Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Background:Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied.Methods:The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line.Results:The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis.Conclusions:Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)

Antiproliferative activity of marine stingray Dasyatis sephenvenom on human cervical carcinoma cell line

Background Venoms comprise mixtures of numerous bioactive compounds that have a wide range of pharmacologic actions. Toxins from venomous animals have attracted the attention of researchers because of their affinity for primary sites responsible for lethality and their efficacy at extremely low concentrations. The venoms of marine stingrays have not been extensively studied and limited data is available on them. The present study aims to evaluate the antiproliferative and biochemical properties of the venom obtained from a species of marine stingray (Dasyatis sephen) on human cervical cancer cell line HeLa.MethodsThe antiproliferative effect of D. sephen venom was determined by MTT assay, and the oxidative stress was determined by lipid peroxidation method along with assessment of changes in the enzymatic and non-enzymatic antioxidant status. We observed intracellular reactive oxygen species (ROS) levels by DCFH-DA method, mitochondrial membrane potential alterations by rhodamine 123 staining and apoptotic morphological changes by acridine orange/ethidium bromide dual staining method.ResultsD. sephen venom enhances lipid peroxidative markers such as thiobarbituric acid reactive substance, conjugated diene, and lipid hydroperoxide in HeLa cell lines. Stingray venom enhances the ROS levels, which is evidenced by the increased 27-diacetyl dichlorofluorescein fluorescence. Further, D. sephen venom treatment altered the mitochondrial membrane potential in HeLa cells. Additionally, we observed increased apoptotic morphological changes in D. sephen venom-treated groups. ConclusionsDasyatis sephen venom exhibits potent antiproliferative effect on HeLa cell line and upon further purification it could be a promising antiproliferative agent.(AU)

Antiproliferative activity of marine stingray Dasyatis sephenvenom on human cervical carcinoma cell line

Background Venoms comprise mixtures of numerous bioactive compounds that have a wide range of pharmacologic actions. Toxins from venomous animals have attracted the attention of researchers because of their affinity for primary sites responsible for lethality and their efficacy at extremely low concentrations. The venoms of marine stingrays have not been extensively studied and limited data is available on them. The present study aims to evaluate the antiproliferative and biochemical properties of the venom obtained from a species of marine stingray (Dasyatis sephen) on human cervical cancer cell line HeLa.MethodsThe antiproliferative effect of D. sephen venom was determined by MTT assay, and the oxidative stress was determined by lipid peroxidation method along with assessment of changes in the enzymatic and non-enzymatic antioxidant status. We observed intracellular reactive oxygen species (ROS) levels by DCFH-DA method, mitochondrial membrane potential alterations by rhodamine 123 staining and apoptotic morphological changes by acridine orange/ethidium bromide dual staining method.ResultsD. sephen venom enhances lipid peroxidative markers such as thiobarbituric acid reactive substance, conjugated diene, and lipid hydroperoxide in HeLa cell lines. Stingray venom enhances the ROS levels, which is evidenced by the increased 27-diacetyl dichlorofluorescein fluorescence. Further, D. sephen venom treatment altered the mitochondrial membrane potential in HeLa cells. Additionally, we observed increased apoptotic morphological changes in D. sephen venom-treated groups. ConclusionsDasyatis sephen venom exhibits potent antiproliferative effect on HeLa cell line and upon further purification it could be a promising antiproliferative agent.

Araneísmo

Existem mais de 30 mil espécies de aranhas descritas no mundo, sendo agrande maioria peçonhenta. As aranhas de interesse veterinário no Brasil pertencem aos gêneros Loxosceles e Phoneutria.

Acidente crotálico

As serpentes do gênero Crotalus, conhecidas popularmente como cascavel, boicininga e maracamboia(Fig. 1), são robustas, não agressivas e pouco ágeis.Possuem fosseta loreal,dentição solenóglifa e um chocalho ou guizo na extremidade da cauda(Fig. 2).

Acidente crotálico

As serpentes do gênero Crotalus, conhecidas popularmente como cascavel, boicininga e maracamboia(Fig. 1), são robustas, não agressivas e pouco ágeis.Possuem fosseta loreal,dentição solenóglifa e um chocalho ou guizo na extremidade da cauda(Fig. 2).

Araneísmo

Existem mais de 30 mil espécies de aranhas descritas no mundo, sendo agrande maioria peçonhenta. As aranhas de interesse veterinário no Brasil pertencem aos gêneros Loxosceles e Phoneutria.

Acidente Botrópico

O gênero Bothrops continha mais de 60 espécies, distribuídas desde o norte do México até o sul da Argentina, mas várias espécies foram redistribuídas em outros sete gêneros. O Brasil possui a maioria das espécies remanescentes do gênero Bothrops, mas apenas duas espécies do gênero Bothriopsis (B. bilineata e B. taeniata) e duas de Bothrocophias (B. hyoprora e B. microphtalmus).

Acidente Botrópico

O gênero Bothrops continha mais de 60 espécies, distribuídas desde o norte do México até o sul da Argentina, mas várias espécies foram redistribuídas em outros sete gêneros. O Brasil possui a maioria das espécies remanescentes do gênero Bothrops, mas apenas duas espécies do gênero Bothriopsis (B. bilineata e B. taeniata) e duas de Bothrocophias (B. hyoprora e B. microphtalmus).

Apidismo

As abelhas Apis mellifera são insetos sociais utilizados, mas não domesticados,pelo homem há centenas de anos. Possuem grande importância econômica pela produção de mel, pólen, própolise geleia real e pela polinização de plantas frutíferas cultivadas. Inicialmente,foram introduzidas no Brasil variedades europeias,que se caracterizam por baixa agressividade.No entanto, colônias de abelhas da variedade africana(A. mellifera scutellata), que possuem maior agressividade que as variedades europeias, escaparam de colmeias experimentais da cidade de Rio Claro, SP,em 1957, e gradativamente se difundiram pelo continente americano, cruzando com as variedades europeias. Atualmente,as abelhas A. melliferacriadas no Brasil são híbridas africanizadas.

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Os escorpiões são artrópodes quelicerados da ordem Scorpiones, sendo conhecidos aproximadamente 1.500 espécies agrupadas em 20 famílias e 165 gêneros, sendo a família Buthidaea de maior importância toxicológica.Estão distribuídos por todos os continentes,exceto a Antártida, mas a maior concentração está nas regiões tropicais e subtropicais. Habitam diversificados ambientes, incluindo desertos,cavernas profundas e altitudes superiores a 4.200m, como nos Andes e no Himalaia.

Apidismo

As abelhas Apis mellifera são insetos sociais utilizados, mas não domesticados,pelo homem há centenas de anos. Possuem grande importância econômica pela produção de mel, pólen, própolise geleia real e pela polinização de plantas frutíferas cultivadas. Inicialmente,foram introduzidas no Brasil variedades europeias,que se caracterizam por baixa agressividade.No entanto, colônias de abelhas da variedade africana(A. mellifera scutellata), que possuem maior agressividade que as variedades europeias, escaparam de colmeias experimentais da cidade de Rio Claro, SP,em 1957, e gradativamente se difundiram pelo continente americano, cruzando com as variedades europeias. Atualmente,as abelhas A. melliferacriadas no Brasil são híbridas africanizadas.

Acidentes por sapos

A classe Amphibia é composta pelasordens Anura (sapos, rãs e pererecas),Caudata (salamandras) e Gymnophiona(cobras-cegas). A ordem Anura é formada por 5.602 espécies, sendo 804 pertencentes à fauna brasileira.Todos os anfíbios produzem veneno, com grande variabilidade de toxicidade, sendo as espécies mais letais aquelas do gênero Phyllobates.

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O acidente elapídicoé causado por serpentes da família Elapidae,gêneros Micrurus e Leptomicrurus. No Brasil, há 19 espécies de Micrurus e três de Leptomicrurus (Quadro 1). As espécies mais importantes são Micrurusfrontalis (cobra-coral,coral), Micrurus corallinus(cobra-coral, coral--verdadeira, boicorá)e Micrurus ibiboboca(coral-venenosa, coral--verdadeira). São serpentes de tamanho mediano(300-1.200mm),que apresentam anéis corporais de colorações pretas, amarelas, brancas e vermelhas. Os olhos são pequenos com pupilas arredondadas.Possuem dentição proteróglifa, então têm fosseta loreal. Uma característica marcante é o comportamento de levantarem a cauda, de forma semienrolada (engodocaudal), quando se sentem ameaçadas.

Acidentes por sapos

A classe Amphibia é composta pelasordens Anura (sapos, rãs e pererecas),Caudata (salamandras) e Gymnophiona(cobras-cegas). A ordem Anura é formada por 5.602 espécies, sendo 804 pertencentes à fauna brasileira.Todos os anfíbios produzem veneno, com grande variabilidade de toxicidade, sendo as espécies mais letais aquelas do gênero Phyllobates.

Mad, bad and dangerous to know: the biochemistry, ecology and evolution of slow loris venom

Only seven types of mammals are known to be venomous, including slow lorises (Nycticebus spp.). Despite the evolutionary significance of this unique adaptation amongst Nycticebus, the structure and function of slow loris venom is only just beginning to be understood. Here we review what is known about the chemical structure of slow loris venom. Research on a handful of captive samples from three of eight slow loris species reveals that the protein within slow loris venom resembles the disulphide-bridged heterodimeric structure of Fel-d1, more commonly known as cat allergen. In a comparison of N. pygmaeus and N. coucang, 212 and 68 compounds were found, respectively. Venom is activated by combining the oil from the brachial arm gland with saliva, and can cause death in small mammals and anaphylactic shock and death in humans. We examine four hypotheses for the function of slow loris venom. The least evidence is found for the hypothesis that loris venom evolved to kill prey. Although the venom's primary function in nature seems to be as a defense against parasites and conspecifics, it may also serve to thwart olfactory-orientated predators. Combined with numerous other serpentine features of slow lorises, including extra vertebra in the spine leading to snake-like movement, serpentine aggressive vocalisations, a long dark dorsal stripe and the venom itself, we propose that venom may have evolved to mimic cobras (Naja sp.). During the Miocene when both slow lorises and cobras migrated throughout Southeast Asia, the evolution of venom may have been an adaptive strategy against predators used by slow lorises as a form of Müllerian mimicry with spectacled cobras.(AU)

Aspectos clinicopatológicos e laboratoriais do envenenamento experimental por Bothrops moojeni e Bothropoides neuwiedi em ovinos / Clinicopathological and laboratory aspects of experimental poisoning by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep

O envenenamento ofídico espontâneo, ou acidente ofídico, é descrito como causa de morte em animais domésticos. No entanto, dados concretos relativos ao gênero e espécie de serpente envolvida, à evolução do quadro clínico, e às alterações clinicopatológicas desenvolvidas, são escassos. Assim sendo, este trabalho teve como objetivo determinar as alterações clinicopatológicas e laboratoriais provocadas pelo veneno de Bothrops moojeni e Bothropoides neuwiedi em ovinos no intuito de fornecer informações adicionais referentes a acidentes ofídicos em animais de produção, auxiliando o estabelecimento do diagnóstico dessa condição. Os venenos liofilizados foram diluídos em 1 ml de solução fisiológica e administrados a quatro ovinos por via subcutânea na face direita, nas doses de 0,41mg/kg e 0,82mg/kg do veneno de B. moojeni em dois ovinos, e de 1,0mg/kg do veneno de B. neuwiedi em dois ovinos. Apenas o ovino que recebeu a menor dose (0,41mg/kg) sobreviveu, apesar de ter desenvolvido quadro clínico muito severo e semelhante aos demais. Os sinais clínicos iniciaram nos primeiros 10 minutos após a inoculação em todos os ovinos. O período de evolução variou de dois a quatro dias. O quadro clínico dos quatro ovinos caracterizou-se por apatia, acentuado aumento de volume da face, da porção ventral do pescoço e do peito, leve aumento de volume da porção proximal dos membros anteriores, tempo de sangramento aumentado, taquicardia, mucosas pálidas e grande quantidade de sangue não digerido nas fezes. Ao exame laboratorial observou-se principalmente redução das proteínas plasmáticas e aumento de creatinaquinase em todos os ovinos. À necropsia, foram observados extensos hematomas nas áreas correspondentes ao aumento de volume subcutâneo. Observaram-se petéquias, equimoses e sufusões leves a moderadas na serosa de diversos órgãos e acúmulo de sangue em meio às fezes na porção final do reto. Além de hemorragias, a principal alteração histopatológica observada foi necrose das fibras musculares esqueléticas e da parede de vasos, nas áreas próximas à inoculação do veneno. Nos ovinos deste estudo o aumento de volume, observado na face, pescoço, peito e membros, era constituído por sangue.(AU)

Spontaneous envenoming by snake bite is described as a cause of death in domestic animals. However, there are just few information about the species of snake involved, course, and clinicopathological and laboratory findings. Thus, this research aimed to determine the clinicopathological and laboratory changes induced by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep, in order to provide additional information regarding snakebites in farm animals and to help establish the diagnosis of this condition. The lyophilized snake venoms were dissolved in 1mL saline solution and administered subcutaneously into the right face of four sheep, at doses of 0.41mg/kg and 0.82mg/kg of B. moojeni venom for two sheep, and 1.0mg/kg of B. neuwiedi venom for two other sheep. Only the sheep which had received the lowest dose (0.41mg/kg) survived, but developed severe clinical signs, similar to the others. First clinical signs were observed about 10 minutes after inoculation in all sheep. The course varied from 2 to 4 days. The clinical findings in all sheep were characterized by apathy, marked swelling of the face, the ventral neck and esternal region, and mild swelling of the proximal portion of the forelimbs, as well as increased bleeding time, tachycardia, pale mucous membranes, and large quantity of undigested blood in the intestinal lumen. Laboratory exams showed mainly a reduction in serum protein and increased creatine kinase in all sheep. At necropsy, extensive hematomas were observed in the subcutaneous tissue of the swollen areas. Also petechiae, bruises and mild to moderate hemorrhagic suffusions on the serosa of various organs, and blood within the intestinal contents of the distal rectum were observed. In addition to hemorrhages, the main histopathological changes were necrosis of skeletal muscle fibers and blood vessel walls next to the inoculation site. The swollen areas on face, neck, sternum and limbs of the sheep were due the hematomas.(AU)

Biochemical and morphological analysis of cell death induced by Egyptian cobra (Naja haje) venom on cultured cells

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.(AU)