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Behavioral effects of type II pyrethroid cyhalothrin in rats
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Toxicology and Applied Pharmacology 191 (2003) 167–176
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Behavioral effects of type II pyrethroid cyhalothrin in rats D. Abbud Righi and J. Palermo-Neto* Applied Pharmacology and Toxicology Laboratory, School of Veterinary Medicine, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil, 05508-900 Received 23 September 2002; accepted 10 May 2003
Abstract Synthetic pyrethroids such as cyhalothrin are extensively used in agriculture for the control of a broad range of ectoparasites in farm animals. It has been suggested that type II pyrethroids might induce anxiogenic-like effects in laboratory animals. The present study was undertaken to investigate a possible anxiogenic-like outcome of cyhalothrin in rats. Adult male rats were orally dosed for 7 days with 1.0, 3.0, or 7.0 mg/kg/day of cyhalothrin, present in a commercial formulation (Grenade Coopers do Brazil S.A.). The neurobehavioral changes induced by cyhalothrin as well as those produced on corticosterone serum levels were measured 24 h after the last treatment. Picrotoxin (1.0 mg/kg) was also acutely used as a positive control for anxiety. Results showed that cyhalothrin: (1) induced some signs and symptoms of intoxication that included salivation, tremors, and liquid feces; (2) reduced total locomotor activity in the open-field; (3) reduced the percentage of time spent in open-field central zones; (4) increased immobility time in the open-field; (5) reduced the percentage of time spent in plus-maze open arms exploration; (6) reduced the time spent in social interactions, and (7) increased the levels of serum corticosterone. The behavioral changes reported for cyhalothrin (3.0 mg/kg/day) were similar of those induced by picrotoxin. The no effect level dose obtained for cyhalothrin in this study was 1.0 mg/kg/day. These results provide experimental evidence that cyhalothrin induces anxiety-like symptoms, with this effect being dose-related. Thus, anxiety must be included among the several signs and symptoms of pesticide intoxication. © 2003 Elsevier Inc. All rights reserved. Keywords: Cyhalothrin; Type II pyrethroid; Insecticides; Picrotoxin; Anxiety; Stress; Open-field; Plus-maze; Social interactions; Corticosterone
Introduction Synthetic pyrethroid insecticides can be structurally distinguished by the absence (type I) or presence (type II) of an ␣-cyano group substituent at the ␣-carbon of the phenoxybenzyl moiety (Vershoyle and Aldridge, 1980; Soderlund et al. 2002). Cyhalothrin is a type II pyrethroid used predominantly on cattle and sheep and to a lesser extent in pigs and goats for the control of a broad range of ectoparasites, including flies, lice, and ticks. As with other type II compounds, cyhalothrin acts primarily on the CNS (Barnes and Verschoyle, 1974; Lawrence and Casida, 1982). Specifically, these pyrethroids reversibly after sodium channels in
* Corresponding author. Faculdade de Medicina Veterina´ria e Zootecnia, Laborato´rio de Farmacologia Aplicada e Toxicologia, Universidade de Sa˜o Paulo, Av. Prof Dr. Orlando Marques de Paiva, n° 87, CEP: 05508-900 Cidade Universita´ria, Sa˜o Paulo, SP, Brasil. Fax: ⫹55-11-30917829. E-mail address: [email protected] (J. Palermo-Neto). 0041-008X/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0041-008X(03)00236-9
the nerve membrane, causing persistent prolongation of the transient increase in the permeability of the neuronal membrane to this ion during excitation (Vijverberg et al., 1982; Narahashi, 1986; Aldridge, 1990; Vijverberg and van den Bercken, 1990). Type II pyrethroids depress resting chloride conductance, thereby amplifying any effects of sodium or calcium (Ray, 1991). Whereas type I pyrethroids induce tremors in humans and animals (T syndrome) at high doses, the toxic signs of type II pyrethroids comprise pawing and burrowing, profuse salivation, and coarse tremors that might progress to choreoathetosis and sometimes to clonic seizures, i.e., they produce the so-called CS syndrome (Gammon et al., 1981; Vershoyle and Aldridge, 1980). Deltamethrin, a type II pyrethroid, was also reported to produce hyperglycemia and elevated plasma levels of adrenaline and noradrenaline, most probably as a consequence of stimulation of the sympathoadrenal medullary system by deltamethrin (Cremer and Seville, 1982). Indeed, low doses of deltamethrin in-
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duce autonomic and neuroendocrine responses, suggesting that they might cause high levels of stress (Boer et al., 1988). In addition, fenvalerate, another type II pyrethroid, was reported to produce behavioral effects in rats that are similar to those produced by anxiogenic-like drugs (Spinosa et al., 1999), most probably by acting on ␥-aminobutyric acid (GABAA) receptor sites within the CNS (Crofton and Reiter, 1984; Bloomquist et al., 1986). Despite the relevance of stress and/or anxiety states for human and animal health and the fact that residues of cyhalothrin might be found in milk and tissues collected from cyhalothrin-treated animals, only few studies have been conducted to analyze the possible behavioral effects of type II pyrethroids in laboratory animals. In this respect, the Acceptable Daily Intake Dose (ADI) established for cyhalothrin by the FAO/WHO joint Expert Committee on Food Additives (JECFA) was considered temporary because cyhalothrin was reported to belong to a class of substances that are characterized by their toxicity to the CNS (FAO/WHO, 2000). ADI is the amount of residue of a chemical substance, expressed on a body weight basis, that can be ingested daily by humans over a lifetime without appreciable health risk. Therefore, neurobehavioral effects in laboratory animals were taken by the JECFA members as the most sensitive indicator of the toxicity of these compounds (FAO/WHO, 2000). Thus, the present study was undertaken to investigate the effects of a cyhalothrin formulation, Grenade, in rats. Open-field, plus-maze, and social interaction tests were performed due to their high efficacy in providing evidence of increased stress or anxiety levels in laboratory animals (File and Johnston, 1989; Pellow et al. 1985). To better characterize a possible anxiogenic-like effect of cyhalothrin in rats, picrotoxin was used in some experiments as a positive control; corticosterone serum levels were also measured after pesticide treatment.
Materials and methods Animals Two hundred male Wistar rats from our own colony weighing 250 –300 g and approximately 2 months old were used. These animals were housed under conditions of controlled temperature (22⫾2°C), humidity (65–70%), and artificial lighting (12-h light/12-h dark, lights on at 7:00 a.m.) with free access to rodent chow and water. The experiments were performed in a different room with similar housing conditions, to which the animals were transferred and maintained in their home cages 7 days before the beginning of the experiments. Animals were housed and used in accordance with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the School of Veterinary Medicine, University of Sa˜ o Paulo, Brazil.
Drugs Pesticide. A commercial formulation of Cyhalothrin (Grenade, Coopers Brazil LTDA; 45 g/L, 92.7% purity of active compound) was used. The samples used consist of four cyhalothrin isomers that comprise two pairs of enantiomers, A and B, in a 60:40 ratio; within each pair, the enantiomers exist in equal amounts. Literature data showed that the peak blood concentration of 14C-cyhalothrin was reached approximately 7 h after a 1 or 25 mg/kg dose. Cyhalothrin metabolism in rats is unaffected by the dose used and its major inactive metabolites found in rats were clopropyl carboxylic acid and 3-(4⬘hydroxyphenoxy) benzoic acid (FAO/WHO, 2000). In the present study, doses of 1.0, 3.0, and 7.0 mg/kg of cyhalothrin were prepared in distilled water and administered to the rats by gavage, once daily, for 7 days. Doses were corrected for the lower percentage of the active compound in the samples used. Theses cyhalothrin doses were at least 20 times smaller than those reported for DL50 in rats, i.e., 140 mg/kg diluted in corn oil (FAO/WHO, 2000), and therefore adequate for the search of a behavioral no effect level dose (NOEL). Previous pilot studies conducted in our laboratory suggested that subacute cyhalothrin treatment (3.0 mg/kg/day for 7 days) increased corticosterone serum levels in rats analyzed 24 h after the last dose. The vehicle employed for cyhalothrin solubilization in Grenade (Monilfenol etoxilate, 9,5 EO (RENEX) plus calcium sulfonate and AROL 3700) was used as the control solution, being administered by the same route, volume (1.0 ml/kg), and period. Behavioral analyses and serum corticosterone determinations were performed 24 h after the last drug or vehicle administration. Picrotoxin. Picrotoxin (Sigma), a noncompetitive GABAA receptor antagonist (Dalvi and Rodgers, 2001), diluted in 0.9% NaCl, was given subcutaneously to rats in a single dose of 1.0 mg/kg; 0.9% NaCl (1.0 ml/kg) was used as control. This dose is in the range of those reported to induce anxiogenic effects across a wide range of animal models (Prado de Carvalho et al., 1983; Corda and Biggio, 1986; Corbelt et al., 1991; Dalvi and Rodgers, 2001; Spinosa et al., 2002). Behavioral effects induced by picrotoxin were analyzed 30 minutes after its administration. Clinical observations The observational grid proposed by Irwin (1968) for toxicological screening was used. Thus, control and cyhalothrin-treated rats were observed daily (between 9:00 and 10:00 a.m.) i.e., immediately after each cyhalothrin treatment (or control solution), for the following overt signs and symptoms of intoxication: mortality, sedation, excitation, stereotypy, aggressiveness, piloerection, salivation, muscle tone, coarse tremors, convulsions, reactivity to touch, sleep, motor incoordination, gait, respiration, and quality of feces. Each symptom was assessed either by observing the spon-
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taneous behavior of the rats in their home cage or by subjecting the animals to standardized manipulations such as bilateral pressure on their flanks (muscle tone). Body weight was measured in animals of all groups immediately before the treatments on experimental days 1, 3, 5, and 7. Behavioral studies Open-field and plus-maze tests. Open-field and plus-maze tests were performed in sequence, starting with the openfield and immediately followed by the plus-maze. The openfield employed consisted of a round wooden arena (96 cm in diameter, 32.5-cm-high walls) painted gray and virtually divided on a computer (Ethovision; Noldus Information Technology, Leesburg, VA) into three zones: central, intermediary, and peripheral. For the observations, each rat was individually placed in the center of the apparatus and observed for 5 min for total locomotor activity (covered distance in cm) and for locomotor activity and time (s) spent by rats in each of the three open-field zones. The number of rearings (number of times the animal stood on its hind legs) and immobility time (number of seconds without movement) were also recorded. A video camera mounted 150 cm above the arena was used to collect data that were subsequently analyzed through the Ethovision System software (Noldus Information Technology) installed on an IBM-compatible computer placed in an adjacent room. The method used to calibrate the Ethovision system assumes the existence of a linear correlation between distances covered in the real world and distances reproduced in a digitized image. Thus, in order to calibrate this system, 35 points were set in the open-field arena; the distance (in cm) between these calibration points was used as real-world coordinates for system calibration as suggested by Noldus (2001). During observations, the system is programmed to “reconstruct” and to calculate in centimeters the total locomotor activity (in the whole arena) and the locomotion in each of the three open-field zones. Rearing activity and immobility time were manually recorded during Ethovision data acquisition with the help of an event recorder, i.e., by pressing and/or holding down two different keys assigned in the computer keyboard. Behavioral analyses in the plus-maze were performed immediately after the end of the open-field test; each rat was individually placed in the central square of a plus-maze apparatus and allowed 5 min of free exploration. Previous results from this and other laboratories have shown that testing animals in an open-field or in a hole board immediately before the plus-maze significantly elevates its basal activity in this apparatus, i.e., the total number of open and closed arm entries (Lister, 1987; File and Briley, 1991; Palermo-Neto and Guimara˜ es, 2000), resulting in easier analysis of plus-maze data. The plus-maze used in this study was made of wood and has two open arms (50 ⫻ 10 cm) and two enclosed arms of the same size with 40-cm high walls. The apparatus is painted gray, elevated 50 cm above the
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ground, and virtually divided, by the same software described above, into five zones: two closed arms, two open arms, and one central square. Behavioral recordings were made with the aid of this system. Calibration was performed as described above for the open-field. Briefly, 24 calibration points were evenly distributed within the apparatus, thus allowing the recording of following parameters: (1) number of entries into the plus-maze open and closed arms and (2) time spent in plus-maze open and closed arms exploration. The measurements that reflect anxiety levels in this test are the percentage of entries into open arms versus closed arms and the percentage of time spent in open arms versus closed arms (Pellow et al., 1985). Thus, the following formulas were employed to analyze the data: % open arm entries ⫽ open arm entries/(open arm entries ⫹ closed-arm entries) ⫻ 100; % time in the open arms ⫽ time in the open arms/(time in the open arms ⫹ time in the closed arms) ⫻ 100; % closed arm entries ⫽ closed arm entries/(open arm entries ⫹ closed arm entries) ⫻ 100, and % time in the closed arms ⫽ time in the closed arms/ (time in open arms ⫹ time in closed arms) ⫻ 100. To minimize the influence of possible circadian changes on open-field and plus-maze behaviors, control and experimental animals were alternated for observations, conducted at the same time of the day (between 8:00 a.m. and 1:00 p.m.). The experimental devices used were wiped with ethanol (5% in water) before introducing each animal to eliminate possible biasing effects due to odor cues left by previous rats. Social interaction. This test was performed in the same open-field described above, according to the methods proposed by File (1980) and File and Hide (1979). Briefly, rats were housed singly for 5 days prior to testing, being matched to form pairs according to their weight (no more than a 10-g difference), and submitted to two 7.5-min familiarization sessions in the test arena 48 and 24 h before the test. On the day of the social interaction test, stopwatches were used to manually record the total time in seconds spent by each pair of rats in active social interaction (sniffing, following, grooming, kicking, boxing, biting, and crawling under or over the partner) scored during a 7.5-min test session. To minimize possible biasing effects due to odor cues left by previous rats, the apparatus was wiped with ethanol in water before each behavioral test. Control and experimental pairs of rats were alternated and observed always between 8:00 a.m. and 1:00 p.m. Serum corticosterone determination Corticosterone is the most abundant circulating steroid secreted by rats and is considered a good indicator of adrenocortical function in this species (Sutanto and de Kloet, 1987; Sutanto et al., 1988). This hormone was determined in serum using commercially available kits (Coat-a-Count, DPC). This procedure is based on a solid-phase radioim-
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munoassay in which 125I-labeled corticosterone competes for a fixed time with the corticosterone present in the rat sample for antibody sites. Serum samples were assayed directly without extraction or purification. To decrease data variability on serum corticosterone levels, the rats were handled daily (5 min/day) for habituation to the experimental conditions of enthanasia and blood collection. Animals were enthanized decaptation after deep CO2 anesthesia. To minimize the reported circadian variations on serum glucocorticoid levels (Albers et al., 1985), control and cyhalothrin-treated animals were intermixed for enthanasia over just 1 day, i.e., 24 h after the last drug and/or control solution administration, i.e., between 8:00 a.m. and 9:00 a.m. Experimental design Five experiments were done in accordance with GLP protocols and quality assurance methods. In the first experiment, 40 rats were divided randomly and equally into four groups: three experimental groups (E1, E2, and E3) and one control group (C). The animals of groups E1, E2, and E3 received 1.0, 3.0, and 7.0 mg/kg of cyhalothrin, respectively, once daily for 7 days whereas those of group C were given the same number and volume (1.0 ml/kg) of the vehicle of cyhalothrin. Animals from all groups were observed daily (between 8:00 and 9:00 a.m.) for the presence or absence of overt clinical signs and symptoms of intoxication, as described above. Twenty-four hours after the last administration of cyhalothrin or vehicle, these rats were observed first for locomotor activity in the open-field and subsequently for the free exploration of a plus-maze apparatus, as described above. In the second experiment, 64 rats were divided and treated as stated above for the first experiment. Twenty-four hours after the last treatment, pairs of rats were placed in the open-field arena for social interaction measured as described above. Twenty-seven rats were used in the third and fourth experiments. In each experiment, the animals were divided randomly and equally into three groups: two experimental groups (P and E2) and one control group (C2). Animals from group P received a single dose of 1.0 mg/kg picrotoxin while those of group E2 received cyhalothrin (3.0 mg/kg/ day for 7 days); rats of group C2 were given a single injection of 1.0 ml/kg of 0.9% NaCl. Twenty-four hours after the last cyhalothrin administration and 30 min after 0.9% NaCl or picrotoxin, the animals were evaluated in the third experiment for open-field behavior (total locomotion, percentage of time spent in each open-field zone, rearing frequency, and immobility time) and plus-maze activity. In the fourth experiment the time spent in social interaction was recorded as described above. In the final and fifth experiment, 42 rats were divided randomly and equally into three groups: two experimental groups (E1 and E2) and one control group (C). The animals
of groups E1 and E2 received 1.0 and 3.0 mg/kg of cyhalothrin once daily for 7 days, respectively, whereas those of group C were given 1.0 ml/kg of the vehicle of cyhalothrin. Twenty-four hours after the last administration of drug or control solution, control and experimental animals were alternated for enthansia and blood collection for determination of serum corticosterone levels, performed as described above. Data analysis Bartlett’s test showed that the data were parametric (p⬍0.05). Thus, data were analyzed by one-way ANOVA (SYSTAT 7.01 for WINDOWS) followed by the Tukey– Kramer post-hoc test. In all experiments, the probability of p⬍0.05 was taken as an indication of statistical significance. Data are presented as means ⫾ SD.
Results Experiment 1 Administration of cyhalothrin (1.0 and 3.0 mg/kg/day) for 7 days did not induce overt clinical signs and symptoms of intoxication in rats. Indeed, differences were not found among rats of groups C, E1, and E2 concerning several items of the observational grid used to analyze toxicity. However, a similar treatment with a 7.0 mg/kg dose induced moderate signs of intoxication that included salivation, tremors, and liquid feces; these three symptoms started to appear in 60% of group E3 animals on treatment day 4. However, clinical signs and symptoms of toxicity were not observed in 40% of the rats treated with this dose of cyhalothrin even after the last day of treatment (day 7). Differences in weight were not found, with each group mean after the last treatment for this parameter being as follows: C ⫽ 274.3 ⫾ 16.8; E1 ⫽ 281.8 ⫾ 10.7; E2 ⫽ 270.6 ⫾ 18.2; E3 ⫽ 268.6 ⫾ 15.7. One rat of group E2 died during the experiment due to erroneous cyhalothrin administration. Open-field data of cyhalothrin-treated rats are depicted in Fig. 1. Cyhalothrin treatment reduced the total locomotion (F(3,35) ⫽ 7.498; p ⫽ 0.0005), the locomotion in the central open-field zone (F(3,35) ⫽ 6.779; p ⫽ 0.001), and the rearing frequency (F(3,35) ⫽ 3.098; p ⫽ 0.0392) of rats. No differences were found among the four groups concerning locomotor activity performed in the open-field peripheral (F(3,35) ⫽ 0.4303; p ⫽ 0.7326) and intermediary (F(3,35) ⫽ 1.376; p ⫽ 0.2663) zones. An increment in the immobility time (F(3,35) ⫽ 3.382; p ⫽ 0.0289) was also observed after cyhalothrin (3.0 mg/kg/day). However, no differences were found in this parameter between rats of groups C (control) and E3 (dosed with 7.0 mg/kg/day of cyhalothrin); animals treated with the highest dose of cyhalothrin displayed some nonambulatory activities within the open-field such as chewing, head bobbing, and tremors. The
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Fig. 1. Effects of cyhalothrin administration (once daily during 7 days) on total locomotion (A), locomotion in peripheral (B), intermediary (C), and central (D) open-field zone as well as on rearing frequency (E) and immobility time (F). Data are means ⫾ SD, for 10 rats per group for the groups vehicle, 1.0, and 7.0, and 9 rats for the group 3.0 mg/kg of cyhalothrin, respectively. ⽧p ⬍ 0.01 and 多p ⬍ 0.05 compared to group vehicle, ⴙp ⬍ 0.05 compared to group 1.0 mg/kg of cyhalothrin. ANOVA and Tukey–Kramer test.
NOEL for cyhalothrin effects on open-field behavior of rats was 1.0 mg/kg/day. Data in Table 1 indicate that cyhalothrin treatment (3.0
mg/kg) reduced the percentage of time spent by rats in the plus-maze open arms exploration (F(3,35) ⫽ 4.176; p ⫽ 0.0125) and increased the percentage of time spent in plus-
Table 1 Effects of cyhalothrin on behavior of rats observed in a plus-maze apparatus Treatment
Dose
% EOA
% ECA
% TOA
% TCA
Vehicle (ml/kg)
1.0 1.0 3.0 7.0
45.2 ⫾ 8.5 48.0 ⫾ 6.6 41.5 ⫾ 15.1 48.4 ⫾ 7.4
54.8 ⫾ 8.5 52.0 ⫾ 6.6 58.5 ⫾ 15.1 51.6 ⫾ 7.4
27.8 ⫾ 7.4 24.3 ⫾ 6.9 19.3 ⫾ 4.5* 28.3 ⫾ 5.4
72.1 ⫾ 7.4 75.7 ⫾ 6.9 80.7 ⫾ 4.5* 71.7 ⫾ 5.4
Cyhalothrin (mg/kg)
Note. Data are reported as means ⫾ SD for 10 rats per group for groups vehicle, 1.0, and 7.0, and 9 rats for the group 3.0 mg/kg cyhalothrin. Cyhalothrin was given once daily during 7 days. % EOA, percentage of entries in open arms, % ECA, percentage of entries in plus-maze closed arms; % TOA, percentage of time spent in open arms exploration; % TCA, percentage of time spent in closed arms exploration. * p ⬍ 0.05 compared to groups vehicle and 7.0, ANOVA and Tukey–Kramer test.
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Fig. 2. Effects of cyhalothrin (A) and picrotoxin (B) on the time spent by rats on social interaction within an open-field. Data are reported as means ⫾ SD for five to eight pairs per group. *p ⬍ 0.05 compared to group vehicle, #p ⬍ 0.05 and ##p ⬍ 0.001 compared to group 0.9% NaCl. ANOVA and Tukey–Kramer test.
maze closed arms (F(3,35) ⫽ 4.176; p ⫽ 0.0125). However, at this dose, this pyrethroid did not alter the percentage of entries into plus-maze open (F(3,35) ⫽ 0.9666; p ⫽ 0.4194) and closed arms (F(3,35) ⫽ 0.9666; p ⫽ 0.4194). Further analyses showed no differences between plus-maze data of vehicle and cyhalothrin (7.0 mg/kg)-treated rats. However, as stated above for the open field, rats treated with 7.0 mg/kg (group E3) presented some signs and symptoms of intoxication also within the plus-maze. The NOEL for cyhalothrin effects on plus-maze was 1.0 mg/kg/day. Experiment 2 Fig. 2A shows that cyhalothrin treatment at doses of 3.0 and 7.0 mg/kg/day for 7 days reduced the amount of time spent by rats in social interaction. The 1.0 mg/kg/day dose of cyhalothrin induced no effects (F(3,28) ⫽ 4.950; p ⫽ 0.0070), thus making it the NOEL.
tion of rats in the open-field (F(2,24) ⫽ 15.071; p ⬍ 0.0001); thus, data of cyhalothrin (3.0 mg/kg) obtained in experiment 1 were replicated. Further analysis showed that both picrotoxin and cyhalothrin decreased the percentage of time spent by rats in the central (F(2,24) ⫽ 7.267; p ⫽ 0.0034) open-field zone. No differences were found among the three groups concerning the percentage of time spent in the intermediary zone (F(2,24) ⫽ 2.211; p ⬎ 0.10). Although both treatments increased the percentage of time spent in open-field peripheral zone (C ⫽ 89.96 ⫾ 4.53; P ⫽ 94.81 ⫾ 3.78; E2 ⫽ 94.08 ⫾ 2.836), statistical significance was detected only for picrotoxin (F(2,24) ⫽ 4.303; p ⫽ 0.0253). Furthermore, picrotoxin and cyhalothrin data were not different (p ⬍ 0.05). A decrease in rearing frequency (F(2,24) ⫽ 18.996; p ⬍ 0.0001) and an increment in the immobility time of rats were also observed after picrotoxin or cyhalothrin treatment (F(2,24) ⫽ 13.348; p ⬍ 0.001). Data in Table 2 indicate that picrotoxin (1 mg/kg single dose) and cyhalothrin (3.0 mg/kg/day for 7 days) reduced the percentage of time spent by rats in the plus-maze open arms exploration (F(2,24) ⫽ 6.542; p ⫽ 0.0054) and increased the percentage of time spent in plus-maze closed arms (F(2,24) ⫽ 6.542; p ⫽ 0.0054). However, neither treatment was able to change the percentage of entries into plus-maze open (F(2,24) ⫽ 0.9847; p ⫽ 0.3882) and closed arms (F(2,24) ⫽ 0.9847; p ⫽ 0.3882). Thus, data on cyhalothrin (3.0 mg/kg) obtained in experiment 1 were replicated. Experiment 4 As it can be seen in Fig. 2B, picrotoxin (1 mg/kg) or cyhalothrin (3.0 mg/kg/day for 7 days) reduced the amount of time spent by rats in social interactions (F(2,12) ⫽ 13.403; p ⫽ 0.0009). Again, and importantly, data obtained previously for cyhalothrin (3.0 mg/kg) were replicated. Experiment 5 Biochemical analysis of serum corticosterone levels showed that cyhalothrin (3.0 mg/kg/day for 7 days; group E2) increased (F(2,36) ⫽ 4.169; p⬍ 0.05) the levels of this hormone compared to those measured in animals of the control group (C ⫽ 101.00 ⫾ 53.97 ng/ml; E1 ⫽ 112.00 ⫾ 68.24; and E2 ⫽ 173.25 ⫾ 79.53 ng/ml). The NOEL for cyhalothrin effects on serum corticosterone levels was 1.0 mg/kg/day.
Discussion Experiment 3 Open-field data of picrotoxin- (1.0 mg/kg single dose) and cyhalothrin- (3.0 mg/kg/day for 7 days) treated rats are depicted in Fig. 3. Both treatments decreased total locomo-
The NOEL obtained here for the behavioral effects of cyhalothrin is 1.0 mg/kg/day. This dose was unable to change not only the behavioral parameters analyzed in the open-field and plus-maze apparatuses, but also the amount of time spent
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Fig. 3. Effects of cyhalothrin (3 mg/kg for 7 days) and a single dose of picrotoxin (1.0 mg/kg) on total locomotion (A), percentage of time spent in the peripheral (B), intermediary (C), and central (D) open-field zones; as well as rearing frequency (E) and immobility time (F). Data are reported as means ⫾ SD, for nine rats per group. #p ⬍ 0.001; ⽧p ⬍ 0.01; *p ⬍ 0.05 compared to group 0.9% NaCl. ANOVA and Tukey–Kramer test.
by rats in social interaction. Conversely, the highest dose of cyhalothrin used (7.0 mg/kg/day for 7 days) induced signs and symptoms of intoxication that included salivation, tremors, and
liquid feces. Rats treated with this dose (group E3) also presented decreased locomotion and rearing within the open-field and spent less time in social interaction.
Table 2 Effects of cyhalothrin and picrotoxin on behavior of rats observed in a plus-maze apparatus Treatment
Dose
% EOA
%ECA
% TOA
%TCA
Control (ml/kg) Picrotoxin (mg/kg) Cyhalothrin (mg/kg)
1.0 1.0 3.0
40.5 ⫾ 9.6 34.4 ⫾ 19.7 43.9 ⫾ 12.0
59.5 ⫾ 9.6 65.6 ⫾ 19.7 56.1 ⫾ 12.0
22.8 ⫾ 10.8 8.5 ⫾ 5.2# 11.3 ⫾ 9.7*
77.2 ⫾ 10.8 91.5 ⫾ 5.2# 88.7 ⫾ 9.7*
Note. Data are reported as means ⫾ SD for nine rats per group. Control is 0.9% NaCl. Cyhalothrin was given once daily during 7 days and picrotoxin was given as a single 1.0 mg/kg dose. %EOA, percentage of entries in open arms; %ECA, percentage of entries in plus-maze closed arms; %TOA, percentage of time spent in open arms exploration, %TCA, percentage of time spent in closed arms exploration. * p ⬍ 0.05 and # p ⬍ 0.01 compared to control. ANOVA and Tukey–Kramer test.
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According to Crofton and Reiter (1984), type II pyrethroids decrease ambulatory behavior and induce nonambulatory activities such as chewing, face washing, and head bobbing in the open-field. Although these activities were not quantified here, they were also observed in this experiment after cyhalothrin (7.0 mg/kg), being responsible for the lack of changes reported for this pyrethroid on openfield immobility time and plus-maze open arms data. Indeed, signs and symptoms of intoxication were also detected here in rats treated with cyhalothrin (7.0 mg/kg/day for 7 days) within the plus-maze. Thus, 3.0 mg/kg/day of cyhalothrin for 7 days is the only regimen that can be used to analyze a possible stressor and/or anxiogenic-like effect for this type II pyrethroid in rats. Within the context of this discussion, stress is defined as a complex process by which animals respond to challenging or dangerous events (Gatchell and Baum, 1983; O’Leari, 1990). Thus, the stressful stimuli are referred to as “stressors” and the response as the “stress response.” In addition, “anxiety level” is operationally inferred as suggested elsewhere (Palermo-Neto et al., 2001; Palermo-Neto and Guimara˜ es, 2000; File, 1991; Dunn and File 1987; Pellow et al., 1985), i.e., as the response to a situation in which behavior is influenced by two opposing motivational forces (e.g., a natural curiosity to investigate unexplored or novel areas versus an aversion to open areas). Thus, when animals are introduced into an open-field, they are inclined to explore mainly the peripheral zone of the apparatus, i.e., they present a so-called positive thigmotaxis (Grossen and Kelley, 1973; Simon et al. 1994). According to Kelley (1993), stressors and/or anxiogenic drugs increase the amount of time spent by rodents in the peripheral zone of the open-field (closest to its walls), therefore decreasing their permanence in the central zones. As reported above, cyhalothrin (3.0 mg/kg/day for 7 days) induced a decrease not only in the total locomotor activity of rats observed in the open-field, but also, and importantly, in the percentage of time they spent exploring the open-field central zone. Furthermore, this cyhalothrin dose increased immobility in rats. In this respect, benzodiazepines (Meert et al. 1997; File, 1991) and 5-HT2 antagonists such as ritanserin (Meert et al. 1997), which are known to have anxiolytic effects, have been reported to cause increments in locomotor activity in different situations. Our present data also showed that cyhalothrin (3.0 mg/ kg/day for 7 days) decreased the percentage of time spent by rats in plus-maze open arms exploration, increasing at the same time the amount of time spent in closed arms exploration. Increments in the number of entries or in the time spent by animals in plus-maze open arm exploration are commonly taken as indicative of the presence of decreased levels of anxiety in these animals (Pellow et al., 1985; File, 1991). Indeed, anxiolytic drugs such as diazepam increase (Pellow et al., 1985) and yohimbine, pentylenetetrazole, caffeine, and amphetamine decrease the percentage of time spent by rats in plus-maze open arms exploration (Pellow et
al., 1985; Zangrossi and File, 1992; Pellow and File, 1986). Thus, since a reduction of locomotion in open-field central zones (Kelley, 1993) and/or in plus-maze open arm exploration (Comissaris, 1993; File, 1991; Pellow et al., 1985) are good indexes of both a stress response and/or anxiety levels, it seems possible to suggest that cyhalothrin (3.0 mg/kg/day for 7 days) might have an anxiogenic-like effect in rats. The data now obtained on social interaction reinforce the present assumption for anxiogenic-like effects for cyhalothrin (3.0 mg/kg/day for 7 days), in agreement with data reported elsewhere for fenvalerate, another type II pyrethroid insecticide (Spinosa et al., 1999). Indeed, anxiogenic drugs decrease (Baldwin et al., 1989; Bhattacharya and Mitra, 1992) while anxiolytic drugs increase (File and Pellow, 1985) the time spent by rats on social interaction in an open-field arena and, as observed in this study, cyhalothrin (3.0 mg/kg/day for 7 days) was shown to decrease this behavior. A possible anxiogenic-like effect of cyhalothrin treatment (3.0 mg/kg/day for 7 days) is strengthened by our data with picrotoxin. Indeed, as demonstrated here, cyhalothrin (3.0 mg/kg/day for 7 days) effects on open-field and plusmaze behaviors and on social interaction of rats were in the same direction of those induced by picrotoxin treatment. As expected, picrotoxin, a classic and well-known anxiogenic drug (Dalvi and Rodgers, 2001), not only decreased the total locomotion and increased immobility time of rats observed in an open-field, but also decreased the amount of time spent in the open-field central zone, increasing, at the same time, the percentage of time spent in the open-field peripheral zone. In this respect, and importantly, the data described here for picrotoxin concerning immobility time and the percentage of time spent by rats in open-field zones, as well as the data obtained from plus-maze and social interaction tests, were not statically different from those induced by cyhalothrin (3.0 mg/kg/day for 7 days). Finally, our results showed that cyhalothrin (3.0 mg/kg/ day for 7 days) increased corticosterone serum levels in rats. Accordingly, deltamethrin, another type II pyrethroid, was shown to increase corticosterone serum levels in rats (Boer et al., 1988), an effect also reported after picrotoxin (1.0 mg/kg) treatment in rats (Lakic et al., 1986). In this respect, it has been demonstrated that activation of the hypothalamic–pituitary–adrenocortical axis by stressors results in readily discernible elevation in plasma corticosterone levels (Axelrod and Reisene, 1984). Altogether, the behavioral and biochemical data reported here show that cyhalothrin (3.0 mg/kg/day for 7 days) might have an anxiogenic-like effect in rats. Most probably, these effects rely on cyhalothrin actions on GABAA receptor sites within the CNS, as already suggested for other type II synthetic pyrethroids (Lawrence and Casida, 1983; Gammon et al., 1982). It should be considered, however, that this anxiogenic-like effect of cyhalothrin is dose related and, as such, must be included among the several signs and symptoms of pesticide intoxication, i.e., they do not allow stating
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that cyhalothrin has an anxiogenic-like profile. In this respect, anxiety is a common symptom reported during the course of several kinds of seizures (Szyndler et al., 2001). Cyhalothrin is widely used in the control of a broad range of ectoparasites in food-producing animals; thus, our data might raise concerns about the possible effects of cyhalothrin residues in food. Moreover, the safety aspects of the use of cyhalothrin in veterinary medicine were considered at the 54th and 58th meetings of JECFA that identified a temporary ADI of 0 – 0.002 mg/kg of body wt per day for the induction of liquid feces in dogs, in a 26-week study (FAO/WHO, 2000). A high safety factor was used to account for the absence of a NOEL for liquid feces in dogs and the absence of a NOEL for neurobehavioral effects. Indeed, the Committee considered that the production of liquid feces might be a consequence of neurotoxic properties of cyhalothrin and further identified the pesticide-induced neurobehavioral effects as being a possible critical toxicological endpoint for synthetic pyrethroids (FAO/ WHO, 2000). The 1.0 mg/kg/day for 7 days NOEL, reported here for a cyhalothrin formulation, through the use of behavioral screening procedures, is coincident with the LOEL reported by the FAO/WHO expert members for the induction of liquid feces in dogs by cyhalothrin (FAO/ WHO, 2000). Thus, the behavioral data reported here for cyhalothrin might be relevant to the final statements on both ADI and MRLs for this synthetic pyrethroid insecticide. However, it should not be forgotten that some motor effects of deltamethrin (another type II pyrethroid) were reported to depend on the route of administration and/or vehicle employed (Crofton et al., 1995). Thus, although the vehicle of cyhalothrin used in these experiments produced no behavioral effects per se, it might be relevant to the pharmacokinetics and metabolization of cyhalothrin and therefore for the data now being reported for this type II pyrethroid. Acknowledgments The authors express sincere thanks to Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo (FAPESP) Foundation (Processes 99/04228-7 and 00/14937-4) and Conselho Nacional de Pesquisa (CNPQ) (Process 520050/ 97-5Nv) for the support given for this study. They also thank Professor Doctor Maria Martha Bernardi and Evelise de Souza Monteiro Fonseca from the Department of Pathology, School of Veterinary Medicine, University of Sa˜ o Paulo, for contributions made to this study. Finally, they express gratitude to Schering Plough Animal Health for donating Grenade and its vehicle. References Albers, H.E., Yogev, L., Todd, R.B., Goldman, B.D., 1985. Adrenal corticoids in hamsters: role in circadian timing. Am. J. Physiol. 248, 434 – 438.
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Behavioral effects of type II pyrethroid cyhalothrin in rats D. Abbud Righi and J. Palermo-Neto* Applied Pharmacology and Toxicology Laboratory, School of Veterinary Medicine, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil, 05508-900 Received 23 September 2002; accepted 10 May 2003
Abstract Synthetic pyrethroids such as cyhalothrin are extensively used in agriculture for the control of a broad range of ectoparasites in farm animals. It has been suggested that type II pyrethroids might induce anxiogenic-like effects in laboratory animals. The present study was undertaken to investigate a possible anxiogenic-like outcome of cyhalothrin in rats. Adult male rats were orally dosed for 7 days with 1.0, 3.0, or 7.0 mg/kg/day of cyhalothrin, present in a commercial formulation (Grenade Coopers do Brazil S.A.). The neurobehavioral changes induced by cyhalothrin as well as those produced on corticosterone serum levels were measured 24 h after the last treatment. Picrotoxin (1.0 mg/kg) was also acutely used as a positive control for anxiety. Results showed that cyhalothrin: (1) induced some signs and symptoms of intoxication that included salivation, tremors, and liquid feces; (2) reduced total locomotor activity in the open-field; (3) reduced the percentage of time spent in open-field central zones; (4) increased immobility time in the open-field; (5) reduced the percentage of time spent in plus-maze open arms exploration; (6) reduced the time spent in social interactions, and (7) increased the levels of serum corticosterone. The behavioral changes reported for cyhalothrin (3.0 mg/kg/day) were similar of those induced by picrotoxin. The no effect level dose obtained for cyhalothrin in this study was 1.0 mg/kg/day. These results provide experimental evidence that cyhalothrin induces anxiety-like symptoms, with this effect being dose-related. Thus, anxiety must be included among the several signs and symptoms of pesticide intoxication. © 2003 Elsevier Inc. All rights reserved. Keywords: Cyhalothrin; Type II pyrethroid; Insecticides; Picrotoxin; Anxiety; Stress; Open-field; Plus-maze; Social interactions; Corticosterone
Introduction Synthetic pyrethroid insecticides can be structurally distinguished by the absence (type I) or presence (type II) of an ␣-cyano group substituent at the ␣-carbon of the phenoxybenzyl moiety (Vershoyle and Aldridge, 1980; Soderlund et al. 2002). Cyhalothrin is a type II pyrethroid used predominantly on cattle and sheep and to a lesser extent in pigs and goats for the control of a broad range of ectoparasites, including flies, lice, and ticks. As with other type II compounds, cyhalothrin acts primarily on the CNS (Barnes and Verschoyle, 1974; Lawrence and Casida, 1982). Specifically, these pyrethroids reversibly after sodium channels in
* Corresponding author. Faculdade de Medicina Veterina´ria e Zootecnia, Laborato´rio de Farmacologia Aplicada e Toxicologia, Universidade de Sa˜o Paulo, Av. Prof Dr. Orlando Marques de Paiva, n° 87, CEP: 05508-900 Cidade Universita´ria, Sa˜o Paulo, SP, Brasil. Fax: ⫹55-11-30917829. E-mail address: [email protected] (J. Palermo-Neto). 0041-008X/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0041-008X(03)00236-9
the nerve membrane, causing persistent prolongation of the transient increase in the permeability of the neuronal membrane to this ion during excitation (Vijverberg et al., 1982; Narahashi, 1986; Aldridge, 1990; Vijverberg and van den Bercken, 1990). Type II pyrethroids depress resting chloride conductance, thereby amplifying any effects of sodium or calcium (Ray, 1991). Whereas type I pyrethroids induce tremors in humans and animals (T syndrome) at high doses, the toxic signs of type II pyrethroids comprise pawing and burrowing, profuse salivation, and coarse tremors that might progress to choreoathetosis and sometimes to clonic seizures, i.e., they produce the so-called CS syndrome (Gammon et al., 1981; Vershoyle and Aldridge, 1980). Deltamethrin, a type II pyrethroid, was also reported to produce hyperglycemia and elevated plasma levels of adrenaline and noradrenaline, most probably as a consequence of stimulation of the sympathoadrenal medullary system by deltamethrin (Cremer and Seville, 1982). Indeed, low doses of deltamethrin in-
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duce autonomic and neuroendocrine responses, suggesting that they might cause high levels of stress (Boer et al., 1988). In addition, fenvalerate, another type II pyrethroid, was reported to produce behavioral effects in rats that are similar to those produced by anxiogenic-like drugs (Spinosa et al., 1999), most probably by acting on ␥-aminobutyric acid (GABAA) receptor sites within the CNS (Crofton and Reiter, 1984; Bloomquist et al., 1986). Despite the relevance of stress and/or anxiety states for human and animal health and the fact that residues of cyhalothrin might be found in milk and tissues collected from cyhalothrin-treated animals, only few studies have been conducted to analyze the possible behavioral effects of type II pyrethroids in laboratory animals. In this respect, the Acceptable Daily Intake Dose (ADI) established for cyhalothrin by the FAO/WHO joint Expert Committee on Food Additives (JECFA) was considered temporary because cyhalothrin was reported to belong to a class of substances that are characterized by their toxicity to the CNS (FAO/WHO, 2000). ADI is the amount of residue of a chemical substance, expressed on a body weight basis, that can be ingested daily by humans over a lifetime without appreciable health risk. Therefore, neurobehavioral effects in laboratory animals were taken by the JECFA members as the most sensitive indicator of the toxicity of these compounds (FAO/WHO, 2000). Thus, the present study was undertaken to investigate the effects of a cyhalothrin formulation, Grenade, in rats. Open-field, plus-maze, and social interaction tests were performed due to their high efficacy in providing evidence of increased stress or anxiety levels in laboratory animals (File and Johnston, 1989; Pellow et al. 1985). To better characterize a possible anxiogenic-like effect of cyhalothrin in rats, picrotoxin was used in some experiments as a positive control; corticosterone serum levels were also measured after pesticide treatment.
Materials and methods Animals Two hundred male Wistar rats from our own colony weighing 250 –300 g and approximately 2 months old were used. These animals were housed under conditions of controlled temperature (22⫾2°C), humidity (65–70%), and artificial lighting (12-h light/12-h dark, lights on at 7:00 a.m.) with free access to rodent chow and water. The experiments were performed in a different room with similar housing conditions, to which the animals were transferred and maintained in their home cages 7 days before the beginning of the experiments. Animals were housed and used in accordance with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the School of Veterinary Medicine, University of Sa˜ o Paulo, Brazil.
Drugs Pesticide. A commercial formulation of Cyhalothrin (Grenade, Coopers Brazil LTDA; 45 g/L, 92.7% purity of active compound) was used. The samples used consist of four cyhalothrin isomers that comprise two pairs of enantiomers, A and B, in a 60:40 ratio; within each pair, the enantiomers exist in equal amounts. Literature data showed that the peak blood concentration of 14C-cyhalothrin was reached approximately 7 h after a 1 or 25 mg/kg dose. Cyhalothrin metabolism in rats is unaffected by the dose used and its major inactive metabolites found in rats were clopropyl carboxylic acid and 3-(4⬘hydroxyphenoxy) benzoic acid (FAO/WHO, 2000). In the present study, doses of 1.0, 3.0, and 7.0 mg/kg of cyhalothrin were prepared in distilled water and administered to the rats by gavage, once daily, for 7 days. Doses were corrected for the lower percentage of the active compound in the samples used. Theses cyhalothrin doses were at least 20 times smaller than those reported for DL50 in rats, i.e., 140 mg/kg diluted in corn oil (FAO/WHO, 2000), and therefore adequate for the search of a behavioral no effect level dose (NOEL). Previous pilot studies conducted in our laboratory suggested that subacute cyhalothrin treatment (3.0 mg/kg/day for 7 days) increased corticosterone serum levels in rats analyzed 24 h after the last dose. The vehicle employed for cyhalothrin solubilization in Grenade (Monilfenol etoxilate, 9,5 EO (RENEX) plus calcium sulfonate and AROL 3700) was used as the control solution, being administered by the same route, volume (1.0 ml/kg), and period. Behavioral analyses and serum corticosterone determinations were performed 24 h after the last drug or vehicle administration. Picrotoxin. Picrotoxin (Sigma), a noncompetitive GABAA receptor antagonist (Dalvi and Rodgers, 2001), diluted in 0.9% NaCl, was given subcutaneously to rats in a single dose of 1.0 mg/kg; 0.9% NaCl (1.0 ml/kg) was used as control. This dose is in the range of those reported to induce anxiogenic effects across a wide range of animal models (Prado de Carvalho et al., 1983; Corda and Biggio, 1986; Corbelt et al., 1991; Dalvi and Rodgers, 2001; Spinosa et al., 2002). Behavioral effects induced by picrotoxin were analyzed 30 minutes after its administration. Clinical observations The observational grid proposed by Irwin (1968) for toxicological screening was used. Thus, control and cyhalothrin-treated rats were observed daily (between 9:00 and 10:00 a.m.) i.e., immediately after each cyhalothrin treatment (or control solution), for the following overt signs and symptoms of intoxication: mortality, sedation, excitation, stereotypy, aggressiveness, piloerection, salivation, muscle tone, coarse tremors, convulsions, reactivity to touch, sleep, motor incoordination, gait, respiration, and quality of feces. Each symptom was assessed either by observing the spon-
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taneous behavior of the rats in their home cage or by subjecting the animals to standardized manipulations such as bilateral pressure on their flanks (muscle tone). Body weight was measured in animals of all groups immediately before the treatments on experimental days 1, 3, 5, and 7. Behavioral studies Open-field and plus-maze tests. Open-field and plus-maze tests were performed in sequence, starting with the openfield and immediately followed by the plus-maze. The openfield employed consisted of a round wooden arena (96 cm in diameter, 32.5-cm-high walls) painted gray and virtually divided on a computer (Ethovision; Noldus Information Technology, Leesburg, VA) into three zones: central, intermediary, and peripheral. For the observations, each rat was individually placed in the center of the apparatus and observed for 5 min for total locomotor activity (covered distance in cm) and for locomotor activity and time (s) spent by rats in each of the three open-field zones. The number of rearings (number of times the animal stood on its hind legs) and immobility time (number of seconds without movement) were also recorded. A video camera mounted 150 cm above the arena was used to collect data that were subsequently analyzed through the Ethovision System software (Noldus Information Technology) installed on an IBM-compatible computer placed in an adjacent room. The method used to calibrate the Ethovision system assumes the existence of a linear correlation between distances covered in the real world and distances reproduced in a digitized image. Thus, in order to calibrate this system, 35 points were set in the open-field arena; the distance (in cm) between these calibration points was used as real-world coordinates for system calibration as suggested by Noldus (2001). During observations, the system is programmed to “reconstruct” and to calculate in centimeters the total locomotor activity (in the whole arena) and the locomotion in each of the three open-field zones. Rearing activity and immobility time were manually recorded during Ethovision data acquisition with the help of an event recorder, i.e., by pressing and/or holding down two different keys assigned in the computer keyboard. Behavioral analyses in the plus-maze were performed immediately after the end of the open-field test; each rat was individually placed in the central square of a plus-maze apparatus and allowed 5 min of free exploration. Previous results from this and other laboratories have shown that testing animals in an open-field or in a hole board immediately before the plus-maze significantly elevates its basal activity in this apparatus, i.e., the total number of open and closed arm entries (Lister, 1987; File and Briley, 1991; Palermo-Neto and Guimara˜ es, 2000), resulting in easier analysis of plus-maze data. The plus-maze used in this study was made of wood and has two open arms (50 ⫻ 10 cm) and two enclosed arms of the same size with 40-cm high walls. The apparatus is painted gray, elevated 50 cm above the
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ground, and virtually divided, by the same software described above, into five zones: two closed arms, two open arms, and one central square. Behavioral recordings were made with the aid of this system. Calibration was performed as described above for the open-field. Briefly, 24 calibration points were evenly distributed within the apparatus, thus allowing the recording of following parameters: (1) number of entries into the plus-maze open and closed arms and (2) time spent in plus-maze open and closed arms exploration. The measurements that reflect anxiety levels in this test are the percentage of entries into open arms versus closed arms and the percentage of time spent in open arms versus closed arms (Pellow et al., 1985). Thus, the following formulas were employed to analyze the data: % open arm entries ⫽ open arm entries/(open arm entries ⫹ closed-arm entries) ⫻ 100; % time in the open arms ⫽ time in the open arms/(time in the open arms ⫹ time in the closed arms) ⫻ 100; % closed arm entries ⫽ closed arm entries/(open arm entries ⫹ closed arm entries) ⫻ 100, and % time in the closed arms ⫽ time in the closed arms/ (time in open arms ⫹ time in closed arms) ⫻ 100. To minimize the influence of possible circadian changes on open-field and plus-maze behaviors, control and experimental animals were alternated for observations, conducted at the same time of the day (between 8:00 a.m. and 1:00 p.m.). The experimental devices used were wiped with ethanol (5% in water) before introducing each animal to eliminate possible biasing effects due to odor cues left by previous rats. Social interaction. This test was performed in the same open-field described above, according to the methods proposed by File (1980) and File and Hide (1979). Briefly, rats were housed singly for 5 days prior to testing, being matched to form pairs according to their weight (no more than a 10-g difference), and submitted to two 7.5-min familiarization sessions in the test arena 48 and 24 h before the test. On the day of the social interaction test, stopwatches were used to manually record the total time in seconds spent by each pair of rats in active social interaction (sniffing, following, grooming, kicking, boxing, biting, and crawling under or over the partner) scored during a 7.5-min test session. To minimize possible biasing effects due to odor cues left by previous rats, the apparatus was wiped with ethanol in water before each behavioral test. Control and experimental pairs of rats were alternated and observed always between 8:00 a.m. and 1:00 p.m. Serum corticosterone determination Corticosterone is the most abundant circulating steroid secreted by rats and is considered a good indicator of adrenocortical function in this species (Sutanto and de Kloet, 1987; Sutanto et al., 1988). This hormone was determined in serum using commercially available kits (Coat-a-Count, DPC). This procedure is based on a solid-phase radioim-
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munoassay in which 125I-labeled corticosterone competes for a fixed time with the corticosterone present in the rat sample for antibody sites. Serum samples were assayed directly without extraction or purification. To decrease data variability on serum corticosterone levels, the rats were handled daily (5 min/day) for habituation to the experimental conditions of enthanasia and blood collection. Animals were enthanized decaptation after deep CO2 anesthesia. To minimize the reported circadian variations on serum glucocorticoid levels (Albers et al., 1985), control and cyhalothrin-treated animals were intermixed for enthanasia over just 1 day, i.e., 24 h after the last drug and/or control solution administration, i.e., between 8:00 a.m. and 9:00 a.m. Experimental design Five experiments were done in accordance with GLP protocols and quality assurance methods. In the first experiment, 40 rats were divided randomly and equally into four groups: three experimental groups (E1, E2, and E3) and one control group (C). The animals of groups E1, E2, and E3 received 1.0, 3.0, and 7.0 mg/kg of cyhalothrin, respectively, once daily for 7 days whereas those of group C were given the same number and volume (1.0 ml/kg) of the vehicle of cyhalothrin. Animals from all groups were observed daily (between 8:00 and 9:00 a.m.) for the presence or absence of overt clinical signs and symptoms of intoxication, as described above. Twenty-four hours after the last administration of cyhalothrin or vehicle, these rats were observed first for locomotor activity in the open-field and subsequently for the free exploration of a plus-maze apparatus, as described above. In the second experiment, 64 rats were divided and treated as stated above for the first experiment. Twenty-four hours after the last treatment, pairs of rats were placed in the open-field arena for social interaction measured as described above. Twenty-seven rats were used in the third and fourth experiments. In each experiment, the animals were divided randomly and equally into three groups: two experimental groups (P and E2) and one control group (C2). Animals from group P received a single dose of 1.0 mg/kg picrotoxin while those of group E2 received cyhalothrin (3.0 mg/kg/ day for 7 days); rats of group C2 were given a single injection of 1.0 ml/kg of 0.9% NaCl. Twenty-four hours after the last cyhalothrin administration and 30 min after 0.9% NaCl or picrotoxin, the animals were evaluated in the third experiment for open-field behavior (total locomotion, percentage of time spent in each open-field zone, rearing frequency, and immobility time) and plus-maze activity. In the fourth experiment the time spent in social interaction was recorded as described above. In the final and fifth experiment, 42 rats were divided randomly and equally into three groups: two experimental groups (E1 and E2) and one control group (C). The animals
of groups E1 and E2 received 1.0 and 3.0 mg/kg of cyhalothrin once daily for 7 days, respectively, whereas those of group C were given 1.0 ml/kg of the vehicle of cyhalothrin. Twenty-four hours after the last administration of drug or control solution, control and experimental animals were alternated for enthansia and blood collection for determination of serum corticosterone levels, performed as described above. Data analysis Bartlett’s test showed that the data were parametric (p⬍0.05). Thus, data were analyzed by one-way ANOVA (SYSTAT 7.01 for WINDOWS) followed by the Tukey– Kramer post-hoc test. In all experiments, the probability of p⬍0.05 was taken as an indication of statistical significance. Data are presented as means ⫾ SD.
Results Experiment 1 Administration of cyhalothrin (1.0 and 3.0 mg/kg/day) for 7 days did not induce overt clinical signs and symptoms of intoxication in rats. Indeed, differences were not found among rats of groups C, E1, and E2 concerning several items of the observational grid used to analyze toxicity. However, a similar treatment with a 7.0 mg/kg dose induced moderate signs of intoxication that included salivation, tremors, and liquid feces; these three symptoms started to appear in 60% of group E3 animals on treatment day 4. However, clinical signs and symptoms of toxicity were not observed in 40% of the rats treated with this dose of cyhalothrin even after the last day of treatment (day 7). Differences in weight were not found, with each group mean after the last treatment for this parameter being as follows: C ⫽ 274.3 ⫾ 16.8; E1 ⫽ 281.8 ⫾ 10.7; E2 ⫽ 270.6 ⫾ 18.2; E3 ⫽ 268.6 ⫾ 15.7. One rat of group E2 died during the experiment due to erroneous cyhalothrin administration. Open-field data of cyhalothrin-treated rats are depicted in Fig. 1. Cyhalothrin treatment reduced the total locomotion (F(3,35) ⫽ 7.498; p ⫽ 0.0005), the locomotion in the central open-field zone (F(3,35) ⫽ 6.779; p ⫽ 0.001), and the rearing frequency (F(3,35) ⫽ 3.098; p ⫽ 0.0392) of rats. No differences were found among the four groups concerning locomotor activity performed in the open-field peripheral (F(3,35) ⫽ 0.4303; p ⫽ 0.7326) and intermediary (F(3,35) ⫽ 1.376; p ⫽ 0.2663) zones. An increment in the immobility time (F(3,35) ⫽ 3.382; p ⫽ 0.0289) was also observed after cyhalothrin (3.0 mg/kg/day). However, no differences were found in this parameter between rats of groups C (control) and E3 (dosed with 7.0 mg/kg/day of cyhalothrin); animals treated with the highest dose of cyhalothrin displayed some nonambulatory activities within the open-field such as chewing, head bobbing, and tremors. The
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Fig. 1. Effects of cyhalothrin administration (once daily during 7 days) on total locomotion (A), locomotion in peripheral (B), intermediary (C), and central (D) open-field zone as well as on rearing frequency (E) and immobility time (F). Data are means ⫾ SD, for 10 rats per group for the groups vehicle, 1.0, and 7.0, and 9 rats for the group 3.0 mg/kg of cyhalothrin, respectively. ⽧p ⬍ 0.01 and 多p ⬍ 0.05 compared to group vehicle, ⴙp ⬍ 0.05 compared to group 1.0 mg/kg of cyhalothrin. ANOVA and Tukey–Kramer test.
NOEL for cyhalothrin effects on open-field behavior of rats was 1.0 mg/kg/day. Data in Table 1 indicate that cyhalothrin treatment (3.0
mg/kg) reduced the percentage of time spent by rats in the plus-maze open arms exploration (F(3,35) ⫽ 4.176; p ⫽ 0.0125) and increased the percentage of time spent in plus-
Table 1 Effects of cyhalothrin on behavior of rats observed in a plus-maze apparatus Treatment
Dose
% EOA
% ECA
% TOA
% TCA
Vehicle (ml/kg)
1.0 1.0 3.0 7.0
45.2 ⫾ 8.5 48.0 ⫾ 6.6 41.5 ⫾ 15.1 48.4 ⫾ 7.4
54.8 ⫾ 8.5 52.0 ⫾ 6.6 58.5 ⫾ 15.1 51.6 ⫾ 7.4
27.8 ⫾ 7.4 24.3 ⫾ 6.9 19.3 ⫾ 4.5* 28.3 ⫾ 5.4
72.1 ⫾ 7.4 75.7 ⫾ 6.9 80.7 ⫾ 4.5* 71.7 ⫾ 5.4
Cyhalothrin (mg/kg)
Note. Data are reported as means ⫾ SD for 10 rats per group for groups vehicle, 1.0, and 7.0, and 9 rats for the group 3.0 mg/kg cyhalothrin. Cyhalothrin was given once daily during 7 days. % EOA, percentage of entries in open arms, % ECA, percentage of entries in plus-maze closed arms; % TOA, percentage of time spent in open arms exploration; % TCA, percentage of time spent in closed arms exploration. * p ⬍ 0.05 compared to groups vehicle and 7.0, ANOVA and Tukey–Kramer test.
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Fig. 2. Effects of cyhalothrin (A) and picrotoxin (B) on the time spent by rats on social interaction within an open-field. Data are reported as means ⫾ SD for five to eight pairs per group. *p ⬍ 0.05 compared to group vehicle, #p ⬍ 0.05 and ##p ⬍ 0.001 compared to group 0.9% NaCl. ANOVA and Tukey–Kramer test.
maze closed arms (F(3,35) ⫽ 4.176; p ⫽ 0.0125). However, at this dose, this pyrethroid did not alter the percentage of entries into plus-maze open (F(3,35) ⫽ 0.9666; p ⫽ 0.4194) and closed arms (F(3,35) ⫽ 0.9666; p ⫽ 0.4194). Further analyses showed no differences between plus-maze data of vehicle and cyhalothrin (7.0 mg/kg)-treated rats. However, as stated above for the open field, rats treated with 7.0 mg/kg (group E3) presented some signs and symptoms of intoxication also within the plus-maze. The NOEL for cyhalothrin effects on plus-maze was 1.0 mg/kg/day. Experiment 2 Fig. 2A shows that cyhalothrin treatment at doses of 3.0 and 7.0 mg/kg/day for 7 days reduced the amount of time spent by rats in social interaction. The 1.0 mg/kg/day dose of cyhalothrin induced no effects (F(3,28) ⫽ 4.950; p ⫽ 0.0070), thus making it the NOEL.
tion of rats in the open-field (F(2,24) ⫽ 15.071; p ⬍ 0.0001); thus, data of cyhalothrin (3.0 mg/kg) obtained in experiment 1 were replicated. Further analysis showed that both picrotoxin and cyhalothrin decreased the percentage of time spent by rats in the central (F(2,24) ⫽ 7.267; p ⫽ 0.0034) open-field zone. No differences were found among the three groups concerning the percentage of time spent in the intermediary zone (F(2,24) ⫽ 2.211; p ⬎ 0.10). Although both treatments increased the percentage of time spent in open-field peripheral zone (C ⫽ 89.96 ⫾ 4.53; P ⫽ 94.81 ⫾ 3.78; E2 ⫽ 94.08 ⫾ 2.836), statistical significance was detected only for picrotoxin (F(2,24) ⫽ 4.303; p ⫽ 0.0253). Furthermore, picrotoxin and cyhalothrin data were not different (p ⬍ 0.05). A decrease in rearing frequency (F(2,24) ⫽ 18.996; p ⬍ 0.0001) and an increment in the immobility time of rats were also observed after picrotoxin or cyhalothrin treatment (F(2,24) ⫽ 13.348; p ⬍ 0.001). Data in Table 2 indicate that picrotoxin (1 mg/kg single dose) and cyhalothrin (3.0 mg/kg/day for 7 days) reduced the percentage of time spent by rats in the plus-maze open arms exploration (F(2,24) ⫽ 6.542; p ⫽ 0.0054) and increased the percentage of time spent in plus-maze closed arms (F(2,24) ⫽ 6.542; p ⫽ 0.0054). However, neither treatment was able to change the percentage of entries into plus-maze open (F(2,24) ⫽ 0.9847; p ⫽ 0.3882) and closed arms (F(2,24) ⫽ 0.9847; p ⫽ 0.3882). Thus, data on cyhalothrin (3.0 mg/kg) obtained in experiment 1 were replicated. Experiment 4 As it can be seen in Fig. 2B, picrotoxin (1 mg/kg) or cyhalothrin (3.0 mg/kg/day for 7 days) reduced the amount of time spent by rats in social interactions (F(2,12) ⫽ 13.403; p ⫽ 0.0009). Again, and importantly, data obtained previously for cyhalothrin (3.0 mg/kg) were replicated. Experiment 5 Biochemical analysis of serum corticosterone levels showed that cyhalothrin (3.0 mg/kg/day for 7 days; group E2) increased (F(2,36) ⫽ 4.169; p⬍ 0.05) the levels of this hormone compared to those measured in animals of the control group (C ⫽ 101.00 ⫾ 53.97 ng/ml; E1 ⫽ 112.00 ⫾ 68.24; and E2 ⫽ 173.25 ⫾ 79.53 ng/ml). The NOEL for cyhalothrin effects on serum corticosterone levels was 1.0 mg/kg/day.
Discussion Experiment 3 Open-field data of picrotoxin- (1.0 mg/kg single dose) and cyhalothrin- (3.0 mg/kg/day for 7 days) treated rats are depicted in Fig. 3. Both treatments decreased total locomo-
The NOEL obtained here for the behavioral effects of cyhalothrin is 1.0 mg/kg/day. This dose was unable to change not only the behavioral parameters analyzed in the open-field and plus-maze apparatuses, but also the amount of time spent
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Fig. 3. Effects of cyhalothrin (3 mg/kg for 7 days) and a single dose of picrotoxin (1.0 mg/kg) on total locomotion (A), percentage of time spent in the peripheral (B), intermediary (C), and central (D) open-field zones; as well as rearing frequency (E) and immobility time (F). Data are reported as means ⫾ SD, for nine rats per group. #p ⬍ 0.001; ⽧p ⬍ 0.01; *p ⬍ 0.05 compared to group 0.9% NaCl. ANOVA and Tukey–Kramer test.
by rats in social interaction. Conversely, the highest dose of cyhalothrin used (7.0 mg/kg/day for 7 days) induced signs and symptoms of intoxication that included salivation, tremors, and
liquid feces. Rats treated with this dose (group E3) also presented decreased locomotion and rearing within the open-field and spent less time in social interaction.
Table 2 Effects of cyhalothrin and picrotoxin on behavior of rats observed in a plus-maze apparatus Treatment
Dose
% EOA
%ECA
% TOA
%TCA
Control (ml/kg) Picrotoxin (mg/kg) Cyhalothrin (mg/kg)
1.0 1.0 3.0
40.5 ⫾ 9.6 34.4 ⫾ 19.7 43.9 ⫾ 12.0
59.5 ⫾ 9.6 65.6 ⫾ 19.7 56.1 ⫾ 12.0
22.8 ⫾ 10.8 8.5 ⫾ 5.2# 11.3 ⫾ 9.7*
77.2 ⫾ 10.8 91.5 ⫾ 5.2# 88.7 ⫾ 9.7*
Note. Data are reported as means ⫾ SD for nine rats per group. Control is 0.9% NaCl. Cyhalothrin was given once daily during 7 days and picrotoxin was given as a single 1.0 mg/kg dose. %EOA, percentage of entries in open arms; %ECA, percentage of entries in plus-maze closed arms; %TOA, percentage of time spent in open arms exploration, %TCA, percentage of time spent in closed arms exploration. * p ⬍ 0.05 and # p ⬍ 0.01 compared to control. ANOVA and Tukey–Kramer test.
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According to Crofton and Reiter (1984), type II pyrethroids decrease ambulatory behavior and induce nonambulatory activities such as chewing, face washing, and head bobbing in the open-field. Although these activities were not quantified here, they were also observed in this experiment after cyhalothrin (7.0 mg/kg), being responsible for the lack of changes reported for this pyrethroid on openfield immobility time and plus-maze open arms data. Indeed, signs and symptoms of intoxication were also detected here in rats treated with cyhalothrin (7.0 mg/kg/day for 7 days) within the plus-maze. Thus, 3.0 mg/kg/day of cyhalothrin for 7 days is the only regimen that can be used to analyze a possible stressor and/or anxiogenic-like effect for this type II pyrethroid in rats. Within the context of this discussion, stress is defined as a complex process by which animals respond to challenging or dangerous events (Gatchell and Baum, 1983; O’Leari, 1990). Thus, the stressful stimuli are referred to as “stressors” and the response as the “stress response.” In addition, “anxiety level” is operationally inferred as suggested elsewhere (Palermo-Neto et al., 2001; Palermo-Neto and Guimara˜ es, 2000; File, 1991; Dunn and File 1987; Pellow et al., 1985), i.e., as the response to a situation in which behavior is influenced by two opposing motivational forces (e.g., a natural curiosity to investigate unexplored or novel areas versus an aversion to open areas). Thus, when animals are introduced into an open-field, they are inclined to explore mainly the peripheral zone of the apparatus, i.e., they present a so-called positive thigmotaxis (Grossen and Kelley, 1973; Simon et al. 1994). According to Kelley (1993), stressors and/or anxiogenic drugs increase the amount of time spent by rodents in the peripheral zone of the open-field (closest to its walls), therefore decreasing their permanence in the central zones. As reported above, cyhalothrin (3.0 mg/kg/day for 7 days) induced a decrease not only in the total locomotor activity of rats observed in the open-field, but also, and importantly, in the percentage of time they spent exploring the open-field central zone. Furthermore, this cyhalothrin dose increased immobility in rats. In this respect, benzodiazepines (Meert et al. 1997; File, 1991) and 5-HT2 antagonists such as ritanserin (Meert et al. 1997), which are known to have anxiolytic effects, have been reported to cause increments in locomotor activity in different situations. Our present data also showed that cyhalothrin (3.0 mg/ kg/day for 7 days) decreased the percentage of time spent by rats in plus-maze open arms exploration, increasing at the same time the amount of time spent in closed arms exploration. Increments in the number of entries or in the time spent by animals in plus-maze open arm exploration are commonly taken as indicative of the presence of decreased levels of anxiety in these animals (Pellow et al., 1985; File, 1991). Indeed, anxiolytic drugs such as diazepam increase (Pellow et al., 1985) and yohimbine, pentylenetetrazole, caffeine, and amphetamine decrease the percentage of time spent by rats in plus-maze open arms exploration (Pellow et
al., 1985; Zangrossi and File, 1992; Pellow and File, 1986). Thus, since a reduction of locomotion in open-field central zones (Kelley, 1993) and/or in plus-maze open arm exploration (Comissaris, 1993; File, 1991; Pellow et al., 1985) are good indexes of both a stress response and/or anxiety levels, it seems possible to suggest that cyhalothrin (3.0 mg/kg/day for 7 days) might have an anxiogenic-like effect in rats. The data now obtained on social interaction reinforce the present assumption for anxiogenic-like effects for cyhalothrin (3.0 mg/kg/day for 7 days), in agreement with data reported elsewhere for fenvalerate, another type II pyrethroid insecticide (Spinosa et al., 1999). Indeed, anxiogenic drugs decrease (Baldwin et al., 1989; Bhattacharya and Mitra, 1992) while anxiolytic drugs increase (File and Pellow, 1985) the time spent by rats on social interaction in an open-field arena and, as observed in this study, cyhalothrin (3.0 mg/kg/day for 7 days) was shown to decrease this behavior. A possible anxiogenic-like effect of cyhalothrin treatment (3.0 mg/kg/day for 7 days) is strengthened by our data with picrotoxin. Indeed, as demonstrated here, cyhalothrin (3.0 mg/kg/day for 7 days) effects on open-field and plusmaze behaviors and on social interaction of rats were in the same direction of those induced by picrotoxin treatment. As expected, picrotoxin, a classic and well-known anxiogenic drug (Dalvi and Rodgers, 2001), not only decreased the total locomotion and increased immobility time of rats observed in an open-field, but also decreased the amount of time spent in the open-field central zone, increasing, at the same time, the percentage of time spent in the open-field peripheral zone. In this respect, and importantly, the data described here for picrotoxin concerning immobility time and the percentage of time spent by rats in open-field zones, as well as the data obtained from plus-maze and social interaction tests, were not statically different from those induced by cyhalothrin (3.0 mg/kg/day for 7 days). Finally, our results showed that cyhalothrin (3.0 mg/kg/ day for 7 days) increased corticosterone serum levels in rats. Accordingly, deltamethrin, another type II pyrethroid, was shown to increase corticosterone serum levels in rats (Boer et al., 1988), an effect also reported after picrotoxin (1.0 mg/kg) treatment in rats (Lakic et al., 1986). In this respect, it has been demonstrated that activation of the hypothalamic–pituitary–adrenocortical axis by stressors results in readily discernible elevation in plasma corticosterone levels (Axelrod and Reisene, 1984). Altogether, the behavioral and biochemical data reported here show that cyhalothrin (3.0 mg/kg/day for 7 days) might have an anxiogenic-like effect in rats. Most probably, these effects rely on cyhalothrin actions on GABAA receptor sites within the CNS, as already suggested for other type II synthetic pyrethroids (Lawrence and Casida, 1983; Gammon et al., 1982). It should be considered, however, that this anxiogenic-like effect of cyhalothrin is dose related and, as such, must be included among the several signs and symptoms of pesticide intoxication, i.e., they do not allow stating
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that cyhalothrin has an anxiogenic-like profile. In this respect, anxiety is a common symptom reported during the course of several kinds of seizures (Szyndler et al., 2001). Cyhalothrin is widely used in the control of a broad range of ectoparasites in food-producing animals; thus, our data might raise concerns about the possible effects of cyhalothrin residues in food. Moreover, the safety aspects of the use of cyhalothrin in veterinary medicine were considered at the 54th and 58th meetings of JECFA that identified a temporary ADI of 0 – 0.002 mg/kg of body wt per day for the induction of liquid feces in dogs, in a 26-week study (FAO/WHO, 2000). A high safety factor was used to account for the absence of a NOEL for liquid feces in dogs and the absence of a NOEL for neurobehavioral effects. Indeed, the Committee considered that the production of liquid feces might be a consequence of neurotoxic properties of cyhalothrin and further identified the pesticide-induced neurobehavioral effects as being a possible critical toxicological endpoint for synthetic pyrethroids (FAO/ WHO, 2000). The 1.0 mg/kg/day for 7 days NOEL, reported here for a cyhalothrin formulation, through the use of behavioral screening procedures, is coincident with the LOEL reported by the FAO/WHO expert members for the induction of liquid feces in dogs by cyhalothrin (FAO/ WHO, 2000). Thus, the behavioral data reported here for cyhalothrin might be relevant to the final statements on both ADI and MRLs for this synthetic pyrethroid insecticide. However, it should not be forgotten that some motor effects of deltamethrin (another type II pyrethroid) were reported to depend on the route of administration and/or vehicle employed (Crofton et al., 1995). Thus, although the vehicle of cyhalothrin used in these experiments produced no behavioral effects per se, it might be relevant to the pharmacokinetics and metabolization of cyhalothrin and therefore for the data now being reported for this type II pyrethroid. Acknowledgments The authors express sincere thanks to Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo (FAPESP) Foundation (Processes 99/04228-7 and 00/14937-4) and Conselho Nacional de Pesquisa (CNPQ) (Process 520050/ 97-5Nv) for the support given for this study. They also thank Professor Doctor Maria Martha Bernardi and Evelise de Souza Monteiro Fonseca from the Department of Pathology, School of Veterinary Medicine, University of Sa˜ o Paulo, for contributions made to this study. Finally, they express gratitude to Schering Plough Animal Health for donating Grenade and its vehicle. References Albers, H.E., Yogev, L., Todd, R.B., Goldman, B.D., 1985. Adrenal corticoids in hamsters: role in circadian timing. Am. J. Physiol. 248, 434 – 438.
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