Graphical abstract

Fluoroacetate (FA) is a tasteless, odorless, water-soluble metabolic poison with severe toxicological effects. Characterized in the mid-1900s, it has been used as a rodenticide but is comparably lethal to all mammals. Many countries have restricted its use, and modern-day accidental human exposures are rare, but recently, concerns have been raised about its application as a chemical weapon with no known antidote. A combined treatment of methylene blue (MB), an antioxidant, and monosodium glutamate (MSG), a precursor of the citric acid cycle substrate alphaketoglutarate, has been recommended as an effective countermeasure; however, no peer-reviewed articles documenting the efficacy of this therapy have been published. Using a rodent model, we assessed the effects of MB and MSG on the neurologic, cardiac, and pulmonary systems. Transcriptomic analysis was used to elucidate inflammatory pathway activation and guide bioassays, which revealed the advantages and disadvantages of these candidate countermeasures. Results show that MB and MSG can reduce neurologic signs observed in rats exposed to sodium FA and improve some effects of intoxication. However, while this strategy resolved some signs of intoxication, ultimately it was unable to significantly reduce lethality.

Fluoroacetate, a severely toxic metabolic poison, has historically been used to control rodent and predator populations. Due to safety concerns, its use is highly restricted in the United States; however, other countries have fewer regulations. Although the mechanism of action is known, no countermeasures have been identified, and accidental or intentional ingestion by humans is often fatal. Case studies documenting exposures describe a variety of nonspecific signs and symptoms, including cardiac dysfunction, pulmonary edema, and neurological symptoms. In cellulo, fluoroacetate is metabolically converted to fluorocitrate, a tightly binding competitive inhibitor of mitochondrial aconitase. Blocking this early step in the citric acid cycle leads to mitochondrial and cellular dysfunction. Characterizing the cellular effects of fluorocitrate allows researchers to strategically select possible therapeutics for screening. Data presented herein recommend activation of alternative energy pathways to support cellular metabolism when glucose oxidation has been blocked. Glutamine and fatty acid metabolism avoid the bottleneck in the citric acid cycle caused by fluorocitrate. Metabolically active immortalized cardiac myocytes model dose‐dependent and time‐dependent responses to fluorocitrate exposure. Past work has established fluorocitrate toxicity as a better model in in vitro systems because of the slow rate of conversion from fluoroacetate to fluorocitrate in cells. Metabolic pathway dependence, flexibility, and capacity were assessed for glucose, glutamine, and fatty acid oxidation over 24 hours in cells exposed to 200 μM fluorocitrate. At the 2‐hour time point, cellular metabolism had begun to shift from glucose oxidation to glutamine oxidation. At the 6‐hour time point, the increase in dependence on glutamine metabolism became significant (p < 0.05) vs. control, while dependence on glucose oxidation continued to decrease. Total glutamine metabolic capacity also increased in response to fluorocitrate exposure, possibly suggesting changes in gene expression to further support this pathway. Complementary data tracking fatty acid metabolism showed an early increase in metabolic flexibility after 1 hour of exposure. These results foreshadowed the increase in dependency observed at the 6‐hour time point. Cells exposed to fluorocitrate for 24 hours were no longer sufficiently metabolically active to be evaluated. These data show cells exposed to a mito‐toxin can shift their metabolic profile to avoid compromised pathways and utilize alternative energy sources. In response to fluorocitrate exposure, inhibited glucose metabolism is countered by increasing metabolic dependence on glutamine and fatty acid oxidation. These data recommend a targeted treatment strategy which supports alternative energy pathways by providing cells with substrates that enter glutamine or fatty acid metabolism. Support or Funding Information This research was supported by an interagency agreement between the NIH and USAMRICD and by the ORISE Program a...

Sodium fluoroacetate (1080) is a colorless, odorless, tasteless, water‐soluble metabolic poison. In the body, fluoroacetate is converted to fluorocitrate (FC) which blocks the citric acid cycle and drastically decreases ATP production. Symptoms of exposure include nausea, vomiting, abdominal pains, salivation, irrational fear, weakness, tachypnea, cyanosis, sweating, increased temperature, and death. Signs and symptoms of 1080 poisoning are nonspecific, complicating diagnosis. 1080 is tightly regulated within the United States, but it is commonly used in Australia, New Zealand, Mexico, Japan, South Korea, and Israel as a rodenticide to control invasive and predatory species. An eco‐terrorist scare in New Zealand (2015) and findings reported by the CIA (2007) have led 1080 to being identified as a potential terrorist weapon. Saccharomyces cerevisiae is a useful model to assess the impact of FC on cellular respiration and to screen therapeutics. In response to FC, yeast shows depressed mitochondrial activity as measured by oxygen consumption rate (OCR). Meclizine, an antihistamine used to treat nausea, vomiting, and dizziness, shows promise as a potential treatment in this model. Toxicity of meclizine was also assessed. Assays were performed in yeast extract peptone galactose (YPG) media, a metabolically restrictive media. To assess the effect of FC on OCR, cells were exposed to 50 μM, 100 μM, and 200 μM FC in liquid culture for 1, 4, or 24 hours. After incubation, mitochondrial respiration was assessed via the MitoXpress assay (Agilent), a fluorescence‐based assay which measures OCR. Once established as a viable model, yeast was then used to screen meclizine as a therapeutic to improve cellular respiration. Yeast was co‐incubated with fluorocitrate (200 μM) and meclizine (1 μM, 5 μM, and 15 μM) for 4 hours, and the effects on mitochondrial respiration were assessed via the MitoXpress assay. OCR decreased in yeast exposed to FC. Treatment with meclizine rescued mitochondrial function as demonstrated by an increase in OCR. Higher concentrations of meclizine (15 μM) improved mitochondrial activity. However, studies analyzing the effect of meclizine on yeast growth showed high concentrations of meclizine (15μM) inhibit growth, suggesting a toxic effect. Lower concentrations of meclizine (1μM) improved growth compared to untreated yeast exposed to FC. Future studies will continue to characterize the effectiveness of meclizine as a treatment and to use yeast as a screening method for other potential treatments for sodium fluoroacetate. Support or Funding Information DISCLAIMER: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.N.A.A., M.C.R., and M.K.A. were supported in part by an appointment to the Internship/Research Participation Program for USAMRICD, administered by the Oak Ridge Institute for Science and Education through an agreement between the US Department of Energy and the USAMRDC.

Carfentanil (CRF) is a potent synthetic opioid primarily used in anaesthetizing large animals. Exposure to CRF can cause both respiratory and central nervous system depression. Respiratory depression can lead to hypoxia, which may lead to cardiac damage and ultimately death. Myocardial damage has been observed in ferrets exposed to aerosolized CRF. Troponin is a protein complex that plays an integral part in muscle contractions within the myocardium and is used as an indicator of myocardial damage. An increase in blood levels of troponin in exposed ferrets would suggest underlying cardiac damage. Select animals were treated with naloxone (NX), an opioid antagonist, to investigate whether reversing opioid effects altered cardiac function or troponin levels. Male telemetrized ferrets were exposed to aerosolized CRF in a whole‐body plethysmography chamber. Cardiac parameters and respiratory dynamics were collected before, during, and after CRF exposure. Ferrets received no treatment, an oral or intramuscular (i.m.) dose of NX (0.375, 0.75, 1.5, or 3 mg/mL), or water 30 minutes post‐exposure. Euthanasia occurred 24 hours post‐exposure, and blood was collected via descending aorta or heart stick. Serum was separated from blood and analyzed using a high sensitivity rat cardiac troponin‐I enzyme‐linked immunosorbent assay (ELISA). Average troponin levels for all animals ranged from 0.00 – 6.30 ng/mL. Troponin levels were not statistically different across treatment groups, though we observed an increase in mean arterial pressure (MAP) and increased incidents of premature junction complexes for animals exposed to CRF, indicating potential cardiac distress. The increase in MAP could be a compensatory mechanism to overcome hypoxia due to decreased respiration induced by CRF. Both control and exposed animals showed an increase in MAP following treatment; however, sham‐exposed animals had no rise in MAP post‐treatment. This rise in MAP could be due to a combination of NX treatment and a response to being handled for the injection. Overall, our results suggest that CRF does not directly cause myocardial damage, nor do surgically implanted cardiac telemetry devices cause an explicit, permanent increase in troponin levels. Further investigation is needed to determine if cardiac abnormalities observed in ferrets are caused via hypoxia or other unknown mechanisms. Support or Funding Information Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. This research complied with the Animal Welfare Act and implementing Animal Welfare Regulations and adhered to the principles noted in The Guide for the Care and Use of Laboratory Animals. This research was supported by DTRA RD‐CB. E.P., W.Y.T., M.R. S.P., A.D., and V.D.C. were supported in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagenc...