Influence of Dimethoate on Acetylcholinesterase Activity

Influence of Dimethoate on Acetylcholinesterase Activity

Environmental Toxicology and Chemistry, Vol. 24, No. 3, pp. 603–609, 2005 q 2005 SETAC Printed in the USA 0730-7268/05 $12.00 1 .00 INFLUENCE OF DIMETHOATE ON ACETYLCHOLINESTERASE ACTIVITY AND LOCOMOTOR FUNCTION IN TERRESTRIAL ISOPODS ELIZABETH L. ENGENHEIRO,†‡ PETER K. HANKARD,‡ JOSÉ P. SOUSA,† MARCO F. LEMOS,†§ JASON M. WEEKS,\ and AMADEU M.V.M. SOARES*§ †Instituto do Ambiente e Vida, Departamento de Zoologia, Universidade de Coimbra, Coimbra, Largo Marques de Pombal, 3004-517 Coimbra, Portugal ‡CEH Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE28 2LS, United Kingdom §Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal \WRc-NSF, Henley Road, Medmendham, Bucks, SL7 2HD, United Kingdom ( Received 3 March 2004; Accepted 12 August 2004) Abstract—Locomotor behavior in terrestrial organisms is crucial for burrowing, avoiding predators, food seeking, migration, and reproduction; therefore, it is a parameter with ecological relevance. Acetylcholinesterase (AChE) is a nervous system enzyme inhibited by several compounds and widely used as an exposure biomarker in several organisms. Moreover, changes in energy reserves also may indicate an exposure to a stress situation. The aim of this study is to link biomarkers of different levels of biological organization in isopods exposed to increasing doses of dimethoate in semifield conditions. Locomotor parameters, AChE activity, and energy reserves (lipid, glycogen, and protein contents) were evaluated in the isopod Porcellio dilatatus after 48-h and 10-d exposure to dimethoate-contaminated soil. Results showed a clear impairment of both locomotor and AChE activity during the entire study, although effects were more pronounced after 48 h. Most locomotor parameters and AChE activity showed a clear dose-response relationship. By contrast, no clear trend was observed on energetic components. A positive and significant relationship was found between AChE activity and those locomotor parameters indicating activity, and the opposite was observed with those locomotor parameters indicating confusion and disorientation. The results obtained in this study enhance the importance of linking biochemical responses to parameters with ecological relevance at individual level, the value of locomotor behavior as an important marker to assess effects of toxicants, and also the usefulness and the acquisition of ecological relevance by AChE as a biomarker, by linking it with ecologically relevant behavioral parameters. Keywords—Biomarker Dimethoate Acetylcholinesterase Energy reserves Locomotor behavior eral species and assays for their quantification also have been optimized [4]. Although the development of biomarkers has been rapid in the last two decades, the use of biomarkers per se, without correlating to population or community effects, lacks ecological relevance. When assessing effects of contaminants, it is now recognized that, if the focus is only on community change, subtle or chronic biological effects that result in irreversible longterm changes could be occurring in apparently healthy ecosystems, but initially would not be detected [5,6]. Another problem with measuring changes only at the population level is that little inference can be made with regard to the cause of the population decline. On the other hand, biomarkers have been identified that indicate that organisms have been exposed to general stress [7] and, in some cases, even to more specific stressors: Metallothionein responses following metal exposure [8] and acetylcholinesterase (AChE) inhibition following exposure to organophosphorous pesticides [7]. Though signaling that an exposure has taken place, such biomarkers contribute little to the prediction of the direct consequences for the organism or population in question. For this to be possible, a particular biomarker response should be related to a degree of impairment of growth, reproductive output, or metabolic function, which directly affects the survival of the organism and can be attributed to exposure to a known amount of the specific toxicant [9]. Acetylcholinesterase is a nervous system enzyme with the function to breakdown the neurotransmitter acetylcholine that INTRODUCTION In the last decades there has been an increased concern about the terrestrial environment and the effects of agricultural and industrial practices that lead to the contamination of soils by xenobiotics. The use and dissemination of artificial substances in nature, such as pesticides, pharmaceuticals, and other chemicals, may have effects on both target and nontarget organisms. The use of insecticides, therefore, may pose a risk for isopods and other key soil-dwelling organisms [1]. New biomarkers and toxicity tests have been developed to study and assess the effects of the toxic chemicals on soil biota. Biomarkers in terrestrial invertebrates have been widely used as tools to quantify exposure to, and effects of environmental pollutants. The Soil Ecotoxicological Risk Assessment System, an European network to promote the protection of the health of the soil environment, and the European Union (EU) funded project SECOFASE (Development, Improvement, and Standardization of Test Systems for Assessing Sublethal Effects of Chemicals on Fauna in the Soil Ecosystem), which focused on the development and standardization of toxicity tests with terrestrial invertebrates [2,3]. However, projects like BIOPRINT and BIOPRINT II, also EU-funded, have focused on the development of techniques for detection of new biomarkers to assess toxic effects of toxicants under laboratory and field conditions. Biomarkers have been identified in sev* To whom correspondence may be addressed (asoares@bio.ua.pt). 603 604 Environ. Toxicol. Chem. 24, 2005 generates postsynaptic potentials. In the absence of AChE, acetylcholine continues to stimulate the postsynaptic neuron to fire, and to do it in a disorganized way. Inhibition of AChE has been used widely as a biomarker to indicate contamination with organophosphates in beneficial invertebrates such as insects and isopods [10,11] and vertebrates such as rodents [12,13] and birds [14]. Locomotor behavior studies with isopods that assess the effects of dimethoate and several heavy metals also have been performed [15–18], but none of them relates these behavioral responses with biomarkers at the biochemical level. To our knowledge, only one study with the carabid beetle Pterostichus cupreus has been carried out that shows a clear quantitative relationship between AChE inhibition and altered locomotor behavior [19]. In organisms like isopods, which depend mostly on locomotion to perform all activities that enable them to find food, reproduce, and avoid predators, it especially is important to assess the effects of chemicals on these two parameters. The AChE inhibition and locomotor behavior allow us to relate the effects of this specific chemical with effects at the physiological and individual levels, which reflect the fitness of the population and community. In this study we have tried to link these biomarkers of two different levels of biological organization: AChE at the physiological level and locomotor parameters at the behavioral level. The objectives of the present study were to characterize the effects of sublethal exposure to dimethoate in semifield conditions on the locomotor behavior of isopods, and to analyze the time course and possible recovery of AChE activity and to link this to locomotor behavior effects. The hypothesis states that the expected AChE inhibition, induced by exposure to dimethoate, would originate changes in locomotor behavior in isopods, namely a decrease in activity and increase in disorientation. Moreover, if occurring, these effects are dose-related. The experiment involved monitoring locomotor behavior of Porcellio dilatatus Brandt, with a video tracking system following sublethal exposure to dimethoate. MATERIALS AND METHODS Woodlice, food, and culture procedures The isopods used in this experiment (P. dilatatus) were obtained from laboratory cultures kept at 228C with 16:8-h light:dark. Animals were kept in plastic boxes covered with a layer of sterilized sand. Ventilation was ensured by holes made in the box cover and periodic spraying with deionized water ensured adequate moisture levels. The isopods were fed ad libitum with alder leaves (Alnus glutinosa) previously oven dried, at 608C, for 2 d. Alder leaves were chosen mainly due to their higher nitrogen content [20]. Experimental setup The soil used in this semifield experiment was a black silt loam soil from Stuffins, United Kingdom. It was stored at the Center for Ecology and Hydrology, Monks Wood station (Abbots Ripton, Huntingdon, Cambridgeshire, UK) and it was transferred to microcosms within the heated mesocosms (see below) before chemical application. The soil parameters were 28% of organic matter content (measured as loss of ignition after 16 h, at 3758C), pH 5 7.2, and a density of 746 g/L (wet wt), after 2-mm sieving. The experiment was carried out in a greenhouse and the experimental bed was monitored for temperature and humidity values after installing the test chambers (mesocosms) and be- E.L. Engenheiro et al. Fig. 1. Schematic representation of the greenhouse bed experimental design (treatment doses were distributed randomly). fore starting the test itself. A single test chamber was composed of a 12-L mesocosm filled with test soil and having six tubes (microcosms) covered with a mesh to prevent animals from escaping (Fig. 1). The remaining space between each test chamber in the experimental bed was filled with sand. After chemical application (see below) a single isopod (previously weighed) and 1 g of oven-dried alder leaves were placed randomly inside each microcosm. A total of 144 animals were used (6 treatments, 12 animals per treatment, and 2 sampling times). Temperature and humidity were monitored throughout the test using Tiny Talk II Dataloggers (Gemini Dataloggers, Chichester, UK). Chemical application The field application rate for dimethoate in the United Kingdom is 340 g active ingredient per hectare [21]. We used 1 to 60 times the field application rate in order to obtain a concentration-effect range. The half-life of 1 kg/ha of dimethoate varies from 4 d under drought conditions to 2.5 d in rainfall and this time can be longer in soils under sterile conditions [22]. The dimethoate used was in the pure form (99% dimethoate) and it was applied to the mesocosms on an area basis (taking into account the spraying area and soil density), but in order to obtain the following doses in the top 5 cm of soil: 1 mg/g, 10 mg/g, 20 mg/g, 30 mg/g, and 60 mg/g of soil. Each mesocosm received one dose of dimethoate only, meaning that animals from the six microcosms within one mesocosm were exposed to the same treatment. Dimethoate is highly soluble in water, and it adsorbs only very weakly to soil particles, so it may be subject to considerable leaching [23,24]. To reduce this effect and to increase the adsorption of dimethoate to soil particles, the chemical was sprayed in a fume hood using a carrier of acetone:olive oil (19:1); the carrier was used as a control. Measurement of locomotor parameters The locomotor behavior of the animals was quantified following 48-h and 10-d incubation in the test chambers. These Environ. Toxicol. Chem. 24, 2005 Link AChE activity with locomotor function in woodlice sampling times were selected after carrying out a preliminary experiment in the laboratory using different times of exposure (0–20 d) with the same doses of dimethoate and assessing the AChE activity at each time. Locomotor behavior was evaluated using special test arenas and a computer-automated video tracking system. Parameters measured were total path length (total distance moved during the 2 h of tracking, expressed in m), average active velocity (average velocity while active, excluding rest periods, expressed in mm/s), number of stops per path (total number of times stopped per distance moved, expressed as number/m), total active time (total duration of time in activity, expressed in min), turning rate (total degrees turned as a function of path length, expressed as degrees/mm), and turning bias (absolute difference in degrees turned left and turned right as a function of path length, expressed as degrees/ mm). Before tracking, animals were weighed and after tracking they were frozen at 2708C until used for further measurements of AChE activity and energy reserves (protein, lipid, and glycogen contents). Test arena and computer-automated video tracking system After exposure, animals were acclimatized in the arenas for 1 h before being tracked individually for 2 h. A Wick-system described by Bayley [17] was created for the six arenas in order to maintain humidity at a constant level. The arenas, with a diameter of 20.3 cm, were set up with a substrate made of a mix of plaster of Paris and distilled water. Holes were incised in these arenas and strings of cotton were pulled through them to absorb the water from the second arena placed underneath. The Wick-system was prepared by using a 25.4cm arena underneath the 20.3-cm arena and by adding distilled water to it. The top arenas were left to dry overnight and the rims were surrounded by fluon (polyetrafluorethylene, ICI Chemicals and Polymers, Wilton, UK) in order to prevent woodlice from escaping. The temperature in the tracking room was kept stable at 218C and humidity at approximately 40%. The computer-aided video tracking system used was described previously by Baatrup and Bayley [25] and Bayley [17]. The image of the test system was captured by a video camera (JVC TK-1070E Colour Camera with a Cosmica 8– 48 1:10 Zoom Lens, New York, NY, USA) and digitized by a frame-grabber, which was interfaced with a desktop PC computer by the software package GIPSTRA (Image House, Copenhagen, Denmark). By using GIPSTRA, raw files were readable using MOTIO software (Institute of Biological Sciences, University of Aarhus, Aarhus, Denmark). Energy reserves measurements Frozen animals were decapitated and the heads were used to determine AChE activity (see below). The bodies were then homogenized in 1 ml of 0.15 M NaCl, using an electrical homogenizer, and centrifuged for 3 min at 8,000 rpm. Samples (homogenates) were kept on ice throughout the homogenization process to avoid protein denaturation and stored at 2708C until the energy reserves (protein, lipids, and glycogen) were determined. For protein determination, samples were diluted 1:10 (vol/ vol) with the buffer used to prepare the homogenates. Total protein was quantified following the method of Bradford [26] adapted to microplate [27,28]: 250 ml of diluted color reagent (Bio-Rad, Hercules, CA, USA) was added to 20 ml of all samples and to standards of bovine serum albumin. After 15 min, the absorbance was read at 595 nm. 605 The lipid fraction in the samples was extracted using a modification of the Blight and Dyer method [29] described by Ribeiro et al. [11]. Total lipids were determined using a modification of the method described by Ahlgren and Uppsala [30] based on the sulfophosphovanilline method of Zöllner and Kirsch [31], also described by Ribeiro et al. [11]. Absorbance was measured at 528 nm. Standards ranging from 0 to 100 mg of linoleic acid were prepared from a stock solution of 0.1% linoleic acid and received the same treatment as the samples. Glycogen was determined by using a modification of the method described by Ribeiro et al. [11]. The sulfuric acid method was then used to quantify glycogen in the samples. The absorbance was measured at 490 nm. Standards ranging from 0 to 100 mg of glucose were prepared from a stock solution of 0.1% glucose and received the same treatment as the samples. AChE analysis The heads of the frozen animals were homogenized in an eppendorf vial with 1 ml of phosphate buffer (0.1 M, pH 7.2), using an electrical homogenizer, and centrifuged for 3 min at 5,000 rpm. Acetylcholinesterase analyses were performed following Ellmans’ method [32] and the absorbance values read with a microplate reader [27,28]. Enzyme activity was expressed as nmol/ml/mg of protein/min. Data analysis All data were analyzed using Sigmastat (SPSS Science, Chicago, IL, USA) and STATISTICA (StatSoft, Tulsa, OK, USA) software packages. Comparisons between treatments on locomotor behavior, energy reserves, and enzymatic activities were done using one-way analysis of variance. Significant differences in relation to the control treatment were evaluated by the Dunnet test. The Log transformation was used to achieve normality for average velocity, number of stops, and active time for the 48-h sampling [33]. The Pearson Product-Moment Correlation was calculated between AChE activity and each locomotor parameter for both sampling times. A significance level of 0.05 was used for all statistical tests. Median effective concentration (EC50) calculations were done, when possible, using a sigmoid model [34]. RESULTS Survival After 48 h of incubation, exposure to dimethoate only affected survival of isopods on the two highest treatment doses (Fig. 2). This effect was more pronounced after 10 d of exposure, with 50% or more of animals dying after being exposed to 20 mg/g or higher of soil (Fig. 2). Locomotor behavior Exposure to dimethoate clearly influenced locomotor behavior after 48 h of exposure. A clear dose-response relationship was observed in most of the parameters measured after this time (Fig. 3) and this trend was maintained even after 10 d (Fig. 4). After 48 h of exposure, a significant decrease with increasing chemical doses was observed for path length (Fig. 3: F 5 6.58, p , 0.001) and active time (Fig. 3: F 5 6.11, p , 0.001). A similar trend was observed also for the average velocity (Fig. 3), but no significant differences between the control and treatments were observed (F 5 1.89, not significant [NS]). As expected, an inverse trend was observed for the number of 606 Environ. Toxicol. Chem. 24, 2005 E.L. Engenheiro et al. Fig. 2. Survival rates (%) for isopods exposed to dimethoate over two exposure times: 48 h (black) and 10 d (white). Ct 5 control, C1 5 5 mg a.i./g soil, C2 5 10 mg a.i./g soil, C3 5 20 mg a.i./g soil, C4 5 30 mg a.i./g soil, C5 5 60 mg a.i./g soil. stops per path (Fig. 3); in this case, significant differences between the control and treatments also were observed (F 5 6.12, p , 0.01). For both turning rate and turning bias (Fig. 3), no clear dose-response nor significant differences between exposed and nonexposed animals were observed (F 5 0.88, NS and F 5 1.15, NS, respectively). Similar trends in all the parameters were observed after 10 Fig. 4. Locomotor parameters (average 6 standard error) of Porcellio dilatatus after 10 d of exposure: Path length, average velocity, number of stops per path, active time, turn rate, and turn bias. Treatment codes are given in Figure 2. Asterisks indicate significant differences in relation to the control. d of exposure (Fig. 4), although some changes on the significance of effects of the chemical were observed when comparing with the responses obtained after 48 h. In this case, only for the number of stops per path (Fig. 4: F 5 5.15, p , 0.01) significant differences with the control values were observed. For both path length and active time (Fig. 4), despite the clear dose-response relationship, no significant differences were observed between exposed and control animals (F 5 1.31, NS and F 5 1.24, NS, respectively). AChE activity Acetylcholinesterase activity in isopods exposed to dimethoate was lower than in the control group and followed a clear dose-response relationship on both exposure times (Fig. 5). Significant differences between the control group and all the other treatments also were observed…

Save your time - order a paper!

Get your paper written from scratch within the tight deadline. Our service is a reliable solution to all your troubles. Place an order on any task and we will take care of it. You won’t have to worry about the quality and deadlines

Order Paper Now