Acute oral toxicity in mice of a new palytoxin analog: 42-Hydroxy-palytoxin

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300-1697 μg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD₅₀ of 651 μg/kg (95% confidence limits: 384-1018 μg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 μg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥848 μg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424-1200 μg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure.Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC₅₀ = 7.6 and 13.2 × 10⁻¹² M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na⁺/K⁺-ATPase as one of the key cellular targets of this toxin.     

Carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase: Biological activities and selective toxicities

Toxoplasma gondii adenosine kinase (EC 2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Several 6-benzylthioinosines have already been identified as subversive substrates of the T. gondii but not human adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not its host. In the present study, we report the testing of the metabolism of several carbocyclic 6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the carbocyclic 6-benzylthioinosine analogues were metabolized to their 5′-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the carbocyclic 6-benzylthioinosine analogues showed a selective anti-toxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. These results indicate that the oxygen atom of the sugar is not critical for substrate binding. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were carbocyclic 6-(p-methylbenzylthio)inosine (IC₅₀ = 11.9 μM), carbocyclic 6-(p-methoxybenzylthio)inosine (IC₅₀ = 12.1 μM), and carbocyclic 6-(p-methoxycarbonylbenzylthio)inosine (IC₅₀ = 12.8 μM). These compounds have about a 1.5-fold better efficacy relative to their corresponding 6-benzylthioinosine analogues (Rais et al., Biochem Pharmacol 2005;69:1409-19 [29]). The results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that carbocyclic 6-benzylthioinosines are potential anti-toxoplasmic agents.     

In vivo biological activity of rocket extracts (Eruca vesicaria subsp. sativa (Miller) Thell) and sulforaphane

Eruca is thought to be an excellent source of antioxidants like phenolic compounds, carotenoids, glucosinolates and their degradation products, such as isothiocyanates. Sulforaphane is one of the most potent indirect antioxidants of Eruca isolated until the date. In this work we investigate: (i) the safety and DNA protective activity of Eruca extracts and sulforaphane (under and without oxidative stress) in Drosophila melanogaster; and (ii) the influence on D. melanogaster life span treated with Eruca extracts and sulforaphane. Our results showed that among the four concentrations of Eruca extracts tested (from 0.625 to 5 mg/ml), intermediate concentrations of the Es2 accession (1.25 and 2.5 mg/ml) exhibited no genotoxic activity, as well as antigenotoxic activity (inhibition rate of 0.2–0.6) and the lowest concentration of Es2 and Es4 accessions (0.625 mg/ml) also enhanced the health span portion of the live span curves. Sulforaphane presented a high antigenotoxic activity in the SMART test of D. melanogaster and intermediate concentrations of this compound (3.75 μM) enhanced average healthspan. The results of this study indicate the presence of potent antigenotoxic factors in rocket, which are being explored further for their mechanism of action.     

Effects of the mycotoxin destruxin A on Locusta migratoria visceral muscles

Destruxins, a family of cyclic peptides, are produced by various species of entomopathogenic fungi. These peptides have been shown to influence calcium-dependent processes in insect cell lines and tissues, such as skeletal muscles. To better understand the mechanism of action of these peptide toxins on insect muscular tissues, we have evaluated the effects of destruxin A on the contractions of oviducts and hindgut of Locusta migratoria. In oviducts, destruxin A increased the frequency of spontaneous contractions and induced a dose-dependent tonic contraction; the EC₅₀ for lower lateral and upper lateral oviducts was 0.7 μM and 8.7 μM, respectively. In hindgut, destruxin A also caused an increase in the frequency of spontaneous contractions; the EC₅₀ was 3.2 μM. The action of destruxin A was abolished in Ca²⁺-free saline or when the Ca²⁺ channel blocker CoCl₂ was added to the incubation saline. Likewise, the presence of 50 μM nifedipine or 100 μM verapamil in the medium reduced the magnitude of destruxin A′s effect, particularly in hindgut. The depolarization of muscle membranes by 100 mM K⁺ saline prevented the action of destruxin A. Preincubation of lower lateral oviducts in the intracellular Ca²⁺ antagonist TMB-8 did not have any effect on destruxin A action; however, preincubation in the calmodulin inhibitor trifluoperazine greatly reduced the effect of destruxin A. Taken together, these results show that destruxin A has an excitatory effect on contractions of insect visceral muscles of L. migratoria. Destruxin A-induced contractions appear to be dependent on extracellular, but not on intracellularly-released Ca²⁺, which suggest that this peptide toxin might be acting on insect visceral muscle by facilitating an influx of extracellular Ca²⁺.     

Validation of in vitro screening models for progestagenic activities: Inter-assay comparison and correlation with in vivo activity in rabbits

The PR CALUX® cell line is a stably transfected human U2-OS cell line expressing the human PR and a luciferase reporter construct containing three progesterone-responsive elements coupled to a minimal promoter. The validity of this assay has been studied as an alternative to the McPhail assay in rabbits, an in vivo assay to detect progestins. The PR CALUX assay was characterized by its stable expression of PR protein which leads to induction of endogenous PR target genes by progestins. It was found to have a highly selective response to low levels of different progestins, as well as an insignificant response to other nuclear hormone receptor ligands. As an important step in their validation, the PR CALUX bioassay was compared with another earlier described in vitro bioassay, a Chinese Hamster Ovary (CHO) cell-based PR-CHO reporter gene assay as well as with an in vitro PR-binding (PR-BIN) assay, and the in vivo McPhail assay. This was done using 35 (with the most accurate potency determinations in all tests) and 50 (with less reliable potency determinations in some tests) compounds tested in all assays. The correlation scores between PR CALUX and PR-CHO were r² = 0.77, and 0.93, respectively; between PR CALUX and PR-BIN r² = 0.69 and 0.80. Comparison between either the PR CALUX or the PR-CHO transactivation assay and the in vivo McPhail assay revealed very good correlations of r² = 0.68 (n = 35), and 0.85 (n = 50). The transactivation assays can discriminate very potent, from potent, weak and inactive compounds rather easily. Besides testing the biological activity of pure chemicals and pharmaceuticals in vitro, the PR CALUX and PR-CHO transactivation assays proved to be relatively good predictors of in vivo progestagenic activity, allowing the use of these assays as prescreening methods or in vitro alternatives.     

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: Involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A₂ (PLA₂) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca²⁺-induced Ca²⁺ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na⁺ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA₂ contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA₂ by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca²⁺ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na⁺ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.     

Bradykinin-related peptides in the venom of the solitary wasp Cyphononyx fulvognathus

Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine⁶-bradykinin (Thr⁶-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr⁶-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B₂ receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr⁶-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B₁ receptor antagonist, while the effect of both cyphokinin and Thr⁶-BK was reversed by B₂ antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.     

Blockade by lobeline of potassium exchange in skeletal muscle

Summary Lobeline (0.05 to 2.5 mM) caused depolarization of muscle fibers in frog sartorius muscle bathed in chloride-free solution. When the extent of depolarization was plotted against the log of the concentration of lobeline, the relationship was described by a straight line with a slope of 57 mV for a 10-fold change in the concentration of lobeline (0.2 to 2.0 mM). Like lobeline, butacaine (0.1 to 0.5 mM) caused muscle depolarization with a 57 mV change in membrane potential for a 10-fold change in the concentration of the local anesthetic. Lobeline (0.05 to 0.15 mM) depressed the depolarization of the muscle fibers caused by the elevation of (K⁺]₀ over a range of 2.5 to 50 mM. ⁴²K-exchange in resting muscle was also depressed by lobeline (0.05 to 0.25 mM). The blockade by lobeline of ⁴²K-efflux followed a typical dose-response relationship with 0.06 mM lobeline required to cause a 50% decrease of ⁴²K-efflux. These findings show that lobeline in concentrations not much greater than those required to block neuromuscular transmission, has a direct action on ion exchange in skeletal muscle. These actions on electrogenic mechanisms may contribute to the anti-nicotinic action of lobeline and to the desensitization of endplate acetylcholine receptors caused by lobeline.This study was supported by a grant from AMA-ERF.     

The marine polyether gambierol enhances muscle contraction and blocks a transient K current in skeletal muscle cells

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 μM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K⁺ channels prolongs the duration of action potentials, enhances the extent and time course of Ca²⁺ release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K⁺ current that is responsible for action potential prolongation in Xenopus skeletal myocytes.     

Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes

Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES^®) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES^® and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study.Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.     

Prevention of in vitro hepatic stellate cells activation by the adenosine derivative compound IFC305

We have previously shown that adenosine and the aspartate salt of adenosine (IFC305) reverse pre-established CCl₄-induced cirrhosis in rats. However, their molecular mechanism of action is not clearly understood. Hepatic stellate cells (HSC) play a pivotal role in liver fibrogenesis leading to cirrhosis, mainly through their activation, changing from a quiescent adipogenic state to a proliferative myofibrogenic condition. Therefore, we decided to investigate the effect of IFC305 on primary cultured rat HSC. Our results reveal that this compound suppressed the activation of HSC, as demonstrated by the maintenance of a quiescent cell morphology, including lipid droplets content, inhibition of α-smooth muscle actin (α-SMA) and collagen α1(I) expression, and up-regulation of MMP-13, Smad7, and PPARγ expression, three key antifibrogenic genes. Furthermore, IFC305 was able to repress the platelet-derived growth factor (PDGF)-induced proliferation of HSC. This inhibition was independent of adenosine receptors stimulation; instead, IFC305 was incorporated into cells by adenosine transporters and converted to AMP by adenosine kinase. On the other hand, addition of pyrimidine ribonucleoside as uridine reversed the suppressive effect of IFC305 on the proliferation and activation of HSC, suggesting that intracellular pyrimidine starvation would be involved in the molecular mechanism of action of IFC305. In conclusion, IFC305 inhibits HSC activation and maintains their quiescence in vitro; these results could explain in part the antifibrotic liver beneficial effect previously described for this compound on the animal model.     

Pseudomonas exotoxin kills Drosophila S2 cells via apoptosis

Pseudomonas exotoxin A (PE) is cytotoxic for eukaryotic cells because it enters cells by receptor-mediated endocytosis, translocates to the cell cytosol and ADP-ribosylates elongation factor 2 (EF2). However, the interaction of this toxin with eukaryotic cells and the mechanism of PE-mediated cell death have not been extensively characterized. The feasibility of carrying out a genome-wide RNAi screen, makes Drosophila melanogaster S2 cells as a good model system to identify essential genes in PE-mediated cytotoxicity, provided a suitable multi-well assay is developed. Here, using the alamarBlue^® viability assay, we show that Drosophila S2 cells are sensitive to PE at picomolar concentrations and that toxin treatments provoke an increase in caspase activity. This prompted us to use RNAi to characterize the mechanism of cell death. Results indicated that PE-mediated death of S2 cells was dependent on the presence of diphthamide, the post translational modification of EF2, and on the presence of Drice, the terminal caspase of insect cells. RNAi to drice or chemical inhibition of caspase action by z-VAD-fmk protected cells from PE-mediated death. Protection from death by RNAi or z-VAD-fmk did not interfere with toxin delivery to the cytosol leading to inhibition of protein synthesis. Using a convenient alamarBlue^® assay, our data confirms the cytotoxicity of PE for S2 cells and establishes apoptosis as the mode of PE-mediated death. This confirms the suitability of Drosophila cells as a convenient and simple model to elucidate the role of specific genes and proteins required for PE action.     

Some effects on muscle and nerve of crude venom from the gastropod Conus striatus

The venom of Conus striatus elicits spontaneous activity in nerves and in skeletal muscles. In frog sartorius muscle the venom lowers resting potentials of muscle fibres and triggers action potentials. The action potentials are of smaller amplitude and much longer duration than normal. Skeletal musculature and nerve ultimately become refractory to electrical stimulation if exposed to adequate concentrations of venom. Spontaneous twitches with amplitudes much greater than normal occur in isolated guinea-pig atria exposed to the venom. The venom has a spasmogenic action on the musculature of rat ilea which can be blocked by the 5-hydroxytryptamine antagonist 2-brom-d-lysergic acid diethylamide bitartrate. Active material in the venom is dialysable. The activity of the venom is modified by heating at 100°C. The pharmacological activity of the venom of C. striatus is similar to but not identical with that of the venom of the piscivorous species C. magus.     

Chlorella vulgaris restores bone marrow cellularity and cytokine production in lead-exposed mice

Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit⁺Lin⁻), granulocyte-macrophage progenitors (Gr1⁺Mac1⁺) and total bone marrow cellularity. In mice gavage-treated daily with 50 mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.     

Toxicity assessment of Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata (Dinophyta) isolated from the northern Ionian Sea (Mediterranean Sea)

In many coastal areas the abundant proliferation of microalgae producing biotoxins determines the occurrence of Harmful Algal Blooms (HABs). Their presence in temperate waters is well documented and often associated with marine toxin-derived disease. The occurrence and toxicity of three harmful microalgae (Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata) from the northern Ionian Sea (Mediterranean Sea) is hereby reported. The three dinoflagellates were sampled both on macroalgae and water and their morphology and occurrence were compared to those of other Mediterranean sites. The toxicity of the three cultured strains was tested by Artemia salina and hemolysis tests and their effects on the first stages of the sea urchin development was also evaluated. The contemporary presence of the three species inhibited the in vitro sea urchin embryonic development. But this action could be ascribed to the sole Ostreopsis as the addition of the single species to the sea urchins embryos evidenced no effects in presence of Amphidinium or Coolia cells, and an irregular segmentation in presence of Ostreopsis. In particular, this latter species exerted a cytotoxic effect in a dose-dependent manner, with a production of deformed embryos even at very low cell concentration (42 cells mL⁻¹). Nevertheless, when algal cell lysate was added, some effects on the sea urchin development was detected for each dinoflagellate, and also in this case Ostreopsis has proved to be the most toxic species. However, the lysate of Amphidinium and Ostreopsis strongly affects the A. salina nauplii vitality, while the hemolytic activity was very low for Amphidinium and Coolia lysate and very strong for Ostreopsis.Our results highlight the importance to monitoring the presence of these dinoflagellates whose effects may also be reflected on the early life stages of marine organisms, especially those species that are important from both an ecological and economic point of view, as the sea urchins are.     

A bioanalytical investigation on the exquisitely strong in vitro potency of the EGFR-DNA targeting type II combi-molecule ZR2003 and its mitigated in vivo antitumour activity

ZR2003 is a type II of combi-molecule designed to target DNA and the epidermal growth factor receptor (EGFR) without requirement for hydrolysis. In human tumour cell lines cultured as monolayers, it showed 6.5-35 fold greater activity than Iressa. Further evaluation in 3D organ-like multilayer aggregates showed that it could block proliferation at submicromolar level. However, despite the superior potency of ZR2003 over Iressa in vitro, its activity xenograft models was not significantly different from that of Iressa. To rationalize these results, we determined the tumour concentration of both ZR2003 and Iressa in vivo and more importantly in vitro in multicellular aggregates. The results showed that in A431 and 4T1 xenografts, the level of ZR2003 absorbed in the tumours were consistently 2-fold less than those generated by Iressa. Likewise, in the multicellular aggregates model, the penetration of ZR2003 was consistently lower than Iressa. In serum containing media, the level of extractable or free ZR2003 was also inferior to those of Iressa. The results from this bioanalytical study, suggest that the discrepancy between the in vitro and in vivo potency of ZR2003 when compared with Iressa, may be imputed to its significantly lower tumour concentration.     

Selectively preparative purification of aristolochic acids and aristololactams from Aristolochia plants

A novel method has been developed for the purification of aristolochic acids and aristololactams compounds from Aristolochia plants, a kind of typically toxic traditional Chinese medicine. In this method, Oligo (ethylene glycol) separation column which has “clustering function” for compounds in TCMs was used to produce the fractions containing the compounds with similar structures. A four-channel parallel preparative HPLC with C18 separation column was employed to purify the target compounds. The extraction sample of the blending of Radix Aristolochiae, Fructus Aristolochiae and Caulis Aristolochiae Manshuriensis was used to develop the method. Then, four aristolochic acids and three aristololactams were obtained using this method and the chemical identification was confirmed by Q-TOF-MS, ¹H NMR and ¹³C NMR. Thus, this method can deal with more than one traditional Chinese medicine simultaneously. Additionally, the results demonstrated that this method was an effective way to purify target compounds selectively from TCMs.     

Isolation and characterisation of P-EPTX-Ap1a and P-EPTX-Ar1a: Pre-synaptic neurotoxins from the venom of the northern (Acanthophis praelongus) and Irian Jayan (Acanthophis rugosus) death adders

The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/‘pseudo’ irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined Acanthophis praelongus and Acanthophis rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719 Da) and P-EPTX-Ar1a (40,879 Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, α, β1 and β2. The two toxins displayed similar levels of PLA₂ activity which was almost solely attributed to the α subunit in both toxins. P-EPTX-Ap1a (20-100 nM) and P-EPTX-Ar1a (20-100 nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the α subunit of both toxins (300 nM) displayed neurotoxic activity. Inhibition of PLA₂ activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity.     

Sea nettle (Chrysaora quinquecirrha) toxin on electrogenic and chemosensitive properties of nerve and muscle

Rat and frog nerves and skeletal muscles and identified neurons of Aplysia californica were exposed in vitro to the toxin of Chrysaora quinquecirrha (sea nettle). All tissues examined were depolarized by the toxin. In skeletal muscle the postsynaptic depolarization had a delayed onset. The effect of the toxin was not accompanied by any alteration of the action potential generating mechanism, delayed rectification, or quantal release of transmitter. The postsynaptic depolarization was followed by a transient increase in miniature endplate potential frequency. Action potential amplitude, overshoot and rate of rise in both rat skeletal muscle and the amplitude and conduction velocity of frog sciatic nerve were reduced by the toxin-induced depolarization but spike generation in rat skeletal muscle was restored by hyperpolarizing the fibers. The depolarizing effects of the toxin were not altered by tetrodotoxin, d-tubocurarine or by changing the external Ca²⁺ level, although lowering the Ca²⁺ level delayed the toxin effect on transmitter release. Heating the toxin to 100°C destroyed its action. In Aplysia central neurons, the depolarization was immediate, accompanied by an increase in spontaneous synaptic activity, and an alteration of resting membrane conductance; the toxin effects were reversible. Neither hyperpolarizations induced by histamine or by acetylcholine nor synaptic cholinergic inhibitory potentials were affected by the toxin. The toxin appears to induce a non-specific membrane depolarization although an increase in sodium conductance resistant to tetrodotoxin cannot be ruled out.