Journal of Toxicology and Pharmacology

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1 Journal of Toxicology and Pharmacology Research Article Effects of Chlorpyrifos or Methyl Parathion on Regional Cholinesterase Activity and Muscarinic Receptor Subtype Binding in Juvenile Rat Brain Shirley X. Guo-Ross, Edward C. Meek, Janice E. Chambers and Russell L. Carr* Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, USA *Corresponding author: Russell L. Carr, Received: 31 August 2017; Accepted: 25 December 2017; Published: 30 December 2017 Abstract The effects of developmental exposure to two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (machr) binding were investigated in preweanling rat brain. Animals were orally gavaged daily with low, medium, and high dosages of the insecticides using an incremental dosing regimen from postnatal day 1 (PND1) to PND20. On PND12, PND17 and PND20, the cerebral cortex, corpus striatum, hippocampus, and medulla-pons were collected for determination of ChE activity, total machr density, and the density of the individual machr subtypes. ChE activity was inhibited by the medium and high dosages of CPF and MPS at equal levels in all four brain regions at all three ages examined. Exposure to both compounds decreased the levels of the M1, M2/M4, and M3 subtypes and the total machr level in all brain regions, but the effects varied by dosage group and brain region. On PND12, only the high dosages induced receptor changes while on PND17 and PND20, greater effects became evident. In general, the effects on the M1 subtype and total receptor levels appeared to be greater in the cerebral cortex and hippocampus than in the corpus striatum and medulla-pons. This did not appear to be the case for the M2/M4 and M3 subtypes effects. The differences between CPF and MPS were minimal even though in some cases, CPF exerted statistically greater effects than MPS did. In general, repeated exposure to organophosphorus insecticides can alter the levels of the various machr subtypes in various brain regions which could induce perturbation in cholinergic neurochemistry during the maturation of the brain regions. Keywords: Organophosphate; Chlorpyrifos; Methyl parathion; Cholinesterase; Muscarinic acetylcholine receptor Introduction Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothionate or CPF] and methyl parathion [O,O-dimethyl- 4-nitrophenyl phosphorothionate or MPS] are two major organophosphorus insecticides (OPs) that were used extensively in agricultural, domestic and industrial settings [1]. Despite the voluntary restrictions that eliminated CPF for residential use in the US, it is still widely used in agriculture as is MPS. Barr et al. [2] reported that one of the metabolites of chlorpyrifos, 3,5,6-trichloro-2-pyridinol (TCP) was detected in 96% of urine samples from people aged 6-59 years in the US. Moreover, significantly higher urinary concentrations of TCP was detected in children aged 6-11 years (3.7 μg/l) than adolescents (2.1 μg/l), and adolescents had significantly higher TCP concentrations than adults (1.6 μg/l). Dietary ingestion has been suggested to be the predominant exposure route to CPF in children and urinary TCP Open Access levels were demonstrated to be higher with increased dietary intake of fresh fruit [3,4]. In fact, urinary residues of OP insecticides have been reported in children in many different areas of the world [5,6]. Thus, there is significant potential for childhood exposure. Because infants and children have higher exposure to OPs, an immature detoxication system, and a developing central nervous system, they are much more vulnerable to the adverse effects of these neurotoxicants [7-15]. It has also been demonstrated in animal models that neonatal and juvenile rats are more sensitive to the neurotoxic effects of CPF and MPS than adult rats, although their ChE activity tends to recover faster than adult rats following OP exposure [16-18]. Acetylcholine, cholinesterase (ChE) and the acetylcholine muscarinic receptors (machr) are components of the cholinergic system that play important roles in neural development, neurological function, and behavior [19-22]. CPF, MPS and their active metabolites could cause neurotoxicity primarily through the inhibition of ChE activity and the accumulation of the neurotransmitter acetylcholine in the synapses [20]. The mechanisms of adverse effects of OPs include: direct inhibition of ChE activity [20], reduction of acetylcholine receptor levels [23], and interactions with the G-protein/adenylyl cyclase signaling cascade [24]. However, CPF has also been suggested to cause adverse neurodevelopmental effects through non-cholinergic pathways in rats exposed at various times ranging from the embryonic period through the postnatal stage [25], such as inducing oxidative stress and DNA damage [26-28], altering serotonergic systems [29-35], and targeting of neurotrophic factors [36-38]. MPS, a Class I insecticide with a rat oral LD50 of mg/kg [39], is more acutely toxic than CPF, a Class II insecticide with a rat oral LD50 of mg/kg [40]. However, chlorpyrifos-oxon, the active metabolite of CPF, is a more potent in vitro inhibitor of ChE compared to methyl paraoxon, the active metabolite of MPS [41]. In addition, CPF and MPS have different rates of bioactivation and detoxication [42], and the rates of reactivation of ChE differ following inhibition by the oxons of CPF and MPS [43]. The effects of these two compounds in the developing brain of rats have been previously compared. We have reported a reduction in the number of surface machr with CPF, but not with MPS [44,45]. We have also reported the changes in ChE activity and machr levels in the anterior and posterior brain regions and medulla-pons at PND4 and PND8 that result from postnatal treatments of CPF and MPS [46]. In the current study, we have extended our previous findings by determining the effects of continued postnatal exposure to CPF and MPS in rat pups. On PND12, PND17 and PND20, the changes in ChE activity, M1, M2/M4, and M3 machr subtype levels, and total machr levels in the cerebral cortex, corpus striatum, hippocampus, and medulla-pons was determined following exposure to low, medium and high dosages of CPF or MPS. Our study was designed to yield comparable levels of ChE inhibition from both CPF and MPS treatment. Materials and Methods Chemicals Analytical grade CPF and MPS (>99% purity) were generously provided by Dr. Howard Chambers (Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University). AF-DX 384, [2,3-dipropylamino- 3 H]- (120 Ci/mmol), 4-DAMP, [N-methyl- 3 H ]- (80 Ci/mmol), pirenzepine [N-methyl- 3 H ]- (86 Ci/mmol), and quinuclidinyl benzilate, L-[benzilic-4,4-3 H]- (QNB, 42 Ci/mmol) were purchased from PerkinElmer, Inc. (Boston, MA). Microscint 20 was also obtained from PerkinElmer. All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Copyright 2017 The Authors. Published by Scientific Open Access Journals LLC.

2 Table 1: Dosages administered in the incremental dosing regimen. Treatment PND1-5 PND6-13 PND14-20 CPF Low 1.0 mg/kg 1.0 mg/kg 1.0 mg/kg CPF Medium 1.0 mg/kg 2.0 mg/kg 4.0 mg/kg CPF High 1.5 mg/kg 3.0 mg/kg 6.0 mg/kg MPS Low 0.2 mg/kg 0.2 mg/kg 0.2 mg/kg MPS Medium 0.2 mg/kg 0.4 mg/kg 0.6 mg/kg MPS High 0.3 mg/kg 0.6 mg/kg 0.9 mg/kg Experimental design Animals were housed in a temperature-controlled room at 22 ± 2 o C with a 12:12 hour light/dark cycle in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility. Rats were provided standard laboratory chow and tap water ad libitum. All procedures were approved by the Mississippi State University Institutional Animal Care and Use Committee. Adult male and female Sprague-Dawley rats (CD IGS; Harlan Laboratories, Indianapolis, IN) were used as breeders. Following parturition, male and female rat pups within the same litter were assigned to different treatment groups. There was a representative control animal present for each sex in each treatment group. Dosing regimen The insecticides were dissolved in corn oil and, using an incremental dosing regimen, were administered by oral gavage daily to Sprague- Dawley rats from PND1 (the day of birth is PND0) to PND12, PND17 or PND20 as described in Table 1. Control animals received corn oil daily. Oral gavage was performed 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 dosing paradigm was a continuation of the paradigm used in our previous study [46] and is also similar, although using lower dosages, to previously published studies [45,47]. The incremental dosing paradigm and dosages used were implemented to avoid mortality and overt signs of toxicity in the rat pups but still induce comparable levels of inhibition of ChE activity between the two insecticides. On PND12, PND17 or PND20, rats were sacrificed four hours after the last dose of CPF, MPS or corn oil. All brains were rapidly removed, dissected on ice to obtain the cerebral cortex, corpus striatum, hippocampus, and medulla-pons regions, frozen, and maintained at -80 C until assay. ChE assay ChE activities were measured in homogenates from cerebral cortex, corpus striatum, hippocampus, and medulla-pons brain regions using a continuous spectrophotometric assay (modification of Ellman et al. [48]) with acetylthiocholine as the substrate and 5, 5 -dithiobisnitrobenzoic acid as the chromagen [49]. machr binding assay Membrane preparations from cerebral cortex, corpus striatum, hippocampus, and medulla-pons were prepared as previously described [46]. The receptor binding assays were conducted by using the modified method of Araujo et al. [50]. Membrane preparations were incubated with either 3 H-pirenzepine, 3 H-AF-DX 384, 3 H-4-DAMP, or 3 H-QNB to measure the Bmax of the M1, M2/M4, M3, or total machr binding, respectively. The specific binding was determined by total binding minus non-specific binding (incubated with each individual [ 3 H]- labeled ligand in the presence of 10 μm atropine). Protein determination Protein concentration was determined in all tissue preparations by the method of Lowry et al. [51] using bovine serum albumin as the standard. Statistical analysis Statistical analysis was performed by analysis of variance (ANOVA) using the Mixed procedure [52] followed by separation of means using Least Significant Means. An overall ANOVA for weight gain was performed to identify differences in the main effects (age, sex, and treatment) and all possible interactions. For weight gain, significant main effects identified included age, sex, and treatment but no significant interactions. An overall ANOVA for each ligand and for ChE activity was performed to identify differences in the main effects (age, brain region, sex, and treatment) and all possible interactions. For each machr ligand, significant main effects included age, region, and treatment and significant interactions for region treatment, age treatment, and age region. For ChE, significant main effects included age, region, and treatment and significant interactions for region treatment, age treatment, age region, sex region, and age sex region. Therefore, for subsequent analysis to identify treatment effects and any significant sex treatment interactions, analysis for each ligand in each brain region was performed separately for each age. No significant sex treatment interactions were observed for either the ligands or ChE activity. Therefore, data from males and females were pooled for statistical analysis. All analyses included litter and sex treatment litter as random effects. The criterion for significance for all parameters was set at p Results No signs of overt functional toxicity of OP-treatment were observed in the rats. With respect to body weight gain (Figure 1), data are presented with sexes separated. The high dosage of CPF significantly reduced weight gain in both females and males at all ages. No other dosages had effects on PND12 (Figure 1A). On PND17, the medium dosage of CPF and the high dosage of MPS significantly reduced weight gain in males but not females (Figure 1B). On PND20, the medium dosage of CPF significantly reduced weight gain in males (Figure 1C). In the control animals, the brain region with the highest ChE activity was the corpus striatum followed by the medulla-pons, then by the cerebral cortex and hippocampus which were similar (Table 2). The ChE activity increased from PND12 to PND17, but remained unchanged from PND17 to PND20 in all brain regions except the medulla-pons which had similar activity at all three ages (Table 2). Reduction in ChE activity was observed at each time point for the medium and high dosages of both CPF and MPS in the corpus striatum, medulla-pons, cerebral cortex, and hippocampus (Figures 2A, 2B, 2C, and 2D, respectively). The level of inhibition was similar between the medium dosages of CPF and MPS and between the high dosages of CPF and MPS. The low dosage of CPF significantly inhibited ChE activity in the medulla-pons, cerebral cortex, and hippocampus on PND17 and PND20 but did not inhibit ChE activity in the corpus striatum. The low dosage of MPS significantly inhibited ChE activity in the corpus striatum and hippocampus on PND12, in the cerebral cortex and hippocampus on PND17, and in the medulla-pons and hippocampus on PND20 M1-mAChR subtype levels were measured by the binding of 3 H-pirenzepine in all brain regions. In control animals, the M1-receptor levels in the corpus striatum, cerebral cortex, and hippocampus were significantly greater than that in the medulla-pons regardless of age (Table 2). The greatest developmental increase in M1-receptor levels in the cerebral cortex and hippocampus occurred between PND12 and PND17 with little change thereafter. However, the greatest developmental increase in the corpus striatum occurred between PND17 and PND20. The M1-receptor levels in the medulla-pons remained at similar levels at all ages. With respect to treatment effects, a significant decrease was observed with the high dosage of MPS at all three ages and with the high dosage of CPF on PND20 in the corpus striatum (Figure 3A). There was a significant difference in corpus striatum Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

3 Figure 1: Body weight gain of female and male rats treated with low, medium or high dosages of chlorpyrifos (CPF) or methyl parathion (MPS) on (A) PND 12, (B) PND 17 and (C) PND 20. Values are expressed as mean ± SE (n = 12-27). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*). Table 2: Specific Activity of cholinesterase (ChE) and B max of muscarinic acetylcholine receptor (machr) subtypes in control rat brain regions on postnatal days 12, 17 and 20. Parameter PND Striatum Corpus Pons Medulla-Cortex Cerebral Hippocampus ChE Activity ± 7.69 B,a ± 4.98 A,b ± 4.39 B,c ± 2.37 B,c ± A,a ± 6.07 A,b ± 1.49 A,c ± 1.95 A,c ± B,a ± 4.50 A,b 44.3 ± 2.73 B,c ± 1.68 B,c M1 machr ± 3.62 A,a ± 1.23 A,b ± 6.34 B,c ± 7.14 B,c ± 6.63 A,a ± 1.20 A,b ± 4.12 A,a ± 5.79 A,a ± B,a ± 1.20 A,b ± B,a ± 5.75 B,a M2/M4 machr ± 4.80 A,a ± 5.18 B,a ± 4.79 B,a ± 5.60 A,a ± 3.22 A,a 56.5 ± 3.61 A,a 50.7 ± 2.12 A,a ± 3.46 A,a ± 8.33 B,a ± 2.55 B,b ± 9.68 C,a ± 4.75 B,a M3 machr ± 3.93 A,a ± 0.56 A,b ± 3.12 A,a ± 2.22 A,a ± 3.09 A,a 9.15 ± 0.95 B,b ± 5.00 B,c ± 5.49 A,ac ± 7.03 B,a 9.61 ± 0.66 B,b ± 3.92 C,a ± 2.83 B,a Total machr ± A,a ± 4.10 A,b ± 8.12 A,a ± 7.85 A,c ± A,a ± 3.91 A,b ± 9.56 B,c ± A,d ± B,a ± 3.65 A,b ± 6.85 C,a ± B,a 1 Specific activity = nmoles product min -1 mg protein -1. Data presented as mean ± SEM. 2 B max = fmol/mg protein. Data presented as mean ± SEM. Within each brain region and within each parameter, different capital letters indicate significant differences between ages. With each row, different lower case letters indicate significant differences between brain regions. Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

4 Guo-Ross et al. Volume 1, Issue 3 Figure 2: Specific activity of cholinesterase (ChE) of rats treated with low, medium or high dosages of CPF or MPS on PND 12, PND 17 and PND 20 in the (A) corpus striatum, (B) medulla/pons, (C) cerebral cortex, and (D) hippocampus. Values are expressed as mean ± SE (n = 8-10). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*). Percent decrease from control at each age for each statistically significant value is presented inside of the corresponding bar. Figure 3: M1-subtype machr binding of rats treated with low, medium or high dosages of CPF or MPS on PND 12, PND 17 and PND 20 in the (A) corpus striatum, (B) medulla-pons, (C) cerebral cortex, and (D) hippocampus. Values are expressed as mean ± SE (n = 8-14). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*) and values connect by brackets are significantly different from one another. Percent decrease from control at each age for each statistically significant value is presented inside of the corresponding bar. Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

5 M1-receptor levels between the medium dosages of CPF and MPS on PND17 but neither was significantly different from control. In the medulla-pons (Figure 3B), significant reductions were only seen with the high dosages of CPF and MPS on PND20. In the cerebral cortex (Figure 3C), significant decreases of M1-receptor levels were observed with the high dosages of CPF and MPS at all three ages. In addition, the medium dosage of CPF also reduced M1 levels on PND17 and PND20. In the hippocampus (Figure 3D), significant reductions were observed with the high dosage of CPF at all three ages, the medium dosage of CPF and the high dosage of MPS on PND17 and PND20, and the medium dosage of MPS on PND20. On PND20, the medium dosage of CPF induced a significantly greater decrease in M1-receptor levels than did the medium dosage of MPS in both the cerebral cortex and hippocampus. M2/M4-mAChR subtype levels were measured by the binding of 3 H-AF-DX 384 in all brain regions. In control animals, the M2/M4- receptor levels were similar between regions on PND12 and PND17 but by PND20, the medulla-pons had lower levels compared to the other three regions (Table 2). The greatest developmental increase in M2/M4-receptor levels in the corpus striatum and hippocampus occurred between PND17 and PND20 while the greatest increase in the medulla-pons occurred between PND12 and PND17. The levels of the M2/M4-receptor in the cerebral cortex continuously increased from PND12 through PND20. With respect to treatment effects, significant reductions in M2/M4-receptors were observed in the medium and high dosages of CPF and the high dosage of MPS at both PND17 and PND20 in the corpus striatum (Figure 4A). On PND17, the medium dosage of CPF induced a significantly greater decrease in corpus striatum M2/M4-receptor levels than did the medium dosage of MPS. In the medulla-pons (Figure 4B), significant reductions were observed with the high dosages of CPF and MPS on PND17 and PND20 and with the medium dosage of CPF on PND20. In the cerebral cortex (Figure 4C), significant reductions were observed with the high dosage of MPS at all three ages, with the medium and high dosages of CPF on PND17 and PND20, and with the medium dosage of MPS on PND20. On PND17, the medium and high dosages of CPF induced a significantly greater decrease in cerebral cortex M2/M4-receptor levels than did the medium and high dosages of MPS, respectively. In the hippocampus (Figure 4D), significant decreases were observed with the high dosages of both CPF and MPS at all three ages and with the medium dosage of CPF on PND17 and PND20. M3-mAChR subtype levels were measured by the binding of 3 H-4-DAMP in all brain regions. In control animals, the M3-receptor levels in the corpus striatum, cerebral cortex, and hippocampus were significantly greater than that in the medulla-pons regardless of age (Table 2). The greatest developmental increase in M3-receptor levels in the corpus striatum and hippocampus occurred between PND17 and PND20, while in the medulla-pons, a decrease occurred between PND12 and PND17. The levels in the cerebral cortex continuously increased from PND12 through PND20. With respect to treatment effects, significant reductions were observed with the high dosages of CPF and MPS on PND17 and PND20 and with the medium dosages of CPF and MPS and low dosage of MPS on PND20 in the corpus striatum (Figure 5A). There was a significant difference in corpus striatum M3-receptor levels between the medium dosages of CPF and MPS on PND17 but neither was significantly different from control. In the medulla-pons (Figure 5B), significant reductions were observed in the high dosages of CPF and MPS at all three ages and with the medium dosage of CPF on PND17 and PND20. In the cerebral cortex (Figure 5C), significant reductions were observed with the high dosage of CPF at all three ages, with the medium dosage of CPF and the high dosage of MPS on PND17 and PND20, and with the medium dosage of Figure 4: M2/M4-subtype machr binding of rats treated with low, medium or high dosages of CPF or MPS on PND 12, PND 17 and PND 20 in the (A) corpus striatum, (B) medulla-pons, (C) cerebral cortex, and (D) hippocampus. Values are expressed as mean ± SE (n = 8-14). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*) and values connect by brackets are significantly different from one another. Percent decrease from control at each age for each statistically significant value is presented inside of the corresponding bar. Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

6 Figure 5: M3-subtype machr binding of rats treated with low, medium or high dosages of CPF or MPS on PND 12, PND 17 and PND 20 in the (A) corpus striatum and (B) medulla-pons, (C) cerebral cortex, and (D) hippocampus. Values are expressed as mean ± SE (n = 8-14). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*) and values connect by brackets are significantly different from one another. Percent decrease from control at each age for each statistically significant value is presented inside of the corresponding bar. MPS on PND20. On PND17 and PND20, the medium dosage of CPF induced a significantly greater decrease in cerebral cortex M3-receptor levels than did the medium dosage of MPS. This significantly greater decrease was also present between the high dosages on PND20. In the hippocampus (Figure 5D), significant reductions were observed with the high dosages of CPF and MPS at all three ages, with the medium dosage of CPF on PND17 and PND20, and with the medium dosage of MPS on PND20. Total machr levels were measured by the binding of 3 H-QNB in all brain regions. In control animals, total receptor levels in the corpus striatum, cerebral cortex, and hippocampus were significantly greater than that in the medulla-pons regardless of age (Table 2). The greatest developmental increase in total receptor levels in the corpus striatum and hippocampus occurred between PND17 and PND20 while the levels continuously increased in the cerebral cortex from PND12 through PND20. Total receptor levels in the medulla-pons remained at similar levels at all ages. With respect to treatment effects, significant reductions were observed with the high dosage of CPF on PND17 and PND20, with the medium dosage of CPF on PND17, and with the high dosage of MPS on PND20 in the corpus striatum (Figure 6A). On PND17, the medium and high dosages of CPF induced a significantly greater decrease in corpus striatum total receptor levels than did the medium and high dosages of MPS, respectively. In the medulla-pons (Figure 6B), significant reductions were observed with the medium and high dosages of CPF and high dosage of MPS on PND17 and PND20. On PND17 and PND20, the medium dosage of CPF induced a significantly greater decrease in medulla-pons total receptor levels than did the medium dosage of MPS. This significantly greater decrease was also present between the high dosages on PND20. In the cerebral cortex (Figure 6C), significant reductions were observed with the high dosages of CPF and MPS at all three ages and with the low and medium dosages of CPF on PND17 and PND20. In the hippocampus (Figure 6D), significant reductions were observed with the high dosages of CPF and MPS at all three ages, with the medium dosage of CPF on PND17 and PND20, and with the medium dosage of MPS on PND20. On PND17 and PND20, the medium and high dosages of CPF induced a significantly greater decrease in cerebral cortex and hippocampus total receptor levels than did the medium and high dosages of MPS, respectively. Discussion Previous studies have reported age-related changes in neurochemical parameters similar to the parameters measured in this study. In most of those previous studies, the parameters were measured at ages and in specific regions that did not exactly match the ages and specific regions utilized in the present study. However, the patterns of development of the parameters reported in these studies are comparable to the patterns we observed. Most studies measuring ChE activity during the late preweanling period also observed a rise in activity similar to what we observed in the various brain regions in the present study [44,45,47,53-55]. In addition, the increase in total machr binding data observed in the various brain regions in the present study is similar to that reported by other studies measured either both whole brain preparations [44,45,47] or regional analysis using autoradiography [56,57]. Although the regions measured in the autoradiography studies were not identical to the regions measured in the present study, the pattern of changes in the specific machr subtype levels reported in those studies closely resembled the patterns of changes that we observed in this present study [56,57]. With respect to weight gain, the CPF high dosage group exerted the greatest effect on weight gain in both sexes throughout the study. Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

7 Figure 6: Total machr binding of rats treated with low, medium or high dosages of CPF or MPS on PND 12, PND 17 and PND 20 in the (A) corpus striatum, (B) medulla-pons, (C) cerebral cortex, and (D) hippocampus. Values are expressed as mean ± SE (n = 8-14). Within each age, values significantly different from control (p < 0.05) are indicated with an asterisk (*) and values connect by brackets are significantly different from one another. Percent decrease from control at each age for each statistically significant value is presented inside of the corresponding bar. Statistically significant differences from the control group were observed in the male pups but not female pups exposed to the CPF medium dosage on PND17 and PND20. This suggests a greater sensitivity of males to OP exposure. Similarly, the only effect on weight gain observed following MPS exposure occurred in males on PND17. However, the amount of weight gained by the CFP medium treated males and females was statistically identical. Several studies have reported that chlorpyrifos can exert sexually dimorphic effects following developmental exposure [31,58-60]. However, we did not observe any sex differences in any of the biochemical parameters that were measured. It was hypothesized that CPF would yield more severe effects than MPS because its metabolite chlorpyrifos-oxon (CPO) more potently inhibits ChE activity than methyl paraoxon (MPO) in vitro. However, MPS is more toxic than CPF in acute in vivo studies. ChE inhibition caused by CPF is more persistent than by MPS based on the reactivation rate of phosphorylated ChE by CPO and MPO. In this study, the degree of inhibition of ChE activities in all four brain regions were comparable following CPF and MPS treatments and was dose dependent. Combined with our previous study [46], the level of ChE inhibition induced by CPF and MPS treatment gradually increased from PND4 to PND17 with small decreases by PND20. This trend parallels the pattern of ChE inhibition reported earlier [44,45,47]. Both the medium and high dosages of CPF and MPS induced significant inhibition in all brain regions. Effects of the low dosages were observed in the medulla-pons, cerebral cortex, and hippocampus on PND17 and PND20 but only in the corpus striatum on PND12, which is prior to the developmental increase in ChE activity levels. Richardson et al. [47] reported that at the latter ages, the majority of ChE enzyme inhibited by an OP would be aged and therefore refractory to undergo spontaneous reactivation. The lack of significant inhibition of ChE in the corpus striatum at the latter ages with the low dosages could be due to greater potential of the corpus striatum to synthesize new ChE to replace the inhibited enzyme. The other regions, which have lower ChE activities, may not possess a similar de novo synthesis capacity. In our previous study on machr binding [46], we observed that the effects of CPF and MPS on machr subtype levels were minimal at PND4, but began to appear by PND8. Our current data yields the same pattern with significant reductions in machr binding as the postnatal age increases. However, the pattern differs with respect to brain region, receptor subtype, and compound. Within receptor subtypes, the pattern of reduction in binding was similar between the cerebral cortex and hippocampus. Although there are some slight differences, all subtypes of machrs in these two regions were sensitive to both CPF and MPS treatment. Some effects with the highest dosages of CPF and MPS were present on PND12 and as the treatment persisted, effects of the lower dosages were also detected. The effects were as well comparable between subtypes in these two brain regions. In the corpus striatum, the earliest effect observed was on the M1 subtype with the highest dosage of MPS on PND12 which persisted through PND20. The high dosage of CPF altered M1 binding only on PND20. Both M2/M4 and M3 subtypes were reduced on PND17 and PND20 with the greatest effect occurring on the M3 subtype with MPS on PND20. In the medulla-pons, the earliest effect observed was with both CPF and MPS on the M3 subtype on PND12. This was followed by effects with both compounds on the M2/M4 subtype on PND17. By PND20, effects were observed on all three subtypes but the largest decrease was on the M1 subtype with the high dosages of both compounds. However, the machrs in the medulla-pons were generally less sensitive than in other regions. Possible mechanisms of OP-induced developmental neurotoxicity Muscarinic Receptor Subtype Binding in Juvenile Rat Brain..

8 may include OPs indirectly affecting machr levels by inhibiting ChE activity, or alternatively, by directly inducing down-regulation of machr [61]. By altering the densities of the machrs subtypes, CPF and MPS can modify two different second messenger systems depending on the receptor subtype involved [62]. By decreasing M1 and M3-subtype receptors, a stimulatory G-protein-mediated phosphoinositide hydrolysis could be disrupted. Likewise, by reducing M2/M4-subtype receptors, the inhibition of adenylyl cyclase-catalyzed camp formation through an inhibitory G-protein could be disrupted. Muscarinic receptor-mediated phosphoinositide hydrolysis is present and increases during the first three postnatal weeks, whereas muscarinic receptor-mediated inhibition of camp formation develops during the third postnatal week [63]. Disturbance of receptor levels during the period of development/maturation of the appropriate signaling capacities could result in persistent deficiencies in cholinergic synaptic functions resulting in long lasting effects [58,64]. Previous work has reported altered adenylyl cyclase signaling induced by exposure to OP insecticides using both in vitro models of development and following in vivo developmental exposures [24,65-67]. However, the direct mechanism by which OP exposure alters adenylyl cyclase signaling is not known. It is well known that developing animals are more sensitive than adults to the cholinergic toxicity caused by OP insecticides [18,61,68]. Both acetylcholine (ACh) and ChE play important morphogenic roles during neural development, such as the regulation of neuronal growth and differentiation, axonal outgrowth, and synapse formation [19,21]. Brimijoin and Koenigsberger [20] first suggested that any perturbation in acetylcholine levels or ChE activity during neural development would cause long term deficits in the architectural development and physiological function of the nervous system. Using an in vitro model, Yang et al. [69] reported that the disruption of the establishment of appropriate neuronal connectivity by developmental OP exposure may be partially the result of interference with the morphogenic activity of ChE. It has also been reported that exposure to OP insecticides differentially altered the levels of the ChE variants and suggested that this alteration may play a role in the developmental neurotoxicity of OPs [70]. In fact, repeated in vitro exposure of septal SN56 basal forebrain cholinergic neurons lead to differential levels of the ChE variants and it was hypothesized that the increased cell death observed was partially mediated by this change [71]. Chronic dietary exposure to chlorpyrifos induced cognitive and emotional disorders in adult rats and these changes were suggested to be related to the changes in the ChE variants [72]. Previously, Meshorer et al. [73] demonstrated that exposure of mice to either stress or repeated exposure to the OP diisoprpylfluorophosphonate (DFP) resulted in a ChE variant shift in a similar pattern. Although the DFP mice were not tested, the ChE variant shift in stressed mice was accompanied by persistent neuronal hypersensitivity detected in hippocampal slices stimulated with anticholinesterase one month after the initial stress. This persistent neuronal hypersensitivity was blocked by muscarinic antagonists suggesting the shift in ChE variants, whether it be induced by stress or exposure to an OP, disrupts the normal function of the cholinergic system. In conclusion, the mechanism for the effects of OP insecticides in developmental neurotoxicity is still unclear. The changes observed in this study indicate that developmental exposure to OP insecticides alters both ChE activity and muscarinic receptor levels. However, it is not known if exposure to OP insecticides during development is actually damaging the biochemical components resulting in persistent deficits or merely slowing growth such that it disrupts the timing of specific windows of development. Regardless, both effects could lead to long term neurological effects. Acknowledgments The authors wish to express our gratitude to Dr. Howard Chambers for providing chlorpyrifos and methyl parathion and Mr. Shane Bennett for helping with treating animals. This research is supported by National Institute of Health (R01 ES 10386), the Mississippi Agriculture and Forestry Experiment Station (MAFES), and the College of Veterinary Medicine, Mississippi State University. This paper is Center for Environmental Health Sciences publication # 170. References 1. Grube A, Donaldson D, Kiely T, Wu L. 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