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Amphibians

Red-eyed tree frog, not poisonous but certainly attractive

Bombina

Effects of Bombina are extremely mild in simple dermal contact, with dermitis being rare. Contact with mucous membranes can be more severe with the potential for severe ocular effects. This genus, however, has yielded some very interesting substances that may prove to be quite useful.

Bombinans are the germicidal proteins indentified from Bombina orientalis (Oriental fire-bellied toad). These are proteins of 28 amino acids that have significant antimicrobial and antifungal properties, the molecular basis for this activity is presumed to result from the predicted amphipathic alpha-helical structures (Mor, Hani et al. 1994). This activity may result in the use of these compounds, or derivates of, as germicidal agents in the treatment against opportunistic infection byfilamentous fungi or pathogenic bacteria in cases involving immunodeficiencies.

Bombina variegata cutaneous venom (Oriental yellow-bellied toad), has been shown to inhibit the proliferation of human leukaemic cell line HL 60 in a dose dependent manner (Balboni, Bernabei et al. 1992). The contact time needed was low, under 3 min, and the effects were not reversed with subseqeunt washing of the cells. This does not demonstrate any real potential for therapeutic use but rather effectively demonstrates the cytotoxic nature of hte toxins. Venom also contains an unnamed bacterial toxin of molecular weight 6,700 that was shown to ahve antibacterial effect against both gram positive and negative bacteria (Mastromei, Barberio et al. 1991). This polypeptide, as may other amphibian antibacterial agents, may have potential for use in clinical settings as more and more antibiotics are rendered useless due to plasmid exchange amongst bacteria.

Bufo

The major clinical effects by members of the bufo genus, particularily the large and ubiquitous B. marinus (Marine toad), are primarily cardioactive due to digoxin-like cardiotonic steroids present in the venom. The clinical symptoms, besides dermatitis, are hypotension accompanied by potentially severe arrythmia. A significant degree of the clinical effects may be due to assimilated plant alkaloides that have been put to use in the defense system(Radford, Gillies et al. 1986). In a case where two Japanese women tried to commit suicide using the traditional Chinese medicine Kyushin, of which toad venom is the primary active component, the clinical effects were a significant elevation of a serum digoxin-like immunoreactive substance (2.35 and 1.84 ng/ml) and symptoms of nausea, vomiting and general malaise. Their blood biochemistry and electrolytes were normal. In one patient, an electrocardiogram revealed a second degree Wenckebach atrioventricular block and T-wave change (Lin, Lin et al. 1989).

Bufo marinus

Two potently antimicrobial proteins, buforin-I and II, have been isolated from Bufo bufo gargarizans. Buforin-I is a 39 amino acid peptide with broad-spectrum antimicrobial activity and was found to be more potent than magainin 2 (Park, Kim et al. 1996). Buforin-II - a 21-amino acid derivative of buforin I, shows even more potent antimicrobial activities than buforin I (Park, Kim et al. 1996).

Kyushin is an interesting traditional Chinese medicine frequently taken without prescription in Japan that has demonstrated digoxin like effects (Fushimi, Koh et al. 1990). The major problem with the traditional Chinese medicines is that the quantity and quality of compounds is greatly varialble (Hong, Chan et al. 1992). This creates a hazardous situation with the potential for grave overdoses being very high. The cardiotonic activity is due to a toad venom component known Chan'su. This venom component has a mian component identified as bufalin and other, slightly less bioactive components such as cinobufagin.

Bufalin, the active principle of Chan'su, is a Na+/K+-ATPase inhibitor and binds with higher affinity than ouabain on the binding sites on the cell membrane (Jing, Watabe et al. 1994; Fushimi and Amino 1995). This has been shown, in controlled studies of hepatic cancerous pain, to have analgesic effects (Wang, Sun et al. 1994) The hypothesis was that the drug would increase hepatic blood circulation which, in decreasing the amount of stagnating blood, would along with directly anagesic compounds reduce pain. The researchers described the results as satisfactory (Wang, Sun et al. 1994). In addition, Bufalin has been shown to have anti-cancer properties in leukemia as well as melanoma cells. In human leukemia cells differentiation is induced in a broad range of human cell lines with apoptosis specifically in human leukemia cells from expression alteration apoptotic genes c-myc and bcl-2 (Jing, Ohizumi et al. 1994; Masuda, Kawazoe et al. 1995; Watabe, Masuda et al. 1996). This is accomplished through the modulation of several protein kinase activities in a distinct way from RA and 1 alpha, 25(OH) 2D3. This effect of bufalin on the cell cycle of leukemia cells is similar to that of topoisomerase inhibitors (Jing, Watabe et al. 1994) The concentration needed for inducing apoptosis of HL-60 cells was 10(-8) M, which is comparable to that of camptothecin but lower that those of other antitumor drugs such as cisplatin, VP16 and all-trans retinoic acid (Jing, Ohizumi et al. 1994). In melanoma cells, the rate of cell proliferation of mouse melanoma clone B16-F10 cells decreased with a concomitant stimulation of expression of its melanotic phenotype (Zhang, Yoshida et al. ). The effect of bufalin on melanogenesis included stimulation of tyrosinase activity and increase of cellular melanin content. (Zhang, Yoshida et al. )

Cinobufagin, the second major component of Chan'su, has been used successfully in high doses in attenuation and treatment of infection and granulocytopenia during combined chemotherapy (Hirai, Morishita et al. 1992).. In high doses, infection was significantly decreased without a significant change in the number of before and after the treatment. This potential use of this compound to significantly reduce the risk of infection and degree and duration of granulocytopenia associated with chemotherapy of patients with malignant blood diseases. (Yue 1992).

Other Bufo toad venom derived cardiotonic steroids, isolated from B. marinus venom, are bufotalin, marinobufagen, marinoic acid, and resibufogenin, all of which inhibit Na+,K(+)-ATPase activity. Bufotalin has a higher potency for inhibition of myocardial Na+,K(+)-ATPase activity than digoxin increasing the myocardial contractile force but did not affecting the heart rate (Hirai, Morishita et al. 1992). Marinobufagenin, similar to ouabain, possesses potent vasoconstrictive activity with higher digoxin-like immunoreactivity than ouabain (Bagrov, Roukoyatkina et al. 1995). Marinoic inhibits Na+, K+, -ATPase enzymatic activity, and 3H ouabain binding to the digitalis receptor set on Na+, K(+) ATPase. However, marinoic acid was less effective inhibitor than typical bufadienolides (Matsukawa, Akizawa et al. 1996). Resibufogenin, 3-hydroxy-14,15 -epoxy-20,22-dienolide, has the same structure as bufalin except for one H+ and is also a potent sodium pump inhibitor (Pamnani, Chen et al. 1994) The elecrophysiological characteristics and electrophysiological properties of RBG are similar to that of acetylstrophanthidin (AS), suggesting that RBG belongs to the family of digitalis-like drugs (Xie, Cunningham et al. 1994)

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Poison Arrow/Dart frogs

 Dendrobates  azureus 

The venom of the poison arrow frogs (Dendrobates, Epipedobates, Minyobates, and Phyllobates) and similar genera (Mantella, Melanophryniscus and Pseudophryne,) contains some of the most powerful cardiotoxins known and symptoms are reflective of this. Clinical effects - The venoms contained are neurotoxic as well as myotoxic with pathology being representative of this. The venom is curare like in effect, causing irreversible blockage of neuromuscular signal transmission. Many of the toxins of this, and other amphibians, have been shown to be alkaloid in structure which is indicative of the origin from plants. The frogs obtain them in their diet and store these powerful toxins for use in chemical defense(Daly, Secunda et al. 1994). This has been demonstrated numerous times by poison arrow frogs losing all or most of their toxicity within the first few months of captivity. (Daly, Secunda et al. 1992). The major classes of dendrobatid alkaloids batrachotoxin decahydroquinolines, the histrionicotoxins, indolizidines, the pumiliotoxins, pyrrolizidines, quinolizidines, and tetrodotoxin. Significnat variations in quantity and structure are found and are specific not only to specie but also to populations of a particular specie (Daly, Secunda et al. 1992).

Dendrobates tinctorius

Certain genera contain alkaloids found only within that genus and sometimes specific to only one specie. D. histrionicus (Columbian Arrow poison frog) contains two main alkaloids unique to this specie: gephyrotoxin and histrionicotoxin. Gephyrotoxin is a novel tricyclic alkaloid that blocks the voltage-sensitive potassium conductance of the indirectly elicited muscle twitch in a concentration-dependent manner (Souccar, Varanda et al. 1984). This neurotoxin reacts with the acetylcholine receptor complex, resulting in a blockage of its channel, primarily in the open conformation (Souccar, Varanda et al. 1984). The target of histrionicotoxin is the ion channel of the nicotinic acetylcholine receptor (Rapier, Wonnacott et al. 1987). This neurotoxin interacts with the ion channel of the nicotinic acetylcholine receptor-ion channel complex in the central nervous system (Aronstam, King et al. 1985; Rapier, Wonnacott et al. 1987). Epipedobates tricolor contains what may turn out to be the most significant compound isolated from amphibian, epibatidine. This compound is a neuronal nicotinic acetylcholine receptor analgesic agent that has been shown to be 120 times more potent that nicotine in analgesic effect (Qian, Li et al. 1993; Muller 1996). Synthetic versions of this have been produced and sucessfully tested. (Okabe and Natsume 1994). The Australian frogs of the genus Pseudophryne also contain unique substances, the most significant being the pseudophrynamine class (Daly, Garraffo et al. 1990)

Dendrobates lehmani

Batrachotoxin, first isolated from Dendrobates pumilo among, is one of a group of potent lipid- soluble neurotoxins which binds voltage-sensitive sodium channels (Casebolt and Brown 1993). Batrachotoxin is the most powerful animal venom known, being 250 times more powerful than strychnine and is present in extreme amounts in Phyllobates terribilus (Daly, Myers et al. 1980). Pumiliotoxins are alkaloids that increase the rate of sodium channel opening and closing, thus producing spontaneous activity of the nerve (Rao, Warnick et al. 1987; Sheridan, Deshpande et al. 1991). Variations are found in species of Dendrobates, Epipedobates, Mantella, Melanophryniscus, Minyobates, Phyllobates. Pseudophryne (Daly, Myers et al. 1980). Dendrobates pumilo was the first specie from which this copound was isolated.

The frogs, like the toads, contain antimicrobial and antifungal compounds in their skin secretions that may prove to be of therepeutic use. Central American tree-frogs in the Phyllomedusa genus contain proteins known as dermaseptins. These polypeptides are 27-34 amino acids in length, having antimicrobial activity as well as inhibiting growth of pathogenic filamentous fungi. Similar compounds are found in Rana and Xenopus. Rana brevipoda yeilds brevins: calcium dependent actin- binding and F-actin-severing anitimicrobial peptides (Gailly, Lejeune et al. 1990). Ranalexin, fromR. catesbeiana (Bullfrog), is structurally related to the bacterial antibiotic polymyxin with an amino acid sequence of NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met- Ile-Cys- Ala-Val-Thr- Lys-Lys - Cys-COOH, containing a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule (Clark, Durell et al. 1994). Xenopus: (African clawed frog possesses two peptides: magainin and xenonin from X. laevis skin glands, a 23 -residue peptide belonging to a broad class of lipid bilayer interacting alpha-helical peptides. Little is currently known concerning the nature of this peptide/lipid interaction on the molecular level. Xenonin-3, 66 Amino Acids with 4 ssbonds, shows similarity to snake venom cytotoxins and short neurotoxins (Kolbe, Huber et al. 1993)

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Zhang, L., T. Yoshida, et al. "Stimulation of melanin synthesis of B16-F10 mouse melanoma cells by bufalin." .