The Effects of Repeated Oral Exposures to Methyl Parathion on Rat Brain Cholinesterase and Muscarinic Receptors during Postnatal Development

Jun Tang1, Russell L. Carr and Janice E. Chambers2

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

Received June 12, 2003; accepted September 10, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dimethyl phosphorylated cholinesterase (ChE) is known to be more rapidly reactivated, spontaneously, and have a higher aging rate than diethyl phosphorylated ChE. This may result in differences in toxic signs and tolerance development after treatment of juvenile rats with methyl parathion (MPS), a dimethyl phosphorothionate, than after treatment with chlorpyrifos (CPS), a diethyl phosphorothionate. The effects of repeated MPS exposures on brain ChE activity and surface and total muscarinic acetylcholine receptor (mAChR) density were studied in postnatal rats gavaged daily from postnatal day 1 (PND1) through PND 21. Results of this study were compared to our recent report with CPS ( Tang et al., 1999, Toxicol. Sci. 51, 265–272[Abstract]). Rats received MPS daily starting at 0.3 mg/kg and increasing gradually to 0.6 mg/kg (for the medium-dosage groups) and then to 0.9 mg/kg (for the high-dosage group). ChE activity was assayed in brain homogenates. Synaptosomal mAChR densities, surface, and total were assayed using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB), respectively, as ligands. Developmental increases in brain ChE activities and mAChR densities were observed from PND 6 through PND 22. On PND 22, inhibition of ChE activity was observed in the low (26%)-, medium (42%)-, and high (55%)-dosage groups. Significant inhibition was still present on PND 30 (16–24%) and PND 40 (12–14%), which were 9 and 19 days after the last treatment, respectively. Densities of 3H-NMS and 3H-QNB binding sites in treated groups were significantly reduced by PND 22, 1 day following cessation of treatment, and were significantly increased during the recovery period. After MPS exposure, the initial recovery of phosphorylated ChE was more rapid and the density of 3H-NMS binding sites was less readily reduced than following CPS exposure. The lesser effects on surface mAChR may explain why more severe signs appeared after each treatment with the high dosage of MPS than were observed previously with CPS, indicating little or no tolerance had developed to MPS.

Key Words: methyl parathion; cholinesterase inhibition; organophosphorous insecticides; muscarinic acetylcholine receptors; developmental neurotoxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic insecticides have been among the most useful weapons for human beings in controlling insect pests. Because of similarities in physiological mechanisms across species, nontarget organisms such as wildlife and human beings are also threatened by insecticides, especially organophosphorus (OP) insecticides, which possess relatively high toxicity levels toward mammals. It has been well established that OP insecticides exert their toxicity through inhibition of cholinesterase (ChE), the subsequent accumulation of the neurotransmitter acetylcholine, and the resulting hyperexcitation of the nervous system (Fukuto, 1990Go).

Methyl parathion (O,O-dimethyl-4-nitrophenyl phosphorothionate; MPS) is a broad spectrum OP insecticide and is used widely on cotton as well as on some food crops. The rat oral LD50 for MPS is 14–24 mg/kg (Gaines, 1960Go). Because of this high mammalian toxicity, MPS is registered and recommended in the United States for agricultural use only and is subject to special restrictions in many states that regulate the use of pesticides (U.S. EPA, 1975Go). New restrictions on its food usages have been applied in recent years. However, because MPS was easy to obtain and was relatively inexpensive, massive illegal applications of MPS for household pest control have occurred in the recent past and were the subject of much public health concern and household remediation (Landrigan et al., 1999Go; New York Times, 1996Go).

Like other organophosphorothionate insecticides such as chlorpyrifos (CPS), the mechanism of acute toxicity of MPS is attributed to the inhibition of ChE activity. MPS can be bioactivated to its active metabolite, methyl paraoxon (MPO), which is a more potent inhibitor of ChE than is MPS (Chambers and Chambers, 1991Go; Fukuto, 1990Go). Similar to CPS, MPS is more toxic to juvenile than to adult rats (Benke and Murphy, 1975Go; Pope et al., 1991Go). However, MPS is less lipophilic than CPS (Chambers and Carr, 1993Go), suggesting that MPS may not induce prolonged effects, as does CPS (Bushnell et al., 1993Go). Compared to the active metabolite of CPS, chlorpyrifos-oxon, MPO is relatively poorly detoxified in vivo (Chambers et al., 1990Go; Chambers and Carr, 1993Go), which is one of the factors that make MPS highly toxic. Dimethyl phosphorylated ChE displays a relatively brief half-life of spontaneous reactivation (about 2 h) compared with diethyl phosphorylated ChE (about 60 h) (Wilson et al., 1992Go), suggesting that recovery of ChE activity after each single exposure to MPS may be more rapid than after each single exposure to CPS. However, dimethyl phosphorylated ChE has a higher aging rate than diethyl phosphorylated ChE (Wilson et al., 1992Go), suggesting that recovery of ChE activity after repeated exposures to MPS may be slower than after repeated exposures to CPS. This is because aged phosphorylated ChE cannot be reactivated and has to be replaced by de novo synthesis.

A common response to the accumulation of acetylcholine following repeated exposure to an OP insecticide is the reduction in density (down-regulation) of the muscarinic acetylcholine receptors (mAChR) (reviewed in Hoskins and Ho, 1992Go). Previous work has demonstrated that down-regulation occurs following exposure to both CPS and MPS in adults and juveniles. Liu et al.(1999)Go compared neurochemical effects of daily subcutaneous (sc) exposures to 1.5 or 3.0 mg/kg MPS or 5 or 10 mg/kg CPS in juvenile (8, 15, and 22 days of age) and adult (90 days of age) rats. They observed that repeated sc exposure to either CPS or MPS induced reductions of ChE activity, total muscarinic acetylcholine receptor (mAChR) density, and M2 subtype mAChR density in the cortex and striatum of both juveniles and adults. The focus of the study identified differences in the responses of juvenile and adult rats with exposure to MPS inducing greater reductions of ChE activity and mAChR density in juveniles than in adults, and CPS inducing similar degrees of neurochemical changes in both age groups. We previously studied the effects of repeated oral administration of CPS (Tang et al., 1999Go) during the first three weeks postpartum, which comprises an important developmental period of the cholinergic system in the rat (Coyle and Yamamura, 1976Go; Kiss and Patel, 1992Go). Following CPS exposure, differential modulation of surface and total mAChR were observed using the radioligands 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB). NMS is hydrophilic, will label cell surface receptors only, and is indicative of sequestration of receptors from the synaptic membrane. QNB is lipophilic, will label both cell surface and internal receptors, and is indicative of either degradation of sequestered receptors and/or delayed synthesis of receptors. An incremental dosing protocol was developed to achieve a dose-response relationship in ChE inhibition. This dosing protocol was necessary because of the rapid development of the cholinergic system during the first three postnatal weeks. Considering the higher toxicity and shorter period of ChE reactivation demonstrated by MPS compared to CPS, a different response pattern could result between these two OP compounds.

Therefore, using the same methods and a similar dosing protocol as were used in the CPS study (Tang et al., 1999Go), these experiments were conducted to determine the persistence of inhibition of brain ChE and any compensatory reduction of surface and total brain muscarinic receptor density in juvenile rats after repeated oral exposures to MPS. In this study, in order to maintain a similar level of inhibition of brain ChE as was present in the CPS study, we administered daily treatment with MPS instead of treatment every other day, because of the shorter period of ChE reactivation demonstrated by MPS compared to CPS. The results of the present study were used for comparison with those reported in the CPS study (Tang et al., 1999Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Analytical grade MPS was supplied by Dr. Howard Chambers (Department of Entomology and Plant Pathology, Mississippi State University). 3H-NMS (82 Ci/mmol) and 3H-QNB (48 Ci/mmol) were purchased from Amersham Life Science, Inc. (Arlington Heights, IL). All other chemicals, if not specified, were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and treatments.
Adult male and female Sprague-Dawley rats [Crl; CD(SD)BR] from Charles River Laboratories were used as breeders. All animals were kept in a temperature-controlled (22 ± 2°C) room with a 12:12 h alternating light/dark cycle in an AAALAC-accredited facility. Animals were allowed free access to food (standard laboratory rodent feed) and water. All procedures were previously approved by the Mississippi State University Institutional Animal Care and Use Committee. Following parturition, litters containing greater than 10 pups were randomly assigned to a treatment and all male and female pups in a single litter received the same treatment.

Rat pups were gavaged daily using an animal feeding needle (Popper and Sons, Inc., New Hyde Park, NY) and syringe with MPS in corn oil at a volume of 0.5-ml/kg body weight from postnatal day 1 (PND 1) (the day of birth was counted as PND 0) through PND 21 (21 doses total). The treatment groups were: (1) Control: administered the corn oil vehicle; (2) Low-dosage: administered MPS at 0.3 mg/kg daily from PND 1 through PND 21; (3) Medium-dosage: administered MPS at 0.3 mg/kg daily from PND 1 through PND 5, and then 0.6 mg/kg daily from PND 6 through PND 21; and (4) High-dosage: administered MPS at 0.3 mg/kg daily from PND 1 through PND 5, 0.6 mg/kg daily from PND 6 through PND 13, and then 0.9 mg/kg daily from PND 14 through PND 21. Male and female pups were sacrificed by decapitation on PND 6, 14, 22, 25, 30, or 40. At preweanling ages (PND 6 and 14), two pups were sacrificed from each litter of each treatment group.

Tissue samples.
The brain was removed from the skull and rapidly dissected. The left hemisphere of the brain was frozen at -70°C in an ultra-low-temperature freezer for later enzyme assay. The right hemisphere of the brain was used fresh to prepare crude synaptosomes for muscarinic receptor assays, as described below.

Cholinesterase assay.
Whole brain (excluding cerebellum) ChE was assayed spectrophotometrically using acetylthiocholine as the substrate and 5,5'-dithio-bis(nitrobenzoic acid) (DTNB) as the chromogen with 10-5 M eserine sulfate (a carbamate inhibitor of ChE) used in the blanks to correct for non-ChE-mediated substrate hydrolysis (Chambers et al., 1988Go; Ellman et al., 1961Go).

Muscarinic receptor binding assay.
Crude synaptosomal fractions of brain (excluding cerebellum) were prepared using the method of Gray and Whittaker (1962)Go, as described previously (Tang et al., 1999Go). Muscarinic receptor binding sites in brain crude synaptosomal preparations were determined with the specific ligands 3H-NMS and 3H-QNB, similar to the method of Yamamura and Snyder (1974)Go as described previously (Tang et al., 1999Go). Six different concentrations (0.004–3 nM) of 3H-NMS or 3H-QNB were used to perform Scatchard analysis using QuattroPro software.

Protein determination.
Protein concentrations were quantified for all preparations by the method of Lowry et al.(1951)Go using bovine serum albumin as the standard.

Statistics.
Statistical analysis was performed using SAS on a personal computer. Overall analysis of each parameter studied indicated that age and treatment were significant but sex was not. Therefore, males and females were pooled for analysis between treatment groups at each age. Differences among ages in the control group were also determined. Data were analyzed by ANOVA using the SAS General Linear Model (GLM) on a personal computer. Significant main effects were further evaluated using the Student-Newman-Keuls post hoc test. Statistical significance is reported for the p <= 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There was no mortality among the MPS-treated pups. Pups showed tremors and convulsions within 2 h after each treatment with the medium and high dosages of MPS; signs were extremely severe in the high-dosage group. However, all signs of intoxication disappeared before each subsequent treatment. It appeared that the high-dosage group had reduced weight gain but there were no significant differences from control (data not shown).

Brain ChE activity in the control group increased significantly (p <= 0.05) from PND 6 through PND 22 and did not change significantly after PND 22 (Fig. 1Go). Brain ChE activities were inhibited in all MPS-treatment groups. ChE inhibition in the low-dosage group was consistent at 25, 24, and 26% on PND 6 (at 18 h after 5th dose), PND 14 (at 18 h after 13th dose), and PND 22 (at 18 h after 21st dose), respectively. Percent inhibition of ChE in the medium- and high-dosage groups increased with incremental dosages. The maximal percent inhibition in the medium-dosage group occurred on PND 22 with 42% inhibition, and in the high-dosage group on PND 22 with 55% inhibition. After the termination of treatments on PND 21, ChE activities in treated groups demonstrated gradual recovery, but the activities were still significantly lower than those of the controls on PND 30 and PND 40, i.e., the 9th and 19th days after the last treatment, respectively (Fig. 1Go). At PND 40, there was a similar reduction in ChE activity in all three treatment groups.



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FIG. 1. Brain cholinesterase activity following exposure to 3 dosages of methyl parathion from postnatal day 1 (PND 1) through PND 21, as described in Materials and Methods. The arrow indicates PND 21, the day of the last treatment. Values are expressed as the mean ± SE (n = 4–6, except low-dosage animals on PND 6, n = 16, and medium-dosage ones on PND 14, n = 12). Percent inhibition for each treatment group at each age is presented in the oval overlaying the corresponding bar. Within each age, bars not labeled with similar letters are significantly different (p <= 0.05).

 
Binding of 3H-NMS (Fig. 2Go) and 3H-QNB (Fig. 3Go) to rat brain synaptosomal fractions (Bmax) increased significantly (p <= 0.05) from PND 6 to PND 22 in the control group. The Kd values were not significantly different across ages (Figs 2 Goand 3Go). Significant reductions of both 3H-NMS and 3H-QNB binding sites (i.e., Bmax) were observed on PND 22 in the medium and high dosage groups. A significant decrease in Bmax for 3H-NMS was also observed on PND 30 in the high dosage group but not on PND 25. Interestingly, Bmax was significantly increased for both 3H-NMS and 3H-QNB in the low-dosage group on PND 30. On PND 40, Bmax was significantly increased for 3H-QNB in the medium- and high-dosage groups. On PND22, the Kd for 3H-NMS binding was significantly lower in the low-dosage group (Fig. 2Go) and the Kd for 3H-QNB binding was significantly lower than control for all dosage groups (Fig. 3Go).



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FIG. 2. Brain 3H-methylscopolamine binding (Bmax) following exposure to 3 dosages of methyl parathion from PND 1 through PND 21, as described in Materials and Methods. The arrow indicates PND 21, the day of the last treatment. Values are expressed as the mean ± SE (n = 4–6, except for low-dosage group on PND 6, where n=16, and medium-dosage on PND 14, where n = 12). The Kd (nM) for each treatment group at each age is presented in the oval overlaying the corresponding bar. Within each age, bars or Kd’s labeled with an asterisk are significantly different from control (p <= 0.05).

 


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FIG. 3. Brain 3H-quinuclidinyl benzilate binding (Bmax), following exposure to 3 dosages of methyl parathion from PND 1 through PND 21, as described in Materials and Methods: The arrow indicates PND 21, the day of the last treatment. Values are expressed as the mean ± SE (n = 4–6, except for the low-dosage animals on PND6, n = 16, and the medium-dosage group on PND 14, where n = 12). The Kd (nM) for each treatment group at each age is presented in the oval overlaying the corresponding bar. Within each age, bars or Kd’s labeled with an asterisk are significantly different from control (p <= 0.05).

 
To compare the brain ChE inhibition in the high dosage groups of MPS and CPS at 2 h (when the severity of intoxication signs reached their peak) and about 1 day after treatment (comparable to the remainder of the study), the previously reported dosing protocol for the high-dosage group of CPS was used (Tang et al., 1999Go), i.e., CPS was administered every other day at a dosage of 3 mg/kg from PND 1 through PND 5, 6 mg/kg from PND 7 through PND 13, and 12 mg/kg from PND 15 through PND 21. At 2 h after treatment, inhibition of brain ChE activity was similar between MPS and CPS treatments on PND 5 (Fig. 4AGo), PND 13 (Fig. 4BGo), and PND 21 (Fig. 4CGo). However, at one day after treatment, inhibition of brain ChE activity was significantly lower following MPS treatment than with CPS treatment on PND 6 (Fig. 4AGo), PND 14 (Fig. 4BGo), and PND 22 (Fig. 4CGo).



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FIG. 4. Brain cholinesterase (ChE) activity at 2 h and 1 day following exposure to chlorpyrifos or methyl parathion. The dosages and schedule of the methyl parathion-treated animals are identical to those of the high-dosage group as described in Materials and Methods. The dosages and schedule of the chlorpyrifos-treated animals are as described in Results. Corresponding control groups received corn oil at equal volume. Values are expressed as the mean ± SE (n = 2–6). Percentage inhibition for each treatment group at each time is presented in the oval overlaying the corresponding bar. Capital letters indicate significant differences between time points within the same compound (p <= 0.05). Asterisks indicate significant differences between compounds within the same time point (p <= 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The observed age-dependent increase in brain ChE activity has been previously reported (Atterberry et al., 1997Go; Carr et al., 2001Go; Fiedler et al., 1987Go; Hrdina et al., 1975Go; Kristt, 1983Go; Tang et al., 1999Go). In addition, the observed age-dependent increase in muscarinic receptor binding (Ben-Barak and Dudai, 1979Go; Fiedler et al., 1987Go; Large et al., 1986Go) and specifically, 3H-QNB and 3H-NMS binding (Tang et al., 1999Go) has been previously reported.

Although methyl parathion (MPS) is a widely used OP insecticide, its effects on juvenile animals have not been studied sufficiently. One of the possible reasons for this lack of investigation in juveniles is that MPS is strictly registered for use in agriculture and, therefore, infants and children should not be exposed to it in residential pesticide applications. However, in the wake of incidences of misuse in recent years, MPS became a public health concern (Environmental Health Perspective, 1997Go; Rehner et al., 2000Go). It has been suggested that the massive illegal applications of MPS for household pest control, which left significant long-lasting residues (Clark et al., 2002Go), created a greater exposure risk for children (Rubin et al., 2002Go) because of the normal behavior of children (e.g., playing close to the ground and hand to mouth behavior), their tendency to explore their environment orally, and the greater amount of time spent indoors (Eskenazi et al. 1999Go). It has also been proposed that the exposure level increases as infants mature into the more mobile toddlers (Eskenazi et al., 1999Go). This experiment was designed to determine whether exposure to constant or increasing levels of MPS during the period of intense brain growth and cholinergic system development results in changes in cholinergic neurochemistry. This period of brain growth is the early postnatal period in the rat and is equivalent to the third trimester of gestation and early postnatal period in humans (Dobbing and Sands, 1979Go).

The dosing regimen in this study resulted in three different levels of brain ChE inhibition on PND 22 (Fig. 1Go). The dosages employed in the medium- and high-dosage groups were incremental with age to prevent high mortality in neonates, which are more sensitive to MPS, presumably because of the immaturity of the detoxication mechanisms (Atterberry et al., 1997Go; Benke and Murphy, 1975Go; Mortensen et al., 1996Go) and perhaps the immaturity of the cholinergic nervous system (Coyle and Yamamura, 1976Go; Tang et al., 1999Go). Corresponding to these three levels of ChE inhibition, discernible signs of intoxication were observed in the medium- and high-dosage groups but not in the low-dosage group. In the medium- and high-dosage groups, tremors and convulsions appeared within two h after each treatment and did not subside substantially with subsequent treatments. This observation of no discernible tolerance, was different from what we described recently with chlorpyrifos (CPS) treatments, where these severe signs of intoxication were observed within two h after only the first treatment of the high dosage, with the severity of signs decreasing rapidly with subsequent treatments (Tang et al., 1999Go).

The more severe intoxication signs observed with MPS treatment than with CPS might have resulted from greater inhibition of brain ChE with MPS. One possible explanation is that the more severe intoxication signs might have been induced by a greater initial ChE inhibition with MPS, which was not detected at 18 h after treatments because the activity following MPS might have recovered more rapidly than following CPS, as would be expected of a dimethyl (MPS) compared to a diethyl (CPS) OP compound (Chambers, 1992Go). When we compared the brain ChE inhibition in the high-dosage group of MPS and CPS at 2 h (when the severity of intoxication signs reached their peak) and about 1 day after treatment (comparable to the remainder of the study) (Fig. 4Go), the data indicated that the greater initial ChE inhibition did not occur at these time points after MPS treatment than after CPS treatment. However, the data also indicated a more rapid recovery of brain ChE activity after MPS treatment than after CPS treatment, which is consistent with the relative reactivation rates reported for diethyl compared to dimethyl phosphorylated ChE (Chambers, 1992Go; Wilson et al., 1992Go). Therefore, the more severe signs of intoxication after MPS treatment did not result from greater ChE inhibition when compared to CPS treatment.

More rapid recovery of brain ChE activity after MPS treatment than occurred after CPS treatment has been reported previously (Chambers and Carr, 1993Go; Pope et al., 1991Go). However, the more rapid initial recovery does not mean the more rapid overall recovery of ChE activity after repeated exposures to MPS. The brain ChE inhibition on PND 22 was 26, 42, and 55% for the low, medium, and high MPS dosages, respectively. Activity ranged from 16 to 25% below control activity on PND 30, i.e., 9 days after the last treatment, and, interestingly, was similar among treatments, being 12 to 15% below control activity on PND 40, i.e., 19 days after the last treatment (Fig. 1Go). A probable explanation is that although dimethyl phosphorylated ChE has a higher spontaneous reactivation rate than diethyl phosphorylated ChE, the former also has a higher aging rate than the latter (Wilson et al., 1992Go). The fraction of ChE activity below control activity at PND 40 may represent phosphorylated and aged ChE that had yet to be replaced by de novo synthesis. Given that the activity of the low-dosage group remained in this range of depression from PND 30 through PND 40 suggests that the replacement of the ChE by de novo synthesis is a slow process. Significant depression of ChE activity was also observed in the medium- and high-CPS dosages in the CPS study cited earlier (Tang et al., 1999Go) on PND 40. While this depression of ChE activity following the medium and high dosages of CPS on PND 40 may represent phosphorylated ChE that had yet to be reactivated spontaneously, the low dosage of CPS, which was 29% below control activity on PND 22, had completely recovered to control levels. The fact that the activity on PND 22 with the low dosage of MPS was only 26% below control activity but did not fully recover by PND 40, as did the low dosage of CPS, strongly implicates aging of the phosphorylated ChE as the reason for the decreased ChE activity at PND 40 in the MPS-treated rats. A possible alternate explanation for this long-term ChE inhibition is that a developmental delay or permanent reduction in the cholinergic system has been incurred by early exposures to these OP compounds with one compound (MPS) possibly having a greater effect than the other (CPS); this possibility is currently under investigation in our laboratory.

As stated earlier, reduction in the density of muscarinic receptors is believed to be one of the mechanisms underlying the tolerance to the toxicity of repeated exposures to anticholinesterases (reviewed in Hoskins and Ho, 1992Go). We used both 3H-NMS and 3H-QNB to monitor changes in muscarinic receptors. A significant reduction of 3H-QNB binding sites (Bmax) was observed on PND 22 in MPS treatment groups; this is similar to what we observed previously in CPS-treated animals (Tang et al., 1999Go). However, significant reductions of 3H-NMS binding sites (Bmax) on PND 6 and PND 14, observed in CPS-treated animals (Tang et al., 1999Go), was not observed here with MPS treatment. Due to its hydrophilic character, 3H-NMS only binds to the surface mAChR (Cioffi and el-Fakahany, 1988Go; Galper et al., 1982Go; Harden et al., 1985Go; Jett et al., 1993Go), which are more physiologically important than internal mAChR, but reduction of the surface mAChR can dampen the activity in the cholinergic system elicited by inhibition of ChE and, therefore, can contribute to the development of tolerance. The different pattern of reduction in the surface mAChR after CPS and MPS treatments may explain the difference in intoxication signs observed. This differential modulation of surface mAChR by MPS and CPS may result from the different patterns in ChE inhibition. The reduction in mAChR density is thought to be agonist-induced. When ChE inhibition is more persistent, more acetylcholine molecules can theoretically accumulate in synapses. Reduction in mAChR density may be less readily induced by MPS treatment than by CPS because of the more rapid spontaneous reactivation of the dimethyl phosphorylated ChE, and, therefore, less acetylcholine molecules accumulating in the synapse. Because of the limited amount of receptor down-regulation, behavioral tolerance to the signs of intoxication did not occur. However, the reduction in QNB binding sites suggests a delayed synthesis of mAChR, possibly reflecting a developmental delay.

Liu et al.(1999)Go reported that the reduction in mAChR induced by repeated subcutaneous (sc) exposures to MPS was more profound than that to CPS in both adult and neonatal rats. The toxicokinetics would be expected to be different between the sc and oral routes used in the present study. Xenobiotics administered orally will undergo the first-pass effect in the liver, whereas xenobiotics administered by the sc route can escape massive and relatively rapid metabolism in the liver and reach target sites directly. More parent compounds (MPS and CPS) via the sc exposure route can reach the nervous system, where slow activation may occur (Chambers and Chambers, 1989Go). In addition, the sc route would allow the compound to be deposited as a depot for release over a more extended period of time, and therefore, would be expected to induce neurochemical compensation more readily than would the oral route. This longer residence time for the sc route as compared to the oral route has been demonstrated with other drugs (Alvinerie et al., 1998Go). The oral route would be expected to result in greater fluctuations in circulating levels of anticholinesterase, which would be less likely to induce down-regulation of receptors than a more consistent level of ChE inhibition resulting from sc administration. In addition, MPS, when administered dermally, yields more sustained MPS concentrations than when administered orally (Kramer et al., 2002Go). Therefore, the difference in toxicokinetics may be one of the reasons for the differences in observations between the present study and that of Liu et al.(1999)Go.

In summary, following repeated exposures of rat pups to MPS, brain ChE activity was persistently decreased through PND 40 (19 days following the last treatment). mAChR densities were reduced only on PND 22. During the recovery period, there was an increase in total mAChR density, but it is not clear if these changes are permanent. Compared to the previously reported chlorpyrifos (CPS) treatment, phosphorylated ChE recovered more rapidly initially and the reduction of surface mAChR occurred less readily after MPS treatment. These neurochemical results corresponded to the intoxication signs observed after treatments with MPS and CPS.


    ACKNOWLEDGMENTS
 
The authors appreciate the gifts of MPS and CPS from Dr. Howard Chambers, Department of Entomology and Plant Pathology, Mississippi State University. The authors gratefully acknowledge the support of NIH R01 ES04394 and R03 ES08531. J.E.C. was supported by the Burroughs Wellcome Toxicology Scholar Award. This research was supported by the Mississippi Agricultural and Forestry Experiment Station (MAFES) under project number MISV-701030 and the College of Veterinary Medicine, Mississippi State University. This article is MAFES publication number J-9629 and the Center for Environmental Health Sciences publication number 96.


    NOTES
 
1 Present address: U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Back

2 To whom correspondence should be addressed at the College of Veterinary Medicine, Box 6100, Mississippi State University, Mississippi State, MS 39762-6100. Fax: (662) 325-1031. E-mail: chambers{at}cvm.msstate.edu. Back


    REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
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