RESUMEN
Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.
Asunto(s)
Bufanólidos , Bufonidae/metabolismo , Locusta migratoria/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Animales , Bufanólidos/química , Bufanólidos/toxicidad , Cromatografía Líquida de Alta Presión , Femenino , Corazón/efectos de los fármacos , Oviductos/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
Rhinella icterica is a poisonous toad whose toxic secretion has never been studied against entomotoxic potential. Sublethal doses of Rhinella icterica toxic secretion (RITS) were assayed in Nauphoeta cinerea cockroaches, in order to understand the physiological and behavioral parameters, over the insect central and peripheral nervous system. RITS (10⯵g/g) injections, induced behavioral impairment as evidenced by a significant decrease (38⯱â¯14%) in the distance traveled (pâ¯<â¯.05), followed by an increase (90⯱â¯6%) of immobile episodes (pâ¯<â¯.001, nâ¯=â¯28, respectively). In cockroaches semi-isolated heart preparations, RITS (16⯵g/200⯵l) induced a significant irreversible dose-dependent negative chronotropism, reaching ~40% decrease in heart rate in 20â¯min incubation. In in vivo cockroach neuromuscular preparations, RITS (20, 50 and 100⯵g/g of animal weight) induced a time-dependent inhibition of twitch tension that was complete for 20⯵g/g, in 120â¯min recordings. RITS (10⯵g/g) also induced a significant increase in the insect leg grooming activity (128⯱â¯10%, nâ¯=â¯29, pâ¯<â¯.01), but not in the antennae counterparts. The RITS increase in leg grooming activity was prevented in 90% by the pretreatment of cockroaches with phentolamine (0.1⯵g/g). The electrophysiological recordings of spontaneous neural compound action potentials showed that RITS (20⯵g/g) induced a significant increase in the number of events, as well as in the rise time and duration of the potentials. In conclusion, RITS showed to be entomotoxic, being the neuromuscular failure and cardiotoxic activity considered the main deleterious effects. The disturbance of the cockroaches' behavior together with the electrophysiological alterations, may unveil the presence of some toxic components present in the poison with inherent biotechnological potentials.
Asunto(s)
Bufonidae/fisiología , Cucarachas/efectos de los fármacos , Octopamina/farmacología , Piel/metabolismo , Toxinas Biológicas/toxicidad , Potenciales de Acción/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Cucarachas/metabolismo , Relación Dosis-Respuesta a Droga , Aseo Animal/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Unión Neuromuscular/efectos de los fármacos , Octopamina/metabolismo , Fentolamina/farmacología , Toxinas Biológicas/metabolismoRESUMEN
The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heartÌs tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.