Neurochemical Effects of Chronic Dietary and Repeated High-Level Acute Exposure to Chlorpyrifos in Rats
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《毒物学科学杂志》
Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Departments of Pharmacology, Neurology
Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ABSTRACT
Very little is known about the effects of chronic exposure to relatively low levels of anticholinesterase insecticides or how the effects of chronic exposure compare to those of higher, intermittent exposure. To that end, adult male rats were fed an anticholinesterase insecticide, chlorpyrifos (CPF), for 1 year at three levels of dietary exposure: 0, 1, or 5 mg/kg/day (0+oil, 1+oil, and 5+oil). In addition, half of each of these groups also
Key Words: chlorpyrifos; rat; chronic; cholinesterase; muscarinic; dopaminergic.
INTRODUCTION
The acute toxicity of organophosphorus insecticides is well known and has been extensively characterized. As inhibitors of cholinesterase (ChE) activity, organophosphorus insecticides produce overstimulation of cholinergic neurotransmission, both centrally and peripherally, eliciting signs and symptoms such as increased salivation, sweating, changes in blood pressure and heart rate, nausea, diarrhea, confusion, headache, muscle tremor, and, in high-dose situations, breathing difficulty, convulsions, and death. It is generally assumed that the toxicity of currently available organophosphorus insecticides is limited to the acute effects as described above and, consequently, that organophosphorus insecticides do not precipitate lasting adverse effects. In fact, however, very little is known about chronic, persistent effects of organophosphorus insecticides. There is growing concern that long-term, low level exposure to organophosphorus insecticides may have protracted or permanent effects (reviewed in Hoppin and Kamel, 2004; Ray and Richards, 2001).
Chlorpyrifos was the highest volume organophosphorus insecticide used in the United States until 2000, when the home and garden use of chlorpyrifos and its use on many food crops was severely restricted (http://www.epa.gov/pesticides/announcement6800.htm; last update Oct. 13, 2004). There has been widespread human exposure, as shown by the prevalence of chlorpyrifos metabolites in urine samples (Buck et al., 2001; Lemus et al., 1997), and human health effects have been attributed to chlorpyrifos exposure (Kaplan et al., 1993; Steenland et al., 2000). Although there have been many chronic studies of chlorpyrifos toxicity in animals (Breslin et al., 1996; McCollister et al., 1974; Sherman and Herrick, 1973; Yano et al., 2000), none of these studies included an in-depth assessment of neurochemical changes, only ChE measurements. Indeed, a subset of people (e.g., pesticides applicators) may have been exposed to additional chlorpyrifos, and there have been no studies exploring the profile of such higher, intermittent, exposures. Therefore, the present study investigated both long-term chronic exposure to chlorpyrifos and intermittent higher level exposure, using multiple neurochemical (present paper) and behavioral end points (Moser et al., 2005; Samsam, et al., 2005). Specifically, adult male Long-Evans rats were fed chlorpyrifos for a year at two dosage levels, with and without a bolus dose of chlorpyrifos every 2 months. This experimental design was constructed to answer the following questions about the toxic effects of long-term chlorpyrifos-induced ChE inhibition. (1) Is there (a) downregulation of CNS muscarinic receptors, (b) any change in striatal dopaminergic tone, or (c) any difference in neurochemical parameters after a challenge dose of chlorpyrifos if only blood and peripheral ChE activity are inhibited by chlorpyrifos, accompanied by no inhibition of ChE activity in the brain (2) Does inhibition of brain ChE activity by about 50% for a year cause changes in muscarinic receptor density or changes in dopaminergic parameters (3) Does a high bolus dose of chlorpyrifos produce different effects on (a) downregulation of CNS muscarinic receptors, (b) striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos as compared to approximately the same total dose fed at a lower rate over a much longer period of time— i.e., Does the pattern of exposure matter (4) If a high bolus dose of chlorpyrifos is given to animals whose ChE activity is already inhibited, is the inhibition dampened because the animals are already "tolerant" to the toxic effects or is it exacerbated by the low level of existing ChE activity (5) After a year of dosing, will the animals recover after the chlorpyrifos dosing is stopped
The present article reports the assessment of the neurochemical end points in the animals at two time points during the dosing (6 and 12 months) and again at 3 months after dosing ceased (15 months). Cholinesterase activity was monitored in blood, smooth muscle, and central nervous system (CNS) to assess both exposure and effect. Moreover, muscarinic receptor density was assessed because it is well-known that continual ChE inhibition in general and chlorpyrifos treatment, in particular, can produce tolerance that is manifested biochemically by downregulation of the muscarinic receptors (Pope et al., 1992; Moser and Padilla 1998; Nostrandt et al., 1997; Zhang et al., 2000, 2002). The biochemistry of the dopamine system in the striatum was also monitored, as there have been suggestions in the literature that pesticide exposure may be correlated with parkinsonism or Parkinson's disease (Bhatt et al., 1999; Davis et al., 1978; Hsieh et al., 2001; Joubert and Joubert, 1988; Senanayake and Sanmuganathan, 1995). It is also recognized that the cholinergic and dopaminergic systems are finely balanced in the mammalian CNS (Graybiel, 1990; Pisani et al., 2001; Van Woert, 1979), and that cholinergic drugs may cause signs reminiscent of Parkinson's disease (Clough et al., 1984; Mori, 2002). Therefore, dopamine and dopamine metabolite levels were monitored to assess the dopaminergic status of the striata of animals chronically fed chlorpyrifos. Moreover, the density of dopamine transporters (which can be a good indicator of dopamine terminal status [Araki et al., 1998; Berger et al., 1991; Javitch et al., 1984; Puschban et al., 2000; Shimizu and Prasad, 1991]) using mazindol binding also was assessed. Together, these end points were chosen to provide data to address the questions posed above.
MATERIALS AND METHODS
For pragmatic reasons, it was necessary to conduct this study with two cohorts of rats (n = 220/cohort). The cohorts were treated identically. All studies were approved in advance and conducted in accordance with the guidelines of the NHEERL Institutional Animal Care and Use Committee at the National Health and Environmental Effects Research Laboratory. Other studies conducted with these animals are presented in two additional articles (Moser et al., 2005 and Samsam et al., 2005).
Treatment and sample collection.
(See Fig. 1 for a summary). Male Long-Evans rats, weight maintained at 350 g, were fed a diet containing 0, 1, or 5 mg/kg/day of chlorpyrifos for 6 or 12 months. The body weight was maintained at 350 g for two reasons: (1) so that the animals' body mass composition remained as constant as possible, thereby helping to insure a consistent dosage over the course of the study, and (2) behavioral assessment used food as a reward, and the animals needed to be motivated to work for that reinforcement. The food was prepared by Bio-Serv (Frenchtown, NJ) from Diet #F0165 (Rodent Grain Base Diet, manufactured in-house) to which appropriate concentrations of chlorpyrifos were added. The food was formed into wafers that were analyzed by high-performance liquid chromatography (HPLC) to assure that the proper dose of chlorpyrifos was delivered (see below for details). After 2 months on this diet, half of each feeding group
In summary, there were six treatment groups: control (0+oil), control+spike (0+CPF), 1 mg/kg/day diet (1+oil), 1 mg/kg/day diet+spike (1+CPF), 5 mg/kg/day diet (5+oil), and 5 mg/kg/day diet+spike (5+CPF). Twenty-four hours after the spike dosage at 6 (i.e., third spike) or 12 months (i.e., sixth spike), rats (n = 5–8) were anesthetized with CO2 and killed by decapitation. Subgroups (n = 5) of these same 6 treatment groups (termed the "recovery" group), were given control wafers (containing no chlorpyrifos) for 3 additional months and then killed for analysis.
Tissues (brain, retina, and diaphragm) were collected, and immediately placed on dry ice. Trunk blood was collected in a heparinized tube. An aliquot of the whole blood was diluted (1:10; 1 part blood to 9 parts 0.1 M sodium phosphate buffer, pH 8.0 containing 1% Triton X-100) for ChE assay. The remainder of the whole blood was separated by centrifugation at 1000 x g for 10 min. After removal of the plasma, an aliquot was taken of both plasma and red cell fractions (the latter diluted 1 part red blood cells to 24 parts 0.1 M sodium phosphate buffer, pH 8.0, containing 1% Triton X-100). All blood components and tissues were stored at –80°C until analysis. Before any biochemical analyses, the pons and striatum were dissected out of the frozen brain; therefore the "brain" is actually all brain tissue (cerebrum and cerebellum) minus the pons and striatum (rostral of the optic chiasm).
Cholinesterase activity.
All tissues were diluted with 0.1 M sodium phosphate buffer, pH 8.0, containing 1% Triton X-100. The tissue dilutions were as follows: pons 1:200 (initial to final; vol:vol); striatum 1:100; brain 1:50; retina 1:133; and diaphragm 1:25. Plasma samples remained undiluted, whereas blood (1:10) and red blood cells (1:25) had already been diluted in the same buffer and frozen prior to their analysis. All tissues were homogenized on ice in a Polytron (Polytron PT3100, probe 3012/2TM, 20,000 rpm, Brinkman Industries, Westbury, NY) for 15-s periods (with 10 s between each homogenization interval) until no particulates were visible in the homogenate. Total ChE activity was determined for pons, striatum, brain, retina, blood, plasma, red blood cells, and diaphragm with a Hitachi 911 Automatic Analyzer (Boehringer Mannheim Corp., Indianapolis, IN) according to a method described by Hunter and coworkers (Hunter et al., 1997). The automated analyzer measures ChE activity according to a variation of the Ellman method (Ellman et al., 1961).
QNB binding.
Muscarinic receptor binding assays were performed on the pons, retina, and brain. Tissues were homogenized in 0.05 M sodium phosphate buffer, pH 7.4, at a dilution of 25 mg wet weight/ml for pons, 15 mg/ml for retina, and 50 mg/ml for brain. Receptor density was determined by binding of saturating amounts of 3H-quinuclidinyl benzilate (QNB) as described by Yamamura and Snyder (1974). Briefly, homogenized tissues were centrifuged (34,000 x g for 10 min at 5°C), the pellet was washed twice and then incubated at 37°C with a saturating amount of QNB with and without atropine (final concentration, 5 μM) for 1 h.
3H-Mazindol binding.
Each striatum was homogenized on ice in 50 volumes of cold assay buffer (50 mM Tris-HCl, 120 mM NaCl, and 5 mM KCl pH 7.4) with a Brinkman PT-3100 Polytron at setting 8.5 for 20 s, and then centrifuged at 40,000 x g for 15 min. The wash procedure was repeated, and the final pellet was re-suspended at a concentration of 20 mg wet wt/ml. Transporter density was measured according to the method of Javitch and coworkers (1984) with slight modification. In each assay, 100 μl of striatal membrane preparation was incubated in duplicate at 0°C for 4 h with 40 nM 3H-mazindol in a final volume of 500 μl. The reaction was terminated via addition of 3 ml cold assay buffer, and rapid filtration over Whatman GF/F glass-fiber filters presoaked in 0.5% polyethyleneimine (PEI). Specific binding was determined by subtracting nonspecific binding from total binding. Nonspecific binding was determined using 10 μM nomifensine.
Catecholamine analysis.
Striata were dissected quickly on an ice-chilled surface using a "brain block" (Heffner et al., 1980), and stored in microcentrifuge tubes at –80°C until analysis. Cold mobile phase and internal standard (isoproterenol) were added to the samples just prior to analysis. Samples were vortexed and sonicated (Branson Cell Disruptor) to release catecholamines, and then centrifuged at 14,000 x g for 18 min. A 200-μl aliquot of the supernatant was placed in an autosampler tube for injection onto the column. Dopamine (DA), and selected metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were quantified using reverse-phase ion pair chromotography with electrochemical detection. A Metachem Technologies (Metasil) reversed-phase column (4.6 mm inner diameter x 100 mm long) was used in the separation. The mobile phase consisted of 0.05 M sodium phosphate, 0.03 M citric acid, 0.1 mM EDTA, 2 mM sodium octyl sulfate, and 14–18% HPLC-grade methanol at pH 3.4. The flow rate was 0.8 ml/min. Neurotransmitters were detected with a glossy carbon electrode set at +0.65 V. The lower limit of 20 fmol of DA in a 20 μl sample was consistently detected. Concentrations of DA, DOPAC, and HVA, in striatal tissue, were calculated by linear regression based on a standard curve generated using a PE Nelson 500 series Interface and Turbochrom software.
Analysis of rat food.
Each lot of rat food was analyzed for chlorpyrifos concentration using HPLC. Five wafers were selected randomly from each lot, and weighed individually to verify that the wafer weight was as specified (i.e., within 10%; 3 ± 0.3 g). The wafers were pulverized individually in a Micro-Mill grinder (Bel-Art Products, Pequannock, NJ) and approximately 80 mg of each sample was weighed into a 15-ml conical glass extraction tube. After the addition of an internal standard (chlorpyrifos methyl), 4 ml of ethyl acetate was added, the samples were vortexed for 5 min (setting 7, multitube vortexer, Dade International, Miami, FL), and then centrifuged for 10 min at 1000 x g (HNS-centrifuge, Damon, Needham Heights, MA). The ethyl acetate layer was removed to a clean glass collection tube, and the extraction procedure was repeated twice more, with all ethyl acetate layers being added to the initial collection tube. The ethyl acetate collected was evaporated under a gentle stream of nitrogen, and the sample was reconstituted in 200 μl of mobile phase and filtered, after which 50 μl was injected into the HPLC.
Analyses were made with a Waters HPLC system, consisting of a 996 PDA detector, a 600 Solvent Delivery System, and a 717plus Autosampler. The mobile phase consisted of 80% acetonitrile and 20% water (Millipore Corporation) containing glacial acetic acid (0.01%) with a flow rate of 0.6 ml/min. Separation was made on a Waters Symmetry Shield RP18 column (5 μm, 3.9 x 150 mm), with the eluent monitored at 290 nm.
Spiking of control samples indicated that chlorpyrifos recovery was 82.5%. Food lots were accepted if the chlorpyrifos concentration was within 10% of the specified level. Each lot of control diet also was analyzed to verify the absence of chlorpyrifos.
Statistical analyses.
The 6- and 12-month data were analyzed together because they were collected from the same cohort, whereas the 15-month data, derived from the other cohort, were analyzed separately. Feed and bolus dose were between-subject factors, with repeated measures for tissues taken within same subject; time was also included as a factor in the 6- and 12-month data. Multivariate analyses of variance (ANOVA) were conducted for: central ChE activity (brain, pons, retina, striatum); peripheral ChE activity (whole blood, red blood cells, plasma, diaphragm); QNB binding (brain, pons, retina); and mazindol binding (striatum only). Where significant interactions occurred, step-down analyses were conducted; otherwise the data were collapsed to identify treatment effects.
RESULTS
Overt Toxicity
Because the animals' weights were maintained at 350 g, overt toxicity could not be assessed with weight gain. All rats maintained their weight at the target 350 g, requiring approximately 12–17 g of food per day. A companion paper (Moser et al., 2005) reports in detail the data from functional observational battery and motor activity assessment in identically treated rats from both cohorts.
Biochemical End Points
The general study design for the determination of all the biochemical end points was to first collect all the samples from all dosage groups from all time points. Then, each end point (e.g., plasma ChE or pons QNB binding) was analyzed according to a pseudo-random design. In other words, for each assay, all samples from all time points from all dosage groups were analyzed together in a structured, but mixed, manner. If all samples for each individual tissue and assay could not be analyzed on the same day, the samples were divided so that each group for analysis contained representatives from all dosage groups and all time points. See Table 1 for a summary of all exposure scenarios and results.
Control levels of ChE activity (Figs. 2 and 3), muscarinic receptor density (Fig. 4), dopamine transporter density (Fig. 5), and striatal dopamine and metabolite levels (data not shown) remained constant throughout the 15 months of the study. As mentioned above, the study was conducted in two cohorts, and the data presented in this report are from both cohorts: the 15-month ("recovery") group was from the first cohort, and the 6- and 12-month animals were from the second cohort. The fact that the control levels were not different across time points is an indication both that biochemical aging is not significant during a 15-month of the study and that the two cohorts are comparable.
Cholinesterase inhibition during the first 12 months.
The ChE activity end points for the 6- and 12-month data were not different (i.e, there was not a dose x time interaction), so the data were combined for all subsequent analyses. In general, this dosing regimen achieved the goal of separating the central and peripheral ChE inhibition in the two diet-only (i.e., no spike) dosage groups. Specifically, in the 1+oil group, ChE activity was inhibited in the blood components (whole blood, plasma, and red blood cells) (Fig. 2), but not in the diaphragm (Fig. 2), CNS, or retina (Fig. 3). In the animals that
In the groups that
Cholinesterase inhibition at 15 months ("recovery").
When examined 3 months after cessation of chlorpyrifos dosing, ChE levels in all groups were not different from control (Figs. 2 and 3).
Receptor binding during the first 12 months.
The muscarinic receptor density in the brain, pons, and retina did not change over time (i.e., there was no dose x time interaction), so the 6-month and 12-month data were combined for all subsequent analyses (Fig. 4). QNB binding density in the brain was decreased in the animals receiving the highest dosages of chlorpyrifos: both the high-dose (5+oil) diet group and the high-dose diet plus spike group (5+CPF) showed decreased muscarinic receptor density (10–20% decrease). The muscarinic receptor density in the retina and the pons did not change in any of the dosage groups.
For the mazindol binding in the striatum (Fig. 5) there was a dose x time interaction, so all three time points were analyzed separately. Mazindol binding, an indicator of the density of presynaptic dopamine transporters, showed an interaction with spike after 6 months of dosing; examination of Figure 5 shows an increase in mazindol binding in the striata of all three groups that had
Receptor binding at 15 months ("recovery").
At 15 months, 3 months after the last dose of chlorpyrifos, there were no measurable effects on either QNB binding in the brain, pons, and retina, or on mazindol binding in the striatum.
Dopamine and metabolite levels (data not shown).
There were no changes in concentrations of dopamine or its metabolites, HVA and DOPAC. Control levels (ng/mg wet weight, mean ± SEM) were (all three time points combined): dopamine = 9.64 ± 0.64; DOPAC = 2.15 ± 0.11; HVA = 0.90 ± 0.04.
DISCUSSION
The dosing levels used in this study represented a broad range of exposures. Calculations indicate that the total exposure of a 350-g rat in the 0+CPF group was approximately 100 mg chlorpyrifos during the 12-month exposure period (see Table 1). Analogous estimates for the other groups would be as follows: 1+oil group = 128 mg; 1+CPF group = 228 mg; 5+oil group = 639 mg; and 5+CPF group = 739 mg. Thus, the design used provided a wide range of chlorpyrifos exposure, as well as a varied pattern of exposure. One group, 0+CPF, had only intermittent ChE inhibition (i.e., every 2 months after the spike dose of chlorpyrifos). The fact that ChE inhibition was not greater at the 12-month time point than at the 6-month time point indicates that progressive inhibition did not occur during the dosing. This observation would seem to indicate that there was complete recovery of ChE inhibition between each spike dose. Also in support of this contention is the observation that our earlier studies with adult male rats given even higher chlorpyrifos dosages showed substantial, if not total, recovery within 2 weeks after dosing in every compartment except red blood cells (Moser and Padilla, 1998). It is therefore not unreasonable to expect total recovery in the 2 months between each spike dose of chlorpyrifos. Another group—1+oil—only had small amounts of ChE activity in the blood components, contrasted with no ChE inhibition in either the CNS or the diaphragm. The second feed group, 5+oil, experienced more than 50% inhibition in all tissues (except diaphragm, which showed only about 30% inhibition) and blood for the duration of the dosing. In related studies (Breslin et al., 1996; Szabo et al., 1988 as cited in Mattsson et al., 1996) a very similar pattern of CNS inhibition was noted. Rats fed 1 mg/kg/day chlorpyrifos showed no brain ChE inhibition, but at 5 mg/kg/day, there was approximately 50% inhibition in the brain. The 1+CPF spike group, like the 0+CPF group, showed CNS cholinesterase inhibition only when given the bolus dose of chlorpyrifos every 2 months. The 5+CPF spike group experienced extensive ChE inhibition throughout the entire dosing duration, and the 5+oil group experienced 60–70% inhibition in the brain, pons, striatum, and retina during the entire dosing period.
There are three general conclusions that can be gleaned from these data.
There was no change in ChE inhibition or muscarinic receptor density between 6 and 12 months, indicating that the animals had reached a steady state with regard to changes in cholinergic biochemistry. To our knowledge, no one has ever assessed muscarinic receptor density during a chronic dosing study, but ChE inhibition has been assessed over time in chronic studies with results that mirror the present data. For example, hens fed chlorpyrifos took less than 4 weeks to reach steady state inhibition of plasma ChE (Sherman and Herrick, 1973). Moreover, rats fed chlorpyrifos at 1 mg/kg had no greater ChE inhibition in the brain, plasma, or red blood cells after 2 years than after 13 weeks of feeding (Yano et al., 2000).
Given the high levels of ChE inhibition in all the spike groups, as well as in the 5+oil group, there were few changes in muscarinic receptor density, with only the 5+oil and 5+CPF groups showing downregulation. The two dosage groups, 0+CPF and 1+CPF, that had high levels of intermittent brain ChE inhibition but not steady brain ChE inhibition, did not show muscarinic receptor downregulation. An examination of the literature shows, in general, that high dosages of chlorpyrifos were required in adult animals to decrease muscarinic receptor density. Muscarinic receptor downregulation was only seen after oral dosages much higher than those used in the present study (Moser and Padilla, 1998; Nostrandt et al., 1997). Moreover, for subcutaneous dosages, it appears that 90% ChE inhibition is associated with changes in muscarinic receptor density in adult male rats (Bushnell et al., 1993; Chakraborti et al., 1993; Huff et al., 2001; Zhang et al., 2000, 2002).
There were no persistent effects on any of the biochemical end points measured, such that by 15 months all biochemical indices had returned to control levels. It appears that there is no published study assessing the recovery of this spectrum of end points in rats after chronic dosing with chlorpyrifos, but there is relevant previous work noting complete recovery in various end points by 2–8 weeks after cessation of dosing. One study assessing the recovery of red blood cell and brain ChE activity after chronic treatment with 1 or 3 mg/kg/day chlorpyrifos noted normal ChE activity within 8 weeks after cessation of dosing (McCollister et al., 1974). Another study assessed the recovery of plasma ChE in hens treated with chlorpyrifos, noting that plasma ChE activity returned to normal within a couple of weeks (Sherman and Herrick, 1973). Using another organophosphorus compound, diisopropyl fluorophosphate, other investigators reported that recovery of brain ChE activity and muscarinic receptor density was complete by 4 weeks in young Sprague-Dawley rats (Pintor et al., 1990).
Although previous articles have reported decreased striatal dopamine in animals treated with dichlorvos (Ali et al., 1980), or increased striatal DOPAC concentration and decreased dopamine synaptosomal reuptake in rats treated with three subcutaneous dosages of 100 mg/kg chlorpyrifos (Karen et al., 2001), the present results found no changes in striatal dopamine or DOPAC concentrations in any dosage group at any time either during or after chlorpyrifos exposure. Moreover, no decrease in dopamine transporters was noted. Instead, dopamine transporter density was increased transiently, but only in the animals receiving the spike dosages of chlorpyrifos. Increases in dopamine transporters have been noted in adult animals treated with heptachlor (Miller et al., 1999), but no previous study used ChE inhibitors. Other studies have explored the interaction of nicotine treatment and dopamine transporters. A recently published article reports increased striatal dopamine transporters in adolescent (but not adult) rats treated repeatedly with nicotine (Collins et al., 2004), whereas an earlier report notes that nicotine treatment slows the age-related decline in dopamine transporters (Prasad et al., 1994). These observations, in concert with the present data, reinforce the connection between the stimulation of acetylcholine receptors, whether nicotinic or muscarinic, and regulation of dopamine uptake in rat striatum.
It is useful to return to the original five questions posed in the Introduction and consider the answers in light of the present results (see Table 1).
Is there (a) downregulation of CNS muscarinic receptors, (b) changes in striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos if only blood and peripheral ChE activity are inhibited by chlorpyrifos, accompanied by no inhibition of ChE activity in the brain There were no changes in muscarinic receptor density or striatal dopamine transporters in the one group—1+oil—that did not experience brain ChE inhibition. Moreover, consumption of that level of chlorpyrifos did not change the response to a spike dose of chlorpyrifos: the neurochemical responses of the 0+oil and the 1+oil groups were indistinguishable.
Does inhibition of brain ChE activity by about 50% for a year cause changes in muscarinic receptor density or changes in dopaminergic parameters Brain muscarinic receptor density was decreased significantly with this constant level of ChE inhibition, whereas none of the striatal dopaminergic parameters were altered in the 5+oil group.
Does a high bolus dose of chlorpyrifos produce different effects on (a) downregulation of CNS muscarinic receptors, (b) changes in striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos as compared to approximately the same total dose fed at a lower rate over a much longer period of time i.e., Does the pattern of exposure matter One way to consider this question is to compare the toxicity profile of the two groups in which the total dosage of chlorpyrifos over the year was similar but the pattern of exposure differed (Table 1): the 0+CPF group (100 mg total) may be compared to 1+oil group (128 mg total), and the 5+oil group (639 mg total) may be compared to the 5+CPF group (739 mg total). Comparison of the 0+CPF and 1+oil groups reveals that the 0+CPF group experienced brain and diaphragm ChE inhibition, whereas the 1+oil group did not; both groups experienced whole blood, plasma, and red blood cell ChE inhibition, but the 0+CPF group experienced intermittently higher levels of inhibition in whole blood and plasma. These results indicate that a more protracted exposure (as opposed to an acute, high-dose exposure) to chlorpyrifos tends to limit the amount of brain and muscle ChE inhibition. Neither the 0+CPF nor 1+oil group experienced changes in muscarinic receptor density, but the 0+CPF group did experience increased dopamine transporters in the striatum, but only at the 6 month time point. The 5+oil and the 5+CPF groups showed, in general, the same qualitative changes for ChE inhibition and muscarinic receptor density: all tissues showed some level of inhibition or downregulation. The differences between the two groups emerged with the degree of inhibition or downregulation, with the group (5+CPF) that
If a high bolus dose of chlorpyrifos is given to animals whose ChE activity is already inhibited, is the inhibition dampened because the animals are already "tolerant" to the toxic effects or exacerbated by the low level of existing ChE activity Using the previous literature as a guide, one might expect a dampened response in the animals already receiving chlorpyrifos in their diet, given that rats receiving one dose of chlorfenvinphos showed less of a response to the second dose, presumably because of depression of the activation of the parent compound (Ikeda et al., 1990, 1991, 1992). In this case one would compare the animals that did not receive chlorpyrifos in their feed but did receive the bolus dosages of chlorpyrifos (i.e., 0+CPF) with either the 1+CPF or the 5+CPF groups. None of the biochemical end points were dampened because of previous exposure to chlorpyrifos in the feed. In fact, in many instances, there was a greater effect if the animal had been receiving chlorpyrifos in the feed, but only at the highest dosage (5 mg/kg/day).
After a year of dosing, will the animals recover biochemically after the chlorpyrifos dosing is stopped Comparison of all the biochemical end points reveals that by 3 months after cessation of dosing, all had returned to control levels.
The present work was designed not only to answer the above questions regarding chlorpyrifos toxicity but to also set the biochemical backdrop for other investigations of the behavioral toxicity of chlorpyrifos in the same groups of animals. Those results are presented in two additional papers (Moser et al., 2005, and Samsam et al., 2005).
ACKNOWLEDGMENTS
Chronic feeding studies require an exceptional amount of oversight and effort from the animal care staff. We thank the dedicated and hardworking staff who cared for the animals used in this study: Kim Howell, Jennifer Kinson and Jenelle Dunn. We also thank Drs. Philip Bushnell and Suzanne McMaster for careful and thoughtful reviews of earlier versions of this manuscript.
This research 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 necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Departments of Pharmacology, Neurology
Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ABSTRACT
Very little is known about the effects of chronic exposure to relatively low levels of anticholinesterase insecticides or how the effects of chronic exposure compare to those of higher, intermittent exposure. To that end, adult male rats were fed an anticholinesterase insecticide, chlorpyrifos (CPF), for 1 year at three levels of dietary exposure: 0, 1, or 5 mg/kg/day (0+oil, 1+oil, and 5+oil). In addition, half of each of these groups also
Key Words: chlorpyrifos; rat; chronic; cholinesterase; muscarinic; dopaminergic.
INTRODUCTION
The acute toxicity of organophosphorus insecticides is well known and has been extensively characterized. As inhibitors of cholinesterase (ChE) activity, organophosphorus insecticides produce overstimulation of cholinergic neurotransmission, both centrally and peripherally, eliciting signs and symptoms such as increased salivation, sweating, changes in blood pressure and heart rate, nausea, diarrhea, confusion, headache, muscle tremor, and, in high-dose situations, breathing difficulty, convulsions, and death. It is generally assumed that the toxicity of currently available organophosphorus insecticides is limited to the acute effects as described above and, consequently, that organophosphorus insecticides do not precipitate lasting adverse effects. In fact, however, very little is known about chronic, persistent effects of organophosphorus insecticides. There is growing concern that long-term, low level exposure to organophosphorus insecticides may have protracted or permanent effects (reviewed in Hoppin and Kamel, 2004; Ray and Richards, 2001).
Chlorpyrifos was the highest volume organophosphorus insecticide used in the United States until 2000, when the home and garden use of chlorpyrifos and its use on many food crops was severely restricted (http://www.epa.gov/pesticides/announcement6800.htm; last update Oct. 13, 2004). There has been widespread human exposure, as shown by the prevalence of chlorpyrifos metabolites in urine samples (Buck et al., 2001; Lemus et al., 1997), and human health effects have been attributed to chlorpyrifos exposure (Kaplan et al., 1993; Steenland et al., 2000). Although there have been many chronic studies of chlorpyrifos toxicity in animals (Breslin et al., 1996; McCollister et al., 1974; Sherman and Herrick, 1973; Yano et al., 2000), none of these studies included an in-depth assessment of neurochemical changes, only ChE measurements. Indeed, a subset of people (e.g., pesticides applicators) may have been exposed to additional chlorpyrifos, and there have been no studies exploring the profile of such higher, intermittent, exposures. Therefore, the present study investigated both long-term chronic exposure to chlorpyrifos and intermittent higher level exposure, using multiple neurochemical (present paper) and behavioral end points (Moser et al., 2005; Samsam, et al., 2005). Specifically, adult male Long-Evans rats were fed chlorpyrifos for a year at two dosage levels, with and without a bolus dose of chlorpyrifos every 2 months. This experimental design was constructed to answer the following questions about the toxic effects of long-term chlorpyrifos-induced ChE inhibition. (1) Is there (a) downregulation of CNS muscarinic receptors, (b) any change in striatal dopaminergic tone, or (c) any difference in neurochemical parameters after a challenge dose of chlorpyrifos if only blood and peripheral ChE activity are inhibited by chlorpyrifos, accompanied by no inhibition of ChE activity in the brain (2) Does inhibition of brain ChE activity by about 50% for a year cause changes in muscarinic receptor density or changes in dopaminergic parameters (3) Does a high bolus dose of chlorpyrifos produce different effects on (a) downregulation of CNS muscarinic receptors, (b) striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos as compared to approximately the same total dose fed at a lower rate over a much longer period of time— i.e., Does the pattern of exposure matter (4) If a high bolus dose of chlorpyrifos is given to animals whose ChE activity is already inhibited, is the inhibition dampened because the animals are already "tolerant" to the toxic effects or is it exacerbated by the low level of existing ChE activity (5) After a year of dosing, will the animals recover after the chlorpyrifos dosing is stopped
The present article reports the assessment of the neurochemical end points in the animals at two time points during the dosing (6 and 12 months) and again at 3 months after dosing ceased (15 months). Cholinesterase activity was monitored in blood, smooth muscle, and central nervous system (CNS) to assess both exposure and effect. Moreover, muscarinic receptor density was assessed because it is well-known that continual ChE inhibition in general and chlorpyrifos treatment, in particular, can produce tolerance that is manifested biochemically by downregulation of the muscarinic receptors (Pope et al., 1992; Moser and Padilla 1998; Nostrandt et al., 1997; Zhang et al., 2000, 2002). The biochemistry of the dopamine system in the striatum was also monitored, as there have been suggestions in the literature that pesticide exposure may be correlated with parkinsonism or Parkinson's disease (Bhatt et al., 1999; Davis et al., 1978; Hsieh et al., 2001; Joubert and Joubert, 1988; Senanayake and Sanmuganathan, 1995). It is also recognized that the cholinergic and dopaminergic systems are finely balanced in the mammalian CNS (Graybiel, 1990; Pisani et al., 2001; Van Woert, 1979), and that cholinergic drugs may cause signs reminiscent of Parkinson's disease (Clough et al., 1984; Mori, 2002). Therefore, dopamine and dopamine metabolite levels were monitored to assess the dopaminergic status of the striata of animals chronically fed chlorpyrifos. Moreover, the density of dopamine transporters (which can be a good indicator of dopamine terminal status [Araki et al., 1998; Berger et al., 1991; Javitch et al., 1984; Puschban et al., 2000; Shimizu and Prasad, 1991]) using mazindol binding also was assessed. Together, these end points were chosen to provide data to address the questions posed above.
MATERIALS AND METHODS
For pragmatic reasons, it was necessary to conduct this study with two cohorts of rats (n = 220/cohort). The cohorts were treated identically. All studies were approved in advance and conducted in accordance with the guidelines of the NHEERL Institutional Animal Care and Use Committee at the National Health and Environmental Effects Research Laboratory. Other studies conducted with these animals are presented in two additional articles (Moser et al., 2005 and Samsam et al., 2005).
Treatment and sample collection.
(See Fig. 1 for a summary). Male Long-Evans rats, weight maintained at 350 g, were fed a diet containing 0, 1, or 5 mg/kg/day of chlorpyrifos for 6 or 12 months. The body weight was maintained at 350 g for two reasons: (1) so that the animals' body mass composition remained as constant as possible, thereby helping to insure a consistent dosage over the course of the study, and (2) behavioral assessment used food as a reward, and the animals needed to be motivated to work for that reinforcement. The food was prepared by Bio-Serv (Frenchtown, NJ) from Diet #F0165 (Rodent Grain Base Diet, manufactured in-house) to which appropriate concentrations of chlorpyrifos were added. The food was formed into wafers that were analyzed by high-performance liquid chromatography (HPLC) to assure that the proper dose of chlorpyrifos was delivered (see below for details). After 2 months on this diet, half of each feeding group
In summary, there were six treatment groups: control (0+oil), control+spike (0+CPF), 1 mg/kg/day diet (1+oil), 1 mg/kg/day diet+spike (1+CPF), 5 mg/kg/day diet (5+oil), and 5 mg/kg/day diet+spike (5+CPF). Twenty-four hours after the spike dosage at 6 (i.e., third spike) or 12 months (i.e., sixth spike), rats (n = 5–8) were anesthetized with CO2 and killed by decapitation. Subgroups (n = 5) of these same 6 treatment groups (termed the "recovery" group), were given control wafers (containing no chlorpyrifos) for 3 additional months and then killed for analysis.
Tissues (brain, retina, and diaphragm) were collected, and immediately placed on dry ice. Trunk blood was collected in a heparinized tube. An aliquot of the whole blood was diluted (1:10; 1 part blood to 9 parts 0.1 M sodium phosphate buffer, pH 8.0 containing 1% Triton X-100) for ChE assay. The remainder of the whole blood was separated by centrifugation at 1000 x g for 10 min. After removal of the plasma, an aliquot was taken of both plasma and red cell fractions (the latter diluted 1 part red blood cells to 24 parts 0.1 M sodium phosphate buffer, pH 8.0, containing 1% Triton X-100). All blood components and tissues were stored at –80°C until analysis. Before any biochemical analyses, the pons and striatum were dissected out of the frozen brain; therefore the "brain" is actually all brain tissue (cerebrum and cerebellum) minus the pons and striatum (rostral of the optic chiasm).
Cholinesterase activity.
All tissues were diluted with 0.1 M sodium phosphate buffer, pH 8.0, containing 1% Triton X-100. The tissue dilutions were as follows: pons 1:200 (initial to final; vol:vol); striatum 1:100; brain 1:50; retina 1:133; and diaphragm 1:25. Plasma samples remained undiluted, whereas blood (1:10) and red blood cells (1:25) had already been diluted in the same buffer and frozen prior to their analysis. All tissues were homogenized on ice in a Polytron (Polytron PT3100, probe 3012/2TM, 20,000 rpm, Brinkman Industries, Westbury, NY) for 15-s periods (with 10 s between each homogenization interval) until no particulates were visible in the homogenate. Total ChE activity was determined for pons, striatum, brain, retina, blood, plasma, red blood cells, and diaphragm with a Hitachi 911 Automatic Analyzer (Boehringer Mannheim Corp., Indianapolis, IN) according to a method described by Hunter and coworkers (Hunter et al., 1997). The automated analyzer measures ChE activity according to a variation of the Ellman method (Ellman et al., 1961).
QNB binding.
Muscarinic receptor binding assays were performed on the pons, retina, and brain. Tissues were homogenized in 0.05 M sodium phosphate buffer, pH 7.4, at a dilution of 25 mg wet weight/ml for pons, 15 mg/ml for retina, and 50 mg/ml for brain. Receptor density was determined by binding of saturating amounts of 3H-quinuclidinyl benzilate (QNB) as described by Yamamura and Snyder (1974). Briefly, homogenized tissues were centrifuged (34,000 x g for 10 min at 5°C), the pellet was washed twice and then incubated at 37°C with a saturating amount of QNB with and without atropine (final concentration, 5 μM) for 1 h.
3H-Mazindol binding.
Each striatum was homogenized on ice in 50 volumes of cold assay buffer (50 mM Tris-HCl, 120 mM NaCl, and 5 mM KCl pH 7.4) with a Brinkman PT-3100 Polytron at setting 8.5 for 20 s, and then centrifuged at 40,000 x g for 15 min. The wash procedure was repeated, and the final pellet was re-suspended at a concentration of 20 mg wet wt/ml. Transporter density was measured according to the method of Javitch and coworkers (1984) with slight modification. In each assay, 100 μl of striatal membrane preparation was incubated in duplicate at 0°C for 4 h with 40 nM 3H-mazindol in a final volume of 500 μl. The reaction was terminated via addition of 3 ml cold assay buffer, and rapid filtration over Whatman GF/F glass-fiber filters presoaked in 0.5% polyethyleneimine (PEI). Specific binding was determined by subtracting nonspecific binding from total binding. Nonspecific binding was determined using 10 μM nomifensine.
Catecholamine analysis.
Striata were dissected quickly on an ice-chilled surface using a "brain block" (Heffner et al., 1980), and stored in microcentrifuge tubes at –80°C until analysis. Cold mobile phase and internal standard (isoproterenol) were added to the samples just prior to analysis. Samples were vortexed and sonicated (Branson Cell Disruptor) to release catecholamines, and then centrifuged at 14,000 x g for 18 min. A 200-μl aliquot of the supernatant was placed in an autosampler tube for injection onto the column. Dopamine (DA), and selected metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were quantified using reverse-phase ion pair chromotography with electrochemical detection. A Metachem Technologies (Metasil) reversed-phase column (4.6 mm inner diameter x 100 mm long) was used in the separation. The mobile phase consisted of 0.05 M sodium phosphate, 0.03 M citric acid, 0.1 mM EDTA, 2 mM sodium octyl sulfate, and 14–18% HPLC-grade methanol at pH 3.4. The flow rate was 0.8 ml/min. Neurotransmitters were detected with a glossy carbon electrode set at +0.65 V. The lower limit of 20 fmol of DA in a 20 μl sample was consistently detected. Concentrations of DA, DOPAC, and HVA, in striatal tissue, were calculated by linear regression based on a standard curve generated using a PE Nelson 500 series Interface and Turbochrom software.
Analysis of rat food.
Each lot of rat food was analyzed for chlorpyrifos concentration using HPLC. Five wafers were selected randomly from each lot, and weighed individually to verify that the wafer weight was as specified (i.e., within 10%; 3 ± 0.3 g). The wafers were pulverized individually in a Micro-Mill grinder (Bel-Art Products, Pequannock, NJ) and approximately 80 mg of each sample was weighed into a 15-ml conical glass extraction tube. After the addition of an internal standard (chlorpyrifos methyl), 4 ml of ethyl acetate was added, the samples were vortexed for 5 min (setting 7, multitube vortexer, Dade International, Miami, FL), and then centrifuged for 10 min at 1000 x g (HNS-centrifuge, Damon, Needham Heights, MA). The ethyl acetate layer was removed to a clean glass collection tube, and the extraction procedure was repeated twice more, with all ethyl acetate layers being added to the initial collection tube. The ethyl acetate collected was evaporated under a gentle stream of nitrogen, and the sample was reconstituted in 200 μl of mobile phase and filtered, after which 50 μl was injected into the HPLC.
Analyses were made with a Waters HPLC system, consisting of a 996 PDA detector, a 600 Solvent Delivery System, and a 717plus Autosampler. The mobile phase consisted of 80% acetonitrile and 20% water (Millipore Corporation) containing glacial acetic acid (0.01%) with a flow rate of 0.6 ml/min. Separation was made on a Waters Symmetry Shield RP18 column (5 μm, 3.9 x 150 mm), with the eluent monitored at 290 nm.
Spiking of control samples indicated that chlorpyrifos recovery was 82.5%. Food lots were accepted if the chlorpyrifos concentration was within 10% of the specified level. Each lot of control diet also was analyzed to verify the absence of chlorpyrifos.
Statistical analyses.
The 6- and 12-month data were analyzed together because they were collected from the same cohort, whereas the 15-month data, derived from the other cohort, were analyzed separately. Feed and bolus dose were between-subject factors, with repeated measures for tissues taken within same subject; time was also included as a factor in the 6- and 12-month data. Multivariate analyses of variance (ANOVA) were conducted for: central ChE activity (brain, pons, retina, striatum); peripheral ChE activity (whole blood, red blood cells, plasma, diaphragm); QNB binding (brain, pons, retina); and mazindol binding (striatum only). Where significant interactions occurred, step-down analyses were conducted; otherwise the data were collapsed to identify treatment effects.
RESULTS
Overt Toxicity
Because the animals' weights were maintained at 350 g, overt toxicity could not be assessed with weight gain. All rats maintained their weight at the target 350 g, requiring approximately 12–17 g of food per day. A companion paper (Moser et al., 2005) reports in detail the data from functional observational battery and motor activity assessment in identically treated rats from both cohorts.
Biochemical End Points
The general study design for the determination of all the biochemical end points was to first collect all the samples from all dosage groups from all time points. Then, each end point (e.g., plasma ChE or pons QNB binding) was analyzed according to a pseudo-random design. In other words, for each assay, all samples from all time points from all dosage groups were analyzed together in a structured, but mixed, manner. If all samples for each individual tissue and assay could not be analyzed on the same day, the samples were divided so that each group for analysis contained representatives from all dosage groups and all time points. See Table 1 for a summary of all exposure scenarios and results.
Control levels of ChE activity (Figs. 2 and 3), muscarinic receptor density (Fig. 4), dopamine transporter density (Fig. 5), and striatal dopamine and metabolite levels (data not shown) remained constant throughout the 15 months of the study. As mentioned above, the study was conducted in two cohorts, and the data presented in this report are from both cohorts: the 15-month ("recovery") group was from the first cohort, and the 6- and 12-month animals were from the second cohort. The fact that the control levels were not different across time points is an indication both that biochemical aging is not significant during a 15-month of the study and that the two cohorts are comparable.
Cholinesterase inhibition during the first 12 months.
The ChE activity end points for the 6- and 12-month data were not different (i.e, there was not a dose x time interaction), so the data were combined for all subsequent analyses. In general, this dosing regimen achieved the goal of separating the central and peripheral ChE inhibition in the two diet-only (i.e., no spike) dosage groups. Specifically, in the 1+oil group, ChE activity was inhibited in the blood components (whole blood, plasma, and red blood cells) (Fig. 2), but not in the diaphragm (Fig. 2), CNS, or retina (Fig. 3). In the animals that
In the groups that
Cholinesterase inhibition at 15 months ("recovery").
When examined 3 months after cessation of chlorpyrifos dosing, ChE levels in all groups were not different from control (Figs. 2 and 3).
Receptor binding during the first 12 months.
The muscarinic receptor density in the brain, pons, and retina did not change over time (i.e., there was no dose x time interaction), so the 6-month and 12-month data were combined for all subsequent analyses (Fig. 4). QNB binding density in the brain was decreased in the animals receiving the highest dosages of chlorpyrifos: both the high-dose (5+oil) diet group and the high-dose diet plus spike group (5+CPF) showed decreased muscarinic receptor density (10–20% decrease). The muscarinic receptor density in the retina and the pons did not change in any of the dosage groups.
For the mazindol binding in the striatum (Fig. 5) there was a dose x time interaction, so all three time points were analyzed separately. Mazindol binding, an indicator of the density of presynaptic dopamine transporters, showed an interaction with spike after 6 months of dosing; examination of Figure 5 shows an increase in mazindol binding in the striata of all three groups that had
Receptor binding at 15 months ("recovery").
At 15 months, 3 months after the last dose of chlorpyrifos, there were no measurable effects on either QNB binding in the brain, pons, and retina, or on mazindol binding in the striatum.
Dopamine and metabolite levels (data not shown).
There were no changes in concentrations of dopamine or its metabolites, HVA and DOPAC. Control levels (ng/mg wet weight, mean ± SEM) were (all three time points combined): dopamine = 9.64 ± 0.64; DOPAC = 2.15 ± 0.11; HVA = 0.90 ± 0.04.
DISCUSSION
The dosing levels used in this study represented a broad range of exposures. Calculations indicate that the total exposure of a 350-g rat in the 0+CPF group was approximately 100 mg chlorpyrifos during the 12-month exposure period (see Table 1). Analogous estimates for the other groups would be as follows: 1+oil group = 128 mg; 1+CPF group = 228 mg; 5+oil group = 639 mg; and 5+CPF group = 739 mg. Thus, the design used provided a wide range of chlorpyrifos exposure, as well as a varied pattern of exposure. One group, 0+CPF, had only intermittent ChE inhibition (i.e., every 2 months after the spike dose of chlorpyrifos). The fact that ChE inhibition was not greater at the 12-month time point than at the 6-month time point indicates that progressive inhibition did not occur during the dosing. This observation would seem to indicate that there was complete recovery of ChE inhibition between each spike dose. Also in support of this contention is the observation that our earlier studies with adult male rats given even higher chlorpyrifos dosages showed substantial, if not total, recovery within 2 weeks after dosing in every compartment except red blood cells (Moser and Padilla, 1998). It is therefore not unreasonable to expect total recovery in the 2 months between each spike dose of chlorpyrifos. Another group—1+oil—only had small amounts of ChE activity in the blood components, contrasted with no ChE inhibition in either the CNS or the diaphragm. The second feed group, 5+oil, experienced more than 50% inhibition in all tissues (except diaphragm, which showed only about 30% inhibition) and blood for the duration of the dosing. In related studies (Breslin et al., 1996; Szabo et al., 1988 as cited in Mattsson et al., 1996) a very similar pattern of CNS inhibition was noted. Rats fed 1 mg/kg/day chlorpyrifos showed no brain ChE inhibition, but at 5 mg/kg/day, there was approximately 50% inhibition in the brain. The 1+CPF spike group, like the 0+CPF group, showed CNS cholinesterase inhibition only when given the bolus dose of chlorpyrifos every 2 months. The 5+CPF spike group experienced extensive ChE inhibition throughout the entire dosing duration, and the 5+oil group experienced 60–70% inhibition in the brain, pons, striatum, and retina during the entire dosing period.
There are three general conclusions that can be gleaned from these data.
There was no change in ChE inhibition or muscarinic receptor density between 6 and 12 months, indicating that the animals had reached a steady state with regard to changes in cholinergic biochemistry. To our knowledge, no one has ever assessed muscarinic receptor density during a chronic dosing study, but ChE inhibition has been assessed over time in chronic studies with results that mirror the present data. For example, hens fed chlorpyrifos took less than 4 weeks to reach steady state inhibition of plasma ChE (Sherman and Herrick, 1973). Moreover, rats fed chlorpyrifos at 1 mg/kg had no greater ChE inhibition in the brain, plasma, or red blood cells after 2 years than after 13 weeks of feeding (Yano et al., 2000).
Given the high levels of ChE inhibition in all the spike groups, as well as in the 5+oil group, there were few changes in muscarinic receptor density, with only the 5+oil and 5+CPF groups showing downregulation. The two dosage groups, 0+CPF and 1+CPF, that had high levels of intermittent brain ChE inhibition but not steady brain ChE inhibition, did not show muscarinic receptor downregulation. An examination of the literature shows, in general, that high dosages of chlorpyrifos were required in adult animals to decrease muscarinic receptor density. Muscarinic receptor downregulation was only seen after oral dosages much higher than those used in the present study (Moser and Padilla, 1998; Nostrandt et al., 1997). Moreover, for subcutaneous dosages, it appears that 90% ChE inhibition is associated with changes in muscarinic receptor density in adult male rats (Bushnell et al., 1993; Chakraborti et al., 1993; Huff et al., 2001; Zhang et al., 2000, 2002).
There were no persistent effects on any of the biochemical end points measured, such that by 15 months all biochemical indices had returned to control levels. It appears that there is no published study assessing the recovery of this spectrum of end points in rats after chronic dosing with chlorpyrifos, but there is relevant previous work noting complete recovery in various end points by 2–8 weeks after cessation of dosing. One study assessing the recovery of red blood cell and brain ChE activity after chronic treatment with 1 or 3 mg/kg/day chlorpyrifos noted normal ChE activity within 8 weeks after cessation of dosing (McCollister et al., 1974). Another study assessed the recovery of plasma ChE in hens treated with chlorpyrifos, noting that plasma ChE activity returned to normal within a couple of weeks (Sherman and Herrick, 1973). Using another organophosphorus compound, diisopropyl fluorophosphate, other investigators reported that recovery of brain ChE activity and muscarinic receptor density was complete by 4 weeks in young Sprague-Dawley rats (Pintor et al., 1990).
Although previous articles have reported decreased striatal dopamine in animals treated with dichlorvos (Ali et al., 1980), or increased striatal DOPAC concentration and decreased dopamine synaptosomal reuptake in rats treated with three subcutaneous dosages of 100 mg/kg chlorpyrifos (Karen et al., 2001), the present results found no changes in striatal dopamine or DOPAC concentrations in any dosage group at any time either during or after chlorpyrifos exposure. Moreover, no decrease in dopamine transporters was noted. Instead, dopamine transporter density was increased transiently, but only in the animals receiving the spike dosages of chlorpyrifos. Increases in dopamine transporters have been noted in adult animals treated with heptachlor (Miller et al., 1999), but no previous study used ChE inhibitors. Other studies have explored the interaction of nicotine treatment and dopamine transporters. A recently published article reports increased striatal dopamine transporters in adolescent (but not adult) rats treated repeatedly with nicotine (Collins et al., 2004), whereas an earlier report notes that nicotine treatment slows the age-related decline in dopamine transporters (Prasad et al., 1994). These observations, in concert with the present data, reinforce the connection between the stimulation of acetylcholine receptors, whether nicotinic or muscarinic, and regulation of dopamine uptake in rat striatum.
It is useful to return to the original five questions posed in the Introduction and consider the answers in light of the present results (see Table 1).
Is there (a) downregulation of CNS muscarinic receptors, (b) changes in striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos if only blood and peripheral ChE activity are inhibited by chlorpyrifos, accompanied by no inhibition of ChE activity in the brain There were no changes in muscarinic receptor density or striatal dopamine transporters in the one group—1+oil—that did not experience brain ChE inhibition. Moreover, consumption of that level of chlorpyrifos did not change the response to a spike dose of chlorpyrifos: the neurochemical responses of the 0+oil and the 1+oil groups were indistinguishable.
Does inhibition of brain ChE activity by about 50% for a year cause changes in muscarinic receptor density or changes in dopaminergic parameters Brain muscarinic receptor density was decreased significantly with this constant level of ChE inhibition, whereas none of the striatal dopaminergic parameters were altered in the 5+oil group.
Does a high bolus dose of chlorpyrifos produce different effects on (a) downregulation of CNS muscarinic receptors, (b) changes in striatal dopaminergic tone, or (c) differences in neurochemical parameters after a challenge dose of chlorpyrifos as compared to approximately the same total dose fed at a lower rate over a much longer period of time i.e., Does the pattern of exposure matter One way to consider this question is to compare the toxicity profile of the two groups in which the total dosage of chlorpyrifos over the year was similar but the pattern of exposure differed (Table 1): the 0+CPF group (100 mg total) may be compared to 1+oil group (128 mg total), and the 5+oil group (639 mg total) may be compared to the 5+CPF group (739 mg total). Comparison of the 0+CPF and 1+oil groups reveals that the 0+CPF group experienced brain and diaphragm ChE inhibition, whereas the 1+oil group did not; both groups experienced whole blood, plasma, and red blood cell ChE inhibition, but the 0+CPF group experienced intermittently higher levels of inhibition in whole blood and plasma. These results indicate that a more protracted exposure (as opposed to an acute, high-dose exposure) to chlorpyrifos tends to limit the amount of brain and muscle ChE inhibition. Neither the 0+CPF nor 1+oil group experienced changes in muscarinic receptor density, but the 0+CPF group did experience increased dopamine transporters in the striatum, but only at the 6 month time point. The 5+oil and the 5+CPF groups showed, in general, the same qualitative changes for ChE inhibition and muscarinic receptor density: all tissues showed some level of inhibition or downregulation. The differences between the two groups emerged with the degree of inhibition or downregulation, with the group (5+CPF) that
If a high bolus dose of chlorpyrifos is given to animals whose ChE activity is already inhibited, is the inhibition dampened because the animals are already "tolerant" to the toxic effects or exacerbated by the low level of existing ChE activity Using the previous literature as a guide, one might expect a dampened response in the animals already receiving chlorpyrifos in their diet, given that rats receiving one dose of chlorfenvinphos showed less of a response to the second dose, presumably because of depression of the activation of the parent compound (Ikeda et al., 1990, 1991, 1992). In this case one would compare the animals that did not receive chlorpyrifos in their feed but did receive the bolus dosages of chlorpyrifos (i.e., 0+CPF) with either the 1+CPF or the 5+CPF groups. None of the biochemical end points were dampened because of previous exposure to chlorpyrifos in the feed. In fact, in many instances, there was a greater effect if the animal had been receiving chlorpyrifos in the feed, but only at the highest dosage (5 mg/kg/day).
After a year of dosing, will the animals recover biochemically after the chlorpyrifos dosing is stopped Comparison of all the biochemical end points reveals that by 3 months after cessation of dosing, all had returned to control levels.
The present work was designed not only to answer the above questions regarding chlorpyrifos toxicity but to also set the biochemical backdrop for other investigations of the behavioral toxicity of chlorpyrifos in the same groups of animals. Those results are presented in two additional papers (Moser et al., 2005, and Samsam et al., 2005).
ACKNOWLEDGMENTS
Chronic feeding studies require an exceptional amount of oversight and effort from the animal care staff. We thank the dedicated and hardworking staff who cared for the animals used in this study: Kim Howell, Jennifer Kinson and Jenelle Dunn. We also thank Drs. Philip Bushnell and Suzanne McMaster for careful and thoughtful reviews of earlier versions of this manuscript.
This research 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 necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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