Ivo Lebrun

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Behavioral, Electroencephalographic, and Histopathologic Effects of a Neuropeptide Isolated From Tityus serrulatus Scorpion Venom in Rats

The effects of intrahippocampal administration of a neuropeptide (TS-8F toxin) isolated from Tityus serrulatus scorpion venom have been determined on behavior, limbic seizures, and neuronal degeneration in rats. Behavioral observation showed orofacial automatism, wet dog shakes, and myoclonus. Concomitantly, the electroencephalographic record showed high-frequency and high-voltage spikes that evolved to seizure activity in the hippocampus and cortex. Seven days after TS-8F toxin microinjection, neuronal damage was observed in CA1 and CA2 pyramidal cells and in granular cells of the dentate gyrus. The results suggest that TS-8F toxin may be responsible, at least in part, by the epileptic effects observed with the crude venom. Thus, this toxin may be a useful tool in the study of some neurobiological process.

A new bradykinin-potentiating peptide (peptide P) isolated from the venom of Bothrops jararacussu (jararacuçu tapete, urutu dourado)

Several bradykinin potentiators were identified in the venom of Bothrops jararacussu by chromatographic techniques and biological assays. One of them which was isolated inhibited the angiotensin-converting enzyme in vitro and potentiated the bradykinin-induced lowering of the arterial pressure in the rat.

Immunomodulatory effects of the Tityus serrulatus venom on murine macrophage functions in vitro.

Tityus serrulatus scorpion venom (TSV) consists of a very complex mixture of molecules and demonstrates significant immunomodulatory activities capable of stimulating immune functions in vivo. The purpose of this study was to compare the crude TSV with fractionated toxins extracted from this venom in order to determine which toxin(s) presented immunomodulatory effects on peritoneal macrophages. TSV was fractionated using gel filtration chromatography resulting in 5 heterogeneous fractions. The effects of these different fractions were analysed in vitro using detection by means of cytokines, oxygen intermediate metabolites (H2O2), and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed: tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity in L929 cells, and other cytokines were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages exposed to different fractions. In vitro studies revealed that all fractions studied here presented an increment in H2O2, NO, and cytokines levels. The more pronounced increments were observed in macrophage cultures exposed to fraction FII which demonstrated that (a) the highest levels of IL-1α, IL-β, and TNF were observed after 12 hours and that (b) the maximum levels of IFN-γ and NO were observed after 72 hours. Taken together, these data indicate that fractions have a differential immunomodulating effect on macrophage secretion, and that FII is a potent activator of TNF production of macrophages.

A microdialysis study of glutamate concentration in the hippocampus of rats after TsTX toxin injection and blockade of toxin effects by glutamate receptor antagonists

Scorpion toxins act on ionic channels changing the release of neurotransmitters. In the present study, we investigated the glutamatergic release evoked by intrahippocampal injection of TsTX toxin isolated from Tityus serrulatus scorpion venom in male Wistar rats and the blockade of the toxin effect by glutamatergic antagonists. Microdialysis for neurotransmitter level quantification, electroencephalographic recording, and histopathological analysis were performed. The microdialysis method revealed enhanced levels of extracellular glutamate in the hippocampal area. The toxin injection preceded by injection of the glutamate receptor antagonists dizolcipine maleate (MK-801), D(-)2-amino-5-phosphonopentanoic acid (AP-5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), L(+)-2-amino-3-phosphonopropionic acid (AP-3), and (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) demonstrated that MK-801 and AP-5 fully blocked the electrographic alterations and the CA1 cell loss induced by the toxin. CNQX, AP-3, and MCPG partially blocked the epileptiform discharges and no hippocampal damage was observed. Thus, we conclude that the toxin evokes glutamate release and that glutamate receptor antagonists can partially or totally block the toxin effect.

Central effects of Tityus serrulatus and Tityus bahiensis scorpion venoms after intraperitoneal injection in rats.

A great number of studies on scorpion venoms associate their effects to the autonomic nervous system, and few data are available about their action on the central nervous system (CNS). The aim of this work was to evaluate some central effects after intraperitoneal injection of Tityus serrulatus or T. bahiensis scorpion venoms. The hippocampal concentration of some neurotransmitters and their metabolites were determined. Electroencephalographic and behavioral observations were performed, and all brains were removed for histopathological analysis of hippocampal areas. Both venoms induced electrographic and behavioral alterations despite T. bahiensis venom affects less the electrographic activity than T. serrulatus venom. Neurochemical analysis demonstrated no alteration in the extracellular levels of almost all the neurotransmitters evaluated, at least in the hippocampus, and no neuronal loss in this area was observed. Meanwhile, extracellular concentration of HVA increased up to 10 times in approximately 1/3 of the animals of both groups. Scorpion venoms seem to exert a small but important central effect. More studies in this field are necessary because they may be useful in developing new strategies to reduce the damage caused by scorpion stings.

TsTx Toxin Isolated from Tityus serrulatus Scorpion Venom‐Induced Spontaneous Recurrent Seizures and Mossy Fiber Sprouting

Summary:  Purpose: TsTx is a scorpion α‐type toxin that binds to site 3 of the Na+ channels in a voltage‐dependent mode, slowing or blocking the inactivation mechanism of these channels (Possani et al., Eur J Biochem 1999). This binding increases depolarization time of the channel and consequently induces excessive neurotransmitter release. Previously we reported that hippocampal injection of TsTx induces clonic convulsions, electrographic seizures, and hippocampal damage. This investigation was designed to characterize the long‐term behavioral, electroencephalographic (EEG), and histopathologic features after a single TsTx injection into the rat hippocampus.

Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo

Mastoparan is an α-helical and amphipathic tetradecapeptide obtained from the venom of the wasp Vespula lewisii. This peptide exhibits a wide variety of biological effects, including antimicrobial activity, increased histamine release from mast cells, induction of a potent mitochondrial permeability transition and tumor cell cytotoxicity. Here, the effects of mastoparan in malignant melanoma were studied using the murine model of B16F10-Nex2 cells. In vitro, mastoparan caused melanoma cell death by the mitochondrial apoptosis pathway, as evidenced by the Annexin V-FITC/PI assay, loss of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species, DNA degradation and cell death signaling. Most importantly, mastoparan reduced the growth of subcutaneous melanoma in syngeneic mice and increased their survival. The present results show that mastoparan induced caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the mice against tumor development.

Neurotoxic effects of fractions isolated from Tityus bahiensis scorpion venom (Perty, 1834).

Tityus serrulatus and Tityus bahiensis are considered to be the most venomous scorpions in Brazil and are responsible for most of the accidents that occur in our country. The main toxic agents in scorpion venoms are small basic polypeptides that act as neurotoxins. They cause a derangement of ion channels that result in abnormal release of neurotransmitters. In the present study we fractionated the venom of Tityus bahiensis and studied the effects of fractions P2, P3, P4, P5, P6 and P7, on the mammalian central nervous system. Intravenous injection of P5, P6 and P7 in rats induced spontaneous convulsion, intrahippocampal injection caused behavioural seizures, and P5 and P6 induced electrographic seizures. P5 caused neuronal damage in the CA1 area and P6 in the CA1, CA3 areas and hilus of the dentate gyrus (DG) of the hippocampus. Injection of P3 in the hippocampus did not induce convulsions or lesions. However, when injected intravenously in mice, this fraction reduced behavioural activity in an open field test. Unilateral injection of P4 in the hippocampus caused neuronal damage in the contralateral CA3, but not in the ipsilateral hippocampus. These results suggest that scorpion toxins present in the venom are able to act directly on the central nervous system promoting behavioural and histopathological effects.