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In situ subcellular imaging of copper and zinc in contaminated oysters revealed by nanoscale secondary ion mass spectrometry Nanyan Weng, Haibo Jiang, and Wen-Xiong Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05090 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017
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Environmental Science & Technology
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In situ subcellular imaging of copper and zinc in contaminated oysters revealed by nanoscale secondary ion mass spectrometry
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Nanyan Weng1, Haibo Jiang2∗, Wen-Xiong Wang1,3,* 1
Marine Environmental Laboratory, HKUST Shenzhen Research Institute, Shenzhen 518057, China 2
Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth WA 6009, Australia 3
Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clearwater Bay, Kowloon, Hong Kong
*Corresponding authors. E-mail:
[email protected],
[email protected] 1
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Abstract
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Determining the in situ localization of trace elements at high lateral resolution levels in the
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biological system is very challenging, but critical for our understanding of metal
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sequestrationa and detoxification. Here, the cellular and subcellular distributions of Cu and
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Zn in contaminated oysters of Crassostrea hongkongensis were for the first time mapped
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using nanoscale secondary ion mass spectrometry (nanoSIMS). Three types of
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metal-containing cells were revealed in the gill and mantle of oysters, including Cu-specific
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hemocytes, Cu and Zn-containing granular hemocytes, and Cu and Zn-containing calcium
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cells. Obvious intercellular distribution of Cu was found in the gill tissue, indicating the
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potential role of hemolymph in the transportation of Cu in oysters. The distribution of Cu
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showed a strong co-localization with sulfur and nitrogen in Cu-specific hemocyte and
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intercellular hemolymph. In the Cu and Zn-containing granular hemocytes and calcium cells,
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the co-occurrence of Cu and Zn with phosphorous and calcium was also found. Different
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relationships of distributions between Cu/Zn and macronutrient elements (nitrogen, sulfur
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and phosphorous) implied the differential metal complexation in oysters. Interestingly,
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quantitative analysis of the ratios of 32S–/12C14N– and 31P–/12C14N– of metal-deposited sites
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suggested the dynamic process of transfer of Cu and Zn from the metabolized protein pool to
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a more thermodynamically stable and detoxified form.
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ABSTRACT GRAPHIC
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KEYWORDS: NanoSIMS; Crassostrea hongkongensis; Copper; Zinc; Subcellular
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localization; Dynamic detoxification
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Introduction Oysters are the well-known hyper-accumulators of copper (Cu) and zinc (Zn), and
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contaminated oysters with green soft tissue caused by Cu pollution have been found in many
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coastal and estuarine environments around the world 1-4. More recently, oysters (Crassostrea
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hongkongensis) with extremely high accumulation of Cu and Zn were discovered in estuaries
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of South China. The soft tissue of these oysters presented a distinct blue colour, and Cu and
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Zn concentration in these blue-colored oysters reached up to 1.9% and 2.4%, respectively 5,6.
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Oysters are the key species in estuarine environments and also one of the major seafoods for
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human consumption. It is thus important to study how the excessive accumulated metals are
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detoxified and what is the distribution pattern of metals in these contaminated oysters. There are very few reports of the localization of trace metals in marine bivalves, and in
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situ visualization of metal distribution in oysters at high-resolution levels has not yet been
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achieved. However, such spatial information is essential for a comprehensive understanding
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of the mechanisms of transport and sequestration of metals in these hyperaccumulators.
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Using subcellular fractionation method, Wang et al.5 reported that most Cu and Zn in
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blue-colored oysters of C. hongkongensis were distributed in the cellular debris. Such
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technique based on operational definition does not provide precise information of metals at
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subcellular scale. George et al.4, using electron-probe X-ray microanalysis, showed that Cu
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and Zn in green-colored oysters of Ostrea edulis were immobilized in membrane-limited
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vesicles in two types of granular amoebocytes. Similar results were also found in the oysters
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Crassostrea gigas 7. Increased number of hemocytes was observed in the mantle of
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blue-colored oysters C. hongkongensis, and ultra-structure observation suggested many
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membrane-limited vesicles with high electron density in these cells 8. These earlier studies
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only provided independent confirmation of elemental distribution in one specific cell type of
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amebocytes, while the employed analytical techniques did not have the required sensitivity to
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detect relatively low metal concentrations. Moreover, evidence suggested that a large
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proportion of Cu in contaminated oyster O. edulis was present in some diffusible compounds
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. The traditional chemical fixation or gradient centrifugation methods used in earlier
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studies may lead to loss or redistribution of these mobile metals, and resulted in unreliable
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conclusions.
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Nanoscale secondary ion mass spectrometry (nanoSIMS) is recognized as an excellent
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technique to visualize the in situ distributions of trace elements in biological systems, owing
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to its high analytical sensitivity (µg/g, detection limit) and high spatial resolution (
