title: Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.
abstract: Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.
authors.affiliation: a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.
authors.forename: Ambbar
authors.initials: A
authors.lastname: Aballay-Gonzalez
authors.affiliation: a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.
authors.forename: Viviana
authors.initials: V
authors.lastname: Ulloa
authors.affiliation: a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.|b Sur-Austral COPAS Program , University of Concepcion , Concepción , Chile.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Rivera
authors.affiliation: c Natural Products Chemistry Laboratory, Botanic Department, Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.
authors.forename: Víctor
authors.initials: V
authors.lastname: Hernández
authors.affiliation: a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.
authors.forename: Macarena
authors.initials: M
authors.lastname: Silva
authors.affiliation: d Axon Guidance Laboratory, Department of Cell Biology, Faculty of Biological Sciences , University of Concepcion , Concepción , Chile.
authors.forename: Teresa
authors.initials: T
authors.lastname: Caprile
authors.affiliation: e Laboratory of Marine Toxins and Mycotoxins, Food Chemistry Section, Environmental Health Department , Institute of Public Health Chile , Ñuñoa , Chile.
authors.forename: Lorena
authors.initials: L
authors.lastname: Delgado-Rivera
authors.affiliation: a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and Oceanographic Sciences , University of Concepcion , Concepción , Chile.|b Sur-Austral COPAS Program , University of Concepcion , Concepción , Chile.
authors.forename: Allisson
authors.initials: A
authors.lastname: Astuya
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D049872:Bivalvia / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002677:Chile; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005506:Food Contamination / Q000517:prevention & control; D005513:Food Inspection / Q000379:methods*; D057166:High-Throughput Screening Assays; D059625:Liquid-Liquid Extraction; D008387:Marine Toxins / Q000032:analysis* / Q000096:biosynthesis / Q000633:toxicity; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D015203:Reproducibility of Results; D012530:Saxitoxin / Q000032:analysis / Q000096:biosynthesis / Q000633:toxicity; D012758:Shellfish / Q000009:adverse effects / Q000032:analysis*; D057096:Shellfish Poisoning / Q000209:etiology / Q000473:pathology / Q000517:prevention & control; D013045:Species Specificity; D014020:Tissue Extracts / Q000032:analysis / Q000302:isolation & purification / Q000633:toxicity
chemical_list: D008387:Marine Toxins; D014020:Tissue Extracts; D012530:Saxitoxin
keywords: Cell-based assay; matrix effect; paralytic shellfish toxins