Elsevier

Neurotoxicology and Teratology

Volume 32, Issue 3, May–June 2010, Pages 329-335
Neurotoxicology and Teratology

Behavioral sequelae following acute diisopropylfluorophosphate intoxication in rats: Comparative effects of atropine and cannabinomimetics

https://doi.org/10.1016/j.ntt.2009.12.006Get rights and content

Abstract

The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague–Dawley rats were treated with vehicle or DFP (2.5 mg/kg, sc), immediately post-treated with either vehicle, atropine (16 mg/kg), URB597 (3 mg/kg), URB602 (10 mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6–8 and 27–29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29 days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.

Introduction

Organophosphorus insecticides (OPs) are a major class of pesticides that elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for the degradation of the neurotransmitter acetylcholine in the central and peripheral nervous systems. Acetylcholine accumulation following extensive AChE inhibition increases/prolongs the stimulation of cholinergic muscarinic and nicotinic receptors on postsynaptic neurons, myocytes and autonomic ganglia and end-organs, leading to an acute cholinergic syndrome characterized by autonomic dysfunction, involuntary movements, muscle fasciculations, respiratory distress, seizures and other signs.

The U.S. Environmental Protection Agency estimates that 65–90 million pounds of OPs are used each year in the United States, and they remain commonly used worldwide [24]. In addition to the well known acute toxicity, a number of epidemiological studies have also reported prolonged neurological changes following OP intoxication [48], [52], [58], [62]. These studies indicate that individuals intoxicated by OPs can exhibit long-term deficits in cognitive and motor processes including abstraction, dexterity, memory, problem solving, visual attention and visuomotor speed. Interestingly, a prospective study of Nicaraguan patients showed that these neurological deficits tended to abate over a two year period and were replaced by an increase in neuropsychiatric symptoms including anxiety, depression, irritability and restlessness [10]. In addition, Sanchez-Amate et al. [49] reported that rats given the OP chlorpyrifos, at doses that failed to elicit overt signs of cholinergic toxicity, exhibited increased time in the open arms of an elevated plus maze, suggesting that OPs may affect anxiety-like behavior. Furthermore, higher rates of anxiety and depression have been reported in agricultural areas [46], [51], [57], and depression has consistently been identified as an important risk factor for suicide [5], [20]. London et al. [27] and Jaga and Dharmani [21] proposed that exposure to OPs could have a causal relationship in the number of suicide attempts worldwide by inducing changes in affect.

The basic principles of therapeutic intervention in cases of acute OP intoxication have not changed substantially over the last half century [31]. In fact, all countries worldwide use the same treatment strategy: an anticholinergic drug (e.g., atropine) to counteract the acute cholinergic syndrome, an oxime (e.g., pralidoxime) to reactivate AChE and an anticonvulsant (e.g., diazepam) to treat seizures and prevent neuropathology. Substantial clinical evidence questions the effectiveness of pralidoxime when given in combination with atropine [8]. In addition, the highest incidence of persistent neuropsychological symptoms in patients with prior OP intoxication is reported in cases requiring hospitalization, and thus almost certainly treated with the first line of defense, atropine [6]. While atropine effectively blocks postsynaptic muscarinic receptors to alleviate some clinical signs elicited by acetylcholinesterase inhibition, it can also block presynaptic muscarinic receptors mediating the adaptive inhibition of acetylcholine release, thus potentially contributing to more prolonged persistence of acetylcholine and potentially increased activation of nicotinic receptors. Thus, alternative therapeutic strategies may lead to more effective long-term recovery following acute intoxications.

Recent studies in our laboratory suggest that the enhancement of endocannabinoid (eCB) signaling can reduce the acute signs of toxicity following exposure to OP anticholinesterases [38], [39]. Endocannabinoids (e.g., anandamide (AEA) and 2-arachidonylglycerol (2-AG)) are neuromodulators produced by neuronal depolarization that act in a retrograde fashion via presynaptic cannabinoid type 1 (CB1) receptors to inhibit the release of various neurotransmitters including acetylcholine [9], [15], [60], dopamine [4], [59], glutamate [26], [55], GABA [34], norepinephrine [53] and serotonin [37]. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for AEA is mediated by fatty acid amide hydrolase (FAAH) and for 2-AG primarily by monoacylglycerol lipase (MAGL) (reviewed in [14], [47]). Kathuria et al. [23] reported that URB597, a FAAH inhibitor [13], elicited anxiolytic behavior in rats. Moreover, URB597 both decreased immobility in rats in the forced swimming test and increased struggling in mice in the tail suspension test, two endpoints widely used for assessing antidepressant activity [17]. In both of these studies, the behavioral effects of URB597 were prevented by the pre-administration of SR141716A, a selective CB1 receptor antagonist/inverse agonist. Thus, we hypothesized that the enhancement of eCB signaling would block neurobehavioral changes following acute OP intoxication. We compared the effects of atropine with indirect cannabinomimetics (URB597; URB602, a MAGL inhibitor [25]; and a combination of these two drugs) on functional/neurobehavioral changes following acute diisopropylfluorophosphate (DFP) intoxication in rats.

Section snippets

Treatments

DFP was purchased from Sigma-Aldrich Co. (St. Louis, MO), dissolved in peanut oil and administered (2.5 mg/kg, sc) in a volume of 1 ml/kg. Atropine was purchased from Sigma-Aldrich Co. (St. Louis, MO), dissolved in saline and administered (16 mg/kg, ip) in a volume of 1 ml/kg. URB597 (3′-carbamoyl-biphenyl-3-y-cyclohexylcarbamate) and URB602 (biphenyl-3-yl-carbamic acid cyclohexyl ester) were purchased from Cayman Chemical Company (Ann Arbor, MI), dissolved in vehicle (2% cremophor/2% dimethyl

Functional signs of toxicity

Fig. 1 shows body weight changes following DFP exposure. Compared to control animals (PNO/VEH), DFP (DFP/VEH) caused a significant reduction in body weight at 1 and 6 days after treatment. Compared to the DFP/VEH group, no significant differences in body weight were observed in the DFP/ATR, DFP/URB597, DFP/URB602 or DFP/COMBO groups. No significant treatment-related changes in body weight were noted at 29 days after dosing, however.

Fig. 2 shows the influence of atropine, URB597, URB602 or the

Discussion

Organophosphorus insecticides are used ubiquitously throughout the world to control insect pests. While acute toxicity from these insecticides has been well recognized and characterized, long-term neurological consequences following acute intoxication are reported but much less understood. The anticholinergic drug atropine has been used for decades as the first line of treatment for acute OP intoxication (reviewed in [12]). However, its effects on long-term neurological sequelae following acute

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was partially supported by research grant R01 ES009119 from the National Institute of Environmental Health Sciences, NIH (C.N.P.) and by the Oklahoma State University Board of Regents. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS.

References (62)

  • A. Nallapaneni et al.

    Modulation of paraoxon toxicity by the cannabinoid receptor agonist WIN 55, 212-2

    Toxicology

    (2006)
  • A. Nallapaneni et al.

    Pharmacological enhancement of endocannabinoid signaling reduces the cholinergic toxicity of diisopropylfluorophosphate

    Neurotoxicology

    (2008)
  • S. Pellow et al.

    Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat

    J. Neurosci. Methods

    (1985)
  • G.B. Quistad et al.

    Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides

    Toxicol. Appl. Pharmacol.

    (2001)
  • G.B. Quistad et al.

    Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications

    Toxicol. Appl. Pharmacol.

    (2002)
  • R.E. Roberts et al.

    Occupation and the prevalence of major depression, alcohol, and drug abuse in the United States

    Environ. Res.

    (1993)
  • L. Rosenstock et al.

    Chronic central nervous system effects of acute organophosphate pesticide intoxication. The Pesticide Health Effects Study Group

    Lancet

    (1991)
  • H. Schulz et al.

    Behavioral effects of subchronic intoxication with parathion-methyl in male Wistar rats

    Neurotoxicol. Teratol.

    (1990)
  • L. Stallones et al.

    Pesticide poisoning and depressive symptoms among farm residents

    Ann. Epidemiol.

    (2002)
  • P. Adamczyk et al.

    Activation of endocannabinoid transmission induces antidepressant-like effects in rats

    J. Physiol. Pharmacol.

    (2008)
  • C.A. Bowden et al.

    Clinical applications of commonly used contemporary antidotes. A US perspective

    Drug Saf.

    (1997)
  • J.F. Cheer et al.

    Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation

    J. Neurosci.

    (2007)
  • A.T. Cheng et al.

    Psychosocial and psychiatric risk factors for suicide. Case-control psychological autopsy study

    Br. J. Psychiatry.

    (2000)
  • D. Cota et al.

    The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis

    Clin. Invest.

    (2003)
  • H.J. de Silva et al.

    Does pralidoxime affect outcome of management in acute organophosphorus poisoning?

    Lancet

    (1992)
  • A. Degroot et al.

    CB1 receptor antagonism increases hippocampal acetylcholine release: site and mechanism of action

    Mol. Pharmacol.

    (2006)
  • E. Delgado et al.

    Central nervous system effects of acute organophosphate poisoning in a two-year follow-up

    Scand. J. Work Environ. Health

    (2004)
  • M.J. Detke et al.

    Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants

    Psychopharmacology

    (1995)
  • D. Fegley et al.

    Characterization of the fatty acid amide hydrolase inhibitor cyclohexyl carbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597): effects on anandamide and oleoylethanolamide deactivation

    J. Pharmacol. Exp. Ther.

    (2005)
  • A.N. Gifford et al.

    Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

    J. Pharmacol. Exp. Ther.

    (1996)
  • J. Glowinski et al.

    Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain

    J. Neurochem.

    (1966)
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