Toxic profile of marinobufagin from poisonous Amazon toads and antitumoral effects on human colorectal carcinomas.

ETHNOPHARMACOLOGICAL RELEVANCE: South Americans natives have extensively used the toad "kururu" to reduce/treat skin infections, cutaneous lesions and sores. They release secretions rich in bufadienolides, polyhydroxy steroids with well-documented cardiotonic and antiproliferative actions, but in vivo antitumoral evaluations in mammals are rare, and toxicological safety has been left in second place. AIMS OF THE STUDY: This investigation used in silico, in vitro and in vivo tools to evaluate acute and subacute toxic effects of marinobufagin and the anticancer action in tumor-bearing mice models. MATERIALS AND METHODS: Initially, in silico toxic predictions were performed, followed by in vitro assays using human and murine normal and tumor lines. Next, acute and subacute studies on mice investigated the behavior, hematological and intestinal transit profile and antitumoral activity of marinobufagin in sarcoma 180- and HCT-116 colorectal carcinoma-transplanted mice for 7 and 15 days, respectively. Ex vivo and in vivo cytogenetic assays in Sarcoma 180 and bone marrow cells and histopathological examinations were also executed. RESULTS: In silico studies revealed ecotoxicological effects on crustaceans (Daphnia sp.), fishes (Pimephales promelas and Oryzias latipes), and algae. A 24-h marinobufagin-induced acute toxicity included signals of central activity, mainly (vocal frenzy, absence of body tonus, increased ventilation, ataxia, and equilibrium loss), and convulsions and death at 10 mg/kg. The bufadienolide presented effective in vitro cytotoxic action on human lines of colorectal carcinomas in a similar way to ouabain and tumor reduction in marinobufagin-treated SCID-bearing HCT-116 heterotopic xenografts. Animals under subacute nonlethal doses exhibited a decrease in creatinine clearance with normal levels of blood urea, probably as a result of a marinobufagin-induced renal perfusion fall. Nevertheless, only minor morphological side effects were identified in kidneys, livers, hearts and lungs. CONCLUSIONS: Marinobufagin has in vitro and in vivo anticancer action on colorectal carcinoma and mild and reversible alterations in key metabolic organs without direct chemotherapy-induced gastrointestinal effects at subacute exposure, but it causes acute ataxia, equilibrium loss, convulsions and death at higher acute exposure.

Dehydrobufotenin extracted from the Amazonian toad Rhinella marina (Anura: Bufonidae) as a prototype molecule for the development of antiplasmodial drugs

he resistance against antimalarial drugs represents a global challenge in the fight and control of malaria. The Brazilian biodiversity can be an important tool for research and development of new medicinal products. In this context, toxinology is a multidisciplinary approach on the development of new drugs, including the isolation, purification, and evaluation of the pharmacological activities of natural toxins. The present study aimed to evaluate the cytotoxicity, as well as the antimalarial activity in silico and in vitro of four compounds isolated from Rhinella marina venom as potential oral drug prototypes. Methods: Four compounds were challenged against 35 target proteins from P. falciparum and screened to evaluate their physicochemical properties using docking assay in Brazilian Malaria Molecular Targets (BraMMT) software and in silico assay in OCTOPUS® software. The in vitro antimalarial activity of the compounds against the 3D7 Plasmodium falciparum clones were assessed using the SYBR Green I based assay (IC50). For the cytotoxic tests, the LD50 was determined in human pulmonary fibroblast cell line using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Results: All compounds presented a ligand-receptor interaction with ten Plasmodium falciparum-related protein targets, as well as antimalarial activity against chloroquine resistant strain (IC50 = 3.44 µM to 19.11 µM). Three of them (dehydrobufotenine, marinobufagin, and bufalin) showed adequate conditions for oral drug prototypes, with satisfactory prediction of absorption, permeability, and absence of toxicity. In the cell viability assay, only dehydrobufotenin was selective for the parasite. Conclusions: Dehydrobufotenin revealed to be a potential oral drug prototype presenting adequate antimalarial activity and absence of cytotoxicity, therefore should be subjected to further studies.(AU)

Bufadienolides from amphibians: A promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+/K+-ATPase inhibition.

Amphibians present pharmacologically active aliphatic, aromatic and heterocyclic molecules in their skin as defense against microorganisms, predators and infections, such as steroids, alkaloids, biogenic amines, guanidine derivatives, proteins and peptides. Based on the discovered bioactive potential of bufadienolides, this work reviewed the contribution of amphibians, especially from members of Bufonidae family, as source of new cytotoxic and antitumor molecules, highlighting the mechanisms responsible for such amazing biological potentialities. Bufonidae species produce bufadienolides related to cholesterol through the mevalonate-independent and acidic bile acid pathways as polyhydroxy steroids with 24 carbons. In vitro antitumor studies performed with skin secretions and its isolated components (specially marinobufagin, telocinobufagin, bufalin and cinobufagin) from Rhinella, Bufo and Rhaebo species have shown remarkable biological action on hematological, solid, sensitive and/or resistant human tumor cell lines. Some compounds revealed higher selectivity against neoplastic lines when compared to dividing normal cells and some molecules may biochemically associate with Na+/K+-ATPase and there is structural similarity to the digoxin- and ouabain-Na+/K+-ATPase complexs, implying a similar mechanism of the Na+/K+-ATPase inhibition by cardenolides and bufadienolides. Some bufadienolides also reduce levels of antiapoptotic proteins and DNA synthesis, cause morphological changes (chromatin condensation, nuclear fragmentation, cytoplasm shrinkage, cytoplasmic vacuoles, stickiness reduction and apoptotic bodies), cell cycle arrest in G2/M or S phases, mitochondrial depolarization, PARP [poly (ADPribose) polymerase] and Bid cleavages, cytochrome c release, activation of Bax and caspases (-3, -9, -8 and -10), increased expression of the Fas-Associated protein with Death Domain (FADD), induce topoisomerase II inhibition, DNA fragmentation, cell differentiation, angiogenesis inhibition, multidrug resistance reversion, and also regulate immune responses. Then, bufadienolides isolated from amphibians, some of them at risk of extinction, emerge as a natural class of incredible chemical biodiversity, has moderate selectivity against human tumor cells and weak activity on murine cells, probably due to structural differences between subunits of human and mice Na+/K+-ATPases.