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.

Dietary exposure to mycotoxins in the French infant total diet study.

The present study evaluated the exposure of children aged from one to 36 months to seven groups of mycotoxins, in the context of the infant French Total Diet Study (iTDS). Exposure was then compared to the health-based guidance values (HBGVs) for each mycotoxin. The value of the 90th percentile of exposure to nivalenol, patulin, fumonisins and zearalenone was less than 40% of the HBGV considered relevant for children. On the other hand, a risk could not be excluded for ochratoxin A and aflatoxins as exposure was close to the HBGV for ochratoxin A and the margin of exposure was much lower than the critical margin of 10,000 for aflatoxins. The HBGVs for toxins T2 and HT2, and for deoxynivalenol (DON) and its acetylated compounds were exceeded. Five percent to 10% of the children aged 5-12 months exceeded the HBGV considering the lower bound hypothesis for toxins T2 and HT2 and 7.5%-27% of the children aged 5 months and above exceeded the HBGV for DON. Consequently, the exposure of young children raises safety concerns for T2/HT2 and DON. Efforts should therefore be pursued to decrease their exposure to these molecules.

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.

Risks to human and animal health related to the presence of moniliformin in food and feed.

Moniliformin (MON) is a mycotoxin with low molecular weight primarily produced by Fusarium fungi and occurring predominantly in cereal grains. Following a request of the European Commission, the CONTAM Panel assessed the risk of MON to human and animal health related to its presence in food and feed. The limited information available on toxicity and on toxicokinetics in experimental and farm animals indicated haematotoxicity and cardiotoxicity as major adverse health effects of MON. MON causes chromosome aberrations in vitro but no in vivo genotoxicity data and no carcinogenicity data were identified. Due to the limitations in the available toxicity data, human acute or chronic health-based guidance values (HBGV) could not be established. The margin of exposure (MOE) between the no-observed-adverse-effect level (NOAEL) of 6.0 mg/kg body weight (bw) for cardiotoxicity from a subacute study in rats and the acute upper bound (UB) dietary exposure estimates ranged between 4,000 and 73,000. The MOE between the lowest benchmark dose lower confidence limit (for a 5% response - BMDL05) of 0.20 mg MON/kg bw per day for haematological hazards from a 28-day study in pigs and the chronic dietary human exposure estimates ranged between 370 and 5,000,000 for chronic dietary exposures. These MOEs indicate a low risk for human health but were associated with high uncertainty. The toxicity data available for poultry, pigs, and mink indicated a low or even negligible risk for these animals from exposure to MON in feed at the estimated exposure levels under current feeding practices. Assuming similar or lower sensitivity as for pigs, the CONTAM Panel considered a low or even negligible risk for the other animal species for which no toxicity data suitable for hazard characterisation were identified. Additional toxicity studies are needed and depending on their outcome, the collection of more occurrence data on MON in food and feed is recommended to enable a comprehensive human risk assessment.

Risk to human and animal health related to the presence of 4,15-diacetoxyscirpenol in food and feed.

4,15-Diacetoxyscirpenol (DAS) is a mycotoxin primarily produced by Fusarium fungi and occurring predominantly in cereal grains. As requested by the European Commission, the EFSA Panel on Contaminants in the Food Chain (CONTAM) assessed the risk of DAS to human and animal health related to its presence in food and feed. Very limited information was available on toxicity and on toxicokinetics in experimental and farm animals. Due to the limitations in the available data set, human acute and chronic health-based guidance values (HBGV) were established based on data obtained in clinical trials of DAS as an anticancer agent (anguidine) after intravenous administration to cancer patients. The CONTAM Panel considered these data as informative for the hazard characterisation of DAS after oral exposure. The main adverse effects after acute and repeated exposure were emesis, with a no-observed-adverse-effect level (NOAEL) of 32 µg DAS/kg body weight (bw), and haematotoxicity, with a NOAEL of 65 µg DAS/kg bw, respectively. An acute reference dose (ARfD) of 3.2 µg DAS/kg bw and a tolerable daily intake (TDI) of 0.65 µg DAS/kg bw were established. Based on over 15,000 occurrence data, the highest acute and chronic dietary exposures were estimated to be 0.8 and 0.49 µg DAS/kg bw per day, respectively, and were not of health concern for humans. The limited information for poultry, pigs and dogs indicated a low risk for these animals at the estimated DAS exposure levels under current feeding practices, with the possible exception of fattening chicken. Assuming similar or lower sensitivity than for poultry, the risk was considered overall low for other farm and companion animal species for which no toxicity data were available. In consideration of the similarities of several trichothecenes and the likelihood of co-exposure via food and feed, it could be appropriate to perform a cumulative risk assessment for this group of substances.

Risks to human and animal health related to the presence of deoxynivalenol and its acetylated and modified forms in food and feed.

Deoxynivalenol (DON) is a mycotoxin primarily produced by Fusarium fungi, occurring predominantly in cereal grains. Following the request of the European Commission, the CONTAM Panel assessed the risk to animal and human health related to DON, 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and DON-3-glucoside in food and feed. A total of 27,537, 13,892, 7,270 and 2,266 analytical data for DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside, respectively, in food, feed and unprocessed grains collected from 2007 to 2014 were used. For human exposure, grains and grain-based products were main sources, whereas in farm and companion animals, cereal grains, cereal by-products and forage maize contributed most. DON is rapidly absorbed, distributed, and excreted. Since 3-Ac-DON and 15-Ac-DON are largely deacetylated and DON-3-glucoside cleaved in the intestines the same toxic effects as DON can be expected. The TDI of 1 µg/kg bw per day, that was established for DON based on reduced body weight gain in mice, was therefore used as a group-TDI for the sum of DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside. In order to assess acute human health risk, epidemiological data from mycotoxicoses were assessed and a group-ARfD of 8 µg/kg bw per eating occasion was calculated. Estimates of acute dietary exposures were below this dose and did not raise a health concern in humans. The estimated mean chronic dietary exposure was above the group-TDI in infants, toddlers and other children, and at high exposure also in adolescents and adults, indicating a potential health concern. Based on estimated mean dietary concentrations in ruminants, poultry, rabbits, dogs and cats, most farmed fish species and horses, adverse effects are not expected. At the high dietary concentrations, there is a potential risk for chronic adverse effects in pigs and fish and for acute adverse effects in cats and farmed mink.

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection.

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.

Interlaboratory study of a multiresidue gas chromatographic method for determination of organochlorine and pyrethroid pesticides and polychlorobiphenyls in milk, fish, eggs, and beef fat.

An interlaboratory study was conducted to validate a gas chromatographic (GC) method for determination of 21 organochlorine pesticides, 6 pyrethroid pesticides, and 7 polychlorobiphenyl (PCB) congeners in milk, beef fat, fish, and eggs. The method was performed at low contamination levels, which represent relevant contents in food, and is an extension of the European standard (method NF-EN-1528, Parts 1-4). It enlarges the applicable scope of the reference EN method to pyrethroid pesticides and proposes the use of solid-phase extraction (SPE) as a cleanup procedure. Cryogenic extraction was made, and SPE cleanup was performed with 2 successive SPE cartridges: C18 and Florisil. After injection of the purified extract onto a GC column, residues were measured by electron capture detection. Food samples (liquid milk, beef fat, mixed fish, and mixed eggs) were prepared, tested for homogeneity, and sent to 17 laboratories in France. Test portions were spiked with 27 pesticides and 7 PCBs at levels from 26 to 45, 4 to 27, 31 to 67, and 19 to 127 ng/g into milk, eggs, fish, and fat, respectively. Based on results for spiked samples, the relative standard deviation for repeatability ranged from 1.5 to 6.8% in milk, 3 to 39% in eggs, 4.5 to 12.2% in fish, and 7 to 13% in fat. The relative standard deviation for reproducibility ranged from 33 to 50% in milk, 29 to 59% in eggs, 31 to 57% in fish, and 30 to 62% in fat. This method showed acceptable intra- and interlaboratory precision data, as corroborated by HORRAT values at low levels of pesticide and PCB contamination. The statistical evaluation of the results was performed according to the International Organization for Standardization (ISO; ISO 3534 standard) and 5725-2 Guideline.

Optimized simultaneous determination of several elements in human intestinal Caco-2 TC7 cells by inductively coupled plasma-mass spectrometry after closed vessel microwave digestion.

A rapid procedure, based on closed vessels microwave digestion and inductively coupled plasma-mass spectrometry (ICP-MS), was evaluated to ascertain the effect of chronic exposure to cadmium on intracellular accumulation of minor and essential trace elements in cultured epithelial cells (Caco-2 TC7). For all measurements, the method of external calibration was used and 3 elements (Be, Sc, In) were selected as internal standards. Optimization procedures are discussed and results are presented for the total determination of 9 key analytes (Na, Mg, Ca, Cr, Mn, Cu, Zn, Mo, and Cd) in certified reference materials (CRMs) and 20 samples of Caco-2 TC7 cells long-term exposed to Cd. The performance characteristics of the analytical system were evaluated by calibration and linearity, limits of detection and quantitation, accuracy with spiking, trueness and repeatability with available CRMs. As a complement to the ICP-MS determinations, both available CRMs and cell samples were analyzed either by electro thermal- or flame atomic absorption spectrometry. The results were in good agreement with the ICP-MS results.

Dietary exposure to mycotoxins and health risk assessment in the second French total diet study.

Mycotoxins are produced in plants by micro-fungi species, and naturally contaminated the food chain. In the second French total diet study (TDS), mycotoxins were analyzed in 577 food samples collected in mainland France to be representative of the population diet and prepared ((as consumed)). Highest mean concentrations were found in wheat and cereal-based products (bread, breakfast cereals, pasta, pastries, pizzas and savoury pastries…). Exposure of adult and child populations was assessed by combining national consumption data with analytical results, using lowerbound (LB) and upperbound (UB) assumptions for left-censorship management. Individual exposures were compared with available health-based guidance values (HBGV). Only the exposure to deoxynivalenol (DON) and its acetylated derivatives was found to significantly exceed the HBGV in LB in adults (0.5% [0.1; 0.8]) and children (5% [4; 6]). HBGV was exceeded in UB only for T-2 and HT-2 toxins by, respectively, 0.2% [0.02; 0.05] and 4% [3; 5] of adults, and 11% [9; 12] and 35% [32; 37] of children. Although the exposures assessed were generally lower than the previous French TDS, the results indicated a health concern for trichothecenes and a need to reduce dietary exposure as well as analytical limits.

New considerations regarding the risk assessment on Tartrazine An update toxicological assessment, intolerance reactions and maximum theoretical daily intake in France.

Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. Since the last assessment carried out by the JECFA in 1964, many new studies have been conducted, some of which have incriminated tartrazine in food intolerance reactions. The aims of this work are to update the hazard characterization and to revaluate the safety of tartrazine. Our bibliographical review of animal studies confirms the initial hazard assessment conducted by the JECFA, and accordingly the ADI established at 7.5mg/kg bw. From our data, in France, the estimated maximum theoretical intake of tartrazine in children is 37.2% of the ADI at the 97.5th percentile. It may therefore be concluded that from a toxicological point of view, tartrazine does not represent a risk for the consumer. It appears more difficult to show a clear relationship between ingestion of tartrazine and the development of intolerance reactions in patients. These reactions primarily occur in patients who also suffer from recurrent urticaria or asthma. The link between tartrazine consumption and these reactions is often overestimated, and the pathogenic mechanisms remain poorly understood. The prevalence of tartrazine intolerance is estimated to be less than 0.12% in the general population. Generally, the population at risk is aware of the importance of food labelling, with the view of avoiding consumption of tartrazine. However, it has to be mentioned that products such as ice creams, desserts, cakes and fine bakery are often sold loose without any labelling.

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model.

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 micromol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 micromol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 micromol Cd/l exposure, and no variation was observed with copper.

First evidence on occurrence of gymnodimine in clams from Tunisia.

Among several batches of clams harvested in Tunisia and imported to France, a small number of them were found to be neurotoxic to mice (intraperitoneal injection) as determined by the diarrhetic shellfish poisoning (DSP) bioassay developed by Yasumoto et al. (1978). The present study was conducted to confirm the nature of the toxic agent, suspected to be gymnodimine. Liquid chromatography tandem mass spectrometry analyses unequivocally revealed the presence of gymnodimine in the shellfish, making Tunisia the second country, after New Zealand, where shellfish contamination with this phycotoxin is reported. Gymnodimine B and C analogues were not detected in the clam samples. Gymnodimine preferentially accumulates in the digestive gland of the Tunisian clams, although substantial amounts are also found in the meat.