A Perspective on the Toxicology of Marine Toxins - American

Perspective pubs.acs.org/crt

A Perspective on the Toxicology of Marine Toxins Luis M. Botana* Department Farmacología, Fac. Veterinaria-USC, 27002 Lugo, Spain ABSTRACT: Although there has been much progress with regard to marine toxins from dinoflagellates, much remains to be done. Because these compounds are a seafood consumer risk, the demands cover from legislative to scientific aspects. Legislation is required for all new toxins that appear in the coasts. On the other hand, it is important to understand the toxicity of the different analogues, in terms of both the relative toxicity to reference compounds and the mechanism of toxicity itself, both acute and long-term. For this, a uniform approach to do toxic studies is necessary, especially acute toxicity. The need for pure standards in sufficient supply and the understanding of the mode of action of some of the compounds (such as yessotoxin or azaspiracids) will help the development of another important field, the use of marine toxins as drug leads, and the chemistry around them.

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CONTENTS

Food Safety Concerns and Legal Issues Mechanistic Studies Analytical Needs Toxicological Studies Author Information References

evidence of their risk. A recent series of opinions published by the European Food Safety Agency are evidence that additional toxicological information is urgently needed to understand the role of marine toxins in food safety.8−20 Currently, the European legislation defined a 3 year transition period (starting in 2011) to replace the mouse bioassay by the use of liquid chromatography coupled to mass spectrometry for the monitoring of marine toxins.21−23 The validity of this approach has been questioned, as several biases may be identified with regard to technology and methodology.24 The limited information about toxic equivalent factors for each toxin analogues in the different group is another limitation of mass spectrometry, since analytical results need to be converted to equivalent toxic values of a reference compound in each group.25 From a toxicological point of view, toxic equivalent factors are urgently needed in terms of food safety monitoring. The scarcity of toxins is a fundamental problem, as today there is no commercial source of some important compounds such as ostreocins, ciguatoxins, or many analogues of each of the groups. An additional food safety problem brought about by the legal change from bioassay to an analytical method is the de facto increase, by several fold, in total levels allowed, since an analysis quantifies each toxin group individually, while the mouse bioassay would respond to the combined toxicicity of the different compounds in the sample.26 To date, the controversy over the toxic nature or not of yessotoxin, pectenotoxin, or cyclic imines makes it difficult to draw conclusions with regard to legal consequences. It is therefore important to assume the lack of legal monitoring requirements for some compounds, such as cyclic imines (spirolides, pinnatoxins), palytoxins, and ostreocins, ciguatoxins, or tetrodotoxins, while on the other hand yessotoxins and pectenotoxins are included in the

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arine toxins are a very large and complex group of compounds produced by algae, bacteria, cyanobacteria, or sponges, with defensive or undefined biological roles, which fit into the flexible concept of secondary metabolites.1 Marine toxins from dinoflagellates are a fascinating group of compounds that have resisted through the years the scrutiny of science. This perspective will focus on the research perspectives of marine toxins produced by dinoflagellates, which accumulate in filter feeding bivalves and are a cause of concern for food safety authorities. There are several toxin groups, each one represented by a reference compound: domoic acid, saxitoxin, okadaic acid, ciguatoxin, palytoxin, pectenotoxin, yessotoxin, azaspiracid, spirolide, tetrodotoxin, maitotoxin, and brevetoxin.2,3 Each group contains several analogues, such as gonyautoxins (saxitoxin), dinophysistoxins (okadaic acid),4 ostreocins (palytoxin),5 and gymnodimines and pinnatoxins, which belong, with spirolides, to the cyclic imine family of toxins.6 Palytoxin and maitotoxin are unique in their chemical diversity, their size, and their potency, as they are the largest and most potent single compounds in nature.7

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FOOD SAFETY CONCERNS AND LEGAL ISSUES Marine toxins are esentially a food safety and an international trade problem, and the legislation that requires the control of the presence of these compounds in seafood is based on the scientific © 2012 American Chemical Society

Received: April 26, 2012 Published: June 1, 2012 1800

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studies. This lack of standard affects both pure toxins to be used as analytical calibrants and toxicological studies. Pectenotoxins were considered for some time diarrheic toxins because initial studies used samples contaminated with okadaic acid and dinophysistoxins.58,59 Both pectenotoxins and okadaic acid and its analogues, the dinophysistoxins, are produced by the same organism, Dinophysis spp.,60 which are phosphatase inhibitors61 that cause intense diarrheas. Nowadays, pectenotoxins are not considered diarrheic compounds. Two different articles just published on the toxicology of spirolides in Swiss albino mice provide evidence on the need for a unified approach.62,63 The ip LD50 for 13-desmethyl spirolide was reported to be 6.9 and 27.9 μg/kg body weight in refs 63 and 62, respectively, while 20methyl spirolide G was reported to have an ip LD50 of 8 μg/kg body weight,63 and no death was reported in ref 62 with up to 63.5 μg/kg. The stability of the compounds needs to be documented; that is, the stability of spirolides, azaspiracids, or pectenotoxins is very dependent on pH.64 Therefore, the nature of the toxins used may be in some cases a source of error, as certified toxic material is very scarce, and only two commercial sources are available (NRC in Canada and CIFGA in Spain). The presence on toxin congeners in trace amounts should not be included in risk assessment; that is, dozens of yessotoxin derivatives have been isolated, some of which are present at only a minute fraction of that of yessotoxin itself. There is little chance that such compounds will contribute to overall toxicity, and studies like these do not contribute to risk assessment. It has been recommended not to consider congeners present at less than 5% of the concentration of the parent toxin.65 The toxicological information with regard to the method used is also very important. Intraperitoneal injection to mice provides information that not always is paralleled by oral toxicity. A good example is yessotoxin, which is very toxic by ip but shows no effect if given orally.66

legislation. The conflicting nature of the information about some of these compounds (cyclic imines, yessotoxin, and pectenotoxin) is due to the fact that although they are toxic to rodents, they were never recorded as toxic to humans. On the other hand, palytoxins and ostreocins, ciguatoxins, or tetrodotoxins are known to be toxic to humans. Nowadays, it is important to understand that all of these toxins are present in Europe, and some of them already have been identified as the cause of serious human intoxications, such as tetrodotoxin from gasteropods27 or ciguatoxins from fish.28 Whether or not new toxins are related to climate change remains to be established.

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MECHANISTIC STUDIES The mechanistic effect of cyclic imines, such as pinnatoxins of spirolides, suggests that they could pose a long-term risk to consumers. Pinnatoxin is a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype,29 while spirolides and gymnodimine are blockers of the muscle type α12βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors30 and irreversible blockers of the muscarinic M3 receptors.31 How chronic subtoxic doses of these compounds may affect humans remains to be investigated, but today, it is difficult to find shellfish without trace levels of cyclic imines (spirolides, pinnatoxin, and gymnodimine) in many markets in Europe.32,33 Moreover, some of the marine toxins identified as dangerous, such as palytoxin in shellfish34 or tetrodotoxin in gasteropods, already have been reported.27 Ciguatoxins are usually classified based on their origin as Caribbean, Pacific, or Indian,35,36 and they have different analogues in each group, with different pharmacological profiles.37,38 The intoxication in Europe seems to be due to a mixture of analogues, and this complicates the perspective of functional assays, as several now unavailable toxins will be needed for the development or implentation detection methods.28 Although the mechanism of action of some toxins is well identified, such as saxitoxin as a sodium channel blocker39 or pectenotoxin as an actin inhibitor,40 the mode of action of some toxin groups remains elusive, as it is the case of azaspiracids or yessotoxins. For azaspiracids, several targets were proposed, from binding proteins41,42 to lipid metabolism43 or apoptotic kinases,44,45 and for yessotoxin, the proposed targets vary from phosphodiesterases46 to cadherin.47 Nevertheless, to date, no clear target has been proposed to explain the mechanism of action of yessotoxin or azaspiracids. The lack of defined targets for certain toxins makes it difficult to design functional detection methods based on the in vitro use of their specific receptors.48 Although many methods are available for the fast detection of marine toxins, the vast majority relies on the use of antibodies.49−51 Because marine toxins are a complex group of chemical analogues, the cross-reactivity of antibodies hardly covers the complete detection of each toxin group, although there are antibodies with cross-reactivity that matches the congeners toxicity of a group.52 For those toxin groups with well-defined receptor targets, functional methods are available and provide useful tools for monitoring.53−56 A recent review describes the difficulties and opportunities of moving away from the mouse testing system to alternative detection methods for biotoxins.57

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TOXICOLOGICAL STUDIES In general, toxicologists agree that the oral route to mice is the option that best resembles the human response. Voluntary consumption by feeding is preferred over gavage, as recommended by Codex Alimentarius 67 and OECD,68 but the scarcity of toxins makes sometimes voluntary feeding a very expensive or impossible task, especially on chronic studies. The problems associated with gavage, such as stress, need for a skilled operator, material improperly deposited in lungs, and limit of dosed volume, make it difficult to rely solely on this source of toxicological information.69 On the other hand, the vehicle for voluntary feeding may be a source of variation, as shown by use of dry food versus cheese. To some authors, for acute experiments, where the test material must be delivered to the animal in a matter of seconds, a thick paste of powdered mousefood in water is adequate, as the test compound can easily be mixed into the paste. Gavage of test materials to rodents does not adequately replicate the human situation, since the contents of the rodent stomach are physically quite different to those of the human stomach. Human stomach contents are liquid, and if one were to administer a substance to humans as a solution by gavage, it would mix with the contents and become uniformly distributed throughout. It would then be slowly released through the pylorus into the small intestine, which is the primary site of absorption.70 Fasted mice may not be the best model for seafood toxins, since seafood is usually eaten as part of a meal.71 Therefore, it is necessary to give the mice some foodstuff that they readily eat. Cream cheese or peanut butter mix work well, but the mice need

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ANALYTICAL NEEDS To date, one of the most conflicting sources of information on marine toxins is the lack of a unified standard for toxicological 1801

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Phytotoxins, Chemistry and Biochemistry (Botana, L. M., Ed.) pp 319− 335, Blackwell Publishing, Ames, IA. (7) Nicolaou, K. C., Frederick, M. O., and Aversa, R. J. (2008) The continuing saga of the marine polyether biotoxins. Angew. Chem. 47, 7182−7225. (8) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010) Statement on further elaboration of the consumption figure of 400 g shellfish meat on the basis of new consumption data. EFSA J. 8, 1706−1726. (9) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010) Scientific Opinion on marine biotoxins in shellfishCyclic imines (spirolides, gymnodimines, pinnatoxins and pteriatoxins). EFSA J. 8, 1628−1667. (10) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2010) Scientific Opinion on marine biotoxins in shellfishEmerging toxins: Ciguatoxin-group toxins. EFSA J. 8, 1627−1665. (11) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009) Marine biotoxins in shellfishSummary on regulated marine biotoxins. EFSA J. 1306, 1−23. (12) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009) Scientific Opinion on marine biotoxins in shellfish Pectenotoxin group. EFSA J. 1109, 1−47. (13) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009) Scientific Opinion on marine biotoxins in shellfishDomoic acid. EFSA J. 1181, 1−61. (14) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2009) Scientific Opinion on marine biotoxins in shellfishPalytoxin group. EFSA J. 7, 1393−1431. (15) Panel, E. C. (2009) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfishSaxitoxin group. EFSA J. 1019, 1−76. (16) Panel, E. C. (2009) Marine toxins in shellfish. Summary on regulated marine biotoxins. Scientific Opinion of the Panel on Contaminants in the Food chain. EFSA J. 1306, 1−23. (17) Panel, E. C. (2008) Influence of processing on the levels of lipophilic marine biotoxins in bivalve molluscs. Statement of the Panel on Contaminants in the Food Chain. EFSA J. 1016, 1−10. (18) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2008) Scientific Opinion on marine biotoxins in shellfishYessotoxin group. EFSA J. 907, 1−62. (19) Panel, E. C. (2008) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfishAzaspiracids. EFSA J. 723, 1−52. (20) Panel, E. C. (2008) Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfishOkadaic acid and analogues. EFSA J. 589, 1−62. (21) (2011) Regulation C. Commission Regulation (EU) No 15/2011 of 10 January 2011 amending Regulation (EC) No 2074/2005 as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs. Off. J. Eur. Commun. L, 3−4. (22) van den Top, H. J., Gerssen, A., McCarron, P., and van Egmond, H. P. (2011) Quantitative determination of marine lipophilic toxins in mussels, oysters and cockles using liquid chromatography-mass spectrometry: Inter-laboratory validation study. Food Addit. Contam., Part A 28, 1745−1757. (23) These, A., Klemm, C., Nausch, I., and Uhlig, S. (2011) Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography-tandem mass spectrometry. Part I: collaborative study. Anal. Bioanal. Chem. 399, 1245−1256. (24) Otero, P., Alfonso, A., Alfonso, C., Rodriguez, P., Vieytes, M. R., and Botana, L. M. (2011) Effect of uncontrolled factors in a validated liquid chromatography-tandem mass spectrometry method question its use as a reference method for marine toxins: Major causes for concern. Anal. Chem. 83, 5903−5911.

to be trained to eat the food. If they are offered small quantities twice a day for a week, they readily consume it as soon as it is offered and will eat 200 mg in a matter of seconds. Either vehicle is satisfactory for mixing with the test substance, and a comparison of cream cheese and the peanut butter mix for delivery shows no significant difference in toxicity in the case of pinnatoxins (Rex Munday, personal communication). The very limited information that the EFSA panel could use with regard to chronic toxicity is striking, and what is the tolerable daily intake for most of the toxins, as there is no information available. Chronic intake of spirolides or domoic acid will probably cause effects, especially in sensitive groups such as young children or pregnant women, but no information is available. Even with all of these considerations, the mice model is not the best to identify toxicity to humans. Clearly, the best source of information is provided by accidental human intoxications, where enough material can be analyzed to calculate the amounts ingested and their circunstances. However, experience says that these conditions are very scarce, and tracability is not always possible; hence, risk assessment is difficult, and false conclusions can be obtained. A recent study on human intoxicacions with diarrheic toxins in France72 has proven threshold toxic values that match the conclusions reported by the EFSA working group.20 Although unrelated to toxicology, but still in need of deep mechanistic research, the potential of marine toxins as drug leads is another area that will lead to major advances in the future, as some works show the help and advances in organic synthesis.73 In summary, research on marine toxins needs a comprehensive integration of consumer surveys, toxicological information, and harmonized standards, analytical progress toward detection methods and toxins availability, and an understanding of their mode of action.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +34 982 8 22233. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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