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Toxicology Letters 200 (2011) 19–23 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase Franz Worek a,, Nadine Aurbek a , Timo Wille a , Peter Eyer b , Horst Thiermann a a Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany b Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Goethestrasse 33, 80336 Munich, Germany article info Article history: Received 24 September 2010 Received in revised form 13 October 2010 Accepted 14 October 2010 Available online 29 October 2010 Keywords: Organophosphorus compound Paraoxon Acetylcholinesterase Oximes Kinetics Species differences abstract Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhi- bition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to deter- mine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Organophosphorus (OP) compounds are a heterogeneous group of chemicals, which present a pertinent toxicological problem and therapeutic challenge (Kwong, 2002). Highly toxic OP-based nerve agents were used during military conflicts and terrorist attacks (MacIlwain, 1993; Nagao et al., 1997) and the extensive use of OP pesticides results in up to 200,000 fatalities by self-poisoning per year in developing countries (Eddleston et al., 2008). OP act primarily by covalent binding to the amino acid serine at the base of a deep gorge of the enzyme acetylcholinesterase (AChE) resulting in the inhibition of the physiological function of the enzyme, i.e. hydrolysis of the neurotransmitter acetylcholine (Taylor et al., 1995; Aldridge and Reiner, 1972). This causes cholin- ergic overflow and may lead to life threatening impairment of vital body functions and finally to central and peripheral respiratory arrest and death (Holmstedt, 1959). The treatment of OP poisoning is primarily directed to coun- teract cholinergic signs and symptoms by reducing acetylcholine action at muscarinic receptors by the reversible antagonist atropine Corresponding author. Tel.: +49 89 3168 2930; fax: +49 89 3168 2333. E-mail address: [email protected] (F. Worek). and to remove the OP residue from the active site of AChE by nucle- ophilic attack (Kwong, 2002). Hence, AChE reactivating oximes may provide a causal treatment and a number of compounds, e.g. obidoxime, pralidoxime and TMB-4, are used in human OP poison- ing (Eyer and Worek, 2007). Despite convincing in vitro data with isolated AChE demonstrating the ability of oximes to reactivate OP- inhibited AChE the value of oximes in OP poisoning is still a matter of debate (Worek et al., 2010). Since the first use of oximes (pralidoxime) in humans in the 1950s several thousand oximes have been synthesized in order to obtain more effective reactivators against OP poisoning (Worek et al., 2007). The limitations performing controlled clinical tri- als with pesticide poisoned patients and ethical constraints on the investigation of experimental oximes in humans exposed to nerve agents require the use of animal models for testing. Previ- ously, substantial species differences in the reactivating efficiency of oximes were recorded with nerve agent-inhibited AChE, espe- cially between human and guinea pig AChE (Worek et al., 2002; Luo et al., 2007, 2008; Clement and Erhardt, 1994). In view of the established species differences with nerve agent- inhibited AChE it was tempting to investigate the interactions between the pesticide paraoxon, serving as representative of the important diethyl-OP group of pesticides, different oximes and human, Rhesus monkey, swine, rabbit rat and guinea pig AChE in 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.10.009

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Page 1: Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

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Toxicology Letters 200 (2011) 19–23

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

inetic analysis of interactions of paraoxon and oximes with human, Rhesusonkey, swine, rabbit, rat and guinea pig acetylcholinesterase

ranz Woreka,∗, Nadine Aurbeka, Timo Willea, Peter Eyerb, Horst Thiermanna

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, GermanyWalther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Goethestrasse 33, 80336 Munich, Germany

r t i c l e i n f o

rticle history:eceived 24 September 2010eceived in revised form 13 October 2010ccepted 14 October 2010vailable online 29 October 2010

eywords:rganophosphorus compoundaraoxon

a b s t r a c t

Previous in vitro studies showed marked species differences in the reactivating efficiency of oximesbetween human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerveagents. These findings provoked the present in vitro study which was designed to determine the inhi-bition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, servingas a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 withhuman, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results wereobtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pigAChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory

cetylcholinesteraseximesineticspecies differences

potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences ofthe reactivation constants between human and animal AChE were determined. Obidoxime was by far themost effective reactivator with all tested species. Only minor species differences were found for the agingand spontaneous reactivation kinetics. The results of the present study underline the necessity to deter-mine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningfultherapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of

animal data to humans.

. Introduction

Organophosphorus (OP) compounds are a heterogeneous groupf chemicals, which present a pertinent toxicological problem andherapeutic challenge (Kwong, 2002). Highly toxic OP-based nervegents were used during military conflicts and terrorist attacksMacIlwain, 1993; Nagao et al., 1997) and the extensive use of OPesticides results in up to 200,000 fatalities by self-poisoning perear in developing countries (Eddleston et al., 2008).

OP act primarily by covalent binding to the amino acid serinet the base of a deep gorge of the enzyme acetylcholinesteraseAChE) resulting in the inhibition of the physiological function ofhe enzyme, i.e. hydrolysis of the neurotransmitter acetylcholineTaylor et al., 1995; Aldridge and Reiner, 1972). This causes cholin-rgic overflow and may lead to life threatening impairment of vitalody functions and finally to central and peripheral respiratory

rrest and death (Holmstedt, 1959).

The treatment of OP poisoning is primarily directed to coun-eract cholinergic signs and symptoms by reducing acetylcholinection at muscarinic receptors by the reversible antagonist atropine

∗ Corresponding author. Tel.: +49 89 3168 2930; fax: +49 89 3168 2333.E-mail address: [email protected] (F. Worek).

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.10.009

© 2010 Elsevier Ireland Ltd. All rights reserved.

and to remove the OP residue from the active site of AChE by nucle-ophilic attack (Kwong, 2002). Hence, AChE reactivating oximesmay provide a causal treatment and a number of compounds, e.g.obidoxime, pralidoxime and TMB-4, are used in human OP poison-ing (Eyer and Worek, 2007). Despite convincing in vitro data withisolated AChE demonstrating the ability of oximes to reactivate OP-inhibited AChE the value of oximes in OP poisoning is still a matterof debate (Worek et al., 2010).

Since the first use of oximes (pralidoxime) in humans in the1950s several thousand oximes have been synthesized in order toobtain more effective reactivators against OP poisoning (Woreket al., 2007). The limitations performing controlled clinical tri-als with pesticide poisoned patients and ethical constraints onthe investigation of experimental oximes in humans exposed tonerve agents require the use of animal models for testing. Previ-ously, substantial species differences in the reactivating efficiencyof oximes were recorded with nerve agent-inhibited AChE, espe-cially between human and guinea pig AChE (Worek et al., 2002;Luo et al., 2007, 2008; Clement and Erhardt, 1994).

In view of the established species differences with nerve agent-inhibited AChE it was tempting to investigate the interactionsbetween the pesticide paraoxon, serving as representative of theimportant diethyl-OP group of pesticides, different oximes andhuman, Rhesus monkey, swine, rabbit rat and guinea pig AChE in

Page 2: Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

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rder to provide a kinetic basis for the extrapolation of animal datao humans.

. Materials and methods

.1. Materials

Acetylthiocholine iodide (ATCh), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) andralidoxime chloride (2-PAM) were supplied by Sigma (Deisenhofen, Germany) andbidoxime dichloride by Merck (Darmstadt, Germany). HI 6 dichloride monohydrateas kindly provided by Dr. Clement (Defence Research Establishment Suffield, Ral-

ton, Alberta, Canada) and MMB-4 dichloride was made available by Prof. FusekPurkyne Military Medical Academy, Hradec Kralove, Czech Republic). Paraoxon wasrom Dr. Ehrenstorfer GmbH (Augsburg, Germany) and was cleaned of disturbing-nitrophenol as described in detail elsewhere (Kiderlen, 2004).

All other chemicals were from Merck Eurolab GmbH (Darmstadt, Germany) athe purest grade available.

The concentration of paraoxon stock solutions in 2-propanol (10 mM) washecked photometrically (Mast, 1997). Paraoxon solutions were stored at 4 ◦C andere appropriately diluted in distilled water just before use. Oximes (200 mM) wererepared in distilled water, stored at −80 ◦C and diluted as required in distilled watert the day of experiment.

All solutions were kept on ice until the experiment. If not otherwise stated theuffer consisted of 0.1 M sodium phosphate, pH 7.4.

.2. Blood samples

Heparinized human, Rhesus monkey (kindly supplied by Dr. Guy Lallement,RSSA, La Tronche, France) and German landrace pig (obtained from the local slaugh-erhouse) whole blood as well as heparinized blood from New Zealand white rabbits,unkin-Hartley guinea pigs and Wistar rats (purchased from Charles River, Sulzfeld,ermany) was centrifuged at 3000 rpm and 4 ◦C for 10 min, the plasma was collectednd the erythrocytes were washed five times with an approximately three-foldolume of phosphate buffer.

The packed erythrocytes were used for preparation of haemoglobin-free ery-hrocyte ghosts as AChE source (Worek et al., 2002). Aliquots of the erythrocytehosts with an AChE activity adjusted to that found in whole blood of this species,ere stored at −80 ◦C. Prior to use, aliquots were homogenized on ice with a

onoplus HD 2070 ultrasonic homogenator (Bandelin Electronic, Berlin, Germany),hree-times for 5 s with 30 s intervals, to achieve a homogeneous matrix for theinetic studies.

Plasma was inhibited by soman (100 nM) for 30 min at 37 ◦C to ensure completenhibition and aging of butyrylcholinesterase (BChE). The treated plasma was dia-yzed against phosphate buffer overnight at 4 ◦C to adjust the pH and to remove anyesidual inhibitor. The soman-treated plasma was used to stabilize AChE activityuring long-term experiments at 37 ◦C (Worek et al., 1999a).

.3. Enzyme assays

AChE activities were measured spectrophotometrically (Cary 3Bio, Varian,armstadt) with a modified Ellman assay (Worek et al., 1999b; Ellman et al., 1961;yer et al., 2003) using polystyrol cuvettes and 0.45 mM ATCh as substrate and.3 mM DTNB as chromogen in 0.1 M phosphate buffer (pH 7.4).

For the determination of the Michaelis–Menten kinetics of Rhesus monkey, rab-it, guinea pig and rat AChE erythrocyte ghost samples were assayed with differentTCh concentrations ranging from 0.025 to 1.0 mM.

All experiments were performed at 37 ◦C and pH 7.4. All concentrations refer tonal concentrations.

.4. Inhibition kinetics of paraoxon with AChE

The inhibition kinetics was determined in the presence of substrate as describedefore (Aurbek et al., 2006). In brief, 10 �l erythrocyte ghosts and 5 �l dilutedaraoxon (8 different concentrations) were added to a cuvette containing phosphateuffer, DTNB and ATCh (final volume 3.165 ml), the resultant paraoxon concen-rations were 0.1–5.0 �M. ATCh hydrolysis was continuously monitored for up to0 min. The recorded curves were analyzed by non-linear regression analysis andsed for the further determination of the bimolecular reaction constant (Hart and’Brien, 1973) ki = k2/Kd (Eq. (1)).

�t = Kd ∗ 1 + 1(1)

�ln v k2 [IX](1 − ˛) k2

ith Kd: dissociation constant; k2: unimolecular phosphylation rate constant; [IX]:araoxon concentration; ˛: [S]/(Km + [S]) where [S] is substrate concentration andm is the species specific Michaelis constant. All experiments were performed inuplicate.

tters 200 (2011) 19–23

2.5. Rate constants for aging (ka) and spontaneous reactivation (ks) ofparaoxon-inhibited AChE

Paraoxon-inhibited AChE was prepared by incubating ghosts with 100 nMparaoxon for 30 min at 37 ◦C resulting in an inhibition of >95% of control activity. Inorder to remove excess paraoxon after inhibition the samples were dialyzed againstphosphate buffer, at 4 ◦C for 16 h and the absence of inhibitory activity was testedby incubation of paraoxon-treated and control enzyme (15 min, 37 ◦C) followed bythe measurement of residual AChE activity. Paraoxon-treated samples were storedin aliquots at −80 ◦C until use.

Paraoxon-treated erythrocyte ghosts were mixed with equal volumes of soman-treated plasma to prevent denaturation of AChE during long-term experimentsat 37 ◦C (Worek et al., 1999a). Aliquots were taken after various time intervalsfor determination of AChE activity (“spontaneous reactivation”) and the decreaseof oxime-induced reactivation (“aging”). Hereby, paraoxon-treated samples wereincubated with 500 �M obidoxime (30 min). Experiments were performed in dupli-cate and data were related to control activities. The pseudo first-order rate constantsks (spontaneous reactivation) and ka (aging) were calculated by a non-linear regres-sion model (Worek et al., 2004).

2.6. Reactivation kinetics of paraoxon-inhibited AChE

Depending on the kinetic properties of the oximes the reactivation rate con-stants of obidoxime, 2-PAM, HI 6 and MMB-4 were determined by a continuousor discontinuous procedure (Worek et al., 2004). For the continuous procedure10 �l paraoxon-inhibited AChE was added to a cuvette containing phosphate buffer,DTNB, ATCh and specified oxime concentrations (final volume 3.16 ml). ATChhydrolysis was continuously monitored over 10 min. Activities were individuallycorrected for oxime-induced substrate hydrolysis. Here, the final oxime concen-tration during assay was limited to 100 �M obidoxime, 2-PAM and MMB-4 and to50 �M HI 6.

The discontinuous procedure allowed the use of higher oxime concentrations(up to 5 mM). 60 �l paraoxon-inhibited AChE was incubated with 2 �l oxime solu-tion (100–4000 �M final concentration) and 1 �l ATCh (0.45 mM). 10 �l aliquotswere transferred to cuvettes after specified time intervals (1–30 min).

8–10 different oxime concentrations were used for the determination of thereactivation rate constants in duplicate.

The constant KD, which approximates the dissociation constant being inverselyproportional to the affinity of the oxime for the inhibited enzyme, and kr, indicatingthe reactivity of the oxime, were calculated as described before (Worek et al., 2004).The hybrid reactivation rate constant kr2 was calculated from the ratio of kr and KD;the dimension resembles a second-order rate constant, but has a different meaning.

2.7. Data analysis

Processing of experimental data for the determination of the different kineticconstants was performed by non-linear regression analysis using curve fitting pro-grams provided by PrismTM Vers. 4.0 (GraphPad Software, San Diego, CA). All dataare shown as means of n = 2. Coefficient of variation was <10% for aging, spontaneousand oxime-induced reactivation kinetics.

3. Results

3.1. Inhibition kinetics

The bimolecular reaction constants (ki) of paraoxon with humanand animal AChE are summarized in Table 1. The inhibition kinet-ics was determined in the presence of the substrate ATCh. For thecorrection of the effect of substrate on the inhibition by paraoxon(cf. Eq. (1)) the following Km values were used: human 95.4 �M(Mast, 1997), Rhesus monkey 80.9 �M, swine 67.2 �M (Woreket al., 2008), rabbit 326.8 �M, rat 145.6 �M, guinea pig 69.2 �M.

With the exception of Rhesus monkey AChE, paraoxonwas less potent with animal AChE compared to human AChE(Table 1). The inhibitory potency decreased in the orderRhesus > human > rabbit > rat > swine > guinea pig AChE. Paraoxonshowed an almost 10-fold higher inhibitory potency with Rhesus

monkey AChE compared to guinea pig AChE.

A 10- and 26-fold species difference of the rate constant k2 andthe dissociation constant KD, respectively, was recorded (Table 1).Hereby, differences between species were in most cases related toa difference in both constants.

Page 3: Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

F. Worek et al. / Toxicology Letters 200 (2011) 19–23 21

Table 1Constants for the inhibition of AChE by paraoxon.

Species ki (M−1 min−1) k2 (min−1) KD (�M) Ratio ki

Human 3.3 × 106a (3.2–3.4 × 106) 3.1 (2.5–3.9) 0.9 (0.8–1.2)Rhesus monkey 5.0 × 106 (4.6–5.5 × 106) 0.7 (0.6–0.9) 0.1 (0.1–0.2) 1.53Swine 1.1 × 106a (0.9–1.3 × 106) 2.9 (0.8–4.5) 2.6 (0.6–4.4) 0.34Rabbit 2.1 × 106 (1.9–2.5 × 106) 0.3 (0.2–0.5) 0.1 (0.1–0.2) 0.65Guinea pig 0.5 × 106 (0.52–0.56 × 106) 0.6 (0.5–0.7) 1.1 (0.9–1.3) 0.16Rat 1.5 × 106 (1.3–2.3 × 106) 1.1 (0.4–2.2) 0.7 (0.2–1.0) 0.45

Data are given as means of duplicate measurements and with 95% confidence intervals in brackets. The ratio of inhibition constants, ki , is given relative to human AChE.a From Worek et al. (2008).

Table 2Rate constants for the spontaneous dealkylation (aging; ka) and reactivation ofparaoxon-inhibited AChE (ks).

Species ka (h−1) ks (h−1)

Humana 0.022 0.022Rhesus monkey 0.016 (1.32) 0.021 (1.01)Swinea 0.021 (1.01) 0.020 (1.07)Rabbit 0.023 (0.96) 0.020 (1.09)Guinea pig 0.021 (1.02) 0.025 (0.84)Rat 0.021 (1.01) 0.015 (1.49)

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he ratio of aging and spontaneous reactivation rate constants is shown relative touman AChE and is given in brackets.a From Worek et al. (2008).

.2. Aging and spontaneous reactivation of paraoxon-inhibitedChE

Aging and spontaneous reactivation of paraoxon-inhibited AChEollowed first-order kinetics (Table 2). With all enzyme speciesconcomitant aging and spontaneous reactivation was observed.ereby, the species differences were rather small (Table 2). Thealf-time of aging and spontaneous reactivation ranged from 30 to3 h and from 28 to 46 h, respectively.

.3. Reactivation kinetics of paraoxon-inhibited AChE

The determination of reactivation rate constants of obidoxime,-PAM, HI 6 and MMB-4 with paraoxon-inhibited AChE resulted

n part in marked differences between the different species and

ximes (Table 3). The most remarkable difference was recorded forhe ratio of the reactivation rate constant kr2 between obidoximend the other oximes (Fig. 1). Here, the reactivating efficiency of-PAM, HI 6 and MMB-4 was substantially lower, primarily due tolower affinity of the oximes towards paraoxon-inhibited AChE.

able 3eactivation rate constants for oxime-induced reactivation of paraoxon-inhibited AChE.

Species Constant Obidoxime

Humanakr (min−1) 0.81KD (�M) 32.2kr2 (mM−1 min−1) 25.1

Rhesusmonkey

kr (min−1) 0.64KD (�M) 25.9kr2 (mM−1 min−1) 24.6

Swineakr (min−1) 0.55KD (�M) 39.7kr2 (mM−1 min−1) 13.9

Rabbitkr (min−1) 0.16KD (�M) 29.3kr2 (mM−1 min−1) 5.5

Guinea pigkr (min−1) 0.69KD (�M) 27.8kr2 (mM−1 min−1) 25.2

Ratkr (min−1) 0.93KD (�M) 16.4kr2 (mM−1 min−1) 57.9

a Data for obidoxime, 2-PAM and HI 6 from Worek et al. (2008).

Fig. 1. Ratio of reactivation rate constants of oximes. The hybrid reactivation rateconstants of 2-PAM, HI 6 and MMB-4, kr2, relative to obidoxime, are shown forhuman, Rhesus monkey, swine, rabbit, guinea pig and rat AChE.

With regard to differences between human and animal AChE thekr2 values of animal AChE were in most cases equivalent or lowercompared to human AChE (Fig. 2). Paraoxon-inhibited rabbit AChEwas much more resistant to reactivation by all tested oximes whileHI 6 showed a more than 5-fold higher reactivating efficiency withrat AChE compared to human AChE.

4. Discussion

Species differences in the toxicity of OP in vivo are mostlya matter of different velocities in toxification and detoxification(Chambers and Carr, 1995) while differences in the inhibitorypotency of the oxons are probably a result of small but functionally

2-PAM HI 6 MMB-4

0.17 0.20 0.36187 548 264

0.89 0.37 1.370.13 0.14 0.51

279 450 5310.45 0.32 0.960.49 0.08 0.36

263 324 3391.89 0.26 1.10.05 0.09 0.39

243 616 10610.22 0.16 0.370.09 0.08 0.29

273 493 1950.34 0.15 1.470.29 0.38 0.26

206 189 3181.45 2.0 0.83

Page 4: Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

22 F. Worek et al. / Toxicology Le

Fig. 2. Ratio of reactivation rate constants. The hybrid reactivation rate constantsoRr

rft1eiApcdccpmfat

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Jandorf, B.J., Michel, H.O., Schaffer, N.K., Egan, R., Summerson, W.H., 1955. The mech-

f obidoxime, 2-PAM, HI 6 and MMB-4, kr2, relative to human AChE, are shown forhesus monkey, swine, rabbit, guinea pig and rat AChE. The dashed line indicates aatio of 1.

elevant structural difference of the AChE (Moralev et al., 2001). Inact, previous studies showed species-related differences of inhibi-ion rate constants with nerve agents and pesticides (Jandorf et al.,955; Kemp and Wallace, 1990; Wang and Murphy, 1982; Aurbekt al., 2006). Wang and Murphy determined the inhibition kinet-cs of paraoxon with Cynomolgus monkey, rat and guinea pig brainChE and determined ki values in the order rat > monkey � guineaig. In our study, the inhibition kinetics of paraoxon with erythro-yte AChE was determined in the presence of substrate. Hence, theerived bimolecular reaction constant ki (Hart and O’Brien, 1973)annot be directly compared to the apparent second-order rateonstants as determined previously (Mast, 1997). Nevertheless,araoxon resulted in a more than two-fold higher ki with Rhesusonkey and human AChE compared to rat AChE and an almost 10-

old higher ki compared to guinea pig AChE (Table 1). Hence, humannd monkey AChE is more susceptible to inhibition with paraoxonhan swine, rabbit, rat and guinea pig AChE (Table 1).

The ability of oximes to reactivate OP-inhibited AChE is the cru-ial factor for the assessment of the therapeutic efficacy of oximes.n a recent study the reactivation kinetics of obidoxime, 2-PAM, HI

and HLö 7 were determined with human, rabbit, rat and guineaig AChE inhibited by sarin, cyclosarin and VX (Worek et al., 2002).ll oximes exhibited a lower reactivating efficiency with animalChE and there was an outstanding difference between human anduinea pig AChE with HI 6 and HLö 7. A subsequent study confirmedhese findings and found a similar relationship with tabun- and VR-nhibited human and guinea pig AChE (Luo et al., 2007). In part hugeifferences between human and swine AChE were determined forhe oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 with the-agents VX, VR and CVX (Aurbek et al., 2006). Previously, Luo ando-workers determined the reactivation kinetics of 2-PAM, HI 6,Lö 7 and MMB-4 with human, Rhesus, Cynomolgus and Africanreen monkey AChE inhibited by cyclosarin or VR and found inart marked differences between human and monkey enzyme (Luot al., 2008).

In the present study almost comparable hybrid reactivationate constants kr2 of obidoxime, 2-PAM, HI 6 and MMB-4 witharaoxon-inhibited human and Rhesus monkey AChE were deter-ined (Fig. 2). The comparison of human and swine, rabbit, rat

r guinea pig AChE revealed mostly moderate differences. Inter-

stingly, with paraoxon-inhibited AChE the difference betweenuman and guinea pig AChE regarding the kr2 with HI 6 was onlywo-fold and much lower than with nerve agent-inhibited AChEWorek et al., 2002).

tters 200 (2011) 19–23

Post-inhibitory reactions of OP-inhibited AChE, i.e. aging andspontaneous reactivation, are important determinants of oximeefficacy in vivo (Eyer, 2003). Aging results in the formation of areactivation-resistant OP-AChE complex and is a major problemin soman poisoning (Fleisher and Harris, 1965) and also limits theefficacy of oximes in humans exposed to dimethyl-OP pesticides(Thiermann et al., 2009; Eyer, 2003). in vitro studies revealed sub-stantial differences in the aging kinetics between soman-inhibitedhuman, monkey, rat and guinea pig AChE (Shafferman et al.,1996; Talbot et al., 1988) and a several-fold difference betweenV-agent-inhibited human and swine AChE (Aurbek et al., 2006).In contrast, comparable aging and spontaneous reactivation kinet-ics of paraoxon-inhibited human, monkey, swine, rabbit, rat andguinea pig AChE were found (Table 2).

The results of the present study on the interaction of paraoxonand oximes with AChE from different species in conjunction withprevious findings on nerve agents underline the necessity to deter-mine the inhibition, aging and reactivation kinetics in vitro forindividual OPs, oximes and AChE from relevant species as a basis forthe development of meaningful therapeutic animal models, for theproper assessment of in vivo animal data and for the extrapolationof animal data to humans.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The study was funded by the German Ministry of Defence. Theauthors are grateful to T. Hannig and L. Windisch for expert tech-nical assistance.

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