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.

中文翻译：

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鱼肝亚细胞金属分离方法的优化：均质化，亚细胞分离和核材料的试验分离

水生生物中金属的亚细胞分隔反映了金属吸收后的内部行为，并可提供有关其潜在毒性的重要信息。通常，用于确定水生生物中亚细胞金属分配的分馏方案包括机械均质化组织，通过差速离心和热变性步骤将亚细胞成分分离为级分，并确定与每个级分相关的金属量。然而，亚细胞成分的准确分离是具有挑战性的，并且很少评估操作定义的亚细胞级分的性质和纯度。但是，在没有这种类型的验证的情况下，亚细胞金属分离结果的解释可能会受到影响。本研究的目的是针对田间采集的鱼的肝脏调整亚细胞分离方案，并使用分数特异性酶标记物测试调整后的方案。总体而言，我们的结果表明，在研究新物种或器官时，需要优化分级分离程序。在这项研究的过程中，根据获得的酶促结果修改了某些级分的分类，以产生更可靠的亚细胞级分分配方案。此外，进行了旨在从细胞碎片中分离核材料的试验，使用DNA作为核材料的标记。经测试的方案未能分离出细胞核，结果表明细胞核可能被细胞膜破坏而被困住。