Pinnatoxins' Deleterious Effects on Cholinergic Networks: From Experimental Models to Human Health.

Pinnatoxins (PnTXs) are emerging neurotoxins that were discovered about 30 years ago. They are solely produced by the marine dinoflagellate Vulcanodinium rugosum, and may be transferred into the food chain, as they have been found in various marine invertebrates, including bivalves. No human intoxication has been reported to date although acute toxicity was induced by PnTxs in rodents. LD50 values have been estimated for the different PnTXs through the oral route. At sublethal doses, all symptoms are reversible, and no neurological sequelae are visible. These symptoms are consistent with impairment of central and peripheral cholinergic network functions. In fact, PnTXs are high-affinity competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Moreover, their lethal effects are consistent with the inhibition of muscle nAChRs, inducing respiratory distress and paralysis. Human intoxication by ingestion of PnTXs could result in various symptoms observed in episodes of poisoning with natural nAChR antagonists. This review updates the available data on PnTX toxicity with a focus on their mode of action on cholinergic networks and suggests the effects that could be extrapolated on human physiology.

Corrected speciation and gyromitrin content of false morels linked to ALS patients with mostly slow-acetylator phenotypes.

A case-control study of sporadic amyotrophic lateral sclerosis (ALS) in a mountainous village in the French Alps discovered an association of cases with a history of eating wild fungi (false morels) collected locally and initially identified and erroneously reported as Gyromitra gigas. Specialist re-examination of dried specimens of the ALS-associated fungi demonstrated they were members of the G. esculenta group, namely G. venenata and G. esculenta, species that have been reported to contain substantially higher concentrations of gyromitrin than present in G. gigas. Gyromitrin is metabolized to monomethylhydrazine, which is responsible not only for the acute oral toxic and neurotoxic properties of false morels but also has genotoxic potential with proposed mechanistic relevance to the etiology of neurodegenerative disease. Most ALS patients had a slow- or intermediate-acetylator phenotype predicted by N-acetyltransferase-2 (NAT2) genotyping, which would increase the risk for neurotoxic and genotoxic effects of gyromitrin metabolites.

Reduced growth of Listeria monocytogenes in two model cheese microcosms is not associated with individual microbial strains.

Two model antilisterial microbial communities consisting of two yeasts, two Gram positive and two Gram negative bacteria, and originating from Livarot cheese smear were previously designed. They were used in the present study to analyse the impact of microbial population dynamics on growth of Listeria monocytogenes in cheese microcosm. Specific culture media and PCR primers were developed for simultaneous culture-dependent and real-time PCR quantification of strains belonging to Marinomonas sp., Paenibacillus sp., Staphylococcus equorum, Arthrobacter arilaitensis, Pseudomonas putida, Serratia liquefaciens, Candida natalensis, and Geotrichum candidum, in cheese microcosms. All strains were enumerated after 3, 5, 8 and 14 days at 15 °C. They established well at high counts in all cheese microcosms. Growth dynamics for all strains in presence of L. monocytogenes WSLC 1685 were compared to those of microbial communities obtained by omitting in turn one of the six members of the initial community. The growth of the microbial strains was neither markedly disturbed by Listeria presence nor by the removal of each strain in turn. Furthermore, these communities had a significant reducing effect on growth of L. monocytogenes independently of pH, as confirmed by mathematical modelling. A barrier effect was observed, that could be explained by specific competition for nutrients.

Ciguatera Fish Poisoning in the Caribbean Sea and Atlantic Ocean: Reconciling the Multiplicity of Ciguatoxins and Analytical Chemistry Approach for Public Health Safety.

Ciguatera is a major circumtropical poisoning caused by the consumption of marine fish and invertebrates contaminated with ciguatoxins (CTXs): neurotoxins produced by endemic and benthic dinoflagellates which are biotransformed in the fish food-web. We provide a history of ciguatera research conducted over the past 70 years on ciguatoxins from the Pacific Ocean (P-CTXs) and Caribbean Sea (C-CTXs) and describe their main chemical, biochemical, and toxicological properties. Currently, there is no official method for the extraction and quantification of ciguatoxins, regardless their origin, mainly due to limited CTX-certified reference materials. In this review, the extraction and purification procedures of C-CTXs are investigated, considering specific objectives such as isolating reference materials, analysing fish toxin profiles, or ensuring food safety control. Certain in vitro assays may provide sufficient sensitivity to detect C-CTXs at sub-ppb levels in fish, but they do not allow for individual identification of CTXs. Recent advances in analysis using liquid chromatography coupled with low- or high-resolution mass spectrometry provide new opportunities to identify known C-CTXs, to gain structural insights into new analogues, and to quantify C-CTXs. Together, these methods reveal that ciguatera arises from a multiplicity of CTXs, although one major form (C-CTX-1) seems to dominate. However, questions arise regarding the abundance and instability of certain C-CTXs, which are further complicated by the wide array of CTX-producing dinoflagellates and fish vectors. Further research is needed to assess the toxic potential of the new C-CTX and their role in ciguatera fish poisoning. With the identification of C-CTXs in the coastal USA and Eastern Atlantic Ocean, the investigation of ciguatera fish poisoning is now a truly global effort.

Early-onset, conjugal, twin-discordant, and clusters of sporadic ALS: Pathway to discovery of etiology via lifetime exposome research.

The identity and role of environmental factors in the etiology of sporadic amyotrophic lateral sclerosis (sALS) is poorly understood outside of three former high-incidence foci of Western Pacific ALS and a hotspot of sALS in the French Alps. In both instances, there is a strong association with exposure to DNA-damaging (genotoxic) chemicals years or decades prior to clinical onset of motor neuron disease. In light of this recent understanding, we discuss published geographic clusters of ALS, conjugal cases, single-affected twins, and young-onset cases in relation to their demographic, geographic and environmental associations but also whether, in theory, there was the possibility of exposure to genotoxic chemicals of natural or synthetic origin. Special opportunities to test for such exposures in sALS exist in southeast France, northwest Italy, Finland, the U.S. East North Central States, and in the U.S. Air Force and Space Force. Given the degree and timing of exposure to an environmental trigger of ALS may be related to the age at which the disease is expressed, research should focus on the lifetime exposome (from conception to clinical onset) of young sALS cases. Multidisciplinary research of this type may lead to the identification of ALS causation, mechanism, and primary prevention, as well as to early detection of impending ALS and pre-clinical treatment to slow development of this fatal neurological disease.

Correction: Lagrange, E.; Vernoux, J.-P. Warning on False or True Morels and Button Mushrooms with Potential Toxicity Linked to Hydrazinic Toxins: An Update. Toxins 2020, 12, 482.

The authors wish to make corrections to their paper [...].

Integrating Selection and Risk Assessment of Chemical Mixtures: A Novel Approach Applied to a Breast Milk Survey.

BACKGROUND: One of the main challenges of modern risk assessment is to account for combined exposure to the multitude of various substances present in food and the environment. OBJECTIVE: The present work proposes a methodological approach to perform chemical risk assessment of contaminant mixtures across regulatory silos regarding an extensive range of substances and to do so when comprehensive relevant data concerning the specific effects and modes of action of the mixture components are not available. METHODS: We developed a complete step-by-step approach using statistical methods to prioritize substances involved in combined exposure, and we used a component-based approach to cumulate the risk using dose additivity. The most relevant toxicological end point and the associated reference point were selected from the literature to construct a toxicological threshold for each substance. DISCUSSION: By applying the proposed method to contaminants in breast milk, we observed that among the 19 substances comprising the selected mixture, ∑DDT, ∑PCBi, and arsenic were main joint contributors to the risk of neurodevelopmental and thyroid effects for infants. In addition, ∑PCCD/F contributed to the thyroid effect and ∑aldrin-dieldrin to the neurodevelopmental effect. Our case study on contaminants in breast milk demonstrated the importance of crossing regulatory silos when studying mixtures and the importance of identifying risk drivers to regulate the risk related to environmental contamination. Applying this method to another set of data, such as human biomonitoring or in ecotoxicology, will reinforce its relevance for risk assessment. https://doi.org/10.1289/EHP8262.

Reversible sub-acute motor neuron syndrome after mushroom intoxication masquerading as amyotrophic lateral sclerosis.

A 56-year-old man presented with rapidly evolving/sub-acute upper and lower motor neuron syndrome in 2015 with significant weakness in the four limbs and the bulbar region. Amyotrophic lateral sclerosis functional rating scale-revised (ALSFRS-r) was rated 34/48. On electromyography, there was a diffuse and active denervation in the four limbs and the tongue. A diagnosis of definite ALS according to international criteria was made. Six months later the patient stopped worsening. In the following years he progressively recovered. ALSFRS-r score improved to reach 48/48 in 2021. His neurological examination is normal and electromyography shows no denervation. Inquiry revealed that he presented a few months and, again a few days before onset, a mushroom poisoning. He was used to eating false morels either crude or undercooked and developed muscles cramps, nausea and vertigo. The relationships between this reversible sub-acute motor neuron syndrome and mushroom intoxication are discussed in the light of the recently described cluster in the Alps with a high incidence of ALS cases. Epidemiological investigations showed that all patients, but not their spouses, used to eat crude or undercooked false morels. Such a mushroom contains hydrazines, a known neurotoxic agent. We are not aware of another case of ALS reversal in that cluster area. We propose that a potential mushroom poisoning be thoroughly searched for when facing with a patient with sub-acute or rapidly worsening ALS syndrome.

Shellfish and residual chemical contaminants: hazards, monitoring, and health risk assessment along French coasts.

In this review, we address the identification of residual chemical hazards in shellfish collected from the marine environment or in marketed shellfish. Data, assembled on the concentration of contaminants detected, were compared with the appropriate regulatory and food safety standards. Moreover, data on human exposure and body burden levels were evaluated in the context of potential health risks.Shellfish farming is a common industry along European coasts. The primary types of shellfish consumed in France are oysters, mussels, king scallops, winkles,whelks, cockles, clams, and other scallops. Shellfish filter large volumes of water to extract their food and are excellent bioaccumulators. Metals and other pollutants that exist in the marine environment partition into particular organs, according to their individual chemical characteristics. In shellfish, accumulation often occurs in the digestive gland, which plays a role in assimilation, excretion, and detoxification of contaminants. The concentrations of chemical contaminants in bivalve mollusks are known to fluctuate with the seasons.European regulations limit the amount and type of contaminants that can appear in foodstuffs. Current European standards regulate the levels of micro-biological agents, phycotoxins, and some chemical contaminants in food. Since 2006, these regulations have been compiled into the "Hygiene Package." Bivalve mollusks must comply with maximum levels of certain contaminants as follows:lead (1.5 mg kg-1), cadmium (1 mg kg-1), mercury (0.5 mg kg-1), dioxins (4 pg g-1 and dioxins + DL-PCBs 8 pg g-1), and benzo[a]pyrene (10 µp.g kg-1).In this review, we identify the levels of major contaminants that exist in shellfish(collected from the marine environment and/or in marketed shellfish). The follow-ing contaminants are among those that are profiled: Cd, Pb, Hg, As, Ni, Cr, V,Mn, Cu, Zn, Co, Se, Mg, Mo, radionuclides, benzo[a]pyrene, PCBs, dioxins and furans, PAHs, TBT, HCB, dieldrin, DDT, lindane, triazines, PBDE, and chlorinated paraffins.In France, the results of contaminant monitoring have indicated that Cd, but not lead (< 0.26 mg kg-1) or mercury (< 0.003 mg kg-1), has had some non-compliances. Detections for PCBs and dioxins in shellfish were far below the regulatory thresholds in oysters (< 0.6 pg g-l), mussels (< 0.6 pg g-1), and king scallops (< 0.4 pg g-1). The benzo[a]pyrene concentration in marketed mussels and farmed shellfish does not exceed the regulatory threshold. Some monitoring data are available on shellfish flesh contamination for unregulated organic contaminants.Of about 100 existing organo stannic compounds, residues of the mono-, di-, and tributyltin (MBT, DBT, and TBT) and mono-, di-, and triphenyltin (MPT, DPT,and TPT) compounds are the most frequently detected in fishery products. Octyltins are not found in fishery products. Some bivalve mollusks show arsenic levels up to 15.8 mg kg-1. It seems that the levels of arsenic in the environment derive less from bioaccumulation, than from whether the arsenic is in an organic or an inorganic form. In regard to the other metals, levels of zinc and magnesium are higher in oysters than in mussels.To protect shellfish from chemical contamination, programs have been established to monitor water masses along coastal areas. The French monitoring network(ROCCH) focuses on environmental matrices that accumulate contaminants. These include both biota and sediment. Example contaminants were studied in a French coastal lagoon (Arcachon Bay) and in an estuary (Bay of Seine), and these were used to illustrate the usefulness of the monitoring programs. Twenty-one pesticidal and biocidal active substances were detected in the waters of Arcachon Bay during the summers from 1999 to 2003, at concentrations ranging from a few nanograms per liter to several hundred nanograms per liter. Most of the detected substances were herbicides, including some that are now banned. Organotin compounds have been detected in similarly semi-enclosed waters elsewhere (bays, estuaries, and harbors).However, the mean concentrations of cadmium, mercury, lead, and benzo[a]pyrene,in transplanted mussels, were below the regulatory limits.In 2007, the mean daily consumption of shellfish in the general French population was estimated to be 4.5 g in adults; however, a wide variation occurs by region and season (INCA 2 study). Tabulated as a proportion of the diet, shellfish consumption represents only 0.16% of overall solid food intake. However, the INCA 2 survey was not well suited to estimating shellfish consumption because of the small number of shellfish consumers sampled. In contrast, the mean consumption rate of bivalve mollusks among adult high consumers of fish and seafood products, i.e., adults who eat fish or seafood at least twice a week, was estimated to be 153 g week-1 (8 kg yr-1). The highest mean consumption is for king scallops (39 g week-1), followed by oysters (34 g week-1) and mussels (22 g week-1). Thus, for high seafood consumers, the contribution of shellfish to inorganic contaminant levels is 1-10% TWI or PTWI for Cd, MeHg, and Sn (up to 19% for Sn), and the arsenic body burden is higher for 22% of individuals studied.The human health risks associated with consuming chemical contaminants in shellfish are difficult to assess for several reasons: effects may only surface after long-term exposure (chronic risk), exposures may be discontinuous, and contamination may derive from multiple sources (food, air, occupational exposure, etc.).Therefore, it is not possible to attribute a high body burden specifically to shellfish consumption even if seafood is a major dietary contributor of any contaminant, e.g.,arsenic and mercury.The data assembled in this review provide the arguments for maintaining the chemical contaminant monitoring programs for shellfish. Moreover, the results presented herein suggest that monitoring programs should be extended to other chemicals that are suspected of presenting a risk to consumers, as illustrated by the high concentration reported for arsenic (in urine) of high consumers of seafood products from the CALIPSO study. In addition, the research conducted in shellfish-farming areas of Arcachon Bay highlights the need to monitor TBT and PAH contamination levels to ensure that these chemical pollutants do not migrate from the harbor to oyster farms.Finally, we have concluded that shellfish contamination from seawater offers a rather low risk to the general French population, because shellfish do not constitute a major contributor to dietary exposure of chemical contaminants. Notwithstanding,consumer vigilance is necessary among regular shellfish consumers, and especially for those residing in fishing communities, for pregnant and breast-feeding women,and for very young children.

Warning on False or True Morels and Button Mushrooms with Potential Toxicity Linked to Hydrazinic Toxins: An Update.

Recently, consumption of the gyromitrin-containing neurotoxic mushroom Gyromitra sp. (false morel), as gourmet food was hypothesized to play a role in sporadic amyotrophic lateral sclerosis genesis. The present review analyses recent data on edibility and toxicity of false and true morels and Agaricus spp. Controversy about the toxic status of Gyromitra esculenta was due to variable toxin susceptibility within consumers. We suggest that Verpa bohemica, another false morel, is also inedible. We found a temporary neurological syndrome (NS) with cerebellar signs associated with high consumption of fresh or dried true morels Morchella sp. After ingestion of crude or poorly cooked fresh or dried morels, a gastrointestinal "haemolytic" syndrome was also observed. Agaritine, a water soluble hydrazinic toxin closely related to gyromitrin is present along with metabolites including diazonium ions and free radicals, in Agaricus spp. and A. bisporus, the button mushroom, and in mice after ingestion. It is a potential weak carcinogen in mice, but although no data are available for humans, a lifetime low cumulative extra cancer risk in humans can be estimated to be about 10-5. To conclude, a safety measure is to avoid consuming any true morels or button mushrooms when crude or poorly cooked, fresh or dried.

Health risk assessment related to pinnatoxins in French shellfish.

Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 µg/kg bw per day has been derived from an oral acute toxicity study in mice. Based on this value and a large shellfish meat portion size of 400g, a concentration lower than 23 µg PnTX G/kg shellfish meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for shellfish production and identified data gaps to refine health risk assessment.

Purification, Characterization and Thermodynamic Assessment of an Alkaline Protease by Geotrichum Candidum of Dairy Origin.

BACKGROUND: Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations. OBJECTIVES: The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi. MATERIAL AND METHODS: In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step method: ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease. RESULTS: The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/ml maximum extracellular protease. Protease revealed that Vmax and Km was 26.25 U.ml-1.min-1 and 0.05 mg.mL-1, respectively using casein as substrate. The enzyme was stable at a temperature range (25-45 °C) and pH (8-9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 °C, respectively. CONCLUSIONS: Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.

Beneficial lactobacilli in food and feed: long-term use, biodiversity and proposals for specific and realistic safety assessments.

Lactobacilli have played a crucial role in the production of fermented products for millennia. Their probiotic effects have recently been studied and used in new products. Isolated cases of lactobacillemia have been reported in at-risk populations, but lactobacilli present an essentially negligible biological risk. We analyzed the current European guidelines for safety assessment in food/feed and conclude that they are not relevant for the Lactobacillus genus. We propose new specific guidelines, beginning by granting a 'long-standing presumption of safety' status to Lactobacillus genus based on its long history of safe use. Then, based on the available body of knowledge and intended use, only such tests as are useful will be necessary before attributing 'qualified presumption of safety' status.

Incidence and clinical characteristics of ciguatera fish poisoning in Guadeloupe (French West Indies) between 2013 and 2016: a retrospective cases-series.

This retrospective case study analysed the incidence and symptoms of ciguatera fish poisoning (ciguatera) in Guadeloupe (French West Indies) between 2013 and 2016. Cases attending the emergency departments of the two public hospitals and the reports received by the regional health authority in charge of monitoring (ARS) were compiled. Two hundred and thirty-four cases of poisoning were observed, with a mean annual incidence of 1.47/10,000 (95% CI): 1.29-1.66), i.e 5 times higher than the previously reported incidence (1996-2006). The main species described as being responsible for poisoning were fish from the Carangidae family (n = 47) (jack), followed by fish from the Lutjanidae family (n = 27) (snapper), Serranidae family (n = 15) (grouper), Sphyraenidae family (n = 12) (barracuda), and Mullidae family (n = 12) (goatfish). One case of lionfish ciguatera was observed. 93.9% of patients experienced gastrointestinal symptoms, 76.0% presented neurological signs (mainly paresthesia, dysesthesia and pruritus) and 40.3% presented cardiovascular symptoms (bradycardia and/or hypotension). A high frequency (61.4%) of hypothermia (body temperature <36.5 °C) was observed. This study reports for the first time the relatively high frequency of cardiac symptoms and low body temperature. The monitoring of ciguatera poisoning throughout the Caribbean region must be improved, notably after reef disturbance due to Irma and Maria major cyclones.

Rough and smooth morphotypes isolated from Lactobacillus farciminis CNCM I-3699 are two closely-related variants.

This study focused on a pleomorphic strain Lactobacillus farciminis CNCM I-3699 known as probiotic for animal applications. On plating, this strain was characterized by the presence of rough and smooth morphotypes depending on experimental conditions. Dominant smooth (S) form, bright white, having smooth edges with moist, ropy, and creamy along with rough (R) form, pale white, having irregular edges and a dry and granular aspect were always obtained from the parent strain under aerobic culture conditions. In anaerobic conditions, only S form growth was observed. Biochemical dosage of capsular exopolysaccharides showed a significant difference between S and R forms (p<0.01), in agreement with a ropy or non ropy phenotype for the S or R form, respectively. These differences were confirmed by transmission electronic microscopy. The auto-aggregation profile revealed major differences in cultural behaviors. The R morphotype presented a highly auto-aggregative ability contrary to the S morphotype. However, biochemical and molecular analyses revealed that R and S morphotypes: 1) shared the same sugar fermentation pattern; 2) belonged to L. farciminis species using 16S rDNA sequencing; 3) had identical PFGE patterns using NotI and ApaI endonucleases; and 4) had identical CRISPR loci but different from those of other L. farciminis strains. Furthermore, the novelty and uniqueness of CRISPR spacer sequences in CNCM I-3699 provides a genetic support for the development of a molecular tracking tool for CNCM I-3699 and its variants. In conclusion, L. farciminis CNCM I-3699 is a pleomorphic strain giving reproducibly rise to two phenotypically distinct morphotypes R and S. This phenomenon may explain survival and growth abilities in in vitro fluctuating aerobic-anaerobic conditions along with modulation of exopolysaccharide synthesis and autoaggregation profile.

Muscarinic effects of the Caribbean ciguatoxin C-CTX-1 on frog atrial heart muscle.

The effects of Caribbean ciguatoxin (C-CTX-1) isolated from horse-eye jack (Caranx latus) on the electrical and mechanical activities of frog auricle were studied. C-CTX-1 (1 pM-50 nM) dose-dependently shortened the duration of the plateau and the repolarizing phase of the action potential (AP). The AP shortening induced by C-CTX-1 (50 nM) was suppressed or prevented either by tetrodotoxin (TTX; 0.6 nM) or by atropine (0.1mM). C-CTX-1 (50 nM) prolonged the TTX (0.6 nM)-sensitive electrical response of the vagus nerve branches, which innervate the auricle. The C-CTX-1 (50 nM)-induced shortening of the plateau and of the repolarization phase were prevented or reversed by gallamine (20 microM) and pirenzepine (0.5 microM), respectively. C-CTX-1 (50 nM) decreased the amplitude of the peak contraction and shortened its duration. In the presence of gallamine (20 microM), C-CTX-1 decreased the amplitude of the peak contraction and shortened its duration in the presence of pirenzepine (0.5 microM). C-CTX-1 (50 nM) decreased the time constant of the relaxation phase of the peak contraction suggesting that it increased the Na(+)/Ca(2+) exchange activity. Acetylcholine (ACh; 1 pM) shortened APD, decreased the peak contraction and mimics the effects of C-CTX-1. In conclusion, the presented data show that C-CTX-1 released ACh from atrial cholinergic nerve terminals which activated M(1) and M(2) muscarinic receptors subtype (mAChR). Our findings suggest that M(1) and M(2) mAChR are present in frog atrial tissue and play a previously unrecognized role in the modulation of the AP duration and of the mechanical activity of cardiac tissue.

Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry.

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M+H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M+H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M+H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels <50% and were subjected to large losses of activity on purification indicating that unstable ciguatoxins were present. A possible conversion of C-CTX-1 into C-CTX-1a was identified when flesh was cooked, without changes in toxicity. In conclusion, HPLC/MS characterised 12 C-CTXs accumulated by C. latus at variable levels.

Isolation and characterisation of Indian Ocean ciguatoxin.

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding.

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for > 90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and < 900 Da were suspected to contribute to the overall toxicity.

Numbers and strains of lactobacilli in some probiotic products.

The numbers and types of Lactobacillus strains in probiotic feed or food products for sale in Europe were compared with the information stated on the product labels. The labels of all 10 products gave information on the genus and species of Lactobacillus, and seven gave information on the numbers of a specified strain(s). Lactobacilli were recovered using a recently validated method, developed as part of the European Community Project SMT4 CT98-2235 for the official control of probiotic microorganisms used as feed additives. Polymerase chain reaction (PCR)-based methods were used to assess the accuracy of labelling with regard to genus and species, and pulsed-field gel electrophoresis (PFGE) was used to identify strains. Five products were mislabelled with respect to the numbers and three with respect to species of lactobacilli. In four cases, the specified strains were not detected. Four fermented milks sold under three trademarks contained the same strain, which was named differently on each label. As safety and functionality of probiotics are strain dependent, these results demonstrated the need to control lactobacilli present in commercially probiotic human food products, not only at the species but also at the strain level, to ensure their quality and protect the consumer. The usefulness of the official methods developed for animal feed additives to identify lactobacilli in probiotic foods for humans was demonstrated.