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Optimization of a subcellular metal fractionation method for fish liver: Homogenization, subcellular separation, and trial isolation of nuclear materials.

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Urien, Nastassia; Caron, Antoine; Lerquet, Marc; Couture, Patrice; Campbell, Peter G. C. (9999). Optimization of a subcellular metal fractionation method for fish liver: Homogenization, subcellular separation, and trial isolation of nuclear materials. Limnology and Oceanography: Methods . DOI: 10.1002/lom3.10371. (Sous Presse)

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Résumé

The subcellular compartmentalization of metals within aquatic organisms reflects their internal behavior after metal uptake and can provide important information about their potential toxicity. Commonly, the fractionation protocol used to determine subcellular metal partitioning in aquatic organisms consists of mechanically homogenizing the tissue, separating subcellular components into fractions by differential centrifugation and heat‐denaturation steps, and determining the amount of metals associated with each fraction. However, the accurate separation of subcellular cell components is challenging and the nature and purity of the operationally defined subcellular fractions are rarely assessed. In the absence of this type of validation, however, the interpretation of subcellular metal fractionation results could be compromised. The aim of the present study was to adjust a subcellular fractionation protocol for the liver of field‐collected fish and to test the adjusted protocol using fraction‐specific enzyme markers. Overall, our results illustrate the need to optimize fractionation procedures when studying a new species or organ. In the course of this study, the categorization of some fractions was revised in accordance with the enzymatic results obtained, in order to yield a more credible subcellular fraction distribution scheme. In addition, trial assays aimed at isolating nuclear materials from the cellular debris were conducted, using DNA as a marker for nuclear material. Tested protocols failed to isolate the nuclei and results suggested that nuclei were probably trapped by disrupted cellular membranes. Recommendations on how to improve future subcellular fractionation studies on freshwater fish are discussed.

Type de document: Article
Mots-clés libres: poisson; limnologie;
Centre: Centre Eau Terre Environnement
Date de dépôt: 25 sept. 2020 21:08
Dernière modification: 25 sept. 2020 21:08
URI: http://espace.inrs.ca/id/eprint/10343

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