The Cloning and Characterization of a Three-Finger Toxin Homolog (NXH8) from the Coralsnake Micrurus corallinus That Interacts with Skeletal Muscle Nicotinic Acetylcholine Receptors.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve-diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Cordia salicifolia and Lafoensia pacari plant extracts against the local effects of Bothrops jararacussu and Philodryas olfersii snake venoms.

Philodryas olfersii produces similar local effects to Bothrops jararacussu snakebite, which can induce misidentification and bothropic antivenom administration. Antivenom therapy is effective, but has its limitations regarding local damage. Since plants are used in folk medicine to treat snakebite victims, we evaluated the protective properties of Cordia salicifolia and Lafoensia pacari extracts against Philodryas olfersii and Bothrops jararacussu venoms. Preparations pretreated with both extracts inhibited > 90% the B. jararacussu venom-induced neuromuscular blockade, and 52% to 81% the P. olfersii venom-induced blockade. C. salicifolia inhibited the myonecrosis promoted by both venoms; however, L. pacari prevented only the myofilaments hypercontraction. Regarding haemorrhagic activity, C. salicifolia was more effective against B. jararacussu venom, while L. pacari was more effective against P. olfersii venom. On the other hand, for oedema-forming activity the results were the opposite. Considering that both extracts prevented (to different levels) the main manifestations of both snakebites (local symptoms), we endorse further studies involving these plants as coadjuvant in snakebite therapeutics.

New record of Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) from Brazil with an illustrated key to species for the genus.

The cheyletid mites that parasitize mammals have been neglected for a long time in Brazil, although they can be common on pets and cause injury to their hosts. Recently, Cheyletiella parasitivorax was found parasitizing a rabbit in Brazil which represents a new host and distribution record for the mite species. An illustrated dichotomous key for the identification of the species in this genus and data from the literature are provided.

New record of Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) from Brazil with an illustrated key to species for the genus / Novo registro de Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) para o Brasil, com uma chave ilustrada das espécies deste gênero

The cheyletid mites that parasitize mammals have been neglected for a long time in Brazil, although they can be common on pets and cause injury to their hosts. Recently, Cheyletiella parasitivorax was found parasitizing a rabbit in Brazil which represents a new host and distribution record for the mite species. An illustrated dichotomous key for the identification of the species in this genus and data from the literature are provided.

New record of Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) from Brazil with an illustrated key to species for the genus / Novo registro de Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) para o Brasil, com uma chave ilustrada das espécies deste gênero

The cheyletid mites that parasitize mammals have been neglected for a long time in Brazil, although they can be common on pets and cause injury to their hosts. Recently, Cheyletiella parasitivorax was found parasitizing a rabbit in Brazil which represents a new host and distribution record for the mite species. An illustrated dichotomous key for the identification of the species in this genus and data from the literature are provided.

Os ácaros da família Cheyletidae que parasitam mamíferos são negligenciados há muito tempo no Brasil, embora eles sejam comuns em animais domésticos. Considerando as dificuldades morfológicas para diagnosticar as espécies dessa família que infestam mamíferos, este estudo refere-se a uma nova ocorrência de Cheyletiella parasitivorax incluindo os poucos registros de literatura. Além disso, também está sendo apresentada uma chave dicotômica ilustrada para identificação de espécies desse gênero.

Impact of Moderate Aerobic Training on Physical Capacities of Hypertensive Obese Elderly.

The association of old age and chronic conditions, such as hypertension and obesity, can lead to larger decreases in the physical capacities of elderly, compared with their healthy counterparts. Physical exercise has been demonstrated to be efficient in postponing this phenomenon, mainly strength training. However, little is known about the effect of aerobic training on this condition. The aim of this work was to investigate the impact of 12 weeks of moderate-intensity aerobic training on the physical capacities of hypertensive obese older women. Aerobic power, lower limb muscle power, upper limb muscle strength, endurance, and flexibility of 19 hypertensive obese elders were evaluated. Afterward, patients were blindly randomized into control group (CG) and exercise group (EG). EG underwent three sessions/week of 60 min of moderate-intensity aerobic training, during 12 weeks. EG showed increases in VO2max compared with CG (p = .03) and increases in flexibility compared with basal moment (+21.6%; p = .01) after 12 weeks, whereas CG did not show any significant alterations. Moderate aerobic training is capable of inducing increases in maximal aerobic power and flexibility in hypertensive obese elderly. However, other essential physical capacities associated with independence in elderly people (i.e., muscle power and strength) were not responsive to this kind of protocol.

Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom.

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30 µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3 µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10 µg/ml) caused neuromuscular blockade in PND preparations within ~ 10 min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10 µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3 µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10 µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom.

Effects of Two Fractions of Swietenia macrophylla and Catechin on Muscle Damage Induced by BothropsVenom and PLA2.

Plant natural products can attenuate the myonecrosis caused by Bothrops snake venom and their phospholipases A2 (PLA2). In this study, we evaluated the effects of two fractions (F4 and F6) from Swietenia macrophylla and purified catechin on the muscle damage caused by a myotoxic PLA2 from Colombian Bothrops asper venom (BaColPLA2) in mice and by Bothrops marmoratus venom from Brazil in mouse phrenic nerve-diaphragm muscle (PND) preparations in vitro. Male mice were injected with PLA2 (50 µg) in the absence or presence of F4, F6, and catechin, in the gastrocnemius muscle and then killed 3, 7, 14, and 28 h later for histopathological analysis of myonecrosis, leukocyte infiltration, and the presence of collagen. Fractions F4 and F6 (500 µg) and catechin (90 µg) significantly reduced the extent of necrosis at all-time intervals. These two fractions and catechin also attenuated the leukocyte infiltration on day 3, as did catechin on day 14. There was medium-to-moderate collagen deposition in all groups up to day 7, but greater deposition on days 14 and 28 in the presence of F6 and catechin. Bothrops marmoratus venom (100 µg/mL) caused slight (~25%) muscle facilitation after 10 minutes and weak neuromuscular blockade (~64% decrease in contractile activity after a 120-minute incubation). Pre-incubation of venom with F4 or F6 abolished the facilitation, whereas catechin, which was itself facilitatory, did not. All three fractions attenuated the venom-induced decrease in muscle contractions. These findings indicate that fractions and catechin from S. macrophylla can reduce the muscle damage caused by Bothrops venom and PLA2. These fractions or their components could be useful for treating venom-induced local damage.

Atividade antiofídica de Camellia sinensis contra a ação tóxica de venenos botrópicos em preparação neuromuscular de ave / Antiophidic activity of Camellia sinensis against the toxic action of bothropic venoms in bird neuromuscular preparations

Introdução: venenos de serpentes do gênero Bothrops são capazes de induzir bloqueio neuromuscular em preparações isoladas de mamífero e ave. O tratamento específico é a soroterapia, porém, nem sempre se mostra eficaz contra a toxicidade induzida pelo veneno. Por outro lado, compostos vegetais têm sido estudados com finalidade antiofídica, como é o caso da Camellia sinensis (chá verde). Objetivo: o objetivo foi de avaliar a atividade do extrato alcoólico de Camellia sinensis contra a ação de diferentes venenos botrópicos em preparações neuromusculares de ave. Métodos: foram utilizadas preparações neuromusculares de biventer cervicis de pintainho (BC) que foram incubadas com: venenos de diferentes serpentes do gênero Bothrops (B. jararaca, B. jararacussu, B. alternatus e B. neuwiedi, 50-100 µg/mL), extrato alcoólico de Camellia sinensis (Cs) 100 µg/ml ou pré-tratadas com Cs (30min) seguida da adição dos diferentes venenos botrópicos. Resultados: ao final do experimento, foi observado bloqueio da resposta contrátil em 69,7 ± 4,3% (B. jararaca), 83,1 ± 10% (B. jararacussu), 88,1 ± 4,5% (B. alternatus) e 92,4 ± 5,6% (B. neuwiedi). Quando as preparações foram pré incubadas com o extrato de Cs, as proteções foram de: 62%, 89,2%, 75,1% e 78,2%, respectivamente. Conclusão: conclui-se que o extrato de Camellia sinensis se mostrou eficaz contra a neurotoxicidade induzida por venenos botrópicos

Introduction: venoms from snakes of the Bothrops genus are capable of inducing neuromuscular blockade in preparations isolated from mammals and birds. The specific treatment is serum therapy, but it is not always effective against the toxicity induced by the venom. On the other hand, some plant compounds have been studied for antiophidic purposes, as is the case with Camellia sinensis (green tea). Objective: evaluate the activity of the alcoholic extract from Camellia sinensis against the action of various bothropic venoms in bird neuromuscular preparations. Methods: Chick biventer cervicis (BC) neuromuscular preparations were used which were incubated with venoms from various snakes of the Bothrops genus (B. jararaca, B. jararacussu, B. alternatus and B. neuwiedi, 50-100 µg/ml), Camellia sinensis (Cs) alcoholic extract, 100 µg/ml, or previously treated with Cs (30 min), followed by the addition of the various bothropic venoms. Results: blockade of the contractile response was observed in 69.7 ± 4.3% (B. jararaca), 83.1 ± 10% (B. jararacussu), 88.1 ± 4.5% (B. alternatus) and 92.4 ± 5.6% (B. neuwiedi). When the preparations were previously incubated with Cs extract, results were 62%, 89.2%, 75.1% and 78.2%, respectively. Conclusion: Camellia sinensis extract was found to be effective against neurotoxicity induced by bothropic venoms.

Moderate Aerobic Training Decreases Blood Pressure but No Other Cardiovascular Risk Factors in Hypertensive Overweight/Obese Elderly Patients.

Hypertension and obesity are prevalent diseases in elderly people, and their combination can cause deleterious effects on physiological system. Moderate intensity aerobic training (MIAT) seems to be a beneficial approach to control and treat these diseases separately. However, few studies have investigated the impact of MIAT on cardiovascular risk factors associated with these conditions (i.e., elevated blood pressure values, blood markers, and body composition). Therefore, the present study was designed to investigate the effects of MIAT on blood pressure, blood markers, and body composition in hypertensive overweight/obese elderly patients. Twenty-four hypertensive overweight/obese elderly patients were randomized into control group (CG) and training group (TG), submitted to 12 weeks of MIAT of 50 min, 3 days per week, at 60% of maximal HR (heart rate). There was a decrease in diastolic blood pressure (-10.1 ± 3.3; p = .01; effect size = 1.29) and mean arterial pressure (MAP; -8.2 ± 3.7; p = .04; effect size = 0.94) following 12 weeks of training in the TG as compared with baseline. There was an increase in triacylglycerol levels in the TG (+0.1 ± 0.0; p = .02). There were no significant changes in body composition for both groups. The present study revealed that 12 weeks of MIAT can decrease blood pressure in hypertensive obese elderly patients, with no significant modifications in blood markers and body composition.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

Pharmacological Properties of Vochysia Haenkeana (Vochysiaceae) Extract to Neutralize the Neuromuscular Blockade Induced by Bothropstoxin-I (Lys49 Phospholipase A2) Myotoxin.

Purpose: Bothrops snakes are responsible for more than 70 % of snakebites every year in Brazil and their venoms cause severe local and systemic damages. The pharmacological properties of medicinal plants have been widely investigated in order to discover new alternative treatments for different classes of diseases including neglected tropical diseases as envenomation by snakebites. In this work, we have investigated the ability of Vochysia haenkeana stem barks extract (VhE) to neutralize the neuromuscular effects caused by Bothropstoxin-I (BthTX-I), the major phospholipase A2 (PLA2) myotoxin from B. jararacussu venom. Methods: The biological compounds of VhE were analysed under thin layer chromatography (TLC) and its neutralizing ability against BthTX-I was assessed through twitch-tension recordings and histological analysis in mouse phrenic nerve-diaphragm (PND) preparations. The antimicrobial activity of VhE was assessed against S. aureus, E. coli and P. aeruginosa strains. The aggregation activity of VhE was analysed under protein precipitation assay. Results: VhE showed the presence of phenolic compound visualized by blue trace under TLC. VhE abolished the neuromuscular blockade caused by BthTX-I applying the pre-toxin incubation treatment and partially neutralized the BthTX-I action under post-toxin incubation treatment; VhE contributed slightly to decrease the myotoxicity induced by BthTX-I. The neutralizing mechanism of VhE may be related to protein aggregation. VhE showed no antimicrobial activity. Conclusion: V. haenkeana extract which has no antimicrobial activity exhibited neutralizing ability against the neuromuscular blockade caused by BthTX-I and also contributed to decrease its myotoxicity. Protein aggregation involving phenolic compounds may be related in these protective effects.

Hippocampal and cerebellar histological changes and their behavioural repercussions caused by brain ischaemic hypoxia experimentally induced by sodium nitrite.

INTRODUCTION: Brain ischaemic hypoxia can produce severe neurological damage that leads to behavioural disorders. This research analysed the hippocampal and cerebellar histological alterations caused by brain ischaemic hypoxia experimentally induced by sodium nitrite (NaNO2) and possible direct repercussions of this hypoxia on behaviour. METHODOLOGY: An experimental study was carried out by administering 60mg/kg NaNO2 to 10 Wistar rats at 3 months of age for 15 consecutive days. Ten control rats did not receive NaNO2. To assess behavioural repercussions, the animals were evaluated in Open Field, Elevated Plus-Maze (EPM), and Forced Swim tests before and after injury to evaluate locomotion, anxiety, and depression, respectively. Markers of stress were evaluated by measuring the blood levels of cortisol, glucose, cholesterol, and lactate. The presence of hippocampal lesions was verified by histologically studying the CA1-CA4 areas. Sections of the cerebellum were also evaluated because Purkinje cells are highly sensitive to ischaemic hypoxia and may serve as markers for this process. RESULTS: The number of neurons with lesions was significantly higher in animals exposed to NaNO2 in the hippocampus areas CA2, CA3, and CA4. The cerebellum was also very vulnerable to hypoxia, presenting extensive lesion áreas. These results are correlated with the parameters of the anxiety and depression tests. CONCLUSION: NaNO2 promoted brain damage due to ischaemic hypoxia in rats. Intoxicated animals showed decreased brain weights; damage in hippocampus and cerebellum; and anxiogenic and depressive behaviour.

Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom.

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA2) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA2 activity inhibition: 24 °C bath temperature, Ca2+ - Sr2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca2+ or Sr2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 µg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA2, proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca2+ - Sr2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca2+ Tyrode solution at 37 °C (usual conditions); on Sr2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca2+ Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA2 (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA2 activity; Ca2+ - Sr2+ replacement reduced only the PLA2 activity (to 15.3%). We observed a strict relation between the inhibition of PLA2 activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA2 or other venom components, such as metalloproteinases.

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa).

Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A2 (PLA2) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 µg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 µM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 µg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 µg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets.

Biological activities of Leptodeira annulata (banded cat-eyed snake) venom on vertebrate neuromuscular preparations.

The physiological properties of colubrid snake venoms are largely unknown and less frequently investigated. In this study, we assessed the enzymatic properties and biological activities of Leptodeira annulata (banded cat-eyed snake) venom, an opistoglyphous snake from Colombia. The proteolytic, phospholipase A2 and amidolytic activities are assessed using colorimetric assays and the biological activities were analyzed in avian and mammalian neuromuscular preparations. L. annulata venom caused neuromuscular blockade in chick biventer cervicis (BC) preparations (40± 15% and 50± 3% of twitch reduction for 30 and 100 µg/ml, respectively; p < 0.05) following 120 incubation; 10 µg/ml of venom did not induce blockade. There was a mild reduction in contracture response to exogenous acetylcholine (110 µM) in BC preparations exposed to 10 and 30 µg of venom/ml (∼4% and ∼32% of reduction, respectively, p > 0.05, n = 4) compared to basal values whereas the highest concentration (100 µg/ml) abolished it after 120 min. The venom caused a significant reduction in contracture response elicited by KCl (∼58 and ∼90 of reduction for 30 and 100 µg/ml, respectively, p < 0.05, n = 4). In mouse phrenic nerve-diaphragm (PND) preparations, L. annulata venom induced a progressive muscle membrane depolarization [from -85.9 ± 1.6 mV (t0) to -72.2 ± 2.9 mV (t120), p < 0.05, n = 4); the postsynaptic receptors remained functional as shown by carbachol-induced depolarization. The morphological analyses showed a concentration-dependent number of pathological states in muscle fibers from both BC and PND preparations pre-exposed to venom. The venom showed high proteolytic activity and low phospholipase A2 activity; there was no evidence for serine protease activity. These results indicate that the neuromuscular effect induced by L. annulata venom resulted from damaged muscle fibers that lead to the blockade of twitches response. The findings suggest that the myotoxicity might be related to the presence of metalloproteases in this venom.

No cytotoxicity or genotoxicity of graphene and graphene oxide in murine lung epithelial FE1 cells in vitro.

Graphene and graphene oxide receive much attention these years, because they add attractive properties to a wide range of applications and products. Several studies have shown toxicological effects of other carbon-based nanomaterials such as carbon black nanoparticles and carbon nanotubes in vitro and in vivo. Here, we report in-depth physicochemical characterization of three commercial graphene materials, one graphene oxide (GO) and two reduced graphene oxides (rGO) and assess cytotoxicity and genotoxicity in the murine lung epithelial cell line FE1. The studied GO and rGO mainly consisted of 2-3 graphene layers with lateral sizes of 1-2 µm. GO had almost equimolar content of C, O, and H while the two rGO materials had lower contents of oxygen with C/O and C/H ratios of 8 and 12.8, respectively. All materials had low levels of endotoxin and low levels of inorganic impurities, which were mainly sulphur, manganese, and silicon. GO generated more ROS than the two rGO materials, but none of the graphene materials influenced cytotoxicity in terms of cell viability and cell proliferation after 24 hr. Furthermore, no genotoxicity was observed using the alkaline comet assay following 3 or 24 hr of exposure. We demonstrate that chemically pure, few-layered GO and rGO with comparable lateral size (> 1 µm) do not induce significant cytotoxicity or genotoxicity in FE1 cells at relatively high doses (5-200 µg/ml). Environ. Mol. Mutagen. 57:469-482, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.