Serafim A, Bebianno M J
CIMA, University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal.
Environ Res. 2009 May;109(4):390-9. doi: 10.1016/j.envres.2009.03.001. Epub 2009 Apr 3.
In order to clarify the role of metallothioneins (MT) in copper (Cu) toxicity, this work aimed to assess the involvement of this protein in the accumulation and elimination strategies of Cu in the clam Ruditapes decussatus exposed to two sublethal concentrations (25 and 50 microgCul(-1)). The behaviour of MT in three different tissues of clams during the accumulation and depuration processes was also followed by gel-filtration chromatography to assess if Cu was bound to MT or to other cytosolic components. The 96 h LC50 for water-borne copper was 715 microgL(-1) in R. decussatus. The Cu accumulation pattern was dependent on Cu exposure concentrations. In clams exposed to 25 microgl(-1), total Cu accumulation in the three tissues increased linearly during the exposure period, while in those exposed to 50 microgl(-1) it followed the first order kinetic model. The greatest amount of Cu accumulated in all tissues is associated to the low molecular weight cytosolic fraction (>50%). The chromatographic assay indicated that Cu in the cytosolic fraction is bound to MT and MT levels increase with the increase of Cu exposure confirming the binding affinity of Cu to MT in all tissues. However, a smaller percentage of Cu seems to be bond to other ligands, such as GSH. Copper was exponentially eliminated (only studied in clams exposed to 25 microgl(-1)) and the estimated half-life was tissue dependent (9, 5 and 14 days for the gills, digestive gland and remaining tissues, respectively). Copper bound to the thermostable compounds was eliminated more quickly (t(1/2)=4-7 days) in all tissues than those bound to the thermolabile compounds (t(1/2)=7-18 days). Interestingly, MT is rapidly degraded (t(1/2)=7 and 18 days), suggesting that this protein is actively involved in the elimination of this metal, through the Cu-MT complex since MT and Cu are turning over simultaneously. Therefore, when Cu exposure is low, the clam can cope efficiently with the excess of Cu levels by increasing MT induction as well as rapidly eliminating this metal via the MT-Cu complex. Copper toxicity in the clam R. decussatus is associated to the limited capacity of MT induction at higher and environmental unrealistic Cu exposures especially in the gills and remaining tissues.
为了阐明金属硫蛋白（MT）在铜（Cu）毒性中的作用，本研究旨在评估该蛋白在暴露于两种亚致死浓度（25和50μgCu l⁻¹）的波纹巴非蛤中铜的积累和消除策略中的参与情况。在积累和净化过程中，还通过凝胶过滤色谱法跟踪了MT在蛤的三种不同组织中的行为，以评估铜是与MT结合还是与其他细胞溶质成分结合。波纹巴非蛤中水体铜的96小时LC50为715μg l⁻¹。铜的积累模式取决于铜的暴露浓度。在暴露于25μg l⁻¹的蛤中，在暴露期间三种组织中的总铜积累呈线性增加，而在暴露于50μg l⁻¹的蛤中，其遵循一级动力学模型。所有组织中积累的铜的最大量与低分子量细胞溶质部分相关（>50%）。色谱分析表明，细胞溶质部分中的铜与MT结合，并且MT水平随着铜暴露的增加而增加，这证实了铜在所有组织中与MT的结合亲和力。然而，似乎有较小百分比的铜与其他配体结合，如谷胱甘肽（GSH）。铜呈指数级消除（仅在暴露于25μg l⁻¹的蛤中进行研究），估计的半衰期因组织而异（鳃、消化腺和其余组织分别为9、5和14天）。与热不稳定化合物结合的铜在所有组织中比与热稳定化合物结合的铜消除得更快（t₁/₂ = 4 - 7天）（t₁/₂ = 7 - 18天）。有趣的是，MT迅速降解（t₁/₂ = 7和18天），这表明该蛋白通过Cu - MT复合物积极参与这种金属的消除，因为MT和铜同时周转。因此，当铜暴露较低时，蛤可以通过增加MT诱导以及通过MT - Cu复合物迅速消除这种金属来有效应对过量的铜水平。波纹巴非蛤中的铜毒性与在较高和环境中不现实（尤其是在鳃和其余组织中）的铜暴露下MT诱导的有限能力有关。
