Learning and Memory of Rats after Long-Term Administration of Low Doses of Parathion

TOXICOLOGICAL SCIENCES ARTICLE NO.

46, 101–111 (1998)

TX982508

Learning and Memory of Rats after Long-Term Administration of Low Doses of Parathion I. A. Ivens,* G. Schmuck,† and L. Machemer† *Preclinical Research, Bayer Corporation, Berkeley, California 94710; and †Institute of Toxicology, Bayer AG, Wuppertal, Germany Received September 20, 1996; accepted May 19, 1998

Learning and Memory of Rats after Long-Term Administration of Low Doses of Parathion. Ivens, I. A., Schmuck, G., and Machemer, L. (1998). Toxicol. Sci. 46, 101–111. A set of four learning and memory tests (Morris Maze I for reference memory, Morris Maze II for working memory, one-way active avoidance, and passive avoidance) were employed to address the questions whether parathion impaired cognitive functions after low, long-term exposure and could cause persistent changes in cognition. Motor activity and general behavior were investigated in a functional observational battery. Parathion was administered in rat food in low doses which caused no clinical symptoms and no or borderline brain acetylcholinesterase inhibition. Parathion doses of 0.5, 2, or 8 ppm in rat food produced the averaged uptake of 24, 100, or 400 mg/kg body weight per group per day in male rats and 36, 152, or 550 mg/kg per day in female rats in week 13. Learning tests were performed in weeks 1 to 4 and 10 to 14, as well as 30 to 34 weeks after the end of treatment, when the male and female rats were about 13 months old. Low doses of parathion given daily for 13 weeks had no cumulative or adverse effects on learning and memory, either during treatment or after the extended treatment-free period, in any of the tests. A significant improvement of learning compared to control observed in the Morris Water Maze I during the first week of treatment (males dose group 0.5 ppm) shows that parathion can improved cognitive functions in rats. Results of the study indicate that adverse effects changing learning and memory in animals may occur only at higher doses of organophosphates, at which the peripheral and brain acetylcholinesterases are inhibited to a greater extent than those in the present study. © 1998 Society of Toxicology. Key Words: organophosphate; parathion; long-term exposure; rat; cognition; learning; reference memory; working memory; Morris Maze; active avoidance; passive avoidance.

Human exposure to organophosphates may result from improper handling during agricultural use, occasional suicide attempts, or sheep dipping. The resulting peripheral and central nervous system effects can be grouped into three classes: acute cholinergic effects, paralytic intermediate effects, and delayed neuropathy (organophosphate-induced delayed neuropathy, OPIDN), specific only for some organophosphates (Jamal, 1995; Lotti, 1992; Pope et al., 1993). Besides these well 101

characterized effects it was recently speculated that high acute exposure or chronic low-level exposure may cause long-term effects, including cognitive changes in humans (for review see: D’Mello, 1993; Ecobichon, 1994; Jamal, 1995; Karczmar, 1984; Levin and Rodnitzky, 1976; Schaumburg and Berger, 1993). However, reports of organophosphate action on mental or cognitive functions in humans are contradictory (Gershon and Shaw, 1961; Karademir et al., 1990; Levin and Rodnitzky, 1976; Brown, 1993; Rodnitzky et al., 1978). Variability in the way of exposure, doses, and duration of exposure of the reported cases makes it difficult to receive a persistent picture of actual changes in mental and cognitive parameters in humans. In addition there are few high-quality epidemiological studies which specifically addressed these questions (Jamal, 1995). Effects after low doses of organophosphate given over long periods are not investigated in animal experiments to the same extent as high acute or repeated doses which have shown that inhibiting brain acetylcholinesterase by greater than 60% can influence performance as well as learning and memory in rats (Bushnell et al., 1991, 1994; Chambers and Chambers, 1989; Costa and Murphy, 1982; Llorens et al., 1993; McDonald et al., 1988; Overstreet et al., 1974; Raffaele et al., 1990) or mice (Reiter et al., 1973). Experiments using repeated dosing regimens can induce tolerance to certain organophosphates, reducing the number and severity of clinical symptoms with ongoing administrations. Tolerance was related to a reduction of muscarinic receptors in the brain (Chaudhuri et al., 1993; Costa and Murphy, 1982; Costa et al., 1982; Jett et al., 1993, 1994). The present study was undertaken to investigate the scenario of long-term administration of low doses of parathion which cause no or borderline brain acetylcholinesterase inhibition to provide data for a realistic human exposure situation. Parathion (E 605; O,O-diethyl-O-(4-nitrophenyl)phosphorothionate) is a compound of the organophosphothionate class and its properties are well investigated (for summary on parathion: compare Gallo and Lawryk, 1991; Grob et al., 1950). Toxicity studies as well as guideline neurotoxicity studies are generally performed for this type of compound. Parathion does not cause delayed OPIDN (WHO, 1986). It has to be activated metabolically to paraoxone to inhibit acetylcholinesterase. To assess a spectrum of learning and memory functions, a 1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.

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battery of tests is needed (Green and Stanton, 1989; Peele, 1989; Peele and Vincent, 1989; Schenk and Morris, 1985) since one test usually evaluates only a limited spectrum of behavior. The battery of tests used in the present study includes four different learning tests. In addition, a functional observational battery (FOB) with grip strength and motor activity measurements was used to distinguish changes in learning and memory from noncognitive changes such as changes in arousal, general malaise, or muscular weakness (Heise, 1983). These tests were selected, since it was not known which type of learning and memory may be influenced by the repeated administration of parathion, although it was shown that changes in the central cholinergic system can be detected in the Morris Maze test (Becker et al., 1980; Beninger et al., 1989; Fibiger, 1991; Lydon and Nakajima, 1992; Moss and Deutsch, 1975; Upchurch and Wehner, 1987). Tests needed to be applicable during a routine subchronic toxicological study in which the test compound was given in animal food ad libitum up to 15 weeks. This type of test compound administration excludes cognitive tests with food deprivation and food reward. Also tests which require excessive training of the animals were not included in the battery, since the present study should be comparable to a routine type of toxicity study in rats where the test compound administration is started before the age of 8 weeks. Tests needed to be relatively short so that they could be performed several times during the study. The Morris Water Maze (for types of Morris Maze tests compare Brandeis et al., 1989; Lindner et al., 1992; Means and Dent, 1991; Means et al., 1992; Morris, 1984) was selected to assess spatial discrimination learning, since no food deprivation was necessary, 1 week of testing was sufficient, variation between rats of the same age was low, and data recording could be automated to allow testing of large numbers of animals. Two types of Morris Maze tasks were used consecutively, one to evaluate spatial reference memory and one to address spatial working memory. New sets of rats were used for each time point of investigation during the study, because it is well known that rats remember previous experiences with a test for up to 26 months and that this influences a later reacquisition (Beatty et al., 1985; Bierley et al., 1986; Caprioli et al., 1991). With naive animals the investigation of the full learning curve for each cognitive test was possible.

beginning of the treatment period rats were 7 to 8 weeks old. They were housed individually in type III Makrolon cages. Tap water and food were provided ad libitum. The animal room was maintained on a 12-h light/dark cycle (lighting from 7 AM to 7 PM) with a temperature of 22 6 2°C and a relative air humidity of 40 –70%. Study Design Before the treatment period, 240 rats were randomized by a computer-based randomization procedure into three sets of animals, each represented by four groups of 10 male and 10 female rats. Each set received 0, 0.5, 2, and 8 ppm of parathion in their food. The first set of animals (groups 1 to 4, each group with 10 male and 10 female rats) was tested in the behavioral tests during weeks 1 to 4 of the administration period (see Table 1). Blood samples were collected in week 4 and animals were euthanized in week 5. The second set of rats (groups 5 to 8) was treated for 15 weeks and tested only during weeks 10 to 14 at the end of the administration period. Animals were euthanized in week 15. The third set of rats (groups 9 to 12) received the test compound for 13 weeks and thereafter food without any test compound. They were tested during study weeks 45 to 49. The FOB and motor activity measurements were performed between 8 AM and 12 PM and Morris Maze and active and passive avoidance between 8 AM and 3:30 PM. Food consumption was recorded weekly. From the individual food consumption the consumption of parathion/kg body weight per day was calculated. Doses were chosen based on the data of plasma, erythrocyte, and brain cholinesterase inhibition in a pilot study with a feeding period of 2 weeks so that the highest dose of 8 ppm induced only weak (up to 20%) or no acetylcholinesterase inhibition in the brain. Cholinesterase Determinations After each set of rats was tested in the behavioral tests, blood from the venous plexus of the eye of was taken for plasma and erythrocyte cholinesterase determinations (plasma acetylcholinesterase, AChE/P: EC 3.1.1.7; method modified according to Ellman et al., 1961; erythrocytes, CHE/E: EC 3.1.1.7; method modified according to Okabe et al., 1977). Thereafter the animals were euthanized and the left brain was obtained for brain acetylcholinesterase determinations (AChE/B: EC 3.1.1.7; preparation of the samples: modified according to Ho and Ellman, 1969; determination of activity: modified according to Ellman et al., 1961). Parathion administration was continued until the time of euthanization for sets one and two. Functional Observational Battery FOB was applied to all animals of a respective set after the motor activity measurement (Table 1). The battery included cage side observations, observations during handling, investigations of sense organs, and autonomic sings including body temperature, open field observations, and grip strength measurements. The FOB adapted the tests described by Moser (1989, 1990), Edwards and Parker (1977), and Meyer et al. (1979).

MATERIALS AND METHODS Automated Measurement of Spontaneous Motor Activity Test Chemical Parathion (O,O-diethyl-O-(4-nitrophenyl)phosphorothionate; E 605; CAS Registry No. 56-38-2; 97.5% pure; Bayer AG, Leverkusen, Germany) was mixed with 1% peanut oil (DAB 10) and then distributed homogeneously in powdered rat food (Altromin GmbH, Lage, Germany) according to target doses of 0.5, 2, and 8 ppm. Analytical investigations revealed that the test compound was stable for a feeding period of 1 week.

Rats were tested in activity monitors [Omnitech digiscan analyzer, Model RXYZCM(16), 16 3 16 infrared beams plus 16 vertical beams] in acrylic cages (41 3 41 cm) for 40 min with sample intervals of 10 min according to the schedule seen in Table 1. The horizontal and vertical activity was calculated for the respective sample period: horizontal activity (HA): the total number of beam interruptions in the horizontal sensors per sample period; vertical activity (VA): the total number of beam interruptions in the vertical sensor per sample period (corresponds to rearing activity).

Test Animals and Husbandry Sets of male and female rats (SPF-bred Wistar rats, strain Hsd Cpb:WU bred by Harlan-Winkelmann, Borchen; Germany) were treated for 4, 13 (recovery groups), or 15 weeks, while the complete study lasted 49 weeks. At the

Morris Maze Computer-Aided Video Tracking of Swimming Behavior The Morris Maze procedure employed here was performed according to Van der Staay (Van der Staay et al., 1990; Van der Staay and de Jonge, 1993). Rats

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TABLE 1 Time Schedule for Different Behavioral Tests Performed with Three Different Sets of Animals Each Containing Three Dose Groups and Control Groups 1 to 4 * W4

W1

Groups 5 to 8 * W14

W1 Groups 9 to 12 W1

* W49

W13

*

Treatment period Period of behavioral testing Treatment-free period Sacrifice and brain tissue sampling Week

Groups

Morris Maze I (M, F)

Morris Maze II (M)

Motor activity, FOB (M, F)

Active avoidance (M)

Passive avoidance (F)

1 to 4 5 to 8 9 to 12

1 11 45

2 12 46

3 10 47

4 13 48

— 14 49

Note. M, test performed in male rats; F, test performed in female rats; W, week. were tested in black tanks (Landbauwerkzeuge Jean Spons, Withuis 5, 6245 KA Eysden, The Netherlands; diameter at upper rim 154 cm, at the bottom 141 cm, black escape platform diameter 10.8 cm, height 43.5 cm, covered by 1 cm clear tap water at 20 to 22°C). Due to the lighting and the black escape platform in the black tank, the platform could not be seen by the rats. The tank was divided into four equal quadrants labeled N (north), O (east), S (south), and W (west). Their order of use was randomized daily. A trial was started by placing the rat into the pool close to the rim, facing the wall of the tank into one of the four quadrants. Each trial lasted maximally 90 s or until the rat found the submerged platform and was separated from the next trial by a 30-s intertrial interval spent on the platform. Rats that did not find the platform within 90 s were placed on the platform. The order in which the rats were tested was randomized daily across treatment groups. Extramaze cues were provided by the furniture and fixtures in the room and posters on the wall. The video tracking system VTMAS (Noldus Information Technology bv Wageningen, The Netherlands, version 1.40) was used to record and store the swimming track per test time for each rat. From this information the parameters escape latency, traveled distance, speed, and distance to point were determined by the software program according to the following definitions: Escape latency: the elapsed time (in seconds) from the start of the observation until the animal’s center of gravity is first detected in the middle of the platform (time is started at the time the rat is introduced into the water and terminated if the rat reaches the platform or after 90 s); traveled distance: the total distance (calculated in cm from the recorded video track of each rat) traveled during each trial; speed: speed (in cm/s) equals the traveled distance in each trial divided by the duration of the trial (maximally 90 s); distance to point: average distance (in cm) between 2 objects (straight line through middle of platform and rat) during the trial. For the Morris Water Maze type I test groups of rats were tested according to the time schedule given in Table 1. Each rat received four daily trials on 5 consecutive days. The following week the same rats were tested in the Morris Maze II task. For the Morris Water Maze type II test male (weeks 2 and 12)

or female rats (week 45) were tested for 5 consecutive days. Animals received four pairs of trials per day. Trials of a pair were spaced by 30 s. Immediately after the first trial of the pair trial 2 was started from the same starting position as trial 1. When all rats had completed their first trial pair the second pair was given, etc. until all rats had received four trials per day. Trial pairs were spaced by approximately 100 min. The escape platform was in the east quadrant on day 1 and was then shifted daily to south, west, north, and east (day 5). One-Way Active Avoidance Male rats were tested in acrylic shuttle boxes (36 3 16 3 22 cm; Coulborn, U.S.A.) divided by an automated retractable door into two equal chambers. The floor was a standard metal grid floor. On the first test day before the first trial each rat was introduced into the shuttle box while the door between both chambers was open and the rat could move freely for 2 min to get used to the new environment. To start a trial a rat was introduced into the right shuttle box chamber (shock chamber). The door opened and a light and tone stimulus (2.9 kHz) was started for 9.5 s (conditioned stimuli, CS). After the end of the stimulus time a foot shock was given to the right chamber floor for 8 s or until the rat crossed into the second chamber (unconditioned stimulus, UCS: ca. 500 mA). No shock was initiated if the rat had already crossed into the second chamber during the light and tone stimulus. Instead the door closed after the rat. The rat was returned to the shock compartment by hand to stay for the 30-s intertrial interval while the door was closed. Each trial was repeated 10 times per day per rat for 5 days. A crossing of the rat into the second chamber during the light and tone and before the start of the shock was counted as avoidance. Crossing during foot shock was recorded as escape. The total number of avoidances or escapes per animal per day was counted. Passive Avoidance For passive avoidance only female rats were tested. The test was started by introducing the rat into the illuminated chamber of a shuttle box (compare active

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avoidance). The second chamber, was dark with black walls. Rats were allowed to enter the dark side for 180 s. As soon as the rat entered the dark chamber with all paws, the door was closed and a foot shock was given (days 1 and 2). At 48 h after training day 1, the same procedure was repeated and the number of rats not entering the dark chamber for the test time of 180 s was recorded. Rats reentering on the second test day were given a footshock and tested again the following, third, day. The time between the opening of the door and the rat’s entry with all paws into the dark chamber was recorded for all days.

TABLE 2 Parathion Consumption/kg Body Weight/Day (Group Mean plus Standard Deviation) for the Three Dose Groups (n 5 10 rats) Week Dose (ppm)

1

2

4

13

36 (2.7) 148 (8.4) 568 (18)

24 (2.3) 100 (7.7) 400 (25)

Males (mg/kg body wt)

Statistical Analysis Arithmetic means and standard deviation of body weights per dose and sex were calculated by the in-house computerized toxicology data system used for guideline studies. Results of the treatment groups were compared with the control groups using the significance test (U test) as described by Mann and Whitney (1947) and by Wilcoxon (1945) at the significance a 5 5% or a 5 1% (two-sided tests). Significant differences to control groups are indicated by * for p # 0.05 and ** for p , 0.01 in the tables. Data from cholinesterase determinations were evaluated with the Adjusted Welch Test (Welch, 1947; Holm, 1979). Motor activity. Arithmetic mean and standard error (SE) of motor activity data were calculated for male and female rats of the respective groups per time of 10 min for the 40 min investigated. The data were analyzed in a two-factor (treatment 3 time) analysis of variance (ANOVA) with repeated measure over time using SAS statistical subroutines. An LSD post-hoc analysis (SAS; t test; p , 0.05) was used to evaluate group differences in more detail. Analyses for male and female rats were performed separately without comparing both sexes. Morris Maze I. In the Morris Maze I test the arithmetic mean of four trials per animal per day were calculated for the four parameters escape latency, traveled distance, speed, and distance to point. These data (mean of four daily trials per animal) were analyzed in a two-factor ANOVA with repeated measure over time using SAS statistical subroutines (general linear model, GLM). An LSD post-hoc analysis (SAS: t test; p , 0.05) was used to evaluate group differences in more detail. Group means with SE were calculated per day for the 10 male and 10 female rat of the respective groups (SAS statistical subroutines). Morris Maze II. For each parameter of the Morris Water Maze test the arithmetic mean of the first trial of each trial pair per rat per day and the mean of the second trial of each trial pair per rat per day (trial 1: (session1 1 session2 1 session3 1 session4)/4; trial 2: (session1 1 session2 1 session3 1 session4)/4) were calculated. These data were analyzed further in SAS in a three-factorial analysis of variance (ANOVA with repeated measures over time; treatment 3 day 3 trial) using SAS statistical subroutines (GLM) comparing trial 1 with trial 2. An LSD post-hoc analysis (t test; p , 0.05) was used to evaluate group differences in more detail. Active avoidance. The percentage of avoidances per rat per day was calculated in relation to the total of 10 possible avoidances. The percentage of escapes per rat per day was calculated as follows: (number of escapes per rat per day/10 trials per day 2 number of avoidances) 3 100. These data were analyzed in a two-factor ANOVA with repeated measure over time using SAS statistical subroutines (GLM). An LSD post-hoc analysis (SAS: t test; p , 0.05) was used to evaluate group differences in more detail. Passive avoidance. The time the rats needed to enter the second chamber was recorded. Group means and standard deviations were calculated (Microsoft EXCEL). For the first day of test, data proved to be normally distributed. Data were analyzed by one-way ANOVA. The data of the following days were not normally distributed and analyzed by Kruskal–Wallis one-way ANOVA on ranks (SigmaStat, Jandel Scientific).

RESULTS

0.5 2.0 8.0

45 (2.7) 184 (7.9) 720 (13.6)

40 (2.0) 168 (12.6) 648 (46)

Females (mg/kg body wt) 0.5 2.0 8.0

50 (5.5) 224 (39.0) 816 (122)

52 (7.8) 224 (44.0) 760 (104)

48 (9.2) 206 (31.8) 672 (148)

36 (5.9) 152 (17.2) 552 (60)

food during the treatment or recovery periods (data not shown). No clinical signs or symptoms related to organophosphate effects were observed at any time. Food and water intakes were normal. The mean parathion consumption calculated for weeks 1, 2, 4, and 13 are summarized in Table 2. Due to the decline in food intake/kg body weight over the growth period of the animals the test compound intake decreased over the treatment period. Female rats had a higher test compound consumption/kg body weight than male rats due to higher food intake/kg body weight. The difference becomes more pronounced toward the end of the study when body weight differences between male and females are largest. Cageside observations, observations during handling, investigations of sense organs, autonomic signs, and open field observations during the FOB, including grip strength, did not show any signs or symptoms indicating organophosphate-induced changes (data not shown). Cholinesterase Activity The results of the measurements of cholinesterase in plasma, erythrocytes, and brain homogenates measured in treatment weeks 5 and 15 are summarized in Table 3. Esterase inhibition was higher in erythrocytes than in plasma. The brain acetylcholinesterase was marginally but statistically significantly (p # 0.05) inhibited in females of the highest dose in week 5. Otherwise, no significant changes in brain acetylcholinesterase were measured. In general, females were slightly more affected than males, probably due to higher intake of the test compound resulting from higher food intake/kg body weight. Esterase activities determined in plasma, erythrocytes, and brain of animals kept without treatment for at least 31 weeks showed no persisting changes in enzyme activities (data not shown).

Physiological Parameters

Spontaneous Motor Activity Measurements

The body weights of male and female rats were not influenced by treatment with doses of 0.5, 2, and 8 ppm parathion in animal

Figure 1 shows the horizontal activity for male and female rats obtained in weeks 3, 10, and 47 for three different sets of

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TABLE 3 Cholinesterase Activity in Plasma, Erythrocytes, and Brain (Mean Values and Standard Deviation) for Male and Female Rats Treated for 5 or 15 Weeks with Parathion Cholinesterase activity Week 5

Dose ppm

ChE/P (n 5 5) (mmol/ml per min)

AChE/E (n 5 5) (mmol/ml per min)

Week 15 AChE/B (n 5 5) (mmol/g per min)

CHe/P (n 5 10) (mmol/ml per min)

AChE/E (n 5 10) (mmol/ml per min)

AChE/B (n 5 5) (mmol/g per min)

0.41 (0.05) 0.48 (0.11) 0.42 (0.05) 0.23** (0.06)

1.02 (0.19) 1.09 (0.18) 0.66** (0.15) 0.15** (0.13)

11.53 (0.78) 12.29 (0.39) 12.53 (0.74) 11.86 (0.68)

2.24 (0.61) 2.16 (0.33) 1.81 (0.59) 1.07** (0.18)

1.00 (0.13) 0.91 (0.24) 0.56** (0.17) 0.15** (0.09)

12.21 (0.77) 11.91 (0.72) 11.26 (0.78) 12.33 (0.22)

Male 0 0.5 2 8

0.46 0.57 0.47 0.30*

(0.11) (0.10) (0.07) (0.05)

1.10 (0.37) 1.00 (0.11) 0.69 (0.24) 0.14** (0.08)

12.28 11.62 11.76 12.50

(0.54) (0.50) (0.42) (0.39) Female

0 0.5 2 8

1.83 (0.68) 2.00 (0.37) 1.60 (0.16) 0.67** (0.13)

1.35 (0.22) 0.99* (0.11) 0.44** (0.33) 0.11** (0.08)

12.84 (0.72) 11.75 (0.71) 12.19 (0.66) 11.53* (0.43)

Note. The test compound was administered until the time point of sample collection. ChE/P, cholinesterase activity in plasma; AChE/E, acetylcholinesterase activity in erythrocytes; AChE/B, acetylcholinesterase activity in brain. Statistical significance: *p # 0.05; **p # 0.01.

animals. The activity of male rats decreased with age, while female rats did not change in their overall activity. The horizontal activity was not significantly different from control in any of the treatment groups in male rats. The occasional deviations of some of the groups were not statistically significant nor dose related. In female rats there was no significant change after 10 and 47 weeks. In week 3 the repeated measure ANOVA indicated an effect of dose (F(3,36) 5 2.90, p 5 0.048) but not an effect of dose 3 time (p 5 0.40) in females. The post-hoc analysis showed that the highest dose group was different from control in the general linear mean analysis, while the highest dose group was different than the 2-ppm group in the post-hoc quadratic trend analysis. From Fig. 1 it can be seen that the highest dose group in week 3 was marginally lower than control, while the 2-ppm middle dose was marginally higher than control. Because changes are small, opposite in direction over a close dose range, and not significant in the repeated measure analysis, they are not regarded as treatment related but probably due to spontaneous variations between groups. The vertical activity was not influenced and did not show significant changes in male or female rats in any of the time points investigated (data not shown). Morris Water Maze Type I This standard type of water escape task evaluates mainly the spatial reference memory of rats with the platform position always in the same place (N, north). The escape latency decreased over the test days (Figs. 2 to 6, escape latency). This

showed that the rats learned the location of the platform during the 5 test days. The parameters traveled distance, speed, and distance to point were recorded additionally to avoid interpretation of confounding changes of behavior (i.e., increased or reduced swimming speed or adoption of a specific search strategy as effects on learning). Morris Water Maze I, First Measurement (Week 1) Figures 2 (males) and 3 (females) summarize the results of the water maze test of reference memory in rats treated for 4 days (corresponding to test days 1 to 5). On the first test day rats had not received any test compound. The difference between groups on this day (males: longer latency of the highest dose group, reduced speed of the 2-ppm dose group, differences in traveled distance) are caused by biological variation between animals. Male rats of the lowest dose learned to find the platform (compare escape latency) in a time of about 20 s on day 3 and females on day 4, while all other groups needed 1 or 2 days longer to reach this low level of escape latency. In males there was a significant dose 3 day interaction in the repeated measure ANOVA (F(12,144) 5 3.28, p , 0.01). Post-hoc analysis revealed that the lowest dose was significantly different from control. In females the effect observed was not significant. The swimming speed did not change over the 5 days investigated. The only difference seen was a low but not statistically different swimming speed in males treated with 2 ppm (Fig. 2, speed) which was already seen without parathion on day 1. The parameter traveled distance decreased with

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small but statistically significant differences between the control and treatment groups in female rats (speed: treatment 3 dose interaction: (F(12,144) 5 1.98, p , 0.05), although only days 2 and 3 showed a larger deviation from control (compare Fig. 5). There was no interaction of dose 3 day seen for these parameters. The effects may represent an improvement of learning but need further investigation. Male rats did not show changes in the four parameters investigated. Morris Water Maze I, Third Measurement (Week 45) The Morris Maze test was repeated with a third set of rats 31 weeks after termination of treatment. In male rats the number of animals that did not learn (three trials of four on day 5 without finding the platform) was 3 (control), 7 (0.5 ppm), 5 (2 ppm), and 4 (8 ppm). In females 1 (control), 1 (0.5 ppm), 3 (2 ppm), and 1 (8 ppm) were unable to reach the set criteria. This resulted in a large variation of data, mainly in male rats. Statistical testing revealed that there was no effect of dose on the number of rats not learning (x2 test, SigmaStat; Jandel Scientific). These rats (Fig. 6) were not included in the statistical evaluation. The escape latency of the control group on days 2 and 3 was marginally above that of the three other groups and on days 4 and 5 marginally below. This is not

FIG. 1. Horizontal activity of male and female rats was measured in intervals of 10 min for in total 40 min. Counts represent interruptions of infrared light beams by the rats. Data (mean per 10 min with standard error (SE)) of 10 male and 10 female rats per dose and sex were obtained in weeks 3 (top graphs), 10 (middle graphs), and 47 (bottom graphs) of the study with different sets of rats. Males and females investigated in week 3 were treated with 0, 0.5, 2, or 8 ppm of parathion in their food for up to 3 weeks. Rats of the groups measured in week 10 were treated with parathion for up to 15 weeks, while the rats measured in week 47 were treated with parathion for up to 13 weeks and then kept without test compound for 32 weeks (open circle, control; filled square, 0.5 ppm; triangle, 2 ppm; diamond, 8 ppm).

time, parallel to the reduction in escape latency. Distance to point decreased during the test days without a difference between dose groups. These findings (reduction in escape latency, no increase in speed, decrease in traveled distance) indicate an improvement of learning in the lowest dose group receiving 0.5 ppm of parathion shortly after the start of the treatment period. Morris Water Maze I, Second Measurement (Week 11) The decrease in escape latency (Fig. 5) of all dose groups of female rats compared to control was statistically significant (repeated measure ANOVA; effect of dose only: (F(3,36) 5 3.42, p , 0.05). Post-hoc analysis showed that the control was different from all treatment groups. There was also a dosedependent reduction in traveled distance (not statistically significant) and distance to point (significant effect of dose: (F(3,36) 5 3.89, p , 0.05). The parameter speed showed also

FIG. 2. Morris Maze I (spatial reference memory test) group means plus SE of escape latency (seconds), speed (cm/s), traveled distance (cm), and distance to point (cm) of 10 male rats per dose treated with parathion for 4 days (week 1 of the study). On day 1 rats were without treatment (open circle, control; filled square, 0.5 ppm; triangle, 2 ppm; diamond, 8 ppm).

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indicated no difference between treatment and control groups. No effect of treatment on the working memory seen as reduction in latency between trials 1 and 2 was observed in this test. Active Avoidance No significant difference in the number of avoidances in weeks 4, 13, or 48 was observed in the ANOVA repeated measure analysis (treatment 3 day: week 4: (F(12,144) 5 1.31, p 5 0.22; week 13: F(12,140) 5 1.04, p 5 0.42; week 48: F(12,132) 5 1.23, p 5 0.27). No significant differences in the repeated measure ANOVA were seen for escapes. Passive Avoidance No statistically significant effect was seen on day 1 in week 14 or after the treatment-free period in week 49 (week 14: between treatment groups: (F(3) 5 0.0657, p 5 0.978: week 49: (F(3) 5 0.178, p 5 0.910). No effect of treatment was observed for week 14 on the second day, when rats had to remember the shock situation. In week 49 the Kruskal–Wallis one-way ANOVA indicated a significant effect for day 2. Comparing groups (Dunn’s method: pairwise multiple comparison procedure) showed that the medium and high treatment groups were different (p , 0.5), but no difference from control existed. No effect was seen from the data of the rats still tested on day 3. FIG. 3. Morris Maze I (spatial reference memory test) group means plus SE of escape latency (seconds), speed (cm/s), traveled distance (cm), and distance to point (cm) of 10 female rats per dose treated with parathion for up to 4 days. On day 1 rats were measured without treatment (open circle, control; filled square, 0.5 ppm; triangle, 2 ppm; diamond, 8 ppm).

regarded as an effect of the test compound. There was no significant difference in any of the investigated parameters compared to control and no effect of the 13 weeks of treatment with parathion was observed after 30 weeks without treatment. Morris Water Maze II In addition to the reference memory version of the Morris Maze, the same rats (one sex only) previously tested in the Morris Maze I were evaluated in the spatial working memory task (Van der Staay and de Jonge, 1993; Whishaw, 1985, 1987). Working memory is indicated in this test by a reduction in escape latency from trial 1 of a trial pair to trial 2. In Fig. 7 the results of the escape latency of rats tested in weeks 2, 12, and 46 are shown. The group means for trial 1 are represented as bars and the means for trial 2 as lines over the five test days. In week 2, a significant effect was recorded for distance to point (day 3 trial interaction: (F(4,288) 5 4.50, p , 0.01, data not shown) but post-hoc analysis indicated no difference between treatment and control groups. In week 46 female rats were tested since 19 of 40 male rats were unable to learn the Morris Maze I task the previous week. In week 46 the parameters traveled distance and averaged speed (data not shown) were significantly different (traveled distance: day 3 trial interaction: (F(4,280) 5 15.45, p , 0.01); averaged speed: day 3 trial interaction: (F(8,276) 5 2.59, p , 0.01)). Post-hoc analysis

FIG. 4. Group means plus SE of escape latency (seconds), speed (cm/s), traveled distance (cm), and distance to point (cm) of 10 male rats per dose treated with parathion for 11 weeks obtained in the Morris Maze I (open circle, control; filled square, 0.5 ppm; triangle, 2 ppm; diamond, 8 ppm).

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higher test compound ingestion resulted in a marginally more pronounced esterase inhibition in female rats. Results of the Morris Maze I documented that parathion did not influence learning and memory adversely at any of the three time points tested. Interestingly, rats at the lowest dose (0.5 ppm, corresponding to about 45 to 50 mg/kg body weight parathion per day) showed improvement in learning indicated by a decrease in escape latency without an increase in swimming speed. In week 11 the swimming speed of female rats was higher on 2 of 5 days tested, while a decreases in escape latency, in traveled distance, and distance to point were observed over the whole test period of 5 days. Since the control group deviated from all treatment groups and no clear dose response was seen, this may represent a variation in the control animals instead of improved learning. Further investigations are necessary to evaluate this finding. In the working memory test (Morris Maze II) no treatment-related effects were seen at any of the time points investigated. No effects were seen in one-way active avoidance or in the passive avoidance test at any of the three time points tested in the present study. The investigations 31 to 35 weeks after the termination of parathion exposure were chosen to test for effects with late

FIG. 5. Group means plus SE of escape latency (seconds), speed (cm/s), traveled distance (cm), and distance to point (cm) of 10 female rats per dose treated with parathion for 11 weeks obtained in the Morris Maze I task (open circle, control; filled square, 0.5 ppm; triangle, 2 ppm; diamond, 8 ppm).

DISCUSSION

The present study was conducted to provide safety data for possible nervous system effects during and after low-dose organophosphate exposure given over an extended period of time. This type of exposure is not well documented in animal studies and reports of human exposure (Gershon and Shaw, 1961; Jamal, 1995; Karademir et al., 1990; Levin and Rodnitzky, 1976; Brown, 1993; Rodnitzky et al., 1978) indicating a need for these investigations. Parathion was chosen as test compound since it is widely used as insecticide. A treatment-free period of at least 7.5 months was included in the study after 3 months of continuous parathion exposure for some of the rats to extend the observation to about half of the life span of this species. The FOB did not indicate any signs or symptoms of parathion neurotoxicity or any other toxicological effect at the doses used. This observation was important for the evaluation of the cognitive tests since impaired physiological functions or changed motor performance may conflict with learning and memory tests. Due to higher food consumption/kg body weight female rats ingested more of the test compound. This and the decline of the test compound ingestion with growth of the animals is a common finding in studies with a constant amount of test compound in food used for males and females throughout the study period. The

FIG. 6. Group means plus SE of the of escape latency (seconds), speed (cm/s), traveled distance (cm), and distance to point (cm) of female rats per dose treated with parathion for 15 weeks and then kept for additional 30 weeks without any treatment obtained in the Morris Maze I task in week 45 of the study (open circle, control (n 5 9), filled square, 0.5 ppm (n 5 6; one animal died); triangle, 2 ppm (n 5 9); diamond, 8 ppm). Animals that did not learn up to day 5 (day 5: three of four trials: escape latency 5 90 s) were not included.

LEARNING AND MEMORY IN RATS AFTER PARATHION

FIG. 7. Group means of the parameter escape latency (s) for trial 1 (bar graphs) and trial 2 (line graphs) of rats treated with 0, 0.5, 2, and 8 ppm parathion obtained in the Morris Maze II task (test for spatial working memory). The top graph (males; n 5 10 rats per group) represents the test done in the second week of treatment, the middle graph was obtained in week 12 (males; n 5 10 rats per group), and the bottom graph represents the test performed in week 46 after a treatment-free period of 30 weeks (females: control, n 5 9; 0.5 ppm, n 5 9; 2 ppm, n 5 7; 8 ppm, n 5 9). The first bar per bar group represents the data obtained in the control group for trial 1; lines, trial 2: diamond, control; square, 0.5 ppm; triangle, 2 ppm; asterisk, 8 ppm. The difference in bar height compared to the data point (line graph) of the respective dose group of the same day represents the improvement of finding the escape platform in the second trial of a trial pair. It can be seen that the variation of data increased with age, while the difference of trials 1 and 2 decreased between weeks 2 and 46.

onset, effects which may not be reversible or may be facilitated by aging. Data showed that no adverse effects were detected in the rats age 12.5 months, although it was observed that male

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rats of all groups including controls had difficulties learning the Morris Maze situation. Several reports have shown that cholinesterase inhibitors or cholinergic compounds can improve learning and memory (Brandeis et al., 1990; Dawson et al., 1991). Recently it was shown that heptylphysostigmine, a new cholinesterase inhibitor, is able to antagonize scopolamine induced amnesia in rats tested in the eight-arm radial maze (Braida et al., 1996) with a U-shaped dose response. Interestingly, Rodnitzky et al. (1978) reported an improvement of cognitive functions with low doses of parathion given to human volunteers. The improvement of brain functions by cholinesterase inhibitors is also well known from investigations of treatment of Alzheimer’s disease (Giacobini and Cuadra, 1994; Canal and Imbimbo, 1996). Disruption of learning was observed at higher parathion doses (6 mg/kg) given to mice shortly before the acquisition of a passive avoidance task. Brain acetylcholinesterase activity was reduced to about 50% (Reiter et al., 1973). Llorens et al. (1993) studied behavioral effects of repeated exposure to disulfoton given ip over 30 days using the Morris maze for spatial reference memory and passive avoidance testing. Acetylcholinesterase was inhibited significantly in different brain areas, with increasing inhibition over time, and muscarinic receptors were downregulated. Animals became tolerant only to clinical cholinergic signs, but not to decreases in motor activity. Retention of a passive avoidance response and acquisition in a Morris Water Maze test were not impaired by the exposure. In the present study only the highest dose inhibited the brain acetylcholinesterase marginally and between weeks 5 and 15 no increase in inhibition was recorded. The use of different organophosphates, different routes of administration, and differences in duration of treatment (4 weeks vs 13–15 weeks) makes it difficult to compare the studies, specifically biochemical parameters. Influences of tolerance to treatment were not investigated in the current study and doses were chosen at a level where no cholinergic symptoms or effects in motor activity were seen. Several studies investigating animal behavior were performed with acute or short-term administration of doses of organophosphates inhibiting the brain acetylcholinesterase significantly, usually to more than 50%, which is considerably higher than in the present study. Pope et al. (1992) reported that motor activity was reduced for the first 2 weeks after treatment with chlorpyrifos, after which it returned to normal. When the animals were challenged with scopolamine, the treated rats had a higher activity than the controls in weeks 2, 4, 6, 8, and 12. The results were interpreted to indicate persistent changes in the cholinergic system. Bushnell et al. (1994) studied the effects of sc chlorpyrifos repeatedly administered to rats. The highest dose induced tremor, working memory impairment, and motor slowing in daily delayed matching-toposition/visual discrimination tests. Prendergast et al. (1997) observed an impairment in performance in the spatial test of working memory in rats for up to 21 days postdosing (sc

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administration of 250 mg/kg DFP for 14 days). Acetylcholinesterase was suppressed to about 43 to 50% depending on the brain region investigated 3 days after termination of treatment. The AChE activity had returned to control levels 21 days after completion of treatment. It can be concluded that the impairment of working memory may not be directly correlated to the levels of AChE inhibition after termination of treatment. Determination of exposure of orchard workers to parathion (Wolfe et al., 1967) showed that their mean dermal and respiratory exposure was 19 and 0.02 mg/h, respectively, without protective clothing. Since dermal absorption of parathion is low (mean absorption of liquid formulation: 1.23%; Durham et al., 1972) this results in a theoretical dermal uptake (usually main uptake route) of 0.003 mg/kg body weight per hour. Assuming 6 h of work this results in about 0.018 mg/kg per day, which would be in the range of the lowest does of 0.5 ppm administered in the present study. It can be concluded that low doses of parathion given daily for 13 weeks had no cumulative and/or adverse effect on learning and memory during treatment and after a long treatment-free period in the behavioral tests employed in this study. Adverse effects on learning and memory in animals may occur only at higher doses of organophosphates, at which the peripheral and brain acetylcholinesterases are inhibited to a greater degree than in the present study (Bushnell et al., 1994; Chambers and Chambers, 1989; Costa and Murphy, 1982; Llorens et al., 1993; McDonald et al., 1988; Reiter et al., 1973). The present study provides indications that humans with long-term low-level exposure to parathion with no or only borderline brain acetylcholinesterase inhibition should not be at risk of an impairment of cognitive or other CNS functions. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Heike Rieger and Martian Quiel in the performance of these experiments and Dr. Van der Staay and Dr. Blokland for their help in implementing the Morris Water Maze experiments.

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