Introduction

Amphibians started trolling the landmasses of earth about 363 million-years-ago, with Acanthostega and Ichthyostega probably being the earliest of known amphibians (Evans et al 1998). Fossil records elucidate that ancestors of modern amphibians like Frogs, Toads, Caecilians and Salamanders probably evolved about 200 million years ago during the Triassic period. A very interesting observation here states that, fossils of these ancestral forms dating back to Triassic, tend to exhibit most phenotypic features of their living relatives (Wilson et al 1974), implying that they haven't “evolved” much since then, owing to a structure, which seems to suite perfectly with their habitats. Amphibians evolved from fishes and during this transition various evolutionary advances were produced in amphibians. While this transition produced many advanced systems like legs, it also produced specialized alveolar or tubular glands in amphibian skins, which is sometimes considered as amphibian's chief evolutionary advance over that of fish-integuments (Noble 1931). These specialized glands had many functions, including secretion of substances having poisonous properties for repelling or killing predators as well as microbial pathogens. This evolutionary advance made amphibians one of many other organisms in the animal kingdom, which indulged in chemical wars (Hoiberg et al 2002; Daly et al 2005) in order to survive in the battle for survival (See Table.1.). In the following sections various properties, effects and perspectives regarding toad skin-secretions have been discussed.

Figure 1

Table 1: Animals indulging in Chemical Warfare Race

Specialized glands of Toad-skin

Amphibians like toads possess two types of alveolar glands in inner-layer of stratified epidermis of their skin: mucous glands and granular glands (Noble 1931; Hickman et al 1995). Mucous glands are scattered all over the body and secrete a transparent mucus secretion acting as lubricant in water and also helping in keeping skin moist on land. This mucus may contain many glycoproteins like mucins, mucinigen and carbohydrate-residues like galactose, fucose and sialic acid (Williams et al 2000). Granular glands on the other hand are “serous” type (Hickman et al 1995) having centrally placed nucleus and secreting acrid poisons or toxins, which help in providing protection from predators like birds, mammals, snakes, crocodiles who try to eat them (Storer 1925; Awasthi 2006). Granular glands usually require considerable stimulation to produce their milky poisonous secretions (Noble 1931) and may be found arranged in form of clustered pads e.g. Parotid glands of common toads. Secretions of mucous glands stain with basic dyes and lack granular appearance while that of granular glands has granular appearance and stain with plasma dyes. Toad granular gland secretions generally induce very serious inflammations of eyes or digestive tract, unpleasant experience and vomiting sensations in toad predators (Pough et al 1999; Biedermann 1930). Cane toad (Bufo marinus) is probably the most venomous of all other toads. Instances of snakes or domestic animals found dead with cane toad in their mouths or guts have been reported. Cane toads have also been found to influence the population of monitor lizards and various other frogs or toads (Hinkley 1962). Such is the impact of these venomous creatures that some animals have evolved strategies to avoid these toads e.g. two snake species have been reported to develop smaller heads so that they can no longer eat venomous cane toad in Australia (Awasthi 2006; Aldous 2004). Even human beings have been reported to die after consumption of mixture of toad skin-secretion components (Barry et al 1996). Toad skin toxin's potency is so profound that hedgehogs (Insectivora, Erinaceidae) use them for enhancing their own mechanical anti-predatory adaptations (Brodie Jr. 1977). Hedgehogs take these secretions into their mouths and lick it on their spines. Hedgehogs use them since presence of such substances in fresh or dried form on their spines probably increases the pain or potential of infection in its predators.

But toads don't enjoy the merit of being the most poisonous amphibians; rather this title is a proud property of poison-dart frogs (Phyllobates terribilis). These frogs secrete a neurotoxin called batrachotoxin, which is so fatal that South-American Indians use it to cover the tips of their arrows for hunting (Daly et al 1992). However in regions like Europe where such frogs aren't found toad may be the most poisonous, so much so that in medieval Europe toad's skin-gland extracts were employed in witchcraft (Hofrichter 2000).

Toad-toxin's Chemistry

Toad secretions of both mucous and granular glands can sometimes be poisonous (Phisalix 1918) however granular glands produce more toxic secretions than mucous glands. Granular gland secretions in toads contain chemicals that can be broadly classified into four categories: (1) Biogenic amines, (2) Bufadienolides, (3) alkaloids and steroids and (4) peptides and proteins (Clarke 1997). Chemically, other than biogenic amines and peptides, granular gland secretions of toads may contain nearly 86 types of Bufadienolides (Steyn and Heerden 1998) along with other components like Bufotoxin, Bufagin and Bufotenine (Morris 1992; Hoiberg et al 2002; Hirai et al 1992). Bufalin, Bufogenin, Bufotalin, Cinobufagin, Marinobufagin, Resibufagin are some of the most important bufadienolides. Major bufadienolides isolated from skin secretions of Bufo species are listed in Table 2. Biogenic amines present in toad-secretions include serotonin (5-hydroxytryplanime), histamine, bradykinin etc. (Basir et al 2000).

Figure 2

Table 2: Certain Bufadienolides isolated from skin secretions of species

Bufadienolide is regarded as derivative of steroids with two double-bonds present in bufanolide side-chain such that it's known as bufotoxin when in combined-state with nitrogenous compounds like arginine and bufogenin when in free-state (Hoiberg et al 2002). Bufadienolides occur both in nonconjugated as well as in conjugated form (Steyn and Heerden 1998). Conjugation may be present at carbon number 3 to give rise to sulfates, dicarboxylic esters and amino-acid dicarboxylic acid esters. Various substitutions in the side-chain or side groups of bufadienolides can give rise to many types of derivatives and such substitutions may vary from specie-to-specie. Bufadienolides are also responsible for imparting a milky appearance to the skin secretions of toad. Toad skin glands actually secrete a compound called bufonin, which is a weaker poison but as soon as it comes in contact with air it gets oxidized a to form a bufadienolide called Bufotalin [C₃₄H₄₆O₁₀] which imparts a milky appearance to the toad secretions (Noble 1931). Bufadienolides or compounds with similar structures are not only found in toads but also in many plant species, fireflies (Photinus sp.), Snakes (Rhabdophis sp.) and mammals (Steyn and Heerden 1998).

Apart from bufadienolides, other components of toad skin secretions like bufotenine and bufogen have also been well characterized. Bufotenine [C₁₂H₁₆N₂O] a crystalline solid, which is insoluble in water but soluble in alcohol (Morris 1992), while Bufogen [C₁₈H₂₄O₄] as well as bufotalin resemble the drug digitalis in their action mechanisms (Abel and Macht 1912).

Alkaloids, steroids and bufadienolides are major constituents of toad skin secretions followed by proteins. Toad secretions contain many proteins which exhibit antimicrobial properties, enzymatic properties and help in repair of bruises or abrasions. Proteins that exhibit antimicrobial properties have been discussed in details in later sections. Other than normal proteins that are found in skin secretions of all toads there exists a distinct family of proteins that are secreted only by the toad genus called Bombina. These proteins, which also possess germicidal activity, have been identified as Bombinans, and were first found in Oriental fire-bellied toad (Bombina orientalis). These proteins have about 28 amino acids and possess an amphipathic alpha-helical structure (Mor et al 1994). BLP-1, BLP-2 and BLP-3 are three bombinin-like peptides isolated from skin of B. orientalis (Gibson et al 1991). All three peptides shared considerable homology. BLP-1 is the most abundant member of the trio. All these BLPs have also been found to consist of high helical content (63-69% alpha-helix) in their chemical structure.

Toad-toxins: Physiological Effects

Toxins secreted by toad skins exhibit wide spectrum of effects and their composition as well as effects differ from specie-to-specie (Wright 1914; Pough et al 1999). These toxins are primarily meant to act like venoms for protecting toad from predators however they also assist in protection from microbe-laden hostile habitat (Zasloff 2002). These toxins defend naked skin against microorganisms as well as assist in wound repair (Simmaco et al 1998). Some amphibians warn about their poisonous nature by virtue of bright skin-colors but, such colors might not always give an idea regarding virulence of secretions in case of toads, e.g. Large Leptodactylus pentadactylus has bright thighs but lacks highly poisonous secretions of drab-colored Bufo marinus (Brazil and Vellard 1926).

Toad-toxins act by inducing various physiological effects on higher as well as lower vertebrates. Granular gland secretions when entered into stomachs of higher-vertebrates cause nausea, weakening of respiration and muscular paralysis, while in contact with eyes they produce serious inflammations (Abel and Macht 1912). Toad secretions may also boast of adrenalin, which is a result of chemical change within mature secretion (Shipley and Wislocki 1915). Clinical aspects of toad-toxins have been studied particularly in Bufo marinus. Secretions of B. marinus are cardioactive, due to activity of bufogen and bufotalin exhibiting clinical symptoms like dermatitis, hypotension and severe arrythmia (Radford and Gillies 1986). Toad-secretions have also been found to show second-degree Wenckebach atrioventricular block and T-wave change (Lin and Lin 1989). Other than heart associated activity, some toad secretions have also been found to exert effects on neurological activities. Skin extracts of Bufo melanostictus (common Indian toad) have been shown to contain sleep inducing factors (SIF) which induce sleep probably by alteration of brain biogenic amine levels, monoamine oxidase (MAO) and tryptophan hydroxylase (TH) activity (Dasa et al 2000).

Toad skin secretions show cardioactive effect since they interact with the enzyme Na ⁺ /K ⁺ ATPase. Normally in organisms like humans, inhibitors of Na ⁺ /K ⁺ ATPase are involved in water and electrolyte homeostasis as well as in the genesis of vasoconstriction in volume-dependent forms of hypertension (Alexei et al 1998). While such inhibitors are endogenous in other cases, amphibians especially those belonging to family Bufonidae have been shown to secrete compounds that inhibit Na ⁺ /K ⁺ ATPase activity as well as antagonize the binding of ouabain to the enzyme from their skin (Flier et al 1980). These compounds play important part in defense against predators. Bufalin, an active component of toad-secretions is a potent Na ⁺ /K ⁺ ATPase inhibitor, which binds to cell membrane with higher affinity then ouabain (Jing, Watabe et al. 1994). Bufotalin inhibits myocardial Na ⁺ /K ⁺ ATPase activity, thereby increasing myocardial contractile force without affecting the heart-rate (Hirai et al 1992). Marinobufagenin (3, 5-dihydroxy-14, 15-epoxy bufadienolide), marinoic acid and resibufogenin show Na ⁺ /K ⁺ ATPase inhibiting properties (Bagrov et al 1995; Matsukawa et al 1996; Pamnani et al 1994). While marinobufagenin is also a potent vasoconstrictor, resibufogenin (3-hydroxy-14, 15-epoxy-20, 22-dienolide) shows electrophysiological properties similar to acetylstrophanthidin (AS), which suggests similarity to family of digitalis-like drugs (Xie et al 1994).

Bufotenine on the other hand, has been found to be an Indole-hallucinogen, which can block action of serotonin (Hoiberg et al 2002). It can also constrict blood-vessels. Bufotenine-like compounds have also been isolated from Amantia muscaria (mushroom) and Piptadenia peregrina (plant). But, bufotenine isn't the most potent hallucinogen found in toad skin secretions; this title is possessed by 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT), an active component of skin secretions of Bufo alvarius (Weil and Davis 1994). While these skin secretions of B. alvarius are fatal when consumed orally yet they may be safely smoked to experience their psychoactive power. Due to this property ancient peoples of Mesoameria probably used these toads as ritual intoxicants.

Toad-toxins: Antimicrobial Effects

Vertebrates and other organisms boast of distinct groups of broad-spectrum antimicrobial substances in addition to immune-systematic responses and toads are no exception to this evolutionary creation (Boman 1995). The only difference though can be that, while other organisms and mammals like humans secrete such antimicrobial chemicals inside their body's coelomic-systems in form of ?-defensins or azurocidin (Nathan 1987), toads secrete them both inside their body as well as outside their body into the environment through skin-pores. Toad-toxins secreted from such skin pores assist toads to survive in habitats full of pathogenic microorganisms which may cause diseases like Red Leg Syndrome, Mycobacteriosis, Salmonellosis, Chromomycosis and Saprolegniasis (Fox et al 1984).

Research on antimicrobial effects of toad skin secretions started mainly in 1980s. Since then only certain bufadienolides and proteins have been found to show antimicrobial properties. Search for more such compounds still continues. Bufadienolides have only been recently found to exhibit antimicrobial properties. Bufadienolides like telocinobufagin and marinobufagin isolated from skin secretions of the Brazilian toad Bufo rubescens have been shown to exhibit anti-microbial activity. These compounds were shown to have inhibitory action over Staphylococcus aureus and Escherichia coli (Cunha-Filho et al 2005). As compared to bufadienolides, toad skin secretions seem to possess more diverse type of proteins, which act as antibiotics. Antimicrobial peptides released by skins of toads are capable of causing lysis of many pathogenic bacteria, viruses, gram-positive and gram-negative bacteria, protozoa, yeasts, and fungi. Such antimicrobial peptides have characteristic chemical properties such as relatively small size (20-46 amino acid residues), basic nature (lysine- or arginine-rich), and amphipathic properties (Nicolas and Mor 1995). Proteins called Bombinans and BLPs (bombinin-like proteins) were probably first antibiotic proteins to be isolated from toad skin secretions. They were isolated from skin of Bombina orientalis. Bombinans secreted by Bombina orientalis have significant antimicrobial and antifungal property, attributed to its amphipathic ?-helix (Mor et al 1994). An unnamed toxin of molecular weight 6,700 isolated from skin secretions of Bombina sp. has been shown to exert antibacterial effects against both gram-positive and gram-negative bacteria (Mastromei et al 1991). On the other hand, BLPs were found to be more potent then magainin 2 (antimicrobial peptide isolated from Xenopus laevis) in their effectiveness to kill bacterial cells (Gibson et al 1991). In members of genus Bufo, antimicrobial activity has been found to be mediated by lectin like proteins. A protein called β-galactoside binding lectin obtained from skin secretions of Bufo arenarum has been found to exhibit bacteriostatic activity (Riera et al 2003). This protein, which is also found in ovary, oocytes and embryos of B. arenarum (Elola et al 1998) has been shown to exhibit bacteriostatic activity against Escherichia coli K12 4100, wild strains of Escherichia coli, Proteus morganii, and Enterococcus faecalis.

While anti-microbial activity of toad secretions is evident from above discussion, these secretions have been recently found to contain anti-viral activity. A 63 kDa heme-binding protein called BAS-AH isolated from Bufo andrewsi has been shown to exhibit anti-HIV activity (Zhao et al 2005). At concentrations that have little effect on cell viability, it has been shown to inhibit recombinant HIV-1 reverse transcriptase activity. Research on anti-fungal and anti-parasitic effects of toad skin toxins is still under way.

Exhibition of microbicidal effect of the protein could be a result of its ability to form channels or pores within the microbial membrane causing permeation and lysis of the cell. While mechanisms by which bufadienolides exert their bactericidal effect are still debatable the effect of antimicrobial peptides present in these secretions seems to be understood. Since these peptides are generally cationic, they have been proposed to mostly act by disrupting and permeabilizing the target cell membrane (Shai 2002) which thereby prevents the target organism from developing resistance to this defense mechanism, implying a potential therapeutic use of these peptides (Jacob and Zasloff 1994). Gene families coding for such antimicrobial peptides show gene diversification resembling gene families coding for immunoglobulins (Hughes 1997) or venom-derived toxins (Ohno et al 1998). Antimicrobial peptides isolated from these organism's skins differ considerably in their sequences from one toad to another or even amongst amphibians (Simmaco et al 1998). Such divergence within species suggests that there may be existing about 100,000 different proteins secreted through dermal-glands of nearly 5000 different anurans (Duellman and Trueb 1994).

Toad-toxins: Pharmacological and Therapeutic Significance

Pharmacological potential of toad secretions was known to certain ancient medical-practitioners since some Chinese and Indian traditional medicinal procedures have been known to make use of Toad secretions. “Kyushin” is traditional Chinese medicine making use of toad-secretions and also exhibiting digoxin-like effects (Fushimi et al 1990). This cardiotonic effect is due to toad-toxin component “Chan'su”, a Chinese term for mixture of various toad-toxins specially, bufalin and cinobufagin. Problem with such medicines are that the quality and quantity of compounds shows great variability, which could be hazardous when in overdose (Hong et al 1992). Ayurveda, the traditional-medicinal practice of India has prescribed rubbing of live toad on swollen areas of animal (cattle) throat for healing and removal of throat obstruction arising due to swelling (Tiwari and Pande 2004). This is related to Agnikarma practice of Ayurveda. Toad skin secretions are source of diverse kind of pharmacologically and therapeutically significant compounds. These secretions contain components that can be used for production of painkillers, antimicrobial drugs, anti-viral drugs and even anti-cancerous drugs. These compounds could also prove to be useful in treatment of cardiovascular ailments and neurological problems.

Toad skin secretions have been shown to contain components, which exhibit analgesic and pain killing effects. A component of toad-toxin called bufalin, which is a Na ⁺ /K ⁺ ATPase inhibitor has in controlled studies of hepatic-cancerous pain, shown analgesic effects (Wang et al 1994). Experimental results on bufalin action have shown that, it increases hepatic-blood circulation, which decreases amount of stagnating blood thereby decreasing pain (Wang et al 1994). At present aspirin and morphine are drugs that are frequently used as analgesics. But aspirin has certain side effects, which include bleeding in stomach, Reye's disease, urticaria and asthmatic attacks (Brown 2001). Morphine also has many ill effects. In this scenario, bufalin can be of potential usage in pain-relief, anxiety diminishing and sleep induction. It may also substitute role of aspirin in treatment of rheumatic fever and arthritis. Other than bufalin, sleep-inducing factors (SIF) isolated from Bufo melanostictus can also be used for sleep induction (Dasa et al 2000).

As discussed in previous sections, certain components of toad skin secretions which resemble digitalin and digitoxin in their structure show effects on cardiovascular system as well. These components can be used in place of digitalis-like drugs to increase strength of heartbeat and are thus useful in cases where cardiac rhythm is abnormal especially in atrial-fibrillation (Brown 2001). Such compounds can also be used in treatment of hypertension and congestive heart failure (Oldfield 1995). The most interesting aspect in therapeutic usage of components present in toad skin secretions other than their antimicrobial effect is their anti-cancerous activity. While multi-drug resistant bacteria are potential candidates against which these toad toxins can be used, another big problem, which would probably be solved by these toxins, could be cancer.

A number of bufadienolides have been recently shown to exhibit anti-cancerous properties in vitro. Components like Bufalin have been shown to exert anti-cancerous properties on leukemia cells lines. Bufalin was found to induce apoptosis in human-leukemia cells by altering expression of apoptotic genes c-myc and bcl-2 (Jing, Ohizumi et al 1994; Masuda et al 1995; Watabe et al 1996). This effect of bufalin on cell cycle of leukemia cell resembles effect of topoisomerase inhibitors (Jing, Watabe et al 1994). The concentration at which bufalin induced apoptosis in HL-60 cells was 10 ^-8 M, which is comparable to drug camptothecin but lower than other anti-tumor drugs like cisplatin, VP16 and all-trans retinoic acid (Jing, Ohizumi et al 1994). Cinobufagin, another bufadienolide has been shown to work in treatment of infection and granulocytopenia during combined chemotherapy (Hirai et al 1992). It reduced the risk of infection and degree as well as duration of granulocytopenia associated with malignant blood diseases (Yue 1992). Other than bufalin and cinobufagin certain other toad skin-secretion components like 3-formyloxyresibufogenin, 19-oxobufalin, 19-oxodesacetylcinobufagin, 6-hydroxycinobufagin and 1-hydroxybufalin have been recently found to exert inhibitory effects on KB, HL-60 and MH-60 cancer cell lines (Nogawa et al 2001). Similarly, Bufogenin derivatives from skin secretions of Bufo bufo gargarizans as well as several semi synthetic derivatives of 20,21-epoxy-resibufogenin have been found to exhibit interleukin-6 (IL-6) antagonistic activity due to their growth-inhibitory activities on IL-6-dependent MH-60 cells (Enomoto et al 2004). However, anti-cancerous properties of toad skin-secretions can be debatable especially those on skin cancers because while they seem to inhibit cancer proliferation in vitro, toad themselves have been reported to be affected by skin cancers (Manskikh 2003). This raises doubts on anti-cancerous activity of these secretions in vivo.

Conclusion

Toad skin-secretions are potent source of drugs. It's probably the only such source in nature from where we can get nearly six types of drugs possessing analgesic, painkiller, antibiotic, anti-viral and anti-cancerous properties as well as possessing potential of treating cardiovascular diseases. We know that cancer, RNA virus causing pandemics/epidemics and multi-drug resistant bacteria are prime problems of 21 ^st century and toad secretions have been shown to inhibit growth of cancer, HIV as well as super-bugs like Staphylococcus aureus. Since potency of these toxins differs from specie to specie among Anurans, we may have about 1, 00,000 such compounds still waiting to be discovered. But, we may loose more than 50% of them in next 20 years itself, if we don't stop destroying the habitats of these Anurans. In recent decades, amphibian populations around the world have experienced dramatic declines, with some species showing high rates of deformities and others simply disappearing. In fact some scientists fear that forces that threaten amphibian population may be fueling an increase in prevalence of infectious diseases like SARS and Lyme disease (Kiesecker et al 2004). We must save fledging population of amphibians as well as other animals or else, we are not only loosing the integrity of our ecosystem but also potential sources of drugs. This is significant since many types of exotic viruses and super bugs are still unknown to humankind.