Accumulation of gliotoxin, a cytotoxic mycotoxin from Aspergillus fumigatus, in blue mussel (Mytilus edulis).

A strain of Aspergillus fumigatus has been isolated from sediments of a mussel bed. When cultured in hyper saline conditions (with sea-water), it produces a cytotoxic and immunosuppressive toxin, gliotoxin, which is excreted in an exudate. In order to know if this toxin could represent a risk for shellfish consumers, an experiment of bioaccumulation of gliotoxin in mussel has been carried out. After 6 days of contamination, toxin was accumulated in the meat of the mussels, at a level up to 2.9 microg/mg of extract weight, with a mode of contamination different to the classical digestive process described for a majority of marine toxins, but similar to the contamination mode of domoic acid.

Ion trap MS(n) for identification of gliotoxin as the cytotoxic factor of a marine strain of Aspergillus fumigatus Fresenius.

When cultured in a marine solid medium, a strain of Aspergillus fumigatus (Fresenius) isolated from a shellfish-farming area in the Loire estuary (France) produced a highly cytotoxic exudate. To identify the origin of this activity, a cytotoxicity test on KB cells was used to monitor the purification of the exudate, together with electrospray/ion trap/mass spectrometry (ESI/IT/MS(n)) to detect and identify the toxic compound. After three purification stages, a comparison of fullscan analyses of the last six fractions showed that a monocharged compound at m/z 349 was present only in the active fraction, corresponding to the sodium adduct of gliotoxin [C(13)H(14)N(2)O(4)S(2)+Na](+). Isotopic distribution determination showed that the m/z 349 product possessed two sulphur atoms and multi-stage fragmentation confirmed the hypothesis. MS/MS analysis exhibited the characteristic gliotoxin loss of the disulphide intracyclic bridge. MS(3) analysis revealed four main ions and confirmed the identity of the m/z 349 ion. This study points out that the combined use of a KB cells bioassay and ESI/IT/MS(n) allows a fast and very specific detection and elucidation of unidentified cytotoxic products in natural samples. This method does not require total purification, and it allowed us to report the first detection of gliotoxin production in marine conditions.

Combined use of LC/MS and a biological test for rapid identification of marine mycotoxins produced by Trichoderma koningii.

Trichoderma koningii Oudemans, a strain isolated from a shellfish farming area, was selected for its high frequency in samples and its ability to produce metabolites when cultured in natural seawater. Combined use of LC/MS and a biological test on blowfly larvae allowed the characterization of four compounds after purification in only two steps (VLC and HPLC). ESI/MS, a powerful tool for rapid identification and sequence determination of peptides, confirmed that these compounds were peptide, alpha-aminoisobutyric acid and amino alcohol (peptaibols), the usual metabolites of Trichoderma.