Brevetoxin Metabolism and Physiology – a Freshwater
BREVETOXIN METABOLISM AND PHYSIOLOGY – A FRESHWATER
MODEL OF MORBIDITY IN ENDANGERED SEA TURTLES
by
Courtney Christine Cocilova
A Dissertation Submitted to the Faculty of
The Charles E. Schmidt College of Science
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
Florida Atlantic University
Boca Raton, FL
May 2017 Copyright by Courtney Christine Cocilova 2017
ii Courtney Christine Cocilova ACKNOWLEDGEMENTS
I would like to begin by thanking my advisor Dr. Sarah Milton for her guidance,
unwavering support and long hours spent working with me throughout my dissertation
work. Words cannot express my gratitude for believing in me from the very beginning. I
am also thankful for my collaborators and the organizations that made this project
possible: Dr. Leanne Flewelling and April Granholm at Florida Fish and Wildlife
Conservation Commission, Dr. Gregory Bossart at Georgia Aquarium, Dr. Catherine
Walsh at Mote Marine Laboratory, Dr. Charles Manire at Loggerhead Marinelife Center, and to my undergraduate student Morgayne Leech at Florida Atlantic University. To my committee members Dr. John Baldwin, Dr. Gregory Bossart, and Dr. Ken Dawson-
Scully, I appreciate the knowledge and direction you have provided me with during my
time in the Ph.D. program. I would also like to acknowledge National Oceanic and
Atmospheric Administration (NOAA) and their Ecology and Oceanography of Harmful
Algal Bloom (ECOHAB) program for funding this project, the multiple fellowship and
scholarship foundations that supported me and my research during my doctoral
dissertation, and to the conference travel awards that allowed me to travel both nationally
and internationally to present my research and network with others.
iv ABSTRACT Author: Courtney C. Cocilova
Title: BREVETOXIN METABOLISM AND PHYSIOLOGY – A FRESHWATER MODEL OF MORBIDITY IN ENDANGERED SEA TURTLES
Institution: Florida Atlantic University
Dissertation Advisor: Dr. Sarah Milton
Degree: Doctor of Philosophy
Year: 2017
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
v 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.
vi BREVETOXIN METABOLISM AND PHYSIOLOGY – A FRESHWATER
MODEL OF MORBIDITY IN ENDANGERED SEA TURTLES
LIST OF TABLES ...... xi
LIST OF FIGURES ...... xii
CHAPTER I: INTRODUCTION ...... 1
Algal blooms ...... 1
Dinoflagellates ...... 3
Brevetoxin (PbTx) ...... 3
Normal synaptic transmission ...... 4
Brevetoxin exposed neurons ...... 5
Calcium ...... 5
Human Impacts ...... 6
Sea turtle impacts ...... 7
Freshwater turtles ...... 8
Diamondback terrapins ...... 9
Goals of this Study ...... 9
CHAPTER II: BREVETOXIN’S MODE OF ACTION IN THE FRESHWATER
TURTLE NEURONS ...... 18
Introduction ...... 18
Materials and methods ...... 21
Animals ...... 21
vii Cell culture preparation ...... 21
Cytotoxicity assays ...... 22
Intracellular Ca2+ monitoring ...... 22
Drugs and chemicals ...... 23
Statistics ...... 24
Results ...... 24
Turtle neurons are highly resistant to PbTx-3 toxicity ...... 24
PbTx-3 increases intracellular Ca2+ in turtle neurons ...... 24
Increases in intracellular Ca2+ are abrogated by TTX, MK-801, CNQX and
TET ...... 25
Discussion ...... 27
Conclusion ...... 33
References ...... 43
CHAPTER III. TISSUE UPTAKE, DISTRIBUTION AND EXCRETION OF
BREVETOXIN-3 AFTER ORAL AND INTRATRACHEAL
EXPOSURE IN THE FRESHWATER TURTLE TRACHEMYS
SCRIPTA AND THE DIAMONDBACK TERRAPIN MALACLEMYS
TERRAPIN ...... 52
Introduction ...... 52
Materials and methods ...... 53
Experimental Animals ...... 53
Brevetoxin ...... 54
Tissue and fluid collection ...... 55
viii Brevetoxin analysis ...... 56
Histopathology ...... 58
Statistics ...... 58
Results ...... 59
Neurological and muscular deficits post PbTx-3 exposure ...... 59
PbTx-3 Tissue Distribution ...... 59
Rates of toxin clearance ...... 60
Histopathological findings ...... 62
Discussion ...... 63
References ...... 79
CHAPTER IV. INTRAVENOUS LIPID EMULSION AS AN EFFECTIVE
TREATMENT PLAN FOR SEA TURTLES EXPOSED TO PBTX-3 ...... 85
Introduction ...... 85
Material and methods ...... 88
Experimental Animals ...... 88
Brevetoxin ...... 89
Treatments ...... 89
Tissue collection ...... 91
Behavioral analysis ...... 92
Statistics ...... 93
Results ...... 93
Symptoms of brevetoxin exposure ...... 93
Bile clearance post cholestyramine treatment ...... 94
ix Symptoms post ILE treatment ...... 94
Clearance of PbTx-3 from the tissues and excretion systems with ILE
treatment ...... 95
Discussion ...... 96
References ...... 112
CHAPTER V. CELLULAR RESPONSES IN TURTLES EXPOSED TO PBTX-3 ..... 115
Introduction ...... 115
Materials and methods ...... 123
Animals ...... 123
Brevetoxin ...... 123
Tissue collection ...... 124
Protein extraction ...... 124
Western blotting ...... 124
Statistics ...... 125
Results ...... 125
Hsp72 expression in tissues post PbTx-3 exposure ...... 125
Discussion ...... 126
References ...... 134
CHAPTER VI. CONCLUSIONS AND FUTURE DIRECTIONS ...... 146
References ...... 150
x LIST OF TABLES
Table 1. Exposure time points post PbTx-3 ...... 73
Table 2. Postmortem log ...... 74
Table 3. Dose curve for IT and oral PbTx-3 exposures ...... 75
Table 4. Distribution of PbTx-3 in T. scripta post oral exposure ...... 76
Table 5. Distribution of PbTx-3 in T. scripta post IT exposure ...... 77
Table 6. Distribution of PbTx-3 in M. terrapin post oral exposure ...... 78
Table 7. Behavioral symptoms post IT PbTx-3 ...... 103
Table 8. Behavioral symptoms post cholestyramine treatment ...... 104
Table 9. Behavioral symptoms pre- and post-intralipid treatment ...... 105
xi LIST OF FIGURES
Figure 1: PbTx-3 binding schematic ...... 35
Figure 2: Cell death increases in turtle neurons post PbTx-3 exposure ...... 36
Figure 3: Intracellular calcium increases post PbTx-3 exposure ...... 37
Figure 4: Intracellular calcium increases post glutamate and ouabain exposure ...... 38
Figure 5: Increases in intracellular calcium are abrogated by antagonists ...... 39
Figure 6: Mean fluorescence data (0-200 min) reflecting intracellular calcium levels .... 40
Figure 7: Mean fluorescence data (80-200 min) reflecting intracellular calcium
levels ...... 41
Figure 8: Fluorescence images following PbTx-3 ...... 42
Figure 9. Distribution of PbTx-3 in the excretion and detoxification systems post oral
exposure ...... 71
Figure 10. Distribution of PbTx-3 in the excretion and detoxification systems post IT
exposure ...... 72
Figure 11. Distribution of PbTx-3 in tissues and fluids 1h and 24h post exposure ...... 106
Figure 12. Distribution of PbTx-3 post cholestyramine treatment ...... 107
Figure 13. Distribution of PbTx-3 1h post exposure + pre- and post-ILE treatment ..... 108
Figure 14. Distribution of PbTx-3 24h post exposure + pre- and post-ILE treatment ... 109
Figure 15. Distribution of PbTx-3 (1h) post ILE treatment ...... 110
Figure 16. Distribution of PbTx-3 (24h) post ILE treatment ...... 111
Figure 17. PbTx-3 exposure induces Hsp72 expression in the brain ...... 132
xii CHAPTER I: INTRODUCTION
Algal blooms
Marine animals, such as manatees, dolphins, sea turtles, and sea birds are faced with many environmental threats and stressors that can be detrimental to survival. These threats may include climate change, disease, habitat loss, and pollutants. When excess nutrients are present in the water, for example from land run-off and/or fertilizers, algal blooms may occur resulting from the rapid reproduction of algal species. Algal blooms occur worldwide (Brand and Compton, 2007; Anderson et al., 2008) and may consist of non-toxic or toxin-producing algae, depending on the species. Algal blooms occur in freshwater or marine environments and can accumulate in mass quantities creating algal mats, which deplete the water of oxygen and create hypoxic zones. This adds stress to the ecosystem, and therefore can injure animals (Hallegraeff, 1993). Algal blooms may contaminate the local water systems and managing the nutrient loads that disperse into aquatic ecosystems will not only protect animals and prevent a decline in biodiversity, but will also ensure the water is safe for human consumption.
Some algal species are considered harmful because they produce and release potent toxins that can cause fatalities among animals that encounter them. In spring of
2002, 34 endangered Florida manatees died off the coast of southwest Florida from a toxic bloom (Flewelling et al., 2005), and in 2004, 107 bottlenose dolphins were stranded
1 dead along the panhandle in an unusual event that was later determined to be caused by a
harmful algal bloom (Twiner et al., 2012). Loggerhead sea turtles have been impacted, with more than 109 deaths in 2005, plus an additional 70 in 2006 also attributed to toxin exposure released during a bloom (Walsh et al., 2010). Harmful algal blooms (HABs,
Red tides) are dependent on the conditions of the water. Typically an overload of nutrients, such as nitrogen and phosphorus (Hardison et al., 2013), an increase in water temperature, changes in salinity, and available sunlight combine to trigger the rapid reproduction of algal species, known as dinoflagellates. Some dinoflagellates release toxins that have varying effects on wildlife populations depending on the route of intoxication, the degree and length of exposure in relation to the size of the animal, as
well as diet. Bioaccumulation and biomagnification are both likely to play a role in the
harmful effects marine animals face during blooms; toxin levels reported in the stomach
contents and tissues of stranded bottlenose dolphins following an algal bloom were
similar to results reported from a 2004 mortality event during a non-bloom (Fire et al.,
2008). A recent study has also reported toxins in the blood of nesting female sea turtles
one year after a major harmful algal bloom occurred (Perrault et al., 2016) confirming
that the toxin persists in the environment. Toxins in the water column and in lower
trophic organisms may thus result in nearly continuous exposure for marine animals in
areas prone to HAB outbreaks, such as the near annual blooms now occurring in the Gulf
of Mexico. Even if the exposure does not result in immediate mortalities, animals may be
more susceptible to disease or to other additional stressors due to immune suppression
(Perrault et al., 2014; Walsh et al., 2015, 2010, 2005).
2 Dinoflagellates
Toxic HABs are generally caused by dinoflagellates: unicellular, eukaryotic, protist
organisms that arose about ~400 MYA (Fensome et al., 1999) and still prosper today.
These microscopic, planktonic, flagellated organisms make up the Phylum
Dinoflagellata. Many dinoflagellates are photosynthetic and are typically found in marine
habitats, but may be found in freshwater as well. Some species of dinoflagellates are
capable of releasing potent neurotoxins such as the saxitoxins and brevetoxins that can
accumulate in shellfish leading to shellfish poisoning in humans, and the ciguatoxins
which accumulate in large fish, leading to food borne illnesses in humans after
consumption. When marine animals are exposed to toxins, they may exhibit a wide range
of behavioral changes that can lead to death. A well-studied dinoflagellate known as
Karenia brevis, common in the Gulf of Mexico, is generally dormant, but when environmental conditions are ideal, they rapid reproduce and upon die off, release potentially fatal toxins, jointly referred to as the brevetoxins.
Brevetoxin (PbTx)
The brevetoxins (PbTx’s) are a family of cyclic, polyether, lipophilic molecules that can easily pass through cell membranes, and cross the buccal mucosa and blood brain barrier. Brevetoxins are classified into two groups, brevetoxin A and brevetoxin B, based on differences in their chemical backbone; brevetoxin A has 10 rings and brevetoxin B has 11 rings (Poli et al., 1986) and is more abundant in the environment (Lin et al., 1981).
Different derivatives of brevetoxins form when there are alterations to the molecular side chains (Baden et al., 2005) and these are found in at least 10 different forms (PbTx-1-10), with PbTx-2 being the most common along with its metabolic derivative PbTx-3.
3 Brevetoxins specifically bind to and activate voltage-gated sodium channels (VGSCs) in electrically excitable cells like nerves and muscles (Berman & Murray, 1999) which causes neurological and muscular deficits in animals that become exposed. A cascade of intracellular pathological events occur that cause cell destruction and programmed cell death, or apoptosis. Apoptosis is a genetically regulated, finely tuned process that is involved in the regulation of cell populations, but can be upregulated by noxious events
(Kerr, et al., 1972).
Normal synaptic transmission
Because brevetoxins binds to VGSCs in cells like nerves, this toxin interferes
with normal neuronal function. Under normal physiological conditions, when a stimulus
occurs in a neuron, VGSCs become activated, causing an influx of sodium into the cell
and changing the membrane potential to a more positive state. Upon reaching threshold,
an action potential will be generated in the presynaptic neuron that travels down the
length of the axon, causing voltage-gated Ca2+ channels (VGCCs) to become activated.
The rise in cytosolic Ca2+ induces the release of the neurotransmitter (Davison, 1989) from vesicles into the synapse. Glutamate is a key excitatory neurotransmitter in the brain and when released, results in the secondary activation of N-methyl-D-aspartate (NMDA)
and/or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on the
postsynaptic neuron permitting the flow of cations through the channels. The entry of
calcium and sodium ions into the post synaptic cell leads to depolarization and interaction
with signal transducing pathways that ultimately elicits a response. When there is not a
continuous stimulus present, the cells return back to their resting state and restore their
homeostatic ion balance.
4 Brevetoxin exposed neurons
At high concentrations, brevetoxins can directly affect excitable cells including
skeletal and cardiac systems leading to neuronal deficits, resulting in lack of muscular
coordination, convulsions, paralysis, and behavior changes (Dechraoui et al., 2006;
Dechraoui and Ramsdell, 2003),. When a cell has been exposed to brevetoxin, the toxin binds to the VGSCs, causing a conformational change that allows the channels to remain open. The continuous influx of sodium into the cell results in depolarization and subsequent Ca2+ influx into the presynaptic cell, which greatly elevates levels of
glutamate in the synapse. Upon glutamate binding to postsynaptic N-methyl-D-aspartate
(NMDA) and/or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors, a cascade of excitotoxic events is triggered, resulting from the excess entry of
calcium and sodium into the postsynaptic cell and/or the release of intracellular calcium
stores. An increase in free radical production and oxidative stress may occur, leading to
pro-apoptotic pathway activation (Sattler & Tymianski 2000). Calcium plays a big role in
intracellular signaling cascades and there is a delicate balance of ions, inside and outside
of the cell, required for homeostasis. Ion imbalances, due to the increased release of
excitatory neurotransmitters into the synapse, causes synaptic transmission between
neurons to be disrupted leading to destruction of the cell; activation of pro-apoptotic pathways may be triggered to enhance cell survival.
Calcium
Calcium (Ca2+) is an essential signaling molecule that acts as a secondary messenger, is involved in muscular contraction and hormone secretion, and plays a role in neurotransmitter release from neurons. Intracellularly, calcium is found in the
5 cytoplasm, in the two major Ca2+ regulating organelles: mitochondria (Rossi and
Lehninger, 1964) and endoplasmic reticulum (Meldolesi and Pozzan, 1998), and is also
present extracellularly. Maintaining homeostatic levels of calcium, both intracellularly
and extracellularly, is tightly controlled and vital for the body to function normally.
When there is an imbalance of calcium, for instance too much calcium entering the cell due to elevated levels of glutamate caused by over-excitation, the cell may undergo a loss of ion balance, cell swelling and therefore apoptosis (Choi, 1987). High levels of intracellular calcium may also trigger enzymatic molecules like proteases, lipases and endonucleases causing neuronal injury (Kass and Orrenius, 1999).
Human Impacts
In addition to effects on marine life, brevetoxin can impact people. Brevetoxin exposure in humans can occur through the consumption of contaminated shellfish (such as clams, mussels, and oysters) resulting in neurotoxic shellfish poisoning (NSP) which results in neurological and gastrointestinal problems. Symptoms can include mild to severe nausea, vomiting, numbness, tingling in the lips and hands, and ataxia (Watkins et al., 2008). Exposure via inhalation of ocean aerosols leading to respiratory complications, particularly among asthmatic patients, is also common (Kalaitzis et al., 2010; Backer et al., 2005; Fleming et al., 2007; Kirkpatrick et al., 2006). There have been no reported fatalities in humans, however gastrointestinal hospitalizations have been reported to increase by 40% during algal blooms; supportive care is the main form of treatment
(Watkins et al., 2008). Supportive care is also the primary treatment of brevetoxin exposed animals such as sea turtles, because brevetoxin’s mode of action and
6 physiological impacts in marine animals have not been previously determined, which makes it difficult to develop focused treatment plans.
Sea turtle impacts
Seven species of sea turtles are inhabit our oceans worldwide and nearly all are listed as either threatened or endangered. These animals are faced with many threats including climate change, fisheries bycatch, light and ocean pollution, habitat destruction, and eutrophication which leads to HABs. Sea turtles play important roles in our ecosystem by sustaining sea grass beds and coral reefs during foraging, contributing to the food web, and creating symbiotic relationships with other organisms (Goatley et al.,
2012; Majewska et al., 2015). Five out of the 7 species of sea turtles are native to and nest and/or forage around the state of Florida, where HABs arise almost annually on the
SW coast. Juvenile and sub-adult turtles that reside and forage near areas where HABs frequently occur are at high risk for toxin exposure. Sea turtles are very robust and physically can withstand a variety of injuries, but are surprisingly sensitive to chemical insults (Milton and Lutz, 2003). They are air breathers with a large tidal volume that permits long dives, though this could in turn increase the degree of exposure for brevetoxin inhalation via ocean aerosols. Not only are animals at risk for toxicity via inhalation (Bossart et al., 1998) and ingestion, but exposure may be greater due to bioaccumulation and biomagnification (Fauquier et al., 2013); seagrasses, crustaceans, and fish accumulate toxins on or in their tissues and may impact animals that consume them, sometimes long after a bloom has occurred (Flewelling et al., 2005b; Naar et al.,
2007; Perrault et al., 2016). Both loggerheads that prey on filter-feeding invertebrates
(Bjorndal, 1996) and seagrass-grazing green sea turtles may thus be vulnerable to the
7 effects of brevetoxin bioaccumulation. Brevetoxin studies on these animals have only been performed through animal necropsies or from blood samples in turtles brought into rehabilitation facilities, which provides little data on initial organ impacts or toxin clearance rates post exposure. As sea turtles are listed as endangered or threatened, however, experimental determinations of toxin impacts cannot be conducted, which has thus led me to develop a commonly used model species, the freshwater turtle, for these studies.
Freshwater turtles
Freshwater turtles inhabit a variety of waters all over the globe and are widely used in scientific studies. They have been used as model systems for investigations into unique evolutionary adaptations like anoxia survival, hibernation, and the ability to dive for long periods of time. The freshwater turtle, Trachemys scripta, has been widely utilized in studies and there is a great deal of physiological data on this species (Robin et al., 1981; Lutz and Leone-Kabler, 1995; Jackson and Ultsch, 2010; Milton and Prentice,
2007; Ultsch and Jackson, 1982; Milton et al., 2006; Nayak et al., 2016; Kesaraju et al.,
2009; Larson et al., 2014). This animal was thus selected as a model system for brevetoxin exposure studies to investigate toxin impacts in order to design treatment strategies applicable for sea turtles exposed to brevetoxin. Despite the fact that freshwater turtles reside in different habitats than sea turtles, they are both long-lived, breath- holding, diving reptiles, suggesting that freshwater turtles are a suitable model system for brevetoxin exposures. An estuarine (brackish water) turtle, the diamondback terrapin,
(Malaclemys terrapin) was also used for a comparative model as its physiology may be
8 closer to sea turtles than T.scripta, but as it is listed as a species of special concern in
Florida, a large comparative study was not possible.
Diamondback terrapins
The diamondback terrapins are turtles native to inshore areas and estuaries of the
eastern United States. Since these turtles are mostly found in brackish environments, they
were utilized as a second comparative model organism. We limited the use of this animal
however, due to its protected status in Florida that allowed for only a small groups of
experimental animals to be studied.
Goals of this Study
The overarching goal of this study was to understand brevetoxin metabolism and
determine the impacts associated with toxin exposure in turtles. Since all nearly all
species of sea turtles are either threatened or endangered, the freshwater turtle was used
as a primary model system to investigate behavioral changes, initial organ system
impacts, uptake, distribution and elimination, rates of clearance, and the physiological
effects caused by brevetoxin exposure. Investigation of brevetoxin’s mode of action in
turtle neurons allowed for an understanding of the cellular mechanisms involved in
brevetoxin binding leading to cell activation and apoptosis; these data was compared to
the known mechanism of PbTx-3 determined in previous mammalian studies.
Examination of a variety of tissues and fluids at different time points post PbTx-3
exposure gave insight into what organ systems were affected and main routes of
elimination. The terrapins were part of this novel study looking into PbTx-3 organ system impacts and were compared to the freshwater turtle data.
9 A further study investigated the impacts of PbTx-3 on stress proteins at the cellular level to determine if pro-survival pathways are upregulated. I hypothesized that expression of Heat Shock Protein 72 (Hsp72), a molecular chaperone protein involved in the stabilization of proteins, would increase in tissues, protecting the cell from stress- activated destruction. Investigation into the expression of various MAP kinases, involved in regulating cell functions, is necessary determine the pro- and anti-apoptotic pathways triggered post toxin exposure. The ultimate goal of this research was to design appropriate treatments targeted to sea turtles exposed to red tides, following a more complete understanding of the distribution, clearance, and effects of brevetoxin in turtles.
This research is needed as HABs are becoming more frequent and severe, and affect not only wildlife but humans as well.
10 References
Anderson, D.M., Burkholder, J.M., Cochlan, W.P., Glibert, P.M., Gobler, C.J., Heil,
C.A., Kudela, R.M., Parsons, M.L., Rensel, J.E.J., Townsend, D.W., Trainer, V.L.,
Vargo, G.A., 2008. Harmful algal blooms and eutrophication: Examining linkages
from selected coastal regions of the United States. Harmful Algae 8, 39–53.
doi:10.1016/j.hal.2008.08.017.
Backer, L.C., Kirkpatrick, B., Fleming, L.E., Cheng, Y.S., Pierce, R., Bean, J.A., Clark,
R., Johnson, D., Wanner, A., Tamer, R., Zhou, Y., Baden, D.G., 2005. Occupational
exposure to aerosolized brevetoxins during Florida red tide events: effects on a
healthy worker population. Environ. Health Perspect. 113, 644–649.
doi:10.1289/ehp.7502.
Bjorndal, K.A. 1996. Foraging ecology and nutrition of sea turtles. In: Lutz PL, Musick
JA (eds) The biology of sea turtles. CRC Press, Boca Raton, FL, p 199–232
Bossart, G.D., Baden, D.G., Ewing, R.Y., Roberts, B., Wright, S.D., 1998.
Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996
epizootic: gross, histologic, and immunohistochemical features. Toxicol.Pathol 26,
276–282. doi:10.1177/019262339802600214.
Brand, L.E., Compton, A., 2007. Long-term increase in Karenia brevis abundance along
the Southwest Florida Coast. Harmful Algae 6, 232–252.
doi:10.1016/j.hal.2006.08.005.
Choi, D.W., 1987. Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7, 369–379.
Davison, A., 1989. Basic Neurochemistry: Molecular, Cellular, and Medical Aspects. J.
Neurol. Neurosurg. Psychiatry 52, 1021. doi:10.1212/WNL.39.3.460-b.
11 Dechraoui, M.Y., Ramsdell, J.S., 2003. Type B brevetoxins show tissue selectivity for
voltage-gated sodium channels: Comparison of brain, skeletal muscle and cardiac
sodium channels. Toxicon. 41, 919–927. doi:10.1016/S0041-0101(03)00088-6
Dechraoui, M.Y., Wacksman, J.J., Ramsdell, J.S., 2006. Species selective resistance of
cardiac muscle voltage gated sodium channels: Characterization of brevetoxin and
ciguatoxin binding sites in rats and fish. Toxicon. 48, 702–712.
doi:10.1016/j.toxicon.2006.07.032
Fauquier, D. a, Flewelling, L.J., Maucher, J., Manire, C. a, Socha, V., Kinsel, M.J., Stacy,
B. a, Henry, M., Gannon, J., Ramsdell, J.S., Landsberg, J.H., 2013. Brevetoxin in
blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia
brevis blooms in central west Florida. J. Zoo Wildl. Med. 44, 364–75.
Fensome, R.A., Saldarriaga, J.F., Fensome, T., 1999. Dinoflagellate phylogeny revisited:
reconciling morphological and molecular based phylogenies. Grana 38, 66–80.
doi:10.1080/00173139908559216.
Fire, S.E., Flewelling, L.J., Wang, Z., Naar, J., Henry, M.S., Pierce, R.H., Wells, R.S.,
2008. Florida red tide and brevetoxins: Association and exposure in live resident
bottlenose dolphins (Tursiops truncatus) in the eastern Gulf of Mexico, U.S.A. Mar.
Mammal Sci. 24, 831–844. doi:10.1111/j.1748-7692.2008.00221.x.
Fleming, L.E., Kirkpatrick, B., Backer, L.C., Bean, J.A., Wanner, A., Reich, A., Zaias, J.,
Cheng, Y.S., Pierce, R., Naar, J., Abraham, W.M., Baden, D.G., 2007. Aerosolized
red-tide toxins (brevetoxins) and asthma. Chest 131, 187–194. doi:10.1378/chest.06-
1830.
12 Flewelling, L.J., Naar, J.P., Abbott, J.P., Baden, D.G., Barros, N.B., Bossart, G.D.,
Bottein, M.-Y.D., Hammond, D.G., Haubold, E.M., Heil, C. a, Henry, M.S.,
Jacocks, H.M., Leighfield, T. a, Pierce, R.H., Pitchford, T.D., Rommel, S. a, Scott,
P.S., Steidinger, K. a, Truby, E.W., Van Dolah, F.M., Landsberg, J.H., 2005a. Red
tides and marine mammal mortalities. Nature 435, 755–756.
doi:10.1038/nature435755a.
Flewelling, L.J., Naar, J.P., Abbott, J.P., Baden, D.G., Barros, N.B., Bossart, G.D.,
Bottein, M.-Y.D., Hammond, D.G., Haubold, E.M., Heil, C.A., Henry, M.S.,
Jacocks, H.M., Leighfield, T.A., Pierce, R.H., Pitchford, T.D., Rommel, S.A., Scott,
P.S., Steidinger, K.A., Truby, E.W., Van Dolah, F.M., Landsberg, J.H., 2005b.
Brevetoxicosis: red tides and marine mammal mortalities. Nature 435, 755–756.
doi:10.1038/nature435755a.
Goatley, C.H.R., Hoey, A.S., Bellwood, D.R., 2012. The role of turtles as coral reef
macroherbivores. PLoS One 7. doi:10.1371/journal.pone.0039979.
Hallegraeff, G.M., 1993. On the global increase of harmful algal bloom. Wetl. 12, 2–15.
Hardison, D.R., Sunda, W.G., Shea, D., Litaker, R.W., 2013. Increased Toxicity of
Karenia brevis during Phosphate Limited Growth: Ecological and Evolutionary
Implications. PLoS One 8. doi:10.1371/journal.pone.0058545.
Kerr, J.F.R., Wyllie, A.H., Currie. A.R., 1972. Apoptosis: A Basic Biological
Phenomenon With Wide- Ranging Implications In Tissue Kinetics. J. Intern. Med.
258, 479–517. doi:10.1111/j.1365-2796.2005.01570.x.
13 Jackson, D.C., Ultsch, G.R., 2010. Physiology of hibernation under the ice by turtles and
frogs. J. Exp. Zool. Part A Ecol. Genet. Physiol. 313 A, 311–327.
doi:10.1002/jez.603.
Kalaitzis, J.A., Chau, R., Kohli, G.S., Murray, S.A., Neilan, B.A., 2010. Biosynthesis of
toxic naturally-occurring seafood contaminants. Toxicon 56, 244–258.
doi:10.1016/j.toxicon.2009.09.001.
Kass, G.E.N., Orrenius, S., 1999. Calcium signaling and cytotoxicity. Environ. Health
Perspect. doi:10.1289/ehp.99107s125.
Kesaraju, S., Schmidt-Kastner, R., Prentice, H.M., Milton, S.L., 2009. Modulation of
stress proteins and apoptotic regulators in the anoxia tolerant turtle brain. Journal of
Neurochem. 109, 1413–1426. doi:JNC6068 [pii]10.1111/j.1471-4159.2009.06068.x.
Kirkpatrick, B., Fleming, L.E., Backer, L.C., Bean, J.A., Tamer, R., Kirkpatrick, G.,
Kane, T., Wanner, A., Dalpra, D., Reich, A., Baden, D.G., 2006. Environmental
exposures to Florida red tides: Effects on emergency room respiratory diagnoses
admissions. Harmful Algae 5, 526–533. doi:10.1016/j.hal.2005.09.004.
Larson, J., Drew, K.L., Folkow, L.P., Milton, S.L., Park, T.J., 2014. No oxygen? No
problem! Intrinsic brain tolerance to hypoxia in vertebrates. Journal of Experimental
Biology. 217, 1024–39. doi:10.1242/jeb.085381.
Lutz, P.L., Leone-Kabler, S.L., 1995. Upregulation of the GABAA/benzodiazepine
receptor during anoxia in the freshwater turtle brain. Am. J. Physiol. 268, R1332–5.
14 Majewska, R., Santoro, M., Bolaños, F., Chaves, G., De Stefano, M., 2015. Diatoms and
other epibionts associated with Olive Ridley (Lepidochelys olivacea) sea turtles
from the Pacific Coast of Costa Rica. PLoS One 10.
doi:10.1371/journal.pone.0130351.
Meldolesi, J., Pozzan, T., 1998. The endoplasmic reticulum Ca2+ store: A view from the
lumen. Trends Biochem. Sci. doi:10.1016/S0968-0004(97)01143-2.
Milton, S.L., Nayak, G., Lutz, P.L., Prentice, H.M., 2006. Gene transcription of
neuroglobin is upregulated by hypoxia and anoxia in the brain of the anoxia-tolerant
turtle Trachemys scripta. J. Biomed. Sci. 13, 509–514. doi:10.1007/s11373-006-
9084-8.
Milton, S.L., Prentice, H.M., 2007. Beyond anoxia: the physiology of metabolic
downregulation and recovery in the anoxia-tolerant turtle. Comp Biochem Physiol A
Mol Integr Physiol 147, 277–290. doi:S1095-6433(06)00382-5
[pii]10.1016/j.cbpa.2006.08.041.
Naar, J.P., Flewelling, L.J., Lenzi, A., Abbott, J.P., Granholm, A., Jacocks, H.M.,
Gannon, D., Henry, M., Pierce, R., Baden, D.G., Wolny, J., Landsberg, J.H., 2007.
Brevetoxins, like ciguatoxins, are potent ichthyotoxic neurotoxins that accumulate in
fish. Toxicon 50, 707–723. doi:10.1016/j.toxicon.2007.06.005
Nayak, G., Prentice, H.M., Milton, S.L., 2016. Lessons from nature: Signaling cascades
associated with vertebrate brain anoxic survival. Exp. Physiol.
doi:10.1113/EP085673.
15 Perrault, J.R., Bauman, K.D., Greenan, T.M., Blum, P.C., Henry, M.S., Walsh, C.J.,
2016. Maternal transfer and sublethal immune system effects of brevetoxin exposure
in nesting loggerhead sea turtles (Caretta caretta) from western Florida. Aquat.
Toxicol. 180, 131–140. doi:10.1016/j.aquatox.2016.09.020.
Perrault, J.R., Schmid, J.R., Walsh, C.J., Yordy, J.E., Tucker, A.D., 2014. Brevetoxin
exposure, superoxide dismutase activity and plasma protein electrophoretic profiles
in wild-caught Kemp’s ridley sea turtles (Lepidochelys kempii) in southwest Florida.
Harmful Algae 37, 194–202. doi:10.1016/j.hal.2014.06.007.
Poli, M. a, Mende, T.J., Baden, D.G., 1986. Brevetoxins, unique activators of voltage-
sensitive sodium channels, bind to specific sites in rat brain synaptosomes. Mol.
Pharmacol. 30, 129–35.
Robin, E.D., Robin, D.A., Ackerman, R., Lewiston, N., Hance, A.J., Caligiuri, M.,
Theodore, J., 1981. Prolonged diving and recovery in the freshwater turtle,
Pseudemys scripta-I. Lung and blood gases, pH, lactate concentrations and “cation”
gap. Comp. Biochem. Physiol. Part A Physiol. 70, 359–364. doi:10.1016/0300-
9629(81)90190-0.
Twiner, M.J., Flewelling, L.J., Fire, S.E., Bowen-Stevens, S.R., Gaydos, J.K., Johnson,
C.K., Landsberg, J.H., Leighfield, T.A., Mase-Guthrie, B., Schwacke, L., van Dolah,
F.M., Wang, Z., Rowles, T.K., 2012. Comparative analysis of three brevetoxin-
associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida
Panhandle region (USA). PLoS One 7. doi:10.1371/journal.pone.0042974.
16 Ultsch, G.R., Jackson, D.C., 1982. Long-Term Submergence At 3 ° C of the Turtle ,
Chrysemys Picta Bellii , in Normoxic and Severely Hypoxic Water. J. Exp. Biol. 96,
11–28.
Walsh, C.J., Butawan, M., Yordy, J., Ball, R., Flewelling, L., De Wit, M., Bonde, R.K.,
2015. Sublethal red tide toxin exposure in free-ranging manatees (Trichechus
manatus) affects the immune system through reduced lymphocyte proliferation
responses, inflammation, and oxidative stress. Aquat. Toxicol. 161, 73–84.
doi:10.1016/j.aquatox.2015.01.019.
Walsh, C.J., Leggett, S.R., Carter, B.J., Colle, C., 2010. Effects of brevetoxin exposure
on the immune system of loggerhead sea turtles. Aquat. Toxicol. 97, 293–303.
doi:10.1016/j.aquatox.2009.12.014.
Walsh, C.J., Luer, C.A., Noyes, D.R., 2005. Effects of environmental stressors on
lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris. Vet.
Immunol. Immunopathol. 103, 247–256. doi:10.1016/j.vetimm.2004.09.026.
Watkins, S.M., Reich, A., Fleming, L.E., Hammond, R., 2008. Neurotoxic shellfish
poisoning. Mar. Drugs. doi:10.3390/md6030431.
17 CHAPTER II: BREVETOXIN’S MODE OF ACTION IN THE FRESHWATER
TURTLE NEURONS
Introduction
Harmful algal blooms (HABs, Red tides) occur when increased oceanic nutrient
loads and a rise in temperature trigger the rapid reproduction of certain single-celled
protists (dinoflagellates) (Anderson et al., 2008; Michalak et al., 2013); these blooms
occur worldwide and are increasing in scope and frequency (Van Dolah, 2000). Many
dinoflagellates produce and release potent neurotoxins that may impact both humans and
animal life, including saxitoxin, ciguatoxin, and brevetoxin. The brevetoxins (PbTx) are a
family of cyclic, lipophilic, polyether molecules produced by Karenia brevis, the
dinoflagellate most associated with red tides in the Gulf of Mexico (Flewelling et al.,
2005). These compounds are classified based on differences in their chemical backbone
and are found in at least 10 different forms (Baden et al., 2005). The three most abundant
brevetoxins in blooms occurring off the coast of SW Florida are PbTx-1 and PbTx-2, and
PbTx-3, the primary product of PbTx-2 reduction (Pierce and Henry, 2008). While PbTx-
2 is the primary intracellular brevetoxin in K. brevis, PbTx-3 is released by the dinoflagellates and may persist in the water after cell counts have decreased below detectable levels (Pierce and Henry, 2008). When brevetoxin is released into the surrounding waters, it easily passes through the cell membranes of other organisms, including the buccal mucosa (Mehta et al., 1991), gut, and the blood-brain barrier.
18 Florida red tides are a complex mix of brevetoxins (Cheng et al., 2005), but all of the
compounds specifically bind to site 5 on the α-subunit of voltage-gated sodium channels
(VGSCs) (Baden et al., 2005; Poli et al., 1986), causing a cascade of pathological events, which may eventually lead to cell death (Baden et al., 2005; Deshpande et al., 1993;
Murrell and Gibson, 2009). In the brain, the opening of VGSCs triggers the release of
excitatory neurotransmitters, including glutamate, and the secondary activation of N-
methyl-D-aspartate (NMDA) receptors (Berman and Murray, 1999) (Fig. 1). The
subsequent Ca2+ influx through these glutamate receptors triggers a cascade of events
leading to activation of proteases and lipases, oxidative stress and cellular apoptosis
(Sattler and Tymianski, 2000). At high concentrations, brevetoxins can thus directly
affect skeletal and cardiac systems and cause neuronal deficits (Dechraoui et al., 2006;
Dechraoui and Ramsdell, 2003).
In humans, brevetoxicosis occurs both through the consumption of contaminated shellfish resulting in neurotoxic shellfish poisoning (NSP), and via inhalation of ocean aerosols, leading to respiratory complications, particularly among asthmatic patients
(Kalaitzis et al., 2010; Backer et al., 2005; Fleming et al., 2007; Kirkpatrick et al., 2006).
Inhalation or ingestion of the toxin also affects animal health, with large blooms resulting
in mass strandings and die-offs of fish, marine birds (Kreuder et al., 2002), mammals
(Flewelling et al., 2005; Twiner et al., 2012), and sea turtles (Fauquier et al., 2013), with
additional longer-term impacts resulting from bioaccumulation and biomagnification up
the food chain (Walsh et al., 2010; Bossart et al., 1998). Animals exposed to brevetoxin
exhibit symptoms that may include ataxia, head bobbing, muscle twitching, long term
behavioral changes, and partial to complete paralysis, depending on the amount of
19 brevetoxin consumed (Baden and Mende, 1982). Depression of pulmonary and cardiac
function, immune suppression, induced inflammation, and possible genomic effects have
also been reported (Abraham et al., 2005; Borison et al., 1985; Baden et al., 2005).
Large numbers of marine animal mortalities have thus been linked to
dinoflagellate blooms. In 2005, more than 109 loggerhead sea turtles deaths were
attributed to brevetoxin exposure with over 70 sea turtle deaths in 2006 (Fauquier et al.,
2013). Die-offs associated with red tides primarily affect juvenile and sub-adult turtles
that are resident in nearshore waters (Fauquier et al., 2013). Symptoms of brevetoxicosis
in sea turtles admitted to rehabilitation facilities include muscle twitching, jerky body
movements, circling, and unresponsiveness (Fauquier et al., 2013), which suggests that
PbTx acts on the nervous and muscular systems in a similar manner as reported in mammalian studies. However, as ectothermic, diving reptiles, the susceptibility of sea turtles to PbTx may be quite different to that of laboratory animals; while generally physically quite robust, sea turtles are surprisingly susceptible to biological and chemical insults (Lutz and Milton, 2004). Since all species of sea turtles are listed as either
threatened or endangered, however, the effects of brevetoxin exposure cannot be
experimentally addressed in these animals. We have thus developed the freshwater turtle
(Trachemys scripta) as a model system to study the effects of toxin exposure in turtles; with a better understanding of the effects of brevetoxin in turtles we can work to develop targeted treatments for affected animals. In this study, primary neuronal cell cultures derived from T. scripta were used to determine the toxicity of PbTx-3 and its mode of action in turtle neurons using a variety of agonists and antagonists along the pathway from VGSC activation to the opening of NMDA channels (Fig. 1). Calcium influx into
20 the cells was monitored by fluorescent microscopy using the Ca2+ sensitive dye fluo-3 acetoxymethylester (Fluo-3 AM) as a secondary indicator of depolarization (Berman and
Murray, 2000). Results from pharmacological intervention with the PbTx-3 pathway suggest that the mechanism of action is similar in turtles and mammals, but that turtles are more resistant to the toxin at the cellular level.
Materials and methods
Animals
All animal experiments were approved by the Florida Atlantic University
Institutional Animal Care and Use Committee. Unsexed juvenile red eared sliders,
(Trachemys scripta), approximately 7-10 cm in length, were obtained from a commercial supplier (Niles Biological, Inc., Sacramento, CA) and maintained in tanks at room temperature (22oC +/- 3oC, 50% humidity +/- 4%) on a 12h day/night cycle. Turtles were cleaned and fed (commercial aquatic turtle food, to satiety) 3x weekly.
Cell culture preparation
The isolation and maintenance of freshwater turtle primary neuronal cell cultures using a density centrifugation technique has been described previously (Milton et al.,
2007). Briefly, turtles were euthanized via decapitation and the brain extracted. Finely minced cortical tissue was digested with collagenase (25U/ml, Gibco/Invitrogen, Grand
Island, NY), dispase (0.32U/ml, Gibco/Invitrogen) and hyaluronidase (1300U/ml, Sigma-
Aldrich, St. Louis, MO) in 4ml of minimal essential media (MEM) supplemented with
10% FBS (Gibco/Life Technologies, Grand Island, NY) and 1% penicillin (Gibco/Life
Technologies). Tissues were gently rocked for 4 hours followed by centrifugation at 4500
RPM for 15 minutes at 22oC using Opti-prep (Sigma-Aldrich) density gradient. The
21 neuronal fraction was plated into 24 well plates (~1 x 105 cells per mL). Neurons were
o maintained in FBS and penicillin media at 30 C in a 5% CO2 incubator for 4 weeks. The
media in each well was changed on the third day post-plating and weekly thereafter
(50/50 change) until cells reached 4 weeks of age.
Cytotoxicity assays
Primary neuronal cultures were exposed to PbTx-3 in Locke’s Buffer (in mM:
154 NaCl, 5.6 KCl, 1.0 MgCl2, 2.3 CaCl2, 8.6 HEPES, 5.6 glucose, 0.1 glycine, pH 7.4) over a range of concentrations from 10nM to 2000nM in order to assess neuronal cell death. Cells were gently rocked for 2 hours with PbTx-3 at either 22oC or 37oC. Neurons
were stained with propidium iodide (PI, 3uM, excitation ~535 nm), to evaluate dead cells
and acridine orange (AO, 3.3uM, excitation ~500 nm) to view live cells, and observed via
inverted fluorescent microscopy (Zeiss). Cell death was calculated as a ratio of dead
neurons to total neurons averaged from 3 samples per well with plates derived from a
minimum of 4 animals.
Intracellular Ca2+ monitoring
Since PbTx binding results in increased levels of intracellular calcium
downstream of the opening of VGSCs (Berman and Murray, 2000), we utilized the Ca2+
indicator, Fluo-3 AM to monitor depolarization. Primary neuronal cell cultures were preloaded with Fluo-3 AM (6uM) in Locke’s Buffer for 30 minutes at room temperature
(22oC). Cells were washed three times with Locke’s buffer and remained in the buffer for
an additional 30 minutes. PbTx-3 was then added to 500ul of Locke’s buffer medium
(1750nM PbTx-3 final concentration, determined from preliminary assays to promote strong calcium influx) and incubated at either 22oC (turtle/room temperature) or at 37oC
22 (for comparison to mammalian data) for 45 minutes. Cells were excited at 488 nm and
Ca2+ -bound Fluo-3 AM emission of ~525 nm examined via fluorescent microscopy.
Digital images were taken at intervals using Q capture and AmScope software. Series images were taken every 10 minutes for PbTx-3 treated cells versus control cells to monitor and compare calcium changes over time (190 total minutes). Images were analyzed by selecting a region of interest (ROI) around fluorescent neurons and measuring the intensity of the pixels in that area over a period of time. Background fluorescence was subtracted from control and treated neurons. Images were analyzed for mean fluorescence using NIH Image J Software and data presented as a change in fluorescence over control (ΔF/F0) (Raw data and representative images are shown in Figs.
6, 7 and 8. Data were graphed using Sigma Plot 11.0.
Drugs and chemicals
Brevetoxin (PbTx-3) was purchased from LKT Laboratories (St. Paul, Minnesota) and was dissolved in ethanol and mixed with Locke’s buffer. Tetrodotoxin (TTX, 2uM) and the NMDA receptor blocker (MK-801, 50uM) were purchased from Abcam
(Cambridge, MA). Ca2+ indicator, Fluo-3 AM was purchased from Thermo Fisher
Scientific (Waltham, Massachusetts), and L-Glutamic acid monosodium salt (glutamate,
200uM), ouabain (mM), tetanus toxin (TET, 25nM) and 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX, 200uM) were all purchased from Sigma-Aldrich Co. (St. Louis, Missouri).
All drugs were mixed in appropriate solvent and stored until use. Control neurons were treated with 0.16% ethanol (final concentration) in Locke’s Buffer.
23 Statistics
Data were analyzed using one-way analysis of variance (ANOVA) followed by
Holm-Sidak Test (Holm-Sidak) pairwise comparison test or by Student’s t-test using
Sigma Plot 11.0 (Systat Software, Inc, San Jose, California). Significant differences are
indicated by asterisks where one asterisk (*) represents p≤0.05 and three asterisks (***)
represents p≤0.001.
Results
Turtle neurons are highly resistant to PbTx-3 toxicity
We investigated cell death in primary neuronal cell cultures in the presence of
PbTx-3 in Locke’s buffer. Originally, we tested concentrations based on a previous
mammalian study conducted by Berman and Murray (1999), which reported
concentration dependent cell death in rat cerebellar granule neurons (CGNs) for PbTx-3
concentrations ranging from 10-100nM. However, cell death did not increase in treated
turtle neurons compared to controls cells for PbTx-3 doses within this range. For
concentrations ranging from 100-2000nM, neuronal cell death did increase in a dose-
dependent manner (Fig. 2). Cells incubated at 22oC were less sensitive to PbTx-3 than
o o cells exposed at 37 C. The EC50 was ~1350nM for 2h exposure to PbTx-3 at 22 C,
whereas the EC50 was approximately 800nM for cells incubated 2h at the mammalian temperature of 37oC.
PbTx-3 increases intracellular Ca2+ in turtle neurons
2+ 2+ To determine the effect of PbTx-3 on intracellular Ca ([Ca ]i) levels, we added
Fluo-3 AM to the medium prior to PbTx-3 treatment (as described above) and changes in fluorescence were monitored over time. PbTx-3 was added to the medium following 90
24 2+ minutes of baseline monitoring, with [Ca ]i becoming significantly different from
2+ control within 20 minutes; the subsequent increase in [Ca ]i was most pronounced over
2+ the period ranging from 40-100 minutes following addition of PbTx-3. Changes in [Ca ]i
in the untreated control cells plateaued over the same period (Fig. 3). L-Glutamic acid
(glutamate, 200uM), known to bind to NMDARs and cause Ca2+ influx, was used as a
2+ positive control. While [Ca ]i increased slowly in untreated control cells over 190
minutes, both glutamate and PbTx-3 caused a significantly greater increase over the same
time frame (Fig. 4). We also used the Na+/K+-ATPase pump inhibitor ouabain as a positive control to induce depolarization directly (Pék-Scott and Lutz, 1998), which resulted in similar increases in fluorescence as glutamate treatments (Fig. 4).
Increases in intracellular Ca2+ are abrogated by TTX, MK-801, CNQX and TET
To determine if brevetoxins cause an acute autocrine excitotoxicity in primary turtle neuronal cultures as has been reported in rat CGNs (Berman and Murray, 1999), downstream effects of PbTx-3-induced depolarization were investigated via pharmacological interventions in the presence of a fixed concentration of PbTx-3
(1750nM at 22oC). We tested the putative mechanism of PbTx-3 action wherein opening
VGSCs results in depolarization-induced glutamate release and the subsequent stimulation of NMDA receptors (Fig. 1). Tetrodotoxin (TTX), a non-competitive antagonist of VGSCs, has been shown to counteract brevetoxin and hinder sodium influx in mammalian cells, thus preventing cellular depolarization and calcium influx (Huang et al., 1984). As in mammalian studies, in the presence of PbTx-3, TTX (2uM) abrogated
2+ the Ca influx into the turtle neurons; ΔF/F0 was similar to control cells (Fig. 5). There were no statistical differences between PbTx-3 + drug groups compared to sham controls.
25 Opening of VGSCs and cellular depolarization results in the release of
neurotransmitters; tetanus toxin (TET) acts to inhibit vesicular release by cleaving
SNARE proteins involved in vesicular transport and thus blocks the release of glutamate
into the synaptic cleft (Link et al., 1992). Addition of TET (2.5nM-25nM) in the presence of PbTx-3 resulted in a dose-dependent decrease in Ca2+ influx with the greatest
response seen at 25nM. At that concentration, the change in fluorescence in response to
PbTx-3 and TET was significantly reduced compared to PbTx-3 alone and was similar to
that of PbTx-3 plus TTX (Fig. 5).
We next assessed the role of the glutamate NMDA receptor by treating the neurons with the non-competitive NMDA receptor antagonist MK-801 (50uM, Shin and
Buck, 2003), which blocks neurotransmitter binding to postsynaptic receptors and
prevents depolarization in mammalian CGNs (Berman and Murray, 1999). As above,
neurons incubated with PbTx-3 for a period of 90 minutes showed a significant increase
in intracellular Ca2+ levels compared to controls; MK-801 treatment decreased Ca2+
influx such that ∆F/F0 was not significantly different than controls (Fig. 5). As glutamate
may bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors
(AMPAR) as well as NMDARs, we also examined the effect of the AMPA receptor antagonist CNQX (200uM, Pamenter and Buck, 2008) when added to the medium with
PbTx-3 (Dravid et al., 2004). As was seen with the NMDA antagonist MK-801, Ca2+
influx was significantly reduced but was not fully abrogated by CNQX in the presence of
PbTx-3 (Fig. 5). When MK-801 + CNQX were added simultaneously to the medium with
PbTx-3, there was no difference in ΔF/F0 between experimental and control cells,
suggesting the complete blockade of glutamate binding. Addition of MK-801 or CNQX
26 along with TTX to brevetoxin treated cells likewise prevented any change to intracellular
calcium levels.
Discussion
In this study, we utilized primary cultures derived from the freshwater turtle T.
scripta to examine brevetoxin’s effects on turtle neurons. Mammalian studies have shown
that the pharmacological target of brevetoxin is site 5 on VGSCs in electrically excitable
cells (Catterall and Risk, 1981), where it binds with high affinity and prevents channel inactivation (Huang et al., 1984; Jeglitsch et al., 1998; Poli et al., 1986). This prolongs mean open time and causes cellular depolarization, release of the neurotransmitter glutamate, and thus activation of Ca2+ sensitive channels (Berman and Murray, 2000,
1999). These events affect excitable cells and trigger the neurological and muscular
symptoms of brevetoxicosis (Templeton et al., 1989), and may lead to cell death (Berman
and Murray, 2000; Murrell and Gibson, 2009; Walsh et al., 2008). Similar neurological
effects have been reported in sea turtles exposed to red tides (Fauquier et al., 2013), and
this study demonstrates that the mode of action of PbTx-3 in turtle neurons is likely to be the same as that reported in mammalian neurons. The downstream calcium influx triggered by PbTx-3 binding to VGSCs could be completely antagonized by TTX, which binds non-competitively to VGSCs (Huang et al., 1984). Tetanus toxin, which inhibits neurotransmitter release on presynaptic neurons by degrading a key molecule involved in vesicular release (Link et al., 1992), also abrogated increases in intracellular calcium.
Upon depolarization and glutamate release, Ca2+ channels like the AMPA and NMDA receptors become activated. As with the direct inhibition of VGSCs and of neurotransmitter release, application of the NMDA channel antagonist MK-801 and the
27 2+ AMPA channel antagonist CNQX prevented increases in [Ca ]i in the presence of PbTx-
3, with the AMPA blockade having slightly less of an effect than NMDAR blockade.
NMDA channels thus appear to contribute to a greater flow of Ca2+ ions than AMPA
receptors; in rat CGNs NMDA receptors were also responsible for the greater part of
calcium influx in PbTx-1 treated cells (Berman and Murray, 2000)
While basic neuronal function is similar between reptiles and mammals (Schlegel
and Kriegstein, 1987), the ectothermic brain could be either more or less vulnerable to
toxin effects due to lower basal metabolic rates but also differences in the density and
activity of membrane ion channels and neurotransmitter receptors (Lutz and Leone-
Kabler, 1995; Sakurai et al., 1993; Xia and Haddad, 2001). While PbTx-3 exposure led to
increased cell death in a concentration-dependent manner, turtle neurons were
surprisingly resistant to the toxin compared to results reported in mammalian studies. We
initially tested PbTx-3 concentrations ranging from 10-100nM based on work that was
done by Berman and Murray (1999), which showed murine cerebellar granule neurons
(CGNs) incubated with PbTx-3 for 2h at 37°C had an EC50 of ~54nM. The same
concentrations, when applied to turtle neuronal cultures, resulted in little to no cell death,
and the final cytotoxicity curve ranged from 100-2000nM. Freshwater turtle neurons are thus clearly more resistant to PbTx-3 toxicity compared at least to rat CGNs with
o o significant differences in the EC50 at both 22 C and 37 C.
In addition to requiring higher doses than mammalian cells to trigger the
downstream effects of depolarization, the turtle neurons in this study also may have
responded more slowly. While Berman and Murray (2000) reported a peak of
fluorescence within 25-50 sec in rat CGNs that plateaued at an elevated level by 3
28 minutes, that study utilized a fluorescent plate reader (FLIPR) technique that can record very rapid changes in fluorescence. In this study, while increased fluorescence was visible by 2 minutes after toxin administration, changes were not significant until 20 minutes post-treatment. There are several possibilities that may explain this difference between rat CGNs and the results reported here. For one, while the turtle neurons grow in culture and form synaptic vesicles, they are not all contiguous in culture and may not have formed synaptic connections. Thus the large volume of medium needed in 24 well plates (500 µl vs 100-150 µl in mammalian cell culture studies performed in 96 well plates (Dravid et al., 2005), would require time for both diffusion of chemicals into the cells, and then the diffusion of neurotransmitters to nearby cells. The longer time for a clear effect to be seen may also be due to an inherent resistance of the neurons to depolarization, as is suggested by the larger doses of PbTx-3 required compared to mammalian cells. During anoxia, turtle neurons undergo a mild depolarization (Pamenter and Buck, 2008) and are able to inhibit the release of excitotoxic neurotransmitters
(Milton et al., 2002a; Milton and Lutz, 2005; Hogg et al., 2014), in part by decreasing the activity of ion channels (Buck, 2004; Pék-Scott and Lutz, 1998) including NDMA
(Bickler et al., 2000; Pamenter et al., 2008b) and AMPA receptors (Pamenter et al.,
2008a). This effect, called “channel arrest” may also be occurring in response to brevetoxin, and though such a possibility was beyond the scope of this study it may be an interesting area for further investigation. It is interesting that concentrations of the
Na+/K+-ATPase blocker ouabain (2mM) that are effective in mammalian cells (Aizman et al., 2001) are also effective in the turtle (Wilkie et al., 2008), but that considerably higher concentrations of PbTx-3 are required to depolarize the cell. This further suggests that
29 the turtle neurons are inherently resistant to toxin-induced VGSC depolarization, as in
anoxia. On the other hand, Berman and Murray (1999) also reported large differences in
toxicity in different neuronal cell types, at least for PbTx-2. While the EC50 for PbTx-2
was 80.5nM in CGNs, neocortical neurons were much less sensitive, requiring 24h at
3uM to induce significant toxic effects. While mammalian neonatal cells in general are
more resistant to cellular depolarization and glutamate release (Cherici et al., 1991;
Pérez-Pinzón et al., 1993), the authors suggested that the toxicity differences between cell
types could be due to differences in neurotransmitter levels, as CGNs are primarily
glutamatergic whereas neocortical neurons have significant levels of GABA (Berman and
Murray, 2000). Elevated GABA levels could then activate cell survival elements that
would counteract the cell death pathways activated by PbTx. Berman and Murray (1999)
did not report the EC50 for PbTx-3 in neocortical neurons, though in vivo EC doses are much lower for PbTx-3 than for PbTx-2 (Baden and Mende, 1982), as are EC50’s for
CGNs in vitro (Berman and Murray, 1999). In vivo, acute toxin exposures that led to behavioral symptoms of brevetoxicosis resulted in brain concentrations of brevetoxins ranging from 2.12nM (Benson et al., 1999) to 6.8nM (Cattet and Geraci, 1993) which are
sufficient to produce significant fractional occupancy (42–70%) of VGSCs (Poli et al.,
1986) and result in neuronal deficits (Yan et al., 2006).
As with CGNs vs. neocortical neurons in rats, a similar tilt of the balance towards
cell survival could also play a role in the resistance of turtle neurons to PbTx-3. As diving
reptiles with extended hibernation periods underwater, T. scripta are highly anoxia
tolerant (reviewed in Larson et al., 2014). When deprived of oxygen, mammalian cells
lose ion homeostasis in the face of decreased ATP supplies, resulting in neuronal
30 2+ depolarization, glutamate release, and increased [Ca ]i that activates apoptotic pathways
(Milton and Prentice, 2007). Turtles like T. scripta and the western painted turtle
Chrysemys picta, however, instead maintain ion gradients, in part by downregulating ion
channels (Pék and Lutz, 1997; Pérez-Pinzón et al., 1992); decrease NMDAR and
AMPAR currents (Buck and Bickler, 1998; Pamenter et al., 2008a; Shin and Buck,
2003); and increase GABA release (Nilsson and Lutz, 1993); receptor density (Lutz and
Leone-Kabler, 1995); and GABA currents (Pamenter et al., 2011).
Extracellular glutamate levels remain low through a combination of decreased release and continued reuptake through neuronal and glial transport mechanisms (Milton et al., 2002; Thompson et al., 2007). Constitutive cellular protective mechanisms may also play a role in cell survival, including detectable basal levels of Hsp72 (Kesaraju et
al., 2014; Prentice et al., 2004). Cellular stress induces a further upregulation of heat
shock proteins (Kesaraju et al., 2009) and MAP kinases including extracellular regulated kinase (ERK 1/2) (Nayak et al., 2016, 2011). While neurotransmitter levels and intracellular responses were not examined in this study, Dravid et al. (2004) noted that
PbTx-2 exposure in rat neocortical neurons increased phosphorylated ERK levels, and that this activation was inhibited by MK-801. Phospho-ERK is generally considered to be a pro-survival protein (Avolio et al., 2014; Xia et al., 1995), and pharmacological
blockade of ERK activation increases anoxic cell death in turtle neurons (Nayak et al.,
2016). Changes to intracellular signaling pathways in response to PbTx-3 are currently
under investigation in our laboratory.
The higher resistance of T. scripta neurons to PbTx-3 toxicity compared to rat
neurons is unlikely to be due to differences in VGSC affinity for brevetoxin. Edwards et
31 al., (1989) demonstrated that the kinetic and pharmacological characteristics of PbTx-3
binding to VGSCs were similar in rat and turtle brain-derived synaptosomes. While the
maximum binding capacity for the turtle was only about 1/3rd that of the rat, the apparent
lower density of sodium channels did not result in different rates of normoxic oxygen
consumption when measured at the same temperature (Edwards et al., 1989; Suarez et al.,
1989) and they concluded the difference in channel density was not sufficient to affect
ion pumping costs. While studies have reported binding affinities in some fish about 1/3rd
that of mammals (Lewis, 1992; Stuart and Baden, 1988; Yotsu-Yamashita et al., 2000), a
study by Dechraoui et al., (2006) reported no differences in PbTx-3 binding to brain or
skeletal muscle between the rat and a marine fish (Black Seabass Centropristis striata),
though interestingly PbTx-3 binding in rat hearts showed lower affinity than in brain or
muscle, but there was no difference between organs in the fish (Dechraoui et al., 2006).
Thus while PbTx-3 binding is different between some ectotherms and endothermic
vertebrates, the differences are not so great as to explain the high resistance of turtle
neurons to the toxin. It would be of interest to determine if PbTx-3 exposure leads to a downregulation of VGSCs or other ion channels or neurotransmitter receptors, as occurs in T. scripta neurons in anoxia. Studies performed on anoxic turtle neurons shows that the
NMDAR activity decreases by about 70% over a 90 minute period of anoxia, decreasing the probability of Ca2+ influx (Bickler et al., 2000); the ability to reduce NMDAR activity
may explain in part why turtle neurons in culture are significantly more resistant to
glutamate toxicity than their mammalian counterparts even under normoxic conditions
(Nilsson and Lutz, 1991).
32 Conclusion
In this study we examined the effects of PbTx-3 in turtle neurons, with the mode of action predicted to be the same as has been reported in mammalian neurons: PbTx-3 binds to and opens VGSCs, causing depolarization of presynaptic neurons. Continuous depolarization triggers excess glutamate release which in turn leads to the activation of
NMDA and AMPA receptors on the postsynaptic neurons. Increased intracellular calcium then triggers a cascade of destructive events resulting in cell death. VGSCs are critical to the normal functioning of excitable cells, and are the target not just of brevetoxin but numerous other toxins as well, with different binding sites and modes of action. This study to confirm PbTx-3’s mode of action in turtle neurons is one part of ongoing work in our laboratory looking at brevetoxin uptake, tissue distribution and excretion, and rates of clearance from different organ systems, as well as physiological impacts. While sea turtles are less anoxia tolerant than T. scripta, they are still ectothermic breathhold divers, able to remain submerged for hours (Hochscheid et al.,
2007, 2005; Moon et al., 1997), and can avoid brain depolarization for at least several hours of anoxia (Nilsson, pers. comm.). If the high resistance of T. scripta neurons to brevetoxicosis also holds true for sea turtles, this suggests that the animals admitted to rehabilitation centers after HAB exposure were likely to have been subjected to quite large doses of toxins, or for long periods of time, to generate the clear neuronal deficits they exhibit. In animals admitted to one facility after red tides in 2005-2006, plasma brevetoxin values ranged as high as 107 ng brevetoxin-3 eq/g (Fauquier et al., 2013), although of course levels in the brain could not be determined. Elevated toxin levels in vivo can occur through both biomagnification and bioaccumulation, and red tide
33 mortalities may occur at times far removed from dinoflagellate blooms. In 2002 and
2004, for example, there were unusual mortalities of Florida manatees and bottlenose dolphins which were attributed to brevetoxin. At the time of those mortalities, K. brevis concentrations in the water were low but animals tested had up to 1,136 ng per g toxin in the stomach contents which was ingested through vectors still carrying the toxin, mainly seagrass (with up to 1,263 ng/g PbTx) and fish (Flewelling et al., 2005). Similarly, PbTx-
3 was detected in blood samples from Kemp’s ridley sea turtles up to 9 months after a
Florida red tide bloom (Perrault et al., 2014), while in another study, brevetoxin persisted in the fish food web for a year after a K. brevis bloom occurred (Naar et al., 2007).
The overarching goal of these studies is to develop targeted therapies aimed at ameliorating the neurological impacts of red tide exposure in sea turtles as well as increasing toxin clearance, utilizing T. scripta as a model species. One such compound that has the potential to reduce neurological deficits is memantine, an NMDA receptor antagonist being tested for cerebral disorders (Takahashi et al., 2015). Some studies have also suggested brevenal, an antagonist of VGSCs that is also produced by K. brevis, as a possible therapy for brevetoxin toxicity (Sayer et al., 2006), though few studies have utilized it in vivo. Brevenal reduces cell death caused from PbTx in vitro (Gold et al.,
2013), and decreases mortality in brevetoxin exposed fish (Bourdelais et al., 2005). At this time, HAB-affected sea turtles are offered primarily supportive care (Fauquier et al.,
2013), including dehydration therapy and anti-inflammatories (Manire et al., 2013).
34 Figure 1: PbTx-3 binding schematic
Schematic describing the effects of PbTx-3 exposure in neurons. Under normal
circumstances, VGSCs on the presynaptic neuron open briefly and then inactivate,
allowing for signal propagation down the axon. When PbTx-3 binds to site 5 on VGSCs,
the channels remains open and cannot inactivate, causing continuous depolarization,
excess glutamate release and binding to NMDA and AMPA receptors, Ca2+ influx into
the postsynaptic cell, and excitotoxic cell death. The use of antagonists (TTX, MK-801,
CNQX, and tetanus toxin) and agonists (glutamate) suggests the mode of action of PbTx-
3 in turtle neurons begins with the binding of PbTx-3 to VGSC’s, leading to the activation of NMDA and AMPA receptors.
35 Figure 2: Cell death increases in turtle neurons post PbTx-3 exposure
[PbTx-3] (nM)
PbTx-3 increases cell death in primary cultured turtle neurons in a dose-dependent manner. Cells were exposed to PbTx-3 for 2h at 22oC or 37oC. The dotted line represents
o o the EC50 which at 22 C is ~1350nM and at 37 C is ~800nM (down arrows). Error bars
represent standard error of the mean and significance was determined using a Student’s t-
test. For each concentration of PbTx-3, cell death at 37oC was significantly higher than
cell death at 22oC (p≤0.001).
36 Figure 3: Intracellular calcium increases post PbTx-3 exposure
PbTx-3 increases intracellular Ca2+ in a time-dependent manner, (22oC). PbTx-3 was
added after a 90 minute control period as denoted by the arrow and changes in
intracellular Ca2+ levels were recorded. Ca2+ influx is pronounced over a period of 40-100 minutes following addition of PbTx-3 cells but plateaus in control cells. Error bars represent standard error of the mean and significance was determined using a Student’s t-
test where *** p≤0.001 and * p ≤0.05 and n≥8.
37 Figure 4: Intracellular calcium increases post glutamate and ouabain exposure
Glutamate, ouabain, and PbTx-3 treatments increase intracellular Ca2+ levels.
Intracellular changes were measured by changes in fluorescence with Fluo-3AM 90
minutes after the addition of glutamate, ouabain or PbTx-3, (22oC). Error bars represent standard error of the mean and significance was determined using one-way ANOVA where *** p≤0.001, * p≤0.05 and n≥30.
38 Figure 5: Increases in intracellular calcium are abrogated by antagonists
Increases in intracellular Ca2+ are abrogated when antagonists TTX, MK-801, CNQX and
TET are added 30 minutes prior to the addition of PbTx-3. Changes in fluorescence were measured at 90 minutes post PbTx-3 exposure (22oC). Error bars represent standard error
of the mean and significance was determined using one-way ANOVA where ***
p≤0.001 and n≥38.
39 Figure 6: Mean fluorescence data (0-200 min) reflecting intracellular calcium levels
Raw data indicating changes in intracellular calcium levels with time following PbTx-3 treatment, showing background fluorescence. Mean fluorescence images were taken every 10 minutes over the course of 190 minutes. Error bars represent standard error of the mean and significance was determined using a Student’s t-test where *** p≤0.001 and * p ≤0.05 and n≥8.
40 Figure 7: Mean fluorescence data (80-200 min) reflecting intracellular calcium levels
Raw fluorescence data following PbTx-3 addition to the medium at minute 90 (22oC), with background fluorescence subtracted from control and treated cells. Error bars represent standard error of the mean and significance was determined using a Student’s t- test where *** p≤0.001 and * p ≤0.05 and n≥8.
41 Figure 8: Fluorescence images following PbTx-3
Representative images showing changes in fluorescence following PbTx-3 exposure.
Images taken with AmScope Camera and Software. Fluo-3 AM was added to the medium
1h prior to the addition of PbTx-3.
42 References
Aizman, O., Uhlén, P., Lal, M., Brismar, H., Aperia, a, 2001. Ouabain, a steroid
hormone that signals with slow calcium oscillations. Proc. Natl. Acad. Sci. U. S. A.
98, 13420–13424. doi:10.1073/pnas.221315298.
Avolio, E., Mahata, S.K., Mantuano, E., Mele, M., Alò, R., Facciolo, R.M., Talani, G.,
Canonaco, M., 2014. Antihypertensive and neuroprotective effects of catestatin in
spontaneously hypertensive rats: interaction with GABAergic transmission in
amygdala and brainstem. Neuroscience 270, 48–57.
doi:10.1016/j.neuroscience.2014.04.001.
Baden, D.G., Bourdelais, A.J., Jacocks, H., Michelliza, S., Naar, J., 2005. Natural and
derivative brevetoxins: historical background, multiplicity, and effects. Environ.
Health Perspect. 113, 621–625. doi:10.1289/ehp.7499.
Baden, D.G., Mende, T.J., 1982. Toxicity of two toxins from the Florida red tide marine
dinoflagellate, Ptychodiscus brevis. Toxicon 20, 457–461. doi:10.1016/0041-
0101(82)90009-5.
Benson, J.M., Tischler, D.L., Baden, D.G., 1999. Uptake, tissue distribution, and
excretion of brevetoxin 3 administered to rats by intratracheal instillation. J. Toxicol.
Environ. Health. A 57, 345–355. doi:10.1080/009841099157656.
Berman, F.W., Murray, T.F., 2000. Brevetoxin-induced autocrine excitotoxicity is
associated with manifold routes of Ca2+ influx. J Neurochem 74, 1443–1451.
doi:10.1046/j.1471-4159.2000.0741443.x.
Berman, F.W., Murray, T.F., 1999. Brevetoxins cause acute excitotoxicity in primary
cultures of rat cerebellar granule neurons. J. Pharmacol. Exp. Ther. 290, 439–444.
43 Bickler, P.E., Donohoe, P.H., Buck, L.T., 2000. Hypoxia-induced silencing of NMDA
receptors in turtle neurons. J. Neurosci. 20, 3522–3528.
Buck, L.T., 2004. Adenosine as a signal for ion channel arrest in anoxia-tolerant
organisms. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 139, 401–14.
doi:10.1016/j.cbpc.2004.04.002.
Buck, L.T., Bickler, P.E., 1998. Adenosine and anoxia reduce N-methyl-D-aspartate
receptor open probability in turtle cerebrocortex. J. Exp. Biol. 201, 289–297.
Catterall, W.A., Risk, M., 1981. Toxin T46 from Ptychodiscus brevis (formerly
Gymnodinium breve) enhances activation of voltage-sensitive sodium channels by
veratridine. Mol. Pharmacol. 19, 345–348.
Cattet, M., Geraci, J.R., 1993. Distribution and elimination of ingested brevetoxin (PbTx-
3) in rats. Toxicon 31, 1483–1486. doi:10.1016/0041-0101(93)90214-4.
Cheng, Y.S., McDonald, J.D., Kracko, D., Irvin, C.M., Zhou, Y., Pierce, R.H., Henry,
M.S., Bourdelaisa, A., Naar, J., Baden, D.G., 2005. Concentration and particle size
of airborne toxic algae (brevetoxin) derived from ocean red tide events. Environ.
Sci. Technol. 39, 3443–3449. doi:10.1021/es048680j.
Cherici, G., Alesiani, M., Pellegrini-Giampietro, D.E., Moroni, F., 1991. Ischemia does
not induce the release of excitotoxic amino acids from the hippocampus of newborn
rats. Brain Res. Dev. Brain Res. 60, 235–40.
Dechraoui, M.Y., Ramsdell, J.S., 2003. Type B brevetoxins show tissue selectivity for
voltage-gated sodium channels: Comparison of brain, skeletal muscle and cardiac
sodium channels. Toxicon 41, 919–927. doi:10.1016/S0041-0101(03)00088-6.
44 Dechraoui, M.Y., Wacksman, J.J., Ramsdell, J.S., 2006. Species selective resistance of
cardiac muscle voltage gated sodium channels: Characterization of brevetoxin and
ciguatoxin binding sites in rats and fish. Toxicon 48, 702–712.
doi:10.1016/j.toxicon.2006.07.032.
Deshpande, S.S., Adler, M., Sheridan, R.E., 1993. Differential actions of brevetoxin on
phrenic nerve and diaphragm muscle in the rat. Toxicon 31, 459–470.
doi:10.1016/0041-0101(93)90181-H.
Dravid, S.M., Baden, D.G., Murray, T.F., 2005. Brevetoxin augments NMDA receptor
signaling in murine neocortical neurons. Brain Res. 1031, 30–8.
doi:10.1016/j.brainres.2004.10.018.
Dravid, S.M., Baden, D.G., Murray, T.F., 2004. Brevetoxin activation of voltage-gated
sodium channels regulates Ca dynamics and ERK1/2 phosphorylation in murine
neocortical neurons. J. Neurochem. 89, 739–49. doi:10.1111/j.1471-
4159.2004.02407.x.
Edwards, R.A., Lutz, P.L., Baden, D.G., 1989. Relationship between energy expenditure
and ion channel density in the turtle and rat brain. Am. J. Physiol. 257, R1354–8.
Fauquier, D.A, Flewelling, L.J., Maucher, J., Manire, C.A, Socha, V., Kinsel, M.J.,
Stacy, B.A, Henry, M., Gannon, J., Ramsdell, J.S., Landsberg, J.H., 2013.
Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to
Karenia brevis blooms in central west Florida. J. Zoo Wildl. Med. 44, 364–75.
Hogg, D.W., Hawrysh, P.J., Buck, L.T., 2014. Environmental remodelling of GABAergic
and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain. J.
Therm. Biol. 44, 85–92. doi:10.1016/j.jtherbio.2014.01.003.
45 Huang, J.M., Wu, C.H., Baden, D.G., 1984. Depolarizing action of a red-tide
dinoflagellate brevetoxin on axonal membranes. J. Pharmacol. Exp. Ther. 229, 615–
21.
Jeglitsch, G., Rein, K., Baden, D.G., Adams, D.J., 1998. Brevetoxin-3 (PbTx-3) and its
derivatives modulate single tetrodotoxin-sensitive sodium channels in rat sensory
neurons. J. Pharmacol. Exp. Ther. 284, 516–25.
Kesaraju, S., Nayak, G., Prentice, H.M., Milton, S.L., 2014. Upregulation of Hsp72
mediates anoxia/reoxygenation neuroprotection in the freshwater turtle via
modulation of ROS. Brain Res. 1582, 247–56. doi:10.1016/j.brainres.2014.07.044.
Kesaraju, S., Schmidt-Kastner, R., Prentice, H.M., Milton, S.L., 2009. Modulation of
stress proteins and apoptotic regulators in the anoxia tolerant turtle brain. J
Neurochem 109, 1413–1426. doi:JNC6068 [pii]10.1111/j.1471-4159.2009.06068.x.
Larson, J., Drew, K.L., Folkow, L.P., Milton, S.L., Park, T.J., 2014. No oxygen? No
problem! Intrinsic brain tolerance to hypoxia in vertebrates. J. Exp. Biol. 217, 1024–
39. doi:10.1242/jeb.085381.
Lewis, R.J., 1992. Ciguatoxins are potent ichthyotoxins. Toxicon 30, 207–11.
Link, E., Edelmann, L., Chou, J.H., Binz, T., Yamasaki, S., Eisel, U., Baumert, M.,
Sudhof, T.C., Niemann, H., Jahn, R., 1992. Tetanus toxin action: Inhibition of
neurotransmitter release linked to synaptobrevin proteolysis. Biochem. Biophys.
Res. Commun. 189, 1017–1023. doi:10.1016/0006-291X(92)92305-H.
Lutz, P.L., Leone-Kabler, S.L., 1995. Upregulation of the GABAA/benzodiazepine
receptor during anoxia in the freshwater turtle brain. Am. J. Physiol. 268, R1332–5.
46 Milton, S.L., Lutz, P.L., 2005. Adenosine and ATP-sensitive potassium channels
modulate dopamine release in the anoxic turtle (Trachemys scripta) striatum. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 289, R77–83.
doi:10.1152/ajpregu.00647.2004.
Milton, S.L., Nayak, G., Kesaraju, S., Kara, L., Prentice, H.M., 2007. Suppression of
reactive oxygen species production enhances neuronal survival in vitro and in vivo
in the anoxia-tolerant turtle Trachemys scripta. J. Neurochem. 101, 993–1001.
doi:10.1111/j.1471-4159.2007.04466.x.
Milton, S.L., Prentice, H.M., 2007. Beyond anoxia: the physiology of metabolic
downregulation and recovery in the anoxia-tolerant turtle. Comp Biochem Physiol A
Mol Integr Physiol 147, 277–290. doi:S1095-6433(06)00382-5
[pii]10.1016/j.cbpa.2006.08.041.
Milton, S.L., Thompson, J.W., Lutz, P.L., 2002. Mechanisms for maintaining
extracellular glutamate levels in the anoxic turtle striatum. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 282, R1317–23. doi:10.1152/ajpregu.00484.2001.
Murrell, R.N., Gibson, J.E., 2009. Brevetoxins 2, 3, 6, and 9 show variability in potency
and cause significant induction of DNA damage and apoptosis in Jurkat E6-1 cells.
Arch. Toxicol. 83, 1009–1019. doi:10.1007/s00204-009-0443-x.
Nayak, G., Prentice, H.M., Milton, S.L., 2016. Lessons from nature: Signaling cascades
associated with vertebrate brain anoxic survival. Exp. Physiol.
doi:10.1113/EP085673.
47 Nayak, G.H., Prentice, H.M., Milton, S.L., 2011. Neuroprotective signaling pathways are
modulated by adenosine in the anoxia tolerant turtle. J Cereb Blood Flow Metab 31,
467–475. doi:jcbfm2010109 [pii]10.1038/jcbfm.2010.109.
Nilsson, G.E., Lutz, P.L., 1993. Role of GABA in hypoxia tolerance, metabolic
depression and hibernation--possible links to neurotransmitter evolution. Comp.
Biochem. Physiol. C. 105, 329–36.
Nilsson, G.E., Lutz, P.L., 1991. Release of inhibitory neurotransmitters in response to
anoxia in turtle brain. Am. J. Physiol. 261, R32–R37.
Pamenter, M.E., Buck, L.T., 2008. Neuronal membrane potential is mildly depolarized in
the anoxic turtle cortex. Comp. Biochem. Physiol. - A Mol. Integr. Physiol. 150,
410–414. doi:10.1016/j.cbpa.2008.04.605.
Pamenter, M.E., Hogg, D.W., Ormond, J., Shin, D.S., Woodin, M.A., Buck, L.T., 2011.
Endogenous GABA(A) and GABA(B) receptor-mediated electrical suppression is
critical to neuronal anoxia tolerance. Proc. Natl. Acad. Sci. U. S. A. 108, 11274–9.
doi:10.1073/pnas.1102429108.
Pamenter, M.E., Shin, D.S.-H., Buck, L.T., 2008. AMPA receptors undergo channel
arrest in the anoxic turtle cortex. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294,
R606–13. doi:10.1152/ajpregu.00433.2007.
Pamenter, M.E., Shin, D.S.-H., Cooray, M., Buck, L.T., 2008b. Mitochondrial ATP-
sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western
painted turtle. J. Physiol. 586, 1043–58. doi:10.1113/jphysiol.2007.142380.
Pék, M., Lutz, P.L., 1997. Role for adenosine in channel arrest in the anoxic turtle brain.
J. Exp. Biol. 200, 1913–1917.
48 Pék-Scott, M., Lutz, P.L., 1998. ATP-sensitive K+ channel activation provides transient
protection to the anoxic turtle brain. Am. J. Physiol. 275, R2023–7.
Pérez-Pinzón, M.A., Nilsson, G.E., Lutz, P.L., 1993. Relationship between ion gradients
and neurotransmitter release in the newborn rat striatum during anoxia. Brain Res.
602, 228–33.
Pérez-Pinzón, M.A., Rosenthal, M., Sick, T.J., Lutz, P.L., Pablo, J., Mash, D., 1992.
Downregulation of sodium channels during anoxia: a putative survival strategy of
turtle brain. Am. J. Physiol. 262, R712–5.
Poli, M. a, Mende, T.J., Baden, D.G., 1986. Brevetoxins, unique activators of voltage-
sensitive sodium channels, bind to specific sites in rat brain synaptosomes. Mol.
Pharmacol. 30, 129–35.
Prentice, H.M., Milton, S.L., Scheurle, D., Lutz, P.L., 2004. The upregulation of cognate
and inducible heat shock proteins in the anoxic turtle brain. J. Cereb. Blood Flow
Metab. 24, 826–8. doi:10.1097/01.WCB.0000126565.27130.79.
Sakurai, S.Y., Lutz, P.L., Schulman, A., Albin, R.L., 1993. Unchanged [3H]MK-801
binding and increased [3H]flunitrazepam binding in turtle forebrain during anoxia.
Brain Res 625, 181–185. doi:0006-8993(93)91057-Y [pii].
Sattler, R., Tymianski, M., 2000. Molecular mechanisms of calcium-dependent
excitotoxicity. J. Mol. Med. (Berl). 78, 3–13. doi:10.1007/s001090000077.
Schlegel, J.R., Kriegstein, A.R., 1987. Quantitative autoradiography of muscarinic and
benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta. J. Comp.
Neurol. 265, 521–9. doi:10.1002/cne.902650406.
49 Shin, D.S.-H., Buck, L.T.,. Effect of anoxia and pharmacological anoxia on whole-cell
NMDA receptor currents in cortical neurons from the western painted turtle.
Physiol. Biochem. Zool. 76, 41–51. doi:10.1086/374274.
Stuart, A.M., Baden, D.G., 1988. Florida red tide brevetoxins and binding in fish brain
synaptosomes. Aquat. Toxicol. 13, 271–279. doi:10.1016/0166-445X(88)90148-8.
Suarez, R.K., Doll, C.J., Buie, A.E., West, T.G., Funk, G.D., Hochachka, P.W., 1989.
Turtles and rats: a biochemical comparison of anoxia-tolerant and anoxia-sensitive
brains. Am. J. Physiol. 257, R1083–8.
Templeton, C.B., Poli, M.A., LeClaire, R.D., 1989. Cardiorespiratory effects of
brevetoxin (PbTx-2) in conscious, tethered rats. Toxicon 27, 1043–1049.
doi:10.1016/0041-0101(89)90155-4.
Thompson, J.W., Prentice, H.M., Lutz, P.L., 2007. Regulation of extracellular glutamate
levels in the long-term anoxic turtle striatum: coordinated activity of glutamate
transporters, adenosine, K (ATP) (+) channels and GABA. J. Biomed. Sci. 14, 809–
17. doi:10.1007/s11373-007-9190-2.
Walsh, C.J., Leggett, S.R., Strohbehn, K., Pierce, R.H., Sleasman, J.W., 2008. Effects of
in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T
cell line (Jurkat). Mar. Drugs 6, 291–307. doi:10.3390/md20080014.
Wilkie, M.P., Pamenter, M.E., Alkabie, S., Carapic, D., Shin, D.S.H., Buck, L.T., 2008.
Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius
auratus). Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 148, 355–62.
doi:10.1016/j.cbpc.2008.06.004.
50 Xia, Y., Haddad, G.G., 2001. Major difference in the expression of delta- and mu-opioid
receptors between turtle and rat brain. J. Comp. Neurol. 436, 202–10.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing
effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–31.
Yan, X., Benson, J.M., Gomez, A.P., Baden, D.G., Murray, T.F., 2006. Brevetoxin-
induced neural insult in the retrosplenial cortex of mouse brain. Inhal. Toxicol. 18,
1109–16. doi:10.1080/08958370600945804.
Yotsu-Yamashita, M., Nishimori, K., Nitanai, Y., Isemura, M., Sugimoto, A., Yasumoto,
T., 2000. Binding properties of (3)H-PbTx-3 and (3)H-saxitoxin to brain membranes
and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary
structure of a voltage-gated Na(+) channel alpha-subunit (fMNa1) from skeletal
muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403–12.
doi:10.1006/bbrc.1999.1974.
51 CHAPTER III. TISSUE UPTAKE, DISTRIBUTION AND EXCRETION OF
BREVETOXIN-3 AFTER ORAL AND INTRATRACHEAL EXPOSURE IN THE
FRESHWATER TURTLE TRACHEMYS SCRIPTA AND THE DIAMONDBACK
TERRAPIN MALACLEMYS TERRAPIN
Introduction
Harmful algal blooms (HABs, red tides) threaten marine life due to the overload of nutrients in the ocean which cause a rapid reproduction of dinoflagellates. When these organisms rapidly reproduce they may release potent species-specific neurotoxins.
Karenia brevis is a well-known dinoflagellate native to the gulf coast of Mexico that releases brevetoxins (PbTx’s). K. brevis has been associated with elevated morbidity and mortality in marine animals, including large dolphin die-offs, mass manatee mortalities, and hundreds of sea turtle deaths (Flewelling et al., 2005; Colbert et al., 1999; Twiner et al., 2012; Walsh et al., 2010). Animals are at risk for toxin exposure via ingestion, inhalation and toxin accumulation over time. Seagrasses, crustaceans and fish are common in the diet of sea turtles, so bioaccumulation may occur with continued consumption of contaminated prey items. Behavioral symptoms of brevetoxicosis include but are not limited to head bobbing, muscle twitching, ataxia, and partial to complete paralysis. Despite the recent mortality events, not much is known about how the toxin impacts endangered sea turtles at the physiological or cellular level. Since all sea turtles are endangered or threatened, it is not possible to perform experiments investigating these factors. We have utilized the freshwater turtle as a model system to examine toxin effects 52 systemically with the ultimate goal of designing treatment strategies that can be used to
improve rehabilitation outcomes for sea turtles exposed to red tides. Current
rehabilitation efforts have been directed at supportive care (Fauquier et al., 2013),
including dehydration therapy and anti-inflammatories (Manire et al., 2013). Treatments will be targeted to helping reduce neurological symptoms and eliminate PbTx-3 more rapidly. We also addressed these concerns utilizing a second comparative model
organism, the diamondback terrapin (Malaclemys terrapin), which is an estuarine
species. As marine organisms it is possible that responses in sea turtles may differ from
the freshwater turtle model, therefore we also did a limited comparative study using the
estuarine species, Malaclemys terrapin. Since no significant differences were found we
limited the use of this species as it is listed as protected in Florida.
Materials and methods
Experimental Animals
All work was approved by the Florida Atlantic University Institutional Animal
Care and Use Committee (IACUC) Fifty-seven male and female juvenile freshwater turtles (Trachemys scripta), approximately 15 – 20 cm straight carapace length and weighing 0.40 – 1.10 kg, were obtained from a commercial supplier (Niles Biological
Inc., Sacramento, CA). Animals were acclimated to the laboratory for two weeks prior to any dosing experiments. Turtles were maintained in aquaria at room temperature (22°C
+/- 3°C, 50% relative humidity +/- 4%) on a 12h day/night cycle. Aquaria were cleaned according to standard husbandry methods and fed (commercial aquatic food, to satiety)
3× weekly. Twelve male diamondback terrapins (Malaclemys terrapin) approximately 10
cm straight carapace length and weighing 0.16 – 0.26 kg were captured by hand from a
53 near shore barrier island off of Apalachicola, Florida, under a Florida FWC scientific
collecting permit. Only males were collected to reduce impacts on the breeding
population. Animals were brought back to Florida Atlantic University where they were
maintained in brackish water. Animal tanks were cleaned and they were fed frozen
shrimp and fish to satiety 3x/week. All animals were given individual identification
numbers and randomly assigned to an experimental group.
Brevetoxin
Brevetoxin (PbTx-3) was purchased from LKT Laboratories (St. Paul, Minnesota)
and was dissolved in ethanol and mixed with 0.9% NaCl saline to a final concentration of
0.05µg/µl. Turtles were restrained by hand and administered PbTx-3 orally (33.48µg/kg,
3x/week for two weeks for a total of 7 doses) via esophageal tube to mimic ingestion or
by intratracheal instillation (IT) (10.53µg/kg, 3x/week for four weeks for a total of 12
doses). For intratracheal doses, the toxin was administered ~1.5cm into the tracheal
opening at the base of the tongue, and a rubber bulb and pipette tip were used to inflate
the lungs 3 times following instillation of the toxin to mimic inhalation. Appropriate
doses were determined using a dosage curve ranging from 22.32µg/kg to 33.48µg/kg for
oral dosing and 3.12µg/kg to 13.16µg/kg for IT exposures; the highest IT dose increased
mortality and so the next lower dose was selected (Table 3). The initial dose curves were based on previous mammalian studies using 18.6 µg/kg for oral exposure in rats and 6.6
µg/kg or lower for intratracheal instillation exposures (Benson et al., 1999; Cattet and
Geraci, 1993; Tibbetts et al., 2006). While our initial dose curve started much lower based on differences in metabolic rates between mammals and reptiles, at the lower doses no behavioral or pathological effects were observed and tissue concentrations were below
54 the detection limit except in the kidney, liver and feces. Thus, the toxin was increased until noted neurological effects were present. Terrapins were orally dosed (30.13µg/kg, a
10% decrease due to smaller size) 3×/ week for two weeks for a total of 7 doses. Control animals received sham doses of physiological saline solution mixed with ethanol to a final concentration to 0.1% EtOH (Cocilova et al., 2016) to mimic the treatment solution, and sham exposures were conducted as in toxin exposures.
Tissue and fluid collection
All turtles were euthanized by decapitation and the following tissues and fluids were collected for ELISA assays: kidney, liver, intestines, bile, brain, heart, spleen, lung, trachea, and plasma. Whole blood was collected by exsanguination, feces were removed from the large intestine, and fluids were collected directly from the organ with a syringe post mortem. While the urine, feces and fat were collected for T. scripta experiments we were unable to obtain adequate material for these samples from the terrapins due to their smaller size. Tissue and fluid samples were collected for both oral and IT post exposure for time points 1h, 24h, 48h, and 1wk post final PbTx-3 exposure to examine rates of clearance (Table 1). Tissues samples were divided and one part flash frozen in liquid nitrogen and stored at -80°C. Plasma was extracted from whole blood by centrifugation and the plasma and other fluid samples were frozen at -80°C. Tissue and fluid samples were sent to Florida Fish and Wildlife Research Institute overnight on dry ice and remained frozen at -80°C until processing. The remaining solid tissue sample portions were placed in 10% neutral buffered formalin and shipped to Dr. Gregory Bossart for histopathology.
55 Brevetoxin analysis
Extraction
A competitive ELISA was used to detect brevetoxins in turtle tissues and
biological fluids as previously described (Fauquier et al., 2013). Solid tissues were
thawed prior to extraction and homogenized in the presence of 80% aqueous methanol (4
ml/g tissue). Homogenates were centrifuged for 10 min at 3000×g and the supernatants
were transferred to clean 50 ml centrifuge tubes. The pellets were extracted a second time
in the same manner and the supernatants were pooled. The combined extracts were then
partitioned once with 100% hexane (1:1, v:v), and the methanol fractions were retained at
-20°C until analyzed. Brevetoxin in bile was extracted by liquid-liquid extraction. In 15 ml centrifuge tubes, bile (0.5 ml) was combined with ethyl acetate (1 ml) and vortexed for 1 min. After centrifuging for 5 min at 3000×g, the top ethyl acetate fraction was transferred to a clean 15 ml centrifuge tube, the bottom bile fraction was extracted two more times in the same manner, and the ethyl acetate fractions were combined. Deionized water (3 ml) was then added to the combined ethyl acetate fraction, the sample was vortexed for 1 min, centrifuged for 5 min at 3000×g and the top ethyl acetate fraction was transferred to a clean disposable glass test tube. The ethyl acetate was evaporated to dryness in a Speedvac (Thermo Scientific Savant) and the residue was re dissolved in 1 ml of 80% aqueous methanol. Urine and plasma were not extracted. They were thawed and diluted in phosphate buffered saline (pH 7.4) containing 0.05% Tween 20 and 0.5% gelatin immediate prior to analysis.
56 ELISA
Brevetoxins and brevetoxin-like compounds were quantified in all samples
extracts using a competitive enzyme linked immunosorbent assay (ELISA; Marbionc,
Wilmington NC) performed according to Naar et al., (2002) with modifications described
in Flewelling (2008). Toxin concentrations were calculated using a PbTx-3 standard curve, and results are reported in PbTx-3 equivalents. The limit of detection as described was approximately 1-2 ng/ml of plasma or urine, 10 ng/ml of bile, and 10 ng/g of feces or tissue.
LCMS
A subset of samples (primarily feces and some urine) were analyzed for PbTx-3 and two common brevetoxin metabolites for which reference material was available (S- desoxy-BTX-B2 and BTX-B2) using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prior to analysis, a 0.5 g equivalent of feces extract diluted to
25% methanol or 1 ml of urine was applied to a pre-conditioned Strata-X cartridge
(Phenomenex; 60 mg, 3 ml). The column was washed with 6 ml of 20% methanol and toxins were eluted with 4 mL of 100% methanol. The methanol extract was then evaporated to dryness and re-dissolved in 0.5 ml of 100% methanol. LC-MS/MS analyses were performed using an Acquity UPLC system coupled to a Quattro micro™ API triple quadrupole mass spectrometer as described in Sunda et al., (2013). Toxins were quantified using a 6-point calibration of a mixed standard of pure brevetoxins purchased from Marbionc (Wilmington, NC). Control extracts of feces and urine were spiked with multiple concentrations of PbTx-3 to provide matrix-matched standards for quantifying
PbTx-3.
57 Histopathology
Multiple tissue sections from freshwater turtles (Trachemys scripta) including brain, heart, kidney, liver, spleen, lung, trachea and intestine (and some adrenal gland, pancreas, ovary and testicle) and from diamond backed terrapins (M. terrapin) including liver, kidney, lung, trachea, intestine (and some testicle, epididymis and stomach) were collected and fixed in 10% neutral buffered formalin for histopathology. Brain, heart and spleen of M. terrapin could not be utilized for histopathology due to their small size; the full tissue was needed for ELISA. Formalin-fixed tissues were routinely processed, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for examination by light microscopy as previously described (Benson et al., 2005; Bossart et al., 1998; Kreuder et al., 2002). The tissues were initially evaluated blindly followed by a review of the experimental protocol for each group.
Statistics
Data were analyzed using one-way analysis of variance (ANOVA) followed by
Holm-Sidak Test (Holm-Sidak) pairwise comparison test using Sigma Plot 11.0 (Systat
Software, Inc, San Jose, California). Samples in which brevetoxin was not measurable were given a value of half the level of detection (2.5 ng per g tissue or ml bile or 0.5 ng per ml fluid) to calculate mean. Significant differences are indicated by asterisks where one asterisk (*) represents p≤0.05 and three asterisks (***) represents p≤0.001. PbTx-3 concentrations were compared 1h post exposures to the 24h, 48h, and 1wk exposures.
58 Results
Neurological and muscular deficits post PbTx-3 exposure
All experimental animals exposed to final concentrations of PbTx-3 either orally or intratracheally exhibited muscular and neurological symptoms consistent with brevetoxicosis including head bobbing, muscle twitching, ataxia, swimming in circles, partial to complete paralysis, penile prolapse in males, edema, and in some cases apparent coma. The onset of symptoms in the oral experimental groups began ~30 min post PbTx-
3 exposure and in the intratracheally exposed animals ~2-5 min post PbTx-3 exposure; symptoms were similar and did not become more or less severe with subsequent doses.
Clinical signs were recorded every hour for the first 8h following each exposure and daily thereafter. The clinical neurological symptoms declined over a 24h period and lethargy was the primary symptom that persisted for longer than 24-48h as well as decreased appetite for the duration of the experiment. Animals that were unresponsive for up to 4h following PbTx-3 exposure were euthanized (Table 2). The terrapin experimental groups exhibited neuronal and muscular deficits including muscle twitching, jerky body movements, and unresponsiveness consistent with those observed in T. scripta. Animals exposed to lower doses of PbTx-3 showed reduced symptoms of brevetoxicosis (Table
3). No behavioral changes were observed in sham treated animals.
PbTx-3 Tissue Distribution
PbTx-3 was distributed to all T. scripta organs systems sampled in both oral and
IT treatment groups. By 1h post oral toxin exposure concentrations were highest in the detoxification and excretion systems: the kidney, liver, and feces (Table 4). IT exposed animals had the highest PbTx-3 concentrations in the trachea, liver, and bile 1h post toxin
59 exposure (Table 5). Lower PbTx-3 concentrations were found in the brain, lung, fat, urine, intestine and plasma for all animal groups. The oral exposure animals showed higher initial toxin levels overall in their tissues and fluids as significantly higher concentrations had to be administered orally to achieve the same symptoms compared to the IT experiments. By 24h post exposure, all tissues and fluids other than the feces and bile contained mean toxin levels of less than 55 ng/g (ng/ml). The tissue distribution of
toxin in M. terrapin similarly distributed to all organ systems, although toxin levels in the terrapins were highest in the kidneys, and the liver concentrations were similar to other organs including the brain, spleen, and heart (Table 6).
Rates of toxin clearance
PbTx-3 rapidly clears from each organ system over a period of 24-48h for both
oral and IT exposures, and in both T. scripta and M. terrapin, which coincides with the
decline in clinical symptoms over the same time period. The highest PbTx-3
concentrations in T. scripta were initially present in the liver and kidney 1h after oral
exposure while bile levels rose from 24h post exposure to 48h, though the increase was
not statistically significant (Fig. 9). Similarly, kidney and liver levels were high following
IT exposures but rapidly declined (Fig. 10). By 1wk, PbTx-3 concentrations in the
tissues and fluids were near or below the ELISA detection limits for both oral and IT
exposures. The results for both routes of exposure are consistent, reflecting a rapid
decrease in toxin concentration over time with increases in the bile and feces; despite
high initial toxin levels in the kidney, urine levels remained relatively low. Though the rapid appearance by 1h of elevated levels in the bile and feces meant that further increases at 24 and 48h were not statistically significant, the results clearly suggest that
60 removal via the bile and feces is the primary route of excretion, though in two animals where LC-MS/MS was run, the amount of PbTx-3 present in the urine was greater than
that in the feces.
The metabolic pathways and products of brevetoxin in turtles are not fully
characterized, and analytical standards are unavailable even for known metabolites;
however LC-MS/MS analyses were done on a subset of samples to further examine metabolism and excretion. Samples analyzed were primarily feces, covering a range of post-intratracheal exposure time points (24h to 1 month). For four turtles exposed orally
(n=2) or by intratracheal instillation (n=2), both urine and feces collected within 24 hours of exposure were analyzed. Of PbTx-3 and the two common brevetoxin metabolites tested for, only PbTx-3 was observed. Brevetoxin is known to metabolize extensively in some animals, but the lack of analytical standards prevented us from identifying more metabolites. However, the degree of metabolism of PbTx-3 by T. scripta can be inferred by calculating the difference between toxin concentrations measured by ELISA and levels of PbTx-3 quantified by LC-MS/MS analysis. In turtles exposed via intratracheal instillation, after 24 hours, the majority of the toxin eliminated through the feces was still in the form of PbTx-3. At 48 hours and one week post exposure, PbTx-3 was either not detected or accounted for a minority (19%-47%) of the toxin concentration measured by
ELISA. After two weeks, PbTx-3 was mainly not detectable. In paired urine and feces samples, PbTx-3 accounted for the highest proportion of the toxin measured in the urine
(37%-70%) compared to the feces (6%-13%), suggesting toxin cleared through the feces had metabolized more extensively.
61 In M. terrapin, PbTx-3 was also cleared out of nearly all tissues within 24h post
toxin administration (Table 6). Toxin levels fell below ELISA detection limits ( within a week. Results show significant changes in the liver, kidney, brain, heart, lung and plasma from 24h to a 1wk compared with the 1h post exposures. Histopathological findings The T. scripta PbTx-3 exposure studies demonstrated a similar pattern of pathologic findings in both IT and orally exposed turtles, involving primarily inflammatory changes of the respiratory tract (trachea and lungs) and to a lesser extent the small intestine, meninges and endocardium/myocardium. Importantly, similar lesions were found in some control animals. Other findings such as acute tubular necrosis of the kidney and pancreatic inflammation occurred occasionally in animals but could not be attributed specifically to brevetoxin exposure. Inflammatory lesions of the respiratory tract consisted of tracheitis, often with associated mucosal hyperplasia and pneumonia. Hemorrhage, edema and intralesional coccoid bacteria were occasionally associated with the respiratory lesions. The severity, distribution and timing (i.e. acute, chronic-active, chronic) of the inflammatory lesions observed varied within and between experimental groups. Lesion severity generally tended to decrease with increased time post-PbTx exposure suggesting a recovery phase associated with toxin elimination. Additionally, while orally-exposed turtles had a similar pattern of inflammatory lesions, the lesions were typically less common than those in the IT exposed groups. In contrast to the findings in T. scripta, the respiratory and enteric lesions observed in M. terrapin (oral exposure only) were usually minimal to mild (except for respiratory lesions present in 1 control animal) and probably did not result in any significant organ compromise. 62 Parasites were common in both control and experimental T. scripta and included helminths consistent with spirorchid trematode ova and acanthocephalans. The spirorchid trematode ova were the most commonly observed parasites but occurred in varying numbers by tissue and case. The acanthocephalans were always present in intestinal lumina and were not usually associated with a host inflammatory response. In contrast to the findings in T. scripta, the respiratory and enteric lesions observed in M. terrapin were usually minimal to mild and probably did not result in any significant organ compromise. Discussion Brevetoxin metabolism and its physiological impacts are difficult to characterize in sea turtles due to their status as threatened or endangered animals, thus comparative model systems such as the freshwater turtle and diamondback terrapin must be used in order to determine toxin impacts in living organisms under controlled laboratory conditions. Results of this study suggest that turtles are highly resistant to PbTx-3, as the doses required either by oral or IT administration were significantly higher than those reported effective in rat studies. Our initial toxin dose curve was based on mammalian studies where dosages of 18.6 µg/kg (oral) and 2.6 - 6.6 µg/kg administered intratracheally had significant impacts in rats (Benson et al., 2005, 1999; Cattet and Geraci, 1993; Leighfield et al., 2014; Tibbetts et al., 2006). While we expected to need less drug due to the lower reptilian standard metabolic rate (typically about 1/10th of mammalian basal metabolic rates), this proved not to be the case as final doses were approximately 2× higher for oral exposures and up to 5× higher in IT exposures in the turtles. In the rat studies, the common clinical signs of brevetoxicosis were typically not present post PbTx exposure with these lower doses, however PbTx did show to distribute 63 to all organ systems (Cattet and Geraci, 1993). In our turtle experiments, PbTx-3 exposure at a low dose as used in mammalian studies (3.12µg/kg) did not result in clinical signs, but unlike those studies we also did not see toxin distribution to tissues which suggests the turtles exhibit more resistance to toxicity. Due to the low standard metabolic rate in turtles compared to mammals, we hypothesized that they would be more sensitive to the toxin, however this proved not to be the case. It is also been shown that PbTx-3 binding affinity is similar between rats and turtles, though the maximum binding capacity for the turtles was only about 1/3 that of a rat; the lower density of sodium channels did not results in differences in oxygen consumption (Edwards et al., 1989). No muscular or behavioral deficits resulting from brevetoxicosis were observed at doses effective in mammals, and PbTx-3 concentrations were minimally detectable by ELISA after 24h. Similarly, we recently reported that T. scripta neurons in cell culture are also resistant to PbTx-3, with EC50’s from 16- to ~26-fold higher than reported in rats (Cocilova and Milton, 2016). In this related in vivo portion of the study, though, the required effective doses were only 60-80% higher per dose than has been used in rat studies, though also administered repeatedly over 2-4 weeks to mimic the longer-term exposures that could be expected to occur in the wild. Higher or more frequent doses resulted in increased mortality. Once an appropriate sublethal dose was determined that resulted in evident neuronal and muscular deficits, PbTx-3 administration to T. scripta resulted in similar clinical symptoms as have been documented in laboratory studies, in wildlife studies (Kreuder et al., 2002) and at rehabilitation facilities where sea turtles were known to have been exposed to brevetoxin (Fauquier et al., 2013; Manire et al., 2013). The most severe 64 neurological deficits for both IT and oral routes of exposure were observed 1-6+h after each PbTx-3 exposure and diminished over a 24h time period, but animals remained somewhat lethargic for the duration of the 2-4 week exposure regime. Symptoms were similar after each administration and did not intensify with successive doses. The similarity in symptoms is unsurprising as PbTx-3 acts on T. scripta neurons by binding to and opening voltage-gated sodium channels, triggering depolarization and over-excitation (Cocilova and Milton, 2016), as also occurs in the mammalian brain (Berman and Murray, 1999, 2000) and is likely to act in a similar manner in muscle tissue. Once administered, PbTx-3 distributes widely to all organ systems sampled in both T. scripta and M.terrapin, as occurs in rats (Benson et al., 1999; Poli et al., 1990) and mice (Tibbetts et al., 2006), with the liver and kidney showing the highest immediate concentrations. Studies in laboratory animals (Benson et al., 1999), fish (Washburn et al., 1994), necropsied manatees (Bossart et al., 1998), and dolphins (Fire et al., 2008; Twiner et al., 2012) similarly show that brevetoxin concentrates into the excretory systems, especially the liver. Cattet and Geraci, (1993) likewise demonstrated in rats that brevetoxin is concentrated in the liver and is excreted in the feces and urine. In this study toxin was also rapidly cleared out of the system over a 24 – 48h period of time. There were significant decreases of PbTx-3 concentrations from the 1h to 24h post exposure times in the kidney, liver, brain, heart and spleen for both oral and IT exposures (Tables 2-4). Toxin concentrations were further reduced in most tissues within 48h and largely not detectable by 1wk post-exposure. This rapid clearance was somewhat surprising as it is similar to the rapid clearance reported in mammalian studies; in a study by Benson et al. (1999), 3H-PbTx-3 administered intratracheally in rats was rapidly cleared from the 65 lung, liver and kidneys with only 20% of the toxin remaining in the tissues by 7 days post exposure (Benson et al., 1999). In other studies on ectotherms, the toxin was cleared far more slowly; in mullet exposed to brevetoxin-containing seawater, blood toxin levels decreased by only 50% over 5 days (Woofter et al., 2005), in Gulf toadfish levels were still high in the bile after 96h, and in stranded sea turtles plasma levels took up to 80 days to decline to below detection limits (Fauquier et al., 2013). Marine animals, however, are exposed to a diverse array of brevetoxins and brevetoxin metabolites present in K. brevis blooms, which may also accumulate in prey and on plants. These can vary in potency and results in different rates of tissue uptake and elimination (Leighfield et al., 2014). Concentrations in necropsied wild animals exposed to K. brevis blooms can also range much higher than we observed here; in sharks and rays from the Gulf coast of Florida, toxin levels ranged from below the limit of detection up to 27,760 ng PbTx-3 eq/g in the liver tissue (Flewelling et al., 2010). In stranded sea turtles, liver PbTx-3 ranged from hawksbill turtle Eretmochelys imbricata), although the mean liver toxin levels in the three species of sea turtle sampled were not that different from the means reported in this study, ranging from 131–297 ng PbTx-3 eq/g (Fauquier et al., 2013). Toxin levels in the liver, kidney, spleen and lung reported in this study were also similar to those reported in necropsied bottlenose dolphins that stranded in 2004-2005 (Twiner et al., 2011) and in 2007-2008 (Fire et al., 2008). Similar liver concentrations of toxin when tissue levels are much higher in wild animals than in our experimental groups may imply a limit on clearance rates by the liver, thus explaining why plasma levels remain so high in sea 66 turtles in rehabilitation facilities removed from further toxin exposure. Decreased health could thus also impact clearance if liver function is affected. Interestingly, brain concentrations at 1h post-administration were higher than most organs other than the excretory systems, but there was no evidence for neurotoxicity as evidenced by histopathologic examination despite clear neurological symptoms. While it has been shown that brevetoxin localizes to the rodent cerebellum after a single injection (Bourdelais et al., 2004) and is cytotoxic in both rat cerebellar granular cells (Berman and Murray, 1999) and turtle neurons in vitro (Cocilova and Milton, 2016), a lack of neuropathology despite clinical symptoms has likewise been noted in rats (Benson et al., 2005). When rats were exposed to high doses of PbTx-3 (up to 100 µg/kg) they experienced signs and symptoms that mimics brevetoxicosis (Templeton et al., 1989) but studies conducted at lower doses (6.6µg/kg) did not have an behavioral changes however PbTx was distributed to all organs systems showing impacts (Benson et al., 1999). In general, the clinical signs observed in this study are consistent with acute brevetoxicosis as described in other species (Van Dolah et al. 2003; Fauquier et al., 2013). Some of the histopathological features detected, including edema in the trachea and lung of PbTx exposed turtles, have also been reported in other wildlife studies. PbTx target organ specificity with respiratory inflammation, pulmonary hemorrhage and nonsuppurative meningitis was reported in Florida manatees (Trichechus manatus latirostris) with inhalational brevetoxicosis (Bossart et al., 1998, 2002, 2004), and mortality-associated inhalational brevetoxicosis is attributed to acute agonal cardiovascular collapse (i.e., acute shock associated with aerosolized intoxication). It is suspected that in manatees, brevetoxicosis may initiate the release of inflammatory mediators that culminate in fatal 67 toxic shock (Bossart et al., 1998). Similar pathologic findings were also reported in cormorants (Phalacrocorax auritus) with suspected brevetoxicosis (Kreuder et al., 2002). Additionally, PbTx impacts the respiratory tract in humans acting as an asthma trigger (Fleming et al., 2009). Brevetoxin exposure may also have significant implications for immune function in loggerhead sea turtles (Walsh et al., 2010), Florida manatees (Walsh et al., 2005), laboratory rats (Benson et al., 2005) and was investigated as well in conjunction with this study (Walsh et al., in review). Related PbTx-associated immunologic perturbations resulting in secondary bacterial infection (postulated in this study) cannot be ruled out as another pathologic mechanism for this biointoxication, although lesions observed in M. terrapin were usually minimal which could reflect a species variation, a dose dependent effect, or represent underlying health status (M. terrapin were wild caught, rather than obtained from a commercial vendor). Importantly, however, in this study the frequency of lesions varied within experimental groups with some turtles having no significant lesions at all, while similar lesions were found in a low number of control turtles suggesting that another common factor(s) could be responsible for the observations. The infestation of both control and experimental animals with acanthocephalans and spirorchid trematodes confounded interpretation of the pathologic, which together with the presence of lesions in control and experimental groups made it difficult to distinguish results from background and thus limited the utility of the pathology data. The parasites are not uncommon in this species (Aho et al., 1992; Divers et al., 2010). Lesion severity did generally tend to decrease with increased time post PbTx exposure suggesting that increased time from PbTx oral 68 exposure is associated with less frequent and severe lesions. A notable exception from this temporal trend was a single T. scripta turtle in the 48 hour post exposure group that had a severe pneumonia with a concurrent meningitis and tracheitis; this animal was one of several over the course of the investigation that were euthanized when it became apparent that they were not recovering from the toxin and would not survive the duration of the experiment (Table 2). The animals were still analyzed for tissue toxin levels; some of these turtles sacrificed 24h or 48h post exposure showed higher toxin concentrations in their tissues than animals in the1h post exposure group, suggesting an inability to quickly clear the toxin. Notably, the toxin concentrations in the bile and feces of animals that were euthanized early were lower than any other treatment groups (data not shown). While anecdotal to some extent, this suggests that poor health strongly impacts ability to clear toxin, and may explain why PbTx levels in the plasma of loggerhead sea turtles in rehabilitation centers following toxic algal blooms in 2005 and 2006 took over 5-80 days to clear (Fauquier et al., 2013); in that study there was also no correlation between the amount of toxin present in the plasma and whether or not they survived. In the wild, red tides could thus trigger a “vicious circle” of alterations to neural and muscular activity that result in poorer body condition, which in turn makes the animal less able to excrete toxin, impacts the immune system, and results in further impacts to health status. Questions still remain as to how much of the toxin turtles in the wild are exposed to and the quantity of the toxin they take in, since evidence from this study indicates that turtles can excrete the toxin rapidly and do so via the same mechanisms as occur in mammals. Bioaccumulation and biomagnification are both likely to play a role in brevetoxicosis in marine animals. Brevetoxin levels reported in the stomach contents and 69 tissues of stranded bottlenose dolphins following algal blooms were similar to results reported from a 2004 mortality event during a non-bloom (Fire et al., 2008). Similarly, a dolphin and manatee mass mortality in Florida in 2002 was related to brevetoxins in fish and on seagrasses, though the mortality event occurred after the dinoflagellate bloom when water toxin levels were low (Flewelling et al., 2005). A recent study has also reported brevetoxin in the blood of nesting female sea turtles outside the time of any algal bloom (Perrault et al., 2016). Toxins in the water column and in lower trophic organisms may result in nearly continuous exposure for marine animals in areas prone to HAB outbreaks, such as the near annual blooms now occurring in the Gulf of Mexico. Even if the exposure does not result in immediate mortalities, they may be more susceptible to disease or to other additional stressors (Perrault et al., 2014; Walsh et al., 2015, 2010). The ultimate goal of this research is to design appropriate treatments targeted to sea turtles exposed to red tides, with a more complete understanding of the distribution, clearance, and effects of brevetoxin in turtles. Current treatments are aimed at supportive care for exposed animals and dehydration if edema is present (Fauquier et al., 2013; Manire et al., 2013). Potential treatment strategies are aimed at clearing the toxin out of the system more quickly and also drawing out the toxin from the tissues by creating an enlarged intravascular lipid pool (Cocilova et al., manuscript in prep). Brevenal, an antagonist of VGSCs that is also produced by K. brevis, may also be a possible therapy for brevetoxin toxicity (Bourdelais et al., 2005; Sayer et al., 2006), though few studies have utilized it in vivo. 70 Figure 9. Distribution of PbTx-3 in the excretion and detoxification systems post oral exposure Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the excretion tissues and fluids of T.scripta over 1 week post oral exposures. Error bars represent standard error of the mean and significance was determined using a one-way ANOVA where *** p≤0.001 and * p≤0.05 compared to 1h post-exposure samples, n≥4. Samples in which brevetoxin was not detectable were given a value of half the level of detection (2.5 ng per g tissue or ml bile or 0.5 ng per ml fluid) to calculate mean. Control (sham) exposures were all 71 Figure 10. Distribution of PbTx-3 in the excretion and detoxification systems post IT exposure Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the excretion tissues and fluids of T.scripta over 1 week post intratracheal (IT) exposures. Error bars represent standard error of the mean and significance was determined using a one-way ANOVA where *** p≤0.001 and * p≤0.05 compared to 1h post-exposure samples, n≥4. Samples in which brevetoxin was not detectable were given a value of half the level of detection (2.5 ng per g tissue or ml bile or 0.5 ng per ml fluid) to calculate mean. Control (sham) exposures were all 72 Table 1. Exposure time points post PbTx-3 Experimental details for T.scripta IT and oral groups. PbTx-3 post exposure time points, gender (F refers to females and M refers to males), and number of animals (N) present in each exposure group. There were a total of 57 animals and PbTx-3 post exposure time points ranged from 1h-1wk. All tissues and fluids were collected for a minimum of 5 animals per group; in some cases additional tissues could be collected from a concurrent study with an identical exposure protocol. Experimental group PbTx-3 post exposure time points Gender Ratio N IT sham control 24h 3F:2M 5 IT 1h 3F:4M 7 IT 24h 7F:2M 9 IT 48h 5F:0M 5 IT 1wk 4F:2M 6 Oral sham control 24h 4F:1M 5 Oral 1h 9F:2M 11 Oral 24h 5F:4M 9 Oral 48h 4F:1M 5 Oral 1wk 5F:0M 5 73 Table 2. Postmortem log Postmortem log of T. scripta that either died (n=5) during the experiment or were euthanized (n=4) due to severe brevetoxicosis. Animals were euthanized if they were experiencing unresponsiveness for up to 4h post toxin administration. Experimental Dose Death # of Behavioral Group Concentration post exposure doses symptoms (ug/kg) IT 3.12 28h 1/12 Head tucked, slow movements, responsive, alert; died IT 7.02 4h 1/12 Ridged limbs; died IT 10.53 24h 6/12 Unresponsive, ridged limbs, paralysis; died IT 10.53 4d 10/12 Unresponsive, dosing stopped; euthanized Oral 33.48 4h 1/7 Unresponsive, paralysis; euthanized Oral 33.48 3h 6/7 Unresponsive, paralysis; euthanized Oral 33.48 3h 6/7 Unresponsive, paralysis; euthanized Oral 33.48 24h 7/7 Unresponsive; died Oral 33.48 24h 2/7 Severe twitching; died 74 Table 3. Dose curve for IT and oral PbTx-3 exposures Dose curve experiments for IT and oral PbTx-3 exposures in T. scripta. IT PbTx-3 doses ranged from 3.12µg/kg to 13.16µg/kg with a final concentration of 10.53µg/kg. Oral PbTx-3 doses ranged from 22.32µg/kg to 33.48µg/kg. Behavioral symptoms were noted after each subsequent PbTx-3 exposure. When present, symptoms for IT exposures occurred within minutes post toxin exposure and oral symptoms occurred ~30 min post. Experimental Dose Group (ug/kg) Behavioral Symptoms IT 3.12 Alert, responsive to touch, no notable signs of brevetoxicosis IT 4.68 Alert, responsive to touch, slight movement in the tanks IT 7.02 Reduced ambulation, mild twitching, slight head bobbing IT 10.53 Significant muscle twitching, swimming in circles, head bobbing, partial to complete paralysis, ataxia IT 13.16 Coma, death Oral 22.32 Responsive to touch, mild head bobbing, slow movements, mild twitching Oral 27.95 Responsive to touch, limb twitching, muscular spasms Oral 33.48 Severe ataxia, head bobbing, partial to complete paralysis, comatose 75 Table 4. Distribution of PbTx-3 in T. scripta post oral exposure Distribution of PbTx-3 in T. scripta tissues and fluids post oral (33.48µg/kg) exposure. Parenthesis indicate the range of PbTx-3 found in each tissue (ng/g) or fluid (ng/ml). Intestines were analyzed with contents. Significant values were determined based on initial toxin concentrations at 1h post exposure and comparing them to 24h, 48h and 1wk time points. ≤ 0.001 and * indicates p ≤ 0.05 using a one-way AVOVA. PbTx-3 ng /g tissue or ng/ml fluid (mean ± SEM) Organ 1h 24h 48h 1wk Kidney 145.7 ± 24.9 52.1 ± 27.5* 13.6 ± 3.8*** 9.5 ± 3.2*** (89.4 – 231.3) (2.5 – 149.9) (2.5 – 26.0) (2.5 – 17.7) Urine 5.9 ± 1.5 7.7 ± 1.9 2.3 ± 0.6 8.6 ± 6.8 (1.7 – 14.5) (4.3 – 17.1) (0.5 – 3.2) (0.5 – 35.4) Liver 177.5 ± 19.7 45.5 ± 23.6* 7.5 ± 3.1*** 2.5 ± 0.0*** (127.6 – 256.6) (2.5 – 120.0) (2.5 – 16.8) (2.5)26 Intestines 26.0 ± 7.3 13.7 ± 6.9 2.5 ± 0.0 2.5 ± 0.0 (11.0 – 59.2) (2.5 – 33.0) (2.5) (2.5) Bile 49.3 ± 7.2 72.1 ± 22.4 77.5 ± 30.3 34.5 ± 10.1 (11.6 – 86.3) (27.7 – 222.7) (2.5 – 148.0) (2.5 – 57.3) Feces 192.9 ± 80.0 220.0 ± 123.8 155.8 ± 90.0 159.4 ± 115.5 (81.1 – 430.2) (2.5 – 367.6) (26.0 – 417.9) (2.5 – 613.7) Brain 71.9 ± 8.1 27.9 ± 16.1* 2.5 ± 0.0*** 2.5 ± 0.0*** (40.5 – 98.0) (2.5 – 79.4) (2.5) (2.5) Heart 61.4 ± 4.8 19.6 ± 11.2*** 2.5 ± 0.0*** 2.5 ± 0.0*** (47.1 – 74.1) (2.5 – 57.6) (2.5) (2.5) Spleen 55.4 ± 6.5 21.1 ± 11.7* 3.7 ± 1.2*** 2.5 ± 0.0*** (41.1 – 76.6) (2.5 – 57.1) (2.5 – 8.7) (2.5) Lung 36.5 ± 5.8 21.4 ± 11.6 2.5 ± 0.0* 2.5 ± 0.0* (22.1 – 59.7) (2.5 – 50.6) (2.5) (2.5) Trachea 23.3 ± 3.8 12.6 ± 6.2 2.5 ± 0.0*** 2.5 ± 0.0*** (10.1 – 38.9) (2.5 – 30.3) (2.5) (2.5) Plasma 12.6 ± 1.5 3.1 ± 1.3*** 0.5 ± 0.1*** 0.5 ± 0.0*** (6.2 – 19.4) (0.5 – 11.5) (0.5 – 0.8) (0.5) Fat 62.1 ± 11.3 23.7 ± 13.8* 6.6 ± 4.1*** 2.5 ± 0.0*** (37.5 – 113.9) (2.5 – 70.1) (2.5 – 23.1) (2.5) 76 Table 5. Distribution of PbTx-3 in T. scripta post IT exposure Distribution of PbTx-3 in T. scripta tissues and fluids post intratracheal instillation (IT) (10.53µg/kg). Parenthesis indicate the range of PbTx-3 found in each tissue (ng/g) or fluid (ng/ml). Intestines were analyzed with contents. Significant values were determined based on initial toxin concentrations at 1h post exposure. PbTx-3 ng /g tissue or ng/ml fluid (mean ± SEM) Organ 1h 24h 48h 1wk Kidney 38.6 ± 4.4 10.8 ± 2.9*** 14.7 ± 4.5*** 4.8 ± 1.4*** (21.8 – 52.6) (2.5 – 29.4) (2.5 – 28.5) (2.5 – 9.1) Urine 5.9 ± 2.1 5.3 ± 2.0 10.1 ± 8.5 2.0 ± 0.5 (2.0 – 13.9) (1.1 – 13.5) (1.1 – 43.9) (1.0 – 2.7) Liver 44.8 ± 3.5 10.5 ± 3.2*** 9.1 ± 4.4*** 4.2 ± 1.7*** (36.3 – 57.3) (2.5 – 23.3) (2.5 – 24.4) (2.5 – 12.5) Intestines 8.0 ± 1.6 2.5 ± 0.0 3.6 ± 1.1* 2.5 ± 0.0* (2.5 – 13.1) (2.5) (2.5 – 8.0) (2.5) Bile 36.1 ± 4.3 34.4 ± 5.9 38.8 ± 6.2 13.1 ± 1.2* (21.8 – 45.5) (23.3 – 80.3) (28.5 – 61.7) (9.6 – 16.7) Feces 39.2 ± 11.1 31.2 ± 8.1 42.4 ± 15.4 28.7 ± 14.8 (15.2 – 8.9) (9.8 – 82.8) (16.6 – 100.7) (2.5 – 71.3) Brain 30.2 ± 3.0 6.7 ± 3.2*** 4.5 ± 2.0*** 2.5 ± 0.0*** (22.6 – 40.7) (2.5 – 21.7) (2.5 – 12.3) (2.5) Heart 20.0 ± 1.2 4.1 ± 1.6*** 3.9 ± 1.4*** 2.5 ± 0.0*** (17.1 – 23.8) (2.5 – 11.8) (2.5 – 9.5) (2.5) Spleen 22.2 ± 2.2 3.6 ± 1.1*** 7.6 ± 3.2*** 4.2 ± 1.7*** (16.7 – 31.4) (2.5 – 12.4) (2.5 – 18.0) (2.5 – 12.8) Lung 20.0 ± 6.0 3.1 ± 0.6* 3.5 ± 1.0 2.5 ± 0.0* (2.5 – 52.2) (2.5 – 8.2) (2.5 – 7.7) (2.5) Trachea 363.3 ± 106.4 11.1 ± 2.7* 11.9 ± 4.5* 10.7 ± 3.6* (72.5 – 805.1) (2.5 – 24.7) (2.5 – 24.2) (2.5 – 26.2) Plasma 4.8 ± 1.0 1.4 ± 0.5*** 1.3 ± 0.5* 0.6 ± 0.1*** (3.5 – 5.1) (0.5 – 5.3) (0.5 – 2.5) (0.5 – 1.0) Fat 15.2 ± 4.0 6.4 ± 2.9 2.5 ± 0.0 2.5 ± 0.0 (2.5 – 26.7) (2.5 – 25.7) (2.5) (2.5) 77 Table 6. Distribution of PbTx-3 in M. terrapin post oral exposure Distribution of PbTx-3 in M. terrapin tissues and fluids post oral (30.13 µg/kg) exposure. Parenthesis indicate the range of PbTx-3 found in each tissue (ng/g) or fluid (ng/ml). Intestines were analyzed with contents. Significant values were determined based on initial toxin concentrations at 1h post exposure. PbTx-3 ng /g tissue or ng/ml fluid (mean ± SEM) Organ 1h 24h 1wk Kidney 76.1 ± 19.5 7.5 ± 2.6* 2.5 ± 0.0* (37.2 – 96.7) (2.5 – 11.1) (2.5) Liver 32.8 ± 3.5 2.5 ± 0.0*** 2.5 ± 0.0*** (26.0 – 37.2) (2.5) (2.5) Intestines 14.1 ± 5.6 2.5 ± 0.0 2.5 ± 0.0 (7.8 – 25.4) (2.5) (2.5) Bile 17.7 ± 4.4 19.0 ± 1.8 10.7 ± 1.5 (12.0 – 26.4) (16.8 – 22.6) (9.2 – 12.1) Brain 41.0 ± 9.8 2.5 ± 0.0* 2.5 ± 0.0* (21.4 – 51.9) (2.5) (2.5) Heart 38.3 ± 8.7 2.5 ± 0.0* 2.5 ± 0.0* (21.3 – 50.2) (2.5) (2.5) Spleen 37.9 ± 14.0 2.5 ± 0.0 2.5 ± 0.0 (12.5 – 60.8) (2.5) (2.5) Lung 13.8 ± 2.8 2.5 ± 0.0* 2.5 ± 0.0* (8.4 – 17.2) (2.5) (2.5) Trachea 9.4 ± 3.6 2.5 ± 0.0 2.5 ± 0.0 (2.5 – 14.8) (2.5) (2.5) Plasma 11.3 ± 1.4 0.63 ± 0.1* 0.5 ± 0.0* (8.5 – 12.6) (0.5 – 0.9) (0.5) 78 References Aho, J.M., Mulvey, M., Jacobson, K.C., Esch, G.W., 1992. Genetic differentiation among congeneric acanthocephalans in the yellow-bellied slider turtle. J. Parasitol. 78, 974– 981. Benson, J.M., Hahn, F.F., March, T.H., McDonald, J.D., Gomez, A.P., Sopori, M.J., Bourdelais, A.J., Naar, J., Zaias, J., Bossart, G.D., Baden, D.G., 2005. Inhalation toxicity of brevetoxin 3 in rats exposed for twenty-two days. Environ. Health Perspect. 113, 626–31. Benson, J.M., Tischler, D.L., Baden, D.G., 1999. Uptake, tissue distribution, and excretion of brevetoxin 3 administered to rats by intratracheal instillation. J. Toxicol. Environ. Health. A 57, 345–355. doi:10.1080/009841099157656. Berman, F.W., Murray, T.F., 2000. Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx. J Neurochem 74, 1443–1451. doi:10.1046/j.1471-4159.2000.0741443.x. Berman, F.W., Murray, T.F., 1999. Brevetoxins cause acute excitotoxicity in primary cultures of rat cerebellar granule neurons. J. Pharmacol. Exp. Ther. 290, 439–444. Bossart, G.D., Baden, D.G., Ewing, R.Y., Roberts, B., Wright, S.D., 1998. Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: gross, histologic, and immunohistochemical features. Toxicol.Pathol 26, 276–282. doi:10.1177/019262339802600214. 79 Bourdelais, A.J., Campbell, S., Jacocks, H., Naar, J., Wright, J.L.C., Carsi, J., Baden, D.G., 2004. Brevenal is a natural inhibitor of brevetoxin action in sodium channel receptor binding assays. Cell. Mol. Neurobiol. 24, 553–563. doi:10.1023/B:CEMN.0000023629.81595.09. Bourdelais, A.J., Jacocks, H.M., Wright, J.L.C., Bigwarfe, P.M., Baden, D.G., 2005. A new polyether ladder compound produced by the dinoflagellate Karenia brevis. J. Nat. Prod. 68, 2–6. doi:10.1021/np049797o. Cattet, M., Geraci, J.R., 1993. Distribution and elimination of ingested brevetoxin (PbTx- 3) in rats. Toxicon 31, 1483–1486. doi:10.1016/0041-0101(93)90214-4. Cocilova, C.C., Milton, S.L., 2016. Characterization of brevetoxin (PbTx-3) exposure in neurons of the anoxia-tolerant freshwater turtle (Trachemys scripta). Aquat. Toxicol. 180, 115–122. doi:10.1016/j.aquatox.2016.09.016. Divers, S.J., Stahl, S.J., Camus, A., 2010. Evaluation of Diagnostic Coelioscopy Including Liver and Kidney Biopsy in Freshwater Turtles (Trachemys scripta). J. Zoo Wildl. Med. 41, 677–687. doi:10.1638/2010-0080.1. Edwards, R.A., Lutz, P.L., Baden, D.G., 1989. Relationship between energy expenditure and ion channel density in the turtle and rat brain. Am. J. Physiol. 257, R1354–8. Fauquier, D.A, Flewelling, L.J., Maucher, J., Manire, C.A, Socha, V., Kinsel, M.J., Stacy, B.A, Henry, M., Gannon, J., Ramsdell, J.S., Landsberg, J.H., 2013. Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia brevis blooms in central west Florida. J. Zoo Wildl. Med. 44, 364–75. 80 Fire, S.E., Flewelling, L.J., Wang, Z., Naar, J., Henry, M.S., Pierce, R.H., Wells, R.S., 2008. Florida red tide and brevetoxins: Association and exposure in live resident bottlenose dolphins (Tursiops truncatus) in the eastern Gulf of Mexico, U.S.A. Mar. Mammal Sci. 24, 831–844. doi:10.1111/j.1748-7692.2008.00221.x. Fleming, L.E., Bean, J.A., Kirkpatrick, B., Cheng, Y.S., Pierce, R., Naar, J., Nierenberg, K., Backer, L.C., Wanner, A., Reich, A., Zhou, Y., Watkins, S., Henry, M., Zaias, J., Abraham, W.M., Benson, J., Cassedy, A., Hollenbeck, J., Kirkpatrick, G., Clarke, T., Baden, D.G., 2009. Exposure and effect assessment of aerosolized red tide toxins (brevetoxins) and asthma. Environ. Health Perspect. 117, 1095–100. doi:10.1289/ehp.0900673. Flewelling, L.J., Adams, D.H., Naar, J.P., Atwood, K.E., 2010. Brevetoxins in sharks and rays (Chondrichthyes, Elasmobranchii) from Florida coastal waters. Mar. Biol. 157. Flewelling, L.J., Naar, J.P., Abbott, J.P., Baden, D.G., Barros, N.B., Bossart, G.D., Bottein, M.-Y.D., Hammond, D.G., Haubold, E.M., Heil, C.A., Henry, M.S., Jacocks, H.M., Leighfield, T.A., Pierce, R.H., Pitchford, T.D., Rommel, S.A., Scott, P.S., Steidinger, K.A., Truby, E.W., Van Dolah, F.M., Landsberg, J.H., 2005. Brevetoxicosis: red tides and marine mammal mortalities. Nature 435, 755–756. doi:10.1038/nature435755a. Kreuder, C., Mazet, J. a K., Bossart, G.D., Carpenter, T.E., Holyoak, M., Elie, M.S., Wright, S.D., 2002. Clinicopathologic features of suspected brevetoxicosis in double-crested cormorants (Phalacrocorax auritus) along the Florida Gulf Coast. J. Zoo Wildl. Med. 33, 8–15. 81 Leighfield, T.A., Muha, N., Ramsdell, J.S., 2014. Tissue distribution of amino acid- and lipid-brevetoxins after intravenous administration to C57BL/6 mice. Chem. Res. Toxicol. 27, 1166–75. doi:10.1021/tx500053f. Manire, C.A., Anderson, E.T., Byrd, L., Fauquier, D.A., 2013. Dehydration as an effective treatment for brevetoxicosis in loggerhead sea turtles (Caretta caretta). J. Zoo Wildl. Med. 44, 447–452. doi:10.1638/2012-0163R.1. Naar, J., Bourdelais, A., Tomas, C., Kubanek, J., Whitney, P.L., Flewelling, L., Karen Steidinger, J.L., Baden, D.G., 2002. A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shelfish, and mamalian body fluid. Environ. Health Perspect. 110, 179–185. doi:10.1289/ehp.02110179. Perrault, J.R., Bauman, K.D., Greenan, T.M., Blum, P.C., Henry, M.S., Walsh, C.J., 2016. Maternal transfer and sublethal immune system effects of brevetoxin exposure in nesting loggerhead sea turtles (Caretta caretta) from western Florida. Aquat. Toxicol. 180, 131–140. doi:10.1016/j.aquatox.2016.09.020. Perrault, J.R., Schmid, J.R., Walsh, C.J., Yordy, J.E., Tucker, A.D., 2014. Brevetoxin exposure, superoxide dismutase activity and plasma protein electrophoretic profiles in wild-caught Kemp’s ridley sea turtles (Lepidochelys kempii) in southwest Florida. Harmful Algae 37, 194–202. doi:10.1016/j.hal.2014.06.007. Poli, M.A., Templeton, C.B., Thompson, W.L., Hewetson, J.F., 1990. Distribution and elimination of brevetoxin PbTx-3 in rats. Toxicon 28, 903–910. doi:10.1016/0041- 0101(90)90020-8. 82 Sayer, A.N., Hu, Q., Bourdelais, A.J., Baden, D.G., Gibson, J.E., 2006. The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro. Food Chem. Toxicol. 44, 1082–1091. doi:10.1016/j.fct.2006.01.002. Sunda, W.G., Burleson, C., Hardison, D.R., Morey, J.S., Wang, Z., Wolny, J., Corcoran, A.A., Flewelling, L.J., Van Dolah, F.M., 2013. Osmotic stress does not trigger brevetoxin production in the dinoflagellate Karenia brevis. Proc. Natl. Acad. Sci. U. S. A. 110, 10223–8. doi:10.1073/pnas.1217716110. Templeton, C.B., Poli, M.A., LeClaire, R.D., 1989. Cardiorespiratory effects of brevetoxin (PbTx-2) in conscious, tethered rats. Toxicon 27, 1043–1049. doi:10.1016/0041-0101(89)90155-4. Tibbetts, B.M., Baden, D.G., Benson, J.M., 2006. Uptake, tissue distribution, and excretion of brevetoxin-3 administered to mice by intratracheal instillation. J. Toxicol. Environ. Health. A 69, 1325–35. doi:10.1080/15287390500360091. Twiner, M.J., Fire, S., Schwacke, L., Davidson, L., Wang, Z., Morton, S., Roth, S., Balmer, B., Rowles, T.K., Wells, R.S., 2011. Concurrent exposure of bottlenose dolphins (Tursiops truncatus) to multiple algal toxins in Sarasota Bay, Florida, USA. PLoS One 6, e17394. doi:10.1371/journal.pone.0017394. Twiner, M.J., Flewelling, L.J., Fire, S.E., Bowen-Stevens, S.R., Gaydos, J.K., Johnson, C.K., Landsberg, J.H., Leighfield, T.A., Mase-Guthrie, B., Schwacke, L., van Dolah, F.M., Wang, Z., Rowles, T.K., 2012. Comparative analysis of three brevetoxin- associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida Panhandle region (USA). PLoS One 7. doi:10.1371/journal.pone.0042974. 83 Walsh, C.J., Butawan, M., Yordy, J., Ball, R., Flewelling, L., De Wit, M., Bonde, R.K., 2015. Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress. Aquat. Toxicol. 161, 73–84. doi:10.1016/j.aquatox.2015.01.019. Walsh, C.J., Leggett, S.R., Carter, B.J., Colle, C., 2010. Effects of brevetoxin exposure on the immune system of loggerhead sea turtles. Aquat. Toxicol. 97, 293–303. doi:10.1016/j.aquatox.2009.12.014. Walsh, C.J., Luer, C.A., Noyes, D.R., 2005. Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris. Vet. Immunol. Immunopathol. 103, 247–256. doi:10.1016/j.vetimm.2004.09.026. Washburn, B.S., Baden, D.G., Gassman, N.J., Walsh, P.J., 1994. Brevetoxin: tissue distribution and effect on cytochrome P450 enzymes in fish. Toxicon 32, 799 – 805. Woofter, R.T., Brendtro, K., Ramsdell, J.S., 2005. Uptake and elimination of brevetoxin in blood of striped mullet (Mugil cephalus) after aqueous exposure to Karenia brevis. Environ. Health Perspect. 113, 11–16. doi:10.1289/ehp.7274. 84 CHAPTER IV. INTRAVENOUS LIPID EMULSION AS AN EFFECTIVE TREATMENT PLAN FOR SEA TURTLES EXPOSED TO PBTX-3 Introduction Extensive mortalities and morbidities in marine life have been documented over many years that have been attributed to harmful algal blooms (HABs, Red tides). HABs occur due to population explosions of microscopic dinoflagellates who naturally may release neurotoxins depending on the species. When toxins are present in the environment humans may contract neurotoxic shellfish poisoning (NSP) from contaminated shellfish or acquire respiratory symptoms due to toxin release via ocean aerosols. In the Gulf of Mexico, Karenia brevis, a neurotoxic dinoflagellate, releases toxins known as brevetoxins. These blooms occur nearly annually and begin around September or October when the waters are warm, and can last anywhere from a few weeks to longer than a year, depending on the severity of the bloom (FWC, 2017). Other factors such as salinity, available sunlight and importantly, an increase in oceanic nutrients trigger these blooms. The Florida Fish and Wildlife Conservation Commission (FWC) updates the red tide status for Florida weekly to increase awareness in the community and to increase conservation efforts for animals that might be stranded or sick due to red tide exposure. Animals such as sea turtles and manatees found suffering from red tide exposure are taken to rehabilitation facilities for treatment. Neurological symptoms of brevetoxicosis, include muscle twitching, ataxia, and paralysis. Current animal treatments are supportive care, placing the animals in a toxin free 85 environment, and maintaining their heads above water to prevent drowning while the toxin clears (Manire et al., 2013). In loggerhead sea turtles experiencing edema, dehydration using furosemide has also proven to be effective (Manire et al., 2013). The severity of the response to brevetoxin exposure can vary by individual, likely due to differences in immune response, diet, body mass, species, and how much of the toxin they have consumed (Fauquier et al., 2013). A study that was conducted in 2013 looked at brevetoxin concentrations in the plasma for sea turtles admitted into rehabilitation experiencing clinical brevetoxicosis. The toxin cleared from their systems within 5-80 days, however, there was no correlation between the brevetoxin levels in the plasma and whether or not the animal survived (Fauquier et al., 2013). In a previous study using dehydration therapy as a method for treating PbTx exposed loggerhead sea turtles, 71% of the animals that were treated showed a decrease in neurological symptoms and therefore survived and were able to be released. Two other treatments that have been attempted for neurotoxin exposures are activated charcoal and mannitol, which have shown limited success in decreasing clinical symptoms (Manire et al., 2013). The ability to design treatment protocols for sea turtles has been limited because of their endangered status and toxin distribution studies have only been performed on necropsied animals which does not show initial impacts upon exposure. Based on our laboratory behavioral data, organ system impact, clearance rates, and histopathology analyses post PbTx-3 exposure, we derived and tested two different potential treatments strategies. The clinical symptoms seen in the laboratory after oral and intratracheal exposures in turtles are similar to those reported in the literature for mammals and for sea turtles taken in to rehabilitation centers, and include muscle 86 twitching and uncoordinated movements, swimming in circles, head bobbing, rigidity of the limbs and partial to complete paralysis (Templeton et al., 1989; Fauquier et al., 2013). In our laboratory studies, neurological symptoms post PbTx-3 appear within 2-5 min after inhalation exposures and approximately 30 minutes after oral exposures. The evident clinical signs last for 6h+ post toxin exposure and disappear within 24 hours; over 24-48h post-PbTx-3 exposure the animals still show reduced activity and lethargy (Cocilova et. al., 2017). The clinical observations mirror data from enzyme-linked immunosorbent assay (ELISA) analysis which confirmed that PbTx-3 distributes to all organ systems and for our relatively acute exposures, is rapidly cleared out the system 24-48 hours post exposure. While PbTx-3 rapidly clears from most organ systems in the turtle, concentrations increase in the bile and feces over the 24-48h period indicating the bile and feces is the primary route of excretion (Cocilova et. al., 2017). The bile plays an important role in the absorption of fat-soluble substances and due to the lipophilic nature of PbTx-3, treatment was targeted in increasing clearance rates of PbTx-3. Based on this information, cholestyramine administration was tested as a potential treatment. Cholestyramine is an anion exchange resin that strongly binds to bile salts to prevent bile reabsorption. It was hypothesized that cholestyramine would become bound to the bile and allow for a more rapid PbTx-3 clearance. Cholestyramine has been used in humans to lower cholesterol levels and several case studies in domestic animals report successful use of the compound in cases of toxin exposure (Shepherd et al., 1980; Rankin et al., 2013), however to my knowledge it has not been tested in reptiles. 87 The second treatment attempted relied on the fact that brevetoxin is a lipid-soluble molecule that easily passes through the cell membranes, thus a high lipid concentration emulsion therapy utilized for treating lipophilic drug poisoning was tested. Intralipid emulsion therapy (Intralipids, ILE) is a lipid compound that is thought to draw toxins out of the fatty membranes of cells by creating a high concentration of lipids in the extracellular compartment (Rothschild et al., 2010). ILE has been reported in several case studies as an antidote in veterinary medicine for animals who have ingested lipophilic toxins (Gwaltney-Brant and Meadows, 2012; Fernandez et al., 2011) In a recent case, a dog who ingested bromethalin, a neurotoxic rodenticide, was successfully treated with ILE therapy; toxin levels in the serum were reduced by 75% post ILE treatment (Kosh et al., 2010). ILE treatment has also been used successfully with two juvenile green sea turtles suffering from domoic acid poisoning, a neurotoxin produced by certain algae (Dr. Charles Manire, pers. comm.). Since ILE therapy has been used in a number of successful cases, I hypothesized that this treatment would reduce the neurological symptoms associated with brevetoxicosis in the turtles. The success of either cholestyramine and/or ILE as treatment for brevetoxicosis in T. scripta would be recommended as a potential treatment in sea turtles (and other animals) undergoing rehabilitation in facilities worldwide to increase survival among populations at risk. Material and methods Experimental Animals A total of 42 T. scripta were used for these experiments and all work was approved by the Florida Atlantic University Institutional Animal Care and Use Committee (IACUC). All animals were acclimated to our laboratory for two weeks prior 88 to conducting any experiments. Male and female freshwater turtles (Trachemys scripta), approximately 12-15 cm straight carapace length and weighing 0.2-0.4 kg were obtained from a commercial supplier (Niles Biological Inc., Sacramento, CA) and maintained in freshwater tanks at room temperature (22°C +/- 3°C, 50% relative humidity +/- 4%) on a 12h day/night cycle. Aquaria were cleaned according to standard husbandry methods and the animals fed commercial aquatic food 3X weekly to satiety. Turtles were given individual identification numbers and randomly assigned to treatment groups. Brevetoxin Brevetoxin (PbTx-3) was purchased from LKT Laboratories (St. Paul, Minnesota) and was dissolved in ethanol and mixed with 0.9% NaCl saline to a final concentration of 0.05µg/µl. Turtles were restrained by hand and administered 10.53 µg PbTx-3/kg body mass by intratracheal instillation (IT) ~1.5cm into the tracheal opening at the base of the tongue, and a rubber bulb and pipette tip were used to inflate the lungs 3 times following instillation of the toxin to mimic inhalation (Cocilova, et al., 2017). Animals received one dose of PbTx-3 either prior to or after the treatment drug. Control animals received sham doses of physiological saline solution mixed with ethanol to a final concentration to 0.1% EtOH (Cocilova and Milton, 2016) to mimic the treatment solution, and sham exposures were conducted as in toxin exposures. Treatments Cholestyramine Cholestyramine powder for oral suspension (4g cholestyramine resin USP, per scoopful) was purchased from PAR Pharmaceutical Companies, Inc. (Spring Valley, NY). Cholestyramine was mixed with DI water to a final concentration of either 20mg/kg 89 or 50mg/kg. Turtles were administered one intratracheal dose of PbTx-3 (10.53µg/kg) prior to receiving oral cholestyramine via an esophageal tube at both 2h and 6h post PbTx-3 exposure. Animal tissues and fluids were collected 24 hours post toxin exposure and tissues and fluids were analyzed via ELISA. Control animals were administered 2 doses of 50 mg/kg of cholestyramine 4 hours apart and sacrificed 24h post cholestyramine. Intralipid emulsion Intralipid® (ILE, Fresenius Kabi, Uppsala, Sweden) is a 20% fat emulsion consisting of 20% soybean oil, 1.2% phospholipids (from powdered egg yolk), 2.25% glycerin, USP, water for injection q.s., with pH 8 (6-8.9) adjusted with sodium hydroxide. Turtles were administered ILE i.v. via the subcarapacial vein prior to or after intratracheal exposure to PbTx-3. ILE must be given i.v. and administered slowly to prevent lipid embolism. Animals were administered one dose of PbTx-3 (10.53 µg/kg) via intratracheal instillation. Intralipids (50 or 100 mg/kg) were administered to animals 30 min pre- or 30 min post- toxin exposure. Animals were monitored every 5 minutes for the first hour, every 30 min for up to 8h, and at 24h to compare brevetoxin symptoms post intralipid treatment versus untreated animals (exposed to PbTx-3 without ILE treatment). Animals were sacrificed either 1h or 24h post toxin exposure and tissues and fluids were collected for to determine PbTx-3 tissue distribution, PbTx-3 concentrations in each tissue/fluid and rates of clearance. Control animals received one dose of ILE (100 mg/kg) and tissues were analyzed via ELISA 24h post toxin exposure. 90 Tissue collection All turtles were euthanized by decapitation and the following tissues and fluids were collected for ELISA: kidney, liver, intestines, bile, brain, heart, spleen, lung, trachea, and plasma. Whole blood was collected by exsanguination, feces were removed from the large intestine, and other fluids were collected directly from the organ with a syringe post mortem. Tissues were flash frozen in liquid nitrogen and stored at -80°C. Plasma was extracted from whole blood by centrifugation and the plasma and other fluid samples were frozen at -80°C. Tissue and fluid samples were shipped overnight to Florida Fish and Wildlife Research Institute on dry ice and remained frozen at -80°C until processing. Extraction A competitive ELISA was used to detect brevetoxins in turtle tissues and biological fluids as previously described (Fauquier et al., 2013). Solid tissues were thawed prior to extraction and homogenized in the presence of 80% aqueous methanol (4 ml/g tissue). Homogenates were centrifuged for 10 min at 3000×g and the supernatants were transferred to clean 50 ml centrifuge tubes. The pellets were extracted a second time in the same manner and the supernatants were pooled. The combined extracts were then partitioned once with 100% hexane (1:1, v:v), and the methanol fractions were retained at -20°C until analyzed. Brevetoxin in bile was extracted by liquid-liquid extraction. In 15 ml centrifuge tubes, bile (0.5 ml) was combined with ethyl acetate (1 ml) and vortexed for 1 min. After centrifuging for 5 min at 3000×g, the top ethyl acetate fraction was transferred to a clean 15 ml centrifuge tube, the bottom bile fraction was extracted two more times in the same manner, and the ethyl acetate fractions were combined. Deionized 91 water (3 ml) was then added to the combined ethyl acetate fraction, the sample was vortexed for 1 min, centrifuged for 5 min at 3000×g and the top ethyl acetate fraction was transferred to a clean disposable glass test tube. The ethyl acetate was evaporated to dryness in a Speedvac (Thermo Scientific Savant) and the residue was re-dissolved in 1 ml of 80% aqueous methanol. Urine and plasma were not extracted. They were thawed and diluted in phosphate buffered saline (pH 7.4) containing 0.05% Tween 20 and 0.5% gelatin immediate prior to analysis. ELISA Brevetoxins and brevetoxin-like compounds were quantified in all sample extracts using a competitive enzyme linked immunosorbent assay (ELISA; Marbionc, Wilmington NC) performed according to Naar et al., (2002) with modifications described in Flewelling (2008). Toxin concentrations were calculated using a PbTx-3 standard curve, and results are reported in PbTx-3 equivalents. The limits of detection as described were approximately 1-2 ng/ml in plasma or urine, 10 ng/ml in bile, and 10 ng/g for feces or tissue. Behavioral analysis Turtles grouped in the 1h post exposure experiments were monitored for clinical symptoms of brevetoxicosis and video recorded immediately and at 5 min, and every 10 min thereafter for one hour post-toxin exposure. Animals that were part of the 24h exposure group were monitored and video recorded at 5 min, then every 10 minutes for the first hour, and every hour after that up to 8h post PbTx-3 and then again at the 24h point. All PbTx-3 control animals were noted as showing severe symptoms of brevetoxicosis (Table 7). 92 Animals that were administered PbTx-3 followed by cholestyramine exhibited clear symptoms of brevetoxin exposure ranging from muscle twitching to unresponsiveness. Turtles were monitored and video recorded immediately after each PbTx-3 treatment and every hour for the first 8h post toxin administration and then at the 24h time point. Animals that were treated with ILE (pre- and post- PbTx-3) were monitored and clinical signs were video recorded 5 min post PbTx-3, and every 10 minutes for the 1h exposures. Animals grouped in the 24h post PbTx-3 exposures were recorded for clinical signs every 30 minutes after 1h and up to 8h post PbTx-3. Turtles were again recorded at the 24h time point. Animal behavioral changes were recorded as having no to severe symptoms post-PbTx-3 exposure depending on experimental group (Tables 8 and 9). Statistics Data were analyzed using one-way analysis of variance (ANOVA) followed by Holm-Sidak Test (Holm-Sidak) pairwise comparison test using Sigma Plot 11.0 (Systat Software, Inc, San Jose, California). Samples in which brevetoxin was not measurable were given a value of half the level of detection (2.5 ng per g tissue or ml bile or 0.5 ng per ml fluid) to calculate mean. Results Symptoms of brevetoxin exposure Turtles that were administered one dose of IT PbTx-3 exhibited muscular and neurological symptoms consistent with brevetoxicosis including head bobbing, muscle twitching, ataxia, swimming in circles and paralysis. The onset of symptoms were within 2-5 minutes post PbTx-3 exposure and lasted over 6h in untreated animals, showing a 93 decline in symptoms over a 24h period. PbTx-3 rapidly clears from tissues 24h post toxin exposure (Fig. 11). In the cholestyramine treated animals post-PbTx-3 exposure, there was not a reduction in clinical signs (Table 8). Bile clearance post cholestyramine treatment The levels of PbTx-3 varied widely in the tissue and fluids of PbTx-3 control animals. We did not see any significant changes in the bile post cholestyramine treatment for either dose of cholestyramine (20mg/kg or 50mg/kg) compared to the PbTx-3 controls (Fig. 12). There was however an overall decrease in the detoxification and excretion systems; the kidney, liver and feces, but there were no significant differences between either cholestyramine treatment groups compared to the PbTx-3 controls. The brain, heart and fat also showed a decrease in PbTx-3 concentration after both dosing treatments with cholestyramine but no significant changes were noted. Symptoms post ILE treatment Animals that received the ILE treatment (100mg/kg) 30 min after PbTx-3 exposure had greatly reduced symptoms of brevetoxicosis within the first 2h compared to animals that did not receive the treatment. The most notable differences between PbTx-3 groups and the ILE treatment groups were at 2h, 6h and 24h post PbTx-3 exposure. At 6h post toxin exposure, untreated animals continued showing the same clinical signs as they did upon initial toxin administration (twitching, limb rigidity, ataxia), whereas ILE treated animals were no longer showing those symptoms and were moving around their tanks actively and in a coordinated manner. We also tested ILE at a concentration of 50mg/kg, and even though symptoms were reduced in comparison to untreated animals, animals responded better to the 100mg/kg dosing regimen, with an earlier and greater 94 reduction in behavioral symptoms of brevetoxicosis. Tissue and fluid levels of PbTx-3 were also lower with the higher ILE dose of 100mg/kg (Figs. 15 and 16). Animals that received the ILE treatment 30 min prior to PbTx-3 showed little to no symptoms of PbTx-3 exposure. Animals initially had slight changes in motor responses but were moving around and appeared clinically normal within the hour. Even though ILE treatment prior to PbTx-3 exposure in the wild is not practicable, this suggests the power of ILE use in cases of fat-soluble toxicity in wildlife. Clearance of PbTx-3 from the tissues and excretion systems with ILE treatment Aside from the reduced clinical signs associated with the ILE treatments, we also investigated the tissue distribution of PbTx-3 in ILE treated animals vs. PbTx-3 only animals. Tissues and fluids showed distribution to most organ systems within the first 1h post-exposure to PbTx-3, with concentrations accumulating higher in the intestines, bile, feces, and urine 24h post PbTx-3 exposure compared to 1h post PbTx-3 (Fig. 11). PbTx-3 concentrations in the kidney, liver, brain, and heart decreased more in the 1h post toxin exposure and higher ILE dose of 100mg/kg compared to the lower dose of 50mg/kg (Figs. 15 and 16). Tissues and fluids show an increase in PbTx-3 in the bile and feces over a 24h period of time post ILE treatment (Fig. 16) which is greater than compared to PbTx-3 control animals, suggesting that the toxin is clearing at a quicker rate post ILE. Similar results were found in the kidney, liver, brain, heart, spleen, lung and fat for 24h post toxin + ILE (100mg/kg) treatments groups (Fig. 16). These results suggest that the higher ILE dose of 100mg/kg is more effective in clearing out PbTx-3 from tissues than the 50mg/kg dose of ILE. Along with reduced to no symptoms of brevetoxicosis, ILE (100mg/kg) administered before brevetoxin resulted in overall lower toxin concentrations 95 in all tissues and fluids tested (Figs. 13 and 14) suggesting the action of ILE is indeed resulting in a lower toxin accumulation in the organ systems. Discussion Brevetoxicosis is a serious environmental hazard for animals that inhabit and nest in areas where HABs are known to occur. Not only are these animals affected by the toxins directly, but impacts on behavior that reduce foraging ability, as well as negative effects on the immune system, may make animals exposed to HABs more susceptible to other diseases. In a previous study looking at the immune system of loggerhead sea turtles exposed to brevetoxins, a number of genes involved in oxidative stress were upregulated, indicating disruption to normal functioning cells and cellular signaling pathways (Walsh et al., 2010). When animals are coming into rehabilitation facilities suffering from brevetoxicosis, they are experiencing the same clinical behaviors that I am seeing in the laboratory, including jerky body movements, ataxia and unresponsiveness. Plasma samples have been collected from live sea turtles during the time spent in rehabilitation to examine rates of clearance. These rates can vary depending on the amount of toxin the animals are coming in with and how long animals have spent in an area where a bloom is occurring. Interestingly, animals that died in rehabilitation had lower brevetoxin levels in the tissues and fluids than animals that survived (Fauquier et al., 2013). These data suggests that the toxin was metabolized back into the animal’s system from the intestinal tract causing continuous intoxication while still in rehabilitation. My previous data showed that brevetoxin concentrates higher in the detoxification and excretions systems, liver, kidney, bile and feces; the highest brevetoxin levels in necropsied sea turtles were found in the feces, stomach and liver (Cocilova et 96 al., 2017; Fauquier et al., 2013). It appears as though biomagnification could be playing a large role in how sea turtles become intoxicated and diet differences between species could play a role in the amount of brevetoxin consumed. Loggerhead sea turtles may be more susceptible than other species because they prey on filter feeding invertebrates that may serve as brevetoxin carriers (Bjorndal, 1996). Histopathology was also conducted on dead sea turtles associated with brevetoxin exposure and similar to the results discussed in chapter III, no definitive pathological lesions were noted post PbTx exposure. It has been shown in mammalian models that they experience similar symptoms, after exposure to high doses of PbTx-2 (up to 100 µg/kg); the time of symptom onset appeared to vary and some animals experienced violent behavioral changes post exposure (Templeton et al., 1989). In the same study, the heart rates were irregular including premature ventricular depolarization, pulse pressure increases, and in some cases complete heart block as well as exhibiting very deep ventilations and decreased body temperatures (Templeton et al., 1989). Mammalian studies have also shown that the mode of action of PbTx in the brain is similar between rats and turtles; PbTx-3 binds and opens to VGSC that leads to increases in intracellular calcium in neighboring cells and could trigger the programmed cell death pathway (Cocilova and Milton, 2016; Berman and Murray, 2000). In addition, air breathing marine animals like sea turtles have large tidal volumes (Lutcavage et al., 1989) which are advantageous for diving and foraging but may increase exposure to toxins aerosolized near the sea surface. The tidal volumes can range from 4 to 187 ml/kg depending on species and sea turtles with the higher tidal volumes are the loggerheads and greens (Lutcavage and Lutz, 1996). Long term blooms typically last for 97 a few months and turtles nesting on the west coast beaches of Florida during blooms can be exposed to ocean aerosols and inhalation. Even after a bloom has come and gone, animals may still become affected due to vectors still carrying the toxin. In a previous mass mortality event in 2004, when a red tide bloom was not present, 34 Florida manatees and 107 bottlenose dolphins were found dead and was later determined to be from vectors still carrying the toxin long after a bloom disappeared (Flewelling et al., 2005). In this study we administered one intratracheal dose of brevetoxin and animals began experiencing severe symptoms of brevetoxicosis within 2-5 minutes that lasted for 6h+ post exposure, but symptoms were dramatically reduced and by 24h and the toxin was cleared quite rapidly from tissues. The length of exposure for animals in the wild is largely unknown and the initial impacts are difficult to address, however necropsied animals have shown PbTx-3 concentrations their tissues of greater than 100 ng/g and even higher in the feces (up to 61,078 mg/g) (Fauquier et al., 2013) supporting the idea that they are exposed to high concentrations in the wild. When sea turtles are brought into rehabilitation facilities with suspected brevetoxicosis, it is hard offer them anything other than supportive care when it is unknown what physiological systems are impacted by the toxin. Thus these studies were performed to determine initial organ system impacts and rates of clearance post PbTx-3 exposure both orally and intratracheally using the freshwater turtles as a model in order to devise appropriate treatment strategies. From our previous studies we have concluded that PbTx-3 distributes to all organ systems, after both oral and intratracheal exposure, with highest concentrations in the liver, kidney, bile and feces; the overall toxin concentrations were higher in the PbTx-3 oral exposed animals as significantly higher concentrations had to be administered orally 98 to achieve the same symptoms compared to the IT experiments. PbTx-3 concentrations in the bile and feces, post oral and IT exposures, increased over a 48h period of time, while other tissues showed a rapid decline in toxin levels. Due to the demonstrated increase of PbTx-3 in the bile over time we first tested cholestyramine as a potential treatment. Cholestyramine binds to bile salts and prevents reabsorption of bile compounds, and is thus used in human medicine to lower cholesterol (Pinto and Zuniga, 2011). We thus hypothesized that the compound would also bind PbTx-3 in the bile and increase the rates of clearance over 24h compared to control animals exposed to only the toxin and not cholestyramine. However, the results from this study did not show any changes in the bile over a 24h period and therefore this hypothesis was rejected. While organs like the kidney, liver, heart, lung, and brain showed a decrease in PbTx-3 over a 24h period of time, there were no noted significant differences observed between the cholestyramine treated group and the PbTx-3 control groups. In this study, though, the turtles were only given one dose of PbTx-3 prior to cholestyramine treatments and the levels in the tissues were not as elevated following a single dose as they were following multiple doses; in the initial studies turtles received 12 doses over a 4 week period (Chapter III). Repeated dosing over a 4 week period would lead to higher final toxin levels in the tissues and fluids after the duration of the experiment which may lead to a more effective examination in PbTx-3 clearance rates over time post cholestyramine. The ELISAs show that PbTx-3 levels in the tissues were not as elevated following a single dose as they are following multiple doses (Fig. 11; Chapter III). In addition, we followed a mammalian protocol as described in Rankin et al., (2013) with either 20mg/kg or 50mg/kg doses given at the same time points (1h and 6h) post toxin exposure, but as the PbTx-3 dose we 99 employed in the turtle studies is significantly higher than is used in laboratory studies in mammals (Chapter III) it is possible that the cholestyramine doses were not high enough to see significance. Animals in the wild are exposed to even higher toxin levels that under our controlled laboratory exposures; PbTx-3 in the liver of red tide stranded sea turtles was found up to a concentration of 1,006 ng/g and up to 61,078 ng/g in the feces (Fauquier et al., 2013). Animals are likely spending longer periods of time in blooms which could be contributing to overall higher toxin loads found in tissues and fluids. Additional doses of cholestyramine, or higher concentration, may prove more effective and should be tested in future studies. Cholestyramine could therefore still be a possible treatment for animals entering rehabilitation facilities suffering from toxin exposure. Turtles exposed to PbTx-3 experimentally in this study show obvious behavioral deficits symptomatic of brevetoxicosis for at least 10-12 hours post-exposure, but recover within 24 hours except for persistent lethargy. As the toxin has been shown to critically affect organs systems such as the brain, liver, kidney, and heart, we also tested the intralipid emulsion as a potential treatment strategy. Since ILE is thought to draw lipid soluble toxins out of tissues and sequester them, I hypothesized that it would be a successful treatment to decrease toxin build up in the tissue and help it clear out of the body more rapidly. ILE has been used successfully in veterinary practices to treat some cases of toxin ingestion from domestic animals; I have shown here that ILE provides successful symptom reduction and increases toxin elimination. In both ILE treatment exposure scenarios, with the emulsion administered pre- or post-toxin, there was significant amelioration of neurological and muscular deficits compared to animals that did not receive ILE. ILE treatment post PbTx-3 proved to be 100 successful in rapidly reducing symptoms of brevetoxin exposure over a 6h period; by 24h post PbTx-3 + ILE the turtles were moving normally within their aquaria. The most notable differences between PbTx-3 groups and ILE post toxin treatment groups were at 2h, 6h and 24h post PbTx-3 exposure. The rapid recovery of ILE treated animals was reflected in the tissues and fluids post ILE treatment as well, which showed an increase in PbTx-3 in the bile and feces over a 24h period of time compared to non-ILE treated animals (Fig. 14), suggesting ILE may be helping the toxin clear at a quicker rate by drawing the toxin out of the tissue and allowing it to be excreted. Similarly, ILE treatment was successfully used in cats exposed to permethrins, which are neurotoxins that also act on sodium channels and cause ataxia, tremors, and seizures; ILE treatment reduced clinical symptoms and vitals were back to normal within a 24h period (DeGroot, 2014). There were notable behavioral differences between the two ILE doses used. The animals that received the higher dose of 100mg/kg showed a greater reduction in symptoms as well as a decrease in PbTx-3 buildup in the tissues. While not practicable from a rehabilitation standpoint, we also tested the efficacy of ILE as a protective agent against PbTx-3 by administering the treatment 30 min prior to the toxin. ILE administered before brevetoxin resulted in overall lower concentrations in all tissues and fluids tested (Figs. 13 and 14), compared to control animals or those receiving ILE after toxin exposure, suggesting that ILE treatment prior to the toxin prevents it from collecting in the tissues in as high concentrations as it does in animals given ILE post PbTx-3. Behavioral symptoms were likewise nearly absent in animals in which ILE was administered prior to the toxin, reinforcing its potential as an aid to detoxification in rehabilitation facilities. We conclude that ILE treatment for lipophilic 101 toxins such as PbTx-3 has the potential to positively impact survival in turtle, and anticipate that this treatment will be implemented at rehabilitation centers for toxin- exposed sea turtles. 102 Table 7. Behavioral symptoms post IT PbTx-3 PbTx-3 was administered to T. scripta one time by intratracheal instillation (IT). Behavioral symptoms were documented 2-5 min post PbTx-3 exposure for each group. N = the number of animals in each experimental group. Gender ratios were also noted. Tissues and fluids were collected 1h and 24 post PbTx-3. Experimental Time point PbTx-3 IT Behavioral symptoms Gende N group post PbTx-3 Dose (µg/kg) r ratio PbTx-3 Control 1h 10.53 Significant muscle 3F:0M 3 twitching, swimming in circles, head bobbing, paralysis, ataxia PbTx-3 Control 24h 10.53 Significant muscle 3F:0M 3 twitching, swimming in circles, head bobbing, paralysis, ataxia 103 Table 8. Behavioral symptoms post cholestyramine treatment PbTx-3 was administered to T. scripta one time by intratracheal instillation (IT) 2h prior to the first dose of cholestyramine, either 20mg/kg or 50mg/kg. Cholestyramine was given again 6h post PbTx-3. Behavioral symptoms were documented 2-5 min post PbTx- 3 exposure. N= the number of animals in each experimental group. Gender ratios were also noted. Tissues and fluids were collected 24 post PbTx-3 exposure. Experimental Cholestyramine Behavioral symptoms Gender N group Dose (mg/kg) ratio Cholestyramine 50 mg/kg Normal Behavior 2F:1M 3 sham control PbTx-3 + 20 mg/kg Significant muscle 3F:2M 5 Cholestyramine twitching, swimming in circles, head bobbing, paralysis, ataxia PbTx-3 + 50 mg/kg Significant muscle 2F:2M 4 Cholestyramine twitching, swimming in circles, head bobbing, paralysis, ataxia 104 Table 9. Behavioral symptoms pre- and post-intralipid treatment PbTx-3 was administered to T. scripta one time by intratracheal instillation and ILE was given 30 min pre- or post-PbTx-3 (50mg/kg or 100mg/kg) and monitored for behavioral changes. Behavioral symptoms for PbTx-3 + ILE groups were recorded for the first 6h post-PbTx-3. ILE + PbTx-3 significant behavioral changes were noted in the first 1-2h post ILE exposure and symptoms were greatly reduced by 6h. Tissues and fluids were collected 1h and 24h post PbTx-3 administration. Experimental Time point ILE Dose Behavioral symptoms Gender N group post PbTx-3 (mg/kg) ratio ILE sham 1h 100 Normal behavior 1F:2M 3 control PbTx-3 + ILE 1h 50 Reduced ambulation, 2F:1M 3 moderate twitching, slight head bobbing PbTx-3 + ILE 1h 100 Reduced ambulation, 3F:1M 4 mild twitching, slight head bobbing ILE + PbTx-3 1h 100 Mild to no twitching 4F:0M 4 PbTx-3 + ILE 24h 50 Reduced ambulation, 2F:0M 2 mild twitching, slight head bobbing PbTx-3 + ILE 24h 100 Reduced ambulation, 3F:1M 4 mild twitching, slight head bobbing ILE + PbTx-3 24h 100 Mild to no twitching 4F:0M 4 105 Figure 11. Distribution of PbTx-3 in tissues and fluids 1h and 24h post exposure 4000 4000 Control PbTx 1h Control PbTx 24h Control 3000 3000 2000 2000 3] ng/g 3] ng/ml - 100 100 - [PbTx [PbTx Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected for sham controls, 1h post-PbTx-3 controls, and 24h post PbTx-3 controls. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg). Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable were given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. 106 Figure 12. Distribution of PbTx-3 post cholestyramine treatment 50 PbTx-3 control 20mg/kg 50mg/kg 40 PbTx-3 ng/ml 30 3] ng/g 3] ng/mL 3] - - 20 PbTx-3 ng/g PbTx-3 [PbTx [PbTx 10 0 Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected 24h PbTx-3 controls and PbTx-3 + cholestyramine treatments, either 20mg/kg or 50 mg/kg. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg) and cholestyramine was given 2h and 6h post PbTx-3. Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable were given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. ILE control exposure were all below the level of detection. 107 Figure 13. Distribution of PbTx-3 1h post exposure + pre- and post-ILE treatment 4000 Control PbTx 1h Control 3000 PbTx+ILE 1h 100mg/kg ILE+PbTx 1h 100 mg/kg 2000 1000 3] ng/g ng/mL 3] - - 100 [PbTx-3]ng/g [PbTx-3]ng/ml [PbTx [PbTx 0 Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected for ILE sham controls, 1h post-PbTx-3 controls, and for 1h post PbTx-3 + ILE and ILE + PbTx-3 where ILE was given at a 100 mg/kg dose. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg). Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable was given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. ILE control exposure were all below the level of detection. 108 Figure 14. Distribution of PbTx-3 24h post exposure + pre- and post-ILE treatment ILE Control 40 PbTx 24h Control PbTx+ILE 24h 100mg/kg ILE+PbTx 24h 100mg/kg 30 3] ng/mL 3] 3] ng/g - - 20 [PbTx-3] ng/g [PbTx-3] [PbTx-3] ng/ml [PbTx-3] [PbTx [PbTx 10 0 Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected for ILE sham controls, 24h post-PbTx-3 controls, and for 24h post PbTx-3 + ILE and ILE + PbTx-3 where ILE was given at a 100 mg/kg dose. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg). Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable was given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. ILE control exposure were all below the level of detection. 109 Figure 15. Distribution of PbTx-3 (1h) post ILE treatment 4000 ILE Control PbTx 1h Control PbTx+ILE 1h 50mg/kg 3000 PbTx+ILE 1h 100mg/kg 2000 3] ng/mL 3] - [PbTx [PbTx-3]ng/g [PbTx-3] ng/ml [PbTx-3] 0 Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected for ILE sham controls, 1h post-PbTx-3 controls and 1h post PbTx-3 + ILE for both ILE concentrations, 50mg/kg and 100 mg/kg. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg). Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable was given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. ILE control exposure were all below the level of detection. 110 Figure 16. Distribution of PbTx-3 (24h) post ILE treatment 50 ILE Control PbTx 24h Control PbTx+ILE 24h 50mg/kg 40 PbTx+ILE 24h 100mg/kg 30 3] ng/mL - 20 PbTx-3 ng/ml PbTx-3 [PbTx-3] ng/g [PbTx-3] [PbTx 10 0 Bile Fat Urine Liver Feces Brain Heart Lung Kidney Spleen Plasma Intestine Trachea Distribution of brevetoxin (ng PbTx-3 eq. per g or ml) in the tissues and fluids of T. scripta collected for ILE sham controls, 24h post-PbTx-3 controls and 24h post PbTx-3 + ILE for both ILE concentrations, 50mg/kg and 100 mg/kg. PbTx-3 was administered one time via intratracheal instillation (10.53µg/kg). Error bars represent standard error of the mean. There was no significance between in the tissue/fluids between the different treatment groups. Samples in which brevetoxin was not detectable was given a value of half the level of detection (2.5ng/g of tissue or ml bile or 0.5 ng/ml fluid) to calculate the mean. ILE control exposure were all below the level of detection. 111 References Berman, F.W., Murray, T.F., 2000. Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx. J Neurochem 74, 1443–1451. doi:10.1046/j.1471-4159.2000.0741443.x. Bjorndal, K.A., 1996. The Foraging Ecology and Nutrition of Sea Turtles. Biol. Sea Turtles, Vol. 1 448. Cocilova, C.C., Milton, S.L., 2016. Characterization of brevetoxin (PbTx-3) exposure in neurons of the anoxia-tolerant freshwater turtle (Trachemys scripta). Aquat. Toxicol. 180, 115–122. doi:10.1016/j.aquatox.2016.09.016. Cocilova, C.C, Flewelling, L.J., Bossart, G.D., Granholm, A.A., Milton, S.L., 2017. Tissue uptake, distribution and excretion of brevetoxin-3 after oral and intratracheal exposure in the freshwater turtle Trachemys scripta and the diamondback terrapin Malaclemys terrapin. Aquat. Toxicol. 187. 29-37. DeGroot, W.D., 2014. Intravenous lipid emulsion for treating permethrin toxicosis in a cat. Can. Vet. J. 55, 1253–1254. Fauquier, D.A, Flewelling, L.J., Maucher, J., Manire, C.A, Socha, V., Kinsel, M.J., Stacy, B.A, Henry, M., Gannon, J., Ramsdell, J.S., Landsberg, J.H., 2013. Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia brevis blooms in central west Florida. J. Zoo Wildl. Med. 44, 364–75. Fernandez, A.L., Lee, J.A., Rahilly, L., Hovda, L., Brutlag, A.G., Engebretsen, K., 2011. The use of intravenous lipid emulsion as an antidote in veterinary toxicology. J. Vet. Emerg. Crit. Care. doi:10.1111/j.1476-4431.2011.00657.x. Flewelling, L.J., Naar, J.P., Abbott, J.P., Baden, D.G., Barros, N.B., Bossart, G.D., 112 Bottein, M.-Y.D., Hammond, D.G., Haubold, E.M., Heil, C.A., Henry, M.S., Jacocks, H.M., Leighfield, T.A., Pierce, R.H., Pitchford, T.D., Rommel, S.A., Scott, P.S., Steidinger, K.A., Truby, E.W., Van Dolah, F.M., Landsberg, J.H., 2005. Brevetoxicosis: red tides and marine mammal mortalities. Nature 435, 755–756. doi:10.1038/nature435755a. Florida Fish and Wildlife Conservation Commission (FWC), 2017. Red Tide Current Status. Retrieved from http://myfwc.com/redtidestatus. Gwaltney-Brant, S., Meadows, I., 2012. Use of Intravenous Lipid Emulsions for Treating Certain Poisoning Cases in Small Animals. Vet. Clin. North Am. - Small Anim. Pract. doi:10.1016/j.cvsm.2011.12.001. Kosh, M.C., Miller, A.D., Michels, J.E., 2010. Intravenous lipid emulsion for treatment of local anesthetic toxicity. Therapeutics and clinical risk management. doi:10.2147/TCRM.S11861. Lutcavage, M.E., Lutz, P.L., Baier, H., 1989. Respiratory Mechanics of the Loggerhead Sea Turtle, Caretta caretta. Respir. Physiol. 76, 10. Manire, C.A., Anderson, E.T., Byrd, L., Fauquier, D.A., 2013. Dehydration as an effective treatment for brevetoxicosis in loggerhead sea turtles (Caretta caretta). J. Zoo Wildl. Med. 44, 447–452. doi:10.1638/2012-0163R.1. Naar, J., Bourdelais, A., Tomas, C., Kubanek, J., Whitney, P.L., Flewelling, L., Karen Steidinger, J.L., Baden, D.G., 2002. A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shelfish, and mamalian body fluid. Environ. Health Perspect. 110, 179–185. doi:10.1289/ehp.02110179. 113 Pinto, X., Zuniga, M., 2011. Treatment of hypercholesterolemia and prevention of cardiovascular diseases through inhibition of bile acid reabsorption with resin cholestyramine. Clin. e Investig. en Arterioscler. 23, 9–16. Rankin, K.A., Alroy, K.A., Kudela, R.M., Oates, S.C., Murray, M.J., Miller, M.A., 2013. Treatment of cyanobacterial (microcystin) toxicosis using oral cholestyramine: Case report of a dog from Montana. Toxins (Basel). 5, 1051–1063. doi:10.3390/toxins5061051. Rothschild, L., Bern, S., Oswald, S., Weinberg, G., 2010. Intravenous lipid emulsion in clinical toxicology. Scand. J. Trauma. Resusc. Emerg. Med. 18, 51. doi:10.1186/1757-7241-18-51. Shepherd, J., Packard, C.J., Bicker, S., Lawrie, T.D., Morgan, H.G., 1980. Cholestyramine promotes receptor-mediated low-density-lipoprotein catabolism. N. Engl. J. Med. 302, 1219–22. doi:10.1056/NEJM198005293022202. Templeton, C.B., Poli, M.A., LeClaire, R.D., 1989. Cardiorespiratory effects of brevetoxin (PbTx-2) in conscious, tethered rats. Toxicon 27, 1043–1049. doi:10.1016/0041-0101(89)90155-4. Walsh, C.J., Leggett, S.R., Carter, B.J., Colle, C., 2010. Effects of brevetoxin exposure on the immune system of loggerhead sea turtles. Aquat. Toxicol. 97, 293–303. doi:10.1016/j.aquatox.2009.12.014. 114 CHAPTER V. CELLULAR RESPONSES IN TURTLES EXPOSED TO PBTX-3 Introduction Freshwater turtles have been used as model systems in many studies to investigate the molecular mechanisms that allow them to remain dormant underwater during hibernation in cold weather, and reduce cellular process like ion pumping to decrease adenosine triphosphate (ATP) consumption, and thus have become an alternative animal models for stroke and age-related diseases in humans (Larson et al., 2014; Krivoruchko and Storey, 2010). The ability to withstand prolonged periods of hypoxia without obvious damage to the brain is an adaptation present in freshwater turtles, seals and whales, naked mole-rats, and arctic ground squirrels (Larson et al., 2014b). However, in most mammals, oxygen is critical for survival and the rapid depletion of oxygen results in excitotoxic events such as ion imbalances, a loss of membrane potential, and a reduction in mitochondrial ATP production (Buja, 2005), leading to irreversible neurological damage (Larson et al., 2014). Early in anoxia the mammalian brain experiences an uncontrolled release of the neurotransmitter glutamate along with failure of glutamate reuptake mechanisms, leading to a buildup of extracellular glutamate and neurotoxic events that brings the cells to apoptosis (Thompson et al., 2007). By contrast, turtles survive extended periods of anoxia, along with subsequent reoxygenation, without neuronal injury due to a significant reduction in their metabolic rates. A study conducted by Herbert and Jackson (1985) demonstrated that the metabolic 115 rate of submerged turtles was only 10-20% of the corresponding aerobic rate when at the same body temperature (Herbert and Jackson, 1985). The molecular mechanisms that control the reduction in basal metabolism are coupled to the suppression of ATP pumps in the mitochondrial membrane and consequently ATP usage is prioritized. The idea of “ion channel arrest” states that there is a decrease in ion leakage across the plasma membrane, reducing the demand for ATP mediated ion pumps (Bickler, 1992) and thus the plasma membrane potential is maintained (Hochachka, 1986). Turtles, in comparison to rats, use glucose at about 1/6th the rate of rats and thus conserve energy stores; the oxidative metabolism in the turtle brain is about two- to three-folds lower than in rat brains even under normoxic conditions (Suarez et al., 1989). Energy is also conserved by the decrease in neurotransmitter release; studies in the turtle brain during anoxic episodes revealed a 30 % decrease in the release of the excitatory neurotransmitter glutamate after 5h of anoxia compared to the normoxic controls (Thompson et al., 2007). Turtles have numerous intracellular mechanisms that allow them to survive varying degrees of environmental changes. Interest in our laboratory is focused on the effects of a naturally occurring environmental toxin and its roles on turtle physiology. My work has been focused on the effects of brevetoxin using the freshwater turtle, Trachemys scripta, as a model system for toxin exposure. We have shown in a previous study that these turtles are 2-5 times more resistant to oral and intratracheal brevetoxin (PbTx-3) toxicity than mammalian species are, which suggests that they may have additional mechanisms in order to cope with cellular stress (Cocilova and Milton, 2016). Differences in sodium channel densities between turtles and rats could be playing a role in the resistance differences; turtles have about 1/3rd the binding capacity as rats, which interestingly does 116 not result in any effect in energy pumping costs (Edwards et al., 1989; Suarez et al., 1989). We demonstrated that neurons from T. scripta are more resistant to PbTx-3 toxicity than cerebral granular neurons in rats; the EC50 in turtles was significantly higher than in rats under similar experimental conditions (Cocilova and Milton, 2016; Berman and Murray, 1999). Taking into consideration this large difference in cell death and the differences noted in PbTx-3 in vivo administrations between the two species, I hypothesize that there are pro-survival pathways triggered to protect the turtle cells from undergoing apoptosis when exposed to PbTx-3. There are a number of neuroprotective pathways in the anoxia tolerant freshwater turtle brain that increase cell survival by stabilizing the mitochondria, decreasing reactive oxygen species (ROS), and therefore decreasing signals that would typically activate the pro-apoptotic pathway (Nayak et al., 2011). T. scripta exhibits modifications to vital molecular processes in response to anoxia including increased expression in heat shock proteins (HSPs), anti-apoptotic factors, the MAP kinases, and antioxidants, which therefore reduce oxidative stress and promote cell survival (Larson et al., 2014). Oxidative stress can impair essential cellular functions, impact the immune system, and become toxic to cells. When animals are exposed to brevetoxin the immune system undergoes reduced immune cells phagocytic activity (Tibbetts et al., 2006), an increase in oxidative stress, and an increase in the antioxidant glutathione-S-transferase (GST), which plays a role in detoxification (Walsh et al, in prep; Walsh et al., 2009). Another molecular adaptation is the upregulation of protective chaperone proteins during stress, which have been shown to contribute to survival in both prokaryotes and eukaryotes by targeting tightly regulated signaling pathways. The HSPs are a family of highly 117 conserved chaperone “housekeeping” proteins that play critical roles in the control of the cell cycle, ensuring proper folding and unfolding of proteins, and neuroprotective properties that help fight against cellular stress that leads to apoptosis (Takayama et al., 2003). HSPs are under the control of a transcription factors (i.e. Hsp1) and when the cell is under stress, Hsp1 becomes bound to DNA consensus sequences thus driving the transcription of HSPs (Stetler et al., 2009). The upregulation of HSPs in turtles under anoxic conditions is accompanied by a fivefold increase in the amount of active heat shock transcription factor 1 (HSF1) in the skeletal muscle (Krivoruchko and Storey, 2010b). HSPs function to recognize and bind to the target proteins and mediate cell survival by protecting the protein from damage or degradation. HSPs are classified based on their molecular weight, for example Hsp72, Hsp73, Hsp90, and can localize and accumulate in the nucleus to assist in protein trafficking and folding, are involved in protection from RNA splicing, and function in preventing apoptosis (Frydman and Hartl, 1996; Yost and Lindquist, 1986; Wang et al., 2014). HSPs have also been involved in posttranslational modifications in adult rats sensory and motor neurons allowing them to survive from peripheral nerve injuries (Benn et al., 2002). Hsp72 or the inducible Hsp70, is one well-studied protein in turtle models that is significantly overexpressed during anoxia. In the anoxic turtle brain, Hsp72 was upregulated in the myocardial cells after exposure to 12h of anoxia, and recovers back to baseline levels after 12h of reoxygenation (Chang et al., 2000). With the knockdown of Hsp72 protein expression in the freshwater turtle under 4h of anoxic conditions led to an increase in ROS production which in turn led to an increase apoptotic inducing factor (AIF) inducing cell death; AIF release from the mitochondria is mediated through ROS production and HSPs play a role 118 in reducing ROS damage demonstrating that Hsp72 is critical for cell survival (Kesaraju et al., 2014). Interestingly, my collaborative studies have shown that oxidative stress is a consequence of intratracheal PbTx-3 exposure; superoxide dismutase (SOD) activity, a measure of oxidative stress, was significantly increased in the plasma of PbTx-3 exposed turtles (Walsh et al., in prep). It is possible that AIF may activated upon PbTx-3 exposure triggering apoptosis, but further studies will need to be performed to investigate this. HSPs are also important anti-apoptotic molecules that may directly interfere with the cell death signaling pathways (Benn et al., 2002). Hsp27 functions as an anti-apoptotic molecule by inhibiting caspase activation (Garrido et al., 1999); triggering the caspase cascade involves proteolysis of hundreds of substrate proteins and inactivation of pro- survival proteins (Logue and Martin, 2008). Hsp72 was also shown to protect against apoptosis by blocking cytochrome c release from the mitochondria (Li et al., 2002) and by the suppression of a stress activating kinase, c-Jun N-terminal kinase (JNK), by inhibiting its signaling pathway (Park et al., 2001). The MAP kinases (MAPK) are a group of proteins directly involved in cellular responses to external stimuli and regulate cell functions such as gene expression, posttranslational modifications, cell survival, oxidative stress and apoptosis (Martindale and Holbrook, 2002). The Ras cellular pathway is an important upstream phosphorylation signaling pathways that it operates the MAPK pathway and mutations to Ras have been known to disrupt normal cell functions and cause malignant cells (Zenonos and Kyprianou, 2013). MAP kinases are all directly activated by phosphorylation in two sites in the activation loop, tyrosine and threonine, and their activation occurs in the absence of a regulatory unit (Pearson et al., 2001). Three major MAP kinase signaling cascades 119 include ERK 1/2, c-Jun N-terminal protein kinase (JNK), and p38; ERK functions in the cell survival pathway for example, during ischemia/reoxygenation enhancing survival in cardiomyocytes (Yue et al., 2000) where as JNK and p38 pathways are stress-activated and drive cells toward apoptosis (Gabai et al., 2000). Stress inducing signals include but are not limited to oxidative stress, environmental stress and toxic chemical insults (Kyriakis et al., 1994). Extracellular-signal regulated kinase (ERK) is a molecule involved in a complex cascade that is initiated by an extracellular cell receptor kinase activation that transmits multiple signals leading to MAPK/ERK activation. When active, ERK can translocate into the nucleus and phosphorylate transcription factors that regulate gene expression (Chang et al., 2003), and is essential for facilitating the mitotic cell cycle involved in DNA synthesis (Dangi et al., 2006). Attention has been drawn to the effects brevetoxin-2 has on rats neuron ERK; brevetoxin’s binding to voltage gated sodium channels (VGSCs) leads to increases in intracellular levels of calcium in neurons, thus triggering the activation of pro-survival ERK 1/2 (Dravid et al., 2004). In the same study, ERK1/2 activation was shown to be mediated by the influx of calcium through NMDA receptors; when these receptors were blocked by MK-801, the expression of ERK 1/2 decreased suggesting calcium contributes to its activation. In vitro studies from the turtle brain revealed that phosphorylated ERK (p-ERK) was upregulated significantly by 1h of anoxia and further increased by 4h, however this likely isn’t the only pathway that leads to an increase in cell survival (Nayak et al., 2011). In the same study the AKT protective pathway was significantly upregulated during anoxia also believed to be contributing to cell survival. It has been reported in some studies that long-term activation of ERK may 120 promote cell death (Wang et al., 2003) and it is very likely that numerous molecular pathways are triggered during cell stress but a greater upregulation of pro-survival pathways may outweigh the pro-death pathways leading to survival. JNK is another subfamily of the MAP kinases that becomes phosphorylated/activated in response to extracellular stimuli such as growth factors (Hibi et al., 1993) and cellular stressors (Cano et al., 1994), and translocates to the nucleus to specifically phosphorylate the transcription factor c-Jun where it can regulate the activity of transcription factors. JNK are proteins involved in cell proliferation, differentiation and apoptosis (Sluss et al., 1994), and activated transcription factors are involved in increasing the expression of pro apoptotic genes and decreasing the expression of pro survival genes (Dhanasekaran and Reddy, 2008). Glutamate toxicity may be involved in JNK’s role in the pro apoptotic pathway and it has been shown by disrupting JNK genes, mice were resistant to glutamate toxicity and therefore neuronal apoptosis was aborted (Yang et al., 1997). This is of interest since brevetoxin binding in turtle neurons is suspected of increasing glutamate levels leading to a subsequent calcium increase in neighboring cells (chapter II), thus the role of JNK in this cellular pathway may be inducing cells to undergo apoptosis. Interestingly, when Hsp72 is increased in the brain during anoxia, JNK becomes dephosphorylated, downregulating its activity, and therefore reducing apoptosis and ischemic injury (Gabai et al., 2000). Lastly, p38 kinase has been shown to activate under extracellular stimuli including heat shock (Rouse et al., 1994), growth factors (Foltz et al., 1997), environmental stress (Gupta et al., 1995), and are involved in cell cycle events. Activated p38 (p38 to Pp38), has the ability to regulate genes such as cytokines, transcription factors and cell surface receptors in complex 121 phosphorylation pathways (Zarubin and Han, 2005) as well as contribute to cell death pathway activation (Xia et al., 1995). p38 is also involved in the regulation of the inflammatory response by activating T cells, part of the innate immune system (Krementsov et al., 2013). It is thought that JNK and p38 work together to induce apoptosis because they have a common regulatory point in the signaling cascades (Cai et al., 2006). The pro-survival and pro- and anti-apoptotic pathways are clearly important in the control of cell survival or death, and are the focal point of numerous studies using a variety of model systems such as worms, flies, turtles, and mice. Attention has been focused on what molecules play a role in critical biological processes that drive cellular homeostasis, cell division, and embryonic development, and how external stimuli stressors like temperature, toxicity, or osmotic stress affect cell survival. As previously stated, PbTx-3 induces neuronal excitability in turtle neurons leading to glutamate release and therefore an increase in intracellular levels of calcium that leads to cell death. We have shown that T. scripta is relatively resistant to the effects of brevetoxin in vivo and in vitro (Cocilova and Milton, 2016, chapters II and III above). It is thus of interest to determine if these molecular signaling pathways contribute to the higher resistance of turtle neurons to brevetoxin, protecting the cell from apoptosis, as the mechanisms of brevetoxicosis are similar to the death cascade of mammalian anoxia. The focus of this study was specifically looking into the expression of Hsp72 in T. scripta post PbTx-3 exposure to gain a better understanding of PbTx-3’s influence in the pro-survival pathway involving molecular chaperones. This data will provide insight into the molecular effects of brevetoxin exposure in turtles. 122 Materials and methods Animals All work was approved by the Florida Atlantic University Institutional Animal Care and Use Committee (IACUC) and all animals were acclimated to the laboratory for two weeks prior to any dosing experiments. Twenty unsexed juvenile freshwater turtles (red eared sliders, Trachemys scripta), approximately 12 – 14 cm straight carapace length and weighing 0.3-0.5 kg, were obtained from a commercial supplier (Niles Biological Inc., Sacramento, CA) and maintained in tanks at room temperature (22°C +/- 3°C, 50% relative humidity +/- 4%) on a 12h day/night cycle. Aquaria were cleaned according to standard husbandry methods and animals were fed (commercial aquatic food, to satiety) 3× weekly. Brevetoxin Brevetoxin (PbTx-3) was purchased from LKT Laboratories (St. Paul, Minnesota) and was dissolved in ethanol and mixed with 0.9% NaCl to a final concentration of 0.05µg/µl. Turtles were restrained by hand and administered PbTx-3 by intratracheal instillation (IT) (10.53µg/kg, 3x/week for four weeks for a total of 12 doses). The toxin was administered ~1.5cm into the tracheal opening at the base of the tongue, and a rubber bulb and pipette tip were used to inflate the lungs 3 times following instillation of the toxin to mimic inhalation. IT dose concentration was determined based on previous findings from PbTx-3 tissue distribution studies (chapter III). Control animals received sham doses of physiological saline solution mixed with ethanol to a final concentration to 0.1% EtOH (Cocilova and Milton, 2016) to mimic the treatment solution, and sham exposures were conducted as in toxin exposures. 123 Tissue collection All turtles were euthanized by decapitation and the following tissues were collected for protein analysis via western blotting: muscle, heart, liver, brain. Tissues were collected at 1h, 24h and 1wk post IT PbTx-3 exposure. Tissues samples were flash frozen in liquid nitrogen and stored at -80°C until extraction. Protein extraction Frozen tissues were defrosted for ~15 min and a sample of approximately 0.30g of tissue was utilized. Proteins were extracted from tissues in RIPA buffer (1M NaCl, 0.5M EDTA, 1% Triton X100, 0.5M Tris-Cl pH 7.4; with added 5M DDT, 0.1M PMSF, 5M metcaptoethanol, 3mM protease inhibitor (PI) diluted 1:1000) using a glass homogenizer at 22°C. The homogenate was centrifuged at 15,000 rpm at 4°C for 10 min and the supernatant was collected for further analysis. Protein concentrations in each tissue sample were determined using a standard BCA assay following manufacturer’s protocol (Pierce Biotechnology, Inc., Rockville, IL). Western blotting Rabbit polyclonal anti-Hsp72 (Enzo, Farmingdale, NY) was diluted at 1:2000 in 5% non-fat milk in TBST (25mM Tris-Cl pH 7.5, 150mM NaCl, 1% Tween®20). Primary antibodies against β-actin (Cell Signaling Technology, Davers, MA) (diluted at 1:3000) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)(United States Biological, Atlanta, GA) (1:2000) were used as loading controls in 5% non-fat dried milk in TSBT. Secondary antibodies were obtained from Southern Biotech (Birmingham, AL) and diluted 1:4000 for anti-rabbit (Hsp72) and 1:3000 for anti-mouse (GAPDH, β-actin). 124 Equal amounts of proteins (20µg/ml) were separated in a 12% SDS- Polyacrylamide gel for 1h at 150 V. The proteins were transferred onto nitrocellulose (Amersham Biosciences, Pittsburgh, PA) for 1h at 0.3A. The membrane was blocked in 5% non-fat dried milk in TBST for 1h at room temperature. Membranes were incubated with the primary antibody rocked gently overnight at 4°C. The membranes were washed 3X 5 min in TBST and probed with corresponding secondary antibody for 1h at room temperature. Immunoactive protein bands were visualized with enhanced chemiluminescence (ECL) (Thermo-Fisher Scientific, Grand Island, NY). Relative density of the bands was determined using Image J software. Results were normalized to percent of β-actin or GAPDH (used as loading controls) and relative changes expressed as a percentage of control. Statistics Data was graphed in Sigma Plot 11.0 (Systat Software, Inc, San Jose, CA) and data were analyzed using one-way analysis of variance (ANOVA). Results Hsp72 expression in tissues post PbTx-3 exposure Hsp72 expression was analyzed in the muscle, heart, liver, and brain extracted from brevetoxin exposed turtle tissues at 1h, 24h, and 1wk post PbTx-3 and compared to sham controls. Preliminary results show the mean expression levels of Hsp72 increased over a 1h period in all tissues tested (Fig. 17). Hsp72 upregulation was most notable in the brain both 1h and 24h post exposure with increases of 32% and 46% over controls respectively and decreased back down to basal levels 1wk post PbTx-3. Hsp72 expression in the heart increased by 12% over controls 24h post exposure and remained 125 above controls 1wk post PbTx-3. The liver and muscle showed a similar trend to each other with only a slight overexpression of Hsp72 1h post PbTx-3 exposure. Although these data were not statistically significant there was still an overall increase in Hsp72 post PbTx-3 exposure. Discussion Freshwater turtles are facultative anaerobes with evolutionary adaptations that suppress their metabolic rates, reduce ATP consumption, upregulate protective pathways and activate signaling molecules to decrease apoptotic cascades. These mechanisms give these animals the capability to dive for prolonged periods of time (Jackson and Ultsch, 2010) and hibernate underwater for months at a time (Robin et al., 1981), thus surviving low oxygen levels without apparent cellular damage (Jackson, 2002). In addition, extensive antioxidant defense systems have been shown to play a role in protecting turtles by minimizing oxidative damage during hibernation (Baker et al., 2007). When intracellular changes occur due to stressful environments including alterations in temperature, ion imbalances, and neurotoxicity, proteins may become misfolded and need the help of molecular chaperone proteins to preserve their structure and therefore their function (Hartl, 1996; Ellis, 1990). In the present study, I hypothesized that there would be an upregulation of the commonly expressed molecular chaperone protein, Hsp72, in the tissues of T. scripta, to aid in protecting the cell again toxic insults. Even though these freshwater species do not reside in areas where HABs are known to occur, with their many adaptations to survive in harsh environments, along with their ability to withstand high toxin levels it is likely that pro-survival pathways are activated and the anti- apoptotic pathways are downregulated. 126 In the present study, the brain showed the highest expression of Hsp72 in comparison to the muscle, liver and heart. Since PbTx-3 is a lipid soluble molecule known to easily pass the blood brain barrier and affect electrically excitable cells (nerves and muscles), it was not surprising that in the brain Hsp72 was overexpressed, as the nervous system is one of the initial targets of PbTx-3 exposure; my previous studies have shown that PbTx-3 binds with high affinity to VGSCs and causes neuronal death, however turtles are more resistant to toxicity than in mammals (Cocilova and Milton, 2016; chapter II). My previous in vivo studies show apparent neurological and muscular symptoms of brevetoxicosis upon inhalation and ingestion (chapter III) which may be leading to neuronal cell death, however the upregulation of protective pathways in tissues like the brain may be allowing them to survive during toxin exposure, as doses were quite high but did not result in animal deaths despite evident and significant neurological deficits. One of the goals of these studies was to investigate the cellular responses triggered post PbTx-3 exposure. Since the turtle proved to be remarkably resistant to PbTx-3 (Cocilova and Milton, 2016, chapter III), and increases in HSP expression levels has been shown to play a role cellular protection (Stetler et al., 2009; Kesaraju et al., 2014; Garrido et al., 1999). Hsp72 (alternative name Hsp70) is an extensively studied protective protein in animals ranging from lizards to lemurs (Ulmasov et al., 1992; (Wu et al., 2015), and including turtles such as T. scripta and Chrysemys picta (Krivoruchko and Storey, 2010; Ramaglia et al., 2004). Extended periods of anoxia result in the increased expression of Hsc73 in the anoxia turtle striatum (Prentice et al., 2004), suggesting a protective role. 127 HSPs have a vital role in enhancing cell survival by modulating reactive oxygen species (ROS) that cause significant damage to cells. ROS is generated from cellular metabolism, primarily from the mitochondria, and its over production affects cell survival by causing damage to macromolecules such as lipids, proteins, and DNA (Freeman and Crapo, 1982; Thannickal and Fanburg, 2000). The knockdown of Hsp72 has been shown to induce ROS and cell death during anoxia and reoxygenation in turtle neurons (Kesaraju et al., 2014). Hsp72 is involved in neuroprotection by modulating ROS production and enhancing cell survival. In contrast, when mammals experience an oxygen deprivation followed by reoxygenation, there is an increase in reactive oxygen species (ROS) and a destruction of normal cell functions such as ion pump failures and electrical hyperexcitability, and the mitochondria become damaged leading to insufficient ATP production. An increase in neuronal excitability may lead to activation of inhibitory neurotransmitters that drive cells to work attempting to restore cellular homeostasis. The anoxic turtle brain avoids excitatory neurotoxicity by decreasing glutamatergic signaling and increasing extracellular Gamma-Aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter that functions to inhibit synapses in the brain to decrease neuronal excitability and the molecular chaperone, Hsc73, may increase its synthesis during long term anoxia (Jin et al., 2003); elevated GABA levels could counteract cell death pathways. In the anoxic turtle brain, glutamate release, generally in excess as a result of neuroexcitability, was decreased by 44% suggesting the control of glutamate release and reuptake abilities persist during anoxic stress therefore, increasing survival (Milton et al., 2002). In my previous studies, I have shown that brevetoxin binding to voltage-gated sodium channels (VGSCs) in the freshwater turtle brain 128 generates an increase in sodium influx, glutamate release, and the activation of NMDA and AMPA receptors, leading to a rise in intracellular calcium levels in the post synaptic cells; studies also show that when PbTx-3 is added to turtle neurons cell death results (Cocilova and Milton, 2016). Even though these animals are strongly affected by brevetoxin toxicity, they are more resistant than mammals, although the mode of action appears to be the same (Cocilova and Milton, 2016; Berman and Murray, 2000). In this current study, the heart and the muscle did not show any significant changes in Hsp72 however, there was a similar trend in Hsp72 expression levels at the 1h and 24h time points post exposure; Hsp72 expression in the heart was still slightly elevated 1 week post PbTx-3 exposure. These data suggests that this molecular chaperone may be contributing to cell survival during toxin exposure as the turtles have proven to be more resistant to toxicity that other species (Cocilova and Milton, 2016). Hsp72 in the heart is increased during anoxia and its expression increases further upon reoxygenation supporting the idea that HSPs may also be acting as a defense mechanism (Ramaglia and Buck, 2004). It has been shown that HSPs significantly increase in expression in rat hearts during ischemia/reperfusion and may protect the heart again heart failure (Nishizawa et al., 1996; Willis and Patterson, 2010; Sammut and Harrison, 2003; Latchman, 2001). Surprisingly, in this study the liver showed the least amount of changes in Hsp72 expression; 24h post exposure Hsp72 levels remained at basal levels and fell slightly below control levels 1 week post exposure. This was unexpected because the liver is the primary route of brevetoxin metabolism and excretion; these results suggest that excretion of the toxin is not a burden in healthy tissues. During a short term dive in Chrysemys picta, there was a decrease in Hsp72 expression which was also a 129 surprising result since all other tissues tested (muscle, heart and brain) were significantly increased during the dive but explanations for these may be attributed to a reduction in protein synthesis by the liver, which has also been shown in other anoxia studies (Ramaglia and Buck, 2004; Scott et al., 2003; Smith et al., 1996). HSPs constitute a large family of proteins and a further investigation of other chaperone HSPs, aside from Hsp72, will be vital in understanding the complex processes that occur and what molecular mechanisms are activated and/or inhibited that drive the cells to survival. It is also may be important to mention the possibility that Src-family kinases (SFKs) could be involved in the cellular processes that respond to environmental stressors and give us more insight into brevetoxin’s affects from a molecular standpoint. SRKs are a family of proteins known to be expressed in the mammalian CNS that regulate the activity of voltage-gated ion channels (Fadool et al., 1997) and neurotransmitter receptors (Wan et al., 1997), and their activity has been investigated in toxicology studies. VGSC activation is involved in the generation of action potentials, essential for signaling between neurons, and when not closely regulated or the ionic homeostasis interrupted, can cause destructive cellular events. Neocortical mice neurons exposed to PbTx-2 showed increased Src activation from an increase in Na+ levels, suggesting NMDA receptors are influenced by Src kinase activity (Cao et al., 2007). I think investigation of the expression of Src kinases in turtles post brevetoxin exposure would be an interesting avenue to explore since brevetoxin’s mode of action influences voltage-gated channels and therefore affects their activation. This chapter has contributed to a greater understanding of Hsp72’s role inside the cell and its response to neurotoxicity. These data suggests that Hsp72 acts as a pro 130 survival protein based on its upregulation in tissues upon stress. There are multiple cell signaling cascades that become activated by various phosphorylation and dephosphorylation events and further investigation looking at the degree to which pro- and anti-apoptotic proteins are expressed is needed. Future studies would include exploration of MAP kinase signaling pathways and the role of ERK 1/2, p38 and JNK post brevetoxin exposure. The ERK signaling cascade has been shown to be involved in and important for neuronal survival by promoting cell survival genes and inactivating the cell death pathway (Bonni, 1999). Voltage-gated calcium channels (VGCCs) and calcium sensitive AMPA receptors in mammals have been shown to regulate ERK 1/2 activation by upregulating genes that promote neuronal survival (Sweatt, 2001). It would also be of interest to study the expression ERK 1/2 in the freshwater turtles by direct VGSC stimulation using PbTx-3. Apoptosis plays a critical role in the proper development of the nervous system in maintaining cellular homeostasis, but when environmental toxicants lead to neurotoxicity, apoptosis is induced. A further understanding of how PbTx-3 exposure influences the stress-activated JNK and p38 pathways will provide a better understanding of cellular function in turtles under toxic stress with additional application to sea turtles who are in the wild exposed to such toxicants like brevetoxins. 131 Figure 17. PbTx-3 exposure induces Hsp72 expression in the brain Tissues were collected from T. scripta exposed to intratracheal PbTx-3 (10.53µg/kg). Proteins were extracted from the muscle, heart, liver and brain. Representative western blot with densitometric analysis is shown. Data shows Hsp72 expression in PbTx-3 exposed animals as % of control for 1h, 24h, and 1wk. The loading control for the muscle and heart was GAPDH and the liver and brain was β- actin. Error bars represent the standard error of the mean (SEM). 132 Figure 17 C 1h 24h24h 1wk C 1h 1h 24h 24h 1wk 1wk CC 1h1h 24h24h 1wk1wk CC 1h 1h 24h 24h 1wk 1wk Hsp72Hsp72 LoadingLoading Control Control MuscleMuscle HeartHeart LiverLiver BrainBrain 250 Control 1h post PbTx-3 24 post-PbTx-3 46% 200 1wk post PbTx-3 32% 150 %of control 100 50 0 Muscle Heart Liver Brain 133 References Baker, P.J., Costanzo, J.P., Lee, R.E., 2007. Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 177, 875–883. doi:10.1007/s00360-007-0185-0. Benn, S.C., Perrelet, D., Kato, A.C., Scholz, J., Decosterd, I., Mannion, R.J., Bakowska, J.C., Woolf, C.J., 2002. Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron 36, 45–56. doi:10.1016/S0896- 6273(02)00941-8. Berman, F.W., Murray, T.F., 2000. Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx. J Neurochem 74, 1443–1451. doi:10.1046/j.1471-4159.2000.0741443.x. Berman, F.W., Murray, T.F., 1999. Brevetoxins cause acute excitotoxicity in primary cultures of rat cerebellar granule neurons. J. Pharmacol. Exp. Ther. 290, 439–444. Bickler, P.E., 1992. Cerebral anoxia tolerance in turtles: regulation of intracellular calcium and pH. Am. J. Physiol. 263, R1298–302. Bonni, A., 1999. Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms. Science (80-. ). 286, 1358– 1362. doi:10.1126/science.286.5443.1358. Buja, L.M., 2005. Myocardial ischemia and reperfusion injury. Cardiovasc. Pathol. doi:10.1016/j.carpath.2005.03.006. Cai, B., Chang, S.H., Becker, E.B.E., Bonni, A., Xia, Z., 2006. p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65. J. Biol. Chem. 281, 25215– 25222. doi:10.1074/jbc.M512627200. 134 Cano, E., Hazzalin, C. a, Mahadevan, L.C., 1994. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol. Cell. Biol. 14, 7352–7362. doi:10.1128/MCB.14.11.7352.Updated. Cao, Z., George, J., Baden, D.G., Murray, T.F., 2007. Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways. Brain Res. 1184, 17–27. doi:10.1016/j.brainres.2007.09.065. Chang, F., Steelman, L.S., Lee, J.T., Shelton, J.G., Navolanic, P.M., Blalock, W.L., Franklin, R.A., McCubrey, J.A., 2003. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 17, 1263–1293. doi:10.1038/sj.leu.2402945. Chang, J., Knowlton, a a, Wasser, J.S., 2000. Expression of heat shock proteins in turtle and mammal hearts: relationship to anoxia tolerance. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R209–R214. Cocilova, C.C., Milton, S.L., 2016. Characterization of brevetoxin (PbTx-3) exposure in neurons of the anoxia-tolerant freshwater turtle (Trachemys scripta). Aquat. Toxicol. 180, 115–122. doi:10.1016/j.aquatox.2016.09.016. Dangi, S., Chen, F.M., Shapiro, P., 2006. Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1. Cell Prolif. 39, 261–279. doi:10.1111/j.1365-2184.2006.00388.x. 135 Dhanasekaran, D.N., Reddy, E.P., 2008. JNK signaling in apoptosis. Oncogene 27, 6245– 51. doi:10.1038/onc.2008.301. Dravid, S.M., Baden, D.G., Murray, T.F., 2004. Brevetoxin activation of voltage-gated sodium channels regulates Ca dynamics and ERK1/2 phosphorylation in murine neocortical neurons. J Neurochem 89, 739–749. doi:10.1111/j.1471- 4159.2004.02407.x\rJNC2407 [pii]. Edwards, R.A., Lutz, P.L., Baden, D.G., 1989. Relationship between energy expenditure and ion channel density in the turtle and rat brain. Am. J. Physiol. 257, R1354–8. Ellis, R.J., 1990. The molecular chaperone concept. Semin. Cell Biol. 1, 1–9. Fadool, D.A., Holmes, T.C., Berman, K., Dagan, D., Levitan, I.B., 1997. Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage- gated potassium channel. J. Neurophysiol. 78, 1563–73. Foltz, I.N., Lee, J.C., Young, P.R., Schrader, J.W., 1997. Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway. J. Biol. Chem. 272, 3296–3301. doi:10.1074/jbc.272.6.3296. Freeman, B.A., Crapo, J.D., 1982. Biology of disease: free radicals and tissue injury. Lab. Invest. 47, 412–426. Frydman, J., Hartl, F.U., 1996. Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. Science 272, 1497–502. doi:10.1126/science.272.5267.1497. 136 Gabai, V.L., Meriin, A.B., Yaglom, J.A., Wei, J.Y., Mosser, D.D., Sherman, M.Y., 2000. Suppression of stress kinase JNK is involved in HSP72-mediated protection of myogenic cells from transient energy deprivation. HSP72 alleviates the stress- induced inhibition of JNK dephosphorylation. J. Biol. Chem. 275, 38088–38094. doi:10.1074/jbc.M006632200. Garrido, C., Bruey, J.M., Fromentin, a, Hammann, a, Arrigo, a P., Solary, E., 1999. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J. 13, 2061–2070. Gupta, S., Raingeaud, J., Gupta, S., Rogers, J.S., Dickens, M., Han, J., Ulevitch, R.J., Davis, R.J., 1995. Pro-inflammatory cytokines and environmental stress cause p38 Mitogen-activated Protein Kinase activation by dual phosphorylation on tyrosine and treonin e. J. Biol. Chem. 270, 7420–7426. doi:10.1074/jbc.270.13.7420. Hartl, F.U., 1996. Molecular chaperones in cellular protein folding. Nature. doi:10.1038/381571a0. Herbert, C. V, Jackson, D.C., 1985. Temperature Effects on the Responses to Prolonged Submergence in the Turtle Chrysemys picta bellii. I. Blood Acid-Base and Ionic Changes during and following Anoxic Submergence. Physiol. Zool. 58, 655–669. doi:10.1086/664584. Hibi, M., Lin, A., Smeal, T., Minden, A., Karin, M., 1993. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 7, 2135–2148. doi:10.1101/gad.7.11.2135. Hochachka, P.W., 1986. Defense strategies against hypoxia and hypothermia. Science 231, 234–41. doi:10.1126/science.2417316. 137 Jackson, D.C., 2002. Hibernating without oxygen: physiological adaptations of the painted turtle. J. Physiol. 543, 731–7. doi:10.1113/jphysiol.2002.024729. Jackson, D.C., Ultsch, G.R., 2010. Physiology of hibernation under the ice by turtles and frogs. J. Exp. Zool. Part A Ecol. Genet. Physiol. 313 A, 311–327. doi:10.1002/jez.603. Jin, H., Wu, H., Osterhaus, G., Wei, J., Davis, K., Sha, D., Floor, E., Hsu, C.-C., Kopke, R.D., Wu, J.-Y., 2003. Demonstration of functional coupling between gamma - aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc. Natl. Acad. Sci. U. S. A. 100, 4293–8. doi:10.1073/pnas.0730698100. Kesaraju, S., Nayak, G., Prentice, H.M., Milton, S.L., 2014. Upregulation of Hsp72 mediates anoxia/reoxygenation neuroprotection in the freshwater turtle via modulation of ROS. Brain Res. 1582, 247–56. doi:10.1016/j.brainres.2014.07.044. Krementsov, D.N., Thornton, T.M., Teuscher, C., Rincon, M., 2013. The emerging role of p38 mitogen-activated protein kinase in multiple sclerosis and its models. Mol Cell Biol 33, 3728–3734. doi:10.1128/MCB.00688-13. Krivoruchko, A., Storey, K.B., 2010a. Forever young: mechanisms of natural anoxia tolerance and potential links to longevity. Oxid. Med. Cell. Longev. 3, 186–198. doi:10.4161/oxim.3.3.4. Krivoruchko, A., Storey, K.B., 2010b. Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans. J. Comp. Physiol. B. 180, 403–414. doi:10.1007/s00360-009-0414-9. 138 Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J., Woodgett, J.R., 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369, 156–160. doi:10.1038/369156a0. Larson, J., Drew, K.L., Folkow, L.P., Milton, S.L., Park, T.J., 2014a. No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates. J. Exp. Biol. 217, 1024– 39. doi:10.1242/jeb.085381. Larson, J., Drew, K.L., Folkow, L.P., Milton, S.L., Park, T.J., 2014b. No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates. J. Exp. Biol. 217, 1024– 39. doi:10.1242/jeb.085381. Latchman, D.S., 2001. Heat shock proteins and cardiac protection. Cardiovasc. Res. doi:10.1016/S0008-6363(01)00354-6. Li, F., Mao, H.P., Ruchalski, K.L., Wang, Y.H., Choy, W., Schwartz, J.H., Borkan, S.C., 2002. Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells. Am. J. Physiol. Cell Physiol. 283, C917–26. doi:10.1152/ajpcell.00517.2001. Logue, S.E., Martin, S.J., 2008. Caspase activation cascades in apoptosis. Biochem Soc Trans 36, 1–9. doi:10.1042/BST0360001. Martindale, J.L., Holbrook, N.J., 2002. Cellular response to oxidative stress: signaling for suicide and survival. J.Cell Physiol 192, 1–15. Milton, S.L., Thompson, J.W., Lutz, P.L., 2002. Mechanisms for maintaining extracellular glutamate levels in the anoxic turtle striatum. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1317–23. doi:10.1152/ajpregu.00484.2001. 139 Nayak, G.H., Prentice, H.M., Milton, S.L., 2011. Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle. J. Cereb. Blood Flow Metab. 31, 467–475. doi:10.1038/jcbfm.2010.109. Nayak, G.H., Prentice, H.M., Milton, S.L., 2011. Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle. J Cereb Blood Flow Metab 31, 467–475. doi:jcbfm2010109 [pii]10.1038/jcbfm.2010.109. Nishizawa, J., Nakai, A., Higashi, T., Tanabe, M., Nomoto, S., Matsuda, K., Ban, T., Nagata, K., 1996. Reperfusion causes significant activation of heat shock transcription factor 1 in ischemic rat heart. Circulation 94, 2185–2192. Park, H.S., Lee, J.S., Huh, S.H., Seo, J.S., Choi, E.J., 2001. Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase. EMBO J. 20, 446–456. doi:10.1093/emboj/20.3.446. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B.E., Karandikar, M., Berman, K., Cobb, M.H., 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 22, 153–183. doi:10.1210/edrv.22.2.0428. Prentice, H.M., Milton, S.L., Scheurle, D., Lutz, P.L., 2004. The upregulation of cognate and inducible heat shock proteins in the anoxic turtle brain. J. Cereb. Blood Flow Metab. 24, 826–8. doi:10.1097/01.WCB.0000126565.27130.79. Ramaglia, V., Buck, L.T., 2004. Time-dependent expression of heat shock proteins 70 and 90 in tissues of the anoxic western painted turtle. J. Exp. Biol. 207, 3775–3784. doi:10.1242/jeb.01211. 140 Ramaglia, V., Harapa, G.M., White, N., Buck, L.T., 2004. Bacterial infection and tissue- specific Hsp72, -73 and -90 expression in western painted turtles. Comp. Biochem. Physiol. - C Toxicol. Pharmacol. 138, 139–148. doi:10.1016/j.cca.2004.06.007. Robin, E.D., Robin, D.A., Ackerman, R., Lewiston, N., Hance, A.J., Caligiuri, M., Theodore, J., 1981. Prolonged diving and recovery in the freshwater turtle, Pseudemys scripta-I. Lung and blood gases, pH, lactate concentrations and “cation” gap. Comp. Biochem. Physiol. -- Part A Physiol. 70, 359–364. doi:10.1016/0300- 9629(81)90190-0. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., Nebreda, A.R., 1994. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037. doi:10.1016/0092-8674(94)90277-1. Sammut, I. a, Harrison, J.C., 2003. Cardiac mitochondrial complex activity is enhanced by heat shock proteins. Clin. Exp. Pharmacol. Physiol. 30, 110–115. Scott, M.A., Locke, M., Buck, L.T., 2003. Tissue-specific expression of inducible and constitutive Hsp70 isoforms in the western painted turtle. J. Exp. Biol. 206, 303– 311. doi:10.1242/jeb.00107. Sluss, H.K., Barrett, T., Dérijard, B., Davis, R.J., 1994. Signal transduction by tumor necrosis factor mediated by JNK protein kinases. Mol. Cell. Biol. 14, 8376–84. Smith, R.W., Houlihan, D.F., Nilsson, G.E., Brechin, J.G., 1996. Tissue-specific changes in protein synthesis rates in vivo during anoxia in crucian carp. Am J Physiol 271, R897–904. 141 Stetler, R.A., Gao, Y., Signore, A.P., Cao, G., Chen, J., 2009. HSP27: mechanisms of cellular protection against neuronal injury. Curr. Mol. Med. 9, 863–72. doi:10.2174/156652409789105561. Suarez, R.K., Doll, C.J., Buie, A.E., West, T.G., Funk, G.D., Hochachka, P.W., 1989a. Turtles and rats: a biochemical comparison of anoxia-tolerant and anoxia-sensitive brains. Am. J. Physiol. 257, R1083–R1088. Suarez, R.K., Doll, C.J., Buie, A.E., West, T.G., Funk, G.D., Hochachka, P.W., 1989b. Turtles and rats: a biochemical comparison of anoxia-tolerant and anoxia-sensitive brains. Am. J. Physiol. 257, R1083–8. Sweatt, J.D., 2001. The neuronal MAP kinase cascade: A biochemical signal integration system subserving synaptic plasticity and memory. J. Neurochem. doi:10.1046/j.1471-4159.2001.00054.x. Takayama, S., Reed, J.C., Homma, S., 2003. Heat-shock proteins as regulators of apoptosis. Oncogene 22, 9041–9047. doi:10.1038/sj.onc.1207114. Thannickal, V.J., Fanburg, B.L., 2000. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L1005–28. doi:10.1164/rccm.2206007. Thompson, J.W., Prentice, H.M., Lutz, P.L., 2007. Regulation of extracellular glutamate levels in the long-term anoxic turtle striatum: coordinated activity of glutamate transporters, adenosine, K (ATP) (+) channels and GABA. J. Biomed. Sci. 14, 809– 17. doi:10.1007/s11373-007-9190-2. Tibbetts, B.M., Baden, D.G., Benson, J.M., 2006. Uptake, tissue distribution, and excretion of brevetoxin-3 administered to mice by intratracheal instillation. J. Toxicol. Environ. Health. A 69, 1325–35. doi:10.1080/15287390500360091. 142 Ulmasov, K.A., Shammakov, S., Karaev, K., Evgen’ev, M.B., 1992. Heat shock proteins and thermoresistance in lizards. Proc. Natl. Acad. Sci. U. S. A. 89, 1666–1670. doi:10.1073/pnas.89.5.1666. Walsh, C.J., Leggett, S.R., Henry, M.S., Blum, P.C., Osborn, S., Pierce, R.H., 2009. Cellular metabolism of brevetoxin (PbTx-2) by a monocyte cell line (U-937). Toxicon 53, 135–145. doi:10.1016/j.toxicon.2008.10.024. Wan, Q., Man, H.Y., Braunton, J., Wang, W., Salter, M.W., Becker, L., Wang, Y.T., 1997. Modulation of GABAA receptor function by tyrosine phosphorylation of beta subunits. J. Neurosci. 17, 5062–5069. Wang, X., Chen, M., Zhou, J., Zhang, X., 2014. HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (review). Int. J. Oncol. doi:10.3892/ijo.2014.2399. Wang, X., Zhu, C., Qiu, L., Hagberg, H., Sandberg, M., Blomgren, K., 2003. Activation of ERK1/2 after neonatal rat cerebral hypoxia-ischaemia. J. Neurochem. 86, 351– 362. doi:10.1046/j.1471-4159.2003.01838.x. Wu, C.-W., Biggar, K.K., Zhang, J., Tessier, S.N., Pifferi, F., Perret, M., Storey, K.B., 2015. Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur, Microcebus murinus. Genomics. Proteomics Bioinformatics 13, 119–26. doi:10.1016/j.gpb.2015.03.004. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., Ahn, N.G., Angel, P., Barrett, G.L., Batistatou, A., Batistatou, A., Blenis, J., Boonstra, J., Borasio, G.D., Borasio, G.D., Buckmaster, E.A., Campbell, J.S., Cobb, M.H., Cowan, W.M., Cowley, S., Davis, R.J., Davis, R.J., Derijard, B., Derijard, B., Dinarello, C.A., 143 Dobrowsky, R.T., Estus, S., Ferrari, G., Freshney, N.W., Frodin, M., Galchevagargova, Z., Gotoh, Y., Greenberg, M.E., Greene, L.A., Greene, L.A., Greene, L.A., Guerrero, I., Gupta, S., Ham, J., Han, J., Hibi, M., Ho, S.N., Hockenbery, D., Hunter, T., Jacobson, M.D., Jacobson, M.D., Jarvis, W.D., Kallunki, T., Koike, T., Kolesnick, R., Kyriakis, J.M., Langecarter, C.A., Lee, J.C., Lin, A., Livingstone, C., Mansour, S.J., Marshall, C.J., Mesner, P.W., Minden, A., Miyasaka, T., Obeid, L.M., Olson, M.F., Oppenheim, R.W., Pittman, R.N., Qiu, M.S., Rabizadeh, S., Raff, M.C., Raingeaud, J., Raingeaud, J., Rapp, U.R., Ray, L.B., Rosen, L.B., Rouse, J., Rukenstein, A., Sanchez, I., Siuss, H.K., Steller, H., Sturgill, T.W., Su, B., Teng, K.K., Thompson, C.B., Togari, A., Vandam, H., Whitmarsh, A.J., Xia, Z., Yan, M.H., Young, S.W., 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–31. doi:10.1126/science.270.5240.1326. Yang, D.D., Kuan, C.Y., Whitmarsh, a J., Rincón, M., Zheng, T.S., Davis, R.J., Rakic, P., Flavell, R. a, 1997. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389, 865–70. doi:10.1038/39899. Yost, H.J., Lindquist, S., 1986. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 45, 185–193. doi:0092-8674(86)90382-X [pii]. 144 Yue, T.L., Wang, C., Gu, J.L., Ma, X.L., Kumar, S., Lee, J.C., Feuerstein, G.Z., Thomas, H., Maleeff, B., Ohlstein, E.H., 2000. Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ. Res. 86, 692–699. doi:10.1161/01.RES.86.6.692. Zarubin, T., Han, J., 2005. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15, 11–18. doi:10.1038/sj.cr.7290257. Zenonos, K., Kyprianou, K., 2013. RAS signaling pathways, mutations and their role in colorectal cancer. World J. Gastrointest. Oncol. 5, 97–101. doi:10.4251/wjgo.v5.i5.97. 145 CHAPTER VI. CONCLUSIONS AND FUTURE DIRECTIONS Not much is known about brevetoxin accumulation, uptake, tissue distribution, excretion and rates of clearance in sea turtles. Due to their threatened and endangered status, it is difficult to conduct the research necessary to determine the impact that toxin inhalation and/or ingestion has on organ systems. Furthermore, treatment methods cannot be devised without a basic understanding of the physiological effects that occur due to toxin exposure in turtles. There is a long record of animal morbidity and mortality that has been associated with harmful algal blooms (HABs), which have been increasing in distribution and frequency worldwide. In southwest Florida, annual HABs can be devastating to the marine animals that inhabit and nest on the beaches; among these are several species of sea turtles. With the development of model organisms that have been used in research for decades, it is now possible to explore in depth the molecular and cellular processes that occur in response to acute and chronic brevetoxin exposure. The freshwater turtle Trachemys scripta is often used as a model species for a variety of research topics due to their evolutionary adaptations and, in this study, have yielded valuable results that are applicable to rehabilitation facilities to conserve sea turtle populations. Karenia brevis is a well-known species of dinoflagellate present in Gulf of Mexico. These organisms rapidly reproduce and release neurotoxins, collectively referred to as brevetoxins. Even at low concentrations, brevetoxin can still target and impair the immune system of many species, including manatees (Walsh et al., 2015) and sea turtles (Walsh et al., 2010), and in a previous rat study, there was a 70% reduction in 146 humoral-mediated immunity when exposed to brevetoxin (Benson et al., 2004). Very little is known about the duration and quantity of toxins animals are exposed to under HAB conditions; therefore, by designing controlled laboratory experiments using in a freshwater turtle model, we have a better understanding of the physiology behind toxin exposure. This project directly address concerns relating to the pathways of toxin metabolism and how the toxin impacts turtles behaviorally. The results from this study have provided insight into brevetoxin’s mode of action in the brain. A series of downstream blockers such as tetrodotoxin (TTX), tetanus toxin (TET), MK-801, and AMPA were utilized to better understand what molecules contribute to and trigger the cell death pathway. I have demonstrated that the mechanism of action of brevetoxin is the same as it appears to be in rats; however, the turtles show much higher resistance to toxicity, suggesting that turtles may have physiological differences in regard to the number of ion channels or rates of channel activation. Additional interesting studies would include investigating the basal extracellular vs intracellular calcium levels during brevetoxin exposure. This would give direct insight into the source of intracellular increase in calcium since calcium is also stored in membrane bound organelles. It is clear that with the use of more advanced technologies more comprehensive methods could be used for detecting intracellular changes. The tissue distribution studies have shown that PbTx-3 rapidly clears from each organ system over a period of 24-48h for both oral and IT PbTx-3 exposures, and in T. scripta, which coincides with the decline in clinical symptoms over the same time period. In addition to the freshwater turtle species, I used the estuarine diamondback terrapin species as a second, environmentally closer model system to better correlate the data with 147 sea turtles. The tissue distribution of PbTx-3 in M. terrapin similarly distributed to all organ systems and suspect that it is much different in sea turtles aside from consumed toxin quantities. Based on this data showing that brevetoxin is lipid soluble and easily passes through cell membranes and distributes to all tissues, I used intralipid emulsion as a treatment. This treatment proved successful in ameliorating symptoms of brevetoxicosis and increasing toxin clearance rates via the excretion and detoxification systems. It is thought that intralipids creates an intravascular lipid layer drawing the toxins out of tissues and therefore increasing clearance rates. Based on these studies I recommend implementation of this treatment at the numerous rehabilitation facilities in the Gulf coast states and worldwide. While better tools and approaches are needed to understand the impact brevetoxin takes in other marine animals such as dolphins and manatees, and to be able to respond quickly when events occur to better manage HABs, we have successfully elucidated important information regarding the physiological response to brevetoxin exposure using turtle models. A far better solution than devising treatment plans for HAB exposure in marine organisms though would be to address the root of the problem: eutrophication. Implementing more research and monitoring programs to spread awareness about the effects of eutrophication and minimizing nutrient overload into our water systems will help protect the environment and potentially reduce the extent, frequency, and duration of these blooms. Increased development of monitoring methods and field related tools for early detection of biotoxins in aquatic systems is currently an area of investigation, and would also contribute to a reduction in deaths caused by toxicity. 148 Future directions should focus on keeping the databases current with all animals that are found stranded or dead, especially ones that are suspected of brevetoxin exposure. There should be continued periodic reviews looking at trends for how and when animals died and correlate it to blooms or other contaminants in the area that may have been contributors to strandings and deaths. The knowledge obtained from this project has already been shared in several different locations around the globe including two international conferences (New Zealand and Brazil) specifically focused on harmful algal blooms and multiple national conferences including sea turtle, integrative biology, and aquatic animal medicine meetings to increase awareness and share the current conservation efforts being implemented for sea turtles. As many other biotoxins accumulate in prey and affect marine life including the saxitoxins, ciguatoxins, domic acid and okadaic acid, there should be ongoing similar research using the freshwater turtle as a model system to examine the impacts of these other toxins. Sea turtles tend to prey on fish, crustaceans, gastropods, while green turtle consume primarily sea grasses; these all become toxin vectors. Increasing our knowledge and awareness of toxins by use of experimental studies in conjunction with environmental conservation efforts will promote population survival. 149 References Benson, J., Hahn, F., March, T., McDonald, J., Sopori, M., Seagrave, J., Gomez, A., Bourdelais, A., Naar, J., Zaias, J., Bossart, G., Baden, D., 2004. Inhalation toxicity of brevetoxin 3 in rats exposed for 5 days. J. Toxicol. Environ. Health. A 67, 1443– 56. doi:10.1080/15287390490483809. Walsh, C.J., Butawan, M., Yordy, J., Ball, R., Flewelling, L., De Wit, M., Bonde, R.K., 2015. Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress. Aquat. Toxicol. 161, 73–84. doi:10.1016/j.aquatox.2015.01.019. Walsh, C.J., Leggett, S.R., Carter, B.J., Colle, C., 2010. Effects of brevetoxin exposure on the immune system of loggerhead sea turtles. Aquat. Toxicol. 97, 293–303. doi:10.1016/j.aquatox.2009.12.014. 150