Dart Poison Frogs and Their Toxins

Jiri Patocka a, Kräuff Schwanhaeuser Wulff b and MaríaVictoria Marini Palomeque c

Department of Toxicology, Military Medical Academy, Hradec Králové, b Medical Faculty, and c Faculty of Natural Sciences, Masaryk University, Brno, Czech Republic.E-mails:patocka@pmfhk.cz, krauff@yahoo.com and victoria@chemi.muni.cz

Introduction

Dart poison frogs, poison-arrow frogs, poison frogs and dendrobatid frogs are all names for small, brightly-colored frogs from Central and South America. These are members of the Dendrobatidae family, which secrete poisons from glands in their skin. The Emberá and Noanamá Chocó Indians use these toxic secretions for poisoning darts used in blowguns [1]. The family Dendrobatidae has eight genera: Dendrobates, Phyllobates, Aromobates, Epidobates, Minyobates, Manophryne, Nephelobates and Colosthetus, with 179 species of dart poison frog known already [1a].

Phyllobates terribilis, probably the most toxic animal of the world. This poison-dart frog lives in rainforests of Colombia.

Dendrobates pumilio lives in Colombia and Ecuador. This full-grown male poison-dart frog is only about 3/4 of an inch (2 cm) long.

Dendrobates auratus lives in rainforests of Surinam.

Dendrobates histrionicus lives in rainforest of Colombia and Ecuador.

Epipedobates tricolor, poison-dart frog from Ecuador, products epipedobatidine - nicotine derivative with very strong analgesic activity. Epipedobatidine is 200 times actrive than morphine.

The secretions from all dendrobatid frogs are poisonous, but only a few are toxic enough to kill a person. The bulk syntheses of many of their toxins are not an overwhelmingly difficult problem; consequently, it is not impossible to imagine the misuse of these toxins as terrorist weapons. The chemical and toxicological classification of the most important frog toxins is the purpose of this paper.

The most important dart poison frog toxins are batrachotoxins, pumiliotoxins, histrionicotoxins, and gephyrotoxins. Of these, the batrachotoxins and pumiliotoxins more toxic. They are usually solid, crystalline compounds, soluble in alcohols and form of water soluble salts. (Reviewer’s note: Interestingly, batrachotoxins have recently been isolated in the skin, feather and striated muscle of the Pitohui birds found in Papua New Guinea.)

Batrachotoxins differ from other dendrobatid toxins in their steroidal ring structure and are much more toxic. The toxicity is due to selective effects on ion permeability, which leads to an irreversible depolarization of nerve and muscle [2,3]. Chemical structures of batrachotoxin and homobatrachotoxin are given in Fig 1. It is evident that both toxins are esters of the same pregnadiene alcohol. Batrachotoxinin A has an epoxide-bridge and side oxazepine ring. The toxicity of batrachotoxinin A is not very high (LD50 for mice, subcutaneously administration is 1 mg/kg), but all esters of this steroidal alcohol are very toxic. The LD50 value of batrachotoxin in mice (subcutaneously) is 0.2 µg/kg, with minimal lethal doses from 0.01 to 0.02 µg/kg). The toxicity of homobatrachotoxin is only slightly less than batrachotoxin, with minimal lethal doses being about 0.04 and 0.06 µg/kg, respectively [1]. Batrachotoxins are found in frogs of genera Phyllobates (P. terribilis, P. bicolor and P. aurotaenia). These are the most toxic of the dart poison frogs [4]. The secretions of the adult frog, P. terribilis, typically contain 700 to 1900 µg of batrachotoxin/homobatrachotoxin.

Batrachotoxin and homobatrachotoxin are among the most potent of all naturally occurring nonprotein poisons. They are strong cardiotoxins, affecting ion permeability, which leads to an irreversible depolarization of nerves and muscles, arrhythmias, fibrillation, and cardiac failure [2]. Observed symptoms in laboratory animals include strong muscle contractions, violent convulsions, salivation, and dyspnoe and death even at doses of less than 0.1 µg [5]. At higher doses, e.g., 1 µg, death occurs in mice within one minute [1].

In a 20-gram white mouse, the minimal lethal dose of batrachotoxin/homobatrachotoxin is about 0.05 µg, when injected subcutaneously. Thus, one frog, P. terribilis, with typical 1100 µg of these compounds, contains enough poison to kill more than 20,000 mice. Although extrapolating toxicity parameters from mouse to man has errors, if we suppose that man is at least as susceptible as mice to these compounds, the lethal dose is about 180 µg for a person. Larger animals are often more susceptible to toxins that smaller organisms, so that the lethal dose for man may be even less. Myers et al. [1] anticipated a lethal dose of batrachotoxin for man of only 2.0 to 7.5 µg, when administered by injection. The oral potency of batrachotoxin is much lower; therefore, Indians can eat animals captured by their darts without of risk of intoxication. In additions, the small amount of poison used is metabolized and the metabolites are not poisonous; most importantly, cooking may also destroy the toxins, although not all toxins are heat labile.

No effective antidote is known, but treatment of batrachotoxin poisoning might best be based on paradigm for agents with similar mechanism of action, as for example aconitine, veratridine or digitalis. One of the few drugs available for this purpose may be DigiBind and a new comparable product, DigiTab, used to treat digitalis and oleander poisoning.

Pumiliotoxins occur in all species groups of Dendrobates and Phyllobates. At present we know more than one hundred of toxins designated as pumiliotoxins, divided to three groups: Pumiliotoxins A, B, and C [6-8]. There are, for example, 2,6-disubstituted piperidines, 3,5-disubstituted indolizidines, derivatives of perhydrochinoline etc. [8,9]. Some selected structures of different pumiliotoxins are given in Fig. 2.

Pumiliotoxins A and B are much more toxic than the pumiliotoxins C. Subcutaneous injection of 100 g of either pumiliotoxins A or B in mice caused locomotor difficulties, partial paralysis of hind limbs, salivation, extensor movements, and finally, clonic convulsions and death in less than 10 minutes [6]. Even at a 20 g dose, one of pumiliotoxin B type caused death in less than 20 min [10]. Lesser doses or less toxic derivatives caused in mice initial hyperactivity with accompanying hypersensitivity to stimuli and locomotor difficulties, but no death [11].

The pharmacology of pumiliotoxins B probably involves calcium and sodium-dependent processes in nerve and skeletal muscle [12], but their mechanism is not yet quite clear [13]. Their cardiotoxic and myotoxic effects, mediated by sodium channel interaction, [14,15] as well as their antagonistic effect to nicotinic acetylcholine receptor has been described [16, 17]. Commonly pumiliotoxins are 100 to 1000 times less toxic than batrachotoxins, but their chemical structures are simple enough to be suitable for chemical syntheses in laboratory. Their toxicities are comparable with nerve organophosphate agents, but no specific antidotal therapy is known.

Dart poison toxins and bioterrorism

Collecting large quantitites of the frog-based alkaloids is difficult. All Dendrobattid frogs are now listed in CITES, which makes trafficking in them subject to documentation. Captive born and raised dendrobatid frogs soon lose their toxins, indicating that their synthesis may be based on environmental or dietary precursors in their natural environment.

For much of the 20th century, the threat of biological warfare has been given a low priority. When the Biological and Toxin Weapons Convention (BTWC) entered into force in 1975, it was generally believed that only one or two countries other than the two superpowers had such weapons. Today, public reports assert that at least dozen countries have such weapons or actively seek them.

Bioterrorist attacks, in theory, could be caused by virtually any appropriate toxin, if it is available in sufficient amount. However, it is important not to ban or list all toxins, which would hamper productive research in new drugs and the use of toxins as potent biochemical research probes. The examples of the dart poison frog toxins are notable in that man has extracted these toxins from frogs, under primitive conditions, and used them to enhance existing weapons, the darts. However, the practicality of using such toxins in weapons of mass destruction is dubious. The toxins are most effective only when injected. However, in a single weapon, such as in a dart or needle, the dart poison frog toxin can be effective, but not nearly as effective as dipping the dart in botulinum toxin.

Acknowledgements

This study was supported by a grant MO 66021299116 from the Ministry of Army of Czech Republic.

References

1. Myers C.W., Daly J.W., Malkin B.: A dangerously toxic new frog (Phyllobate) used by Emberá Indians of Western Colombia, with discussion of blowgun fabrication and dart poisoning.. Bulletin American Museum Nat History, New York, 1978; 161: Article 2, 311-64.

1a. Duellman, .E. 1999. Global Distribution of Amphibians (Chapter I) in: Duellman, W.E.(ed), “Patterns of Distribution of Amphibians-A Global Perspective”. The Johns Hopkins University Press, Baltimore

2. Albuquerque E.X., Daly J.W., Witkop B.: Batrachotoxin: chemistry and pharmacology, Science 1977; 172: 995-1002.

3. Albuquerque E.X., Daly J.W.: Batrachotoxin, a selective probe for channels modulating sodium conductances in electrogenic membranes. In: Cuatrecasas P. (Ed.): The Specificity and Action of Animal, Bacterial and Plant Toxins. Receptors and Recognition. Ser. B. 3. Vol. I. Chapman and Hall, London 1977, p. 297.

4. Daly J.W., Myers C.W., Warnick J.E., Albuquerque E.X.: Levels of batrachotoxin and lack of sensitivity to its action in dart poison fogs (Phyllobates). Science 1980; 208: 1383-5.

5. Märki F., Witkop B.:The venom of the Colombian arrow poison frog Phyllobates bicolor. Experientia 1963;19:329-38.

6. Daly J.W., Myers C.W.: Toxicity of Panamanian poison frogs (Dendrobates): some biological and chemical aspects. Science 1967; 156:970-3.

7. Daly J.W., Tokuyama T., Habermehl G., Karle I.L., Witkop B.: Froschgifte. Isolierung und Structur von Pumiliotoxin C. Justus Liebigs Annal. Chem. 1969; 729: 198-204.

8. Daly J.W., Spande T.F., Whittaker A., Highet R.J., Feigl D., Nishimori A., Tokuyama Z., Myers C.W.: Alkaloids from dendrobatid frogs: structures of two omega-hydroxy congeners of 3-butyl-5-propylindolizidine and occurrence of 2,5-disubstituted pyrrolidines and 2,6-disubstituted piperidine. J. Nat. Prod. 1986;49:265-80.

9. Edwards M.W., Daly J.W., Myers C.W.: Alkaloids from a Panamanian poison frog, Dendrobates speciosus: identification of pumiliotoxin-A and allopumiliotoxin class alkaloids, 3,5-disubstituted indolizidines, 5-substituted 8-methylindolizidines, and a 2-methyl-6-nonyl-4-hydroxypiperidine. J. Nat. Prod. 1988;51:1188-97.

10. Daly J.W., Brown G.B., Mensah-Dwumah M., Myers C.W.: Classification of skin alkaloids from neotropical dart poison frogs (Dendrobatidae). Toxicon 1978; 16:163-88.

11. Mensah-Dwumah  H., Daly J.W.: Pharmacological activity of alkaloids from poison-dart frogs (Dendrobatidae). Toxicon 1997;16:189-94.

12. Rao K.S., Warnick J.E., Daly J.W., Albuquerque E.X.: Pharmacology of the alkaloid pumuliotoxin-B. II. Possible involvement of calcium and sodium-dependent processes in nerve and skeletal muscle. J. Pharmacol. Exp. Ther. 1987;243:775-83.

13. Sheridan R.E., Deshpande S.S., Lebeda F.J., Adler M.: The effect of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons. Brain. Res. 1991;556:53-60.

14. Daly C.W., McNeal E.T., Overman L.E., Ellison D.H.: A new class of cardiotonic agents: structure activity correlations for natural and synthetic analogues of the alkaloid pumiliotoxin B (8-hydroxy-8-methyl-6-alkylidene-1-azabicyclo[4,3,0] nonanes). J. Med. Chem. 1985;4:482-6.

15. Daly C.W., McNeal E.T., Gusovsky F., Ito F., Overman L.E.: Pumiliotoxin alkaloids: relationship of cardiotonic activity to sodium channel activity and phosphatidylinositol turnover. J. Med. Chem. 1988;2:477-80.

16. Warnick J.E., Jessup P.J., Overman L.E., Eldefrawi M.E., Nimit Y., Daly J.W., Albuquerque E.X.: Pumiliotoxin-C and synthetic analogues. A new class of nicotinic antagonists. Mol. Pharmacol. 1982;3:567-73.

17. Erspamer, V. 1994. “Bioactive secretions of the amphibian integument” in: Heatwole, H.(ed): Amphibian Biology-Volume I - The Integument.  Surrey Beatty and Sons, New South Wales.

Figures

Fig 1. Chemical structures of batrachotoxinin A, batrachotoxin a homobatrachotoxin, principal toxic compounds of dart poison frogs genera Phyllobates.

Fig. 2. Chemical structures of pumiliotoxin A, pumiliotoxin B, pumiliotoxin 267C and pumiliotoxin 241D, principal toxic compounds of poison-dart frog Dendrobates pumilio.

 

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