toxins Article
Accumulation and Elimination Dynamics of the Hydroxybenzoate Saxitoxin Analogues in Mussels Mytilus galloprovincialis Exposed to the Toxic Marine Dinoflagellate Gymnodinium catenatum Pedro Reis Costa 1,2, * , Ana Catarina Braga 1,3 and Andrew D. Turner 4 1 2 3 4
*
IPMA—Portuguese Institute for the Sea and Atmosphere, Av. Brasília, 1449-006 Lisbon, Portugal;
[email protected] CCMAR—Centre of Marine Sciences, University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal Biology Department and CESAM, Aveiro University, 3810-193 Aveiro, Portugal Centre for Environment Fisheries and Aquaculture Science (CEFAS), Barrack Road, Weymouth, Dorset DT4 8UB, UK;
[email protected] Correspondence:
[email protected]; Tel.: +351-213027000
Received: 18 September 2018; Accepted: 23 October 2018; Published: 26 October 2018
Abstract: Paralytic shellfish poisoning (PSP) is a severe food-borne illness, caused by the ingestion of seafood containing paralytic shellfish toxins (PST), which are naturally produced by marine dinoflagellates and accumulate in shellfish during algae blooms. Novel PST, designated as hydroxybenzoate analogues (also known as GC toxins), was relatively recently discovered in Gymnodinium catenatum strains worldwide. However, to date, there have been no studies examining their accumulation in shellfish. In this study, mussels (Mytilus galloprovincialis) were exposed to G. catenatum for five days and then exposed to a non-toxic diet for 24 h, to investigate the toxin’s accumulation/elimination dynamics. As determined by UHPLC-HILIC-MS/MS, the hydroxybenzoate analogues, GC1 to GC6, comprised 41% of the algae toxin profile and only 9% in mussels. Elimination of GC toxins after 24 h was not evident. This study highlights that a relevant fraction of PST in mussels are not routinely analysed in monitoring programs and that there is a need to better understand the toxicological potential of the hydroxybenzoate analogues, in order to properly address the risk of G. catenatum blooms. Keywords: saxitoxin; shellfish metabolism; paralytic shellfish poisoning; harmful algal blooms Key Contribution: A step forward to understanding the accumulation of GC toxins in shellfish.
1. Introduction Paralytic shellfish toxins (PST) are potent neurotoxic alkaloids, produced in the marine environment by dinoflagellate species belonging to three genera, namely Alexandrium, Pyrodinium, and Gymnodinium. The toxicity of PST is caused by a high affinity inhibition of voltage-gated sodium channels (Nav ) on the extracellular membranes of nerve cell terminals [1,2]. Structural differences among PST analogues—which were classically divided in three groups, the carbamoyl, dicarbamoyl, and sulfocarbamoyl groups—result in different affinities to the binding sites of Nav , leading to a varying degree of toxicity. A fourth group of PST was described in the early 2000s, in Gymnodinium cateantum strains from Australia, Uruguay, China, Spain, and Portugal [3]. This fourth group has the carbamate side chain replaced with a hydroxybenzoate moiety, as shown in Figure 1. The new toxins were designated as GC toxins (GC1–3), with GC3 corresponding to the 4-hydroxybenzoate ester
Toxins 2018, 10, 428; doi:10.3390/toxins10110428
www.mdpi.com/journal/toxins
not the GC toxins. This may justify why the presence and variability of hydroxybenzoate saxitoxin analogues in shellfish have been poorly investigated. One attempt to quantify GC toxins in shellfish after a G. catenatum bloom was carried out by [4], using a modified and automated version of the HPLC-FLD method. Low levels of GC toxins were determined, suggesting their conversion into decarbamoyl toxins [4]. The conversion would have been mediated by the activity of carbamoylase Toxins 2018, 10, 428 2 of 8 enzymes, resulting in the loss of the benzoate moiety [4]. However, carbamoylase enzyme activity is known in very few species, such as the surf clam (Spisula solida), which typically show a toxin profile derivative of decarbamoylsaxitoxin and GC1 and GC2 to the epimeric sulphate derivatives composed of decarbamoyl toxins (dcSTX), [14]. of GC3PST [3]. are Indeed, Gymnodinium catenatum has been toxins, shown but to produce wide array of PST. generally characterized as hydrophilic with GCatoxins, their less polar In subsequent years, several other hydroxybenzoate have been reported designated hydroxybenzoate substituents may favour GC analogues toxin bioaccumulation and aand reduction in as their p-hydroxybenzoyl, di-hydroxybenzoyl, sulfo-benzoyl derivatives Thedeveloped toxicity ofand GCvalidated toxins elimination rate [3,8]. The aim of thisand study was to determine, via a [4–7]. recently is still poorly known. In vitro studies, using rat brain synaptosomes with enriched sodium channels, UHPLC-HILIC-MS/MS method [15,16], the presence of GC toxins in mussels, Mytilus galloprovincialis, indicate affinity binding Nav [8],and andafter computer that modelled toxicity processes after 5high days’ exposure to G. to catenatum a 24 h simulations elimination period, under controlled laboratory also suggest high affinity to Nav [9]. conditions.
Figure 1. Chemical structure of the four groups of paralytic shellfish toxins (PST). Figure 1. Chemical structure of the four groups of paralytic shellfish toxins (PST).
Accumulation and transfer of PST throughout the marine food web may cause the death of marine 2. Results and Discussion wildlife and human intoxication [10,11]. Paralytic shellfish poisoning (PSP) is an illness characterized by 2.1. neurological issues, from numbness of the fingers and extremities, tingling, nausea, and vomiting, Toxin Profile of Gymnodinium Catenatum Strain Used for Mussel Exposure to muscular paralysis and death by respiratory paralysis and cardiovascular shock [12,13]. In order to most abundant PST, found in the IO-13-04 G. catenatum determinedprogram by HILICprotect The public health from acute intoxication, most coastal countriesstrain, have aasmonitoring UPLC–MS/MS, were the N-sulfocarbamoyl derivatives, followed by the decarbamoyl derivatives. in place that closes shellfish harvesting, whenever PSP toxicity exceeds 800 µg STX (saxitoxin) Small −levels of carbamoyl toxins were also as shown in Table 1. Saxitoxin and 1 in shellfish equiv.kg meat. PSP From 1 January 2019,determined, the EU reference method for determination neosaxitoxin (Neo) were not detected, with GTX3 being the most abundant carbamoyl analogue of PSP toxins—based on pre-column oxidation (pre-cox) high performance liquid-chromatography found. This profile, dominated by C toxins and decarbamoyl derivatives, is typically observed in G. with fluorescence detection (HPLC-FLD)—will come into effect. This method was developed to catenatum strains isolated from the Portuguese coastand [17].sulfocarbamoyl In addition to PST, classic quantify “classic” hydrophilic carbamoyl, dicarbamoyl, butPST, not hydroxybenzoyl the GC toxins. derivatives were detected, representing a significant part of the toxin profile of G. catenatum. GC1 to This may justify why the presence and variability of hydroxybenzoate saxitoxin analogues in shellfish have been poorly investigated. One attempt to quantify GC toxins in shellfish after a G. catenatum bloom was carried out by [4], using a modified and automated version of the HPLC-FLD method. Low levels of GC toxins were determined, suggesting their conversion into decarbamoyl toxins [4]. The conversion would have been mediated by the activity of carbamoylase enzymes, resulting in the loss of the benzoate moiety [4]. However, carbamoylase enzyme activity is known in very few species, such as the surf clam (Spisula solida), which typically show a toxin profile composed of decarbamoyl toxins [14]. PST are generally characterized as hydrophilic toxins, but with GC toxins, their less polar hydroxybenzoate substituents may favour GC toxin bioaccumulation and a reduction in their elimination rate [3,8]. The aim of this study was to determine, via a recently developed and validated UHPLC-HILIC-MS/MS method [15,16], the presence of GC toxins in mussels, Mytilus galloprovincialis, after 5 days’ exposure to G. catenatum and after a 24 h elimination period, under controlled laboratory conditions. 2. Results and Discussion 2.1. Toxin Profile of Gymnodinium Catenatum Strain Used for Mussel Exposure The most abundant PST, found in the IO-13-04 G. catenatum strain, as determined by HILIC-UPLC–MS/MS, were the N-sulfocarbamoyl derivatives, followed by the decarbamoyl derivatives. Small levels of carbamoyl PSP toxins were also determined, as shown in Table 1. Saxitoxin and neosaxitoxin (Neo) were not detected, with GTX3 being the most abundant carbamoyl
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analogue found. This profile, dominated by C toxins and decarbamoyl derivatives, is typically observed in G. catenatum strains isolated from the Portuguese coast [17]. In addition to classic PST, hydroxybenzoyl derivatives were detected, representing a significant part of the toxin profile of G. catenatum. GC1 to GC6 were here identified as previously found [6] in a different G. catenatum strain, isolated from the Portuguese coast, but none of the di-hydroxybenzoyl or sulfobenzoyl GC analogues indicated [4,7] were observed. Table 1. PSP (paralytic shellfish poisoning) toxin profile of the Gymnodinium catenatum culture used to feed mussels. PSP Toxins
Toxin Concentration (fmol/cell)
Molar Fraction (%)
N-sulfocarbamoyl
C1 C2 C3 C4 GTX5 GTX6
2.0 5.6 0.2 1.4 0.2 0.2
10.7 30.1 1.1 7.3 1.2 1.2
Decarbamoyl
dcSTX dcNeo dcGTX2 dcGTX3
0.3 0.2 0.1 0.3
1.4 1.2 0.2 1.4
Carbamoyl
GTX1 GTX2 GTX3 GTX4 STX Neo
0.1 0.1 0.5 0.1
