Brevetoxin Metabolism and Physiology - A Freshwater Model of Morbidity in Endangered Sea Turtles

The dinoflagellate Karenia brevis is one organism responsible for harmful algal blooms (HABs) that severely impact marine life. K. brevis produces a suite of neurotoxins referred to as brevetoxins (PbTx) which bind to voltage-gated sodium channels (VGSCs) in excitable tissues, affecting cellular permeability leading to a suite of symptoms and potentially cell death. Brevetoxicosis is difficult to treat in sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. Due to their threatened and endangered status, experimental exposures cannot be performed to determine the fate of brevetoxin in sea turtle tissues, making it difficult to design appropriate treatments. The freshwater turtle, Trachemys scripta, was utilized as a model for brevetoxin exposure in turtles. Turtles were exposed to intratracheal instillation (10.53μg/kg) or oral dosing (33.48μg/kg) of PbTx-3 3x weekly over a period of 2-4 weeks. Tissues and fluids were collected for ELISA to determine PbTx-3 uptake and distribution, routes of excretion and rates of clearance (1h-1wk post-exposure). Tissues were also preserved for histopathology. Primary turtle neuronal cell cultures were exposed to PbTx-3 in the presence and absence of various agonists and antagonists to determine brevetoxin’s mode of action. PbTx-3 was widely distributed in all tissues and fluids following both intratracheal and oral exposures, but was largely cleared from the system within 24 hours; PbTx-3 moved into the bile and feces over 48h post exposure indicating that this is the main route of excretion. While exposed animals showed clear behavioral symptoms of toxicity including muscle twitching, swimming in circles, and ataxia, there was no evident tissue pathology. Despite the evident behavioral effects, turtle neurons are surprisingly resistant to PbTx-3, with an EC50 significantly higher than is seen in mammalian neurons. While PbTx-3 exposure resulted in significant Ca2+ influx, various antagonists prevented Ca2+ influx when added with PbTx-3 confirming the mechanism of action through VGSCs. Upregulation of Hsp72 in the turtle brain could be enhancing cell survival. Based on results, intralipid treatment post PbTx-3 exposure rapidly decreases symptoms and proves to be a suitable treatment for toxin exposure.