Growing evidence suggests that at least some toxins in the suite found in Poison Dart Frogs comes from assimilation within their food. These small frogs scour the leaf litter within their natural environment looking for a variety of arthropods including beetles, millipedes and flies. In tests where captive bred frogs fed with fruit flies were raised alongside frogs with leaf litter from their native environment only the native frogs incorporated toxins (Daly et al. 1994; Daly et al. 2000). Clearly, the leaf litter in a Dendrobatids home environment can provide some dietary sources for the numerous toxins in this group of animals. Scientists are only now beginning to realise the importance of diet and food selection as a major role in toxin development.
The most important poison dart frog toxins are batrachotoxins, pumiliotoxins, histrionicotoxins and gephyrotoxins. Out of these batrachotoxins and pumiliotoxins are more toxic. These are usually solid; crystalline compounds, soluble in alcohols and form of water-soluble salts; being seen as milky secretions from the frogs.
Batrachotoxins (sodium channel activators) 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 depolarisation of nerve and muscle.
Above (& previous page) are the chemical structures for batrachotoxin and homobatrachotxin. It is evident that both toxins are esters of the same pregnadiene alcohol. Batrachotoxin A has an expoxide bridge and side oxazepine ring. However this form does not show much toxicity, with a LD50
in mice on1 mg/kg being administered subcutaneously. Though the esters of this steroidal alcohol are very toxic, with batrachotoxin having a LD50 of 0.2μg/kg in mice with a lethal minimal dose of about 0.01μg/kg and homobatrachotoxin only being slightly less toxin with a minimal lethal dose of between 0.04 – 0.06μg/kg.
Batrachotoxin and homobatrachotoxin are among the most potent of all naturally occurring nonprotein poisons. The current thoughts behind the mechanism consist of (a) a generation of an action potential in nerve, i.e., depolarisation of the nerve membrane with passive diffusion of sodium ions into the axon (increase sodium permeability), followed by repolarisation due to passive diffusion of potassium ions out of the axon (increase in potassium permeability) The membrane potential with excess sodium ions outside and excess potassium ions inside the cell is maintained by the action of the sodium pump (Na+ K+ activated ATPase) which transports sodium ions out of, and potassium ions into the cytoplasm. (b) A calcium dependant release quantal release of acetylcholine as a result of depolarisation of the membrane of the pre – synaptic terminal. (c) Interaction of acetylcholine with receptors in the muscle endplate resulting in depolarisation of the muscle membrane, generation or a muscle action potential, and a concomitant liberation of calcium ions from the sarcoplasmic reticulum into the muscle cytoplasm. (d) Combination of calcium ions with tropin, which thereby permits the interaction of actin and myosin, the basic process of a muscle contraction. (e) Sequestration of calcium ions is followed by inhibition of actinmyosin interaction by tropinin and muscle relaxation. This complex series of events has been elucidated in large measure through the use of compounds, which specifically interact with one of the steps. A few examples are given in the summarised schematically in Fig 1.
Subsequent work has now demonstrated that batrachotoxin does not affect the action potential generating system or either nerve or muscle and that acetylcholin sensitivity of the muscle endplate is unaffected, suggestive of blockade of transmission in the presynaptic terminal. Batrachotoxin does not inhibit Na+ K+ ATPase, as does ouabain. Instead, instead it appears to cause specific and irreversible increase in the permeability of excitable membranes, especially in the presynaptic terminal, to sodium ions. This increase in sodium permeability results in depolarisation of the presynaptic terminal and a concomitant calcium dependant increase in acetylcholine release. The subsequent block in transmitter release appears to be due to complete depolarisation of the nerve terminal.
The effect of batrachotoxin can be prevented by tetrodotoxin, a compound that blocks passive diffusion of sodium ions through excitable membranes.
The initial muscle contracture caused by batrachotoxin results from muscle depolarisation elicited by an increase in sodium permeability in the muscle membranes, while subsequent sustained contracture appear to be due to release of calcium ions from an osmotically disrupted sarcotubular system.
Batrachotoxins have no effect on cells, which do not posses voltage sensitive sodium channels. They are strong cardio toxins, affecting ion permeability, thus leading to irreversible depolarisation of nerves and muscles, arrhythmias, fibrillation, and cardiac failure. Their irreversibility is due to the lipophilic nature of the compound. Larger animals are often more susceptible to toxins than smaller organisms. The oral potency of batrachotoxin is much lower; therefore native Indians are able to eat animals captured by their darts without risk of intoxication. In additions, the small amount of poison used is metabolised and the metabolites are not poisonous; cooking may also destroy toxins but not all toxins will be heat labile.
Results from studies have demonstrated that batrachotoxin is an extremely important tool for the study of events in muscle, nerve and synapse.
Pumiliotoxins (positive modulators of sodium channels) occur in all species of Phyllobates and Dendrobates, over 100 toxins have been placed in this group. Despite the numerous toxins in this group, much is still unknown about this group of toxins. Divided into three groups, Pumiliotoxins A and B are significantly more toxic than the C group. It appears that this group of toxins affect the transport of calcium ions in the calcium and sodium dependent processes within nerve and skeletal muscles (Myers and Daly 1983; Patocka et al. 1999). For comparisons sake, the pumiliotoxins are often 100 to 1000 times less toxin than their batrachotoxin counterparts (Patocka et al. 1999).
The pharmacology of pumiliotoxin B probably involves calcium and sodium dependant processes in nerve and skeletal muscles, however their mechanism is not quite clear.
Activity is strongly dependant on structure, and appears to require at least three hydroxyl groups for optimal activity.
Histionicotoxins are an unusual set of toxins has the ability to create a blockage of ions between the end-plate channel and the acetylcholine receptor (Myers and Daly 1983), i.e. non-competitive blockers of nicotinic channels. The toxin prevents ions from flowing out of nerve and muscle cells thereby preventing them from returning to the resting state. This in turn lengthens the transmission of nerve muscles and consequently prolongs muscle contraction (Myers and Daly 1983). It is not certain as to where these toxins are derived as of yet. The length and nature of the two side chains influence activity.
A radio labelled perhydrohistrionicotoxin has proved useful in studying the interaction of many compounds, including phencyclidine, quinacrine, chlorpromazine and local anaesthetics, with nicotinic channels blocking them in a similar manner to histrionicotoxin.
Both enantiomers of Epibatidine have proved to be incredibly potent nicotinic agonist with selectivity towards neuronal and ganglionic receptors. Agonist activity at central nicotinic receptors was the basis for the analgetic activity of epibatidine, which is about 200 fold more potent than morphine. Epibatidine now represents a powerful tool for the study of nicotinic receptors and function, and analogs are being developed for possible therapeutic use as analgetics and cognitive enhancers.
The dendrobatid alkaloids have proved to be not only unexpectedly numerous, but also remarkably diverse in structure are pharmacology. Like many plant alkaloids, the dendrobatid toxins clearly have a defensive function as supported by the following observations: (1) the toxins are released in secretions on the slightest damage to the frogs skin. (2) Most of the toxic dendrobatids are brightly coloured, and the bold patterns of some species might make these diurnal frogs recognisable even to predators that lack any colour vision. Following from the above considerations that the toxins probably evolve under strong selective pressures.
Poison Dart frogs contain a variety of toxins. Below is a description of a few of the more common ones. Most of the toxins within Poison Dart frogs affect the contraction of muscle cells or interfere with the transmission nerve impulses. For muscle contraction to occur, it must usually receive an electrical impulse from the surrounding muscle nerves. This impulse is carried down the axon of the neuron and then the signal is passed onto the next neuron across the synaptic cleft (Campbell 1993). For that signal to be carried across neuro transmitters bind with ion channels allowing ions such as sodium, potassium and chlorine to cross the membrane. This change of ions allows a change of membrane potential and ultimately the passing on of the signal to the next neuron (Campbell 1993). Most of the toxins in Poison Dart frogs affect with this transmission of signals towards muscle fibres.
Collecting large quantities of frog-based alkaloids is difficult. All dendrobatid frogs are now listed in CITES, which makes trafficking in them subject to documentation. Frogs born and raised in captivity soon lose their toxins or contain significantly less, indicating that their synthesis may be based on environmental or dietary precursors in their natural environment.
For much of the 20th century, the treat of biological warfare has been given a low given a low priority. When the Biological and Toxin Weapons Conventions (BTWC) entered into force in 1975, it generally felt that not more than one or two countries would have the capabilities to produce such weapons. Today, public reports suggest that at least a dozen countries have such weapons or are actively seeking them.
Bioterroist attacks in theory could be caused by almost any appropriate toxin, if it available in sufficient amounts. However it is important not to ban or list all toxins, which could hamper productive research in new drugs and the use of biological research probes.
The use of poison dart frog toxins in weapons of mass destruction is dubious in that man had extracted these toxins from frogs under primitive conditions, and used them to enhance existing weapons (the darts). These toxins are most effective when injected.
Poisons from other animals have also been looked at closely for possible applications in medicines by design, here are just a few.
The Southern Copperhead snake, which is native to Eastern USA and Mexico, contains a powerful clot buster, which is well known to scientist. The venom contains a protein called contortrostatin; this protein has the ability to retard the growth and metastasis of tumours. It has been found that injections of contortrostatin in mice, not only has the ability to prevent the spread of ovarian and breast tumours but also has been seen to shrink them.
Chlorotoxin is a substance in the venom of the Giant Israeli scorpion; it may offer hope to the thousands suffering glioma, an incurable rapidly spreading form of brain cancer. Chlorotoxin targets glioma cells and blocks their fluid balancing chloride channels, preventing them from shrinking and then migrating to else where in the brain.
The Thai cobra is armed with venom that paralysis nerves and muscles eventually leading to respiratory failure. Currently clinical trials of Immunokine, a drug derived from the venom of the snake are being administered to people with multiple sclerosis (MS). Immunokine seems to prevent immune cells from attacking and destroying the myelin sheath that protects the nerve cell.
The tropical oceans harbour more than 500 species of cone snails, which posses the ability to stab their prey with harpoons loaded with a paralytic poison. Each species of cone snail produces unique venom, which can contain between 50 to 200 pharmacologically active peptides known as conotoxins. These provide potential treatments for neurological and neuromuscular disorders.
There is a possibility that there cure or treatment to man, out there. Being used by a plants or animals for it survival in the natural world. However with countless numbers of organisms still waiting to be discovered, deep down in the oceans or in tropical rain forest. The possibilities of discovering them are evaporating with each passing day.
Tropical rain forests are being destroyed by miles, on a daily basis leading to a direct change in the ecosystem and the habitat of thousands of species of animals. Placing greater
evolutionary pressures on them. Increasing sea temperatures, may initiate the loss of a species or even the being of entire organisms.
This has been seen earlier, with dendrobatid frogs bread in captivity soon loss their toxins or produce considerably less. Environmental or dietary precursors in their natural environment may play an equal role in the synthesis of potential pharmacological active compounds present in organisms; with out one you may have none.
It would appear that amphibians, in their effort to evolve chemical defences against predators, have provided a rich source of novel biologically active compounds not present elsewhere.
In view of the still large number of amphibians, which have not been investigated, it seems likely that further classes of pharmacologically active compounds will be obtained from this natural source.
If all of the frog alkaloids are sequestered from dietary sources, then small, even tiny arthropods may be a virtually untapped source of new pharmacologically active agents from the world’s rain forest.
The unique pharmacological activities and therapeutic potential of frog alkaloids such as batrachotoxins, pumiliotoxins, histrionicotoxins and gephyrotoxins have provided a rich source of possible leads in the further development of potential medicines. Agents that have selective effects n membrane permeability should find wide application in studies of nerve, muscle, and synapse. Providing essential information required by pharmacologist to enable them to produce fast acting specific medicines.
Appendix.
Fig 1.
References.
1. Clinical Toxicology. 1971. Volume 4.
Batrachotoxin, an extremely active cardio- and neurotoxin from the Colombian arrow poison frog.
J. Daly and B. Witkop.
2. Toxicon. 1978. Volume 16.
Pharmacological activity of alkaloids from poison – dart frogs (dendrobatidae).
Monica Mensah – Dwumah.
3. Chemistry, Biology and Pharmacology of new alkaloids from tropical poison - dart frogs
John W. Daly.
4. http://www.iupac.org/symposia/proceedings/phuket97/daly.html
Biodiversity of alkaloids in amphibian skin: A dietary arthropod source.
John W. Daly.
5. Applied Science and Analysis, Inc. The ASA Newsletter. 1999.
Dart poison frogs and their toxins.
Jiri Patocka, Krauff Schwanhaeuser Wulff and Maria Victoria Marini Palomeque.
6. Jama. Medical News & Perspectives. Volume 279.
Nature’s agents help heal humans – some now take steps to reciprocate.
Charles Marwick
7. Time Pacific. January 15, 2001. No. 2
Potions from poisons. Looking for new drugs in unusual places.
Andrea Dorfman.
