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

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

Phosphotriesterase activity identified in purified serum albumins.

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

A stereospecific phosphotriesterase in hen liver and brain.

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

Inhibition and aging of neuropathy target esterase by the stereoisomers of a phosphoramidate related to methamidophos.

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.

Organophosphorus inhibition and heat inactivation kinetics of particulate and soluble forms of peripheral nerve neuropathy target esterase.

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. Pellín (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.

In vivo inhibition by mipafox of soluble and particulate forms of organophosphorus neuropathy target esterase (NTE) in hen sciatic nerve.

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.

Non-calcium dependent activity hydrolysing organophosphorus compounds in hen plasma.

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.

Chiral high-performance liquid chromatography and gas chromatography of the stereoisomers of hexyl 2,5-dichlorophenyl phosphoramidate.

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.

Soluble and particulate organophosphorus neuropathy target esterase in brain and sciatic nerve of the hen, cat, rat, and chick.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of O-hexyl O-2,5-dichlorophenyl phosphoramidate hydrolysing activity in hen plasma.

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.

Effect of some metallic cations and organic compounds on the O-hexyl O-2,5-dichlorophenyl phosphoramidate hydrolysing activity in hen plasma.

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.

Hen liver and plasma can metabolize hexyl-DCP phosphoramidate at a rate comparable to that of rat.

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.