Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs).

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

The PI3K/Akt/mTOR signaling pathway plays a role in regulating aconitine-induced autophagy in mouse liver.

Aconitine, a major aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias and neurological symptoms. One mechanism of aconitine-induced toxic responses is the induction of apoptosis. Apoptosis and autophagy are interconnected processes and the two pathways share critical components. In this study, we investigated the role of autophagy in aconitine-induced toxicity using mouse model. 120 mice were randomly divided into 4 experimental groups (normal saline), low dose group (0.14 µmol/L), medium dose group (0.28 µmol/L) and high dose group (0.56 µmol/ L). 30 mice in each group were administered with aconitine (lavage) for 30 days. The livers were collected for analysis of autophagy-related proteins by Western blotting. The expression of LC3II/LC3I ratio and Beclin 1 were found to increase and then decrease with the highest expression at 10 days and the p62 showed a time-dependent decreases. Autophagy is regulated by the mTOR pathway, we further analyzed the effects of aconitine on this pathway and found aconitine inhibited, phosphorylation of p-PI3K, p-Akt and p-mTOR. The p-p70s6k and p-4EBP1 which are downstream of mTOR were concomitantly decreased. These results suggest that aconitine induce autophagy in mouse liver. The PI3K/Akt/mTOR signaling pathway is involved in the regulation of aconitine-induced autophagy in the liver of mice.

The Snake with the Scorpion's Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus).

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Progressive development of cardiomyopathy following altered autonomic activity in status epilepticus.

Seizures are associated with altered autonomic activity, which has been implicated in the development of cardiac dysfunction and structural damage. This study aimed to investigate the involvement of the autonomic nervous system in seizure-induced cardiomyopathy. Male Sprague-Dawley rats (320-350 g) were implanted with EEG/ECG electrodes to allow simultaneous telemetric recordings during seizures induced by intrahippocampal (2 nmol, 1 µl/min) kainic acid and monitored for 7 days. Seizure activity occurred in conjunction with increased heart rate (20%), blood pressure (25%), and QTc prolongation (15%). This increased sympathetic activity was confirmed by the presence of raised plasma noradrenaline levels at 3 h post-seizure induction. By 48 h post-seizure induction, sympathovagal balance was shifted in favor of sympathetic dominance, as indicated by both heart rate variability (LF/HF ratio of 3.5 ± 1.0) and pharmacological autonomic blockade. Functional cardiac deficits were evident at 7 and 28 days, as demonstrated by echocardiography showing a decreased ejection fraction (14% compared with control, P < 0.05) and dilated cardiomyopathy present at 28 days following seizure induction. Histological changes, including cardiomyocyte vacuolization, cardiac fibrosis, and inflammatory cell infiltration, were evident within 48 h of seizure induction and remained present for up to 28 days. These structural changes most probably contributed to an increased susceptibility to aconitine-induced arrhythmias. This study confirms that prolonged seizure activity results in acute and chronic alterations in cardiovascular control, leading to a deterioration in cardiac structure and function. This study further supports the need for modulation of sympathetic activity as a promising therapeutic approach in seizure-induced cardiomyopathy.

Activity of drugs and components of natural origin in the severe myoclonic epilepsy of infancy (Dravet syndrome).

The sea anemones (Cnidaria) produce neurotoxins, polypeptides active on voltage-gated sodium channels, which induce a non-inactivating condition, with consequent seizures and paralysis in zebrafish (Danio rerio). In humans, severe myoclonic epilepsy of infancy (SMEI) is due to SCN1A gene mutation, which causes a non-inactivating sodium channels condition with seizures. Some symptoms, such as age of first seizure, repetitive events, frequent status epilepticus, scarce responsiveness to antiepileptic drugs (AEDs), may be due to superimposed environmental causes. The authors report a case of SMEI treated for years with benzodiazepines and subsequently with valproate. The attenuation of the frequency of epileptic events and of time in seizing, but no change in burst duration and EEG events was observed. These results are similar to those reported in the literature about zebrafish scn1Lab mutant, which recapitulates the SCN1A symptoms and AED resistance occurring in humans. During seizures the production of polypeptides similar to sea anemones neurotoxins, causing repetitive seizures, status epilepticus, and AED resistance can be hypothesized in subjects with SCN1A mutation.

Cardioprotective effect of grape-seed proanthocyanidins on doxorubicin-induced cardiac toxicity in rats.

CONTEXT: Doxorubicin (Dox) is an anthracycline antibiotic used as anticancer agent. However, its use is limited due to its cardiotoxicity which is mainly attributed to accumulation of reactive oxygen species. OBJECTIVE: This study was conducted to assess whether the antioxidant, proanthocyanidins (Pro) can ameliorate Dox-induced cardiotoxicity in rats. MATERIALS AND METHODS: Male Sprague-Dawely rats were divided into four groups. Group I was control. Group II received Pro (70 mg/kg, orally) once daily for 10 days. Group III received doxorubicin 15 mg/kg i.p. as a single dose on the 7th day and Group IV animals were treated with Pro once daily for 10 days and Dox on the 7th day. The parameters of study were serum biomarkers, cardiac tissue antioxidant status, ECG, and effect on aconitine-induced cardiotoxicity. RESULTS: Cardiac toxicity of doxorubicin was manifested as a significant increase in heart rate, elevation of the ST segment, prolongation of the QT interval and an increase in T wave amplitude. In addition, Dox enhanced aconitine-induced cardiotoxicity by a significant decrease in the aconitine dose producing ventricular tachycardia (VT). Administration of Pro significantly suppressed Dox-induced ECG changes and normalized the aconitine dose producing VT. The toxicity of Dox was also confirmed biochemically by significant elevation of serum CK-MB and LDH activities as well as myocardial MDA and GSH contents and decrease in serum catalase and myocardial SOD activities. Administration of Pro significantly suppressed these biochemical changes. DISCUSSION AND CONCLUSION: These results suggest that proanthocyanidins might be a potential cardioprotective agent against Dox-induced cardiotoxicity due to its antioxidant properties.

Effects of acute administration of ethanol on experimental arrhythmia.

Many studies have shown that the relationship between alcohol consumption and most cardiovascular diseases is U-shaped, with nondrinkers and heavier drinkers having higher risks than moderate drinkers. However, the association between cardiac arrhythmias and acute alcohol consumption is not well understood. We set up several experimental arrhythmia animal models to examine the effects of acute administration of ethanol on arrhythmia. The results showed 0.4, 0.8 and 1.6 g/kg ethanol consumption obviously delayed the onset time of atrial fibrillation (AF) (P < 0.05 or P < 0.01) and increased the survival rates on acetylcholine-CaCl2-induced AF in mice. Ethanol (0.4, 0.8 and 1.6 g/kg) consumption significantly delayed the onset time of ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA) (P < 0.01), and 0.4 and 0.8 g/kg ethanol consumption increased the survival rates on CaCl2-induced arrhythmia in rats. Ethanol (0.4 g/kg) essentially increased the cumulative dosage of aconitine required to CA (P < 0.05), and 0.8 g/kg, 1.6 g/kg ethanol reduced the cumulative aconitine dosage to induce VT, VF and CA (P < 0.05 or P < 0.01) on aconitine-induced arrhythmia in rats. Ethanol (0.4, 0.8 and 1.6 g/kg) consumption remarkably increased the cumulative dosage of deslanoside to induce ventricualr premature contraction (P < 0.01) on deslanoside-induced arrhythmia in guinea pigs. Collectively, our results indicate that low concentrations of ethanol had anti-arrhythmic effect on experimental arrhythmia, and high concentrations of ethanol may aggravated the occurrence of experimental arrhythmia.