Cloning, characterization and specificity of a new aromatic-L-amino-acid decarboxylases from Bufo bufo gargarizans.

5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans (BbgAADC) was cloned from the parotoid gland of B. bufo gargarizans. The recombinant BbgAADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold-BbgAADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins. The enzymological properties of BbgAADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5'-phosphate (PLP) to BbgAADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as BbgAADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan, respectively. The Km of the recombinant protein BbgAADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 µM·min-1 when 5-HTP was used as substrate. The Kcat was 0.0545 min-1, and Kcat/Km was 0.1868 mM-1·min-1. To elucidate the mechanism of BbgAADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for BbgAADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of BbgAADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of BbgAADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.

Mining and Characterization of Indolethylamine N-Methyltransferases in Amphibian Toad Bufo gargarizans.

Strong, psychedelic indolethylamines (IAAs) are typically present in trace amounts in the majority of species, but they build up significantly in the skin of amphibian toads, especially N-methylated 5-hydroxytryptamine (5-HT) analogues. However, there is no pertinent research on the investigation of indoleamine N-methyltransferase (INMT) in amphibians, nor is there any adequate information on the key amino acids that influence the activity of known INMTs from other species. Herein, we focused on Bufo toad INMT (BINMT) for the first time and preliminarily identified BINMT 1 from the transcriptomes of Bufo gargarizans active on tryptamine, 5-HT, and N-methyl-5-HT. We established the enzyme kinetic characteristics of BINMT 1 and identified the essential amino acids influencing its activity via molecular docking and site-directed mutagenesis. Subsequently, we carried out sequence alignment and phylogenetic tree analysis on 43 homologous proteins found in the genome of B. gargarizans with BINMT 1 as the probe and selected seven of them for protein expression and activity assays. It was found that only three proteins possessing the highest similarity to BINMT 1 had INMT activity. Our research unveils the binding residues of BINMT for 5-HT analogues for the first time and initiates the study of INMTs in amphibian toads, serving as a tentative reference for further study of BINMT and providing insight into the comprehension of BINMT's catalytic mechanism and its role in the biosynthesis of 5-HT analogues in Bufo toads. It also contributes to the expansion of the INMT library to help explore and explain interspecies evolution in the future.

Metabolic Adaption of Flexor Carpi Radialis to Amplexus Behavior in Asiatic Toads (Bufo gargarizans).

Amplexus is a type of mating behavior among toads that is essential for successful external fertilization. Most studies have primarily focused on the behavioral diversity of amplexus, and less is known regarding the metabolic changes occurring in amplectant males. The aim of this study was to compare the metabolic profiles of amplectant Asiatic toad (Bufo gargarizans) males in the breeding period (BP group) and the resting males in the non-breeding period (NP group). A metabolomic analysis was conducted on the flexor carpi radialis (FCR), an essential forelimb muscle responsible for clasping during courtship. A total of 66 differential metabolites were identified between the BP and NP groups, including 18 amino acids, 12 carbohydrates, and 8 lipids, and they were classified into 9 categories. Among these differential metabolites, 13 amino acids, 11 carbohydrates, and 7 lipids were significantly upregulated in the BP group compared to the NP group. In addition, a KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis identified 17 significant metabolic pathways, including ABC transporters, aminoacyl-tRNA biosynthesis, arginine biosynthesis, pantothenate and CoA biosynthesis, and fructose and mannose metabolism. These results suggest that amplectant male toads are metabolically more active than those during the non-breeding period, and this metabolic adaptation increases the likelihood of reproductive success.

Size matters either way: Differently-sized microplastics affect amphibian host and symbiotic microbiota discriminately.

Concerns about the implications of microplastics (MPs) on aqueous animals have gained widespread attention. It has been postulated that the magnitude of MPs can influence its toxicity. However, little is known about how MPs toxicity changes with particle size. Amphibians are reliable bioindicators of ecosystem health due to their complex life cycles. In this study, we compared the influences of two sizes nonfunctionalized polystyrene microspheres (1 and 10 µm) on the metamorphosis of Asiatic toad (Bufo gargarizans). Acute exposure to MPs at high concentrations led to bioaccumulation in the digestive track and internal organs (i.e., liver and heart) of tadpoles. Long-term exposure to either size, at environmentally-related concentrations (1 and 4550 p/mL), led to growth and development delay in pro-metamorphic tadpoles. Remarkably, developmental plasticity mitigated these deleterious effects prior to the onset of metamorphic climax without compromising survival rate in later stages. MPs with a diameter of 10 µm dramatically altered the gut microbiota (e.g., abundance of Catabacter and Desulfovibrio) of pro-metamorphic tadpoles, whereas MPs with a diameter of 1 µm induced much more intensive transcriptional responses in the host tissues (e.g., upregulation of protein synthesis and mitochondrial energy metabolism, and downregulation of neural functions and cellular responses). Given that the two MPs sizes induced similar toxic outcomes, this suggests that their principal toxicity mechanisms are distinct. Small-sized MPs can travel easily across the intestinal mucosa and cause direct toxicity, while large-sized MPs accumulate in gut and affect the host by changing the homeostasis of digestive track. In conclusion, our findings indicate that MPs can affect the growth and development of amphibian larvae, but their developmental plasticity determines the ultimate detrimental effects. Multiple pathways of toxicity may contribute to the size-dependent toxicity of MPs. We anticipate that these findings will increase our understanding of the ecological effects of MPs.

Preliminary ionome of the parotoid gland secretion from Rhinella jimi toad.

The cururu toad (Rhinella jimi) is an anuran belonging to the fauna of the Brazilian northeast region, which releases a secretion with toxins from your parotoid glands. Although it has some information about secondary metabolites and proteins, the elemental composition of the released secretion is unknown. Therefore, this is the first report on the ionome of the secretion of the parotoid glands from R. jimi, investigating the influences of abiotic factors such as biome, seasonality, and gender. ICP-MS was used for measurements combined with principal component analysis (PCA). A screening of the secretion sample detected 68 elements which the total concentration of 18 elements was determined. PCA revealed that biome and seasonality factors have a greater influence on the ionomic profile of parotoid secretion. The presence of toxic metals in the secretion samples indicates that the R. jimi toad can be considered a potential bioindicator. These findings may contribute to understanding the metabolism, lifestyle, and interaction of the R. jimi toad with environmental factors as well as open new perspectives to investigate the relationships of the ionome with other biomolecules, for example, metalloproteins and their physiological functions.

Neglected skin-associated microbial communities: a unique immune defense strategy of Bufo raddei under environmental heavy metal pollution.

Amphibians defend against pathogens using skin microbial communities, in addition to innate and adaptive immunity. Despite skin microbial communities play a key role in the immune function of amphibians, few studies have focused on the changes in its composition and function. In the present study, we identified the variation in adaptive immunity, as well as the corresponding changes in skin microbiome of Bufo raddei living in a heavy metal polluted area. The adaptive immunity of B. raddei in heavy metal polluted area was significantly lower than that in relatively unpolluted area. Further, different skin bacterial communities were found in the two areas. In the heavy metal polluted area, Actinobacteria and Microbacterium were the dominant bacteria in the skin microbiome of B. raddei, which showed broad-spectrum antibacterial activity. Besides, the antibiotic synthesis was also increased in metabolic pathways. The present study suggested that the adaptive immunity of B. raddei was weakened under long-term heavy metal stress. However, the toads increased the abundance of bacteriostatic bacteria by regulating the composition of skin microbiome, which released a large number of bacteriostatic metabolites and enhanced the host resistance to external pathogens in turn.

Nitrate and sodium nitroprusside alter the development of Asian black-spined toads' embryos by inducing nitric oxide production.

Nitrate is the most stable and abundant form of inorganic nitrogen in water. However, owing to human activities, the nitrate concentration in aquatic ecosystems has notably increased worldwide. One of the mechanisms underlying nitrate toxicity in vertebrates includes the functional inhibition of the sodium iodide symporter, resulting in thyroid dysfunction. In this study, we aimed to determine the alternative mechanisms underlying the toxicological effects of nitrates on the Asian black-spined toad (Duttaphrynus melanostictus). Embryos of D. melanostictus were exposed to sodium nitroprusside (SNP, positive control) or 100 mg/L nitrate-nitrogen (NO3-N) for 184 h. We observed that both SNP and NO3-N significantly decreased body mass and length and delayed developmental processes. Teratogenic symptoms, including tumors, hyperplasia, and abdominal edema, were also observed in embryos exposed to SNP and NO3-N. Furthermore, SNP and NO3-N significantly increased nitric oxide levels in the embryos, altering the thyroid hormone, nitrogen, cytochrome P450-mediated drug, and xenobiotic metabolism signaling pathways, as well as the pathway involved in chemical carcinogenesis. The similar toxicological effects of SNP and NO3-N suggested that nitrate toxicity resulted from the generation of nitric oxide. Therefore, the present study provides insights into an alternative mechanism underpinning nitrate toxicity, which is useful for the conservation of amphibians in nitrate-rich environments.

Biochemical and body condition markers in Rhinella icterica tadpoles exposed to atrazine, glyphosate, and quinclorac based herbicides in ecologically relevant concentrations.

Amphibians have suffered population decline due to several factors, including exposure to pesticides. In the south of Brazil, rice cultivations use herbicides based on atrazine, glyphosate and quinclorac as the commercial formulations Primoleo®, Roundup® and Facet®, respectively. Rhinella icterica was chosen to evaluate oxidative balance markers and body condition after exposure to three concentrations of herbicides (10, 20 and 40 µg/L of atrazine and quinclorac; 100, 250 and 500 µg/L of glyphosate). These xenobiotics, regardless of the concentrations used, accelerated the development process of animals and seemed to act as modulators of development. We observed no significant variations for any of the oxidative balance markers studied (superoxide dismutase, catalase, TBARS and carbonylated proteins); however, we cannot rule out that other antioxidant system components prevent oxidative stress. In general, atrazine and glyphosate accelerated the development of tadpoles, and quinclorac retards this process, which could impact the survival of these animals.

Comparative study of composition of methanolic extracts of the paratoid gland secretions (PGS) of Rhinella jimi (cururu toad) from northeastern Brazil: Gender, seasonality and geographic occurrence.

Toads belonging to the Bufonidae family have a pair of paratoid glands that store highly toxic a biological secretion with varied chemical composition, that act as a chemical defense against microbial infections and predators. The paratoid gland secretion (PGS) of bufonids is rich in bioactive steroids, alkaloids, proteins, bufadienolides and bufotoxins. In the present investigation we performed a systematic analysis of the chemical profile of PGS obtained from the Bufonidae toad Rhinella jimi ("Cururu" toad) collected at three different regions of Piauí state, Northeastern Brazil. Our aim was to investigate the PGS variation related to the season of animals collection, geographic distribution and gender of the animals. The methanolic extracts of PGS were analyzed by UPLC-QToF-MS/MS. Principal component analysis (PCA) were applied to the data set obtained by the UPLC-QToF-MS/MS analyses. Among 23 compounds identified, dehydrobufotenine, suberoyl arginine, 3-(N-suberoyl-argininyl) telocinobufagin, 3-(N-suberoyl-argininyl) marinobufagin, telocinobufagin, marinobufagin and 3-(N-suberoyl-argininyl) bufalin were detected in all PGS. Minimal variations in the composition of paratoid secretions of R. jimi were observed related to distinct geographical and seasonal parameters. R. jimi female animals presented the most diverse chemical composition in its PGS. With this comparative study, unprecedented for the species, it was possible to observe that the secretions of the paratoid glands produced by R. jimi from different regions of the state of Piauí, at different times of the year, presented consistent chemical composition, with discrete particularities in the number and nature chemistry of its constituents.

Thermal conditions predict intraspecific variation in senescence rate in frogs and toads.

Variation in temperature is known to influence mortality patterns in ectotherms. Even though a few experimental studies on model organisms have reported a positive relationship between temperature and actuarial senescence (i.e., the increase in mortality risk with age), how variation in climate influences the senescence rate across the range of a species is still poorly understood in free-ranging animals. We filled this knowledge gap by investigating the relationships linking senescence rate, adult lifespan, and climatic conditions using long-term capture-recapture data from multiple amphibian populations. We considered two pairs of related anuran species from the Ranidae (Rana luteiventris and Rana temporaria) and Bufonidae (Anaxyrus boreas and Bufo bufo) families, which diverged more than 100 Mya and are broadly distributed in North America and Europe. Senescence rates were positively associated with mean annual temperature in all species. In addition, lifespan was negatively correlated with mean annual temperature in all species except A. boreas In both R. luteiventris and A. boreas, mean annual precipitation and human environmental footprint both had negligible effects on senescence rates or lifespans. Overall, our findings demonstrate the critical influence of thermal conditions on mortality patterns across anuran species from temperate regions. In the current context of further global temperature increases predicted by Intergovernmental Panel on Climate Change scenarios, a widespread acceleration of aging in amphibians is expected to occur in the decades to come, which might threaten even more seriously the viability of populations and exacerbate global decline.

Bufadienolides from the Skin Secretions of the Neotropical Toad Rhinella alata (Anura: Bufonidae): Antiprotozoal Activity against Trypanosoma cruzi.

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16ß-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16ß-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16ß-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1-10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.

Effects of temperature on growth, development and the leptin signaling pathway of Bufo gargarizans.

Climate change is one of the most important causes of the decline in amphibians. Changes in temperature have an important effect on the growth and development and energy metabolism of amphibians. The aim of this study is to unravel the effects of temperature on the leptin signaling pathway of Bufo gargarizans and its molecular mechanisms. Our results showed that high temperature accelerated the development rate of tadpoles, but reduced body size and mass, while low temperature deferred the development of tadpoles, but increased size and mass. Both high temperature and low temperature exposure caused pathological damage of the liver in B. gargarizans. The results of RT-qPCR revealed that the high temperature treatment significantly upregulated the transcript levels of genes related to thyroid hormone (DIO2 (D2), Thyroid Hormone Receptor-α (TRα)) and the leptin signaling pathway (Leptin Receptor (LepR), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Tyrosine kinase 2 (TYK2), Signal Transducer And Activator Of Transcription 3 (STAT3), Signal Transducer And Activator Of Transcription 3.1 (STAT3.1), and Signal Transducer And Activator Of Transcription 6 (STAT6)), while there was a decrease of mRNA expression of these genes (TRα, Thyroid Hormone Receptor-Beta (TRß), LepR, JAK1, and TYK2) in the liver of tadpoles exposed to high temperature compared with the intermediate temperature treatment. Therefore, our results suggested that temperature extremes might interfere with the thyroid and leptin signaling pathways and affect the growth and development of B. gargarizans. Furthermore, tissue injury of the liver could occur due to exposure to temperature extremes. This work promotes public awareness of environmental protection and species conservation needs, also provides valuable experimental data and a theoretical basis for the protection of amphibians.

Cloning and characterization of steroid 5ß-reductase from the venom gland of Bufo bufo gargarizans.

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.

Cadmium induced skeletal underdevelopment, liver cell apoptosis and hepatic energy metabolism disorder in Bufo gargarizans larvae by disrupting thyroid hormone signaling.

Cadmium (Cd) is hazardous to human health and it is also highly detrimental to amphibian life. In this study, Bufo gargarizans larvae were exposed to environmentally relevant Cd concentrations of 5, 100 and 200 µg L-1 from Gosner stage (Gs) 26 to Gs 42 of metamorphic climax about 6 weeks. The results showed thyroid structural injuries and thyroid signaling disruption were induced by high Cd exposure (100 and 200 µg L-1). Moreover, tadpole skeleton including whole body, vertebrata, forelimb and hindlimb was developmentally delayed by high Cd exposure through downregulating the mRNA expressions of genes involved with skeletal ossification and growth pathway. Moreover, liver histopathological injuries were caused by high Cd exposure featured by hepatocytes malformation, nuclear degeneration and increasing melanomacrophage centers. Meanwhile, liver apoptosis rate showed on the rise in a dose-dependent way and Cd stimulated liver apoptosis by upregulating mRNA expressions of genes related to extrinsic and intrinsic apoptosis pathways. Furthermore, high Cd caused hepatic glucometabolism disorder by decreasing the genetic expressions associated with glycolysis and mitochondrial oxidative phosphorylation. In addition, liver lipid metabolism was disrupted by high Cd exposure through downregulating mRNA levels of genes related to fatty oxidation and upregulating mRNA levels of genes related to fatty acid synthesis. We suggested that Cd did great harm to tadpole health by disturbing thyroid function, skeletal growth, liver cell apoptosis signaling and hepatic energy metabolism pathway.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism.

(1) Background: Toad venom (Bufonis Venenum, known as 'Chansu' in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Response Patterns of Biomarkers as Tools to Identify Toxic Effects of Cadmium and Lead on Bufo gargarizans Embryo.

Molecular biomarkers play an increasing crucial role in evaluating and predicting toxicity of metals. Expressions patterns of genes related to oxidative stress, apoptosis, immune and inflammation response in the Bufo gargarizans embryo exhibited a development dependent manner. The genes related to oxidative stress (HSP, GPx and SOD) are the first response in the development of embryo, followed by the apoptosis (Bax, BCLAF1 and TRAIL) and inflammation and immune response (SOCS3, IL-27 and IL-17D), respectively. Then, we have verified the HSP, Bax and SOCS3 IL-27 (expressed highest in their respective processes) exhibited the most significant changes in Cd-Pb mixed group compared with control. In addition, we found exposure of Cd-Pb mixed metals causes greater adverse effects than Cd, Pb alone on development and morphology of embryo. Overall, our results provide a useful tool to use the sensitive molecular biomarkers as indicators of developmental toxicity in amphibian embryo.

Comparative study of the chemical profile of the parotoid gland secretions from Rhaebo guttatus from different regions of the Brazilian Amazon.

Amphibian cutaneous secretion has great potential for bioprospection and is a great tool in the development of bioproducts. Thus, the objective of the present work was to evaluate the comparative study of the chemical profile parotoid gland secretions from Rhaebo guttatus collected in two distinct regions of the Brazilian Amazon. For this, the chemical composition of six methanolic extracts of this species were analyzed by Liquid Chromatography in UV and MS Detection Ultra-Chromatography Systems (UFLC-DAD-micrOTOF). All obtained chromatograms presented two distinct regions; one referring to the more hydrophilic molecules (alkaloids), while the other refers to the more hydrophobic compounds (steroids). The steroid region resembles all samples, regardless of where they were collected. In the alkaloid region, there was a standardized variation for the samples from the southern Brazilian Amazon, but the same was not true for the samples collected in the Amazon-Cerrado transition region. Thus, the data suggest that the environment and diet of R. guttatus may be important in alkaloid production, but do not influence steroid content. These results add new information about the poison of the toad R. guttatus and raises new questions to be further investigated, thus contributing to the knowledge of the anuran fauna of the Brazilian Amazon.

Metabolites from Bufo gargarizans (Cantor, 1842): A review of traditional uses, pharmacological activity, toxicity and quality control.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufo gargarizans (Cantor, 1842) (BGC), a traditional medicinal animal distributed in many provinces of China, is well known for the pharmaceutical value of Chansu and Chanpi. As traditional Chinese medicines (TCMs), Chansu and Chanpi, with their broad-spectrum of therapeutic applications, have long been applied to detoxification, anti-inflammation, analgesia, etc. OVERARCHING OBJECTIVE: We critically analyzed the current evidence for the traditional uses, chemical profiles, pharmacological activity, toxicity and quality control of BGC (Bufonidae family) to provide a scientific basis for future in-depth studies and perspectives for the discovery of potential drug candidates. METHODOLOGY: All of the available information on active constituents and TCMs derived from BGC was obtained using the keywords "Bufo gargarizans", "Chansu", "Chanpi", "Huachansu", or "Cinobufacini" through different electronic databases, including PubMed, Web of Science, Chinese National Knowledge Infrastructure (CNKI), the Wanfang Database, and Pharmacopoeia of China. In addition, Chinese medicine books from different times were used to elucidate the traditional uses of BGC. Electronic databases, including the "IUCN Red List of Threatened Species", "American Museum of Natural History" and "AmphibiaWeb Species Lists", were used to validate the scientific name of BGC. RESULTS: To date, about 118 bufadienolide monomers and 11 indole alkaloids have been identified from BGC in total. The extracts and isolated compounds exhibit a wide range of in vitro and in vivo pharmacological effects. The literature search demonstrated that the ethnomedicinal uses of BGC, such as detoxification, anti-inflammation and the ability to reduce swelling and pain associated with infections, are correlated with its modern pharmacological activities, including antitumor, immunomodulation and attenuation of cancer-derived pain. Bufadienolides and indole alkaloids have been regarded as the main active substances in BGC, among which bufadienolides have significant antitumor activity. Furthermore, the cardiotoxicity of bufadienolides was discussed, and the main molecular mechanism involves in the inhibition of Na+/K+-ATPase. Besides, with the development of modern analytical techniques, the quality control methods of BGC-derived TCMs are being improved constantly. CONCLUSIONS: An increasing number of reports suggest that BGC can be regarded as an excellent source for exploring the potential antitumor constituents. However, the future antitumor research of BGC needs to follow the standard pharmacology guidelines, so as to provide comprehensive pharmacological information and aid the reproducibility of the data. Besides, to ensure the efficacy and safety of BGC-derived TCMs, it is vital to construct a comprehensive quality evaluation model on the basis of clarifying pharmacodynamic-related and toxicity-related compositions.