Absence of a Protective Effect of the Oxime 2-PAM toward Paraoxon-Poisoned Honey Bees: Acetylcholinesterase Reactivation Not at Fault

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

152, 184 –192 (1998)

TO988483

Absence of a Protective Effect of the Oxime 2-PAM toward Paraoxon-Poisoned Honey Bees: Acetylcholinesterase Reactivation Not at Fault Androniki Polyzou,* Marie-The´re`se Froment,† Patrick Masson,† and Luc P. Belzunces* *Institut National de la Recherche Agronomique, Phytopharmacie, Unite´ de Toxicologie, Site Agroparc, 84914 Avignon Cedex 9, France; and †Centre de Recherches du Service de Sante´ des Arme´es, Unite´ d’Enzymologie, BP 87, 38702 La Tronche Cedex, France Received December 3, 1997; accepted May 26, 1998

Absence of a Protective Effect of the Oxime 2-PAM toward Paraoxon-Poisoned Honey Bees: Acetylcholinesterase Reactivation Not at Fault. Polyzou, A., Froment, M.-T., Masson, P., and Belzunces, L. P. (1998). Toxicol. Appl. Pharmacol. 152, 184 –192. We investigated the failure of 2-PAM to protect honey bees against poisoning with paraoxon. The protective effect of the oxime 2-PAM against inhibition of acetylcholinesterase (AChE) by paraoxon was estimated in vitro and in vivo and was correlated with the mortality of paraoxon-treated bees. In vitro, 2-PAM protected 90% of AChE activity in the presence of paraoxon and reactivated more than 90% of inhibited AChE. Minor soluble and major membrane-bound forms of bee AChE presented about similar extents of reactivation, but the first order rate constant of reactivation (kobs) of the soluble form is threefold higher than that of the membrane-bound form. However, this difference did not significantly influence the reactivation kinetics of total AChE; the constant kobs of the membrane-bound form reflected that of total AChE. The linear kinetic profile of total AChE reactivation supported the conclusion that there was a single population of reactivatable species. The bimolecular rate constant of reactivation (kr), the dephosphorylation rate constant (k2), and the dissociation constant (Kd) were 646 M21.min21, 0.84 min21 and 1.30 mM, respectively. In vivo, administration of 2-PAM, after paraoxon exposure, induced a complete protection of AChE activity, but did not elicit any significant effect on mortality in paraoxon-treated bees. The inefficiency of 2-PAM to antagonize paraoxon-induced mortality was not changed by the administration of 2-PAM in pretreatment-therapy and in therapy treatments. These results indicated that the mortality of paraoxon-poisoned honey bees was not due to a lack of AChE reactivation. © 1998 Academic Press

The use of organophosphate (OP) insecticides as agricultural pesticides has resulted in toxicity to nontarget species, including beneficial insect pollinators such as the honey bee Apis mellifera (Barker et al., 1980; Johansen et al., 1983; Fiedler, 1987; Stevenson, 1978). Acetylcholinesterase (AChE, E.C. 3.1.1.7), a key enzyme in cholinergic transmission in the nervous system, is the major target of OP agents through phosphylation of the catalytic serine (Toutant, 1989; Massoulie´ et 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

al., 1993). The toxicity of OP insecticides is caused by a progressive inhibition of AChE in neural tissue, resulting in the accumulation of acetylcholine (ACh) in the synaptic cleft. Subsequently, the overstimulation of ACh receptors leads to neurotoxic symptoms (Corbett et al., 1984; Eldefrawi, 1985; Fournier and Mutero, 1994). In mammals, as in other animals, phosphylated AChE can be reactivated by nucleophilic agents, among which oximes are of pharmacological interest as antidotes against OP poisoning (Bismuth et al., 1992). The effectiveness of oxime reactivators as antidotes was primarily attributed to the nucleophilic displacement of the OP moiety from the inhibited enzyme. The capacity of phosphylated AChE to be reactivated varies with the structure of the bound OP (Caranto et al., 1994; Berkman et al., 1993), the oxime (Worek et al., 1996), and the type of cholinesterase (Ashani et al., 1995; Clement and Erhardt, 1994; Masson et al., 1997; Schwarz et al., 1995). In some cases, the inhibited AChE undergoes dealkylation of a branched alkoxy chain, termed “aging,” which converts it to a form that is resistant to reactivation by oximes (Aldridge and Reiner, 1972). In addition to AChE reactivation, there is evidence that oximes have other beneficial pharmacological effects, e.g., on acetylcholine receptors (Van Helden et al., 1996). Like other insect AChE, honey bee AChE shows soluble and membrane-bound forms that represent, respectively 3– 6% and 94 –97% of total brain AChE activity (Belzunces et al., 1988b). The physicochemical properties and the sensitivity to OPs of these forms are different: e.g., the soluble form is less sensitive to OPs than the membrane-bound form (Belzunces and Debras, 1997). The standard antidotal treatment, after exposure to OP agents, includes administration of a muscarinic receptor antagonist (atropine) to counteract the accumulation of ACh, along with an oxime to reactivate OP-inhibited AChE (Dawson, 1994; Go¨ransson-Nyberg et al., 1995; Lau et al., 1996). The combination of atropine plus monopyridinium oximes, such as 2-PAM, or bispyridinium oximes, such as HI-6, is a satisfactory treatment against OP poisoning in many animals

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(Sanchez-Fortun et al., 1996 Bismuth et al., 1992). However, treatment by atropine with salts of N-methyl pyridinium-2aldoxime (2-PAM), for example, does not lead to a reduction of mortality in bees poisoned by OP and carbamate insecticides (Barker, 1970). The inefficacy of oximes as antidotal treatment has been examined in several studies (Willems and Belpaire, 1992), and different possible causes have been proposed to explain this failure: (1) low solubility in lipids and variable and limited transfer across the blood– brain barrier, (2) formation of phosphoryloximes that are themselves potent anti-ChE compounds, (3) interruption of oxime administration, before the concentration of the toxic agent has declined below toxicologically significant levels, (4) differential reactivity toward OPenzyme conjugates, and (5) aging of OP-enzyme conjugates and nonreactivatability of phosphylated AChE due to steric hindrance (Thompson et al., 1992). Previous studies, however, suggested that honey bee AChE could be reactivated in vitro by oximes (Westlake et al., 1985). The purpose of the present study was to investigate the causes of the inefficacy of 2-PAM as an antidote for OPpoisoned honey bees. For this purpose, the protecting and reactivating actions of 2-PAM against paraoxon-inhibited honey bee AChE were investigated both in vitro and in vivo and related to the mortality of paraoxon-treated bees. Differences in the reactivation of membrane-bound and soluble honey bee AChE were also examined.

MATERIALS AND METHODS Materials. Triton X-100, Triton X-114, aprotinin, antipain, leupeptin, pepstatin A, benzamidin, acetylthiocholine iodide (AcSCh), paraoxon (diethyl p-nitrophenyl phosphate), 5,59-dithiobis-(2-nitrobenzoic acid) (DTNB), and 2-PAM iodide (pyridine-2-aldoxime methiodide) were purchased from Sigma (Saint Louis, MO). Analytical procedures. Bee head AChE was solubilized in a low-salt Triton X-100-containing buffer as described previously (Belzunces et al., 1988a). The AChE concentration in the extract was determined by active site titration using paraoxon with which no significant spontaneous reactivation was observed during 2 days. In the protection and reactivation studies, the AChE concentration was always below 1029 M so that [AChE] ! [2-PAM]. AChE activity was assayed at 25°C by the method of Ellman et al., (1961), in a 1–5-ml medium consisting of 1.5 mM DTNB, 0.3 mM AcSCh, and 100 mM sodium phosphate pH 7.0 (Ellman reagent). At this pH, the spontaneous hydrolysis of AcSCh was considerably reduced, thus allowing for better measurement of low AChE activity. The substrate concentration used corresponded to the maximum concentration at which no inhibition by excess of substrate is observed (Belzunces et al., 1988a). The enzyme activity was corrected for spontaneous hydrolysis of AcSCh and for 2-PAM-induced hydrolysis of AcSCh. The reaction rate was monitored spectrophotometrically by the increase in A412. In vitro protection of AChE by 2-PAM. Honey bee AChE was incubated at 25°C in 100 mM phosphate buffer pH 7.0, with 0.2–10 mM 2-PAM and different concentrations of paraoxon. Residual enzyme activity after different incubation times was determined by diluting the reaction mixture 50-fold in the Ellman reagent. Possible underestimation of oxime protection, due to reversible inhibition of the free enzyme by 2-PAM, was avoided by adding 2-PAM to the control enzyme.

Reactivation kinetics of paraoxon-inhibited AChE. AChE was inhibited with paraoxon. This OP was selected because its AChE adduct does not undergo a significant aging during the time course of inhibition (Aldridge and Reiner, 1972). Inhibition of AChE was performed at 25°C in a 100 mM phosphate buffer pH 7.0, containing 0.5 mM paraoxon. At this paraoxon concentration, a plateau of residual AChE activity (about 5%) was reached after 120-min incubation. The absence of a residual inhibitory activity in paraoxon-treated samples was checked by adding active AChE. The control enzyme was treated under the same conditions except that paraoxon was omitted. Reactivation reactions were initiated by adding 2-PAM in 100 mM phosphate buffer pH 7.0 (final 2-PAM concentration ranged between 0.12 and 5 mM) to the paraoxon-free enzyme solution in 100 mM phosphate buffer pH 7.0 at 25°C. AChE activity, after a given reactivation time (Et), was assayed by diluting 100 ml reactivation mixture samples in 4.9 ml of Ellman reagent (50-fold dilution). Samples without enzyme and with noninhibited enzyme were incubated with 2-PAM and were used for the estimation of the background readings and the control AChE activity (Eo), respectively. Reactivation curves reached a plateau corresponding to Emax in less than 120 min. Spontaneous reactivation of paraoxon-inhibited AChE was assessed by incubation in buffer without oxime. Kinetic analysis. According to Green and Smith (1958a), the oxime reactivation process of phosphylated AChE can be described by the following model: Kd

k2

E~OP! 1 22PAM º E~OP!.22PAM 3 E 1 P,

(Scheme 1)

where E(OP) is the phosphylated enzyme, 2-PAM is the oxime reactivator, E(OP).2-PAM is the Michaelian complex, P represents reaction products, i.e., the phosphylated 2-PAM and its breakdown products (Harvey et al., 1986a,b), and E is the reactivated enzyme. Kd and k2 are the complex dissociation constant and the dephosphylation rate constant, respectively. Since [E(OP)]![2-PAM], the time-dependent change of the phosphorylated enzyme concentration is given by, 1n(1 2 Et/Emax) 5 2kobst, where kobs is the first-order rate constant of reactivation. The kinetic parameters of reactivation (Kd, k2 and kr 5 k2/Kd, the overall bimolecular rate constant of reactivation) were determined (according to Equation 1) by plotting 1/kobs against 1/[2PAM]: Kd 1 1 1 5 3 1 . k obs k 2 @22PAM# k 2

(1)

In vivo effect of 2-PAM. Honey bees, mainly foragers, were reared under controlled conditions excluding poisoning with carbamate or OP agrochemicals. The bees were captured from the honey and pollen combs of hives, placed in observation boxes (20 per box) for 24 h at 28°C, and fed ad libitum with a mixture of honey and sugar. All experiments were carried out at 28°C. Honey bees were anesthetized with carbon dioxide before each treatment. The experimental design (Table 1) used in these experiments was a modification of that described previously (Clement and Erhardt, 1994). After treatment, alive and dead bees were counted and immediately stored at 220°C until analysis. AChE activity was quantitatively determined in the bees individually. The association of prophylactic and therapeutic effects were evaluated in Pre–Post treatments. The AChE activity in this experiment resulted from the protective effect of 2-PAM against inhibition by paraoxon and the immediate reactivation of phosphorylated enzyme. Posttreatment of 2-PAM was used to test its therapeutic effect on paraoxon-poisoned bees and its reactivating action on paraoxon-inhibited AChE. The control 2-PAM treatment made it possible to evaluate the toxicity of 2-PAM in honey bees (Barker, 1970) and its effect on AChE activity (Grosfeld et al., 1996; Mounter and Ellin, 1968). After paraoxon exposure, mortality and AChE inhibition were estimated in paraoxon-treated control bees. Differences in AChE activity between experimental groups were tested using one-way analysis of variance followed by the Scheffe

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TABLE 1 Protocol and Timing Sequence of In Vivo Experiments Timing of treatment (min)

Control

Paraoxon control

2-PAM (Pre–Post)

2-PAM (Post)

2-PAM control

0 10 30 60

PBS PBS 1 acetone PBS Stored (220°C)

PBS PBS 1 paraoxon PBS Stored (220°C)

2-PAM 2-PAM 1 paraoxon 2-PAM Stored (220°C)

PBS PBS 1 paraoxon 2-PAM Stored (220°C)

2-PAM 2-PAM 1 acetone 2-PAM Stored (220°C)

Note. Paraoxon in acetone was applied topically to the thorax at the doses of 0, 50, 100, 150, 200, and 300 ng per bee. 2-PAM, in phosphate-buffered saline (PBS: 150 mM sodium chloride, 10 mM sodium phosphate buffer pH 7.4), was injected in the abdomen. In each injection, 1 ml of freshly prepared 2-PAM solution was administered (50 mg per bee). In this study, Pre–Post described the situation where the 2-PAM was present before, during, and after the exposure to paraoxon. Post described the situation where the 2-PAM was present only after exposure to paraoxon.

test (Ferguson, 1971). A difference with p , 0.01 was considered statistically significant.

RESULTS

Effect of 2-PAM on Paraoxon Potency During in vivo and in vitro protection experiments, the effect of 2-PAM was evaluated in the presence of paraoxon. Under these conditions, 2-PAM may increase the inhibitory potency of paraoxon through the formation of phosphorylated oxime, which may be potentially anticholinesterase or anti-AChE (and not: acetylcholinesterase 5 AChE) (Schoene, 1973; Harvey et al., 1986b). To test this possibility, paraoxon and 2-PAM were preincubated together at 25°C for different periods of time and diluted 10,000-fold. This resulting mixture was used to test the ability of paraoxon to inhibit AChE. As shown in Table 2, the ability of paraoxon to inhibit AChE was not changed after incubation with 2-PAM for up to 5 h. TABLE 2 Effect of the Oxime 2-PAM on the Inhibitory Potency of Paraoxon Toward Honey Bee AChE Incubation time (h) 0 Control Paraoxon Paraoxon 1 2-PAM

1

2.25

3.5

5

100 100 100 100 100 62.0 6 6.4 61.6 6 1.3 61.0 6 0.0 63.5 6 5.5 72.1 6 6.7 62.6 6 6.2 62.6 6 1.5 59.1 6 3.7 65.6 6 3.7 73.4 6 5.7

Note. Values are percentage of residual AChE. Paraoxon, at the concentration of 1 mM, was incubated at 25°C for different times in 100 mM phosphate buffer pH 7.0, containing or not 10 mM 2-PAM. Incubation mixtures were diluted stepwise to obtain final concentrations of 0.1 mM paraoxon and 1 mM 2-PAM, if present, in phosphate buffer containing AChE after the last dilution. The resulting media containing AChE was incubated at 25°C for 1 h and samples of the reaction mixture were assayed (50-fold dilution in Ellman reagent) for AChE activity. Data represented the means 6 SD of AChE activity assayed on three independent samples.

In Vitro Protection of AChE by 2-PAM To simulate the in vivo conditions in which the oxime is present simultaneously with the inhibitor and the target enzyme, we tested the efficacy of 2-PAM to control AChE inhibition in the presence of paraoxon. Figure 1A shows the effect of 2-PAM on in vitro titration of honey bee AChE by paraoxon. In the absence of 2-PAM, paraoxon, at concentrations higher than 1026 M, inhibited more than 95% of AChE in 60 min. The presence of 2-PAM led to an increase in AChE activity irrespective of the paraoxon concentration used. The protective effect of 2-PAM varied with the concentration of paraoxon. At 1026 M paraoxon, 2-PAM (1022 M) permitted the complete protection of AChE, whereas, with 1024 M paraoxon, the protective extent decreased to 36.5%. Moreover, the presence or the absence of 2-PAM strongly affected the susceptibility of bee head AChE, expressed as the inhibitor concentration reducing 50% of the initial activity (IC50); the IC50 was 410-fold higher in the presence of 2-PAM than in the absence (Fig. 1A). The protective effect of 2-PAM against inhibition by paraoxon also depended on the oxime concentration (Fig. 1B). The 2-PAM produced a dose-dependent increase in AChE activity, which was maximum (about 90%) at concentrations above 5 mM. Under our experimental conditions, the protective effect of 2-PAM was rapid and time independent for at least 300 min (Fig. 1C). It should be noted, however, that the maximum level of protected AChE depended on oxime concentration. Complementary experiments proved that the protective effect of 10 mM 2-PAM was not changed after 26 h incubation. This result indicated that reinhibition of AChE by phosphorylated oxime was not significant under these experimental conditions. In Vitro Reactivation of Paraoxon-Inhibited AChE In order to clarify the mechanism of 2-PAM protection, we tested the ability of 2-PAM to reactivate paraoxon-inhibited AChE (Fig. 2). The oxime 2-PAM was capable of reactivating the potential AChE activity in a dose-dependent mode. Para-

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FIG. 2. Time course of reactivation of paraoxon-inhibited AChE (5.6 3 10210 M) by 0.35 (ƒ), 1 (‚), 2 (h), and 5 mM (E) 2-PAM. The percentage of AChE reactivation was calculated by the following formula: 100[(Et 2 Ei)/(Eo 2 Ei)], where Et is the activity at time t, Eo, the control activity, and Ei, the residual activity of the inhibited control. The residual activity corresponded varied to 2–5% of control.

oxon-inhibited AChE was almost completely reactivated (95%) by 5 mM 2-PAM in 3.5 min. This showed that, under these conditions, significant aging did not occur during the phosphorylation process. Since the residual activity of paraoxon-inhibited AChE did not change within the experimental scale of time (data not shown), the spontaneous reactivation of diethylphosphorylated AChE was not significant. Reactivation profiles were linear (r 2 5 0.97– 0.99) (Fig. 3A). This indicated a homogeneity in reactivatable enzyme species and reflected the absence of significant interference from competing reactions, such as reversible inhibition of enzyme by 2-PAM and reinhibition by the phosphorylated oxime. Kinetic parameters of reactivation kr, k2, and Kd were determined by the double reciprocal plot of kobs as a function of 2-PAM concentration (Fig. 3B). Such linear behavior also supports the proposed reactivation process (Scheme 1) under the conditions studied, and, therefore, kr, k2, and Kd constants could serve as adequate parameters to measure the 2-PAM reactivity toward phosphorylated bee AChE. The efficacy of 2-PAM for reactivation of paraoxon-inhibited AChE was also investigated for both membrane-bound and soluble forms (Fig. 4). Soluble AChE displayed an apparent FIG. 1. (A) Effect of 2-PAM on in vitro titration of bee AChE by paraoxon. AChE (5.7 3 10210 M) was incubated at 25°C in the assay medium (100 mM phosphate buffer, pH 7.0) with increasing amounts of paraoxon, in the presence (h) or in the absence (■) of 10 mM 2-PAM, for 60 min. Control samples were incubated with 2-PAM. Data are representative of three independant experiments. The IC50 of paraoxon for bee AChE was 4.5 3 1025 6 1.3 3 1025 M (n 5 3), with 10 mM 2-PAM and 1.1 3 1027 6 0.36 3 1027 M (n 5 3) without 2-PAM. (B) Dose effect of 2-PAM on protection of honey bee AChE against inactivation by paraoxon. Bee AChE (3.3 3 10210 M) was incubated at 25°C, in 100 mM phosphate buffer pH 7.0 with various 2-PAM concentrations for 60 min in the presence (h) or in the absence (■) of 1026 M

paraoxon. The extent of protection was calculated by the following formula: 100[(Er 2 Ei)/(Eo 2 Ei)], where Er is the recovered activity, Ei is the residual activity of the inhibited control, and Eo is the control activity. Control samples were incubated without 2-PAM. (C) Time course of protection by 2-PAM of bee AChE in the presence of paraoxon. The incubation medium (100 mM phosphate buffer pH 7.0) contained bee AChE (4.6 3 10210 M), 1026 M paraoxon, and 0 (F), 1 (Œ), and 10 mM (■) 2-PAM. The noninhibited AChE sample was also incubated with 1 (‚) and 10 mM (h) 2-PAM. Control sample contained neither paraoxon nor 2-PAM.

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termined in a same paraoxon-treated bee, is presented in Fig. 5. After paraoxon exposure, AChE activity was inhibited in a dose-dependent manner (Fig. 5A). The inhibition was significant (about 70% of control) at doses of 200 and 300 ng/bee. The mortality due to poisoning by paraoxon appeared before 50 min following treatment. The mortality also increased with the paraoxon dose and reached the maximum value (100% of control) at the dose of 300 ng/bee (Fig. 5B). The 2-PAM clearly protected bee AChE activity against paraoxon (Fig. 5A). Regardless of the paraoxon dose used, Post or Pre–Post administration of 2-PAM resulted in almost complete recovery (more than 91% of control) of AChE activity 30 and 60 min after 2-PAM injection, respectively, although AChE protection had no significant effect on mortality of paraoxon-treated bees. With Pre–Post or Post 2-PAM treatments, high mortality levels (more than 70%) were observed in paraoxon-treated bees, before 50 min following paraoxon administration (Table 1 and Fig. 5B). Neither 2-PAM injection nor solvent injection produced acute toxic effects or a decrease in AChE activity. DISCUSSION

Previous in vivo experiments have shown that the combination of cholinesterase reactivators and antimuscarinic agents does not reduce mortality of OP-poisoned honey bees (Barker, 1970). Results of the present study show that 2-PAM oxime

FIG. 3. Determination of the kinetic constants Kd, k2, and kr for the reactivation of diethylphosphorylated AChE (5.6 3 10210 M) by 2-PAM. (A) Semilogarithmic plot of the time course of reactivation of paraoxon-inhibited bee AChE by 2-PAM. Concentrations of 2-PAM were 0.25 (h), 0.35 (E), 0.7 (‚), 1 (ƒ), 1.25 (3), and 2 mM (1). (B) Double reciprocal plots of kobs versus reactivator concentration, yielding the values of 2(1/Kd), 1/k2, from x- and y-intercept, respectively, and 1/kr from the slope. The kinetic parameters calculated from these data are Kd 5 1.30 mM, k2 5 0.84 min21, and kr 5 646 M21. min21.

reactivation constant threefold higher than that of membranebound AChE. However, kobs of the membrane-bound form was not significantly different from that of the total AChE extract (p . 0.01), providing evidence that the small amount (3– 6%) of the soluble form did not affect the reactivation kinetics of total AChE. The two molecular forms showed similar maximum reactivation extent: 74.7 6 16.36% (n 5 3) for the soluble form and 72.8 6 2.46% (n 5 4) for the membranebound form, which is close to the reactivation level of total AChE (72.7 6 7.3%, n 5 3). In Vivo Protective Effect of 2-PAM The efficacy of 2-PAM against AChE inhibition was evaluated in relation to the mortality of paraoxon-poisoned bees. The effect of 2-PAM on AChE activity and morality, as de-

FIG. 4. Semilogarithmic plot of the time course of reactivation of different paraoxon-inhibited molecular forms of bee AChE by 1 mM 2-PAM. (E) Soluble form, (h) membrane-bound form, and (‚) total AChE. Soluble AChE (10210 M) was obtained after Triton X-114 phase separation of low-salt extract. Membrane-bound AChE (6.1 3 10210 M) was obtained after a two-step extraction procedure, in low-salt and low-salt Triton X-100-containing buffer, as described previously (Polyzou et al., 1997). Total AChE (8.2 3 10210 M) was obtained by direct extraction in low-salt Triton X-100-containing buffer (Belzunces et al., 1988a). Lines were representative of five to eight independent experiments. The kobs of the soluble form (2.04 6 0.81 min21, n 5 5) was significantly different ( p , 0.01) than that of the membrane-bound form (0.70 6 0.23 min21, n 5 8) and total AChE (0.56 6 0.24 min21, n 5 5). The kobs of the membrane-bound form and total AChE were not significantly different ( p . 0.01).

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189

FIG. 5. In vivo effect of 2-PAM on AChE activity (A) and mortality (B) in paraoxon-treated honey bees. Paraoxon and 2-PAM treatments in each experimental group were described in Materials and Methods. Mortality and AChE activity were considered in a same bee, with 10 –15 individual bees in each experimental group. No mortality was observed in untreated control, solvent-treated control, and 2-PAM control. Tissue AChE activity (mmol/min/g of head) was 9.72 6 3.15 (n 5 52) in untreated control, 8.55 6 2.15 (n 5 57) in solvent-treated control and 8.52 6 1.68 (n 5 43) in 2-PAM control. *Significantly different ( p , 0.01) compared with the other experimental groups, which received the same paraoxon dose. In paraoxon control, Pre–Post, and Post experimental groups, AChE activities of dead bees did not significantly differ from those of surviving bees ( p . 0.01).

protects the target enzyme AChE against inhibition by paraoxon, both in vitro and in vivo (Figs. 1A–C and 5A). Westlake et al. (1985) have also shown that AChE inhibited in vivo by OPs is reactivated in vitro by 2-PAM. The present study, furthermore, demonstrates, that 2-PAM protects AChE in poisoned honey bees (Fig. 5A). The efficacy of 2-PAM against AChE inhibition is similar in pretreatment-therapy (Pre–Post) and in therapy (Post) treatments. In addition, in vitro, 2-PAM is able to reactivate more than 90% of inhibited AChE (Fig. 2). This result suggests that the high recovery rate of AChE activity after in vivo Pre–Post and Post 2-PAM treatment is essentially due to AChE reactivation. The potency of 2-PAM, as a reactivator of paraoxon-inhibited bee AChE, as measured by kr, is slightly higher (646 M21.min21.) than that observed for paraoxon-inhibited human AChE (460 M21.min21, Grosfeld et al., 1996 and 580 M21.min21, Green and Smith, 1958b) but far lower than for electric eel AChE (1.4 3 104 M21.min21, Kitz et al., 1965). Diethylphosphorylated honey bee AChE exhibits lower affinity (Kd 5 1.30

mM) and higher rate of dephosphorylation (k2 5 0.84 min21) than diethylphosphorylated human AChE (0.13 mM and 0.06 min21 respectively, Grosfeld et al., 1996). This finding indicates that the kinetics parameters of AChE reactivation present a great variability between species. In vivo treatments of 2-PAM trigger the complete restoration of bee head AChE activity, but do not change the bee mortality (Figs. 5A and 5B). AChE activity is at its average physiological level 30 and 60 min after Post and Pre–Post 2-PAM administration, respectively (Table 1 and Fig. 5A). However, the bees die before 50 min following the administration of paraoxon, either associated with 2-PAM or not (Table 1 and Fig. 5B). These results indicate that the poor performance of 2-PAM, as an antidote, does not result from the failure to penetrate into the brain or to reactivate in vivo AChE. Mortality due to reinhibition of AChE by diethylphosphorylated 2-PAM or to aging is less likely, because complete and stable levels of reactivation and protection are obtained after incubation with 2-PAM for long periods of time (Figs. 1C and 2). Nevertheless,

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Harvey et al. (1986b), and Grosfeld et al. (1996), have shown that the formation of phosphorylated oxime or the aging process does not change the reactivation profile of diethylphosphorylated AChE. Moreover, it is also less likely that the difference between reactivation rates of the membrane-bound and the soluble forms explain the deficiency in the antidotal 2-PAM treatment. The two molecular forms of bee AChE exhibit different kinetics of reactivation, but the final percentage of reactivation was the same for both molecular forms (higher than 70%). The difference between the reactivation constants of membrane-bound and soluble AChE might reflect differences in accessibility of 2-PAM to the active center or in the overall conformation of the two molecular forms (Velan et al., 1993). However, a possible difference in the architecture of the active site cannot be ruled out. In humans, mutations in the active center of AChE and BuChE changed the kinetics of reactivation by 2-PAM (Grosfeld et al., 1996; Masson et al., 1997). In the honey bee, possible structural differences between soluble and membrane-bound AChE are supported by results showing that soluble and membrane-bound AChE display different kinetic parameters and physico-chemical properties (Belzunces and Debras, 1997). These results are in accordance with studies showing that structural modifications by mutations in the active center of human AChE and BuChE elicit changes in the kinetic parameters of inhibition by paraoxon (Ordentlich et al., 1996; Masson et al., 1997). In the present study, we have shown that 2-PAM can reactivate or protect bee AChE without inducing any effect on bee longevity (Figs. 5A and 5B), in other words, that mortality is not due to failure of 2-PAM to reactivate or protect bee AChE. This is in accordance with experiments evidencing that the AChE-inhibiting potency of OPs does not always correlate with acute in vivo toxicity (Chambers, 1992). Such a differential effect of 2-PAM strongly suggests that OPs have secondary targets of toxicological importance on which 2-PAM is inefficient. Direct effects on muscarinic and nicotinic receptors appear to be involved in the mechanism of OP poisoning. Both parathion and paraoxon, like other OP AChE inhibitors, may bind to mACh receptor (AChR) and act as agonists, affecting the receptor in a different manner than acetylcholine (Van den Beukel et al., 1996; Jett et al., 1991). The authors of these studies suggested that interactions between paraoxon and AChR might play a role in the neurotoxicity of paraoxon. Paraoxon displaces the agonist [3H]oxotremorine from mAChR in rat brain, human brain, and Chinese hamster ovary cells expressing human muscarinic AChR (Van den Beukel et al., 1997). A possible binding of paraoxon to mAChR and its interference with agonists or antidotal mAChR antagonists may explain the inefficiency of atropine in antagonizing acute lethal effects of OP insecticides in honey bees (Barker, 1970). However, studies also strongly suggest that the protective effect of oximes, observed in vertebrates, may not always be linked to AChE reactivation (for review, see Van Helden et al., 1996). The oximes toxogonin, HI-6 and HLo¨-7 elicit a signif-

icant increase in the survival time of rats treated with a lethal dose of the OP crotylsarin, without triggering restoration of AChE activity (Van Helden et al., 1994). This suggests that different pathways may be involved in the protective action of oximes and in the toxicity of OP insecticides. This is supported by experimental results showing that oximes may alter the synthesis and release of acetylcholine and may either stimulate or block nicotinic and muscarinic receptors (Van Helden et al., 1996). Hence, depending on the species, oximes can reinforce or counteract the toxicity of OPs by modulating the cholinergic neurotransmission. Thus, differences in the action of oximes between vertebrates and invertebrates may account for the inefficiency of 2-PAM to protect the honey bee against poisoning by OPs. The possibility that paraoxon mortality could be determined by a balance between cholinergic and noncholinergic mechanisms should be also investigated. This is supported by experiments indicating that paraoxon inhibits the function of g-aminobutyric acidA (GABAA), glycine, N-methyl-D-aspartic acid (NMDA), and nicotinic receptors in cultured hippocampal neurones (Rocha et al., 1996). A noncompetitive blockade, by paraoxon, of postsynaptic GABAA receptors was described in the invertebrate Aplysia californica (Filbert et al., 1992). This suggests that these noncholinergic mechanisms may have a role in the neurotoxicity caused by paraoxon. A noncholinergic mode of lethality following injection of carbamate insecticides was also observed in rabbits (Takahashi et al., 1994). Considering that GABA is the transmitter at inhibitory synapses in many insect muscles (Eldefrawi, 1985), we cannot rule out the possibility that the lethal effect in honey bees is caused partly by the combined effects of paraoxon on gabaergic and cholinergic transmissions. In conclusion, this study demonstrates that the failure of 2-PAM to protect paraoxon-poisoned honey bees is not due to a lack of AChE reactivation by 2-PAM. Paraoxon-induced mortality is accompanied with inhibition of bee head AChE. Although 2-PAM treatments (pre- or postexposure to paraoxon) almost completely restore the activity of bee head AChE, they do not affect the mortality of paraoxon-poisoned bees. In addition, in vitro and in vivo reactivation studies indicate that up to 90% of paraoxon-inhibited AChE is reactivatable and that the reactivation process is not altered by competing reactions such as aging of the OP-enzyme conjugate, formation of a phosphoryloxime, which is itself a potent anti-AChE, and heterogeneous reactivation of AChE molecular forms. Although 2-PAM can reactivate paraoxon-inhibited AChE, its inefficient antidotal effect suggests that other mechanisms, besides AChE inhibition, may contribute to the neurotoxicity of paraoxon or that the beneficial effect of 2-PAM are not limited to AChE reactivation. Suggested causes of 2-PAM inefficacy in paraoxon-intoxicated honey bees can include: (1) secondary OP targets (cholinergic or not cholinergic) of toxicological importance on which 2-PAM is inefficient, and (2) absence of a protective effect of 2-PAM, except

EFFECT OF 2-PAM IN PARAOXON TOXICITY AND AChE INHIBITION

AChE reactivation, in the honey bee cholinergic system, in contrast to vertebrates in which the oximes have other beneficial pharmacological effects (e.g., modulation of muscarinic AChR). ACKNOWLEDGMENTS The authors thank Alain and Rene´ Paris for competent beekeeping, the Institut National de la Recherche Agronomique (INRA) Unit of Zoology for providing honey bees, and Sylvaine Regis-Rolle for her critical reading of the manuscript. This work was supported by INRA, Re´gion Provence-Alpes-Coˆte d’Azur (PACA), a French-Greek PLATON project, and the Greek State Scholarships Foundation.

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