Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells.

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (≤ 3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (≥ 10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of α- and ß-toxins with neuronal sodium channels.

Scorpion venom increases acetylcholine release by prolonging the duration of somatic nerve action potentials.

Scorpionism is frequently accompanied by a massive release of catecholamines and acetylcholine from peripheral nerves caused by neurotoxic peptides present in these venoms, which have high specificity and affinity for ion channels. Tityus bahiensis is the second most medically important scorpion species in Brazil but, despite this, its venom remains scarcely studied, especially with regard to its pharmacology on the peripheral (somatic and autonomic) nervous system. Here, we evaluated the activity of T. bahiensis venom on somatic neurotransmission using myographic (chick and mouse neuromuscular preparations), electrophysiological (MEPP, EPP, resting membrane potentials, perineural waveforms, compound action potentials) and calcium imaging (on DRG neurons and muscle fibres) techniques. Our results show that the major toxic effects of T. bahiensis venom on neuromuscular function are presynaptically driven by the increase in evoked and spontaneous neurotransmitter release. Low venom concentrations prolong the axonal action potential, leading to a longer depolarization of the nerve terminals that enhances neurotransmitter release and facilitates nerve-evoked muscle contraction. The venom also stimulates the spontaneous release of neurotransmitters, probably through partial neuronal depolarization that allows calcium influx. Higher venom concentrations block the generation of action potentials and resulting muscle twitches. These effects of the venom were reversed by low concentrations of TTX, indicating voltage-gated sodium channels as the primary target of the venom toxins. These results suggest that the major neuromuscular toxicity of T. bahiensis venom is probably mediated mainly by α- and ß-toxins interacting with presynaptic TTX-sensitive ion channels on both axons and nerve terminals.

Tb II-I, a Fraction Isolated from Tityus bahiensis Scorpion Venom, Alters Cytokines': Level and Induces Seizures When Intrahippocampally Injected in Rats.

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative β-sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 µg/2 µL) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.

Tityus bahiensis scorpion venom injected to dams during pregnancy affects some cytokines of fetuses.

Due to the high incidence of scorpion stings in Brazil, pregnant women are among the possible victims. Cytokines are important during the pregnancy, and scorpion venoms can change their release. We evaluated the levels of some cytokines in the fetuses after the treatment of pregnant rats with the Tityus bahiensis scorpion venom. The concentration of some of them is altered and can be responsible for the effects previously observed on innate reflexes, and the physical and behavioral development of the offspring.

Mild reproductive effects of the Tityus bahiensis scorpion venom in rats

Scorpion envenoming is a public health problem in Brazil, where Tityus serrulatus and T. bahiensis are considered the most dangerous scorpions. They are well adapted to urbanized environments, and there is an increasing probability of human exposure to these venoms, including during pregnancy. Not much is known about the effects of prenatal exposure to the venom, and no information is available to aid in the rational treatment of victims stung during pregnancy. Thus, this study aimed to investigate whether venom from the scorpion T. bahiensis administered once to pregnant female rats at a dose that causes a moderate envenomation may lead to deleterious effects on the reproductive performance of the dams and on the development of their offspring. This is the first work demonstrating that T. bahiensis venom, when administered experimentally to rats, alters maternal reproductive performance and the morphological development of fetuses. The venom was given to dams on the 5th (GD5) or on the 10th (GD10) gestational day. After laparotomy, on GD21, fetuses and placentas were counted, weighed and externally analyzed. The corpora lutea were counted. The sex and vitality of fetuses were evaluated, and each litter was then randomly divided for visceral or skeletal analyses. Data were analyzed by ANOVA followed by the Tukey-Kramer test and Fisher's exact test. The significance level for all tests was set at p < 0.05.

Mild reproductive effects of the Tityus bahiensis scorpion venom in rats

Scorpion envenoming is a public health problem in Brazil, where Tityus serrulatus and T. bahiensis are considered the most dangerous scorpions. They are well adapted to urbanized environments, and there is an increasing probability of human exposure to these venoms, including during pregnancy. Not much is known about the effects of prenatal exposure to the venom, and no information is available to aid in the rational treatment of victims stung during pregnancy. Thus, this study aimed to investigate whether venom from the scorpion T. bahiensis administered once to pregnant female rats at a dose that causes a moderate envenomation may lead to deleterious effects on the reproductive performance of the dams and on the development of their offspring. This is the first work demonstrating that T. bahiensis venom, when administered experimentally to rats, alters maternal reproductive performance and the morphological development of fetuses. The venom was given to dams on the 5th (GD5) or on the 10th (GD10) gestational day. After laparotomy, on GD21, fetuses and placentas were counted, weighed and externally analyzed. The corpora lutea were counted. The sex and vitality of fetuses were evaluated, and each litter was then randomly divided for visceral or skeletal analyses. Data were analyzed by ANOVA followed by the Tukey-Kramer test and Fisher's exact test. The significance level for all tests was set at p < 0.05.

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

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

Reproductive toxic effects of Tityus serrulatus scorpion venom in rats.

Tityus serrulatus is the most venomous scorpion in Brazil. Little is known about the effect of maternal exposure to the venom on fetal development. We investigated the effect of low to moderate doses of the venom (0.3 or 1.0 mg/kg s.c. on either day 5 or day 10 of gestation) on pregnant rats and on their offspring. For dams, we observed their body weight gain and reproductive parameters. For the offspring, we observed their body weight and weight of internal organs and the number of live and dead fetuses, and we investigated whether the venom caused external, visceral, skeletal or histopathological alterations in the offspring. The offspring were examined on gestational day 21. Injection of the venom on gestational day 5 did not change the reproductive parameters of the dams, their weight or fetuses' weight. Rats that received the high dose of the venom (1.0 mg/kg) on gestational day 10 had heavier placentas and heavier fetuses with heavier lungs. Injections on day 10 of gestation did not alter the reproductive parameters of the dams nor their weight gain at either dose. The venom did not cause malformations of the fetal skeleton or viscera and did not delay fetal development with either dose. In conclusion, subcutaneous administration of 0.3 or 1.0 mg/kg T. serrulatus venom to pregnant Wistar rats at either day 5 or day 10 of gestation did not cause maternal or clear fetal toxicity. Subtle increases in placental weight and fetal body and lung weights observed following treatment with 1.0 mg/kg on day 10 of gestation were not associated with histopathological findings. Whether these observations represent a reaction to treatment and, if so, the underlying mechanisms and their toxicological impact remain to be examined further in future studies.

Dantrolene protects hippocampal cells from damage induced by TsTX, an alpha-scorpion toxin from Tityus serrulatus.

We examined the effects of dantrolene, an inhibitor of intracellular calcium release, on alterations associated with the intrahippocampal injection of the TsTX scorpion toxin. Male Wistar rats (230-250 g) were injected with Ringer solution (1 microl; n = 6); TsTX toxin (1 microg/microl; n = 8); and dantrolene (10.0 mg/kg) plus TsTX toxin (1 microg/microl; n = 6). After injection, electroencephalographic (EEG) recordings and observation of animals behaviour were performed continuously for 4 h. One week later, animals were submitted to histopathological analysis. TsTX caused electrographic seizure expressed by moderate or intense discharges and neuronal loss in hippocampal areas in all injected animals (n = 8). Dantrolene reduced the effect of TsTX. Thus, 67% of rats (four out of six) treated with toxin and dantrolene had electrographic convulsions, but only for 30 min after injection and none of them presented neuronal damage. Dantrolene or Ringer had no effects on the EEG.

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

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

Brazilian scorpion venoms: pharmacological aspects

This review focuses on clinical, epidemiological and therapeutic aspects of envenomation caused by scorpions in Brazil. In addition, it presents studies with the crude venom and neurotoxins isolated from Tityus serrulatus scorpion venom. Such studies show the pharmacological effects of the venom and isolated toxins in experiments that use the scorpion venom and neurotoxins as neurobiological tools to study receptor sites in sodium channels and neurotransmitter release.