* 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, and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
1 To whom correspondence should be addressed at Neurotoxicology Division (B10506), Office of Research and Development, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 4903 Page Road, Durham, NC 27711. FAX: 9195413335. E-mail: Padilla.Stephanie{at}epa.gov.
Received June 23, 2005; accepted July 27, 2005
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ABSTRACT |
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Key Words: chlorpyrifos; rat; chronic; cholinesterase; muscarinic; dopaminergic.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 received either a spike dose of chlorpyrifos in corn oil or corn oil vehicle by gavage (1 mg/kg). Spikes continued every 2 months to a total of six spikes during the 12 months of dietary exposure. The dosage in the first spike was 60 mg/kg, and that in the remaining spikes was 45 mg/kg. One of the 120 animals treated orally with 60 mg/kg of chlorpyrifos died. The dosage of subsequent spikes was reduced to 45 mg/kg to prevent further deaths. Both cohorts were treated exactly alike, so the second cohort also received 60 mg/kg for the first spike dosage (and, again, 1 of 120 animals died) and 45 mg/kg thereafter.
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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 1418% 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.
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RESULTS |
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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.
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In the groups that received the intermittent bolus dosages of chlorpyrifos every 2 months, 24 h after the spike ChE activity was markedly inhibited in the blood, muscle, brain, and retina to approximately 30% of control (Figs. 2 and 3). In general, all spiked groups showed the same level of inhibition in a given blood component or tissue (Figs. 2 and 3). There were two tissues for which this was not the case: in the diaphragm and in the brain, ChE inhibition was greater in the 5+CPF group than in the 0+CPF group. In no case was the ChE inhibition lower in the spiked animals that had been receiving chlorpyrifos in their feed.
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 (1020% 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 received the spike dose of chlorpyrifos 24 h earlier. After 12 months of dosing, however, the effect of spike was not apparent.
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.
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DISCUSSION |
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There are three general conclusions that can be gleaned from these data.
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).
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
).
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ACKNOWLEDGMENTS |
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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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