Further Insights into Brevetoxin Metabolism by de Novo Radiolabeling,

International audienceThe toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-14C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors

Semisynthesis of S-Desoxybrevetoxin-B2 and Brevetoxin-B2, and Assessment of Their Acute Toxicities

animals and people consuming seafood. Brevetoxin-B2 (5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B (1). S-Desoxybrevetoxin-B2 (4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B (1) into S-desoxybrevetoxin-B2 (4), and a simple method for converting 4 into brevetoxin-B2 (5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD50 values for 4, 5, and dihydrobrevetoxin-B (6) by ip injection in mice were 211, 400, and 250 µg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards

Ciguatoxin and ciguatera

Ciguatera is a disease caused by the consumption of fishes from tropical and subtropical waters that have accumulated lipophilic sodium channel activator toxins known as ciguatoxins (CTXs) to levels sufficient to cause human poisoning. Consumption of these temperature-stable, orally active polycyclic ether compounds leads to the activation of neuronal sodium channels that produces a range of characteristic neurological, gastrointestinal, and cardiovascular signs and symptoms that clinically define the illness. Ciguatera is estimated to affect similar to 50,000 people annually worldwide after accounting for misdiagnosis and non-reporting. Currently there are no clinically validated treatments and no routine tests that can cost-effectively detect ciguatoxins prior to consumption, with government bans on capture or personal avoidance of risk fish species providing the only effective means to mitigate the risk currently. A recently developed rapid extraction method for ciguatoxins coupled to LC/MS/MS detection has potential for surveillance and confirmation of ciguatera outbreaks