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Eugenio Vilanova - One of the best experts on this subject based on the ideXlab platform.
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Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain.
Toxicology, 2018Co-Authors: Jorge Estévez, Iris Mangas, Miguel A. Sogorb, Mónica Benabent, Eva Del Río, Verónica Selva, Eugenio VilanovaAbstract: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 (EPα, EPβ, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, Mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα 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 EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.
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Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and Mipafox in human neuroblastoma SH-SY5Y cells.
Toxicology, 2018Co-Authors: Miriam González-gonzález, Eugenio Vilanova, Jorge Estévez, Eva Del Río, Miguel A. SogorbAbstract:The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP Mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by Mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to Mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of Mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.
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Effects of Mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells.
Chemico-biological interactions, 2016Co-Authors: Miguel A. Sogorb, Jorge Estévez, Eva Del Río, Encarnación Fuster, Eugenio VilanovaAbstract: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.
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Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.
Toxicology letters, 2014Co-Authors: Mónica Benabent, Iris Mangas, Eugenio Vilanova, Miguel A. Sogorb, Jorge EstévezAbstract: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 Eα, Eβ and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 μM PMSF for 30 min) 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, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eβ PVase activity (resistant PVase activity to 25 μM Mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 μM Mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.
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Kinetic interactions of a neuropathy potentiator (phenylmethylsulfonyl fluoride) with the neuropathy target esterase and other membrane bound esterases
Archives of Toxicology, 2014Co-Authors: Iris Mangas, Eugenio Vilanova, Jorge EstévezAbstract: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, such as Mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and “ongoing inhibition” (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I _50 (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min^−1). Four esterase components were globally identified considering also previously data with paraoxon and Mipafox: EPα (4–8 %), highly sensitive to paraoxon and Mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38–41 %), sensitive to paraoxon and PMSF, but practically resistant to Mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39–48 %), paraoxon-resistant and sensitive to the micromolar concentration of Mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.
Miguel A. Sogorb - One of the best experts on this subject based on the ideXlab platform.
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Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain.
Toxicology, 2018Co-Authors: Jorge Estévez, Iris Mangas, Miguel A. Sogorb, Mónica Benabent, Eva Del Río, Verónica Selva, Eugenio VilanovaAbstract: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 (EPα, EPβ, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, Mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα 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 EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.
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Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and Mipafox in human neuroblastoma SH-SY5Y cells.
Toxicology, 2018Co-Authors: Miriam González-gonzález, Eugenio Vilanova, Jorge Estévez, Eva Del Río, Miguel A. SogorbAbstract:The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP Mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by Mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to Mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of Mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.
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Effects of Mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells.
Chemico-biological interactions, 2016Co-Authors: Miguel A. Sogorb, Jorge Estévez, Eva Del Río, Encarnación Fuster, Eugenio VilanovaAbstract: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.
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Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.
Toxicology letters, 2014Co-Authors: Mónica Benabent, Iris Mangas, Eugenio Vilanova, Miguel A. Sogorb, Jorge EstévezAbstract: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 Eα, Eβ and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 μM PMSF for 30 min) 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, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eβ PVase activity (resistant PVase activity to 25 μM Mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 μM Mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.
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Genomic and Phenotypic Alterations of the Neuronal-Like Cells Derived from Human Embryonal Carcinoma Stem Cells (NT2) Caused by Exposure to Organophosphorus Compounds Paraoxon and Mipafox
International journal of molecular sciences, 2014Co-Authors: David Pamies, Eugenio Vilanova, Miguel A. Sogorb, Marco Fabbri, Laura Gribaldo, Angelo Collotta, Bibiana Scelfo, Georgina Harris, Anna Bal-priceAbstract:Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, Mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM Mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by Mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.
Jorge Estévez - One of the best experts on this subject based on the ideXlab platform.
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Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain.
Toxicology, 2018Co-Authors: Jorge Estévez, Iris Mangas, Miguel A. Sogorb, Mónica Benabent, Eva Del Río, Verónica Selva, Eugenio VilanovaAbstract: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 (EPα, EPβ, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, Mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα 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 EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.
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Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and Mipafox in human neuroblastoma SH-SY5Y cells.
Toxicology, 2018Co-Authors: Miriam González-gonzález, Eugenio Vilanova, Jorge Estévez, Eva Del Río, Miguel A. SogorbAbstract:The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP Mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by Mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to Mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of Mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.
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Effects of Mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells.
Chemico-biological interactions, 2016Co-Authors: Miguel A. Sogorb, Jorge Estévez, Eva Del Río, Encarnación Fuster, Eugenio VilanovaAbstract: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.
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Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.
Toxicology letters, 2014Co-Authors: Mónica Benabent, Iris Mangas, Eugenio Vilanova, Miguel A. Sogorb, Jorge EstévezAbstract: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 Eα, Eβ and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 μM PMSF for 30 min) 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, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eβ PVase activity (resistant PVase activity to 25 μM Mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 μM Mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.
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Kinetic interactions of a neuropathy potentiator (phenylmethylsulfonyl fluoride) with the neuropathy target esterase and other membrane bound esterases
Archives of Toxicology, 2014Co-Authors: Iris Mangas, Eugenio Vilanova, Jorge EstévezAbstract: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, such as Mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and “ongoing inhibition” (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I _50 (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min^−1). Four esterase components were globally identified considering also previously data with paraoxon and Mipafox: EPα (4–8 %), highly sensitive to paraoxon and Mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38–41 %), sensitive to paraoxon and PMSF, but practically resistant to Mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39–48 %), paraoxon-resistant and sensitive to the micromolar concentration of Mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.
Evelyn Tiffany-castiglioni - One of the best experts on this subject based on the ideXlab platform.
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Degradation of organophosphorus neurotoxicity in SY5Y neuroblastoma cells by organophosphorus hydrolase (OPH).
Journal of toxicology and environmental health. Part A, 2006Co-Authors: Tae-hyeon Cho, Kirby C. Donnelly, James R. Wild, Evelyn Tiffany-castiglioniAbstract:Numerous approaches have been studied to degrade organophosphorus (OP) compounds and ameliorate their toxicity. In the current study, the potential of genetically engineered organophosphorus hydrolase (OPH) enzymes to functionally biotransform OP neurotoxicants was examined by assessing effects of OPH-hydrolyzed OPs on acute and delayed indicators of neurotoxicity. SY5Y human neuroblastoma cells were used as a model test system, as these cells respond distinctly to Mipafox, which produces OP-induced delayed neuropathy, and paraoxon, which does not. Short-term effects of four OPH-treated OPs on acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were measured in retinoic acid-differentiated or undifferentiated cells, and delayed effects of OPH-treated paraoxon or Mipafox on levels of neuronal cytoskeletal proteins in nerve growth factor (NGF)-differentiated cells. The anti-AChE activity of paraoxon (maximum 3 μM) and anti-NTE activity of Mipafox (250 μM) in SY5Y cells were prevented...
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Neurofilament 200 as an Indicator of Differences Between Mipafox and Paraoxon Sensitivity in SY5Y Neuroblastoma Cells
Journal of toxicology and environmental health. Part A, 2004Co-Authors: Tae-hyeon Cho, Evelyn Tiffany-castiglioniAbstract:Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM Mipafox during the last 5 days of neurite extension (experimental set A). However, Mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by Mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to Mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs.
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Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds.
Toxicology and applied pharmacology, 2003Co-Authors: Marjorie S. Hong, Sandra J. Hong, Rola Barhoumi, Robert C. Burghardt, Kirby C. Donnelly, James R. Wild, Vijayanagaram Venkatraj, Evelyn Tiffany-castiglioniAbstract:Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not Mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while Mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though Mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with Mipafox. These results indicate that the decrease in neurite extension that resulted from Mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.
Marion Ehrich - One of the best experts on this subject based on the ideXlab platform.
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Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells
Neurotoxicity Research, 2005Co-Authors: Christiane Massicotte, Bernard S. Jortner, Kristin Knight, Cornelis J. Van Der Schyf, Marion EhrichAbstract:Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and Mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 μM PSP, Mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and Mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds.
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Morphological Effects of Neuropathy-Inducing Organophosphorus Compounds in Primary Dorsal Root Ganglia Cell Cultures
Neurotoxicology, 2003Co-Authors: Christiane Massicotte, Bernard S. Jortner, Marion EhrichAbstract:Abstract Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds. This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then exposed to 1 μM of the OP compounds phenyl saligenin phosphate (PSP) or Mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle. The medium containing these toxicants was removed after 12 h, and cultures maintained for 4–7 days post-exposure. Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with Mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure. Ultrastructural alterations of neurons treated with PSP and Mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria. Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes. This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds.
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Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors
Fundamental and applied toxicology : official journal of the Society of Toxicology, 1995Co-Authors: Marion Ehrich, Bernard S. Jortner, Stephanie PadillaAbstract:Abstract Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors. Ehrich, M., Jortner, B. S., and Padilla, S. (1995). Fundam. Appl. Toxicol. 24, 94-101. Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AChE) activities was compared in brain and spinal cords of adult White Leghorn hens and adult male Long Evan rats 4-48 hr after administration of tri- ortho -tolyl phosphate (TOTP po. 50-500 mg/kg to hens: 300-1000 mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1-2.5 mg/kg to hens; 5-24 mg/kg to rats), Mipafox (3-30 mg/kg ip to hens and rats), diisopropyl phosphorofluoridate (DFP sc, 0.25-1.0 mg/kg to hens; 1-3 mg/kg to rats), dichlorvos (5-60 mg/kg ip to hens; 5-30 mg/kg to rats), malathion (75-300 mg/kg po to hens; 600-2000 mg/kg to rats), and carbaryl (300-560 mg/kg ip to hens; 30-170 mg/kg to rats). Inhibitions of NTE and AChE were dose-related after administration of all compounds to both species. Hens and rats given TOTP, PSP, Mipafox, and DFP demonstrated delayed neuropathy 3 weeks later, with spinal cord lesions and clinical signs more notable in hens. Ratios of NTE/AChE inhibition in hen spinal cord, averaged over the doses used, were 2.6 after TOTP. 5.2 after PSP, 1.3 after Mipafox, and 0.9 after DFP, which contrast with 0.53 after dichlorvos, 1.0 after malathion, and 0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9 after TOTP, 2.6 after PSP, 1.0 after Mipafox, 0.62 after DFP, 1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl. The lower NTE, AChE ratios in rats given dosages of the four organophosphorus compounds that caused delayed neuropathy interferred with survival, an effect that was not a problem in hens. This observation, along with the absence of overt and specific clinical signs and the restricted presence of neuropathological lesions in rats, suggests that the hen remains the animal of choice for testing for organophosphorus-induced delayed neuropathy.
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Interaction of organophosphorus compounds with muscarinic receptors in SH‐SY5Y human neuroblastoma cells
Journal of toxicology and environmental health, 1994Co-Authors: Marion Ehrich, Liliana Intropido, Lucio G. CostaAbstract:Human neuroblastoma cells (line SH‐SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH‐SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phos‐phorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and Mipafox (N,N'‐diiso‐propyl phosphorodiamide fluoridate) that ranged between 10−9 M and 10−3 M (10−2 M for Mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]‐N‐methylscopolamine (3H‐NMS). Concentrations of paraoxon >0.5 × 10−3 M and PSP 1 × 10−3 M significantly inhibited the binding of a saturating concentration of 3H‐NMS. Concentrations of >10−5 M paraoxon or PSP could significantly inhibit the binding of a half‐saturating concentration of 3H‐NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX‐116 for M2, and 4‐DAMP for M3) indicated that SH‐SY5Y cells have m...
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Modification of Mipafox-induced inhibition of neuropathy target esterase in neuroblastoma cells of human origin.
Toxicology and applied pharmacology, 1993Co-Authors: A.c. Nostrandt, Marion EhrichAbstract:A neuroblastoma cell line of human origin was used as an in vitro model system to examine early effects on inhibition of neuropathy target esterase (NTE, also known as neurotoxic esterase) in the presence of agents belonging to classes of chemicals previously demonstrated to modify organophosphorus-induced delayed neuropathy in hens. For this study, differentiated SY-5Y cells were treated for up to 10 min with Mipafox, an organophosphorus compound, and NTE inhibition was determined when cells exposed to Mipafox were also exposed to the carbamate, aldicarb, and to the calcium channel blocker, verapamil. Cells were exposed to aldicarb or verapamil 5 min before, at the same time, or 2 min after Mipafox. Less NTE inhibition was observed when either aldicarb or verapamil was included in the incubation of SY-5Y cells with Mipafox. Effects of aldicarb and verapamil on NTE inhibition in differentiated SY-5Y cells were similar to effects in chicken brain homogenates. These results indicate that NTE inhibition can be detected in neuroblastoma cells, that these cells respond in a manner similar to chicken brain, and that Mipafox-induced inhibition of NTE can be decreased in the presence of aldicarb or verapamil.