mtt-formazan and Diarrhea

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin-2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1-100 nM). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC₅₀ of 27 nM. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2.

Topics: Animals; Bivalvia; Cell Survival; CHO Cells; Chromatography, Liquid; Cricetinae; Cricetulus; Diarrhea; Dose-Response Relationship, Drug; Esters; Food Contamination; Formazans; Humans; Hydrogen-Ion Concentration; Hydrolysis; Inhibitory Concentration 50; Marine Toxins; Mass Spectrometry; Molecular Structure; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Tetrazolium Salts; Tissue Extracts

Tissue culture cells' metabolism and viability are measured by the mitochondrial reduction rate of a yellow tetrazolium salt (MTT) to blue formazan crystals in the MTT-bioassay. Thus the MTT-bioassay is a standardizable and reproducible bioassay for measuring cytotoxicity or cytostimulation. It is shown that the MTT-bioassay is also very suitable for determining bacterial cytotoxins using Escherichia coli's Shiga-like toxins as example. 177 strains of E. coli, isolated from carcasses and organs of cattle, are classified biochemically and tested for cytotoxin production by means of the MTT-bioassay. One of these strains is recognized as producer of Shiga-like toxin 2. 4 Enterohemolysin-producing strains of E. coli are cultivated from a feces sample of a diarrhoeic nubian ibex and identified as Shiga-like toxin 1 producers by help of the MTT-bioassay.

Topics: Animals; Bacterial Toxins; Biological Assay; Cattle; Cell Survival; Cells, Cultured; Cytotoxins; Diarrhea; Escherichia coli; Feces; Formazans; Meat; Ruminants; Shiga Toxin 2; Tetrazolium Salts