Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Evidence that free radical generation occurs during scorpion envenomation
Introduction
Among the heterogeneous mixture of peptides contained in scorpion venom, only a small group of peptides is responsible for the mortality in humans. For the old world scorpions, the major lethal peptides are called alpha-toxins. These toxins prolong the action potential of excitable cells by delaying sodium channel inactivation (Couraud et al., 1982) causing an increase in the presynaptic release of neurotransmitters, including catecholamines (Einhorn and Hamilton, 1977, Lefebvre et al., 1978, Ito et al., 1981), acetylcholine (Gwee et al., 2002) or glutamate (Massensini et al., 1998). The clinical manifestations following scorpion stings clearly demonstrate a dysfunction of the peripheral rather than that of the central nervous system (Clot-Faybesse et al., 2001).
After severe scorpion envenomations, a cholinergic syndrome involving the parasympathetic nervous system is often observed with abdominal pain, mydriasis and exocrine hypersecretion (Goyffon et al., 1982, Ismail, 1995). Neuromuscular manifestations consisting of dystonia, fasciculations and muscle cramps, while other manifestations include agitation, hyperthermia, convulsions or coma, are sometimes observed (Goyffon et al., 1982, Ismail, 1995).
Cardiovascular manifestations include high blood pressure accompanied by arrhythmias and atrio-ventricular conduction block that may lead to cardiovascular collapse due to myocardial involvement (Gueron et al., 1980, Gueron et al., 1992, Gueron and Ovsyshcher, 1987). Although it is now well established that these symptoms, including death, are due to the increase in neurotransmitter release secondary to the binding of toxins to voltage-sensitive sodium channels, the mechanism of tissue damage remains unclear. Necrosis and myocytolysis have been observed in heart, kidneys and lungs (Correa et al., 1997, Daisley et al., 1999); until now, however, there is no evidence linking these effects with the direct cytotoxicity of toxins in the organs. For example, acute pulmonary edema that occurs following scorpion stings may be secondary to the hypertension that occurs with most severe envenomations (Sofer and Gueron, 1990, Gueron et al., 1992) but may be the result of a direct toxic action on cardiac myocytes by the toxins (Teixeira et al., 2001). Nevertheless, the concentration of toxins in the organs is generally low (Devaux et al., 2004) and cannot explain the necrosis or the cytolysis often observed in many organs. Thus, an explanation may be the generation of free radicals in plasma and organs during severe envenomation. In support, it has been reported that free radicals are involved in the mechanism of action of anthrax toxin and that antioxidants protect animals against lethal doses (Hanna et al., 1994). The aims of this study were thus three-fold: Firstly to define the participation of free radicals by evaluating lipid peroxidation products formation in organs after experimental envenomation of rats by fractions of the venom of Androctonus australis Hector, one of the world’s most dangerous scorpions. Increases in lipid peroxidation products would indicate an increase in tissue damage due to free radicals generation (Mihara and Uchiyama, 1978). Secondly we wished to evaluate the potential protective effect of antioxidant agents in experimentally envenomed mice. Finally, we wished to investigate the possible role of nitric oxide (NO) release in venom toxicity. Indeed, it has been reported that NO is released by tissues after severe scorpion envenomation in children (Meki and Mohey El-Dean, 1998) and that venom exhibits nitrergic actions (Gong et al., 1997). Since NO is synthesized from L-arginine by a NO synthase, we determined the effects of ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase, on the survival of experimentally envenomed mice.
Section snippets
Drugs
Adrenaline was from Renaudin Laboratories (Itxassou, France). Ascorbate, α−tocopherol acetate, superoxide dismutase (SOD), N-acetylcysteine (NAC), ω-nitro-l-arginine methyl ester (l-NAME) and all remaining reagents were obtained from Sigma-Aldrich (St Quentin Fallavier, France).
Animals
Approval for this study was obtained from the animal care and use committee (CHU Timone, Marseille). Female Wistar rats (Rattus norvegicus, weighing 240–300 g, mean 270 g) and C57/BL6 mice (Mus musculus, 25–30 g) were
LD50
In rats, the LD50 value of Aah-FG50 was 1 ± 0.1 mg/kg after subcutaneous (sc) injection, whereas in mice it was 300 ± 22 μg/kg (sc) and 1.15 ± 0.09 μg/kg after intracerebroventricular (icv) injection.
In vivo assays
Adrenaline or Aah-FG50 induced a significant increase in mean arterial blood pressure and heart rate compared to control. However, there was no significant difference between effects of adrenaline and Aah-FG50. Means of arterial blood pressure (BP), calculated as 1 / 3 (systolic BP–diastolic BP) + diastolic
Discussion
We chose to test Aah-FG50 instead of whole venom because this fraction accounts for almost all the toxicity of the venom (Rochat et al., 1979) and we sought for a relationship between lethality and free radicals generation. We shall screen the other fractions in further studies.
The principal results of this work can be summarized as follows: i) Experimental envenomation is accompanied by generation of free radicals in several organs (to our knowledge there are no data on the involvement of free
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