Analysis of Chloramphenicol and Two Metabolites in Crab and Shrimp Following Waterborne Exposure.

The use of chloramphenicol (CAP) in aquaculture products is banned in many countries, including the United States, due to human health issues. Very few depletion and metabolism studies of CAP in seafood have been performed. Current detection methods for CAP residues in food are directed toward the parent drug molecule, but rapid elimination following treatment suggests the need for an alternative marker residue. We identified, characterized, and determined the persistence of two CAP metabolites, CAP-base (CAP-B) and CAP-alcohol (CAP-OH), in crab and shrimp. Interday recoveries of CAP, CAP-B, and CAP-OH in muscle fortified ( n = 9) at levels of 0.15 to 0.60 ng/g ranged from 95 to 127% and 101 to 119% for crab and shrimp, respectively, with repeatability ranging from 4 to 19%. The limit of detection for CAP and metabolites in crab and shrimp ranged from 0.05 to 0.11 ng/g. We also monitored the depletion of CAP, CAP-B, and CAP-OH in crab following waterborne exposures. To our knowledge, we present the first CAP depletion and metabolite study following waterborne exposure in crabs, with the aim of identifying alternative marker residues.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,ß-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,ß-unsaturated carbonyl groups and can be expected to behave in a similar manner.

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.

Cytotoxic responses in BC3H1 myoblast cell lines exposed to 1-desulfoyessotoxin.

1-Desulfoyessotoxin (1-dsYTX) is a desulfated polyether compound belonging to the yessotoxin group of marine toxins. This analogue has been detected in mussels. There are so far no reports on the mechanisms of action of 1-dsYTX in in vitro cell systems. This work evaluates cytotoxic responses in BC3H1 cells exposed to 100 nM 1-dsYTX. The toxicity of 1-dsYTX seems to be similar to that of yessotoxin (YTX). 1-Desulfoyessotoxin induced morphological and biochemical traits typical of a non-apoptotic form of cell death resembling paraptosis. Treated BC3H1 cells showed extensive cytoplasmic vacuolation, enlargement of mitochondria and endoplasmic reticulum and lack of DNA fragmentation. Western blotting analysis revealed phosphorylation of the protein kinase p38 and involvement of the heat shock protein Hsp70. This activation suggests involvement of different signalling pathways for programmed cell death.

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation.

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography-mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.

Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4).

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.

Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites.

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.

Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2.

Marine biotoxins from microalgae can accumulate in shellfish and lead to poisoning of human consumers as well as fish, marine mammals and sea birds. Toxicological assessment of the toxins and development of analytical methods require large amounts of high-purity toxins and their metabolites. Although these toxins can be obtained in limited amounts from contaminated shellfish or from microalgal cultures, difficulties arise when the toxin-producing microalga is difficult to culture or its identity is not known. To circumvent this problem, we have developed a large-scale method for solid-phase extraction (SPE) of lipophilic biotoxins from natural microalgal blooms in seawater. To enhance subsequent purification of toxins adsorbed on the column, we included a filtration step to release the toxins from the cells while removing insoluble compounds and cellular debris. The efficacy of the method was illustrated by extraction and purification of okadaic acid and dinophysistoxin-2 from a high-density Dinophysis acuta bloom in Spain and from a mixed bloom containing low densities of D. acuta in Norway. Isolation of the toxins adsorbed on the SPE column was simple and efficient, and results obtained so far indicate that the method is potentially applicable to a wide range of microalgal toxins such as azaspiracids, pectenotoxins, spirolides and microcystins. The method should also be useful for harvesting toxins from large-scale microalgal cultures, and for bioprospecting for and isolation of bioactive natural products from marine and freshwater environments.

Ovine metabolism of lithogenic sapogenins. Synthesis of [2,2,4,4-(2)h(4)]sarsasapogenone, [2,2,4,4-(2)h(4)]sarsasapogenin, and [2,2,4,4-(2)h(4)]episarsasapogenin and evaluation of deuterium retention in a sheep-dosing trial.

The suitability of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), [2,2,4,4-(2)H(4)]sarsasapogenin (2b), and [2,2,4,4-(2)H(4)]episarsasapogenin (3b) as isotopically labeled dosing substrates to determine the levels of free and conjugated sapogenins present in feces from sheep grazing saponin-containing plants implicated in the development of ovine heptagenous photosentization diseases was investigated. A 1:4 mixture of [2,2,4,4-(2)H(4)]sarsasapogenin (2b) and [2,2,4,4-(2)H(4)]episarsasapogenin (3b), obtained by reduction of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), was found to retain 94% of incorporated deuterium, when dosed to one sheep. The recovery of the dosed mixture of genins 2b and 3b was calculated to be 85%. Considerable loss of deuterium and a lower recovery of genin material were observed when [2,2,4,4-(2)H(4)]sarsasapogenone (1b) was dosed.