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 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.
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 compounds12,13,14. 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.
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. Please click here to view a larger version of this figure.
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.
2. Tissue Sample Preparation for AMG Analysis
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.
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. Please click here to view a larger version of this figure.
4. Establishment of the Cetacean Histological Ag Assay (CHAA) by Regression Model
Note: The following analysis is performed in Prism 6.01 for Windows.
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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
5. Estimation of Ag Concentrations by CHAA.
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 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 byinductively coupled plasma mass spectroscopy (ICP-MS): 21.82 μg/g dry weight). (B) The AMG positive signals are brown to black granules of various sizesin 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 thecytoplasm of the proximal renal tubular epithelium (red arrows) (Ko; field code: TC20110722;Ag concentration measured by ICP-MS: 0.05 μg/g dry weight). Please click here to view a larger version of this figure.
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 | |||
Precison 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 | |||
*The regression equations of the CHAA for livers and kidneys were respectively Y = 2.249 × X (adjusted R2 = 0.74) and Y = 0.07288 × X (adjusted R2 = 0.69). |
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.
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 |
*The regression equations of the CHAA for livers and kidneys were respectively Y = 2.249 × X (adjusted R2 = 0.74) and Y = 0.07288 × X (adjusted R2 = 0.69). |
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 of six stranded cetaceans. Gg = Grampus griseus, Ko = Kogia spp., Lh = Lagenodelphis hosei, Sa = Stenella attenuata.
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 species20,29,30,31. 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.
The authors have nothing to disclose.
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 the Forestry Bureau, Council of Agriculture, Executive Yuan for their permit.
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 |