[Cyanobacteria and their toxic potential in dam water content in Northern Tunisia]. / Les cyanobactéries et leurs potentialités toxiques dans les retenues des barrages du Nord de la Tunisie.

In order to get data about toxic cyanobacteria and their potential sanitary risk in 12 waterbodies situated in the north of Tunisia, some taxonomic, ecological and toxicological studies were undertaken since 2001. This paper provides the first screening of the potential toxic species of cyanobacteria as well as their geographical distribution. The microscopic examination of the phytoplankton samples show 42 species of cyanobacteria; 9 are frequently quoted by the literature as being potentially toxic. Among the inventoried cyanobacteria genera there are Pseudanabaena, Planktothrix Phormidium, Lyngbya, Microcystis,... Oscillatoria constitutes the most widespread one. The content of total microcystin (MCYST) was determined by protein phosphatase inhibition assays (PP2A). The total microcystin, detected in dissolved and particulate fractions in all the examined samples is generally low and varies between 2 and 7455 ng/l microcystin-LR equivalent per liter. The highest MCYST concentration is observed in autumn and generally in particulate MCYST concentrations.

Optimization of the (32)P-Postlabeling/Thin Layer Chromatography Assay ((32)P-TLC) for In Vitro Detection of 8-Oxo-Deoxyguanosine as a Biomarker of Oxidative DNA Damage.

During the last years, much attention was focused on the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) as a marker of oxidative DNA damage. Among various analytical techniques, the (32)P-postlabeling assay has been applied in determination of 8-oxo-dG. However, artefactual DNA oxidation could take place during the work-up procedures leading to over-estimate the level of 8-oxo-dG. In the present study, we optimized the (32)P-postlabelling assay with thin layer chromatography to measure 8-oxo-dG in standard samples of 8-oxo-dG, calf thymus DNA and primary cultured rat hepatocytes. The background levels of 8-oxo-dG in calf thymus DNA and in primary cultured rat hepatocytes were lesser than those determined by the previously described (32)P-postlabeling procedures and were in the range of those determined by chromatography methods (GC-MS, HPLC-MS).

A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration.

Protein phosphatase inhibition assays currently used for the detection of cyanobacterial peptide hepatotoxins in drinking water require an enrichment step using C18 cartridges to achieve lower the detection limit. This paper describes a colorimetric and fluorometric protein phosphatase inhibition method for the direct detection of microcystin-LR (MCYST-LR) in drinking water without complex clean-up steps and preconcentration procedures. In this assay three different substrates, p-nitrophenyl phosphate (p-NPP) and two fluorogenic compounds, 4-methylumbelliferyl phosphate (MUP) and 6,8-difluoro-4-methylumbelliferyl phosphate DiFMUP), were tested. The detection limits of the assay are 0.25 and 0.1 microg/l using colorimetric and fluorometric methods, respectively. These levels are well below the provisional guideline value for MCYST-LR of 1 microg/l of drinking water. The detection limit of the fluorometric method is comparable to that of the classical ELISA test. Although both the latter tests allow the detection of MCYST-LR in drinking water directly without pretreatment, the protein phosphatase inhibition assay remain less expensive and therefore more attractive for use in the routine assessment of drinking water contamination by microcystins.

Morphological and toxicological variability of Prorocentrum lima clones isolated from four locations in the south-west Indian Ocean.

Eight clones of the toxic dinoflagellate Prorocentrum lima (Ehrenberg) Dodge from four sites (two clones per site) on the coral reef of La Réunion, Mayotte, Europa, and Mauritius Islands in the SW Indian Ocean were isolated and cultivated under the same conditions. Morphological features of each clone, including cell size and valve and marginal pore numbers, were examined by scanning electron microscopy. The toxic potential of each clone was determined by protein phosphatase type 2A (PP2A) inhibition test and fibroblast cell line FR3T3 bioassay. Scanning electron microscopy showed that variation in morphological features of clones within and between sites was minimal and not significant. However, equivalent okadaic acid content, determined by PP2A assay, was different within and between clones isolated from the four islands. Cytotoxicity bioassay with the FR3T3 cell line confirmed the variation on global toxic potential within and between the eight P. lima clones. This test also suggested the presence of other toxic compounds without PP2A inhibiting activity in crude extracts of some clones.

A new method for determination of maitotoxin by capillary zone electrophoresis with ultraviolet detection.

Capillary zone electrophoresis with UV detection was applied to the rapid and efficient separation of an underivatized phycotoxin, maitotoxin, associated with ciguateric fish poisoning. Highly sensitive detection was obtained by UV absorption at 195 nm. A detection limit of 50 pg of maitotoxin was achieved at this wavelength. Analysis involved using a fused silica capillary coated with a hydrophobic phase, polyvinylalcohols. Confirmation of the electrophoretic peak of maitotoxin was further evaluated by cytotoxicity on a mammalian fibroblastic cell line, BHK 21 C13.

Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection.

Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40-640 pg. OA was detected in mussels spiked with 10 ng/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima.

[Determination of phycotoxins in aquatic medium by capillary electrophoresis]. / Détermination de phycotoxines de milieux aquatiques par électrophorèse capillaire.

Marine phycotoxins present a major public health problem due to their ability to contaminate seafoods. Toxic phytoplankton is not limited, however, to the coastal areas in Europe. There are more and more problems in freshwater reservoirs, in lakes, and in ponds from which grazing animals get their water. Okadaic acid (OA), a polyether toxin from the marine dinoflagellate, Prorocentrum lima, and microcystins (MCYSTs), a potent hepatotoxic cyclic heptapeptides produced by many strains of cyanobacteria have been shown to be a powerful tumor promotor. Thus, considerable effort has been undertaken to find sensitive and easy detection techniques for the determination of phycotoxins from various matrices (microalgae, freshwater, mussels, fish, ...). Different biological and chemical methods have been developed to identify these toxins in shellfish and fish. The mouse bioassay method has been widely used for the detection of toxins such as maitotoxin and ciguatoxin, which are not easily identified by chemical procedures. In this report we describe a capillary electrophoretic method that enables us to detect okadaic acid, microcystins (MCYST-YR, MCYST-LR, MCYST-RR) and maitotoxin in picogram range.

Detection of cyanobacterial toxins (microcystins) in cell extracts by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method with UV detection is described for the rapid and efficient separation of three microcystins: microcystin-YR (MCYST-YR), microcystin-LR (MCYST-LR) and microcystin-RR (MCYST-RR). A detection limit of 7.5 pg for each toxin was achieved. The UV intensities of these toxins measured at 200 nm showed good linearity in the range 7.5-150 pg. The production of MCYST-LR in three cultured strains of cyanobacteria, namely Microcystis aeruginosa strain IP7806, Microcystis aeruginosa strain IP7813 and Oscillatoria agardhii strain IP7805, was evaluated.

Zarzissine, a new cytotoxic guanidine alkaloid from the Mediterranean sponge Anchinoe paupertas.

From a CH2Cl2 extract of the Mediterranean sponge Anchinoe paupertas were isolated and characterized zarzissine [2], a new 4,5-guanidino-pyridazine compound and the known p-hydroxybenzaldehyde [1]. The structure of zarzissine [2] was elucidated by spectroscopic methods, including the application of a number of 2D nmr techniques. Biological activities of compounds 1 and 2 were also determined, with zarzissine exhibiting cytotoxicity against three human and murine tumor cell lines.

Isolation, structural identification and biological activity of two metabolites produced by Penicillium olsonii Bainier and Sartory.

From the culture broth of a fungus, two metabolites have been isolated: bis(2-ethylhexyl)phthalate (DEHP) precedently isolated from Streptomyces sp. and 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) here reported as a natural compound in the (E)-s-cis configuration. The producing organism was identified as a strain of Penicillium olsonii. Culture growth and chemical identification are discussed in the present work.

Cytotoxic diterpenoids from the brown alga Dilophus ligulatus.

A new xenicane-type diterpene, dilopholide [5], and four known diterpenoids, acetoxycrenulide [1], acetylcoriacenone [2], and its epimer isoacetylcoriacenone [3], and hydroxyacetyldictyolal [4], have been isolated from the brown alga Dilophus ligulatus [syn. spiralis (Montagne) Hamel]. The structure of dilopholide [5] was elucidated by spectroscopic methods, including the concerted application of a number of 2D nmr techniques, including 1H-1H COSY, heteronuclear proton-carbon chemical shift correlation (HETCOSY), and long-range HETCOSY. The cytotoxic activity of compounds 1-5 has been examined against several types of mammalian cells: human nasopharynx carcinoma cells (KB), human lung carcinoma cells (NSCLC-N6), murine leukemia cells (P-388), and murine leukemia cells expressing the multi-drug-resistance gene, mdr (P-388/DOX).

Bioactive Diterpenoids Isolated from Dilophus ligulatus.

Nine diterpenoids, acetyldictyolal ( 1), epoxyoxodolabelladiene ( 2), dictyotalide B ( 3), neodictyolactono ( 4), pachylactone ( 5), the acetals 6A and 6B, isoacetoxycrenulatin ( 7), and dictyolactone ( 8) were isolated from the brown alga DILOPHUS LIGULATUS (Kütz.) Feldm. Their structures have been elucidated by comparison of mass, IR, (1)H- and (13)C-NMR spectra with reported literature data. Compounds 1, 2, 4, 7, and 8 exhibited antifungal activity. Cytotoxic activity against several types of mammalian cells (KB, P-388, P-388/DOX, and NSCLCN6-L16) in culture was examined for the 8 diterpenoids. Some of them showed stronger cyto-toxicity than mercaptopurine which was used as a positive control in this study.