Estevez J

General

Full name : Estevez Jorge

First name : Jorge

Mail : University Miguel Hernandez\; Institute Bioenineering\; Avenida de la Universidad\; Elche\; 3202

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City :

Country : Spain

Email : jorge.estevez@umh.es

Phone : +34965222159

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Website : \/\/www.umh.es\/contenido\/Universidad\/:uor_889_29\/datos_es.html

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References (28)

Title : Inhibition with simultaneous spontaneous reactivation and aging of acetylcholinesterase by organophosphorus compounds: Demeton-S-methyl as a model - Estevez_2023_Chem.Biol.Interact_14ChEPon_110789
Author(s) : Estevez J , Pizarro L , Marsillach J , Furlong C , Sogorb MA , Richter R , Vilanova E
Ref : Chemico-Biological Interactions , :110789 , 2023
Abstract : The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus, carbamates and sulfonylfluorides) sometimes cannot yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work kinetic data were obtained for demeton-S-methyl (DSM) with human acetylcholinesterase in two kinds of experiments: (a) time progressive inhibition with a range of concentrations, (b) progressive spontaneous reactivation starting with pre-inhibited enzyme. DSM is an organophosphorus compound used as pesticide and considered a model for studying the dermal exposure of nerve agents such as VX gas. A kinetic model equation was deduced with four different molecular phenomena occurring simultaneously: (1) inhibition; (2) spontaneous reactivation; (3) aging; and (4) ongoing inhibition (inhibition during the substrate reaction). A 3D fit of the model was applied to analyze the inhibition experimental data. The best-fitting model is compatible with a sensitive enzymatic entity. The second-order rate constant of inhibition (ki = 0.0422 microM(-1) min(-1)), the spontaneous reactivation constant (ks = 0.0202 min(-1)) and the aging constant (kg = 0.0043 min(-1)) were simultaneously estimated. As an example for testing the model and approach, it was tested also in the presence of 5 % ethanol (conditions as previously used in the literature), the best fitting model is compatible with two apparent sensitive enzymatic entities (17 % and 83 %) and only one spontaneously reactivates and ages. The corresponding second-order rate constants of inhibition (ki = 0.0354 and 0.0119 microM(-1) min(-1)) and the spontaneous reactivation and aging constants for the less sensitive component (kr = 0.0203 min(-1) and kg = 0.0088 min(-1)) were estimated. The results were also consistent with a significant ongoing inhibition. These parameters were similar to those deduced in spontaneous reactivation experiments of the pre-inhibited samples with DSM in the absence or presence of ethanol. The two apparent components fit was interpreted by an equilibrium between ethanol-free and ethanol-bound enzyme. The consistency of results in inhibition and in spontaneous reactivation experiments was considered an internal validation of the methodology and the conclusions.
ESTHER : Estevez_2023_Chem.Biol.Interact_14ChEPon_110789
PubMedSearch : Estevez_2023_Chem.Biol.Interact_14ChEPon_110789
PubMedID: 37931869

Title : Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity - Estevez_2021_Chem.Biol.Interact__109764
Author(s) : Estevez J , Terol M , Sogorb MA , Vilanova E
Ref : Chemico-Biological Interactions , :109764 , 2021
Abstract : Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site ("PVsite") were deduced. In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.
ESTHER : Estevez_2021_Chem.Biol.Interact__109764
PubMedSearch : Estevez_2021_Chem.Biol.Interact__109764
PubMedID: 34875277

Title : Effects of silver nanoparticles on T98G human glioblastoma cells - Fuster_2020_Toxicol.Appl.Pharmacol_404_115178
Author(s) : Fuster E , Candela H , Estevez J , Arias AJ , Vilanova E , Sogorb MA
Ref : Toxicol Appl Pharmacol , 404 :115178 , 2020
Abstract : Nanotechnology has been well developed in recent decades because it provides social progress and welfare. Consequently, exposure of population is increasing and further increases in the near future are forecasted. Therefore, assessing the safety of applications involving nanoparticles is strongly advisable. We assessed the effects of silver nanoparticles at a non-cytotoxic concentration on the performance of T98G human glioblastoma cells mainly by an omic approach. We found that silver nanoparticles are able to alter several molecular pathways related to inflammation. Cellular repair and regeneration were also affected by alterations to the fibroblast growth factor pathways operating mainly via mitogen-activated protein kinase cascades. It was concluded that, given the relevant role of glia on central nervous system maintenance homeostasis, exposure to silver nanoparticles could eventually lead to severe toxicity in the central nervous system, although current exposure levels do not pose a significant risk.
ESTHER : Fuster_2020_Toxicol.Appl.Pharmacol_404_115178
PubMedSearch : Fuster_2020_Toxicol.Appl.Pharmacol_404_115178
PubMedID: 32739528

Title : Interactions of human butyrylcholinesterase with phenylvalerate and acetylthiocholine as substrates and inhibitors: kinetic and molecular modeling approaches - Estevez_2019_Arch.Toxicol_93_1281
Author(s) : Estevez J , Rodrigues de Souza F , Romo M , Mangas I , Franca TCC , Vilanova E
Ref : Archives of Toxicology , 93 :1281 , 2019
Abstract : Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site ("PV-site"). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.
ESTHER : Estevez_2019_Arch.Toxicol_93_1281
PubMedSearch : Estevez_2019_Arch.Toxicol_93_1281
PubMedID: 30877329

Title : Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain - Estevez_2018_Toxicology_410_73
Author(s) : Estevez J , Benabent M , Selva V , Mangas I , Sogorb MA , Del Rio E , Vilanova E
Ref : Toxicology , 410 :73 , 2018
Abstract : Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In previous studies, in membrane chicken brain fractions, four components (EPalpha, EPbeta, EPgamma and EPdelta) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, mipafox, PMSF). EPgamma is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPalpha PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPalpha is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. In this papers we shows that cholinesterase activities showed inhibition kinetic by PV, which does not fit with a competitive inhibition model when tested for the same experimental conditions used to discriminate the PVase components. Four enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in cholinesterase activity in the membrane fraction according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA. Components CP1 and CP2 could be related to EPalpha as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.
ESTHER : Estevez_2018_Toxicology_410_73
PubMedSearch : Estevez_2018_Toxicology_410_73
PubMedID: 30176330

Title : Butyrylcholinesterase identification in a phenylvalerate esterase-enriched fraction sensitive to low mipafox concentrations in chicken brain - Mangas_2017_Arch.Toxicol_91_909
Author(s) : Mangas I , Radic Z , Taylor P , Ghassemian M , Candela H , Vilanova E , Estevez J
Ref : Archives of Toxicology , 91 :909 , 2017
Abstract : Multiple epidemiological and experimental studies have demonstrated that exposure to organophosphorus compounds (OPs) is associated with a variety of neurological disorders. Some of these exposure symptoms cannot be precisely correlated with known molecular targets and mechanisms of toxicity. Most of the known molecular targets of OPs fall in the protein family of serine esterases. We have shown that three esterase components in the soluble fraction of chicken brain (an animal model frequently used in OP neurotoxicity assays) can be kinetically distinguished using paraoxon, mipafox and phenylmethyl sulfonyl fluoride as inhibitors, and phenyl valerate as a substrate; we termed them Ealpha, Ebeta and Egamma. The Ealpha-component, which is highly sensitive to paraoxon and mipafox and resistant to PMSF, has shown sensitivity to the substrate acetylthiocholine, and to ethopropazine and iso-OMPA (specific inhibitors of butyrylcholinesterase; BChE) but not to BW 284C51 (a specific inhibitor of acetylcholinesterase; AChE). In this work, we employed a large-scale proteomic analysis B with a LC/MS/MS TripleTOF system; 259 proteins were identified in a chromatographic fractionated sample enriched in Ealpha activity of the chicken brain soluble fraction. Bioinformatics analysis revealed that BChE is the only candidate protein identified to be responsible for almost all the Ealpha activity. This study demonstrates the potential information to be gained from combining kinetic dissection with large-scale proteomics and bioinformatics analyses for identification of proteins that are targets of OP toxicity and may be involved in detoxification of phosphoryl and carbonyl esters.
ESTHER : Mangas_2017_Arch.Toxicol_91_909
PubMedSearch : Mangas_2017_Arch.Toxicol_91_909
PubMedID: 26838044

Title : New insights on molecular interactions of organophosphorus pesticides with esterases - Mangas_2017_Toxicology_376_30
Author(s) : Mangas I , Estevez J , Vilanova E , Franca TCC
Ref : Toxicology , 376 :30 , 2017
Abstract : Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the organophosphorus toxicity.
ESTHER : Mangas_2017_Toxicology_376_30
PubMedSearch : Mangas_2017_Toxicology_376_30
PubMedID: 27311923

Title : Phenyl valerate esterase activity of human butyrylcholinesterase - Mangas_2017_Arch.Toxicol_91_3295
Author(s) : Mangas I , Vilanova E , Estevez J
Ref : Archives of Toxicology , 91 :3295 , 2017
Abstract : Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of organophosphorus delayed neuropathy. In previous studies we observed that phenyl valerate esterase (PVase) activity of an enzymatic fraction in chicken brain might be due to a butyrylcholinesterase protein (BuChE), and it was suggested that this enzymatic fraction could be related to the potentiation/promotion phenomenon of the organophosphate-induced delayed neuropathy (OPIDN). In this work, PVase activity of purified human butyrylcholinesterase (hBuChE) is demonstrated and confirms the novel observation that a relationship of BuChE with PVase activities is also relevant for humans, as is, therefore the potential role in toxicity for humans. The KM and catalytic constant (kcat) were estimated as 0.52/0.72 microM and 45,900/49,200 min(-1) respectively. Furthermore, this work studies the inhibition by preincubation of PVase and cholinesterase activities of hBuChE with irreversible inhibitors (mipafox, iso-OMPA or PMSF), showing that these inhibitors interact similarly in both activities with similar second-order inhibition constants. Acethylthiocholine and phenyl valerate partly inhibit PVase and cholinesterase activities, respectively. All these observations suggest that both activities occur in the same active center. The interaction with a reversible inhibitor (ethopropazine) showed that the cholinesterase activity was more sensitive than the PVase activity, showing that the sensitivity for this reversible inhibitor is affected by the nature of the substrate. The present work definitively establishes the capacity of BuChE to hydrolyze the carboxylester phenyl valerate using a purified enzyme (hBuChE). Therefore, BuChE should be considered in the research of organophosphorus targets of toxicity related with PVase proteins.
ESTHER : Mangas_2017_Arch.Toxicol_91_3295
PubMedSearch : Mangas_2017_Arch.Toxicol_91_3295
PubMedID: 28299395

Title : Editorial - Estevez_2016_Chem.Biol.Interact_259_374
Author(s) : Vilanova E , Sogorb MA , Estevez J
Ref : Chemico-Biological Interactions , 259 :49 , 2016
Abstract :
ESTHER : Estevez_2016_Chem.Biol.Interact_259_374
PubMedSearch : Estevez_2016_Chem.Biol.Interact_259_374
PubMedID:

Title : Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors - Estevez_2016_Chem.Biol.Interact_259_374
Author(s) : Estevez J , Selva V , Benabent M , Mangas I , Sogorb MA , Vilanova E
Ref : Chemico-Biological Interactions , 259 :374 , 2016
Abstract :
ESTHER : Estevez_2016_Chem.Biol.Interact_259_374
PubMedSearch : Estevez_2016_Chem.Biol.Interact_259_374
PubMedID: 27507601

Title : Esterases hydrolyze phenyl valerate activity as targets of organophosphorus compounds - Mangas_2016_Chem.Biol.Interact_259_358
Author(s) : Mangas I , Estevez J , Vilanova E
Ref : Chemico-Biological Interactions , 259 :358 , 2016
Abstract : OPs are a large diverse class of chemicals used for several purposes (pesticides, warfare agents, flame retardants, etc.). They can cause several neurotoxic disorders: acute cholinergic toxicity, organophosphorus-induced delayed neuropathy, long-term neurobehavioral and neuropsychological symptoms, and potentiation of neuropathy. Some of these syndromes cannot be fully understood with known molecular targets. Many enzyme systems have the potential to interact with OPs. Since the discovery of neuropathy target esterase (NTE), the esterases that hydrolyze phenyl valerate (PVases) have been of interest. PVase components are analyzed in chicken tissue, the animal model used for testing OP-delayed neurotoxicity. Three enzymatic components have been discriminated in serum, and three in a soluble fraction of peripheral nerve, three in a soluble fraction of brain, and four in a membrane fraction of brain have been established according to inhibitory kinetic properties combined with several inhibitors. The criteria and strategies to differentiate these enzymatic components are shown. In the brain soluble fraction three enzymatic components, namely Ealpha, Ebeta and Egamma, were found. Initial interest focused on Ealpha activity (highly sensitive to paraoxon and spontaneously reactivated, mipafox and resistant to PMSF). By protein separation methods, a subfraction enriched in Ealpha activity was obtained and 259 proteins were identified by Tandem Mass Spectrometry. Only one had the criteria for being serine-esterase identified as butyrylcholinesterase, which stresses the relationship between cholinesterases and PVases. The identification and characterization of the whole group of PVases targets of OPs (besides AChE, BuChE and NTE) is necessary to clarify the importance of these other targets in OPs neurotoxicity or on detoxication pathways. A systematic strategy has proven useful for the molecular identification of one enzymatic component, which can be applied to identify them all.
ESTHER : Mangas_2016_Chem.Biol.Interact_259_358
PubMedSearch : Mangas_2016_Chem.Biol.Interact_259_358
PubMedID: 27087132

Title : Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches - Mangas_2016_Arch.Toxicol_90_603
Author(s) : Mangas I , Taylor P , Vilanova E , Estevez J , Franca TCC , Komives E , Radic Z
Ref : Archives of Toxicology , 90 :603 , 2016
Abstract : The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.
ESTHER : Mangas_2016_Arch.Toxicol_90_603
PubMedSearch : Mangas_2016_Arch.Toxicol_90_603
PubMedID: 25743373

Title : Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity - Sogorb_2016_Chem.Biol.Interact_259_352
Author(s) : Sogorb MA , Pamies D , Estevan C , Estevez J , Vilanova E
Ref : Chemico-Biological Interactions , 259 :352 , 2016
Abstract : Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.
ESTHER : Sogorb_2016_Chem.Biol.Interact_259_352
PubMedSearch : Sogorb_2016_Chem.Biol.Interact_259_352
PubMedID: 27475862

Title : Effects of mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells - Sogorb_2016_Chem.Biol.Interact_259_368
Author(s) : Sogorb MA , Fuster E , Del Rio E , Estevez J , Vilanova E
Ref : Chemico-Biological Interactions , 259 :368 , 2016
Abstract : Chlorpyrifos (CPS) is an organophosphorus compound (OP) capable of causing well-known cholinergic and delayed syndromes through the inhibition of acetylcholinesterase and Neuropathy Target Esterase (NTE), respectively. CPS is also able to induce neurodevelopmental toxicity in animals. NTE is codified by the Pnpla6 gene and plays a central role in differentiation and neurodifferentiation. We tested, in D3 mouse embryonic stem cells under differentiation, the effects of the NTE inhibition by the OPs mipafox, CPS and its main active metabolite chlorpyrifos-oxon (CPO) on the expression of genes Vegfa, Bcl2, Amot, Nes and Jun, previously reported to be under- or overexpressed after Pnpla6 silencing in this same cellular model. Mipafox did not significantly alter the expression of such genes at concentrations that significantly inhibited NTE. However, CPS and CPO at concentrations that caused NTE inhibition at similar levels to mipafox statistically and significantly altered the expression of most of these genes. Paraoxon (another OP with capability to inhibit esterases but not NTE) caused similar effects to CPS and CPO. These findings suggest that the molecular mechanism for the neurodevelopmental toxicity induced by CPS is not based on NTE inhibition, and that other unknown esterases might be potential targets of neurodevelopmental toxicity.
ESTHER : Sogorb_2016_Chem.Biol.Interact_259_368
PubMedSearch : Sogorb_2016_Chem.Biol.Interact_259_368
PubMedID: 27117976

Title : Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain - Benabent_2014_Toxicol.Lett_230_132
Author(s) : Benabent M , Vilanova E , Mangas I , Sogorb MA , Estevez J
Ref : Toxicol Lett , 230 :132 , 2014
Abstract : Organophosphorus compounds (OPs) induce neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, OPs interact with other esterases of unknown biological function. In soluble chicken brain fractions, three components of enzymatic phenylvalerate esterase activity (PVase) called Ealpha, Ebeta and Egamma, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Ealpha PVase activity (resistant PVase activity to 1500muM PMSF for 30min) was tested with different acetylthiocholine concentrations, inhibition was observed. The best-fitting model to the data was the non-competitive inhibition model (Km=0.12, 0.22mM, Ki=6.6, 7.6mM). Resistant acetylthiocholine-hydrolyzing activity to 1500muM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11mM; Ki=1.7, 2.2mM). Ebeta PVase activity (resistant PVase activity to 25muM mipafox for 30min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25muM mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06mM; Ki=0.44, 0.58mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.
ESTHER : Benabent_2014_Toxicol.Lett_230_132
PubMedSearch : Benabent_2014_Toxicol.Lett_230_132
PubMedID: 24576786

Title : Cholinesterase assay by an efficient fixed time endpoint method - Benabent_2014_MethodsX_1_258
Author(s) : Benabent M , Vilanova E , Sogorb MA , Estevez J
Ref : MethodsX , 1 :258 , 2014
Abstract : Many cholinesterase assays are performed to study the inhibition of cholinesterase (ChE) activity. Frequently a large number of samples are processed and Ellman's method [1] is the most commonly used [2,3]. Activity is estimated from the increment in absorbance between two reaction times when the reaction is not stopped. Bellino et al. [4] described a method based on Ellman's method whereby the reaction was stopped with SDS and then the absorbance was measured. In these methods, the chromogen reagent 5,5'dithiobis nitro benzoic acid (DTNB) is added with the substrate and colour is monitored. Some authors pointed that the chromogen can alter cholinesterase activity [5].*A modification of Bellino's method is proposed for acetylcholine-hydrolyzing activity determinations that is based on stopping the reaction after a fixed substrate reaction time using a mixture of detergent SDS and DTNB.*The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.
ESTHER : Benabent_2014_MethodsX_1_258
PubMedSearch : Benabent_2014_MethodsX_1_258
PubMedID: 26150962

Title : Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography - Mangas_2014_Toxicol.Lett_225_167
Author(s) : Mangas I , Vilanova E , Benabent M , Estevez J
Ref : Toxicol Lett , 225 :167 , 2014
Abstract : Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Ealpha, Ebeta and Egamma. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Ealpha, Ebeta and Egamma were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins.
ESTHER : Mangas_2014_Toxicol.Lett_225_167
PubMedSearch : Mangas_2014_Toxicol.Lett_225_167
PubMedID: 24355587

Title : Interactions of neuropathy inducers and potentiators\/promoters with soluble esterases - Estevez_2013_Chem.Biol.Interact_203_245
Author(s) : Estevez J , Mangas I , Sogorb MA , Vilanova E
Ref : Chemico-Biological Interactions , 203 :245 , 2013
Abstract : Organophosphorus compounds (OPs) cause neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, the OPs can potentially interact with other esterases of unknown significance. Therefore, identifying, characterizing and elucidating the nature and functional significance of the OP-sensitive pool of esterases in the central and peripheral nervous systems need to be investigated. Kinetic models have been developed and applied by considering multi-enzymatic systems, inhibition, spontaneous reactivation, the chemical hydrolysis of the inhibitor and "ongoing inhibition" (inhibition during the substrate reaction time). These models have been applied to discriminate enzymatic components among the esterases in nerve tissues of adult chicken, this being the experimental model for delayed neuropathy and to identify different modes of interactions between OPs and soluble brain esterases. The covalent interaction with the substrate catalytic site has been demonstrated by time-progressive inhibition during ongoing inhibition. The interaction of sequential exposure to an esterase inhibitor has been tested in brain soluble fraction where exposure to one inhibitor at a non inhibitory concentration has been seen to modify sensitivity to further exposure to others. The effect has been suggested to be caused by interaction with sites other than the inhibition site at the substrate catalytic site. This kind of interaction among esterase inhibitors should be considered to study the potentiation/promotion phenomenon, which is observed when some esterase inhibitors enhance the severity of the OP induced neuropathy if they are dosed after a non neuropathic low dose of a neuropathy inducer.
ESTHER : Estevez_2013_Chem.Biol.Interact_203_245
PubMedSearch : Estevez_2013_Chem.Biol.Interact_203_245
PubMedID: 23200747

Title : Kinetics of inhibition of soluble peripheral nerve esterases by PMSF: a non-stable compound that potentiates the organophosphorus-induced delayed neurotoxicity - Estevez_2012_Arch.Toxicol_86_767
Author(s) : Estevez J , Barril J , Vilanova E
Ref : Archives of Toxicology , 86 :767 , 2012
Abstract : The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus carbamates and sulfonyl fluorides) is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work, kinetic data were obtained for phenylmethylsulfonyl fluoride (PMSF) tested at different concentrations incubated for up to 3 h with soluble fraction of chicken peripheral nerve. PMSF is a protease and esterase inhibitor causing protection or potentiation of the organophosphorus-induced delayed neuropathy and is unstable in water solution. The target of the promotion effect was proposed to be a soluble esterase not yet identified. A kinetic model equation was deduced assuming a multienzymatic system with three different molecular phenomena occurring simultaneously: (1) inhibition, (2) spontaneous chemical hydrolysis of the inhibitor and (3) ongoing inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied for analyzing the experimental data. The best-fitting model is compatible with a resistant component (16.5-18%) and two sensitive enzymatic entities (both 41%). The corresponding second-order rate constants of inhibition (ki = 12.04 x 10(-)(2) and 0.54 x 10(-)(2) muM(-)(1) min(-)(1), respectively) and the chemical hydrolysis constant of PMSF (kh = 0.0919 min(-)(1)) were simultaneously estimated. These parameters were similar to those deduced in fixed-time inhibition experiments. The consistency of results in both experiments was considered an internal validation of the methodology. The results were also consistent with a significant ongoing inhibition. The proportion of enzymatic components showed in this work is similar to those previously observed in inhibition experiments with mipafox, S9B and paraoxon, demonstrating that this kinetic approach gives consistent results in complex enzymatic systems.
ESTHER : Estevez_2012_Arch.Toxicol_86_767
PubMedSearch : Estevez_2012_Arch.Toxicol_86_767
PubMedID: 22354540

Title : NTE and non-NTE esterases in brain membrane: kinetic characterization with organophosphates - Mangas_2012_Toxicology_297_17
Author(s) : Mangas I , Vilanova E , Estevez J
Ref : Toxicology , 297 :17 , 2012
Abstract : Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets. In this work, we evaluate and characterize the interaction (inhibition, reactivation and "ongoing inhibition") of two model compounds: paraoxon (non-neuropathy-inducer) and mipafox (neuropathy-inducer), with esterases of chicken brain membranes, an animal model, tissue and fractions, where neuropathy target esterase (NTE) was first described and isolated. Four enzymatic components were discriminated. The relative sensitivity of time-progressive inhibition differed for paraoxon and mipafox. The most sensitive component for paraoxon was also the most sensitive component for mipafox (EPalpha: 4.4-8.3% of activity), with I(50) (30 min) of 15-43 nM with paraoxon and 29 nM with mipafox, and it spontaneously reactivated after inhibition with paraoxon. The second most sensitive component to paraoxon (EPbeta: 38.3% of activity) had I(50) (30 min) of 1540 nM, and was practically resistant to mipafox. The third component (EPgamma: 38.6-47.6% of activity) was paraoxon-resistant and sensitive to micromolar concentrations of mipafox; this component meets the operational criteria of being NTE (target of organophosphorus-induced delayed neuropathy). It had I(50) (30 min) of 5.3-6.6 muM with mipafox. The fourth component (EPdelta: 9.8-10.7% of activity) was practically resistant to both inhibitors. Two paraoxon-resistant and mipafox-sensitive esterases were found using the sequential assay removing paraoxon, but only one was paraoxon-resistant and mipafox-sensitive according to the assay without removing paraoxon. We demonstrate that this apparent discrepancy, interpreted as reversible NTE inhibition with paraoxon, is the result of spontaneous reactivation after paraoxon inhibition of a non-NTE component. Some of these esterases' sensitivity to OPs suggests that they may play a role in toxicity in low-level exposure to organophosphate compounds or have a protective effect related with spontaneous reactivation. The kinetic characterization of these components will facilitate further studies for isolation and molecular characterization.
ESTHER : Mangas_2012_Toxicology_297_17
PubMedSearch : Mangas_2012_Toxicology_297_17
PubMedID: 22503708

Title : Phenylmethylsulfonyl fluoride, a potentiator of neuropathy, alters the interaction of organophosphorus compounds with soluble brain esterases - Mangas_2012_Chem.Res.Toxicol_25_2393
Author(s) : Mangas I , Vilanova E , Estevez J
Ref : Chemical Research in Toxicology , 25 :2393 , 2012
Abstract : Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection or potentiation/promotion of organophosphorus delayed neuropathy (OPIDN) depending on whether it is dosed before or after an inducer of delayed neuropathy. The molecular target of promotion has not yet been identified. Kinetic data of esterase inhibition were first obtained for PMSF with a soluble chicken brain fraction and then analyzed using a kinetic model with a multienzymatic system in which inhibition occurred with the simultaneous chemical hydrolysis of the inhibitor and ongoing inhibition (inhibition during the substrate reaction). The best fitting model was a model with resistant fraction, Ealpha (28%), and two sensitive enzymatic entities, Ebeta (61%) and Egamma (11%), with I(50) at 20 min of 70 and 447 muM, respectively. The estimated constant of the chemical hydrolysis of PMSF was kh = 0.23 min(-1). Ealpha, which is sensitive to mipafox and resistant to PMSF, became less sensitive to mipafox when the preparation was preincubated with PMSF. Its Ealpha I(50) (30 min) of mipafox increased with the PMSF concentration used to preincubate it. Egamma is sensitive to both PMSF and mipafox, and after preincubation with PMSF, Egamma became less sensitive to mipafox and was totally resistant after preincubation with 10 muM PMSF or more. The sensitivity of Ealpha to paraoxon (I(50) 30 min from 9 to 11 nM) diminished after PMSF preincubation (I(50) 30 min 185 nM) and showed no spontaneous reactivation capacity. The nature of these interactions is unknown but might be due to covalent binding at sites other than the substrate catalytic center. Such interactions should be considered to interpret the potentiation/promotion phenomenon of PMSF and to understand the effects of multiple exposures to chemicals.
ESTHER : Mangas_2012_Chem.Res.Toxicol_25_2393
PubMedSearch : Mangas_2012_Chem.Res.Toxicol_25_2393
PubMedID: 23009703

Title : Inhibition with spontaneous reactivation of carboxyl esterases by organophosphorus compounds: paraoxon as a model - Estevez_2011_Chem.Res.Toxicol_24_135
Author(s) : Estevez J , Garcia-Perez A , Barril J , Vilanova E
Ref : Chemical Research in Toxicology , 24 :135 , 2011
Abstract : In this work kinetic data were obtained for different paraoxon concentrations incubated with chicken serum and the soluble fraction of chicken peripheral nerve. A kinetic model equation was deduced by assuming a multienzymatic system with three different simultaneously occurring molecular phenomena: (1) inhibition; (2) simultaneous spontaneous reactivation; (3) "ongoing" inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied to analyze the experimental data versus the concentration of the inhibitor and the preincubation time in an inhibition experiment. The best-fitting model in the soluble fraction of chicken peripheral nerve was compatible with a resistant component (22%) and with two sensitive enzymatic entities (37 and 41%). The corresponding second-order rate constants of inhibition (k(i) = 1.8 x 10(-3) and 5.1 x 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.428 and 0.011 min(-1), respectively) were estimated. The best-fitting model in chicken serum was compatible with a resistant component (5.6%) and with two sensitive enzymatic entities (22.1 and 72.3%). The corresponding second-order rate constants of inhibition (k(i) = 5.8 x 10(-2) and 2.0 x 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.0044 and 0.0091 min(-1), respectively) were estimated. These parameters were similar to those observed in spontaneous reactivation experiments with preinhibited paraoxon samples. The consistency of the results of all the experiments is considered an internal validation of the methodology. The results are also consistent with a significant ongoing inhibition. The proportion of enzymatic components shown in this work by the inhibition and reactivation of paraoxon is similar to that previously observed in inhibition experiments with mipafox in both tissues, demonstrating that this kinetic approach provides consistent results in complex enzymatic systems. The high sensitivity (at nanomolar concentrations) of these esterases suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation.
ESTHER : Estevez_2011_Chem.Res.Toxicol_24_135
PubMedSearch : Estevez_2011_Chem.Res.Toxicol_24_135
PubMedID: 21155548

Title : Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system - Mangas_2011_Toxicol.Appl.Pharmacol_256_360
Author(s) : Mangas I , Vilanova E , Estevez J
Ref : Toxicol Appl Pharmacol , 256 :360 , 2011
Abstract : Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and "ongoing inhibition") of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (Ealpha) for paraoxon (11-23% of activity, I(50) (30 min)=9-11 nM) is also the most sensitive for mipafox (I(50) (30 min)=4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (Ebeta, 71-84% of activity; I(50) (30 min)=1216 nM) is practically resistant to mipafox. The third component (Egamma, 5-8% of activity) is paraoxon resistant and has I(50) (30 min) of 3.4 muM with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies.
ESTHER : Mangas_2011_Toxicol.Appl.Pharmacol_256_360
PubMedSearch : Mangas_2011_Toxicol.Appl.Pharmacol_256_360
PubMedID: 21600909

Title : Inhibition with spontaneous reactivation and the ongoing inhibition effect of esterases by biotinylated organophosphorus compounds: S9B as a model - Estevez_2010_Chem.Biol.Interact_187_397
Author(s) : Estevez J , Barril J , Vilanova E
Ref : Chemico-Biological Interactions , 187 :397 , 2010
Abstract : The biotinylated organophosphorus compound 1-(saligenin cyclic phospho)-9-biotinyldiaminononane (S9B) has been used for the detection, labeling and isolation of the membrane-bound neuropathy target esterase (NTE) as it was considered a specific inhibitor of NTE. After incubation with the soluble fraction of chicken peripheral nerve, most of the soluble esterase activity was highly sensitive to S9B, indicating NTE-like esterases. A kinetic model equation was used to assume a multi-enzymatic system with three different simultaneously occurring molecular phenomena; (1) inhibition; (2) simultaneous spontaneous reactivation; and (3) ongoing inhibition (inhibition during the substrate reaction); to fit the data to analyze kinetic behavior. A high "ongoing inhibition" effect was observed in an enzymatic component. A three-dimensional fit of the model was applied. The best fitting model is compatible with three sensitive enzymatic entities (33, 52 and 15%), and only one spontaneously reactivate. The second-order rate constants of inhibition (k(i)=116 x 10(6), 4.6 x 10(6) and 0.28 x 10(6)M(-1)min(-1), respectively) and the spontaneous reactivation constant for the first sensitive component (k(r)=0.0054 min(-1)) were simultaneously estimated. These parameters are similar to those deduced in spontaneous reactivation experiments of the preinhibited samples with S9B. The estimated proportions of enzymatic components are similar to those previously observed in inhibition experiments with mipafox, demonstrating that this kinetic approach offers consistent results.
ESTHER : Estevez_2010_Chem.Biol.Interact_187_397
PubMedSearch : Estevez_2010_Chem.Biol.Interact_187_397
PubMedID: 20493177

Title : Model equations for the kinetics of covalent irreversible enzyme inhibition and spontaneous reactivation: esterases and organophosphorus compounds - Estevez_2009_Crit.Rev.Toxicol_39_427
Author(s) : Estevez J , Vilanova E
Ref : Crit Rev Toxicol , 39 :427 , 2009
Abstract : Type B carboxylesterases (acetylcholinesterases, neuropathy target esterase, serine peptidases), catalyse the hydrolysis of carboxyl-ester substrates by formation of a covalent acyl-enzyme intermediate and subsequent cleavage and release of the acyl group. Organophosphorus compounds, carbamates, and others exert their mechanism of neurotoxicity by permanent covalent organophosphorylation or carbamylation at the catalytic site of carboxylesterases. Classical kinetic studies converted the exponential kinetic equation to a logarithmic equation for graphic analysis. This process, however, does not allow analysing complex situations. In this paper, kinetic model equations are reviewed and strategies developed for the following cases: (a) single enzyme, with classical linear equation; (b) multi-enzymatic system-discriminating several inhibitor-sensitive and inhibitor-resistant components; (c) 'ongoing inhibition'-high sensitive enzymes can be significantly inhibited during the substrate reaction time, the model equations need a correction; (d) spontaneous reactivation (de-phosphorylation)-one or several components can be simultaneously inhibited and spontaneously reactivated; (e) spontaneous reactivation from starting time with the enzyme being partly or totally inhibited; (f) aging-single enzyme can be inhibited, spontaneously reactivated and dealkylating reaction ('aging') simultaneously occurs; and (g) aging and spontaneous reactivation from starting time with the enzyme being partly or totally inhibited. Analysis of data using the suggested equations allows the deduction of inhibition kinetic constants and the proportions of each of the enzymatic components. Strategies for practical application of the models and for obtaining consistent kinetic parameters, using multi-steps approaches or 3D fitting, are presented.
ESTHER : Estevez_2009_Crit.Rev.Toxicol_39_427
PubMedSearch : Estevez_2009_Crit.Rev.Toxicol_39_427
PubMedID: 19514915

Title : The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds - Estevez_2004_Toxicol.Lett_151_171
Author(s) : Estevez J , Garcia-Perez AG , Barril J , Pellin M , Vilanova E
Ref : Toxicol Lett , 151 :171 , 2004
Abstract : In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.
ESTHER : Estevez_2004_Toxicol.Lett_151_171
PubMedSearch : Estevez_2004_Toxicol.Lett_151_171
PubMedID: 15177652

Title : Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition - Garcia-Perez_2003_Toxicol.Lett_142_1
Author(s) : Garcia-Perez AG , Barril J , Estevez J , Vilanova E
Ref : Toxicol Lett , 142 :1 , 2003
Abstract : Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.
ESTHER : Garcia-Perez_2003_Toxicol.Lett_142_1
PubMedSearch : Garcia-Perez_2003_Toxicol.Lett_142_1
PubMedID: 12765233

Title : Peripheral nerve soluble esterases are spontaneously reactivated after inhibition by paraoxon: implications for a new definition of neuropathy target esterase - Barril_1999_Chem.Biol.Interact_119-120_541
Author(s) : Barril J , Estevez J , Escudero MA , Cespedes MV , Niguez N , Sogorb MA , Monroy A , Vilanova E
Ref : Chemico-Biological Interactions , 119-120 :541 , 1999
Abstract : Soluble extracts of chicken peripheral nerve contain detectable amounts of phenyl valerate esterase (PVase) activity (about 2000 nmol/min per g of fresh tissue). More than 95% of this activity is inhibited in assays where substrate has been added to a preincubated mixture of tissue with the non-neuropathic organophosphorus compound (OP) paraoxon (O,O'-diethyl p-nitrophenyl phosphate): residual activity includes soluble neuropathy target esterase (S-NTE) which, by definition, is considered resistant to long-term progressive (covalent) inhibition by paraoxon. However we have previously shown that paraoxon strongly interacts with S-NTE so interfering with its sensitivity to other inhibitors. We now show that, surprisingly, removal of paraoxon by ultrafiltration ('P' tissue) in order to avoid such an interference results in the reappearance of about 65% of total original soluble PVase activity which is inhibited in the presence of this OP. Although a purely reversible non-progressive inhibition might be suspected, kinetic analysis data show a time-progressive inhibition which suggests that such PVase(s) covalently bind paraoxon. Also a time-dependent recovery due to spontaneous reactivation of the PVase activity was observed after dilution of the inhibitor. Gel filtration chromatography of 'P' tissue in Sephacryl S-300 shows that the reactivated activity is associated with proteins of about 100-kDa mass which include S-NTE and an, as yet, unknown number of other PVases. The implications of these findings in the definition of NTE in a target tissue for the so-called organophosphorus-induced delayed polyneuropathy (OPIDP) are discussed.
ESTHER : Barril_1999_Chem.Biol.Interact_119-120_541
PubMedSearch : Barril_1999_Chem.Biol.Interact_119-120_541
PubMedID: 10421493