Esters of okadaic acid and dinophysistoxin-2in Portuguese bivalves related to human

poisonings

Paulo Vale *, Maria Anto nia M. Sampayo

Instituto de Investigac° aÄo das Pescas e do Mar, Av. BrasõÂlia, 1400 Lisbon, Portugal

Received 7 September 1998; accepted 17 November 1998

Abstract

Liquid chromatography (HPLC) was used to search for esters of DSP toxins inPortuguese bivalves. Hexane-soluble derivatives of okadaic acid (OA) anddinophysistoxin-2 (DTX-2) were found. Presumably they are acyl derivatives, globally

known as `dinophysistoxin-3' (DTX-3). In certain instances DTX-3 content surpassed50% of the total amount of DSP toxins. A human diarrhetic poisoning (DSP) incidentwith Donax clams (Donax trunculus) harvested at Fuzeta (Algarve coast) was

con®rmed where the apolar (DTX-3 type) and other esters remaining in the polaraqueous methanol layer were implicated. The percentage of acyl esters of OA wasalways higher than those of DTX-2. An enzymic mechanism for the conversion of OA

and DTX-2 seems to be implicated in some kind of detoxi®cation process because thepercentage of esters increases with the toxin amount ingested by the bivalve and thereis some degree of selectivity as DTX-2 seems more di�cult to acylate. These ®ndingspose a problem for the current assay methods used to detect DSP because mainly

they are able to detect the parent toxins but not their esters. The current bioassaymethod [Le Baut, C., Bardin, B., Bardouil, M., Bohec, M., Masselin, P., Truquet, P.,1990. Etude de la de contamination de moules toxiques. Rapport IFREMER DERO-

90-02 MR. 21 pp.] used in Portugal includes a hexane washing step that preventsinterference from free fatty acids. However, it cannot detect the presence of acylderivatives because they are highly soluble in hexane. # 1999 Elsevier Science Ltd. All

rights reserved.

Toxicon 37 (1999) 1109±1121

0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0041 -0101 (98)00247 -5

PERGAMON

* Corresponding author. Fax: +351-1-301-5948.

1. Introduction

Since 1987 diarrhetic shell®sh poisoning (DSP) toxins have been monitored inPortugal using the mouse bioassay. Since 1994 all bivalves screened for DSPtoxicity are also checked by HPLC using the ¯uorometric method of Lee et al.(1987).

The DSP toxins found were okadaic acid (OA) (Fig. 1) and dinophysistoxin-2(DTX-2) (Vale and Sampayo, 1996). Although OA is very often found associatedwith DSP contamination, DTX-2 was only more recently chemically identi®ed(Hu et al., 1992a). DTX-2 is the predominant diarrhetic toxin in Irish mussels(Carmody et al., 1996) and was also identi®ed in mussels from the Galician region(Spain) (Gago-Martinez et al., 1996).

Dinophysistoxin-2 contamination is mainly derived from the dino¯agellateDinophysis acuta, while Dinophysis acuminata seems to produce mainly OA (Valeet al., 1998). These same two species account for the similar contamination foundin mussels from Ireland (Carmody et al., 1996) and Spain (Blanco et al., 1995).

In bivalve molluscs other toxins derived from these are also found and globallydesignated as `dinophysistoxin-3' (DTX-3). Originally DTX-3 referred to thecomplex mixture of 7-O-acylderivatives of dinophysistoxin-1 (DTX-1), rangingfrom tetradecanoic acid (C14:0) to docosahexaenoic acid (C22:6o3) (Yasumoto etal., 1985). It was the main diarrhetic toxin found in scallops in Japan, while DTX-1 was the main toxin detected in mussels (Yasumoto et al., 1985). Later it wasdemonstrated that either OA, DTX-1 or DTX-2 may be acylated to produce`DTX-3' (Marr et al., 1992). These acylated forms have never been found in

Fig. 1. Structure of lipid-soluble okadaic acid and its known naturally occurring derivatives. Groups I

and II comprise the main toxins responsible for human intoxications. Groups III and IV are found in

the algae of the genera Prorocentrum.

P. Vale et al. / Toxicon 37 (1999) 1109±11211110

marine microalgae and so it is presumed that they originate in the bivalve byacylation (Lee et al., 1989).

In bivalves the main attention has been almost only focused worldwide onokadaic acid (and DTX-1/DTX-2) determination by HPLC ¯uorometric methods.Little attention has been paid to the acyl derivatives of these toxins. Due to theirhigh molecular weight and lipophilicity, they cannot be directly detected by the¯uorometric procedure of Lee et al. (1987), but an alkaline hydrolysis reaction torelease fatty acids from the parent toxins can give an estimation of theirabundance. Their detection by LC±LC after ¯uorescence labelling has beenproposed by Akasaka et al. (1996), but involves a more complicated LC systemand needs standards for all of the toxins involved. In Europe they have alreadybeen found in small amounts in Irish (Marr et al., 1992) and Spanish (Ferna ndezet al., 1996a) mussels.

Being aware of their occurrence, we introduced monitoring for these toxins inthe summer of 1995 in one sampling area: the Aveiro Lagoon. Results obtainedduring the previous year had shown that the Aveiro Lagoon's mussels arecontaminated with diarrhetic toxins for long periods several times throughout theyear (Vale and Sampayo, 1996; Vale et al., 1998).

2. Materials and methods

2.1. Search of free OA and dinophysistoxins-1/2

Normal HPLC analysis was carried out according to the method of Lee et al.(1987) slightly modi®ed using 1 g of fresh hepatopancreas (mussels and oysters)or 5 g of fresh edible tissues (other shell®sh) mixture used for the mousebioassay. This was extracted once with 4 (20)* ml1 methanol:water (80:20),respectively. For analysis in edible tissues a (4-fold) higher amount of extractwas processed due to the lower concentration of toxins. A 1 (2)* ml aliquotwas washed twice with 2 ml of hexane, 0.5 ml of water was added andextracted twice with 2.3 ml of chloroform2. The volume was corrected to 5 ml,and 1 (2)* ml was dried.

2.2. Search for OA and DTX-1/2 esters

Another 1 (2)* ml methanol aliquot was washed twice with 2.3 ml of hexane.The hexane layer was dried. For hydrolysis of esters both the methanolic anddried hexanic layers were mixed with 400 ml of methanolic 1 M NaOH andallowed to react at room temperature for 1 h (alternatively at 408C for half an

1 Numbers marked with an asterisk: the value before the parentheses refers to the digestive glands

and inside the parentheses it refers to the edible tissues.2 In the 1998 analysis, chloroform has been replaced by dichloromethane.

P. Vale et al. / Toxicon 37 (1999) 1109±1121 1111

hour). The methanolic layer was neutralised with 485 ml aqueous 1 M HCl andthe hexane layer with 450 ml. Both were washed twice with 2 ml of hexane; 0.5 mlof water was added and then they were extracted twice with 2.3 ml of chloroform.The volume was corrected to 5 ml, and 1 (2)* ml of the methanol- or hexane-soluble esters was dried. As the hexane-soluble compounds produced cleanerchromatograms, the total 5 ml chloroform can be concentrated to lower thedetection limit.

2.3. Fluorescence labelling and clean-up

Labelling of the toxins was done with 0.1% (w/v) acetone solution of 9-anthryldiazomethane (ADAM, Serva, Heidelberg, No. 13682). A fresh solutionwas prepared for each derivatization batch, ADAM crystals were kept at ÿ408Cfor several months. For the normal method of Lee, 200 ml were used, and for thehydrolysed extracts (Section 2.2) 400 ml were used.

For the 1995 analyses the clean-up step was performed as described by Lee et al.(1987) using Sep-Pak Classic (690 mg) silica cartridges (Waters No. 51900);elution was carried out by gravity. Extracts were dried under a N2 stream andresuspended in 200 ml methanol. For the 1998 analyses Sep-Pak Light (120 mg)silica cartridges (Waters No. 23537) were used. ADAM derivatives wereresuspended with 1 ml dichloromethane and passed through the cartridges,washed with 2 ml dichloromethane/acetone 100:6 and eluted with 2 mldichloromethane/methanol 100:8; dried under vacuum and resuspended in 200 mlmethanol.

2.4. HPLC analysis

Liquid chromatography was performed on a Hewlett-Packard (HP) Model 1050equipped with a quaternary pump, autosampler and the 1046-A ¯uorescencedetector; data collection and results treatment were performed by the HP`Chemstation' software. In the 1995 analyses the ¯uorescent esters were separatedusing 0.9 ml/min of acetonitrile/water (85:15) on a Merck Superspher 100 RP-18(4 mm, 250�4 mm), protected by a guard-column (4�4 mm) packed withLichrospher 100 RP-18 (5 mm). Detection wavelengths were set at lex 365 nm andlem 412 nm. The column temperature was kept at 358C and 20 ml were injectedonto the column. In the 1998 analyses the ¯uorescent esters were separated using0.3 ml/min of acetonitrile/water (81:19) on a Merck Superspher 100 RP-18 (4 mm,250�2 mm), protected by the same guard-column. Detection wavelengths were setat lex 254 nm and lem 412 nm. The column temperature was kept at 308C and 5 mlwere injected onto the column.

Okadaic acid from Boehringer Mannheim (Mannheim) was used as a standard.DTX-2 was previously con®rmed using an Irish contaminated mussel (see Valeand Sampayo, 1996). DTX-1 was purchased from Calbiochem (Novabiochem).

P. Vale et al. / Toxicon 37 (1999) 1109±11211112

Fig. 2. Chromatograms of the ADAM esters of DSP toxins in blue mussels (Mytilus edulis) from the

Aveiro Lagoon (August 16th 1995). (a) Sample extracted according to conditions of the method of Lee

et al. (1987) (OA: 1.1 mg/g, DTX-2: 5.1 mg/g). (b) Combined (double extraction) hexanic layers dried,

hydrolysed with NaOH and extracted 2� with CHCl3. The release of both free OA and DTX-2 from

their apolar ester forms is evident (OA: 5.4 mg/g, DTX-2: 2.9 mg/g). (c) Methanolic fraction after

double washing with hexane, hydrolysed with NaOH and extracted 2� with CHCl3. Peak area is

slightly higher than on chromatogram (a), indicating that some esters were not extracted to the hexane

layer, remaining in the aqueous MeOH (additional OA: 0.7 mg/g, and DTX-2: 0 mg/g).

P. Vale et al. / Toxicon 37 (1999) 1109±1121 1113

3. Results

3.1. The 1995 DSP outbreak in Mytilus edulis from the Aveiro Lagoon

When the hexane layers of DSP-contaminated blue mussels (Fig. 2a) werehydrolysed, two peaks appeared at the retention time of okadaic acid anddinophysistoxin-2 (Fig. 2b). Control hexane layers not submitted to alkalinehydrolysis did not show any of these peaks. In order to be sure that these werenot already present in the hexane layer, amounts of ADAM higher than usualwere used. Higher amounts of ADAM were always used in the hydrolysed layersbecause the hydrolysis released free carboxylic acid groups that react withADAM.

After extracting the methanolic layer with hexane, hydrolysis was also carriedout on the remaining lipids. The peak area increased after hydrolysis (Fig. 2c)showing that some esters had remained in this layer.

The variation of free and esteri®ed DSP toxins is exempli®ed in musselsharvested in the summer of 1995 at the Aveiro Lagoon (Fig. 3). The largemajority of the esters was extracted after the double hexane extraction. Howeversome remained in the aqueous methanol layer. During the period shown in Fig. 2,D. acuta was the dominant species, D. acuminata occurring at a lower density(Vale et al., 1998).

One conspicuous result which was observable in these mussels was that apolaresters from okadaic acid were much more abundant than those resulting from theacylation of dinophysistoxin-2. The OA esters reached 80±90% of the total

Fig. 3. Partition of the OA and DTX-2 content on their free form, conjugated with fatty acids (DTX-3)

that are removed by the hexane extraction (apolar fraction) and esters remaining in the polar layer

(aqueous MeOH). (Results from the Aveiro Lagoon's mussels, 1995.)

P. Vale et al. / Toxicon 37 (1999) 1109±11211114

okadaic acid detected, while DTX-2 esters never surpassed 50% of total DTX-2found. Despite the high values of DSP toxins we received no report ofhuman poisonings because the harvest of mussels was banned. Although thisspecies is widely used as a toxicity indicator, it is not largely consumed as it isfurther north in the Galician region (Spain). In 1996 and 1997 toxic Dinophysisspp. were much less abundant and correspondingly the toxicity levels found werelower. During these two years when only the hexane layers were studied for esters,these were found but in much lower percentages than those found in 1995 (datanot shown).

3.2. The 1998 DSP outbreak at the Algarve

``Eighteen people among co-workers from the Health Centre Unit at LouleÂ(Algarve District, south coast of Portugal) and their family members reported afood poisoning syndrome between 12 and 16 of February 1998. Onset ofsymptoms started 6 h after ingestion and included diarrhoea, vomiting, gastric andabdominal pains, muscular weakness and headaches. The apparent food itemresponsible for the intoxication was Donax clams harvested at Fuzeta (Algarvecoast). The symptoms developed over a four-day period, after which the victimswere all ®t to work. Only one person went to an emergency unit and was treatedwith life support measures. The gravity of the symptoms was directly related tothe amount ingested. Those who ate little had mild symptoms while those whoingested a half kilogram of clams presented the most serious symptoms. Toxicitywas not related to the cooking method. Those who ate raw and those who atecooked shell®sh developed the same symptoms''3.

A sample of these clams (Donax trunculus4) was sent to IPIMAR were it wasanalysed for toxigenic bacteria and DSP toxins. The search for Salmonella spp.gave a negative result in 25 g. Analysis by HPLC revealed a low amount ofokadaic acid (10 mg/100 g edible tissues, Fig. 4a). After alkaline hydrolysis of theMeOH extract 130 mg/100 g of okadaic acid were found (Fig. 4d). At the time,D. acuminata was already occurring in the plankton samples. When a few dayslater this extract was re-analysed in detail to determine the distribution of toxins,the hexane-soluble esters comprised 38% (Fig. 4b) and the esters remaining in themethanolic layer comprised 51% (Figs. 4c and 5). Donax clams are small in sizeand have a very small amount of edible tissue (18 to 20% ratio of tissue/wholeshell®sh). For calculating the maximum dose ingested for those who consumed0.5 kg (according to the above report; for attaining this value 100±200 shell®shmust be eaten so this is only a tentative estimate) a value of 90±100 g edibletissues must have been eaten. This would give a total of 117±130 mg of okadaicacid equivalents. Apolar esters of OA were also determined in additional shell®sh

3 These data are based on the epidemiological report of Dr. Tavares de Sousa, Head of the LouleÂ

Health Centre.4 D. trunculus is the main occurring species of this genera although other species may co-occur.

P. Vale et al. / Toxicon 37 (1999) 1109±1121 1115

Fig. 4. Chromatograms of the ADAM esters of DSP toxins in Donax clams (D. trunculus) harvested at

Fuzeta and responsible for a food poisoning outbreak at Loule (12 February, 1998). (a) Sample

extracted according to conditions of the method of Lee et al. (1987) (OA: 9.5 mg/100 g). (b) Combined

(double extraction) hexanic layers dried, hydrolysed with NaOH and extracted 2� with CH2Cl2. The

release of free OA from its apolar ester forms is evident (OA: 33 mg/100 g). (c) Methanolic fraction

after double washing with hexane, hydrolysed with NaOH and extracted 2� with CH2Cl2. Peak area is

higher than on the upper chromatogram (a), indicating that some esters of higher polarity were not

extracted to the hexane layer, remaining in the aqueous MeOH (additional OA: 44 mg/100 g).

(d) Methanolic fraction without hexane washing, hydrolysed with NaOH and extracted 2� with

CH2Cl2 (total OA: 130 mg/100 g). The sum of OA forms found in (a) through (c) does not add to the

value found in this total DSP analysis which could be explained due to the reported high instability of

DTX-3 (Yasumoto et al., 1985); (b) and (c) analyses were carried out a few days after (a) and (d).

P. Vale et al. / Toxicon 37 (1999) 1109±11211116

including Spisula solida, Ruditapes decussata and Cerastoderma edule, comprisingthe majority of the okadaic acid form found.

After this outbreak of human poisoning, DSP contamination with OA andesters persisted at lower levels until in August the isomer DTX-2 was detected dueto the occurrence of D. acuta in the plankton (Fig. 5). Again, OA-apolar esterscomprised 70±90% of total OA while DTX-2-apolar esters never surpassed 50%of the total DTX-2. The presence in these samples of DTX-2 esters not extractedinto hexane is also suspected (Fig. 5).

4. Discussion

The LC±FLU results do not allow us to determine the precise nature of eachgroup of toxin esters found. Until now the known hexane-soluble esters inshell®sh were those obtained by acylation of toxins with fatty acids which rendersthem apolar. Our apolar esters in mussels from 1995 belong to this category(Quilliam, unpublished results). The double hexane washing may not haveremoved all the acyl derivatives, in particular those having the shorter acyl side

Fig. 5. Partition of the OA and DTX-2 content on their free form, conjugated with fatty acids which

are removed by the hexane extraction (apolar fraction) and esters remaining in the aqueous methanol

(polar layer). Results are from Algarve Donax clams (1998). Total DSP levels found for each sample

were, respectively, from left to right in mg/100 g: 12-Feb: 86; 17-Feb: 46; 13-Jul: 23; 3-Aug (Lavage's

beach): 51 OA and 38 DTX2; 3-Aug (Faro's beach): 53 OA and 42 DTX2; 13-Aug: 14 OA and 8

DTX2.

P. Vale et al. / Toxicon 37 (1999) 1109±1121 1117

chains (C14 for example) that will be less apolar than those with longer chains(C22). So this may explain in part the small percentage of `polar' esters found inmussels. The recent results from Donax clams point to the possible existence ofother type of esters that are not so extractable with hexane. In cultivatedProrocentrum species, several kinds of esters which are precursors of okadaic acidhave been described: lipid-soluble OA-diol esters (Yasumoto et al., 1989; Hu et al.,1992b) and even water-solubles DTX-4, DTX-5a and DTX-5b (Hu et al.,1995a,b). The di�culties in cultivating Dinophysis species have until nowprevented the study of their biochemistry of toxin production. Describing thechemical complexity of DSP toxin pro®les with LC±FLU is becoming anincreasingly di�cult task without more modern techniques like LCÐMS. E�ortmust be made to determine their structure and con®rm whether they originate inDinophysys spp. or result from biotransformation by shell®sh.

Regarding the transformation that occurred in shell®sh, it was found that OAwas preferentially acylated rather than DTX-2, in contrast to the ®rst reportedresults obtained with Irish mussels where OA and DTX-2 were found to beacylated proportionally to their free concentrations (Marr et al., 1992). However,more recently in Spanish mussels the curious disproportion free/acylated OAversus DTX-2 has also been found (Ferna ndez et al., 1998). Another observationhas been that the percentage of esteri®ed OA (and DTX-2 to a lesser extent) mayincrease with increased contamination levels. In Spanish mussels the higherpercentage of hexane-soluble esters was mainly found on the more contaminatedsamples, however their percentages were not so high as those reported here(Ferna ndez et al., 1996a). It is possible that the esteri®cation is an enzymic5

mechanism involved in detoxi®cation that is increasingly more activated at highertoxicity levels and not simply a general lipid metabolism, or else more constantratios of esteri®ed toxins would be expected. The modi®ed toxins, e.g. DTX-3(Takai et al., 1992), diol esters (Hu et al., 1992b) and DTX-4 (Hu et al., 1995a),do not inhibit protein phosphatases in vitro. In contamination experiments whenOA, diol esters and DTX4 were added to the diatom Thalassiosira weiss¯ogiiculture medium new metabolite products of diol esters were found probablyoriginating from a monooxygenase detoxi®cation pathway (Windust et al., 1997).

When the poisoning was ®rst chemically characterised in Japanese mussels,DTX-1 comprised 88% of the toxicity and the hexane-soluble toxins only 4%(Murata et al., 1982). Only in Japanese scallops was DTX-3 the main toxin(Yasumoto et al., 1985). However this did not always hold to be true becausescallops harvested in 1986 and 1987 di�ered in that DTX-1 was predominant(Yasumoto et al., 1989). Preliminary studies on European mussels showed OA tobe the major toxin (Kumagai et al., 1986), with the exception of Norwegianmussels were DTX-1 was more important (Lee et al., 1988). This is the ®rst reportin Europe for DSP ester toxins representing the majority of the toxic fraction.

5 For comparison commercial antibodies for OA have a lower cross-reactivity towards (i.e. are able

to distinguish the subtle di�erence of) DTX-2.

P. Vale et al. / Toxicon 37 (1999) 1109±11211118

These ®ndings pose a problem for certain mouse bioassays, HPLC and other fastdetection techniques like ELISA and protein phosphatase (PP2A) inhibitionassay because they require the prior hydrolysis of DTX-3 to the respective parenttoxins.

This is also the ®rst con®rmation of human intoxications in Europe by okadaicacid esters. Clearly OA could not account by itself for the human poisonings. Ifwe calculate the ingested dose based solely on the free OA content, we obtain amaximum ingested dose of only 9 mg. In the ®rst con®rmed intoxications in Japana dose of 32 mg DTX-1 was determined as the lowest dose to induce symptoms inhumans (Yasumoto et al., 1985). Although Donax are very small bivalves andlarge numbers of them are usually eaten, the maximum uptake of total OA in thisoutbreak was estimated to be around 120±130 mg which is well above the dosenecessary to induce symptoms in humans. The other individuals who reported tohave ingested a smaller number of shell®sh would still have ingested dosesabove the value calculated by the Japanese researchers. DTX-3 is reported tohave as much diarrhetic potency as non-acylated OA or DTX-1 whenadministered orally to mice (Ito and Terao, 1994). Although several toxins lethalto mice by i.p. administration (pectenotoxins and yessotoxins) were foundassociated with the Japanese DSP poisoning outbreaks, until now only theokadaic acid group has been proved to be diarrhetic (Hamano et al., 1985; Oginoet al., 1997).

According to Takagi et al. (1984) certain free fatty acids may be responsiblefor false DSP positive results found seasonally during the year. These fattyacids may be lethal to mice by intraperitonial administration but represent nodanger to man. The modi®cation of Yasumoto's method used at ourlaboratory (Le Baut et al., 1990) consists of the removal of apolar lipids inorder to circumvent this drawback of the mouse bioassay. This kind ofbioassay cannot detect the DTX-3 type of contamination. Regarding otherkinds of bioassays with the 5 h survival criteria (and where the hexanewashing step is not carried out) DSP toxicity would be underestimated becauseDTX-3 has a lower lethality by i.p. injection, due to the peritoneal barrier tonon polar substances (Ito and Terao, 1994). An extension of the 5 h meansurvival time used for regulatory purposes to 24 h was proposed by Ferna ndezet al. (1996b) in order to account for the presence of acyl derivatives becausethey have a lower dose/time response.

There is a regulatory limit of 5 MU (or 20 mg OA)/100 g edible tissues proposedby several countries (Van Egmond et al., 1992). By chemical methods our samplefrom February was below this limit and yet there was a serious outbreak ofdiarrhoea. Clearly the criterion is un®t and should include the total amount ofDSP toxins thus comprising OA, DTX-1/2 and their respective esters. The harvestof Donax spp. was prohibited almost from Spring through Summer 1998 whentotal DSP levels exceeded 20 mg OA equivalents/100 g. No more humanintoxications were reported when levels were around this limit. This new criterioncan prevent diarrhoea in humans and is proposed by us for use in future chemical(HPLC) and biochemical (ELISA, PP2A) assays.

P. Vale et al. / Toxicon 37 (1999) 1109±1121 1119

Acknowledgements

We are indebted to Dr. Valetina Tavares de Sousa, Head of the Health Unit atLoule , who interviewed the intoxicated people and provided us with a copy of herepidemiological report. Madalena Albuquerque, our colleague at IPIMAR-Lisbon,supplied us with the bacteriological results. Thanks are also due to M.A. Quilliam(NRC of Canada, Halifax) for helpful discussions and comments on the manuscript.This work was partially supported by JNICT with contract No. PBIC/C/MAR/1292/95 and grant BIC-854 to the ®rst author, the Programme PROPESCAS/Sanityand Salubrity of Bivalve Molluscs and, PRAXIS XXI with contract No. 2/2.1/MAR/1718/95 and grant No. XXI/BD/11448/97 to the ®rst author.

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