Effects of Sublethal Organophosphate Toxicity and Anti-cholinergics on Electroencephalogram and Respiratory Mechanics in Mice

Abstract

Organophosphates are used in agriculture as insecticides but are potentially toxic to humans when exposed at high concentrations. The mechanism of toxicity is through antagonism of acetylcholinesterase, which secondarily causes excess activation of cholinergic receptors leading to seizures, tremors, respiratory depression, and other physiological consequences. Here we investigated two of the major pathophysiological effects, seizures and respiratory depression, using subcutaneous injection into mice of the organophosphate diisopropylfluorophosphate (DFP) at sublethal concentrations (2.1 mg/Kg) alone and co-injected with current therapeutics atropine (50 mg/Kg) or acetylcholinesterase reactivator HI6 (3 mg/Kg). We also tested a non-specific cholinergic antagonist dequalinium chloride (2 mg/Kg) as a novel treatment for organophosphate toxicity. Electroencephalogram (EEG) recordings revealed that DFP causes focal delta frequency (average 1.4 Hz) tonic spikes in the parietal region that occur transiently (lasting an average of 171 ± 33 min) and a more sustained generalized theta frequency depression in both parietal and frontal electrode that did not recover the following 24 h. DFP also caused behavioral tremors that partially recovered the following 24 h. Using whole body plethysmography, DFP revealed acute respiratory depression, including reduced breathing rates and tidal volumes, that partially recover the following day. Among therapeutic treatments, dequalinium chloride had the most potent effect on all physiological parameters by reducing acute EEG abnormalities and promoting a full recovery after 24 h from tremors and respiratory depression. Atropine and HI6 had distinct effects on EEGs. Co-treatment with atropine converted the acute 1.4 Hz tonic spikes to 3 Hz tonic spikes in the parietal electrode and promoted a partial recovery after 24 h from theta frequency and respiratory depression. HI6 fully removed the parietal delta spike increase and promoted a full recovery in theta frequency and respiratory depression. In summary, while all anticholinergic treatments promoted survival and moderated symptoms of DFP toxicity, the non-selective anti-cholinergic dequalinium chloride had the most potent therapeutic effects in reducing EEG abnormalities, moderating tremors and reducing respiratory depression.

Keywords: EEG; anticholinergic; organophosphate; respiration; seizure.

FIGURE 1

Tremor intensity in response to diisopropylfluorophosphate (DFP) alone or DFP with treatments. Bars indicate average tremor intensity and standard error of means for 1 h immediately after injection (1st bar), and 22–24 h after injection (recovery, 2nd bar). The number of animals scored for each group were: control (saline), 6; DFP alone, 11; DFP with atropine, 10; DFP with HI6, 8; and DFP with dequalinium chloride, 6. Statistical significance was determined with one-way ANOVA between treatment groups, and paired t-test to compare acute tremors and recovery within a treatment group. Significance of p < 0.05, p < 0.01, p < 0.001 is indicated with single, double, and triple asterisks, respectively.

FIGURE 2

Electroencephalogram (EEG) activity in response to injections of diisopropylfluorophosphate (DFP) alone or DFP with treatment. Left panels show example traces before (top traces, A1–G1), immediately after (middle traces, A2–G2), and 22–24 h after DFP or DFP with treatment (bottom traces, A3–G3). Traces are shown for frontal (F) and parietal electrodes (P). Vertical grid lines represent 1 s intervals. Right panels display power analysis across a 15 min sampling period that includes the representative example traces. Power analysis are for control records (black), records immediately following DFP alone or DFP with treatment (blue), or recovery 22–24 h after injection (brown). Right top panel show analysis from frontal electrode 1, and right bottom panel show analysis for parietal electrode 2. Lines below the graph are regions where there was a statistically significant difference (Two-way ANOVA, Tukey multiple comparisons test, P < 0.05) in normalized power between control and acute EEG response (blue line), or control and recovery (black line). Data is for analysis of 11 mice for DFP alone, 10 mice for DFP with atropine, eight mice for DFP with HI6, and six mice for DFP with dequalinium chloride.

FIGURE 3

Timeline of breathing frequency (breaths per minute) and tidal volume (ml) in response to diisopropylfluorophosphate (DFP) alone or DFP with treatments. Left panels (A,D,G,J,M) show example flow traces for mice before (black), acutely after treatment (treatment indicated, blue) and 22–24 h after treatment (brown, recovery). Middle and right panels show averages of tidal volumes (ml volumes, middle panels) and breathing frequency (breathes per minute, right panels) before (control, black), acutely after treatment (treatment, blue), and 22–24 h after treatment (brown, recovery) for saline treatment (B,C), DFP alone (E,F), DFP with Atropine (H,I), DFP with HI6 (K,L), and DFP with dequalinium chloride (N,O). Data is for analysis of 10 mice for saline alone, 10 mice for DFP alone, seven mice for DFP with atropine, eight mice for DFP with HI6, and four mice for DFP with dequalinium chloride. Scale bars in the left panels indicate 1 ml/s flow (vertical) and 1 s time (horizontal).

FIGURE 4

Summary breathing frequency (breaths per minute) and tidal volume (ml) changes in response to diisopropylfluorophosphate (DFP) alone or DFP with treatments. Bar graphs show average breathing frequency (top panels) and tidal volumes (bottom panels) before (control), acutely after DFP or DFP with treatment (middle bar) and 22–24 h after treatment (recovery). Data is the average across the last 5 min of 1 h measurements (data from Figure 3) of breathing frequency and tidal volume, respectively, for saline treatment (A,B), DFP alone treatment (C,D), DFP with Atropine (E,F), DFP with HI6 (G,H), and DFP with dequalinium chloride (I,J). Data is for analysis of 10 mice for saline alone, 10 mice for DFP alone, seven mice for DFP with atropine, eight mice for DFP with HI6, and four mice for DFP with dequalinium chloride. Statistical significance was determined with one-way ANOVA comparison between treatments. Significance of < 0.05, p < 0.01, p < 0.001 is indicated with single, double, and triple asterisks, respectively.