Pharmacological enhancement of endocannabinoid signaling reduces the cholinergic toxicity of diisopropylfluorophosphate

Abstract

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5mg/kg), URB597 (FAAH inhibitor, 3mg/kg), URB602 (MAGL inhibitor, 10mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10mg/kg) on DFP toxicity were then examined. Adult male rats were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24h and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.

Figure 1. Effect of DFP on cholinergic and cannabinergic markers in vitro

Membrane preparations from rat hippocampal tissues were either pre-incubated at 37°C for 30 minutes (ChE, MAGL and FAAH activities) or at room temperature for 15 minutes (CB1 receptor binding) with different concentrations of DFP and residual activity/binding evaluated as described in methods section.

Figure 2. Effects of WIN 55212-2, URB597, AM404 or URB602 on DFP-induced signs of cholinergic toxicity

Adult male rats (n = 5/treatment group) were treated with DFP (2.5 mg/kg, sc, open square) and immediately exposed to either vehicle, WIN 55,212-2 (1.5 mg/kg, closed square), URB597 (3 mg/kg, open triangle), AM404 (10 mg/kg, open circle) or URB602 (10 mg/kg, open diamond) as described in methods. Controls (n = 5) received only peanut oil. Functional signs of cholinergic toxicity (involuntary movements and SLUD signs) were observed for 24 hours and are shown as median scores ± interquartile range (IQR). Figure 2A and Figure 2B represent involuntary movements and SLUD signs, respectively. No signs of toxicity were noted in vehicle controls (data not shown). All four cannabinomimetics significantly reduced involuntary movements elicited by DFP (Figure 2A). WIN 55212-2 had no significant effect, while URB597, URB602 and AM404 significantly reduced DFP-induced SLUD signs (Figure 2B). See Results for details. Note that while all five treatment groups are shown on the figure, some of the curves overlap.

Figure 3. Interactive effects DFP and WIN 55212-2, URB597, AM404 or URB602 on hippocampal neurochemistry

Rats were treated as described above. Tissues were collected 24 hours after treatment for measurement of cholinesterase, fatty acid amide hydrolase, monoacylglycerol lipase and CB1 receptor binding. ChE activity was calculated as mean nmol acetylcholine hydrolyzed/min.mg protein ± SE. FAAH activity was measured as nmol anandamide hydrolyzed/min.mg protein ± SE. MAGL activity was measured as nmol 2-oleoyl glycerol hydrolyzed/min.mg protein ± SE. CB1 receptor binding pmol/mg protein ± SE. All values were shown as percent of control. An asterisk indicates a significant difference from control (Vehicle) whereas a pound sign indicates a significant difference from the DFP only group.