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Abstract: Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by B. asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by B. asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A2, a metalloproteinase and serine proteinases isolated from B. asper venom are also described. [Copyright &y& Elsevier]

Abstract: An animal model of cancer pain induced by injection of Walker 256 carcinoma cells into the plantar surface of rat hind paw is described. Tumor growth and the occurrence of metastasis were investigated by histopathological analysis. Tumor cell growth was also analyzed plethysmographically by the increase in paw volume. For characterization of pain symptoms, hyperalgesia, allodynia, and spontaneous pain were evaluated 5 to 8 days after cell injection. The volume of the inoculated paw started to increase on day 2 after inoculation, being 40% higher on day 5 after injection. At this time, there was a marked proliferation of tumor cells, with the presence of anaplastic and pleomorphic cells, nucleoli, and atypical mitotic features. On days 7 and 8 after injection, histopathological analysis of popliteal lymph nodes showed the presence of tumor cells. The intraplantar injection of Walker 256 cells caused hyperalgesia at day 5 after cell inoculation. Low-threshold mechanical allodynia was significant 2 days after cell injection, being increased on day 5. In addition, inoculation of tumor cells induced gross behavior, characterized by a significant increase in licking and lifting of the injected paw 5 days after injection. The pain-enhancing effect caused by cell inoculation was partially inhibited by indomethacin on day 2 after cell injection, whereas morphine blocked allodynia on days 2 and 5. These results indicate that intraplantar injection of Walker 256 cells cause pain symptoms characteristic of cancer pain. This experimental model can then be used to investigate new analgesic or anti-tumor drugs. Perspective: This article presents a new animal model for studying cancer pain and metastasis. This model could help in understanding the mechanisms involved in cancer pain symptoms and may be used for the investigation of new analgesic or anti-tumor drugs. [Copyright &y& Elsevier]

Previous work has shown that nitric oxide (NO) mediates the antinociceptive effect of Crotalus durissus terrificus venom on carrageenin-induced hyperalgesia. In the present study the role of constitutive neuronal or of inducible form of nitric oxide synthase on venom effect was determined. The rat paw prostaglandin E2 (PGE2)-induced mechanical hyperalgesia model was used for nociceptive evaluation. The venom (200 μg/kg) administered per oz immediately before prostaglandin induced antinociception that persisted for 120 h. The characterisation of the antinociceptive effect of the venom in this model of hyperalgesia showed that κ and δ-opioid receptors are involved in this effect. 7-nitroindazole (7-NI), a neuronal nitric oxide synthase (NOS) inhibitor, but not L-N6-(1-iminoethyl)lysine (L-NIL), an inhibitor of the inducible form of NOS, injected by intraplantar (i.pl.) route, antagonized the antinociceptive effect of the venom. The i.pl. administration of 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a seletive guanylate cyclase inhibitor, blocked antinociception, whereas Rp-cGMP triethylamine, a cGMP-dependent protein kinase inhibitor, partially reversed this effect. These data indicate that peripheral κ- and δ-opioid receptors are involved in the antinociceptive effect of Crotalus durissus terrificus on prostaglandin E2-induced hyperalgesia. Peripheral nitric oxide, generated by neuronal NO synthase, and cGMP/PKc are responsible, at least partially, for the molecular mechanisms of venom effect. [Copyright &y& Elsevier]

Abstract: The mechanisms involved in both local and systemic effects of Loxosceles intermedia (brown spider) venom (LIV) are still poorly understood. We show using rats treated with Evans blue dye (50mg/kg, i.v.) that small doses of the LIV (0.1, 0.3, 1 and 3μg/site) dose-dependently increase the vascular permeability in rats, an effect unchanged by indomethacin (5mg/kg, i.p.), atropine (1mg/kg, i.p.), HOE-140 (2mg/kg, s.c.) or SR140333 (0.3mg/kg, i.p.), but fully avoided by promethazine (15mg/kg, i.p.), methysergide (2mg/kg, i.p.) and compound 48/80 (3mg/kg/day for 3 days). Addition of cumulative concentrations of LIV (0.1–5μg) in phenylephrine-contracted aortic rings resulted in a partial (∼40%) and endothelium-dependent relaxation, inhibited by the nitric oxide synthase inhibitors l-NAME (10μM) and l-NMMA (1mM), and the guanylate cyclase inhibitors methylene blue (100μM) and ODQ (10μM). LIV-induced relaxation was abolished by compound 48/80 (10μM) and pyrilamine (a selective histamine H1 receptor antagonist; 100μM), but not by atropine (1μM) and indomethacin (10μM). Our results disclose that LIV increases vascular permeability and induces vascular relaxation. These effects occur due to its ability to degranulate mast cells and release mediators such as histamine and serotonine. [Copyright &y& Elsevier]

Cnidarians are equipped with nematocysts, which are specialized organelles used to inoculate venom during prey capturing and defense. Their venoms are rich in toxins and a potential source of bioactive compounds, however, poorly explored so far. In this work, the activity of the methanolic extracts from the hydromedusa Olindias sambaquiensis and the cubozoan jellyfish Chiropsalmus quadrumanus were studied in sympathetic neurotransmission. For that, bisected rat vas deferens - a classic model of sympathetic neurotransmission - were incubated with the extracts for further myographic and histopathological analysis. The O. sambaquiensis extract, at 0.1 μg/mL, facilitated the neurogenic contractions of the noradrenergic-rich epididymal portion, while reducing the noradrenaline (NA) potency, which suggests an interaction with postsynaptic α 1 -adrenoceptors. On the other hand, a higher concentration (1 μg/mL) leads to time- and frequency-dependent blockade of nerve-evoked contractions without significantly changing the response to exogenous NA. In turn, the C. quadrumanus extract at 0.1 μg/mL induced blockade of nerve-evoked noradrenergic contractions while reducing the potency to exogenous NA. Both extracts did not affect the purinergic neurotransmission or induce muscle damages. Our results demonstrate that O. sambaquiensis and C. quadrumanus extracts significantly interfere with the noradrenergic neurotransmission without altering purinergic response or smooth muscle structure on rat vas deferens. Such results bring to light the pharmacological potential of O. sambaquiensis and C. quadrumanus molecules for therapeutics focusing on noradrenergic neurotransmission. [Display omitted] • Cnidarian venom has several toxins/molecules with potential to be used for research and therapeutic proposes. • Both extracts show benefits by interfering with noradrenergic neurotransmission without acting on the purinergic response. • At the tested concentrations, both extracts affected sympathetic neurotransmission without damaging the smooth muscle cells. [ABSTRACT FROM AUTHOR]

The use of molecules inspired by natural scaffolds has proven to be a very promising and efficient method of drug discovery. In this work, capsaicin, a natural product from Capsicum peppers with antitumor properties, was used as a prototype to obtain urea and thiourea analogues. Among the most promising compounds, the thiourea compound 6g exhibited significant cytotoxic activity against human melanoma A2058 cells that was twice as high as that of capsaicin. Compound 6g induced significant and dose-dependent G 0 /G 1 cell cycle arrest in A2058 cells triggering cell death by apoptosis. Our results suggest that 6g modulates the RAF/MEK/ERK pathway, inducing important morphological changes, such as formation of apoptotic bodies and increased levels of cleaved caspase-3. Compared to capsaicin, 6g had no significant TRPV1/6 agonist effect or irritant effects on mice. Molecular modeling studies corroborate the biological findings and suggest that 6g , besides being a more reactive molecule towards its target, may also present a better pharmacokinetic profile than capsaicin. Inverse virtual screening strategy found MEK1 as a possible biological target for 6g. Consistent with these findings, our observations suggested that 6g could be developed as a potential anticancer agent. [ABSTRACT FROM AUTHOR]

Noninvasive imaging using positron emission tomography/computed tomography (PET/CT) and single photon emission computed tomography/computed tomography (SPECT/CT) are considered revolutionized approaches to detect bone cancer. Both PET/CT and SPECT/CT technologies have advanced to permit miniaturization, which has provided the advantage of including animals as their own controls in longitudinal studies. The present study was designed to evaluate the potential of PET/CT and SPECT/CT as research tools to detect bone cancer in rats. We used a rat model of bone cancer induced by injecting Walker 256 tumor cells into the femoral cavity. Computed tomography demonstrated that rats presented a solid tumor at 15 days post injection (dpi). However, CT was not an effective method for identifying tumors at an earlier time point (8 dpi), when mechanical hyperalgesia (the most common symptom during bone cancer progression) had already initiated. At this early stage, PET/CT and SPECT/CT analysis detected higher uptake in the injected femur of the tracers 18 F-Fluoride and 99m Tc-Methyl diphosphonate ( 99m Tc-MDP), respectively. These findings demonstrated for the first time that both 18 F-Fluoride PET/CT and 99m Tc-MDP SPECT/CT can detect cancer at early stages in rats and advocates for the PET/SPECT/CT as research tools to evaluate bone cancer in further longitudinal studies involving small animals. [ABSTRACT FROM AUTHOR]

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus , which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA 4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA 4 is a key mediator of the immunosuppressive effects of CTX. [Display omitted] • CTX impairs peripheral lymphocyte recirculation via formyl peptide receptors (FPR). • FPR are involved in CTX-induced leukocyte-endothelium adhesion. • FPR mediates CTX-induced CD11a/CD18 up-regulation. • CTX impairs lymphocyte proliferation and antibody production via FPR. • CTX stimulates lymphocytes to release 15-epi-LXA4. [ABSTRACT FROM AUTHOR]

Abstract: Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1h after treatment and lasted for up to 48h. Intraplantar injection of nor-binaltorphimine (50g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain. [Copyright &y& Elsevier]

Abstract: Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric β-neurotoxin that consists of a weakly toxic basic phospholipase A2 and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure–function studies and investigations that have led to the identification of novel pharmacological activities for the toxin. [Copyright &y& Elsevier]

Abstract: Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine6-bradykinin (Thr6-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr6-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B2 receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr6-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B1 receptor antagonist, while the effect of both cyphokinin and Thr6-BK was reversed by B2 antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms. [Copyright &y& Elsevier]

Abstract: Crotoxin (CTX), a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg, i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). On the other hand, indomethacin (4 mg/kg, i.v.), naloxone (1 mg/kg, i.p.), and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of α-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin. [Copyright &y& Elsevier]

Abstract: We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of κ- and δ-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be

Abstract: Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1–2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A2 (PLA2). PLA2, but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte–endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin. [Copyright &y& Elsevier]

Intravenous administration of antibothropic antivenom (BAv) neutralises the systemic effects, but does not efficiently reverse the local symptoms elicited by the Bothrops jararaca venom (BjV). The mechanisms involved in this poor protection have not been clarified. In this work, intravital microscopy studies were carried out to determine the efficacy of different schedules of BAv treatment on local effects evoked by topical application of BjV in the microcirculatory network of the internal spermatic fascia of Wistar rats. Results demonstrated that BAv administration 15 min before, simultaneously with, or 15 min after BjV application did not totally reverse the local symptoms, represented by disturbances of coagulation, development of haemorrhage lesions, vascular permeability increase and increment on leukocyte–endothelium interactions. This lack of effectiveness neither reflects an inadequate amount of specific antibodies in the antivenom against toxins responsible for local effects nor an insufficient dose of circulating BAv during the assays. Administration of fluorescein isothiocyanate (FITC) labelled-BAv showed the dynamics of distribution of the antivenom in the microcirculatory network. Images obtained from prior and simultaneously treated animals showed that the antivenom remains at luminal side of vessels before venom application, and the latency time to antivenom leakage is coincidental to that for local effects evoked by the venom. In addition, images from posterior treatment demonstrated that the intense alterations in the microcirculatory network impair antivenom distribution at the site of injection. Together, our data show that the lack of effectiveness of antivenom therapy is due to impaired and delayed venom and antivenom interaction at the site of injury. [Copyright &y& Elsevier]