiation stage of OPIDN. Hens are the most commonly used animal models of OPIDN. The spinal cord phospholipidomic profiles of hens treated by TOCP were studied by using HPLC-MS-MS. The results revealed that TOCP induced an increase of PC, LPC, and sphingomyelin (SM) levels and a decrease of GPC, phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), phosphatidylglycerol (PG), and phosphatidylinositol (PI) levels., Levels of DAG and PA were also decreased. Pretreatment with phenylmethylsulfonyl fluoride (PMSF) 24 h before TOCP administration prevented OPIDN and restored the TOCP-induced changes of phospholipids except GPC. Thus, the disruption of ER phospholipid homeostasis may contribute to the initiation of organophosphate-induced delayed neurotoxicity.

Biologic activity of cyclic and caged phosphates: a review.

The recognition in the early 1960s by Morifusa Eto that tri-o-cresyl phosphate (TOCP) is hydroxylated by the cytochrome P450 system to an intermediate that spontaneously cyclizes to a neurotoxic phosphate (saligenin phosphate ester) ignited the interest in this group of compounds. Only the ortho isomer can cyclize and clinically cause Organo Phosphate Induced Delayed Neurotoxicity (OPIDN); the meta and para isomers of tri-cresyl phosphate are not neuropathic because they are unable to form stable cyclic saligenin phosphate esters. This review identifies the diverse biological effects associated with various cyclic and caged phosphates and phosphonates and their possible use. Cyclic compounds that inhibit acetylcholine esterase (AChE), such as salithion, can be employed as pesticides. Others are neurotoxic, most probably because of inhibition of neuropathy target esterase (NTE). Cyclic phosphates that inhibit lipases, the cyclipostins, possibly represent promising therapeutic avenues for the treatment of type 2 diabetes mellitus and/or microbial infections; those compounds inhibiting β-lactamase may prevent bacterial resistance against β-lactam antibiotics. Naturally occurring cyclic phosphates, such as cyclic AMP, cyclic phosphatidic acid and the ryanodine receptor modulator cyclic adenosine diphosphate ribose, play an important physiological role in signal transduction. Moreover, some cyclic phosphates are GABA-antagonists, while others are an essential component of Molybdenum-containing enzymes. Some cyclic phosphates (cyclophosphamide, ifosfamide) are clinically used in tumor therapy, while the coupling of therapeutic agents with other cyclic phosphates (HepDirect® Technology) allows drugs to be targeted to specific organs. Possible clinical applications of these compounds are considered. Copyright © 2016 John Wiley & Sons, Ltd.

Propetamphos enhances chlorpyrifos-induced organophosphate-induced delayed neurotoxicity (OPIDN) in Hens: a role for plasma butyrylcholinesterase

ABSTRACTThis study examined dose-response effects of chlorpyrifos (CPF) and propetamphos (PRO) on producing organophosphorus-induced delayed neurotoxicity (OPIDN) in hens. A single oral dose of 1, 3, 30, or 100 mg/kg of each compound was administered individually, or in combination to groups of 10 hens. Plasma butyrylcholinesterase (BChE) activity was inhibited by all doses of each individual compound. Chlorpyrifos alone or in combination with PRO resulted in decreased acetylcholinesterase (AChE) activity. No dose of CPF or PRO alone resulted in the inhibition of neuropathy target esterase (NTE) or produced OPIDN. In contrast, combined exposure to high doses of CPF and PRO produced diminished NTE activity and produced OPIDN. Brain NTE activity was decreased by 67% and 71% by combined treatment of 100 mg/kg CPF with 30 mg/kg and 100 mg/kg PRO, respectively, consistent with the development of ataxia and paralysis, characteristic of OPIDN. Only the highest dose of CPF 100 mg/kg interacted with the two highest doses of PRO to block NTE and induce OPIDN, whereas no marked interaction occurred at lower doses. Data indicate that PRO enhances the ability of high doses of CPF to produce OPIDN by binding to plasma BChE that appears to act as a buffering system to modulate the toxicity of CPF. In effect, the ability of BChE to bind to CPF is diminished by PRO, resulting in more CPF delivery to the brain, and subsequent development of OPIDN at a dose that does not initiate this effect by itself.

Organophosphorus Flame Retardants (OPFR): Neurotoxicity

Organophosphorus flame retardants (OPFRs) are used as additives in plasticizers, foams, hydraulic fluids, anti-foam agents, and coatings for electronic components/devices to inhibit flames. These chemicals were developed and used as flame retardants because of environmental and health concerns of previously used brominated and chlorinated flame retardants (FRs). OPCFRs are divided into five main groups: organophosphates, organophosphonates, organophosphinates, organoposphine oxide, and organophosphites. Most of OPFRs are organophosphate esters that are further classified into the following five groups: 1. Aliphatic, 2. Brominated aliphatic, 3. Chlorinated aliphatic, 4. Aromatic-aliphatic, and 5. Aromatic phosphates. These OPFRs have the following neurotoxic actions: 1. Cholinergic Neurotoxicity, 2. Organophosphate-Induced Delayed Neurotoxicity (OPIDN), and 3. Organophosphate-Induced Chronic Neurotoxicity (OPICN) in addition to being endocrine disruptors. OPFRs have very low cholinergic neurotoxicity and this effect does not pose significant health hazards to adults or children. On the other hand, some OPFRs have shown to cause OPIDN that is a delayed central-peripheral axonopathy, characterized by neuronal cell death of the lower brain regions, spinal cord and peripheral nervous systems, leading to long-term neuronal injury. OPICN is characterized by neuronal cell death in the cortex, hippocampus campus and cerebellum and spinal cord. Finally, OPCFRs act as endocrine disrupters, that affect many functions of the body such thyroid glands and reproductive functions, and may be involved in the development of diabetes and cancer. Residues of these OPCFRs are widespread in the environment, home and workplaces. These chemicals adversely affect human health, especially for vulnerable population such as the elderly, pregnant women, fetuses, and children. Because some OPFRs cause neuronal cell death in the brain and spinal cord that do not repair as well as act as endocrine disrupters they may lead to permanent functional deficits such obesity, memory impairment, decreased motor skill and even more serious diseases such as diabetes and cancer. Because recent reports have accredited FRs for significant decrease in building fires, it is important to balance the risk and benefits of FRs and to use only the safest available FRs including OPFRs. *Corresponding author: Mohamed B. Abou-Donia, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 277, USA, Tel: +919-68422/ Fax: 919-681-822, E-mail: donia@duke.edu Citation: Abou-Donia, M.B., et al. Organophosphorus Flame Retardants (OPFR): Neurotoxicity. (2016) J Environ Health Sci 2(1): 129. Organophosphorus Flame Retardants (OPFR): Neurotoxicity Mohamed B. Abou-Donia1* Mohamed Salama2, Mohamed Elgamal2, Islam Elkholi2,3, Qiangwei Wang1,4 Received date: July 07, 2015 Accepted date: May 05, 2016 Published date: May 11, 2016 DOI: 10.15436/2378-6841.16.022

Molecular design of O-phosphorylated oximes—Selective inhibitors of butyrylcholinesterase

Anticholinesterase compounds, including organophosphorus inhibitors (OPIs), are used to treat glaucoma, schistosomiasis, Alzheimer’s disease, myasthenia gravis, and other diseases. These compounds arealso widely used in agriculture as insecticides and inindustry [1]. The varied use of such compounds isbased on their common mechanism of action, which isassociated with the inhibition of acetylcholinesterase(AChE, EC 3.1.1.7), the biological role of which is thehydrolysis of the key neurotransmitter acetylcholine.In the treatment of Alzheimer’s disease (AD) underconditions of deficiency of central acetylcholine andreduced cholinergic transmission, the inhibition ofAChE in the brain provides cognition enhancement,which, however, may be accompanied by manifestations of acute toxicity and other side effects associatedwith inhibition of peripheral AChE [2].Many OPIs interact in the body with otheresterases. These interactions may lead to differentpharmacological and toxicological effects. For example, the neuropathy target esterase (NTE, EC 3.1.1.5),which normally catalyzes deacylation of phosphatidylcholine of cell membranes [3], is an “antitarget”responsible for the development of organophosphateinduced delayed neurotoxicity (OPIDN). This phenomenon is due to the fact that its inhibition and subsequent rapid “aging” initiates distal degeneration ofsensory and motor axons in the peripheral nervous system and/or spinal cord, causing paralysis [1]. This disease is characterized by a latent period of up to severalweeks; specific drugs for its treatment are still notknown. Thus, the possibility of initiation of delayedneurotoxicity is an important risk factor in industrialand medical applications of OPIs.Other serine esterases, such as butyrylcholinesterase (BChE, EC 3.1.1.8) and carboxylesterase (CaE,EC 3.1.1.1), function as stoichiometric scavengers,providing protection against the toxic effect of anticholinesterase agents [4]. BChE and CaE of bloodplasma and liver are also in volved in the metabolism ofmany drugs containing ester groups, significantlyaffecting their pharmacokinetics.Recent studies have shown that BChE is alsoinvolved in cholinergic transmission and is able tocompensate for some functions of AChE, optimizingthe cholinergic neurotransmission by hydrolysis ofacetylcholine under conditions when AChE is, forsome reason, unable to perform this function(for example, in severe forms of AD, when the activityof AChE is significantly reduced). It was found thatselective inhibitors of BChE increase the level of acetylcholine in the brain and provide cognition enhancement in rodents, having no side effects of classicalinhibitors of AChE. In this regard, the development ofselective inhibitors of BChE is a new strategy toimprove the quality of life of patients with AD, especially in severe cases, when the activity of AChE in thebrain is depleted along with a decrease in the level ofacetylcholine, whereas the activity of BChE isincreased [2].Within the considered scheme of interaction withthe four serine esterases, the esterase profile of aninhibitor can be described by four activity values(the inhibitory activity with respect to AChE

TOX 21: New Dimensions of Toxicity Testing

On the ground floor of the National Institutes of Health Chemical Genomics Center (NCGC) in Rockville, Maryland, a $10-million automated laboratory spends all day and night screening chemicals at speeds no team of human researchers could ever match. In a week, depending on the nature of the assay, it can yield up to 2.2 million molecular data points derived from thousands of chemicals tested at 15 concentrations each.

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.

Verapamil abolished the enhancement of protein phosphorylation of brainstem mitochondria and synaptosomes from the hens dosed with tri- o-cresyl phosphate

To explore the changes of the endogenous phosphorylation of brainstem mitochondrial and synaptosomal proteins in adult hens dosed with tri- o-cresyl phosphate (TOCP) following the development of organophosphate-induced delayed neurotoxicity (OPIDN). Verapamil (7 mg/(kg day), i.m.) was given for 4 days. A dose of TOCP (750 mg/kg, p.o.) was administrated in second day after verapamil. Phosphorylation of the proteins from brainstem mitochondria and synaptosomes was assayed in vitro by using [γ- 32P]ATP as phosphate donor. Radiolabeled proteins were separated by SDS-PAGE and visualized by autoradiography. The results showed that TOCP administration enhanced the phosphorylation of the cell organelle proteins (mitochondria: 60, 55, 45, and 20 kDa; synaptosomes: 65, 60, and 20 kDa), while verapamil abolished the enhancement induced by TOCP. Additionally, the reaction for the phosphorylation is catalyzed by the calcium/calmodulin protein kinase. Therefore, TOCP can enhance the phosphorylation of the brainstem mitochondrial and synaptosomal proteins from the hens with OPIDN; however, protection from the enhancement of the phosphorylation should be involved in the mechanisms of the amelioration of TOCP-induced delayed neurotoxicity by verapamil.

The L‐Type Calcium Channel Blocker Nimodipine Mitigates Cytoskeletal Proteins Phosphorylation in Dichlorvos‐Induced Delayed Neurotoxicity in Rats

The present investigation was carried out to assess the protective efficacy of nimodipine against dichlorvos-induced organophosphate induced delayed neurotoxicity (OPIDN). Single subcutaneous dose of dichlorvos (200 mg/kg body weight) led to a consistent increase in the activity of both microtubule associated protein kinases viz. Ca2+/Calmodulin-dependent and cAMP dependent protein kinases, at all post exposure intervals (day 7, 15 and 21) as compared to that of controls. Autoradiography followed by microdensitometric studies demonstrated enhanced phosphorylation of 55 kDa and 280 kDa proteins in dichlorvos-exposed animals. These two proteins were confirmed to be tubulin and microtubule associated protein-2 (MAP-2) by western blotting. The hyperphosphorylation of these two proteins was shown to interfere with the assembly of neuronal microtubules as shown by electron microscopic studies that may eventually lead to possible disruption of neuronal cytoarchtecture resulting in axonal degeneration. Administration of nimodipine along with dichlorvos brought about a significant reduction in the activities of both the kinases as well as the extent of microtubule associated protein phosphorylation. This indicates that nimodipine, a centrally acting calcium channel blocker, may contribute to the amelioration of dichlorvos induced neurotoxicity by attenuation of calcium mediated disruption of cytoskeletal proteins and hence, calcium channel blockers like nimodipine have great future as new therapeutic agents for OPIDN.

Toxicogenomic Studies of the Rat Brain at an Early Time Point Following Acute Sarin Exposure

We have studied sarin-induced global gene expression patterns at an early time point (2 h: 0.5 x LD50) using Affymetrix Rat Neurobiology U34 chips and male Sprague-Dawley rats. A total of 46 genes showed statistically significant alterations from control levels. Three gene categories contained more of the altered genes than any other groups: ion channel (8 genes) and calcium channel and binding proteins (6 genes). Alterations were also found in the following gene groups: ATPases and ATP-based transporters (4), growth factors (4), G-protein-coupled receptor pathway-related molecules (3), neurotransmission and neurotransmitter transporters (3), cytoskeletal and cell adhesion molecules (2), hormones (2), mitochondria-associated proteins (2), myelin proteins (2), stress-activated molecules (2), cytokine (1), caspase (1), GABAnergic (1), glutamergic (1), immediate early gene (1), prostaglandin (1), transcription factor (1), and tyrosine phosphorylation molecule (1). Persistent alteration of the following genes also were noted: Arrb1, CaMKIIa, CaMKIId, Clcn5, IL-10, c-Kit, and Plp1, suggesting altered GPCR, kinase, channel, and cytokine pathways. Selected genes from the microarray data were further validated using relative RT-PCR. Some of those genes (GFAP, NF-H, CaMKIIa, Calm, and MBP) have been shown by other laboratories and ours, to be involved in the pathogenesis of sarin-induced pathology and organophosphate-induced delayed neurotoxicity (OPIDN). Induction of both proapoptotic (Bcl2l11, Casp6) and antiapoptotic (Bcl-X) genes, besides suppression of p21, suggest complex cell death/protection-related mechanisms operating early on. Principal component analysis (PCA) of the expression data confirmed that the changes in gene expression are a function of sarin exposure, since the control and treatment groups separated clearly. Our model (based on current and previous studies) indicates that both degenerative and regenerative pathways are activated early and contribute to the level of neurodegeneration at a later time, leading to neuro-pathological alterations.

Effect of tri- o-cresyl phosphate and methamidophos on 45Ca uptake by brain synaptosomes in hens

The effect of tri- o-cresyl phosphate (TOCP) and methamidophos (MET) on potassium-stimulated 45Ca uptake by brain synaptosomes in hens was studied. An in vivo test showed that TOCP increased potassium-stimulated calcium uptake 2 h after its administration, but that verapamil suppressed the enhancement of this calcium uptake. An in vitro test showed that lower concentrations of TOCP stimulated calcium uptake by synaptosomes, but that higher concentrations inhibited the uptake. In contrast, all tested concentrations of MET obviously inhibited calcium uptake; however, since calcium uptake was decreased by the administration of verapamil plus either TOCP or MET, the mechanism by which TOCP affects the voltage-operated calcium channel may be different from that of MET. The disruption of calcium homeostasis may be involved in organophosphate-induced delayed neurotoxicity (OPIDN). Calcium channel blocker may ameliorate OPIDN by maintaining calcium homeostasis in nerve cells.

Neurotoxicity of organophosphorus and dithiocarbamate compounds

The neurotoxicity of organophosphorus compounds (OPs) including leptophos, TOCP and triphenyl phosphite and dithiocarbamate compounds were reviewed in this study. The major neurotoxicities of OPs were acute toxicity produced by the acetylcholine esterase (AChE) inhibiting action of OPs and delayed neurotoxicity produced by such OPs as leptophos and TOCP. The direct action of OP on the muscarinic and/or nicotinic acethylcholine receptors in the synaptic membranes have lately attracted attention in relation to acute toxicity. Delayed neurotoxicity is a delayed onset of prolonged locomotor ataxia resulting from a single or repeated exposure to an OP. Although neurotoxic esterase (NTE) inhibition might be related to the onset of organophosphate-induced delayed neurotoxicity (OPIDN), the precise mode of action is not yet clear. The effect of dithiocarbamates on the nervous system is also mentioned, because the compounds are currently suspected not only for neurotoxicity, but also as endocrine-disrupting chemicals. Although dithiocarbamates showed weak neurotoxicity in adult animals, we need to pay more attention to developmental neurotoxicity.

Possible involvement of dopaminergic neurotransmitter system in dichlorvos induced delayed neurotoxicity.

The present investigation was carried out to elucidate the possible involvement of the dopaminergic neurotransmitter system in the development of dichlorvos induced delayed neurotoxicity in the rat and to assess the protective efficacy of nimodipine against OPIDN (Organophosphate induced delayed neurotoxicity). Single subcutaneous dose of dichlorvos (200 mg/kg body weight) resulted in marked changes in the dopaminergic neurotransmitter system in terms of increased levels of both dopamine and norepinephrine along with significant increase in the activity of both the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase. This increase was accompanied with a concomitant decrease in the activity of the major degradative enzyme, monoamine oxidase. Scatchard plot analysis revealed a significant decrease in both K(d) and B(max) for dopamine D2 receptors. Administration of nimodipine, a centrally acting calcium channel blocker, along with dichlorvos restricted all these alterations to within control values and could also ameliorate certain behavioural deficits by maintaining the dopaminergic neurotransmitter system. The study underlines the importance of alterations in the dopamine system as a possible causative mechanism behind the behavioural and functional changes associated with delayed neurotoxicity.

Possible role of enhanced microtubule phosphorylation in dichlorvos induced delayed neurotoxicity in rat

The effect of a single subcutaneous dose of 200 mg/kg body weight dichlorvos on neuronal microtubule phosphorylation has been studied in rat following the development of organophosphate induced delayed neurotoxicity (OPIDN). Microtubule associated Ca 2+/calmodulin dependent as well as cAMP dependent protein kinases were assayed. Dichlorvos administration led to a consistent increase in the activity of both the kinases at all post exposure intervals (7th, 15th and 21st day) as compared to that of controls. After in vitro phosphorylation using [γ- 32P]ATP, various proteins were resolved on one-dimensional 8% SDS–PAGE, stained with Coomassie Blue and autoradiographed. The amount of 32P incorporated was quantified by microdensitometry. Dichlorvos enhanced the phosphorylation of 55- and 280-kDa proteins. These two proteins were identified as tubulin and microtubule associated protein-2 (MAP-2) by immunoblotting. This study showed that dichlorvos induced hyperphosphorylation of tubulin and MAP-2 which in turn destabilizes microtubule assembly, and may ultimately result in axonal degeneration leading to dichlorvos induced delayed neurotoxicity.

A new approach for determination of neuropathy target esterase activity

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

A stable preparation of hen brain neuropathy target esterase for rapid biochemical assessment of neurotoxic potential of organophosphates

Neuropathy target esterase (NTE) is a molecular target for organophosphate-induced delayed neurotoxicity (OPIDN). This enzyme has proved to be an excellent tool for the assessment of neuropathic potential of organophosphates (OP), in particular by comparison of an OP inhibitory activity in vitro against NTE and acetylcholinesterase. A large-scale OP screening for delayed neurotoxicity was largely prevented by the lack of an available stable preparation of NTE. To obtain a stable NTE preparation the influence of intensive freezing and subsequent lyophilization of paraoxon-preinhibited (P2+P3) hen brain membrane fraction on NTE properties has been studied using two neuropathic OP: mipafox and O,O-dipropyldichlorovinyl phosphate (PrDChVP). It was shown that lyophilization preserved a high NTE specific activity and did not alter the inhibitor characteristics of the enzyme. A long-term storage study showed that lyophilized NTE preparation exhibited inhibitory features actually identical to those of the native enzyme during 1 year and retained rather high specific activity; in this case some loss of NTE specific activity has been observed. Comparative studies of inhibition of the native and lyophilized NTE preparations by a model series of phenyl phosphonates RO(C6H5)P(O)ONCClCH3 (R=alkyl), demonstrated a good correlation between the values pI50 obtained with both enzyme preparations as well as identical structure-activity relationships for the lyophilized and native enzymes. The results allow the conclusion that the obtained NTE preparation can be used as a standard, stable and readily available source of NTE for assessing the anti-NTE activity of OP.

Cholinesterase activity in quails of neuropathy caused by organophosphates.

It is known that some organophosphates produce not only well-known acute toxicity but also characteristic delayed neurotoxicity. Tri-ortho-tolyl phophate (TOTP), which was formerly named Tri-ortho-cresyl phosphaete (TOCP), was first noticed in an incident of poisoning as the compound which produced organophosphate induced delayed neurotoxicity (OPIDN). It is said that triphenyl phosphite (TPP) is also one of the organophosphates which possesses OPIDN. However, it is thought that TPP-induced delayed neurotoxicity (TPP-DN) is not identical with classical OPIDN. An intermediate syndrome was later proposed as the third neurotoxicity caused by organophosphates. We think that TPP is a model chemical of the third neurotoxicity. We compared TOTP with TPP using Japanese quails. We measured cholinesterase (ChE) activity and clearly demonstrated the difference between the two chemicals, that is to say, the activity recovered after 72 hrs from the administration of TPP, whereas the inhibition continued for more than 11 days after the administration of TOTP.

Effect of phenylmethylsulfonyl fluoride administration prior to and following leptophos administration on electrolyte concentration and enzyme activity in hen serum

We observed acute toxicity, delayed neurotoxicity, disappearance of leptophos from tisuues and biochemical changes in four groups of hens: a group given only 30 mg/kg leptophos (iv) as the 'leptophos group', two groups given a treatment of 30 mg/kg phenylmethylsulfonyl fluoride (PMSF) (sc) 24 hr prior to (as the 'pretreated group') and following (as the 'posttreated group') administration of the same dose of leptophos as the leptophos group, and a group given a vehicle only as the 'control group'. All groups other than the control group showed acute toxicity. The scores for organophosphate-induced delayed neurotoxicity (OPIDN) in the posttreated group reached the maximal level on the 16th day after leptophos administration and those in the leptophos group reached the maximal level on the 25th day. Serum acid phosphatase (AcP) activities in the leptophos group and the posttreated group were significantly lower than that in the control group (p<0.05) on the 6th day after leptophos administration and then recovered to the normal level on the 15th day. In these two groups, serum creatine phosphokinase (CPK) activity was significantly higher (p<0.01) and the concentration of serum Ca(2+) was significantly lower (p<0.05) than in the control group on the 15th day after leptophos administration. Serum leucine aminopeptidase (LAP) activity in the posttreated group was significantly lower than that in the control group (p<0.01). As for the significant changes by time interval between the 6th and the 15th days after leptophos administration, CPK activity was elevated and serum Ca(2+) reduced in both the leptophos group and the posttreated group, and LAP activity was also reduced in the posttreated group. The courses of leptophos disappearance in several tissues of these hens were similar in the 3 groups. These results suggest that the treatment by PMSF prior to or following the administration of leptophos can significantly modify not only clinical signs of OPIDN but also changes of several biochemical indices accompanied by OPIDN. Furthermore, it is possible to expect that these biochemical indices can provide some valuable clues for exploring the modification of OPIDN by PMSF treatment.

Possible involvement of a neurotrophic factor during the early stages of organophosphate-induced delayed neurotoxicity ☆

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neurotoxic esterase (NTE). We hypothesized that a trophic factor may be produced in situ shortly after exposure to the OP which participates in the progression of OPIDN. To bioassay for such a growthmodulating factor(s), we treated chickens with the neuropathic agents diisopropylfluorophosphate (DFP) or cyclic phenyl saligenin phosphate (PSP), with or without phenylmethylsulfonyl fluoride (PMSF, a chemical which markedly modifies OPIDN). Soluble extracts of cervical spinal cord (a region of the nervous system which degenerates with OPIDN) were collected 24 h later and these were incubated with human neuroblastoma SY5Y cells in culture. The cells were allowed to grow for another 6 days and observed for changes in morphology and growth. After 3 days in culture, tissue extracts from OP-treated chickens caused SY5Y cells to begin to elongate and extend processes (neurites), similar to cells treated with nerve growth factor (1μg/ml). Extracts from chickens not receiving OP had no or minimal effects on cell morphology. In addition, extracts from chickens in which OPIDN was prevented by pretreatment with PMSF did not cause the marked extension of cell processes exhibited after exposure of SY5Y cells to extracts from chickens given regimens known to cause OPIDN. In parallel-treated animals, DFP and PSP caused clinical dysfunction characteristic of OPIDN, PMSF posttreatment markedly amplified the clinical deficits and PMSF pretreatment prevented OPIDN. In vivo DFP treatment also caused a marked reduction in the activity of the growthrelated enzyme ornithine decarboxylase (ODC) in spinal cord but DFP was without effect on ODC activity in vitro (up to 1 mM final concentration). Characterization of this growth-modulating factor(s) may aid in the elucidation of pathological mechanisms of OPIDN.