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Age-related effects of chlorpyrifos and parathion on acetylcholine synthesis in rat striatum
Neurotoxicology and Teratology 25 (2003) 599 – 606 www.elsevier.com/locate/neutera
Age-related effects of chlorpyrifos and parathion on acetylcholine synthesis in rat striatum Subramanya Karanth*, Carey Pope Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078, USA Received 17 December 2002; received in revised form 28 February 2003; accepted 19 March 2003
Abstract We compared the in vivo effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and parathion (PS) on acetylcholine (ACh) synthesis in neonatal, juvenile and adult rats. Basal levels of ACh synthesis were highest in adult rats, intermediate in juveniles and lowest in neonates. Following high (maximum tolerated dosage) subcutaneous exposure to either insecticide, relatively similar degrees of cholinesterase inhibition were noted, but the time to peak reduction varied among the age groups. CPF had no effect on ACh synthesis in neonates, increased synthesis in juveniles and decreased synthesis in adults, but only in the low dose group. PS had more consistent effects on ACh synthesis, decreasing transmitter synthesis in neonates (24 h after dosing) but increasing synthesis in juveniles and adults at both 4 and 24 h after exposure. Selective changes in neurotransmitter synthesis may contribute to differential age-related toxicity of these agents. D 2003 Elsevier Inc. All rights reserved. Keywords: Organophosphorus; Acetylcholine; Acetylcholinesterase; Age related; Neurotoxicity
1. Introduction Organophosphorus (OP) pesticides are widely used all over the world in the control of agricultural and household pests [1]. OP pesticides exhibit a wide range of toxicity in mammals but typically elicit their adverse effects through inhibition of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) in synapses of the central and peripheral nervous systems (for a review, see Ref. [37]). AChE inhibition leads to accumulation of the neurotransmitter acetylcholine (ACh), resulting in overstimulation of postsynaptic cholinergic receptors and consequent signs of neurotoxicity [10,12]. Immature animals are generally more sensitive than adults to the acute toxicity of OP insecticides including parathion (PS) and chlorpyrifos (CPF) [3,4,35]. Lesser detoxification has often been reported to contribute to higher sensitivity of young animals to OP insecticide toxicity [2,3,29,30]. For example, several studies have reported a relationship between the relative toxicity of PS * Corresponding author. Tel.: +1-405-744-4868; fax: +1-405-7448263. E-mail address: [email protected] (S. Karanth). 0892-0362/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0892-0362(03)00049-7
and CPF and the maturational expression of detoxification processes [2,3,17,30]. Toxicodynamic factors may also contribute to age-related sensitivity to OP toxicants. Some OP anticholinesterases are known to interact with other macromolecular targets including neurotransmitter receptors and components of second messenger signaling pathways [6 –8,15,18,32,40,41,45]. Thus, both toxicokinetic and toxicodynamic processes may contribute to age-related sensitivity to OP insecticides. Earlier reports indicated that several OP toxicants can alter high affinity choline uptake (HACU), the rate-limiting step in ACh synthesis [20,24,25]. HACU on the other hand has been reported to be underdeveloped in immature rat brain, with both the coupling between uptake and ACh synthesis and the degree of activity-dependent regulation changing during maturation [21]. Immature animals also lack presynaptic autoreceptors that can inhibit neurotransmitter release in a feedback manner, possibly influencing the toxic response to extensive AChE inhibition [48]. Interaction of OP anticholinesterases with such presynaptic cholinergic processes and possible modulation of ACh synthesis may therefore be an important contributing factor in the expression of cholinergic toxicity. However, the age-related effects of OP agents on ACh synthesis have not been evaluated.
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In the present study, we investigated the in vivo effects of the commonly used OP pesticide, CPF, and the prototype OP pesticide, PS, on cholinesterase activity (i.e., activity from both AChE and butyrylcholinesterase) and ACh synthesis in neonatal (7 days), juvenile (21 days) and adult (90 days) rats. The results suggest that these OP insecticides may differentially affect ACh synthesis in an age-related manner, potentially contributing to selective differences in toxicity.
2. Materials and methods 2.1. Chemicals CPF (O,O0-diethyl-3,5,6-trichloro-2-pyridinyl-phosphorothioate, 99.5% pure; GC/MS analysis) and PS (O,O0diethyl-p-nitrophenyl-phosphorothioate, 99.5% pure; GC/ MS analysis) were obtained from Chem Service (West Chester, PA). ACh iodide (acetyl-3H, specific activity 82.0 Ci/mmol) and choline chloride (methyl-3H, specific activity 75 Ci/mmol) were purchased from Dupont New England Nuclear (Boston, MA). All other chemicals were purchased from Sigma (St. Louis, MO). 2.2. Animals and treatment Sprague –Dawley rats (7- and 21-day-old of both sexes, and 3-month-old males) were used throughout the experiment. Animals were housed in steel mesh cages with a 12:12-h light–dark illumination cycle and were allowed free access to food (Harlan Tek-Lab diet) and water. All procedures involving animals were in accordance with the protocols established in the NIH/NRC Guide for the Care and Use of Laboratory Animals and approved by the local Institutional Animal Care and Use Committee. OP compounds were dissolved in peanut oil (100% purity; Lou Ana brand, Ventura Foods, Opelousas, LA) and injected subcutaneously (sc) at a volume of 1 ml/kg (except in the case of CPF in adult rats where in an injection volume of 2 ml/kg was used). Maximum tolerated dosages (MTDs; i.e., highest dosages of OP agents which produced no cumulative lethality over a 7-day period) were reported elsewhere [17,35]. MTDs for PS were 2.1, 4.8 and 18 mg/kg and 45, 127 and 279 mg/kg for CPF in neonatal, juvenile and adult rats, respectively. Animals (n = 6/treatment group) were challenged with either vehicle, 0.3 MTD or 1.0 MTD of either OP insecticide and sacrificed at 4, 24 and 96 h after treatment for measurement of neurochemical changes. Body weight and functional signs of toxicity were recorded as described earlier [50]. 2.3. Tissue preparation and biochemical assays Crude synaptosomes were used to measure ACh synthesis essentially by the method of Vogelsberg et al. [44].
Rats were sacrificed and the striatum was dissected rapidly on ice according to the method of Glowinski and Iversen [13]. Tissues were homogenized (1:20 wt/vol) with a PotterElvehjem homogenizer in ice-cold sucrose solution (0.32 M). The homogenates were centrifuged at 1000 g for 10 min in a Beckman J-20I high-speed refrigerated centrifuge and the supernatant was further centrifuged at 17,000 g for 20 min. The resulting pellet was resuspended in ice-cold sucrose solution (original volume) just prior to assay. Aliquots of synaptosomes (about 250– 350 mg protein) were incubated at 0 or 37 C with tritiated choline (2.5 mM, about 300,000 cpm/reaction) for 10 min in oxygenated Krebs bicarbonate buffer (pH 7.4). Physostigmine (50 mM) was included to prevent hydrolysis of ACh. The reactions were terminated by addition of 2-ml ice-cold Krebs bicarbonate buffer and centrifugation for 10 min at 17,000 g. The resulting pellets were resuspended in 130 mL of glycyl – glycine buffer (400 mM, pH 8.3) containing 20 mM MgCl2 and 20 mM ATP. Ten microliters of choline kinase (5 units/ ml) was added and tubes were vortexed and incubated at 37 C for 45 min. The reaction mixtures were transferred to 1.5-ml microcentrifuge tubes; 400 ml of tetraphenyl boron solution in 3-heptanone (15 mg/ml) was added and the tubes were vortexed and centrifuged for 5 min. Aliquots of the organic layer were carefully transferred to scintillation vials for counting using a biodegradable scintillation fluid (‘‘Scintisafe’’ 30%, Fischer Scientific, Pittsburgh, PA). ACh synthesis was reported as cpm/min mg protein. Cholinesterase activity (i.e., total activity from both AChE and butyrylcholinesterase) was measured by the radiometric method of Johnson and Russell [16] using [3H]acetylcholine iodide as the substrate (1 mM final concentration). Preliminary experiments determined assay conditions with incubation time and tissue concentration
Fig. 1. ACh synthesis and cholinesterase activity (inset) in striatum of neonatal, juvenile and adult rats. ACh synthesis was measured in synaptosomes from the conversion of [3H]choline to [3H]acetylcholine and expressed as cpm [3H]acetylcholine formed/min mg protein. ChE activity was measured using [3H]acetylcholine iodide as substrate and expressed as nanomoles ACh hydrolyzed/min mg protein as described in Materials and methods. Data represent mean ± S.E.M., n = 24 – 36/group. Asterisks indicate significant difference ( P < .05) compared to adult rats.
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2.4. Data analysis The JMP package [38] was used for statistical analyses. ACh synthesis and ChE activity data (percent transformations) were tested for statistical significance by a one-way ANOVA, and the Tukey – Kramer analysis was used for post hoc comparisons between the groups. Body weight data were analyzed by repeated measures ANOVA followed by linear contrasts. In all cases, P < .05 was considered significant.
Fig. 2. Effects of 0 MTD, 0.3 MTD or 1 MTD doses PS and CPF on mean body weight in (A) neonatal (PS = 0.63 and 2.1; CPF = 13.5 and 45 mg/kg bodyweight), (B) juvenile (PS = 1.44 and 4.8; CPF = 38.1 and 127 mg/kg bodyweight) and (C) adult (PS = 5.4 and 18; CPF = 83.7 and 279 mg/ kg bodyweight) rats (5 Control; ! PS 0.3 MTD; & PS 1 MTD; 4 CPF 0.3 MTD; . CPF 1 MTD). Data represent mean ± S.E.M., n = 4/ group. Asterisks indicate significant differences ( P < .05) between contemporaneous control and the following groups— (A) neonates: CPF, 1 MTD, (B) juveniles: CPF, 1 MTD and (C) adults: CPF, 1 MTD. Mean body weight on Day 0 was 14.5 ± 0.5; 43 ± 1.2 and 274 ± 2.4 g in neonatal, juvenile and adult rats, respectively.
to produce linear rates of substrate hydrolysis. Activity was reported as nanomole ACh hydrolyzed/min mg protein. Protein content was estimated by the method of Lowry et al. [28] using bovine serum albumin as the standard.
Fig. 3. Effects of PS (0, 0.3 or 1 MTD) on striatal cholinesterase activity in (A) neonatal (0, 0.63 and 2.1 mg/kg), (B) juvenile (0, 1.44 and 4.8 mg/ kg) and (C) adult (0, 5.4 and 18 mg/kg) rats. Rats were exposed to PS and sacrificed at 4, 24 and 96 h as described in Materials and methods. Activity is expressed as percent of contemporaneous control values. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
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3. Results Basal ACh synthesis in striatal synaptosomes from neonatal, juvenile and adult rats is shown in Fig. 1. A marked difference in basal transmitter synthesis was observed among the age groups. Adult rats exhibited markedly higher (about sixfold) synthesis in synaptosomes compared to neonates and about twofold higher synthesis compared to juveniles. For comparison, synaptosomal striatal ChE levels are shown in Fig. 1 inset. While
Fig. 5. Effects of PS on ACh synthesis in the striatum of (A) neonatal, (B) juvenile and (C) adult rats. Rats were treated with either vehicle, 0.3 MTD or 1 MTD of PS (neonatal = 0, 0.63 and 2.1; juvenile = 0, 1.44 and 4.8 and adult = 0, 5.4 and 18 mg/kg) and sacrificed at 4, 24 and 96 h. ACh synthesis was measured as described in Materials and methods. Combined control values for ACh synthesis are shown in Fig. 1. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
Fig. 4. Effects of CPF (0, 0.3 or 1 MTD) on striatal cholinesterase activity in (A) neonatal (0, 13.5 and 45 mg/kg), (B) juvenile (0, 38 and 127 mg/kg) and (C) adult (0, 84 and 279 mg/kg) rats. Rats were exposed to CPF and sacrificed at 4, 24 and 96 h as described in Materials and methods. Activity is expressed as percent of contemporaneous control values. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
markedly higher ChE levels were apparent in both juvenile and adult compared to neonatal tissues, no statistical difference in ChE activity was noted between juveniles and adults. Few overt signs of cholinergic toxicity were noted in any of the treatment groups (data not shown). In contrast, Fig. 2 shows that all three age groups exhibited significant changes in body weight following CPF MTD exposures. CPF affected body weight more in neonates and juveniles than in adult rats. At the respective MTD
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exposures, neonates were particularly sensitive to changes in body weight, with a 20% reduction 2 days after treatment while untreated controls gained about 30% (Fig. 2A). In juveniles, relatively similar reductions (7 – 8%) were noted 24 h after either 0.3 MTD or 1 MTD exposures (Fig. 2B) concurrent with a 10% increase in controls. In adults, the MTD slightly reduced (2%) weight while controls exhibited a 10% increase by 96 h after treatment.
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Age-related changes in ChE activity following PS or CPF exposure are shown in Figs. 3 and 4. Peak ChE inhibition by MTD dosages of PS and CPF was relatively similar (85 – 95% inhibition) among the age groups. In contrast, time to peak reduction in activity varied among the age groups, i.e., maximal reduction in ChE activity occurred earlier in neonates compared to juveniles and adult rats. In neonates, peak ChE inhibition occurred at 24 h after treatment with either PS (Fig. 3A) or CPF (Fig. 4A). In juveniles (Figs. 3B and 4B) and adults (Figs. 3C and 4C), maximal ChE inhibition was noted 96 h after PS and CPF dosing. Effects of PS on ACh synthesis are shown in Fig. 5. PS altered ACh synthesis in all three age groups. In neonatal tissues, PS (MTD) reduced ACh synthesis (25%) at 24 h after dosing (Fig. 5A). In contrast, PS increased ACh synthesis in both juvenile and adult tissues. In juveniles, an increase was observed at 4 (20%) and 24 h (33%) following PS (MTD, Fig. 5B). In adult rats, at 4 h following PS, ACh synthesis was increased by 13% and 20% with 0.3 MTD and 1 MTD exposures, respectively; whereas at 24 h after treatment, a significant increase (23%) was observed only with the higher dosage (Fig. 5C). Effects of CPF on ACh synthesis are shown in Fig. 6. In general, CPF had lesser effects on ACh synthesis. ACh synthesis was not altered at any time point or with either dosage in neonatal tissues (Fig. 6A). As noted with PS, CPF increased synthesis in juveniles 24 h after dosing (Fig. 6B). In contrast, CPF decreased ACh synthesis in adult rats at 4 h after dosing but only in the low dose group (0.3 MTD, Fig. 6C).
4. Discussion
Fig. 6. Effects of CPF on ACh synthesis in the striatum of (A) neonatal, (B) juvenile and (C) adult rats. Rats were treated with either vehicle or 0.3 MTD and 1 MTD of CPF (neonatal = 13.5 and 45; juvenile = 38 and 127; and adult = 84 and 279 mg/kg bodyweight) and sacrificed at 4, 24 and 96 h. ACh synthesis was measured as described in Materials and methods. Combined control values for ACh synthesis are shown in Fig. 1. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
OP insecticides elicit toxicity through inhibition of AChE. A number of OP toxicants have been reported to have additional molecular actions; however, that could potentially influence the expression of cholinergic toxicity [8,18,45]. For example, if some OP agents affect the synthesis or release of ACh [22,26,31], expression of cholinergic toxicity following AChE inhibition could be modified. In addition, marked age-related differences in sensitivity to many of these OP insecticides have been reported [2,6,35]. We evaluated possible age-related and OP-selective actions of PS and CPF, two OP insecticides that exhibit marked age-related differences in toxicity, on ACh synthesis in rat striatum. Basal ACh synthesis in striatal synaptosomes increased markedly during maturation (Fig. 1). Striatal ChE activity was also lowest in neonatal tissues, but no significant differences were noted in ChE activity between adults and juveniles. Relatively similar increases in cholinergic neurochemical markers in the brain during postnatal maturation have been previously reported [9,11,32,34].
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Relatively similar reductions in ChE activity were noted following MTD dosages of either OP insecticide, but the time to peak reduction in ChE activity varied among the age groups. These findings regarding age-related differences in time to peak inhibition following OP insecticide exposure are in general agreement with our earlier studies [35,48,50]. In general, CPF had little effect on striatal ACh synthesis. No changes in ACh synthesis were noted in neonatal tissues at any time point following either dosage of CPF. MTD exposure was associated with an increase in synthesis in juvenile tissues at 24 h after dosing, and a decrease in synthesis was noted in adult striatum following the respective 0.3 MTD dosage. PS elicited more consistent changes in ACh synthesis. ACh synthesis was significantly decreased in neonates at 24 h after dosing. In contrast, there was a significant increase at early time points (4 and 24 h) in tissues from both juvenile and adult rats. Changes in ACh synthesis did not generally coincide with peak reduction in ChE activity. For example, ACh synthesis was increased (20%; Fig. 5B) 4 h after PS exposure in juvenile rats while ChE activity was only moderately inhibited (52%; Fig. 3B). Similarly in adult rats, the low dosage of PS increased ACh synthesis at 4 h (13%; Fig. 5C) while ChE activity was not significantly reduced at this time point (Fig. 3C). These results suggest that changes in ACh synthesis following OP insecticide exposure are not directly coupled to ChE inhibition. It therefore appeared that while CPF and PS elicited relatively similar reductions in striatal ChE activity, ACh synthesis was affected in an OPselective and age-related manner. Exposure to either CPF or PS elicited few overt signs of cholinergic toxicity. These results are somewhat different from previous studies in our laboratory [7,33,36] indicating that subcutaneous MTD exposure to CPF or PS causes substantial body weight reduction in adult male rats, and that MTD PS exposure typically elicits a relatively high incidence of toxicity. The reasons for the relative lack of toxicity in adults treated with the MTDs in the present study are unclear. While lesser in magnitude than noted in our previous studies with adult rats, significant body weight reductions were noted in all three age groups following MTD exposures. In particular, neonatal rats treated with the MTD of CPF exhibited marked reductions in body weight gain compared to controls (Fig. 2A). Our studies were designed to evaluate possible age-related differences in ACh synthesis following ‘‘equitoxic’’ exposures. While the age-specific MTDs for both CPF and PS could reasonably be considered equitoxic when defined on lethality, the substantial differences in body weight reduction noted among the three age groups following CPF exposure argue against an equitoxic comparison across ages with this pesticide. It should be noted, however, that ACh synthesis was relatively unchanged in neonatal rat striatum following CPF dosing even though body weight reduction was most extensive in this treatment group.
The mechanisms underlying changes in ACh synthesis following either PS or CPF exposure are unclear. There are two critical steps in the synthesis of ACh: choline uptake into the presynpatic terminal and catalytic acetylation of choline. HACU is generally considered the rate-limiting step in ACh synthesis (for a review see, Ref. [27]). Differential modulation of HACU could therefore lead to selective alterations in ACh synthesis. Earlier studies from our laboratory using adult female rats demonstrated that both PS and CPF in vivo could reduce HACU in striatum [25]. Considering these previous findings, it is of interest to note that we observed increases in ACh synthesis in juvenile and adult striatum following PS exposure (Fig. 5). This could potentially be due to a number of experimental differences between the studies but could also suggest that HACU may not always reflect transmitter synthesis. Several other OP toxicants including sarin, soman and DFP have been reported to affect HACU in brain [20,23,47]. However, no studies have evaluated age-related changes in HACU following OP anticholinesterase exposures. Interestingly, Kotas and Prince [21] reported that HACU was not exclusively located in cholinergic neurons during early postnatal development in rat brain (4– 8 days of age) but became more cholinergic selective with maturation. Furthermore, these investigators [21] reported that the regulation of HACU was different during maturation, i.e., stimulation of choline uptake by depolarization increased dramatically in both frontal cortex and hippocampus from perinatal to adult periods. If activation of cholinergic neurons occurs early after PS exposure, the maturational expression of activitydependent regulation of HACU could contribute to the observed increase in ACh synthesis in juveniles and adults only. Choline is acetylated to ACh in the presynaptic terminal by the synthetic enzyme choline acetyltransferase (ChAT); thus, alteration of ChAT activity could potentially alter ACh synthesis [43]. High concentrations of PS have previously been reported to inhibit ChAT activity and ACh synthesis in vitro [31]. A recent report [19] indicated an early increase in ChAT activity following sarin exposure while soman inhibited ChAT activity in some brain regions [42]. Changes in ChAT activity were reported to have little effect on ACh synthesis in adult rat hippocampus [39], however, suggesting that changes in synthetic enzyme activity per se are not likely to contribute to OP-mediated alterations in ACh synthesis. ACh release in the mammalian brain is regulated presynaptically by muscarinic autoreceptors [14,26,46,49]. We previously reported that neonatal (7 days of age) rats have essentially no striatal muscarinic autoreceptor function, and therefore lack the ability to inhibit ACh release in a feedback manner [48]. This lack of presynaptic control in the very young animal’s nervous system could potentially contribute to age-related differences in ACh synthesis following OP insecticide exposure. A multitude of biochemical pathways utilize product inhibition as a control measure.
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Presynaptic autoreceptors in cholinergic neurons work analogously, i.e., ACh released after terminal depolarization is a product of presynaptic cell activation and it can act as a feedback signal to prevent further release. In addition to inhibiting ACh release, some evidence suggests that presynaptic muscarinic receptors may also be involved in inhibiting ACh synthesis. Cancela et al. [5] reported that cellular cAMP levels regulate HACU. Vogelsberg et al. [44] confirmed and extended these findings. A feedback mechanism to inhibit short-term (transmitter release) and long-term (transmitter synthesis) signaling would be a logical strategy for controlling overstimulation. As muscarinic M2 and M4 receptors, the putative subtypes of most muscarinic autoreceptors in the mammalian brain [49] are coupled to inhibition of adenylyl cyclase, maturational expression of these receptors may not only influence feedback inhibition of ACh release but may also allow feedback control of transmitter synthesis. Thus, the maturational expression of presynaptic muscarinic receptors could possibly contribute to age-related differences in ACh release and synthesis in response to cholinesterase inhibitors. Further studies are required to elucidate the contribution of changes in ACh synthesis to age-related and OP-selective differences in toxicity. The present findings suggest that an early increase in ACh synthesis following PS exposure in juvenile and adult rats could potentially lead to increased levels of ACh in the synapse and subsequent exacerbation of cholinergic toxicity. This may be an additional contributing factor in the differential age-related toxicity of selected OP toxicants.
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This work was partially supported by research grant R01 ES09119 from the National Institute of Environmental Health Sciences, NIH (C.N.P.) and by the Oklahoma State University Board of Reagents. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.
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S. Karanth, C. Pope / Neurotoxicology and Teratology 25 (2003) 599–606 rat striatum following exposure to chlorpyrifos or parathion, J. Toxicol. Environ. Health, Part A 53 (1998) 531 – 544. J. Liu, T. Chakraborti, C.N. Pope, In vitro effects of organophosphorus anticholinesterases on muscarinic receptor-mediated inhibition of acetylcholine release in rat striatum, Toxicol. Appl. Pharmacol. 178 (2002) 102 – 108. P.R. Lockman, D.D. Allen, The transport of choline, Drug Dev. Ind. Pharm. 28 (2002) 749 – 771. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. S.R. Mortensen, S.M. Chanda, M.J. Hooper, S. Padilla, Maturational differences in chlorpyrifos – oxonase activity may contribute to agerelated sensitivity to chlorpyrifos, J. Biochem. Toxicol. 11 (1996) 279 – 287. V.C. Moser, S.M. Chanda, S.R. Mortensen, S. Padilla, Age and gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities, Toxicol. Sci. 46 (1998) 211 – 222. M. Muramatsu, K. Kuriyama, Effect of organophosphorus compounds on acetylcholine synthesis in brain, Japanese J. Pharmacol. 26 (1976) 249 – 254. K. Olivier Jr., J. Liu, C. Pope, Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats, J. Biochem. Mol. Toxicol. 15 (2001) 263 – 269. K. Olivier Jr., J. Liu, S. Karanth, H. Zhang, D.S. Roane, C.N. Pope, Glucose feeding exacerbates parathion-induced neurotoxicity, J. Toxicol. Environ. Health, Part A 63 (2001) 253 – 271. F. Pedata, J. Slavikova, A. Kotas, G. Pepeu, Acetylcholine release from rat cortical slices during postnatal development and aging, Neurobiol. Aging 4 (1983) 31 – 35. C.N. Pope, T.K. Chakraborti, M.L. Chapman, J.D. Farrar, D. Arthun, Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides, Toxicology 68 (1991) 51 – 61. C.N. Pope, J. Chaudhuri, T.K. Chakraborti, Organophosphate-sensitive cholinergic receptors: possible role in modulation of anticholinesterase-induced toxicity, in: D.M. Quinn, A.S. Balasubramanian, B.P. Doctor, P. Taylor (Eds.), Enzymes of the Cholinesterase Family, Plenum, New York, 1995, pp. 305 – 312.
[37] C.N. Pope, Organophosphorus pesticides: do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health, Part B 2 (1999) 101 – 121. [38] SAS, JMP User’s Guide, Ver. 3, SAS Institute, Carey, NC, 1995. [39] B.M. Schmidt, R.J. Rylett, Basal synthesis of acetylcholine in hippocampal synaptosomes is not dependent upon membrane-bound choline acetyltransferase activity, Neuroscience 54 (1993) 649 – 656. [40] T.A. Slotkin, Developmental cholinotoxicants: nicotine and chlorpyrifos, Environ. Health Perspect. 107 (Suppl. 1) (1999) 71 – 80. [41] S. Song, F.J. Seidler, J.L. Saleh, J. Zhang, S. Padilla, T.A. Slotkin, Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase signaling cascade, Toxicol. Appl. Pharmacol. 145 (1997) 158 – 174. [42] T.L. Thompson, W.E. Thomas, Organophosphate-mediated inhibition of choline acetyltransferase activity in rat brain tissue, Experientia 41 (1985) 1437 – 1438. [43] S. Tucek, Short-term control of the synthesis of acetylcholine, Prog. Biophys. Mol. Biol. 60 (1993) 59 – 69. [44] V. Vogelsberg, N.H. Neff, M. Hadijiconstantinou, Cyclic AMP-mediated enhancement of high affinity choline transport and acetylcholine synthesis in brain, J. Neurochem. 68 (1997) 1062 – 1070. [45] T.R. Ward, W.R. Mundy, Organophosphorus compounds preferentially affect second messenger systems coupled to M2/M4 receptors in rat frontal cortex, Brain Res. Bull. 39 (1996) 49 – 55. [46] M.H. Weiler, Muscarinic modulation of endogenous acetylcholine release in rat neostriatal slices, J. Pharmacol. Exp. Ther. 250 (1989) 617 – 623. [47] C.E. Whaley, T.-M. Smith, Effects of soman and sarin on high affinity choline uptake by rat brain synaptosomes, Brain Res. Bull. 22 (1989) 853 – 858. [48] Y.K. Won, J. Liu, K. Olivier, Q. Zheng, C.N. Pope, Age-related effects of chlorpyrifos on acetylcholine release in rat brain, Neurotoxicology 22 (2001) 39 – 48. [49] W. Zhang, M. Yamada, J. Gomeza, A.S. Basile, J. Wess, Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1 – M5 muscarinic receptor knockout mice, J. Neurosci. 22 (2002) 6347 – 6352. [50] Q. Zheng, K. Olivier, Y.K. Won, C.N. Pope, Comparative cholinergic neurotoxicity of oral chlorpyrifos exposures in preweanling and adult rats, Toxicol. Sci. 55 (2000) 124 – 132.
Age-related effects of chlorpyrifos and parathion on acetylcholine synthesis in rat striatum Subramanya Karanth*, Carey Pope Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078, USA Received 17 December 2002; received in revised form 28 February 2003; accepted 19 March 2003
Abstract We compared the in vivo effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and parathion (PS) on acetylcholine (ACh) synthesis in neonatal, juvenile and adult rats. Basal levels of ACh synthesis were highest in adult rats, intermediate in juveniles and lowest in neonates. Following high (maximum tolerated dosage) subcutaneous exposure to either insecticide, relatively similar degrees of cholinesterase inhibition were noted, but the time to peak reduction varied among the age groups. CPF had no effect on ACh synthesis in neonates, increased synthesis in juveniles and decreased synthesis in adults, but only in the low dose group. PS had more consistent effects on ACh synthesis, decreasing transmitter synthesis in neonates (24 h after dosing) but increasing synthesis in juveniles and adults at both 4 and 24 h after exposure. Selective changes in neurotransmitter synthesis may contribute to differential age-related toxicity of these agents. D 2003 Elsevier Inc. All rights reserved. Keywords: Organophosphorus; Acetylcholine; Acetylcholinesterase; Age related; Neurotoxicity
1. Introduction Organophosphorus (OP) pesticides are widely used all over the world in the control of agricultural and household pests [1]. OP pesticides exhibit a wide range of toxicity in mammals but typically elicit their adverse effects through inhibition of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) in synapses of the central and peripheral nervous systems (for a review, see Ref. [37]). AChE inhibition leads to accumulation of the neurotransmitter acetylcholine (ACh), resulting in overstimulation of postsynaptic cholinergic receptors and consequent signs of neurotoxicity [10,12]. Immature animals are generally more sensitive than adults to the acute toxicity of OP insecticides including parathion (PS) and chlorpyrifos (CPF) [3,4,35]. Lesser detoxification has often been reported to contribute to higher sensitivity of young animals to OP insecticide toxicity [2,3,29,30]. For example, several studies have reported a relationship between the relative toxicity of PS * Corresponding author. Tel.: +1-405-744-4868; fax: +1-405-7448263. E-mail address: [email protected] (S. Karanth). 0892-0362/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0892-0362(03)00049-7
and CPF and the maturational expression of detoxification processes [2,3,17,30]. Toxicodynamic factors may also contribute to age-related sensitivity to OP toxicants. Some OP anticholinesterases are known to interact with other macromolecular targets including neurotransmitter receptors and components of second messenger signaling pathways [6 –8,15,18,32,40,41,45]. Thus, both toxicokinetic and toxicodynamic processes may contribute to age-related sensitivity to OP insecticides. Earlier reports indicated that several OP toxicants can alter high affinity choline uptake (HACU), the rate-limiting step in ACh synthesis [20,24,25]. HACU on the other hand has been reported to be underdeveloped in immature rat brain, with both the coupling between uptake and ACh synthesis and the degree of activity-dependent regulation changing during maturation [21]. Immature animals also lack presynaptic autoreceptors that can inhibit neurotransmitter release in a feedback manner, possibly influencing the toxic response to extensive AChE inhibition [48]. Interaction of OP anticholinesterases with such presynaptic cholinergic processes and possible modulation of ACh synthesis may therefore be an important contributing factor in the expression of cholinergic toxicity. However, the age-related effects of OP agents on ACh synthesis have not been evaluated.
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In the present study, we investigated the in vivo effects of the commonly used OP pesticide, CPF, and the prototype OP pesticide, PS, on cholinesterase activity (i.e., activity from both AChE and butyrylcholinesterase) and ACh synthesis in neonatal (7 days), juvenile (21 days) and adult (90 days) rats. The results suggest that these OP insecticides may differentially affect ACh synthesis in an age-related manner, potentially contributing to selective differences in toxicity.
2. Materials and methods 2.1. Chemicals CPF (O,O0-diethyl-3,5,6-trichloro-2-pyridinyl-phosphorothioate, 99.5% pure; GC/MS analysis) and PS (O,O0diethyl-p-nitrophenyl-phosphorothioate, 99.5% pure; GC/ MS analysis) were obtained from Chem Service (West Chester, PA). ACh iodide (acetyl-3H, specific activity 82.0 Ci/mmol) and choline chloride (methyl-3H, specific activity 75 Ci/mmol) were purchased from Dupont New England Nuclear (Boston, MA). All other chemicals were purchased from Sigma (St. Louis, MO). 2.2. Animals and treatment Sprague –Dawley rats (7- and 21-day-old of both sexes, and 3-month-old males) were used throughout the experiment. Animals were housed in steel mesh cages with a 12:12-h light–dark illumination cycle and were allowed free access to food (Harlan Tek-Lab diet) and water. All procedures involving animals were in accordance with the protocols established in the NIH/NRC Guide for the Care and Use of Laboratory Animals and approved by the local Institutional Animal Care and Use Committee. OP compounds were dissolved in peanut oil (100% purity; Lou Ana brand, Ventura Foods, Opelousas, LA) and injected subcutaneously (sc) at a volume of 1 ml/kg (except in the case of CPF in adult rats where in an injection volume of 2 ml/kg was used). Maximum tolerated dosages (MTDs; i.e., highest dosages of OP agents which produced no cumulative lethality over a 7-day period) were reported elsewhere [17,35]. MTDs for PS were 2.1, 4.8 and 18 mg/kg and 45, 127 and 279 mg/kg for CPF in neonatal, juvenile and adult rats, respectively. Animals (n = 6/treatment group) were challenged with either vehicle, 0.3 MTD or 1.0 MTD of either OP insecticide and sacrificed at 4, 24 and 96 h after treatment for measurement of neurochemical changes. Body weight and functional signs of toxicity were recorded as described earlier [50]. 2.3. Tissue preparation and biochemical assays Crude synaptosomes were used to measure ACh synthesis essentially by the method of Vogelsberg et al. [44].
Rats were sacrificed and the striatum was dissected rapidly on ice according to the method of Glowinski and Iversen [13]. Tissues were homogenized (1:20 wt/vol) with a PotterElvehjem homogenizer in ice-cold sucrose solution (0.32 M). The homogenates were centrifuged at 1000 g for 10 min in a Beckman J-20I high-speed refrigerated centrifuge and the supernatant was further centrifuged at 17,000 g for 20 min. The resulting pellet was resuspended in ice-cold sucrose solution (original volume) just prior to assay. Aliquots of synaptosomes (about 250– 350 mg protein) were incubated at 0 or 37 C with tritiated choline (2.5 mM, about 300,000 cpm/reaction) for 10 min in oxygenated Krebs bicarbonate buffer (pH 7.4). Physostigmine (50 mM) was included to prevent hydrolysis of ACh. The reactions were terminated by addition of 2-ml ice-cold Krebs bicarbonate buffer and centrifugation for 10 min at 17,000 g. The resulting pellets were resuspended in 130 mL of glycyl – glycine buffer (400 mM, pH 8.3) containing 20 mM MgCl2 and 20 mM ATP. Ten microliters of choline kinase (5 units/ ml) was added and tubes were vortexed and incubated at 37 C for 45 min. The reaction mixtures were transferred to 1.5-ml microcentrifuge tubes; 400 ml of tetraphenyl boron solution in 3-heptanone (15 mg/ml) was added and the tubes were vortexed and centrifuged for 5 min. Aliquots of the organic layer were carefully transferred to scintillation vials for counting using a biodegradable scintillation fluid (‘‘Scintisafe’’ 30%, Fischer Scientific, Pittsburgh, PA). ACh synthesis was reported as cpm/min mg protein. Cholinesterase activity (i.e., total activity from both AChE and butyrylcholinesterase) was measured by the radiometric method of Johnson and Russell [16] using [3H]acetylcholine iodide as the substrate (1 mM final concentration). Preliminary experiments determined assay conditions with incubation time and tissue concentration
Fig. 1. ACh synthesis and cholinesterase activity (inset) in striatum of neonatal, juvenile and adult rats. ACh synthesis was measured in synaptosomes from the conversion of [3H]choline to [3H]acetylcholine and expressed as cpm [3H]acetylcholine formed/min mg protein. ChE activity was measured using [3H]acetylcholine iodide as substrate and expressed as nanomoles ACh hydrolyzed/min mg protein as described in Materials and methods. Data represent mean ± S.E.M., n = 24 – 36/group. Asterisks indicate significant difference ( P < .05) compared to adult rats.
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2.4. Data analysis The JMP package [38] was used for statistical analyses. ACh synthesis and ChE activity data (percent transformations) were tested for statistical significance by a one-way ANOVA, and the Tukey – Kramer analysis was used for post hoc comparisons between the groups. Body weight data were analyzed by repeated measures ANOVA followed by linear contrasts. In all cases, P < .05 was considered significant.
Fig. 2. Effects of 0 MTD, 0.3 MTD or 1 MTD doses PS and CPF on mean body weight in (A) neonatal (PS = 0.63 and 2.1; CPF = 13.5 and 45 mg/kg bodyweight), (B) juvenile (PS = 1.44 and 4.8; CPF = 38.1 and 127 mg/kg bodyweight) and (C) adult (PS = 5.4 and 18; CPF = 83.7 and 279 mg/ kg bodyweight) rats (5 Control; ! PS 0.3 MTD; & PS 1 MTD; 4 CPF 0.3 MTD; . CPF 1 MTD). Data represent mean ± S.E.M., n = 4/ group. Asterisks indicate significant differences ( P < .05) between contemporaneous control and the following groups— (A) neonates: CPF, 1 MTD, (B) juveniles: CPF, 1 MTD and (C) adults: CPF, 1 MTD. Mean body weight on Day 0 was 14.5 ± 0.5; 43 ± 1.2 and 274 ± 2.4 g in neonatal, juvenile and adult rats, respectively.
to produce linear rates of substrate hydrolysis. Activity was reported as nanomole ACh hydrolyzed/min mg protein. Protein content was estimated by the method of Lowry et al. [28] using bovine serum albumin as the standard.
Fig. 3. Effects of PS (0, 0.3 or 1 MTD) on striatal cholinesterase activity in (A) neonatal (0, 0.63 and 2.1 mg/kg), (B) juvenile (0, 1.44 and 4.8 mg/ kg) and (C) adult (0, 5.4 and 18 mg/kg) rats. Rats were exposed to PS and sacrificed at 4, 24 and 96 h as described in Materials and methods. Activity is expressed as percent of contemporaneous control values. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
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3. Results Basal ACh synthesis in striatal synaptosomes from neonatal, juvenile and adult rats is shown in Fig. 1. A marked difference in basal transmitter synthesis was observed among the age groups. Adult rats exhibited markedly higher (about sixfold) synthesis in synaptosomes compared to neonates and about twofold higher synthesis compared to juveniles. For comparison, synaptosomal striatal ChE levels are shown in Fig. 1 inset. While
Fig. 5. Effects of PS on ACh synthesis in the striatum of (A) neonatal, (B) juvenile and (C) adult rats. Rats were treated with either vehicle, 0.3 MTD or 1 MTD of PS (neonatal = 0, 0.63 and 2.1; juvenile = 0, 1.44 and 4.8 and adult = 0, 5.4 and 18 mg/kg) and sacrificed at 4, 24 and 96 h. ACh synthesis was measured as described in Materials and methods. Combined control values for ACh synthesis are shown in Fig. 1. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
Fig. 4. Effects of CPF (0, 0.3 or 1 MTD) on striatal cholinesterase activity in (A) neonatal (0, 13.5 and 45 mg/kg), (B) juvenile (0, 38 and 127 mg/kg) and (C) adult (0, 84 and 279 mg/kg) rats. Rats were exposed to CPF and sacrificed at 4, 24 and 96 h as described in Materials and methods. Activity is expressed as percent of contemporaneous control values. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
markedly higher ChE levels were apparent in both juvenile and adult compared to neonatal tissues, no statistical difference in ChE activity was noted between juveniles and adults. Few overt signs of cholinergic toxicity were noted in any of the treatment groups (data not shown). In contrast, Fig. 2 shows that all three age groups exhibited significant changes in body weight following CPF MTD exposures. CPF affected body weight more in neonates and juveniles than in adult rats. At the respective MTD
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exposures, neonates were particularly sensitive to changes in body weight, with a 20% reduction 2 days after treatment while untreated controls gained about 30% (Fig. 2A). In juveniles, relatively similar reductions (7 – 8%) were noted 24 h after either 0.3 MTD or 1 MTD exposures (Fig. 2B) concurrent with a 10% increase in controls. In adults, the MTD slightly reduced (2%) weight while controls exhibited a 10% increase by 96 h after treatment.
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Age-related changes in ChE activity following PS or CPF exposure are shown in Figs. 3 and 4. Peak ChE inhibition by MTD dosages of PS and CPF was relatively similar (85 – 95% inhibition) among the age groups. In contrast, time to peak reduction in activity varied among the age groups, i.e., maximal reduction in ChE activity occurred earlier in neonates compared to juveniles and adult rats. In neonates, peak ChE inhibition occurred at 24 h after treatment with either PS (Fig. 3A) or CPF (Fig. 4A). In juveniles (Figs. 3B and 4B) and adults (Figs. 3C and 4C), maximal ChE inhibition was noted 96 h after PS and CPF dosing. Effects of PS on ACh synthesis are shown in Fig. 5. PS altered ACh synthesis in all three age groups. In neonatal tissues, PS (MTD) reduced ACh synthesis (25%) at 24 h after dosing (Fig. 5A). In contrast, PS increased ACh synthesis in both juvenile and adult tissues. In juveniles, an increase was observed at 4 (20%) and 24 h (33%) following PS (MTD, Fig. 5B). In adult rats, at 4 h following PS, ACh synthesis was increased by 13% and 20% with 0.3 MTD and 1 MTD exposures, respectively; whereas at 24 h after treatment, a significant increase (23%) was observed only with the higher dosage (Fig. 5C). Effects of CPF on ACh synthesis are shown in Fig. 6. In general, CPF had lesser effects on ACh synthesis. ACh synthesis was not altered at any time point or with either dosage in neonatal tissues (Fig. 6A). As noted with PS, CPF increased synthesis in juveniles 24 h after dosing (Fig. 6B). In contrast, CPF decreased ACh synthesis in adult rats at 4 h after dosing but only in the low dose group (0.3 MTD, Fig. 6C).
4. Discussion
Fig. 6. Effects of CPF on ACh synthesis in the striatum of (A) neonatal, (B) juvenile and (C) adult rats. Rats were treated with either vehicle or 0.3 MTD and 1 MTD of CPF (neonatal = 13.5 and 45; juvenile = 38 and 127; and adult = 84 and 279 mg/kg bodyweight) and sacrificed at 4, 24 and 96 h. ACh synthesis was measured as described in Materials and methods. Combined control values for ACh synthesis are shown in Fig. 1. Data represent mean ± S.E.M., n = 6/group/time point. Asterisks indicate significant difference ( P < .05) compared to controls.
OP insecticides elicit toxicity through inhibition of AChE. A number of OP toxicants have been reported to have additional molecular actions; however, that could potentially influence the expression of cholinergic toxicity [8,18,45]. For example, if some OP agents affect the synthesis or release of ACh [22,26,31], expression of cholinergic toxicity following AChE inhibition could be modified. In addition, marked age-related differences in sensitivity to many of these OP insecticides have been reported [2,6,35]. We evaluated possible age-related and OP-selective actions of PS and CPF, two OP insecticides that exhibit marked age-related differences in toxicity, on ACh synthesis in rat striatum. Basal ACh synthesis in striatal synaptosomes increased markedly during maturation (Fig. 1). Striatal ChE activity was also lowest in neonatal tissues, but no significant differences were noted in ChE activity between adults and juveniles. Relatively similar increases in cholinergic neurochemical markers in the brain during postnatal maturation have been previously reported [9,11,32,34].
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Relatively similar reductions in ChE activity were noted following MTD dosages of either OP insecticide, but the time to peak reduction in ChE activity varied among the age groups. These findings regarding age-related differences in time to peak inhibition following OP insecticide exposure are in general agreement with our earlier studies [35,48,50]. In general, CPF had little effect on striatal ACh synthesis. No changes in ACh synthesis were noted in neonatal tissues at any time point following either dosage of CPF. MTD exposure was associated with an increase in synthesis in juvenile tissues at 24 h after dosing, and a decrease in synthesis was noted in adult striatum following the respective 0.3 MTD dosage. PS elicited more consistent changes in ACh synthesis. ACh synthesis was significantly decreased in neonates at 24 h after dosing. In contrast, there was a significant increase at early time points (4 and 24 h) in tissues from both juvenile and adult rats. Changes in ACh synthesis did not generally coincide with peak reduction in ChE activity. For example, ACh synthesis was increased (20%; Fig. 5B) 4 h after PS exposure in juvenile rats while ChE activity was only moderately inhibited (52%; Fig. 3B). Similarly in adult rats, the low dosage of PS increased ACh synthesis at 4 h (13%; Fig. 5C) while ChE activity was not significantly reduced at this time point (Fig. 3C). These results suggest that changes in ACh synthesis following OP insecticide exposure are not directly coupled to ChE inhibition. It therefore appeared that while CPF and PS elicited relatively similar reductions in striatal ChE activity, ACh synthesis was affected in an OPselective and age-related manner. Exposure to either CPF or PS elicited few overt signs of cholinergic toxicity. These results are somewhat different from previous studies in our laboratory [7,33,36] indicating that subcutaneous MTD exposure to CPF or PS causes substantial body weight reduction in adult male rats, and that MTD PS exposure typically elicits a relatively high incidence of toxicity. The reasons for the relative lack of toxicity in adults treated with the MTDs in the present study are unclear. While lesser in magnitude than noted in our previous studies with adult rats, significant body weight reductions were noted in all three age groups following MTD exposures. In particular, neonatal rats treated with the MTD of CPF exhibited marked reductions in body weight gain compared to controls (Fig. 2A). Our studies were designed to evaluate possible age-related differences in ACh synthesis following ‘‘equitoxic’’ exposures. While the age-specific MTDs for both CPF and PS could reasonably be considered equitoxic when defined on lethality, the substantial differences in body weight reduction noted among the three age groups following CPF exposure argue against an equitoxic comparison across ages with this pesticide. It should be noted, however, that ACh synthesis was relatively unchanged in neonatal rat striatum following CPF dosing even though body weight reduction was most extensive in this treatment group.
The mechanisms underlying changes in ACh synthesis following either PS or CPF exposure are unclear. There are two critical steps in the synthesis of ACh: choline uptake into the presynpatic terminal and catalytic acetylation of choline. HACU is generally considered the rate-limiting step in ACh synthesis (for a review see, Ref. [27]). Differential modulation of HACU could therefore lead to selective alterations in ACh synthesis. Earlier studies from our laboratory using adult female rats demonstrated that both PS and CPF in vivo could reduce HACU in striatum [25]. Considering these previous findings, it is of interest to note that we observed increases in ACh synthesis in juvenile and adult striatum following PS exposure (Fig. 5). This could potentially be due to a number of experimental differences between the studies but could also suggest that HACU may not always reflect transmitter synthesis. Several other OP toxicants including sarin, soman and DFP have been reported to affect HACU in brain [20,23,47]. However, no studies have evaluated age-related changes in HACU following OP anticholinesterase exposures. Interestingly, Kotas and Prince [21] reported that HACU was not exclusively located in cholinergic neurons during early postnatal development in rat brain (4– 8 days of age) but became more cholinergic selective with maturation. Furthermore, these investigators [21] reported that the regulation of HACU was different during maturation, i.e., stimulation of choline uptake by depolarization increased dramatically in both frontal cortex and hippocampus from perinatal to adult periods. If activation of cholinergic neurons occurs early after PS exposure, the maturational expression of activitydependent regulation of HACU could contribute to the observed increase in ACh synthesis in juveniles and adults only. Choline is acetylated to ACh in the presynaptic terminal by the synthetic enzyme choline acetyltransferase (ChAT); thus, alteration of ChAT activity could potentially alter ACh synthesis [43]. High concentrations of PS have previously been reported to inhibit ChAT activity and ACh synthesis in vitro [31]. A recent report [19] indicated an early increase in ChAT activity following sarin exposure while soman inhibited ChAT activity in some brain regions [42]. Changes in ChAT activity were reported to have little effect on ACh synthesis in adult rat hippocampus [39], however, suggesting that changes in synthetic enzyme activity per se are not likely to contribute to OP-mediated alterations in ACh synthesis. ACh release in the mammalian brain is regulated presynaptically by muscarinic autoreceptors [14,26,46,49]. We previously reported that neonatal (7 days of age) rats have essentially no striatal muscarinic autoreceptor function, and therefore lack the ability to inhibit ACh release in a feedback manner [48]. This lack of presynaptic control in the very young animal’s nervous system could potentially contribute to age-related differences in ACh synthesis following OP insecticide exposure. A multitude of biochemical pathways utilize product inhibition as a control measure.
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Presynaptic autoreceptors in cholinergic neurons work analogously, i.e., ACh released after terminal depolarization is a product of presynaptic cell activation and it can act as a feedback signal to prevent further release. In addition to inhibiting ACh release, some evidence suggests that presynaptic muscarinic receptors may also be involved in inhibiting ACh synthesis. Cancela et al. [5] reported that cellular cAMP levels regulate HACU. Vogelsberg et al. [44] confirmed and extended these findings. A feedback mechanism to inhibit short-term (transmitter release) and long-term (transmitter synthesis) signaling would be a logical strategy for controlling overstimulation. As muscarinic M2 and M4 receptors, the putative subtypes of most muscarinic autoreceptors in the mammalian brain [49] are coupled to inhibition of adenylyl cyclase, maturational expression of these receptors may not only influence feedback inhibition of ACh release but may also allow feedback control of transmitter synthesis. Thus, the maturational expression of presynaptic muscarinic receptors could possibly contribute to age-related differences in ACh release and synthesis in response to cholinesterase inhibitors. Further studies are required to elucidate the contribution of changes in ACh synthesis to age-related and OP-selective differences in toxicity. The present findings suggest that an early increase in ACh synthesis following PS exposure in juvenile and adult rats could potentially lead to increased levels of ACh in the synapse and subsequent exacerbation of cholinergic toxicity. This may be an additional contributing factor in the differential age-related toxicity of selected OP toxicants.
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This work was partially supported by research grant R01 ES09119 from the National Institute of Environmental Health Sciences, NIH (C.N.P.) and by the Oklahoma State University Board of Reagents. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.
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