Gut dysbiosis following organophosphate, diisopropylfluorophosphate (DFP), intoxication and saracatinib oral administration

Abstract

Organophosphate nerve agents (OPNAs) act as irreversible inhibitors of acetylcholinesterase and can lead to cholinergic crisis including salivation, lacrimation, urination, defecation, gastrointestinal distress, respiratory distress, and seizures. Although the OPNAs have been studied in the past few decades, little is known about the impact on the gut microbiome which has become of increasing interest across fields. In this study, we challenged animals with the OPNA, diisopropylfluorophosphate (DFP, 4mg/kg, s.c.) followed immediately by 2mg/kg atropine sulfate (i.m.) and 25mg/kg 2-pralidoxime (i.m.) and 30 minutes later by 3mg/kg midazolam (i.m.). One hour after midazolam, animals were treated with a dosing regimen of saracatinib (SAR, 20mg/kg, oral), a src family kinase inhibitor, to mitigate DFP-induced neurotoxicity. We collected fecal samples 48 hours, 7 days, and 5 weeks post DFP intoxication. 16S rRNA genes (V4) were amplified to identify the bacterial composition. At 48 hours, a significant increase in the abundance of Proteobacteria and decrease in the abundance of Firmicutes were observed in DFP treated animals. At 7 days there was a significant reduction in Firmicutes and Actinobacteria, but a significant increase in Bacteroidetes in the DFP groups compared to controls. The taxonomic changes at 5 weeks were negligible. There was no impact of SAR administration on microbial composition. There was a significant DFP-induced reduction in alpha diversity at 48 hours but not at 7 days and 5 weeks. There appeared to be an impact of DFP on beta diversity at 48 hours and 7 days but not at 5 weeks. In conclusion, acute doses of DFP lead to short-term gut dysbiosis and SAR had no effect. Understanding the role of gut dysbiosis in long-term toxicity may reveal therapeutic targets.

Keywords: diisopropylfluorophosphate (DFP); gut microbiome; orally administered drugs; organophosphate nerve agent; saracatinib.

FIGURE 1

Experimental design and impact of DFP and SAR on seizures and bodyweight changes. (A, B) Two cohorts of animals were used in this experiment. Both cohorts were challenged with 4mg/kg diisopropylfluorophosphate (DFP, s.c.) followed immediately by 2mg/kg atropine sulfate (ATS, i.m.) and 25mg/kg 2-pralidoxime (2-PAM, i.m.) and one hour later 3mg/kg midazolam (MDZ, i.m.). Two hours after MDZ, saracatinib (SAR) or vehicle (VEH) treatment began. Cohort-1 received 25mg/kg twice a day for the first three days followed by 20mg/kg once a day for four days with fecal collections at 48 hours and 7 days. The second cohort received seven daily doses 20mg/kg SAR and had fecal collections at 5 weeks. (C) Seizure response over time, linear mixed effects model. (D) Number of minutes animals spent in a convulsive seizure (CS), t-test. (E) Bodyweight changes over the treatment period. Linear mixed effects model, *p < 0.05.

FIGURE 2

Impact of DFP and SAR on the phyla level at 48 hours post-exposure. (A) Overall changes and actual abundance in phyla. (B–I) Actual abundance by phyla for Firmicutes (B), Bacteroidetes (C), Proteobacteria (D), Actinobacteria (E), Verricomicrobia (F), Tenericutes (G), Deferrbacteriodietes (H), and Cynanobacteria (I). ANOVA or Kruskal Wallis test, *p<0.05, n=4-5.

FIGURE 3

Impact of DFP and SAR on the genus level at 48 hours post-exposure. (A) LEfSe analysis revealed genera with LDA scores above 2.0. (B) Heatmap clustering by genus. (C-I). Trends in actual abundance of the seven genera with an overall p<0.05, n=6.

FIGURE 4

Impact of DFP and SAR on the phyla level at 7 days post-exposure. (A) Overall changes in actual abundance in phyla. (B-I) Actual abundance by phyla for Firmicutes (B), Bacteroidetes (C), Proteobacteria (D), Actinobacteria (E), Verricomicrobia (F), Tenericutes (G), Deferrbacteriodietes (H). ANOVA or Kruskal Wallis test, *p < 0.05, n=4-5.

FIGURE 5

Impact of DFP and SAR on the genus level at 7 days post-exposure. (A) LEfSe analysis reveals genera with LDA scores above 2.0. (B) Heatmap clustering by genus. (C–L). Trends in actual abundance of the seven genera with an overall p < 0.05, n=4-5.

FIGURE 6

Impact of DFP and SAR on the phyla level at 5 weeks post-exposure. (A) Overall changes in phyla actual abundance. (B–I) Actual abundance by phyla for Firmicutes (B), Bacteroidetes (C), Proteobacteria (D), Actinobacteria (E), Verricomicrobia (F), Tenericutes (G), Deferrbacteriodietes (H). ANOVA or Kruskal Wallis test, n=4-5.

FIGURE 7

Impact of DFP and SAR on the genus level at 5 weeks post-exposure. (A) LEfSe analysis reveals genera with LDA scores above 2.0. (B) Heatmap clustering by genus. (C–F) Trends in actual abundance of the seven genera with an overall p-value<0.05, n=4-5.

FIGURE 8

Alpha Diversity. Alpha diversity was measured using several metrics including observed, chao1, ACE, Shannon, Simpson, and Fisher at 48 hours (A), 7 days (B) and 5 weeks (C) post-exposure, ANOVA or Kruskal Wallis test, *p < 0.05, n=4-5.

FIGURE 9

Beta diversity. Beta diversity was assed using principle coordinate analysis (PCoA) and multidimensional scaling (NMDS) at 48 hours (A), 7 days (B) and 5 weeks (C) post-exposure.