Cholinesterase and carboxylesterase inhibition in Planorbarius corneus exposed to binary mixtures of azinphos-methyl and chlorpyrifos

Aquatic Toxicology 128–129 (2013) 124–134

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Cholinesterase and carboxylesterase inhibition in Planorbarius corneus exposed to binary mixtures of azinphos-methyl and chlorpyrifos Luis Claudio Cacciatore, Noemí Verrengia Guerrero, Adriana Cristina Cochón ∗ Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Nu˜ nez, 1428, Buenos Aires, Argentina

a r t i c l e

i n f o

Article history: Received 23 June 2012 Received in revised form 6 October 2012 Accepted 9 December 2012 Keywords: Cholinesterase Carboxylesterase Invertebrate Pesticides Mixtures

a b s t r a c t Though pesticide mixtures are commonly encountered in fresh water systems, the knowledge of their effects on non-target aquatic species is scarce. The present investigation was undertaken to explore the in vivo inhibition of the freshwater gastropod snail Planorbarius corneus cholinesterase (ChE) and carboxylesterases (CES) activities by the organophosphorus pesticides azinphos-methyl (AZM) and chlorpyrifos (CPF). To this end, snails were exposed for 48 h to different concentrations of AZM and CPF in single-chemical and binary-mixture studies, and ChE and CES activities were measured in the whole soft body tissues and hemolymph. ChE activity was measured with acetylthiocholine as substrate and CES activity was measured with four substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1- and 2-naphthyl acetate. Single-chemical experiments showed that CPF was a more potent inhibitor of ChE activity than AZM (350 and 27 times for the whole soft tissue and hemolymph, respectively). CES were 15 times more sensitive than ChE when the activities were measured in the whole soft tissue of the animals exposed to AZM. Based on a default assumption of concentration addition, P. corneus snails were exposed to mixtures of AZM + CPF designed to yield predicted soft tissue ChE inhibitions of 31% (mixture 1), 50% (mixture 2) and 61% (mixture 3). Results showed that ChE inhibition produced by mixture 1 followed a model of concentration addition. In contrast, the other mixtures showed synergism, both in whole soft tissue and hemolymph. In addition, results obtained when the snails were exposed sequentially to the pesticides showed that the sequence AZM/CPF produced at 48 h a higher ChE inhibition than the sequence CPF/CPF. A range of metabolic pathways and responses associated with bioactivation or detoxification may play important roles in organophosphorus interactions. In particular, the data presented in the present study indicate that CES enzymes would be important factors in determining the effects of the mixtures of OPs on P. corneus ChE activity. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Pesticide mixtures are commonly encountered in aquatic bodies. However, the evaluation of the potential toxic effects and the risks associated with the exposure of non-target aquatic species to mixtures of pesticides is still a challenge for the scientific community (Spurgeon et al., 2010).

Abbreviations: AChE, acetylcholinesterase; AcSCh, acetylthiocholine iodide; AZM, azinphos-methyl; CES, carboxylesterases; ChE, cholinesterase; CPF, chlorpyrifos; DTNB, 5,5 -dithio-2-bis-nitrobenzoate; 1- and 2-NA, 1- and 2-naphthyl acetate; p-NPA, p-nitrophenyl acetate; p-NPB, p-nitrophenyl butyrate; OP, organophosphorus; SDS, sodium dodecil sulfate. ∗ Corresponding author at: Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Departamento de Química Biológica, Ciudad Universitaria, Pab. II, 4to piso, 1428 Buenos Aires, Argentina. Tel.: +54 11 4576 3342; fax: +54 11 4576 3342. E-mail address: [email protected] (A.C. Cochón). 0166-445X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2012.12.005

Organophosphorus (OP) insecticides share a common mechanism, the inhibition of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses. Besides, OP compounds can also inhibit the activity of other B-esterases such as butyryl cholinesterase and carboxylesterases (CES) (Sogorb and Vilanova, 2002). In fact, several studies have reported that CES enzymes may be even more sensitive than ChE enzymes to some OP pesticides (Escartín and Porte, 1997; Kristoff et al., 2012; Wheelock et al., 2005). Azinphos-methyl (O,O-dimethyl S-[4-oxo-1,2,3-benzotriazin3(4H)-yl) methyl]triazin-3-ylmethyl] dithiophosphate, AZM) and chlorpyrifos (O,O-diethyl O-(3,5,6-trichloropyridin-2-yl) thiophosphate, CPF) are broad-spectrum OP insecticides used for pest control on a number of food crops in many parts of the world (Loewy et al., 2011; USEPA, 2001, 2011). Reported concentrations of AZM in surface waters range from 0.06 to 420 ␮g L−1 (0.19 nM to 1.3 ␮M) (Granovsky et al., 1996; Klosterhaus et al., 2003; Loewy et al., 2011; Schulz, 2004; Wan et al., 1995). In the case of CPF, reported

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concentrations vary from less than 0.35 to 26.6 ␮g L−1 (less than 1 nM to 76 nM) (Marino and Ronco, 2005; Otieno et al., 2012). In particular, Loewy et al. (2011) reported that in the Neuquen River Valley, North Patagonian Region of Argentina, the most intensively used pesticide for the control of Carpocapsa pomonella was AZM, often replaced by CPF (Loewy et al., 2011). In that study, coexistence of AZM and CPF was observed in the drainage water (surface and subsurface) originating from flood irrigated areas. The detection frequency levels of both pesticides were higher than 70%. Other compounds detected were methidathion, dimethoate and propoxur, the latter two with a detection frequency under 5% (Loewy et al., 2011). Since OPs inhibit AChE, it is expected that the effects of mixtures of OP pesticides on this activity would follow a model of concentration addition. That is, the concentration of each pesticide may be replaced by an equally effective concentration of one of them (Bosgra et al., 2009). However, there have been some documented cases where mixtures of OP pesticides do not follow this additive model. For example, it has been reported that when salmons were exposed to certain mixtures of OPs, they showed a synergistic inhibition of the brain AChE activity (Laetz et al., 2009). Moreover, these authors observed that several combinations of OPs were lethal at concentrations that were sublethal in single-chemical trials. Also, studies in vitro performed with homogenates of the freshwater snail Planorbarius corneus have shown that several mixtures of the potent oxon metabolites of AZM and CPF resulted in cumulative ChE inhibitions higher than those expected on a concentration addition basis (Cacciatore et al., 2012). These results point out the need for additional investigations to determine more precisely the in vivo effects of mixtures of OPs in non-target aquatic species. A range of metabolic pathways and responses associated with bioactivation or detoxification may play important roles in OP interactions. These include phase I enzymes such as cytochrome P450s (CYPs) and esterases, among others. Since both AZM and CPF have a thiophosphoryl bond (P S) instead of a phosphoryl bond (P O), they possess minimal or no anticholinesterase activity and require metabolic activation to their oxon analogs to inhibit ChE and CES activities (Gupta, 2006). Studies performed with c-DNA expressed human CYPs and human liver microsomes have shown that different isoforms of CYP are involved in the metabolization of OPs (Buratti et al., 2002, 2003; Crane et al., 2012; Tang et al., 2006). For example, at high pesticide concentration, CYP 3A4 showed the main contribution to bioactivation (desulfuration) of AZM and CPF. However, at low pesticide concentration, AZM and CPF were mainly desulfurated by CYP 1A2, 2B6 and 2C19. In addition, CYPs also catalyze a dearylation (detoxification) reaction, which is parallel to the desulfuration reaction. These two reactions, dearylation and desulfuration, compete with one another and different CYP isoforms can catalyze both reactions with different ratios. For example, human CYP2B6 has the highest reported intrinsic clearance (CLi) for bioactivation of CPF while CYP2C19 has the highest reported CLi for CPF detoxification (Buratti et al., 2007; Crane et al., 2012). Besides, the radical S: produced during the desulfuration reaction is capable of inhibiting the CYPs. Therefore, it can be hypothesized that if AZM and CPF compete for the same CYPs, mixtures of these two OPs would probably deviate from a concentration addition model. In addition to the CYP-mediated metabolism of OPs, several authors have concluded that detoxification enzyme activities such as those of A esterases and CES must also be considered when evaluating the effects of combined exposure to OPs (Cacciatore et al., 2012; Jansen et al., 2009; Karanth et al., 2001; Richardson et al., 2001). It has been shown that the A esterases display low affinity for many OPs and oxons and a high affinity for only a few OPs such as CPF (Tang et al., 2006). Besides, CES are important contributors to the stoichiometric detoxification of many oxons, even

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though of those with low affinity for the A esterases. Thus, CES enzymes can reduce the amount of pesticide available for ChE inhibition by direct binding and sequestration of the oxons (Sogorb and Vilanova, 2002).Some species of the Phylum Mollusca have been recommended as suitable biological experimental models to assess sublethal impacts of contaminants and mixtures of contaminants in aquatic ecosystems (Rittschof and McClellan-Green, 2005). Besides, a considerable decrease in the diversity of mollusks as a consequence of anthropogenic activities has been reported (Strong et al., 2008). In particular, freshwater gastropods represent about 20% of recorded mollusk extinctions (Strong et al., 2008). Declines in the gastropod populations may have a relevant negative impact on the ecosystems since they play an important role in trophic chains as grazers and as preys (Wojdak and Trexler, 2010). The gastropod P. corneus is a hermaphroditic snail that usually inhabits small temporary ponds and streams. This species belongs to the Planorbidae family, the largest family of aquatic pulmonate gastropods which is distributed all over the world (Jopp, 2006). P. corneus has already been used as a model organism for toxicity studies (Benstead et al., 2011; Clarke et al., 2009; Otludil et al., 2004; Pavlica et al., 2000). Besides, ChE and CES activities in this species have already been characterized (Cacciatore et al., 2012). The present investigation was undertaken to explore: (1) the in vivo inhibition of P. corneus ChE and CES activities by AZM and CPF, and (2) the likeliness of in vivo non-additive effects of binary mixtures of these two OPs on AChE activity. To this end, 48 h bioassays were performed by exposing snails to different AZM and CPF concentrations, including levels of environmental relevance, in single-chemical and binary-mixture studies. Previous studies performed in another mollusk species have shown that ChE activity was higher in hemolymph than in whole organism soft tissue whilst the reverse was true for CES (Galloway et al., 2002). For this reason, the research described herein focused on the activities of ChE and CES enzymes in soft tissues and hemolymph. 2. Materials and methods 2.1. Chemicals Acetylthiocholine iodide (AcSCh), p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (p-NPB), 1- and 2-naphthyl acetate (1- and 2-NA), 5,5 -dithio-2-bis-nitrobenzoate (DTNB), sodium dodecil sulfate (SDS), Fast Blue RR salt, azinphos-methyl PESTANAL® (CAS No. 86-50-0, 97.2% pure), and chlorpyrifos PESTANAL® (CAS No. 2921-88-2, 99.9% pure) were purchased from Sigma–Aldrich. All other chemicals used were of analytical reagent grade. 2.2. Snails Adult P. corneus snails were purchased from Discus Morón S.R.L., Buenos Aires, Argentina. Afterwards the snails were reared in our laboratory in aerated glass aquaria (17–20 L), at a temperature of 22 ± 2 ◦ C, and under a 14:10 (L:D) h artificial photoperiod regime. Animals were fed lettuce leaves ad libitum. For all the experiments, adult snails of similar size (10 ± 2 mm of shell length, 300 ± 36 mg of wet weight) were used. Water quality characteristics were as follows: total hardness = 67 ± 3 mg CaCO3 L−1 ; alkalinity = 29 ± 2 mg CaCO3 L−1 ; pH 7.0 ± 0.2 and conductivity = 250 ± 17 ␮S cm−1 . 2.3. Azinphos-methyl and chlorpyrifos inhibition studies The experimental design consisted of 1 L glass vessels containing 600 mL of each treatment condition, i.e. different pesticide concentrations, solvent control and solvent-free control. In experiments I,

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II and IV, triplicate vessels were used for each treatment condition. In experiment III, four vessels were used. Six randomly selected snails were placed in each vessel for experiments I and II. In the case of experiments III and IV, 3 snails were placed in each vessel. At the end of the experiments, the hemolymph and whole soft tissues from 3 snails per vessel were pooled together as one hemolymph sample and one soft tissue sample. Therefore, the number of samples per treatment group (n) was: 6, for experiments I and II, 4 for experiment III, and 3 for experiment IV. Both ChE and CES activities were measured in each sample. During the treatments animals were not fed. All bioassays were performed at a temperature of 22 ± 2 ◦ C, and under a photoperiod of 14:10 h light/dark without aeration. No mortality was observed either in control animals or in any of the treatments. Carbon filtered dechlorinated tap water was used as the test medium. Aqueous solutions containing the pesticides AZM and CPF were prepared by dissolving the pesticides in acetone, and diluted with an appropriate amount of dechlorinated tap water. Pesticide concentration was tested using HPLC with UV detector. Reversedphase chromatography was performed on a Shimadzu Class-VP model with isocratic pump (LC-10AT VP), a variable wavelength programmable UV–visible detector (SPD-10A VP) and a Supelcosil LC-18 (250 mm × 4.6 mm, 5 ␮m particle size) column. In the case of AZM, the operating conditions were: mobile phase: acetonitrile/water (45:55, v/v), flow rate: 1 mL min−1 , temperature: 40 ◦ C, run time: 17 min, and detection wavelength: 220 nm. Linearity was observed in the concentration range of 0.17–54.6 mg L−1 with regression equation y = 118,736.73x (mg L−1 ) + 974.10. In the case of CPF, the operation conditions were: mobile phase: acetonitrile/1 mM phosphate buffer (75:25 (v/v), pH 4.5), flow rate: 1 mL min−1 , temperature: 40 ◦ C, run time: 12 min, detection wavelength: 230 nm. Linearity was observed in the concentration range of 0.05–53 mg L−1 with regression equation y = 53,034x (mg L−1 ) + 857.29. With this procedure, we were not able to test all the concentrations used for the exposure experiments due to the limit of quantitation of the method (0.17 mg L−1 and 0.053 mg L−1 for AZM and CPF, respectively). Therefore, the only concentrations tested corresponded to the stock AZM and CPF solutions and to some AZM dilutions. The concentration values measured were always within the range 95–102% of the nominal values. To avoid pesticide degradation during exposure bioassays, test media was renewed every 24 h. The constancy of pesticide concentration during the time period of 24 h was tested in stability studies conducted in our laboratory using several AZM concentrations (0.25, 0.5 and 3 mg L−1 ) and one concentration of CPF (0.1 mg L−1 ). The 24-h average measured pesticide concentration remained in all cases within the range 82–98% of the initial concentration. In all experiments, the concentration of acetone was kept at 0.05% and solvent (acetone 0.05%) and solvent-free (dechlorinated tap water only) controls were included.

2.3.1. Single-chemical studies (experiment I) Snails were exposed for 48 h to nine nominal AZM concentrations (0.01; 0.025; 0.05; 0.1; 0.15; 0.25; 0.5; 1.5; and 3 mg L−1 ) or to six nominal CPF concentrations (0.001; 0.0025; 0.005; 0.01; 0.05; and 0.10 mg L−1 ). Absolute and relative EC50 values of ChE and CES inhibitors were calculated with the 4-parameter logistic model using OriginPro 7.5 (OriginLab, Northampton, MA). This model can be written as an equation that defines the response as a function of concentration and four parameters (A1 , A2 , p, x0 ). The model equation is specified by: y = A2 +

(A1 − A2 ) 1 + (x/x0 )

p

(1)

In this equation, “y” is the enzyme activity (expressed as nmol substrate hydrolyzed min−1 mg protein−1 ) at concentration “x” of the inhibitor. “A1 ” denotes the value of “y” for the maximal curve asymptote (theoretically, the level of response in the absence of pesticide). “A2 ” denotes the value of “y” for the minimal curve asymptote (theoretically, the level of response produced by an infinitely high concentration of the inhibitor). “p” denotes the steepness of the dose–response curve (Motulsky and Christopoulos, 2004). The relative EC50 is the parameter “x0 ” and represents the concentration corresponding to a response midway between the estimates of the top (A1 ) and the bottom (A2 ) plateaus. The four parameters were calculated with the software OriginPro 7.5 as the best-fit values in the model by an iterative procedure. Absolute EC50 is defined as the concentration giving exactly a 50% response (Laguerre et al., 2009) and was calculated from the logistic Eq. (1) considering y = half control activity level. 2.3.2. Binary-mixture studies (experiments II–IV) The effects of several combinations of AZM and CPF on ChE and CES activities were analyzed. In all cases, the concentration of each pesticide in the mixture tests was based on the EC50 values for soft tissue ChE inhibition derived from tests conducted with individual pesticides. In experiment II, exposures to three 50:50 combinations of AZM and CPF were performed: mixture 1: 0.25 EC50 units of AZM (1.3 ␮M = 0.42 mg L−1 ) + 0.25 EC50 units of CPF (3.6 nM = 1.25 ␮g L−1 ), mixture 2: 0.5 EC50 units of AZM (2.7 ␮M = 0.85 mg L−1 ) + 0.5 EC50 units of CPF (7.1 nM = 2.5 ␮g L−1 ), and mixture 3: 0.75 EC50 units of AZM (4 ␮M = 1.27 mg L−1 ) + 0.75 EC50 units of CPF (10.7 nM = 3.75 ␮g L−1 ). At the same time, vehicle control and single pesticide controls consisting of concentrations corresponding to 0.5 EC50 , 1.0 EC50 , and 1.5 EC50 were also performed. Snails were exposed to these mixtures for 48 h and ChE activity in hemolymph and soft tissue homogenates was measured. To analyze whether the effects of mixtures 1, 2 and 3 of the pesticides on ChE activity were additive, antagonistic or synergistic, the method described by Laetz et al. (2009) was used. In brief, the concentration of each pesticide was normalized to the respective EC50 concentration. In other words, each pesticide concentration was divided by the concentration estimated to produce a 50% decrease in ChE activity relative to carrier controls. The EC50 normalized data for the two pesticides were subsequently plotted in the same graph and jointly fit with a single regression to Eq. (1) using OriginPro 7.5 (OriginLab, Northampton, MA). When the software fits a curve with nonlinear regression, it can superimpose on the graph prediction bands. Therefore a single curve with a 95% prediction band was obtained. The 95% prediction band encloses the area that is expected to enclose 95% of future data points. In other words, if one adds one more experiment data point whose independent variable is within the independent variable range of the original dataset, there is 95% chance that the data point will appear within the prediction band. If the concentration–effect curves of the two OPs are parallel, this curve can be used for detecting interactions between them in mixtures. If concentration addition occurs, ChE inhibition for a mixture would fall on the curve or within the 95% prediction band for the regression. Results falling significantly above the curve (less than expected inhibition) would be antagonistic, and results falling significantly below the curve (more than expected inhibition) would be synergistic. In this way, the curve fit to the data from single-chemical trials can be used as a basis for detecting interactions between OP pesticides in mixtures. In experiment III, exposures to 25:75, 50:50 and 75:25 combinations of AZM and CPF were performed. Therefore, mixtures were composed as follows: 0.25 EC50 units of AZM (1.3 ␮M = 0.42 mg L−1 ) + 0.75 EC50 units

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of CPF (10.7 nM = 3.75 ␮g L−1 ), 0.5 EC50 units of AZM (2.7 ␮M = 0.85 mg L−1 ) + 0.5 EC50 units of CPF (7.1 nM = 2.5 ␮g L−1 ), and 0.75 EC50 units of AZM (4 ␮M = 1.27 mg L−1 ) + 0.25 EC50 units of CPF (3.6 nM = 1.25 ␮g L−1 ). At the same time, vehicle control and single pesticide controls consisting of concentrations corresponding to 1.0 EC50 were also performed. Snails were exposed to these mixtures for 48 h and ChE and CES activities in soft tissue homogenates were measured. In experiment IV, snails were exposed for 24 h to 0.2 EC50 units of AZM or CPF (1 ␮M = 0.32 mg L−1 AZM or 2.9 nM = 1 ␮g L−1 CPF) followed by a 24 h period of exposure to 1.0 EC50 units of CPF (14.3 nM = 5 ␮g L−1 ). ChE and CES activities were measured in soft tissues at 24 and 48 h after the first exposure.

were performed and specific activity was calculated using 13.6 and 11.8 mM−1 cm−1 as molar extinction coefficients, respectively.

2.4. Enzymatic determinations

3.1. Concentration dependence of azinphos-methyl and chlorpyrifos inhibition of ChE and CES activities (experiment I)

Animals were placed on ice for 6–8 min. The shells were dried with absorbent tissue paper and carefully removed. The hemolymph was collected with a pipette, placed into Eppendorf tubes and diluted 1:2 with cold 20 mM Tris/HCl buffer, pH 7.5, plus 0.5 mM EDTA. The soft tissues were washed in distilled water, placed on filter paper to drain extra fluids, and weighed. Soft tissues were homogenized (1:10, w:v) in cold 20 mM Tris/HCl buffer, pH 7.5, plus 0.5 mM EDTA. Homogenates were centrifuged at 11,000 × g for 20 min at 4 ◦ C and the supernatants were immediately used as enzymatic source. Protein content was determined according to the method of Lowry et al. (1951), using bovine serum albumin as standard. 2.4.1. ChE activity assay ChE activity was measured in 100 mM phosphate buffer, pH 8.0, 0.2 mM DTNB, and 1.5 mM AcSCh as substrate according to the method of Ellman et al. (1961). Each supernatant or hemolymph dilution was assayed for ChE activity in duplicate. Activity was recorded continuously at 412 nm and specific activity was expressed as nmol min−1 mg protein−1 . The enzymatic activity was corrected for spontaneous hydrolysis of the substrate and non-specific reduction of the chromogen by tissue extracts. 2.4.2. CES activity assay CES activity was determined using four different substrates: p-NPA, p-NPB, 1-NA, and 2-NA. Each supernatant or hemolymph dilution was assayed for CES activity in duplicate. Hydrolysis of p-NPA and p-NPB by CES was determined according to Kristoff et al. (2010). Reactions were performed in 2.5 mL 100 mM phosphate buffer pH 8.0 containing 5% acetone and 1 mM p-NPA or p-NPB. Activity was continuously recorded at 400 nm. Specific activity was calculated using the molar extinction coefficient for p-nitrophenol (18.6 mM−1 cm−1 ). CES activity using 1-NA or 2-NA as substrate was determined according to van Asperen (1962) with modifications. Hemolymph was diluted 1:12.5 and 11,000 × g supernatants of soft tissues were diluted 1:3 in 20 mM Tris/HCl buffer, pH 7.5, plus 0.5 mM EDTA. Reactions were performed in 2.5 mL 40 mM phosphate buffer pH 7.0 containing 5% acetone, 1 mM 1-NA or 2-NA, and sample. Sample volumes of 10 ␮L and 20 ␮L were chosen for the determinations with 1-NA and 2-NA, respectively. After 15 min incubation at 25 ◦ C, reaction was stopped by the addition of 500 ␮L of freshly prepared SDS-Fast Blue solution (2 parts of 1% Fast Blue RR salt in acetone and 5 parts of a 5% solution of SDS in 50% acetone). The solutions were allowed to stand at room temperature for 15 min and the absorbance of the naphthol–Fast Blue RR complex was measured at 600 nm (1-NA) or 550 nm (2-NA) using a Shimadzu UV-160A double-beam UV–visible spectrophotometer (Shimadzu, Kyoto, Japan). Calibration curves for 1- and 2-naphthol

2.5. Statistical analysis Results were expressed as mean ± S.D. Data were analyzed by one-way ANOVA followed by Tukey HSD post-test by using VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html). The level of significance used was 0.05. Prior to ANOVA, data were tested for normality and homogeneity of variance using the Shapiro–Wilk and Levene’s tests, respectively by using OriginPro 7.5. 3. Results

Results of ChE inhibition in soft tissue and hemolymph by increasing concentrations of AZM and CPF are shown in Fig. 1A and B, respectively. To measure ChE activity, AcSCh was used as substrate because previous studies from our laboratory have shown that ChE activity in both tissues was higher with AcSCh than with propionylthiocholine and butyrylthiocholine (Cacciatore et al., 2012; and data not shown). Enzyme activities, expressed as nmol substrate hydrolyzed min−1 mg protein−1 , were fitted to the logistic sigmoid equation y = A2 + (A1 − A2 )/(1 + (x/x0 )p ). The corresponding EC50 values and the parameters of the non-linear regressions are shown in Table 1. Results show that, in all cases, a concentration-dependent inhibition of ChE activity was observed. AZM was a less potent inhibitor of soft tissue and hemolymph ChE activity than CPF. Also, AZM EC50 was approximately one order of magnitude higher in the whole soft tissue than in hemolymph. On the contrary, CPF EC5O values in soft tissue and hemolymph were very similar. Regarding CES activity, it has been reported that not only basal activity but also sensitivity to pesticide inhibition might vary with the substrate used to perform the measurements (Kristoff et al., 2010; Laguerre et al., 2009). Therefore, studies were carried out with four different substrates: 1- and 2-NA, p-NPA, and p-NPB (Fig. 2 ). On the whole, CES activity in soft tissue and hemolymph was highly affected in AZM-exposed snails regardless of the substrate used (Figs. 2A, C, E, and G). In the case of hemolymph, the EC50 values for CES inhibition by AZM were of the same order than the EC50 values for ChE inhibition (Table 1). In contrast, CES activity in soft tissue was more sensitive to AZM inhibition than ChE activity. Regarding CPF exposed snails, CES activities in soft tissue and hemolymph were in most cases either not affected or only partially inhibited (Figs. 2B, D, F, and H). Due to these low inhibition responses, the only EC50 value that could be calculated corresponded to p-NPB hydrolysis in soft tissue, and resulted of the same order than the EC50 value for ChE inhibition (Table 1). 3.2. ChE and CES activities in snails exposed simultaneously to mixtures of azinphos-methyl and chlorpyrifos (experiments II and III) To study the effects of mixtures of the pesticides on ChE activity, the concentration of each pesticide was normalized to the respective soft tissue EC50 concentration (EC50 -normalized) and the results were combined and fitted with a single regression (Fig. 3A). This curve (y = 100/(1 + x1.12 ), r2 = 0.96618) with its 95% prediction band was used as a basis to determine whether specific combinations of the pesticides deviate from concentration addition in soft tissue.

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A Soft tissue Hemolymph

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Fig. 1. Effects of increasing concentrations of (A) azinphos-methyl and (B) chlorpyrifos on cholinesterase (ChE) activity in soft tissues and hemolymph. ChE activity was assayed using acetylthiocholine iodide (AcSCh) as substrate. Data points show means ± S.D. for 6 determinations. ChE activity from the solvent and solvent-free controls did not differ significantly (p > 0.05). Therefore, only data from the solvent control (acetone 0.05%) is shown. The curves are fits of the logistic sigmoid equation y = A2 + (A1 − A2 )/(1 + (x/x0 )p ), to the data using OriginPro version 7.5. The asterisk (*) denotes the values that are statistically different from controls (p < 0.05).

In experiment II, three combinations of AZM and CPF were performed. For experimental conditions see Section 2 and the legend to Fig. 3. The results showed that ChE activity in soft tissues of snails exposed to mixture 1 (Fig. 3 B) or in single pesticide controls (Fig. 3C) fell on the combined curve y = 100/(1 + x1.12 ) shown in Fig. 3A, or within the 95% prediction band. This indicates that for mixture 1 the expected concentration addition occurred. In contrast, mixtures 2 and 3 showed ChE inhibitions higher than the expected inhibitions, since results fell below the curve (Fig. 3B). This indicates that these two mixtures were synergistic. Besides, ChE activity in the hemolymph of snails exposed to mixtures 2 and 3 was more than 85% inhibited whereas in their corresponding single pesticide controls ChE inhibition was less than 60% (Fig. 4). Since mixture 2 (0.5 EC50 units of AZM + 0.5 EC50 units of CPF) showed a synergistic inhibition of ChE activity, it was further investigated whether the relative proportion of each pesticide in the mixture affected the degree of ChE and CES inhibition in soft tissue. To this end, snails were exposed to mixtures consisting of 0.75 EC50 units of AZM + 0.25 EC50 units of CPF or 0.25 EC50 units of AZM + 0.75 EC50 units of CPF and the results were compared to those of mixture 2 (Fig. 5). In all cases, the degree of ChE inhibition was higher in the mixtures than in single pesticide controls (Fig. 5A).

Interestingly, ChE inhibition in the mixture containing 0.25 EC50 units of AZM + 0.75 EC50 units of CPF was higher than in the other two combinations. In the case of CES (Fig. 5B), both 1-NA and pNPB hydrolyzing activities were lower in the snails exposed to the mixtures than in those exposed to CPF only. In contrast, the snails exposed to the mixtures showed similar or lower CES inhibition than AZM controls, with the exception of the mixture containing the lowest AZM concentration. In this last case, 1-NA hydrolyzing activity was slightly but statistically significantly higher than AZM controls.

3.3. ChE and CES activities in snails exposed sequentially to azinphos-methyl and chlorpyrifos (experiment IV) First, snails were exposed for 24 h to a concentration of AZM (0.32 mg L−1 ) that maximally inhibits CES activity while minimally inhibiting ChE activity in soft tissue. Afterwards the snails were exposed for another 24 h to CPF (5 ␮g L−1 ). These concentrations correspond to 0.2 units of EC50 and 1.0 units of EC50 for 48 h ChE inhibition by AZM and CPF, respectively. Besides, another group of snails were only exposed to CPF (1 ␮g L−1 for 24 h followed by

Table 1 Concentrations of azinphos-methyl (AZM) and chlorpyrifos (CPF) causing inhibitions of 50% (EC50 ) on cholinesterase and carboxylesterases activities in soft tissue and hemolymph, and the four parameter values of the non-linear regressions shown in Figs. 1 and 2. Tissue AZM Soft tissue

Hemolymph

CPFb Soft tissue Hemolymph a

Substratea

A1

A2

X0

r2

p

EC50 (mg L−1 )

EC50 (␮M)

AcSCh 1-NA 2-NA p-NPA p-NPB

294 785 940 201 195

± ± ± ± ±

10 28 23 5 7

0 0 0 0 21 ± 4

1.75 0.18 0.16 0.07 0.05

± ± ± ± ±

0.19 0.03 0.02 0.01 0.01

1.10 0.83 0.72 0.44 1.42

± ± ± ± ±

0.16 0.03 0.04 0.03 0.20

0.9817 0.9905 0.9955 0.9928 0.9928

1.75 0.18 0.16 0.07 0.06

± ± ± ± ±

0.19 0.03 0.02 0.01 0.01

5.51 0.57 0.51 0.22 0.19

± ± ± ± ±

0.60 0.10 0.06 0.03 0.03

AcSCh 1-NA 2-NA p-NPA p-NPB

449 926 1039 176 144

± ± ± ± ±

12 36 36 2 7

124 ± 15 0 0 14 ± 4 25 ± 3

0.08 0.05 0.08 0.02 0.11

± ± ± ± ±

0.01 0.01 0.01 0.01 0.03

0.85 0.71 0.60 0.49 0.68

± ± ± ± ±

0.13 0.07 0.05 0.04 0.11

0.9914 0.9902 0.9909 0.9944 0.9777

0.19 0.05 0.08 0.03 0.20

± ± ± ± ±

0.02 0.01 0.01 0.01 0.05

0.60 0.16 0.25 0.10 0.63

± ± ± ± ±

0.06 0.03 0.03 0.03 0.16

AcSCh p-NPB

296 ± 15 182 ± 7

17 ± 8 12 ± 9

0.004 ± 0.001 0.005 ± 0.001

1.22 ± 0.27 1.04 ± 0.18

0.9910 0.9947

0.005 ± 0.001 0.006 ± 0.001

0.014 ± 0.003 0.020 ± 0.010

AcSCh

440 ± 8

199 ± 8

0.003 ± 0.001

2.63 ± 0.41

0.9972

0.007 ± 0.001

0.021 ± 0.003

Cholinesterase activity was assayed using acetylthiocholine (AcSCh) as substrate and carboxylesterase activity was assayed using p-nitrophenyl acetate (p-NPA), pnitrophenyl butyrate (p-NPB), and 1- and 2-naphthyl acetate (1- and 2-NA). b The chlorpyrifos EC50 values for carboxylesterase activity in soft tissues and hemolymph towards 1-NA, 2-NA, p-NPA, and in hemolymph towards p-NPB could not be calculated because of the low inhibition responses observed.

L.C. Cacciatore et al. / Aquatic Toxicology 128–129 (2013) 124–134

B

1-NA

Soft tissues Hemolymph

800

CES activity

* *

600

* *

* * *

(nmol

* 0 0.0

0.5

*

*

*

1.0

1.5

*

(nmol

*

200

* 2.0

2.5

1-NA hydrolyzed min-1 mg proteín-1)

CES activity -1 -1 1-NA hydrolyzed min mg proteín )

A 1000

400

1-NA

1000

* 600

400

* 0 0.000

0.002

Soft tissues Hemolymph

1000

*

CES activity

600

* * * * *

400

* * 0.5

*

*

0 0.0

*

1.0

1.5

(nmol

*

*

200

* 2.0

2.5

2-NA

*

400

*

200

0 0.002

*

*

*

*

*

*

*

0.0

0.5

1.0

1.5

2.0

2.5

CES activity p-NPA hydrolyzed min-1 mg proteín-1)

F Soft tissues Hemolymph

*

0

0.010

0.10

Soft tissues Hemolymph

* *

*

*

100

*

* *

50

* 0 0.000

0.002

0.004

0.006

0.008

0.010

0.10

Chlorpyrifos (mg L-1)

H

150

* * * * *

* *

*

0 0.0

*

*

*

*

0.5

1.0

1.5

* * 2.0

Azinphos-methyl (mg L-1)

2.5

3.0

CES activity p-NPB hydrolyzed min-1 mg proteín-1)

250

Soft tissues Hemolymph

(nmol

p-NPB hydrolyzed min-1 mg proteín-1) (nmol

CES activity

0.008

150

3.0

200

50

0.006

200

G

p-NPB

100

0.004

p-NPA

250

Azinphos-methyl (mg L-1) 250

Soft tissues Hemolymph

600

0.000

(nmol

CES activity

-1 -1 (nmol p-NPA hydrolyzed min mg proteín )

50

0.10

Chlorpyrifos (mg L-1)

150

100

0.010

800

3.0

200

* * * * * * * *

0.008

1000

E

p-NPA

0.006

1200

Azinphos-methyl (mg L-1)

250

0.004

Chlorpyrifos (mg L-1)

D

2-NA

*

*

200

3.0

2-NA hydrolyzed min-1 mg proteín-1)

CES activity -1 (nmol 2-NA hydrolyzed min-1 mg proteín )

C 1200

Soft tissues Hemolymph

800

-1 Azinphos-methyl (mg L )

800

129

Soft tissues Hemolymph

p-NPB 200

150

* *

100

* *

50

* *

0 0.00

0.02

0.04

0.06

0.08

0.10

Chlorpyrifos (mg L-1)

Fig. 2. Effects of increasing concentrations of (A, C, E, G) azinphos-methyl and (B, D, F, H) chlorpyrifos on carboxylesterase (CES) activities in soft tissue. CES activity was assayed using (A and B) 1-naphthyl acetate (1-NA), (C and D) 2-naphthyl acetate (2-NA), (E and F) p-nitrophenyl acetate (p-NPA), and (G and H) p-nitrophenyl butyrate (p-NPB), as substrates. Data points show means ± S.D. for 6 determinations. CES activity from the solvent and solvent-free controls did not differ significantly (p > 0.05). Therefore, only data from the solvent control (acetone 0.05%) are shown. The effects of AZM on 1-NA, 2-NA, p-NPA, and p-NPB hydrolyzing activities and the effects of CPF on p-NPB hydrolyzing activity in soft tissue are fits of the logistic sigmoid equation y = A2 + (A1 − A2 )/(1 + (x/x0 )p ), to the data using OriginPro version 7.5. The asterisk (*) denotes the values that are statistically different from controls (p < 0.05).

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A

2 r = 0.96618

80

60

40

20

100

100

80

80

ChE activity (control %)

ChE activity (control %)

ChE activity (control %)

C

B y = 100/(1+ x1.12)

100

60

40

20

Mixture 1 Mixture 2 Mixture 3

0

0

CPF AZM 0.1

1

40

20

0

CPF AZM -20

-20

-20

60

0.1

10

1

EC50 (normalized)

10

EC50 (normalized)

0.1

1

10

EC50 (normalized)

Fig. 3. Cholinesterase (ChE) inhibition in soft tissue by single pesticides and 50:50 binary mixtures of azinphos-methyl (AZM) and chlorpyrifos (CPF) after normalization to their respective EC50 . (A) Plot of the concentration-response data shown in Fig. 1A and B after normalization to their respective EC50 concentrations and collectively fitted with the logistic sigmoid equation y = 100/(1 + x1.12 ). Data points show means ± S.D. for 6 determinations. The solid line shows the result from non-linear regression and dashed lines are the 95% prediction band. (B) Effects of 50:50 binary mixtures of AZM and CPF on ChE activity. Based on a default assumption of concentration addition, the pairings were predicted to yield soft tissue ChE inhibitions of 31% (0.5 EC50 , mixture 1), 50% (1.0 EC50 , mixture 2), and 61% (1.5 EC50 , mixture 3). Lines show the curve and the 95% prediction band depicted in A. Triangles represent individual determinations (n = 6 for each mixture). (C) Single pesticide reference controls consisting of concentrations corresponding to 0.5 EC50 (0.85 or 0.0025 mg L−1 , for AZM and CPF, respectively), 1.0 EC50 (1.7 or 0.005 mg L−1 , for AZM and CPF, respectively), and 1.5 EC50 (2.55 or 0.0075 mg L−1 , for AZM and CPF, respectively). These controls were performed at the same time as the assays with the binary mixtures shown in B. Lines show the curve and the 95% prediction band depicted in A. Triangles represent individual determinations (n = 6 for each condition). ChE activity from the solvent and solvent-free controls did not differ significantly (p > 0.05). Therefore, the enzyme activity was expressed as percentage of solvent controls.

ChE activity in hemolymph (control %)

AZM CPF AZM+CPF

b

80 70

c e

60 50

a

a

a

a

40 30 20

d f

10 0

MIXTURE 1

MIXTURE 2

MIXTURE 3

Fig. 4. Hemolymph cholinesterase (ChE) inhibition by single pesticides and binary mixtures of azinphos-methyl (AZM) and chlorpyrifos (CPF). The concentration of AZM and CPF in single pesticide controls and in the mixtures are the same as those depicted in Fig. 3. Results are expressed as percentage of un-exposed controls. Bars are means ± S.D. for 4–6 determinations. Means not followed by the same uppercase are significantly different at p < 0.05.

5 ␮g L−1 for another 24 h). ChE and CES activities were measured both in soft tissues and hemolymph at 24 and 48 h (Fig. 6). Results show that at 24 h (Fig. 6A and C), ChE and CES activities in CPF-exposed snails were not statistically inhibited (p > 0.05). In contrast, CES activities in AZM-exposed snails were highly inhibited (71% and 63% for soft tissue and hemolymph, respectively). Besides, ChE activity in AZM-exposed snails was only significantly inhibited in hemolymph (43% of inhibition). At 48 h (Fig. 6B and D), both ChE and CES activities were much more inhibited (p < 0.05) in the snails that were pretreated with AZM than in the snails pretreated with an equipotent concentration of CPF. 4. Discussion In the present investigation P. corneus snails were exposed for 48 h to different concentrations of AZM and CPF in

single-chemical and binary-mixture studies, and ChE and CES activities were measured in the whole organism soft tissues and hemolymph. Single-chemical bioassays (experiment I) showed that AZM produced a concentration-dependent inhibition of ChE and CES activities in both tissues. CPF also produced a concentrationdependent inhibition of ChE activity in both tissues but CES activities were in most cases either not affected or only partially inhibited by this pesticide. In experiment II, P. corneus snails were exposed to three binary mixtures consisting of 50:50 combinations of AZM + CPF. In the mixture with the lowest pesticide concentrations, additive inhibition of P. corneus ChE activity was found. In contrast, in the other two mixtures, synergistic effects were observed both in whole soft tissue and hemolymph. In experiment III, 25:75; 50:50; and 75:25 combinations of AZM and CPF were performed. Results showed that ChE inhibition in the mixture containing the lowest AZM concentration was higher than in the other two combinations. The fact that AZM and CPF produced in vivo inhibition of ChE and CES activities demonstrates the ability of P. corneus to metabolically activate these pesticides to their oxon forms. Besides, CPF was a more potent inhibitor of ChE activity than AZM (350 and 27 times for the whole soft tissue and hemolymph, respectively). Thus, while CPF produced significant inhibition of ChE activity at nanomolar levels, AZM did so at micromolar levels. Similarly, previous in vitro studies performed with the oxon forms of the pesticides showed that CPF-oxon was a more potent inhibitor of P. corneus ChE activity than AZM-oxon (Cacciatore et al., 2012). CPF EC5O values for ChE activity in soft tissue and hemolymph were very similar. In contrast, AZM EC50 was 9.2 times lower in hemolymph than in soft tissues. This higher sensitivity of hemolymph than soft tissues was particularly observed at low AZM concentrations (less than 0.5 mg L−1 ). For example, the exposure of snails to 0.1 mg L−1 AZM, which is an environmentally relevant concentration, did not produce a significant ChE inhibition in soft tissues when compared to controls. In contrast, this same concentration of the pesticide inhibited approximately 44% of ChE activity in hemolymph. In P. corneus, the hemolymph is easy to sample and has a higher specific ChE activity than soft tissues. Therefore, the measurement of ChE activity in this tissue in addition to soft tissues could be a convenient approach for evaluating the toxicity of anticholinesterase compounds in this gastropod. However, it should be

L.C. Cacciatore et al. / Aquatic Toxicology 128–129 (2013) 124–134

70

A

ChE activity (control %)

60

a a

50

AZM + CPF 40 b b

30

20

c

10

0

1-NA CES hydrolysing activity (control %)

AZM

70

CPF

0.75:0.25

0.50:0.50

0.25:0.75

B b

60

50

AZM + CPF 40 a

30

20 a

a

a

10

0

p-NPB CES hydrolysing activity (control %)

AZM

70

CPF

C

0.75:0.25

0.50:0.50

0.25:0.75

b

60

50

AZM + CPF 40

30 a a

20 a

10

a

0 AZM

CPF

0.75:0.25

0.50:0.50

0.25:0.75

AZM (mg L-1)

1.7

-

1.27

0.85

0.42

CPF (μg L-1)

-

5

1.25

2.5

3.75

Fig. 5. Soft tissue cholinesterase (ChE) and carboxylesterases (CES) inhibition by different binary combinations of azinphos-methyl (AZM) and chlorpyrifos (CPF). (A) ChE activity was assayed using acetylthiocholine iodide (AcSCh) as substrate. (B and C) CES activity was assayed using 1-naphthyl acetate (1-NA), and p-nitrophenyl butyrate (p-NPB) as substrates, respectively. Based on a default assumption of concentration addition, the pairings were predicted to yield soft tissue ChE inhibitions

131

taken into account that the sampling of hemolymph in P. corneus is an invasive procedure and that more research is required to support its use for field monitoring. Moreover, to our best knowledge the physiological role of ChE activity in the hemolymph of gastropods is not known. It has been reported that certain CES activities were more sensitive to inhibition by OPs than ChE in some aquatic invertebrates (Basack et al., 1998; Kristoff et al., 2012). In contrast, present results showed that P. corneus CES activities were equally or less sensitive than ChE activities in the hemolymph and the whole soft tissue of snails exposed to CPF. However, the present study also showed that P. corneus CES proved to be much more sensitive than ChE when the activities were measured in the whole soft tissue of the animals exposed to AZM. Therefore, it is suggested that both activities should be jointly measured to a proper assessment of the impact of OP pesticides in this species. The inhibition of some B esterase activities from P. corneus informed herein occurred at environmental relevant concentrations of the two OPs. Inhibition of B-esterases from freshwater gastropods exposed to environmental concentrations of CPF and AZM have also been informed for Potamopyrgus antipodarum (Gagnaire et al., 2008) and Biomphalaria glabrata (Kristoff et al., 2012), respectively. In contrast, Gagnaire et al. (2008) reported that ChE activity from the freshwater gastropod Valvata piscinalis was insensitive to low concentrations of CPF. Based on a default assumption of concentration addition, in experiment II we exposed P. corneus snails to mixtures of AZM + CPF designed to yield predicted soft tissue ChE inhibitions of 31% (mixture 1), 50% (mixture 2) and 61% (mixture 3). It was found that only mixture 1 produced a ChE inhibition in soft tissue and hemolymph that can be explained by the concentration addition model. In contrast, the other mixtures showed synergism in both tissues. Similarly, previous in vitro studies with binary mixtures of the oxon metabolites of AZM and CPF have shown additive effects on P. corneus soft tissue ChE activity at low concentrations and synergistic effects at higher concentrations (Cacciatore et al., 2012). Deviations of ChE inhibition from a concentration addition model have already been reported by Laetz et al. (2009), who determined AChE activity in brains of coho salmons co-exposed for 96 h to binary mixtures of the OPs diazinon (DZN), malathion and CPF. In that study salmons were exposed to mixtures designed to yield predicted brain AChE inhibitions of 10%, 29% and 50%. Their results showed that, with exception of the pair malathion + CPF at the lower exposure concentration, the degree of AChE inhibition in all the other possible pairings yielded rates of enzyme activity significantly lower than would be expected based on concentration addition. Interestingly, the mixtures of OPs designed to yield 30% of ChE inhibition showed additive effects in our study and synergistic effects in the study of Laetz et al. (2009). Several factors can account for these differences e.g. the pesticides assayed, the time of exposition, the species’ sensitivity, and the tissue used for the determination. As mentioned in Section 1, OPs which are thiophosphates have to undergo bioactivation by CYP-mediated desulfuration to their active oxon metabolites. Besides, CYPs also catalyze the OP detoxification via a dearylation reaction. In a study performed in rats, Timchalk et al. (2005) reported the in vivo pharmacokinetic

of 50%. Each bar represents the mean ± S.D. of 4 determinations. Black and white bars are single pesticide controls. Striped bars are the different AZM and CPF combinations assayed. The concentration of the pesticides in the different combinations is depicted in the table below the graphic. Means not followed by the same uppercase are significantly different at p < 0.05.

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ChE

CES First exposition (0-24 h)

First exposition (0-24 h) 110

110

A

90

80

80

70 60

*

50 40 30

50

*

30 20 10 0

AZ M

AZ M

CPF

Second exposition (24-48 h)

Soft tissue Hemolymph

110

CPF

Second exposition (24-48 h) #

110

100

B

D

100

90 80

*#

*#

60

*

30

*

20

CES activity (control %)

90

70

40

*

40

10 0

ChE activity (control %)

70 60

20

50

C

100

90

CES activity (control %)

ChE activity (control %)

100

80

*#

70 60 50

*

40 30

*

20

10

10

0

0

AZM//CPF

CPF//CPF

AZM//CPF

CPF//CPF

Fig. 6. Cholinesterase (ChE) and carboxylesterases (CES) activities in soft tissues and hemolymph of snails exposed sequentially to azinphos-methyl (AZM) and chlorpyrifos (CPF). 15 snails were divided in 5 groups: control (un-exposed), AZM (exposed for 0–24 h to 0.32 mg L−1 AZM), CPF (exposed for 0–24 h to 1 ␮g L−1 CPF), AZM//CPF (exposed for 0–24 h to 0.32 mg L−1 AZM followed by an exposition for another 24 h to 5 ␮g L−1 CPF) and CPF//CPF (exposed for 0–24 h to 1 ␮g L−1 CPF followed by an exposition for another 24 h to 5 ␮g L−1 CPF). (A) ChE and (C) CES activities were measured at 24 h. (B) ChE and (D) CES activities were measured at 48 h. ChE activity was assayed using acetylthiocholine iodide as substrate and CES activity was assayed using p-nitrophenyl butyrate (p-NPB) as substrate. Results are expressed as percentage of un-exposed controls. Bars are means ± S.D. from 3 independent experiments. (*) significantly different from controls (p < 0.05) and (#) significantly different from AZM//CPF group (p < 0.05).

interaction of a mixture of high doses of CPF and DZN. The authors adjudicated this interaction to a competition between CPF and DZN for CYP metabolism. They observed that co-exposure of rats to CPF/DZN at the binary dose of 60/60 mg/kg increased the maximum blood concentration (Cmax ) value and the area under the blood concentration curve and decreased the clearance for both parent compounds. However, a dose of 15/15 mg/kg did not alter the pharmacokinetics of CPF, DZN or their metabolites in blood. There is no information about CYP mediated metabolization of AZM and CPF in P. corneus. However, it can be hypothesized that if AZM and CPF compete for the same CYPs in P. corneus tissues, it is possible that one of them would favor the activation, in detriment of the detoxification, of the other OP. This would give rise to more molecules of oxon with the consequent synergistic effect on ChE inhibition. In addition to the CYP-mediated metabolism of OPs, the oxon metabolites can be hydrolyzed by A-esterases or may be “sequestered” by CES (Tang et al., 2006). As discussed before, AZM

was a potent CES inhibitor in P. corneus, having increased affinity for CES over ChE, whereas CPF was a potent ChE inhibitor. Therefore, the presence of AZM in the mixtures would have led to reduced CPF detoxification by CES with the consequent exacerbation of CPF toxicity. This effect was more relevant in mixtures 2 and 3 than in mixture 1 because the detoxification mechanism of CES enzymes is saturable (Tang et al., 2006). In addition, results obtained when the snails were exposed sequentially to the pesticides (experiment IV) showed that the sequence AZM/CPF produced at 48 h a higher ChE inhibition than the equipotent (based on single compound exposures) sequence CPF/CPF. In the first case, CES activity was highly inhibited during the first 24 h of exposure to low concentrations of AZM. This CES inhibition allowed that, in the second exposure step, more molecules of CPF-oxon were available to inhibit more molecules of ChE. In contrast, in the sequence CPF/CPF, CES activity remained either unaffected (during the first 24 h) or with high levels (at the end of the experiment). These

L.C. Cacciatore et al. / Aquatic Toxicology 128–129 (2013) 124–134

results suggest that CES enzymes are an important factor in determining the interaction between AZM and CPF in P. corneus. On the other hand, since we did not assay A esterase activities, the contribution of these last enzymes to the synergism of the AZM + CPF mixtures cannot be disregarded. However, taking into account that the OP hydrolysis via A esterases is not saturable, the role of these enzymes in the AZM + CPF mixtures would not be as relevant as CES. 5. Conclusions The present study shows the importance of measuring a battery of enzyme biomarkers (such as ChE and CES with different substrates) in different tissues to assess accurately the effect of pesticides in non-target species as gastropods. Using this approach, it was found that the exposure of P. corneus to environmentally relevant concentrations of AZM and CPF produced significant inhibitions of ChE and CES activities in soft tissues and hemolymph, CPF being a more potent inhibitor of ChE activity than AZM. Our results also showed that, depending on the concentration, mixtures of AZM and CPF can produce either additive or synergistic inhibition of P. corneus ChE activity. The occurrence of synergistic effects showed that a risk assessment based solely on single chemical bioassays may not properly protect non-target organisms. The data presented here indicate that CES enzymes would be important factors in determining the effects of the mixtures of OPs on ChE activity. However, other multiple additional factors can modify the cumulative effects of mixed exposures and should be further investigated. Acknowledgement This work was supported by grants from the Universidad de Buenos Aires (grants 20020100100455 and 20020110100070). References Basack, S.B., Oneto, M.L., Fuchs, J.S., Wood, E.J., Kesten, E.M., 1998. Esterases of Corbicula fluminea as biomarkers of exposure to organophosphorus pesticides. Bulletin of Environment Contamination and Toxicology 61, 569– 576. Benstead, R.S., Baynes, A., Casey, D., Routledge, E.J., Jobling, S., 2011. 17 ␤-Oestradiol may prolong reproduction in seasonally breeding freshwater gastropod molluscs. Aquatic Toxicology 101, 326–334. Bosgra, S., van Eijkeren, J.C.H., van der Schans, M.J., Langenberg, J.P., Slob, W., 2009. Toxicodynamic analysis of the combined cholinesterase inhibition by paraoxon and methamidophos in human whole blood. Toxicology and Applied Pharmacology 236, 9–15. Buratti, F.M., Volpe, M.T., Fabrizi, L., Meneguz, A., Vittozzi, L., Testai, E., 2002. Kinetic parameters of OPT pesticide desulfuration by c-DNA expressed human CYPs. Environmental Toxicology and Pharmacology 11, 181–190. Buratti, F.M., Volpe, M.T., Meneguz, A., Vittozzi, L., Testai, E., 2003. CYPspecific bioactivation of four organophosphorothionate pesticides by human liver microsomes. Toxicology and Applied Pharmacology 186, 143– 154. Buratti, F.M., Leoni, C., Testai, L., 2007. The human metabolism of organophosphorothionate pesticides: consequences for toxicological risk assessment. Journal für Verbraucherschutz und Lebensmittelsicherheit 2, 37–44. Cacciatore, L.C., Kristoff, G., Verrengia Guerrero, N., Cochón, A., 2012. Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus. Chemosphere 88, 450–458. Clarke, N., Routledge, E.J., Garner, A., Casey, D., Benstead, R., Walker, D., Watermann, B., Gnass, K., Thomsen, A., Jobling, S., 2009. Exposure to treated sewage effluent disrupts reproduction and development in the seasonally breeding Ramshorn snail (subclass: Pulmonata, Planorbarius corneus). Environmental Science and Technology 43, 2092–2098. Crane, A.L., Klein, K., Zanger, U.M., Olson, J.R., 2012. Effect of CYP2B6*6 and CYP2C19*2 genotype on chlorpyrifos metabolism. Toxicology 293, 115–122. Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95. Escartín, E., Porte, C., 1997. The use of cholinesterase and carboxylesterase activities from Mytilus galloprovincialis in pollution monitoring. Environmental Toxicology and Chemistry 16, 2090–2095.

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