* Center for Research on Occupational and Environmental Toxicology and Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239; and
Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
1 To whom correspondence should be addressed at Oregon Health & Science University, CROET/L606, 3181 SW Sam Jackson Park Rd., Portland, OR 97239. Fax: (503) 494-3849. E-mail: leinp{at}ohsu.edu
Received August 21, 2004; accepted September 29, 2004
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ABSTRACT |
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Key Words: organophosphorus pesticides; asthma; M2 muscarinic receptor; airway hyperreactivity.
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INTRODUCTION |
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In support of epidemiological evidence linking OP exposure to asthma, we have recently established that chlorpyrifos, a widely used OP, induces airway hyperreactivity in a guinea pig model (Fryer et al., 2004). However, our data show that chlorpyrifos potentiates vagally induced bronchoconstriction in the absence of AChE inhibition. Rather, the mechanism involves decreased function of inhibitory M2 muscarinic receptors on the parasympathetic nerves supplying airway smooth muscle. Vagally induced bronchoconstriction normally is limited by these autoinhibitory M2 muscarinic receptors (Coulson and Fryer, 2003
; Fryer and Maclagan, 1984
; Minette and Barnes, 1988
). Loss of M2 receptor function leads to increased release of acetylcholine from the parasympathetic nerves, resulting in potentiation of vagally mediated bronchoconstriction, which contributes to airway hyperreactivity. OP-induced inhibition of M2 receptor function and the consequent airway hyperreactivity are consistent with previous studies demonstrating that neuronal M2 receptors are dysfunctional in animal models of asthma (Fryer and Wills-Karp, 1991
; Gambone et al., 1994
; Jacoby and Fryer, 1991
) and in patients with asthma (Minette et al., 1989
).
There are reports of OPs interacting with muscarinic receptors in brain tissue at doses that do not inhibit AChE (Katz and Marquis, 1989); however, these effects appear not to be consistent across brain regions or between different OPs (Pope, 1999
). Recent in vitro studies using slice cultures from rat striatum indicated that the active metabolite of chlorpyrifos increased acetylcholine release via inhibition of autoinhibitory muscarinic receptors (Liu et al., 2002
), which is consistent with our observations of chlorpyrifos effects on cholinergic neurotransmission in the airway. However, these striatal studies also found that, in the presence of AChE inhibitors, the active metabolites of the OPs parathion and methyl parathion act like muscarinic agonists to inhibit acetylcholine release. These data raise a question of whether observations of chlorpyrifos-induced airway hyperactivity via decreased M2 receptor function can be generalized across the OP class of insecticides. To address this question, we tested two different OPs, diazinon and parathion, which have different profiles of toxicity (Ecobichon, 2001
; Moser, 1995
). Although EPA-mandated restrictions for residential use of diazinon (Spectracide®) have recently been phased in, it remains a commonly used insecticide in the United States (USDA, 2003
; Whitmore et al., 2003
). Moreover, there is evidence of widespread exposure to diazinon in the general population (Barr et al., 2004
; Whyatt et al., 2002
). Parathion was included in this study despite the fact that EPA cancelled all uses of this pesticide in 1992, because its toxicological properties in both humans and animals are well known, and there are previous reports that parathion induces symptoms of asthma in experimental animals (Segura et al., 1999
). Pyrethroids are a widely used class of non-OP pesticides that do not act via cholinesterase inhibition and have been shown to either not affect or increase muscarinic receptor function (Abou-Donia et al., 2004
; Ahlbom et al., 1994
; Eriksson and Nordberg, 1990
; Husain et al., 1994
). For these reasons, and because there is concern that pyrethroids may exacerbate asthma (Landrigan et al., 1999
), we included permethrin in our tests of pesticide effects on airway hyperreactivity.
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MATERIALS AND METHODS |
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Pesticide exposures. Parathion (o,o-diethyl-o-p-nitrophenyl phosphorothioate, 99.5% pure), diazinon (o,o-diethyl-o-[2-isopropyl-4-methyl-6-prydimyl] phosphorothioate, 99.5% pure), and permethrin (3-phenoxybenzyl-(1RS)-cis/trans-3-2,2-dichlorovinyl-2,2-demeth, 20% cis, 78% trans) were purchased from Chem Service (West Chester, PA) and used prior to the expiration date, with interim storage as recommended by the manufacturer. Pesticides dissolved in peanut oil or an equal volume (300 µl) of peanut oil alone were administered to guinea pigs by subcutaneous (sc) injection in the subscapular region. Subcutaneous dosing is commonly used in mechanistic studies of OPs (Bushnell et al., 1991; Chiappa et al., 1995
; Pope et al., 1992
; Stanton et al., 1994
) and is proposed to result in gradual release of the pesticide into the systemic circulation (Pope et al., 1991
), which approximates most human exposures (Gallo and Lawryk, 1991
). The highest doses of diazinon and parathion tested in these studies were determined to be those that caused a 50% inhibition of AChE in guinea pig lungs. The dose of permethrin used in these studies is within one order of magnitude of the amount of permethrin absorbed by guinea pig dermis (40 mg/kg) following a single application of 5% permethrin cream, which is a standard formulation for treating scabies (Franz et al., 1996
), and approximately one-tenth the dermal LD50 reported for rats (approximately 4000 mg/kg) and rabbits (approximately 2000 mg/kg). Animals dosed with parathion or diazinon were monitored for signs of cholinergic intoxication (tremors, altered gait, and excessive excretions) at 1 and 24 h following injections. In addition, effects on physiological parameters (heart rate, blood pressure) under basal conditions were monitored in animals treated with pesticides prior to initiating experiments. Physiological measurements of lung function were carried out 24 h post injection, and since previous studies demonstrated that lung function in guinea pigs treated with peanut oil does not differ from that seen in saline-treated controls (Fryer et al., 2004
), only peanut oil controls are reported herein.
Anesthesia and measurement of pulmonary inflation pressure. Guinea pigs were anesthetized with 1.5 g/kg urethane (ip). Heart rate and blood pressure were measured from the carotid artery. The trachea was cannulated, and the animals were ventilated via a tracheal cannula with a positive pressure constant volume (1 ml per 100 g body weight and 100 breaths/minute). The jugular veins were cannulated, and the nicotinic receptor antagonist succinylcholine (10 µg/kg/min, iv) infused to paralyze the animals. Pulmonary inflation pressure (Ppi) was measured from a side arm at the trachea; bronchoconstriction was measured as the increase in Ppi over the pressure produced by the ventilator as previously described (Fryer and Maclagan, 1984; Fryer and Wills-Karp, 1991
; Jacoby and Fryer, 1991
).
Measurement of vagally induced bronchoconstriction. All animals received guanethidine (10 mg/kg, iv) prior to the start of the experiment to deplete noradrenaline. Both vagus nerves were cut. The distal ends were placed on electrodes under oil and were stimulated at 2-min intervals (0.2 ms, 10 V, 125 Hz, 5-sec duration), producing frequency-dependent bronchoconstriction and bradycardia due to release of acetylcholine onto postjunctional M3 muscarinic receptors in the lungs and postjunctional M2 muscarinic receptors in the heart. Both vagally induced bronchoconstriction and bradycardia could be abolished by atropine (1 mg/kg, iv).
Measurement of neuronal M2 muscarinic receptor function. The function of neuronal M2 receptors was determined by measuring the ability of the muscarinic agonist, pilocarpine, to inhibit bronchoconstriction in response to vagal stimulation at 2 Hz. Pilocarpine is a muscarinic agonist with selectivity for prejunctional M2 versus postjunctional M3 receptors in vivo (Fryer and Maclagan, 1984; Langley, 1878
), thus, pilocarpine inhibits vagally induced bronchoconstriction via stimulation of the neuronal M2 receptors at doses that are 100-fold less than the doses required to cause bronchoconstriction by stimulating postjunctional M3 receptors (Fryer and Maclagan, 1984
). The effect of pilocarpine on vagally induced bronchoconstriction is reported as the ratio of bronchoconstriction in the presence of pilocarpine to bronchoconstriction in the absence of pilocarpine. A shift to the right of the pilocarpine dose-response curve indicates decreased M2 receptor function (Fryer and Maclagan, 1984
; Fryer and Wills-Karp, 1991
; Jacoby and Fryer, 1991
).
Measurement of postjunctional muscarinic receptor function. Intravenous injection of acetylcholine (110 µg/kg,) was used to assess the function of the postjunctional M3 receptors in the lungs and postjunctional M2 receptors in the heart. To determine if AChE inhibition influenced the response to acetylcholine, these experiments were repeated using methacholine (110 µg/kg, iv), an agonist that is less susceptible to hydrolysis by cholinesterases than acetylcholine (Bruning et al., 1996; Norel et al., 1993
). Since muscarinic agonists also initiate a reflex bronchoconstriction (Delpierre et al., 1983
; Wagner and Jacoby, 1999
), these experiments were performed in vagotomized animals.
AChE assay. Since OP inhibition of AChE differs between lung and brain within any given species (Lessire et al., 1996), we measured AChE activity in the lung in addition to AChE activity in the brain and blood, both of which are commonly used biomarkers of OP toxicity. Immediately following the completion of physiological measurements, lungs, brain, and heparinized blood samples were collected for determination of AChE activity via the standard Ellman assay (Ellman et al., 1961
) using 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide (ASChI) as the substrate. Lung and brain samples were homogenized in lysis buffer (0.1 M phosphate, pH 8.0) containing 0.1% Triton using a Dounce homogenizer, centrifuged at 13,400 x g, and the supernatant collected for analysis. Assays were run against blanks containing DTNB. The reaction was started with the addition of ASChI after equilibration for 5 min. Hydrolysis of ASChI was determined by monitoring the change in absorbance at 405 nm. To inhibit pseudocholinesterase activity, 100 µM tetraisopropyl pyrophosphoramide (iso-OMPA) was included in the assay. Data from lung and brain samples were normalized using protein concentration as determined using the BCA assay according to the manufacturer's directions (Pierce, Rockford, IL). AChE activity in blood samples was normalized according to the number of red blood cells (RBC) as determined using a hemacytometer.
Statistics. Data are expressed as mean ± standard error of the mean (SEM). Frequency, pilocarpine, methacholine, and acetylcholine dose-response curves were analyzed using a two-way analysis of variance for repeated measures. Baseline heart rates (beats/min), blood pressures (mmHg), pulmonary inflation pressures (mmH2O), and changes in pulmonary inflation pressure (mmH2O before pilocarpine administration/mmH2O after pilocarpine administration), as well as AChE activity levels (as % of control) were analyzed using analysis of variance (ANOVA; Statview 4.5, Abacus Concepts, Inc., Berkley, CA); a p value 0.05 was considered significant.
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RESULTS |
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Neuronal M2 receptor function was measured using the muscarinic agonist pilocarpine. Prior to administering pilocarpine, simultaneous electrical stimulation of both vagus nerves (2 Hz, 0.2 ms, 515 Volts, 22 sec at 1-min intervals) produced transient bronchoconstriction (measured as an increase in Ppi) that was not different among groups (peanut oil, 33.0 ± 2.6 mmH2O; parathion 100.1 mg/kg, 24.2 ± 2.7 mmH2O, 19.8 ± 3 mm H2O, 22.1 ± 4 mmH2O; diazinon 75 and 0.75 mg/kg, 32.1 ± 3.8 mmH2O, 20.9 ± 5.4 mmH2O). In guinea pigs treated with peanut oil, pilocarpine (1100 µg/kg, iv) decreased vagally induced bronchoconstriction in a dose-dependent manner, demonstrating that the neuronal M2 muscarinic receptors are functional (Fig. 1, open squares). The ability of pilocarpine to decrease vagally induced bronchoconstriction was significantly inhibited in animals treated with 10 or 1.0 mg/kg, but not 0.1 mg/kg, parathion (Fig. 1, left side, filled symbols). Similarly, diazinon, at both 75 mg/kg and at a 100-fold lower dose of 0.75 mg/kg blocked the ability of pilocarpine to decrease vagally induced bronchoconstriction (Fig. 1, right side, filled symbols). Thus, both OPs inhibited the function of autoinhibitory neuronal M2 receptors.
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DISCUSSION |
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Studies of OP neurotoxicity have indicated that acute effects of these compounds are mediated primarily by AChE inhibition (Pope, 1999). Several observations from our studies rule out AChE inhibition as the mechanism underlying the potentiation of vagally induced bronchoconstriction by parathion and diazinon. First, direct measurements of AChE activity in lungs, blood, and brain indicated that parathion and diazinon inhibited AChE in a dose-related manner, but this did not correlate with airway hyperreactivity. Second, although inhibition of AChE by pharmacological cholinesterase inhibitors has been shown to potentiate bronchoconstriction in response to acetylcholine (Colbatch and Halmagyi, 1963
; Daly and Schweitzer, 1951
), neither diazinon nor parathion potentiated bronchoconstriction induced by iv acetylcholine in vagotomized guinea pigs (Fig. 3), even at concentrations that caused 50% or more inhibition of AChE in the lung and blood (Fig. 6). The observation that these OPs induce airway hyperreactivity independent of AChE inhibition is important because it indicates that OP-induced airway hyperreactivity occurs below thresholds of toxic exposure that are currently defined by AChE inhibition.
In contrast, the non-OP insecticide permethrin attenuated vagally induced bronchoconstriction. Although we did not test the ability of permethrin to interact with neuronal M2 receptors, it has been reported that permethrin can increase M2 receptor function (Abou-Donia et al., 2004; Ahlbom et al., 1994
; Eriksson and Nordberg, 1990
; Husain et al., 1994
), which would be consistent with our observations of its effects on airway hyperreactivity. An unexpected finding was that permethrin attenuated acetylcholine-induced bronchoconstriction. The mechanism underlying this effect is not known.
Neuronal M2 muscarinic receptors limit release of acetylcholine from parasympathetic nerves in the lungs (Fryer and Maclagan, 1984). Pharmacological blockade of neuronal M2 receptors increases release of acetylcholine from these nerves (Baker et al., 1992
; Fryer et al., 1996
), which potentiates vagally induced bronchoconstriction (Fryer and Maclagan, 1984
; Fryer and Wills-Karp, 1991
; Jacoby and Fryer, 1991
). Our data show that neuronal M2 receptor function is inhibited by both parathion and diazinon at doses that cause airway hyperreactivity. In contrast, a dose of parathion that does not affect M2 receptor function also does not alter vagally induced bronchoconstriction. Thus, OP-induced inhibition of neuronal M2 receptor function mediates airway hyperreactivity. Loss of neuronal M2 receptor function in the lungs is also associated with other models of airway hyperreactivity including antigen challenge (Fryer and Wills-Karp, 1991
), viral infection (Jacoby and Fryer, 1991
), and exposure to ozone (Gambone et al., 1994
), suggesting that decreased M2 receptor function on airway nerves is a generalized mechanism underlying airway hyperreactivity.
The ability of OPs to inhibit neuronal M2 receptors may not be restricted to the lungs. OPs compete for binding to muscarinic receptors in the brain (Abdallah et al., 1992; Bomser and Casida, 2001
; Huff et al., 1994
; Jett et al., 1993
, 1994
; Katz and Marquis, 1989
, 1992
) and in the heart (Silveira et al., 1990
). In the heart, M2 receptors are present on both cardiac muscle, where they mediate bradycardia (Brodde et al., 2001
; Maeda et al., 1988
), and parasympathetic nerves that supply the heart, where they function to inhibit release of acetylcholine (Manabe et al., 1991
; Oberhauser et al., 2001
). Both parathion (10 mg/kg, sc) and diazinon (75 mg/kg, sc) potentiated bradycardia induced by vagal stimulation but not bradycardia induced by iv administration of acetylcholine or methacholine, suggesting inhibition of prejunctional neuronal M2 receptors, but not postjunctional cardiac M2 receptors. Similarly, the function of the neuronal M2, but not postjunctional M2, receptors in the heart is inhibited by chlorpyrifos (Fryer et al., 2004
) and by systemic administration of double-stranded RNA (Bowerfind et al., 2002
). Thus neuronal M2 receptors appear to be more vulnerable to inhibition than postjunctional M2 receptors.
The interaction of OPs with pre- and postjunctional muscarinic receptors in the brain is complex. OPs have been demonstrated to antagonize muscarinic receptors either via direct effects on the receptors themselves (Fitzgerald and Costa, 1992; Huff et al., 2001
; Katz and Marquis, 1989
; Liu et al., 2002
; Zhu et al., 1991
) or indirectly by decreasing receptor number in response to increased acetylcholine resulting from AChE inhibition (Cioffi and el-Fakahany, 1986
; Zhu et al., 1991
). Conversely, it has also been reported that OPs stimulate neuronal muscarinic receptors either directly (Liu et al., 2002
; Ward and Mundy, 1996
) or indirectly as a result of increased synaptic levels of acetylcholine consequent to AChE inhibition (Kilbinger and Wessler, 1980
; Liu et al., 2002
). Reports of OPs directly stimulating muscarinic receptors were derived from functional studies of striatum (Liu et al., 2002
) and frontal cortex (Ward and Mundy, 1996
). Although there are M2 receptors on presynaptic nerves in both the striatum (Hersch et al., 1994
) and cortex (Levey et al., 1991
), studies in knockout mice suggest that it is the M4, and not the M2 receptors, that are functionally significant in inhibiting acetylcholine release in these brain regions (Zhang et al., 2002
). These observations raise the possibility that earlier reports of direct stimulation of muscarinic receptors by OPs reflect effects on M4 rather than M2 receptors. When considered together with our findings that OPs inhibit neuronal M2 receptor function in the lungs and heart, these data strongly suggest that M2 and M4 receptors are differentially affected by OPs.
The mechanism(s) by which OPs inhibit M2 receptor function are not yet known. Because parathion and diazinon decreased M2 receptor function at doses that did not inhibit AChE, it seems unlikely that either indirect stimulation or decreased expression of M2 receptors secondary to AChE inhibition underlie M2 receptor inhibition. Although it is possible that AChE was acutely inhibited at the time of administration, causing persistent downregulation of M2 receptors 24 h later, this seems unlikely because the function of the postjunctional M2 receptors on the heart (see Fig. 5) was not inhibited 24 h after administration of OPs. Furthermore, the OPs were administered not as a single bolus dose, but rather subcutaneously in oil, a method that allows for gradual release of the OPs in the systemic circulation (Pope et al., 1991). It is also not the case that M4 receptors mediate OP-induced airway hyperreactivity, since M4 receptors are not expressed on parasympathetic nerves in the lungs (Fryer et al., 1996
). Therefore, it seems likely that OPs directly inhibit neuronal M2 receptor function. Whether they do so by downregulation of muscarinic receptor expression (Jett et al., 1993
, 1994
), modulation of ligand binding to M2 receptors (Jett et al., 1991
; Katz and Marquis, 1989
, 1992
), or alteration of signal transduction pathways downstream of muscarinic receptor (Bomser et al., 2002
; Huff et al., 1994
; Schuh et al., 2002
; Ward and Mundy, 1996
) has yet to be determined. A significant difference between these earlier published studies and our findings is that the former reported the potency of OP binding to muscarinic receptors as comparable to that of OP binding to acetylcholinesterase (AChE), whereas our data suggest that OP interactions with neuronal M2 receptors in airways occur at lower doses than those required to inhibit AChE activity in the lung or blood.
Data presented here confirm that the diethyl phosphorothionate OP insecticides cause airway hyperreactivity via a common mechanism of disrupting negative feedback control of cholinergic regulation in the lungs. Thus, we have shown that not only chlorpyrifos (Fryer et al., 2004), but also diazinon and parathion, inhibit neuronal M2 receptor function in the lung at concentrations that do not inhibit AChE. Since the resting vagal tone in guinea pig lungs is approximately 1015 Hz (Myers and Undem, 1996
), our data suggest that loss of M2 receptor function results in increased basal release of acetylcholine. Furthermore, irritation of the lung results in reflex bronchoconstriction that is mediated by the parasympathetic nerves (Carr and Undem, 2003
; Undem and Carr, 2002
), and loss of M2 receptor function also increases reflex bronchoconstriction (Costello et al., 1999
; Evans et al., 2000
). M2 receptors on parasympathetic nerves supplying glands in the airways also regulate mucin secretion in the airways (Ramnarine et al., 1996
; Rogers, 2001
). Thus OPs could potentiate basal tone, reflex bronchoconstriction, and mucus secretion, all of which are characteristics of asthma.
Use of OP insecticides has increased significantly in urban and agricultural settings over the past 30 years (Fenske et al., 2002; Koch et al., 2002
; USDA, 2003
; Wilhoit et al., 1999
), coincident with an increase in asthma (Hartert and Peebles, 2000
; Weitzman et al., 1992
). Children represent a potentially sensitive subpopulation with respect to asthma, and there is evidence of wide-spread exposure of children to OPs. Screens of fetal exposure to OP pesticides have detected chlorpyrifos (8.26 µg/ml), diazinon (13 µg/ml), and parathion (2.3 µg/ml) in meconium (Ostrea et al., 2002
). Recent studies in Seattle found that of 110 preschool children from 96 households of varying cultures, family income, and housing type, all excreted OP metabolites in their urine (Curl et al., 2003
; Lu et al., 2001
). Similarly, in a sample of 84,000 children across the United States, the urinary levels of chlorpyrifos metabolites were above the detection limit 98% of the time, compared to a 4% detection rate for the herbicide atrazine (Adgate et al., 2001
). Not only is there widespread exposure of children to these insecticides, but data collected as part of the most recent National Health and Nutrition Examination Survey (NHANES) indicated that across all racial and ethnic groups, urinary concentrations of OP metabolites in children 611 years of age were consistently significantly higher than in adults (Barr et al., 2004
). Consistent with these conclusions, chlorpyrifos residues have been shown to persist in the home for up to 2 weeks after a single application, with potential exposure to infants and children reaching levels 60120 times greater than the U.S. EPA recommended reference levels (Fenske et al., 1990
; Gurunathan et al., 1998
). The U.S. EPA has determined the dermal and acute dietary NOAEL (no observed adverse effect level) for diazinon to be 1 mg/kg/day and 0.25 mg/kg/day, respectively, based on plasma cholinesterase inhibition (U.S. EPA, 2000
). We observed airway hyperreactivity in response to diazinon (sc) at a dose that did not inhibit plasma cholinesterase, suggesting that diazinon may exert adverse effects on human lung function at doses considered safe under current EPA guidelines. These data suggest that exposure to these compounds may contribute to the observed increase in asthma prevalence over the past 30 years (Hartert and Peebles, 2000
; Weitzman et al., 1992
).
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ACKNOWLEDGMENTS |
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