Cytotoxicity potential of chemical constituents isolated and derivatised from Rhinella marina venom.

Chemical compounds from skin secretions from toads of Bufonidae family have been long-studied. In the search for new molecules with pharmacological action, the 3ß-OH groups of bufadienolides are commonly derivatised using acetyl groups. This work described the isolation and/or structural elucidation of isolated and derivatised compounds from the venom of the Brazilian anuran Rhinella marina, and their evaluation in in vitro assays. In the methanolic extract of the R. marina venom, compound cholesterol (1) was isolated from the CRV-52 fraction by classic column chromatography, dehydrobufotenine (2) by Sephadex LH-20 from the CRV-28 fraction, and a mix of suberoyl arginine (3) and compound 2 was obtained from the CRV-6-33 fraction. The compounds marinobufagin (4), telocionbufagin (5) and bufalin (6) were isolated by classic column chromatography, followed by separation via HPLC in the CRV-70 fraction, and the compound marinobufotoxin (9) was isolated by classic column chromatography in the CRV-6 fraction, here being isolated for the first time in R. marina specimens. Compounds 4 and 5 were submitted for acetylation with acetic anhydride, in the presence of pyridine and 4-dimethyilaminopiridine (DMAP), in order to obtain the compounds 3-acetyl-marinobufagin (7) and 3-acetyl-telocinobufogin (8). The isolated and derivatised compounds were identified by 1H and 13C NMR, and their molecular mass confirmed by mass spectrometry. All compounds (except 1 and 3) were tested in cytotoxic assays by the MTT method and presented cytotoxic potential against human cancer cell lines, as well as against non-tumoral human embryonic kidney HEK-293 cells. With the exception of compound 2, all molecules presented IC50 values < 4 µM, and none caused hemolysis of human erythrocytes, demonstrating a promising cytotoxic potential of natural and chemically-modified bufadienolides. This study presents a detailed contribution of bioactive chemicals from Brazilian Amazon Rhinella species, and indicates promising areas for further studies and pharmaceutical investments.

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. MATERIAL AND METHOD: Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. RESULTS: Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ±â€¯0.005 µg/ml and 0.18 ±â€¯0.007 µg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ±â€¯0.084 µg/ml and 0.32 ±â€¯0.077 µg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. CONCLUSION: Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

Antiproliferative activity and chemical composition of the venom from the Amazonian toad Rhinella marina (Anura: Bufonidae).

Little is known on the composition of Peruvian Amazon toad venoms. The large toad Rhinella marina is common in the cleared tropical forests of the Iquitos region and is regarded as poisonous. The venom from two different populations of R. marina was collected in the Departamento de Loreto, Perú. The samples were assessed for antiproliferative effect and composition. Some 29 compounds were identified or tentatively identified from the venom by spectroscopic and spectrometric means. The main free bufadienolide was marinobufagin 7 while marinobufotoxin 15 and bufalitoxin 9 were the main bufadienolide argininyl diacid derivatives. The alkaloids dehydrobufotenin 28 and bufotenidin 29 were present in both venoms. The main difference in the venoms was the relative ratio of argininyl diacids from bufadienolides to free bufadienolides. The argininyl diacids included derivatives from bufalin, marinobufagin, telocinobufagin, hellebrigenin, resibufogenin and bufotalinin. Four compounds, including undecadienoyl aginine 6 and three argininyl diacids from bufadienolides were tentatively identified for the first time in the samples. The venom showed a strong antiproliferative effect towards MRC-5 normal human lung fibroblasts (0.063-0.247 µg/mL), AGS human gastric adenocarcinoma cells (0.076-0.272 µg/mL), SK-MES-1 human lung cancer cells (0.154-0.296 µg/mL), J82 human bladder carcinoma cells (0.169-0.212 µg/mL), and HL-60 human promyelocytic leukemia (0.071-0.283 µg/mL). The antiproliferative effect is mediated by ROS production and cell cycle arrest in human breast cancer cells (MCF7 and MDA-MB-231). This is the first report on the composition of R. marina venom from the Peruvian Amazon pointing out the need to include different venom samples to get a better picture from the activity and composition of South American toad defense substances.

A New Indole Alkaloid from the Toad Venom of Bufo bufo gargarizans.

A new indole alkaloid named bufobutarginine (1), along with three known bufotenines, namely, serotonin (2), bufotenidine (3), and bufotenine (4), were isolated from the water extract of toad venom. Their structures were elucidated by spectral methods. This is the first time that arginine has been found to be involved in the biosynthesis of bufotenines in parotid of toad. The cytotoxic activities of these compounds have been assayed against A375 and A549 cell lines by the MTT method; however, they showed no cytotoxic activities.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

On frogs, toxins and true friendship: an atypical case report

Abstract The authors report a series of events including the scientific interest for poisonous dendrobates of French Guiana, the human confrontation with the immensity of the evergreen rainforest, the fragility of the best-prepared individuals to a rough life, and the unique and very special manifestation of a solid friendship between two experts and enthusiasts of outdoor life. In the evergreen forest of South America, as in many other scientific field expeditions, everything may suddenly go wrong, and nothing can prepare researchers to accidents that may occur in a succession of uncontrollable errors once the first mistake is done. This is what happened during an expedition in search for dendrobates by an experienced forest guide and naturalist. The authors decided to report the story, considering that it deserved to be brought to the attention of those interested in venomous animals and toxins, in order to illustrate the potential danger of dealing with these organisms.

Bilirubin attenuates bufadienolide-induced ventricular arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated intracellular Na(+) levels.

Bufadienolides, known ligands of the sodium pump, have been shown to inhibit the proliferation of several cancer cell types. However, their development to date as anticancer agents has been impaired by a narrow therapeutic margin resulting from their potential to induce cardiotoxicity. In the present study, we examined the effects of bilirubin, an endogenous antioxidant, on the cardiotoxicity of bufadienolides (derived from toad venom) in guinea-pigs. The results showed that bufadienolides (8 mg/kg) caused ventricular arrhythmias, conduction block, cardiac dysfunction and death in guinea-pigs. Pretreatment with bilirubin (75 and 150 mg/kg) significantly prevented bufadienolide-induced premature ventricular complexes, ventricular tachycardia, ventricular fibrillation and death. Bilirubin also markedly improved the inhibition of cardiac contraction in bufadienolide-treated guinea-pigs as evidenced by increases in left ventricular systolic pressure and decreases in left ventricular diastolic pressure in vivo. Furthermore, bilirubin significantly reduced the intracellular sodium content ([Na(+)]( i )) in ex vivo bufadienolide-stimulated guinea-pig ventricular myocytes loaded with the sodium indicator Sodium Green. An antitumor study showed that bilirubin did not compromise the ability of bufadienolides to inhibit gastric cancer cell MGC-803 proliferation. These results suggested that bilirubin can attenuate bufadienolide-induced arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated [Na(+)]( i ) and may improve bufadienolide therapeutic index in cancer treatment.

Toad poisoning in three dogs: case reports

Toad poisoning is frequent in dogs, but has been infrequently addressed in published case reports and review articles. Dogs can be poisoned when they bite a toad or otherwise ingest the venom. The venom effects manifest soon after the accident, since the toxin is rapidly absorbed by the mucous membrane of the digestive system. Hospital records of three dogs, diagnosed with toad poisoning, were retrospectively reviewed from January 2005 to July 2007. Poisoned dogs may present only local irritation or systemic signs in the gastrointestinal, cardiac and neurological systems. All three cases presented herein had clinical signs of gastrointestinal alterations including vomiting, sialorrhea and diarrhea. Two dogs developed abnormal cardiac rhythm and two exhibited neurological signs. A poisoned animal requires emergency care and symptomatic therapy with intense monitoring of its clinical parameters. Although there have been reports on the low mortality of dogs poisoned by toads, one animal died even after appropriate therapy. The severity of clinical signs and the risk of death must be considered by the veterinarian.

Intoxicação por veneno de sapo em um canino / Toad venom intoxication in a dog

O sapo do gênero Bufo possui nas suas glândulas paratóides uma secreção mucóide contendo toxinas como bufaginas e Bufotoxinas, que são esteróides cardiogênicos. Os cães podem atacar os sapos, entrando em contato com o veneno por meio das mucosas. Um canino, da raça Bulldog Francês, foi encaminhado ao Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (UFRGS) para a necropsia com histórico de provável intoxicação por veneno de sapo. Na necropsia o canino apresentava pulmões aumentados de volume, avermelhados e com edema, e rins de coloração vermelho-escura. As alterações microscópicas indicaram congestão, hemorragia e edema pulmonar. Nos rins, no baço e nos linfonodos foi observada congestão. As análises toxicológicas para os venenos de rotina foram negativas. Porém, a investigação do veneno de sapo a partir de cromatografia por camada delgada e gasosa demonstrou resultado positivo, revelando ser esta a causa da morte do canino.

The toads of the genus Bufo produce, in their parotoid glands, a mucoid secretion containing toxins such as bufagins and Bufotoxins, which are cardiogenic steroids. The mucous membranes of dogs can absorb this venom when they attack the toads. A French bulldog with a history of probable toad venom intoxication was referred to Veterinary Pathology Section of Federal University of Rio Grande do Sul (UFRGS) for necropsy. The necropsy revealed enlarged, reddish, edematous lungs, and kidneys displaying a dark red color. The microscopic alterations indicated the presence of congestion, hemorrhage, and pulmonary edema. Congestion was observed in the kidneys, spleen and lymph nodes. The routine toxicological analyses for venom detection were negative. Nevertheless, the toad venom test result was positive as assessed by thin layer and gas chromatography, indicating that toad venom intoxication was the cause of death.

Ranas venenosas de Colombia / Poisonous frogs of Colombia

Colombia es uno de los países del mundo más rico en biodiversidad. Ocupa el primer lugar en Amphibia, tanto en número de especies como en toxicidad. La familia Dendrobatidae está representada por los géneros Phyllobates, Dendrobates, Epipedobates y Minyobates, todas ellas venenosas. La mayoría de las especies están distribuidas en el occidente colombiano y a lo largo del Pacífico.Palabras claves: Dendrobatidae, batrachotoxinas, histrionicotoxina, pumiliotoxina, epibatidina, lehmizidina.

Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxicity due to more than Na, K-ATPase blocking.

Extracts of Chan Su, a traditional Chinese medication used as a topical anesthetic and cardiac medication, were incubated with cardiomyocytes that had been loaded with a calcium specific fluorescent probe. Calcium transients were measured by real-time fluorescence spectrophotometry following treatment. The transients were rapidly abolished following addition of a moderate concentration of the extract (400 ng/ml), resulting in high levels of intracellular calcium, while the lower amount (40 ng/ml) blocked the sodium-potassium adenosine triphosphatase. Treatments with ouabain and nifedipine were also made, either prior to, or after the addition of the Chan Su, in an attempt to better delineate the site(s) of action. The moderate concentration of Chan Su (400 ng/ml) extract caused the myocytes to cease beating within seconds of addition, even in experiments when saturating concentrations of nifedipine or ouabain had been previously added to the cells. As expected bufalin, the active component of Chan Su has similar effects. Our findings indicate that this compound is extremely cardiotoxic, even in small dose and acts rapidly to alter intracellular calcium stores in cardiomyocytes and possibly acts at sites other than the Na(+)+K(+) ATPase, either directly, or indirectly via changes in calcium concentrations.

Neuropeptide Y release by pumiliotoxin-B in the electrically-stimulated mouse vas deferens: an immunohistochemical study.

Morphologic and immunohistochemical studies were conducted to ascertain whether pumiliotoxin-B (PTX-B), an indolizine alkaloid from the skin of the Neotropical dendrobatid frog, Dendrobates pumilio, affects the anatomic and immunohistochemical features of the electrically stimulated mouse vas deferens preparations. PTX-B, at a concentration of 1 microM, consistently decreased the density pattern of neuropeptide Y (NPY)-immunoreactive nerve fibers contained within the circular muscular layer. The alkaloid also induced striking morphologic changes. It enlarged the lumen of the vasa and relaxed the muscular wall. Pretreatment with prazosin or haloperidol affected neither the release of NPY nor the morphologic changes; pretreatment with tetrodotoxin and guanethidine abolished NPY release and prevented the PTX-B-induced morphologic changes. PTX-B had no appreciable effect on the density and distribution pattern of nerve fibers immunostained for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, enkephalin, pancreatic polypeptide, 5-hydroxy-tryptamine and tyrosine hydroxylase.

The venomous "Sapito minero" (dendrobates leucomelas steindachner 1864) (Dendrobatidae): its medical importance and the phenotypic variations in specimens from two regions of The Amazonas State, Venezuela

El sapito minero (Dendrobates leucomelas) ha sido descrito en el Amazonas venezolano. Una variación fenotípica, la cual ilustramos en color, fue encontrada en la serranía del Cuao, municipalidad de Atures del estado Amazonas y presenta marcadas diferencias en coloración con los especímenes del norte del mismo estado. En estudios muy preliminares de la toxina de estos animales venezolanos hemos empezado a demostrar sus actividades tóxicas sobre ratones inoculados, caracterizados por dificultades locomotoras, parálisis parcial del tren posterior, salivación, convulsiones y muerte en menos de 20 min.

Voltage-dependent block by histrionicotoxin of the acetylcholine-induced current in an insect motoneurone cell body.

Acetylcholine (ACh)-induced currents were recorded from the voltage-clamped cell body of the fast coxal depressor motoneurone of the cockroach Periplaneta americana, at membrane potentials in the range -120 mV to -60 mV. In the presence of histrionicotoxin (HTX) (1.0 X 10(-6) M), ACh currents were blocked. The blocking action of HTX was voltage-dependent, being more effective at more negative membrane potentials.