Changes in Cellular Metabolic Pathways in Response to Fluoroacetate/Fluorocitrate Toxicity

Abstract

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 InformationThis research was supported by an interagency agreement between the NIH and USAMRICD and by the ORISE Program administered through an interagency agreement between the US DoE and USAMRDC. The views expressed are solely those of the authors.