Enzymatic validation of species‐specific protocols for metal subcellular fractionation in freshwater animals

Abstract

The use of fractionation protocols to determine metal subcellular distribution in aquatic organisms has gained much interest over the last 15 yr, however, accurate separations among the different components of cells are challenging. Subcellular fractions separated with such an approach are operationally defined and a potentially significant difference can exist between anticipated and resulting fractions. This study customizes and validates subcellular partitioning protocols, for three different freshwater organisms representing a diversity of challenges for subcellular fractionation: Daphnia magna, Chironomus riparius, and liver of Oncorhynchus mykiss. Several protocols involving different homogenization methods, centrifugation speeds, or conservation conditions were tested, and their efficiencies were assessed using enzymatic biomarker assays. Our work allowed us to identify critical steps to improve separations. First, for D. magna, a crustacean with a reinforced chitinous exoskeleton, the use of a strong homogenization method using a sonicator is necessary. Second, for both invertebrates, we observed the leaking of the mitochondrial matrix during cell fractionation, regardless of the homogenization strength and conservation conditions. Therefore, we propose that the mitochondria fraction should be referred to as the mitochondrial membrane fraction, and the cytosol fraction should be identified as the cytosol and mitochondrial matrix fraction. Third, the presence of a lipid-rich layer during O. mykiss liver fractionation may lead to an overlap between mitochondria and cytosol and must be considered in the protocol development. Finally, lysosomes should not be pooled with the microsomes fraction without prior validation. Overall, this study provides a benchmark for future methodological studies on similar taxa.

Toxicity and Subcellular Fractionation of Yttrium in Three Freshwater Organisms: Daphnia magna, Chironomus riparius, and Oncorhynchus mykiss

“…The mean (±SD) recovery values of Y were 90 ± 11% ( n = 12) for D. magna , 70.0 ± 0.1% ( n = 12) for C. riparius, and 87 ± 22% ( n = 9) for O. mykiss . Note that it was assumed by Cardon et al 16 that around 25% of C. riparius samples were probably not efficiently homogenized with the subcellular fractionation procedure performed on this species. It could explain why we obtained lower recovery values for C. riparius .…”

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

“…This overlap could explain the 30% APHO activity found in the mitochondria in protocol # III, where a centrifugation force of 25,000 × g was used to pellet mitochondria. In a recent study of rainbow trout, Cardon et al (2018) also suggested that lysosomes and mitochondria could be combined in the same fraction.…”

“…Complete subcellular fractionation protocols. It has been recently observed in the literature that a subcellular protocol that works for on species or organ does not necessarily fit for another (Rosabal et al 2014;Cardon et al 2018;Urien et al 2018a;Khadra et al 2019). Consequently, in the present study we chose to test different tools (Potter-Elvehjem tissue grinder, cordless motor mixer, sonication, etc.)…”

“…These results indicate that these organelles were damaged, but as the apparent degree of damage remained approximately constant for all protocols, even for protocol # I, considered to be the gentlest, it is difficult to attribute this damage to the homogenization method used. After their collection, fish tissues were stored at −80 C. In a recent study, Cardon et al (2018) reported that using fresh samples of Chironomus and rainbow trout decreased mitochondria leakage compared to frozen samples (e.g., 70% of CS activity was found in the cytosol in frozen Chironomus vs. 25% in fresh insects). Therefore, in our study, it is reasonable to think that storage at −80 C could have led to weakening of organelle membranes (mitochondria and lysosomes at least).…”

“…Given the abovementioned artifacts that can be encountered during the fractionation protocol, existing subcellular fractionation procedures could clearly benefit from adjustments designed to maximize the separation of the subcellular fractions. Such adjustments would help improve the interpretation of the partitioning results (Cardon et al 2018). However, in the literature, studies assessing the performance of fractionation protocols are surprisingly scarce.…”