Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).
ABSTRACT Brevetoxin uptake and elimination were examined in Eastern oyster (Crassostrea virginica) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated (r(2)=0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well (r(2)=0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well (r(2)=0.65), and were weakly correlated with mouse bioassay (r(2)=0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.
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ABSTRACT: Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes.Marine Drugs 03/2012; 10(3):583-97. · 3.98 Impact Factor
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ABSTRACT: Brevetoxin metabolites were identified and characterized in the hard clam (Mercenaria sp.) after natural exposure to Karenia brevis blooms by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal brevetoxins BTX-1 and BTX-2 produced by K. brevis were not detectable in clams. Metabolites of these brevetoxins found in clams included products of oxidation, reduction, hydrolysis and amino acid/fatty acid conjugation. Of highest abundance were cysteine and taurine conjugates. We also found glutathione, glycine-cysteine, and γ-glutamyl-cysteine conjugates. A series of fatty acid derivatives of cysteine-brevetoxin conjugates were also identified.Toxicon 08/2012; 60(6):1030-40. · 2.92 Impact Factor
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ABSTRACT: The eastern oyster (Crassostrea virginica) and northern quahog (= hard clam, Mercenaria mercenaria) are two species of economic and ecological significance in east coast waters of the United States and the Gulf of Mexico. Commercial industries for these species, especially within the state of Florida, are significant. The current study was undertaken to build upon the already established body of knowledge surrounding effects of the toxic dinoflagellate Karenia brevis on shellfish, to provide an understanding of the kinetics of brevetoxins within shellfish tissues, and to provide an estimate of brevetoxin retention times in these shellfish after a bloom event. Individual clams and oysters were exposed to the toxic dinoflagellate, K. brevis at a bloom concentration of 5 × 105 cells·L-1 for eight days and then transferred to filtered water for depuration. Individuals were sampled periodically to determine depuration rates. Concentrations of brevetoxins (and/or their metabolites measured as PbTx-3 equivalent) in tissues were determined using an Enzyme Linked Immunosorbent Assay (ELISA). After five days of exposure, brevetoxin levels in tissues of both species reached concentrations well above the regulatory limit of 800 ng g-1 (Pb-TX3 equivalent). Averaged concentration of brevetoxins in clams was 1000 ng g-1, while the oysters averaged 1986 ng g-1. After two weeks of depuration, tissue concentrations in both species were below regulatory levels with clams averaging ∼204 ng g-1 and oysters averaging ∼437 ng g-1. Toxins (or their metabolities) remained detectable in both clams (139 days) and oysters (82 days) for the duration of the experiment.Toxicon 02/2013; · 2.92 Impact Factor