Roberta Jeane Bezerra Jorge

Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest.

Bothrops jararaca is a slender and semi-arboreal medically relevant pit viper species endemic to tropical and subtropical forests in southern Brazil, Paraguay, and northern Argentina (Misiones). Within its geographic range, it is often abundant and is an important cause of snakebite. Although no subspecies are currently recognized, geographic analyses have revealed the existence of two well-supported B. jararaca clades that diverged during the Pliocene ~3.8Mya and currently display a southeastern (SE) and a southern (S) Atlantic rainforest (Mata Atlântica) distribution. The spectrum, geographic variability, and ontogenetic changes of the venom proteomes of snakes from these two B. jararaca phylogroups were investigated applying a combined venom gland transcriptomic and venomic analysis. Comparisons of the venom proteomes and transcriptomes of B. jararaca from the SE and S geographic regions revealed notable interpopulational variability that may be due to the different levels of population-specific transcriptional regulation, including, in the case of the southern population, a marked ontogenetic venom compositional change involving the upregulation of the myotoxic PLA2 homolog, bothropstoxin-I. This population-specific marker can be used to estimate the proportion of venom from the southern population present in the B. jararaca venom pool used for the Brazilian soro antibotrópico (SAB) antivenom production. On the other hand, the southeastern population-specific D49-PLA2 molecules, BinTX-I and BinTX-II, lend support to the notion that the mainland ancestor of Bothrops insularis was originated within the same population that gave rise to the current SE B. jararaca phylogroup, and that this insular species endemic to Queimada Grande Island (Brazil) expresses a pedomorphic venom phenotype. Mirroring their compositional divergence, the two geographic B. jararaca venom pools showed distinct bioactivity profiles. However, the SAB antivenom manufactured in Vital Brazil Institute neutralized the lethal effect of both venoms to a similar extent. In addition, immobilized SAB antivenom immunocaptured most of the venom components of the venoms of both B. jararaca populations, but did not show immunoreactivity against vasoactive peptides. The Costa Rican bothropic-crotalic-lachesic (BCL) antivenom showed the same lack of reactivity against vasoactive peptides but, in addition, was less efficient immunocapturing PI- and PIII-SVMPs from the SE venom, and bothropstoxin-I, a CRISP molecule, and a D49-PLA2 from the venom of the southern B. jararaca phylogroup. The remarkable paraspecificity exhibited by the Brazilian and the Costa Rican antivenoms indicates large immunoreactive epitope conservation across the natural history of Bothrops, a genus that has its roots in the middle Miocene. This article is part of a Special Issue entitled: Omics Evolutionary Ecolog.

Antinociceptive and anti-inflammatory activities of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides

BACKGROUND Red and brown algae sulfated polysaccharides (SPs) have been widely investigated as antinociceptive and/or anti-inflammatory agents; however, no description of these biological properties concerning green algae SPs have been reported. Caulerpa curpressoides (Chlorophyta) presents three SPs fractions (Cc-SP1, Cc-SP2, and Cc-SP3). Anticoagulant (in vitro) and anti- and pro-thrombotic (in vivo) effects of Cc-SP2 had been recently reported. We evaluated the effects of Cc-SP2 using models of nociception and acute inflammation in vivo. METHODS Male Swiss mice received Cc-SP2 (iv) 30 min prior to receiving 0.6% acetic acid (10 ml/kg, ip), 1% formalin (20 μl, sc) or were subjected to thermal stimuli (51 ± 1 °C). Cc-SP2 was injected sc to male Wistar rats in a peritonitis model or a paw edema model using carrageenan (ip or ipl, 500 μg). To analyze the systemic effects, Cc-SP2 (27 mg/kg, sc) was administrated to both genders mice before waiting for 14 days. RESULTS Cc-SP2 (3, 9 or 27 mg/kg) reduced (p < 0.05) the number of writhes induced by acetic acid by 57, 89.9 and 90.6%, respectively, the licking time in the first (9 or 27 mg/kg with 42.47 and 52.1%, respectively) and the second (3, 9 or 27 mg/kg with 68.95, 82.34 and 84.61%, respectively) phases. In the hot-plate test, the antinociceptive effect of Cc-SP2 (9 mg/kg) was primarily observed at 60 min (26.7 ± 1.2 s), with its effect reversed by naloxone (8.6 ± 1.3 s), suggesting the involvement of the opioid system. Cc-SP2 (3, 9 or 27 mg/kg, sc, p < 0.05) showed anti-inflammatory effects by decreasing neutrophils migration by 64, 69 and 73%, respectively, and potently reduced the paw edema, especially at the second (0.16 ± 0.02, 0.16 ± 0.03 and 0.12 ± 0.05 ml) and third (0.16 ± 0.03, 0.18 ± 0.02 and 0.14 ± 0.04 ml) hours, respectively. Cc-SP2 did not cause hepatic or renal alterations or affect body mass or the macroscopy of the organs examined (p > 0.05). Histopathological analyses of the liver and kidney showed that both organs were affected by Cc-SP2 treatment, but these effects were considered reversible. CONCLUSION The results indicate that the analgesic and anti-inflammatory effects of Cc-SP2 could be of biomedical applicability as a new, natural tool in pain and acute inflammatory conditions.

Antinociceptive and Anti-Inflammatory Activities of Sulphated Polysaccharides from the Red Seaweed Gracilaria cornea

Seaweeds have attracted special interest as good sources of sulphated polysaccharides (SP) for use in pharmaceutical industries and biotechnology. In this study, we evaluated the effects of SP from the red seaweed Gracilaria cornea (Gc-TSP) in nociceptive and inflammatory models. In mice, Gc-TSP (3, 9 or 27 mg/kg) significantly reduced nociceptive responses, as measured by the number of writhes, at all tested doses. In a formalin test, Gc-TSP significantly reduced licking time in both phases of the test at a dose of 27 mg/kg. In a hot-plate test, the antinociceptive effect was observed only in animals treated with 27 mg/kg of Gc-TSP, suggesting that the analgesic effect occurs through a central action mechanism at the highest dose. Gc-TSP (3, 9 or 27 mg/kg) caused only a slight reduction in neutrophil migration in the rat peritoneal cavity. However, lower doses of Gc-TSP (3 and 9 mg/kg) significantly inhibited paw oedema induced by carrageenan, especially at 3 hr after treatment. Reduction in oedema was confirmed by myeloperoxidase activity in the affected paw tissue. In addition, treatment (s.c.) of animals with different doses of Gc-TSP inhibited paw oedema induced by dextran within the first hour in all doses tested. After 14 consecutive days of intraperitoneal administration of Gc-TSP (9 mg/kg), we measured the wet weight of the liver, kidney, heart, spleen and thymus and performed biochemical, haematological and histopathological evaluations. No systemic damage was found. These results indicate that Gc-TSP possesses analgesic and anti-inflammatory effects and is a potentially important tool worthy of further study.

Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil

The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been appreciated by herpetologists and toxinologists as a general feature of highly adaptable and widely distributed snake species, the five B. erythromelas populations investigated exhibit highly conserved venom proteomes. The overall toxin profile of the Caatinga lanceheads venom explains the local and systemic effects observed in envenomations by B. erythromelas. The five geographic venom pools sampled also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated using different bothropic venoms in the immunization mixtures. The large immunoreactive epitope conservation across genus Bothrops offers promise for the generation of a broad-spectrum bothropic antivenom.

Peripheral antinociception and anti-edematogenic effect of a sulfated polysaccharide from Acanthophora muscoides

BACKGROUND Sulfated polysaccharides from red marine algae have presented a variety of potentially therapeutic biological effects, however, their antinocicpetive and anti-inflammatory properties are not well understood. METHODS Male Swiss mice were pretreated with a sulfated polysaccharidic fraction obtained from the marine alga Acanthophora muscoides (AmII) (1, 3 or 9 mg/kg, iv) 30 min prior to either receiving an injection of 0.8% acetic acid or 1% formalin or prior to a thermal stimulus. AmII (1, 3 or 9 mg/kg, sc) was evaluated on carrageenan-, dextran- bradykinin-, histamine- and serotonin-induced rat paw edema models. AmII (500 μg, sc) was also injected into the paw. Additionally, mice were treated with the total sulfated polysaccharides from A. muscoides (Am-TSP) (20 mg/kg, ip) for 14 days. RESULTS AmII reduced the number of acetic acid-induced writhes and licking time in the second phase of the formalin test, but it did not alter the response latency in the hot plate test, suggesting that its antinociceptive action occurs through a peripheral mechanism. AmII did not reduce carrageenan-induced paw edema and MPO activity. However, it reduced dextran-, histamine- and serotonin-induced paw edemas, but not bradykinin-induced edema, suggesting that histamine is the major target of AmII anti-edematogenic activity. AmII injected into the paw did not evoke local edema. Furthermore, Am-TSP induced no consistent signs of systemic damage, as revealed by body mass, organs wet weight and by biochemical, hematological and histopathological analyses. CONCLUSION AmII has important antinociceptive and anti-inflammatory properties and represents an important therapeutic agent warranting future studies.

Inhibitory effects of sertraline in rat isolated perfused kidneys and in isolated ring preparations of rat arteries.

Objectives  Sertraline is often prescribed to patients suffering with end stage renal disease, but its action on kidney has not been investigated. We aimed to investigate the pharmacological action of sertraline on rat kidney with emphasis on the underlying mechanisms involved in the vascular actions of the drug.

Antiophidic Solanidane Steroidal Alkaloids from Solanum campaniforme

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including 1D and 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A2 activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2.

L-Aminoacid Oxidase from Bothrops leucurus Venom Induces Nephrotoxicity via Apoptosis and Necrosis

Acute renal failure is a common complication caused by Bothrops viper envenomation. In this study, the nefrotoxicity of a main component of B. leucurus venom called L-aminoacid oxidase (LAAO-Bl) was evaluated by using tubular epithelial cell lines MDCK and HK-2 and perfused kidney from rats. LAAO-Bl exhibited cytotoxicity, inducing apoptosis and necrosis in MDCK and HK-2 cell lines in a concentration-dependent manner. MDCK apoptosis induction was accompanied by Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation and mitochondrial dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 μg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by snakebites. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, at least in part, for the nephrotoxicity observed in isolated kidney.

Antinociceptive and Anti-inflammatory Activities of the Lectin from Marine Red Alga Solieria filiformis

Lectins are proteins that bind to specific mono- or oligosaccharides. This study aimed to evaluate the antinociceptive and anti-inflammatory effects of the lectin from the red marine alga Solieria filiformis. The animals (n = 6) were pretreated with S. filiformis lectin 30 min before they were given the nociceptive or inflammatory stimulus. The antinociceptive activity was evaluated in Swiss mice using the abdominal writhing, formalin, and hot plate tests. The anti-inflammatory properties were evaluated in Wistar rats using carrageenan-induced peritonitis and paw edema induced by different phlogistic agents. The S. filiformis lectin toxicity was assayed through its application in mice (7 days). S. filiformis lectin significantly reduced the number of abdominal writhings and reduced the paw licking time in the second phase of the formalin test (p < 0.05), but it did not prolong the reaction time in the hot plate test (p > 0.05). Furthermore, S. filiformis lectin reduced neutrophil migration in a peritonitis model and reduced paw edema induced by carrageenan, dextran, and serotonin (p < 0.05). Additionally, the administration of S. filiformis lectin resulted in no signs of systemic damage. Thus, S. filiformis lectin appears to have important antinociceptive and anti-inflammatory activities and could represent a potential therapeutic agent for future studies.

l-amino acid oxidase from Bothrops marajoensis causes nephrotoxicity in isolated perfused kidney and cytotoxicity in MDCK renal cells.

Renal alterations caused by Bothrops venom and its compounds are studied to understand these effects and provide the best treatment. Previously, we studied the renal effect of the whole venom of Bothrops marajoensis and its phospholipase A2 (PLA2), but these effects could not to be attributed to PLA2. To continue the study, we report in this short communication the effects of l-amino acid oxidase from B. marajoensis venom (LAAOBm) on renal function parameter alterations observed in the same model of isolated perfused kidney, as well as the cytotoxic effect on renal cells. LAAOBm caused a decrease in PP, RVR, UF, GFR, %TNa(+) and %TCl(-), very similar to the effects of whole venom using the same model. We also demonstrated its cytotoxicity in MDCK cells with IC50 of 2.5 μg/mL and late apoptotic involvement demonstrated by flow cytometry assays. In conclusion, we suggested that LAAOBm is a nephrotoxic compound of B. marajoensis venom.