An integrated strategy for marine toxins of cell based bioassays and analytical tools to ensure safe seafood.

摘要

Marine toxins are produced by algae that can accumulate in seafood. Consumption of contaminated products may lead to intoxication such as memory loss, paralysis, diarrhoea or in severe cases death. In order to protect consumers, methods have been developed and put into practice. Within Europe several analytical chemical alternatives for the widely applied mouse bioassay (MBA) are implemented within legislation. Current trends involve rapid LC-MS/MS or targeted screening with LC-hrMS for regulated and unregulated toxins. Despite the current alternatives, the unethical and unreliable test with mice is still being used, as it is capable to detect possible unknown toxins or new risks for consumers. Animal-free in vitro cell based effect assays offer the same opportunity as the mouse bioassay, i.e. to detect unknown toxins and new risks. A neuroblastoma cell assay was optimized at our laboratory for the detection of various toxins in seafood samples. This easy and relatively cheap in vitro cell assay is used as a first screening to differentiate between blank and suspect samples. Only suspect samples are further investigated with analytical chemical tools. For the regulated toxins such targeted LC-MS/MS methods have been established, and can be applied in monitoring programs (e.g. if countries miss laboratories with cell culture facilities). However, the combination of effect screening with LC-MS/MS confirmation is very powerful. If unexplained results are obtained, i.e. suspect samples in the cell assay that cannot be confirmed by LC-MS/MS, a second stage of cell based assays is applied which are more directed to the specific mode of action. For diarrheic marine toxins, these techniques are based on gene expression profiles in human Caco-2 cells, and a multielectrode array with neural cells was set-up for neurotoxic compounds. If suspect samples also show a response in these cell assays, and thus suggest presence of an unknown, analytical tools (LC-hrMS) in combination with library searching and/or statistical- and structure elucidation tools are used for identification. This strategy has been applied to over 100 mainly contaminated samples. The majority of the suspect samples could be confirmed by LC-MS/MS and contained clearly elevated levels of marine toxins. Till now only one false negative was observed. The assay also flagged some as suspect that could not be explained by the LC-MS/MS methods. These samples are currently under investigation. Results indicate that the complete proposed strategy, effect based assays combined with novel analytical tools, will be able to bypass the MBA.