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