Author Report for: Vilanova E

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.

Some organophosphorus compounds (OPs), which are used in the manufacturing of insecticides and nerve agents, are racemic mixtures with at least one chiral center with a phosphorus atom. Acute exposure of humans to these mixtures induces the covalent modification of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) and causes a cholinergic syndrome or organophosphate-induced delayed polyneuropathy syndrome (OPIDP). These irreversible neurological effects are due to the stereoselective interaction of the racemic OPs with these B-esterases (AChE and NTE) and such interactions have been studied in vivo, ex vivo and in vitro, using stereoselective hydrolysis by A-esterases or phosphotriesterases (PTEs) and the PTE from Pseudomonas diminuta, and paraoxonase-1 (PON1) from mammalian serum. PON1 has a limited hydrolytic potential of the racemic OPs, while the bacterial PTE exhibits a significant catalytic activity on the less toxic isomers P(+) of the nerve agents. Avian serum albumin also shows a hydrolyzing capacity of chiral OPs with oxo and thio forms. There are ongoing environmental and bioremediation efforts to design and produce recombinants as bio-scavengers of OPs.

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy. The R(+)-HDCP inhibits and caused the so call "aging reaction" on inhibited-NTE. This enantiomer is not hydrolyzed by Ca(II)-dependent A-esterases in mammal tissues but is hydrolyzed by Cu(II)-dependent chicken serum albumin (CSA). With the aim of identifying HDCP hydrolysis by other vertebrate albumins, we incubated albumin with 400 microM racemic HDCP in the presence of 100 microM copper sulfate. HDCPase activity was assessed by measurement of HDCP with chiral chromatography. Human, sheep, dog, pig, lamprey or cobra serum albumin did not show a significant activity (-10 %). Rabbit and bovine albumins hydrolyzed both enantiomers of HDCP (25% and 50% respectively). Turkey serum albumin had more HDCPase activity (-80 microM remaining) than the chicken albumin (-150 microM remaining). No animal albumins other than chicken showed stereoselective hydrolysis. Preincubation of chicken albumin with 1 mM the histidine modifying agents, 100 microM N-bromosuccinimide (NBS) and Zn(II), inhibited its Cu(II)-dependent R(+)-HDCPase activity, where as other mM aminoacids modifiers had no inhibitory effects. . These results confirm that the stereoselective hydrolysis of (+)-HDCP is a specific A-esterase catalytic property of chicken albumin. The higher HDCPase activity by turkey albumin suggests the amino-terminal sequence of avian albumins (DAEHK) is the active center of this Cu(II)-dependent A-esterase activity.

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.

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.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy in hens. It has been used as a tool to identify new A-esterase activities in animal tissues. This study shows the EDTA-resistant, Cu(2+)- and Zn(2+)-dependent hydrolysis of racemic HDCP in domestic and sea bird serum using UV/Vis spectrophotometry and chiral chromatography. The results clearly show a significant (p < 0.05) Cu(2+)- and Zn(2+)-dependent HDCP hydrolysis in the serum of all bird species versus EDTA, except for the Zn(2+)-dependent HDCPase activity from Yucatecan quail serum. The ratio of Cu(2+)/Zn(2+) hydrolysis varied between 1 and 7 (intraspecies) and 15.6 (interspecies). EDTA affected the Cu(2+)- and Zn(2+)-dependent HDCPase activity in the range of 37-95% and 40-50%, respectively. HDCP hydrolysis activated by Cu(2+) was significantly (p < 0.05) stereoselective (R-(+)-HDCP > S-(-)-HDCP) in chicken and sea bird serum. Its R-(+)-HDCP/S-(-)-HDCP ratios were 6.8 and 1.6-2.8, respectively. EDTA-resistant and zinc-dependent HDCP hydrolysis were not stereospecific in all bird sera tested. The present ex vivo study reinforces the idea that bird sera have HDCPase activity that is sensitive to divalent metals, resistant to EDTA and possibly associated with the protein albumin.

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.

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral analogous compound of the methamidophos insecticide that induces delayed neuropathy, and the R-(+)-HDCP enantiomer is an inhibitor of neuropathy target esterase (NTE). This enantiomer is not hydrolized by Ca(2+)-dependent phosphotriesterases in mammal tissues. Our group had reported R-(+)-HDCP hydrolysis in chicken serum enhanced by 30-250 muM copper in ex vivo assays, which we call "antagonistic stereoselectivity". We checked the hypothesis of the role of cupper binding proteins. Two hundred micrograms of human serum ceruloplasmine or horse kidney methallotionein in 1mL containing 400muM HDCP for 60min showed no significant Cu(2+)-dependent hydrolysis. However under the same conditions, 10muL of chicken serum or 10muL of buffer containing 216mug of chicken serum albumin (CSA) (amount of albumin content in this serum volume) with 100muM Cu(2+) showed the same stereoselectivity and similar levels to the Cu(2+)-dependent R-(+)-HDCP hydrolysis. About 75% of R-(+)-HDCP were hydrolyzed after 120 min in the presence of 100 muM Cu(2+) (inhibited by 5mM EDTA). No effects was observed by divalent cations Cu(2+), Zn(2+), Fe(2+), Ca(2+), Mn(2+) and Mg(2+). These results confirm that albumin is the protein responsible for "antagonistic stereoselectivity" observed in chicken serum.

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.

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.

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.

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.

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.

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.

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.

O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) is a racemic organophosphate compound (OP) that induces delayed neuropathy in vivo. The O-hexyl 2,5-dichlorophenyl phosphoramidate R (R-HDCP) isomer inhibits and ages neuropathic target esterase (NTE) in hen brain. Moreover, human serum paraoxonase-1 (PON1) is a Ca(2+)-dependent enzyme capable of hydrolyzing OPs. The enzymatic activity of PON1 against OPs depends on the genetic polymorphisms present at position 192 (glutamine or arginine). The catalytic efficiency of PON1 is an important factor that determines neurotoxic susceptibility to some OPs. In the present study, we characterized the stereospecific hydrolysis of HDCP by alloforms PON1 Q192R human serum by chiral chromatography. Forty-seven human samples were characterized for the PON1 192 polymorphism. The hydrolysis data demonstrate that the three alloforms of PON1 show an exclusive and significant stereospecific Ca(2+)-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate S isomer (S-HDCP) at 19-127 microM at the concentrations that remain in all the samples. This stereoselective Ca(2+)-dependent hydrolysis of S-HDCP is inhibited by EDTA and is independent of the PON1 Q192R alloform. The present research reinforces the hypothesis that R-HDCP (an isomer that inhibits and causes NTE aging) is the enantiomer that induces delayed neuropathy by this chiral phosphoramidate due to the low hydrolysis level of the R-HDCP observed in this study.

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.

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.

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.

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 microM 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 microM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 microM 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.

Neuropathy target esterase (NTE) is involved in several disorders in adult organisms and embryos. A relationship between NTE and nervous system integrity and maintenance in adult systems has been suggested. NTE-related motor neuron disease is associated with the expression of a mutant form of NTE and the inhibition and further modification of NTE by organophosphorus compounds is the trigger of a delayed neurodegenerative neuropathy. Homozygotic NTE knockout mice embryos are not viable, while heterozygotic NTE knockout mice embryos yields mice with neurological disorders, which suggest that this protein plays a critical role in embryonic development. The present study used D3 mouse embryonic stem cells with the aim of gaining mechanistic insights on the role of Pnpla6 (NTE gene encoding) in the developmental process. D3 cells were silenced by lipofectamine transfection with a specific interference RNA for Pnpla6. Silencing Pnpla6 in D3 monolayer cultures reduced NTE enzymatic activity to 50% 20 h post-treatment, while the maximum loss of Pnpla6 expression reached 80% 48 h postsilencing. Pnpla6 was silenced in embryoid bodies and 545 genes were differentially expressed regarding the control 96 h after silencing, which revealed alterations in multiple genetic pathways, such as cell motion and cell migration, vesicle regulation, and cell adhesion. These findings also allow considering that these altered pathways would impair the formation of respiratory, neural, and vascular tubes causing the deficiencies observed in the in vivo development of nervous and vascular systems. Our findings, therefore, support the previous observations made in vivo concerning lack of viability of mice embryos not expressing NTE and help to understand the biology of several neurological and developmental disorders in which NTE is involved.

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.

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10muM CPF and TClP and 100muM CPO for 12h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20muM CPF and 25muM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.

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.

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.

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.

Several approaches have been proposed to assess impacts on natural assemblages. Ideally, the potentially impacted site and multiple reference sites are sampled through time, before and after the impact. Often, however, the lack of information regarding the potential overall impact, the lack of knowledge about the environment in many regions worldwide, budgets constraints and the increasing dimensions of human activities compromise the reliability of the impact assessment. We evaluated the impact, if any, and its extent of a nuclear power plant effluent on sessile epibiota assemblages using a suitable and feasible sampling design with no 'before' data and budget and logistic constraints. Assemblages were sampled at multiple times and at increasing distances from the point of the discharge of the effluent. There was a clear and localized effect of the power plant effluent (up to 100 m from the point of the discharge). However, depending on the time of the year, the impact reaches up to 600 m. We found a significantly lower richness of taxa in the Effluent site when compared to other sites. Furthermore, at all times, the variability of assemblages near the discharge was also smaller than in other sites. Although the sampling design used here (in particular the number of replicates) did not allow an unambiguously evaluation of the full extent of the impact in relation to its intensity and temporal variability, the multiple temporal and spatial scales used allowed the detection of some differences in the intensity of the impact, depending on the time of sampling. Our findings greatly contribute to increase the knowledge on the effects of multiple stressors caused by the effluent of a power plant and also have important implications for management strategies and conservation ecology, in general.

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.

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.

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 mug/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity.

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.

Neuropathy Target Esterase (NTE) was initially identified as the primary target esterase of some organophosphorus compounds that cause delayed neuropathy. Some studies in vivo suggest that this protein may also perform a function in embryonic development and therefore also in cell differentiation. The aim of this work was to characterize embryonic stem cells (ESC) as cellular model before to approach to the role of NTE in embryotoxicity processes through mechanistic studies. Mouse D3 ESC in monolayer expressed an NTE activity of 23 nmol phenol/min/mg of protein, while mouse R1 ESC showed a specific NTE activity 3 times higher than D3. An increased expression of gene Pnpla6 (that codifies for NTE) was seen during differentiation in both the D3 cells in monolayer and embryonic bodies (EBS). The maximums of the Pnpla6 expression were reached after 30 h and 5 days of differentiation in monolayer and EBS cultures, respectively. This peak of the Pnpla6 expression correlated with the peak of the NTE enzymatic activity in D3 monolayers. NTE activity and Pnpla6 expression returned to basal levels after 48 h (in monolayer cultures) and 10 days (in EBS) of differentiation, respectively. The changes in the Pnpla6 expression did not correlate with changes noted in the expression of two endoderm, two ectoderm and one neuroectoderm gene markers. In conclusion, this manuscript reports about NTE expression in ESC and its variation during first stages of differentiation. Nevertheless, the role of this activity and the meaning of the variations detected during differentiation must be further studied.

Serum albumin displays an esterase activity that is capable of hydrolysing the anti-cholinesterase compounds carbaryl, paraoxon, chlorpyrifos-oxon, diazoxon and O-hexyl, O-2,5-dichlorphenyl phosphoramidate. The detoxication of all these anti-cholinesterase compounds takes place at significant rates with substrate concentrations in the same order of magnitude as expected during in vivo exposures, even when these substrate concentrations are between 15 and 1300 times lower than the recorded K(m) constants. Our data suggest that the efficacy of this detoxication system is based on the high concentration of albumin in plasma (and in the rest of the body), and not on the catalytic efficacy itself, which is low for albumin. We conclude the need for a structure-activity relationship study into the albumin-associated esterase activities because this protein is universally present in vertebrates and could compensate for reduced levels of other esterases, i.e., lipoprotein paraoxonase, in some species. It is also remarkable that the biotransformation of xenobiotics can be reliably studied in vitro, although conditions as similar as possible to in vivo situations are necessary.

Organophosphorus-induced delayed polyneuropathy (OPIDP) is a syndrome induced by certain organophosphorus compounds (OPs) through a mechanism based on the inhibition and further modification (aging) of neuropathy target esterase (NTE). OECD guidelines for testing the capability of OPs to trigger OPIDP include two in vivo tests with hens. Activities of acetylcholinesterase and NTE found in SH-SY5Y human neuroblastoma cells were inhibited by 10 different OPs with kinetics similar to those found with chicken brain enzymes (model system for in vivo and in vitro-ex vivo assays). NTE in SH-SY5Y cells inhibited by these OPs aged and reactivated similarly to that described for hen brain NTE ex vivo. In short, we have developed an alternative methodology for predicting the capability of OPs to induce OPIDP based on the inhibition kinetics of acetylcholinesterase and NTE and on the capability of OPs to age the inhibited NTE from SH-SY5Y cell line. The results obtained always agreed with the previously reported ex vivo results with hen brain. The developed methodology correctly predicted the neuropathic potential of the tested OPs in eight cases. The in vivo-in vitro discrepancies with two of the tested compounds can be explained on the basis of differences between in vivo and in vitro biotransformation.

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) (35)S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.

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.

Marine sponges (Porifera) are ancient and simple eumetazoans. They constitute key organisms in the evolution from unicellular to multicellular animals. We now demonstrated that pure sulfated polysaccharides from marine sponges are responsible for the species-specific cell-cell interaction in these invertebrates. This conclusion was based on the following observations: (1) each species of marine sponge has a single population of sulfated polysaccharide, which differ among the species in their sugar composition and sulfate content; (2) sulfated polysaccharides from sponge interact with each other in a species-specific way, as indicated by an affinity chromatography assay, and this interaction requires calcium; (3) homologous, but not heterologous, sulfated polysaccharide inhibits aggregation of dissociated sponge cells; (4) we also observed a parallel between synthesis of the sulfated polysaccharide and formation of large aggregates of sponge cells, known as primmorphs. Once aggregation reached a plateau, the demand for the de novo synthesis of sulfated polysaccharides ceased. Heparin can mimic the homologous sulfated polysaccharide on the in vitro interaction and also as an inhibitor of aggregation of the dissociated sponge cells. However, this observation is not relevant for the biology of the sponge since heparin is not found in the invertebrate. In conclusion, marine sponges display an ancestor event of cell-cell adhesion, based on the calcium-dependent carbohydrate-carbohydrate interaction.

Human serum albumin was able to hydrolyze the organophosphorus compounds paraoxon, chlorpyrifos-oxon, and diazoxon at toxicologically relevant concentrations. Human serum displayed two paraoxon hydrolyzing activities: the so-called paraoxonase, which is associated with the lipoprotein fraction and is calcium dependent and EDTA sensitive, and the activity associated with albumin, which is EDTA resistant and sensitive to fatty acids. Human serum albumin hydrolyzed these compounds with the same relative efficacy as lipoproteins (chlorpyrifos-oxon > diazoxon > paraoxon). The capability of detoxication of activity associated with human serum albumin was similar or even higher than paraoxonase associated with lipoproteins in the case of paraoxon at concentrations as low as those noted in an acute in vivo intoxication. However, paraoxonase activity associated with lipoprotein was more effective than paraoxonase activity associated with albumin at toxicologically relevant chlorpyrifos-oxon concentrations. These results explain why mice deficient in paraoxonase associated with lipoprotein are not more sensitive to paraoxon than wild animals.

This method simultaneously determines epinephrine, norepinephrine, dopamine and 5-hydroxytryptamine by HPLC coupled to atmospheric pressure chemical ionization mass spectrometry, using bovine chromaffin cells to test xenobiotic neurotoxicity and the secretion alterations of these neurotransmitters as endpoint. Chromatographic separation was developed by injecting the sample without previous treatment into a reversed-phase column. The signal was recorded in selected ion mode. The lowest limit of detection was found for hydroxytryptamine, while the highest limit was for norepinephrine. The feasibility of the proposed method was checked by performing measurements of neurotransmitters during the assessment the effect of mipafox on the basal and potassium-induced secretions of chromaffin cell cultures.

The Ca(2+)-dependent and EDTA-resistant hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and paraoxon was studied in serum and subcellular fractions of liver, kidney and brain of hen, rat and rabbit. HDCP was the best substrate among all the tissues studied, except that of rabbit serum which showed the highest Ca(2+)-dependent paraoxon hydrolysing activity (paraoxonase). High HDCP hydrolysing activity (HDCPase) was detected in the brain tissue of the three species studied, whereas low or no paraoxonase was found. The HDCPase/paraoxonase ratio of Ca(2+)-dependent hydrolysing activities ranged from 0.5 to 83 for tissues of the same species. EDTA-resistant HDCPase activity was more than 50% of the total activities in hen tissues, with an almost undetectable Ca(2+)-dependent paraoxonase activity in most organs. The same response was observed in rat tissues, except for serum where the Ca(2+)-dependent HDCPase and paraoxonase activities were higher (70 and 25% of total activities, respectively). EDTA-resistant HDCPase and paraoxonase activities represented less than 25% of all activities in rabbit tissues. Paraoxon has traditionally been the substrate for measuring organophosphorus hydrolysing activities. However, HDCP could be a good substrate in addition to paraoxon for monitoring other phosphotriesterases in biological tissues.

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.

Chromaffin cells in culture show high neuropathy target esterase (NTE) activity. It is well known that inhibition and specific modification of NTE by some organophosphorus (OPs) compounds induces a neurodegenerative neuropathy. It has been suggested that NTE is responsible for phosphatidylcholine homeostasis, although its role in neuropathy induction remains unclear. The cDNA of human NTE (4.4kbp) was inserted into an adenoviral vector. Bovine chromaffin cells cultured at 50,000 cells/well were incubated with the vector for 2h and after removing the volume of infection, cells were maintained in the incubator. After 24h, NTE activity was 6.8+/-0.5mU/10(6) cells in untreated cells and 14.8+/-1.5mU/10(6) cells, 19.3+/-2.9mU/10(6) cells, 24.8+/-0.9mU/10(6) cells and 30.9+/-1.0mU/10(6) cells in cells incubated with 2, 4, 8 and 16microl of vector, respectively. After 60min of inhibition with mipafox increased concentrations, the calculated I(50) (60min) values were 5.5, 6.2 and 6.6microM for cells infected with 0, 2 and 10microl of vector preparation. We confirm that the adenoviral vector containing the human NTE gene is active in bovine chromaffin cells in culture and that the NTE activity expressed by the vector shows the same inhibition pattern by the neuropathic OP mipafox as the NTE activity of bovine chromaffin cells and cells remained viable after the high NTE activity expression.

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.

The hydrolysis of carbaryl by bovine serum albumin (BSA) was studied at toxicologically relevant concentrations (range 15-300 microM) in order to determine the role of this protein in the detoxication of the carbamate in vivo. The 1-naphthol released during the hydrolysis of carbaryl was monitored using gas chromatography coupled with mass spectrometry. BSA hydrolyzed carbaryl in a time-progressive way. The hydrolysis was also dependent of enzyme (1.0, 2.5, 5.0 and 7.0 mg ml(-1)) and substrate (range between 15 and 1,000 microM) concentration. The estimated turnover number and Michaelis-Menten constant were 1.6 x 10(-4) s(-1) and 430 microM, respectively. Thus, the second order rate constant was 0.37 M(-1) s(-1). At enzyme concentrations of 7.0 mg ml(-1) and substrate concentrations ranging between 50 and 300 microM about 80% of substrate was hydrolyzed in 3 h. At lower substrate concentrations (15 and 30 microM carbaryl) also significant hydrolysis was detected at the highest enzyme concentration, even when these substrate concentrations were 30 and 15 times lower than the Michaelis-Menten constant. Although the efficacy of the enzymatic hydrolysis is low, the extrapolation of our results to the physiological albumin high concentrations (around 40 mg ml(-1)) suggests that the hydrolysis of carbaryl by serum albumins plays a critical role in the detoxication of this carbamate at in vivo toxicologically relevant concentrations.

Organophosphorus and carbamate insecticides inhibit the carboxylesterases found in plasma. Therefore, these carboxylesterases might be used as biomarkers of exposure to these insecticides. This work initiates the characterization of the phenylacetate (PA) and 1-nafthylacetate (NA) hydrolyzing activities (PAase and NAase) in the plasma of 11 different wild bird species and aims to determine their suitability as biomarkers of exposure. PAase activity values, expressed as mumol product/30min/mL plasma, ranged between 38+/-2.3 (black vulture) and 27+/-0.85 (barn owl), while NAase values ranged between 6.0+/-5.2 (griffon vulture) and 38+/-0.85 (barn owl). In all assayed species, NAase was between 1.1 and 2.8 times higher than the corresponding PAase. PAase and NAase of chicken white stork were 1.6 and 1.7 times higher, respectively, than the corresponding activities of adult individuals. Nocturnal raptors, eagle owl and barn owl, exhibited PAase and NAase between 1.3 and 8.0 times higher than activities exhibited by diurnal raptors (Montagu's harrier, common buzzard, booted eagle, Spanish imperial eagle, black kite, griffon vulture and black vulture). Data presented in this work suggest that plasma PAase and NAase of the studied birds might be used as biomarkers of exposure to organophosphorus and carbamate insecticides, although further studies of inhibition of these activities are still needed.

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.

A capillary electrophoresis method was developed to detect interactions between methadone and anti-retroviral compounds. Eight subjects, who underwent methadone maintenance treatment in the Province of Alicante (Spain), consented to participate in the present study. Of those, one subject was followed up for 123 days to detect drug-drug interactions. The enantiomers of methadone and those of its main metabolite were conveniently resolved within 4 min using a chiral electrophoresis buffer mixture which consisted of phosphate buffer, pH 5, plus 0.2% highly sulphated-(beta)-cyclodextrin. The effective mobility of the analytes was in the 0.061-0.140 cm(2)/(kV s) range at pH 5. The R-methadone plasma concentration range for seven patients was 91-318 ng/mL, it decreased from 186 to 46 ng/mL in a patient followed-up on commencement of the anti-retroviral therapy, returning to the previous higher levels after progressive dose increases. We conclude that monitoring R-methadone plasma levels can be a useful tool for the dose adjustment of methadone.

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.

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.

Human serum (HS) and human serum albumin (HSA) were able to hydrolyse the carbamate carbaryl. Carbarylase activity found in HSA was slightly activated by 1 mM Zn2+, Mn2+, Cd2+, Ni2+ and Na+ and by 0.01 mM Pb2+. The organophosphorus compounds paraoxon and O-hexyl O-2,5-dichlorophenyl phosphoramidate, caprylic acid, palmitic acid and the carboxyl ester p-nitrophenyl butyrate inhibited the hydrolysis of carbaryl by HSA, being in the last case a competitive inhibition. Using selective amino acid reagents, we concluded that Cys, Trp, Arg and Tyr seem to play important roles in the carbarylase activity of HSA. In addition, Tyr and Arg seem to be located in the active centre of the enzyme since carbaryl protected the activity from the inhibition. It was concluded that HSA hydrolyses carbaryl by a mechanism similar to that described for rabbit serum albumin based in transient carbamylation of a Tyr residue. The extrapolation of the hydrolysis rate to physiological albumin concentrations suggests that albumin might be playing a critical role in the detoxication of carbaryl.

Organophosphorus compounds (OPs) are being used as insecticides and warfare agents. OP insecticides represent an important problem of public health, causing around 200,000 deaths annually. The World Health Organization has pointed to the necessity to introduce new medical practices that improve the results of classical treatments. Many studies have shown that the administration of phosphotriesterases (enzymes that detoxify OPs through hydrolysis) is a promising treatment of persons poisoned with OPs. Such an enzyme-based treatment might introduce important improvements in the treatment of patients having ingested large amounts of OPs. Phosphotriesterases might also be suitable for prophylactic treatment of persons at risk to be severely exposed. The new experimental treatments do not exhibit the intrinsic neurotoxicity of the classical prophylaxis based on carbamates and antimuscarinic drugs. Experimental data suggest that might be time to initiate clinical trials in order to study the efficacy of phosphotriesterases in the therapy and prophylaxis of OP intoxication.

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.

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.

One of the main detoxification processes of the carbamate insecticides is the hydrolysis of the carbamic ester bond. Carboxylesterases seem to play important roles in the metabolization of carbamates. This study performs a biochemical characterization of the capabilities of rabbit serum albumin (RSA) to hydrolyze the carbamate carbaryl. Rabbit serum albumin was able to hydrolyze carbaryl with a K(cat) of 7.1 x 10(-5) s(-1). The K(m) for this hydrolysis reaction was 240 microM. Human, chicken, and bovine serum albumins were also able to hydrolyze carbaryl. The divalent cation Cu(2+) at 1 mM concentration inhibited around 50% of the hydrolysis of carbaryl by RSA. Other mono- and divalent cations at 1 mM concentration and 5 mM EDTA exerted no significant effects on the hydrolysis of carbaryl by RSA. The inhibition of the carbaryl hydrolysis by sulfydril blocking agents suggests that a cysteine residue plays an important role in the active center of the catalytic activity. Both caprylic and palmitic acids were noncompetitive inhibitors of the carbaryl hydrolysis by RSA. The carboxyl ester p-nitrophenyl butyrate is a substrate of RSA and competitively inhibited the hydrolysis of carbaryl by this protein, suggesting that the hydrolysis of carbaryl and the hydrolysis of carboxyl esters occur in the same catalytic site and through a similar mechanism. This mechanism might be based on the carbamylation of a tyrosine residue of the RSA. Serum albumin is a protein universally present in nontarget species of insecticides; therefore, the capability of this protein to hydrolyze other carbamates must be studied because it might have important toxicological and ecotoxicological implications.

The most employed insecticides for indoor and agriculture purposes belong to carbamates, pyrethroid or organophosphates. The chemical structures of these three groups correspond to carbamic, carboxylic and triphosphoric esters. Technical monographs suggest that the hydrolysis of ester bonds of carbamates and pyrethroids plays an important role in the detoxification of these compounds. However, detailed studies about enzymes hydrolysing carbamates and pyrethroids in vertebrates are not available. Certain carbamate hydrolysing activities are associated to serum albumin. Phosphotriesterases, being of an unknown physiological role, hydrolyse (in some cases stereospecifically) organophosphorus insecticides (OP). Phosphotriesterases have been found in a multitude of species, from mammals to bacteria. A phosphotriesterase activity, EDTA-resistant, has been detected in serum albumin. Phosphotriesterases in serum of mammals display polymorphisms. Phosphotriesterases offer applications in therapy of organophosphorus poisonings, in biodegradation and bioremedation of organophosphates. Similar studies should be developed with enzymes hydrolysing pyrethroids and carbamate insecticides. Such studies will improve the knowledge of the detoxification routes in non-target species and will help to design specific and safer carbamate and pyrethroid insecticides.

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.

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.

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.

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.

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.

Neural carboxylesterases can be discriminated by differential inhibition assays with organophosphorus compounds (OPs), paraoxon (O,O'-diethyl p-nitrophenyl phosphate) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) being the ones used to discriminate esterases that should be either irrelevant or candidates as targets of the mechanism of induction of the organophosphorus-induced delayed polyneuropathy (OPIDP). The brain membrane-bound phenyl valerate esterase (PVase) defined by Dr Johnson in 1969 as neuropathy target esterase (NTE) and recently cloned by Dr Glynn and coworkers is termed here as particulate NTE due to its association to the membrane particulate fraction. It is considered as the target of OPIDP and is the activity measured in standard NTE assays and toxicity tests. Following the same operational criteria in the soluble fraction of sciatic nerve a paraoxon-resistant but mipafox-sensitive PVase activity was described and termed as S-NTE, with an apparent lower sensitivity to some inhibitors than particulate NTE. Two isoforms (S-NTE1 and S-NTE2) were subsequently separated by gel filtration chromatography. In a partly purified S-NTE2 preparation polypeptides were identified in western blots by labelling with S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane], the same biotinylated OP used to label and isolate particulate NTE, but not with anti-particulate NTE antibodies. From sequential inhibition protocols, inhibitor washing-out and time course inhibition studies it is deduced that reversibility of inhibition is a new factor introducing a higher complexity in the identification of the esterases that could be candidates as targets of the mechanisms of induction and/or promotion of neuropathy. We have evidences that in sciatic nerve soluble fraction a high proportion (about 70%) of the activity that is inhibited by paraoxon in the usual concurrent assay is quickly reactivated after removing paraoxon and it is permanently inhibited by mipafox. Under this improved sequential paraoxon/mipafox inhibition procedure S-NTE represents about 50% of total PVases while in the usual concurrent assay it was only apparently about 1-2%. Moreover with such criteria, S-NTE2 isoform(s) represents about 97-99% of total S-NTE, and S-NTE1 is only a marginal amount probably resulting of a partial solubilization from particulate NTE. Fixed time inhibiton curves with variable mipafox concentration failed to discriminate more than one component. However kinetic behaviour of the time progressive inhibition cannot be explained by a simple model with a single exponential mathematical component, indicating that either the possibility of more than one component or a more complex mechanistic model should be considered. Consequently both particulate NTE and S-NTE assay protocols and their role in induction and promotion of neuropathies will need to be reviewed. Data published by Drs Lotti, Moretto and coworkers suggest that particulate NTE cannot be the target of promotion of axonopathies. The proposal that S-NTE2 could be such a target is suggestive and under collaborative biochemical and toxicological studies.

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.

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.

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.

Neuropathy target esterase (NTE) is suggested to be the molecular target for the initiation of the organophosphorus induced delayed polyneuropathy (OPIDP). O,O'-diethyl p-nitrophenyl phosphate (paraoxon) was the non-neurotoxic OP of choice for the standard assay of NTE to block the non-relevant esterases (phenylvalerate hydrolases) because it was supposed not to inhibit the enzymic activity of the target protein while N,N'-diisopropyl phosphorodiamidofluoridate (mipafox) is the neuropathic OP used to inhibit (and so to detect) NTE activity. A soluble form of NTE (S-NTE) had previously been described in peripheral nerve which showed a different inhibitor response from that of the particulate NTE (P-NTE). The use of a sequential type of inhibition protocol revealed the presence of an activity component within S-NTE which was extremely sensitive to different esterase inhibitors. Such a soluble activity component remained hidden under the usual concurrent inhibition procedure with paraoxon and was about one order of magnitude more sensitive than P-NTE to the inhibitors studied in the present article. Our results suggest that paraoxon could produce a strong reversible effect on S-NTE when the concurrent procedure is used so that it interferes with its inhibition by both neuropathy inducers and promoters. As a result S-NTE seems to be much more sensitive, than previously believed, to several esterase inhibitors involved in either the genesis of delayed polyneuropathy and/or axonopathy promotion.

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.

Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Gel filtration chromatography with Sephacryl S-300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S-NTE1 and S-NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1-250 microM range, in the presence of 40 microM paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with I50 (30 min) values of 1.7 and 19 microM, respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components (Vo peak, or S-NTE1; and 100-K peak, or S-NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high-molecular-mass (>700-kDa) peak or S-NTE1 was obtained. It showed an I50 of 5.2 microM in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the Vo peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the Vo peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S-NTE2 should be a different entity than S-NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.

Neuropathy target esterase (NTE) activity is defined operatively as the paraoxon-resistant mipafox-sensitive phenyl valerate esterase activity. A preparation containing a soluble isoform (S-NTE2) has been obtained from sciatic nerve. It was inhibited by the biotinylated organophosphorous ester S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] in a progressive manner showing a second-order rate constant of (3.50 +/- 0.26) x 10(6) M(-1) x min(-1) with an I50 for 30 min of 6.6 +/- 0.4 nM. S-NTE2 was enriched 218-fold by gel filtration followed by strong and weak anion-exchange chromatographies in HPLC. In western blots, this enriched sample showed two bands of endogenous biotinylated polypeptides after treating the blots with streptavidin-alkaline phosphatase complex. When the sample was treated with S9B, another biotinylated band was observed with a molecular mass of approximately 56 kDa, which was not seen when the sample had been pretreated with mipafox before the S9B labeling. It was deduced that this band represents a polypeptide (identified as the S-NTE2 protein) that is bound by both mipafox and S9B and that should be responsible for the progressive S9B inhibition. It is possible that S-NTE2 is the target for attack by compounds that promote delayed neuropathy.

Discrepancies in the aging reaction between neuropathy target esterase (NTE) inhibited in vitro and in vivo by racemic mixtures of O-alkyl O-2,5-dichlorophenyl phosphoramidates have been observed. It suggested the existence of differences in the interactions (inhibition and aging) between NTE and each stereoisomers of the above mentioned compounds. In order to verify this hypothesis, stereoisomers of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) were isolated by chiral column chromatography, followed by the evaluation of NTE inhibition and aging for each stereoisomers. The loss of reactivation capacity by KF was used as criterion of aging. The stereoisomer S-(-)-HDCP inhibited hen brain NTE with an I50 of 7.6 nM for 30 min of incubation, this being similar to the value obtained for the racemic mixture (I50 = 6.2 nM), and much lower than that recorded for R-(+)-HDCP (I50 = 191 nM). NTE inhibited by HDCP racemic mixture and the stereoisomer S-(-)-HDCP was reactivated by KF after 20 h of incubation at 37 degrees C. The NTE inhibited by R-(+)-HDCP could not be fully reactivated after inhibition.

An automatable microassay method developed for phenyl valerate esterase (PVase) activity has been applied to determine the following activities in the soluble fraction of hen sciatic nerve: activity A (total PVase activity), activity B (paraoxon-resistant PVase activity), activity C (PVase activity resistant to 40 microM paraoxon and 250 microM mipafox) and neuropathy target esterase (NTE) activity (resistant to 40 microM paraoxon but sensitive to 250 microM mipafox), operationally defined as activity (B-C). This microassay is based on the technique described by Barril et al. (Toxicology. 1988. 49:107-114). The Automated Biomek 1000 Station was used, which guarantees both inter- and intra-assay reproducibility of the results, and shortens the total assay time. The technical problems involved when processing many samples were thus resolved and with same regards it can also apply manually and using a microplate reader. In the case of activity A, the sensitivity of the method allowed the detection of activity in 1 microgram of protein (0.15 mg fresh sciatic nerve tissue), and the response was linear for different concentrations of 0.15-1.7 mg fresh tissue. For B, C and NTE, sensitivity corresponded to 10 micrograms of protein (1.5 mg fresh tissue in the microassay), with a linear response in the range of 1.5-17 mg fresh tissue. The response was linear versus the time of enzyme-substrate reaction (30-150 min). As tissue concentration increased, the response became nonlinear at shorter time. The procedure may be used to measure other enzymatic activities that yield phenols and chlorophenols as reaction products.

Depolarization induced catecholamine release from chromaffin cells was decreased 28% by N,N'-diisopropyl diamido-phosphorofluoridate (mipafox), an organophosphorus compound (OP) causing neurotoxic effects, while secretion stimulated by nicotinic agonist was inhibited 65%. The reversibility of this effect and the fact that calcium-dependent secretion from digitonin-permeabilized cells was unaffected by mipafox suggest that this compound affects the ionic currents implicated in catecholamine release. Patch-clamp experiments showed that the activity of voltage-dependent calcium channels (VDCC) was inhibited 35% by mipafox being this effect reversible whereas only minor effects were detected on Na(+) and K(+) currents. Finally, we studied the effect of mipafox on nicotinic ionic currents in chromaffin cells. In this case, the OP was able to cause reversible inhibition reaching maximal effects of 50-60%. In conclusion, nicotinic receptors and VDCC should be considered as potential targets in order to understand the neurotoxicity of these chemicals.

Carboxylesterase activities are widely distributed in a great variety of tissues; however, the biological function of these enzymes remains unclear. Some organophosphorus compounds induce a neurodegenarative syndrome related to the covalent modification of a carboxylesterase known as neuropathy target esterase. We investigated the expression of neuropathy target esterase and related carboxylesterase in bovine chromaffin cells with the aim of developing a potential in vitro model for studying the cellular function of carboxylesterase enzymes and toxic effects of organophosphorus compounds. Total phenyl valerate esterase exhibited an activity of 1.27 +/- 0.19 mU/10(5) cells (SD, n = 15). From the phenyl valerate esterase paraoxon and mipafox inhibition curves the following activities have been determined: B-activity (resistant to 40 microM paraoxon), 1.05 +/- 0.08 mU/10(5) cells (n = 8); C-activity (resistant to 40 microM paraoxon plus 250 microM mipafox), 0.12 +/- 0.05 mU/10(5) cells (n = 8); and neuropathy target esterase, calculated by the difference between B- and C-activities, 0.93 +/- 0.08 mU/10(5) cells (n = 8). All of these activities increased linearly with the number of cells and time of incubation with the substrate. Most of the phenol product of the reaction was released and detected in the extracellular medium. None of the components of the reaction were shown to affect cell viability when assessed by trypan blue exclusion. The study shows that bovine chromaffin cells possess carboxylesterase activities and respond to inhibition by paraoxon and mipafox, thus facilitating the discrimination of neuropathy target esterase. In conclusion, bovine chromaffin cells are appropriate as an in vitro cell model for studying toxic effects of organophosphorus compounds.

Neuropathy target esterase (NTE) is the proposed target site for the mechanism of initiation of the so-called organophosphorus-induced delayed polyneuropathy (OPIDP). NTE is operationally defined in this article as the phenylvalerate esterase activity which is resistant to inhibition by 40 microM paraoxon and sensitive to 250 microM mipafox. Soluble (S-NTE) and particulate (P-NTE) forms of NTE had first been identified in hen sciatic nerve [E. Vilanova, J. Barril, V. Carrera, and M. C. Pellin (1990). J. Neurochem., 55, 1258-1265]. P-NTE and S-NTE showed different sensitivities to the inhibition by several organophosphorus compounds over a range of inhibitor concentrations for a 30 or 120 minute fixed inhibition time at 37 degrees C. S-NTE was less sensitive to the inhibition by O,O'-diisopropyl phosphorofluoridate (DFP), hexyl 2,5-dichlorophenyl phosphoramidate (H-DCP), and mipafox than P-NTE and brain NTE, while the opposite was true for O,S-dimethyl phosphoroamidothioate (methamidophos). For each of the four inhibitors assayed, S-NTE showed two components of different sensitivity according to the inhibition curves fitted with exponential models. However, the inhibition of P-NTE by mipafox, DFP, and HDCP did not show the presence of a considerable proportion of a second component. The kinetics of heat inactivation showed that P-NTE inactivated faster and to a greater extent than S-NTE. It is concluded that (1) sciatic nerve S-NTE is more different from brain NTE than P-NTE; (2) P-NTE and S-NTE have different sensitivities to the inhibition by the studied organophosphorous compounds; (3) the inhibition curves suggest that S-NTE has two different enzymatic components while these are not so evident for P-NTE.

Neuropathy target esterase (NTE) activity is operatively defined in this paper as the phenyl valerate esterase activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Molecular exclusion column chromatography with Sephacryl S-300 of the soluble (S) fraction from chick sciatic nerve demonstrated two NTE activity peaks. The first eluted with the front, thus indicating a mol. wt. of over 700 kDa (peak Vo), while the second peak eluted with kd = 0.36, suggesting a mol. wt. of about 100 kDa. The curve of total phenyl valerate (PVase) activity inhibition with paraoxon (0.19-200 microM) shows that at a concentration of 40 microM the esterases highly sensitive to paraoxon are inhibited in the Vo and 100-kDa peaks. The NTE activity in these two peaks in turn represented 31% and 44% of the 40 microM paraoxon resistant activity, respectively. The mipafox inhibition curves (1.0-250 microM) revealed different sensitivities to mipafox, with I50 values (t = 30 min) of approximately 1.47 and 63 microM, for Vo and 100-kDa peaks respectively. Mipafox sensitivity of the Vo and 100-kDa peaks correlates with the two components, that had been deduced from the kinetic properties of the S-fraction.

Neuropathy target esterase (NTE) is a protein suggested to be involved in the initiation mechanism of organophosphorus-induced delayed neuropathy (OPIDP). We previously described two different forms of NTE activity in hen sciatic nerve: a particulate form (P-NTE) representing 40-50% of total NTE activity in sciatic nerve, and a remaining soluble component (S-NTE). In brain tissue on the other hand, more than 90% of NTE activity was recovered as P-NTE. In this work we studied the in vivo inhibition of both NTE forms with different doses of mipafox and the results were compared with sensitivity to mipafox in vitro. The highest dose with no observable neuropathic effects (1.5 mg/kg mipafox p.o.) inhibited 33% P-NTE and 55% S-NTE activity. The difference between P-NTE and S-NTE activity was statistically significant (P < 0.001, n = 9). Higher doses (3 mg/kg) induced neuropathy and inhibited NTE more than 75%, but differences between P- and S-NTE were not significant (P > 0.5). The greater inhibition of S-NTE than P-NTE in vivo contrasts with the observation that S-NTE is less sensitive in vitro.

O-Hexyl O-2,5, dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. The chicken has very low activity of Ca-dependent organophosphorus-hydrolases (OP-hydrolases) such as paraoxonase. HDCP is degraded at a similar rate in rat and hen plasma (16 and 21 nmol/min/microliters plasma, respectively) when measured by the loss of its anti-cholinesterase potency (Diaz-Alejo et al., 1990). The time course of the HDCP hydrolysis was not significantly affected by the following treatments: (a) 0.5-1 mM Ca2+ or 1-10 mM EDTA added at 30 min before starting the reaction at 37 degrees C; (b) preincubation with a carboxylesterase inhibitor 100 microM diisopropyl phosphorosfluoridated (DFP) for 60 min at 37 degrees C; (c) preincubation with 100 microM HDCP for 60 min at 37 degrees C; and (d) the presence of 50 microM DCP. However, the hydrolysis of HDCP was slightly modified by the other product of its hydrolysis. There is no contribution to the HDCP hydrolysis by covalent binding to carboxylesterase proteins. The course of the hydrolysis of HDCP was similar when measured by either the loss of anti-cholinesterase potency or the DCP liberated. HDCP is hydrolysed by an OP-hydrolase which is not Ca-dependent and is present in hen in contrast to the best known OP-hydrolases which are Ca-dependent and are undetectable in birds.

The mechanism by which organophosphorus-induced delayed polyneuropathy is induced relates to the specific inhibition and subsequent modification ("aging") of a protein known as neuropathy target esterase (NTE), operatively defined as paraoxon-resistant and mipafox-sensitive phenyl valerate (PV) esterase activity. This protein has fundamentally been investigated in hen brain, the latter being the habitually employed OPIDP study model. In the present article, a partial characterization is made of the NTE and other related PV esterases in the bovine adrenal medulla and brain; NTE sensitivity to the neurotoxic organophosphorus compound mipafox is investigated, and its subcellular distribution is studied. The NTE activity of the adrenal medulla was found to be the highest of those among the tissues studied to date (5000 +/- 1400 mU/g tissue; +/- SD, n = 12). This activity represented 93% of the PV esterase activity resistant to 40 microM paraoxon in the particulate fraction of the adrenal medulla and approximately 50% of total PV esterase activity. In the bovine brain, these proportions were 72 and 26%, respectively, i.e., similar to those described in hen brain. The mipafox inhibition curve of PV esterase activity resistant to 40 microM paraoxon in the particulate fraction of the adrenal medulla suggests that NTE activity fundamentally comprises a mipafox-sensitive component with an I50 of 6.39 microM at 30 minutes, which is similar to the value reported in hen brain. NTE activity in the bovine adrenal medulla is almost exclusively limited to the particulate fraction, the microsomal fraction, plasma membrane, and chromaffin granule-enriched fractions being the highest in terms of specific activity.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral organophosphorus compound that undergoes enzymatic hydrolysis in the rat and hen. Studies of the stereospecificity of its biodegradation are necessary to establish HDCP toxicity. To this effect, methods have been developed for the analysis of the HDCP stereoisomers by gas chromatography (GC) and high-performance liquid chromatography (HPLC). The best resolution and analysis were obtained by HPLC with UV detection, a OA-4100 Techocel chiral column and the mobile phase: hexane-1,2-dichloroethane-ethanol (92:5:3, v/v/v). The detection limit was 25 microM for HDCP and 5 microM for one of its hydrolytic products: 2,5-dichlorophenol (DCP). The method was reproducible intra o inter die. Moreover, a method is described for the liquid extraction of HDCP and DCP with 1,2-dichloroethane in biological samples, with a yield of (80.3 +/- 9.7)% (n = 10, S.D.) for HDCP and (84.1 +/- 10.0)% (n = 10, S.D.) for DCP. The method is compared with the solid-phase extraction technique with C18 sorbent. The hydrolysis of HDCP by hen plasma is studied.

One of the main detoxification mechanisms of organophosphorus (OP) compounds is hydrolysis by OP hydrolysing enzymes (OP-hydrolases) or phosphoric triester hydrolases. We previously reported an OP-hydrolase from hen plasma which hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP). In this study, a total of 18 cations, as well as several thiol blocking reagents, ethylenediaminetetraacetic acid (EDTA) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) were assayed as activators or inhibitors of the HDCP hydrolysing activity of hen plasma in vitro. Of the 18 inorganic cations only 1 M Na+ caused any inhibition. Most of the cations, including Ca2+, exerted no detectable effect; however, 1 mM Cu2+ was found to produce an activation of up to 263%, with a lesser activation of up to 168% for 1 mM Zn2+. The thiol blocking reagents methyl vinyl ketone (MVK) and N-ethylmaleimide (NEM) inhibited the enzyme in a time-dependent manner, the maximum effect depending upon concentration in the case of NEM, but not in the case of MVK; however, 5,5'-dithiobis (2-nitrobenzoic acid) caused inhibition that was concentration dependent but which was independent of time. Other thiol blocking reagents such as p-hydroxymercuribenzoic acid (sodium salt), phenylmercuric acetate, iodoacetic acid (sodium salt) and iodoacetamide produced only slight inhibition, as did EDTA. Finally, the OP compound mipafox exerted no detectable effect.

The organophosphorus (OP) compound O-hexyl-O-2,5-dichlorophenyl phosphoramidate (H-DCP) is hydrolysed in the plasma, liver and brain of hens and rats. We study in hen plasma the effect of tissue and substrate concentrations and of pH on the hydrolysing activity of H-DCP. The data on each tissue concentration were fitted to a double exponential mathematical model. The kinetics of this activity was not linear; in a first exponential component or fast initial phase (k1 = (1.603 +/- 0.248) x 10(-3) min-1/microliter plasma (n = 4, S.E.)) about 15% of the total compound was hydrolysed, followed by a slow second phase (k2 = (0.144 +/- 0.026) x 10(-3) min-1/microliter plasma (n = 4, S.E.)) in which the remaining 85% was hydrolysed. Both constants increased in value with pH. The hydrolytic activity and rate constants increased with the amount of plasma, but no change in kinetics was observed. The kinetic data are discussed in terms to lend support to the hypothesis of a stereospecific degradation of H-DCP.

Considerable evidence exists suggesting that the so-called neuropathy target esterase (NTE) is involved in the mechanisms responsible for organophosphorus-induced delayed polyneuropathy (OPIDP). Earlier studies in the adult hen, the habitually employed experimental model in OPIDP, have shown that most NTE activity in the brain is centered in particulate fractions, whereas approximately 50% of this activity in the sciatic nerve is encountered in soluble form, with the rest being particulate NTE. In the present work, we have studied the particulate and soluble fractional distribution of paraoxon-resistant phenylvalerate esterase activity (B activity), paraoxon- and mipafox-resistant phenylvalerate esterase activity (C activity), and NTE activity (B-C) according to ultracentrifugation criteria (100,000 g for 1 h). To this effect, two sensitive (adult hen and cat) and two scarcely sensitive (rat and chick) models were used. In all four experimental models, the distribution pattern was qualitatively similar: B activity and total NTE were much greater in brain (900-2,300 nmol/min/g of tissue) than in sciatic nerve (50-100 nmol/min/g of tissue). The proportion of soluble NTE in brain was very low (< 2%), whereas its presence in sciatic nerve was substantial (30-50%). The NTE/B ratio in brain was high for the particulate fraction (> 60%) and low in the soluble fraction (7-30%); in sciatic nerve the ratio was about 50% in both fractions.

NTE inhibitors cause different toxicological consequences (protection, induction or potentiation/promotion of neuropathy) depending on the order of dosing. These effects might be explained in terms of several phosphorylable sites with 'allosteric irreversible' behaviour. Brain neuropathy target esterase (NTE) has been preinhibited with phenylmethylsulphonyl fluoride (PMSF) (0, 5, 10, 15, 30 and 60 microM) or with diisopropylphoshoro fluoridate (DFP) (0, 0.2, 0.5, and 1 microM) at 37 degrees C for 30 min. After washing by centrifugation, tissues were then reinhibited with a range of PMSF (0 to 80 microM) or DFP (0 to 1 microM) concentrations. The slopes of the inhibition curves (log % activity vs. concentration) of pretreated tissues were identical to those of the non-pretreated tissues, with non-distinguishable I50 values. It is concluded that allosteric effects are not likely to be involved in membrane-bound NTE of hen brain.

NTE (neuropathy target esterase) is considered to be the target for organophosphorus-induced delayed polyneuropathy and is operationally measured by radiolabelling or by determining its esteratic activity as the paraoxon-resistant mipafox-sensitive phosphorylable site(s). From electrophoresis and density gradient centrifugation using radiolabelling techniques, several phosphorylable sites have been described in hen brain that are paraoxon-resistant mipafox-sensitive; however, only the majority electrophoresis band (155 kDa) shows properties related with the aging reaction. Kinetic criteria have also suggested two components of brain NTE (NTEA and NTEB). Most brain NTE is recovered in the particulate microsomal fraction and only about 1% in soluble fraction. In sciatic nerve about 50%/50% activity is recovered as soluble (S-NTE) or particulate (P-NTE) forms. A similar distribution were observed in hen, cat, rat and young chick. The fixed time inhibition curves show that P-NTE is more sensitive to mipafox, DFP and hexyl-DCP than S-NTE, while the reverse is true for methamidophos. P-NTE fits properly to one sensitive component while S-NTE fits better to two sensitive component models, except in the case of methamidophos. In vivo, significant differences in the inhibition of P- and S-NTE by mipafox were found only when using low non-neuropathic dosing. The possible significance of different NTE forms are discussed.

Diisopropyl phosphorofluoridate (DFP), mipafox, cresylsaligenyl phosphate, and phenylsaligenyl phosphate were applied to a 1.5-cm segment of the common trunk of the sciatic nerve in adult hens. At doses of 18-182 micrograms mipafox and 9-110 micrograms DFP, inhibition of neuropathy target esterase (NTE) for the treated segment was over 80%, whereas for the adjacent distal and proximal segments inhibition was under 40%, 15 min after application. NTE was not affected in the peripheral distal terminations arising from the common sciatic nerve (peroneal branches), contralateral sciatic nerve, brain, and spinal cord. A 24-hr study suggested a displacement of the activity-free region toward more distal segments of the nerve. All animals treated with 55 and 110 micrograms DFP or 110 micrograms mipafox lost a characteristic avian retraction reflex in the treated leg 9-15 days after dosing, suggesting peripheral neurological alterations. Only hens dosed at the maximum dose in both extremities presented alterations in motility (Grade 1 or 2 on a 0-8 scale), suggesting no significant central nervous system alterations. Electron microscopy of peroneal branches showed axon swelling and accumulation of smooth endoplasmic reticulum similar to animals dosed systemically (s.c.) with 1-2 mg/kg DFP. The branches also contained granular and electron-dense materials, as well as some intraaxonal and intramyelinic vacuolization. Clinical effects were not observed in animals protected with a 30 mg/kg (s.c.) dose of phenylmethanesulphonyl fluoride. It is concluded that the peripheral neurological effects of local dosing correlate with the specific modification of NTE in a segment of sciatic nerve and that the axon is a more likely target than the perikaryon or nerve terminal in the triggering mechanism of this axonopathy.

The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for the n-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose of L(-) methamidophos or of the ethyl or iso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. The n-propyl, n-butyl and n-hexyl compounds caused typical OPIDP at doses causing a peak of 70-95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be greater than 8 times the unprotected LD50 and the D-(+) isomer caused OPIDP at about 5-7 x LD50. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution.

The in vitro and in vivo biochemical properties of O-hexyl, O-dichlorophenyl phosphoramidate (hexyl-DCP) as inhibitor of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) were studied, as well as their neurotoxic effects. The differences found were suggested to be due to biotransformation effects. In this work, the in vitro time-dependent degradation of hexyl-DCP by plasma, liver and brain homogenates of rat and hen at 37 degrees C at pH 7.4 are studied using 100 nM initial concentration. The loss of inhibitory potency against AChE was used as sensor of the biodegradation rate. An approximate estimation of the residual compound was made by comparison with an inhibition calibration curve. The rate of enzymatic degradation was corrected for the spontaneous hydrolysis. Rat tissues showed some higher activities (24, 17, 1 mU/g for plasma, liver, and brain, respectively) than hen (17, 6, 1 mU/g), with activities being highest for plasma and lowest for brain. Hexyl-DCP is a chiral compound. The loss of anti-AChE power could be due to degradation of only one of the two stereoisomers.

The calcium-dependent enzyme activity which hydrolyzes the p-nitrophenyl-O-P bond of paraoxon (paraoxonase) has been studied in several rat and human tissues. Rat plasma and liver showed the highest activities (1.31 +/- 0.19, 0.82 +/- 0.09 nmol/min mg protein +/- SEM, respectively), while other tissues showed less than 2% plasma activity. The Arrhenius plot showed monophasic patterns in both tissues with activation energy values of Ea = 57 +/- 3 and 69 +/- 4 kcal/mol degree K for rat liver and plasma, respectively. Rat plasma and liver paraoxonase lost about 80% activity after 24-hr storage at 27-30 degrees C and was not restored by calcium addition. There was no loss of activity in human serum after 3 days and only 33% after 5 days. The pH optimum for paraoxonase activities was about 7.4 for both rat tissues. It is concluded that plasma paraoxonase is similar to the liver enzyme and is a good mirror for total body detoxifying activity.

Neuropathy target esterase (NTE) is the suggested "target" molecule involved in the initiation of organophosphorus-induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P-NTE) and soluble (S-NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80-100 nmol/min/g tissue from which 50-60% was recovered in the soluble supernatant fraction and the remaining 40-50% in the pellet fraction. About 90% of brain tissue activity (approximately 1,800 nmol/min/g tissue) was recovered as P-NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P-NTE and S-NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose-6-phosphatase, Na+,K(+)-ATPase, 5'-nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P-NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S-NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane-bound portions of a single protein, with P-NTE activity depending on a membrane factor from which it is separated through fractionation at high pH.

The interaction in vivo of four O-alkyl O-2,5-dichlorophenyl phosphoramidates with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) and their ability to cause delayed polyneuropathy in hens has been examined. Previous studies in vitro (Vilanova, Johnson & Vicedo, Pestic. Biochem. Physiol., 28 (1987) 224) had led to the prediction that these compounds would not be neuropathic but, rather, would be prophylactic agents against organophosphorus-induced delayed polyneuropathy. In vivo the effects of these esters on the enzymes differ in 2 respects from effects in vitro: (i) Relative sensitivity of the enzymes was different: thus greater than 50% of brain NTE remained 24 h after an oral dose of 15 mg/kg of the n-hexyl ester while only 10-30% of AChE remained although NTE was the more sensitive enzyme in vitro; (ii) In no case could the inhibited NTE or AChE in autopsy samples from birds dosed with any of the 4 esters be reactivated by treatment with potassium fluoride in vitro: the inhibited enzymes produced by incubation of tissue with the esters in vitro had been reactivatable. Prophylaxis, with therapy in some cases, was required to prevent acute anticholinesterase poisoning when doses were sufficient to cause high inhibition of neural NTE. Inhibition in brain was typically 5-10% more than in spinal cord and 10-15% more than in sciatic nerve. Unambiguous signs of polyneuropathy (Grade 3 or more on an 8-point scale) were not seen in birds observed up to 3 weeks after doses which caused less than 70% inhibition of NTE in brain and spinal cord or less than 60% inhibition in sciatic nerve of pair-dosed birds assayed 24 h after dosing. Doses of 300, 10, 100 and 65 mg/kg, respectively, of the methyl, ethyl, n-butyl and n-hexyl esters caused greater than 70% inhibition of NTE in all 3 neural tissues and neuropathy in the majority of observed birds. Analysis of consolidated dose/response data from 36 assayed and 51 observed birds showed that effects of Grade 3 or more were produced in about 90% of birds when inhibition of NTE was greater than 90% in brain, greater than 85% in spinal cord or greater than 75% in sciatic nerve.

Some organophosphorus compounds (OP) induce a delayed polyneuropathy (OPIDP) which is initiated by the phosphorylation of the so-called neuropathy target esterase (NTE). In this work some aspects of hen sciatic nerve NTE are studied. The assay method is reported and modifications are discussed and a combined method proposed. Proximo-distal distribution showed a significant difference from proximal (100 +/- 10%) to distal (69 +/- 9%) fragments, in accordance with reported data. The time course of in vivo regeneration after a single TOCP dose (200 mg/kg, post oral) showed some differences when compared with hen brain NTE. Sciatic nerve NTE showed a delay of 2-3 days before regeneration but then regenerated faster (74% activity at day 7) than brain NTE (50% activity at day 7). A slower rate of regeneration of distal than proximal segments has been suggested to explain higher sensitivity of distal segments [3], however in this work no significant differences were observed in the rate of regeneration when comparing proximal and distal fragments.

Simultaneous intoxication with hexacarbon solvents and organophosphorus compounds has been considered a possible critical factor in some occupational neuropathies and their interactions proved to cause potentiation effects in hens [1-3]. A high degree of inhibition of neuropathy target esterase (NTE) is needed to develop organophosphorus induced polyneuropathy (OPIDP). In this work, the inhibition of NTE, BCHE and AChE by TOCP on control and n-hexane pretreated (7-15 days, 300 mg/kg per day) hens is studied. Using a single TOCP dose of 200 mg/kg, n-hexane pretreated hens showed synergistic effects, but no significant differences were observed in the inhibition of cholinesterases and NTE in brain or spinal cord. With lower TOCP dose (20 mg/kg) statistically significant differences were observed, which were not drastic but could be important because they involved an increase of inhibition up to critical threshold values (from 40-50% to 60-70% inhibition). However, no clinical effects were observed in these animals. Possible mechanisms of neurotoxic interaction are discussed.

Hens were given a single oral dose (0.235 mg/kg) of tri-o-cresylphosphate (TOCP) during chronic n-hexane treatment (200 mg/kg daily, 5 days/week). They were compared with other animals treated only with n-hexane or only with TOCP. Animals treated with a higher TOCP dose (1 ml/kg) were used as positive controls. The animals treated with both n-hexane and TOCP showed rapid development of severe ataxia. The rate of the ataxia development was similar to that of the positive controls but with earlier onset of the first signs and with less loss of body weight. However, animals treated only with n-hexane, under the same conditions, showed only reversible weakness and sedative effects, and those treated only with TOCP showed slow and slight ataxia development. The n-hexane- and TOCP-treated hens showed axonal swelling with myelin retraction associated with Ranvier's nodes, which is characteristic of long hexacarbon exposure. Some internodal swellings were also observed but less frequently than the paranodal swellings. The time course of the ataxia development was similar to an organophosphorus-induced delayed neuropathy (OPIDN); however, the light microscopy observation more closely resembled hexacarbon neuropathy. The results suggest a potentiation effect of n-hexane and TOCP neurotoxicities which could be related to some human occupational neuropathies.

At 37 C and pH 7.4-8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, ka increased from 7.2 x 102 to 1.0 x 10 5 M-1 min-1 between methyl and n-hexyl S-methyl esters and from 9.3 x 10 3 to 8.9 x 10 5 M-1 min-1 between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 x 10 4 for S-methyl esters, and were 9.7 x 10 4 to 7.8 x 10 6 M-1 min-1 for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75-100% activity without aging of AChE was found after 19 hr incubation at 37 C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.

Cholinesterase (ChE) inhibitors have been described in the distillation residue of hexane and other industrial solvents. The residue of a commercial hexane has been fractionated by preparative chromatography. The anticholinesterase (antiChE) activity was isolated in two fractions (F-5, F-7) which contained only 0.61 and 0.16% respectively of the original dry weight hexane residue. In the former fraction, reversible and irreversible progressive inhibition was observed, and organophosphorus compounds (OPs) were detected colorimetrically and by gas chromatography. This fraction was subfractionated in a second chromatographic step. One subfraction containing the highest antiChE activity and 88% phosphorus of F-5 was isolated. In this subfraction triphenylphosphate and other not definitely identified OP compounds were detected by gas chromatography/mass spectrometry, together with several adipates and phthalates, including di-n-butylphthalate. This phthalate could explain the reversible inhibition of ChE by the hexane residue, and triphenylphosphate and the unidentified OP the irreversible inhibition. A possible toxicological role of these impurities is discussed in relation to occupational neuropathies by exposure to solvents.

Commercial hexane was concentrated by distillation. The distillation residue (0.43 ml/l original solvent) contains material which inhibits human serum cholinesterase (ChE) "in vitro" with a slight effect on acetylcholinesterase. Phosphorus was detected equivalent to 0.33 mumol monophosphorus compound/litre original solvent. The inhibition was progressive with the enzyme-inhibitor preincubation time. A partial reactivation of the inhibited enzyme was obtained by treatment with hydroxylamine and 2-PAM. The results are coherent with a covalent inactivation by more than one inhibitor which acylate (probably phosphorylate) ChE, although it seems likely that a reversible but unstable inhibitor could also be present in the hexane residue. The results are discussed in the context of the known neurotoxic effects of n-hexane and some organophosphorus esters.