Kathleen R. El Said

Confirmation of brevetoxin metabolism in the Eastern oyster (Crassostrea virginica) by controlled exposures to pure toxins and to Karenia brevis cultures

Previously, we analyzed Eastern oysters (Crassostrea virginica) naturally exposed to a Karenia brevis red tide and found that brevetoxins (PbTx) are rapidly accumulated and metabolized. Several metabolites were isolated and later identified, including a cysteine-PbTx conjugate (MH(+): m/z 1018) and its sulfoxide product (m/z 1034). In the present study, we confirm and extend those findings by examining PbTx metabolism and elimination in oysters exposed to pure toxins (PbTx-2 and -3) under controlled conditions. Waterborne PbTx-3 was rapidly accumulated, but not metabolized, in the oyster and was largely eliminated within 2 weeks after exposure. In contrast, PbTx-2 was accumulated and rapidly metabolized. Metabolites of PbTx-2 included the reduction product PbTx-3 (m/z 897), and the cysteine conjugates (m/z 1018 and 1034) isolated previously from the field samples. Levels of the metabolite PbTx-3 in PbTx-2-exposed oysters were highest immediately after exposure and declined at a rate similar to parent PbTx-3 in PbTx-3-exposed oysters. Cysteine-PbTx persisted for 8 weeks after exposure. The same metabolites were confirmed in oysters exposed to laboratory cultures of K. brevis. PbTx metabolites contribute to neurotoxic shellfish poisoning (NSP) and should be included in analytical protocols for monitoring shellfish toxicity after a K. brevis red tide event.

Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).

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.

Residue depletion of nitrofuran drugs and their tissue-bound metabolites in channel catfish (Ictalurus punctatus) after oral dosing.

The depletion of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin and their tissue-bound metabolites [3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AH), respectively] were examined in the muscle of channel catfish following oral dosing (1 mg/kg body weight). Parent drugs were measurable in muscle within 2 h. Peak levels were found at 4 h for furazolidone (30.4 ng/g) and at 12 h for nitrofurazone, furaltadone, and nitrofurantoin (104, 35.2, and 9.8 ng/g respectively). Parent drugs were rapidly eliminated from muscle, and tissue concentrations fell below the limit of detection (1 ng/g) at 96 h. Peak levels of tissue-bound AMOZ and AOZ (46.8 and 33.7 ng/g respectively) were measured at 12 h, and of SC and AH (31.1 and 9.1 ng/g, respectively) at 24 h. Tissue-bound metabolites were measurable for up to 56 days postdose. These results support the use of tissue-bound metabolites as target analytes for monitoring nitrofuran drugs in channel catfish.

Pharmacokinetics, tissue distribution, and metabolism of flumequine in channel catfish (Ictalurus punctatus)

Abstract The pharmacokinetics and metabolism of the fluoroquinolone drug flumequine (FLU) were examined after intravascular (1 mg/kg) or oral (5 mg/kg) dosing in channel catfish. Parent FLU concentrations in plasma declined slowly after intravascular dosing, with a half-life of 25 h. After oral dosing, FLU concentrations in plasma were highest (3.1 μg/ml) at 14 h after dosing; absorption and elimination half-lives were 4.9 and 22 h, respectively. The oral bioavailability of FLU was 44%, based on normalized plasma data. Plasma protein binding of parent FLU was extensive, but saturable (88%–55% bound at 0.125–8.0 μg/ml). After oral dosing with 14C-labeled FLU, radioactive residues were evenly distributed among the major tissues analyzed, with peak concentrations occurring at 12–24 h. Residue concentrations were highest in liver (6.2 μg/g) and lowest in muscle (1.8 μg/g) at 24 h. Only parent FLU was found in muscle and was eliminated with a half-life of 26 h. FLU and its metabolites were recovered in urine and bile. Residues in bile consisted almost entirely of a taurine conjugate of FLU. In urine, the taurine conjugate and hydroxy-FLU metabolites were found, in addition to the parent compound.

Performance evaluation of commercial ELISA kits for screening of furazolidone and furaltadone residues in fish.

Regulatory monitoring for nitrofuran drug residues in aquaculture products has largely focused on LC-MS/MS. In addition, there is a need for facile and high-throughput screening methods for monitoring programs. We evaluated the performance of Ridascreen (R-Biopharm) ELISA kits for nitrofuran drug residues in fish muscle, with verification by LC-MS/MS. Kits were available for 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholino-methyl-2-oxazolidinone (AMOZ) side-chains of furazolidone and furaltadone, respectively. We found good repeatability in fortified and incurred muscle samples, with RSDs ranging from 1.8% to 7.6%. Recoveries of AOZ and AMOZ from muscle fortified at levels of 0.5-2 ng/g ranged from 98% to 114%. Excellent selectivity was demonstrated. The minimum detection limits (MDLs) for AOZ and AMOZ in muscle were 0.05 and 0.2 ng/g, respectively. ELISA data were highly correlated with those of LC-MS/MS. Results of this study support the use of these kits as screening assays for nitrofuran residues in fish muscle.

A rapid assay for the brevetoxin group of sodium channel activators based on fluorescence monitoring of synaptoneurosomal membrane potential

A functional pharmacologically-based assay for the brevetoxin group of sodium channel activators was developed using synaptoneurosomes isolated from the brains of CD1 mice. The assay can detect the depolarizing effect of brevetoxin congeners PbTx-2 and PbTx-3 as enhancements of the veratridine-dependent increase in fluorescence of the voltage-sensitive fluorescent probe rhodamine 6G. The assay is relatively rapid and can detect brevetoxin activity in the nanomolar range. The synaptoneurosomal assay has been used to analyse mussel tissue extracts spiked with PbTx-2, and composite toxicity, expressed as PbTx-3 equivalents in extracts of oysters naturally exposed to brevetoxins. In this latter context, the synaptoneurosomal technique was shown to compare favorably with the cytotoxicity assay, the receptor binding assay and HPLC/MS. Our results support the concept that this membrane potential assay detects brevetoxins based on their interaction with sodium channels.

Characterization of brevetoxin metabolism in Karenia brevis bloom-exposed clams (Mercenaria sp.) by LC-MS/MS

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.

Biomarkers of Neurotoxic Shellfish Poisoning

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

Cyano Metabolite as a Biomarker of Nitrofurazone in Channel Catfish

The use of nitrofuran drugs in food-producing animals continues to attract international concern as a food safety issue. Methods for monitoring nitrofuran residues have been directed to the intact side chain of tissue-bound metabolites. Semicarbazide, the side chain of nitrofurazone (NFZ), can enter food products from non-NFZ sources, suggesting the need for an alternative biomarker for confirmatory purposes. We characterized a cyano derivative as a major metabolite of NFZ in channel catfish (Ictalurus punctatus). The depletion of cyano metabolite was examined in the muscle of channel catfish after oral dosing (10 mg of NFZ/kg of body weight). Parent NFZ was rapidly eliminated in muscle, with a half-life of 6.3 h. The cyano metabolite was detected for up to 2 weeks, with an elimination half-life of 81 h. The cyano metabolite represents an alternative biomarker for confirming the use of NFZ in channel catfish.

Performance Assessment and Comparability of a Commercial Enzyme-Linked Immunosorbent Assay Kit with Liquid Chromatography–Tandem Mass Spectrometry for Chloramphenicol Residues in Crab and Shrimp

Monitoring for chloramphenicol (CAP) in aquaculture products is primarily performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which requires expensive equipment and specialized training. Many laboratories prefer to screen samples with facile and high-throughput enzyme-linked immunosorbent assay (ELISA) kits for CAP residues before submitting samples for LC-MS/MS quantification and confirmation. We evaluated the performance of a Ridascreen (R-Biopharm) ELISA kit for CAP in spiked and incurred crab and shrimp muscle at levels bracketing the minimum required performance level for analysis (0.3 ng/g). The Ridascreen ELISA kit incorporates antibody directed against CAP. Incurred CAP levels in crab and shrimp muscle were verified using LC-MS/MS. We found good repeatability (relative standard deviation) of the ELISA in spiked and incurred crab and shrimp muscle samples, with values ranging from 6.8 to 21.7%. Recoveries of CAP from tissues spiked at 0.15 to 0.60 ng/g ranged from 102 to 107%. Minimal cross-reactivity with blank crab and shrimp muscle matrix components was observed. ELISA data were highly correlated with those of LC-MS/MS for CAP in incurred muscle tissue. We believe this study to be the first evaluation of the performance and comparability of a CAP ELISA kit and LC-MS/MS for determination of CAP residues, as well as their elimination, in crab muscle. Our findings support the use of this ELISA kit for screening purposes and, when used in conjunction with validated instrumental methods, for regulatory monitoring of CAP in these species.