Ecotoxicology and Environmental Safety 100 (2014) 39–43

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Characterization of plasma cholinesterase in rabbit and evaluation of the inhibitory potential of diazinon Ana Lourdes Oropesa n, Marcos Pérez-López, Francisco Soler Toxicology Area, Veterinary Faculty, University of Extremadura, Avda. de la Universidad, s/n, P.O. Box 643, 10003 Cáceres, Spain

art ic l e i nf o

a b s t r a c t

Article history: Received 18 October 2013 Received in revised form 28 November 2013 Accepted 1 December 2013 Available online 20 December 2013

Several studies indicate that more than one cholinesterase form may be present in the blood of mammals. In this study the predominant plasma cholinesterase activity, the physiological cholinesterase activity as well as cholinesterase sex-dependent changes in non-exposed individuals of rabbit have been established. Plasma cholinesterase was characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylthiocholine and butyrylthiocholine, and the predominant enzymatic activity was acetylcholinesterase. Physiological plasma cholinesterase activity was 198.9 75.8 nmol/min/ml for male and 205.2 7 5.0 nmol/min/ml for female using acetylthiocholine as substrate. Thus, sex had no significant effect on the physiological cholinesterase activity (p4 0.05). In addition, the in vivo and in vitro sensitivity of plasma cholinesterase to diazinon was also investigated. In rabbits exposed to single doses of diazinon (25 or 125 mg/kg) the higher inhibitions of plasma cholinesterase were reached 9 h after oral administration (53% and 87% inhibition, respectively). Cholinesterase activity significantly recovered up to values similar to pre-administration between 3 and 7 d depending on the administered dose and sex of the animals. Plasma cholinesterase activity decreased to 24%, 53% and 74% of the initial activity at 9 h of in vitro exposure to 1.25, 3.13 and 6.25 mg/l of diazinon, respectively, and it remained steadily depressed throughout the experimental period (10 d). This study has demonstrated the sensitivity of cholinesterase activity in plasma of rabbits following both in vivo and in vitro exposure to sub-lethal concentrations of diazinon. & 2013 Elsevier Inc. All rights reserved.

Keywords: Biomarker Cholinesterase inhibition Diazinon Rabbits

1. Introduction Diazinon {O,O-diethyl-O-[6-methyl-2-(1-methylethyl)-4pyrimidinyl] phosphorothioate} is a broad-spectrum contact organophosphorus (OP) pesticide used as an insecticide, nematicide and acaricide (Aggarwal et al., 2013). The toxicity of this pesticide is mainly due to the inhibition of acetylcholinesterase (AChE) activity, the enzyme which degrades the neurotransmitter acetylcholine in cholinergic synapses (Eto, 1974). The inhibition of AChE provokes an accumulation of acetylcholine at the nerve synapses and disruption of the nerve function (Peakell, 1992), a toxicity mechanism that may lead to death. It is extensively known that the inhibition of cholinesterases (ChEs) is appropriate for evaluation of exposure to OP pesticides because these are rapidly degraded and excreted from the organisms and therefore they are not easily detectable by chemical analysis (Hill and Fleming, 1982; Hill, 1995; Fairbrother, 1996). Blood cholinesterases, including AChE and a less specialized

n

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enzyme commonly designed as pseudocholinesterase or butyrylcholinesterase (BChE), are also inhibited by these substances being a widely used non-destructive biomarker to diagnose the exposure to anticholinesterase agents (Sánchez et al., 1997). Therefore, for both ethical and conservational reasons, the use of non-destructive biomarkers to investigate the exposure to pesticides in populations of rabbits is mostly adequate since they can provide early indications of toxic effects. Blood is in fact the best biological material for non-destructive biomarker analysis (Fossi et al., 1994). There are a lot of studies in birds reporting the use of blood cholinesterase as a biomarker of exposure to anticholinesterase agents (Westlake et al., 1981a, 1981b; Gard and Hooper, 1993; SolerRodríguez et al., 1998; Parsons et al., 2000; Mayack and Martin, 2003; Rendón-von Osten et al., 2005; Roy et al., 2005; Oropesa et al., 2013). The measurement of plasma esterase inhibition in wild mammalian species has been carried out in order to assess the effects of agricultural chemicals (Westlake et al., 1980, 1982). However, to our knowledge, there are no published data on the use of plasma cholinesterase activity in rabbits to evidence exposure to this group of substances. Diagnosis of exposure to OP is an initial step in assessing the impact of these chemicals on wildlife.

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The use of ChE activity as a biomarker requires of the biochemical characterization of the ChE forms present in the species and in the tissue to be studied because primary ChE substrates for plasma are different among species and between mammals and birds (Cooper et al., 1978). With a view to using the ChE activity of rabbit plasma as a biomarker, the objectives of this work were  To characterize the ChE activity present in plasma of rabbit using different substrates and specific inhibitors.  To determine the in vivo and in vitro effects of the OP diazinon on plasma ChE activity, i.e., the inhibition and recovery of plasma ChE activities following exposure to diazinon.

2. Material and methods 2.1. Chemicals Acetylthiocholine iodide (ASCh), butyrylthiocholine iodide (BSCh), propionylthiocholine iodide (PSCh), iso-OMPA (tetraisopropyl pyrophosphoramide), eserine sulfate, BW284C51 (1,5-bis(4-allyldimethyammoniumphenyl) pentan-3-one dibromide), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and ethanol, were purchased from Sigma-Aldrich Quimica S.A., St. Louis, USA. Diazinon (Purity: 98.3%) was purchased from Fluka, Seelze, Germany.

recommended for mammals by Thomson (1999)) using 0.050 ml of diluted plasma (1/10 in potassium-phosphate buffer, 0.1 M pH ¼ 7.2) and 0.250 ml of the reaction mixture [19.96 ml potassium-phosphate buffer (0.1 M pH ¼ 7.2), 1 ml of reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) 10 mM in phosphate buffer and 5.120 ml of substrate 75 mM] and a wavelength of 412 nm. Four replicates per treatment per each sample of plasma were used. The ChE activity was measured (optical density changes) for 5 min on a microplate reader Power-Wave™ BioTeks Instruments, Inc. The enzyme activity was expressed as nmol of substrate hydrolised per minute per ml of plasma. 2.7. In vitro inhibition of ChE by specific inhibitors and diazinon Eserine sulfate, iso-OMPA and BW284C51 were used in this study as specific inhibitors of all ChEs, BChE and AChE, respectively. Stock solutions of iso-OMPA in ethanol, BW284C51 and eserine sulfate in water were prepared at 0.0078, 0.031, 0.12, 0.5, 2, 8 mM for iso-OMPA; 0.0078, 3.1, 12, 50, 200, 800 μM for BW284C51 and 0.00048, 0.0019, 0.0078, 3.1, 12, 50, 200, 800 μM for eserine sulfate. Diazinon was prepared in ethanol at 1.25, 3.13, 6.25 mg/l. The effect of in vitro specific inhibitors and diazinon on ChE activity was determined after an incubation period of 30 min at 25 1C in the dark as follows: for each chemical, 0.005 ml of each stock solution were added to 0.495 ml of a pool of diluted plasma from non-exposed rabbits. Controls were incubated with 0.005 ml of ultrapure water. Additional controls were incubated with 0.005 ml of ethanol and included when appropriate. For diazinon the activity of ChE was also determined in the same way after 9 h, 1, 3, 7 and 10 d of incubation in order to study the relationship between the in vivo and in vitro inhibition of ChE activity by this pesticide. Four replicates per treatment per each sample of plasma were used. ChE activity was determined immediately after the end of the incubation period as previously indicated.

2.2. Animals and their holding 2.8. Statistical analysis

2.3. In vivo exposure to diazinon Rabbits were exposed to a single oral dose of diazinon. The animals were divided into 3 groups, consisting of 8 animals each (4 males and 4 females): a control group (exposed to ethanol and distilled water) and two experimental groups which received doses of 25 and 125 mg/kg b.w. diazinon diluted in ethanol and then in distilled water administered by oral gavage to achieve different levels of ChE inhibition. These doses corresponded to 0.1 LD50 and 0.5 LD50 (LD50 for rabbits¼ 250 mg/kg; Merck index). The influence of these doses on ChE activity of the plasma rabbits was investigated for 10 d. The assay was carried out in the Service of Animal Facility from University of Extremadura (Registration number: ES 1003700001803). All animal experiments were conducted in accordance with ethical guidelines of the European Union Council (Council Directive 86/609/EEC) and with the approval of the Bioethical Committee from University of Extremadura (Spain). 2.4. Collection and preparations of samples Blood was sampled in the morning to avoid error due to circadian variations in enzyme activities (Thompson, 1999). Whole blood was taken from rabbits through puncture of the auricular vein using a syringe with heparin (25-gauge) at 0 and 9 h, 1, 3, 7 and 10 d after diazinon administration. Plasma was obtained by centrifugation (2000g, for 5 min at 4 1C) and immediately stored at  80 1C until further analysis. 2.5. Characterization and catalytic properties of ChE The substrate preferences of plasma ChE were investigated by determining the activity of ChE in a pool of plasma from non-exposed rabbits (control group: 4 males and 4 females) at increasing concentrations of ASCh, BSCh and PSCh (from 0.02 to 20.48 mM, incubation concentration). The maximum velocity of substrate hydrolysis (Vmax), indicative of total enzyme present, Michaelis–Menten constant (Km) and the ratio (Vmax/Km) that indicates the catalytic efficiency of the enzyme(s) were estimated by the double-reciprocal method of Lineweaver and Burk (1934).

Results are expressed as mean 7 standard error of the mean (S.E.M.). Assumptions of data normality and variance homogeneity were checked using the Kolmogorov–Smirnov and Levene's tests, respectively. As the ANOVA assumptions were not met, the non-parametric Kruskal–Wallis test was used. The Mann– Whitney U-test was subsequently applied for pairwise comparisons between groups (Zar, 1996). The significance level was set at 0.05. All statistical analyses were carried out using the statistical software SPSS v19.0 for Windows.

3. Results 3.1. ChE characterization The ChE activity in plasma of rabbits as a function of different substrates showed a certain degree of preference for PSCh (Fig. 1). So, the maximum activity of ChE was measured with PSCh and the lowest activity was obtained with BSCh. The Km's for ASCh, BSCh and PSCh were 0.17, 0.09 and 0.33 mM, respectively. The substrate concentrations of 75 mM used in the following assays were sufficiently high to achieve zero order kinetics. The ratio Vmax/Km, 500 ASCh

450 ChE activity (nmol/min/ml)

The study was performed with a total of 24 New Zealand white rabbits, 12 males and 12 females. The animals weighed 3.2 7 0.6 kg and were 6 months old. Rabbits were individually housed in cages in an acclimatized room (temperature, 21 1C; photoperiod, 7 a.m.–19 p.m.; free access to rabbit feed and water) for 15 d before starting the treatment and also during the exposure.

BSCh

400

PSCh

350 300 250 200 150 100 50 0 0

5

10

15

20

25

Substrate concentration (mM)

2.6. Enzyme activity ChE activity was determined using the Ellman's technique (Ellman et al., 1961) adapted to microplates following the general procedure indicated by Guilhermino et al. (1996). Briefly, the ChE activity was determined at 37 1C (temperature

Fig. 1. Plasma cholinesterase activity of rabbits (Oryctolagus cuniculus) as a function of the substrates acetylthiocholine iodide (ASCh), S-butyrylthiocholine iodide (BSCh) and propionylthiocholine iodide (PSCh). Values are the mean of pooled plasma from non-exposed rabbits (4 males and 4 females) (four enzymatic determinations per concentration) with corresponding standard error bars.

A.L. Oropesa et al. / Ecotoxicology and Environmental Safety 100 (2014) 39–43

which indicates the enzymatic catalytic efficiency, showed the enzyme preferences: ASCh (2000)4BSCh (1428.2)4PSCh (1250). Eserine sulfate, a strong inhibitor of all the ChE enzymes, significantly inhibited the enzyme activity, with more than 90% of inhibition at 3.13 μM or higher concentrations [Lowest observed effect concentration (LOEC) o 0.048 μM; p o0.05] (Fig. 2(A)). These results indicate that almost all the esterase activity measured in rabbit plasma is from both ChEs (AChE and BChE). Plasma ChE activity was significantly inhibited by BW284C51 from the lowest concentrations of exposure tested, with a LOEC o0.048 μM (p ¼0.05; Fig. 2(A)). On the other hand, plasma ChE activity of

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rabbits showed a significant inhibition regarding the control groups from 0.78 mM of iso-OMPA (p ¼0.05; Fig. 2(B)). Therefore, the no observed effect concentration (NOEC) and LOEC values for iso-OMPA were estimated to be 0.195 and 0.78 mM, respectively. No significant effect of the solvent ethanol on ChE activity was observed. 3.2. ChE activity in non-exposed individuals The mean value of ChE activity in control rabbits was 198.9 75.8 nmol/min/ml for males and 205.2 75.0 nmol/min/ml for females using ASCh as substrate. No significant differences (p 40.05) were found between the ChE activity of the two sexes. 3.3. In vivo and in vitro inhibition of ChE after exposure to diazinon Plasma cholinesterase values in rabbits exposed to oral doses of diazinon as a function of time are shown in Table 1. ChE activities are compared to the ChE activity at t ¼0 min, previous to exposure of diazinon, for each group. Doses of 0.1 LD50 and 0.5 LD50 fast and significantly inhibited the plasma activity in a dose dependent manner. The inhibitions represented 53% and 80% of the activity in males and 52% and 87% of the activity in females up to 9 h post administration (p o0.05), respectively. After this time, the enzyme activity started to recover to a level of 100% or higher 10 d after diazinon administration for both groups. Statistically significant differences, with respect to t ¼0 min, appeared for males at 9 h and 1 d for 0.1 LD50 group and 9 h, 1, 3 and 7 d for 0.5 LD50 group (p o0.05). On the other hand, in females statistically significant differences, with respect to t¼0 min, were observed from 9 h to 1 d in both 0.1 LD50 and 0.5 LD50 groups (po 0.05). Concerning the in vitro experiment, plasma ChE activities significantly decreased (po0.05) to 24%, 53% and 74% of t ¼0 min activity 9 h of exposure to 1.25, 3.13 and 6.25 mg/l of diazinon. These inhibitions were dose-dependent during the time of in vitro exposure to diazinon (Table 2 and Fig. 3). The enzymatic activities were significantly (p o0.05) depressed at a constant level throughout the experimental period (10 d). The trend of inhibition was similar in all exposed plasma samples.

4. Discussion

Fig. 2. Effect of eserine sulfate and BW284C51 (A) and iso-OMPA (B) on plasma cholinesterase activity of rabbits (Oryctolagus cuniculus) using acetylthiocholine as substrate. Values are the mean of pooled plasma from non-exposed rabbits (4 males and 4 females) (four enzymatic determinations per concentration) with corresponding standard error bars. (n) Indicates significant differences from the control group mean (pr0.05).

Plasma ChE is a good biomarker of low levels of exposure to OP pesticides because it is more rapidly inhibited and to a larger degree than brain ChE (Hill and Fleming, 1982; Sánchez et al., 1997; SolerRodríguez et al., 1998). The use of plasma of rabbits allows sampling without killing the animal as well as repeat sampling of plasma using the same individuals. We decided to use plasma instead of red blood

Table 1 Specific activity of cholinesterase in plasma of rabbits (Oryctolagus cuniculus) after 10 d of in vivo exposure to oral single dose of diazinon. Time of measurement

Specific activity (nmol/min/ml) Males

0 min 9h 1 d (24 h) 3 d (72 h) 7 d (168 h) 10 d (240 h)

Females

0.1 LD50

Inhibition (%)

199.1 752.2 93.1 714.0n 145.4 721.7n 200.3 736.2 206.8 724.7 226.3 727.3

– 53 27 – – –

a

0.5 LD50

Inhibition (%)

211.3 7 29.4 41.3 7 3.4n 99.8 7 12.2n 160.5 7 19.5n 188.9 7 14.8n 234.4 7 27.7

– 80 53 24 11 –

a

0.1 LD50

Inhibition (%)a

0.5 LD50

Inhibition (%)a

197.9 7 49.2 95.7 7 14.0n 150.7 7 26.7n 157.17 30.2 176.7 7 22.7 230.5 7 29.3

– 52 24 21 11 –

214.1 722.3 28.8 79.4n 85.2 717.2n 184.5 715.5 200.9 719.8 247.5 725.7

– 87 60 14 6 –

Data are presented as means of four replicates (from 4 males or 4 females per group)7 S.E.M. (standard error of mean). n

a

Significantly different from t ¼ 0 min for each group at p r 0.05. Percentages of inhibition of ChE activity compared to t¼ 0 min for each group.

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Table 2 Specific activity of cholinesterase in plasma of rabbits (Oryctolagus cuniculus) after 10 d of in vitro exposure to 1.25, 3.13 and 6.25 mg/l of diazinon. Time of measurement

0 min 30 min (0.5 h) 9h 1 d (24 h) 3 d (72 h) 7 d (168 h) 10 d (240 h)

Specific activity (nmol/min/ml) Control

1.25 (mg/l)

Inhibition (%)a

3.13 (mg/l)

Inhibition (%)a

6.25 (mg/l)

Inhibition (%)a

263.7 7 6.3 244.5 7 7.8 237.0 7 1.4 240.87 3.2 225.5 7 12.0 227.6 7 2.1 228.1 7 1.7

255.4 7 2.2 211.4 7 1.8n 194.5 7 0.8n 194.3 7 2.0n 198.137 1.1n 179.5 7 1.4n 172.4 7 0.8n

– 17 24 24 22 30 32

249.5 7 6.1 137.17 1.3n 117.4 7 7.8n 108.9 7 3.0n 118.17 11.5n 104.17 1.9n 117.5 7 0.9n

– 45 53 56 53 58 53

242.1 75.2 93.1 71.0n 62.1 70.3n 50.1 70.0n 57.8 70.1n 47.8 710.0n 51.0 73.1n

– 62 74 79 76 81 79

Data are presented as means of four replicates (pooled plasma from non-exposed rabbits—4 males and 4 females) 7 S.E.M. (standarderror of mean). n

a

Significantly different from t ¼0 min for each group at p r 0.05. Percentages of inhibition of ChE activity compared to t¼ 0 min for each group.

Control

300

1.25 mg/l 3.13 mg//l

ChE activity (nmol/min/ml)

250

6.25 mg/l

200

150

100

50

0 0

50

100

150

200

250

300

Time of exposure (h) Fig. 3. In vitro effect of diazinon on plasma cholinesterase activity of rabbits (Oryctolagus cuniculus). Mean values of cholinesterase activity with corresponding standard error bars of pooled plasma from non-exposed rabbits (4 males and 4 females) after 10 d of in vitro exposure to 1.25, 3.13 and 6.25 mg/l of diazinon.

cells for measuring of AChE based on the study of Winters et al. (1997). These authors reported that rabbit plasma ChE is more sensitive to an anticholinesterase agent (diazinon) than red blood cells ChE. Plasma ChE returns to normal activity more quickly than does red blood cells ChE. Ward and Glicksberg (1971) also provided this statement in dogs exposed to dichlorvos. This is due to fact that plasma ChE is synthesized by the liver, and regeneration is dependent on the rate of the enzyme's synthesis. On the contrary, regeneration of blood cells ChE is dependent on the rate of red blood cell turnover which is slower than liver AChE production. ChEs can be characterized by selective substrate specificity and by their reaction with highly selective inhibitors. Thus, enzyme kinetic experiments in plasma were undertaken to characterize the ChEs in plasma. This enzyme showed slight preference for PSCh at the tested concentrations (0.02–20.48 mM). ASCh was hydrolyzed faster than BSCh although the hydrolysis of ASCh and BSCh at the highest concentration was similar. Therefore, at this concentration the two substrates could be used in the analysis of ChE. The contribution of non-specific esterases was estimated using the compound eserine sulfate, which is considered a specific inhibitor of total ChE at low concentrations in the 10  6–10  5 M range (Eto, 1974). In the present study, the enzymatic activity measured in plasma was almost

completely inhibited (more than 90%) by eserine sulfate at 3.13 μM or higher concentrations, which is considered typical for ChE. This result indicates that the activity determined in plasma in our experimental conditions is mainly ChE. Specific inhibition of the ChE enzyme by BW284C51, a selective reversible inhibitor of AChE (Xu and Bull, 1994), reduced total enzyme activity in a significant way from 0.048 μM, whereas specific inhibition with iso-OMPA, an inhibitor of BChE but not of AChE (Barahona and Sánchez-Fortún, 1999), failed up to 0.78 mM. Therefore, the results indicate that plasma ChE activity is principally AChE which is in agreement with the theory of Tecles and Cerón (2001) who indicated that the characterization using specific substrates is less accurate than with specific inhibitors. In addition, these authors pointed that ASCh is the main substrate hydrolyzed in plasma of ruminants. Studies of plasma ChE activity in mammals have shown that there is a strong relationship between ChE activity and phylogenetic classification (Westlake et al., 1983). From an anatomical point of view the skeleton and organs of lagomorphs are similar to those of the even-toed ungulates order (Janečka et al., 2007) which includes the ruminant suborder. Therefore, from these lines of evidence, it seems that lagomorphs and ruminants share the same type of ChE activity in plasma, i.e. AChE activity. In addition, it is important to emphasize that the maximum ChE activity was reached at a lower concentration of ASCh (0.64 mM) than BSCh that is usually used as substrate in the analysis of plasma ChE. Using ASCh as substrate, plasma ChE activity of control rabbits was 198.9 7 5.8 nmol/min/ml for male and 205.2 7 5.0 nmol/min/ ml for female. The resulting levels of ChE in plasma were in the same order of magnitude as those found by Winters et al. (1997) in control rabbits. The baseline level of ChE activity in plasma was similar between males and females. This finding suggests that rabbits of both sexes are useful in assessing anticholinesterase agents' exposure using this potential biomarker. Similar inhibition of plasma ChE was observed in male (53% and 27%) and female (52% and 24%) rabbits 9 h and 1 d after exposure to 25 mg/kg of diazinon (0.1 LD50). Nevertheless, males did not show inhibition in the ChE activity 3 d after exposure while females showed an inhibition of 21 and 11% 3 and 7 d after exposure, respectively, with respect to t¼0 min. For female, our results are comparables with those obtained by Winters et al. (1997) in rabbits after exposure to 25 mg/kg of diazinon. In general, the ChE values recovered in a progressive way from 9 h until 7 d after exposure to 125 mg/kg of diazinon (0.5 LD50) and by 10 d plasma ChE activity had reached values higher than those at t¼0 min. As might be expected, the magnitude of the inhibition was higher in rabbits exposed to 0.5 LD50 of diazinon than in those exposed to 0.1 LD50 of the pesticide. Davies and Holub (1980) indicated that sensitivity to the toxicity of dietary diazinon is higher in female rats than in males as a function of the degree of ChE activity inhibition in blood. In

A.L. Oropesa et al. / Ecotoxicology and Environmental Safety 100 (2014) 39–43

our study there were no differences between sexes in the maximum ChE inhibition at both doses of diazinon although the recovery of the enzyme in females of the 0.1 LD50 group was slower than in males at 3 and 7 d after diazinon administration. However, at the end of the experiment the recovery was the same in both sexes. To our knowledge, there are no published data in rabbits about the influence of sex in the inhibition of plasma ChE after anticholinesterase pesticide exposure. The administration of two independent oral doses of diazinon (0.1 LD50 and 0.5 LD50) showed a dose-dependent inhibition and recovery of the plasma ChE activity in rabbits. According to Fleming (1981), ChEs return to physiological levels if exposure to an inhibitor is discontinued and consequently the animal survives OP poisoning. Analysis of the data showed that the plasma ChE activity got its recovery to physiological levels 7 d after exposure to the lowest tested dose of diazinon. Recovery of ChE activity following OP exposure usually depends on de novo synthesis and liberation of ChE by liver and several authors have reported that the inhibited plasma ChE undergoes a recovery even to values higher than those of controls (Fleming, 1981; Soler-Rodríguez et al., 1998), as occurred in our experiment. Knowledge of the recovery pattern is essential from a diagnostic point of view in the interpretation of ChE data for wildlife exposed to anticholinesterase agents. Results from the present study demonstrate that ingestion of diazinon in rabbits provoked a plasma ChE inhibition certain up to 1 d after exposure. The sensitivity of plasma ChE demonstrates its potential for being used as non-destructive diagnostic tool for assessing the exposure of rabbits to diazinon, providing an opportunity for repeating sampling in the same animal in studies of environmental pollution biomonitoring. The trend of response of plasma ChE activity after in vitro exposure to diazinon (1.25, 3.13 and 6.25 mg/l) was similar between groups, i.e. this enzymatic activity underwent a progressive inhibition up to 9 h followed by a steady inhibition during 10 d. Obviously, the inhibition of ChE was concentration-dependent, irreversible and therefore no recovery was observed along in vitro exposure period because there are no mechanisms of metabolic degradation as happens in the animal. In aqueous solution diazinon might be transformed to diazoxon (Mutch and Williams, 2006) being responsible to in vitro ChE inhibition. It could be concluded that measuring cholinesterase activity can be used as a good biomarker of exposure of rabbits to OP insecticides, and therefore allowing the use of European rabbits in future studies of biomonitoring for agroecosystems to be considered. Acknowledgments Authors wish to thank the “Ministerio de Educación y Ciencia” from Spain (CTM2007-60041) for the economic support. References Aggarwal, V., Deng, X., Tuli, A., Goh, K.S., 2013. Diazinon-chemistry and environmental fate: a California perspective. Rev. Environ. Contam. Toxicol. 223, 107–140. Barahona, M.V., Sánchez-Fortún, S., 1999. Toxicity of carbamates to the brine shrimp Artemia salina and the effect of atropine, BW284C51, iso-OMPA and 2PAM on carbaryl toxicity. Environ. Pollut. 104, 469–476. Cooper, J.R., Bloom, F.E., Roth, R.H., 1978. The Biochemical Basis of Neuropharmacology, 3rd Ed. Oxford University Press, New York p. 80. Council Directive of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC). Davies, D.B., Holub, B.J., 1980. Comparative subacute toxicity of dietary diazinon in the male and female rat. Toxicol. Appl. Pharmacol. 54, 359–367. Ellman, G.L., Courtney, D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Eto, M., 1974. Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland.

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