In Vitro Effects of Chlorpyrifos, Parathion, Methyl Parathion and Their Oxons on Cardiac Muscarinic Receptor Binding in Neonatal and Adult Rats

Toxicology 170 (2002) 1–10 www.elsevier.com/locate/toxicol

In vitro effects of chlorpyrifos, parathion, methyl parathion and their oxons on cardiac muscarinic receptor binding in neonatal and adult rats

Marcia D. Howard a, Carey N. Pope b,*

a Department of Toxicology, College of Pharmacy, The Uni6ersity of Louisiana at Monroe, Monroe, LA 71209, USA b Department of Physiological Sciences, College of Veterinary Medicine, 264 McElroy Hall, Oklahoma State Uni6ersity, Stillwater, OK 74078, USA

Received 7 June 2001; received in revised form 8 August 2001; accepted 15 August 2001

Abstract

Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular the m2 subtype, in tissues from adult rats. As both the density and agonist affinity states of cardiac muscarinic receptors (primarily m2) have been reported to change in an age-related manner, we evaluated the relative in vitro sensitivity of cardiac muscarinic receptors in tissues from neonatal (7–11 days of age) and adult (90 days of age) rats to selected organophosphorus compounds (chlorpyrifos, parathion, methyl parathion and their oxygen analogs or oxons). The effects of the cholinergic agonist carbachol (100 pM–5 mM) or an organophosphorus toxicant (50 pM–10 mM) on muscarinic receptor binding were determined using the 3 3 nonselective muscarinic ligand [ H]quinuclidinyl benzilate or the m2-preferential ligand [ H]oxotremorine-M acetate. Carbachol displaced [3H]oxotremorine labeling in adult and neonatal membranes in a relatively similar manner 3 (IC50 =7–20 nM). The oxons all displaced [ H]oxotremorine binding in a concentration-dependent manner, with chlorpyrifos oxon being the most potent (IC50: neonates, 15 nM; adults, 7 nM) and efficacious (maximum displacement: neonates, 42%; adults, 56%). Interestingly, methyl parathion was an extremely potent displacer of 3 [ H]oxotremorine binding in adult tissues (IC50 =0.5 nM, maximum displacement=37%) but had no effect in neonatal tissues. The displacement of [3H]oxotremorine binding by chlorpyrifos oxon (10 mM) was still apparent after washing the tissues, suggesting the oxon irreversibly blocked agonist binding to the receptor while interaction with MePS appeared reversible. As effective concentrations of the oxons were relatively similar to their anticholinesterase potencies, these findings suggest that direct interaction with cardiac muscarinic receptors by some organophosphorus agents may occur at relevant exposure levels and contribute to cardiac toxicity. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Organophosphates; Cardiac; Age-related; Insecticides; Mechanism; Acetylcholinesterase inhibition

* Corresponding author. Tel.: +1-405-744-6257; fax: +1-405-744-0462. E-mail address: [email protected] (C.N. Pope).

1. Introduction of muscle contraction. While m2 receptors are thought to be the primary subtype of muscarinic

Pesticides are unique contaminants in that they receptors in the heart, all subtypes (m1 –m5) have are intentionally released into the environment to been recently identiﬁed in human heart (Wang et elicit toxicity in certain ‘pest’ species. Unfortu- al., 2001). nately, a lack of selectivity often leads to prob- The OP insecticides examined in this study are lems of toxicity in humans and other non-target phosphorothioate compounds that must be con- species. Organophosphorus (OP) pesticides are verted by cytochrome P450 enzymes to their re- the major chemical class of insecticides used in the spective oxons to elicit cholinergic toxicity world today (Aspelin, 1994; Bardin et al, 1994). (Sultatos, 1994). While all OP insecticides are Coye et al. (1987) reported that OP insecticides thought to act through a common mechanism were the most frequently involved pesticides in initiated by inhibition of AChE, differential ex- non-occupational poisonings in the state of Cali- pression of toxicity following similar changes in fornia. Organophosphorus poisonings continue to acetylcholinesterase activity have been reported be a major cause of morbidity and mortality in (Chaudhuri et al., 1993; Pope et al., 1995; Liu and third world countries (Peter and Cherian, 2000). Pope, 1998). Differential effects in the presence of The primary mode of action for OP pesticides is similar changes in acetylcholinesterase activity initiated through inhibition of acetylcholinesterase could be the result of direct or indirect actions at (AChE, E.C. 3.1.1.7), the enzyme responsible for macromolecular targets other than AChE (Bakry degrading the neurotransmitter acetylcholine et al., 1988; Silveira et al., 1990; Jett et al., 1991; (Saunders and Harper, 1994; Brown and Taylor, Ward et al., 1993; Huff et al., 1994; Huff and 1996; Pope, 1999). Excessive acetylcholine (ACh) Abou-Donia, 1995; Ward and Mundy, 1996; accumulation due to enzyme inhibition results in Pope, 1999). Several studies suggest that some overstimulation of cholinergic receptors and signs OPs can bind directly to the m2 muscarinic recep- of cholinergic toxicity such as salivation, lacrima- tor and function as an agonist (Bakry et al., 1988; tion, urination and defecation (SLUD), bradycar- Silveira et al., 1990; Huff et al., 1994; Ward and dia, miosis, nausea, and respiratory dysfunction Mundy, 1996; Van Den Beukel et al., 1997). As

(Ecobiochon, 1996). Cholinergic receptors can be the heart expresses predominately m2 receptors, divided into nicotinic (nAChR) and muscarinic this organ system could be particularly sensitive (mAChR) subclasses. Five muscarinic receptor to such direct actions of some OP agents (Kellar subtypes, referred to as m1 –m5, have been iden- et al., 1985; Sharma et al., 1997). In contrast to tified through molecular cloning (Brown and Tay- brain, where muscarinic receptor density increases lor, 1996) and four muscarinic receptor subtypes during postnatal maturation, cardiac muscarinic have been pharmacologically identified using receptor density declines with maturation. In ad- [3H]ACh binding (Alberts et al., 1994). dition, age-related differences in the ability of Muscarinic receptors belong to the largest su- cardiac muscarinic receptors to convert from high perfamily of cell-surface receptors, i.e. those to low affinity states have been reported (Baker et linked to G proteins: m1,m3 and m5 subtypes are al. 1985; McMahon, 1989) generally coupled to stimulation of phospholipase Generally, young animals are more sensitive

Candm2 and m4 subtypes are linked to inhibition than adults to acute OP insecticide toxicity of adenylyl cyclase (Alberts et al., 1994; Brown (Brodeur and DuBois, 1963; Benke and Murphy, and Taylor, 1996). The G-protein coupled mus- 1975; Pope et al., 1991; Atterberry et al., 1997; carinic receptors contain seven hydrophobic seg- Zheng et al., 2000). The possibility of higher ments, 20–30 amino acids long, spanning the sensitivity in children to some pesticides in part phospholipid bilayer (Peralta et al., 1987; Bonner, led to the enactment of the US Food Quality 1989; Alberts et al., 1994). Muscarinic receptors in Protection Act (FQPA), requiring an additional the heart are intimately involved in cardiac func- safety factor in the risk assessment process and an tion, dynamically regulating force and frequency explicit determination that tolerances were safe M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10 3 for children (FQPA, 1996). Immature animals are tained on 12-h light:12-h dark cycle and were fed more sensitive to the acute toxicity of chlorpyrifos food and water ad libitum. (CPF), parathion (PS) and methyl parathion (MePS) (Benke and Murphy, 1975; Pope et al., 2.3. Tissue collection, membrane preparation and 1991; Moser et al., 1998). We evaluated possible biochemical assay age-related differences in direct interaction between CPF, PS, MePS and their oxons (CPFO, Following decapitation, hearts were quickly ex- PO and MePO, respectively) and cardiac mus- cised and rinsed in 10 mM Tris buffer, pH 7.4 carinic receptors. Our findings suggest that car- (25 °C) containing 1 mM EDTA (McMahon, diac muscarinic receptors may be particularly 1989). After removal of connective tissue and sensitive to low levels of some OP toxicants, and blood vessels, both atria and ventricles were that these direct interactions between receptors minced and frozen at −70 °C until time of assay. and OP agents could contribute to age-related Cardiac membranes were prepared according to and OP-selective toxicity. the method of Silveira et al. (1990). Briefly, hearts were rinsed in ice-cold saline, blotted dry and homogenized (1:15 in 5 mM Hepes, pH 7.4) four 2. Materials and methods times for 30 s (25 000 rpm, Polytron, Brinkmann Instruments, Westbury, NY), with a 1-min pause 2.1. Chemicals between homogenizations. Homogenized tissues were then centrifuged for 10 min at 1000×g All OP toxicants were purchased from Chem (4 °C) and the resulting supernatant was subse- Service (West Chester, PA; ]97% purity). quently centrifuged for 45 min at 40 000×g.The 3 [ H]oxotremorine-M acetate (specific activity P2 pellet was resuspended with 10 strokes using a 85.8–86.4 Ci/mmol; OXO), and [3H]quinuclidinyl Dounce homogenizer and membranes were used benzilate (specific activity 42.0–49.0 Ci/mmol, immediately for the binding assay. All steps were QNB) were purchased from New England Nu- performed on ice unless otherwise noted. clear (Boston, MA). All other chemicals were Muscarinic receptor binding was evaluated by reagent grade supplied by Sigma Chemical Com- the method of Silveira et al. (1990) with minor pany (St. Louis, MO). Carbachol was prepared in modifications. Hepes buffer (5 mM, pH 7.4) was Hepes buffer (5 mM final concentration, pH 7.4). used for all steps unless otherwise indicated. All OP toxicants were dissolved in 100% dry Membranes (300–350 mg protein per rxn) were ethanol (EtOH) for stock preparation and stored incubated for 90 min at 21 °C in the presence of at −70 °C until day of the assay. Ethanol con- 1nM[3H]OXO or 0.75 nM [3H]QNB. Carbachol centration in the assays was kept constant at (100 ml,100 pM–5 mM) or OP toxicant (10 ml, 50 0.1%. pM–10 mM) was added for a final reaction volume of 1.0 ml. Non-specific binding was deter- 2.2. Animals mined in the presence of atropine (10 mM), with specific binding calculated as difference between Neonatal (7–11 day, male and female) and paired samples in the presence and absence of adult (90 day, male only) Sprague–Dawley rats atropine. The reaction was terminated by vacuum were used throughout all experiments. Un-timed filtration with ice-cold buffer over Whatman GF/ pregnant females were purchased from Harlan B (Brandel Inc., Gaithersburg, MD) filter papers Sprague Dawley (Indianapolis, IN) and caged saturated with 0.05% polyethylenimine immedi- individually in plastic cages. Pups were random- ately prior to use (Van Den Beukel et al., 1997). ized and culled to 10 pups per dam on postnatal Filter disks were soaked overnight in 4.0 ml scin- day 2 (date of birth representing postnatal day 0). tillation cocktail (Scintisafe, FisherBrand, Dallas, Adults were housed individually in plastic cages TX) and counted at 60% efficiency (Wallac Model for 7 days prior to use. All animals were main- 1490, Perkin–Elmer, Gaithersburg, MD). Protein 4 M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10 content was determined by the Lowry method Table 1 3 3 using bovine serum albumin (BSA) as the stan- Saturation analyses for [ H]OXO and [ H]QNB binding in rat cardiac membranes dard (Lowry et al., 1951). OXO QNB 2.4. Re6ersibility of chlorpyrifos oxon and methyl parathion binding to cardiac muscarinic receptors Kd Bmax Kd Bmax 9 9 9 9 3 Adult163 65 46 4 93 8 98 2 To determine if [ H]OXO displacement by an Neonate150924 13194 118910 17494 OP toxicant was reversible, adult membranes were prepared as previously described. CPFO was se- Saturation analyses were conducted using various concentra- lected as it was most effective at displacing spe- tions of [3H]OXO and [3H]QNB in adult and neonatal cardiac cific[3H]OXO binding while MePS was chosen membranes (n=3/age). Membranes were co-incubated with 100 pM–5nM[3H]OXO or 10 pM–3nM[3H]QNB in the due to its age selectivity. Membranes were prein- presence and absence of atropine (10 mM) for 90 min at 21 °C. cubated in the presence of vehicle, CPFO or Values are the means of three separate experiments, Kd is m 9 9 MePS (10 M final concentration) at 21 °C for 30 expressed as pM S.E., Bmax represents fmol/mg protein min. Following pre-incubation, the tissues were S.E. centrifuged (40 000×g, 45 min) and pellets were resuspended in fresh buffer at the original volume. binding was 8990.4% for adults and 9390.5% This washing step was repeated and [3H]OXO for neonates. binding was subsequently evaluated as before. 3.2. Displacement of [ 3H]OXO binding by 2.5. Statistical analyses carbachol Saturation isotherms ([3H]OXO, 100 pM–5 Displacement of [3H]OXO by the cholinergic nM; [3H]QNB, 10 pM–3 nM) were analyzed us- agonist carbachol was evaluated in cardiac mem- ing the GraphPad Prism® software (Graphpad branes from both age groups. As shown in Fig. 1, Software, San Diego, CA, USA, carbachol was relatively similar in potency and www.graphpad.com) non-linear regression to de- efficacy for adult (IC =7; 95% confidence inter- termine K and B estimates. IC values were 50 d max 50 val, 4–12 nM) and neonate (IC =20; 95% confi- estimated by one-site competition using Graph- 50 dence interval, 9–44 nM) membranes. At high Pad Prism. Competition binding data were ana- concentrations, carbachol completely blocked spe- lyzed by the statistical program JMP® using ANOVA and linear contrasts where appropriate. Data from reversibility assays were analyzed by t-test. In all cases, PB0.05 was considered statis- tically significant.

3. Results

3.1. [ 3H]OXO and [ 3H]QNB binding in rat cardiac membranes Fig. 1. In vitro displacement of [3H]OXO binding by carbachol in adult and neonatal cardiac membranes. Membranes were Table 1 shows Kd and Bmax estimates for both 3 ligands in tissues from the two age groups. Spe- co-incubated with 1 nM [ H]OXO and carbachol at the concentrations noted. Assays were conducted in duplicate, with cific binding in adult and neonatal membranes and without atropine (10 mM) to determine non-specific bind- represented 8591 and 9590.3%, respectively of ing. Data represent mean (9S.E.) percent of control from total [3H]OXO binding while specific[3H]QNB three separate experiments. M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10 5

branes. CPFO was the most potent and efﬁca- cious of the oxons at displacing [3H]OXO binding

in both age groups (IC50 =15 nM for neonates and 7 nM for adults; maximum displacement= 56% for adult and 42% for neonatal tissues). Interestingly, MePS potently displaced [3H]OXO in adult membranes at concentrations ]0.5 nM (maximum displacement=37%). In contrast, MePS had no effect on [3H]OXO binding in neonatal tissues. No signiﬁcant effects were noted with any of the OP agents using [3H]QNB as the ligand.

Fig. 2. In vitro displacement of [3H]OXO and [3H]QNB by chlorpyrifos oxon, methyl paraoxon and paraoxon in adult and neonatal cardiac membranes. Membranes were prepared as described in Section 2 (n=3–12/group) and co-incubated with either 1 nM [3H]OXO (open square, neonate; closed square, adult) or 0.75 nM [3H]QNB (open triangle, neonate; closed triangle, adult) and chlorpyrifos oxon (A), methyl paraoxon (B) or paraoxon (C) in the concentrations indicated. Following incubation for 90 min at 21 °C, the reaction was terminated by vacuum filtration and washing of the filters with ice-cold buffer. Specific binding was determined by the inclu- sionof10mM atropine in paired tubes. Data represent percent of control (mean9S.E.). Asterisks represent values significantly different from adult control while pound signs represent values significantly different from neonatal control (PB0.05). cific[3H]OXO binding in tissues from both age Fig. 3. In vitro displacement of [3H]OXO and [3H]QNB by groups. chlorpyrifos, methyl parathion and parathion in adult and neonatal cardiac membranes. Membranes were prepared as described in Section 2 (n=4–13/group) and co-incubated with 3 3 3 either mM [ H]OXO (open square, neonate; closed square, 3.3. [ H]OXO and [ H]QNB displacement by OP adult) or 0.75 nM [3H]QNB (open triangle, neonate; closed toxicants triangle, adult) and chlorpyrifos (A), methyl parathion (B) or parathion (C) in the concentrations indicated. Following incu- Fig. 2 and Fig. 3 show displacement of radioli- bation for 90 min at 21 °C, the reaction was terminated by gand binding in cardiac membranes by the OP vacuum filtration and washing with ice-cold buffer as before. Specific binding was determined by the inclusion of 10 mM toxicants. In general, the oxons were more effec- atropine in paired tubes. Data represent percent of control tive than the parent compounds for radioligand (mean9S.E.). Asterisks represent values significantly different displacement both in adult and neonatal mem- from adult control (PB0.05). 6 M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10

addition to inhibiting acetylcholinesterase, a number of OP toxicants bind directly to muscarinic receptors, with relatively high potency towards

the muscarinic m2 subtype (Silveira et al., 1990; Ward et al., 1993; Ward and Mundy, 1996; Van Den Beukel et al., 1997). A direct interaction between OP toxicants and muscarinic receptors could potentially modulate the toxic response to acetylcholinesterase inhibition or lead to non- cholinergic effects. While it has been reported that AChE sensitivity to OP anticholinesterases in vitro does not change during maturation (Benke and Murphy, 1975; Atterberry et al., 1997; Mortensen et al., 1998), potential age-related differences in sensitivity to direct OP toxicant per receptor interactions have not been evaluated. We Fig. 4. In vitro pre-incubation of adult cardiac membranes with either chlorpyrifos oxon or methyl parathion, followed by therefore compared the in vitro effects of the [3H]OXO binding. Adult cardiac membranes were pre-incu- common organophosphorus insecticides chlor- bated in the presence of 10 mM CPFO (A) or 10 mMMePS(B) pyrifos, parathion, methyl parathion and their for 30 min at 21 °C, and washed by centrifugation (n=4/ oxygen analogs (oxons) on specific binding to the group) and re-suspension in fresh buffer. This washing proce- non-selective muscarinic antagonist dure was repeated and tissues were then incubated in the 3 presence of 1 nM [3H]OXO for 90 min at 21 °C. Paired [ H]quinuclidinyl benzilate or the M2-preferential 3 samples were pre-incubated with OP toxicant but membranes agonist [ H]oxotremorine-M acetate in cardiac were kept on ice until assay (i.e. they were not washed). membranes from neonatal (7–11 days of age) and Receptor binding was terminated by vacuum filtration. Spe- adult (90 days of age) rats. m cific binding was determined by the inclusion of 10 M Muscarinic receptor density (B ) was consid- atropine in paired tubes. Data represent percent of control max (mean9S.E.). Asterisks represent values significantly different erably higher (1.8–2.8 fold) in neonatal heart from respective controls (PB0.05). membranes compared with adults while the binding affinity constant (Kd) was relatively similar Fig. 4 shows the displacement of [3H]OXO between the age groups with both ligands (Table binding by CPFO (4A) and MePS (4B) as influ- 1). A 2-fold age-related decrease in muscarinic enced by washing of the membranes prior to receptor density during postnatal maturation has addition of the radiolabel. As seen in this figure, been previously reported (McMahon, 1989). Ne- CPFO still reduced specific[3H]OXO binding un- doma and Tucek (1986) found no age-related der these conditions. In contrast, no significant changes in affinity but a linear decrease in block of [3H]OXO binding was noted in mem- [3H]QNB binding density in both atrial and ven- branes pre-incubated with MePS and then washed tricular preparations from postnatal day 1 to 3 prior to assay. months of age. Narayanan and Derby (1983) reported a higher density of muscarinic receptors in the atria (but not ventricles) in aged rats (24 months) compared with younger (3–8 months of 4. Discussion age). It appears from these studies that cardiac muscarinic receptor density changes throughout Organophosphorus (OP) insecticides elicit toxi- life, and is generally higher in neonatal than in city through inhibition of acetylcholinesterase, adult heart. The non-selective cholinergic agonist leading to accumulation of acetylcholine in the carbachol displaced [3H]OXO binding in a potent nervous system and consequent signs of choliner- and highly effective manner in tissues from both gic toxicity (Mileson et al., 1998; Pope, 1999). In age groups (Fig. 1). Together, these data suggest M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10 7 that cardiac muscarinic receptor density changes (Mortensen et al., 1998; Atterberry et al., 1997), during maturation but that agonist binding direct interaction with cardiac muscarinic recep- affinity to the receptors is relatively similar be- tors could occur at toxicologically-relevant expo- tween the two age groups. sures. In the case of CPFO, this interaction CPFO, MePO and PO all significantly dis- appears irreversible. As these receptors are cou- placed [3H]OXO binding in adult tissues. Both pled to inhibition of formation of the second CPFO and MePO also blocked [3H]OXO binding messenger cAMP, and considering cAMP’s global in neonatal tissues, while no significant reduction role as an intracellular mediator of cell signaling, in OXO binding in neonatal tissues was noted covalent binding to these receptors could have with PO. Oxons covalently modify the active site long-lasting effects on cAMP levels within the region of acetylcholinesterase to induce long-term cardiac cells potentially inducing long-term inhibition of acetylcholinesterase and consequent changes in the regulation of cardiac function. cholinergic toxicity (Sultatos, 1994). However, The parent compounds generally had little ef- previous studies reporting direct interactions of fect on either [3H]OXO or [3H]QNB binding (Fig. OP pesticides with muscarinic receptors have been 3). MePS, on the other hand, was a potent dis- unclear regarding the nature of the interaction placer of [3H]OXO binding in adult tissues (Fig. (Silveira et al., 1990; Huff and Abou-Donia, 3B). To our knowledge, this is the first evidence 1994). Silveira et al. (1990) suggested that sarin indicating a direct interaction between MePS and and paraoxon both interact competitively and muscarinic receptors. Surprisingly, MePS had no reversibly with cardiac receptors labeled with the similar effect on [3H]OXO binding in neonatal muscarinic agonist [3H]cis-methyldioxolane. In tissues. When adult tissues were preincubated contrast, Huff and Abou-Donia (1994) reported with MePS and then washed by centrifugation that CPFO covalently binds to striatal muscarinic and resuspension, however, no inhibition of receptors, but apparently not at the agonist bind- [3H]oxotremorine binding was noted (Fig. 4B). In ing site. A recent report provided evidence that contrast to findings with CPFO, these data sug- radiolabeled CPFO could diethylphosphorylate gest that MePS reversibly blocks agonist binding. cardiac m2 receptors in vitro (Bomser and Casida, Age-related differences in the membrane mi- 2001). This study also noted that the presence of croenvironment could affect MePS binding. a high affinity agonist did not inhibit [3H]CPFO Yechiel et al. (1985) reported alterations in the labeling, suggesting that CPFO and agonists may membrane lipid composition with aging myocytes bind to different but coupled sites. The apparent in culture. These investigators reported elevated irreversible blockade of [3H]OXO binding in adult cholesterol levels as well as a decrease in phos- heart membranes following preincubation with phatidylcholine (PC) content in cells cultured CPFO (Fig. 4A) provides further support for longer times. Moscona-Amir et al. (1986) ob- covalent binding to muscarinic receptors by some served that in the absence of PC, the non-hy- OP pesticides. drolyzable guanine nucleotide analog, OP agents have been reported to interact with a 5%-guanylylimidodiphosphate (GppNHp) induced number of additional macromolecular targets (for a shift in aged cultures of cardiac myocytes (i.e. review, see Pope, 1999). An important consider- conversion of low affinity state to high affinity ation in the evaluation of relevance of non- state), while only low affinity sites were present in cholinesterase targets for OP compounds is the aged cultures treated with PC and GppNHp. concentration (or dosage) at which the interaction Berstein et al. (1989) showed that brain and atrial occurs. The potency of CPFO at displacing muscarinic receptors exhibited similar affinities [3H]OXO binding in both neonatal and adult for the ligand [3H]QNB, but receptors in brain cardiac membranes reported in our current stud- had a much higher affinity for the M1-selective ies was relatively low (IC50 =7–15 nM). As the in antagonist pirenzepine. Upon partial purification vitro potency of CPFO towards brain acetyl- of the receptors from both tissues, however, rela- cholinesterase is also in the low nanamolar range tively similar affinities for both ligands were 8 M.D. Howard, C.N. Pope / Toxicology 170 (2002) 1–10 noted. Higher affinity of the brain muscarinic Acknowledgements receptor for pirenzepine was again noted when the purified receptors were inserted back into either This work was partially supported by STAR brain or heart membranes (Berstein et al., 1989). research grant R 825811 with the US Environ- Together, these results suggest that membrane mental Protection Agency (Carey N. Pope), RO1 microenvironment may change with maturation ES09119 by National Institute of Environmental and that changes in the lipid environment around Health Sciences, NIH (Carey N. Pope), the Uni- the receptors could modulate OP toxicant/mus- versity of Louisiana System Board of Regents carinic receptor interactions. Support Fund and the Minority Doctoral Schol- It was previously reported that the relationship arship Program at the University of Louisiana of between acetylcholinesterase inhibition and mus- Monroe (M. Howard). The contents of this carinic receptor density in brain following re- manuscript are solely the responsibility of the peated MePS exposures was different between authors and do not necessarily represent the offi- neonatal and adult rats (Liu et al., 1999). While cial views of either the EPA or NIEHS. these studies evaluated muscarinic receptors in different tissues (heart vs. brain), a direct interaction between MePS and brain muscarinic receptors in adult tissues and not neonates could References conceivably contribute to age-related differences in modulation of receptor density with repeated Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Wat- son, J.D., 1994. Cell signaling. In: Robertson, M. (Ed.), exposures. The possible direct interaction between Molecular Biology of the Cell. Garland Publishing, New MePS and brain muscarinic receptors in different York, pp. 721–785. age groups should also be evaluated. Aspelin, A.L., 1994. Pesticide industry sales and usage: 1992 In summary, CPFO, MePO and PO signifi- and 1993 market estimates. Office of Pesticide Programs, cantly reduced muscarinic receptor binding to the US Environmental Protection Agency, EPA, Washington, 3 DC, 733-K-94-001. radioligand [ H]oxotremorine-M acetate in a con- Atterberry, T.T., Burnett, W.T., Chambers, J.E., 1997. Age-re- centration-dependent manner in cardiac mem- lated differences in parathion and chlorpyrifos toxicity in branes from both neonatal (CPFO and MePO) male rats: target and nontarget esterase sensitivity and and adult (CPFO, MePO and PO) rats. Of the cytochrome P450-mediated metabolism. Toxicol. Appl. parent insecticides, only MePS affected [3H]OXO Pharmacol. 147, 411–418. Baker, S.P., Marchand, S., O’neil, E., Nelson, C.A., Posner, binding, and only in tissues from adults. All three P., 1985. Age-related changes in cardiac muscarinic recep- oxons exhibited high potency at displacing tors: decreased ability of the receptor to form a high [3H]OXO binding in adult tissues, and MePS was affinity agonist binding state. J. Gerontol. 40, 141–146. even more potent than the oxons. The interaction Bakry, N.M.S., El-Rashidy, A.H., Eldefrawi, A.T., Eldefrawi, between CPFO and the muscarinic receptor ap- M.E., 1988. Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine peared irreversible, providing further support that receptors. J. Biochem. Toxicol. 3, 235–259. some OP insecticides can covalently modify the Bardin, P.G., van Eeden, S.F., Moolman, J.A., Foden, A.P., muscarinic M2 receptor. In contrast, MePS ap- Joubert, J.R., 1994. Organophosphate and carbamate poi- pears to reversibly block agonist binding. The soning. Arch. Intern. Med. 154, 1433–1441. effective concentrations of these agents suggest Benke, G.M., Murphy, S.D., 1975. The influence of age on the toxicity and metabolism of methyl parathion and that these OP toxicant/muscarinic receptor inter- parathion in male and female rats. Toxicol. Appl. 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