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PubMed1994

Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples.

Paralytic shellfish poison (PSP) has historically been a problem for the shellfish industry. In order to prevent the marketing of contaminated seafood products, governments have implemented monitoring programs where standards of toxins are necessary. The stability of these standard toxins is very important. In this paper we analysed the stability of saxitoxin (STX) and neosaxitoxin in acidic solution and lyophilized samples. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing post-column oxidation of the toxins to form fluorescent derivatives. Our results demonstrate that STX is very stable in solution samples and could be adopted as a reference standard. This toxin can be kept in dilute acidic solutions for 18 months without loss of potency. However, neosaxitoxin is unstable, possibly due to transformation to other toxins.

Animals↗
PubMed1994

Expression of sodium channels with different saxitoxin affinity during rat forebrain development.

This work characterizes the development of the saxitoxin (STX)-sensitive Na+ channels from rat whole forebrain between embryonic day 15 (E15) and postnatal day 90 (P90), both with binding studies and with single channel studies. The Na+ channel total mRNA and the individual mRNAs encoding Na+ channels I, II and III were also determined. The total STX binding rose about 40-fold from E15 to reach a plateau at P30 and its temporal course correlated with the expression of Na+ channel total mRNA. Low affinity and high-affinity STX binding sites, predominant in embryonic and postnatal forebrains, respectively, were found. The single channel studies of batrachotoxin-modified channels also revealed two main populations. In E15 only low-affinity channels (KD = 32.7 nM; 200 mM NaCl) and in P30 only high affinity ones (KD = 1.6 nM) were present. At P0 channels with intermediate affinity (KD range 3-34 nM) were observed. The increase in affinity was due to a gradual increase in the STX association rate.

Animals↗
PubMed1994

Method for the identification of saxitoxin in rat urine.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.

Animals↗
PubMed1993

Binding of [3H]batrachotoxinin A-20-alpha-benzoate and [3H]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes.

1. [3H]Batrachotoxinin A-20-alpha-benzoate ([3H]BTX-B) and [3H]saxitoxin ([3H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes. 2. Specific [3H]BTX-B binding was temperature dependent with highest levels of specific [3H]BTX-B binding observed at 7 degrees C. Specific binding was inversely correlated with assay temperature at temperatures above 7 degrees C. 3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [3H]BTX-B binding at 27 degrees C, but not at 7 degrees C. The dihydropyrazole insecticide RH 3421 inhibited specific [3H]BTX-B binding at 7 degrees C but had no effect on specific binding at 27 degrees C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [3H]BTX-B binding at both 7 degrees C and 27 degrees C. 4. Displacement experiments in the presence of ScV at 27 degrees C gave an equilibrium dissociation constant (KD) for [3H]BTX-B of 710 nM and a maximal binding capacity (Bmax) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 x 10(5) min-1 nM-1) and dissociation (0.0514 min-1) of the ligand-receptor complex. 5. The binding of [3H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a KD of 3.8 nM and a Bmax of 1.9 pmol/mg protein. 6. These studies provide evidence for high affinity, saturable binding sites for [3H]BTX-B and [3H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [3H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [3H]BTX-B binding in mammalian brain preparations had no effect on specific [3H]BTX-B binding in the trout.

Animals↗
PubMed2004

Morphological abnormalities and sensorimotor deficits in larval fish exposed to dissolved saxitoxin.

The dietary uptake of one suite of dinoflagellate-produced neurotoxins, that are commonly called paralytic shellfish poisoning (PSP) toxins, is known to cause acute fish kills. However, little is known about the effects of dissolved phase exposure and the potential sublethal effects of this route of exposure on early developmental stages of fish. Toxin exposure during early development is of particular concern because the embryos and larvae of some marine fish species may be unable to actively avoid the dissolved toxins that algal cells release into the water column during harmful algal blooms. Here we use the zebrafish (Danio rerio) as a model experimental system to explore the sublethal effects of a dissolved PSP toxin, saxitoxin (STX), on early development in fish, including sensorimotor function, morphology, and long-term growth and survival. Aqueous phase exposures of 229 +/- 7 microg STX eq. l(-1) caused reductions in sensorimotor function as early as 48 h postfertilization (hpf) and paralysis in all larvae by 4 days postfertilization (dpf). Rohon-Beard mechanosensory neurons appeared to be more sensitive to STX than dorsal root ganglion neurons at this dose. Additionally, exposure to 481 +/- 40 microg STX eq. l(-1) resulted in severe edema of the eye, pericardium, and yolk sac in all exposed larvae by 6 dpf. The onset of paralysis in STX-exposed larvae was stage-specific, with older larvae becoming paralyzed more quickly than younger larvae (5 h at 6 dpf as compared to 8 and 46 h for 4 and 2 dpf larvae, respectively). When transferred to clean water, many larvae recovered from the morphological and sensorimotor effects of STX. Thus, the sublethal effects of the toxin on larval morphology and behavior were reversible. However, zebrafish exposed to STX transiently during larval development (from 2 to 4 dpf) had significantly reduced growth and survival at 18 and 30 days of age. Collectively, these data show that (1) dissolved phase STX is bioavailable to fish embryos and larvae, (2) the toxin is a paralytic with potencies that are stage-specific for fish larvae, (3) the observed toxicological effects of STX exposure are reversible, and (4) a short-term toxin exposure can negatively impact the survival of fish several weeks later. Dissolved algal toxins may therefore have important sublethal effects on vulnerable species of fish.

Animals↗
PubMed2004

Evidence for differences in the metabolism of saxitoxin and C1+2 toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3.

The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1+2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 microg l(-1). Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 microg l(-1) CAM increased C1+2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1+2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1+2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3.

Agmatine↗
PubMed2004

Liquid chromatography/quadrupole time-of-flight mass spectrometry for determination of saxitoxin and decarbamoylsaxitoxin in shellfish.

Saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) were determined by liquid chromatography with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A shellfish tissue was extracted with 0.1 mol/l HCl under ultrasonication, and cleanup of extract was accomplished by solid-phase extraction with a C18 cartridge. Chromatographic separation was carried out on a C18 column (150 mm x 2.1 mm, 3.5 microm) with gradient elution of MeOH-H2O (20:80) containing 0.05% heptafluorobutyric acid and MeOH-H2O (15:85) containing 0.05% acetic acid. The protonated molecule [M + H]+ ions at m/z 257 for dcSTX and 300 for STX were selected in precursor ion scanning for Q-TOF MS in the positive electrospray ionizaion mode. Average recoveries and relative standard deviations, by analyzing samples spiked at a level of 0.1, 0.8 or 1.6 microg/g, were 84-92 and 8-14%, respectively. Identification of the presence of the toxins in shellfish tissues was based on the structural information offered by Q-TOF MS.

Animals↗
PubMed2004

Gas-phase dissociation reactions of protonated saxitoxin and neosaxitoxin.

The aim of this study was to investigate the behavior of the protonated paralytic shellfish poisons saxitoxin (STX) and neosaxitoxin (NEO) in the gas-phase after ion activation using different tandem mass spectrometry techniques. STX and NEO belong to a group of neurotoxins produced by several strains of marine dinoflagellates. Their chemical structures are based on a tetrahydropurine skeleton to which a 5-membered ring is fused. STX and NEO only vary in their substituent at N-1, with STX carrying hydrogen and NEO having a hydroxyl group at this position. The collision-induced dissociation (CID) spectra exhibited an unusually rich variety and abundance of species due to the large number of functional groups within the small skeletal structures. Starting with triple-quadrupole CID spectra as templates, linked ion-trap MSn data were added to provide tentative dissociation schemes. Subsequent high-resolution FTICR experiments gave exact mass data for product ions formed via infrared multiphoton dissociation (IRMPD) from which elemental formulas were derived. Calculations of proton affinities of STX and NEO suggested that protonation took place at the guanidinium group in the pyrimidine ring for both molecules. Most of the observed parallel and consecutive fragmentations could be rationalized through neutral losses of H2O, NH3, CO, CO2, CH2O and different isocyanate, ketenimine and diimine species, many of which were similar for STX and NEO. Several exceptions, however, were noted and differences could be readily correlated with reactions involving NEO's additional hydroxyl group. A few interesting variations between CID and IRMPD spectra are also highlighted in this paper.

Animals↗
PubMed2004

Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum.

Eleven male and 14 female specimens of a marine puffer Arothron firmamentum were collected from Oita and Iwate Prefectures, Japan. The toxicity assay using mouse showed that only ovary and skin of the female specimens were toxic, the toxicity scores being 5-740 as paralytic shellfish poison and <5-30 MU/g as tetrodotoxin (TTX), respectively. The toxin extracts from the both tissues were then treated with cartridge columns, and subjected to high performance liquid chromatography and liquid chromatography-mass spectral analyses. In the analyses, saxitoxin (STX) and decarbamoylSTX (dcSTX) were identified as the major toxins in the ovary, while the skin contained only TTX.

Animals↗
PubMed2004

Pharmacological effects of the marine toxins, brevetoxin and saxitoxin, on murine frontal cortex neuronal networks.

Brevetoxins and saxitoxins (STXs), which are produced by marine dinoflagellates, are very potent neurotoxins targeting separate sites of the alpha subunit of voltage-dependent sodium channels (VDSCs). An attractive approach for marine toxin detection relies on pharmacological modulation of VDSCs expressed in cells or tissues. While these function-based cellular assays exhibit the required sensitivity, they are typically slow and have limited potential use for field applications. Cultured neuronal networks grown on substrate integrated microelectrode arrays (MEAs) have emerged as a robust and sensitive approach for environmental threat detection. The present work describes the rapid effects of brevetoxin-2 (PbTx-2) and STX on embryonic murine frontal cortex neuronal networks on MEAs. Network recording parameters such as mean spike rate, burst rate, burst duration, number of spikes per burst and spike amplitude were analyzed before and after exposure to the toxins. STX produced fast and reversible inhibition of all electrophysiological parameters with IC(50)s ranging between 1.2 and 2.2nM. Although PbTx-2 also caused inhibition of most of the network electrophysiological parameters, it produced an increase in burst duration at lower concentrations (EC(50)=15+/-2 nM, n=4) followed by inhibition at higher ones (IC(50)=63+/-4 nM, n=4). Exposure of frontal cortex networks to PbTx-2 and STX also caused differential effects on spike amplitude. This work demonstrates that cultured neuronal networks not only could be used for pharmacological characterization of marine toxins but they also provide a tool with unique properties for their detection.

Action Potentials↗
PubMed2006

Distribution of tetrodotoxin, saxitoxin, and their analogs among tissues of the puffer fish Fugu pardalis.

The anatomical distribution of tetrodotoxin (TTX), saxitoxin (STX) and their analogs (TTXs, STXs) in three female and three male specimens of the marine puffer fish Fugu pardalis from Miyagi Prefecture, 2005, Japan, were studied. 5-DeoxyTTX, 11-deoxyTTX, and 5,6,11-trideoxyTTX were quantified by liquid chromatography/mass spectrometry (LC/MS) for the first time, and other TTXs and STXs were determined by liquid chromatography-fluorescent detection (LC-FLD). As a result, 5,6,11-trideoxyTTX was found to be the major TTX analog in all tissues tested, whereas 5-deoxyTTX and 11-deoxyTTX were minor components. Especially, in female (n=3), the ratios of 5,6,11-trideoxyTTX to total of all TTX analogs (mole/mole) in ovaries (mean+/-SD, 0.42+/-0.055) were significantly larger than those in livers (0.17+/-0.025) (P<0.05). In contrary, the ratios of 4,9-anhydroTTX to total of all TTX analogs in livers (0.27+/-0.047) were significantly larger than those in ovaries (0.073+/-0.040) (P<0.01). The ratios of TTX to total of all TTX analogs were not significantly different between ovaries (0.47+/-0.078) and livers (0.55+/-0.067). In male (n=3), all these ratios were not significantly different between livers and testis. 4-S-CysteinylTTX was detected in liver, spleen, gall, and intestine in 1-6mole% of total of all TTX analogs, supporting our previous hypothesis that 4-S-cysteinylTTX is a metabolite of TTX.

Animals↗
PubMed2006

Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel and for this reason, it has been widely used in the study of Na+ channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150-200 g) were injected i.p. with STX (5 and 10 microg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 microg/kg STX dose) and 30 min (for 10 microg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood-brain barrier and is distributed along the whole brain.

Animals↗
PubMed2004

Removal of saxitoxins from drinking water by granular activated carbon, ozone and hydrogen peroxide--implications for compliance with the Australian drinking water guidelines.

In a laboratory-scale trial, we studied the removal of saxitoxins from water by ozone, granular activated carbon (GAC) and H(2)O(2), and considered the implications of residual toxicity for compliance with the Australian drinking water standards. Cell-free extracts of Anabaena circinalis were added to raw, untreated drinking water obtained from a water supply reservoir to provide a toxicity of 30 microg (STX equivalents)l(-1). Ozone alone, or in combination with H(2)O(2), failed to destroy the highly toxic STX and GTX-2/3, and only partially destroyed dc-STX, and the low-toxicity C-toxins and GTX-5. In all cases, the toxicity of the water was reduced by less than 10%. GAC removed all of the STX, dc-STX and GTXs, but only partially removed the C-toxins. However, the residual toxicity was reduced to the suggested Australian drinking water guideline concentration of 3 microg (STX equivalents)l(-1) without O(3) pre-treatment. Modelling the spontaneous chemical degradation of residual C-toxins following treatment shows that residual toxicity could increase to 10 microgl(-1) after 11 d due to formation of dc-GTXs and would then gradually decay. In all, residual toxicity would exceed the Australian drinking water guideline concentration for a total of 50 d.

Anabaena↗
PubMed1984

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.

Animals↗
PubMed1986

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons.

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.

Animals↗
PubMed1986

Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner.

The inhibition by saxitoxin (STX) of single Na channels incorporated into planar lipid bilayers and modified by batrachotoxin (BTX) previously has been shown to be voltage dependent (Krueger, B.K.,J.F. Worley, and R. J. French, 1983, Nature [Lond.], 303:172-175; Moczydlowski, E., S. Hall, S. S. Garber, G. S. Strichartz, and C. Miller, 1984, J. Gen. Physiol., 84:687-704). We tested for such a voltage dependence of STX block of the Na current in voltage-clamped frog nodes of Ranvier. The block by STX of normal Na channels showed no modulation in response to maintained (20 s) changes of the membrane potential or to a train of brief pulses to potentials more positive than the holding potential. However, when the nodal channels were modified by BTX, the train of pulses produced a modulation of the block of the Na current by STX. The modulation of STX block depended on the voltage of the conditioning pulses and this voltage dependence agreed well with that predicted from the single channel studies over the membrane potential range used in those studies. In addition, we found that the voltage dependence of STX block was manifest only at potentials equal to or more positive than required to activate the channels. Most of the apparent differences among data from single channels in bilayers, equilibrium binding studies of STX, and the experiments described here are resolved by the hypotheses that (a) STX binding to open channels is voltage dependent, and (b) the affinities of STX for closed and inactivated channels are independent of voltage, equal, and less than the open channel affinity at potentials less than 0 mV. Whether these hypotheses apply to the STX block of all Na channels or just of BTX-modified channels remains to be determined.

Animals↗
PubMed1991

Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.

Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.

Amino Acid Sequence↗
PubMed1994

Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.

We have probed a cysteine residue that confers resistance to tetrodotoxin (TTX) block in heart Na channels, with membrane-impermeant, cysteine-specific, methanethiosulfonate (MTS) analogs. Covalent addition of a positively charged group to the cysteinyl sulfhydryl reduced pore conductance by 87%. The effect was selectively prevented by treatment with TTX, but not saxitoxin (STX). Addition of a negatively charged group selectively inhibited STX block without affecting TTX block. These results agree with models that place an exposed cysteinyl sulfhydryl in the TTX site adjacent to the mouth of the pore, but do not support the contention that STX and TTX are interchangeable. The surprising differences between the two toxins are consistent with the hypothesis that the toxin-receptor complex can assume different conformations when STX or TTX bound.

Animals↗