An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 µg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 µg/mL) caused irreversible paralysis. Preincubation of TM (200 µg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Effect of the phospholipase A2 inhibitor Varespladib, and its synergism with crotalic antivenom, on the neuromuscular blockade induced by Crotalus durissus terrificus venom (with and without crotamine) in mouse neuromuscular preparations.

The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A2 (PLA2) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom.

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.

Effects of high-dose creatine supplementation on kidney and liver responses in sedentary and exercised rats.

This study evaluated the effects of high-dose of short-term creatine supplementation (5g.kg(-1).day(-1) to 1 week) and long-term creatine supplementation (1g.kg(-1). day(-1) to 4-8 weeks) on kidney and liver structure and function of sedentary and exercised Wistar rats (Exercise sessions consisted of swimming at 80% of maximal work load supported during 5 days per week with daily sessions of 60 minutes throughout the duration of the supplementation). Seventy- two animals (245 ± 5g) were divided into four groups (n = 18): control diet Sedentary (SED), Creatine diet Sedentary (CRE), control diet Exercised (EXE), and Creatine diet Exercised (EXECRE). Histological and blood biochemical studies were performed after one, four, and eight weeks of creatine supplementation and exercise (n = 6). No differences were found when comparing SED, EXE and EXECRE groups for kidney and liver structure and function at one, four and eight weeks. However, the CRE group showed higher levels of creatinine (1.1 ± 0.2 vs. 0.4 ± 0.1 mg.dl(-1); p < 0.05), and urea (37 ± 3 vs. 19 ± 1 mg.dl(-1); p < 0.05) when compared with all others groups at four and eight weeks. At eight weeks, the CRE group presented increased levels of ALT (41 ± 7 vs. 23 ± 7 U.L(-1); p < 0.05), AST (89 ± 6 vs. 62 ± 5 U.L(-1); p < 0.05), GGT (8.0 ± 0.9 vs. 3.9 ± 1.0 U.L(-1); p < 0.05), and AP (125 ± 10 vs. 69 ± 9 U.L(-1); p < 0.05) also when compared with all others groups. Moreover, the CRE group demonstrated some structural alterations indicating renal and hepatic damage at four and eight weeks, respectively. These results suggest that long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats. Key pointsCreatine supplementation is an established ergogenic aid in sports and is now claimed to have therapeutical applications in a variety of diseases.Although acknowledged, this nutritional supplement is rarely monitored precisely about their possible side effects.Previous studies indicated that short-term creatine supplementation associate with the physical exercise may be safe, but the effect of long-term creatine supplementation is still unknown.There is a need for further research to elucidate the controversial points refers to renal and hepatic function after creatine supplementation.The results of the current study indicate that supraphysiological long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

New pharmacological insights of Galactia glaucescens.

Galactia glaucescens leaves are popularly used against snakebites in Brazil. The hydroethanolic extract from aerial parts of G. glaucescens (HEGg) was assayed against the neurotoxicity and myotoxicity induced by Bothrops jararacussu venom. A traditional myographic technique was applied for neurotoxicity and the resulting muscles were treated routinely by light microscopy analysis for myotoxicity. Additionally, the antimicrobial potential of HEGg was evaluated against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains, as well as Rutin was isolated for the first time in this specie using chromatographic and spectroscopic methods and its antiophidian property was assessed. HEGg totally prevents the neurotoxicity and myotoxicity effects caused by B. jararacussu, but did not show any antimicrobial effect. Concluding, HEGg and Rutin were able to counteract the toxic effects of the venom and confirmed the antiophidian potential, but not antimicrobial, of G. glaucescens as an alternative for neutralization of B. jararacussu venom.

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Local and hematological alterations induced by Philodryas olfersii snake venom in mice.

Envenomation by the South American opisthoglyphous snake Philodryas olfersii causes local pain, edema, erythema and ecchymosis; systemic envenomation is rare. In this work, we examined the inflammatory activity of P. olfersii venom (10, 30 and 60 µg) in mouse gastrocnemius muscle 6 h after venom injection. Intramuscular injection of venom did not affect hematological parameters such as red cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The venom caused thrombocytopenia (at all three doses), leukopenia and lymphopenia (both at the two highest doses), as well as neutrophilia (30 µg), monocytosis (30 µg) and basophilia (10 µg). Of the cytokines that were screened [IL-1ß, IL-6, IL-10, IL-13, IL-17, TNF-α, IFN-γ, MIP-2 and KC] and IGF-1, only IGF-1 showed a significant increase in its circulating concentration, seen with 60 µg of venom; there were no significant changes in the cytokines compared to control mice. Histological analysis revealed the presence of edema, an inflammatory infiltrate and progressive myonecrosis. Edema and myonecrosis were greatest with 60 µg of venom, while the inflammatory infiltrate was greatest with 10 µg of venom. All venom doses caused the migration of polymorphonuclear and mononuclear leukocytes into muscle, but with no significant dose-dependence in the response. These findings show that, at the doses tested, P. olfersii venom does not cause hematological alterations and has limited effect on circulating cytokine concentrations. These data also confirm that the principal effects of the venom in mice are local edema, inflammatory cell infiltration and myonecrosis.

Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa).

Bothrops snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA(2) from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA(2), with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA(2) previously isolated from this venom (PA2_BOTIN, SwissProt accession number ) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA(2) showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA(2) from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA(2) that consisted of three alpha-helices, a beta-wing, a short helix and a calcium-binding loop. BinTX-I (30 microg/paw) produced mouse hind paw edema that was maximal after 1h compared to after 3h with venom (10 and 100 microg/paw); in both cases, the edema decreased after 6h. BinTX-1 and venom (40 microg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.

Incidence of systemic joint hypermobility and temporomandibular joint hypermobility in pregnancy.

The purpose of this study was to establish a possible correlation between systemic hypermobility and temporomandibular hypermobility during pregnancy. One hundred (100) healthy pregnant women were evaluated: 7% in the first trimester (1T), 38% in the second trimester (2T), and 55% in the third trimester (3T) of gestation. In the series, the authors analyzed systemic joint hypermobility (SJH), range of mandibular movement (MMR), head and shoulder posture, head lateralization, and the presence of noise, pain, and parafunction in the temporomandibular joint. They observed that pain is present to a mild degree mostly in the head and ears of all pregnant women who presented with pain. Most of the subjects had some type of parafunction, but only 42.8% had noises. Mild SJH was seen in 50% of the 2T and 3T subjects, and in 28.5% of 1T subjects. Mild mandibular hypermobility was found for jaw opening (46%) and lateralization to the right (44%) or to the left (46%). Most of the subjects had hypomobility for jaw protrusion and retraction. The subjects had head protrusion and anterior posture as a result of the change in their center of gravity brought about by pregnancy. The authors found no association between systemic joint hypermobility (SJH) and temporomandibular hypermobility, although hormonal changes and complex factors during pregnancy may represent a risk factor for both types of mobility change.

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom.

Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01-2.5 microg/paw) induced paw oedema which, at high doses (>/=0.5 microg/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H(1) and serotonin 5-HT(2) receptor antagonist), mepyramine (histamine H(1) receptor antagonist), L-NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA(2)) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT(1)/5-HT(2) receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species.

Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada).

Whereas the presynaptic action of Crotalus durissus terrificus venom is well-established, Bothrops venoms have historically been considered to have only postsynaptic and muscular effects. However, some studies have also suggested a presynaptic action for these venoms. In this work, we used chick biventer cervicis preparations to compare the presynaptic actions of two Bothrops venoms (B. insularis and B. neuwiedi) with that of C. d. terrificus venom. At 10 microg/ml, all venoms produced irreversible blockade of the twitch tension responses, with no reduction in acetylcholine (ACh)-induced contractures and only a slight decrease in potassium induced-contractures. The times (in min) required to produce 50% neuromuscular blockade (C. d. terrificus: 16.3+/-0.7, n = 8; B. insularis: 30.0+/-1.9, n = 5; B. neuwiedi: 42.0+/-2.0, n = 8; mean +/- SEM) were significantly different among the venoms (p < 0.01). Lowering the temperature at which the experiments were done (from 37 to 24 degrees C) prevented neuromuscular blockade by the three venoms, indicating that enzyme activity may be involved in this response. At concentrations capable of causing complete neuromuscular blockade, creatine kinase release remained close to levels seen in control preparations incubated with Krebs solution alone (500-1200 IU/l). Commercial crotalic antivenom, but not bothropic antivenom, protected against the neuromuscular blockade caused by B. insularis and B. neuwiedi venoms. These observations indicate that bothropic venoms may contain components which act presynaptically in a manner similar to C. d. terrificus venom, and that at low venom concentrations a direct action on skeletal muscle does not contribute to this presynaptic neurotoxicity.

Effect of Bothrops leucurus venom in chick biventer cervicis preparations.

Bothrops leucurus is a poorly studied pitviper found in northeastern Brazil. We examined the action of B. leucurus venom (5-100 microg/ml) on contractile responses in chick biventer cervicis preparations. Muscle damage was assessed by quantifying the release of creatine kinase (CK) and by histological analysis. B. leucurus venom dose-dependently inhibited the contractile responses of indirectly stimulated preparations, the maximum inhibition with 100 microg of venom/ml being 74.0+/-6.6% (mean+/-SEM) after 120 min. The venom also reduced contractures to exogenous acetylcholine (55 and 110 microM) and K(+) (13.4mM) (85-100% reduction with 100 microg of venom/ml) and increased the release of CK (348+/-139 U/ml in controls vs 1260+/-263 U/ml with 20 microg of venom/ml after 120 min, p<0.05). The accompanying morphological changes included multivacuolated, swollen, amorphous fibers and agglutinated myofibrils. These results indicate that B. leucurus venom can adversely affect neuromuscular transmission and produce muscle damage in avian preparations.

Analgesic Effect of Light-Emitting Diode (LED) Therapy at Wavelengths of 635 and 945 nm on Bothrops moojeni Venom-Induced Hyperalgesia.

Envenoming induced by Bothrops snakes is characterized by drastic local tissue damage involving hemorrhage, myonecrosis and proeminent inflammatory and hyperalgesic response. The most effective treatment is antivenom therapy, which is ineffective in neutralizing the local response. Herein, it was evaluated the effectiveness of light-emitting diode (LED) at wavelengths of 635 and 945 nm in reducing inflammatory hyperalgesia induced by Bothrops moojeni venom (BmV) in mice, produced by an subplantar injection of BmV (1 µg). Mechanical hyperalgesia and allodynia were assessed by von Frey filaments at 1, 3, 6 and 24 h after venom injection. The site of BmV injection (1.2 cm(2) ) was irradiated by LEDs at 30 min and 3 h after venom inoculation. Both 635 nm (110 mW, fluence of 3.76 J/cm(2) and 41 s of irradiation time) and 945 nm (120 mW, fluence of 3.8 J/cm(2) and 38 s of irradiation time) LED inhibited mechanical allodynia and hyperalgesia of mice alone or in combination with antivenom treatment, even when the symptoms were already present. The effect of phototherapy in reducing local pain induced by BmV should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snake bites.

Neuromuscular activity of Bothrops fonsecai snake venom in vertebrate preparations.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160µg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160µg/ml, respectively) and attenuated the contractures to 110µM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200µg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200µg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.