1. BTX
(Batrachotoxin)
Jenny Johnston, Mat Ley, Michael Miller, Tony Nguyen

2. Outline What is Batrachotoxin? History Sources of BTX
Uses of Batrachotoxin
Absorption of BTX
BTX Effects
Action Potentials
Synthesis and Mechanism
BTX Treatments
BTX and other Toxins
Evolution
Mimicry and Batrachotoxins
Exogenous vs. Endogenous Acquisition

3. CLICK HERE! Hooded Pitohui (Pitohui dichrous)
A bird with batrachotoxins? Watch the video below to learn how the Pitohui bird’s toxic feathers were discovered by Dr. Jack Dumbacher. (California Academy of Sciences)
CLICK HERE!
Hooded Pitohui (Pitohui dichrous)

4. What is Batrachotoxin? BTX is a neurotoxin
BTX is a class of extremely toxic steroidal alkaloids
Lipid solubility greatly increases the toxicity of BTX
BTX has an LD50 of approximately 2 µg/kg (in mice)
BTXs are found across a wide range of organisms
BTX is a neurotoxin
Irreversible activator of Na+ channels
Affects both nerve and muscle cells
Stabilizes the open conformation of voltage gated ion channels

5. History of Batrachotoxin
BTX was first isolated from frogs
“batrachos” = frog (Greek)
BTX is found in several species of frogs from the genus Phyllobates and Dendrobates
Phyllobates bicolor (seen right)

6. History of Batrachotoxin
Indigenous people from parts of South and Central America used the toxic frogs to make poisonous blow darts
Much like the toxin curare which also a neurotoxin

7. Batrachotoxin in New Guinea
BTX is also found in passerine birds of New Guinea
6 species of birds mostly from the genus Pitohui express BTX
Birds express BTX mostly in the skin and feathers

8. Batrachotoxin in New Guinea
Indigenous people in New Guinea do not hunt/eat the birds
Simply handling the birds causes respiratory irritation
The meat is reportedly bitter tasting and nauseating

9. Source of Batrachotoxin
Phyllobates lugubris
Neither frogs nor birds are believed to be able to synthesize BTX de novo
The source of BTX is most likely dietary
Frogs raised in captivity do not produce BTX
Birds show varying levels of BTX congruent with dietary variation

10. Uses of Batrachotoxin
BTX has been used to experimentally probe and characterize Na+ channels
Research has revealed that some mutations (point mutations and deletions) can reduce or increase susceptibility to BTX

11. Absorption The common pathways for absorption are: Ingestion
Inhalation
Tissue Contact
Skin Contact

12. Absorption by Inhalation
Its been reported by the indigenous people of New Guinea that handling the Pitohui birds will cause respiratory irritation because the skin particles and feathers of these birds contain BTX.

13. Absorption by Ingestion
Of these absorption pathways, ingestion is the most common and is most represented when predators of poison dart frogs succumb to the toxic effects of BTX when attempting to prey upon these frogs. The same can be said of the Pitohui birds and natives noted these birds as non-consumable.

14. Absorption by Tissue Contact
Indigenous people of Central and South America often coat their hunting darts with toxins obtained from poison dart frogs and use them to hunt. The darts will puncture the skin of the animal and thereby distributing the toxin within the animal by exposing muscle tissue to this toxin.

15. Absorption by Skin Contact
Because tissue contact with BTX causes numbness any contact with BTX by skin in small amounts can result in numbness of the skin as well is the senses within the area affected by BTX.

16. BTX Effects
BTX’s bind with high affinity to voltage gated sodium channels in nerve and muscle membranes, locking them in an open state.(1)
Highly toxic to cardiac muscle tissue.
(BTX) toxin causes muscle and nerve depolarization, fibrillation, arrhythmias, and heart failure(2)
Binding is irreversible, suggesting a chemical change in the Na+ channel that locks it open.
Since binding only occurs in open sodium channels, “it has been hypothesized that batrachotoxin binds within the pore of the channel.”(3)
This binding causes disruption of action potentials across the membrane.

17. Action Potential Overview
An action potential is a rapid, temporary change in electrical potential across a membrane, from negative to positive back to negative.(4)
Created by an unequal distribution of ions.
Depolarization
caused by voltage gated Na+ channels opening and an influx of Na+ . (event 1)
Causes an action in the cell(such as muscle contraction, or nerve impulse)
During depolarization, K+ channels open and k+ exits the cell. (event 2)
Figure 1 (5)

18. Action Potential Overview (Cont.)
Repolarization
Begins with closing of voltage gated Na+ channels.(event 3)
The now open K+ channels allow for efflux of K+ ions(event 4).
As the charge nears the resting potential, K+ channels close(event 5) and the cell returns to its resting potential.
Excess K+ diffuses away(event 5)
All events occur in a very short time(<1 millisecond)
Figure 1 (5)

19. BTX Effects (Cont.)
The permanent opening of the Na+ channels would cause permanent depolarization of the cell. Figure 2 to figure 3.
This would lead to paralysis, and abnormal heart function.
Figure 4 shows the effect of BTX on Na+ channels.

20. BTX and Other Toxins
BTX is similar to other neurotoxins in function. One example is tetrodotoxin.
Both affect voltage gated sodium channels. Causing paralysis among other effects.
BTX however is more toxic than many other toxins.
The ld50 in mice is 2 micrograms/kg. Convulsions and death after 8 min at 10 micrograms/kg.(6) This means that an amount equivalent to a grain of salt could kill an average human.
Tetrodotoxin
Batrachotoxin

21. Synthesis (7)
A complete synthesis of a Batrachotoxinin A was discovered in 1998.
The synthesis of Batrachotoninin A is considered a synthesis of Batrachotoxin because it is readily converted into Batrachotoxin.
Link to complete synthesis H
Batrachotoxinin A
Batrachotoxin

22. Synthesis Summary Ketone Synthesis Synthesis of Diels-Alder Precursor
Note: Stereo-selectivity of product varies.
Synthesis of Diels-Alder
Precursor

23. Synthesis Summary (Cont.)
Synthesis of Methyl Ketal
Final Steps

24. BTX Treatment Options
There is currently no safe and effective treatment for batrachotoxin. But tetrodotoxin seems to be the most likely candidate for treatment.
Tetrodotoxin targets the same voltage-gated sodium channels, but instead of permanently opening them and creating a huge influx of ions, it shuts them off and prevents the movement of ions.\
Although it is not an ideal antitoxin it serves to slow down the effects of BTX and more research is being done to understand neurotoxins and their effects before any type of treatment can be approved.

25. Evolution of Batrachotoxins
Both the Poison Dart Frogs and Pitohiu birds have acquired there toxicity from outside sources. These organisms obtain the toxins fully synthezised in their diet. Once ingested, batrachotoxin is secreted by glands on the skin.
This is called the “Diet-Toxicity” Hypothesis
Specic species of ants and beetles producebatrachotoxins that ultimately end up on the frogs skin. (Darst, 2005)
Poison Dart Frog (family Dendrobatidae) use coloration a warning of their toxicity. The frog’s coloring and use of batrachotoxin are believed to have to coevolved.
Toxicity is an anti-predator defense. Predators learn to avoid prey of certain colors to minimize the chances of eating toxic prey. (Summers, 2001)
A Pitohui bird next to a Choresine pulchra. This beetle is believed to be responsible for the bird’s acquisition of batrachotoxins.

26. Blue Poison Dart Frog (Dendrobates azureus)

27. Mimicry and Batrachotoxins
Pitohui birds of New Guinea contain batrachotoxins in their feathers. Highest concentrations of the toxins are found in feathers on the stomach, legs and breast of the bird. (Dumbacher, 2000)
The Pitohui birds and poison dart frogs display Mullerian mimicry. The animals send an honest signal to predators to make them aware they are not edible. Other animals that are palatable mimic the coloration of the Pitohui birds or poison dart frogs in order to avoid predation (called Batesian mimicry).
Can you identify the harmless species from the picture above?
We recommend a taste test to figure this one out. :)

28. Homobatrachotoxins as a Lice Repellant
Dr. John Dumbacher of the California Academy of Sciences found that lice preferred feathers of birds without homobatrachotoxins.
Lice living on feathers containing the toxin also had a reduced life span.
This suggests that the evolution of the toxin on the Pitohui’s feathers may protect the bird from lice infestation. Selective pressures for birds without lice may have attributed to the evolution of the toxin. (Dumbacher, 1999)
Vs.

29. ¿Una Pregunta?
Why is batrachotoxin presence in poison dart frogs and Pitohui birds unique?

30. ¿Una Pregunta?
Why is batrachotoxin presence in poison dart frogs and Pitohui birds unique?
There is no de novo synthesis of batrachotoxin within the organism. Many amphibians utilize toxins, but very few acquire the toxin exogenously. Most amphibians acquire monomers from their diet and synthesize the toxin within their body (endogenous acquisition).

31. Toxins Comparison Name Toxic Compound Defense Exogenous or Endogenous
Warning Coloration?
Dendrobatids, Phyllobates (Poison Dart Frogs), and Pitohui Birds
Batrachotoxins
(a neurotoxin)
Secreted by glands on the skin. Prevents closing of sodium channels in nerve cells
Exogenous
Yes - Bright body coloration
Rough-Skinned Newt (Taricha granulosa)
Tetrodotoxin (a neurotoxin)
When ingested, results in manipulation of sodium channels
Believed to be Endogenous
Yes - Bright coloration on belly
Marine Toad
(Bufo marinus)
Bufotoxin
(a cardiotoxin)
Secreted by glands on skin. Manipulation of Sodium-Potassium Channels in myocardial cells
Endogenous No

32. Works Cited
Daly, John W., Myers, Charles W. and Borys Malkin. “A Dangerously Toxic New Frog (Phyllobates) used by Embera Indians of Western Colombia with Discussion of Blowgun Fabrication and Dart Poisoning.” Bulletin of the American Museum of Natural History (1978): ).
Darst, Catherine, Pablo Menendez-Guerrero, Luis Coloma, and David Cannatella. "Evolution of Dietary Specialization and Chemical Defense in Poison Frogs (Dendrobatidae): A Comparative Analysis." The American Naturalist (2005):
"Dr. Jack Dumbacher: California Academy of Sciences." California Academy of Sciences - San
Francisco Museum and Planetarium - Bay Area Natural History Museum. Web. 17 Mar
<
Duellman, William, and Linda Trueb. Baltimore: John Hopkins UP, 1986.
Dumbacher, J. P., T. F. Spande, and J. W. Daly. "Batrachotoxin Alkaloids from Passerine Birds:
A Second Toxic Bird Genus (Ifrita Kowaldi) from New Guinea." Proceedings of the National
Academy of Sciences of the United States of America (2000):
Dumbacher, John P. "Evolution of Toxicity in Pitohuis: I. Effects of Homobatrachotoxin on
Chewing Lice (Order Phthiraptera)." The Auk (1999):
Hanifin, Charles, Edmund III Brodie, and Edmund Jr. Brodie. "Tetrodotoxin Levels of the Rough-skin Newt, Taricha Granulosa, Increase in Long-term Captivity." Toxicon 40 (2002):
Hilgris, R "Bufo marinus" (On-line), Animal Diversity Web. Accessed April 17, 2010 at
Lorenz, A. and J. Harding "Taricha granulosa" (On-line), Animal Diversity Web. Accessed April 17, 2010 at
Summers, Kyle, and Mark Clough. "The Evolution of Coloration and Toxicity in the Poison Frog Family (Dendrobatidae)." Proceedings of the National Academy of Sciences of the United States of America (2001):
Albuquerque, E. X., et. all. (1971) science 172,
Bosmans, F. et. all. The poison Dart frog's batrachotoxin modulates Nav1.8. FEBS Lett. (2004), 577,
Hadid, D. et. all. The Batrachotoxin Receptor on the Voltage-Gated Sodium Channel is Guarded by the Channel Activation Gate. Mol. Pharmacol. (2002), 61,
Freeman, Scott. Biological Science. (2005), 2nd edition, G1. Pearson Education, London.