Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2).

Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model.

Stereospecific hydrolysis of a phosphoramidate as a model to understand the role of biotransformation in the neurotoxicity of chiral organophosphorus compounds.

Calcium-dependent and EDTA-resistant hydrolyses of R and S isomers of O-hexyl O-2,5-dicholorophenyl phosphoramidate (HDCP) were observed in serum and subcellular fractions of liver, kidney and brain from hen, rat and rabbit. In serum, the Ca(2+)-dependent hydrolysis was much higher in rabbit than in other species. Liver showed a higher activity than kidney and brain. The S-HDCP isomer was hydrolysed to a higher extent than the other isomer. The fact that this stereospecificity favours the S-isomer is more clearly observed in rabbit serum, and in rat and rabbit liver particulate fractions. In such tissues and species, the EDTA-resistant hydrolysis was not stereospecific. Soluble fractions of rat brain and of hen liver, kidney and brain, showed a lower total activity but with a higher proportion of EDTA-resistant activity and a higher hydrolysis of the R-HDCP isomer. The Ca(2+)-dependent stereoselective biodegradation of S-HDCP is dominant in the most active tissues in rabbit and rat. It can therefore be concluded that S-HDCP would be biodegraded faster than R-HDCP. Furthermore, R-HDCP is the isomer that will remain at a higher proportion to be available for interaction with the target of neurotoxicity.

Comparison of chromaffin cells from several animal sources for their use as an in vitro model to study the mechanism of organophosphorous toxicity.

It had been observed that the chromaffin cells of bovine adrenal medulla contain high levels of neuropathy target esterase (NTE), the esterase whose inhibition and aging is associated with induction of the organophosphorous induced delayed neuropathy. In this study, total esterase and NTE activities, and their inhibition kinetics by OPs are characterized in adrenal medulla of several species in order to find the best source for chromaffin cells. Total esterase activity in membrane fraction of bovine, equine, porcine, ovine and caprine were 6100+/-840, 4200+/-270, 5000+/-120, 28800+/-3000, and 10800+/-2400mU/gtissue, respectively (mean+/-S.D., n=3-4). NTE represented around 70%, 24%, 58%, 10% and 24% of the total esterases in the same tissues, respectively. It was deduced that NTE represents between 69% and 89% of the "B-activity" (activity resistant to 40microM paraoxon) in the membrane fraction of all species. The mipafox I(50) calculated for 30-min inhibition of NTE at 37 degrees Celsius ranged between 7.4 and 12microM. These values are in the range of that for brain NTE in hen (the usual model for testing OP delayed neurotoxicity). Considering that bovine adrenal medulla contains high NTE activity, that it represents a high proportion of total activity, it is easier to dissect than adrenal medulla from equine, caprine or ovine, and is more readily available than species cited previously, and that its inhibitory properties are similar to the classical hen brain model, it is deduced that bovine adrenal medulla is the most appropriate source of chromaffin cells to study OP toxicity, with porcine as the second alternative. The kinetic properties of chromaffin cell cultures from bovine and porcine were in accordance with their properties in homogenate and subcellular fractions, and they displayed an appropriate stability and viability of the primary culture to be used in in vitro toxicological studies for both mechanistic and testing purposes.

La docencia de la toxicología en el Espacio Europeo de Educación Superior. Una aproximación desde la asignatura de biotoxinas / The teaching of toxicology in the european higher education system. An approach through the subject of biotoxins

Durante los tres últimos cursos académicos la asignatura de Biotoxinas de la Universidad Miguel Hernández de Elche ha sido impartida utilizando un sistema que combinó metodologías docentes expositivas clásicas con otras centradas principal-mente en el trabajo del estudiante. Así, inicialmente los profesores presentaron en forma de lección magistral participativa los temas esenciales para el desarrollo de la asignatura. Posteriormente el profesor tutorizó la elaboración de trabajos por parte de los alumnos que voluntariamente accedieron a ello. Dichos trabajos fueron presentados posteriormente al resto de la clase en forma de seminario. El trabajo elaborado y su presentación constituyeron los principales elementos empleados en la evaluación del aprendizaje del alumno. Los alumnos que no elaboraron trabajo fueron evaluados mediante un examen tradicional. Los resultados obtenidos demuestran una gran aceptación por parte de los alumnos, ya que se registró un progresivo aumento del número de matrículas así como una excelente media de asistencia a clase y a los seminarios. Los resultados académicos también fueron satisfactorios, ya que la tasa de éxito fue siempre del 100% y la de rendimiento siempre fue superior al 80%. Además de los buenos resultados obtenidos la metodología empleada tiene la ventaja de que se adapta a los requerimientos del futuro Espacio Europeo de Educación Superior

During the last three academic years the subject called Biotoxins has been taught at the Universidad Miguel Hernández de Elche using a system that combines classical expositive methodologies with others that are mainly focused on the work of the student. Initially, the teachers performed magisterial lessons the most important themes necessary for the understanding of the subject. Afterwards, the teachers supervised the development of individual academic work voluntarily carried out by the students. These works were then presented to the class in seminary format and together with the presentation of the seminary, they were the main elements employed in the learning evaluation of each student. Students that did not perform the voluntary work were evaluated through a classical exam. The obtained results splay a good acceptance of the subject among the students as the number of students who signed up for the course increased every academic year. The number of students that regularly attended the teaching sessions was also very high. The academic results were also very satisfactory as the success ratio was always 100%, while the yield ratio was always higher than 80%. In addition to the good obtained results, this methodology also has the additional advantage that it meets the requirements of the European Higher Education System

Bovine chromaffin cell cultures as model to study organophosporus neurotoxicity.

Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophosphorus neurotoxicity. Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modification by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox and sensitive to paraoxon). Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of bovine adrenal medulla were 5200+/-35, 5000+/-280 and 1700+/-260 mU/g tissue, respectively. Cultured chromaffin cells displayed a total hydrolysing activity of 41+/-5 mU/10(6) cells. Homogenates of bovine adrenal medulla displayed only about 6% of activity sensitive to paraoxon. Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy inducing compound) was found in particulate fraction. Cultured chromaffin cells displayed kinetics of inhibition by mipafox similar to the kinetics displayed by homogenates of bovine adrenal medulla. We conclude that NTE could be assayed in this system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox). Also, the proposal is supported of using chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced polyneuropathy.

Distribution of serum paraoxon hydrolyzing activity in a large Spanish population using a routine automized method in clinical laboratory.

This work was performed to adapt the manual laboratory method of measuring serum paraoxonase activity using a routine automatized method in the clinical laboratory and to study the distribution of paraoxonase activity in a large population from Alcoy, a region of Spain. The serum samples for the study were obtained from extractions of blood from 2891 individuals, distributed by sex and age groups, in a routine check in a primary care facility of Alcoy. Paraoxonase activity was assayed by measuring the release of p-nitrophenol according to a previously published method adapted to an automatized analyzer. The mean paraoxonase activity recorder was 70.2 +/- 16.5 IU/L. Paraoxonase activity in children (both males and females) was significantly lower (p < 0.0005) than in older individuals. Paraoxonase activity detected in males and females older than 56 was slightly lower than that detected in younger individuals, although in this case the difference was not statistically significant. The paraoxonase activity shows higher mean values in females than in males (p < 0.0005). Human paraoxonase activity shows a unimodal distribution pattern in the studied population, which is in contrast with other studies showing bimodal distribution.

Importancia de la hidrólisis estereoespecífica en la evaluación de riesgos tóxicos de insecticidas fosforamidatos / The importance of stereospecific hydrolysis in the risk assessment of phosphoramidate insecticides

Los principales efectos tóxicos de los insecticidas organofosforados derivan de la inhibición y envejecimiento de la acetilcolinesterasa y la esterasa diana de la neuropatía. Las fosfotriesterasas son esterasas capaces de hidrolizar insecticidas organofosforados. En este trabajo se estudia en varios tejidos de conejo, rata y gallina la estereoespecificidad de las fosfotriesterasas implicadas en la hidrólisis del insecticida O-hexil O-2,5-diclorofenil fosforamidato. También se ha caracterizado la capacidad de los isómeros R y S de dicho fosforamidato para inhibir y envejecer la esterasa diana de neuropatía. Entre todas las especies estudiadas los tejidos de conejo mostraron los mayores niveles de actividades hidrolizantes de insecticida, siendo la gallina la especie con menores niveles de esta actividad. En conejo y rata la mayoría de las fosfotriesterasas de los tejidos estudiados mostraron preferencia por la hidrólisis del isómero S del compuesto. Sin embargo, no se pudieron extraer conclusiones claras respecto a la estereoespecificidad de la hidrólisis en gallina. El isómero S del O-hexil O-2,5-diclorofenil fosforamidato inhibió la esterasa diana de la neuropatía de cerebro de gallina con I50 de 7.6 nM, siendo 190 nM el valor para la inhibición por el correspondiente isómero R. La citada esterasa inhibida por el compuesto de configuración S pudo ser reactivada por KF transcurridas 22 horas de la inhibición, reactivación que no ocurrió cuando el inhibidor fue el compuesto de configuración R. Así pues, los niveles de fosfotriesterasas y su estereoespecificidad van a determinar qué isómero alcanza las dianas tóxicas y en qué concentración y por tanto cuáles serán los efectos tóxicos que se manifiestan. Se concluye que se deben considerar los niveles de fosfotriesterasas y su estereoespecificidad en la evaluación de riesgos tóxicos de insecticidas fosforamidatos (AU)

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase.

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues.

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

Dichlorophenyl phosphoramidates as substrates for avian and mammalian liver phosphotriesterases: activity levels, calcium dependence and stereospecificity.

The present study shows the existence of both Ca2+-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca2+-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca2+-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca2+-dependent one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.

Peripheral nerve soluble esterases are spontaneously reactivated after inhibition by paraoxon: implications for a new definition of neuropathy target esterase.

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.

The role of phosphotriesterases in the detoxication of organophosphorus compounds.

The enzymes that hydrolyze organophosphorus compounds are called phosphotriesterases. The presence of phosphotriesterases has been described in a variety of tissues. The physiological role of these enzymes is not known, although a clear correlation exists between the levels of phosphotriesterases and susceptibility of the species to the toxic effects of organophosphorus compounds. Thus, mammals that possess high levels of phosphotriesterases in serum and liver are more tolerant to the toxic effects of these compounds than birds and insects - these being species considered lacking of phosphotriesterases. Because most of these enzymes are not well characterized, they are usually differentiated according to their different patterns of response to activators and/ or inhibitors. Phosphotriesterases usually depend of divalent cations and therefore EDTA usually inhibits them. A peculiar EDTA-resistant phosphotriesterase has been described in serum albumin. The biotechnological and therapeutical applications of phosphotriesterases are currently subject to study.

EDTA-resistant and sensitive phosphotriesterase activities associated with albumin and lipoproteins in rabbit serum.

Phosphotriesterase (PTE) activities in mammalian serum are typically found in the lipoprotein fraction. This PTE requires Ca++ for activity and is consequently inactivated by ethylenediaminetetraacetic acid (EDTA). There is also a little known PTE in mammal serum that is resistant to EDTA inactivation. In this work, the PTE activities for the substrates O-hexyl O-2, 5-dichlorophenyl phosphoramidate (HDCP) and O,O'-diethyl p-nitrophenyl phosphate were purified from rabbit serum by ultracentrifugation, molecular exclusion, and anion exchange chromotography. Rabbit serum produced two PTE activities. One was sensitive and the other was resistant to EDTA inhibition. The EDTA-resistant HDCP hydrolyzing activity and paraoxonase activities of rabbit serum were purified to homogeneity. These activities copurified and were associated to albumin. This EDTA-resistant activity exhibited no stereoselectivity in the hydrolysis of HDCP. The EDTA-sensitive activity was isolated in the lipoprotein fraction and stereoselectively hydrolyzed the S-HDCP over the R-HDCP. Other differences between the EDTA-sensitive paraoxonase and HDCP hydrolyzing activity were discovered in response to p-nitrophenylbutkyrate, 5,5-dithio-bis(2-nitrobenzoic acid), caprylic acid, sodium ions, and ammonium ions. This work demonstrates the existence of two well differentiated PTE activities in rabbit serum. One is sensitive to EDTA, stereoselective, and found in the lipoprotein fraction, and the other is resistant to EDTA inhibition and nonstereospecific.

Enzyme concentration as an important factor in the in vitro testing of the stereospecificity of the enzymatic hydrolysis of organophosphorus compounds.

A report is made of important differences in the Ca(2+)-dependent hydrolysis of the chiral phosphoramidate O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) when recorded using different quantities of hen liver microsomes. In a colorimetric microassay using the microsomes from 5mg tissue in the presence of HDCP stereoisomers and 2.5mM calcium, the R-HDCP isomer was hydrolysed at a rate similar to or slightly faster than S-HDCP isomer (14% v. 11%), while the S-HDCP stereoisomer was hydrolysed faster than R-HDCP (17% v. 25% and 21% v. 43%) when HDCP isomers hydrolysis was assayed in the presence of the microsomes from 10 or 20mg, respectively. This stereospecific hydrolysis was verified assaying racemic HDCP and quantities of liver microsomes from 10 to 80mg of tissue, using a chiral chromatographic method; thus, the increase in the ratio of remaining R-HDCP/S-HDCP was dependent on the amount of liver microsomes (range one- to threefold). This study demonstrates that the concentration of the subcellular fraction in in vitro assays is a critical factor to be taken into account in securing a more realistic approximation to the stereospecific enzymatic processes occurring in biological systems. Our data concerning the hydrolysis of HDCP by liver microsomes at high enzyme concentrations afford a better fit to the in vivo toxicological response with HDCP than assays performed with the most commonly used highly diluted preparations.

Chicken serum albumin hydrolyzes dichlorophenyl phosphoramidates by a mechanism based on transient phosphorylation.

The hydrolyzing activities of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and p-nitrophenyl butyrate (p-NPB) in chicken serum had been found to copurify in the same protein, identified as albumin. The hydrolyzing activities of both chicken serum and commercial serum albumins from different species were inhibited in a dose-dependent manner by short chain fatty acids. On simultaneous incubation of chicken serum with HDCP and p-NPB, a competitive interaction was detected between the two substrates. This behavior suggests that both are hydrolyzed in the same albumin active site. When chicken serum was preincubated with one of the substrates, and the latter were withdrawn by large dilution, the hydrolyzing activities with both substrates were found to be reduced. This reduction was in turn dependent upon the time of preincubation with the first substrate. These results suggest that HDCP and p-NPB are hydrolyzed by the same albumin active site, via a mechanism based on transient phosphorylation/acylation of the active site. The proposed hydrolysis mechanism would account for the hydrolytic kinetics of both substrates.

Phosphotriesterase activity identified in purified serum albumins.

The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/ PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 +/- 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate (p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.

A stereospecific phosphotriesterase in hen liver and brain.

O-Hexyl, O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. This compound is hydrolyzed by a phosphotriesterase known as HDCPase in hen and rat plasma, liver and brain. We studied the stereospecificity of HDCPase in hen tissues and in human and rabbit plasma employing a chromatographic method for analysis and quantification of HDCP stereoisomers. Hen and human plasma HDCPases were not stereospecific. However, rabbit plasma showed a remarkable stereospecificity to S-(-)-HDCP. High levels of stereospecific HDCPase were found in the particulate fraction of hen liver, where S-(-)-HDCP is hydrolyzed faster than R-(+)-HDCP. However, in hen brain the stereospecificity was found in the soluble fraction, where R-(+)-HDCP is hydrolyzed faster than S-(-)-HDCP. It is concluded that liver particulate fraction must be the main tissue responsible for the HDCP stereospecific biotransformation in hens. In an oral administration, the steroisomer R-(+)-HDCP would survive after passing through the liver and would interact with acetylcholinesterase and neuropathy target esterase in the nervous system.

Discrimination of carboxylesterases of chicken neural tissue by inhibition with a neuropathic, non-neuropathic organophosphorus compounds and neuropathy promoter.

Carboxylesterases are enzymes present in neural and other tissues that are sensitive to organophosphorus compounds. The esterase activity in particulate forms, resistant to paraoxon and sensitive to mipafox have been implicated in the initiation of organophosphorus-induced delayed polyneuropathy (OPIDP) and is called neuropathy target esterase (P-NTE). Certain esterases inhibitors such as phenylmethylsulfonyl fluoride (PMSF), can also irreversibly inhibit P-NTE and by this mechanism PMSF 'protects' from further effect of neuropathic OPs. However, if PMSF is dosed after a low non-neuropathic dose of a neuropathic OP, its neurotoxicity is 'promoted', causing severe neuropathy. The molecular target of promotion has not yet been identified and it has been shown that it is unlikely to be the P-NTE. In order to discriminate the different esterases, we used non-neuropathic (paraoxon), and neuropathic organophosphorus compounds (mipafox, DFP) and a neuropathy promoter (PMSF). They were used alone or in concurrent inhibition to study particulate and soluble fractions of brain, spinal cord and sciatic nerve of chicken. From the experimental data, a matrix was constructed and equations deduced to estimate the proportions of the different potential activity fractions that can be discriminated by their sensitivity to the tested inhibitors. It was deduced that only combinations of up to three inhibitors can be used for the analysis with consistent results. In all tissues, inside the paraoxon sensitive activity, most of the activity was sensitive either to mipafox, to PMSF or both. In all fractions, except brain soluble fractions, within the paraoxon resistant activity, a mipafox sensitive component was detected that is operationally considered NTE (P-NTE and S-NTE in particulate and soluble fractions, respectively). Most of this activity was also sensitive to PMSF, and this should be considered the target of organophosphorus inducing neuropathy and of PMSF protective effect. Either in brain and spinal cord, a significant amount of the activity resistant to 40 microM paraoxon and 250 microM mipafox (usually called 'C' activity) is sensitive to PMSF. It could be a good candidate to contain the target of the promotion effect of PMSF as well as the S-NTE activity that is also PMSF sensitive.