Tetrodotoxin/Saxitoxins Selectivity of the Euryhaline Freshwater Pufferfish Dichotomyctere fluviatilis.

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7-6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.

Binding and Pharmacokinetics of the Sodium Channel Blocking Toxins (Saxitoxin and the Tetrodotoxins).

Tetrodotoxin (TTX) found in diverse variety of animals including puffer fishes, some newts, frogs and limited number of non-vertebrate species (6 different phyla). The saxitoxin (STX) and the TTX are small molecules composed of 7,8,9 guanidinium and 1,2,3 guanidinium groups, respectively in their structures. These groups provide positive charge to the molecules and are believed to interact with negatively charged Glu755 and Asp400 residues in domain II and I of the sodium channel strongly. The pharmacokinetic studies (absorption, distribution and accumulation) reported on Takifugu rubripes, Takifugu pardalis, Takifugu niphobles, Takifugu vermicularis, Takifugu snyderi, etc. revealed that higher concentration of TTX is accumulated in liver than in the skin or other tissues. Although TTX is also accumulated in the skin of various marine species (secretory glands) and the excess of TTX are emitted through skin which acts as a defence agent for those species. STX showed high toxicity on crab and other animals, due to its accumulation in the tissues and resistance to the sodium channel proteins. It concluded that TTX and STX based toxicities are developed on the species by the absorption, distribution and accumulation of toxins in tissues. Also the ingestion of these species (marine species) as food may allow transferring toxin to the human being.

Voltage-gated Na+ channel ß1B: a secreted cell adhesion molecule involved in human epilepsy.

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ß1 and ß1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ß1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ß1B. We show that wild-type ß1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ß1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ß1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ß1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ß1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ß1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ß1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.

Transcriptional up-regulation of cell surface Na V 1.7 sodium channels by insulin-like growth factor-1 via inhibition of glycogen synthase kinase-3ß in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion.

Insulin-like growth factor-1 (IGF-1) plays important roles in the regulation of neuronal development. The electrical activity of Na(+) channels is crucial for the regulation of synaptic formation and maintenance/repair of neuronal circuits. Here, we examined the effects of chronic IGF-1 treatment on cell surface expression and function of Na(+) channels. In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, chronic IGF-1 treatment increased cell surface [(3)H]saxitoxin binding by 31%, without altering the Kd value. In cells treated with IGF-1, veratridine-induced (22)Na(+) influx, and subsequent (45)Ca(2+) influx and catecholamine secretion were augmented by 35%, 33%, 31%, respectively. Pharmacological properties of Na(+) channels characterized by neurotoxins were similar between nontreated and IGF-1-treated cells. IGF-1-induced up-regulation of [(3)H]saxitoxin binding was prevented by phosphatydil inositol-3 kinase inhibitors (LY204002 or wortmannin), or Akt inhibitor (Akt inhibitor IV). Glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl, valproic acid, SB216763 or SB415286) also increased cell surface [(3)H]saxitoxin binding by ∼ 33%, whereas simultaneous treatment of IGF-1 with GSK-3 inhibitors did not produce additive increasing effect on [(3)H]saxitoxin binding. IGF-1 (100 nM) increased Ser(437)-phosphorylated Akt and Ser(9)-phosphorylated GSK-3ß, and inhibited GSK-3ß activity. Treatment with IGF-1, LiCl or SB216763 increased protein level of Na(+) channel α-subunit; it was prevented by cycloheximide. Either treatment increased α-subunit mRNA level by ∼ 48% and accelerated α-subunit gene transcription by ∼ 30% without altering α-subunit mRNA stability. Thus, inhibition of GSK-3ß caused by IGF-1 up-regulates cell surface expression of functional Na(+) channels via acceleration of α-subunit gene transcription.

Comparison of neosaxitoxin versus bupivacaine via port infiltration for postoperative analgesia following laparoscopic cholecystectomy: a randomized, double-blind trial.

BACKGROUND AND OBJECTIVES: Wound infiltration with available local anesthetics generally provides analgesia for less than 8 hrs. The site 1 sodium-channel toxin neosaxitoxin (neoSTX) produced analgesia for over 24 hrs in animals and human volunteers. In this randomized, double-blind trial, we examined the postoperative course of patients undergoing laparoscopic cholecystectomy under a standardized general anesthesia with wound infiltration using either neoSTX or bupivacaine. We hypothesized that neoSTX would reduce pain compared with bupivacaine at 12 hrs postoperatively. METHODS: Patients received preincisional infiltration of laparoscope entry sites with 20 mL containing either neoSTX (total dose, 100 µg) or bupivacaine 0.25% (total dose, 50 mg). The primary outcome measure was the visual analog pain score at 12 hrs postoperatively. Secondary outcomes included repeated pain scores at rest and with movement,analgesic use, functional recovery, and adverse effects. Groups were compared using Mann-Whitney U tests for pain scores, Fisher exact test for proportions of patients with severe pain and complete analgesia, and Kaplan-Meier curves for time to full recovery. RESULTS: Among 137 subjects, 69 were randomized to neoSTX and 68 to bupivacaine. Median pain scores at rest and with movement 12 hrs postoperatively were lower in the neoSTX group compared with the bupivacaine group (P<0.01). Additional pain measures and recovery parameters also favored neoSTX. No serious adverse events occurred,and no adverse events were more frequent in the neoSTX group. CONCLUSIONS: NeoSTX shows promise as a long-acting local anesthetic. Future studies will examine dose response, combination formulations, and safety with dose escalation.

First report about saxitoxins in freshwater fish Hoplias malabaricus through trophic exposure.

Cyanobacterial waterblooms, such as the saxitoxin (STX) producer Cylindrospermopsis raciborskii, have been a worldwide concern in environmental health. However, the bioaccumulation of this neurotoxin in the trophic chain is not completely known. The aim of the present work was to evaluate STX bioaccumulation through chemical analyses and the toxic and trophic effects using biomarkers in the tropical freshwater fish Hoplias malabaricus. They were fed once every five days with Astyanax sp. before being subjected to intraperitoneal inoculation with STX extract (0.08 µg/100 g) obtained by lysis of toxic C. raciborskii strain (T3). After 20 days the brain was collected for acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione (GSH), lipoperoxidation (LPO), protein carbonylation (PCO), and comet assay analysis. The muscle was collected for STX chemical analysis. The activities of SOD and concentrations of PCO and LPO increased. The CAT, GST, and GPx activities decreased. Genotoxicity was observed in the experimental group. STX was not detected in muscle samples. Thus, an oxidative stress was observed in the brain, leading to the damage of lipids, proteins, and DNA. The mechanism of action of the neurotoxin in this subchronic exposure suggests an apoptotic cellular process.

Kinetic properties of saxitoxin in Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua).

The disposition of STX in Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) was studied after intraperitoneal (IP) injection (5 microg STX/kg bm and 3.43 microg (3)H-STXeq/kg bw respectively), intravenous (IV) injection (5 microg STX/kg bm, only salmon) and waterborne exposure (50 microg STXeq/L, only salmon). Plasma concentrations in salmon were quantified using a receptor binding assay and cod tissues were analyzed using scintillation counting of tissue extracts and autoradiography of whole fish slices. The estimated elimination half-life (T(1/2)) after IV administration of STX in salmon was 102.6 min. The volume of distribution (Vz) was observed to be 467.2 mL/kg and the total body clearance (Cl(T)) was 3.2 mL/min/kg. Waterborne exposure clearly showed that salmon absorbed PSP toxins directly from the water. In cod, (3)H-STX was observed in gills, muscle, brain, liver and posterior kidney from 30 to 480 min. The lowest concentrations of (3)H-STX were found in brain and muscle, whereas posterior kidney contained the majority of the toxin. Autoradiograms confirmed the high levels of (3)H-STX in the kidneys, indicating that renal excretion was the main elimination route. Buildup of harmful levels in edible tissue is not very likely due to the low concentrations accumulated in muscle tissue and rapid excretion.

Conduction block in PMP22 deficiency.

Patients with PMP22 deficiency present with focal sensory and motor deficits when peripheral nerves are stressed by mechanical force. It has been hypothesized that these focal deficits are due to mechanically induced conduction block (CB). To test this hypothesis, we induced 60-70% CB (defined by electrophysiological criteria) by nerve compression in an authentic mouse model of hereditary neuropathy with liability to pressure palsies (HNPP) with an inactivation of one of the two pmp22 alleles (pmp22(+/-)). Induction time for the CB was significantly shorter in pmp22(+/-) mice than that in pmp22(+/+) mice. This shortened induction was also found in myelin-associated glycoprotein knock-out mice, but not in the mice with deficiency of myelin protein zero, a major structural protein of compact myelin. Pmp22(+/-) nerves showed intact tomacula with no segmental demyelination in both noncompressed and compressed conditions, normal molecular architecture, and normal concentration of voltage-gated sodium channels by [(3)H]-saxitoxin binding assay. However, focal constrictions were observed in the axonal segments enclosed by tomacula, a pathological hallmark of HNPP. The constricted axons increase axial resistance to action potential propagation, which may hasten the induction of CB in Pmp22 deficiency. Together, these results demonstrate that a function of Pmp22 is to protect the nerve from mechanical injury.

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.

Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam (Callista chione).

At the western Mediterranean coast of Morocco, the cockle (Acanthocardia tuberculatum) contained persistent high levels of paralytic shellfish toxins for several years, while other bivalve molluscs such as sweet clam (Callista chione) from the same vicinity were contaminated seasonally to a much lesser extent. In order to understand the causes of this prolonged contamination, a comparative study on PSP decontamination between sweet clam and cockle was conducted from November 2001 until June 2002. PSP toxicity was analysed by automated pre-column oxidation (Prechromatographic oxidation and LC-FD) in several organs of both species, namely digestive gland, foot, gill, mantle, muscle and siphon for sweet clams. The results showed that cockle sequester PSP toxins preferably in non-visceral organs (Foot, gill and mantle) contrary to sweet clam that sequester them in visceral tissues (digestive gland). The toxin profile of cockle organs indicated dominance of dcSTX, whereas sweet clam tissues contained especially C-toxins. Substantial differences in toxin profile between cockle and sweet clam, from the same area as well as from the composition of PSP toxin producer, Gymnodinium catenatum, confirm the bioconversion of PSP toxins in cockle.

Accumulation of paralytic shellfish toxins (PST) from the cyanobacterium Aphanizomenon issatschenkoi by the cladoceran Daphnia magna.

In order to access the effects of Paralytic Shellfish Toxins (PST) in freshwater environment, the accumulation of PST produced by the cyanobacteria Aphanizomenon issatschenkoi in juvenile Daphnia magna was investigated. D. magna was exposed to A. issatschenkoi cells (1.2 x 10(6) cells ml(-1)) for 6, 8, 12, 24 and 30 h and also to lyophilised material (1 mg ml(-1)) for 24h. Survival and somatic growth of the juvenile D. magna was investigated, as was the activity of the biotransformation enzyme system glutathione-S-transferases (GSTs). Between 643+/-65.35 and 1170+/-51.72 pmol PST ml(-1) were detected by HPLC-FLD in D. magna culture medium containing cells and 2745+/-64.61 pmol PST toxin ml(-1), in the medium containing lyophilised material. PST were detected in D. magna tissues in cells exposure (between 6.51 x 10(-2)+/-1.37 x 10(-2) and 3.78 x 10(-1)+/-1.15 x 10(-2)pmol PST animal(-1)). In D. magna exposed to lyophilised material the mean (+/-SD) PST concentration was found to be 6.96 x 10(-3) (+/-3.84 x 10(-3)) pmol PST animal(-1). Following exposure to 1.2 x 10(6) cells ml(-1)A. issatschenkoi fresh cells growth and survival of D. magna were reduced. D. magna exposed to the two A. issatschenkoi treatments (fresh cells and lyophilised material), showed a reduction in activity of the cytosolic glutathione-S-transferases (cGSTs). The results of this study indicate that D. magna can accumulate PST toxins and that the cyanobacterium A. issatschenkoi affects both the fitness and growth potential of juvenile D. magna.

Permeability of human jejunal segments to gonyautoxins measured by the Ussing chamber technique.

The aim of this work was to study the mechanisms involved in intestinal permeability of gonyautoxins. For this purpose, the influence on transmucosal resistance of gonyautoxins and their permeability was investigated in excised human jejunal segments. To evaluate these events, the isolated mucosa was mounted in Ussing chambers for electrophysiological characterization. The organic gonyautoxin cations were applied to the mucosal side and samples collected on the serosal side. The permeability of gonyautoxins measured at 37 degrees C was 4.3-fold greater than at 4 degrees C, indicative of high cation selective transcellular permeability. In order to characterize the permeability of gonyautoxins, the effects of choline, ouabain, phlorizin and fluorescein were studied. The inhibition by these compounds was expressed as percent inhibition of the maximal flux of gonyautoxins at 120 min. Replacement of sodium ion by choline, showed the highest inhibition (85.5% from control). Ouabain, fluorescein and phlorizin inhibit the gonyautoxins flux by 53.9, 41.0 and 9.64%, respectively. The inhibition of gonyautoxins' permeability produced by ouabain and phlorizin go in parallel with an increase in the transmucosal electrical resistance (TER). This study shows that permeability of gonyautoxin cations occurred predominantly by the transcellular pathway (76%) when toxins were applied in the mucosal-serosal direction. The paracellular pathway of gonyautoxins was 24% of total permeability when compared with [3H] mannitol permeability. These findings suggests that permeability of gonyautoxins depends on temperature and processes involving sodium ion. Replacing sodium ions by choline ions showed a marked effect on TER.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea.

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean = 1.4 x 10(9) cells1(-1), S.D. = 0.29 x 10(9) cellsl(-1)) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PSTl(-1), S.D. = 9.9 nmol PSTl(-1)). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus. Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 microg PST100 g(-1) by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi. Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day(-1)) and those fed with A. falcatus (8.2% day(-1)) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

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.

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.

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.

Kinetics of accumulation and transformation of paralytic shellfish toxins in the blue mussel Mytilus galloprovincialis.

Mussels (Mytilus galloprovincialis) were fed cultures of the Paralytic Shellfish Poisoning agent Alexandrium minutum (Strain AL1V) for a 15-day period and, for the next 12 days, they were fed the non-toxic species Tetraselmis suecica, in order to monitor the intoxication/detoxification process. The toxin content in the bivalve was checked daily throughout the experiment. During the time-course of the experiment, the toxin profile of the bivalves changed substantially, showing increasingly greater differences from the proportions found in the toxigenic dinoflagellate used as food. The main processes involved in the accumulation of toxins and in the variation of the toxic profiles were implemented in a series of numerical models and the usefulness of those models to describe the actual intoxication/detoxification kinetics was assessed. Models that did not include transformations between toxins were unable to describe the kinetics, even when different detoxification rates were allowed for the toxins involved. The models including epimerization and reduction provided a good description of the kinetics whether or not differential detoxification was allowed for the different toxins, suggesting that the differences in detoxification rates between the toxins are not an important factor in regulating the change of the toxic profile. The implementation of Michaelis-Menten kinetics to describe the two reductive transformations produced a model that had a poorer fit to the data observed than the model that included only a first order kinetics. This suggests that, it is very unlikely that any enzymatic reaction is involved in the reduction of the hydroxycarbamate (OH-GTXs) to carbamate (H-GTXs) gonyautoxins.

Effects of toxic dinoflagellates and toxin biotransformation in bivalves.

Attempts were made to elucidate the different responses of shellfish to paralytic shellfish poison (PSP) and the PSP donor Alexandrium minutum T1. Five species of edible bivalves (Crassostrea gigas, Meretrix lusoria, Mytilus edulis, Ruditapes philippinarum, and Soletellina diphos) were collected and examined for susceptibility to PSP and PSP donor. It was determined that all five bivalves had low susceptibility to PSP following an intramuscular injection (> 300 MU/20 g). The abnormal effects on bivalves were species-specific and varied with the concentration of A. minutum T1. Judging from the LC50 data (medium lethal concentration), the resistance of bivalves to the toxic dinoflagellate was as follows (least to most resistant): C. gigas < R. philippinarum < M. lusoria < M. edulis, S. diphos. With the exception of S. diphos, the bivalves accumulated very little toxin (< 2 MU/g edible tissue) when they were exposed to 10(7) cells/L of A. minutum for four days. The toxin levels in S. diphos increased with exposure time to the toxic dinoflagellates and accumulated primarily in the digestive gland (88-100%), followed by the gill (0-10%), and other organs (0-8%). Although the concentrations of toxin components in the digestive gland were found to be variable during the exposure period, the toxin profile in the digestive gland of S. diphos during the early exposure period was similar to that of A. minutum. Moreover, toxin components in the gills and in other organs were retained at near constant concentrations during the exposure period.

Effects of age and gene dose on skeletal muscle sodium channel gating in mice deficient in myotonic dystrophy protein kinase.

Myotonic muscular dystrophy (DM) is characterized by abnormal skeletal muscle Na channel gating and reduced levels of myotonic dystrophy protein kinase (DMPK). Electrophysiological measurements show that mice deficient in Dmpk have reduced Na currents in muscle. We now find that the Na channel expression level is normal in mouse muscle partially or completely deficient in Dmpk. Reduced current amplitudes are not changed by age or gene dose, and the reduction is not due to changes in macroscopic or microscopic gating kinetics. The mechanism of abnormal membrane excitability in DM may in part be silencing of muscle Na channels due to Dmpk deficiency.