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

Summary

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.

Abstract

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.

Introduction

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their great antimicrobial effects^1,2. Therefore, the production of AgNPs and the number of AgNP-containing products are increased over time^3,4. However, AgNPs may be released into the environment and accumulate in the ocean^5,6. They have become the major source of Ag contamination, and the public awareness of the environmental toxicity of Ag is increasing.....

Protocol

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), 2 Kogia spp. (Ko), 2 Lagenodelphis hosei (Lh), 1 Stenella attenuata (Sa). Each stranded cetacean had a field number for ind....

Results

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.......

Discussion

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.......

Materials

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