Skin Secretions Of Toads Research Articles

Identifying toxic effects and metabolic perturbations of Duttaphrynus melanostictus skin extracts in human erythrocytes.

Skin secretions of toads are widely used in medicine all over the world for their antiviral, anti-infective, and cardiotonic properties. Because these secretions are mostly employed to combat blood parasite infection, it is important to understand their potential toxic effects on human erythrocytes. Therefore, the objective of the current investigation was to elucidate the effects of Duttaphrynus melanostictus (Schneider) skin extracts on the physiology of human erythrocytes. Toads captured from their natural habitat were separated into three groups according to their body size. Hydroalcoholic extracts of toad skin were prepared by reflux heating. These extracts were then evaluated for their hemolytic and hemoglobin denaturation potential. The effects of the extracts on cytosolic and membrane-bound enzymes of human erythrocytes were assessed. The hemolysis and hemoglobin denaturation caused by these extracts correlated positively with the respective toad sizes. Extracts from medium and large toads led to increased osmotic fragility even at near iso-osmotic concentrations. Biochemical analysis of hemolysate showed that the treatment induced a shift of metabolic flux toward the glutathione pathway. Analysis of membrane-bound enzymes revealed a significant decrease in the activity of Na+/K+ ATPase and acetylcholinesterase. SDS-PAGE analysis of the erythrocyte membrane did not show the band of tropomodulin for the cells treated with 1000 𝜇g/ml extract from large toads. In conclusion, the present study demonstrates that the toxicity of toad skin secretions aggravates with the size of the animal and interferes with the physiology of human erythrocytes, leading to their membrane disruption and rapid lysis.

Hellebrigenin triggers death of promyelocytic leukemia cells by non-genotoxic ways

Bufadienolides are digitalis-like aglycones mainly found in skin secretions of toads. Among their biological properties, the mechanisms of antiproliferative action on tumor cells remain unclear for many compounds, including against leukemia cells. Herein, it was evaluated the mechanisms involved in the antiproliferative and genotoxic actions of hellebrigenin on tumor cell lines and in silico capacity to inhibit the human topoisomerase IIa enzyme. Firstly, its cytotoxic action was investigated by colorimetric assays in human tumor and peripheral blood mononuclear cells (PBMC). Next, biochemical and morphological studies were detailed by light microscopy (trypan blue dye exclusion), immunocytochemistry (BrdU uptake), flow cytometry and DNA/chromosomal damages (Cometa and aberrations). Finally, computational modelling was used to search for topoisomerase inhibition. Hellebrigenin reduced proliferation, BrdU incorporation, viability, and membrane integrity of HL-60 leukemia cells. Additionally, it increased G2/M arrest, internucleosomal DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization in a concentration-dependent manner. In contrast to doxorubicin, hellebrigenin did not cause DNA strand breaks in HL-60 cell line and lymphocytes, and it interacts with ATPase domain residues of human topoisomerase IIa, generating a complex of hydrophobic and van der Waals interactions and hydrogen bonds. So, hellebrigenin presented potent anti-leukemic activity at concentrations as low as 0.06 μM, a value comparable to the clinical anticancer agent doxorubicin, and caused biochemical changes suggestive of apoptosis without genotoxic/clastogenic-related action, but it probably triggers catalytic inhibition of topoisomerase II. These findings also emphasize toad steroid toxins as promising lead antineoplasic compounds with relatively low cytotoxic action on human normal cells.

In-silico analysis of bufadienolides as potential inhibitors of Trypanosoma nucleoside hydrolase

Trypanosomiasis is one of the neglected tropical diseases which curtails the productivity of the victim and causes mortality in extreme cases. Non-availability of vaccines, low therapeutic index of trypanocidal drugs and development of resistance lead to the need for research focused on developing alternative treatment options against this disease. The aim of the current work is to use computer-assisted drug discovery techniques to examine the anti-trypanosomal action of bufadienolides, which are commonly detected in the skin secretions of toads. A library of 16 compounds from the bufadienolides family was tested for toxicity and docked against the Trypanosoma brucei hydrolase. The oral bioavailability and good suitability of the compounds for transdermal delivery was also indicated by the ADMET study. The findings based on molecular docking and dynamics simulation along with toxicity assessment showed that three compounds, marinobufagin, resibufogenin and resibufaginol, have the lowest toxicity and maximum binding energy against Trypanosoma brucei hydrolase. According to the results obtained by molecular docking interactions between drugs and target proteins are observed and contain a number of hydrogen bonds near the residues in the active pocket of the enzyme. The results of this study contribute to the understanding of the SAR of bufadienolides and provide a foundation for further research and drug development efforts. The computational analysis, including the density functional theory study, the knowledge of inhibitory mechanisms and aids in the design and optimization of novel anti-trypanosomal drugs.

Antimicrobial Compounds from Skin Secretions of Species That Belong to the Bufonidae Family.

Skin secretions of toads are a complex mixture of molecules. The substances secreted comprise more than 80 different compounds that show diverse pharmacological activities. The compounds secreted through skin pores and parotid glands are of particular interest because they help toads to endure in habitats full of pathogenic microbes, i.e., bacteria, fungi, viruses, and protozoa, due to their content of components such as bufadienolides, alkaloids, and antimicrobial peptides. We carried out an extensive literature review of relevant articles published until November 2022 in ACS Publications, Google Scholar, PubMed, and ScienceDirect. It was centered on research addressing the biological characterization of the compounds identified in the species of genera Atelopus, Bufo, Duttaphrynus, Melanophryniscus, Peltopryne, Phrynoidis, Rhaebo, and Rhinella, with antibacterial, antifungal, antiviral, and antiparasitic activities; as well as studies performed with analogous compounds and skin secretions of toads that also showed these activities. This review shows that the compounds in the secretions of toads could be candidates for new drugs to treat infectious diseases or be used to develop new molecules with better properties from existing ones. Some compounds in this review showed activity against microorganisms of medical interest such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Coronavirus varieties, HIV, Trypanosoma cruzi, Leishmania chagasi, Plasmodium falciparum, and against different kinds of fungi that affect plants of economic interest.

A pore-forming protein drives macropinocytosis to facilitate toad water maintaining

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporin, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

Proteins from Rhinella jimi parotoid gland secretion: A comprehensive analytical approach

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L−1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g−1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Hellebrigenin anti-pancreatic cancer effects based on apoptosis and autophage.

Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Hellebores and Kalanchoe genera. It has been shown to be active against Leishmania chagasi promastigotes and Trypanosoma cruzi trypomastigotes and also reported to play an anti-tumor effect on several cancer cell lines in vitro, including pancreatic cancer. This study is aimed to investigate the effects of Hellebrigenin on pancreatic carcinoma cells, SW1990 and BxPC-3 in vitro and its molecular mechanism involved in antitumor activities. Our results showed that Hellebrigenin effectively inhibited the proliferation of SW1990 and BxPC-3 cells in dose- and time-dependent manner. Flow cytometry results showed that Hellebrigenin induced the G0/G1 arrest in both of SW1990 and BxPC-3 cells and promoted cell early apoptosis and autophagy according to morphological observation. Immunofluorescence staining results further confirmed that cell apoptosis and autophagy also increased upon the Hellebrigenin treatment. Moreover, higher dose of Hellebrigenin further increased the cell apoptosis rate while decrease the mitochondrial membrane potential 24 h after treatment. The autophagy rate increased 48 h after treatment with significant difference (P < 0.05). Western blot analysis showed that the expression of caspase 3, 7, cleaved caspase 7, Atg 12, LC3 proteins were increased in SW1990 cell after treatment with Hellebrigenin. In addition, increasing expression of caspase 3, 7, 9, PARP, cleaved caspase 3, 7, 9, PARP, the sub basic protein of the PI3K family, Beclin-1, LC 3, Atg 3, 5, 12, 16 L were also observed after BxPC-3 cells treated with Hellebrigenin. In summary, this study reported for the first time that Hellebrigenin effectively induced autophagy and apoptosis especially the early apoptosis in SW1990 and BxPC-3 cells.

Global Metabolomic and Lipidomic Analysis Reveal the Synergistic Effect of Bufalin in Combination with Cinobufagin against HepG2 Cells.

Chansu, which is prepared from the skin secretions of toad (Bufo bufo gargarizans Cantor), is widely used in traditional Chinese medicine (TCM). Being the principal bioactive constituents of Chansu, bufalin (BFL) and cinobufagin (CBF) have been shown to possess anticancer properties. TCM confers bioactivities through the synergistic effect between potential active ingredients, so as to interfere with the development of the disease, and ultimately achieve the therapeutic effect. We found that the anticancer effect was significantly potentiated by cotreatment with BFL and CBF compared to monotreatment, suggesting their synergistic interaction. To reveal their synergistic mechanisms, metabolomic and lipidomic profiling based on liquid chromatography-mass spectrometry (LC-MS) was utilized to delineate the responses in HepG2 cells after treatment with BFL and CBF individually or in combination. Metabolic pathways, including methionine metabolism, energy metabolism, lipid metabolism, and amino acid metabolism, were modulated and subsequently led to apoptosis and cell cycle arrest of HepG2 cells. In particular, the discrepant regulation of methionine metabolism between the monotreatment and cotreatment with BFL and CBF may account for their synergistic effect. Our study provided novel insights into the mechanistic links between cellular metabolism and the synergistic effect, which may ultimately lead to better treatments for hepatoma.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin.

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

Analysis and Prediction of some Histone-derived Antimicrobial Peptides from Toads Duttaphrynus melanostictus and Phyrinoidis asper

Antimicrobial peptides in skin secretions of toads is a promising methods to combat a wide spectrum of bacteria. Histone H2A is a type of DNA-binding protein that acts as a precursor for several antimicrobial peptides. In toads (family Bufonidae) buforin I and buforin II are examples of antimicrobial peptides that derived from histone H2A. This study investigated the genetic diversity and phylogenetic analysis and in silico prediction of antimicrobial peptides derived from histone H2A of Duttaphrynus melanostictus and Phyrinoidis asper , which were collected from Bogor Agricultural University’s campus area. A new set of primers (Buf_fwd and Buf_rev) were designed by using PrimerBLAST, to amplify 393 nucleotides of the histone H2A gene that codes 131 amino acids. Haplotype diversity of both species are very low. Phylogenetic analysis shows the sample D. melanostictus and P. asper are separated to each other in two different clades. Several short predicted peptides from histone H2A show a potential as an antimicrobial peptides based on in silico prediction. Psychochemical characteristics and 3D structure of potent antimicrobial peptides are described.

Biological characterization of compounds from Rhinella schneideri poison that act on the complement system.

BackgroundThe skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity of Rhinella schneideri poison (RsP) components on the complement system.MethodsThe components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.ResultsThe fractionation protocol was able to isolate the component S5 from the RsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b-9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.ConclusionsThis is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.

Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt

Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser1981), p-Chk2 (Tyr68), p-CDK1 (Tyr15) and Cyclin B1, and down-regulation of p-CDC25C (Ser216). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer.

Toad skin-secretions: Potent source of pharmacologically and therapeutically significant compounds

Toad skin-secretions: Potent source of pharmacologically and therapeutically significant compounds

Antimicrobial activity of skin secretions isolated from Indian toad, Bufo melanostictus Schneider 1799

AbstractAmphibians like toads have been known to secrete antimicrobial secretions outside their body into their environments, through skin pores and parotid glands. Toad skin-secretions contain four types of compounds namely, biogenic amines, bufadienolides, alkaloids &amp; steroids and peptides &amp; proteins. Bulk of research relating to amphibian antimicrobial secretions has been done on frogs. In toads, such research has only been done in South America, Europe and China. Antimicrobial secretions vary considerably from specie-to-specie and drastically across various biomes. This prompted us to examine and confirm presence of antimicrobial activity (if any) in Indian Common Toad (Bufo melanostictus Schneider 1799) skin secretions since; no such analysis had been previously done on this toad which is found all across the South-east Asia. The antibacterial potency of toad skin secretions was tested against the bacteria, Escherichia coli. After running preliminary antibacterial analysis assays, we found that these cutaneous secretions retrieved from Indian Common Toad possessed potential bactericidal activity. The results that we got confirmed that some unexplored bactericidal components were present in skin secretions of these toads. These conclusions call for further research into biochemistry and molecular characterization of these components.

Antimicrobial activity of skin secretions isolated from Indian toad, Bufo melanostictus Schneider 1799

Amphibians like toads have been known to secrete antimicrobial secretions outside their body into their environments, through skin pores and parotid glands. Toad skin-secretions contain four types of compounds namely, biogenic amines, bufadienolides, alkaloids & steroids and peptides & proteins. Bulk of research relating to amphibian antimicrobial secretions has been done on frogs. In toads, such research has only been done in South America, Europe and China. Antimicrobial secretions vary considerably from specie-to-specie and drastically across various biomes. This prompted us to examine and confirm presence of antimicrobial activity (if any) in Indian Common Toad (Bufo melanostictus Schneider 1799) skin secretions since; no such analysis had been previously done on this toad which is found all across the South-east Asia. The antibacterial potency of toad skin secretions was tested against the bacteria, Escherichia coli. After running preliminary antibacterial analysis assays, we found that these cutaneous secretions retrieved from Indian Common Toad possessed potential bactericidal activity. The results that we got confirmed that some unexplored bactericidal components were present in skin secretions of these toads. These conclusions call for further research into biochemistry and molecular characterization of these components.