Scholarly article on topic 'Assessing the exposure to nanosilver and silver nitrate on fathead minnow gill gene expression and mucus production'

Assessing the exposure to nanosilver and silver nitrate on fathead minnow gill gene expression and mucus production Academic research paper on "Environmental engineering"

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Abstract of research paper on Environmental engineering, author of scientific article — Natàlia Garcia-Reyero, Cammi Thornton, Adam D. Hawkins, Lynn Escalon, Alan J Kennedy, et al.

Abstract Silver exposure is toxic to fish due to disturbances of normal gill function. A proposed toxicity mechanism of silver nanoparticles (AgNP) is derived from the release of silver ions, similar to silver nitrate (AgNO3). However, some datasets support the fact that AgNP can have unique toxic effects that are mediated at the gill. To determine if differences between AgNO3 and AgNP toxicities exist, fathead minnows were exposed to 20nm PVP- or citrate-coated silver nanoparticles (50.3μg/L PVP-AgNP; 56.0μg/L citrate-AgNP) or 3.81μg/L AgNO3 for 96h. These concentrations were applied to approximate the dissolved fraction of Ag in the AgNP suspensions. Mucus production in the water was measured. While mucus production was initially significantly increased in the first 4h of exposure in all silver treatments compared to control, a decrease in mucus production was observed following 24–96h of exposure. To determine which genes/pathways are driving this shift in mucus production, gills were dissected and microarray analysis was performed. Hierarchical clustering of differentially expressed genes revealed that all samples distinctly clustered by treatment. There were 109 differentially expressed genes shared among all Ag treatments compared to controls. However, there were 185, 423, and 615 differentially expressed genes unique to AgNO3, PVP-AgNP, and citrate-AgNP, relative to control. While functional analysis indicated several common enriched pathways, such as aryl hydrocarbon receptor signaling, this analysis also indicated some unique pathways between nanosilver and AgNO3. Our results show that AgNO3, PVP-AgNP, and citrate-AgNP exposure affected mucus production in fish gills and also lead to common and unique transcriptional changes.

Academic research paper on topic "Assessing the exposure to nanosilver and silver nitrate on fathead minnow gill gene expression and mucus production"

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Environmental Nanotechnology, Monitoring &

Management

journal homepage www.elsevier.com/locate/enmm

Assessing the exposure to nanosilver and silver nitrate on fathead minnow gill gene expression and mucus production

Natalia Garcia-Reyeroa b *, Cammi Thorntonc, Adam D. Hawkinsc, Lynn Escalon3, Alan J Kennedy2, Jeffery A. Steevensa, Kristine L. Willettc

a US Army Engineer Research and Development Center, Vicksburg, MS 39180, USA

b Institute for Genomics Biocomputing and Biotechnology, Mississippi State University, Starkville, MS 39759, USA

c Department ofBioMolecular Sciences and Environmental Toxicology Research Program, School ofPharmacy, University ofMississippi, University, MS 38677, USA

CrossMark

ARTICLE INFO

Article history: Received 17 December 2014 Received in revised form 26 May 2015 Accepted 1 June 2015

Keywords: Microarrays Silver nanoparticles Gene expression Fathead minnows Gills

Mucus production

ABSTRACT

Silver exposure is toxic to fish due to disturbances of normal gill function. A proposed toxicity mechanism of silver nanoparticles (AgNP) is derived from the release of silver ions, similar to silver nitrate (AgNO3). However, some datasets support the fact that AgNP can have unique toxic effects that are mediated at the gill. To determine if differences between AgNO3 and AgNP toxicities exist, fathead minnows were exposed to 20 nm PVP- or citrate-coated silver nanoparticles (50.3 ^g/L PVP-AgNP; 56.0 ^g/L citrate-AgNP) or 3.81 ^g/L AgNO3 for 96 h. These concentrations were applied to approximate the dissolved fraction of Ag in the AgNP suspensions. Mucus production in the water was measured. While mucus production was initially significantly increased in the first 4 h of exposure in all silver treatments compared to control, a decrease in mucus production was observed following 24-96 h of exposure. To determine which genes/pathways are driving this shift in mucus production, gills were dissected and microarray analysis was performed. Hierarchical clustering of differentially expressed genes revealed that all samples distinctly clustered by treatment. There were 109 differentially expressed genes shared among all Ag treatments compared to controls. However, there were 185,423, and 615 differentially expressed genes unique to AgNO3, PVP-AgNP, and citrate-AgNP, relative to control. While functional analysis indicated several common enriched pathways, such as aryl hydrocarbon receptor signaling, this analysis also indicated some unique pathways between nanosilver and AgNO3. Our results show that AgNO3, PVP-AgNP, and citrate-AgNP exposure affected mucus production in fish gills and also lead to common and unique transcriptional changes.

Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Engineered nanomaterials are being increasingly manufactured worldwide. Of those, silver nanoparticles (AgNP) are of particular interest due to the antimicrobial properties of silver. Nanosilver production has been estimated at 2.8-20 tons per year in the United States alone (Hendren et al., 2011). As a result, AgNP are appearing in an increasing number of commercial products such as textiles, athletic equipment, medical devices, keyboards, baby bottles, stuffed animals, and food containers (Seltenrich, 2013).

Abbreviations: AgNP, silver nanoparticles; DEG, differentially expressed genes; FHM, fathead minnows; NP, nanoparticles; PVP, polyvinylpyrrolidone.

* Corresponding author at: Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Starkville, MS, USA.

E-mail address: nvinas@igbb.msstate.edu (N. Garcia-Reyero).

These products can release both silver ions and AgNP into the environment creating potential toxicity to aquatic organisms (Fabrega et al., 2011). This is of substantial concern, especially given the high toxicity of silver to aquatic organisms, second only to mercury among metals (Seltenrich, 2013). Despite many recent studies suggesting that AgNP have the potential to cause toxicity in humans and wildlife (Christen et al., 2013; Fabrega et al., 2011; Garcia-Reyero et al., 2014; Hadrup et al., 2012; McCarthy et al., 2013; Powers et al., 2011a, 2010; Suliman et al., 2013; Xu et al., 2013), there is still significant uncertainty in both the scientific and regulatory communities regarding the specific biological impacts of such substances. Furthermore, while the potential adverse effects of nanoparticles (NP) in the environment at the population or ecosystem levels have been minimally explored, recent studies suggest that the impact could be more significant than previously suspected (Colman et al., 2013; Levard et al., 2012; Oberdorster et al., 2005; Pokhrel and Dubey, 2012; Unrine et al., 2012), stress-

http://dx.doi.org/10.1016/j.enmm.2015.06.001

2215-1532/Published by Elsevier B.V. This is an open access article underthe CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ing the need to understand the adverse effects of both ionic silver and NP in the environment. However, it also must be understood that the bioavailability of silver can be altered by transformations, photooxidation and interactions with environmental ligands (Kennedy et al., 2014). The reactivity of NP depends on complex physico-chemical properties such as size, agglomeration state, capping agent, dissolution kinetics, polydispersity, zeta potential, specific surface area or coating material. While toxicity due to AgNP is generally thought to be due to the dissolution of the silver from NP (Groh et al., 2014; Kennedy et al., 2010; Newton et al., 2013; van Aerle et al., 2013), several studies suggest that it cannot be explained exclusively by the effects of ionic silver (Garcia-Reyero et al., 2014,b; Powers et al., 2011a,b; Shaw and Handy, 2011). Differences may also relate to the extent to which ions are released, organism type and acute versus chronic exposure duration (Yang et al., 2012). Recent studies suggest that AgNP could be converted to more stable silver sulfides in certain oxygen-free environments such as wastewater plants. This conversion would significantly reduce the particles' ability to release silver ions and kill bacteria (Kaegi et al., 2013). Other studies also show the size or coating-dependent toxicity ofNP (Ahn et al., 2014; Ivasket al., 2014; Shang et al., 2014), or even how the exposure medium, such as content of chloride ions (Groh et al., 2014), or dissolved organic carbon (Kennedy et al., 2012) can influence NP toxicity. This information stresses the need to understand the potential specific toxicity of the NP either due to their shape, coating, or to their ability to release silver ions.

The anatomy of fish gills reflects their primary function as gas-exchange, osmoregulatory, and excretory organ (Di Giulio and Hinton, 2008). The physiological and anatomical features of fish gills that promote efficient respiration also contribute to the uptake of xenobiotics and other compounds directly from water (Di Giulio and Hinton, 2008). Fish then use mucus hypersecretion as a response to the stress (Shephard, 1994) provoked by toxicants and irritants like metals (McDonald and Wood, 1992), carbon nano-tubes (Smith et al., 2007), TiO2 NP (Federici et al., 2007), AgNO3 and AgNP (Bilberg et al., 2012; Bilberg et al., 2010; Bilberg et al., 2012, 2010; Hawkins et al., 2014b). Mucus hypersecretion protects not only by trapping and sloughing chemicals, but also by bringing innate immune proteins to pathogens (Mallatt, 1985). Nevertheless, concerns about the potential ability of mucus to eventually enhance toxicity have also been raised. If the mucus layer becomes more rigid and/or if chemical stressors such as NP are concentrated at the gill surface (Lichtenfels et al., 1996), toxicity could be increased. Alternatively, altered mucus homeostasis could adversely affect ionic regulation and a gas exchange.

Here we exposed fathead minnows (Pimephales promelas, FHM) to polyvinylpyrrolidone (PVP)-coated AgNP, citrate-coated AgNP, or silver nitrate (AgNO3) in order to examine whether observed effects were due to AgNP, dissolved silver, or a combination of the two and to compare the effects of the two different coatings. We examined the mucus production after initial chemical insult (4 and 28 h) and transcriptional changes in gill after 96 h of exposure using microarrays. Microarrays have the potential to give information about mechanism of action for specific classes of chemicals and provide what is known as compound signature. The parallel analysis of multiple biochemical pathways at the mRNA level can provide a systems-wide understanding of toxicity that can be correlated to phenotypic changes (reviewed in (Denslow et al., 2007)).

2. Material and methods

2.1. Fish source, care and handling

Male fathead minnows (P. promelas) at eight months old were obtained from Aquatic Bio Systems (Fort Collins, CO, USA) and

Table 1

Linear regression of mucus production for 1-4 h post dosing. Slopes within the same column with different letters are statistically different (n = 5).

Treatment Day 0 Day 1

Slope R2 Slope R2

Control 0.1093±0.0053a 0.948 0.1623 ±0.0101a 0.918

3.8 AgNOa 0.3403±0.0157b 0.953 0.0127±0.0029b 0.454

50 PVP 0.3369±0.0163b 0.949 0.0402±0.0042c 0.796

56 Citrate 0.3664±0.0104b 0.982 0.0081 ±0.0008b 0.835

cultured according to University of Mississippi IACUC approved conditions. The fish were allowed to acclimate in glass chambers containing 1.5 L of moderately hard water (MHW) prepared according to U.S. EPA guidelines (Technology, 2002) for four days prior to the exposure. During acclimation, water was changed daily and fish were fed daily with Tetramin flakes.

2.2. Silver

PVP-AgNP and citrate-AgNP were obtained from Nano Com-posix (San Diego, CA, USA) at a concentration of 1 mg/ml and a nominal size of 20 nm. Stock preparation and particle characterization have been previously described by (Hawkins et al., 2014a). Briefly, primary particle size was determined by transmission electron microscopy and image analysis of particle diameter and hydrodynamic diameter was determined by dynamic light scattering (DLS) and field flow fractionation (FFF). Silver nitrate was obtained from Sigma-Aldrich (St. Louis, MO, USA) prepared in distilled deionized water and diluted to a working stock with a nominal concentration of 10 |ig/ml.

2.3. Exposure

Fathead minnows were exposed to control, PVP-AgNP and citrate-AgNP at 50.3 or 56.0 |ig/L, respectively, or silver nitrate (AgNO3) at 3.81 |g/L for 96h (measured concentrations; n = 5 jars/treatment; 3 fish/jar; 1.5 L water/jar). The selected doses were targeted to provide a similar dissolved Ag concentration in the PVP-AgNP and citrate-AgNP treatments, when compared to the concentration of AgNO3. Exposure water was changed and redosed daily. The fish were fed once at 48 h, 30 min before water change. Water quality was 289 ±2 |S/cm, 231 ±1 ppm TDS, 26.4 ±0.8 °C, 151 ± 1 ppm salinity, and pH 8.09±0.3. At 96h, the fish were euthanized with buffered MS-222. Body weight and length were recorded. Gills were removed, preserved in RNA later and stored at -80 °C until analysis.

2.4. Mucus water concentrations

The estimation of mucus water concentration was done by using the phenol sulfuric acid assay protocol established by (DuBois et al., 1956) and modified for this application by (Parrish and Kroen, 1988). Glucose was used as a standard curve to represent the mucus carbohydrate content. Details on this assay have been reported by (Hawkins et al., 2014b). Briefly, water samples (n = 5 tanks/treatment/day) were collected on day 0 (at 0-4 h) and day 1 (immediately following water change and redosing; at 24-28 h) of the exposure.

2.5. Water sampling and ¡CP-MS analysis

Water samples were taken immediately following dosing to confirm Ag concentration (n = 5/treatment/collection). Details on water sampling for total and dissolved Ag and ICP-MS analysis have been described in (Hawkins et al., 2014a).

-m- Control b

-m- 3.8 AgNO3

50 PVP

56 Citrate c /b

"b —•

Fig. 1. Mucus water concentration. Amount of mucus present in water after exposing FHM to Ag at 0,1, 2,3, and 4 hours post dosing on day 0 and day 1.

2.6. Microarray analysis

Total RNA was isolated from individual gill samples using RNA extraction kits (RNeasy, Qiagen, Valencia, CA, USA). Microfluidic gel electrophoresis was used to assess RNA degradation (Agilent 2100 Bioanalyzer, Agilent, Santa Clara, CA, USA) and quantity was determined using a Nanodrop® ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA was stored at -80°C until analyzed. All treatments had 5 biological replicates except for the PVP treatment, which had 4 samples, and the citrate treatment, which only had 3 available samples. One of those 3 samples was hybridized twice to serve as a method control. RNA was analyzed using custom fathead minnow 60,000 gene arrays (GPL15775) (Garcia-Reyero et al., 2014). One |g total RNA was used for all hybridizations. Probe labeling, amplification and hybridization were performed using kits following the manufacturer's protocols (Quick Amp Labeling kit and one-color microarray hybridization protocol, v6.5; Agilent) and scanned with a highresolution microarray scanner (Agilent). Data were resolved from microarray images using Agilent Feature Extraction software v10.7 (Agilent). Raw microarray data from this study have been deposited at the Gene Expression Omnibus website (http://www.ncbi.nlm. nih.gov/geo/; GSE64259).

2.7. Bioinformatics

Raw microarray data was imported into the analysis software GeneSpring version GX11 (Agilent Technologies, Santa Clara, CA), and normalized using quantile normalization followed by median scaling across all samples. One-way Analysis of Variance (ANOVA) was performed followed by pair-wise comparison (p<0.05) to identify differentially expressed genes. Hierarchical clustering was performed with GeneSpring. Functional analysis and identification of upstream regulators of pathways was performed using the human ortholog genes of fathead minnow differentially expressed genes and the software Ingenuity Pathway Analysis (IPA, Redwood City, CA). Venn diagrams were constructed using Venny (http:// bioinfogp.cnb.csic.es/tools/venny/index.html).

2.8. Real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) was performed on the same samples used for microarray analysis. Briefly, 800 ng of total RNA was used to synthesize DNase-treated cDNA in a 20 |L reaction containing 250 ng of random primers and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The qPCR assays were per-

formed on an ABI Sequence Detector 7900 (Applied Biosystems, Foster City, CA). Each 20 |L reaction was run in duplicate and contained 6 |L of synthesized cDNA template along with 2 |L of each forward and reverse primer (5 |M/|L) and 500 nM SYBR Green PCR Master Mix (Applied Biosystems). Cycling parameters were 95 °C for 15 min, 40 cycles of 95 °C for 15 s, and 60°C for 1 min. Primers were designed using Primer Express (Applied Biosystems) and synthesized by Operon Biotechnologies (Huntsville, AL, USA) and have been published elsewhere (Garcia-Reyero et al., 2014). Results were normalized to 18S rRNA and analyzed using the AACt method (Applied Biosystems, Foster City, CA, USA) (Bobe et al., 2009; Filby, 2005; Garcia-Reyero et al., 2009; Martyniuk et al., 2013; Rime et al., 2010).

3. Results

3.1. Nanoparticle and exposure characterization

Nanoparticles characterization has been previously reported (Hawkins et al., 2014a). The primary particle size and hydro-dynamic size of the nominally 20 nm PVP-AgNPs were 22 ±2 by TEM and 34.8 ±0.4 by DLS. The primary particle size and hydrodynamic size of the citrate-AgNPs were 21 ± 4 by TEM and 29.5 ± 0.1 nm by DLS, respectively. Total and dissolved silver water concentrations for all treatments are shown in Supplementary Information Fig. 1. The total AgNP concentrations were not significantly different for PVP (50 ±4.7 |g/L) and citrate-NPs (56 ± 2.9 | g/L) but they were significantly higher than the AgNO3 concentration (3.8 ± 0.23 | g/L). None of the dissolved Ag fraction concentrations (range 0.99-1.3 |g/L) were significantly different between the three silver treatments.

3.2. Mucus production

The mucus concentration in water for all treatments is shown in Fig. 1. After 1-4 h of exposure, mucus concentrations significantly increased in all silver treatments when compared to control. However, after 25-28 h of exposure, silver-treated fish had significantly lower mucus release compared to controls Table 1. Unfortunately, we were not able to measure mucus concentrations at 96 h, but we would expect these concentrations to also be decreased compared to control, as it was shown in a similar exposure with the same NPs (Hawkins et al., 2014b).

Fig. 2. Hierarchical clustering of DEGs (p <0.05) in gill of FHM exposed to PVP-AgNPs, citrate-AgNP or AgNO3. Legend shows colors corresponding to intensity.

3.3. Gene expression

Transcriptional analysis was performed on five fish for the control and AgNO3 treatments, four fish for the PVP-AgNP treatment, and three fish for the citrate-AgNP treatment (due to quality of the samples, therefore one of the samples was hybridized twice). Identified differentially expressed genes are shown in Supplemental Material Tables S1-S3. Hierarchical analysis of all samples showed very distinct gene expression patterns effects for all treatments in gills of exposed fish (Fig. 2). A total of 462 genes were differentially expressed after AgNO3 exposure, 740 were differentially expressed after PVP-AgNP exposure, and 880 were differentially expressed after citrate-AgNP exposure compared to control. Of those, 109 were common among the three treatments. PVP-AgNP and AgNO3 treatments had the most differentially expressed genes (DEGs) in common (219), followed by 207 common genes between the two types of NP, and only 167 were common between AgNO3 and citrate-AgNP (Fig. 3a). Among the common genes, we found hsp90a1 (heat-shock protein 90a1), cyp26a1 (cytochrome p450 26 a1), cyp27a7, ormap2k7 (mitogen-activated protein kinase kinase 7). Real-time qPCR results can be found in Table 2.

Table 2

Real-time quantitative PCR results. Values are fold change relative to control. Only significant values are shown (p < 0.05).

Gene name and symbol

Cytochrome p450 1A (cyp1a)

Cytochrome p450 3A (cyp3a)

Hypoxia inducible factor 1 (hif1a) Flotilin (flot) Signal transducers and activators of transcription family 1 (stat1)

Signal transducers and activators of transcription family 3 (stat3)

Tumor protein p53 (tp53)

Superoxide dismutase 3 (sod3)

PVP-AgNP

Citrate-AgNP

1.4 1.4

2.6 1.7

Fig. 3. (a) Common and unique number of differentially expressed genes among all treatments (p <0.05). (b) Common and unique number of significant pathways among all treatments (p < 0.05).

Analysis of pathways, which are generally more common between treatments because they are composed of several genes, indicated that 27 pathways were common between all treatments (Fig. 3b). Those include some signaling pathways such as aryl hydrocarbon receptor (AhR), ATM, p53, apoptosis, or GADD45 signaling, as well as some pathways related to immune response, such as regulation of 1L-2 expression in lymphocytes, or role of 1L-17F in allergic inflammatory airway diseases (see Supplementary information, Tables S4-S6 for details). Twenty-eight pathways were unique in the AgNO3 exposure, including several related to immune response. Twenty pathways were only significantly enriched after PVP-AgNP exposure, and 63 after citrate-AgNP exposure, including ERK/MAPK signaling and several related to estrogen signaling, such as estrogen receptor signaling. All treatments had a transcriptional effect on FHM gills. However, citrate-AgNP had the most impact at the transcriptional level.

The analysis of toxicity lists, another categorization developed by 1PA, identified 10 common toxicity lists among all treatments (Fig. 4, Supplementary Information Tables S7-S9). Interestingly, this categorization also identified similar signaling pathways such as AhR and p53 signaling, and lists related to PPARa activation. Both a pathway and a toxicity list related to hypoxia were significantly enriched in both the AgNO3 and the PVP-AgNP treatments, but not the citrate-AgNP. Seven toxicity lists were unique in the AgNO3 exposure, including NRF2-mediated oxidative stress and TGF-P signaling. Three toxicity lists were unique in each of the NP exposures, including pro-apoptosis for PVP-AgNP and RAR activation or NF-kB signaling in the citrate-AgNP exposure.

4. Discussion

The fish gill is a complex organ where many interconnected physiological processes take place. These processes are essential to maintaining homeostasis in the case of changing internal or environmental conditions. 1n recent years, the fast development of molecular techniques has greatly improved our understanding of specific pathways and mechanisms involved in these processes, many of which are similar to those found in mammals. However, important differences do exist. The general morphometric and irrigation/perfusion constraints of gas exchange in fish are mediated by structures and processes similar to those in mammals, although they have been adapted to the specifics of aquatic environment and mediated by gills rather than lungs (Evans, 2005). The gill is also a major receptor of the damage produced by environmental pollut-

ants, and it is believed to be a site for metabolism and/or excretion of toxins (Evans, 2005; Olson, 1998). We therefore explored mucus production after 4 and 28 h of exposure to AgNO3, PVP-AgNP, and citrate-AgNP. In order to better understand the molecular changes after a longer exposure that could lead to potential adverse effects, we also analyzed transcriptional changes in gills after 96 h of expo-

4.1. Mucus production

Mucus production was increased by all treatments after 1-4 h of exposure, (Fig. 1 ). However, by 25 h of exposure mucus production significantly decreased. This is consistent with a parallel exposure (Hawkins et al., 2014b), where mucus production increased in response to initial stress by all treatments (AgNO3, PVP-AgNP, and citrate-AgNP), but by day three of the exposure the high dose PVP-AgNP and citrate-AgNP mucus production rates were only 26 and 29% of the rates in the first four hours of the exposure. Mucus production remained low after 96 h of exposure (Hawkins et al., 2014b). Therefore, while we were unable to test mucus production after 96 h of exposure, we would expect it to be decreased by all treatments. (Hawkins et al., 2014b) suggested that reduced mucus concentration could be the result of the formation of a thickened mucus layer after silver exposure, which would make it more difficult for fish to slough and renew, resulting in accumulation of chemicals and microorganisms. They also observed that after 96 h of silver exposure, mucus-secreting goblet cells were degenerated (Hawkins et al., 2014a), and therefore, hypothetically were less capable of mucus production which in turn could prevent the gills from removing silver and thus increase toxicity.

Another study (Bilberg et al., 2010) showed that the critical oxygen tension (Pcrit) of perch (Perca fluviatilis) was greatly increased after exposure to AgNO3 and PVP-AgNP, suggesting a respiratory disturbance and seriously impairing the hypoxia tolerance of perch. This would be consistent with an initial mucus hypersecretion, as increased gill mucus production increases the oxygen water-blood diffusion distance and elevates Pcrit (Ultsch and Gros, 1979). Additionally, AgNP and AgNO3 could inhibit oxygen diffusion by precipitating on the perch gills, as AgNO3 has been shown to accumulate in rainbow trout gills (Morgan and Wood, 2004). Mucus hypersecretion, increased ventilation rate, and increased surface respiration have also been observed in zebrafish (Danio rerio) exposed to PVP-AgNP (Bilberg et al., 2012).

Fig. 4. Common and unique number of significantly enriched Toxicity lists (p < 0.05).

4.2. Gene expression

Many of the altered genes and pathways, including the most enriched pathway in all treatments, cell cycle control of chromosomal replication, were related to cell cycle, apoptosis, and cell proliferation. When these pathways are considered with our previously performed FHM studies at the same time point and similar exposure concentrations, these altered pathways are consistent with the histopathological alterations in the gill including regressive alterations such as mucous cell degeneration and epithelial desquamation and progressive alterations including hypertrophy and new cell generation in hyperplasia (Hawkins et al., 2014a). Gene expression analysis showed that while some genes and functional categories were specific for each treatment, there was a high percentage of genes and pathways common to the three treatments (Fig. 3). The common pathways and toxicity lists included AhR signaling, p53 signaling, GADD45 signaling, role of IL-17F in allergic inflammatory airway diseases, genes up-regulated in response to proteinuria-induced oxidative stress in renal proximal tubule cells, or renal necrosis/cell death (Supplementary Tables 4-9). Despite the fact that fish have functional kidneys, the gill actually performs most of the functions controlled by pulmonary and renal processes in mammals. As a consequence, many of the pathways that mediate these processes in mammalian renal epithelial are expressed in the gill, and many of the modulators of these processes are also found in fish endocrine tissues and the gill itself (Evans, 2005). Other renal-related pathways or toxicity lists (such as increases renal proliferation or acute renal failure panel) were significant in the AgNO3 treatment only. Lung and kidney-related significant pathways in the AgNP treatments included persistent renal ischemia-reperfusion injury, renal proximal tubule toxicity biomarker panel, renal cell carcinoma signaling, small cell lung cancer signaling, or renal proximal tubule toxicity biomarker panel. Another common pathway

in all treatments was cell cycle: G2/M DNA damage checkpoint regulation. Interestingly, AgNPs intensify DNA damage and G2/M cell cycle arrest in human renal epithelial cells (Kang et al., 2012).

AhR signaling and hypoxia-related pathways and toxicity lists were also significantly enriched in all treatments. The AhR is a ligand-activated transcription factor that mediates many of the responses to environmental pollutants. In order to regulate gene expression, AhR requires the co-activator ARNT (AhR nuclear translocator), which is also required by the hypoxia-inducible factor 1 a (HIF1 a), a crucial regulator in hypoxic conditions. The role of ARNT in both pathways establishes the foundation for a crosstalk between these two crucial pathways. Thus, hypoxic environments that affect HIF1 a and activation of the AhR signaling pathway, could interfere with HIF1a-mediated responses (Vorrink and Domann, 2014). Consistent with the above-mentioned effects, a previous study showed that mammalian HIF1 a was activated in vitro in a transcriptional activation assay by PVP-AgNP and the hypoxia-inducible factor signaling pathway was activated in vivo by AgNO3 and PVP-AgNP (Garcia-Reyero et al., 2014). Other researchers have shown the transcriptional activation of HIF1 a after exposure to NP (Lim et al., 2009; Pietruska et al., 2011). Another study analyzing the effects of AgNP on an ovalbumin-induced murine model of allergic airway disease found that increased levels of HIF1 a, vascular endothelial growth factor (VEGF), phosphatidylinositol (PI3K) and phosphorylated Akt levels were significantly decreased by AgNP. They showed that AgNP substantially suppressed mucus hypersecretion and PI3K/HIF1 a/VEGF signaling pathways, suggesting a link between the hypoxia signaling pathways and the decreased mucus production detected on fathead minnows after 48 h of exposure (Park et al., 2012). The crosstalk between AhR and HIF1 a could also potentially lead to increased toxicity if the effects on hypoxia-signaling pathways alter the ability of the AhR signaling pathway to mediate chemical toxicity.

As mentioned earlier, the gills are a site for metabolism and excretion of toxins (Evans, 2005; Olson, 1998), and cytochrome p450 enzymes (CYPs) have been localized in gills (Evans, 2005; Miller et al., 1989). CYP enzymes catalyze oxidative transformation that leads to activation or inactivation of chemicals, endogenous and exogenous, with consequences for normal physiology and health (Goldstone et al., 2010). Several CYPs were found differentially expressed in all treatments, mostly up-regulated except for one (cypldl in the citrate-AgNP exposure). The only CYP up-regulated in all treatments was cyp27a7, while cyp27a1 was up-regulated in both AgNP treatments. The Cyp27 family is involved in vitamin D3 metabolism in mammals and seven distinct cyp27 genes have been identified in zebrafish (Goldstone et al., 2010). Moreover, cyp27 has been identified in mammals as an element of monocyte-macrophage transition, is induced during macrophage development, and is transcriptionally regulated by both retinoids and PPAR ligands (Nagy et al., 2012; Szanto et al., 2004). These facts would be consistent with AgNO3 and AgNP effects on immune response and activation of PPAR/RXR signaling pathways. Consistently, another study (Gagné et al., 2013) showed that exposure of the freshwater mussel Elliptio complanata to AgNO3 and AgNP resulted in immunotoxicity and increased phagocytosis.

Enrichment of p53 signaling pathways was consistent with previous studies that also showed up-regulation of that pathway in FHM liver after AgNO3 and PVP-AgNP exposure (Garcia-Reyero et al., 2014). Another major effect of dissolved silver is the production of reactive oxygen species (ROS) which has been linked to oxidative stress (Chae et al., 2009; Cortese-Krott et al., 2009; Foldbjerg et al., 2012; Handy et al., 2008; Lim et al., 2012). Increased ROS activates MAPK signaling pathways leading to a number of cellular responses including increased proliferation, differentiation, inflammatory responses, and apoptosis (Son et al., 2011). These data are consistent with the fact that many of the enriched functional categories were related to cancer/apoptosis and immune response.

Another common pathway differentially impacted by all silver treatments was PPARa/RXRa activation. This is consistent with previous results that showed that pathway was activated in FHM exposed to AgNO3 and PVP-AgNP. Furthermore, PVP-AgNP directly interacted with the mammalian PPARa nuclear receptor in vitro (Garcia-Reyero et al., 2014). The agreement of effects between mammalian in vitro assays and significantly enriched pathways provides strong evidence that PVP-AgNP can directly interact with transcription factors and receptors and cause biological effects.

Several pathways common among all treatments were related to DNA damage and DNA repair, consistent with the effects other researchers saw on zebrafish gills (Griffitt et al., 2013). While in this work we mostly focused on common pathways by the three treatments, there were also some clear and distinct transcriptional effects by each treatment, consistent with the hypothesis that not all effects are due to the release of ionic silver. Fig. 4 shows the common and distinct toxicity pathways enriched by treatment. At this functional level, a high percentage of the toxicity lists affected were common either by all treatments, or shared by at least two treatments. However, each treatment had specific and unique enriched toxicity lists.

Some of the pathways significantly enriched only after AgNO3 exposure include NRF2-mediated oxidative stress, TGF-P signaling, or increased renal proliferation. A major effect of dissolved silver is the production of reactive oxygen species (ROS), which has been linked to oxidative stress (Cortese-Krott et al., 2009; Foldbjerg et al., 2009). Consistently, silver has been shown to induce NRF2 oxida-tive stress in a cell type-dependent manner in mammalian cells (Simmons et al., 2011). Increased ROS activates MAPK signaling pathways, which would lead to a number of cellular responses such

as proliferation, differentiation, and inflammatory responses (Son et al., 2011). This would be consistent with many of the unique pathways in the AgNO3 exposure related to inflammation and the immune system as well as proliferation. Interestingly, two MAPK were differentially expressed in the AgNO3 exposure (mapklO, and mapkll), three in the citrate-AgNP exposure (mapk3, mapklO, and mapk13), and none in the PVP-AgNP exposure, suggesting that the release of silver from each NP might be different, or that citrate-AgNP might be activating the MAPK transcription by other alternate mechanisms. Nevertheless, some of the unique pathways enriched after PVP-AgNP exposure are related to cancer, apoptosis, and immune system, including endometrial cancer signaling, complement system, and Myc-mediated apoptosis signaling.

The unique pathways in the citrate-AgNP exposure include circadian rhythm signaling, calcium signaling, and several estrogen receptor-related pathways such as estrogen receptor (ER) signaling. Citrate-AgNP did not directly affect the transcription of the ERs in the gills, but had an overall effect on estrogen receptor-related pathways. Exposure of the freshwater mudsnail Potamopyrgus antipodarum to AgNP decreased its reproduction and led to increased estrogenic effects of ethinylestradiol (EE2), an ER agonist, at concentrations that had no influence on reproduction when applied in absence of nanosilver (Volker et al., 2014). This information would suggest that there might be some effect of AgNP on ER signaling, which might be dependent on the tissue, species, and particle size or coating.

Real-time qPCR of genes previously shown to be affected by either AgNO3 or PVP-AgNP in fathead minnow are shown in Table 2. Interestingly, only one of the measured genes is changed in the AgNO3 exposure, while the most changes are in the AgNP exposures. Data also confirm that transcriptional changes can also vary by tissue, as previous exposures have shown (Garcia-Reyero et al., 2014). Flotillin l, a protein previously proposed to be involved in NP transport into cells (Poynton et al., 2012), was significantly increased only in the PVP-AgNP exposure. Hifla was significantly up-regulated in both NP exposures, but not in the AgNO3. These results differ from previously shown data where hif1a was up-regulated in both silver and PVP-AgNP exposures, further suggesting that the effects of AgNO3 and AgNP at the transcriptional level may differ by tissue (Garcia-Reyero et al., 2014).

5. Conclusions

Our results show that AgNO3, PVP-AgNP, and citrate-AgNP exposure affected mucus production on FHM gills. All treatments behaved in a similar way: increasing mucus production in the initial 4 hr of exposure, but significantly decreasing it after 28 h. Silver treatments also lead to common and unique transcriptional changes on the gills of FHM, suggesting that the effects may not be solely related to ionic silver release from NP. Common functional categories affected by all exposures were AhR signaling, p53 signaling, and PPARa/RXRa activation. These data combined suggest somewhat different mechanisms of toxicity associated with inflammation responses at the gill following exposure to dissolved Ag and AgNP.

Acknowledgements

This work was funded by the US Army Environmental Quality Research Program (including BAA 11-4838 to NGR and #W912HZ-09-C-0033 to KLW). Permission was granted by the Chief of Engineers to publish this information.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enmm.2015.06. 001

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