Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins

Abstract

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Towards tailored assays for cell-based approaches to toxicity testing

“…Past eC legislation required the use of mouse bioassays for the detection of some lipophilic toxins in contaminated shellfish to protect consumer health (EU, 2005). The lack of specificity of these bioassays and the ethical concerns linked with the extensive use of animals for analytical purposes have been the major driver for the development of cell-based assays (Rossini, 2005;Hess et al, 2006;Schindler et al, 2006Schindler et al, , 2009. the pyrogen test in rabbits, in place for more than 40 years, was largely replaced by a test with correlative tors for estrogens in their target tissues (Jensen and Jacobson, 1962), paving the way for the clarification of the molecular mechanism of action of a large superfamily of trans-acting transcription factors and their ligands.…”

“…efforts in this area are supported by the recognition that naturally contaminated materials often include many distinct analogues (MacKenzie et al, 2002;Aasen et al, 2005;Dell'Aversano et al, 2008;Rehmann et al, 2008) of varying potency, pinpointing the need to quantify the full series of compounds with toxicological relevance. A second line of intervention recognizes that contamination of seafood by microalgal toxins has been evolving over the years, and novel or atypical contaminations have been detected (Moore and Scheuer, 1971;Hu et al, 1995;Deeds and Schwartz, 2010;ledreux et al, 2012). thus, cell-based assays, which have the potential to detect toxicologically relevant components of both characterized toxins and novel agents, have been sought.…”

“…A second case refers to unexplained toxicity detected in some shellfish samples from the Arcachon Bay, France since 2005 (Butler, 2006;ledreux et al, 2012). the issue was raised when the testing of shellfish extracts in the mouse bioassay gave positive results, i.e., mouse death within 24 hours of extract administration, but none of the already characterized classes of toxins was detected in those extracts, indicating that those materials most likely contained substances of atypical toxicity (ledreux et al, 2012).…”

Smartphone-based portable biosensing system using cell viability biosensor for okadaic acid detection

“…The preparation of shellfish extracts are carried out according to the protocol offered by Ledreux et al [39]. Meanwhile, the detection system starts to monitor the cell viability curves.…”

The CHROMEVALOA Database: A Resource for the Evaluation of Okadaic Acid Contamination in the Marine Environment Based on the Chromatin-Associated Transcriptome of the Mussel Mytilus galloprovincialis

“…Consequently, a very important effort has been devoted to OA biomonitoring in estuarine areas by using sentinel organisms, most notably bivalve molluscs [9,11]. These studies have progressively transitioned from traditional biomonitoring methods (based on physicochemical and physiological parameters) to more sensitive molecular probes [12,13,14,15]. Given the role of chromosomal proteins in the modulation of chromatin structure and DNA metabolism (including DNA repair) [16], the study of chromatin-associated biomarkers constitutes a powerful and sensitive approach for the evaluation of genotoxicity.…”