Subcellular partitioning of metals and metalloids (As, Cd, Cu, Se and Zn) in liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a mining operation

Abstract

In the present study, we examined the subcellular distribution of metals and metalloids (As, Cd, Cu, Se and Zn) in the liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a metal mining operation (exposure area) and in a reference area. Metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins (HDP), mitochondria and microsomes) and potentially biologically detoxified fractions (heat-stable proteins (HSP) and metal-rich granules) within cells was determined after differential centrifugation, NaOH digestion and heat-denaturation steps. Metal-handling strategies between liver and gonads, and between sexes, were examined. Hepatic metal concentrations were significantly higher in exposed compared to reference fish, especially for Se (14x), Cd (5x) and Cu (3x), and did not vary between sexes. In contrast, gonadal Cd, Cu, Se and Zn concentrations were consistently lower in testes than in ovaries; marked differences in Cd and Se concentrations between exposed and reference fish were observed for both sexes. Overall, metal-handling strategies were similar in both liver (male and female pooled) and female gonads, but differed from those in male gonads, likely due to the different functions assigned to ovaries and testes. Subcellular partitioning of As, Cd and Cu showed that the HSP fraction was most responsive to increased metal exposure, presumably reflecting Cu regulation, and possibly Cd and As detoxification. Zinc concentrations were tightly controlled and mainly found in the HDP fraction. Interestingly, changes in Cd-handling strategy in female gonads were particularly evident, with Cd shifting dramatically from the metal-sensitive HDP fraction in reference fish to the metal-detoxified HSP fraction in exposed fish. It seems that Cd detoxification in female gonads was not fully induced in the less contaminated fish, but became more effective above a threshold Cd concentration of 0.05 nmol/g dry weight. Partitioning of Se was different, with the largest contributor to the total liver and gonad Se burdens being the putative metal-sensitive HDP fraction, suggesting that excess Se in this fraction in exposed fish may lead to Se-related stress. The present subcellular partitioning results demonstrate that metal handling strategies vary among metals, between organs and (in some cases) as a function of metal exposure. They also show promise in identifying metals of potential concern in a risk assessment context.

Cytosolic Distribution of Metals (Cd, Cu) and Metalloids (As, Se) in Livers and Gonads of Field-Collected Fish Exposed to an Environmental Contamination Gradient: An SEC-ICP-MS Analysis

“…Comparisons between total [Cu] and [Cu] HSP indicate that the contribution of the HSP fraction to the total metal burden was above 60%; only for the testes was this contribution lower (≈20%). Similar relative contributions were previously reported in Urien et al [10].…”

“…In a study on juvenile yellow perch (Perca flavescens) collected along an environmental contamination gradient, Giguère et al [9] observed that Cd and Cu present in fish livers were mainly associated with the HSP fraction, suggesting a detoxification response by the binding of these two metals to MT and/or MTLP. In our recent work, assessing subcellular metal and metalloid partitioning in the liver and gonads of field-collected white suckers (WS) from a reference lake and a contaminated lake, similar observations were reported for Cd and Cu; more than 60% of the total Cd and Cu burdens were associated with the HSP fraction [10]. In the same study, As was also found to be associated with the HSP fraction (close to 50%), whereas the Se burden was associated with both the HSP and HDP fractions, the contribution of the HSP fraction to the total hepatic Se burden being more important in exposed, as compared to reference, fish, suggesting the possible induction of a detoxification response in the fish.…”

“…In summer 2016, mature white suckers, calibrated by length, were collected from a lake located downstream from a metal mining effluent and exhibiting a polymetallic (As, Cd, Cu, Ni, Pb, Se and Zn) contamination (n = 14 females and 6 males) and at a reference site (n = 7 females and 12 males) (for details about the metal exposure gradient, see Urien et al [10]). Upon capture (using gill nets), white suckers were sacrificed quickly by concussion, and once fish length (±1 mm) and weight (±0.01 g) had been recorded, fish were dissected to collect liver and gonads, and sex was determined (the capture and sampling protocol was approved by the INRS (Institut National de la Recherche Scientifique) animal-care committee).…”

“…HSP fractions were isolated by applying a subcellular fractionation protocol similar to that employed by Urien et al [10]. Briefly, tissues (≈0.5 g wet weight) were individually gently homogenized in a fresh ammonium acetate solution (100 mM, pH 7.4) with a motorized Potter-Elvehjem homogenizer equipped with a Teflon pestle (Fisher Scientific, Whitby, ON, Canada) (ratio wet weight tissue: volume of ammonium acetate solution: 1:3 for liver; 1:4 for gonads).…”

“…However, in the case of less studied elements, such as Se or As, evidence for MT or MTLP involvement in detoxification is not obvious. In the light of the results from our recent work on white suckers [10] and to answer the question "Are the elements present in the HSP fraction truly handled by MT and MTLP? ", the nature of the metal-binding biomolecules in this cytosolic fraction (HSP) was investigated.…”