Effects of Chronic Dietary and Repeated Acute Exposure to Chlorpyrifos on Learning and Sustained Attention in Rats
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《毒物学科学杂志》
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
Cognitive and motor impairment often follow acute poisoning with an organophosphorous (OP) pesticide. However, the persistence of these effects and the conditions necessary for their appearance are not clear: two specific concerns are whether symptomatic poisoning is necessary for persistent effects, and whether inhibition of cholinesterase (ChE) activity is a protective metric of OP exposure. This study examined the effects of chronic dietary and repeated high-level acute exposure to the pesticide chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) on learning and attention. Beginning at 3 months of age, male Long-Evans rats
Key Words: attention; learning; dietary exposure; acute effect; organophosphate; persistent effect; signal detection.
INTRODUCTION
Epidemiological studies of exposure to cholinesterase-inhibiting pesticides suggest that persistent cognitive deficits can follow from overtly symptomatic poisoning with these agents (e.g., Rosenstock et al., 1991; Savage et al., 1988; Stallones and Beseler, 2002; Steenland et al., 1994; see also reviews by Jamal et al., 2002a,b). It is far less certain whether permanent impairment follows prolonged "sub-clinical" exposure—i.e., exposure that does not induce signs or symptoms of poisoning (see Bushnell and Moser, in press; Kamel and Hoppin, 2004). For example, some studies have reported persistent cognitive deficits in farmers and termiticide applicators repeatedly exposed to organophosphates (OPs) but symptom-free (Dick et al., 2001; Steenland et al., 2000; Stephens et al., 1995, 1996), whereas other studies observed no effects in orchard workers whose exposure to pesticides was low and well controlled (Daniell et al., 1992; Karr et al., 1992). Thus, epidemiological studies alone provide inconclusive evidence to determine whether chronic, low-level exposure to OPs, without acute poisoning episodes may result in persistent deficits in occupationally exposed humans (Kamel and Hoppin, 2004). Similarly, evidence for such effects in the general population is entirely lacking. The limitations of epidemiological studies suggest the need for animal models to address the potential long-term effects of subsymptomatic exposure to these compounds.
Animal models of OP intoxication have revealed deficits in cognitive and motor functions in adult rats, both after acute administration and during repeated exposures. For example, working memory was impaired for several weeks after a single subcutaneous injection of 60 or 125 mg/kg of chlorpyrifos (CPF) in male rats performing a delayed matching-to-position (DMTP) task (Bushnell et al., 1993). The pattern of effects suggested a primary effect of CPF on attention, which was subsequently confirmed (Bushnell et al., 2001). Learning deficits in a repeated-acquisition task have also been observed during repeated daily oral doses of 12.5 mg/kg of CPF (Cohn and MacPhail, 1997).
However, it is rare to find studies of CPF that report cognitive and motor deficits persisting beyond the period during which cholinesterase (ChE) activity is inhibited. For example, acute subcutaneous CPF decreased trial completion, choice accuracy and response speed in an operant delayed matching-to-position (DMTP) task for about a month after dosing, and functional recovery paralleled recovery of ChE activity in blood and muscarinic receptor density in brain (Bushnell et al., 1993). Similar patterns of behavior change followed by recovery after a single dose have been reported for motor activity and behavior assessed with a functional observational battery (Mattsson et al., 1996; Moser et al., 1997; Moser and Padilla, 1998) and with a visual signal detection task (Bushnell et al., 2001). In addition, repeated dosing studies with CPF in adult animals generally have not yielded evidence for persistent effects (Bushnell et al., 1994; Cohn and MacPhail, 1997; Mattsson et al., 1996; Maurissen et al., 2000; Terry et al. 2003). Nevertheless, one study reported impaired spatial navigation in the water maze and a lack of sensitivity to amphetamine-induced place preference a year after the second of two acute treatments with CPF (Sánchez-Santed et al., 2004), as well as a number of negative results.
Despite the facts (1) that the predominant route of public exposure to OPs is dietary and (2) that until recently CPF was the most commonly-used OP insecticide in the U.S., no previous studies have looked for cognitive effects of chronic ingestion of CPF, and few have sought to determine whether effects of oral CPF persist after termination of exposure. In addition, nondietary exposure to higher doses of CPF may occur when, for example, it is used locally to treat lawns and gardens for insects. Therefore, in reality, exposure typically involves combinations of long-term, chronic ingestion accompanied by periodic acute exposures to higher concentrations. Thus, the goals of this study were (1) to evaluate the effects of chronic dietary exposure to CPF; (2) to evaluate the effects of intermittent, acute oral doses of CPF given by gavage; (3) to determine potential interactions between the two dosing scenarios during exposure to the compound; (4) to determine whether these treatments caused effects that persisted beyond the termination of exposure; and (5) to identify the factors of dosing that contributed to these effects.
Doses for this study were selected to cause specific degrees of inhibition of ChE (Padilla et al., 2005), rather than to reflect estimated public or occupational exposures to CPF. This approach was taken as a way to determine whether setting regulatory standards on the basis of ChE inhibition provides adequate protection against adverse functional effects of exposure to OP pesticides. That is, if functional deficits occur at doses that do not inhibit ChE activity, then a more protective marker of effect is needed. In contrast, if functional deficits occur only in the presence of significant inhibition of ChE, then ChE inhibition provides a protective marker of exposure to these compounds. By selecting doses of CPF that inhibit ChE activity only in the blood, or in both blood and brain, it is possible to judge the protectiveness of both of these markers of exposure. Based on pilot studies, a low chronic dose of CPF (nominally 1 mg/kg/day) was chosen to inhibit ChE activity in the blood, but not in the brain of rats, and a high chronic dose (nominally 5 mg/kg day) was chosen to inhibit ChE activity in both blood and in brain. Rats
We report here the effects of these exposure scenarios in adult male rats on the acquisition and performance of a visual signal detection task designed to assess sustained attention. This task was selected because of its demonstrated sensitivity to acute CPF (Bushnell et al., 2001) and to cholinergic drugs (Bushnell et al., 1997; Rezvani and Levin, 2003). The rats began training to perform the task after 6 months of dietary exposure to CPF. This strategy enabled us to assess the effects of both chronic and acute high-level CPF exposure on the processes engaged by learning and performance of the task, starting after a steady state of ChE inhibition had been established. Testing continued until the animals reached asymptotic performance on the task (2 months after termination of dosing), to determine the persistence of any effects of CPF. Details of the CPF exposure and its neurochemical effects are reported elsewhere (Padilla et al., 2005).
MATERIALS AND METHODS
Subjects.
Male Long-Evans rats (440 total for the entire experiment) were
All rats were weighed once per week and maintained at 350 g ± 10 g by scheduled daily feeding throughout the study. Body weights obtained on Mondays were used to determine the amount of food to be fed each rat for the remaining weekdays; rats were fed a maintenance amount of 15 g of food per day on the weekends and holidays. Chronic CPF dosing began 1 month after arrival (Fig. 1; Padilla et al., 2005).
Beginning 4 weeks prior to training (at 8.5 months of age and after 6 months of treatment), the 48 rats assigned to this study were moved to a different holding room and weighed daily (Monday through Friday). Their weights continued to be maintained at 350 g ± 10 g by scheduled daily feedings calculated according to an algorithm developed to maintain constant body weights (Ali et al., 1992). As with the larger study, 15 g of food was provided per rat per day on weekends and holidays.
All rats were fed the control diet during the first 30 days, experimental CPF diets for 1 year (see below), and regular rodent chow (PMI Nutrition International, Richmond, IN) thereafter. Behavioral training and testing occurred during the light phase of the diurnal cycle, and feeding occurred in the home cage after behavioral testing. Two rats died prior to training: one rat died during acute dosing, while the cause of death of the second rat is unknown (dose groups were 1-CPF and 5-CPF, respectively).
Chlorpyrifos dosing.
Beginning 30 days after arrival, rats were fed CPF incorporated into nutritionally-complete, grain-based, 3-g wafers (BioServ, Frenchtown, NJ) at concentrations intended to deliver doses of 0, 1, or 5 mg/kg body weight per day. The 1 mg/kg/day dose was designed to inhibit cholinesterase in the blood only, whereas the 5 mg/kg/day dose was designed to inhibit cholinesterase in both blood and brain (Padilla et al., 2005). Each lot of 3-g wafers was assayed using HPLC to determine its actual CPF concentration, and lots were accepted if the CPF concentration was within 10% of the specified level. The control diet was also analyzed to verify the absence of CPF. Thus the 3-g wafers contained CPF at nominal concentrations of 0, 23.33, and 116.66 mg CPF per kg food, in the 0, 1, and 5 mg/kg dietary groups, respectively. Methods and results of the assays of 3-g wafers are detailed elsewhere (Padilla et al., 2005).
In addition, half of the rats in each feeding group
Analysis of 45-mg pellets for chlorpyrifos.
Behavioral testing required the use of 45-mg nutritionally complete pellets, which were manufactured from the same premix as the 3-g wafers (BioServ, Frenchtown, NJ). These pellets were assayed identically for CPF concentration using HPLC methods (Padilla et al., 2005). Five groups of 20 pellets each were randomly selected from each lot and weighed; the average weight per pellet was calculated to verify that the pellet weights were as specified (45 mg ± 10%). Each group of pellets was pulverized in a Micro-Mill grinder (Bel-Art Products, Pequannock, NJ) and assayed as in Padilla et al. (2005).
Each lot of control diet was also analyzed to verify the absence of chlorpyrifos.
Behavioral apparatus.
Twelve standard operant conditioning chambers (Model E1010, Coulbourn Instruments, Lehigh Valley, PA) were each assembled as previously described (Bushnell, 1999) with a house light, an incandescent signal light, a 5-cm cone loudspeaker, and two retractable levers, one on each side of a lighted food cup with a hinged clear plastic door. Background white noise (60 dB(A)) was generated in each chamber. In half of the boxes, the left lever was designated the "signal" lever, and the right lever as the "blank" lever; the converse designations were made in the other boxes. The signal light was located directly above the signal lever for initial training and was moved to above the food cup after all rats reached a criterion accuracy of 80% correct.
A signal consisted of an increase in the brightness of the signal light, from a background intensity (1.22 lux) to signal intensities of 1.52, 1.91, 2.37, 2.96, 3.61, 4.54 or 5.63 lux, above the ambient illumination of the houselight (0.097 lux). The sound intensities were measured using a Bruel & Kjaer (Nrum, Denmark) sound level meter (Model #2235), and the light intensities were measured using a photometer (Model 450 with cosine probe, EG & G, Salem, MA). All aspects of stimulus presentation and data collection were accomplished using SKED-11 software (State Systems, Kalamazoo, MI) running under RSX-11M-plus on a PDP11/73 computer (Digital Equipment, Maynard, MA).
Behavioral procedures.
Operant training began at 10 months of age, after 34 weeks of chronic treatment, and 6 weeks after the third acute dose had been administered (Fig. 1). The rats earned most of their daily food allotment as 45-mg pellets in the operant chambers and
The training schedule required that a series of behavioral tests be administered in a specific sequence. The timing of the training and dosing schedules resulted in the following conditions. All but the final two months of training were conducted during chronic dosing; autoshaping tests preceded acute dose 4; acute dose 5 was given during visual discrimination training; and acute dose 6 was given after three weeks of signal detection task (SDT) training. These last two acute doses provided an opportunity to assess the effect of acute inhibition of ChE activity on learning and performance of the SDT. Behavioral methods for each training phase are detailed below.
Autoshaping.
All rats began training with an autoshaping-operant method, as previously described (Bushnell, 1999; Davenport, 1974), in which the retraction of a lever beneath a visual signal is paired with the delivery of food. The lever was inserted and the signal light illuminated for 15 s on each of 50 trials in a test session. If the rat pressed the lever, the lever retracted immediately, the signal light was extinguished, and food was delivered. If the rat did not press the lever within 15 s, the lever was retracted, the signal light was extinguished, and food was delivered. Lever presses were counted in each of five 10-trial blocks in each of three test sessions. Hand-shaping was used to train rats that did not consistently press the lever under these contingencies; all rats eventually learned the task.
Visual discrimination training.
After each rat pressed the lever reliably, training continued with 100-trial sessions in which rats were required to report during each trial the presence or absence of the signal (a single, brief illumination of the signal light) by a single press on one of the two levers. Two trial types, "signal" and "blank," were presented in equal number in each test session in a pseudorandom sequence. Signal and blank trials differed only in that no signal was presented during a blank trial. Each correct response (a press on the signal lever during a signal trial, "hit," or a press on the blank lever during a blank trial, "correct rejection") was followed by the delivery of a food pellet into the illuminated food cup. After each incorrect response (a press on the signal lever during a blank trial, "false alarm," or a press on the blank lever during a signal trial, "miss") or response failure, the houselight was turned off for 3 s (timeout), and no food was delivered. The criterion for advancement at each stage of training was 80% correct responding.
Signal detection task.
At the end of training, rats performed daily 300-trial sessions that lasted approximately 60 min. The signal duration was 300 ms and its intensity was varied randomly within each session across seven intensities (see above). A variable signal period (range, 7 to 13 s) was used to make the occurrence of the signal temporally unpredictable. Correct responses were followed by illumination of the food cup (2 s), and a food pellet was delivered after 80% of the correct responses to prevent satiation during the test session and weight gain during the study. Each incorrect response and response failure was followed by a timeout (3 s). A trial was not repeated after a response failure. These temporal parameters yielded an event rate of approximately five trials per minute.
Data analysis.
To quantify sustained attention in the signal detection task, the number of hits, misses, correct rejections, and false alarms were recorded for each session. The proportion of hits [P(hit) = (number of hits) / (number of hits + number of misses)] was calculated for each signal intensity, while the proportion of false alarms [P(fa) = (number of false alarms) / (number of false alarms + number of correct rejections)] was calculated for each entire session, because no signal was present on these trials. These measures served to quantify performance accuracy.
All statistical analyses were performed using SAS 6.12 (SAS Institute, Cary, NC) and included two between-groups factors, Chronic (3 levels: 0, 1, and 5 mg/kg/day) and Acute (2 levels: Oil and CPF). These variables were crossed with within-subject variables appropriate for each assessment. For autoshaping, the number of lever presses emitted by each rat during each of the five 10-trial blocks in each of the three test sessions of autoshaping training was counted and analyzed by categorical modeling (CATMOD: SAS, 1990). For the visual discrimination training, the proportion of correct responses for each rat in each test session was analyzed by a mixed ANOVA (GLM: SAS, 1990) with test session as a repeated measure. For the assessment of asymptotic SDT performance, P(hit) values for each rat at each signal intensity were averaged across four sessions, and these averages were analyzed similarly with signal intensity as a repeated measure (GLM: SAS, 1990). The P(fa) values for each rat were averaged across the four sessions, and these averages were analyzed (GLM: SAS, 1990) with no repeated measure. Step-down ANOVAs were performed to characterize significant interactions among the independent variables in each assessment. After significant Chronic by Acute interactions, one-way step-down ANOVAs for dose were performed for each acute condition. In all analyses, Huynh-Feldt degree of freedom (df) corrections were used to reduce the effects of asymmetrical variance-covariance matrices for repeated measures. The criterion for significance was set at 0.05 for all tests.
RESULTS
Analysis of 45-mg CPF Pellets
Although made from the same premix as the 3-g wafers, the pellets did not contain the same CPF concentrations. Assay of the pellets yielded concentrations of 0, 16.37, and 82.24 mg CPF per kg of food in the 0, 1, and 5 mg/kg dietary groups respectively (concentrations in the wafers were within 10% of their nominal values of 0, 23, and 117 mg/kg, respectively). When multiplied by daily food consumption (apportioned between 45-mg pellets and 3-g wafers each day) and divided by the body weights of the rats, these concentrations yielded the daily doses of CPF shown in Table 1.
Autoshaping
The groups differed in acquisition of the lever press response. Whereas the main effects of Chronic and Acute were not significant, the main effect of Block [2 (11) = 103.81, p = 0.0000] and the interactions of Chronic by Block [2 (22) = 42.60, p = 0.0053], Acute by Block [2 (11) = 21.49, p = 0.0287], and Chronic by Acute by Block [2 (22) = 84.15, p = 0.0000] were significant. In the groups not acutely dosed with CPF, acquisition of the response was facilitated by chronic CPF in a dose-related fashion (Fig. 2). In those groups that were acutely dosed with CPF, the 5-CPF group learned the lever press response, while the 0-CPF and 1-CPF groups did not learn the response during the three sessions of autoshaping (Fig. 2).
Signal Detection Task
The effects of dietary CPF during autoshaping did not transfer to learning the subsequent visual discrimination needed for the SDT, as there were no significant differences among the groups during this period between acute doses (Fig. 3).
The last acute dose of CPF was given at the end of the year of chronic dosing. Prior to this dose, baseline accuracy did not differ between the groups. Acute CPF reduced P(hit) in all dosed groups (Fig. 4) [Acute main effect F(1, 38) = 12.92, p = 0.0009; Acute by Day interaction F(3, 114) = 19.76, = 0.7480, p = 0.0001]; the Chronic effect and the Acute by Chronic interactions were not significant. P(fa) was significantly increased in all acute groups [Acute main effect F(1, 38) = 11.13, p = 0.0019; Acute by Day interaction F(3, 114) = 9.34, = 0.9036, p = 0.0001] (Fig. 4). The Chronic effect and the Acute by Chronic interactions were not significant for P(fa).
Asymptotic performance of the SDT was achieved 2 months after all dosing had stopped. Examination of functions relating P(hit) to intensity at this stage showed no significant differences related to either chronic CPF or to the acute doses (Figs. 5A, 5B). In contrast, P(fa) was differentially affected by acute and chronic treatments [Chronic by Acute interaction F(2,38) = 4.61, p = 0.0162]. The step-down analysis revealed that P(fa) was significantly higher in the 5-CPF group compared to the 0-CPF and 1-CPF groups, whereas there was no difference in P(fa) among the 0-Oil, 1-Oil, and 5-Oil groups.
DISCUSSION
Four major conclusions can be drawn from these results: First, chronic exposure to CPF at doses that inhibited ChE activity in blood (to 10% of control) and brain (to 50% of control) (McCollister et al., 1974; Padilla et al., 2005), but caused no visible signs of intoxication (Moser et al., 2005), did not affect learning or sustained attention in rats, either during or after a year of treatment. Second, acute doses of CPF that were sufficient to induce signs of cholinergic toxicity (Moser et al., 2005) impaired learning and sustained attention. Third, a subtle deficit in the accuracy of signal detection was seen two months after termination of dosing in the group of rats most highly exposed to CPF (5 mg/kg/day plus six acute doses). Fourth, the facilitative effects of ongoing chronic exposure to CPF on learning were reversed by acute exposure to CPF.
The first conclusion does not support epidemiological evidence (Dick et al., 2001; Steenland et al., 2000; Stephens et al., 1995) that chronic, subclinical exposure to OP pesticides leads to later cognitive deficits. Rather, this observation is more consistent with reports that such exposure does not lead to measurable cognitive sequelae (e.g., Daniell et al., 1992).
This conclusion also speaks to the issue of using ChE inhibition as a marker of exposure to ChE-inhibiting pesticides. Both the acute doses and the 5 mg/kg chronic dose inhibited ChE activity in the brains of other rats in this study, whereas the 1 mg/kg chronic dose inhibited ChE activity in blood but not in brain (Padilla et al., 2005). The absence of behavioral effects of the 1 mg/kg chronic CPF dose (present data and Moser et al., 2005) suggests that this level of exposure is insufficient to affect cognitive function in rats, and that using blood ChE activity as a marker of exposure may provide a conservative estimate of exposure for these effects. In contrast, inhibition of autoshaping was observed 6 weeks after the third of three acute doses of 45–60 mg/kg (Fig. 2), and impaired sustained attention was seen 2 months after exposure to six acute doses and the 5 mg/kg chronic dose (Fig. 5). These observations suggest that persistent, detrimental sequelae may follow a CPF exposure scenario that includes repeated acute doses that inhibit ChE activity in blood to less than 10% of control (Padilla et al., 2005) and cause overt signs of intoxication (Moser et al., 2005), with or without chronic inhibition of ChE activity. However, because ChE activity was not inhibited when the last behavioral effects were noted (Fig. 5; Moser and Padilla, 1998; Padilla et al., 2005), explanation of observed effects cannot rely solely on current measurements of ChE activity, but require knowledge of the exposure history as well.
Second, the effects of acute high-level doses are entirely consistent with previous studies of the acute effects of CPF (Bushnell et al., 1993, 2001; Mattsson et al., 1996; Moser and Padilla, 1998), which demonstrate behavioral disruption in parallel with inhibition of ChE activity. These observations confirm that the tests used in this study are sensitive to the acute effects of high doses of CPF and show that they are not sensitive to chronic dietary exposure at the doses administered here. It should also be noted that the parameters of the test at this stage of training (one signal intensity) did not permit us to evaluate potential shifts in visual threshold associated with CPF-induced miosis, as we have previously observed (Bushnell et al., 2001). Indeed, the pattern of deficit in Figure 4 (increased false alarms along with decreased hits) is more consistent with a deficit in attention than with a change in threshold (Bushnell, 1998).
Third, the subtle deficit observed in the 5-CPF group 2 months after termination of CPF exposure involved a slight decrease in P(hit) and a significant increase in P(fa). In terms of the theory of signal detection (Green and Swets, 1974), these changes reflect a reduction in sensitivity and a relaxation of the response criterion; that is, more events—both "signal" and "noise" events—were reported as signals by the rats in the 5-CPF group, thus increasing their incorrect reports that a signal had occurred. To the extent that discriminating signals from noise requires attention to the test conditions, this pattern can be interpreted as an impairment of sustained attention (Bushnell, 1998). This change very likely represents a persistent effect of treatment, because previous work has shown that effects of acute CPF on signal detection recover within 8 days after 50 mg/kg of CPF orally, and 4 weeks after 250 mg/kg subcutaneously (Bushnell et al., 2001). Further, effects of acute oral CPF on unconditioned behaviors and neurochemical indices of intoxication returned to normal within 2 weeks after doses of 20 or 80 mg/kg (Moser et al., 1998; Moser and Padilla, 1998).
Fourth, the effects of CPF on acquisition of the lever-press response in the autoshaping test suggested that chronic and acute exposures of CPF exerted opposite effects on learning. That is, chronic exposure to dietary CPF facilitated the acquisition of the lever-press response in a dose-related manner. In contrast, three previous acute high-level doses of CPF, 2 months apart and 1 to 5 months prior to the autoshaping test sessions, suppressed acquisition of the response in control rats and in those rats receiving chronic dietary CPF at 1 mg/kg/day. Each of these three acute doses of CPF had inhibited ChE activity to about 10% of control in blood and 25% of control in brain. These prior treatments did not, however, prevent acquisition of the lever press response in rats receiving dietary CPF at 5 mg/kg/day. This pattern suggests that a history of intermittent acute doses of oral CPF blocked an enhancing effect of chronic dietary CPF on the behavioral processes engaged by autoshaping.
The possibility that the facilitation of autoshaping by chronic CPF was mediated by inhibition of ChE activity in the CNS cannot be discounted, given the cognitive enhancement afforded by other ChE inhibitors in animals (e.g., van der Staay and Bouger, 2005) and the therapeutic benefit provided by ChE inhibitors in the treatment of human dementia (e.g., Doody, 2003; Giacobini, 2003). On the other hand, facilitation of autoshaping has previously been associated with exposure to other toxicants, including p-xylene (Bushnell, 1988), colchicine (Nanry et al., 1989), ethanol (Tomie et al., 1998), and Aroclor 1254 (Geller et al., 2001). In the case of p-xylene, the facilitation was eliminated by increasing the force required to press the response lever, providing evidence that poor motor control increased the likelihood that the intoxicated rats operated the lever while exploring it early in training. In this scenario, the intoxicated rats experienced the contingency between movement of the lever and food reward after fewer trials than did unintoxicated rats and, once this contingency was experienced, acquired the press response at a normal rate (i.e., the slopes of the acquisition curves were not affected by treatment) (Bushnell, 1988). A similar pattern was observed in this study, in that the effects of CPF appeared as a change in the number of trials given before pressing began, whereas the rate of acquisition was not altered (Fig. 2). This interpretation is also consistent with other reports. For example, Nanry et al. (1989) suggested that an "increase in locomotor activity or reactivity to the experimental stimuli" contributed to a facilitated acquisition in colchicine-treated rats. Similarly, Tomie et al. (1998) attributed facilitation of autoshaping by ethanol to a drug-induced enhancement of impulsiveness, causing the treated rats to press the lever more freely and thus acquire the response earlier than controls.
Alterations in motor function, including reduced locomotor activity (Mattsson et al., 1996; Moser, 1996; Moser et al., 1997, Pope et al., 1992), slowing of responding (Bushnell et al., 1993, 1994; Maurissen et al., 2000), and reduced response rates (Cohn and MacPhail, 1997), have been associated with CPF treatment in rats. These effects of CPF, together with the effects of other neurotoxicants on autoshaping (Bushnell, 1988; Geller et al., 2001; Nanry et al., 1989; Tomie et al., 1998), suggest the possibility that the facilitation of lever-press acquisition observed here may also have been mediated by CPF-induced motor dysfunction, though direct evidence for this supposition was not obtained in this study.
The fact that dietary CPF did not affect the subsequent training of visual discrimination is consistent with the hypothesis of chronic CPF-induced motor dysfunction. Acquisition of the discrimination necessary for the SDT does not involve learning new motor skills, but rather involves associating the presence or absence of the signal light with the location of the reinforced response. This lack of effect on associative learning is also consistent with observations of rats exposed to p-xylene, who were not impaired in automaintained reversal learning, despite facilitated autoshaping (Bushnell, 1988). Nevertheless, acute high-level oral exposure to CPF in this study did disrupt signal detection in a manner similar to that previously observed (Bushnell et al. 2001). That is, P(hit) was reduced, P(fa) was increased, and response times were prolonged for several days after the last acute dose. This behavioral pattern reflects a deficit in sustained attention, as we have proposed elsewhere (Bushnell, 1998; Bushnell et al., 1997).
The lower concentration of CPF in the 45-mg pellets fed to these rats during the behavioral tests reduced the daily dose of CPF from the nominal values of 1 and 5 mg/kg/day to 0.7 and about 3.6 mg/kg/day, depending on the group (Days 224–408 in Table 1). This exposure very probably reduced the degree of inhibition of ChE activity in these rats relative to the inhibition measured after the first 6 months of exposure in other rats, and relative to inhibition in the rats maintained on 3-g wafers throughout the study (Padilla et al., 2005). The degree of inhibition of brain ChE activity expected from the reduced dose can be estimated from a study by McCollister et al. (1974), who dosed rats daily with CPF for 2 years and reported that CPF at 3 mg/kg/day inhibited brain ChE to 44% of control after 12 months of dosing. This value falls between those observed in the 1 and 5 mg/kg/day groups, at the end of this study (Padilla et al., 2005), and suggests that the brain ChE activity in the 5-Oil and 5-CPF groups in this report may be estimated at about 50% during the 6 months of treatment and behavioral testing. Because Padilla et al. saw no significant inhibition of brain ChE activity in rats dosed daily at 1 mg/kg of CPF, it is very unlikely that brain ChE was inhibited in the 1-Oil and 1-CPF groups either.
In conclusion, these data suggest that mild cognitive deficits might be expected to follow chronic exposure to CPF, if the dose level is high enough to inhibit ChE activity in the brain (to about 50% of control), and if this chronic exposure is also accompanied by intermittent exposure to doses that cause signs of intoxication. The fact that these observations were made 2 months after termination of exposure indicates that they persisted beyond the inhibition of ChE activity, because biochemical indices of exposure to oral CPF in rats recover within 2 weeks after dosing (Moser and Padilla, 1998; Moser et al., 1998). The fact that P(fa) was not increased in either the 0-CPF or the 5-Oil group indicates that neither repeated acute exposure nor chronic exposure alone was sufficient to cause this effect, and that the effects of the two treatments were somehow additive. Conversely, the interaction of chronic and acute dosing on autoshaping suggests antagonistic effects of the two dose scenarios, with acute high-level exposure impairing acquisition of the lever-press response, and chronic exposure facilitating it.
Summary
Three acute, high-level oral doses of CPF (one of 60 mg/kg, two of 45 mg/kg) administered during the first 6 months of chronic exposure impaired acquisition of a lever-press response for food that began 1 month after the third acute dose. In the same test, chronic dietary CPF (1 and 5 mg/kg/day) facilitated acquisition of the lever press response; the 5mg/kg chronic dose also ameliorated the effect of the three acute doses on lever-press acquisition. Later in training, the sixth acute oral dose of CPF (45 mg/kg) transiently reduced the accuracy of visual signal detection, and this effect was not ameliorated by chronic CPF. The combination of the 5 mg/kg/day chronic dose and the six acute doses caused a subtle deficit in asymptotic performance of the signal detection task during tests conducted two months after all CPF exposure had stopped.
NOTES
Portions of this research were presented at the Society of Toxicology meeting, March 2000.
This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
We thank Renée S. Marshall for her diligence in managing this project; Wendy M. Oshiro, Katherine L. McDaniel, and Pamela M. Phillips for technical assistance; Kay Rigsbee for animal care; Charles Hamm for engineering support; and Drs. Stephanie Padilla, Suzanne McMaster, and William Boyes for thoughtful reviews of the manuscript.
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ABSTRACT
Cognitive and motor impairment often follow acute poisoning with an organophosphorous (OP) pesticide. However, the persistence of these effects and the conditions necessary for their appearance are not clear: two specific concerns are whether symptomatic poisoning is necessary for persistent effects, and whether inhibition of cholinesterase (ChE) activity is a protective metric of OP exposure. This study examined the effects of chronic dietary and repeated high-level acute exposure to the pesticide chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) on learning and attention. Beginning at 3 months of age, male Long-Evans rats
Key Words: attention; learning; dietary exposure; acute effect; organophosphate; persistent effect; signal detection.
INTRODUCTION
Epidemiological studies of exposure to cholinesterase-inhibiting pesticides suggest that persistent cognitive deficits can follow from overtly symptomatic poisoning with these agents (e.g., Rosenstock et al., 1991; Savage et al., 1988; Stallones and Beseler, 2002; Steenland et al., 1994; see also reviews by Jamal et al., 2002a,b). It is far less certain whether permanent impairment follows prolonged "sub-clinical" exposure—i.e., exposure that does not induce signs or symptoms of poisoning (see Bushnell and Moser, in press; Kamel and Hoppin, 2004). For example, some studies have reported persistent cognitive deficits in farmers and termiticide applicators repeatedly exposed to organophosphates (OPs) but symptom-free (Dick et al., 2001; Steenland et al., 2000; Stephens et al., 1995, 1996), whereas other studies observed no effects in orchard workers whose exposure to pesticides was low and well controlled (Daniell et al., 1992; Karr et al., 1992). Thus, epidemiological studies alone provide inconclusive evidence to determine whether chronic, low-level exposure to OPs, without acute poisoning episodes may result in persistent deficits in occupationally exposed humans (Kamel and Hoppin, 2004). Similarly, evidence for such effects in the general population is entirely lacking. The limitations of epidemiological studies suggest the need for animal models to address the potential long-term effects of subsymptomatic exposure to these compounds.
Animal models of OP intoxication have revealed deficits in cognitive and motor functions in adult rats, both after acute administration and during repeated exposures. For example, working memory was impaired for several weeks after a single subcutaneous injection of 60 or 125 mg/kg of chlorpyrifos (CPF) in male rats performing a delayed matching-to-position (DMTP) task (Bushnell et al., 1993). The pattern of effects suggested a primary effect of CPF on attention, which was subsequently confirmed (Bushnell et al., 2001). Learning deficits in a repeated-acquisition task have also been observed during repeated daily oral doses of 12.5 mg/kg of CPF (Cohn and MacPhail, 1997).
However, it is rare to find studies of CPF that report cognitive and motor deficits persisting beyond the period during which cholinesterase (ChE) activity is inhibited. For example, acute subcutaneous CPF decreased trial completion, choice accuracy and response speed in an operant delayed matching-to-position (DMTP) task for about a month after dosing, and functional recovery paralleled recovery of ChE activity in blood and muscarinic receptor density in brain (Bushnell et al., 1993). Similar patterns of behavior change followed by recovery after a single dose have been reported for motor activity and behavior assessed with a functional observational battery (Mattsson et al., 1996; Moser et al., 1997; Moser and Padilla, 1998) and with a visual signal detection task (Bushnell et al., 2001). In addition, repeated dosing studies with CPF in adult animals generally have not yielded evidence for persistent effects (Bushnell et al., 1994; Cohn and MacPhail, 1997; Mattsson et al., 1996; Maurissen et al., 2000; Terry et al. 2003). Nevertheless, one study reported impaired spatial navigation in the water maze and a lack of sensitivity to amphetamine-induced place preference a year after the second of two acute treatments with CPF (Sánchez-Santed et al., 2004), as well as a number of negative results.
Despite the facts (1) that the predominant route of public exposure to OPs is dietary and (2) that until recently CPF was the most commonly-used OP insecticide in the U.S., no previous studies have looked for cognitive effects of chronic ingestion of CPF, and few have sought to determine whether effects of oral CPF persist after termination of exposure. In addition, nondietary exposure to higher doses of CPF may occur when, for example, it is used locally to treat lawns and gardens for insects. Therefore, in reality, exposure typically involves combinations of long-term, chronic ingestion accompanied by periodic acute exposures to higher concentrations. Thus, the goals of this study were (1) to evaluate the effects of chronic dietary exposure to CPF; (2) to evaluate the effects of intermittent, acute oral doses of CPF given by gavage; (3) to determine potential interactions between the two dosing scenarios during exposure to the compound; (4) to determine whether these treatments caused effects that persisted beyond the termination of exposure; and (5) to identify the factors of dosing that contributed to these effects.
Doses for this study were selected to cause specific degrees of inhibition of ChE (Padilla et al., 2005), rather than to reflect estimated public or occupational exposures to CPF. This approach was taken as a way to determine whether setting regulatory standards on the basis of ChE inhibition provides adequate protection against adverse functional effects of exposure to OP pesticides. That is, if functional deficits occur at doses that do not inhibit ChE activity, then a more protective marker of effect is needed. In contrast, if functional deficits occur only in the presence of significant inhibition of ChE, then ChE inhibition provides a protective marker of exposure to these compounds. By selecting doses of CPF that inhibit ChE activity only in the blood, or in both blood and brain, it is possible to judge the protectiveness of both of these markers of exposure. Based on pilot studies, a low chronic dose of CPF (nominally 1 mg/kg/day) was chosen to inhibit ChE activity in the blood, but not in the brain of rats, and a high chronic dose (nominally 5 mg/kg day) was chosen to inhibit ChE activity in both blood and in brain. Rats
We report here the effects of these exposure scenarios in adult male rats on the acquisition and performance of a visual signal detection task designed to assess sustained attention. This task was selected because of its demonstrated sensitivity to acute CPF (Bushnell et al., 2001) and to cholinergic drugs (Bushnell et al., 1997; Rezvani and Levin, 2003). The rats began training to perform the task after 6 months of dietary exposure to CPF. This strategy enabled us to assess the effects of both chronic and acute high-level CPF exposure on the processes engaged by learning and performance of the task, starting after a steady state of ChE inhibition had been established. Testing continued until the animals reached asymptotic performance on the task (2 months after termination of dosing), to determine the persistence of any effects of CPF. Details of the CPF exposure and its neurochemical effects are reported elsewhere (Padilla et al., 2005).
MATERIALS AND METHODS
Subjects.
Male Long-Evans rats (440 total for the entire experiment) were
All rats were weighed once per week and maintained at 350 g ± 10 g by scheduled daily feeding throughout the study. Body weights obtained on Mondays were used to determine the amount of food to be fed each rat for the remaining weekdays; rats were fed a maintenance amount of 15 g of food per day on the weekends and holidays. Chronic CPF dosing began 1 month after arrival (Fig. 1; Padilla et al., 2005).
Beginning 4 weeks prior to training (at 8.5 months of age and after 6 months of treatment), the 48 rats assigned to this study were moved to a different holding room and weighed daily (Monday through Friday). Their weights continued to be maintained at 350 g ± 10 g by scheduled daily feedings calculated according to an algorithm developed to maintain constant body weights (Ali et al., 1992). As with the larger study, 15 g of food was provided per rat per day on weekends and holidays.
All rats were fed the control diet during the first 30 days, experimental CPF diets for 1 year (see below), and regular rodent chow (PMI Nutrition International, Richmond, IN) thereafter. Behavioral training and testing occurred during the light phase of the diurnal cycle, and feeding occurred in the home cage after behavioral testing. Two rats died prior to training: one rat died during acute dosing, while the cause of death of the second rat is unknown (dose groups were 1-CPF and 5-CPF, respectively).
Chlorpyrifos dosing.
Beginning 30 days after arrival, rats were fed CPF incorporated into nutritionally-complete, grain-based, 3-g wafers (BioServ, Frenchtown, NJ) at concentrations intended to deliver doses of 0, 1, or 5 mg/kg body weight per day. The 1 mg/kg/day dose was designed to inhibit cholinesterase in the blood only, whereas the 5 mg/kg/day dose was designed to inhibit cholinesterase in both blood and brain (Padilla et al., 2005). Each lot of 3-g wafers was assayed using HPLC to determine its actual CPF concentration, and lots were accepted if the CPF concentration was within 10% of the specified level. The control diet was also analyzed to verify the absence of CPF. Thus the 3-g wafers contained CPF at nominal concentrations of 0, 23.33, and 116.66 mg CPF per kg food, in the 0, 1, and 5 mg/kg dietary groups, respectively. Methods and results of the assays of 3-g wafers are detailed elsewhere (Padilla et al., 2005).
In addition, half of the rats in each feeding group
Analysis of 45-mg pellets for chlorpyrifos.
Behavioral testing required the use of 45-mg nutritionally complete pellets, which were manufactured from the same premix as the 3-g wafers (BioServ, Frenchtown, NJ). These pellets were assayed identically for CPF concentration using HPLC methods (Padilla et al., 2005). Five groups of 20 pellets each were randomly selected from each lot and weighed; the average weight per pellet was calculated to verify that the pellet weights were as specified (45 mg ± 10%). Each group of pellets was pulverized in a Micro-Mill grinder (Bel-Art Products, Pequannock, NJ) and assayed as in Padilla et al. (2005).
Each lot of control diet was also analyzed to verify the absence of chlorpyrifos.
Behavioral apparatus.
Twelve standard operant conditioning chambers (Model E1010, Coulbourn Instruments, Lehigh Valley, PA) were each assembled as previously described (Bushnell, 1999) with a house light, an incandescent signal light, a 5-cm cone loudspeaker, and two retractable levers, one on each side of a lighted food cup with a hinged clear plastic door. Background white noise (60 dB(A)) was generated in each chamber. In half of the boxes, the left lever was designated the "signal" lever, and the right lever as the "blank" lever; the converse designations were made in the other boxes. The signal light was located directly above the signal lever for initial training and was moved to above the food cup after all rats reached a criterion accuracy of 80% correct.
A signal consisted of an increase in the brightness of the signal light, from a background intensity (1.22 lux) to signal intensities of 1.52, 1.91, 2.37, 2.96, 3.61, 4.54 or 5.63 lux, above the ambient illumination of the houselight (0.097 lux). The sound intensities were measured using a Bruel & Kjaer (Nrum, Denmark) sound level meter (Model #2235), and the light intensities were measured using a photometer (Model 450 with cosine probe, EG & G, Salem, MA). All aspects of stimulus presentation and data collection were accomplished using SKED-11 software (State Systems, Kalamazoo, MI) running under RSX-11M-plus on a PDP11/73 computer (Digital Equipment, Maynard, MA).
Behavioral procedures.
Operant training began at 10 months of age, after 34 weeks of chronic treatment, and 6 weeks after the third acute dose had been administered (Fig. 1). The rats earned most of their daily food allotment as 45-mg pellets in the operant chambers and
The training schedule required that a series of behavioral tests be administered in a specific sequence. The timing of the training and dosing schedules resulted in the following conditions. All but the final two months of training were conducted during chronic dosing; autoshaping tests preceded acute dose 4; acute dose 5 was given during visual discrimination training; and acute dose 6 was given after three weeks of signal detection task (SDT) training. These last two acute doses provided an opportunity to assess the effect of acute inhibition of ChE activity on learning and performance of the SDT. Behavioral methods for each training phase are detailed below.
Autoshaping.
All rats began training with an autoshaping-operant method, as previously described (Bushnell, 1999; Davenport, 1974), in which the retraction of a lever beneath a visual signal is paired with the delivery of food. The lever was inserted and the signal light illuminated for 15 s on each of 50 trials in a test session. If the rat pressed the lever, the lever retracted immediately, the signal light was extinguished, and food was delivered. If the rat did not press the lever within 15 s, the lever was retracted, the signal light was extinguished, and food was delivered. Lever presses were counted in each of five 10-trial blocks in each of three test sessions. Hand-shaping was used to train rats that did not consistently press the lever under these contingencies; all rats eventually learned the task.
Visual discrimination training.
After each rat pressed the lever reliably, training continued with 100-trial sessions in which rats were required to report during each trial the presence or absence of the signal (a single, brief illumination of the signal light) by a single press on one of the two levers. Two trial types, "signal" and "blank," were presented in equal number in each test session in a pseudorandom sequence. Signal and blank trials differed only in that no signal was presented during a blank trial. Each correct response (a press on the signal lever during a signal trial, "hit," or a press on the blank lever during a blank trial, "correct rejection") was followed by the delivery of a food pellet into the illuminated food cup. After each incorrect response (a press on the signal lever during a blank trial, "false alarm," or a press on the blank lever during a signal trial, "miss") or response failure, the houselight was turned off for 3 s (timeout), and no food was delivered. The criterion for advancement at each stage of training was 80% correct responding.
Signal detection task.
At the end of training, rats performed daily 300-trial sessions that lasted approximately 60 min. The signal duration was 300 ms and its intensity was varied randomly within each session across seven intensities (see above). A variable signal period (range, 7 to 13 s) was used to make the occurrence of the signal temporally unpredictable. Correct responses were followed by illumination of the food cup (2 s), and a food pellet was delivered after 80% of the correct responses to prevent satiation during the test session and weight gain during the study. Each incorrect response and response failure was followed by a timeout (3 s). A trial was not repeated after a response failure. These temporal parameters yielded an event rate of approximately five trials per minute.
Data analysis.
To quantify sustained attention in the signal detection task, the number of hits, misses, correct rejections, and false alarms were recorded for each session. The proportion of hits [P(hit) = (number of hits) / (number of hits + number of misses)] was calculated for each signal intensity, while the proportion of false alarms [P(fa) = (number of false alarms) / (number of false alarms + number of correct rejections)] was calculated for each entire session, because no signal was present on these trials. These measures served to quantify performance accuracy.
All statistical analyses were performed using SAS 6.12 (SAS Institute, Cary, NC) and included two between-groups factors, Chronic (3 levels: 0, 1, and 5 mg/kg/day) and Acute (2 levels: Oil and CPF). These variables were crossed with within-subject variables appropriate for each assessment. For autoshaping, the number of lever presses emitted by each rat during each of the five 10-trial blocks in each of the three test sessions of autoshaping training was counted and analyzed by categorical modeling (CATMOD: SAS, 1990). For the visual discrimination training, the proportion of correct responses for each rat in each test session was analyzed by a mixed ANOVA (GLM: SAS, 1990) with test session as a repeated measure. For the assessment of asymptotic SDT performance, P(hit) values for each rat at each signal intensity were averaged across four sessions, and these averages were analyzed similarly with signal intensity as a repeated measure (GLM: SAS, 1990). The P(fa) values for each rat were averaged across the four sessions, and these averages were analyzed (GLM: SAS, 1990) with no repeated measure. Step-down ANOVAs were performed to characterize significant interactions among the independent variables in each assessment. After significant Chronic by Acute interactions, one-way step-down ANOVAs for dose were performed for each acute condition. In all analyses, Huynh-Feldt degree of freedom (df) corrections were used to reduce the effects of asymmetrical variance-covariance matrices for repeated measures. The criterion for significance was set at 0.05 for all tests.
RESULTS
Analysis of 45-mg CPF Pellets
Although made from the same premix as the 3-g wafers, the pellets did not contain the same CPF concentrations. Assay of the pellets yielded concentrations of 0, 16.37, and 82.24 mg CPF per kg of food in the 0, 1, and 5 mg/kg dietary groups respectively (concentrations in the wafers were within 10% of their nominal values of 0, 23, and 117 mg/kg, respectively). When multiplied by daily food consumption (apportioned between 45-mg pellets and 3-g wafers each day) and divided by the body weights of the rats, these concentrations yielded the daily doses of CPF shown in Table 1.
Autoshaping
The groups differed in acquisition of the lever press response. Whereas the main effects of Chronic and Acute were not significant, the main effect of Block [2 (11) = 103.81, p = 0.0000] and the interactions of Chronic by Block [2 (22) = 42.60, p = 0.0053], Acute by Block [2 (11) = 21.49, p = 0.0287], and Chronic by Acute by Block [2 (22) = 84.15, p = 0.0000] were significant. In the groups not acutely dosed with CPF, acquisition of the response was facilitated by chronic CPF in a dose-related fashion (Fig. 2). In those groups that were acutely dosed with CPF, the 5-CPF group learned the lever press response, while the 0-CPF and 1-CPF groups did not learn the response during the three sessions of autoshaping (Fig. 2).
Signal Detection Task
The effects of dietary CPF during autoshaping did not transfer to learning the subsequent visual discrimination needed for the SDT, as there were no significant differences among the groups during this period between acute doses (Fig. 3).
The last acute dose of CPF was given at the end of the year of chronic dosing. Prior to this dose, baseline accuracy did not differ between the groups. Acute CPF reduced P(hit) in all dosed groups (Fig. 4) [Acute main effect F(1, 38) = 12.92, p = 0.0009; Acute by Day interaction F(3, 114) = 19.76, = 0.7480, p = 0.0001]; the Chronic effect and the Acute by Chronic interactions were not significant. P(fa) was significantly increased in all acute groups [Acute main effect F(1, 38) = 11.13, p = 0.0019; Acute by Day interaction F(3, 114) = 9.34, = 0.9036, p = 0.0001] (Fig. 4). The Chronic effect and the Acute by Chronic interactions were not significant for P(fa).
Asymptotic performance of the SDT was achieved 2 months after all dosing had stopped. Examination of functions relating P(hit) to intensity at this stage showed no significant differences related to either chronic CPF or to the acute doses (Figs. 5A, 5B). In contrast, P(fa) was differentially affected by acute and chronic treatments [Chronic by Acute interaction F(2,38) = 4.61, p = 0.0162]. The step-down analysis revealed that P(fa) was significantly higher in the 5-CPF group compared to the 0-CPF and 1-CPF groups, whereas there was no difference in P(fa) among the 0-Oil, 1-Oil, and 5-Oil groups.
DISCUSSION
Four major conclusions can be drawn from these results: First, chronic exposure to CPF at doses that inhibited ChE activity in blood (to 10% of control) and brain (to 50% of control) (McCollister et al., 1974; Padilla et al., 2005), but caused no visible signs of intoxication (Moser et al., 2005), did not affect learning or sustained attention in rats, either during or after a year of treatment. Second, acute doses of CPF that were sufficient to induce signs of cholinergic toxicity (Moser et al., 2005) impaired learning and sustained attention. Third, a subtle deficit in the accuracy of signal detection was seen two months after termination of dosing in the group of rats most highly exposed to CPF (5 mg/kg/day plus six acute doses). Fourth, the facilitative effects of ongoing chronic exposure to CPF on learning were reversed by acute exposure to CPF.
The first conclusion does not support epidemiological evidence (Dick et al., 2001; Steenland et al., 2000; Stephens et al., 1995) that chronic, subclinical exposure to OP pesticides leads to later cognitive deficits. Rather, this observation is more consistent with reports that such exposure does not lead to measurable cognitive sequelae (e.g., Daniell et al., 1992).
This conclusion also speaks to the issue of using ChE inhibition as a marker of exposure to ChE-inhibiting pesticides. Both the acute doses and the 5 mg/kg chronic dose inhibited ChE activity in the brains of other rats in this study, whereas the 1 mg/kg chronic dose inhibited ChE activity in blood but not in brain (Padilla et al., 2005). The absence of behavioral effects of the 1 mg/kg chronic CPF dose (present data and Moser et al., 2005) suggests that this level of exposure is insufficient to affect cognitive function in rats, and that using blood ChE activity as a marker of exposure may provide a conservative estimate of exposure for these effects. In contrast, inhibition of autoshaping was observed 6 weeks after the third of three acute doses of 45–60 mg/kg (Fig. 2), and impaired sustained attention was seen 2 months after exposure to six acute doses and the 5 mg/kg chronic dose (Fig. 5). These observations suggest that persistent, detrimental sequelae may follow a CPF exposure scenario that includes repeated acute doses that inhibit ChE activity in blood to less than 10% of control (Padilla et al., 2005) and cause overt signs of intoxication (Moser et al., 2005), with or without chronic inhibition of ChE activity. However, because ChE activity was not inhibited when the last behavioral effects were noted (Fig. 5; Moser and Padilla, 1998; Padilla et al., 2005), explanation of observed effects cannot rely solely on current measurements of ChE activity, but require knowledge of the exposure history as well.
Second, the effects of acute high-level doses are entirely consistent with previous studies of the acute effects of CPF (Bushnell et al., 1993, 2001; Mattsson et al., 1996; Moser and Padilla, 1998), which demonstrate behavioral disruption in parallel with inhibition of ChE activity. These observations confirm that the tests used in this study are sensitive to the acute effects of high doses of CPF and show that they are not sensitive to chronic dietary exposure at the doses administered here. It should also be noted that the parameters of the test at this stage of training (one signal intensity) did not permit us to evaluate potential shifts in visual threshold associated with CPF-induced miosis, as we have previously observed (Bushnell et al., 2001). Indeed, the pattern of deficit in Figure 4 (increased false alarms along with decreased hits) is more consistent with a deficit in attention than with a change in threshold (Bushnell, 1998).
Third, the subtle deficit observed in the 5-CPF group 2 months after termination of CPF exposure involved a slight decrease in P(hit) and a significant increase in P(fa). In terms of the theory of signal detection (Green and Swets, 1974), these changes reflect a reduction in sensitivity and a relaxation of the response criterion; that is, more events—both "signal" and "noise" events—were reported as signals by the rats in the 5-CPF group, thus increasing their incorrect reports that a signal had occurred. To the extent that discriminating signals from noise requires attention to the test conditions, this pattern can be interpreted as an impairment of sustained attention (Bushnell, 1998). This change very likely represents a persistent effect of treatment, because previous work has shown that effects of acute CPF on signal detection recover within 8 days after 50 mg/kg of CPF orally, and 4 weeks after 250 mg/kg subcutaneously (Bushnell et al., 2001). Further, effects of acute oral CPF on unconditioned behaviors and neurochemical indices of intoxication returned to normal within 2 weeks after doses of 20 or 80 mg/kg (Moser et al., 1998; Moser and Padilla, 1998).
Fourth, the effects of CPF on acquisition of the lever-press response in the autoshaping test suggested that chronic and acute exposures of CPF exerted opposite effects on learning. That is, chronic exposure to dietary CPF facilitated the acquisition of the lever-press response in a dose-related manner. In contrast, three previous acute high-level doses of CPF, 2 months apart and 1 to 5 months prior to the autoshaping test sessions, suppressed acquisition of the response in control rats and in those rats receiving chronic dietary CPF at 1 mg/kg/day. Each of these three acute doses of CPF had inhibited ChE activity to about 10% of control in blood and 25% of control in brain. These prior treatments did not, however, prevent acquisition of the lever press response in rats receiving dietary CPF at 5 mg/kg/day. This pattern suggests that a history of intermittent acute doses of oral CPF blocked an enhancing effect of chronic dietary CPF on the behavioral processes engaged by autoshaping.
The possibility that the facilitation of autoshaping by chronic CPF was mediated by inhibition of ChE activity in the CNS cannot be discounted, given the cognitive enhancement afforded by other ChE inhibitors in animals (e.g., van der Staay and Bouger, 2005) and the therapeutic benefit provided by ChE inhibitors in the treatment of human dementia (e.g., Doody, 2003; Giacobini, 2003). On the other hand, facilitation of autoshaping has previously been associated with exposure to other toxicants, including p-xylene (Bushnell, 1988), colchicine (Nanry et al., 1989), ethanol (Tomie et al., 1998), and Aroclor 1254 (Geller et al., 2001). In the case of p-xylene, the facilitation was eliminated by increasing the force required to press the response lever, providing evidence that poor motor control increased the likelihood that the intoxicated rats operated the lever while exploring it early in training. In this scenario, the intoxicated rats experienced the contingency between movement of the lever and food reward after fewer trials than did unintoxicated rats and, once this contingency was experienced, acquired the press response at a normal rate (i.e., the slopes of the acquisition curves were not affected by treatment) (Bushnell, 1988). A similar pattern was observed in this study, in that the effects of CPF appeared as a change in the number of trials given before pressing began, whereas the rate of acquisition was not altered (Fig. 2). This interpretation is also consistent with other reports. For example, Nanry et al. (1989) suggested that an "increase in locomotor activity or reactivity to the experimental stimuli" contributed to a facilitated acquisition in colchicine-treated rats. Similarly, Tomie et al. (1998) attributed facilitation of autoshaping by ethanol to a drug-induced enhancement of impulsiveness, causing the treated rats to press the lever more freely and thus acquire the response earlier than controls.
Alterations in motor function, including reduced locomotor activity (Mattsson et al., 1996; Moser, 1996; Moser et al., 1997, Pope et al., 1992), slowing of responding (Bushnell et al., 1993, 1994; Maurissen et al., 2000), and reduced response rates (Cohn and MacPhail, 1997), have been associated with CPF treatment in rats. These effects of CPF, together with the effects of other neurotoxicants on autoshaping (Bushnell, 1988; Geller et al., 2001; Nanry et al., 1989; Tomie et al., 1998), suggest the possibility that the facilitation of lever-press acquisition observed here may also have been mediated by CPF-induced motor dysfunction, though direct evidence for this supposition was not obtained in this study.
The fact that dietary CPF did not affect the subsequent training of visual discrimination is consistent with the hypothesis of chronic CPF-induced motor dysfunction. Acquisition of the discrimination necessary for the SDT does not involve learning new motor skills, but rather involves associating the presence or absence of the signal light with the location of the reinforced response. This lack of effect on associative learning is also consistent with observations of rats exposed to p-xylene, who were not impaired in automaintained reversal learning, despite facilitated autoshaping (Bushnell, 1988). Nevertheless, acute high-level oral exposure to CPF in this study did disrupt signal detection in a manner similar to that previously observed (Bushnell et al. 2001). That is, P(hit) was reduced, P(fa) was increased, and response times were prolonged for several days after the last acute dose. This behavioral pattern reflects a deficit in sustained attention, as we have proposed elsewhere (Bushnell, 1998; Bushnell et al., 1997).
The lower concentration of CPF in the 45-mg pellets fed to these rats during the behavioral tests reduced the daily dose of CPF from the nominal values of 1 and 5 mg/kg/day to 0.7 and about 3.6 mg/kg/day, depending on the group (Days 224–408 in Table 1). This exposure very probably reduced the degree of inhibition of ChE activity in these rats relative to the inhibition measured after the first 6 months of exposure in other rats, and relative to inhibition in the rats maintained on 3-g wafers throughout the study (Padilla et al., 2005). The degree of inhibition of brain ChE activity expected from the reduced dose can be estimated from a study by McCollister et al. (1974), who dosed rats daily with CPF for 2 years and reported that CPF at 3 mg/kg/day inhibited brain ChE to 44% of control after 12 months of dosing. This value falls between those observed in the 1 and 5 mg/kg/day groups, at the end of this study (Padilla et al., 2005), and suggests that the brain ChE activity in the 5-Oil and 5-CPF groups in this report may be estimated at about 50% during the 6 months of treatment and behavioral testing. Because Padilla et al. saw no significant inhibition of brain ChE activity in rats dosed daily at 1 mg/kg of CPF, it is very unlikely that brain ChE was inhibited in the 1-Oil and 1-CPF groups either.
In conclusion, these data suggest that mild cognitive deficits might be expected to follow chronic exposure to CPF, if the dose level is high enough to inhibit ChE activity in the brain (to about 50% of control), and if this chronic exposure is also accompanied by intermittent exposure to doses that cause signs of intoxication. The fact that these observations were made 2 months after termination of exposure indicates that they persisted beyond the inhibition of ChE activity, because biochemical indices of exposure to oral CPF in rats recover within 2 weeks after dosing (Moser and Padilla, 1998; Moser et al., 1998). The fact that P(fa) was not increased in either the 0-CPF or the 5-Oil group indicates that neither repeated acute exposure nor chronic exposure alone was sufficient to cause this effect, and that the effects of the two treatments were somehow additive. Conversely, the interaction of chronic and acute dosing on autoshaping suggests antagonistic effects of the two dose scenarios, with acute high-level exposure impairing acquisition of the lever-press response, and chronic exposure facilitating it.
Summary
Three acute, high-level oral doses of CPF (one of 60 mg/kg, two of 45 mg/kg) administered during the first 6 months of chronic exposure impaired acquisition of a lever-press response for food that began 1 month after the third acute dose. In the same test, chronic dietary CPF (1 and 5 mg/kg/day) facilitated acquisition of the lever press response; the 5mg/kg chronic dose also ameliorated the effect of the three acute doses on lever-press acquisition. Later in training, the sixth acute oral dose of CPF (45 mg/kg) transiently reduced the accuracy of visual signal detection, and this effect was not ameliorated by chronic CPF. The combination of the 5 mg/kg/day chronic dose and the six acute doses caused a subtle deficit in asymptotic performance of the signal detection task during tests conducted two months after all CPF exposure had stopped.
NOTES
Portions of this research were presented at the Society of Toxicology meeting, March 2000.
This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
We thank Renée S. Marshall for her diligence in managing this project; Wendy M. Oshiro, Katherine L. McDaniel, and Pamela M. Phillips for technical assistance; Kay Rigsbee for animal care; Charles Hamm for engineering support; and Drs. Stephanie Padilla, Suzanne McMaster, and William Boyes for thoughtful reviews of the manuscript.
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