The Effect of Chlorpyrifos and Chlorpyrifos-Oxon on Brain Cholinesterase, Muscarinic Receptor Binding, and Neurotrophin Levels in Rats Following Early Postnatal Exposure

Angela M. Betancourt and Russell L. Carr1

Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 39762

Received August 7, 2003; accepted September 23, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorpyrifos (CPS) is a widely used diethyl organophosphorus insecticide in agricultural settings. Household and urinary residue analysis has suggested that children in agricultural communities are at risk of exposure to diethyl organophosphorus insecticides. The effects of repeated postnatal exposure to CPS and its metabolite chlorpyrifos-oxon (CPO) on total muscarinic acetylcholine receptor (mAChR) binding, nerve growth factor (NGF) levels, and brain derived neurotrophic factor (BDNF) levels in the forebrain of neonatal rats were investigated. Peak inhibition of brain cholinesterase (ChE) for CPS and CPO was determined after acute exposure to dosages of each compound (a low and a high for each), which produced similar degrees of initial ChE inhibition. Pups were administered CPS (1.5 or 3.0 mg/kg), CPO (0.25 or 0.35 mg/kg), or the corn oil vehicle by daily gavage from postnatal day 1 (PND 1) through PND 6. This exposure paradigm resulted in persistent ChE inhibition by CPS but only transient inhibition by CPO, suggesting that, even though the initial ChE inhibition is similar between compounds, the effects of repeated exposure differ significantly. Forebrain mAChR density, as measured by the binding of 3H-QNB, and NGF levels were significantly reduced on PND 4 and 7 after CPS but not on PND 12. No effects on mAChR density or NGF levels were observed with CPO. No effects on BDNF levels were observed with either compound. The data suggest that the persistent ChE inhibition and decreased mAChR binding may play a role in the decreased NGF levels following CPS exposure.

Key Words: chlorpyrifos; chlorpyrifos-oxon; neonatal; cholinesterase inhibition; neurotrophins; muscarinic receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate], one of the highly used organophosphorus (OP) insecticides, controls a broad spectrum of insects in both agricultural and urban settings. Despite recent regulatory decisions eliminating its residential use in the United States, chlorpyrifos (CPS) continues to be widely used in agriculture and commercial settings in the United States.

The OP insecticides exert their toxicity through the inhibition of acetylcholinesterase in the brain cholinergic synapses and neuromuscular junctions. CPS has little capacity to inhibit cholinesterase (ChE) in vivo and must undergo oxidative desulfuration to its active metabolite chlorpyrifos-oxon (CPO), which is a potent ChE inhibitor. This inhibition leads to the accumulation of acetylcholine in the synaptic cleft, which overstimulates the acetylcholine receptors, causing excessive activation of both muscarinic and nicotinic receptors in the peripheral and central nervous system (Ecobichon, 1996Go). The buildup of acetylcholine in the synaptic cleft can also alter pharmacodynamic patterns of receptor binding, density, and choline uptake kinetics (Padilla, 1995Go).

It is generally considered that children living in agricultural worker households or living in close proximity to pesticide-treated farmland would have increased exposures compared to other children living in the same community. Household residue analysis of OP insecticides has supported this idea (Lu et al., 2002Go). However, the presence of similar levels of diethyl OP residues in the urine of children regardless of age, parental occupation, or residential proximity to fields suggests that pesticide spraying in an agricultural region can increase children’s exposure in the absence of parental work contact with pesticides or residential proximity to OP-treated farmland (Koch et al., 2002Go). Thus, all children living in agricultural communities may be of risk of pesticide exposure. Besides the concern of greater possibility for exposure to CPS in children, studies have also reported a marked susceptibility of young animals to the toxic effects of CPS (Moser, 2000Go; Pope et al., 1991Go). Although it has been suggested that differences in sensitivity may exist only at high dose levels, Moser (2000)Go reported that, even at lower doses, age-related differences in ChE inhibition and behavioral alterations reflected the same magnitude of differences as the maximum tolerated doses.

At the cellular level, the neurotoxic effects of CPS have been observed at dosages below the threshold for systemic toxicity. These included reduction of DNA and protein synthesis (Dam et al., 1998Go; Whitney et al., 1995Go), decreased cell numbers (Campbell et al., 1997Go), inhibition of adenylyl cyclase activity (Auman et al., 2000Go; Song et al., 1997Go), decreased RNA levels (Johnson et al., 1998Go), decreased choline acetyltransferase activity (Dam et al., 1999Go), and disruption of the binding of transcription factors to DNA (Crumpton et al., 2000Go). The relationship of CPS-induced cell damage and inhibition of ChE is not completely clear in the developing brain. It has been suggested that at early postnatal ages, the effects of CPS in the brain may be mediated by noncholinesterase mechanisms (Auman et al., 2000Go; Campbell et al., 1997Go; Crumpton et al., 2000Go; Dam et al., 1998Go, 1999Go; Johnson et al., 1998Go; Song et al., 1997Go; Whitney et al., 1995Go). However, the inhibition of ChE has to be considered as an important component in the evaluation of the developmental toxicity of CPS.

Postnatal brain development is a highly coordinated process in which neurotrophins play an essential role. Neurotrophins, such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), are a family of chemically related proteins that have trophic effects on a variety of peripheral and central neurons, including the cholinergic neurons of the basal forebrain that project into the hippocampus and cerebral cortex (for review, see Dreyfus, 1998Go; Hohmann and Berger-Sweeney, 1998Go; Lindsay et al., 1994Go). For example, NGF effects on the cholinergic neurons of the basal forebrain include increased activity and expression of choline acetyltransferase, increased levels and expression of the high affinity NGF receptors (TrkA), synthesis and release of acetylcholine and the expression of the vesicular acetylcholine transporter, and promotion of neuronal differentiation and survival (Auld et al., 2001Go; Chen et al., 1997Go; Holtzman et al., 1992Go; Li et al., 1995Go; Lorenzi et al., 1992Go; Oosawa et al., 1999Go). Although less potent than NGF, BDNF also stimulates increased choline acetyltransferase activity and acetylcholine release in the cholinergic neurons of the basal forebrain (Alderson et al., 1990Go; Auld et al., 2001Go).

In vivo studies have shown that alterations in cholinergic innervation during early postnatal development can change various features of cortical ontogeny. In particular, neonatal lesions to basal forebrain cholinergic afferents result in delayed cortical neuronal development and permanently altered cortical cytoarchitecture and cognitive behaviors (for review, see Hohmann, 2003Go). If the levels of neurotrophins are altered during development, their trophic function could be impacted such that negative effects on brain development would occur. CPS has been reported to alter brain development and neuronal morphogenesis in the absence of significant ChE inhibition (Campbell et al., 1997Go; Das and Barone, 1999Go). Thus, it is possible that the neurotoxic effects of CPS in young animals could be related to alterations in the levels of the neurotrophic factors, such as NGF and BDNF, which could disrupt the establishment of normal neuronal processes. Therefore, this project was designed to investigate the effects of exposure to CPS or its metabolite CPO during the critical postnatal developmental period (PND 1–6) on muscarinic acetylcholine receptor density and on the endogenous levels of NGF and BDNF in the forebrain of neonatal rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Chlorpyrifos and 3,5,6-trichloro-2-pyridinol, for synthesis of chlorpyrifos-oxon, were a generous gift from DowElanco Chemical Company (Indianapolis, IN). Analytical grade chlorpyrifos-oxon was synthesized by Dr. Howard Chambers (Department of Entomology and Plant Pathology, Mississippi State University) as previously described (Chambers and Chambers, 1989Go). 3H-Quinuclidinyl benzilate (QNB; 48 Ci/mmol) and MicroScint 20® were purchased from Perkin Elmer (Boston, MA). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO).

Animal treatments.
Adult male and female Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were used for breeding. All animals were housed in an AAALAC-accredited facility in a temperature controlled environment (24 ± 2°C) with a 12-h dark:light cycle with lights on between 0700 and 1900 h and provided food (LabDiet rodent chow) and water (tap) ad libitum throughout the experimentation. All procedures were previously approved by the Mississippi State University Institutional Animal Care and Use Committee. Following parturition, male and female rat pups within the same litter were assigned to different treatment groups. There was always a control animal present in each litter. The size of the litter was adjusted as much as possible in order to obtain litters of the same size (8–10 pups) and even distribution of male and female pups within each litter. For CPS and CPO, 26 and 27 litters were used, respectively. All chemicals were dissolved in corn oil and administered by oral gavage at a volume of 0.5 ml/kg. Upon sacrifice by decapitation, all brains were rapidly removed, dissected on ice to obtain the forebrain (anterior to the optic chiasma), frozen immediately in liquid nitrogen, and maintained at -70°C until assay. For ChE activity and mAChR binding, the entire forebrain of each animal was used for determination of each respective parameter. For NGF and BDNF levels, the forebrain was divided longitudinally, with the left half being used for NGF and the right half being used for BDNF.

Chlorpyrifos was administered every day from postnatal day 1 (PND 1) through PND 6 by oral gavage (the day of birth was considered as PND 0). Oral gavage was performed by using a 25-µl tuberculin syringe equipped with a 1-inch 24-gauge straight intubation needle (Popper and Sons, Inc., New Hyde Park, NY) to deliver the solution to the back of the throat. The treatment groups were: corn oil (control); 1.5 mg/kg CPS (low CPS); and 3 mg/kg CPS (high CPS). Rat pups were sacrificed on PND 4 and PND 7 (24 h after the last exposure to CPS), and on PND 12 (6 days after the last exposure to CPS), and the forebrain was collected. ChE activity, total mAChR binding, and NGF and BDNF levels were then determined, as described below.

Once the CPS experiment was complete, low and high dosages of CPO that would yield the same percentage of inhibition of ChE in the forebrain as the low and high dosages of CPS, respectively, were determined in order to attempt to compare between CPS and CPO. To determine the time and amount of maximal (peak) inhibition of ChE activity following CPS exposure, rat pups were administered the low and high dosages of CPS described above by oral gavage on PND 1 and were sacrificed at various time intervals (4, 8, 10, 12, and 24 h) following exposure. The forebrain was collected, ChE activity was determined as described below, and percentage of inhibition was calculated. Following determination of the time and amount of peak inhibition of ChE activity with CPS, the time of peak inhibition was determined for CPO. The two dosages of CPO initially selected were 0.20 mg/kg and 0.30 mg/kg. As with CPS, rat pups were administered CPO on PND 1 and were sacrificed at various time intervals (0.5, 1, 2, and 3 h) following exposure. The forebrain was collected, ChE activity was determined as described below, and percentage of inhibition was calculated. Once the time of peak inhibition of ChE was determined for CPO, two dosages of CPO that yielded inhibition of ChE similar to the two dosages of CPS were determined. These dosages were 0.25 mg/kg (low CPO) and 0.35 mg/kg (high CPO).

Rats were repeatedly administered either the control vehicle or one of the two dosages of CPO determined above every day from PND 1 through PND 6, which is similar to the repeated dosing scheme with CPS. Rat pups were sacrificed on PND 4 and PND 7 (24 h after the last exposure to CPO), and on PND 12 (6 days after the last exposure to CPO), and the forebrain was collected. ChE activity, total mAChR binding, and NGF and BDNF levels were then determined, as described below. Although these dosages of CPO yielded similar levels of ChE inhibition as those with CPS on PND 1, the level of ChE inhibition with CPO on PND 4 (24 h after last exposure on PND 3) was much lower than that obtained with CPS. To ensure that significant ChE inhibition was present following dosing on PND 3 with CPO, rat pups were repeatedly exposed to CPO from PND 1 through 3 and were sacrificed on PND 3 at the time of peak inhibition of ChE for CPO (1 h). The forebrain was collected, ChE activity was determined as described below, and percentage of inhibition was calculated.

Cholinesterase analysis.
The forebrains were homogenized at 40 mg/ml in cold 0.05 M Tris-HCl buffer (pH 7.4 at 37°C) in a glass mortar using a Wheaton motorized tissue grinder and a Teflon pestle. The activity of ChE was measured spectrophotometrically using a modification (Chambers et al., 1988Go) of Ellman et al.(1961)Go. Protein concentration of the forebrain homogenate was quantified with the Folin phenol reagent using bovine serum albumin as a standard (Lowry et al., 1951Go). Specific activity of ChE was calculated and expressed as nmoles product produced per min-mg protein.

Muscarinic acetylcholine receptor binding.
Total mAChR binding in the forebrain of neonatal rats was investigated using a modified method of Araujo et al.(1991)Go. Briefly, the forebrains were homogenized in cold Tris-HCl (50 mM, pH 7.4) buffer (containing 120 mM NaCl; 5 mM KCl; 1 mM CaCl2) in a glass mortar using a Wheaton motorized tissue grinder and a Teflon pestle. The homogenate was centrifuged under refrigeration for 10 min at 48,000 x g, the supernatant was discarded, and the pellet was washed twice by resuspending the pellet in fresh cold buffer and centrifuging again. The final membrane pellet was resuspended in cold buffer, and mAChR binding was determined with the specific ligand 3H-QNB, using a method similar to that of Yamamura and Snyder (1974)Go as described by Tang et al.(1999)Go, with a single saturating concentration (12 nM) of 3H-QNB. Protein concentration was measured by the method of Lowry et al.(1951)Go using bovine serum albumin as a standard. The specific binding was calculated as the total binding (incubated without 10 µM atropine sulfate) minus nonspecific binding (incubated with atropine) and expressed as fmol/mg protein.

Neurotrophin quantification.
The forebrains were suspended in 300 µl of cold lysis buffer (137 mM NaCl, 20 mM Tris HCl, 1% NP40, 10% glycerol, 1mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mM sodium vanadate). Tissues were homogenized in a glass mortar using a Wheaton motorized tissue grinder and a Teflon pestle. The tissue homogenate was centrifuged under refrigeration at 1500 x g for 20 min. The supernatant fraction was removed, placed in microcentrifuge tubes, and maintained at -70°C until assayed. NGF and BDNF levels were measured using the NGF and BDNF Emax ImmunoAssay System (Promega Corporation, Madison, WI), respectively. Protein concentration of the supernatant was determined using the Bradford Protein Assay (Standard Procedure for Microtiter Plates; Bio-Rad Laboratories Hercules, CA). The concentrations of NGF and BDNF were calculated and expressed as pg NGF or BDNF per mg protein.

Statistical analysis.
Statistical analysis was performed by analysis of variance (ANOVA) using the general linear model (GLM) procedure followed by separation of means using Least Significant Difference (LSD). No significant sex differences in either controls or treated groups were present in any parameter determined. Therefore, males and females were pooled for statistical analysis. The criterion for significance was set at p <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following a single exposure to low CPS (1.5 mg/kg), the time of peak inhibition occurred at 12 h (58% inhibition), with minor recovery afterwards through 24 h (49% inhibition) (Fig. 1AGo). Following a single exposure to high CPS (3.0 mg/kg), the time of peak inhibition occurred at 4 h (82% inhibition), with substantial recovery by 24 h (54% inhibition) (Fig. 1AGo). Following a single exposure to CPO, the time of peak inhibition was 1 hour with both 0.2 mg/kg (45% inhibition) and 0.3 mg/kg (63% inhibition) (Fig. 1BGo). There was recovery by 3 h, with only 28% and 48% inhibition remaining for 0.2 and 0.3 mg/kg CPO, respectively. As described in the Materials and Methods, the time of peak inhibition (1 h) for CPO was used to determine two dosages of CPO that would yield the same percentage of inhibition of forebrain ChE as the two dosages of CPS. At 1 h, inhibition with the low dosage of CPO (0.25 mg/kg) was 57 ± 3.8%, and inhibition with the high dosage of CPO (0.35 mg/kg) was 81 ± 4.8%. These dosages were used throughout the remainder of the study.



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FIG. 1. Time course of forebrain cholinesterase inhibition in 1-day-old rat pups following an acute oral gavage of (A) either 1.5 mg/kg or 3.0 mg/kg chlorpyrifos (CPS) or (B) either 0.2 mg/kg or 0.3 mg/kg chlorpyrifos-oxon (CPO), as described in Materials and Methods. Values are expressed as mean percentage of control ± SE (n = 3–4). Time of peak inhibition was 12 and 4 h for 1.5 and 3.0 mg/kg CPS, respectively, and was 1 h for both 0.2 and 0.3 mg/kg CPO. All values are significantly different from control (p <= 0.05).

 
Repeated exposure to CPS produced no signs of overt toxicity and no signs of cholinergic hyperstimulation. In contrast, repeated exposure to high CPO (0.35 mg/kg), but not low CPO (0.25 mg/kg), exerted significant signs of cholinergic hyperstimulation (tremors) at the time of peak inhibition following every treatment, but these signs had disappeared by 2 h.

Following administration of CPS (PND 1–6), body weights were significantly different from controls on PND 4 and PND 7 with high CPS but not low CPS (Fig. 2AGo). No significant differences were present on PND 12. Forebrain ChE activity in the control group increased significantly with age (Fig. 2BGo), as did control total mAChR levels, as measured by 3H-quinuclidinyl benzilate (QNB) binding, (Fig. 3Go). ChE activity was significantly reduced following repeated exposure to low and high CPS on PND 4 and PND 7 (Fig. 2BGo). ChE was not significantly reduced with low CPS on PND 12 (13% inhibition), although there was a tendency toward significance (p <= 0.061). ChE activity was significantly reduced with high CPS on PND 12, but substantial recovery of ChE activity was evident compared to PND 4 and PND 7. In a similar pattern, total mAChR levels were significantly reduced following repeated exposure to both low and high CPS on PND 4 and PND 7 (Fig. 3Go). By PND 12, mAChR had returned to control levels with an apparent rebound effect.



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FIG. 2. (A) Body weights and (B) specific activity of forebrain cholinesterase (ChE) of rat pups exposed daily by oral gavage to either 1.5 mg/kg or 3.0 mg/kg chlorpyrifos (CPS), as described in Materials and Methods. Body weights were measured 24 h following the last exposure and values are expressed as mean body weight ± SE (n = 29–53). ChE values are expressed as mean specific activity ± SE (n = 10–18). Control values with different uppercase letters are statistically significant (p <= 0.05) from one another. Bars within each age with different lowercase letters are statistically significant (p <= 0.05) from one another. Percentage decrease from control at each age for each statistically significant value is presented in the oval overlaying the corresponding bar.

 


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FIG. 3. Total muscarinic acetycholine receptor (mAChR) binding in the forebrain of rat pups repeatedly exposed to either 1.5 or 3.0 mg/kg chlorpyrifos (CPS) from postnatal day 1 through 6, as described in Materials and Methods. Values are expressed as mean receptor binding ± SE (n = 6–11). Control values with different upper case letters are statistically significant (p <= 0.05) from one another. Bars within each age with different lower case letters are statistically significant (p <= 0.05) from one another. Percentage decrease from control levels at each age for each statistically significant value is presented in the oval overlaying the corresponding bar.

 
Following repeated administration of CPO, no effects in body weight were observed (Fig. 4AGo). ChE activity was significantly reduced with low and high CPO on PND 4 but not PND 7 (Fig. 4BGo). Considering the significant decrease in ChE activity observed following exposure to a single administration of these same dosages of CPO, the small amount of inhibition observed on PND 4 following repeated exposure was surprising. However, when ChE activity was measured 1 h (on PND 3) after repeated exposure to these dosages, a substantially greater amount of ChE inhibition was present with both the low (47% inhibition) and high (59% inhibition) dosages of CPO than was present 23 h later on PND 4. Despite the significant amount of ChE inhibition, no effects of CPO on total mAChR levels were observed (data not shown).



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FIG. 4. (A) Body weights and (B) specific activity of forebrain cholinesterase (ChE) of rat pups exposed daily by oral gavage to either 0.25 mg/kg or 0.35 mg/kg chlorpyrifos-oxon (CPO), as described in Materials and Methods. Body weights were measured 24 h following the last exposure and values are expressed as mean body weigh ± SE (n = 13–25). ChE values are expressed as mean specific activity ± SE (n = 7–18). Bars within each age with different lower case letters are statistically significant (p <= 0.05) from one another. Percentage decrease from control at each age for each statistically significant value is presented in the oval overlaying the corresponding bar.

 
Control NGF levels increased significantly with age (Fig. 5AGo). NGF levels were dose-dependently reduced following repeated exposure to CPS on PND 4 and PND 7, but the reduction on PND 12 (10% reduction, both dosages) only approached significance (p <= 0.054). Control BDNF levels were similar between PND 4 and PND 7 but increased significantly by PND 12. There were no effects of CPS exposure on BDNF levels on PND 4, 7, or 12 (Fig. 5BGo). There were no significant effects of CPO exposure on forebrain NGF or BDNF levels at any age studied (data not shown).



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FIG. 5. (A) Nerve growth factor (NGF) and (B) brain derived neurotrophic factor (BDNF) levels in the forebrain of rat pups repeatedly exposed to either 1.5 or 3.0 mg/kg chlorpyrifos (CPS) from postnatal day 1 through 6, as described in Materials and Methods. Values are expressed as mean NGF or BDNF concentration ± SE (n = 11–15). Control values with different upper case letters are statistically significant (p <= 0.05) from one another. Bars within each age with different lower case letters are statistically significant (p <= ±0.05) from one another. Percentage decrease from control levels at each age for each statistically significant value is presented in the oval overlaying the corresponding bar.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Similar to previous studies (Song et al., 1997Go; Whitney et al., 1995Go), the dosages of CPS used in this study produced changes in neurochemistry in the developing rat brain without eliciting any signs of systemic toxicity. With CPS, we observed persistent inhibition of ChE and decreased mAChR binding. This decreased mAChR binding paralleled the decrease in the levels of NGF. With CPO, ChE inhibition was not persistent, and no changes in mAChR or NGF were detected. This suggests that the decreased NGF levels may be the result of the persistent effects of CPS on the cholinergic system.

Following acute exposure to CPS on PND 1, forebrain ChE was initially inhibited in a dose-related manner, but by 24 h inhibition was similar with both dosages. The time of peak inhibition with 3.0 mg/kg CPS observed here is similar to that observed in other studies with neonatal rats (Moser and Padilla, 1998Go; Won et al., 2001Go). This level of inhibition for 3.0 mg/kg CPS in the neonatal forebrain (87%) is also similar to the results obtained by Zheng et al.(2000)Go. However, the peak inhibition for 1.5 mg/kg CPS did not occur until 12 h after exposure, and the recovery by 24 h was minimal. This suggests that the pattern of inhibition and recovery of ChE activity may vary depending on the dosage of CPS administered. One possible explanation is that, with the higher dosage of CPS, the cytochrome P450(s) that activate CPS to CPO were effectively saturated with substrate (CPS), and maximal production of CPO was occurring. This high production of CPO allowed the saturation of what few protective enzymes were present and allowed the inhibition of brain ChE to occur in a shorter time frame. With the lower dosage of CPS, the rate of oxon production was much slower, so a greater amount of time was required to saturate the protective enzymes present, thus prolonging the time required to achieve maximal inhibition of brain ChE.

Following acute exposure to CPO on PND 1, forebrain ChE was also inhibited in a dose- related manner. However, unlike the pattern with the two dosages of CPS, the peak inhibition of ChE occurred one hour after exposure with both dosages of CPO, followed by some recovery of activity by 3 h. This faster time to peak effect with CPO than with CPS is similar to that reported by others using CPO with adult rats (Chambers and Carr, 1993Go; Huff et al., 1994Go). The observed signs of cholinergic hyperstimulation with high CPO coincided with the time of peak inhibition of ChE, and these signs disappeared as ChE recovered. Thus, the signs of cholinergic hyperstimulation observed with high CPO can be attributed to the faster inhibition of ChE following CPO exposure, since it does not require metabolic activation prior to entering the bloodstream, as does CPS. Even though the level of ChE inhibition with CPS eventually reached the same level as CPO, the greater amount of time required to do so could have allowed the exposed rat to undergo some physiological compensation such that signs of cholinergic hyperstimulation were not observed.

Body weight changes occurred following repeated exposure to the high dosage of CPS but not to the low dosage. These data are similar to that of Zheng et al.(2000)Go, who reported that the No Observed Effect Level (NOEL) for decreased body weights in neonates was 1.5 mg/kg/day CPS. One possible scenario is that the significant decrease in body weight with the high dosage may be related to the higher degree of ChE inhibition with this dosage as compared to the low dosage, such that the ability of the rats to nurse may have been impaired in the high dosage. The lack of effect on body weight with CPO is probably related to the rapid recovery of ChE activity following CPO exposure. Our data suggest that CPO appears to be present in the body for only a very short time; thus it has only a transient effect on ChE levels and may not interfere with the ability of rats to nurse. Thus, while similar ChE inhibition was present with both CPS and CPO following acute exposure, ChE inhibition following repeated exposure is significantly different between the two compounds.

Consistent with previous reports (Atterberry et al., 1997Go; Carr et al., 2001Go; Fiedler et al., 1987Go; Tang et al., 1999Go), there was a steady increase with age of control forebrain ChE-specific activity. Following repeated exposure to CPS, the percentage of ChE inhibited is constant from PND 4 through 7 in both dosages, even though the specific activities in these treated groups increased. By PND 12, which is 6 days after the last treatment, there was still significant inhibition of ChE with high CPS, although some recovery of activity had occurred. It has been demonstrated that, following repeated exposures to CPS, neonatal animals exhibit a greater amount of recovery of ChE activity as compared to adult animals following each exposure (Zheng et al., 2000Go). This rapid recovery of brain ChE activity following exposure could be due either to spontaneous reactivation of the phosphorylated enzyme or to de novo protein synthesis of new enzyme (Chakraborti et al., 1993Go). This rapid recovery is most evident in the animals repeatedly treated with CPO, in that when ChE activity is assessed at 1 and 24 h after the last dosage on PND 3, the amount of recovery of activity is dramatic. The faster recovery of ChE in neonatal rats treated with CPO compared with CPS may reflect differences in toxicokinetic properties (i.e., CPO being much less lipophilic than CPS) (Chambers and Carr, 1993Go), contributing to the former compound having a much shorter half-life in the body than the latter.

The persistent inhibition of ChE with CPS leads to a reduction in mAChR. Reductions in the total number of mAChR, as measured by the binding of the lipophilic antagonist 3H-quinuclidinyl benzilate (QNB), have been reported following repeated exposures of adult rats to CPS (Chakraborti et al., 1993Go; Liu et al., 1999Go; Nostrandt et al., 1997Go). A reduction in mAChR levels following repeated exposure to a similar dosage of CPS has previously been observed in neonatal animals (Tang et al., 1999Go). Thus, regardless of age, reduction of mAChR levels can be considered to be a compensatory mechanism underlying tolerance to OP compounds and directly related to persistent inhibition of ChE. CPO exposure did not result in persistent inhibition, which is probably the reason there was no reduction in mAChR levels.

Although the maximum amount of ChE inhibition following the initial exposure to CPS and CPO were similar, differences in NGF levels were observed following CPS exposure but not CPO exposure. These differences in effect may be explained based in the degree and duration of the inhibition of ChE after repeated CPS or CPO exposure and the decrease in mAChR levels. Previous experiments have demonstrated that the synthesis and release of NGF is upregulated by neuronal activity. Expression and secretion of NGF have been shown to be increased by muscarinic and nicotinic receptor signaling (Blöchl and Thoenen, 1995Go; da Penha Berzaghi et al., 1993Go; Knipper et al., 1994Go). CPS induced a significant decrease in both NGF levels and mAChR binding on PND 4 and PND 7, but not on PND 12. If NGF synthesis is dependent to a degree on mAChR signaling, then the reduction of the mAChR could have the potential to decrease the levels of NGF. The rapid recovery of ChE-specific activity after CPO exposure and the lack of any change in the cholinergic receptor levels may explain the lack of effects of CPO on the levels of NGF.

Some evidence for the potential regulation of NGF level by mAChR has also been reported in the literature. In neonatal and adult brain, stimulation of the mAChR receptors with the M1/M3 agonist pilocarpine increased the levels of NGF and BDNF mRNA (da Penha Berzaghi et al., 1993Go). In addition, the neurotrophin-mediated survival of ganglionic retinal cells has been shown to be disrupted by the addition of the M1/M3 mAChR receptor antagonist telepizine (Pereira et al., 2001Go). Since the M2/M4 mAChR are not yet effectively coupled to their signal transduction mechanisms at these early ages but the M1/M3 mAChR are (Lee et al., 1990Go), it is possible that the disruption of the levels of the M1/M3 mAChR could have the potential to alter NGF concentrations. Further investigation on the relationship between M1/M3 mAChR and NGF levels during development is needed.

In agreement with previous reports (Friedman et al., 1991Go; Katoh-Semba et al., 1997Go; Zhou et al., 1996Go), BDNF forebrain control levels increased two-fold from PND 4 to PND 12. However, it has been reported that BDNF expression is low in developing regions of the brain and only increases as these regions mature, while the level of NGF expression varies during the development of discrete regions (Maisonpierre et al., 1990Go). Our BDNF data seem to agree with this report, since we observed no increase between PND 4 and PND 7 but a significant increase by PND 12. This first postnatal week is when the rat forebrain is still undergoing significant development and is equivalent to the third trimester of gestation and early postnatal period in humans (Dobbing and Sands, 1979Go). During the first postnatal week, it has been reported that BDNF plays an important role in differentiation of cholinergic neurons but not their survival (Nonomura and Hatanaka, 1992Go). Also, BDNF does not prevent apoptotic death of developing septal cholinergic neurons following NGF withdrawal (Kew and Sofroniew, 1997Go). Thus, the role of BDNF during development may differ significantly from that of NGF. In addition, while the muscarinic agonist pilocarpine has been shown to increase both NGF and BDNF mRNA (da Penha Berzaghi et al., 1993Go), the actual relationship between mAChR and BDNF in brain development is not clear. Since BDNF levels do not increase during the first postnatal week in a manner similar to NGF and mAChR, their relationships in development may be different. Thus, it is not clear if they both would be impacted in a similar fashion following CPS exposure. Further investigation in this area is needed.

Overall, cholinergic afferents innervate the cerebral cortex and other brain structures such as the hippocampus during the first two postnatal weeks, which is the most dynamic period of neuronal differentiation and synapse formation. NGF seems to have several functions at this early stage of development of the forebrain cholinergic system. A number of in vivo studies have shown that alterations in cholinergic innervation during early postnatal development can change various features of cortical ontogeny. The results of this study indicate that neonatal exposure to CPS, even at doses that do not cause systemic toxicity or any overt signs of intoxication, reduces NGF levels in the forebrain. The changes in NGF appear to be associated with the effects of CPS on the cholinergic system and dependent on the persistence of the inhibition of ChE and the decreased mAChR levels. Thus, it may be that the alteration of brain development associated with neonatal exposure to CPS may, in part, be the result of transiently decreased NGF levels.


    ACKNOWLEDGMENTS
 
This study was funded in part by National Institutes of Health grant #1 R01 ES 10386-01A1. Research was also partially supported by the Mississippi Agricultural and Forestry Experiment Station (MAFES) under MAFES project #MISV-339010 and the College of Veterinary Medicine, Mississippi State University. This paper is MAFES publication #J-10393 and Center for Environmental Health Sciences publication #105.


    NOTES
 
1 To whom correspondence should be addressed at Center for Environmental Health Sciences, College of Veterinary Medicine, Box 6100, Mississippi State University, Mississippi State, MS 39762-6100. Fax: (662) 325-1031. E-mail: rlcarr{at}cvm.msstate.edu. Back


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