In Vitro Assessment of Cytoprotective Effects of CANOVA against Cell Death Induced by the Anti-malarial Artesunate - A Preliminary Experiment.

BACKGROUND: Artesunate (ATS) is a semi-synthetic compound derived from artemisinin, which is widely accepted in the treatment of malaria. However, there is evidence that ATS, under certain in vitro conditions, induces several impairments to normal cell functions. Canova (CA) is a Brazilian homeopathic formulation indicated for patients with depressed immune system. CA shows both in vitro and in vivo protective effects against mutagenic/carcinogenic compounds. Therefore, we aimed to assess in vitro the cytoprotective effects of CA against the cytotoxicity of ATS in Vero cells. METHODS: Viability of Vero cells exposed to ATS was assessed by MTT assay, whereas the anti-cytotoxic effect of CA was evaluated by apoptosis and necrosis quantification with fluorescent dyes. RESULTS: After 24 hours of ATS treatment, a reduction in cell viability was observed at 32 and 64 µg/mL, the latter being statistically significant (p < 0.05) in relation to the negative control. The concentration of 64 µg/mL was chosen for the subsequent experiments. ATS significantly induced both apoptosis and necrosis in Vero cells in relation to controls (p < 0.01). We also observed a statistically significant decrease in the number of apoptotic cells observed in the CA 16% + ATS co-treatment compared with ATS treatment (p < 0.01). Treatment with CA alone also had no influence on either type of cell death. CONCLUSION: Our results demonstrated that ATS is cytotoxic in the assessed conditions. However, such cytotoxicity was attenuated when the cells were treated simultaneously with ATS and CA.

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo.

Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.

Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 µM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 µM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.

Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 µM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 µM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 µM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 µM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 µM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 µM for 6-24 h.

The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells.

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Enhancement of the citrulline-nitric oxide cycle in astroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom.

The biological activity of the proline-rich decapeptide Bj-PRO-10c, a processing product of the C-type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensin-converting enzyme activity with subsequent anti-hypertensive effect. However, recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide. Here we show that Bj-PRO-10c enhances and sustains the generation of nitric oxide (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline-NO cycle. Bj-PRO-10c-mediated effects not restricted to the cardiovascular system, since NO production was also induced in cells of astroglial origin. Bj-PRO-10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline-NO cycle cells in a dose-dependent fashion. In view of that, astroglial cells function as L-arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj-PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS. Moreover, proliferation of astroglial cells was reduced in the presence of Bj-PRO-10c; however, cell death was not induced. Since NO donors have been studied for the treatment of solid cancers, Bj-PRO-10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide's ability to augment NO production.

Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis.

BACKGROUND: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. CONCLUSIONS/SIGNIFICANCE: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

Distribution of (125)I-labeled crotamine in mice tissues.

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with (125)I using chloramine T method, and separated by gel filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6)cpm/mouse) of (125)I native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain.

Pulmonary hemorrhage induced by jararhagin, a metalloproteinase from Bothrops jararaca snake venom.

Jararhagin is the most important hemorrhagic component in the venom of the snake Bothrops jararaca, a species of medical importance in South America. It is a P-III zinc-dependent metalloproteinase comprising catalytic, disintegrin-like, and cysteine-rich domains. Jararhagin injected intravenously into mice induced rapid and prominent bleeding in the lungs, whereas other organs were devoid of overt hemorrhagic manifestations. This action depends on the proteolytic activity of jararhagin, since it was abrogated by the synthetic inhibitor batimastat. There were conspicuous ultrastructural alterations in cells at the alveolo-capillary unit, i.e., capillary endothelial cells and type I pneumocytes, with a characteristic pattern of "regional alveolar damage" associated with extravasation. These pathological effects were observed under conditions in which the whole blood clotting time, bleeding time, and fibrinogen levels were not affected. 125I-labeled jararhagin is concentrated mainly in liver and kidneys after iv injection, with little radioactivity observed in the lungs, thereby indicating that the predominance of pulmonary microvascular damage is not due to a preferential concentration of this enzyme in the lungs. Despite the fact that jararhagin is complexed by plasma proteins after iv injection, its hemorrhagic activity was not inhibited by the plasma proteinase inhibitor alpha(2)-macroglobulin, and was only partially reduced by normal mouse serum, suggesting that resistance to inhibition may contribute to its ability to cause pulmonary hemorrhage.

Purification and characterization of a novel metalloproteinase, acurhagin, from Agkistrodon acutus venom.

Acurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anion-exchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aalpa chain of fibrinogen, followed by Bbeta chain, while gamma chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37 degrees C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrin-like and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of platelet-rich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro.

Barbourin is a 73 amino acid venom protein that inhibits platelet aggregation. Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahistidine (H6) tags. BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP. BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin alphaIIbbeta3. The terminal catabolic half-life of BLAH6 approached that of RSAH6 [3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 +/- SD)], but was substantially increased relative to that of BARH6 [0.15 +/- 0.03 days (n = 3 +/- SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.

Rocha e Silva, the snake venom and 50th anniversary of bradykinin discovery

In this article we emphasize the importante of the snake venom toxins for the development of the autopharmacology concept (Sir Henry Dale, 1933) and how they led to the discovery of bradykinin and to the development of the first active-site directed inhibitor of the angiotensin converting enzyme. We also describe the most recent development concerning the possible role of bradykinin potentiating peptides from Bothrops jararaca venom for the cardiovascular pharmacology. A fundamental step leading to what can be considered one of the most significant contributions given by Brazilian scientists to biomedical sciences, was originated in the efforts of Rocha e Silva to identify and characterize the actors involved in anaphylaxis. His main scientific motivation and efforts which influenced a whole generation of prominent Brazilian scientists, should be reminded when we are commemorating the 50th anniversary of the bradykinin discovery. One important consequence of the use of the Bothrops jararaca venom leading to the discovery of bradykinin was the development of captopril, the most successful drug used by milions of people all over the world to treat arterial hypertension. The purpose of this article is to dissect some of the historical background which aim to stress the need for the adequate scientific environment allowing the privileged scientific minds to uncover secrets of Nature for the benefit of mankind.

In vitro platelet binding compared with in vivo thrombus imaging using alpha(IIb)beta3-targeted radioligands.

Radioligands for the alpha(IIb)beta3 integrin on platelets are being studied for their ability to image venous thrombi and pulmonary emboli. One such radioligand, 123I-bitistatin, was previously shown to have higher thrombotic uptake in an animal model than other disintegrins, but the reason for this difference was not clear. The purpose of this study was to evaluate three labeled disintegrins, bitistatin, kistrin and barbourin, to look for in vitro differences in platelet binding which could explain the in vivo behavior. Disintegrins labeled with 121I were compared in vitro for extent of binding to platelets and rates of binding and dissociation. These findings were related to organ distribution and image quality for imaging thrombotic lesions, following administration of 123I-disintegrins in an animal model. Fibrinogen at 8.8 micromol/l was able to displace 125I-barbourin and 125I-kistrin more rapidly from ADP-stimulated platelets, with half-times of 3.5 and 10.7 min, compared with 125I-bitistatin (31.6 min). At equivalent concentrations in whole blood, a higher percentage of bitistatin bound to platelets compared with the other two. In vivo, kistrin and barbourin had significantly lower thrombus:muscle and pulmonary embolus:lung ratios in images compared with bitistatin. There was evidence of more metabolic deiodination of labeled kistrin and barbourin in vivo compared with bitistatin. A surprising finding was that conventional in vitro platelet binding studies did not predict the relative in vivo behavior of labeled disintegrins. The results suggest that labeled bitistatin has improved targeting of thrombi because it is less easily displaced from stimulated platelets, permitting longer lesion retention. It also appears to have a greater association with resting platelets in the blood, which may increase bioavailability and delay metabolic breakdown.

Distribution of 131I-labeled Bothrops erythromelas venom in mice

Bothrops erythromelas is responsible for many snake bites in northeastern Brazil. In the present study we determined the in vivo distribution of the venom following its subcutaneous injection into mice. B. erythromelas venom and albumin were labeled individually with I by the chloramine T method, and separated in a Sephacrylr S-200 column. The efficiency of labeling was 68 percent. Male Swiss mice (40-45 g), which had been provided with drinking water containing 0.05 percent KI over a period of 10 days prior to the experiment, were inoculated dorsally(sc) with 03. ml (2.35 x 10(5) cpm/mouse) of I-venom (N=42), I-albumin or I (controls, N=28 each). Thirty minutes and 1, 3, 6, 12, 18 and 24 h after inoculation, the animals were perfused with 0.85 percent NaCl and skin and various organs were collected in order to determine radioactivity content. There was a high rate of venom absorption in the skin (51 percent) within the first 30 min compared to albumin (20.1 percent) and free iodine (8.2 percent). Up to the third hour after injection there was a tendency for venom and albumin to concentrate in the stomach (3rd h), small intestine (3rd h) and large intestine (6th h). Both control groups had more radioactivity in the digestive tract, especially in the stomach, but these levels decreased essentially to baseline by 12-18 h postinjection. In the kidneys, the distribution profiles of venom, albumin and iodine were similar. Counts at 30 min postinjection were low in all three groups (1.37, 1.86 and 0.77, respectively), and diminished to essentially 0 percent by 12-18 h. Albumin tended to concentrate in muscle until the 3rd h postinjection (1.98 percent). There was a low binding of labeled venom is the liver (<0.54 percent), thyroid (<0.11 percent) and lungs (<0.08 percent), and no iodinated venom was detected in brain, heart, diaphragm, spleen or bladder. The low venom binding observed in most internal organs, comparable to that of albumin, suggests that B. erythromelas venom does not specifically target most internal organs. That is, the systemic effects of envenomation are mainly due to an indirect action.