Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan.

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.     

High affinity for the rat brain sodium channel of newly discovered hydroxybenzoate saxitoxin analogues from the dinoflagellate Gymnodinium catenatum.

The paralytic shellfish poison family has been recently extended by the discovery of several analogues possessing a hydoxybenzoate moiety instead of the carbamoyl group one finds in saxitoxin, the parent molecule of this toxin family. We have investigated the potency of these new analogues on a representative isoform of the pharmacological target of these toxins, the voltage gated sodium channel. These toxins were found to have K1's in the low nanomolar range, only slightly less potent than saxitoxin. The hydroxybenzoate group may increase the lipophilicity of these toxins and improve their ability to pass through epithelia and therefore its uptake and elimination in both intoxication victims and animals that bioaccumulate paralytic shellfish toxins.     

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.     

Differential effects of bupivacaine enantiomers,ropivacaine and lidocaine on up-regulation of cell surface voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, (+/-)-bupivacaine inhibited veratridine-induced 22Na(+) influx (IC(50) 6.8 microM). The IC(50) of (+)-bupivacaine (2.8 microM) was 6.2-, 7.4-, and 17.1-fold lower than those of (-)-bupivacaine (17.3 microM), (-)-ropivacaine (20.6 microM), and lidocaine (47.8 microM). Chronic (i.e. 3-h) treatment of cells with (+/-)-bupivacaine increased cell surface [3H]saxitoxin ([3H]STX) binding capacity by 48% (EC(50) of 233 microM; t(1/2)=7.4 h), without changing the K(d) value. Treatment for 24 h with either (+)- or (-)-bupivacaine, or (-)-ropivacaine elevated [3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [3H]STX binding by (+/-)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (+/-)-bupivacaine did not increase Na(+) channel alpha- and beta(1)-subunit mRNA levels. In cells subjected to (+/-)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na(+) influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na(+),K(+)-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na(+) influx by 2.3-fold in the (+/-)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (-)-ropivacaine acutely inhibit Na(+) channel gating, whereas its chronic treatment up-regulates cell surface expression of Na(+) channels via translational and externalization events.     

Route of metabolization and detoxication of paralytic shellfish toxins in humans

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria.Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver.In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with β-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation.The apparent V_max and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V_max formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8 ± 1.9 × 10⁻³; 8.3 ± 2.8 × 10⁻³ and 9.7 ± 2.8 × 10⁻³ pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2 ± 0.12; 27.06 ± 0.23 and 32.02 ± 0.64 μM respectively).In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.     

Paralytic shellfish poisoning: Seafood safety and human health perspectives

Paralytic shellfish poisoning (PSP) is the foodborne illness associated with the consumption of seafood products contaminated with the neurotoxins known collectively as saxitoxins (STXs). This family of neurotoxins binds to voltage-gated sodium channels, thereby attenuating action potentials by preventing the passage of sodium ions across the membrane. Symptoms include tingling, numbness, headaches, weakness and difficulty breathing. Medical treatment is to provide respiratory support, without which the prognosis can be fatal. To protect human health, seafood harvesting bans are in effect when toxins exceed a safe action level (typically 80 μg STX eq 100 g⁻¹ tissue). Though worldwide fatalities have occurred, successful management and monitoring programs have minimized PSP cases and associated deaths. Much is known about the toxin sources, primarily certain dinoflagellate species, and there is extensive information on toxin transfer to traditional vectors - filter-feeding molluscan bivalves. Non-traditional vectors, such as puffer fish and lobster, may also pose a risk. Rapid and reliable detection methods are critical for toxin monitoring in a wide range of matrices, and these methods must be appropriately validated for regulatory purposes. This paper highlights PSP seafood safety concerns, documented human cases, applied detection methods as well as monitoring and management strategies for preventing PSP-contaminated seafood products from entering the food supply.     

A neurophysiological method of rapid detection and analysis of marine algal toxins

We have examined the effectiveness of the in vitro rat hippocampal slice preparation as a means of rapidly and specifically detecting the marine algal toxins saxitoxin, brevetoxin, and domoic acid and have identified toxin-specific electrophysiological signatures for each. Brevetoxin (PbTX₃, 50–200 nM) produced a significant reduction in orthodromic population spike amplitude which was quick to reverse during a 50 min wash-out, while antidromic population spikes and field EPSPs exhibited only slight reductions, and fibre spiof orthodrokes showed no change at all. Domoic acid (100 nM) produced a robust, reversible increase in amplitude mic spikes, and the appearance of multiple spikes (i.e., epileptiform activity) within minutes of toxin wash-in. Other notable features of the domoic acid signature included a significant decrease in amplitude of the field EPSPs, and a complete absence of effect on either antidromic or fibre spikes. Fifty nanomolar saxitoxin (PSP) abolished all responses in all slices. Only antidromic spikes showed any recovery during wash-out. Field EPSP and fiber spike analysis further demonstrated that the preparation is capable of reliably detecting saxitoxin in a linearly responsive fashion at toxin concentrations of 25–200 nM, and tests of naturally contaminated shellfish confirmed the utility of this assay as a screening method for PSP. Our findings suggest that the in vitro hippocampal slice preparation has potential in the detection and analysis of three marine algal toxins important to the shellfish industry.     

Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve

Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na⁺. Relief of TTX blockage was also observed by adding 100 mM chloride salts of Li⁺, hydrazine⁺, guanidine⁺ and K⁺ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.     

Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line

Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN-1A cells. Peak sodium currents had a half-maximal activation, V_m0.5, of −22.6 ± 1.0 mV with a voltage dependence, K_m, of 7.28 ± 0.39 mV⁻¹. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with V_h0.5 = −93.8 ± 1.2 mV and k_h = −6.8 ± 0.4 mV⁻¹. The second type of sodium channel inactivated w V_h0.5 = −44.6 ± 1.5 mV and k_h = −7.3 ± 0.4 mV⁻¹. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent IC50s of 22 nM and 5.3 μM. STX was more potent, with apparent IC₅₀s of 1.6 nM and 1.2 μM. There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types.