Chemico-Biological Interactions xxx (2013) xxx–xxx

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Chemico-Biological Interactions

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Investigation of kinetic interactions between approved oximesand human acetylcholinesterase inhibited by pesticide carbamates

0009-2797/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.cbi.2013.08.004

⇑ Corresponding author. Address: Bundeswehr Institute of Pharmacology andToxicology, Neuherbergstrasse 11, 80937 Munich, Germany. Tel.: +49 89 31682305; fax: +49 89 3168 2333.

E-mail address: timowille@bundeswehr.org (T. Wille).

Please cite this article in press as: T. Wille et al., Investigation of kinetic interactions between approved oximes and human acetylcholinesterase inby pesticide carbamates, Chemico-Biological Interactions (2013), http://dx.doi.org/10.1016/j.cbi.2013.08.004

Timo Wille ⇑, Lisa Kaltenbach, Horst Thiermann, Franz WorekBundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:Human acetylcholinesteraseCarbamateOximesCarbamoylationDecarbamoylationKinetics

Carbamates are widely used for pest control and act primarily by inhibition of insect and mammalian ace-tylcholinesterase (AChE). Accidental or intentional uptake of carbamates may result in typical signs andsymptoms of cholinergic overstimulation which cannot be discriminated from those of organophospho-rus pesticide poisoning. There is an ongoing debate whether standard treatment with atropine and oxi-mes should be recommended for human carbamate poisoning as well, since in vitro and in vivo animaldata indicate a deleterious effect of oximes when used in combination with the N-methyl carbamate car-baryl. Therefore, we performed an in vitro kinetic study to investigate the effect of clinically used oximeson carbamoylation and decarbamoylation of human AChE. It became evident that pralidoxime and obi-doxime in therapeutic concentrations aggravate the inhibition of AChE by carbaryl and propoxur, withobidoxime being substantially more potent compared to 2-PAM. However, obidoxime had no impacton the decarbamoylation kinetics. Hence, the administration of 2-PAM and especially of obidoxime toseverely propoxur and carbaryl poisoned humans cannot be recommended.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Carbamates (Table 1) are widely used for pest control in differ-ent countries and act primarily by covalent binding to the activesite serine of insect and mammalian acetylcholinesterase (AChE)[1]. Carbamates are moderate, reversible AChE inhibitors [2,3]but accidental or intentional uptake of high doses results in typicalsigns and symptoms of cholinergic overstimulation. In the absenceof additional information a discrimination between carbamate andorganophosphate poisoning is a major challenge [4]. In view of thisdifficulty, it is mandatory to know whether the standard treatmentof poisoning by organophosphorus pesticides and nerve agents, i.e.atropine and an oxime [5,6], is at least without harm in carbamatepoisoning. In fact, there is an ongoing debate whether oximesshould be administered to carbamate poisoned patients and anumber of authors do not recommend oximes in carbamate poi-soning [6–8]. This assumption is mainly based on in vitro andin vivo data from animal experiments with the carbamate carbaryl.There is also one case report of a fatal human carbaryl poisoningtreated with pralidoxime (2-PAM) [9]. However, the patient diedobviously from lung edema and it remains unclear whether the

oxime treatment or an insufficient atropinization was causativefor the fatal outcome [4].

In vivo studies in rats and mice showed that treatment with 2-PAM and obidoxime increased the carbaryl toxicity. When given incombination with atropine, oximes reduced the therapeutic effi-cacy of this antimuscarinic compound [10–12]. Dawson investi-gated the carbamoylation of AChE by carbaryl with bovine andhuman erythrocyte AChE and reported an aggravation in the pres-ence of 2-PAM and obidoxime [13,14].

Thus, a promising approach was to investigate kinetic interac-tions of human AChE, currently used carbamates for pest controlwith obidoxime or 2-PAM in order to provide an in vitro base forthe evaluation of the effect of clinically used oximes in human car-bamate poisoning.

2. Materials and methods

2.1. Materials

Aldicarb, carbaryl, carbofuran, methomyl and propoxur (Table 1)were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Ger-many). Obidoxime dichloride (Fig. 1) was obtained from Merck(Darmstadt, Germany). Pralidoxime chloride (2-PAM; Fig. 1), 5,50-dithiobis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide(ATCh) were supplied by Sigma–Aldrich (Taufkirchen, Germany).All other chemicals were from Merck (Darmstadt, Germany).

hibited

Pralidoxime (2-PAM)Obidoxime

2 Cl-

Cl-

OHNN+

ON+

NOH

N+

NOH

Fig. 1. Chemical structures of obidoxime dichloride and pralidoxime chloride (2-PAM).

2 T. Wille et al. / Chemico-Biological Interactions xxx (2013) xxx–xxx

Carbamate stock solutions (50 mM) were prepared in acetoni-trile and stored at 20 �C. Oxime stock solutions (200 mM) wereprepared in distilled water and stored at�80 �C. Working solutionswere appropriately diluted in distilled water just before the exper-iment and were kept on ice until use.

Freshly drawn heparinized human whole blood was centrifugedat 3000 rpm for 10 min. Plasma was removed by suction and thered blood cells were washed three times with an approximatelythreefold volume of 0.1 M phosphate buffer. Then, hemoglobin-free human erythrocyte ghosts were prepared as the AChE sourceand stored at �80 �C [15].

2.2. Inhibition kinetics

The second-order inhibition rate constants (ki) of carbaryl weredetermined spectrophotometrically in the presence of substrate asdescribed before [16,17]. In brief, cuvets were filled with 3.0 mlphosphate buffer (0.1 M, pH 7.4) and 0.1 ml DTNB (10 mM) andwere temperature equilibrated at 37 �C. Then, 10 ll human eryth-rocyte ghosts and 5 ll carbamate (at least 8 concentrations) wereadded followed by 50 ll ATCh (28.4 mM). The absorbance changewas continuously monitored for 20 min. The recorded curves wereanalyzed by non-linear regression analysis and used for the subse-quent determination of ki [18]. The second-order inhibition rateconstants of carbamates were determined in the absence and pres-ence of 10 lM obidoxime or 2-PAM as representatives for clinicallyrelevant oxime plasma concentrations in the low micromolarrange [19,20].

2.3. Decarbamoylation kinetics

Erythrocyte ghosts were incubated with 5 lM (aldicarb, carba-ryl, carbofuran, propoxur) or 10 lM (methomyl) carbamate for30 min at 37 �C to achieve an AChE inhibition of 90–95% of control.Then, inhibited AChE was diluted 300-fold in phosphate buffer(containing 0.1% gelatin) in the presence or absence of 10 lMobidoxime.

Aliquots were taken to determine AChE activity (0–120 min).The enzyme activity was corrected for concentration-and sub-stance-dependent oximolysis and was referred to identically trea-ted control samples to calculate the % reactivation. The rateconstants of spontaneous decarbamoylation were calculated bynon-linear regression analysis (Prism 5.0, GraphPad, San Diego,USA) [21].

Fig. 2. Inhibition kinetics of carbaryl with human AChE. The concentration-dependent inhibition rate constants k1 were calculated and 1/k1 was plottedagainst 1/[CAR](1 � a) (C). [CAR] is the carbamate concentration, a stands for [S]/(Km + [S]) where [S] is substrate concentration and Km is the Michaelis constant. Thesecond-order inhibition constant ki was calculated according to [16].

2.4. AChE assay

AChE activity was measured spectrophotometrically at 412 nm(Cary3Bio, Varian, Darmstadt) with a modified Ellman assay [22].The assay mixture contained 0.45 mM acetylthiocholine (ATCh)as substrate and 0.3 mM DTNB as chromogen in 0.1 M phosphate

Please cite this article in press as: T. Wille et al., Investigation of kinetic interacby pesticide carbamates, Chemico-Biological Interactions (2013), http://dx.doi

buffer (pH 7.4). All experiments and assays were run at 37 �C(n = 4).

2.5. Statistical analysis

Statistical analysis of data obtained from two groups was per-formed with the Mann Whitney u-test and comparisons amongmultiple groups were performed using an analysis of variance (AN-OVA) with Bonferroni post hoc comparisons. A p-value of <0.05was considered significant.

3. Results

3.1. Inhibition kinetics of carbaryl with human AChE

The incubation of human AChE with all tested carbamates re-sulted in a concentration-dependent inhibition of the enzyme(for carbaryl as an example see Fig. 2). Inhibition kinetics deter-mined in the presence of 2-PAM or obidoxime (both 10 lM) andthe carbamates aldicarb, carbofuran and methomyl, did not differsignificantly from the respective second order inhibition rate con-stant ki of the carbamates in the absence of oximes (Table 1). Thecombination of propoxur with 2-PAM and obidoxime resulted ina moderate (2-fold and 1.5-fold) increase. The combination of2-PAM with carbaryl resulted in a similar moderate increase.However, if AChE was incubated with carbaryl in combination withobidoxime the increase of ki was more than tenfold (Fig. 2,Table 1).

3.2. Decarbamoylation kinetics

The determination of the decarbamoylation kinetics of humanAChE inhibited by the various N-monomethyl carbamates resultedin comparable decarbamoylation rate constants kdecarb (Table 2).Incubation of carbamate-inhibited AChE with obidoxime (10 lM)during the decarbamoylation phase had no significant effect onthe decarbamoylation kinetics of any of the investigated carba-mates (Table 2).

4. Discussion

The analysis of kinetic interactions between human AChE, car-bamates used for pest control and oximes demonstrated that theinhibitory potency of carbaryl and propoxur towards human AChEwas significantly increased in the presence of the clinically used

tions between approved oximes and human acetylcholinesterase inhibited.org/10.1016/j.cbi.2013.08.004

Table 1Inhibition kinetics of carbamates with human AChE.

Carbamate Oxime ki (M�1 min�1)

Aldicarb Ø 4.18 ± 0.32⁄104

+2-PAM 5.20 ± 2.20⁄104

+Obidoxime 5.30 ± 1.14⁄104

Carbaryl Ø 7.64 ± 0.28⁄104

+2-PAM 12.11 ± 2.20⁄104#

+Obidoxime 80.73 ± 4.02⁄104#

Carbofuran Ø 29.23 ± 0.50⁄104

+2-PAM 32.13 ± 1.52⁄104

+Obidoxime 32.78 ± 1.74⁄104

Methomyl Ø 8.51 ± 1.28⁄104

+2-PAM 11.98 ± 0.97⁄104

+Obidoxime 10.60 ± 1.21⁄104

Propoxur Ø 16.00 ± 1.02⁄104

+2-PAM 34.83 ± 6.74⁄104#

+Obidoxime 27.18 ± 4.07⁄104#

Data are given as means ± SD (n = 4).# p < 0.05 Compared to the respective carbamate without pralidoxime or obidoxime (both 10 lM).

Table 2Decarbamylation kinetics of carbamate-inhibited human AChE.

Inhibition Decarbamylation kdecarb (min�1)

Aldicarb Ø 0.045 ± 0.001Obidoxime 0.047 ± 0.002

Carbaryl Ø 0.037 ± 0.002Obidoxime 0.036 ± 0.002

Carbofuran Ø 0.039 ± 0.001Obidoxime 0.042 ± 0.001

Methomyl Ø 0.032 ± 0.002Obidoxime 0.032 ± 0.001

Propoxur Ø 0.031 ± 0.001Obidoxime 0.034 ± 0.002

Human AChE was inhibited by 5 lM (aldicarb, carbaryl, carbofuran and propoxur)or 10 lM (methomyl) carbamate for 30 min. After extensive dilution (300-fold)decarbamylation was recorded in the absence or presence of 10 lM obidoxime.Data are given as means ± SD (n = 4).

T. Wille et al. / Chemico-Biological Interactions xxx (2013) xxx–xxx 3

oximes 2-PAM and obidoxime which is in agreement with previousfindings obtained with bovine erythrocyte AChE [13,14]. Hence, itmay be assumed that similar to animal experiments [10–12] thetreatment of severely propoxur or carbaryl poisoned humans withobidoxime and 2-PAM may further increase the inhibition of AChEand may in certain cases lead to an aggravation of poisoning.

Such a synergistic effect of propoxur and carbaryl with oximeswill depend on the incorporated dose and the toxicokinetics of thecarbamate. Toxicokinetic studies in rats indicate that after oral car-baryl administration the residence time in plasma is rather short.Herr and co-workers dosed rats with 30 mg/kg carbaryl and thecarbaryl plasma concentration was below the limit of quantitationafter 2 h [23]. Propoxur showed a moderate inhibition of cholines-terases in human volunteers after oral administration of 1.5 mg/kgwith complete recovery of enzyme inhibition after 2 h and an ele-vated excretion of metabolites in the urine up to 7 h [24].

Please cite this article in press as: T. Wille et al., Investigation of kinetic interacby pesticide carbamates, Chemico-Biological Interactions (2013), http://dx.doi

Due to the moderate inhibitory potency of carbamates towardshuman AChE, severe poisoning will predominantly occur afterintentional intake of large doses. This will result in a high bodyload and agent may be redistributed from tissue depots into thesystemic circulation resulting in prolonged toxicologically relevantlevels and almost completely inhibited AChE [25]. Hence, adminis-tration of oximes to severely propoxur and carbaryl poisoned pa-tients could result in a further aggravation of AChE inhibition.

The determination of the decarbamoylation kinetics of inhibitedhuman AChE showed that therapeutic concentrations of obidoxime– although resulting in a higher inhibition if administered togetherwith carbaryl or propoxur – it did not have a significant effect onthe spontaneous reactivation of the inhibited enzyme which agreeswith previous results determined with bovine erythrocyte AChE[13,14] and with human AChE inhibited by the N-methyl carba-mate physostigmine [21]. Hence, it cannot be expected that theseoximes accelerate the spontaneous decarbamoylation of inhibitedAChE in human carbaryl poisoning. Likewise, from a therapeuticalview, the available data provide no evidence that oximes activelyreactivate carbamate-inhibited AChE.

As potential mechanisms for an aggravated inhibition caused bythe combination of propoxur or carbaryl with an oxime the bindingof an oxime to the peripheral or allosteric binding site [14], the for-mation of a carbamoyl-oxime, which similar to phosphyloximes[26] could exhibit a substantially higher inhibitory potency [11]have been discussed.

Obidoxime und 2-PAM are weak reversible inhibitors of humanAChE [27,28] and binding to peripheral or allosteric binding sitescould contribute to the enhanced inhibition by propoxur and espe-cially carbaryl. However, the results of the present study cannotprovide a definitive explanation for the strong oxime effect on car-baryl inhibition of human AChE.

In conclusion, the kinetic analysis of interactions between hu-man AChE, carbamates and the clinically used oximes, 2-PAM

tions between approved oximes and human acetylcholinesterase inhibited.org/10.1016/j.cbi.2013.08.004

4 T. Wille et al. / Chemico-Biological Interactions xxx (2013) xxx–xxx

and obidoxime, demonstrated that these oximes aggravate theinhibition of AChE by carbaryl and propoxur at therapeutic concen-trations, with obidoxime being extremely potent when adminis-tered together with carbaryl. In addition, the tested oximes didnot accelerate the spontaneous decarbamoylation of the inhibitedAChE which would present a therapeutic benefit. Thereby, thepresent study confirms previous in vitro and in vivo animal studiesand administration of 2-PAM and of obidoxime to severely propo-xur or carbaryl poisoned humans cannot be recommended.

Conflict of interest statement

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, J. Letzelter and L. Binder for ex-pert technical assistance.

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tions between approved oximes and human acetylcholinesterase inhibited.org/10.1016/j.cbi.2013.08.004