Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

Entomotoxic activity of Rhinella icterica (Spix, 1824) toad skin secretion in Nauphoeta cinerea cockroaches: An octopamine-like modulation.

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 µg/g) injections, induced behavioral impairment as evidenced by a significant decrease (38 ±â€¯14%) in the distance traveled (p < .05), followed by an increase (90 ±â€¯6%) of immobile episodes (p < .001, n = 28, respectively). In cockroaches semi-isolated heart preparations, RITS (16 µg/200 µl) induced a significant irreversible dose-dependent negative chronotropism, reaching ~40% decrease in heart rate in 20 min incubation. In in vivo cockroach neuromuscular preparations, RITS (20, 50 and 100 µg/g of animal weight) induced a time-dependent inhibition of twitch tension that was complete for 20 µg/g, in 120 min recordings. RITS (10 µg/g) also induced a significant increase in the insect leg grooming activity (128 ±â€¯10%, n = 29, p < .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 µg/g). The electrophysiological recordings of spontaneous neural compound action potentials showed that RITS (20 µg/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.

Modulation of the cholinergic pathway induced by skin secretion of Phyllomedusa iheringii Boulenger, 1885 in a vertebrate model.

Amphibians represent one of the main natural sources of bioactive molecules of interest to biotechnological research. The Phyllomedusidae family has several species occurring in Brazil and some studies demonstrate the biological potential of poisons of these species, however many still need to be characterized. Phyllomedusa iheringii is endemic in Brazilian and Uruguayan Pampa Biome and has little data in the literature regarding the action of its poison on experimental organisms. Thus, the present work evaluates the biological activity of P. iheringii secretion on the central and peripheral nervous system of a vertebrate model. The skin secretions of P. iheringii (SSPI) were collected through manual compression and electrical stimulation of the animal's bodies. The resulting content was used in neurobiological tests searching for modulatory effects on the main pathways involved in the neurotoxicity mechanism of vertebrates. SSPI affected the contraction force of the chick biventer cervicis muscle (Gallus gallus domesticus) at some concentrations used (5, 10, and 12 µg/mL). In slices from the cerebral cortex of G. gallus domesticus an increase in cell viability was observed after treatment with SSPI (10 µg/mL) and a neuroprotective effect when treated simultaneously with hydrogen peroxide (H2O2), Neostigmine (NEO) and Trichlorfon (TRI). The cholinergic pathway is possibly the main pathway modulated by SSPI since assays with the cerebral cortex and biventer cervicis muscle demonstrated the increased activity of the enzyme acetylcholinesterase (AChE) (SSPI 10 µg/mL and 12 µg/mL, respectively). SSPI (10 µg/mL) also prevented the modulation of NEO and TRI, two recognized anticholinesterase agents, in AChE activity in slices of the cerebral cortex. Therefore, our results have demonstrated the unpublished biotechnological potential of P. iheringii over the vertebrate model and its modulation on the nervous system, with apparent action on the cholinergic pathway.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s 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.