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Two new unusual C25 steroid epimers, 24-O-methyl-24-epi-cyclocitrinol (1) and 24-O-methylcyclocitrinol (2), together with the known oxylipin α-dimorphecolic acid (3), were isolated from the extract of cultures of a Penicillium ubiquetum MMS330 strain sampled from the blue mussel Mytilus edulis. The structures of the new compounds were established based on 1D and 2D NMR spectral analyses and CP3 conformation analysis. LC–MS dereplication of other C25 steroids in culture extracts was also performed on the basis of their HRMS/MS fragmentation patterns, allowing to detect other analogues. Biological activities of compounds 1 and 2 were evaluated as a mixture on the two human cancer cell lines KB and MCF-7.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 μM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 μM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

The wide polarity range and highly polar compounds of two selected red seaweed, Grateloupia turuturu and Palmaria palmata were extracted using two different types of solvent, dichloromethane/methanol and methanol/water. Monthly in vitro antibacterial activities were studied using the microplate method against the marine bacteria Vibrio harveyi strain ORM4, known to infect abalone. Inhibition, slowdown and delay of Vibrio harveyi growth were investigated. Polar compounds of seaweed showed an activity against the abalone pathogen. The best activity was recorded from P. palmata collected in spring, with an inhibition of 7.9% of the bacterial growth. Preliminary 1H NMR profiles identified the differences between the extracts.

International audience; Vibrio harveyi is the main pathogen of the European abalone Haliotis tuberculata, and recently caused important mortalities at the production sites of this marine gastropod in France. In the present work, the monthly antibacterial activity of two red seaweed species from the French Atlantic coast, the native Palmaria palmata and the introduced Grateloupia turuturu, were investigated against the abalone pathogen Vibrio harveyi strain ORM4. Water-soluble extracts were screened using the microplate method. Grateloupia turuturu showed an antibacterial activity with a maximal growth inhibition in spring of around 16%. In contrast, Palmaria palmata was inactive, as further growth of the bacteria was observed. Preliminary one-dimensional proton nuclear magnetic-resonance (H-1 NMR) profiles identified the differences between the two water-soluble extracts.

In order to assess the putative toxigenic risk associated with the presence of fungal strains in shellfish-farming areas, Penicillium strains were isolated from bivalve molluscs and from the surrounding environment, and the influence of the sample origin on the cytotoxicity of the extracts was evaluated. Extracts obtained from shellfish-derived Penicillia exhibited higher cytotoxicity than the others. Ten of these strains were grown on various media including a medium based on mussel extract (Mytilus edulis), mussel flesh-based medium (MES), to study the influence of the mussel flesh on the production of cytotoxic compounds. The MES host-derived medium was created substituting the yeast extract of YES medium by an aqueous extract of mussel tissues, with other constituent identical to YES medium. When shellfish-derived strains of fungi were grown on MES medium, extracts were found to be more cytotoxic than on the YES medium for some of the strains. HPLC-UV/DAD-MS/MS dereplication of extracts from Penicillium marinum and P. restrictum strains grown on MES medium showed the enhancement of the production of some cytotoxic compounds. The mycotoxin patulin was detected in some P. antarcticum extracts, and its presence seemed to be related to their cytotoxicity. Thus, the enhancement of the toxicity of extracts obtained from shellfish-derived Penicillium strains grown on a host-derived medium, and the production of metabolites such as patulin suggests that a survey of mycotoxins in edible shellfish should be considered. Significance and Impact of the Study Penicillium strains isolated from bivalve molluscs produce extracts exhibiting a higher cytotoxicity than extracts from Penicillium strains isolated from the surrounding marine environment. The use of a mussel-based medium for cultures of some shellfish-derived strains enhances the cytotoxicity of extracts when compared with classical media. The production of cytotoxic compounds and of the mycotoxin patulin on such a host-derived medium highlights a potential health risk for shellfish consumers.

In order to enhance the knowledge of the putative toxinic risk linked to mycotoxin excretion in shellfish farming areas, the influence of seawater salinity was studied on 2 marine-derived Aspergillus fumigatus strains. This fungal species produces gliotoxin, an epipolythiodioxopiperazine immunosuppressive mycotoxin that can be accumulated in the meat of cultured blue mussel (Mytilus edulis), and could be responsible for disease when ingested. Two marine strains were grown in vitro both on a non-saline and a saline culture media and were compared with 13 terrestrial strains to observe the effects of seawater on fungal growth and gliotoxin excretion in the exudate produced. Daily measurement of the colony areas showed that the seawater salinity significantly reduced the rate of growth of all the strains. Marine and terrestrial strains appeared to be almost similar as regards the appearance, growth and gliotoxin excretion, but the marine strains exudation seemed to be less influenced by seawater salinity than the terrestrial strains. Seawater salinity, however, enhanced exudation and gliotoxin excretion by A. fumigatus, and thus seems to be an aggravating factor for the toxicity of this species in the marine environment.

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 [C13H14N2O4S2+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. D 2002 Elsevier Science B.V. All rights reserved.

International audience

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.

International audience

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of “fast-acting toxins”, its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL−1 and 0.19 µg mL−1 were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL−1). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

A marine strain of Penicillium waksmanii Zaleski was isolated from a sample of seawater from shellfish-farming area in the Loire estuary (France). The in vitro marine culture showed an important antifungal activity. Bioassay-guided fractionation was used to purify the crude extract. Dereplication by electrospray-ion trap/mass spectrometry (ESI-IT/MS) afforded the identification of the antifungal compound, after a semi-purification consisting of two stages. A comparison of the ionic composition between the active and the non-active fractions allowed the detection of a monocharged ion at m/z 353 containing a chlorine atom, which could be attributed to the antifungal griseofulvin [C17H17ClO6+H](+). Multi-stage fragmentation (MSn) confirmed the identity of the m/z 353 ion of the antifungal fraction as griseofulvin. It is the first description of griseofulvin production by a strain of P. waksmanii and the first chemical study of a strain of this species isolated from marine temperate cold water.

This review concerns substances produced by fungi isolated from the marine environment (seas, estuaries, brackish waters, salt marshes) which are capable of reproducing in a salt medium. Enzymatic activities and known primary or secondary metabolites are discussed in chemical, biochemical and biological terms. The potential industrial and pharmaceutical applications of these substances are considered as well as their potential toxic incidence for aquaculture and public health.

Several clones of Prorocentrum minimum were isolated from various French Mediterranean and English Channel sites. Mouse tests performed using methanolic extracts from cultures revealed a neurotoxic activity in four clones. The water-soluble toxin detected worked rapidly, killing mice within a few minutes at high doses. Toxin production in cultures was observed during the phase of decline and appeared to be weak or nil during the growth phase. Toxin production was stimulated by associated bacteria in the culture, but a clone rendered axenic remained able to produce toxins. The potential risks of human poisoning from consumption of shellfish harvested during or after toxic blooms of P.minimum are discussed.

La contribution fournit tout d'abord un cadre général pour analyser l'ensemble des dommages d'une marée noire. Il est ensuite appliqué au cas de l'Erika, ce qui conduit à discuter les différentes méthodes d'évaluation selon que ces dommages relèvent ou non de la sphère marchande. La question très délicate de la dynamique des dommages est ensuite abordée. Elle débouche sur la détermination de l'équilibre optimal entre efforts de nettoyage et restauration naturelle des écosystèmes. Enfin cette contribution se focalise sur l'évaluation du dommage écologique en présentant différentes approches possibles. Leurs fondements économiques et leur acceptabilité par la société sont discutés en détail.