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Poisoning with Soman, an Organophosphorus Nerve Agent,Alters Fecal Bacterial Biota and Urine Metabolites: a Case forNovel Signatures for Asymptomatic Nerve Agent Exposure
Derese Getnet,a Aarti Gautam,a Raina Kumar,a,b Allison Hoke,a,c Amrita K. Cheema,d Franco Rossetti,e
Caroline R. Schultz,f Rasha Hammamieh,a Lucille A. Lumley,g Marti Jetta*
aIntegrative Systems Biology Program, U.S. Army Center for Environmental Health Research, Fort Detrick,Maryland, USA
bAdvanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Fort Detrick,Maryland, USA
cThe Geneva Foundation, U.S. Army Center for Environmental Health Research, Fort Detrick, Maryland, USAdDepartments of Oncology and Biochemistry, Molecular and Cellular Biology, Georgetown University MedicalCenter, Washington, DC, USA
eClinical Research Management, Silver Spring, Maryland, USAfEdmond Scientific Company, Aberdeen Proving Ground, Maryland, USAgU.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland, USA
ABSTRACT The experimental pathophysiology of organophosphorus (OP) chemicalexposure has been extensively reported. Here, we describe an altered fecal bacterialbiota and urine metabolome following intoxication with soman, a lipophilic G classchemical warfare nerve agent. Nonanesthetized Sprague-Dawley male rats were sub-cutaneously administered soman at 0.8 (subseizurogenic) or 1.0 (seizurogenic) of the50% lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratifiedbased on seizing activity to evaluate effects of soman exposure on fecal bacterial bi-ota and urine metabolites. Soman exposure reshaped fecal bacterial biota by alter-ing Facklamia, Rhizobium, Bilophila, Enterobacter, and Morganella genera of the Firmi-cutes and Proteobacteria phyla, some of which are known to hydrolyze OP chemicals.However, analogous changes were not observed in the bacterial biota of the ileum,which remained the same irrespective of dose or seizing status of animals after so-man intoxication. However, at 75 days after soman exposure, the bacterial biota sta-bilized and no differences were observed between groups. Interestingly, in consider-ing just the seizing status of animals, we found that the urine metabolomes weremarkedly different. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, nor-epinephrine, and aldosterone were excreted at much higher rates at 72 h in seizinganimals, consistent with early multiorgan involvement during soman poisoning.These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial bi-ota in combination with urine metabolome alterations as forensic evidence for pre-symptomatic OP exposure temporally to enable administration of neuroprotectivetherapies of the future.
IMPORTANCE The paucity of assays to determine physiologically relevant OP expo-sure presents an opportunity to explore the use of fecal bacteria as sentinels incombination with urine to assess changes in the exposed host. Recent advances insequencing technologies and computational approaches have enabled researchersto survey large community-level changes of gut bacterial biota and metabolomicchanges in various biospecimens. Here, we profiled changes in fecal bacterial biotaand urine metabolome following a chemical warfare nerve agent exposure. The sig-nificance of this work is a proof of concept that the fecal bacterial biota and urinemetabolites are two separate biospecimens rich in surrogate indicators suitable for
Received 1 May 2018 Accepted 7 August2018
Accepted manuscript posted online 14September 2018
Citation Getnet D, Gautam A, Kumar R, Hoke A,Cheema AK, Rossetti F, Schultz CR,Hammamieh R, Lumley LA, Jett M. 2018.Poisoning with soman, an organophosphorusnerve agent, alters fecal bacterial biota andurine metabolites: a case for novel signaturesfor asymptomatic nerve agent exposure. ApplEnviron Microbiol 84:e00978-18. https://doi.org/10.1128/AEM.00978-18.
Editor Andrew J. McBain, University ofManchester
This is a work of the U.S. Government and isnot subject to copyright protection in theUnited States. Foreign copyrights may apply.
Address correspondence to Marti Jett,firstname.lastname@example.org.
* Present address: Marti Jett, U.S. Army Centerfor Environmental Health Research, FortDetrick, Maryland, USA.
D.G., A.G., and R.K. contributed equally to thisarticle.
November 2018 Volume 84 Issue 21 e00978-18 aem.asm.org 1Applied and Environmental Microbiology
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monitoring OP exposure. The larger value of such an approach is that assays devel-oped on the basis of these observations can be deployed in any setting with moder-ate clinical chemistry and microbiology capability. This can enable estimation of theaffected radius as well as screening, triage, or ruling out of suspected cases of expo-sures in mass casualty scenarios, transportation accidents involving hazardous mate-rials, refugee movements, humanitarian missions, and training settings when cou-pled to an established and validated decision tree with clinical features.
KEYWORDS soman, gut microbiome, 16S rRNA gene, urine metabolome
Despite the serious health threat posed to communities, organic derivatives ofphosphorus (organophosphorus [OP])-containing acids have a wide range ofapplications in modern society (13). OP-containing products are in excessive useworldwide for the control of agricultural or household pests. OP-containing pesticidesaccount for almost 38% of all pesticides used across the globe, leading to nearly 3million poisonings, over 200,000 deaths annually, and the contamination of numerousecosystems (4). In addition, application of OP derivatives as agents of war and terrorismin the form of nerve agents poses a significant threat to both civilians and thewarfighter. Exposure to OP leads to various degrees of neurotoxicity due to cholinergicreceptor hyperactivity, mediated primarily by the inhibition of acetylcholinesterase(AChE) (5). The excessive accumulation of acetylcholine leads to severe physiologicalcomplications that may manifest as muscarinic symptoms (e.g., lacrimation, salivation,diarrhea, miosis, and bradycardia), nicotinic symptoms (e.g., tachycardia, hypertension,convulsions, and paralysis of skeletal and respiratory muscles), and death (13, 6, 7).
Soman (pinacolyl methylphosphonofluoridate or GD [German agent D]) is one of theG class nerve agents (volatile agents associated with inhalation toxicity) that inhibitAChE much more rapidly but less specifically than V class nerve agents (viscous agentsassociated with transdermal toxicity) (2, 6, 8). Whole-body autoradiography studies inmice revealed that intravenously administered tritiated soman ([3H]soman) spreadsthrough the entire body in less than 5 min (9). High levels of accumulation were notedin the lungs, skin, gallbladder, intestinal lumen, and urine during the first 24 h.[3H]pinacolyl methylphosphoric acid ([3H]PMPA), a hydrolyzed acid and primary me-tabolite of soman, was found to be concentrated in specific organs such as lungs, heart,and kidneys within minutes of [3H]soman administration, which reflected the highlyreactive (i.e., rapidly aging) nature of soman in vivo (10). Significant amounts of somanwere also detected in red blood cells, a major esterase depot, compared to the plasma.In addition, those studies revealed that the common route of excretion for PMPA, amajor soman metabolite, was via urine and the intestinal lumen content (9, 11).Interestingly, only trace amounts of [3H]soman, [3H]PMPA, or [3H]methylphosphonicacid (hydrolyzed PMPA) were observed in the central nervous system (CNS). Currentclinical nerve agent exposure assessments are primarily based on overt physiologicalreactions such as convulsions, loss of consciousness, and salivation for high-doseexposures or pupil constriction and respiratory distress for low-dose exposures (12, 13).Recent studies have also demonstrated the feasibility of identifying OP hydrolysisproducts in hair and nail clippings to verify nerve agent exposure after 30 days (13, 14).Hence, monitoring asymptomatic exposure or verifying suspected exposure during thepresymptomatic phase using minimally invasive and rapid molecular methods repre-sents an ideal approach and capability. Thus, identification of new surrogate biomark-ers of toxicity and/or exposure to soman and other OP chemicals is essential from botha clinical and a public health standpoint, especially for triaging population-level expo-sures.
Using an omics approach, we have assessed the potential value of correlationsbetween changes in fecal bacterial biota or urine metabolites and OP exposure todetermine if these biospecimens are suitable for diagnostic use for exposure surveil-lance and monitoring in a rat model of soman exposure. More importantly, we filled ina knowledge gap regarding how OP exposure directly or indirectly impacts bacterial
Getnet et al. Applied and Environmental Microbiology
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communities of the mammalian gut and alters the global urine metabolic profile. Forover 20 years, specific species of the Bacteroidetes and Proteobacteria phyla have beenimplicated in enhanced biodegradation of OP pesticides in the bioremediation f