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Organophosphorus and other inhibitors of brain 'neurotoxic esterase' and the development of delayed neurotoxicity in hens.

ABSTRACT: 1. The delayed neurotoxic effects of some organophosphorus compounds are associated with phosphorylation of the active site of a nervous-tissue enzyme capable of hydrolysing phenyl phenylacetate. 2. Neurotoxic organophosphorus compounds and some carbamates and sulphonyl fluorides progressively inhibit the enzyme, attaching a substituent covalently at the active site. 3. Prolonged inhibition of the enzyme by phenyl N-benzyl-N-methylcarbamate or phenylmethane-sulphonyl fluoride does not lead to neurotoxic effects. 4. Prior inhibition of the enzyme by carbamates or sulphonyl fluorides in vivo prevents the neurotoxic effects of several organophosphorus compounds. 5. After dosage of hens with protective compounds, protection lasts until about 70% of the enzyme site again becomes available for phosphorylation. 6. Reaction of all the inhibitors at the active site of the enzyme leads to the same inhibitory effect with respect to hydrolysis of phenyl phenylacetate but does not in all cases lead to delayed neurotoxicity. It is concluded that the nature of the group substituted at the enzyme active site determines the toxic response.

SUBMITTER: Johnson MK

PROVIDER: S-EPMC1179633 | biostudies-other | 1970 Dec

REPOSITORIES: biostudies-other

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Neuropathy target esterase (NTE/PNPLA6) and organophosphorus compound-induced delayed neurotoxicity (OPIDN).

Project description:Systemic inhibition of neuropathy target esterase (NTE) with certain organophosphorus (OP) compounds produces OP compound-induced delayed neurotoxicity (OPIDN), a distal degeneration of axons in the central nervous system (CNS) and peripheral nervous system (PNS), thereby providing a powerful model for studying a spectrum of neurodegenerative diseases. Axonopathies are important medical entities in their own right, but in addition, illnesses once considered primary neuronopathies are now thought to begin with axonal degeneration. These disorders include Alzheimer's disease, Parkinson's disease, and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Moreover, conditional knockout of NTE in the mouse CNS produces vacuolation and other degenerative changes in large neurons in the hippocampus, thalamus, and cerebellum, along with degeneration and swelling of axons in ascending and descending spinal cord tracts. In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness. Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders. This chapter defines NTE and OPIDN, presents an overview of OP compounds, provides a rationale for NTE research, and traces the history of discovery of NTE and its relationship to OPIDN. It then briefly describes subsequent studies of NTE, including practical applications of the assay; aspects of its domain structure, subcellular localization, and tissue expression; abnormalities associated with NTE mutations, knockdown, and conventional or conditional knockout; and hypothetical models to help guide future research on elucidating the role of NTE in OPIDN.

| S-EPMC7271139 | biostudies-literature

The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase.

Project description:1. Organophosphorus compounds that produce a delayed neurotoxic effect in hens phosphorylate a specific site in the brain soon after administration. 2. Phosphorylation of the specific site by di-isopropyl [(32)P]phosphorofluoridate in vitro is blocked by the prior addition of phenyl phenylacetate. 3. A small proportion of the total activity of hen brain hydrolysing phenyl phenylacetate in vitro was shown to be due to an enzyme different from two others previously described. 4. This enzyme is only slightly inhibited in vitro by concentrations of tetraethyl pyrophosphate and paraoxon (diethyl 4-nitrophenyl phosphate) up to 64mum and is completely inhibited by 6mum-di-isopropyl phosphorofluoridate and 128mum-mipafox. 5. It is also inhibited in vivo by effective doses of neurotoxic organophosphorus compounds but not by high doses of non-neurotoxic analogues. 6. It is deduced that the active site of this enzyme is the phosphorylation site associated with the genesis of delayed neurotoxicity.

| S-EPMC1184957 | biostudies-other

A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds.

Project description:1. It is proposed that part of a neurotoxic dose of di-isopropyl phosphorofluoridate will be covalently bound in vivo to a specific component in the brain and spinal cord as the initial biochemical event in the genesis of the lesion. 2. A test system in vitro was devised that removes many di-isopropyl phosphorofluoridate-binding sites and indicates that the specific component may be a protein present in brain at a concentration comparable with that of the cholinesterases. 3. The site was found to be present and capable of binding di-isopropyl phosphorofluoridate in vitro in brain samples taken from either normal hens or those dosed with organophosphorus esterase inhibitors that are not neurotoxic. 4. Very little of the specific binding activity was found in brain samples from hens pre-dosed with a variety of neurotoxic organophosphorus compounds. 5. A solubilized preparation of the active brain component was obtained, suitable for further purification and study.

| S-EPMC1187568 | biostudies-other

Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters.

Project description:1. It was proposed [Johnson (1974) J. Neurochem. 23, 785--789] that both inhibition of neurotoxic esterase of nervous tissue and subsequent 'aging' of the inhibited esterase are necessary events in the pathogenesis of organophosphate-induced delayed neuropathy: aging has now been demonstrated with a number of neurotoxic compounds. 2. Reactivation by KF was observed for hen brain neurotoxic esterase inhibited by 14 organophosphates and phosphonates, and time-dependent loss of reactivatibility (aging) occurred in every case. 3. For five other compounds no reactivation occurred and aging could not therefore be established, but independent evidence for two compounds suggests that aging was rapid. 4. Half-lives of aging of neurotoxic esterase inhibited by phosphates ranged from less than 1 min to 10 min, and for phosphonates the range was 3--600 min. 5. The relationship of these findings to the mechanism of toxicity and to the prospects of therapy are considered. 6. Aging occurred rapidly with aryloxy and linear alkoxy groups attached to phosphorus and slowly with a highly branched alkoxy substituent: these effects seem incompatible with an SN1 (dealkylation) mechanism.

| S-EPMC1161452 | biostudies-other

Characterization of [3H]di-isopropyl phosphorofluoridate-binding proteins in hen brain. Rates of phosphorylation and sensitivity to neurotoxic and non-neurotoxic organophosphorus compounds.

Project description:The experiments described in this paper were designed to isolate [3H]di-isopropyl phosphorofluoridate-binding proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for the purpose of characterizing and identifying potential initiation sites for organophosphorus-compound-induced delayed neurotoxicity. The major Paraoxon-insensitive Mipafox-sensitive binding protein (Mr 160 000) was found to be identical with one previously identified as neurotoxic esterase, an enzyme that has been proposed to be the target site for organophosphorus-compound-induced delayed neurotoxicity. However, two other binding proteins with suitable binding characteristics were also found in smaller amounts, one of which has not been detected previously. Di-isopropyl phosphorofluoridate was found to phosphorylate all three of these proteins at rates similar to the rate at which neurotoxic esterase is inhibited by di-isopropyl phosphorofluoridate. Varying the concentration of di-isopropyl phosphorofluoridate or the time of incubation produced similar increases in binding to each of the labelled proteins. This suggests that the reaction rates of di-isopropyl phosphorofluoridate with proteins may be described by first-order kinetics, and the concentration of the Michael is complex formed during binding is minimal for all the phosphorylated proteins. The recovery of the binding activity in the 160 000-Mr band was found to be similar to the recovery of neurotoxic esterase activity, lending further support to the contention that this band is identical with neurotoxic esterase.

| S-EPMC1145020 | biostudies-other

Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase.

Project description:We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

| S-EPMC1137116 | biostudies-other

Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate.

Project description:The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

| S-EPMC1163375 | biostudies-other

Analysis of the neurotoxic effects of neuropathic organophosphorus compounds in adult zebrafish.

Project description:Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN.

| S-EPMC5859099 | biostudies-literature

Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate.

Project description:1. It was proposed [Johnson (1974) J. Neurochem.23, 785-789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with (3)H and (32)P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37 degrees C occurred with a half-life of about 2-4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred (3)H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.

| S-EPMC1186405 | biostudies-other

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

Project description:The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.

| S-EPMC1152820 | biostudies-other

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