國立臺灣大學獸醫專業學院獸醫學研究所 博士論文
Graduate Institute of Veterinary Medicine School of Veterinary Medicine
National Taiwan University Doctoral Dissertation
台灣擱淺鯨豚組織銀濃度與
奈米銀對鯨豚免疫細胞影響的活體外研究
Silver Tissue Contamination in Taiwanese Stranded Cetaceans and Effects of Silver Nanoparticles on
Cetacean Immune Cells in Vitro
李文達 Wen-Ta Li
指導教授： 鄭謙仁 博士 共同指導教授：楊瑋誠 博士 Advisor: Chian-Ren Jeng Ph.D.
Co-advisor: Wei-Chang Yang Ph.D.
中華民國 107 年 7 月 July, 2018
摘要
奈米銀因其光學性質，電子性質，良好的抗微生物活性，催化活性和磁性活 性而被廣泛應用於各類商品，也成為環境銀汙染的重要來源。在齧齒類及魚類等 實驗動物的研究顯示，奈米銀可由呼吸道及消化道進入血液循環並沉積在各臟器 中 (特別是腦組織和睪丸)。奈米銀已經被證實可以在上述這些動物模式引起細胞 氧化壓力上升、去氧核苷核酸的損傷和細胞凋亡，並對藻類、海洋無脊椎動物和 魚類具有毒性。鯨豚是海洋高階掠食者，也是最可能因奈米銀汙染而受到影響的 動物之一。但目前卻沒有任何鯨豚相關的奈米銀毒性研究被發表，因此，評估奈 米銀對鯨豚的健康影響是亟需進行。本研究開發輔助方法以定位銀在次器官 (suborgan) 層級的位置 (第二章)，藉由鯨豚組織學銀濃度分析技術 (cetacean histological Ag assay; CHAA)，估算鯨豚組織的銀濃度 (第二章和第三章)，並藉上 述方法進行研究，建立鯨豚可能的銀代謝途徑之假說，並證明銀可能對鯨豚健康 引起系統性而非器官特定性的負面影響 (第三章)。此外，本研究也揭示奈米銀對
鯨豚白血球的細胞毒性和免疫毒性（第四章和第五章）。以上結果皆證實銀/銀化合
物和奈米銀對鯨豚健康的負面影響，也顯示其在海洋環境中的潛在生態毒性。
Abstract
Silver nanoparticles (AgNPs), an important source of silver contamination, have been widely used in many commercial products due to their optical properties, electronic properties, antimicrobial activity, catalytic activity, and magnetic activity. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect the animals of high trophic level via food-web chain, such as cetaceans and humans. Several rodent and fish studies have demonstrated AgNPs can enter the blood circulation via alimentary/respiratory tracts and deposit in multiple organs especially brain and testis. AgNPs have been reported to induce cellular oxidative stress, DNA damage and apoptosis in these animal models, and cause toxic effects on algae, marine invertebrates, and fishes. Cetaceans, as the top predators of ocean, may have been negatively affected by AgNPs, but no toxicity study of AgNPs in cetaceans has been reported. Therefore, it is urgent to investigate the possible negative effects of AgNPs on the health of cetacean. The current study presented an adjuvant method to localize the Ag distribution at suborgan levels (Chapter II), estimated the Ag concentrations of various tissues by cetacean histological Ag assay (CHAA) (Chapters II and III), provided a presumptive metabolic pathway of Ag in cetaceans, demonstrated the possible systemic rather than organ-targeting negative health effects caused by Ag in cetaceans (Chapter III), and revealed the cytotoxicity and immunotoxicity caused by AgNPs on the leukocytes of cetaceans (Chapters IV and V). All the data have demonstrated the negative effects of Ag/Ag compounds and AgNPs on the health of cetaceans and their potential ecotoxicity in marine environment.
Table of Contents
摘要 ... i
Abstract ... ii
Table of Contents ... iii
Chapter I: General Introduction ... 1
Section 1. Nanotechnology and Silver Nanoparticles (AgNPs) ... 1
Section 2. An Emerging Contaminant– AgNPs ... 1
Section 3. The Biodistribution and Bioavailability of AgNPs ... 3
Section 4. The Toxicity of AgNPs... 4
Section 5. The Ecotoxicology of AgNPs ... 8
Section 6. Summary and Objectives ... 9
Chapter II: Use of Autometallography to Localize and Semi-quantify Silver in Cetacean Tissues (Manuscript in Submission) ... 12
Chapter III: Investigation of Silver (Ag) Deposition in Tissues from Stranded Cetaceans by Autometallography (AMG) Environmental Pollution, 2018, 235: 534-545 ... 32
Chapter IV: Immunotoxicity of Silver Nanoparticles (AgNPs) on the Leukocytes of Common Bottlenose Dolphins (Tursiops truncatus) Scientific Reports, 2018, 8:5593 . 45 Chapter V: Th2 Cytokine Bias Induced by Silver Nanoparticles (AgNPs) in Peripheral Blood Mononuclear Cells (PBMCs) of Common Bottlenose Dolphins (Tursiops truncatus) (Manuscript in Submission) ... 58
Chapter VI: General Discussion ... 89
References ... 93
Chapter I: General Introduction
Section 1. Nanotechnology and Silver Nanoparticles (AgNPs)
Nanoparticles (NPs) are defined as a naturally, incidentally or artificially generated materials, which contain particles as individual or aggregated/agglomerated particles, and the size distribution of the particles ranges 1 to 100 nm (McGillicuddy et al., 2017).
Nowadays, NPs have been widely used in biomedical products, electronic equipment, and energy production due to their special physicochemical characteristics, such as melting point, wettability, electrical/thermal conductivity, catalytic activity, and antimicrobial activity, which lead to an enhanced performance over their bulk counterparts (Jeevanandam et al., 2018; McGillicuddy et al., 2017). Although the applications of nanotechnology in different areas provide lots of business opportunities, the potential threats of NPs to humans, animals and environment should not be overlooked (Handy et al., 2008; McGillicuddy et al., 2017). Silver nanoparticles (AgNPs), different from other engineered nanomaterials, are most often used in the commercial products, such as water filters, textiles, cosmetics, food packaging and health care items, mainly due to their strong antimicrobial properties (McGillicuddy et al., 2017). Besides, its unique physicochemical properties, such as high electrical and thermal conductivities, also lead to an increased application of AgNPs in electronic devices and medical imaging (Ajmal et al., 2016; Ge et al., 2014). The production of AgNPs and the number of AgNP- containing products have dramatically increased in the past few years and are expected to increase over time (Hansen et al., 2016; Vance et al., 2015).
Section 2. An Emerging Contaminant– AgNPs
Previous studies have demonstrated that AgNPs in the environment are not all artificially produced (i.e. not produced by humans)(Gomez-Caballero et al., 2010; Wen
et al., 1997). Humic acids (HAs) are ubiquitous and natural reducing agents in the environment, and they are composed of many functional groups, including quinines, ketones, aldehydes, phenolic and hydroxyls, which enable them to reduce Ag ions to form AgNPs (Akaighe et al., 2011; Sal'nikov et al., 2009). Early investigation has showed that AgNPs or copper NPs can be generated from bulk objects, such as silver wire and eating utensils (Glover et al., 2011). Furthermore, some organisms such as algae, plants, and bacteria have the capability to take up Ag ions and thereby form AgNPs (Mukunthan et al., 2011; Yang et al., 2010). These findings suggest that macroscaled Ag containing objects are a potential source of AgNPs in the environment and AgNPs can be produced by natural substances or organisms. However, the increasing use of AgNPs has triggered notable interest in developing methods to produce different types of AgNPs (Yu et al., 2013). AgNPs can be released during the production, transport, decay, use, and/or disposal of AgNP-containing products, draining into the surface water, and then accumulating in the marine environment (Farre et al., 2009; Walters et al., 2014). The increased production and use of AgNPs may eventually elevate the deposition of AgNPs in the environment, and thus AgNPs have been considered as a potential source of Ag contamination (McGillicuddy et al., 2017). The fate of AgNPs in the aquatic environment is complicated and changeful. Previous studies have found that AgNPs in the aquatic environment can remain as individual particles in suspension, aggregate, dissolve, react with different chemical species, or be regenerated from Ag+ ions (McGillicuddy et al., 2017; Yu et al., 2013). Wang et al. (2014) have demonstrated that Ag/Ag compounds and AgNPs can precipitate in marine sediments, be ingested by benthic organisms (such as benthic invertebrate species), and thereby enter the food chain in the marine environment.
Several previous studies have indicated that AgNPs can be transferred from one trophic level to the next via the food chain and cause negative effects on the animals at different
trophic levels (Buffet et al., 2014; Farre et al., 2009; Gambardella et al., 2015; Huang et al., 2016b; Wang et al., 2014). The extensive use and growing production of AgNP- containing products may aggravate the environmental contamination level of AgNPs.
Therefore, aquatic animals and marine environment will suffer from the negative impacts caused by Ag/Ag compounds and AgNPs, which further raises concern about the environmental toxicity of their deposition.
Section 3. The Biodistribution and Bioavailability of AgNPs
Concerning about the environmental toxicity of Ag/Ag compounds and AgNPs, the interaction between Ag and biological systems is drawing a serious attention. There are numerous studies investigating the biodistribution and bioavailability of Ag/Ag compounds and AgNPs by using different laboratory animal models. Jung et al. (2014) demonstrated that the Japanese medaka (Oryzias latipes) could uptake AgNPs through gills and gastrointestinal tract, and AgNPs could enter blood circulation and mainly accumulated in the liver. When laboratory rats were exposed to 3.0 x 106 particle/cm3 AgNPs via inhalation pathway, Ag can be detected in the lung and liver tissues after 2 hours of exposure (Takenaka et al., 2001). Furthermore, central nervous system (CNS) can be targeted by airborne AgNPs, and the possible mechanism is the deposition of AgNPs compounds on the olfactory mucosa of the nasopharyngeal region of the respiratory tract and subsequent translocation via the olfactory nerve to the CNS (Ji et al., 2007; Oberdorster et al., 2004; Sung et al., 2009). On the other hand, when laboratory rats were orally exposed to AgNPs and AgNO3 for 28 days, the results showed that Ag could be detected in the blood, feces, liver, spleen, kidney, brain, ovary and testis (Lee et al., 2013; van der Zande et al., 2012). It was also reported that 1) the highest Ag concentrations were generally found in the liver and spleen of laboratory animals after exposure, 2) the Ag concentrations of blood, feces, liver, spleen, kidney, and ovary were
significantly decreased after oral exposure of AgNPs and AgNO3, which suggested that the Ag can be metabolized after ingestion, 3) the Ag concentrations of brain and testis were not decreased after 4 months of recovery period (an obstruction in clearance of accumulated Ag), suggesting that biological barriers, such as the blood–brain barrier (BBB) and blood-testis barrier (BTB), may play an important role in the Ag clearance from these tissues (Lee et al., 2013; van der Zande et al., 2012).
In summary, previous studies conducted in laboratory animals, including fishes, mice and rats, have demonstrated that AgNPs can enter the blood circulation through oral and inhalation exposure, accumulate in multiple organs, and be metabolized through liver and kidney. Besides, AgNPs can penetrate BBB and BTB and persistently accumulate in brain and testis.
Section 4. The Toxicity of AgNPs
Since previous studies have demonstrated the biodistribution and bioavailability of AgNPs in a variety of animals, the toxicity of AgNPs is of interest.
1. In vivo studies
The in vivo studies on the toxicity of AgNPs have been conducted by using laboratory animals, including rats, mice, invertebrates, and fishes, and demonstrated that AgNPs are toxic to all tested animals in a dose-dependent manner (Buffet et al., 2014;
Kim and Ryu, 2013; Myrzakhanova et al., 2013). The exposure methods in rats and mice include oral ingestion, inhalation, intraperitoneal injection, and intravenous injection;
those in invertebrates (such as mussels and oyster), fishes, and fish embryos are mainly immersing (Buffet et al., 2014; Gagne et al., 2013; Kim and Ryu, 2013; Myrzakhanova et al., 2013).
For laboratory rats and mice, the concentrations and treatment time periods of AgNPs were ranged, respectively, from 1 to 1000 mg/kg and 3 to 90 days in the oral
exposure experiments; the concentrations and treatment time periods of AgNPs were ranged from 1.32 to 1.91 x 107 particles/cm3 and 14 ~ 19 days in the inhalation exposure experiments, respectively (Hadrup and Lam, 2014; Kim and Ryu, 2013; Sung et al., 2009).
Previous studies of inhalation toxicity of AgNPs in laboratory rats found several negative health effects, including 1) significantly increased numbers of goblet cells and the amount of mucin of respiratory tract (1.32 x 106 particles/cm3, 1.98 to 64.9 nm AgNPs; 6 hours/day, 5 times a week for 4 weeks)(Hyun et al., 2008), 2) pulmonary inflammation, decreased pulmonary function, and bile duct hyperplasia (2.9 x 106 particles/cm3, 2 to 65 nm AgNPs; 6 hours/day, 5 times a week for 13 weeks)(Sung et al., 2009), 3) alteration of gene expression in the brain with motor neuron disorders, neurodegenerative disease, and altered immune cell function (1.91 x 107 particles/cm3, 22.18 ± 1.72 nm AgNPs; 6 hours/day, 5 times a week for 2 weeks)(Lee et al., 2010). On the other hand, early studies of oral toxicity of AgNPs in laboratory rats found several negative health effects, including 1) weight loss (5 mg/kg) and damage to intestinal epithelial cells (20 mg/kg) (3 to 20 nm AgNPs for 21 days) (Shahare and Yashpal, 2013), 2) weight loss, pigmentation in ileum, increases in alkaline phosphatase (ALKP) activity and serum cholesterol level, and bile duct hyperplasia (56 ± 1.46 nm AgNPs for 90 days) (Kim et al., 2008; Kim et al., 2010). Apart from previous studies with high concentrations of AgNPs exposure, there were some studies conducted of relatively low concentrations of AgNPs. Sardari et al.
(2012) had found that rats orally exposed to 1 to 2 mg/kg/days of 70 nm AgNPs for 30 days could induce hepatitis, necrosis of glomerular cells/proximal tubular epithelial cells, changes in normal splenic architecture. In addition, significantly increased ALKP and aspartate transaminase (AST) activities and changes in the levels of cytokines, and mild inflammation of renal cortex in the mice exposed to 1 mg/kg/days of 42 nm AgNPs for 28 days by oral administration (Park et al., 2010a). As above, the toxicity caused by
AgNPs in laboratory animals included weight loss, damage to the alimentary/hepatobiliary system, abnormalities in hematology/biochemistry, genotoxicity, neurotoxicity, and immunotoxicity.
In previous studies of invertebrates (such as mussels and oyster), fishes, and fish embryos, the exposure concentrations and exposure time periods of AgNPs were ranged, respectively, from 0.0008 to 50 mg/L and 1 to 14 days. These studies demonstrated that AgNPs could cause disruption of the ionic regulation/steroidogenesis, histological alterations of gills (telangiectasia, circulatory disturbances, epithelial lifting, epithelial desquamation, deformed lamellae, and epithelial hyperplasia), neurotoxicity, immunotoxicity, cytotoxicity, and genotoxicity (Degger et al., 2015; Gagne et al., 2013;
Hawkins et al., 2015; Kim and Ryu, 2013; Kwok et al., 2016; Thummabancha et al., 2016;
Wu and Zhou, 2013).
2. In vitro studies
Numerous studies of in vitro toxicity of AgNPs have been published, which include human cancer cell lines (such as skin carcinoma cells [A431], human lung adenocarcinoma cells [A549], human hepatoma cells [HepG2]), rainbow trout hepatocyte, rainbow trout gill cells, and Japanese medaka fibroblast cells (OLHNI2) (Kim and Ryu, 2013; Zhang et al., 2014; Zhang et al., 2016). Although the possible mechanisms of toxic effects caused by AgNPs are not fully understood, previous studies have suggested that the AgNPs can cause cytotoxicity (i.e. mitochondrial dysfunction and apoptosis) and genotoxicity (i.e. DNA damage, formation of micronuclei, cell cycle arrest) via reactive oxygen species (ROS)-dependent pathway and ROS-independent pathway (Kim and Ryu, 2013; Zhang et al., 2014; Zhang et al., 2016). An early study has found that AgNPs can penetrate through the cell membrane, be ionized in the cytoplasm, and then induce negative effects, which is considered a Trojan-horse type mechanism (Park et al., 2010b).
Furthermore, recent studies have demonstrated that AgNPs cause cytotoxicity through the disruption of normal autophagic flux (Mao et al., 2016; Mishra et al., 2016).
2.1 ROS-dependent pathway
Reactive oxygen species (ROS) are by-products of cellular oxygen metabolism and mainly produced during mitochondrial respiration in eukaryotic cells. When there are increased amounts of ROS accumulating in cells, this condition is known as oxidative stress (Kim and Ryu, 2013). It is reported that the AgNPs can induce oxidative stress by increasing the amount of intracellular ROS with glutathione depletion, lipid peroxidation enhancement, DNA damage, cell cycle alteration, and cell proliferation inhibition, and these changes ultimately lead to cell death (Kim and Ryu, 2013; Zhang et al., 2014).
2.2 ROS-independent pathway
A previous study using HepG2 and Caco2 cell lines showed that AgNPs could cause damages to the DNA and mitochondria without increasing oxidative stress (Sahu et al., 2014). Farkas et al. (2010) demonstrated that the integrity of cell membrane and the metabolic activity of rainbow trout hepatocytes were significantly decreased without increased oxidative stress. These findings suggest that the cytotoxicity caused by AgNPs may not be associated with a ROS-dependent pathway.
3. AgNPs and Ag ion
Because Ag ions are consistently dissolved from AgNPs, the toxicity of AgNPs is actually due to the dissolved Ag ions and/or AgNPs is still controversial. Some studies suggested that the dissolved Ag ions are the cause of toxicity induced by AgNPs (Lubick, 2008) or AgNPs may act as the Trojan-horse to bring Ag ions into the cells and induce toxic effects (Farkas et al., 2010). However, there were several studies indicating that 1) the toxicity of AgNPs was higher than that of pure Ag ions, 2) only AgNPs could cause formation of micronuclei, and 3) the alteration on gene expression induced by AgNPs was
different from that by pure Ag ions (Kim et al., 2009a; Piao et al., 2011; Sahu et al., 2014).
Therefore, the toxic mechanisms of AgNPs and Ag ions may be different.
4. Factors Affecting the Toxicity of AgNPs
The toxicity of AgNPs is influenced by not only cell type and exposure time/concentration, but also the different coatings and sizes of AgNPs (Kim and Ryu, 2013; Riaz Ahmed et al., 2017; Zhang et al., 2014; Zhang et al., 2016)
5. The effect of AgNPs on immune system
According to the previous in vivo studies, AgNPs would enter the blood circulation through alimentary/respiratory administrations, and thus the negative effects caused by AgNPs to the leukocytes should be of concern. Several studies on the negative effects of AgNPs on human leukocytes have demonstrated that AgNPs could cause several effects in human neutrophils, including morphological alterations, cytotoxicity, atypical cell death, inhibition of de novo protein synthesis, increased production of the CXCL8 chemokine (IL-8), and impaired lysosomal activity (Liz et al., 2015; Poirier et al., 2014;
Poirier et al., 2016; Soares et al., 2016). Cytotoxicity and inhibition of lymphocyte proliferation in lymphocytes and macrophages were also revealed (Huang et al., 2016a;
Shin et al., 2007). However, the effects of AgNPs on the functional activities of human neutrophils and lymphocytes are still poorly understood.
Section 5. The Ecotoxicology of AgNPs
Due to the extensive use of AgNP-containing product and increased production of AgNPs, AgNPs have been considered as potential sources of Ag contamination. The environmental contamination level of Ag (including Ag, Ag compounds, and AgNPs) have dramatically increased in the past few years and are expected to increase over time.
Therefore, it raises the public concern about the environmental toxicity of Ag. The current knowledge regarding ecotoxicological data of AgNPs in the marine ecosystem is still
scarce, and only limited data on the potential toxicity of AgNPs to marine organisms at different trophic levels has been reported, including alga, jellyfish (Aurelia aurita), arthropod, oyster (Crassostrea virginica), sea urchin (Paracentrotus lividus), endobenthic species, and marine medaka (Oryzias melastigma) (Buffet et al., 2014; Gambardella et al., 2015; Garcia-Alonso et al., 2014; Huang et al., 2016b; Moreno-Garrido et al., 2015;
Ringwood et al., 2010; Wang et al., 2014). The results have demonstrated that AgNPs are toxic to all the tested marine organisms and suggested that the AgNPs may cause negative effects on the marine organisms at different trophic levels. Recent study has indicated that the AgNPs in marine environment will aggregate/precipitate into the sediments, be ingested by filter feeders and sediment-dwelling organisms, and transferred from one trophic level to the next level via the food chain (Buffet et al., 2014; Farre et al., 2009;
Wang et al., 2014). The environmental contamination level of Ag is expected to increase greatly in the near future, and cetaceans as the top predators in the ocean may suffer from the negative health effects caused by Ag/Ag compounds and AgNPs.
Section 6. Summary and Objectives
Silver nanoparticles (AgNPs) have been extensively used in numerous commercial products, including textiles, cosmetics, health care items, and electronic devices/medical images. AgNPs can be released during the production, transport, erosion, washing, and/or disposal of AgNP-containing products, subsequently draining into the aquatic environment, and ultimately accumulating in the ocean. Therefore, AgNPs have been considered as potential sources of Ag contamination, which has raised the public concern about the environmental toxicity of Ag. Ag can be transferred from one trophic level to the next via the food chain and may cause negative effects on the animals at higher trophic levels. The environmental contamination level of Ag is expected to increase greatly in the near future, and aquatic animals and marine environment may suffer the potentially
negative impacts caused by Ag. However, the ecotoxicological studies on AgNPs and Ag are still sparse. Cetaceans, as the top predators in the ocean, may endure the negative health impact by long-term exposure to and accumulation of Ag/Ag compounds in their bodies.
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 requirement for tissue storage/preparation. Considering the difficulties in measuring Ag concentrations by ICP-MS in most stranded cetaceans, it is valuable to develop a histological method using formalin-fixed and paraffin-embedded (FFPE) tissue samples to localize Ag and estimate the Ag concentrations in the cetacean liver and kidney tissues.
On the other hand, the toxic effects caused by AgNPs may be different from those caused by Ag/Ag compounds, and AgNPs can enter blood circulation after alimentary exposure.
Therefore, it is necessary to investigate the negative effects caused by AgNPs by using the cetacean leukocytes (consistent exposure). Most importantly, cetaceans as well as humans are mammals; and the negative health impact caused by Ag/Ag compounds and AgNPs in cetaceans may also occur in humans. In other words, cetaceans are good sentinel animals for the condition of marine environment and human health.
As above, the current study aimed to 1) develop a histological method to evaluate the tissue distribution of Ag and to estimate the Ag concentrations in the liver and kidney of cetaceans (Chapter II); 2) determine the metabolic pathway of Ag, estimate Ag concentrations of cetacean liver and kidney tissues by CHAA, and investigate the histopathological lesions possibly caused by Ag in cetaceans (Chapter III); 3) investigate the immunotoxicity of AgNPs on the leukocytes of cetaceans (Chapter IV); and 4) evaluate the changes in in vitro cytokine profile of cetacean peripheral blood mononuclear
cells (cPBMCs) exposed to AgNPs (Chapter V).
Chapter II: Use of Autometallography to Localize and Semi- quantify Silver in Cetacean Tissues (Manuscript in Submission)
AUTHORS & AFFILIATIONS:
Wen-Ta Lia, Bang Yeh Lioua, Wei-Cheng Yangb, Meng-Hsien Chenc, Hui-Wen Changa, Hue-Ying Chioud, Victor Fei Panga, and Chian-Ren Jenga*
aGraduate Institute of Molecular and Comparative Pathobiology, National Taiwan University, Taipei, Taiwan; bCollege of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan; cDepartment of Oceanography and Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan; dGraduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan
CORRESPONDING AUTHOR: Chian-Ren Jeng
KEYWORDS:
Autometallography (AMG); Cetacean; Cetacean Histological Ag Assay (CHAA);
ImageJ; Inductively Coupled Plasma Mass Spectroscopy (ICP-MS); Silver (Ag);
Quantitative Analysis
SHORT ABSTRACT:
A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.
LONG 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 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 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 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 (< 0.025 μg/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 compounds12-14. 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
microscopy18-21. 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.
[Place Figure 1 here]
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 individual identification.
The tissue sample preparation for ICP-MS analysis followed the method established in M.H. Chen's lab, and M.H. Chen's lab conducted the ICP-MS analysis11,13,25.
1.1. Collect liver and kidney tissues for ICP-MS analysis from stranded cetaceans and store them at −20 °C until use.
1.2. Collect pair-matched liver and kidney tissues from the same stranded cetaceans for AMG analysis (please see step 2).
1.3. Trim the outer layer of the tissue samples collected for ICP-MS analysis with a stainless-steel scalpel. Cut the inner part of the tissue samples into small cubes (about 1 cm3) and place them in zip lock plastic bags. Normally, each bag contains 10 g of the tissues.
1.4. Store the plastic bags containing tissue samples at −20 °C for subsequent procedures.
1.5. Put the 1 cm3 cubes samples in a freeze dry system (-50°C, Vacuum pump with a displacement of at least 98 L/min, 0.002 mBar) for at least 72 h till completely dried by weighting to the constant.
1.6. Homogenize the dried cubes into powder by homogenizer for subsequent tissue digestion.
1.7. Weigh 0.3 g of homogenized freeze-dried samples in 30 mL polytetrafluoroethylene (PTFE) bottles and mix them with 10 mL of 65% w/w nitric acid.
1.8. Put closures on the PTFE bottles, but leave the closures untightened.
Note: it allows the brown fume to be formed in the PTFE bottles and reflux inside the bottle for digestion till the brown fume disappear and turn clear.
1.9. Heat the digested samples with a hot plate, from 30 °C to 110/120 °C (according to the brown fume forming condition) in the PTFE bottles for 2 to 3 weeks until the brownish gas in the PTFE bottles becomes colorless and the liquid in the PTFE bottles becomes translucent greenish pale yellow or completely clear.
Note: Perform the heating process in chemical fume hood.
1.10. Heat the digested samples at 120 °C to evaporate the nitric acid in the PTFE bottles until only 0.5 to 1 mL remains.
Note: Perform the heating process in a chemical fume hood, and always monitor the temperature increase to ensure that no brownish gas leaks from the PTFE bottles’
closures.
1.11. Tighten the closures and cool them at room temperature for about one hour.
1.12. Place the funnels with filter papers on 25 mL volumetric flasks and wash the remaining liquid with 1 M HNO3 to a final volume of 25 mL.
Note: Wash the bottle for at least three times and the closure twice.
1.13. Validate the analytical quality of ICP-MS analysis by using the standard reference materials, including DOLT-2 (dogfish liver) and DORM-2 (dogfish muscle).
1.14. Use duplicates of each analytical sample and triplicates of standard reference materials for ICP-MS analysis.
1.15. Average the Ag concentrations of each analytic samples and present the data as dry weight basis concentration (μg/g dry weight).
2. Tissue Sample Preparation for AMG Analysis
2.1. Collect pair-matched liver and kidney tissues for AMG analysis from a stranded cetacean and fix them in 10% neutral buffered formalin until use.
Note: Store the tissue samples in plastic bottles in 10% neutral buffered formalin (NBF, pH 7.0) for 24 to 48 hours. The volume of NBF should be at least 10 times greater than the tissue volume.
2.2. Trim the formalin fixed liver and kidney tissues with stainless steel disposable microtome blades and put the trimmed tissue sections in cassettes with labels.
Note: The size of each tissue sections should be approximately 2 x 1 cm and the thickness of each tissue section should not exceed 3 mm. Put the liver and kidney tissues from the same individual in the same cassette.
2.3. Dehydrate the trimmed tissue sections with a tissue processor through a series of graded ethanol (70% for 1 h, 80% for 1 h, 95% for 1 h, 95% for 2 h, 100% for 1 h x 2 staining dishes, and 100% for 2 h), non-xylene (for 1 h and 2 h in different staining dishes), and immerse the dehydrated tissue samples in paraffin (for 1 h and 2 h in different staining dishes).
2.4. Place the dehydrated tissue samples in the bottoms of steel histology molds and embed the dehydrated tissue samples with paraffin.
2.5. Chill the formalin fixed paraffin-embedded (FFPE) tissue blocks at −20 °C. Trim the FFPE blocks with the microtome until the tissue surface is exposed.
2.6. Chill the FFPE blocks at −20 °C again. Section the FFPE blocks at 5 µm by microtome.
2.7. Fill a water bath with double-distilled water at 45 °C. Lift the ribbons of tissue sections and make them float on the surface of the warm water by using tweezers and brushes.
2.8. Separate the ribbons of tissue sections with tweezers. Place a section onto a microscope slide.
2.9. Place the microscope slides on a slide warmer and allow sections to dry overnight at 37 °C.
2.10. Put the microscope slides in slide racks and deparaffinize them by soaking them in 3 different staining dishes of pure non-xylene (approximately 200 to 250 mL) for 8, 5, and 3 min.
2.11. Hydrate the tissue sections in slide racks by soaking them in different staining dishes of graded ethanol solutions (100% ethanol twice, 90% ethanol once and 80%
ethanol once [1 min each]), and rinse them in double-distilled water.
Note: These solutions are approximately 200 to 250 mL in different staining dishes.
2.12. Rinse the tissue sections in phosphate-buffered saline (PBS) with 0.5% Triton X-100, wash them with PBS for several times, and then rinse them in double-distilled water.
Note: These solutions are approximately 200 to 250 mL in different staining dishes.
2.13. Prepare equal amounts of the three components (initiator, moderator, and activator) provided by silver enhancement kit in dark and mix them thoroughly.
Note: The solutions of moderator and activator are sticky, so please use pipette with wide tip openings (or cut the tips to create wider openings). For each slide, 300 μL of the mixed solution (depending on the size of the tissue section) is usually enough.
Therefore, if 10 slides are used, the amount of each component (initiator, moderator, and activator) is 1000 μL (the mixed solution is 3000 μL for 10 slides).
2.14. Incubate the tissue sections in the mixed solution for 15 min in dark at room temperature. Fully cover the tissue sections on the slides with the mixed solution. A longer incubation time may lead to false-positive AMG signals.
2.15. Wash the slides with double-distilled water and stain them in hematoxylin for 10 s as a counterstain.
2.16. Wash the slides with running tap water, dry them, and mount them with mounting medium.
2.17. Examine the slides under a light microscope.
2.18. Randomly capture ten histological images with a 40X objective lens from each tissue section by using a connected digital camera with computer imaging software.
3. Semi-Quantitative Analysis for AMG Positive Values of Histological Images Note: AMG positive value means the percentage of the area with AMG positive signals.
3.1. Use image analysis software (ImageJ) to analyze the histological images.
3.2. Open the histological image by pressing File | Open.
3.3. Split the chosen picture into three color channels (red, blue, and green) by pressing Image | Type | RGB Stack.
3.4. Quantify the AMG positive signals by using the blue channel. Nuclear false positive signals are usually decreased under the blue channel when hematoxylin stain is applied for nuclear counterstain (Figure 2).
3.5. Measure the percentage of the area with AMG positive signals in each histological image with the threshold tool (Image | Adjust | Threshold).
3.6. Manually adjust the cut-off value of the threshold for each histological image (from 90 to 110) based on the presences of false positive areas in nuclei and/or red blood cells.
Note: In default setting, the AMG positive signals should be highlighted in red.
3.7. Press Analyze | Set Measurements, and check the box of Area Fraction to specify that the area fraction is recorded.
3.8. Press Analyze | Measure. The positive percent area of each histological image is displayed in the column of %Area of the Result window.
3.9. Average the positive percent areas of 10 histological images from each tissue section and define the result as the AMG positive value for each tissue section.
[Place Figure 2 here]
4. Establishment of the Cetacean Histological Ag Assay (CHAA) by Regression Model
Note: The following analysis is performed in Prism 6.01 for Windows.
4.1. Evaluate the correlation between the results of ICP-MS and AMG positive values.
4.2. Open the Prism software, create a new prism project file, and choose XY and Correlation.
4.3. Input data including the results of ICP-MS and AMG positive values.
4.4. Press Analysis and choose Correlation under the category XY Analysis to analyze the strength of association between the results of the ICP-MS and AMG positive values by Pearson correlation analysis.
Note: The results of the ICP-MS and AMG positive values have to be positively correlated with each other; otherwise, the subsequent regression model should not be developed.
4.5. Statistically compare the regression models, including linear regression, quadratic regression, cubic regression, and linear regression through origin, through statistics software12,26,27.
Note: If the regression model generates an unrealistic Ag concentration, the regression model should be abandoned12.
4.6. Go back to the Data Table (left panel) and press Analysis | Nonlinear regression (curve fit) under the category XY Analysis | OK.
4.7. In the window Parameters: Nonlinear regression, choose different regression model in the page Fit and then compare different regression models in the page Compare.
4.8. In the page Compare, choose the comparison methods, including the extra sum-of- squares F test and Akaike's information criterion (AIC). According to the results of the comparison methods, use a relatively appropriate regression model in the CHAA.
4.9. Estimate Ag concentrations of the cetacean liver and kidney tissues with unknown Ag concentrations by using the CHAA.
4.10. Evaluate the accuracy and precision of the CHAA for liver and kidney tissues.
The difference between precision and accuracy is illustrated in Figure 3.
4.11. Accuracy: Calculate the mean standard deviation (SD) from differences between known and estimated Ag concentrations.
4.12. Precision: Perform repeated measurement (at least triplicate) of AMG positive values of serial sections from the same FFPE tissues. Calculate the mean SD of measurements from liver or kidney tissues from differences between known and estimated Ag concentrations
Note: The methods of evaluating the accuracy and precision are depicted in Figure 4.
[Place Figures 3 and 4 here]
5. Estimation of Ag Concentrations by CHAA.
5.1. Collect the liver and kidney tissues from stranded cetaceans and fix them in 10%
neutral buffered formalin.
5.2. Process the formalin-fixed tissues routinely (please see step 2).
5.3. Estimate the Ag concentrations of the cetacean liver and kidney tissues with unknown Ag concentrations by CHAA (please see steps 3 and 4).
REPRESENTATIVE 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 correlation between the results of ICP-MS and AMG
positivity values in liver and kidney tissues, and linear regression through origin is preferred according to the extra sum-of-squares F test and AIC12,26,27. In the accuracy test, the mean SDs of the CHAA for liver and kidney are 3.24 and 0.16, respectively. In the precision test, the mean SDs of the CHAA for liver and kidney are 2.8 and 0.35, respectively. The raw data of the accuracy and precision tests are summarized in Table 1. The AMG positive values, Ag concentrations estimated by CHAA, and Ag
concentrations measured by ICP-MS from the liver and kidney tissues of these six stranded cetaceans are summarized in Table 2.
FIGURE AND TABLE LEGENDS:
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.
Figure 2: The presence of nuclear false positive signals under different color channels (counterstain: hematoxylin stain). Representative nuclear false positive signals are indicated by yellow arrows. PPA = positive percentage of areas.
Figure 3: The difference between accuracy and precision. Accuracy means how close the measurement is to the true value (i.e., Ag concentration determined by ICP- MS); precision means the repeatability of the measurement (i.e., the consistency among the repeated measurements of AMG positive values from the triplicate tissue sections).
Figure 4: The scheme depicting the methods of evaluating the accuracy and precision. CHAA = cetacean histological Ag assay; FFPE = Formalin-fixed, paraffin- embedded; ICP-MS = inductively coupled plasma mass spectroscopy; Ai = Each of the Ag concentrations determined by ICP-MS of each pair-matched tissue sample; Bi = Each of the Ag concentrations estimated by CHAA of each pair-matched tissue sample;
Ci, Di, and Ei = Each of The Ag concentrations estimated by CHAA of triplicate samples from each pair-matched tissue sample; i = 1 to n. Please see raw data of the accuracy and precision tests in the section of representative results.
Figure 5: Representative histological images of the AMG positive signals in the liver and kidney tissues of cetaceans (counterstain: hematoxylin stain). (A) The
AMG positive signals in cetacean liver tissue are evenly distributed (Grampus griseus (Gg); field code: TP20111116; Ag concentration measured by inductively coupled plasma mass spectroscopy (ICP-MS): 21.82 μg/g dry weight). (B) The AMG positive signals are brown to black granules of various sizes in the cytoplasm of hepatocytes (red arrows) and Kupffer cells (red arrow heads) (Gg; field code: TP20111116). (C) A few AMG positive signals of brown to black granules are shown in the cytoplasm of hepatocytes (red arrows) (Kogia spp. (Ko); field code: TC20110722; Ag concentration measured by ICP-MS: 3.86 μg/g dry weight). (D) The AMG positive signals in cetacean kidney tissue are mainly located in the renal cortex (Gg; field code: TP20111116; Ag concentration measured by ICP-MS: 0.42 μg/g dry weight). The black dashed line is placed on the junction between the renal cortex and medulla. (E) Higher magnification of Figure 5D (red dashed rectangle). The AMG positive signals in the renal cortex are brown to black granules of various sizes in the cytoplasm of the proximal renal tubular epithelium (red arrows). Amorphous golden yellow to brown AMG positive signals are shown in the lumens (red arrow head) and basement membrane (yellow arrow head) of some proximal renal tubules. No to minimal AMG positive signals are shown in the glomeruli (green arrow) and distal renal tubules (green arrow head)(Gg; field code:
TP20111116). (F) Scattered brown granules of various sizes are shown in the cytoplasm of the proximal renal tubular epithelium (red arrows) (Ko; field code: TC20110722; Ag concentration measured by ICP-MS: 0.05 μg/g dry weight).
Table 1: The representative results of the accuracy and precision tests for cetacean histological Ag assay (CHAA). CHAA = cetacean histological Ag assay, ICP-MS = inductively coupled plasma mass spectroscopy, SD = standard deviation.
Accuracy test
Field number Liver Kidney
CHAA* ICP-MS SD CHAA* ICP-MS SD
TP20111116 16.82 21.82 4.99 0.64 0.42 0.22 TC20110611 10.12 2.77 0.96 0.11 0.05 0.35 TC20110722 2.70 3.86 1.15 0.01 0.05 0.04 TD20110608 0.76 0.06 7.35 0.02 0.05 0.06 TP20110830 13.97 14.93 4.28 0.69 1.04 0.24 IL20110101 6.00 1.73 0.72 0.38 0.14 0.03
Mean SD 3.24 Mean SD 0.16 Precision test
Field number Liver Kidney
CHAA* ICP-MS SD CHAA* ICP-MS SD
TP20111116
20.90
21.82 4.08
0.21
0.42 0.44
16.11 0.22
17.75 0.14
TD20110608
1.52
0.06 1.71
0.00
0.05 0.02
2.40 0.00
1.12 0.00
TP20110830
13.12
14.93 2.70
0.45
1.04 0.59
12.50 0.26
11.35 0.33
Mean SD 2.83 Mean SD 0.35
Table 2: The AMG positive values, Ag concentrations (μg/g, dry weight) estimated by cetacean histological Ag assay (CHAA), and Ag concentrations (μg/g, dry weight) measured by ICP-MS from the liver and kidney tissues ofsix stranded cetaceans. Gg = Grampus griseus, Ko = Kogia spp., Lh = Lagenodelphis hosei, Sa = Stenella attenuata.
Field number Species Liver Kidney
AMG CHAA* ICP-MS AMG CHAA* ICP-MS TP20111116 Gg 7.48 16.82 21.82 8.82 0.64 0.42 TC20110611 Ko 4.50 10.12 2.77 1.52 0.11 0.05 TC20110722 Ko 1.20 2.70 3.86 0.11 0.01 0.05 TD20110608 Lh 0.34 0.76 0.06 0.21 0.02 0.05 TP20110830 Lh 6.21 13.97 14.93 9.43 0.69 1.04 IL20110101 Sa 2.67 6.00 1.73 5.26 0.38 0.14
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) estimation of Ag concentrations by CHAA.
In this study, the data of ICP-MS were significantly and positively correlated with those of AMG positive values, suggesting that the Ag concentration in cetacean tissues can be estimated by the AMG positive value. Therefore, the CHAA, which is based on the AMG positive value and regression model, has been developed for estimating the Ag concentrations in the liver and kidney tissues of cetaceans. Generally, a regression model with more parameters (i.e., a more complex regression model) fits well into the data, but it is undetermined that the more complex one is actually better than the simpler one. Therefore, the best regression model must be chosen by statistical analysis26,27. The results of the statistical analysis indicate that the linear regression model is sufficient to estimate the Ag concentration based on the AMG positive value12.
In CHAA for kidney tissue, the mean SD (0.35) of the precision test was larger than that of the accuracy test (0.16). Conversely, in CHAA for liver tissue, the mean SD (2.8) of the precision test was smaller than that of the accuracy test (3.24). Based on this result, it is suggested that the uneven distribution of the AMG positive signals and the relatively low Ag concentrations in cetacean kidney tissue interfere negatively with the precision of CHAA for kidney tissue. Therefore, the CHAA for kidney tissue may be accurate but imprecise. However, the even distribution of the AMG positive signals and the relatively high Ag concentrations in cetacean liver tissues suggest that the CHAA for liver tissue is a reliable method to estimate the Ag concentrations in cetacean liver tissues. Furthermore, if more tissues with known Ag concentrations determined by ICP- MS are available, a more accurate and precise regression model can be developed to estimate the Ag concentration.
Although the current protocols provide an adjuvant method to investigate Ag in animal tissues, some limitations on the AMG method should be noted. First, false- positive AMG signals may present due to interference from other heavy metals, such as mercury, bismuth and zinc28. Therefore, the results of the AMG method have to be interpreted with other specific methods, such as ICP-MS, to monitor the actual composition of heavy metals28. Second, it is difficult to detect a homogenously distributed heavy metal because it may generate brighter amorphous AMG positive
signals, which may not be identified by visualization under microscopic examination.
Furthermore, the amorphous and brighter AMG positive signals are difficult to analyze with image analysis software because the color of the AMG positive signals may be similar to that of the background (e.g., the amorphous AMG positive signals found in the lumen of proximal renal tubules). Therefore, the AMG positive signals cannot be highlighted after the adjustment of the cut-off value of the threshold in the image analysis software. Third, because the AMG positive values are based on the percentage of the area of AMG positive signals, it is possible that the values of highly concentrated heavy metals may be underestimated.
FFPE samples are relatively easy to collect and store, and our previous study has demonstrated that the current AMG method can successfully amplify FFPE samples stored for over 15 years12. The mechanism of AMG is not affected by different animal species, for it has been wildly used in various animal species20,29-31. Although the current article is focused on the cetaceans, the protocols described here may also be used in different animal species. In addition, the cost of the AMG method with ICP-MS is relatively low (as compared to laser ablation-ICP-MS), and thus the current protocols are valuable for researchers or countries lacking sufficient research funding to
investigate the distribution and concentration of heavy metals in animal tissues. In conclusion, the use of AMG with quantitative analysis to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.
ACKNOWLEDGMENTS:
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 & Cetacean Research Center, National Cheng- Kung University. We also thank Forestry Bureau, Council of Agriculture, Executive Yuan for their permit. This study is partially supported by the Ministry of Science &
Technology, Taiwan under Grant MOST 106-2313-B-002-054-.
DISCLOSURES:
The authors have nothing to disclose.
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Chapter III: Investigation of Silver (Ag) Deposition in Tissues from Stranded Cetaceans by Autometallography (AMG) Environmental Pollution, 2018, 235: 534-545
Wen-Ta Lia, Hui-Wen Changa, Meng-Hsien Chenb, Hue-Ying Chiouc, Bang Yeh Lioua, Victor Fei Panga, Wei-Cheng Yangd* and Chian-Ren Jenga*
aGraduate Institute of Molecular and Comparative Pathobiology, National Taiwan University, Taipei, Taiwan; bDepartment of Oceanography and Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan; cGraduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan; dCollege of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan
*Correspondence: Wei-Cheng Yang. Email: [email protected]; Chian-Ren Jeng.
Email: [email protected]
