Methylene Blue - A Potential Therapeutic Treatment for Sodium Fluoroacetate Poisoning.

Sodium fluoroacetate (1080) is a toxic metabolic poison with no known antidote. Its use is strictly regulated within the United States, but it is commonly used as a pesticide in Australia, Israel, Japan, Mexico, New Zealand, and South Korea. 1080 has been identified as a possible terrorist weapon, and exposure is extremely difficult to diagnose given its overlapping symptoms with other common diseases. An abandoned U.S. patent indicated that methylene blue (MB) could be used as a potential countermeasure to 1080 poisoning when combined with monosodium glutamate (MSG). In this study, MB was used as a stand-alone treatment to determine if it is capable of rescuing cellular metabolism or decreasing 1080 lethality. Male Sprague-Dawley rats were exposed to increasing concentrations of 1080 (0.5, 1.1, 2.1, 4.2, and 8.4 mg/kg) based on a LD50 logarithmic scale. A low dose of MB (11 mg/kg) was administered via intraperitoneal injection (i.p.) at 0.5 hr and 2 hr post-exposure. Euthanasia occurred 24 hours post-exposure and blood was collected from the descending aorta. Serum was separated after collection and stored at -80 °C. Bronchoalveolar lavage fluid (BALF) was also collected from the right lung using phosphate buffered saline (PBS) and then stored at -80 °C. Gas chromatography-mass spectrometry (GCMS) was performed on serum samples to identify potential metabolic compounds of interest. From the GCMS data, L-carnitine and methylmalonic acid (MMA) were proposed as potential markers of 1080 exposure. To quantitate circulating levels, an L-carnitine colorimetric/fluorometric assay and a methylmalonic acid enzyme-linked immunosorbent assay (ELISA) were performed. Based on previous data indicating cardiopulmonary effects of 1080, a high sensitivity rat cardiac troponin-I ELISA was used to determine cardiac damage, and BALF was tested for the presence of protein and hemoglobin. The coenzyme-A linked form of MMA is converted into succinyl-CoA for use in the TCA cycle. MMA levels were relatively low and unchanged across all groups, except for rats receiving a 1.1 mg/kg dose of 1080. L-carnitine is a compound involved in metabolism via the carnitine shuttle system, and is activated when ATP concentration decreases, allowing transport of fatty acid chains into the mitochondria to help replenish the ATP supply. Carnitine levels varied across the exposure concentrations in the groups treated with MB, but at the 2.1 mg/kg exposure dose, treatment with MB appeared to decrease the amount of circulation carnitine. Hemorrhagic pulmonary edema is a symptom of 1080 poisoning, and MB was able to significantly reduce the presence of hemoglobin in BALF for 0.5, 1.1, and 2.1 mg/kg groups. Treatment with MB was able to shift the LD50 curve of 1080 to 3.29 mg/kg from 2.18 mg/kg, giving a protective ratio of 1.53. MB was also able to increase the probability of survival of animals at the 8.4 mg/kg exposure dose. Overall, while MB has shown some promise as treatment for 1080, further testing is needed to determine how viable it is and identify any likely biomarkers for screening to lead to an accurate diagnose.