Methylene blue and monosodium glutamate improve neurologic signs after fluoroacetate poisoning

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 alpha-ketoglutarate, 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.

Keywords: 1080; TCA cycle; antioxidant; fluorocitrate; metabolic toxin; rat models.

Figure 1.

Metabolic blood panel. The concentration of ALT, AST, and BUN 24 h after exposure to 1 × LD₅₀ of 1080. Male rats were exposed to 1 × LD₅₀ of 1080 and then treated with MB and MSG or sham injection. Treated animals were given MB (5 mg/kg, SC) and MSG (250 mg/kg, IP) twice at 0.5 and 2 h after exposure. Significant increases in all three makers were observed in the double sham-treated animals, and treatment with MB and MSG appears to restore alanine aminotransferase (A) and aspartate transaminase (B) concentrations to control levels. BUN levels were also elevated in exposed animals, but treatment did not appear to be of any benefit (C). Dash = average, n = 6–10, *P < 0.05 with a one-way ANOVA followed by Dunnett’s multiple comparison test versus sham control.

Figure 2.

Respiratory parameters following 1080 exposure. Minute volume was measured for animals exposed to 1 × LD₅₀ of 1080 (red) and control animals (blue). Baseline respiration (B) and the first 23 h are shown. As relevant, MB (5 mg/kg, SC) and MSG (250 mg/kg, IP) were administered at either 0.5 (A) or 0.5 and 2 h (B) postexposure. Sham animals were treated with sterile water. All exposed animals showed a marked reduction in minute volume compared with the control animals starting around 7 h postexposure. Treatment with MB and MSG showed no effect on respiration. Error bars indicate standard deviation, n = 9–10.

Figure 3.

Determination of heme in BALF. Animals were exposed to 1 × LD₅₀ of 1080, and treated animals were given MB (5 mg/kg, SC) and MSG (250 mg/kg, IP) either once at 0.5 h (A) or at both 0.5 and 2 h (B). BALF was collected 24 h after exposure. In both cases, treating with MB and MSG was able to reduce the amount of absorbance at 540 nm, indicating a reduction in Hb in BALF. Error bars indicate standard deviation with average marked, n = 5–8, * P < 0.05 with a one-way ANOVA followed by Dunnett’s multiple comparison test versus sham control.

Figure 4.

Protein content in BALF. Animals were exposed to 1 × LD₅₀ of 1080. Treated animals were given MB (5 mg/kg, SC) and MSG (250 mg/kg, IP) either at 0.5 h (A) or at 0.5 and 2 h (B). BALF was collected 24 h after exposure and total protein content was analyzed. Exposure to 1080 caused an increase in protein in the lungs, consistent with the development of pulmonary edema. A single administration of MB and MSG was unable to reduce the amount of protein in BALF (A), but 24 h after two administrations, the elevated protein concentration was no longer significant (B). Error bars indicate standard deviation with average marked, n = 5–10, **P < 0.01 with a one-way ANOVA followed by Dunnett’s multiple comparison test versus sham control.

Figure 5.

Transcriptomic pathway analysis. Analysis of the acute phase response pathway in cardiac tissue at 2 (A) and 3 (B) h after 1080 exposure. Downregulated genes are shown in green and upregulated genes are in red. The P value and fold change, respectively, of significantly altered genes are shown adjacent to the affected genes. The acute phase response pathway was predicted to be activated at 2 h postexposure (Z-score: 2.31) but not at 3 h (Z-score: 1.79).

Figure 6.

White blood cell (WBC) count. Treated animals were given MB (5 mg/kg, SC) and MSG (250 mg/kg, IP). Injections were administered at 0.5 and 2 h after exposure. From animals that survived to 24 h, blood was collected from the descending aorta, and a complete blood count was performed with an automated hematology analyzer. Overall, the WBC counts were decreased in exposed animals (A), which was driven largely from a reduction in lymphocytes (C). Neutrophils were elevated in both exposed and treated control animals (B), while monocytes remained largely unchanged (D). Dash = average, n = 6–110, *P < 0.05 with a one-way ANOVA followed by Dunnett’s multiple comparison test versus sham control.