The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase.
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ABSTRACT:
SUBMITTER: Johnson MK
PROVIDER: S-EPMC1184957 | biostudies-other | 1969 Oct
REPOSITORIES: biostudies-other
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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
Project description: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.
| S-EPMC1179633 | biostudies-other
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
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
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
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
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.
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Project description:The standard methods for toxicity testing using rodent models cannot keep pace with the increasing number of chemicals in our environment due to time and resource limitations. Hence, there is an unmet need for fast, sensitive, and cost-effective alternate models to reliably predict toxicity. As part of Tox21 Phase III's effort, a 90-compound library was created and made available to researchers to screen for neurotoxicants using novel technology and models. The chemical library was evaluated in zebrafish in a dose-range finding test for embryo-toxicity (ie, mortality or morphological alterations induced by each chemical). In addition, embryos exposed to the lowest effect level and nonobservable effect level were used to measure the internal concentration of the chemicals within the embryos by bioanalysis. Finally, considering the lowest effect level as the highest testing concentration, a functional assay was performed based on locomotor activity alteration in response to light-dark changes. The quality control chemicals included in the library, ie, negative controls and replicated chemicals, indicate that the assays performed were reliable. The use of analytical chemistry pointed out the importance of measuring chemical concentration inside embryos, and in particular, in the case of negative chemicals to avoid false negative classification. Overall, the proposed approach presented a good sensitivity and supports the inclusion of zebrafish assays as a reliable, relevant, and efficient screening tool to identify, prioritize, and evaluate chemical toxicity.
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Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.
2015-06-10 | E-GEOD-69712 | biostudies-arrayexpress
Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes.
2015-06-10 | GSE69712 | GEO