Selective Effects of Carbamate Pesticides on Rat Neuronal Nicotinic Acetylcholine Receptors and Rat Brain Acetylcholinesterase Chantal J.G.M
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Available online at www.sciencedirect.com R Toxicology and Applied Pharmacology 193 (2003) 139–146 www.elsevier.com/locate/taap Selective effects of carbamate pesticides on rat neuronal nicotinic acetylcholine receptors and rat brain acetylcholinesterase Chantal J.G.M. Smulders,a Tjerk J.H. Bueters,b Regina G.D.M. Van Kleef,a and Henk P.M. Vijverberga,* a Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands b TNO Prins Maurits Laboratory, P.O. Box 45, NL-2280 AA Rijswijk, The Netherlands Received 14 May 2003; accepted 31 July 2003 Abstract Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit ␣44 nicotinic receptors is fenoxycarb Ͼ EPTC Ͼ carbaryl, bendiocarb Ͼ propoxur Ͼ aldicarb with IC50 values ranging from 3 M for fenoxycarb to 165 M for propoxur and Ͼ1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb Ͼ propoxur, aldicarb Ͼ carbaryl ϾϾ EPTC, fenoxycarb with IC50 values ranging from 1 M for bendiocarb to 17 M for carbaryl and ϾϾ1 mM for EPTC and fenoxycarb. The ␣42, ␣34, and ␣32 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for ␣44 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the ␣32 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of ␣ and  subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure. © 2003 Elsevier Inc. All rights reserved. Keywords: Carbamate pesticides; Neuronal nicotinic acetylcholine receptor; Rat brain acetylcholinesterase; Xenopus oocytes; Two-microelectrode voltage clamp Introduction are generally caused by inhibition of the enzyme acetylcho- linesterase (AChE, EC 3.1.1.7), which leads to accumula- Carbamates, all derived from the basic structure of car- tion of acetylcholine (ACh) (for review see Ecobichon, bamic acid, represent a broad variety of compounds, which 2001). Studies on chronic exposure to carbamate insecti- have a number of applications. They are widely applied as cides and case reports of long-term exposure give equivocal insecticides, herbicides, and fungicides. Many of these results. Some show no effects after chronic exposure chemicals are potential neurotoxicants, particularly follow- (Risher et al., 1987), whereas others describe memory im- ing occupational, accidental, or intentional exposure. Acute pairment (De´si et al., 1974; Ruppert et al., 1983; Ecobichon, toxic symptoms of carbamate poisoning, e.g., miosis, uri- 2001), degenerative polyneuropathy (Umehara et al., 1991), nation, diarrhea, diaphoresis, lacrimation, salivation, and neurobehavioral effects (Moser, 1999), and other neurolog- excitation of the central nervous system (O’Malley, 1997), ical disorders (De´si et al., 1974; Dickoff et al., 1987). An extensive survey of the toxicology of the common insecti- cide carbaryl reports a variety of reversible neurobehavioral * Corresponding author. Fax:ϩ31-30-2535077. and neurotoxic effects in vertebrates, all associated with E-mail address: [email protected] (H.P.M. Vijverberg). acute poisoning symptoms (Cranmer, 1986). Overall, it ap- 0041-008X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2003.07.011 140 C.J.G.M. Smulders et al. / Toxicology and Applied Pharmacology 193 (2003) 139–146 pears that at least some carbamate esters may initiate neu- investigated effects of a range of carbamate pesticides on rological and behavioral changes at dose levels that produce neuronal nAChRs heterologously expressed in Xenopus lae- few overt signs of acute toxicity or significant reduction in vis oocytes. The potencies of carbamate effects on the nervous tissue AChE activity (Ecobichon, 2001). Mecha- nAChRs are compared to the potencies of rat brain AChE nisms involved in these changes, however, remain to be inhibition and the differential sensitivities of specific sub- elucidated. types of ganglionic and brain neuronal nAChRs have been Previous research showed evidence of carbamates inter- investigated. The combined results demonstrate that several acting with receptors of the cholinergic system. The car- of the carbamates investigated more potently inhibit neuro- bamate physostigmine and related cholinesterase inhibitors nal nAChRs than brain AChE and that ganglionic and brain 3 displace the muscarinic ACh receptor agonist [ H]ox- nAChRs are inhibited by carbamates in a compound- and otremorine-M from its receptors in rat cortex and brain stem receptor-specific way. homogenates (Van den Beukel et al., 1997; Lockhart et al., 2001). Additional evidence has been presented to suggest that some of these cholinesterase inhibitors are muscarinic ACh receptor agonists (Volpe et al., 1985; Lockhart et al., Methods 2001). Agonistic, antagonistic, potentiating, and inhibitory effects of physostigmine and related cholinesterase inhibi- Chemicals. ACh (acetylcholine chloride), collagenase type tors on nicotinic ACh receptors (nAChRs) have also been I, DMSO (ACS reagent), MS-222 (3-amino benzoic acid described. Low concentrations of physostigmine and ethyl ester, methane sulfonate salt), NaCl, and neomycin neostigmine agonize or potentiate, whereas high concentra- solution (10 mg neomycin/ml in 0.9% NaCl) were obtained tions of these drugs block neuronal nAChR channels from Sigma (St. Louis, MO). Aldicarb (2-methyl-2-(meth- (Storch et al., 1995; Nagata et al., 1997; Van den Beukel et ylthio)propionaldehyde O-(methylcarbamoyl)oxime), ami- al., 1998). High concentrations of physostigmine also block nocarb (4-(dimethylamino)-3-methylphenolmethylcarbam- the ion channel of muscle-type nAChRs (Wachtel, 1993). ate ester), bendiocarb (2,2-dimethyl-1,3-benzodioxol-4-ol Various cholinesterase drugs with similar potentiating and methylcarbamate), carbaryl (1-naphthalenol methylcarba- inhibitory actions on neuronal nAChRs appear to be com- mate), carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofura- petitive ligands at the agonist recognition sites of muscle nol methylcarbamate), dioxacarb (2-(1,3-dioxolan-2-yl)phenyl and neuronal types of nAChR (Sherby et al., 1985; Svens- methylcarbamate), EPTC (S-ethyl N,N-dipropylthiocarbam- son and Nordberg, 1997; Zwart et al., 2000). For physostig- ate), fenoxycarb (ethyl[2-(4-phenoxyphenoxy)ethyl]car- mine the mechanism involves a competitive interaction with bamate), methomyl (N-[[(methylamino)carbonyl]oxy]etha- the agonist recognition site of the nAChR (Zwart et al., nimidothioic acid methyl ester), oxamyl (2-(dimethylamino)- 2000). The potentiation occurs when low concentrations of N-[[(methylamino)-carbonyl]oxy]-2-oxoethanimidothioic acid ACh and physostigmine are combined, i.e., when a fraction methyl ester), pirimicarb (dimethylcarbamic acid 2-(dimethyl- of the nAChRs is occupied by a single ACh and a single amino)-5,6-dimethyl-4-pyrimidinyl ester), and propoxur (2-(1- physostigmine molecule simultaneously. At high concentra- methylethoxy)phenol methylcarbamate) were purchased from tions physostigmine blocks the response by occupying both Riedel de Hae¨n (Seelze, Germany). CaCl (1 M solution), agonist recognition sites as well as by noncompetitive ion 2 MgCl (1 M solution), MgSO , NaHCO , and NaOH were channel block. The degrees of potentiation and inhibition 4 3 purchased from BDH Laboratory Supplies (Poole, England). were shown to differ between drugs and to depend on Ca(NO ) , Hepes, and KCl were from Merck (Darmstadt, receptor subunit composition (Zwart et al., 1999, 2000). 3 2 Germany). For AChE assays, acetylthiocholine iodide, 5,5Ј- Some carbamate insecticides, e.g., the cholinesterase in- hibitors aminocarb, aldicarb, and carbaryl tested at a con- dithio-bis-(2-nitro)benzoic acid, ethopropazine, Triton X-100, centration of 100 M, displace [3H]ACh from muscle-type physostigmine, and EDTA were from Sigma. NaCl, K2HPO4, nAChRs in Torpedo electric organ membranes (Eldefrawi and KH2PO4 were bought from Merck and Tris (2-amino-2- and Eldefrawi, 1983). Like neostigmine, carbaryl causes a (hydroxymethyl)-1,3-propanediol) was obtained from Boehr- concentration-dependent potentiation and inhibition of neu- inger (Mannheim, Germany). ronal nAChR channels in rat pheochromocytoma PC12 cells Saline solutions for electrophysiology were prepared (Nagata et al., 1997). An observed enhancement of cholin- with distilled water and solutions for AChE assays were ergic transmission in guinea pig myenteric plexus prepara- prepared with Milli-Q filtered water (Millipore SA, Mol- tions has been proposed to be associated with an effect of sheim, France). Stock solutions (0.1 M) of aldicarb, amino- the dithiocarbamate fungicide propineb on ganglionic and carb, bendiocarb, carbaryl, carbofuran, dioxacarb, EPTC, not on muscle-type