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Chemico-Biological Interactions 187 (2010) 397–402

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

nhibition with spontaneous reactivation and the “ongoing inhibition” effect ofsterases by biotinylated organophosphorus compounds: S9B as a model

orge Estévez ∗, José Barril, Eugenio Vilanovanidad de Toxicología, Instituto de Bioingeniería, Universidad Miguel Hernández, Elche (Alicante), Spain

r t i c l e i n f o

rticle history:vailable online 20 May 2010

eywords:9Bsterasesnhibitionngoing inhibitionrganophosphoruspontaneous reactivation

a b s t r a c t

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 targetesterase (NTE) as it was considered a specific inhibitor of NTE. After incubation with the soluble fractionof chicken peripheral nerve, most of the soluble esterase activity was highly sensitive to S9B, indicatingNTE-like esterases. A kinetic model equation was used to assume a multi-enzymatic system with threedifferent simultaneously occurring molecular phenomena; (1) inhibition; (2) simultaneous spontaneousreactivation; and (3) ongoing inhibition (inhibition during the substrate reaction); to fit the data to ana-lyze kinetic behavior. A high “ongoing inhibition” effect was observed in an enzymatic component. Athree-dimensional fit of the model was applied. The best fitting model is compatible with three sensi-

tive enzymatic entities (33, 52 and 15%), and only one spontaneously reactivate. The second-order rateconstants of inhibition (ki = 116 × 106, 4.6 × 106 and 0.28 × 106 M−1 min−1, respectively) and the spon-taneous reactivation constant for the first sensitive component (kr = 0.0054 min−1) were simultaneouslyestimated. These parameters are similar to those deduced in spontaneous reactivation experiments of thepreinhibited samples with S9B. The estimated proportions of enzymatic components are similar to thosepreviously observed in inhibition experiments with mipafox, demonstrating that this kinetic approach offers consistent results.

. Introduction

The biotinylated organophosphorus compound 1-(saligeninyclic phospho)-9-biotinyldiaminononane (S9B) was considered

specific inhibitor of neuropathy target esterase (NTE) whenested against the membrane-bound esterases in chicken brain1]. NTE is considered the main target that triggers the so-calledrganophosphorus-induced polyneuropathy (OIDP). Thanks to thispecificity, S9B has been used for the detection, labeling, isolationnd purification of NTE for its cloning and molecular genomic char-cterization [1,2].

After incubation with the soluble fraction of chicken peripheralerve however, most soluble esterase activity was inhibited at a

ow concentration (nanomolar levels), indicating that highly S9Bensitive esterases are present in the soluble fraction of the periph-ral nerve. The toxicological role of these highly sensitive soluble

sterases has not yet been elucidated. This work analyzes the inter-ction of S9B with the soluble fraction of hen peripheral nerve,haracterizes its inhibitory sensitivity and kinetic behavior, andeduces the existence of esterases with a different kinetic behavior.

∗ Corresponding author. Tel.: +34 965222159; fax: +34 965222033.E-mail address: jorge.estevez@umh.es (J. Estévez).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.05.008

© 2010 Elsevier Ireland Ltd. All rights reserved.

The formation of a reversible non-covalent Michaelis-likeintermediate is not considered in this paper as the low sol-ubility does not allow the concentrations to cause significantsaturation [3]. The kinetic behavior is described for a direct phos-phorylation: E + I → E − P + X. The situation of possible reversibleinteractions with a second site in the enzyme is not considered[3].

In vitro experiments involve pre-incubating the enzyme prepa-ration with an inhibitor concentration (I) during the inhibitiontimes (t), and then incubating with a substrate during theenzyme–substrate reaction time (ts) to measure residual enzymeactivity (E) (Fig. 1). During the substrate reaction, inhibition is notusually significant given the dilution of the inhibitor and the protec-tive effect of the substrate. However some inhibition may occur forthe highly potent inhibitors. In this paper, this is known as “ongo-ing inhibition” and may be taken into account in the kinetic model[4].

The spontaneous reactivation of organophosphorus-inhibitedesterases has been observed in both the chicken peripheral nerve

soluble fraction and chicken serum [5,6]. The precise analysisof the kinetics of multi-enzymatic systems with “spontaneousreactivation-inhibition” has always been hindered by both thesystems’ complex mathematical performance and difficult inter-pretations.

398 J. Estévez et al. / Chemico-Biological Interactions 187 (2010) 397–402

Fig. 1. Typical time course in an inhibition experiment. Enzyme preparation is treated with an inhibitor (first vertical arrow) and is pre-incubated during the “inhibitiont rrow)s d a cou e buth

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Fig. 2 shows the spontaneous reactivation in the peripheralnerve soluble fraction preinhibited with S9B once the inhibitorhad been removed by ultrafiltration and after allowing a longreactivation time. Around 10–20% of total activity was lost in theultrafiltration process and further manipulation. The inhibited non-

ime” (ti) (continuous horizontal line). Then the substrate is added (second vertical aubstrate–enzyme reaction. Finally, the reaction is stopped (third vertical arrow) ansually stops inhibition due to dilution and competitive protection by the substratigh sensitive inhibitors.

In this work, a kinetic model has been used considering: (1) aomplex multi-enzymatic system, (2) the inhibition process (phos-horylation), (3) the reactivation process and ongoing inhibitionuring substrate reaction for the most sensitive enzymatic com-onent, and (4) no significant aging reaction occurs during thexperimental time. These assumptions are based on the observedxperimental behavior. The theoretical kinetic models shown bystévez and Vilanova [4] are applied to this real example to analyzehe peripheral soluble fraction esterases inhibition by S9B.

. Materials and methods

.1. Chemicals, tissue preparation and enzymatic assay

1-(Saligenin cyclic phospho)-9-biotinyldiaminononane (S9B)as made and supplied by P. Glynn and D. Read from the MRC Tox-

cology Unit (Carshalton, Surrey, U.K.). The preparation of chickenciatic nerve soluble fraction, the phenylvalerate esterase activityssay, the preparation and use of the substrate phenylvalerate, andhe reagents for stopping color formation with the product (phenol)btained are those described previously [7,8].

.2. Inhibition procedure

S9B inhibition kinetic was analyzed in a peripheral nerve solubleraction at 37 ◦C. The samples containing the soluble fraction from0 mg fresh tissue/ml were pre-incubated with several inhibitoroncentrations and inhibition times (see Fig. 1). The substratehenylvalerate (PV) was added and the phenylvalerate esterasePVase) assay proceeded as previously described [7,8], with the onlyxception that PV was incubated with the sample for 10 min insteadf 30 min. The results are expressed as % activity (E/E0 × 100) overhe control without an inhibitor, and as the plotted line versus timet) for each dataset for the respective inhibitor concentration.

.3. Kinetics of reactivation after removing S9B by ultrafiltration

.3.1. Inhibition and removal procedureA volume of 1.5 ml, containing the soluble fraction correspond-

ng to 20 mg fresh tissue/ml, was incubated with 100 nM of S9B for0 min at 37 ◦C in a total volume of 1.65 ml. Controls were incu-ated only with the buffer and the sample was incubated with9B. Both the non-inhibited control and the preinhibited samplesere diluted to 15 ml with buffer. Then they were subjected to twoashes through Millipore ultrafree-15 biomax 50 K (15 ml) filters

y centrifuging at 1500–2000 × g at 4 ◦C. Centrifugation contin-ed until the sample volume reduced to 0.15 ml. Next concentrates

ere diluted to 15 ml with buffer and filtered as before. At the end

f the process, buffer was added to obtain the sample concentrationsed in the PVase assay (the soluble fraction from 2 mg fresh tis-ue/ml). Residual S9B concentrations were estimated to be around.0011 nM.

and incubated during the “substrate time” (ts) (dashed horizontal line) to allow thelor reagent is added before measuring the absorbance. The addition of the substratesome inhibition may continue (“ongoing inhibition”) during substrate reaction for

2.3.2. ReactivationAfter diluting, the ultrafiltrated samples were incubated at 37 ◦C,

and aliquots of 1 ml were taken at 0, 30, 60, 120 and 180 min toallow reactivation. PVase activity was assayed as described above.The reaction time with the substrate was 60 min. The results areexpressed as % activity (E/E0 × 100) over the control without theinhibitor.

Inhibited non-ultrafiltrated controls were prepared. A volume of1 ml, containing the soluble fraction corresponding to 20 mg freshtissue/ml, was incubated with 100 nM of S9B for 30 min at 37 ◦C in atotal volume of 1.1 ml. Controls were incubated with only the bufferand inhibited non-ultrafiltrated controls were incubated with S9B.

2.4. Computerized estimation of the kinetic parameters

Model equations were fitted to the experimental inhibitionkinetic data by a non-linear computerized method based on theleast squares principle using the Sigma Plot software, version 8.

2.5. Model equation for inhibition and the reactivation process

The model equation deduced to describe the inhibition for asystem with several enzymatic components is described in Estévezand Vilanova [4].

3. Results

3.1. Spontaneous reactivation of S9B-inhibited esterases in theperipheral nerve soluble fraction

Fig. 2. Kinetics of the spontaneous reactivation after inhibition by S9B. Three inde-pendent preparations were inhibited by preincubation with 100 nM S9B for 30 min.Then after cooling the inhibitor was removed by two washing cycles of ultrafiltra-tion and dilution, and was then incubated at 37 ◦C for reactivation (See Section 2).E1, E2 and E3 are different enzymatic components.

J. Estévez et al. / Chemico-Biological Interactions 187 (2010) 397–402 399

Fig. 3. Activity at the pre-inhibition zero time. The data correspond to the inhi-bition experiment depicted in Fig. 4. Residual activity in presence of S9B at theptTB

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re-inhibition zero time is plotted versus the S9B concentration. The curve line ishe best fit of an exponential component, plus a constant component (E0·e−ka′ ·x + ER).he parameters of this fit are E0 = 36.0%, ka′ = 0.097 nM−1, ER = 64.0% (see Table 1, line). This figure shows the inhibition during the reaction time with the substrate.

ltrafiltrated controls displayed 10% PVase activity. The inhibitedltrafiltrated samples showed around 10% of initial PVase activity.he activity showed a time-progressive reactivation up to a level ofround 30% at overnight (Fig. 2). This confirms that a spontaneouseactivation takes place which should be considered in the kineticodel.

.2. S9B inhibition curves in the peripheral nerve soluble fraction

Inhibition of peripheral nerve soluble PVase activity with S9Bevealed a time-progressive inhibition which is coherent with aovalent irreversible inactivation. Fig. 4 presents the curves cor-esponding to the best fit according to the F-test of the individualurve for each S9B concentration used.

Although each curve seems to graphically fit the experimentalata, no consistent values of the kinetic constants, numbers and theroportion of the components have been obtained.

For a long inhibition time, the curve of the low concentration ofnhibitor tended to show a parallel line. These observations suggesthe existence of a spontaneous and simultaneous reactivation.

.3. Evaluation of ongoing inhibition during the substrateeaction

An analysis of the inhibition during the substrate reaction isrovided in Fig. 3 by plotting the percentage of activity for the prein-ubation zero time as a function of the inhibitor concentration. Thisndicates that the ongoing inhibition during the substrate incuba-ion under the assayed conditions is significant up to 36% at theighest inhibitor concentration. Therefore ongoing inhibition dur-

ng the substrate incubation needs to be considered in the model;hat is, at least the most sensitive components. Exponential decay

odels were used to fit the data.The best model according to the F-test was E0·e−ka′ ·x + ER. Param-

ter ka′ represents the apparent first-order kinetic constant of thebserved inhibition without the preincubation caused by inhibitionuring the substrate reaction time applied to measure activity.

The obtained parameters (Table 1, line B) show that the propor-ion of enzyme sensitive to ongoing inhibition is around 36% of theotal PVase activity, while around 64% is resistant to ongoing inhi-ition, which justifies that the ongoing inhibition factor should be

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400 J. Estévez et al. / Chemico-Biological Interactions 187 (2010) 397–402

Fig. 4. Kinetics of the time-progressive inhibition with different S9B concentrations.A preparation of soluble fraction of 20 mg fresh tissue/ml was pre-incubated 0, 1, 5,10, 20, 35 and 50 nM of S9B (upper to lower plots) for the indicated time. Then theetwa

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Fig. 5. Inhibitory surface obtained by fitting the 3D model equation to the data

nzymatic activity was assayed with phenylvalerate for 10 min. Percentages refer tohe activity of those samples pre-incubated with 0 nM S9B at each time. Each curveas fitted with exponential model equations by selecting the best fitting equation

ccording to the F-test.

.4. A kinetic model considering ongoing inhibition and theimultaneous spontaneous reactivation process in the peripheralerve soluble fraction

.4.1. Study of the time-progressive reactivation after removinghe inhibitor by ultrafiltration

Three independent preparations were inhibited with 100 nM9B for 30 min and the inhibitor was thoroughly removed after sev-ral ultrafiltration steps (See Section 2 and Fig. 2). The estimatedesidual concentration of S9B was 0.0011 nM. The residual concen-ration of S9B must be considered in the kinetic model becausehis concentration can have an effect in the reactivated enzymaticomponent during long time (25 h). The inhibited non-ultrafiltratedontrols showed 10% PVase activity. The diagram showing this pro-ess and the resulting kinetic equation are described in Estévez andilanova [4].

Mathematical models corresponding to the one, two or morenhibited enzymes which had spontaneously reactivated weresed. The best fit according to the F-test was a model with onlyne reactivated component:

= EP0 · kr

k · I + kr− EP0 · kr

k · I + kr· e−(k·I+kr)·t + R (1)

here kr is the reactivation constant, EP0 is the proportion (ampli-ude) of the initial inhibited enzymatic component, and R is thenzymatic fraction resistant to inhibition.

The deduced kinetic parameters values are shown in Table 1,ine C, and the curve is plotted in Fig. 2. The I50 (30 min) values forach component (Ei) were obtained by approximation by applyinghe following equation:

activity (Ei) =[

kr · 100ki · I + kr

]+

[ki · I · 100ki · I + kr

]· e−(ki·I+kr)·30 (2)

The previously estimated kinetic constants were fixed. Then suc-essive iterations with a different I in an electronic spreadsheetere carried out to obtain the I value for the % activity equal to

0 ± 0.1%.

.4.2. Study of the inhibition experimentThe profile of the inhibition curve in Fig. 4 and the evidence

f reactivation (Fig. 2) both suggest that a complex kinetic model

corresponding to S9B inhibition shown in Fig. 4. The surface reflects the result of thebest model according to the F-test and corresponds to a model with three sensitiveenzymatic components of which one was simultaneously inhibited and reactivatedwith significant ongoing inhibition.

should be used to fit the data and that it should include bothinhibition and the reactivation process for several enzymatic com-ponents. The evidences shown in Fig. 4 suggest that a correction forthe ongoing inhibition should also be included.

It is reasonable to assume that ongoing inhibition is only sig-nificant for the most sensitive enzymatic component. Thereforean additional exponential factor [e−ka′ ·I] should be added in themost sensitive component, E1 (see Eq. (3)). This constant has to beconsidered an operational empiric constant, and not a real kineticconstant.

The model which considers inhibition with the simultaneousand spontaneous reactivation was used with one, two or threeenzymatic components by considering the factor for the “ongo-ing inhibition” effect in the most sensitive component. The assayof reactivation suggests that only one component is reactivated.The best fitting model (according to the F-test) consisted of threeenzymatic entities, of which one was inhibited and spontaneouslyreactivated, and two were only inhibited. This mathematic modelis shown as follows:

E = [e−ka′ ·I] ·{[

kr1 · E10

k1 · I + kr1

]+

[k1 · I · E10

k1 · I + kr1

]· e−(k1·I+ kr1)· t

}

+E20 · e−(k2·I·t) + E30 · e−(k3·I·t) (3)

where k1, k2, k3 are the inhibition constants, kr1 is the reactivationconstant, E10, E20, E30 are the proportions (amplitude) of enzy-matic components E1, E2, E3, respectively and ka′ is the exponentialconstant of the ongoing inhibition [4].

For the purpose of obtaining a coherent solution in the inter-active computing estimation, some restrictions were applied: (1)all the parameters (rate constants and amplitudes) should be pos-itive values (>0); (2) component 1 is the most sensitive, thereforek1 > k2 and k2 > k3; (3) the following complementary restrictionwas also applied: E10 + E20 + E30 = 100%. A three-dimensional fit-ting (% PVase activity versus t and I) was performed with the data

described in Fig. 4. The results are provided in Table 1, and thededuced 3D surface is plotted in Fig. 5. The system allows more thanone solution, although this depends on the initial value in the inter-active computing estimation. The results were accepted only if thereactivation constants were of the same order of magnitude as in

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J. Estévez et al. / Chemico-Biolog

he reactivation experiment, as shown later. The consistency of theesults improved if a correction factor for the ongoing inhibitionuring the substrate reaction was included in the most sensitiveomponent. Table 1, line A, shows the resulting parameters.

The I50 (30 min) values for the most sensitive component (E1)ere obtained by approximation and by applying the following

quation:

activity (Ei) =[

kr · 100ki · I + kr

]+

[ki · I · 100ki · I + kr

]· e−(ki·I+kr)·30 (4)

rom the previously estimated kinetic constants and then by car-ying out successive iterations with a different I in an electronicpreadsheet to obtain the I value for the % activity equal to 50 ± 0.1%.he I50 (30 min) values for the less sensitive components (E2 and3) were obtained by applying the following equation:

50 (30 min) = Ln(1/2)−ki · 30

(5)

rom the previously estimated kinetic constants.

. Discussion

This paper provides evidence that nanomolar concentrationsf S9B, a chiral biotinylated organophosphorus compound thatas considered specific inhibitor of the membrane-bound NTE,

re able to inhibit about 85% of total soluble esterases in chickeneripheral nerve in a time-progressive manner, suggesting cova-

ent irreversible phosphorylation. The kinetic behavior is coherentith two highly sensitive esterase components existing and with

he majority interaction of one stereoisomer.After preincubation with 100 nM S9B for 30 min at 37 ◦C and

emoving the inhibitor by ultrafiltration, the residual activityas about 10% and it was similar to the residual activity of

he preinhibited non-ultrafiltrated controls. The activity was notmmediately reactivated, suggesting irreversible covalent phos-horylation (Fig. 2). A slow, time-progressive reactivation wasbserved for a fraction of activity up to around 30% after washingut the inhibitor by ultrafiltration. The spontaneous reactiva-ion phenomenon of the preinhibited carboxyl esterases takinglace with different inhibitors has been described in other works5,6,10,11]. The time-progressive spontaneous reactivation sug-ests that it is not intermediated by a reversible complex buthat it is a dephosphorylation reaction and it is significant duringhe inhibition reaction and should, therefore, be considered in the

athematical kinetic model.We concluded that the esterase fraction that is reactivated is the

omponent E1 detected in the inhibition experiment based on theollowing reasons: (a) the inhibition kinetics suggest two highlyensitive components: E1 and E2 which represent about 20–30%nd 50–70% of total activity, respectively, and a low sensitivity oresistant component (E3). In the reactivation experiment the pro-ortion of reactivated fraction is similar to the E1 component; (b)imilar inhibition and reactivation constants are obtained if reac-ivation is considered for E1 in the inhibition experiment and ifnhibition by residual inhibitor is considered in the reactivationxperiment.

Therefore, we concluded that in the reactivation experiment were observing an almost complete reactivation of component E1fter 25 h while component E2 is not reactivated at all, becausehis stable inhibition might be due to an aging reaction or becausehe phosphorylated enzyme is stable and resistant to spontaneous

eactivation.

The “ongoing inhibition” effect is considered for the high sensi-ive enzymatic component, just as Fig. 3 shows. The proportion ofctivity showing ongoing inhibition adequately corresponds withhe amplitude of the most sensitive component (E1) estimated

teractions 187 (2010) 397–402 401

in the time-progressive inhibition experiment. Moreover, the ka′

constants estimated in the fixed-time inhibition curve at the pre-inhibition zero time and in the time-progressive inhibition aresimilar (Table 1). These constants have to be considered opera-tional empiric constants, and not real kinetic constants. Carringtonand Abou-Donia also used a similar deduced exponential factor toanalyze the effect of inhibition during the substrate reaction time[9].

A 3D fit is the best tool to fit the data of inhibition in this com-plex model [8]. The 3D fit enables the simultaneous inclusion ofall the data in the same fit. The model applied was that in Eq. (3)with the indicated restriction (see Section 2) which assumes threeenzymatic components: E1, displaying both ongoing inhibition andreactivation at sub-nanomolar S9B, E2, showing progressive inhi-bition at few nanomolar S9B and E3 that is practically resistantto S9B inhibition. Spontaneous reactivation and ongoing inhibitioneffect is considered for E1, the highest sensitive component butnot for E2 and E3. The consistency of the estimated parameterswas checked by comparing the results of the inhibition experi-ment with those obtained with the reactivation experiment. Thekinetic model was the same, but the starting conditions differedfor inhibition and reactivation. Therefore, different mathematicalequations were applied. The comparison is presented in Table 1(lines A, B and C). The reactivation constant (kr) and the inhibi-tion constant (ki) related to E1 are similar in the inhibition andreactivation experiments.

These results are also consistent with the same number of inhi-bition components and similar relative amplitudes (proportion)obtained in the inhibition experiments previously published withmipafox [8].

However, the relative sensitivity of the time-progressive inhi-bition differs for mipafox and S9B. The minor sensitive component(E1 in this study) is the most sensitive for S9B, while the majorcomponent (E2 in this study) is the most sensitive for mipafox. Inany case, both components prove to be very sensitive enzymes I50in the order of nanomolar if compared with other esterases (i.e.,neuropathy target esterases bound to brain membranes) with I50for 30 min in the order of micromolar [12–15].

S9B was used for the detection, labeling and isolation of NTEfrom brain membranes as it was considered a specific NTE inhibitorthat distinguishes NTE from the other esterases in brain membranes[1]. However most esterase activity in a soluble fraction of periph-eral nerve has been observed to be inhibited by S9B An S9B-labeledprotein, which was also bound by mipafox, has been isolated andcalled “soluble NTE” (S-NTE2) [16,17], but it has not been molecu-larly and genomically characterized.

It has been concluded that a soluble fraction of peripheral nervecontains two S9B-highly sensitive components of 33% (E1) and 52%(E2) (I50 = 0.24 and 6 nM for 30 min, respectively), and that 15%(E3) of total activity is of low sensitivity or is almost resistant tothe highest tested concentration (50 nM). Due to high sensitivityof E1, ongoing inhibition should be considered for this component.E1 simultaneously undergoes a spontaneous reactivation (dephos-phorylation) while the second component (E2) is permanentlyinhibited. The steady inhibition in E2 component might be due toan aging reaction or because the phosphorylated enzyme in E2 israther stable.

Conflict of interest

None declared.

Acknowledgments

We are most grateful to A.G. García-Pérez, and M.A. Sogorb fromour laboratory for their scientific discussions and advice. To P. Glynn

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nd D. Read from the MRC Toxicology Unit (Carshalton, Surrey,.K., currently in University of Leicester) for the supply of S9B. Thisork has been partly supported by the project A051/2007/3.14.4

f the Spanish Ministry of the Environment and by the institutionalupport from the University Miguel Hernández, Elche.

eferences

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[2] P. Glynn, D.J. Read, M.J. Lush, J. Li Yand Atkins, Molecular cloning of neuropathytarget esterase (NTE), Chem. Biol. Interact. 14 (1999) 513–517.

[3] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates, North-Holland Pub-lishing Company, Amsterdam, 1972.

[4] J. Estévez, E. Vilanova, Model equations for the kinetics of covalent irreversibleenzyme inhibition and spontaneous reactivation: esterases and organophos-phorus compounds, Crit. Rev. Toxicol. 39 (5) (2009) 427–448.

[5] J. Barril, J. Estévez, M.A. Escudero, M.V. Céspedes, N. Níguez, M.A. Sogorb, A.Monroy, E. Vilanova, Peripheral nerve soluble esterases are spontaneouslyreactivated after inhibition by paraoxon: implication for a new definition ofneuropathy target esterase, Chem. Biol. Interact. 119–120 (1999) 541–550.

[6] A.G. García-Pérez, J. Barril, J. Estévez, E. Vilanova, Properties of phenyl valerate

esterase activities from chicken serum are comparable with soluble esterasesof peripheral nerves in relation with organophosphorus compounds inhibition,Toxicol. Lett. 142 (April (1–2)) (2003) 1–10.

[7] M.K. Johnson, Improved assay of neurotoxic esterase for screening organophos-phates for delayed neurotoxicity potential, Arch. Toxicol. 37 (1977)113–115.

[

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[8] J. Estévez, A.G. García-Pérez, J. Barril, M. Pellín, E. Vilanova, The inhibition of thehigh sensitive peripheral nerve soluble esterases by mipafox: a new mathemat-ical processing for the kinetics of inhibition of esterases by organophosphoruscompounds, Toxicol. Lett. 151 (2004) 171–181.

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