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Prenatal exposure to dichlorvos: physical and behavioral effects on rat offspring
Neurotoxicology and Teratology 26 (2004) 607 – 614 www.elsevier.com/locate/neutera
Prenatal exposure to dichlorvos: physical and behavioral effects on rat offspring C.A. Lazarinia,*, R.Y. Limab, A.P. Guedesc, M.M. Bernardic a
Faculdade de Medicina de Marı´lia Disciplina de Farmacologia Av. Monte Carmelo, 800 Marı´lia, SP, CEP 17.519-030 Brazil b Bandeirantes University-Sa˜o Paulo, SP, Brazil c Department of Pathology, School of Veterinary Medicine and Zootechny, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Received 8 October 2003; received in revised form 10 March 2004; accepted 11 March 2004 Available online 6 May 2004
Abstract The effects of prenatal exposure to dichlorvos (DDVP), an organophosphate (OP) pesticide, on pups’ physical and neurobehavioral developments were investigated. Forty pregnant rats were treated by gavage with 8.0 mg/kg DDVP or its vehicle (1 ml/kg) from the 6th to the 15th day of pregnancy. At birth, pups were weighed, the litters culled to eight animals (four male and four female), and then observed for physical (pinna detachment, incisor eruption, eye opening, testes descent, and vaginal opening) and neurobehavioral developments (palmar grasp, surface righting, negative geotaxis, and open-field behaviors). As adults, open-field, apomorphine-induced stereotypy, and passive avoidance behaviors were also assessed. Results showed no differences between the body weight of DDVP and control-treated groups. No differences were observed on the measures of physical and neurobehavioral development. Locomotor activity of male pups at 21 days of age was decreased by DDVP exposure. Adult experimental offspring showed a decreased locomotor frequency and an increased immobility duration on open-field behavior in relation to control animals; the apomorphine-induced stereotyped behavior was decreased by the pesticide exposure as well as performance on the passive avoidance task. These data suggest that prenatal DDVP exposure was able to decrease offspring motor function (adolescence and adults) and conditioned response learning, probably by interference with the cholinergic – dopaminergic balance of activity involved with the control of motor function as well as the cholinergic system that modulates learning process. D 2004 Published by Elsevier Inc. Keywords: Dichlorvos; Prenatal; Behavior; Neurobehavioral development; Rat
1. Introduction Organophosphate (OP) insecticides are commonly used for small animals as flea and tick powders, sprays, foggers, shampoos and dips, flea collars, and formerly, as systemic insecticides. They are also frequently used as household, garden, and farm insecticides. They are highly toxic to all animals, including pets, livestock, and humans, although some are far more toxic than others. All OP insecticides are fat soluble and therefore are easily absorbed through the skin and then transported throughout the body. These chemicals kill insects and cause poisoning in animals by inhibiting the enzyme acetylcholinesterase (AChE) that normally functions to degrade acetylcholine (ACh) in nerve
* Corresponding author. Tel./Fax: +55-14-423-4344. E-mail address: [email protected] (C.A. Lazarini). 0892-0362/$ – see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.ntt.2004.03.006
synapses. Inhibition of AChE in the nerves results in a buildup of ACh and overstimulation of both muscarinic and nicotinic ACh receptors. Overstimulation of muscarinic receptors gives rise to the characteristic SLUDD signs of OP poisoning: salivation, lacrimation, urination, defecation, and dyspnea (due to increased bronchial secretions and bronchoconstriction), plus bradycardia and miosis. Overstimulation of nicotinic ACh receptors produces muscular fasiculations and tremors initially followed by flaccid paralysis. Death in acute poisonings is frequently due to respiratory failure resulting from inhibition of central (medullary) respiratory drive, excessive bronchial secretions, and bronchospasms coupled with depolarizing blockade at neuromuscular junctions (diaphragm and intercostals). Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) is an OP insecticide and antihelmintic agent with widespread use. Dichlorvos has a high acute toxicity; showing oral LD50 in rats between 56 and 108 mg/kg. It
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is classified by the WHO as a ‘highly hazardous’ agent of the IB class [18]. The dermal toxicity is similar to oral toxicity, and dermal exposure is a cause for concern [18]. DDVP vaporizes quickly and exposure by inhalation after its use in nonventilated or poorly ventilated areas has been reported to be one of the principal causes of human poisoning [13]. Splashing of concentrated formulations onto the skin is another cause of poisoning and failure to remove the splash has proved fatal. Prompt removal has resulted in initial symptoms of intoxication with a full recovery after treatment [13]. Some studies have shown several toxic perinatal effects in offspring exposed to DDVP. These effects include a delay in the onset of the first estrous cycle [33], decreased AChE activity in several brain areas and an increase of cholinesterase activity in the blood plasma [22] and brain hypoplasia [23]. There is some evidence shown that prenatal exposure to OP insecticides induces neurotoxic effects. Oral exposure to 1.0 mg/kg methamidophos during gestational organogenesis of rats did not promote evidence of maternal toxicity or affect body weight gain of the dams and offspring but interfered with the offspring’s physical and maturational development landmarks according to age. The behavioral performance of the offspring with or without a pharmacological challenge was tested at different postnatal days in an open-field apparatus or in the swimming behavior, but no overt signs were observed [11]. The organophosphorus compound trichlorfon was administered by stomach tube (100 mg/kg) to pregnant guinea pigs at two different stages of gestation (Days 36 – 38 and 51 –53). The pups developed locomotor disturbances, and postmortem examination revealed significantly decreased weights of the total brain and the cerebellum, as compared to controls. There was also a significant weight reduction particularly of the medulla oblongata, and also of the hippocampus, the thalamus, and the colliculi. Histological examination of the cerebellum revealed reduction of the external granular layer and the molecular layer, and regional absence of Purkinje cells. The activities of the neurotransmitter enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in the cerebellum were reduced as compared to the control values [2]. Rat pups which had been exposed to chlorpyrifos prenatally demonstrated significant behavioral neurotoxicity in the rotarod test compared to time-matched salinetreated litters [24]. In addition, developmental neurotoxicity of this OP pesticide is thought to involve both neurons and glia, thus producing a prolonged window of vulnerability [16], a reversible interference with the cholinergic system development of pups [28], and widespread deficiencies in catecholaminergic synaptic function that persist into adulthood [30]. Bigbee et al. [3] show that AChE, the enzyme that hydrolyzes the neurotransmitter ACh at cholinergic synapses and neuromuscular junctions, has an extrasynaptic, noncholinergic role during
neural development. Their findings suggest a morphogenic role of AChE as a cell adhesive function during neural development. The main objective of this study was to examine the long-lasting effects of prenatal exposure to a maximal nontoxic dose of DDVP, administered during the organogenic period of the rats’ pregnancy, on offspring physical and behavioral parameters observed during infancy and at adult age. Because changes in cholinergic activity would alter general activity [19,25], learning, and memory processes [26,27], as well as the motor behavior of rats [5,12], open-field activity, passive avoidance, and apomorphineinduced stereotypy, these behaviors were assessed in this study. The DDVP dose selected for use in this study was the maximal dose that did not induce signs of toxicity when administered during 10 consecutive days to female rats from our laboratory.
2. Methods 2.1. Animals Male and female Wistar rats, weighing 250– 270 g, about 90 days of age, housed under controlled temperature (22 – 24 jC), with a 12L:12D light schedule and free access to food and water, were used. These experiments followed the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. 2.2. Drug Dichlorvos (dichloro-vinyl-phosphate; DDVP) from Quimio-Ind. Quı´mica, was administered by the oral route (gavage). The DDVP vehicle (formulation not revealed, 1:50, w/v) was used as control solution. 2.3. Procedure 2.3.1. Reproductive parameters and maternal data Forty nulliparous female rats were distributed into groups of three and placed overnight with one young male rat, previously determined to be fertile. The onset of pregnancy was confirmed by the presence of spermatozoa in vaginal smears on the following morning, designated as gestation day 0 (GD0). On GD6, the dams were divided into two groups. One group (DDVP group, n=20) was treated once daily from GD6 to GD15 with 8.0 mg/kg of DDVP. The other group (control group, n=20) was identically treated with the DDVP vehicle (1 ml/kg, 1:50 solution, w/v). These pregnant rats were weighed at GD1, GD5, GD6, GD15, and GD21. The length of gestation, litter size, and sex ratio were also assessed. All the pregnant rats were allowed to litter normally and nurture their offspring. Parturition was considered to be postnatal day 0 (PND0).
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On PND1, all litters were examined externally, and sexed. The litters were culled to eight pups, four males and four females and the remaining pups were discarded. Litters from 20 rats (DDVP=10 and control=10) were used to study physical and neurobehavioral development, and open-field behavior until PND21. The other 20 litters (DDVP=10 and control=10) were used to open-field behavior, apomorphine-induced stereotypy, and one-way passive avoidance tests.
with no movement). Hand-operated counters and stopwatches were employed to score these behaviors. To minimize the influence of possible circadian changes on open-field behaviors, all animals were observed at the same time of day in each session. (between 1:00 and 5:00 p.m.). The device was washed with a 5% alcohol– water solution before testing each animal to obviate possible biasing effects due to odor clues left by previous rats.
2.3.2. Physical and neurobehavioral development One rat from each litter (DDVP=10, control=10) was weighed at PND1, PND2, PND7, PND14, and PND21. The following physical parameters were assessed in one male and one female of each litter: pinna detachment (the opening of ear channel, beginning on Day 2), incisor eruption (observation of superior and inferior teeth, beginning on Day 6), eye opening (opening of both eyes, beginning on Day 10), testes descent (descent of both testes to scrotum, beginning on Day 15), and vaginal opening (opening of vaginal channel, beginning on Day 30). The following reflexes were assessed in one male and one female of each litter: surface righting reflex (the animal was placed on its back and allowed to assume the normal upright position with all four feet on the table, beginning on Day 1; the animals had 15 s to show this response), negative geotaxis (to turn at least 180j after being placed faced down on a platform inclined 45j, beginning on Day 5; the animals had 30 s. to show this response), and palmar grasp reflex (grasps a paper clip with forepaws if stroked, beginning on Day 2). Pups were observed daily, between 9:00 and 9:30 a.m., and removed from mothers for observation and immediately returned to their home cage. The mean day of appearance of each of the above parameters was calculated. Data were analyzed considering the litter as the smallest unit. On PND21, the offspring were weaned and the littermates were housed together but separated by sex.
2.3.4. Apomorphine-induced stereotypy Rats were randomly and equally distributed into two groups of 10 animals per group (control and experimental group, respectively). The animals of both groups were distributed into two groups of five animals per group and injected with 0.3 or 0.6 mg/kg of apomorphine and observed for apomorphine-induced stereotypy in their cages. Stereotypy behavior was quantified every 10 min for 120 min after apomorphine, using the scoring proposed by Setler et al. [29]. Briefly, scores varying from 0 (asleep or stationary) to 6 (continuous licking and gnawing of cage grids) were assigned by an observer unaware of the drug treatment. Interobserver agreement was r=+0.98 between the scores from two different observers (Pearson’s correlation=0.98). However, the criteria are still subjective, although there is a high agreement between observers. The sum total of stereotypy behavior scores obtained in the first and second hours after apomorphine treatment was used to evaluate stereotypy behavior intensity.
2.3.3. Open-field studies The open-field behaviors of both male and female offspring were measured at weaning. Only male offspring were tested as adults. The devices were similar to that described by Broadhurst [6], i.e., a round arena [50 cm (for pups, PND21) and 97 cm (for adults) round surface surrounded by a 20-cm high enclosure] painted white and subdivided into 25 parts for pups (PND21), and 19 parts for adults, painted black. During the experiments, a 40-W white light bulb located 74 cm from the floor provided continuous illumination of the arena. For the observations, each animal was individually placed in the center of the arena and the following parameters were measured during a 5-min period: locomotion frequency (number of floor units entered with all paws), rearing frequency (number of times the animals stood on their hind legs), and immobility time (total number of seconds
2.3.5. One-way passive avoidance test Ten rats prenatally exposed to DDVP and 10 vehicle control rats constituted the experimental and control groups for the one-way passive avoidance test. The animals were trained and tested for one-way passive avoidance learning in the apparatus consisting of two boxes provided with a manual guillotine door placed between the two modular testing chambers. One chamber was illuminated by a 25-W light, while the other remained dark. Footshocks were applied through the grid floor in the dark chamber. In the training session, each animal was placed in the illuminated compartment with the sliding door closed. After 10 s, the door was opened and as soon as the animal stepped into the shock compartment, the door was closed and a footshock (0.5 mA, for 5 s) was delivered. Immediately after the footshock, the animal was removed from the apparatus. The maximum time for an animal to cross into the dark compartment was 300 s. In the test session, performed 7 days after the training session, the rat was placed in the illuminated compartment and after 10 s, the door was opened. The latency (maximum of 300 s) to enter the dark compartment (all four paws) was measured. No shock was administered during the test session. Those animals which had memory retention avoided entering or had a high latency time to enter the dark compartment, and this change
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in behavior was termed a passive avoidance response. The apparatus was washed with a 5% alcohol– water solution before testing each animal. Control and experimental rats were intermixed and the observations were made between 2:00 and 6:00 p.m. 2.4. Statistical analysis Results are expressed as litter meansFS.E. as the maternal unit to avoid litter effects. Two-factor ANOVA was used to analyze the data of control and experimental groups with sex and treatment as the factors. We employed an ANOVA followed by the Tukey post hoc test to compare homoscedastic data presenting interaction. To analyze data without interaction, the Student’s t test was used to compare groups with parametric data. For nonparametric data, the Mann – Whitney U test was employed. In all cases, results were considered significant for P<0.05.
3. Results Maternal weight gain (Fig. 1A and B) as well as offspring body weight (Fig. 1C and D) of animals exposed to DDVP during pregnancy were not modified by the insecticide. Two-factor ANOVA showed no differences in both physical development and reflexes (Table 1) between con-
Table 1 Effects of prenatal exposure to DDVP on physical and neurobehavioral development of rats Parameters
Pinna detachment Incisor eruption Eye opening Testes descent Vaginal opening Palmar grasp Surface righting Negative geotaxis
Male
Female
Control
DDVP
Control
DDVP
4.0F0.2 11.2F0.3 15.2F0.2 15.1F0.01 – 2.0F0.01 4.6F0.4 6.7F0.3
3.7F0.4 11.3F0.3 15.8F0.5 15.3F0.3 – 2.0F0.01 3.3F0.4 7.3F0.3
4.3F0.2 11.5F0.2 15.1F0.1 – 39.5F0.8 2.0F0.01 4.0F0.7 7.0F0.01
4.0F0.2 11.0F0.2 15.2F0.2 – 37.2F1.6 2.0F0.01 4.3F0.3 7.0F0.5
DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD6 to GD 15 of pregnancy. The values are represented as meansFS.E. days of age that each parameter occurred. n=10 for each group.
trol and experimental male and female offspring. Mann – Whitney U test showed no differences between control and experimental male on testes descent. Student’s t test showed no differences in vaginal opening between control and experimental females. In relation to the offspring open-field behavior (Fig. 2), the two-factor ANOVA showed difference in locomotor frequency [locomotion frequency: treatment: F(1,79)=12.13, P=0.0008; sex: F(1,79)=1.54, P=0.21; interaction: F(1,79)=0.02, P=0.89)]. Because no interaction was observed, the test was applied to analyze this
Fig. 1. Effects of maternal weight gain (A=Days 1 – 5 of pregnancy and B=Days 6 – 15 of pregnancy) and offspring corporea weight (C=Day 1 of being alive, and D=Day 21 of live) of rats. DDVP (8.0 mg/kg) or vehicle solution (control) were administered from GD 6 to GD 15. The values are represented as meansFS.E. n=20 for both groups.
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Fig. 2. Effects of prenatal exposure to DDVP on open field behavior of rats (21 days of age). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD 6 to GD 15. *P<0.05, compared to the control group (Student’s t-test). The values are represented as meansFS.E. n=10 for both groups.
parameter. Thus, this analysis showed a decreased locomotor activity in male pups of DDVP group in relation to control group. In relation to rearing frequency and
immobility duration, the two-factor ANOVA showed no differences between groups (Fig. 2). In addition, no differences were detected between sex, meaning no
Fig. 3. Effects of prenatal exposure to DDVP on open field behavior of rats (adulthood). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD6 to GD15. *P<0.05, compared to the control group (Student’s t-test). The values are represented as meansFS.E. n=10 for both groups.
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Fig. 4. Effects of prenatal exposure to DDVP on SB (0.3 mg/kg=A and B; 0.6 mg/kg=C and D) of rats (adulthood). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD 6 to GD 15. *P<0.05, compared to the control group (Mann – Whitney U-test). The values are represented as median. n=10 for both groups.
sexual dimorphism interference, probably in consequence of the pups’ age. In adults, experimental male offspring showed a decreased locomotor frequency and an increased immobility duration in relation to control animals (Fig. 3). The intensity of stereotypy behavior induced by 0.6 mg/kg apomorphine was also reduced by prenatal DDVP exposure at 60 and 120 min; no differences were observed between the stereotypy behavior of groups after the 0.3 apomorphine dose (Fig. 4A and B). The time-response curves showed that the 0.6 mg/kg apomorphine induced a lower level of stereotypy behavior as well as reducing the stereotypy behavior duration (Fig. 4C and D). Animals exposed to the pesticide during gestation also had a decreased performance in the passive avoidance task as adults (Table 2). Table 2 Mean and standard error of latency (s) to cross on pretest and test of passive avoidance task of male rats (adulthood) exposed to DDVP (8.0 mg/kg) from GD 6 to GD 15 of pregnancy Groups
Section Pretest
Test
Control DDVP
23.3F5.6 10.9F1.9
298.1F1.7 141.1F47.7*
Values are represented as meansFS.E. n=10 for both groups. *P<0.05, compared to the control group (Student’s t-test).
4. Discussion Previous work of our laboratory studied the effects, the possible embryotoxic effects, of 8 mg/kg DDVP dose administered during the organogenic period (unpublished data). This dose is the maximal dose that did not induce any sign of toxicity when administered during 10 consecutive days to pregnant dams (NOAEL); thus, in the present study, we evaluated the neurotoxic effect to this regimen of dose during the same period of pregnancy. The present data suggest that prenatal exposure to 8.0 mg/kg DDVP did not promote maternal or fetal toxicity, because no differences were observed between maternal or offspring body weights. The lack of increased pup mortality or changes in birthweights suggests that the DDVP did not induce severe pup toxicity. Prenatal DDVP exposure did not change the time course of male and female growth and physical development, including testes descent and vaginal opening. In addition, prenatal DDVP did not modify the development of dynamic postural adjustments of male and female pups because no differences were observed between palmar grasp, surface righting, and negative geotaxis reflexes (Table 1). These results suggest a lack of effects of prenatal DDVP exposure in these parameters of development. At weaning, prenatal exposure to DDVP decreased locomotor activity in the open field (Fig. 2); in adult
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DDVP-exposure animals, we found a decreased locomotor activity and a greater level of immobility (Fig. 3). Gupta et al. [17] and Bird et al. [4] found reduced exploratory and motor activities after prenatal exposure to mathylparathion and DDVP, respectively. Prenatal administration of sumithion, an OP insecticide, resulted in doserelated decreases in open-field activity and motor coordination in the offspring. Long-lasting alterations in the acquisition and extinction of a conditioned escape response, as well as increased social interactions, were observed in the adult offspring [20]. In relation to the open-field data, we observed reduced locomotor activity and an increased immobility duration, meaning a motor alteration. The present result suggests that prenatal exposure to a low DDVP dose reduced exploratory activity in adolescence and motor activity in adult age. The administration of 0.3 mg/kg apomorphine did not modify the intensity and the duration of stereotyped behavior of the animals prenatally exposed to DDVP (Fig. 4A and B). On the other hand, we observed a reduced stereotyped response to 0.6 mg/kg apomorphine at 60 and 120 min of observation (Fig. 4C and D). The time-effect curve shows a reduced stereotypy behavior duration and intensity. The apomorphine-stereotyped behavior is involved directly with the activation of postsynaptic striatal dopaminergic receptors [14,15,32]. Some evidence has demonstrated the existence of a dynamic interaction between the cholinergic and dopaminergic systems [1,9,10,12] where the cholinergic system inhibits the dopaminergic system. There are several reports showing that prenatal exposure to OP insecticide induces neurotoxic effects mainly in cholinergic pathways [7]. Thus, this could affect the cholinergic –dopaminergic imbalance and, in consequence, the apomorphine-stereotyped behavior expression. Our results suggest a possible precocious stimulation of muscarinic cholinergic receptors for the ACh during the organogenic period, promoting alterations in the development of the cholinergic and dopaminergic imbalance on striatal systems, resulting in a reduced effectiveness of apomorphine on the dopaminergic system. The prenatal exposure to DDVP modifies the passive avoidance behavior of the rats (Table 2). The OP pesticides interfere with the learning in OP perinatally exposed animals [20,21,34]. They damage the learned responses in several such tasks as in the T maze [8], Morris water maze [31], etc. The passive avoidance behavior was modified by the prenatal exposure to the OP, suggesting that the pathways linked to conditioned aversive response were damaged during the animals’ development. The performance on passive avoidance depends, partly, on the motor activity of the animals. Because we observed a reduced motor activity in open field in DDVP-exposed animals observed in adult age, and a low latency to cross to the dark compartment, no motor impairment could be related to the reduced retention of passive avoidance. In addition, the performance of experimental rats was similar to those of control group in the training section.
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Taken as a whole, the present data show that exposure to a low dose of DDVP during the organogenic period induces long-lasting effects on motor and learning behaviors.
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Prenatal exposure to dichlorvos: physical and behavioral effects on rat offspring C.A. Lazarinia,*, R.Y. Limab, A.P. Guedesc, M.M. Bernardic a
Faculdade de Medicina de Marı´lia Disciplina de Farmacologia Av. Monte Carmelo, 800 Marı´lia, SP, CEP 17.519-030 Brazil b Bandeirantes University-Sa˜o Paulo, SP, Brazil c Department of Pathology, School of Veterinary Medicine and Zootechny, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Received 8 October 2003; received in revised form 10 March 2004; accepted 11 March 2004 Available online 6 May 2004
Abstract The effects of prenatal exposure to dichlorvos (DDVP), an organophosphate (OP) pesticide, on pups’ physical and neurobehavioral developments were investigated. Forty pregnant rats were treated by gavage with 8.0 mg/kg DDVP or its vehicle (1 ml/kg) from the 6th to the 15th day of pregnancy. At birth, pups were weighed, the litters culled to eight animals (four male and four female), and then observed for physical (pinna detachment, incisor eruption, eye opening, testes descent, and vaginal opening) and neurobehavioral developments (palmar grasp, surface righting, negative geotaxis, and open-field behaviors). As adults, open-field, apomorphine-induced stereotypy, and passive avoidance behaviors were also assessed. Results showed no differences between the body weight of DDVP and control-treated groups. No differences were observed on the measures of physical and neurobehavioral development. Locomotor activity of male pups at 21 days of age was decreased by DDVP exposure. Adult experimental offspring showed a decreased locomotor frequency and an increased immobility duration on open-field behavior in relation to control animals; the apomorphine-induced stereotyped behavior was decreased by the pesticide exposure as well as performance on the passive avoidance task. These data suggest that prenatal DDVP exposure was able to decrease offspring motor function (adolescence and adults) and conditioned response learning, probably by interference with the cholinergic – dopaminergic balance of activity involved with the control of motor function as well as the cholinergic system that modulates learning process. D 2004 Published by Elsevier Inc. Keywords: Dichlorvos; Prenatal; Behavior; Neurobehavioral development; Rat
1. Introduction Organophosphate (OP) insecticides are commonly used for small animals as flea and tick powders, sprays, foggers, shampoos and dips, flea collars, and formerly, as systemic insecticides. They are also frequently used as household, garden, and farm insecticides. They are highly toxic to all animals, including pets, livestock, and humans, although some are far more toxic than others. All OP insecticides are fat soluble and therefore are easily absorbed through the skin and then transported throughout the body. These chemicals kill insects and cause poisoning in animals by inhibiting the enzyme acetylcholinesterase (AChE) that normally functions to degrade acetylcholine (ACh) in nerve
* Corresponding author. Tel./Fax: +55-14-423-4344. E-mail address: [email protected] (C.A. Lazarini). 0892-0362/$ – see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.ntt.2004.03.006
synapses. Inhibition of AChE in the nerves results in a buildup of ACh and overstimulation of both muscarinic and nicotinic ACh receptors. Overstimulation of muscarinic receptors gives rise to the characteristic SLUDD signs of OP poisoning: salivation, lacrimation, urination, defecation, and dyspnea (due to increased bronchial secretions and bronchoconstriction), plus bradycardia and miosis. Overstimulation of nicotinic ACh receptors produces muscular fasiculations and tremors initially followed by flaccid paralysis. Death in acute poisonings is frequently due to respiratory failure resulting from inhibition of central (medullary) respiratory drive, excessive bronchial secretions, and bronchospasms coupled with depolarizing blockade at neuromuscular junctions (diaphragm and intercostals). Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) is an OP insecticide and antihelmintic agent with widespread use. Dichlorvos has a high acute toxicity; showing oral LD50 in rats between 56 and 108 mg/kg. It
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is classified by the WHO as a ‘highly hazardous’ agent of the IB class [18]. The dermal toxicity is similar to oral toxicity, and dermal exposure is a cause for concern [18]. DDVP vaporizes quickly and exposure by inhalation after its use in nonventilated or poorly ventilated areas has been reported to be one of the principal causes of human poisoning [13]. Splashing of concentrated formulations onto the skin is another cause of poisoning and failure to remove the splash has proved fatal. Prompt removal has resulted in initial symptoms of intoxication with a full recovery after treatment [13]. Some studies have shown several toxic perinatal effects in offspring exposed to DDVP. These effects include a delay in the onset of the first estrous cycle [33], decreased AChE activity in several brain areas and an increase of cholinesterase activity in the blood plasma [22] and brain hypoplasia [23]. There is some evidence shown that prenatal exposure to OP insecticides induces neurotoxic effects. Oral exposure to 1.0 mg/kg methamidophos during gestational organogenesis of rats did not promote evidence of maternal toxicity or affect body weight gain of the dams and offspring but interfered with the offspring’s physical and maturational development landmarks according to age. The behavioral performance of the offspring with or without a pharmacological challenge was tested at different postnatal days in an open-field apparatus or in the swimming behavior, but no overt signs were observed [11]. The organophosphorus compound trichlorfon was administered by stomach tube (100 mg/kg) to pregnant guinea pigs at two different stages of gestation (Days 36 – 38 and 51 –53). The pups developed locomotor disturbances, and postmortem examination revealed significantly decreased weights of the total brain and the cerebellum, as compared to controls. There was also a significant weight reduction particularly of the medulla oblongata, and also of the hippocampus, the thalamus, and the colliculi. Histological examination of the cerebellum revealed reduction of the external granular layer and the molecular layer, and regional absence of Purkinje cells. The activities of the neurotransmitter enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in the cerebellum were reduced as compared to the control values [2]. Rat pups which had been exposed to chlorpyrifos prenatally demonstrated significant behavioral neurotoxicity in the rotarod test compared to time-matched salinetreated litters [24]. In addition, developmental neurotoxicity of this OP pesticide is thought to involve both neurons and glia, thus producing a prolonged window of vulnerability [16], a reversible interference with the cholinergic system development of pups [28], and widespread deficiencies in catecholaminergic synaptic function that persist into adulthood [30]. Bigbee et al. [3] show that AChE, the enzyme that hydrolyzes the neurotransmitter ACh at cholinergic synapses and neuromuscular junctions, has an extrasynaptic, noncholinergic role during
neural development. Their findings suggest a morphogenic role of AChE as a cell adhesive function during neural development. The main objective of this study was to examine the long-lasting effects of prenatal exposure to a maximal nontoxic dose of DDVP, administered during the organogenic period of the rats’ pregnancy, on offspring physical and behavioral parameters observed during infancy and at adult age. Because changes in cholinergic activity would alter general activity [19,25], learning, and memory processes [26,27], as well as the motor behavior of rats [5,12], open-field activity, passive avoidance, and apomorphineinduced stereotypy, these behaviors were assessed in this study. The DDVP dose selected for use in this study was the maximal dose that did not induce signs of toxicity when administered during 10 consecutive days to female rats from our laboratory.
2. Methods 2.1. Animals Male and female Wistar rats, weighing 250– 270 g, about 90 days of age, housed under controlled temperature (22 – 24 jC), with a 12L:12D light schedule and free access to food and water, were used. These experiments followed the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. 2.2. Drug Dichlorvos (dichloro-vinyl-phosphate; DDVP) from Quimio-Ind. Quı´mica, was administered by the oral route (gavage). The DDVP vehicle (formulation not revealed, 1:50, w/v) was used as control solution. 2.3. Procedure 2.3.1. Reproductive parameters and maternal data Forty nulliparous female rats were distributed into groups of three and placed overnight with one young male rat, previously determined to be fertile. The onset of pregnancy was confirmed by the presence of spermatozoa in vaginal smears on the following morning, designated as gestation day 0 (GD0). On GD6, the dams were divided into two groups. One group (DDVP group, n=20) was treated once daily from GD6 to GD15 with 8.0 mg/kg of DDVP. The other group (control group, n=20) was identically treated with the DDVP vehicle (1 ml/kg, 1:50 solution, w/v). These pregnant rats were weighed at GD1, GD5, GD6, GD15, and GD21. The length of gestation, litter size, and sex ratio were also assessed. All the pregnant rats were allowed to litter normally and nurture their offspring. Parturition was considered to be postnatal day 0 (PND0).
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On PND1, all litters were examined externally, and sexed. The litters were culled to eight pups, four males and four females and the remaining pups were discarded. Litters from 20 rats (DDVP=10 and control=10) were used to study physical and neurobehavioral development, and open-field behavior until PND21. The other 20 litters (DDVP=10 and control=10) were used to open-field behavior, apomorphine-induced stereotypy, and one-way passive avoidance tests.
with no movement). Hand-operated counters and stopwatches were employed to score these behaviors. To minimize the influence of possible circadian changes on open-field behaviors, all animals were observed at the same time of day in each session. (between 1:00 and 5:00 p.m.). The device was washed with a 5% alcohol– water solution before testing each animal to obviate possible biasing effects due to odor clues left by previous rats.
2.3.2. Physical and neurobehavioral development One rat from each litter (DDVP=10, control=10) was weighed at PND1, PND2, PND7, PND14, and PND21. The following physical parameters were assessed in one male and one female of each litter: pinna detachment (the opening of ear channel, beginning on Day 2), incisor eruption (observation of superior and inferior teeth, beginning on Day 6), eye opening (opening of both eyes, beginning on Day 10), testes descent (descent of both testes to scrotum, beginning on Day 15), and vaginal opening (opening of vaginal channel, beginning on Day 30). The following reflexes were assessed in one male and one female of each litter: surface righting reflex (the animal was placed on its back and allowed to assume the normal upright position with all four feet on the table, beginning on Day 1; the animals had 15 s to show this response), negative geotaxis (to turn at least 180j after being placed faced down on a platform inclined 45j, beginning on Day 5; the animals had 30 s. to show this response), and palmar grasp reflex (grasps a paper clip with forepaws if stroked, beginning on Day 2). Pups were observed daily, between 9:00 and 9:30 a.m., and removed from mothers for observation and immediately returned to their home cage. The mean day of appearance of each of the above parameters was calculated. Data were analyzed considering the litter as the smallest unit. On PND21, the offspring were weaned and the littermates were housed together but separated by sex.
2.3.4. Apomorphine-induced stereotypy Rats were randomly and equally distributed into two groups of 10 animals per group (control and experimental group, respectively). The animals of both groups were distributed into two groups of five animals per group and injected with 0.3 or 0.6 mg/kg of apomorphine and observed for apomorphine-induced stereotypy in their cages. Stereotypy behavior was quantified every 10 min for 120 min after apomorphine, using the scoring proposed by Setler et al. [29]. Briefly, scores varying from 0 (asleep or stationary) to 6 (continuous licking and gnawing of cage grids) were assigned by an observer unaware of the drug treatment. Interobserver agreement was r=+0.98 between the scores from two different observers (Pearson’s correlation=0.98). However, the criteria are still subjective, although there is a high agreement between observers. The sum total of stereotypy behavior scores obtained in the first and second hours after apomorphine treatment was used to evaluate stereotypy behavior intensity.
2.3.3. Open-field studies The open-field behaviors of both male and female offspring were measured at weaning. Only male offspring were tested as adults. The devices were similar to that described by Broadhurst [6], i.e., a round arena [50 cm (for pups, PND21) and 97 cm (for adults) round surface surrounded by a 20-cm high enclosure] painted white and subdivided into 25 parts for pups (PND21), and 19 parts for adults, painted black. During the experiments, a 40-W white light bulb located 74 cm from the floor provided continuous illumination of the arena. For the observations, each animal was individually placed in the center of the arena and the following parameters were measured during a 5-min period: locomotion frequency (number of floor units entered with all paws), rearing frequency (number of times the animals stood on their hind legs), and immobility time (total number of seconds
2.3.5. One-way passive avoidance test Ten rats prenatally exposed to DDVP and 10 vehicle control rats constituted the experimental and control groups for the one-way passive avoidance test. The animals were trained and tested for one-way passive avoidance learning in the apparatus consisting of two boxes provided with a manual guillotine door placed between the two modular testing chambers. One chamber was illuminated by a 25-W light, while the other remained dark. Footshocks were applied through the grid floor in the dark chamber. In the training session, each animal was placed in the illuminated compartment with the sliding door closed. After 10 s, the door was opened and as soon as the animal stepped into the shock compartment, the door was closed and a footshock (0.5 mA, for 5 s) was delivered. Immediately after the footshock, the animal was removed from the apparatus. The maximum time for an animal to cross into the dark compartment was 300 s. In the test session, performed 7 days after the training session, the rat was placed in the illuminated compartment and after 10 s, the door was opened. The latency (maximum of 300 s) to enter the dark compartment (all four paws) was measured. No shock was administered during the test session. Those animals which had memory retention avoided entering or had a high latency time to enter the dark compartment, and this change
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in behavior was termed a passive avoidance response. The apparatus was washed with a 5% alcohol– water solution before testing each animal. Control and experimental rats were intermixed and the observations were made between 2:00 and 6:00 p.m. 2.4. Statistical analysis Results are expressed as litter meansFS.E. as the maternal unit to avoid litter effects. Two-factor ANOVA was used to analyze the data of control and experimental groups with sex and treatment as the factors. We employed an ANOVA followed by the Tukey post hoc test to compare homoscedastic data presenting interaction. To analyze data without interaction, the Student’s t test was used to compare groups with parametric data. For nonparametric data, the Mann – Whitney U test was employed. In all cases, results were considered significant for P<0.05.
3. Results Maternal weight gain (Fig. 1A and B) as well as offspring body weight (Fig. 1C and D) of animals exposed to DDVP during pregnancy were not modified by the insecticide. Two-factor ANOVA showed no differences in both physical development and reflexes (Table 1) between con-
Table 1 Effects of prenatal exposure to DDVP on physical and neurobehavioral development of rats Parameters
Pinna detachment Incisor eruption Eye opening Testes descent Vaginal opening Palmar grasp Surface righting Negative geotaxis
Male
Female
Control
DDVP
Control
DDVP
4.0F0.2 11.2F0.3 15.2F0.2 15.1F0.01 – 2.0F0.01 4.6F0.4 6.7F0.3
3.7F0.4 11.3F0.3 15.8F0.5 15.3F0.3 – 2.0F0.01 3.3F0.4 7.3F0.3
4.3F0.2 11.5F0.2 15.1F0.1 – 39.5F0.8 2.0F0.01 4.0F0.7 7.0F0.01
4.0F0.2 11.0F0.2 15.2F0.2 – 37.2F1.6 2.0F0.01 4.3F0.3 7.0F0.5
DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD6 to GD 15 of pregnancy. The values are represented as meansFS.E. days of age that each parameter occurred. n=10 for each group.
trol and experimental male and female offspring. Mann – Whitney U test showed no differences between control and experimental male on testes descent. Student’s t test showed no differences in vaginal opening between control and experimental females. In relation to the offspring open-field behavior (Fig. 2), the two-factor ANOVA showed difference in locomotor frequency [locomotion frequency: treatment: F(1,79)=12.13, P=0.0008; sex: F(1,79)=1.54, P=0.21; interaction: F(1,79)=0.02, P=0.89)]. Because no interaction was observed, the test was applied to analyze this
Fig. 1. Effects of maternal weight gain (A=Days 1 – 5 of pregnancy and B=Days 6 – 15 of pregnancy) and offspring corporea weight (C=Day 1 of being alive, and D=Day 21 of live) of rats. DDVP (8.0 mg/kg) or vehicle solution (control) were administered from GD 6 to GD 15. The values are represented as meansFS.E. n=20 for both groups.
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Fig. 2. Effects of prenatal exposure to DDVP on open field behavior of rats (21 days of age). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD 6 to GD 15. *P<0.05, compared to the control group (Student’s t-test). The values are represented as meansFS.E. n=10 for both groups.
parameter. Thus, this analysis showed a decreased locomotor activity in male pups of DDVP group in relation to control group. In relation to rearing frequency and
immobility duration, the two-factor ANOVA showed no differences between groups (Fig. 2). In addition, no differences were detected between sex, meaning no
Fig. 3. Effects of prenatal exposure to DDVP on open field behavior of rats (adulthood). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD6 to GD15. *P<0.05, compared to the control group (Student’s t-test). The values are represented as meansFS.E. n=10 for both groups.
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Fig. 4. Effects of prenatal exposure to DDVP on SB (0.3 mg/kg=A and B; 0.6 mg/kg=C and D) of rats (adulthood). DDVP (8.0 mg/kg) or vehicle solution (control) were administered to dams from GD 6 to GD 15. *P<0.05, compared to the control group (Mann – Whitney U-test). The values are represented as median. n=10 for both groups.
sexual dimorphism interference, probably in consequence of the pups’ age. In adults, experimental male offspring showed a decreased locomotor frequency and an increased immobility duration in relation to control animals (Fig. 3). The intensity of stereotypy behavior induced by 0.6 mg/kg apomorphine was also reduced by prenatal DDVP exposure at 60 and 120 min; no differences were observed between the stereotypy behavior of groups after the 0.3 apomorphine dose (Fig. 4A and B). The time-response curves showed that the 0.6 mg/kg apomorphine induced a lower level of stereotypy behavior as well as reducing the stereotypy behavior duration (Fig. 4C and D). Animals exposed to the pesticide during gestation also had a decreased performance in the passive avoidance task as adults (Table 2). Table 2 Mean and standard error of latency (s) to cross on pretest and test of passive avoidance task of male rats (adulthood) exposed to DDVP (8.0 mg/kg) from GD 6 to GD 15 of pregnancy Groups
Section Pretest
Test
Control DDVP
23.3F5.6 10.9F1.9
298.1F1.7 141.1F47.7*
Values are represented as meansFS.E. n=10 for both groups. *P<0.05, compared to the control group (Student’s t-test).
4. Discussion Previous work of our laboratory studied the effects, the possible embryotoxic effects, of 8 mg/kg DDVP dose administered during the organogenic period (unpublished data). This dose is the maximal dose that did not induce any sign of toxicity when administered during 10 consecutive days to pregnant dams (NOAEL); thus, in the present study, we evaluated the neurotoxic effect to this regimen of dose during the same period of pregnancy. The present data suggest that prenatal exposure to 8.0 mg/kg DDVP did not promote maternal or fetal toxicity, because no differences were observed between maternal or offspring body weights. The lack of increased pup mortality or changes in birthweights suggests that the DDVP did not induce severe pup toxicity. Prenatal DDVP exposure did not change the time course of male and female growth and physical development, including testes descent and vaginal opening. In addition, prenatal DDVP did not modify the development of dynamic postural adjustments of male and female pups because no differences were observed between palmar grasp, surface righting, and negative geotaxis reflexes (Table 1). These results suggest a lack of effects of prenatal DDVP exposure in these parameters of development. At weaning, prenatal exposure to DDVP decreased locomotor activity in the open field (Fig. 2); in adult
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DDVP-exposure animals, we found a decreased locomotor activity and a greater level of immobility (Fig. 3). Gupta et al. [17] and Bird et al. [4] found reduced exploratory and motor activities after prenatal exposure to mathylparathion and DDVP, respectively. Prenatal administration of sumithion, an OP insecticide, resulted in doserelated decreases in open-field activity and motor coordination in the offspring. Long-lasting alterations in the acquisition and extinction of a conditioned escape response, as well as increased social interactions, were observed in the adult offspring [20]. In relation to the open-field data, we observed reduced locomotor activity and an increased immobility duration, meaning a motor alteration. The present result suggests that prenatal exposure to a low DDVP dose reduced exploratory activity in adolescence and motor activity in adult age. The administration of 0.3 mg/kg apomorphine did not modify the intensity and the duration of stereotyped behavior of the animals prenatally exposed to DDVP (Fig. 4A and B). On the other hand, we observed a reduced stereotyped response to 0.6 mg/kg apomorphine at 60 and 120 min of observation (Fig. 4C and D). The time-effect curve shows a reduced stereotypy behavior duration and intensity. The apomorphine-stereotyped behavior is involved directly with the activation of postsynaptic striatal dopaminergic receptors [14,15,32]. Some evidence has demonstrated the existence of a dynamic interaction between the cholinergic and dopaminergic systems [1,9,10,12] where the cholinergic system inhibits the dopaminergic system. There are several reports showing that prenatal exposure to OP insecticide induces neurotoxic effects mainly in cholinergic pathways [7]. Thus, this could affect the cholinergic –dopaminergic imbalance and, in consequence, the apomorphine-stereotyped behavior expression. Our results suggest a possible precocious stimulation of muscarinic cholinergic receptors for the ACh during the organogenic period, promoting alterations in the development of the cholinergic and dopaminergic imbalance on striatal systems, resulting in a reduced effectiveness of apomorphine on the dopaminergic system. The prenatal exposure to DDVP modifies the passive avoidance behavior of the rats (Table 2). The OP pesticides interfere with the learning in OP perinatally exposed animals [20,21,34]. They damage the learned responses in several such tasks as in the T maze [8], Morris water maze [31], etc. The passive avoidance behavior was modified by the prenatal exposure to the OP, suggesting that the pathways linked to conditioned aversive response were damaged during the animals’ development. The performance on passive avoidance depends, partly, on the motor activity of the animals. Because we observed a reduced motor activity in open field in DDVP-exposed animals observed in adult age, and a low latency to cross to the dark compartment, no motor impairment could be related to the reduced retention of passive avoidance. In addition, the performance of experimental rats was similar to those of control group in the training section.
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Taken as a whole, the present data show that exposure to a low dose of DDVP during the organogenic period induces long-lasting effects on motor and learning behaviors.
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