Toxicology 224 (2006) 91–99

Analysis of inhibition, reactivation and aging kinetics of highlytoxic organophosphorus compounds with

human and pig acetylcholinesterase

N. Aurbek a, H. Thiermann a, L. Szinicz a, P. Eyer b, F. Worek 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

Received 13 March 2006; received in revised form 11 April 2006; accepted 14 April 2006Available online 27 April 2006

Abstract

Organophosphorus compounds (OP) are in wide spread use as pesticides and highly toxic OP may be used as chemical warfareagents (nerve agents). OP inhibit acetylcholinesterase (AChE), therefore, standard treatment includes AChE reactivators (oximes) incombination with antimuscarinic agents. In the last decades, the efficacy of oximes has been investigated in various animal models,mostly in rodents. However, extrapolating animal data to humans is problematical because of marked differences between rodentsand humans concerning the toxicokinetics of nerve agents, the pharmacokinetics of antidotes and the AChE enzyme kinetics. In

order to improve the understanding of species differences and to enable a more reliable extrapolation of animal data to humans astudy was initiated to investigate the effect of highly toxic nerve agents, i.e. VX, Russian VX (VR) and Chinese VX (CVX), withhuman and pig erythrocyte AChE. Hereby, the rate constants for the inhibition of AChE by these OP (ki) and for the spontaneousdealkylation (ka) and reactivation (ks) of OP-inhibited AChE as well as for the oxime-induced reactivation of OP-inhibited AChEby the oximes obidoxime, 2-PAM, HI 6, HLo 7 and MMB-4 were determined. Compared to human AChE pig AChE showed alower sensitivity towards the investigated OP. Furthermore, a slower spontaneous dealkylation and reactivation of pig AChE wasrecorded. The potency of the investigated oximes was remarkably lower with OP-inhibited pig AChE. These data may contributeto a better understanding of species differences and may provide a kinetic basis for extrapolation of data from pig experiments tohumans.© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Acetylcholinesterase; Pig; Human; Organophosphorus compounds; Oximes; Inhibition; Reactivation; Aging

Abbreviations: AChE, acetylcholinesterase (E.C. 3.1.1.7); BChE, butyrylcholinesterase (E.C. 3.1.1.8); ATCh, acetylthiocholine iodide;BTCh, S-butyrylthiocholine iodide; DTNB, 5,5′-dithio-bis(-2-nitrobenzoic acid), soman, pinacolylmethylphosphonofluoridate; VX, O-ethyl-S-[2-(diisopropylamino)ethyl]methylphosphonothioate; CVX, O-n-butyl-S-[2-(diethylamino)ethyl]methylphosphonothioate; VR, O-isobutyl-S-[2-(di-ethylamino)ethyl]methylphosphonothioate; obidoxime, 1,1′-(oxy-bis-methylene)bis[4-(hydroxyimino)methyl]pyridinium dichloride; pralidoxime,2-[hydroxyimino methyl]-1-methylpyridinium chloride; HI 6, 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate; HLo 7, 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl]pyridinium di-methanesulfonate; MMB-4, 1,1′-methylene-bis[4-(hydroxyimino)methyl]pyridinium dibromide

∗ Corresponding author. Tel.: +49 89 3168 2930; fax: +49 89 3168 2333.E-mail address: FranzWorek@Bundeswehr.org (F. Worek).

0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2006.04.030

92 N. Aurbek et al. / Toxicology 224 (2006) 91–99

1. Introduction

In spite of great efforts to abandon chemical war-fare agents large stocks of highly toxic organophospho-rus compounds (OP; nerve agents) are still available.In view of the use of nerve agents against civilians inIraq (MacIlwain, 1993) and Japan (Nagao et al., 1997)and the currently constant increasing threat of terroristattacks with chemical warfare agents the developmentof an effective medical treatment regimen is indispens-able. The intoxication with OP causes a generalizedcholinergic crisis due to an irreversible inhibition ofcholinesterases by phosphylation (denotes phosphory-lation and phosphonylation) of their active site serine(Marrs, 1993; Taylor et al., 1995; MacPhee-Quigleyet al., 1985) and successive accumulation of the neu-rotransmitter acetylcholine. This results in overstim-ulation of cholinergic receptors and finally paralysisof neuromuscular function. Therefore, standard treat-ment of OP-poisoning includes a muscarinic antago-nist, e.g. atropine, and a reactivator of OP-inhibitedAChE (oxime) (Volans, 1996). Obidoxime and prali-doxime (2-PAM), the two presently commercially avail-able oximes, are considered to be rather ineffectiveagainst certain nerve agents, e.g. soman and cyclosarin(Sidell, 1992; Worek et al., 1998a,b). In the last decadesseveral compounds were synthesized and tested for theirefficacy as reactivating agents. Two newer oximes, HI6 and HLo 7, are, according to the available data,considered to be promising antidotes against soman-,

and a more reliable extrapolation of animal data tohumans a study was initiated to investigate the effectof the highly toxic nerve agents VX, Russian VX (VR;Maxwell et al., 1997) and Chinese VX (CVX; Weber,2000; Kireev et al., 2002) with human and pig ery-throcyte AChE. The rate constants for inhibition ofAChE by these OP, for spontaneous dealkylation (aging)and reactivation of OP-inhibited AChE as well as forthe oxime-induced reactivation of OP-inhibited AChEby the oximes obidoxime, 2-PAM, HI 6, HLo 7 andMMB-4 were determined. These data may serve as akinetic database for the extrapolation of data from pigexperiments to humans. Erythrocyte AChE was used asenzyme source assuming that it is an adequate surrogateparameter for synaptic AChE (Ballantyne and Marrs,1992).

2. Materials and methods

2.1. Materials

Acetylthiocholine iodide (ATCh), S-butyrylthiocholineiodide (BTCh), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB)and pralidoxime chloride (2-PAM) were obtained fromSigma. Obidoxime dichloride (obidoxime) was purchasedfrom Duphar. HI 6 was kindly provided by Dr. Clement(Defence Research Establishment Suffield, Ralston, Alberta,Canada). MMB-4 was provided by Prof. Fusek (Purkyne Mil-itary Medical Academy, Hradec Kralove, Czech Republic).HLo 7 was a custom synthesis by J. Braxmeier. VX, CVX, VR(Fig. 1) and soman (>98% by GC–MS, 1H NMR and 31P NMR)

sarin- and cyclosarin-poisoning (Worek et al., 1998a;Bismuth et al., 1992; Eyer et al., 1992), whereas dataregarding the reactivating potency of another oxime,MMB-4, are ambiguous (Shih, 1993; Harris et al.,1990).

The efficacy of antidotal agents cannot be investi-gated in humans for ethical reasons. Hence, the ther-apeutic effectiveness of oximes has been evaluated inanimal experiments mainly with rodent species in thelast decades. However, due to substantial species dif-ferences in the affinity and reactivity of oximes withOP-inhibited AChE (Berry, 1971; Clement and Erhardt,1994; Worek et al., 2002) the extrapolation of animaldata to humans may pose a problem. Consequently,the exact determination and evaluation of species dif-ferences is necessary for the assessment of oxime effi-cacy in humans. Previous data showed marked differ-ences between rodent species and humans concerningthe reactivation kinetics of sarin-, cyclosarin- and VX-inhibited AChE (Worek et al., 2002). Due to the assump-tion that experiments with bigger mammalian speciesallow an extensive monitoring of different parameters

were made available by the German Ministry of Defence. Allother chemicals were from Merck Eurolab GmbH (Darmstadt,Germany).

Stock solutions of soman, VX, CVX and VR (0.1% v/v)were prepared weekly in 2-propanol, stored at 4 ◦C and wereappropriately diluted in distilled water just before the exper-iment. Oximes (50 and 200 mM) were prepared in distilledwater, stored at −60 ◦C and diluted as required in distilledwater at the day of the experiment. All solutions were kept onice until the experiment.

Human blood samples were provided by volunteers fromour lab and pig blood samples were gathered at the localslaughter house. Hemoglobin-free erythrocyte ghosts wereprepared according to (Dodge et al., 1963) with minor mod-ifications (Worek et al., 2002). In brief, heparinized humanand pig blood was centrifuged (3000 × g, 10 min) and theplasma removed. The erythrocytes were washed three timeswith two volumes of phosphate buffer (0.1 M, pH 7.4). Then,the packed erythrocytes were diluted in 20 volumes of hypo-tonic phosphate buffer (6.7 mM, pH 7.4) to facilitate hemolysisfollowed by centrifugation at 50.000 × g (30 min, 4 ◦C). Thesupernatant was removed and the pellet re-suspended in hypo-tonic phosphate buffer. After two additional washing cyclesthe pellet was re-suspended in phosphate buffer (0.1 M, pH

N. Aurbek et al. / Toxicology 224 (2006) 91–99 93

Fig. 1. Structures of organophosphates used in this study.

7.4) and the virtually hemoglobin-free erythrocyte ghosts wereconcentrated by centrifugation at 100.000 × g (30 min, 4 ◦C).Finally, the AChE activity was adjusted to the original activity(i.e. 4–5 U/ml) by appropriate dilution with phosphate buffer(0.1 M, pH 7.4). Aliquots of the erythrocyte ghosts were storedat −60 ◦C until use. Prior to use, aliquots were homogenizedon ice with a Sonoplus HD 2070 ultrasonic homogenator (Ban-delin electronic, Berlin, Germany), three-times for 5 s with30 s intervals, to achieve a homogeneous matrix for the kineticstudies.

In order to prevent AChE denaturation during long-termexperiments at 37 ◦C AChE was stabilized by addition ofhuman or pig plasma with totally blocked BChE (Woreket al., 1999a). Plasma was obtained as described aboveand inhibited by soman (100 nM) for 30 min at 37 ◦C toensure complete inhibition and aging of BChE. The inhib-ited plasma was dialyzed (phosphate buffer, 0.1 M, pH 7.4)overnight at 4 ◦C to adjust pH and to remove residualinhibitor.

2.2. Enzyme assays

AChE and BChE activities were measured spectrophoto-metrically (Cary 3Bio, Varian, Darmstadt) with a modifiedEllman assay (Worek et al., 1999b; Eyer et al., 2003). Theassay mixture (3.16 ml) contained 0.45 mM ATCh (AChE) or1.0 mM BTCh (BChE) as substrate and 0.3 mM DTNB as chro-mogen in 0.1 M phosphate buffer (pH 7.4). Assays were run at37 ◦C.

2.3. Determination of inhibition rate constants (ki)

t(tolo(

where Kd is the dissociation constant, k2 the unimolecu-lar phosphylation rate constant, [IX] the OP-concentration,α = [S]/(Km + [S]) where [S] is substrate concentration and Km

is the Michaelis constant. Under the conditions employed α

was 0.833.

tion) as indicated, ATCh hydrolysis was continuously monitored (A).The slopes of the tangents (k1) were calculated. 1/k1 was plotted against1/[IX](1 − α) (B). [IX] is the inhibitor concentration, where α standsfor [S]/(Km + [S]) where [S] is substrate concentration and Km is theMichaelis constant. The second-order inhibition constant ki was cal-culated according to Forsberg and Puu (1984).

10 �l human or pig AChE and 5 �l diluted OP were addedo a cuvette containing phosphate buffer, DTNB and ATChfinal volume 3.165 ml), resultant OP concentrations consti-uted 2–75 nM. ATCh hydrolysis was continuously monitoredver up to 30 min. The recorded curves were analyzed by non-inear regression analysis and used for the further determinationf ki = k2/KD based on Eq. (1) according to Forsberg and Puu1984) (Fig. 2):

�t

�ln v= Kd

k2× 1

[IX](1 − α)+ 1

k2(1)

Fig. 2. Inhibition kinetics of human AChE inhibited by VX. Enzymewas incubated with buffer, substrate and VX (in nM final concentra-

94 N. Aurbek et al. / Toxicology 224 (2006) 91–99

The analysis of the data was performed with PrismTM Ver-sion 4.00 (GraphPad Software, San Diego, CA, USA).

2.4. Determination of rate constants for aging (ka) andspontaneous reactivation (ks)

OP-inhibited AChE was prepared by incubating ghosts withappropriate OP concentrations for 15 min at 37 ◦C resulting inan inhibition of 95–98% of control activity. In order to removeexcess OP after inhibition by VX, CVX and VR the sampleswere dialyzed and the absence of inhibitory activity was testedby incubation of treated and control enzyme (15 min, 37 ◦C).OP-treated samples were stored in aliquots at −60 ◦C until use.

OP-treated human and pig ghosts were mixed with equalvolumes of soman-treated human or pig plasma to preventdenaturation of AChE during long-term experiments at 37 ◦C.Aliquots were taken after various time intervals for determina-tion of AChE activity (spontaneous reactivation) and of thedecrease of oxime-induced reactivation (aging). Data werereferred to control activities and the percentage reactivation(% react) was calculated (de Jong and Wolring, 1978). Thepseudo-first-order rate constants ks (spontaneous reactivation)and ka (aging) were calculated by a non-linear regression model(Worek et al., 1999a; Skrinjaric-Spoljar et al., 1973; Worek etal., 2004) (Fig. 3).

2.5. Oxime reactivation of OP-inhibited AChE

The ability of oximes to reactivate OP-inhibited AChEwas tested in pilot experiments by adding oxime (10, 100,1000 �M) to enzyme samples and measuring the residual activ-ity at specified time intervals (1–30 min). In case of expectedhigh reactivating potency the reactivation kinetics were deter-

Fig. 3. Spontaneous reactivation (�) and aging (�) of human (A) andpig (B) CVX-inhibited AChE. The curves were fitted to the data pointsby assuming two pseudo-first-order reactions.

AChE, [EPOX] the Michaelis-type phosphyl–AChE–oximecomplex, [OX] the reactivator, [E] the reactivated enzymeand [POX] the phosphylated oxime. KD is equal to the ratio[EP] × [OX]/[EPOX] and approximates the dissociation con-stant which is inversely proportional to the affinity of the oximeto [EP], and kr the rate constant for the displacement of thephosphyl residue from [EPOX] by the oxime, indicating thereactivity. KD and kr were calculated according to Worek et al.(2004).

3. Results

3.1. Inhibition kinetics of OP with human and pigerythrocyte AChE

The rate constants for the inhibition of human and pigAChE (ki) by VX (Fig. 2), Russian VX (VR) and ChineseVX (CVX) are summarized in Table 1. The three OPtested had a comparable inhibitory potency with humanAChE, VR being the most active inhibitor. Compared tohuman AChE pig AChE was found to be somewhat lesssensitive towards all three tested OP.

mined with the continuous procedure presented by Kitz and co-workers (Kitz et al., 1965; Worek et al., 2004) (Fig. 4A and B).Hereby, 10 �l OP-inhibited AChE was added to a cuvette con-taining phosphate buffer, DTNB, ATCh and specified oximeconcentrations (final volume 3.16 ml). ATCh hydrolysis wascontinuously monitored over 10 min. Activities were individu-ally corrected for oxime-induced hydrolysis of ATCh. In orderto reduce the effect of oxime-induced AChE inhibition the max-imum concentration was 40 �M (HI 6, HLo 7) and 100 �M(obidoxime, pralidoxime, MMB 4), respectively.

In case of low reactivating potency a discontinuous pro-cedure was applied (Fig. 4C and D) (Wang and Braid, 1967;Worek et al., 1999a) which allowed use of higher oxime con-centrations (up to 5 mM). 60 �l OP-inhibited AChE was incu-bated with 2 �l oxime solution (different concentrations) and1 �l ATCh (450 �M final concentration) and 10 �l aliquotswere transferred to cuvettes after specified time intervals(1–9 min).

Eight to 10 different oxime concentrations were used forthe determination of the reactivation rate constants.

2.6. Kinetics of oxime reactivation

Oxime reactivation of OP-inhibited AChE proceeds accord-ing to Scheme 1. In this scheme [EP] is the phosphylated

N. Aurbek et al. / Toxicology 224 (2006) 91–99 95

Fig. 4. Reactivation kinetics of CVX-inhibited human (A and B) and pig AChE (C, D) by HI 6. (A) Continuous recording of absorption changein the Ellman assay after addition of HI 6 (1–40 �M, as indicated). (C) Single data points indicate calculated AChE activities after the designatedtime of reactivation with 50 �M HI 6 (�), 75 �M HI 6 (�), 100 �M HI 6 (�), 150 �M HI 6 (©), 200 �M HI 6 (�) and 300 �M HI 6 (�). kobs wascalculated according to Worek et al. (2004). Secondary plot of kobs vs. [HI 6] for human (B) and pig AChE (D), note the different dimension of theabscissa.

Scheme 1.

3.2. Aging and spontaneous reactivation ofOP-inhibited AChE

Aging and spontaneous reactivation kinetics of VX-,VR- and CVX-inhibited human and pig AChE followedfirst-order kinetics. The investigation demonstratedmarked differences concerning aging and spontaneousreactivation half-times depending on the species andthe inhibitor used (Table 2). Aging occurred without

exception whereas aging half time ranged from 32to 139 h (human) and 85–265 h (pig), respectively.VX-, VR- and CVX-inhibited human and pig AChEwere subject to spontaneous reactivation. However,spontaneous reactivation proceeded substantially fasterwith human AChE when compared to pig enzyme. Mostobvious was the difference with CVX-inhibited AChE.Here, the half-time was approximately 4 h with humanand approximately 112 h with pig AChE (Fig. 3).

3.3. Reactivation kinetics of OP-inhibited humanand pig AChE by oximes

The reactivation rate constants of obidoxime, 2-PAM,HI 6 (Fig. 4), HLo 7 and MMB-4 with VX-, VR- andCVX-inhibited human and pig erythrocyte AChE are

Table 1Rate constant for the inhibition of human and pig AChE by VX, VR and CVX (ki)

Inhibitor Human Pig Ratio, ki (human vs. pig)

k2 (min−1) KD (M) ki (M−1 min−1) k2 (min−1) KD (M) ki (M−1 min−1)

VX 8.08 8.15 × 10−8 9.91 × 107 2.94 6.64 × 10−8 4.43 × 107 2.24VR 5.42 1.18 × 10−8 4.6 × 108 2.60 1.38 × 10−8 1.88 × 108 2.45CVX 5.99 1.96 × 10−8 3.06 × 108 3.04 2.09 × 10−8 1.46 × 108 2.10

96 N. Aurbek et al. / Toxicology 224 (2006) 91–99

Table 2Rate constants for the spontaneous dealkylation (ka) and reactivationof OP-inhibited AChE (ks)

Inhibitor Human, ka (h−1) Pig, ka (h−1) Ratio (human vs. pig)

VX 0.019a 0.0081 2.35VR 0.005a 0.0026 1.91CVX 0.022 0.0050 4.28

Inhibitor Human, ks (h−1) Pig, ks (h−1) Ratio (human vs. pig)

VX 0.021a 0.0086 2.43VR 0.039a 0.0073 5.37CVX 0.171 0.0062 27.50

a From Worek et al. (2004).

shown in Table 3. These data indicate substantial dif-ferences in the reactivating potency depending on theinhibitor, oxime and species. With human and pig AChEHLo 7 had the highest affinity (1/KD) throughout, inde-pendent of the inhibitor. In contrast, the rank order ofreactivity (kr) of the oximes was different depending onthe inhibitor. Obidoxime had the highest reactivity withVX-inhibited, MMB-4 with VR- and CVX-inhibitedhuman and pig-AChE. The tested oximes showed amarkedly lower affinity towards OP-inhibited pig AChEwhen compared to human AChE while there where onlymoderate differences concerning the reactivity betweenhuman and pig AChE (Fig. 5A and B).

Comparison of the second-order rate constants (kr2)(Table 3) demonstrates the superiority of HLo 7 as the

most potent reactivator of the VX-, VR- and CVX-inhibited human and pig AChE (with the exception ofVX-inhibited pig AChE). With VR- and CVX-inhibitedhuman and pig AChE the rank order was HLo 7 > HI6 > MMB4 ≥ obidoxime > 2-PAM. Relative to humanAChE the second-order rate constant was lower withpig AChE for all OP and oximes tested (Fig. 5C). Thespecies differences of the specific reactivity were pro-nounced with HLo 7 and HI 6 due to their low affinitywith pig AChE.

4. Discussion

The determination of inhibition, reactivation andaging kinetics of human and pig AChE with VX, VR andCVX showed remarkable species differences. In order todevelop an animal model which allows a reliable extrap-olation of animal data to humans and due to substantialspecies differences between humans and rodent speciesin the ability of oximes to reactivate OP-inhibited AChE(Worek et al., 2002; Clement and Erhardt, 1994; Berry,1971) the pig was chosen for this study to provide akinetic basis for further experiments. The nerve agentsVX, Russian VX (VR) and Chinese VX (CVX) wereselected for this study due to their high percutaneoustoxicity. VX- and VR-inhibited AChE were shown to besusceptible towards reactivation by oximes (Worek et al.,2004) while no data are presently available for CVX inopen literature.

Table 3Reactivation constants of OP-inhibited human and pig erythrocyte acetylchol

Inhibitor Reactivator Human

kr (min−1) KD (�M) kr2 (mM−

VX Obidoxime 0.89a 27.35a 32.72-PAM 0.22a 28.07a 7.7HI 6 0.24a 11.53a 21.0HLo 7 0.49a 7.76a 63.2MMB-4 0.33 241.9 1.4

VR Obidoxime 0.63a 105.9a 6.02-PAM 0.06a 30.65a 1.9HI 6 0.71a 9.15a 77.8

8.75.9

5.50.559.34.96.0

HLo 7 0.84a 5.28a 15MMB-4 0.85 144.3

CVX Obidoxime 0.26 46.922-PAM 0.08 139.1HI 6 0.48 16.41 2HLo 7 0.26 3.94 6MMB-4 1.02 169.7

a From Worek et al. (2004).

inesterase (AChE)

Pig

1 min−1) kr (min−1) KD (�M) kr2 (mM−1 min−1)

0.45 31.2 14.40.23 239 0.960.25 379 0.660.29 90.8 3.20.36 402 0.90

0.14 374 0.380.16 994 0.230.43 226 1.90.50 147 3.430.62 341 1.83

0.15 295 0.510.08 366 0.230.36 156 2.30.43 95.9 4.50.57 287 2.0

N. Aurbek et al. / Toxicology 224 (2006) 91–99 97

Fig. 5. Reactivation kinetics of VX (right hatched columns), VR (blackcolumns) and CVX (left hatched columns) inhibited pig AChE. Thereactivation constants kr (A), KD (B) und kr2 are given relative to humanAChE.

4.1. Inhibition of human and pig AChE by VX, VRand CVX

The examination of inhibition kinetics of V-agentswith human and pig erythrocyte AChE showed a highinhibitory potency, being up to 10 times higher whencompared to sarin (Worek et al., 2004). Human AChEwas somewhat more sensitive when compared to pig

AChE (Table 1). Despite of the substantial structural dif-ferences between the investigated OP (cf. Fig. 1) the inhi-bition rate constants were comparable. The rank orderof the inhibitory potency was VR > CVX > VX in bothspecies.

4.2. Aging and spontaneous reactivation of VX-,VR-, and CVX-inhibited human and pig AChE

Phosphylated AChE may be subject to spontaneousdealkylation (aging) and dephosphylation (reactivation)(Aldridge and Reiner, 1972). Previous data indicate thataging kinetics is dependent on the structure of the phos-phylated AChE, being most pronounced with branchedalkyl groups (Berry and Davies, 1966; Shafferman et al.,1996). However, this assumption cannot be confirmedwith the present data since the aging half-time of VR-inhibited AChE, bearing an O-i-butyl group, was muchlonger when compared to VX (O-ethyl) and CVX (O-n-butyl) in both species (Table 2). Marked differencesin the aging half-times between human and pig AChEwere recorded, aging being substantially slower with pigenzyme. Nevertheless, with aging half-times being in therange of 31–138 h for human AChE and of 85–266 h forpig AChE it cannot be expected that aging would havea substantial impact on the effectiveness of immediatepost-exposure oxime treatment.

Comparable to aging the spontaneous reactivationof VX-, VR- and CVX-inhibited human AChE wasseveral-fold faster when compared to pig AChE. With

the exception of VX-inhibited AChE spontaneous reacti-vation proceeded markedly faster than aging with humanAChE. Due to this fact and in the absence of free inhibitormore than 80% of VR- and CVX-inhibited human AChE(Fig. 3) would be regenerated.

4.3. Oxime-induced reactivation of VX-, VR-, andCVX-inhibited human and pig AChE

In general, human AChE inhibited by VX, VRand CVX could be easily reactivated by oximes (cf.Table 3). Compared to tabun-inhibited human AChE,which showed to be rather resistant towards oxime-induced reactivation (Worek et al., 2004), the second-order reactivation rate constants were up to 1000-foldhigher (HLo 7). However, marked differences concern-ing affinity and reactivity were recorded dependingon the inhibitor and the oxime. The newer oximesHLo 7 and HI 6 were superior to the 4-pyridiniumoximes obidoxime and MMB-4 while pralidoxime wasa weak reactivator of inhibited AChE. The wide differ-ences between the oximes were mainly due to different

98 N. Aurbek et al. / Toxicology 224 (2006) 91–99

affinities of the oximes to phosphonylated AChE. Inter-estingly, MMB-4 showed a high reactivity with VR- andCVX-inhibited AChE. However, this advantage was lostby the poor affinity resulting in rather low second-orderrate constants. Thus, MMB-4 had comparable kr2 val-ues to obidoxime with VR- and CVX-inhibited AChEbut reactivating potency was substantially lower com-pared to HLo 7 and HI 6 with all tested inhibitors. Thereactivating potency of the tested oximes with VX-, VR-and CVX-inhibited pig AChE was markedly lower com-pared to human AChE. This was mainly due to a pooraffinity of the oximes. The difference in reactivatingpotency between human and pig AChE was especiallydistinct with HLo 7 and HI 6, while less differenceswere recorded for obidoxime, 2-PAM and MMB-4. Sim-ilar findings were obtained previously with human androdent AChE inhibited by sarin, cyclosarin, VX andsoman (Worek et al., 2002).

5. Conclusions

The presented data on interactions between humanand pig AChE, OP and oximes provide a kinetic basisfor the evaluation of oxime efficacy and effective oximeconcentrations in humans and pigs. Although markeddifferences in inhibition, aging and reactivation kinet-ics between human and pig AChE were observed, theavailable data can be useful for defining equieffectiveoxime doses at different scenarios of nerve agent poi-

Clement, J.G., Erhardt, N., 1994. In vitro oxime-induced reactivation ofvarious molecular forms of soman-inhibited acetylcholinesterasein striated muscle from rat, monkey and human. Arch. Toxicol. 68,648–655.

de Jong, L.P.A., Wolring, G.Z., 1978. Effect of 1-(ar)-alkyl-hydroxyiminomethyl-pyridinium salts on reactivation and agingof acetylcholinesterase inhibited ethyldimethylphosphoramido-cyanidate (tabun). Biochem. Pharmacol. 27, 2229–2235.

Dodge, J.T., Mitchell, C., Hanahan, D.J., 1963. The preparation andchemical characteristics of hemoglobin-free ghosts of human ery-throcytes. Arch. Biochem. Biophys. 100, 119–130.

Eyer, P., Hagedorn, I., Klimmek, R., Lippstreu, P., Loffler, M., Oldiges,H., Spohrer, U., Steidl, I., Szinicz, L., Worek, F., 1992. HLo 7dimethanesulfonate, a potent bispyridinium-dioxime against anti-cholinesterases. Arch. Toxicol. 66, 603–621.

Eyer, P., Worek, F., Kiderlen, D., Sinko, G., Stuglin, A., Simeon-Rudolf, V., Reiner, E., 2003. Molar absorption coefficients forthe reduced Ellman reagent: reassessment. Anal. Biochem. 312,224–227.

Forsberg, A., Puu, G., 1984. Kinetics for the inhibition of acetyl-cholinesterase from the electric eel by some organophosphates andcarbamates. Eur. J. Biochem. 140, 153–156.

Harris, L.W., Anderson, D.R., Lennox, W.J., Woodard, C.L., Paste-lak, A.M., Vanderpool, B.A., 1990. Evaluation of severaloximes as reactivators of unaged soman- inhibited whole bloodacetylcholinesterase in rabbits. Biochem. Pharmacol. 40, 2677–2682.

Kireev, A.F., Rybal’chenko, I.Y., Savchuk, V.I., Suvorkin, V.N.,Tipukhov, I.A., Khamidi, B.A., 2002. Qualitative chromato-graphic–mass spectrometric analysis of highly toxic alkyl phos-phonate and alkyl thiophosphonate derivatives. J. Anal. Chem. 57,708–716.

Kitz, R.J., Ginsburg, S., Wilson, I.B., 1965. Activity–structure relation-ships in the reactivation of diethylphosphoryl acetylcholinesteraseby phenyl-1-methyl pyridinium ketoximes. Biochem. Pharmacol.

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