We thank the Taiwan Cetacean Stranding Network for sample collection and storage, including the Taiwan Cetacean Society, Taipei; the Cetacean Research Laboratory (Prof. Lien-Siang Chou), the Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei; the National Museum of Natural Science (Dr. Chiou-Ju Yao), Taichung; and the Marine Biology &#38; Cetacean Research Center, National Cheng-Kung University. We also thank the Forestry Bureau, Council of Agriculture, Executive Yuan for their permit.

The study was performed in accordance with international guidelines, and the use of cetacean tissue samples was permitted by the Council of Agriculture of Taiwan (Research Permit 104-07.1-SB-62).
1. Tissue Sample Preparation for ICP-MS Analysis
Note: The liver and kidney tissues were collected from freshly dead and moderately autolyzed stranded cetaceans24, including 6 stranded cetaceans of 4 different species, 1 Grampus griseus (Gg), ...

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The purpose of the article study is to establish an adjuvant method to evaluate the Ag distribution at suborgan levels and to estimate Ag concentrations in cetacean tissues. The current protocols include 1) Determination of Ag concentrations in cetacean tissues by ICP-MS, 2) AMG analysis of pair-matched tissue samples with known Ag concentrations, 3) Establishment of the regression model (CHAA) for estimating the Ag concentrations by AMG positive values, 4) Evaluation of the accuracy and precision of CHAA, and 5) Estimat...

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their great antimicrobial effects1,2. Therefore, the production of AgNPs and the number of AgNP-containing products are increased over time3,4. However, AgNPs may be released into the environment and accumulate in the ocean5,6. They have become the major source of Ag contamination, and the public awareness of the environmental toxicity of Ag is increasing.
The status of AgNPs and Ag in the marine environment is complicated and constantly changing. Previous studies have indicated that AgNPs can remain as particles, aggregate, dissolve, react with different chemical species, or be regenerated from Ag+ ions7,8. Several types of Ag compounds, such as AgCl, have been found in marine sediments, where they can be ingested by benthic organisms and enter the food chain9,10. According to a previous study conducted in the Chi-ku Lagoon area along the southwestern coast of Taiwan, the Ag concentrations of marine sediments are extremely low and similar to the crustal abundance, and those of fish liver tissue are usually below the detection limit (&#60; 0.025 &#956;g/g wet/wet)11. However, previous studies conducted in different countries have demonstrated relatively high Ag concentrations in the livers of cetaceans12,13. The Ag concentration in the livers of cetaceans is age-dependent, suggesting that the source of Ag in their bodies is most likely their prey12. These findings further suggest the biomagnification of Ag in animals at higher trophic levels. Cetaceans, as the apex predators in the ocean, may have suffered negative health impacts caused by Ag/Ag compounds12,13,14. Most importantly, like cetaceans, humans are mammals, and the negative health impacts caused by Ag/Ag compounds in cetaceans may also occur in humans. In other words, cetaceans could be sentinel animals for the health of marine environment and humans. Therefore, the health effects, the tissue distribution, and concentration of Ag in cetaceans are of great concern.
Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost (instrument and maintenance) and the requirements for tissue storage/preparation12,15. In addition, it is usually difficult to collect comprehensive tissue samples in all investigations of stranded cetacean cases due to logistical difficulties, a shortage of manpower, and a lack of related resources12. The frozen tissue samples for ICP-MS analysis are not easily stored because of limited refrigeration space, and frozen tissue samples may be discarded due to broken refrigeration equipment12. These aforementioned obstacles hamper investigations of contamination levels in cetacean tissues by ICP-MS analysis using frozen tissue samples. In contrast, formalin fixed tissue samples are relatively easy to collect during the necropsy of dead-stranded cetaceans. Therefore, it is necessary to develop an easy to use and inexpensive method to detect/measure the heavy metals in cetacean tissues by using formalin fixed tissue samples.
Although the suborgan distributions and concentrations of alkali and alkaline earth metals may be altered during the formalin-fixed, paraffin-embedded (FFPE) process, only lesser effects on transition metals, such as Ag, have been noted16. Hence, FFPE tissue has been considered as an ideal sample resource for metal localization and measurements16,17. Autometallography (AMG), a histochemical process, can amplify heavy metals as variably sized golden yellow to black AMG positive signals on FFPE tissue sections, and these amplified heavy metals can be visualized under light microscopy18,19,20,21. Hence, the AMG method provides information on the suborgan distributions of heavy metals. It can provide important additional information for studying the metabolic pathways of heavy metals in biological systems because ICP-MS can only measure the concentration of heavy metals at the organ level18. Furthermore, digital image analysis software, such as ImageJ, has been applied to the quantitative analysis of histological tissue sections22,23. The variably-sized golden yellow to black AMG positive signals of FFPE tissue sections can be quantified and used to estimate the concentrations of heavy metals. Although the absolute Ag concentration cannot be directly determined by the AMG method with image quantitative analysis, it can be estimated by a regression model based on the data obtained from the image quantitative analysis and ICP-MS, which is named cetacean histological Ag assay (CHAA). Considering the difficulties in measuring Ag concentrations by ICP-MS analysis in most stranded cetaceans, CHAA is a valuable adjuvant method to estimate Ag concentrations in cetacean tissues, which cannot be determined by ICP-MS analysis due to the lack of frozen tissue samples. This paper describes the protocol of a histochemical technique (AMG method) for localizing Ag at the suborgan level and an assay named CHAA to estimate the Ag concentrations in the liver and kidney tissues of cetaceans.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58232/58232fig1.jpg" /> 
Figure 1: Flowchart depicting the establishment and application of cetacean histological Ag assay (CHAA) for estimating Ag concentrations. CHAA = cetacean histological Ag assay, FFPE = Formalin-fixed, paraffin-embedded, ICP-MS = inductively coupled plasma mass spectroscopy. <a href="https://www.jove.com/files/ftp_upload/58232/58232fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>HQ Silver enhancement kit</td>
			<td>Nanoprobes</td>
			<td>#2012</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgipath Paraplast</td>
			<td>Leica Biosystems</td>
			<td>39601006</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>4026</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Xylene</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>4328</td>
			<td></td>
		</tr>
		<tr>
			<td>Silane coated slide</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>511614</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (25 x 50 mm)</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>24501</td>
			<td></td>
		</tr>
		<tr>
			<td>Malinol</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>20092</td>
			<td></td>
		</tr>
		<tr>
			<td>GM Haematoxylin Staining</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>3008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin solution</td>
			<td>Chin I Pao Co., Ltd</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip (1000 &#956;L)</td>
			<td>MDBio, Inc.</td>
			<td>1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN Classic P1000</td>
			<td>Gilson, Inc.</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tube</td>
			<td>GeneDireX, Inc.</td>
			<td>PC115-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dogfish liver</td>
			<td>National Research Council of Canada</td>
			<td>DOLT-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dogfish muscle</td>
			<td>National Research Council of Canada</td>
			<td>DORM-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma mass spectrometry (ICP-MS)</td>
			<td>PerkinElmer Inc.</td>
			<td>PE-SCIEX ELAN 6100 DRC</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeZone 6 liter freeze dry system</td>
			<td>Labconco</td>
			<td>7752030</td>
			<td>For freeze drying</td>
		</tr>
		<tr>
			<td>BRAND&#174; SILBERBRAND volumetric flask</td>
			<td>Merck</td>
			<td>Z326283</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL standard vial, flat interior with 33 mm closure</td>
			<td>Savillex Corporation</td>
			<td>200-030-12</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>Nitric acid, superpur&#174;, 65.0%</td>
			<td>Merck</td>
			<td>1.00441</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>Hot Plate/Stirrers</td>
			<td>Corning&#174;</td>
			<td>PC-220</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>High Shear lab mixer</td>
			<td>Silverson</td>
			<td>SL2T</td>
			<td>For homogenization</td>
		</tr>
		<tr>
			<td>Sterile polypropylene sample jar (250mL)</td>
			<td>Thermo Scientific&#8482;</td>
			<td>6186L05</td>
			<td>For homogenization</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Nikon Corporation</td>
			<td>DS-Fi2</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Nikon Corporation</td>
			<td>ECLIPSE Ni-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon&#8482; Finesse&#8482; 325 manual microtome</td>
			<td>Thermo Scientific&#8482;</td>
			<td>A78100001H</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu-Cut&#174; SRM&#8482; 200 rotary microtome</td>
			<td>Sakura</td>
			<td>1429</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade S35</td>
			<td>FEATHER&#174;</td>
			<td>207500000</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide staining dish and cover</td>
			<td>Brain Research Laboratories</td>
			<td>#3215</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel staining rack</td>
			<td>Brain Research Laboratories</td>
			<td>#3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon embedding center</td>
			<td>Thermo Scientific&#8482;</td>
			<td>S-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Citadel&#174; tissue processor</td>
			<td>Thermo Scientific&#8482;</td>
			<td>69800003</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide warmer</td>
			<td>Lab-Line Instruments</td>
			<td>26005</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shandon Capshaw</td>
			<td>3964</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merck</td>
			<td>1541-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 6.01 for windows</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Statistic software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel tissue embedding mould</td>
			<td>Shenyang Roundfin Trade Co., Ltd</td>
			<td>RD-TBM003</td>
			<td>For paraffin emedding</td>
		</tr>
	</tbody>
</table>

use of autometallography to localize and semi-quantify silver in cetacean tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

Representative images of the AMG positive signals in the cetacean liver and kidney tissues are shown in Figure 5. The AMG positive signals include variably-sized brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. There is a positive correlat...

Watch this Scientific Journal Video about Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues at JoVE.com

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

This method can help answer key questions in the field of environmental toxicology about sub-organ distributions and concentration of heavy metals within biological systems. The main advantage of this technique is that AMG is an easy to use and inexpensive methodology, that is a valuable adjuvant for investigating heavy metals in tissues. We use the tissues from dolphins and whales to demonstrate the protocol.However, we believe the protocol could be used in a variety of animal species. Demonstrating the procedure with Wen-Ta Li will be Shun Theng, a clinician from our institute. Begin by collecting pair-matched liver and kidney tissues for AMG analysis from a stranded cetacean, and fixing the tissue in 10 times the volume of 10%neutral buffered formalin for 24 to 48 hours.Next, use disposable stainless steel microtome blades to trim the formalin-fixed liver and kidney tissues into two-centimeter by one-centimeter, three-millimeter sections, and place both liver and kidney sections from the same individual into the same labeled cassettes. Dehydrate the sections in a tissue processor through a series of graded ethanol and xylene immersions, followed by one-hour and two-hour paraffin immersions. After the two-hour paraffin immersion, transfer the tissues to the bottoms of steel histology molds and embed the dehydrated tissue samples with fresh melted paraffin.Chill the formalin-fixed, paraffin-embedded tissue blocks on a cold plate, until the paraffin solidifies, and trim each block at the microtome until the tissue surface is exposed. Chill the samples at minus 20 degrees Celsius for 10 minutes, and obtain five-micrometer tissue sections on the microtome. Using tweezers and brushes, lift the ribbons of tissue section as they are acquired, and float them on the surface of a 45 degree Celsius double-distilled water bath, until they can be separated, and place onto individual microscope slides.Then, place the slides on a slide warmer for one hour, at 60 degrees Celsius. Then, place the slides in slide racks, and de-paraffinize the sections in three different staining dishes containing 200 to 250 milliliters of pure non-xylene for eight-five-and three-minute incubations. Hydrate the de-parrafinized samples in different staining dishes of graded ethanol solutions, followed by rinsing in double-distilled water.Rinse the sections one time in PBS supplemented with 5%Triton X 100, several times in PBS alone, and one time in fresh double-distilled water for 30 seconds per wash. After the last wash, label each sample with 300 microliters of mixed silver enhancement kit solution, for no more than 15 minutes in the dark at room temperature. At the end of the incubation, wash the slides with double-distilled water, and counter-stain the sections in 300 microliters of hematoxylin per slide, for 10 seconds.Wash the slides with running tap water. Dry them and mount the sections with mounting medium. Then, capture 10 random histological images of each tissue section, under the 40X objective of a light microscope.To analyze the autometallography, or AMG, positivity of the samples, open the images in an appropriate image analysis software program, and select image, type, and RGB stack, to split the first image into the red, blue, and green color channels. In the blue channel, select image, adjust, and threshold, to measure the percentage of the area with AMG-positive signals in each image, manually adjusting the cut-off value for the threshold of each image based on the presence of false-positive areas in the nuclei, or red blood cells. Click analyze, and set measurements, and check the area fraction, to specify that the area fraction is recorded.Click analyze, and measure, to display the positive percent area of each histological image percent area column of the result window. To evaluate the correlation between the results of inductively-coupled plasma mass spectroscopy and AMG-positive values, open the appropriate graphing software. Create a new project file, and select XY and correlation.Input the results of the inductively-coupled plasma mass spectroscopy and AMG analyses, and select analysis, and correlation, to analyze the strength of association between the results of the analysis by Pearson correlation analysis. In the parameters, non-linear regression window, select a different regression model on the fit page, and select the comparison methods on the compare page. Include the extra sum of squares F test, and Akaike's Information Criterion.According to the results of the comparison methods, select a relatively appropriate regression model in the cetacean histological silver assay, and use the assay to estimate the silver concentrations of the cetacean liver and kidney tissues, with unknown silver concentrations. AMG-positive signals include variably-sized, brown to black granules in the cytoplasm of hepatocytes and Kupffer cells, and occasional amorphous, golden yellow to brown AMG-positive signals in the lumen and basement membrane of some proximal renal tubules. There is a positive correlation between the results of inductively-coupled plasma mass spectroscopy and AMG-positivity values in the analyzed liver and kidney tissues, with a preferred linear regression through the origin, according to the extra sum of squares F test, and Akaike's Information Criterion.While attempting this procedure, it's important to remember that the AMG method is an adjuvant method, and must be used with other specific method, such as ICPMS, for monitoring the actual composition of heavy metals in tissues.

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7.1K ビュー.  National Taiwan University. この方法は、生体内の重金属のサブ器官分布と濃度に関する環境毒性学の分野における重要な質問に答える助けとなる。この技術の主な利点は、AMGが使いやすく安価な方法論であり、組織中の重金属を調査するための貴重なアジュバントであるということです。私たちは、プロトコルを実証するためにイルカやクジラからの組織を使用しています。しかし、このプ?...