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.

Detection of griseofulvin in a marine strain of Penicillium waksmanii by ion trap mass spectrometry.

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.

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.

Bioaccumulation of mycotoxins by shellfish: contamination of mussels by metabolites of a Trichoderma koningii strain isolated in the marine environment.

To determine whether toxic metabolites produced by fungi could cause shellfish toxicities, mussels were contaminated in laboratory conditions by sterile filtrates of a liquid culture of a strain of the fungus Trichoderma koningii previously isolated from a shellfish, the cockle (Cerastoderma edule). Mussels were kept in aerated natural seawater and fed with a culture of the microalga Isochrysis galbana, to which a filtrate of liquid fungal culture was added. Mussels were exposed to contamination for 7 days at 16 or 20 degrees C and extractions were then performed and their activity tested on blowfly larvae. The same toxicity was found in the fungal filtrate and the shellfish, indicating bioaccumulation. The digestive gland was the most toxic part of the mussel, confirming contamination by filtration. Treated mussels produced a mucus which appeared to be a means of eliminating toxic metabolites.

Combined use of analytical high-performance liquid chromatography and cell morphology transformation assay to detect new protein phosphatase inhibitors of okadaic acid type.

The search for new protein phosphatase inhibitors in shellfish contaminated by toxin-producing dinoflagellates generally relies on preliminary separation techniques followed by biological tests. To detect such substances without purifying them initially, we developed an approach based on a correlation of the results of two different analytical techniques applied to toxic extracts: high-performance liquid chromatography after derivation of the toxins and the cell morphology transformation assay on KB cells. Application of this protocol to stored frozen mussels showed a decrease in okadaic acid concentration during storage, with formation of degradation derivatives, some of which possessed notable protein phosphatase inhibition activity.

Specificity of the test based on modification of cell morphology for detection of lipophilic inhibitors of protein phosphatases.

The test based on morphological changes in KB cells was assayed with different toxins. Only lipophilic inhibitors of protein phosphatases, such as okadaic acid or calyculin A, induced visible changes in cell morphology. The activity of contaminated mussel extracts on KB cells was evaluated comparatively by direct interpretation of morphological changes and by a colorimetric method estimating the number of viable cells after staining. The latter technique revealed interferences (not detected by the former) with mussel cytotoxins. These results show that the technique, based on determination of the minimal active concentration of toxic extracts inducing morphological changes, is more specific, faster and preferable to the determination of IC50 for the detection of protein phosphatase inhibitors in shellfish.

Antiinflammatory mechanism of cordiachromene A action.

The mechanism of antiinflammatory action of cordiachromene A, isolated from the chloromethylenic extract of the ascidian Aplidium antillense or chemically synthesized, was studied using different in vivo and in vitro inhibition tests on enzymes of the cyclooxygenase cascade. Cordiachromene A inhibits prostacyclin synthesis and arachidonic acid metabolism but not phospholipase A2 and peroxidase. The mechanism of action, already known to be stereospecific, operates by cyclooxygenase inhibition.

Studies on the detection of okadaic acid in mussels: preliminary comparison of bioassays.

Diarrheic toxins, especially okadaic acid, are detected nearly every year in mussels on French coasts. The monitoring network determines the toxicity of these shellfish by using a mouse test now considered unsatisfactory from an ethical point of view. Two alternative methods have been investigated: the daphnia test, for which there is a standardized method used routinely in ecotoxicology, and a cytotoxicity test on the KB cell line developed for this study. Using the same samples, the results of these two tests were compared with those obtained by chemical analysis (HPLC okadaic acid assay) or the mouse test. Linear regression studies showed that results for the two bioassays were well correlated with those for HPLC or the mouse test.

In vivo study of vinorelbine associations with cisplatin, 5-fluorouracil or actinomycin D relative to a non-small-cell lung carcinoma (NSCLCN6-L16).

Vinorelbine (NavelbineR) is a new antitumor agent chemically related to the Vinca alkaloid group, but differentiated by its in vivo activity relative to non-small-cell lung carcinoma (NSCLC). Studies in the NSCLC-bearing nude mouse of vinorelbine associations with drugs currently used clinically in phase III (cisplatin, 5-fluorouracil and actinomycin D) showed that such associations are ineffective or even of negative value and that it would be preferable to use vinorelbine alone in a single dose at the maximum tolerated concentration.

Short-time cytotoxicity of mussel extracts: a new bioassay for okadaic acid detection.

Okadaic acid (OA), the main toxin responsible for diarrhoeic shellfish poisoning (DSP) has high cytotoxicity for KB cell cultures (apparent after 3 hr of contact), facilitating rapid detection in contaminated mussels. We developed a method to determine the minimal active concentration (MAC) based on direct microscopic study of toxin-induced changes in cell morphology. A high correlation was found between the MAC of tested extracts and corresponding OA concentrations in mussel hepatopancreas as measured by high performance liquid chromatography. This technique is rapid and reproducible and does not require the use of living animals.

Marine bioactive compounds: stereospecific anti-inflammatory activity of natural and synthetic cordiachromene A.

A new synthesis is proposed for cordiachromene A (CCA), a bioactive component of the ascidian Aplidium antillense Gravier, using a method producing a racemic mixture. The anti-inflammatory activities of a natural extract and a chemically synthetic form of CCA were assessed in vivo by carrageenan-induced rat-paw edema. The activity of synthetic CCA was confirmed by a test on kaolin-induced granuloma in the rat. Strong activities were measured for both CCA, but comparison of results of the first test suggests that only the natural optically active isomer has an anti-inflammatory effect. CCA is similar to indomethacin in its effect on carrageenan-induced rat-paw edema and ten times as active as phenylbutazone.