Abstract in English:

Abstract Background: Spiders of medical importance in the Amazon region belong to the genera Phoneutria, Loxosceles and Latrodectus. Natural history data show that Phoneutria spp. occur in both periodically flooded forest areas (igapós) and non-flooded areas (terra firme), as well as in commercial plantations in the Amazon. Negative interactions with wandering spiders (Phoneutria spp.) can occur along forest trails, leading to homes, schools and workplaces. Harmful species, such as Loxosceles amazonica and Latrodectus aff. curacaviensis, are mainly associated with accidents in rural settings. Methods: To understand the dynamics of spider accidents in the Brazilian Amazon, we conducted a search for scientific articles in five databases (Google Scholar, PubMed/MEDLINE, Scopus by Elsevier and SciELO). In addition, we analyzed the content of four reference books on the ecological aspects of Amazonian spiders. All told, we identified 64 eligible studies, including six regional surveys published between 1996 and 2016. Results: From 2015 to 2022, a total of 25 human lives were lost to spider envenomation in the Brazilian Amazon. An analysis of the data revealed that many riverside families engage in agricultural practices that expose them to venomous animals. Hospital data reveal that most patients bitten by spiders come from impoverished rural communities, which rely on public hospitals of Brazil’s Unified Health System (SUS) for medical treatment. The results indicate that spider bites in the Amazon represent a neglected public health problem, especially in locations far from capital cities. Conclusion: Amerindian and non-Amerindian communities living in areas at high risk of venomous animal attacks do not receive adequate attention in health policies. Given the wide dispersion of rural populations vulnerable to venomous animal incidents in the Amazon, the establishment of new referral medical centers is an essential strategy, especially for riverside communities with limited access to health services.

Abstract in English:

Abstract Background: Fish venoms have been poorly characterized and the available information about their composition suggests they are uncomplicated secretions that, combined with epidermal mucus, could induce an inflammatory reaction, excruciating pain, and, in some cases, local tissue injuries. Methods: In this study, we characterized the 24-hour histopathological effects of lionfish venom in a mouse experimental model by testing the main fractions obtained by size exclusion-HPLC. By partial proteomics analysis, we also correlated these in vivo effects with the presence of some potentially toxic venom components. Results: We observed a strong lesion on the skin and evident necrosis in the skeletal muscle. None of the tissue-damaging effects were induced by the fraction containing cytolysins, membrane pore-forming toxins ubiquitously present in species of scorpionfish, stonefish, and lionfish, among others. On the contrary, injuries were associated with the presence of other components, which have remained practically ignored so far. This is the case of an abundant protein, present in venom, with homology to a Golgi-associated plant pathogenic protein 1-like (GAPR1), which belongs to the same protein superfamily as venom CRISPs and insect allergens. Conclusion: This GAPR1-like protein and the hyaluronidase are probably responsible for the hemostasis impairment and hemorrhagic lesions observed in mouse skin, whereas muscle injuries can be indirectly caused by a combination of inflammatory and hemorrhagic events. More information is required to establish the components accountable for the myonecrotic effect.

Abstract in English:

Abstract Background: Non-small cell lung cancers (NSCLC) represent the primary cause of cancer-related deaths worldwide. Rhopalurus junceus venom has been shown to exert cytotoxic effects against a panel of epithelial cancer cells in vitro and suggested that NSCLC was the subtype most susceptible to the treatment. Methods: This study evaluated the effect of Rhopalurus junceus scorpion venom on cell viability, in non-cancerous (MRC-5, lung; CHO-K1, ovary) and NSCLC (A549; NCI-H460) cell lines. The effects on cell cycle, apoptosis, and cell signaling-related proteins were determined by flow cytometry and WB. Protein fractions responsible for the observed effect were identified using HPLC. Results: Scorpion venom was more effective against NSCLC than non-cancerous cells. Emax values were 20.0 ± 5.8% and 22.47 ± 6.02% in A549 and NCI-H460 cancer cells, respectively, as compared to 50 ± 8.1% in MRC-5 and 54.99 ± 7.39% in CHO-K1 cells. It arrested NSCLC cells in the G2/M phase, while non-cancerous cells were arrested in the S (MRC-5) or G0/G1 (CHO-K1) phases. No changes were observed in the Bax/Bcl-2 or the cleaved-caspase 3/Total caspase 3 ratios in cells treated with venom. Likewise, the scorpion venom treatment did not affect p-ERK, p-AKT, or p-38MAPK protein levels. In contrast, scorpion venom treatment increased the cytosolic apoptosis-inducing factor (AIF) in A549 cells, indicating caspase-independent apoptosis. Additionally, combined etoposide/venom exposure provoked G2/M arrest and apoptosis in NSCLC more strongly than either substance alone. Furthermore, upon crude venom fractioning through RP-HPLC, we found two soluble fractions with high cytotoxic effects. Conclusion: The present study concludes that a specific fraction of Rhopalurus junceus venom reduces cell viability of NSCLC cells. The AIF protein plays a key role in mediating caspase-independent apoptotic cell death. These findings suggest that Rhopalurus junceus venom enhances the anticancer effect of etoposide in vitro by causing cell cycle arrest and caspase-independent apoptosis.

Abstract in English:

Abstract Background: Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately seven million individuals worldwide, with the highest number of cases in Latin America. CD has two phases, of which the chronic phase is characterized by reduced efficacy in drug therapies. This and other factors make developing new strategies that aim to identify molecules capable of becoming alternatives to or complement current chemotherapy vitally important. Methods: Cruzain and AsCystatin were obtained recombinantly through expression in E. coli. Bioinformatic assays were conducted with both molecules, followed by in vitro enzyme inhibition assays. Subsequently, in silico studies allowed for the design of peptides, which were then assessed for molecular interactions with cruzain. The designed peptides were synthesized, and their inhibitory potential on cruzain and their trypanocidal and cytotoxic effects in vitro were finally assessed. Results: AsCystatin, a potential inhibitor of cysteine proteases, was identified from previously published scientific literature. In silico assays suggested that AsCystatin interacts with key regions of cruzain, and was subsequently produced through heterologous expression, obtaining a protein with a high degree of purity. Next, the inhibition of AsCystatin on the activity of cruzain was assessed, observing that approximately 20 µM of cystatin could inhibit 50% of the catalytic activity of the recombinant enzyme. Based on the in-silico analysis performed previously, original, and modified peptides were designed and tested, which allowed for identifying four peptides with inhibitory capacity on the enzymatic activity of cruzain. Finally, three of these peptides showed trypanocidal activity on epimastigote forms of T. cruzi in in vitro models. Conclusion: It was possible to identify AsCystatin and four peptides derived from this protein with inhibitory activity on cruzain, highlighting the trypanocidal effect of these peptides observed in in vitro assays.

Abstract in English:

Abstract Background: Temperature regulation is essentially important for survival of poikilotherms such as snakes. Body temperature is regulated by snakes through behavioral and physiological responses. The global-warming crisis, combined with the need to house large population of snakes in limited spaces, increases the likelihood of exposing snakes to high ambient temperature (HTa), requiring it reliance on physiological responses. This study aimed to study the effect of HTa exposure on physiological responses and venom production, which have rarely been studied. Methods: Eleven adult monocled cobras (Naja kaouthia Lesson, 1831) were divided into two groups. The concurrent control group was housed in a temperature-controlled room, and the heat exposed group was housed in the same room with gradually increasing temperatures (25°C-35°C) for 4 h on four consecutive days. Data were collected 3 days before the experiment as the baseline and then compared with day 1 and day 4 after HTa exposure data representing immediate and prolonged effects. Body temperature, body weight, water intake, heart rate, hematology, plasma biochemistry, body-fluid compartments, hormonal response, heat shock protein expression and venom production were measured. Results: In response to HTa exposure, body temperature and heart rate increased, plasma volume significantly decreased, but water intake increased. Hematocrit and plasma protein progressively decreased in the latter stages of experimentation, but HTa diminished this effect. HTa only increased plasma corticosterone on day 1. Exposure to HTa increased venom protein concentration on day 4 and diminished the decreased proportion effect of frequent venom collection on phospholipase A2 component. Conclusion: Increased heart rate and fluid shift from the intravascular compartment appeared to be the underlying mechanism for heat dissipation during HTa exposure. Under the study condition, HTa caused heat stress, but the snake could adapt after continued exposure. Additionally, HTa increased venom protein concentration in N. kaouthia, particularly phospholipase A2 component.

Abstract in English:

Abstract Background: Scorpion envenomation poses a significant health threat in endemic regions, eliciting complex immune responses in affected individuals. Recent research suggests that the timing of envenomation - whether it occurs during the day or night - may influence the host inflammatory response and subsequent organ damage. This study investigates the impact of envenomation timing on host inflammatory and oxidative responses using an experimental scorpion envenomation model. Methods: Mice were divided into two groups, corresponding to their resting phase (day) and activity phase (night), and were monitored for twenty-four hours post-envenomation. We analyzed systemic inflammatory markers, hormonal changes within the hypothalamic-pituitary-adrenal (HPA) axis, and assessed liver toxicity. Results: Our findings reveal that the release of the myeloperoxidase enzyme, along with the pro-inflammatory cytokines IL-6 and IL-17, varied significantly based on the timing of envenomation. Notably, envenomation occurring during the nighttime resulted in elevated levels of these mediators. We also observed a pronounced imbalance in oxidative stress, characterized by a higher presence of prooxidant species during the daytime and enhanced antioxidant activities during the nighttime. This diurnal variation highlights the dynamic nature of the inflammatory and oxidative processes. Importantly, our analysis points to the probable involvement of corticosterone, the final effector of the HPA axis, in modulating these variations in the inflammatory response. By influencing both the intensity of the immune response and the degree of oxidative stress, corticosterone appears to play a pivotal role in the overall pathophysiology of scorpion envenomation. Conclusion: This study provides valuable insights into how the timing of scorpion envenomation influences inflammatory responses and organ-specific toxicity, offering potential implications for the treatment and management of envenomation cases.

Abstract in English:

Abstract Background: Leishmaniasis is a neglected disease that mainly affects impoverished populations and receives limited attention from governments and research institutions. Current treatments are based on antimonial therapies, which present high toxicity and cause significant side effects, such as cardiotoxicity and hepatotoxicity. This study proposes using crotapotin, isolated from Crotalus durissus terrificus venom, as a potential inhibitor of the enzyme trypanothione reductase from Leishmania braziliensis (LbTR). Methods: In silico assays were conducted to evaluate the interaction of crotapotin with LbTR using molecular docking and molecular dynamics techniques. Recombinant LbTR was expressed in E. coli, and its enzymatic activity was confirmed. The inhibitory action of crotapotin on LbTR was then tested in enzymatic assays. Results: The stability of these interactions was confirmed over 200 ns molecular dynamics simulations, with a clustering analysis using the GROMACS method revealing a total of 12 distinct clusters. The five most representative clusters showed low RMSD values, indicating high structural stability of the LbTR-crotapotin complex. In particular, cluster 1, with 3,398 frames and an average RMSD of 0.189 nm from the centroid, suggests a dominant stable conformation of the complex. Additional clusters maintained average RMSD values between 0.173 nm and 0.193 nm, further reinforcing the robustness of the complex under physiological conditions. Recombinant LbTR expression was successful, yielding 4.8 mg/L with high purity, as verified by SDS-PAGE. In the enzymatic assays, crotapotin partially inhibited LbTR activity, with an IC50 of 223.4 μM. Conclusion: The in silico findings suggest a stable and structured interaction between crotapotin and LbTR, with low structural fluctuation, although the inhibition observed in in vitro assays was moderate. These results indicate the potential of crotapotin as a promising basis for developing specific LbTR inhibitors, contributing to the bioprospecting of new antiparasitic agents.

Abstract in English:

Abstract Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer, on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels. Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described. Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC50 value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC50 value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability. Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability.