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A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis

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A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis
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  A European perspective on progressin moving away from the mousebioassay for marine-toxin analysis Katrina Campbell, Natalia Vilarin˜o, Luis M. Botana, Christopher T. Elliott This review considers the ethical and technical problems currently associated with employing mouse bioassays for marine-toxinanalysis and the challenges and the difficulties that alternative methods must overcome before being deemed applicable forimplementation into a regulatory monitoring regime. We discuss proposed alternative methods, classified as functional, imm-unological and analytical, for well-established European toxins as well as emerging toxins in European waters, highlighting theiradvantages and disadvantages. We also consider emerging tools and technologies for future toxin analysis.Even though regulatory bodies have recently recommended analytical methods for a number of toxins, there is still scope forfunctional and immunological methods in rapid screening and detecting emerging toxins. Future developments foreseen in theanalysis of marine biotoxins are multiplex-based analysis, miniaturization and portability for on-site testing. However, thelongstanding lack of reference materials and standards continues to pose a severe limitation on progress in development,validation and therefore implementation of any alternative method based on the criteria stipulated by European Union legislation. ª  2010 Elsevier Ltd. All rights reserved. Keywords:  Analytical method; Antibody; Biosensor; Functional assay; Immunology; Marine toxin; Mouse bioassay; On-site testing; Rapid screening;Receptor 1. Introduction Marine biotoxins are naturally-occurringpoisonous substances synthesized bymicroscopic toxin-producing algae or theirassociated bacteria, though normally innon-harmful quantities. However, a com-bination of increased temperatures, sun-light and nutrient-rich waters is believedto cause rapid algal reproduction andthereby lead to potentially ‘‘harmful algalblooms’’. Worldwide, increasing occur-rences of toxic blooms are thought to belinked to climate change, increased oceaneutrophication and commercial shipping[1]. These toxins transfer through thetrophic chain into shellfish and fish. Mol-luscan shellfish are bivalve-filter feedersand ingest the algae, whereupon toxinsmay increase to levels that are potentiallylethal to humans or other consumers (e.g.,marine mammals and birds). Hence, asthis has major implications for publichealth, seafood destined for human con-sumption is routinely monitored by regu-latory bodies worldwide and is deemed fitfor consumption based on regulatory lim-its and methods established to preventacute poisoning [2–7]. The monitoring of marine toxins is vital to the aquacultureindustry, as these toxins may cause sub-stantial ecological damage and economiclosses through frequent or prolongedcontamination and closure of harvestingsites [8].Marine biotoxins detected worldwide,but particularly in European waters, weresrcinally classified based on their acutesymptomatic effect in humans followingintoxification. The three main groupsmonitored in the European Union (EU)are:   Paralytic Shellfish Poisoning (PSP) tox-ins;   Diarrheic Shellfish Poisoning (DSP) tox-ins; and,   Amnesic Shellfish Poisoning (ASP).However, as alternative detection meth-ods are considered, classification isbeginning to focus more on chemicalstructures and properties of the toxins. DSPtoxins have in recent times become knownas lipophilic toxins incorporating oka-daic acid, dinophysistoxins, azaspiracids, Katrina Campbell*,Christopher T. Elliott Institute of Agri-Food and LandUse, School of BiologicalSciences, Queen  s UniversityBelfast, BT9 5AG, UK Natalia Vilarin˜o,Luis M. Botana Departamento deFarmacologı´a, Facultad deVeterinaria, USC, 27002 Lugo,Spain * Corresponding author.Tel.: +44 (0)28 9097 6796;E-mail: katrina.campbell@qub.ac.uk Trends in Analytical Chemistry, Vol. 30, No. 2, 2011 Trends 0165-9936/$ - see front matter ª  2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.10.010  239  pectenotoxins and yessotoxins with the last two notproved to cause diarrheic symptoms following intoxica-tion. For each of these three main toxin groups and sub-groups, the occurrence of the toxins, their chemicalcharacteristics, toxicokinetic evaluations, human-expo-sureassessmentsanddetailedreviewofpotentialmethodsof analysis have in recent years been published by theEuropean Food Safety Authority (EFSA) as scientificopinions[9–14].Thediversityofthenumerousanaloguesor natural enzymatic metabolites of marine biotoxins hasbeen described [15]. Fig. 1 highlights the structure of the parent or reference toxin within each group and an indi-cation of the number of relative analogues or naturalenzymatic metabolites. Table 1 lists the producers of thetoxin, mechanism of action and effects in humans inaddition to the current European Union (EU) referencemethodsofanalysisandregulatorylimitsinshellfishmeatapplied in the monitoring regimes.Currently, EU regulations stipulate that the referencemethods for the detection of marine biotoxins are twodistinct animal bioassays based on the hydrophilic [16]and lipophilic [17] solvents used for the extractionprocedure. The detection of domoic acid is an excep-tion where the reference method is high-performanceliquid chromatography with ultraviolet detection(HPLC-UV) [3,5]. HPLC with fluorimetric detection(HPLC-FLD) for saxitoxin and analogues [4] and anenzyme-linked immunosorbent assay (ELISA) for do-moic acid [5] are officially accepted as screeningmethods but the reference methods for these toxins arethe aforementioned.However, this review also includes prospectiveemerging toxins to European waters [e.g., cyclic imines,palytoxin, tetrodotoxin, maitotoxin, ciguatoxins andneurotoxin-poisoning brevetoxins (Fig. 2)], as theiroccurrence could have severe implications with regardsto seafood safety [18]. In addition, as the shellfish tradeexpands globally with increased exports and imports toand from regions of the world where these toxins areprevalent, effective monitoring methods will need to bein place within the EU. Although EFSA has publishedscientific opinions for emerging toxins (Table 2) [19–22], with the exception of tetrodotoxin, these toxins are notspecified by the current EU regulations. At present, theirdetection is coincidental, as some co-extract with DSP orPSP toxins using the specified sample-preparation pro-tocols for the EU-approved animal bioassays. However,in many other regions of the world, animal bioassays arethe method of choice for monitoring these phycotoxinsin various seafoods. This review discusses the problems (a) PSP toxins (> 30 analogues) [85] NNR 1 NHHNNH 2+ OHR 3+ H 2 NR 4 R 2 OH  N  -Sulfocarbamoyl toxins Decarbamoyl (dc) toxins Deoxydecarbamoyl (do) toxins R 1  R 2 R 3 R 4 : OCONH 2 R 4 : OCONHSO 3- R 4 : OH  R 4 : HH H H Saxitoxin (STX) B1 (GTX 5) dc-STX do-STX H H OSO 3-  Gonyautoxin (GTX) 2 C1dc-GTX 2 do-GTX 2 H OSO 3-  H GTX 3 C2 dc-GTX 3 do-GTX 3 OH H H Neosaxitoxin (NEO) B2 (GTX 6) dc-NEO OH H OSO 3- GTX 1 C3 dc-GTX 1 OH OSO 3-  H GTX 4 C4 dc-GTX 4 Carbamate toxins Figure 1.  Chemical structure of the parent/reference toxin(s) for marine biotoxin groups regulated by the European Union. Trends Trends in Analytical Chemistry, Vol. 30, No. 2, 2011 240  http://www.elsevier.com/locate/trac  associated with animal bioassays and the progress anddifficulties in finding alternative methods. 2. The problems with bioassays The animal bioassays for marine biotoxins, discussed ina previous review [23], are widely considered to beantiquated, cruel methods that involve injecting shellfishextract into a mouse or rat and observing if the animallives or dies over a defined time frame as well as theresulting symptoms. At least two animals, and oftenthree, are used per sample tested and are sacrificed at theend of the test, irrespective of the toxicity outcome. Themajor drawbacks of these assays are that there isgrowing recognition that they are both unethical, due to (ii)Azaspiracid (~20 analogues) [88,89] Azaspiracid-1 (iii)Pectenotoxin (~13 analogues) [90] Pectenotoxin-2 (iv)Yessotoxin (~36 analogues) [91] (b) DSP / Lipophilic toxins (i)Okadaic acid and dinophysistoxins (DTXs) (>10 analogues and esters) [86,87] OOCH 3 CH 3 OOOCH 2 OHOR 6  CH 3 OOOOR 2 R 3 H 3 C OHR 1 OR 4 R 5 Toxin analogue R 1 R 2  R 3  R 4 R 5  R 6 Okadaic Acid H H CH 3  H H H DTX-1 H HCH 3  CH 3  H H DTX-2 H H H H CH 3  H DTX-3 (Acylated forms of OA, DTX-1 and DTX-2) H Fatty acid H / CH 3  H / CH 3  H / CH 3  H Fig 1.  (continued)  Trends in Analytical Chemistry, Vol. 30, No. 2, 2011 Trends http://www.elsevier.com/locate/trac  241  the suffering and the sacrifice of laboratory animals, andare technically inadequate.A conflict of interest between two EU Directives, 91/492 for public health control of marine biotoxinsimplementing the animal bioassays [24] and 86/609 onthe protection of laboratory animals [25], has in morerecent years increased the pressure within Europeancountries to move away from animal bioassays. From atechnical perspective, the methods are costly to performwith the animals requiring housing and restricted toweight conditions. Furthermore, they lack sensitivitywith limits of detection approaching the regulatorylimits, suffer from non-specificity (whereby toxins cannotbe identified or individually quantified) and are prone toinaccuracies in detection [26–28].If the recent EFSA opinion on marine biotoxins is tobe implemented [29] and lower regulatory limits areestablished, these bioassays will not be fit for purpose.Conversely, these bioassay methods have been appliedfairly effectively for over 70 years for PSP toxins and30 years for DSP toxins in protecting the consumer,even though they lack adequate validation by today  sstandards. The aquaculture industry is generallyreluctant to change. More sensitive assays may trans-late to a greater number or longer unnecessary closuresof harvesting sites. However, there is increasing con-cern that the effects of toxin consumption at sub-reg-ulatory levels over more chronic, long-term exposurewere not taken into account when the risks were beingdefined. Irrespective, with increasing toxic occurrencesand new toxins presenting in established risk areas andwell-known toxins occurring in new locations, thesebioassays are becoming unsustainable, due in part tothe large numbers of mice required and the perfor-mance of the procedures.There is a worldwide trend towards expansion of theshellfish industries, so the frequency of sampling andtesting is rising, exacerbated by the increasing numbersof short-term blooms, which increase the potential forlarge-scale shellfish poisoning episodes (e.g., those thatoccurred over the past decade, particularly for DSPtoxins) [30]. These episodes have an immense detri-mental effect on the industry. 3. Challenges facing alternative methods In order to protect consumer health, the recent EFSAopinion on marine biotoxins [29] recommends, with theexception of yessotoxin, the implementation of substan-tially lower regulatory limits for toxin contents in sea-food destined for human consumption. For regulatoryauthorities to be in a position to comply with the EFSAopinion, although not a draft for legislation, access tosuitable alternative detection methods will therefore beessential.These new methods must be reliable and have ade-quate sensitivity in order to detect the presence of thetoxins and analogues at not only the current regulatorylevels but also lower levels that are likely to result fromthe recommendations of EFSA and other regulatorybodies worldwide. Alternative methods with improvedsensitivity, compared to the mouse bioassay, have beendeveloped to replace the bioassays over the past30 years, but a series of drawbacks and limitations haveprevented their full implementation as referencemethods in routine-monitoring programs. In order toprotect the health of the consumer and the aquacultureindustry, regulatory authorities have establishedstringent performance criteria that must be realized byan alternative method before the mouse bioassay is dis-continued. The most challenging requirement is thatintra-laboratory and inter-laboratory validation must beperformed to internationally-recognized standards [3].The complexity of toxin analogues and their limitedavailability as standard material, particularly fromcompetitive commercial sources, not only restrictsmethod development but also proves validation to bepractically impossible to such accredited levels.Standards for marine toxins are generally producedfollowing their extraction and purification from large-scale culturing of toxic algal or from contaminatedshellfish material harvested from regions following anoutbreak [31]. Specialist laboratories able to performsuch work are rare worldwide, as they are expensive toestablish and to maintain. Explicit, dedicated expertise inchemical separation and purification techniques is re-quired in addition to a regular supply of contaminated  (c) ASP toxins – Domoic acid (~10 analogues) [92] Fig 1.  (continued)  Trends Trends in Analytical Chemistry, Vol. 30, No. 2, 2011 242  http://www.elsevier.com/locate/trac  Table 1.  Predominant toxins covered by European Union legislation, including action and effect and regulatory methods employed Toxin group Reference toxin (numberof analogues)Algal species derivedfromAction and effectin humansCurrent EURegulatory limitsRegulation (EC) No.853/2004 ( l g/kg of shellfish meat)Current EU referencemonitoring methodLimit of detection(LOD)/Limit of quantification (LOQ)[29]Standardized method Paralyticshellfishpoisoningtoxins[12]Saxitoxin(>30 analogues) [85] Alexandrium   species Gymnodinium   species Pyrodinium   speciesBlockage of site 1 of the voltage-gatedsodium channelcausingcardiorespiratoryfailure and death800 STX Eq  Mouse bioassay with 0.1M HCL (15 min) Regulation (EC) No 2074/ 2005LOD: 370  l g STX Eq/ kgAOAC method 959.08 HPLC-FLD (Lawrencemethod) (For screening purposes)Regulation (EC) No 1664/ 2006amending (EC) No 2074/ 2005LOQ: 10–80  l g STXEq/kg for individualanaloguesAOAC method 2005.06Diarrheicshellfishpoisoningtoxins[9–11,13]Okadaic acid anddinophysistoxins (>10analogues)[86,87] Dinophysis   species Prorocentrum lima Inhibit proteinphosphatases bybinding to PP1 andPP2a receptor sitescausing diarrhea.160 OA Eq  Mouse Bioassay or RatBioassay  with acetoneextraction (24 h)Regulation (EC) 2074/2005Unknown for eachtoxin. These bioassaysare incapable of detecting these toxinsat their currentregulatory limit with100% certainty.For okadaic acid theprobability of detection at theregulatory limit is aslow as 40%.NoPectenotoxin-2(  13 analogues) [90] Dinophysis   species  In vitro   disruption of actin cytoskeleton anddiarrheic effects are indispute160 OA EqAzaspiracid-1(  20 analogues)[88,89] Azadinium spinosum   Action is stillunknown but causesdiarrhea andneurotoxic effects160 AZA EqYessotoxin(  36 analogues) [91] Protoceratium reticulatum Lingulodinium polyedrum Gonyaulax spinifera Action not fullyknown but interactswithphosphodiesteraseenzymes and diarrheiceffects are beingquestioned1000 YTX EqAmnesicshellfishpoisoningtoxins [14]Domoic acid(  10 analogues) [92] Pseudo-nitzschia species Chondria armata Interacts with kainitereceptors causingneurological damage,memory loss anddeath20 000 DA Eq  HPLC-based methods Regulation (EC) No 1244/ 2007amending (EC) No 2074/ 2005LOD: 0.2–1 mg DA/kgLOQ: 1–2.5 mg DA/kgAOAC method 991.26CEN method 14176 Antibody-basedmethods(ELISA) (For screening purposes)Regulation (EC) No1244/ 2007amending (EC) No 2074/ 2005LOD: 0.003 mg DA/kgLOQ: 0.01 mg DA/kgAOAC method 2006.02LC-FLD,Liquidchromatography-fluorescencedetection;HPLC,High-performanceliquidchromatography;Eq,Equivalents;ELISA,Enzyme-linkedimmunosorbentassay;CEN,EuropeanCommitteeforStandardization.  T  r  e n d   s  i    n A  n a l      y t    i    c a l     C  h   e m i    s  t    r   y  , V  o l     . 3  0   , N o . 2   , 2  0  1  1  T  r  e n d   s   h   t    t     p :   /    /    w w w . e l     s  e v i    e r  . c o m /    l     o c a t    e /    t    r  a c  2   4   3  
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