In Situ Subcellular Imaging of Copper and Zinc in Contaminated


Nov 27, 2017 - In Situ Subcellular Imaging of Copper and Zinc in Contaminated Oysters Revealed by Nanoscale Secondary Ion Mass Spectrometry. Nanyan We...
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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]

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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 (