Yves-François Pouchus

Toxigenic saprophytic fungi in marine shellfish farming areas.

Toxigenic saprophytic fungi were isolated from samples of shellfish, sediment and seawater obtained from marine shellfish farming areas. The 456 strains identified included 12 different genera, with a clear predominance (68%) of Penicillium, Aspergillus, Trichoderma and Cladosporium. To assess the risk of poisoning due to the presence of these fungi in shellfish farming areas, the strains were cultured in liquid medium, filtered, and tested on larvae of Artemia salina, a small crustacean highly sensitive to mycotoxins. Thirty-five point five percent of the strains proved active with this test. This study confirms the existence of fungi in shellfish farming areas, as suggested by our earlier work showing that filter-feeding shellfish accumulate toxic metabolites of fungal origin. The presence of fungi in the marine environment represents a real risk of poisoning through the consumption of contaminated shellfish.

Toxicity of French strains of the dinoflagellate Prorocentrum minimum experimental and natural contaminations of mussels

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.

Cytotoxicity and mycotoxin production of shellfish-derived Penicillium spp., a risk for shellfish consumers

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.

Ion trap MSn 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.

Cytotoxicity, Fractionation and Dereplication of Extracts of the Dinoflagellate Vulcanodinium rugosum, a Producer of Pinnatoxin G

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.

Marine Fungal Substances

Abstract 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.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

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.

Métabolites cytotoxiques d'une souche d'Aspergillus fumigatus isolée de zones aquacoles: toxicité, bioaccumulation, isolement

lineaire, effectuee entre ces valeurs et les CMI obtenues par Ia methode de reference, a montre que Ia CMI pour Ia methode utilisant Ia CMF pouvait etre definie comme Ia CI80. La deuxieme partie de notre etude a ete consacree a lespece c. lusitaniae. Des reSIStances a lamphotericine 8 ont ete frequemment decrites chez cette espece, mais Ia detection de ces souches par Ia methode NCCLS est aleatoire, meme en utilisant le milieu antibiotique n° 3 recommande par certains auteurs. Nous avons done choisi une seconde methode de reference : le Etest (methode par diffusion en gelose). 59 souches de C. lusitaniae, dont six considerees comme resistantes a lamphotericine 8 sur Ia base de donnees obtenues in vivo et/ou in vitro, ont ete testees avec les trois methodes. Les coefficients de correlation CMF/NCCLS et CMF/Etest sont respectivement de 0,63 et de 0,93 (P < 0,01 ; n = 59). Pour les souches resistantes Ia methode utilisant Ia CMF et le Etest donnent des CMI plus elevees que les CMI obtenues avec Ia methode NCCLS. Celles-ci sont souvent proches du seuil de sensibilite et done delicates a interpreter. La methode CMF/DiOC5 (3) permet de detecter les souches resistantes a lamphotericine 8 de fac;on extremement rapide. Elle serait particulierement utile au choix du traitement et au suvi therapeutique dans les infections a Candida, en particulier a C. /usitaniae.