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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.

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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.

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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.

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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.

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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 (Díaz-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.

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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.

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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.

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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.

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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.

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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.

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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.

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