Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus g

RESEARCH ARTICLE

Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis

Younes Bouallegui, Ridha Ben Younes, Faten Turki and Ridha Oueslati

Research Unit for Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of Bizerte, University of Carthage, Bizerte, Tunisia

ABSTRACT Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanopar- ticles (AgNP: <50 nm and <100nm) on the frequency of hemocytes subpopulations as immunomodula- tion biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over dif- ferent durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant varia- tions in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that dif- fering internalization mechanisms could be key factors impacting on the potential for NP in the environ- ment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.

ARTICLE HISTORY Received 13 February 2017 Revised 6 May 2017 Accepted 24 May 2017

KEYWORDS Silver nanoparticles; endocytosis; hyalinocytes; granulocytes; Pappenheim panoptical staining

Introduction

Nanoparticles (NP) are defined as materials with all dimensions in nanoscale [1–100 nm] (Luoma 2008). Silver nanoparticles (AgNP) have become the fastest growing product category in nanotechnology due to their thermoelectrical conductivity, cata- lytic activity and nonlinear optical behavior and have great value in the formulation of inks, microelectronic products and biomed- ical facilities (i.e. imaging devices) (Tiede et al. 2009; Katsumiti et al. 2015). Their exceptional broad-spectrum bactericidal prop- erties and biocompatibility (i.e. as drug delivery agent) have also made AgNP extremely useful in a diverse range of consumer goods (Luoma 2008; Rainville et al. 2014; Cozzari et al. 2015; Katsumiti et al. 2015; Marisa et al. 2016).

Worldwide AgNP production is estimated at � 55 tonne/yr (Piccinno et al. 2012). However, release of AgNP into aquatic environs can happen through wastewaters generated during AgNP synthesis and/or incorporation into goods and consumer products (Canesi et al. 2012; Matranga & Corsi 2012; Katsumiti et al. 2015; Marisa et al. 2016). As such, AgNP have emerged as potential stressors that might enter marine environment (Luoma 2008). A lack of appropriate tools to evaluate effective NP (of AgNP in particular) levels in aquatic environments make selection of appropriate testing levels a major problem in risk assessment of engineered NP. As a result, predicted environmen- tal concentrations for AgNP are often set at a level of � 0.01 lg/L (Tiede et al. 2009; Katsumiti et al. 2015). Even so,

levels much lower than that have commonly been used in aquatic species ecotoxicity tests (1–100 lg/L) (Tiede et al. 2009; Canesi & Corsi 2016), including those with mollusk models.

In the mussel Mytilus galloprovincialis (filter-feeding organ- ism), hemocytes are hemolymph cells responsible for immune defence and serve as a first line of defence against foreign substan- ces (Gosling 2003; Parisi et al. 2008; Giron-Perez 2010; Matozzo & Bailo 2015). Immune defences carried out by hemocytes constitute important targets for potential NP toxicity (Canesi et al. 2012; Canesi & Prochazkova 2013; Katsumiti et al. 2015).

Several studies have shown that different NP types, that is, car- bon black, C60 fullerenes, TiO2, SiO2, ZnO, CeO2, Cd-based, Au- based and Ag-based, are rapidly taken up by hemocytes. Internalization of these NP subsequently impacted on morpho- logic/functional characteristics including immune responses (Canesi et al. 2008, 2010a, b, 2012; Katsumiti et al. 2015; Marisa et al. 2016). Various mussel hemocyte parameters, including total hemocyte count (THC), differential hemocyte count (DHC), hemocyte viability, phagocytic activity and lysosomal membrane stability, have been used as a tool for screening of immunomodu- latory effects of differing NP (Matozzo et al. 2007; Parisi et al. 2008; Hoher et al. 2013; Matozzo & Bailo 2015; Canesi & Corsi, 2016; Marisa et al. 2016). Specifically, hyalinocytes and granulo- cytes have been assessed for morphological changes among hemo- cytes in Mytilus galloprovincialis (Pipe et al. 1997; Chang et al. 2005; Garcia-Garcia et al. 2008).

CONTACT Younes Bouallegui [email protected] Research Unit of Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of Bizerte, Zarzouna 7021, Bizerte, Tunisia � 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF IMMUNOTOXICOLOGY, 2017 VOL. 14, NO. 1, 116–124 https://doi.org/10.1080/1547691X.2017.1335810

While granulocytes are large ovoid-shaped cells with a small eccentric nucleus and granulated cytoplasm (low nucleus/cyto- plasm [N/C] ratio) that are able to spread out and produce pseudo- podia), hyalinocytes are small round cells with an agranular (zero- few granules) small cytoplasm surrounding a large nucleus (high N/C ratio) (Carballal et al. 1997; Parisi et al. 2008; Cima 2010; Matozzo & Bailo 2015). Overall, hemocytes can be classified into two types, granulocytes and hyalinocytes (so-called agranulocytes), based on morphological characteristics (the presence/absence of granules in cytoplasm). Staining of the cytoplasm by certain dyes allows for sub-distinguishing of acidophils from basophils among the granulocytes. Ultimately, the basophils of M. edulis appear as granulocytes with small granules, while acidophilic granulocytes contain large granules. In comparison to the granulocytes, hyalino- cytes in bivalve have only basophilic properties. Thus, in earlier studies that described hemocyte subpopulations, the author indi- cated that basophilic cells (hyalinocytes þ basophils) made up about 40% of the total hemocyte pool in bivalves/mussels while eosinophils accounted for the remaining � 60% of all hemocytes (Chang et al. 2005; Garcia-Garcia et al. 2008).

Cellular uptake by endocytosis (clathrin- or caveolae-mediated routes) are crucial for a variety of cellular and physiological activities (i.e. nutrient uptake, immune defence) (Haucke 2006; Sandvig et al. 2011); each has also been identified as potential means for NP entry into cells (Moore 2006; dos Santos et al. 2011; Khan et al. 2015). Clathrin-dependent endocytosis involves formation of a clathrin (protein)-coated pit used in enzymatic destruction of internalized contents. Caveolae-dependent endo- cytosis occurs via cell-surface flask-shaped invaginations enriched with caveolin (cholesterol-binding proteins) (Nichols & Lippincott-Shwartz 2001; Razani & Lisanti 2002) that permit sub- cellular movements of ingested materials through a series of endosomal compartments of increasing acidity allowing for hydrolytic breakdown (Moore 2006; Puthenveedu & von Zastrow 2006; Doherty & McMahon 2009). Each route can be modified with inhibitors (Moore 2006; Ivanov 2008; dos Santos et al. 2011; Khan et al. 2015). Clathrin-mediated endocytosis could be inhib- ited by the antiviral amantadine through disruption of the cla- thrin coat, while antibiotic nystatin can impact on cholesterol- rich microdomains of caveolae-mediated endocytosis (Ivanov 2008; Khan et al. 2015).

In this context, the present study aimed to record the vari- ation in the percentages of circulating subpopulations of hemo- cytes, using as method differential hemocytes count [DHC] after Pappenheim’s panoptical staining [MGG] to: (1) assess effects of AgNP on circulating hemocyte sub-populations; (2) establish a relationship linking length of exposure to different size AgNP and variations in sub-populations [DHC]; and (3) evaluate the role of uptake pathways (clathrin- and caveolae-dependent endo- cytosis) – as well as changes in their function – in the effect of NP on circulating hemocyte subpopulations.

Material and methods

Silver nanoparticles (AgNP) source and characterization

Poly-vinyl-pyrrolidone (PVP)-coated AgNP of <100 nm (99.5% pure) were purchased from Sigma (Steinheim, Germany). PVP- coated AgNP <50 nm were produced by a modified process wherein AgNO3 (Sigma) was dissolved in ethylene glycol (EG) solvent (ACROS Organics, 98%, Geel, Belgium) in the presence of PVP (K30, Sigma) as a capping agent (Mezni et al. 2014a,b).

A stock solution of each AgNP size was suspended in artificial seawater (ASW; 58.5% NaCl; 26.5% MgCl2; 9.8% Na2SO4; 2.8%

CaCl2; 1.65% KCl; 0.5% NaHCO3; 0.24% KBr; 0.07% H3BO3; 0.0095% SrCl2; 0.007% NaF (Pinsino et al. 2015)). Prior to use, each AgNP stock was mixed several times and an aliquot removed as a working solution that was sonicated 15 min in alternating cycles (2 � 30 s) in an ultrasonic bath (VWR, Strasbourg, France). Primary physicochemical properties of each AgNP was confirmed by transmission electron microscopy (TEM) coupled with a micro- analysis characterization (TECNAI G20, Ultra-Twin, FSB, Bizerte, Tunisia) and ultraviolet-visible (UV-Vis) spectroscopy (T60; PG- Instruments, Leicestershire, UK). X-ray diffraction (XRD) charac- terization was performed using a D8 Advance diffracto-meter (Bruker, Bizerte), with analyses performed in Bragg–Brentano con- figuration at 40 kV and 40 mA.

Endocytotic internalization blockers

A stock solution of amantadine (3 mg/mL; Sigma, Steinheim, Germany) was prepared in ultrapure water. Nystatin (Sigma) stock solution (5 mg/mL; Sigma) was prepared in dimethyl sulf- oxide (DMSO) vehicle (Sigma); the final concentration of DMSO in all Nystatin exposures was 0.05% (v/v). Exposures to vehicle alone or in the presence of AgNP of differing sizes were con- ducted to assure effects were not caused by any carrier modula- tion of NP behavior or by the carrier itself. Effective concentration ranges used were chosen based on previous study by Khan et al. (2015).

Sampling and experimental design

Mature mussels (M. galloprovincialis) of average shell length 75 [±5] mm were collected from Bizerte lagoon (Tunisia) and main- tained in oxygenated ASW (35% salinity, pH 8.0; as for local nat- ural seawater) in static tanks under standard conditions (aeration, 12/12 h photoperiod, 16 �C). Animals used for exposure experiments were acclimated for 1–3 days (Canesi et al. 2010b) and were not fed during either acclimation or exposure. Exposure in each tank was 1 mussel/0.5 L ASW in all studies. As only predicted environmental concentrations (PEC) were avail- able in literature, the chosen dose of 100 lg AgNP/L was selected as the test concentration; this dose is usually used in ecotoxicity tests on aquatic species and would be effective in producing adverse effects that could be correlated with outcomes of previ- ous in vitro studies (Katsumiti et al. 2015; Canesi & Corsi 2016).

Mussels (n ¼ 10/group) were separately exposed to AgNP <50 nm (AgNP50) or AgNP <100 nm (AgNP100) for 3, 6 and 12 h with/without initial treatment with the pharmaceutical inhibitors. For inhibitor-treated groups, mussels were incubated for 3 h with 100 lM amantadine (AMA), then placed in AgNP exposure solutions (without AMA) for the required times. For nystatin (NYS), mussels were exposed with 50 lM NYS for 1 h before and then continuing over into the AgNP exposure time- frames (Ivanov 2008; Angel et al. 2013; Khan et al. 2015). Control groups (n ¼ 10) of mussels were maintained in oxygen- ated tanks of only ASW and/or ASW with the inhibitors exactly as above with the AgNP treatments. All exposures were done in triplicate.

Pappenheim’s panoptical staining (MGG) and differential hemocyte counts (DHC)

At the completion of the given exposure period, hemolymph samples were quickly withdrawn (to minimize stress inflicted)

JOURNAL OF IMMUNOTOXICOLOGY 117

from the adductor muscles of each animal, using nn 18-G needle fitted onto a 3-mL syringe. All samples were collected at 16 �C. For each sample, hemolymph of all 10 individuals/treatment regi- men was pooled; the material was then filtered through 1-mm2

mesh sterile gauze into a 5-mL tube at 4 �C to avoid aggregation (Canesi et al. 2010a). After mixing, 40 lL aliquots were deposited onto glass slides; after 15 min, the attached cells were fixed with methanol and then the hemocytes were stained with May- Gr€unwald solution (Bio-optica, Milan, Italy). Slides were then counterstained with 5% Giemsa, air-dried and then mounted using a mounting medium (Entellan Neu, Merck, Darmstadt, Germany) and cover slipped. Slides were then evaluated using a GX-10 light microscope (Olympus, Tokyo, Japan); differential hemocyte counts were made from counts of stained cells in 10 different fields/slide. A minimum of 350 cells/slide was counted. Ten slides/experimental condition were evaluated.

Statistical analysis

All results are expressed as percentages (±SD) of total hemocytes. Normal distribution and homogeneity of variance were tested using Shapiro–Wilk and Bartlett tests prior to statistical analysis. Statistical analysis of absolute percentages was performed using a one-way analysis of variance (ANOVA) with a Tukey’s HSD post hoc test. Modulation in the percentages of hemocyte subpopula- tions were compared to those of controls (untreated mussels). Correlation tests were used to determine relationships among modulated hemocyte subpopulations. Significance overall and within any correlation (confirmed by linear regression test) was accepted at p < 0.05.

Results

Source and characterization of AgNP

Purchased AgNP (<100 nm; AgNP100) were characterized; charac- terizations met the manufacturer supplied valued (99.5% trace metal basis). Representative TEM showed homogeneous spherical characteristics with an approximate primary size of 90 nm (Figure 1(A)); size distribution histograms revealed a median size of 85.0 [±32.6] nm (Figure 1(C)). Representative TEM of synthesized AgNP (<50 nm; AgNP50) demonstrated homogeneous spherical characteristics with an approximate size of 50 nm (Figure 1(B)); size distribution histograms revealed a median size of 41.6 [±18.8] nm (Figure 1(D)). Analyses of each sample indicated that the level of particles <50 nm within the AgNP100 mixture was � 1.38/each 100 particles from AgNP mixture (i.e. <1.5%).

The XRD pattern recorded from a representative batch of sil- ver powder is shown in Figure 1(E). The crystalline nature of the AgNP was demonstrated by diffraction peaks that matched the face-centered cubic (fcc) phase of silver. The absorption max- imum of the measured UV-vis spectrum of the colloidal solution provides information on the average particle size, whereas its full width at half-maximum (fwhm) can be used to estimate particle dispersion as demonstrated by Leopold and Lendl (2003). Agglomeration status analyses performed prior to exposure was confirmed by absorbance spectra measures at kmax ¼ 400 nm (Figure 1(F)) that clearly indicated the AgNP had a homogenous dispersion in aqueous solutions.

Determination of hemocyte subpopulations

Evaluations based on cytoplasmic granules (presence or absence) and stained granule color (Figure 2) showed that levels of

circulating hemocytes from mussels exposed to AgNP suspen- sions at the same dose (100 lg/L) varied as a function of differing particle size. For example, when exposed to AgNP50 for only 3 h, mussels evinced a significant increase in acidophilic granulocytes (acidophils) (78.93 [±6.29]%) compared to levels in controls (60.28 [±8.63]%); however, the AgNP100 at this timepoint imparted no significant effect. Conversely, exposure to either size AgNP led to a significant decrease in basophilic granulocyte (basophils) levels in the same timeframes (i.e. 10.76 [±2.78]% for AgNP50 and 13.43 [±0.90]% for AgNP100) vs. control (19.77 [±2.89]%).

No significant variations were noted in levels of hyalinocytes (10.30 [±3.68]% AgNP50, 10.37 [±3.33]% AgNP100, 19.94 [±5.77]% control). Conversely, when exposed to AgNP50 for 6 h, mussel levels of hyalinocytes displayed a significant increase (16.21 [±3.69]%) versus control values (7.48 [±3.43]%). No other significant variations were recorded for basophils (16.24 [±2.49]% AgNP50, 14.27 [± 1.97]% AgNP100, 15.32 [±1.82]% con- trol) or acidophils (67.54 [±6.07]% AgNP50, 77.49% [±2.69]% AgNP100, 77.19 [±4.21]% control) in the same timeframe. For the 12-h exposure, no significant variations in hemocyte sub-popula- tions were noted with either AgNP [hyalinocytes ¼16.63 [±5.37] % AgNP50, 18.02 [±3.52]% AgNP100, 20.33 [±1.44]% control; basophils ¼ 24.11 [±7.03]% AgNP50, 19.62 [±2.33]% AgNP100, 17.58 [±0.96]% control; acidophils ¼59.20 [±12.30]% AgNP50, 62.35 [±2.23]% AgNP100, 62.07 [±0.52]% control) (Figure 3(A)).

Effect of uptake pathway on circulating hemocytes

Clathrin-mediated endocytosis inhibition (amantadine [AMA])

Significant increases in basophils were seen [16.02 [±1.62] % vs. AMA at 12.00 [±0.90] %) in hosts exposed to AgNP100 for 3 h but not to AgNP50 [15.03 [±1.99] %). No significant variations were recorded with any 6-h exposures (hyalinocytes: 15.49 [±0.93]% AMA, 13.8 [±2.09]% AMA þ AgNP50, 18.12 [±1.10] % AMA þ AgNP100; basophils: 15.12 [±0.95]% AMA, 15.62 [±4.09]% AMA þ AgNP50, 14.79 [±2.11]% AMA þ AgNP100; acidophils: 69.37 [±1.88] % [AMA], 70.57 [±6.15] % AMA þ AgNP50, 67.07 [±3.21] % AMA þ AgNP100). At 12 h, acidophil levels were significantly increased in hosts exposed to either AgNP [74.23 [±2.81] % AgNP50, 73.85 [±0.77] % AgNP100, 68.28 [±0.63] % AMA. Conversely, basophil levels were signifi- cantly decreased in mussels exposed for 12 h to AgNP100 with clathrin path blocking (14.51 [±0.15]% vs. AMA at 19.29 [±1.33]%) but not to AgNP50 (19.29 [±1.33]%). Hyalinocyte lev- els were also significantly reduced in mussels exposed for 12 h to AgNP50 with clathrin path blocking (8.76 [±0.12] % vs. AMA at 11.79 [±1.03] %); AgNP100 imparted no significant effect (11.63 [±0.76] %) (Figure 3(B)).

Caveolae-mediated endocytosis inhibition

Effect of exposure to AgNP in presence of DMSO (Vehicle)

Percentages of circulating hemocytes in mussels exposed to DMSO (0.05%) alone for 3, 6 or 12 h were not significantly changed from levels in untreated mussels (control) (Figure 4(A)). However, in the presence of AgNP50 or AgNP100, only a signifi- cant decrease in basophil levels was noted at the 6-h timepoint (13.46 [±3.78]% and 12.07 [±2.65]%, respectively) as compared to in hosts exposed only to DMSO (18.25 [±9.06]%). No other significant changes due to either form of AgNP at all other time- points was noted (Figure 4(B)).

118 Y. BOUALLEGUI ET AL.

Effect of exposure to AgNP in presence of nystatin (NYS; caveolae blocker)

No significant changes in circulating hemocytes sub-populations were evident for either size AgNP with 3 h of exposure in the

presence of NYS (hyalinocytes: 13.91 [±3.64]% AgNP50, 10.39 [±2.31]% AgNP100, 10.13 [±3.37]% NYS). In contrast, exposure to AgNP50 for 6 h in the presence of NYS caused only a sig- nificant decrease in acidophils [79.15 [±1.02]% vs. NYS at 84.51 [±2.14]%) and a significant increase in basophils

Figure 1. (A) TEM image of AgNP shows homogenous distribution in size (average size � 50 nm). (B) Histogram of size (diameter) distribution for AgNP <50 nm. (C) XRD pattern of AgNP powder. (D) Size UV-Vis absorption spectra of PVP-coated AgNP dissolved in MiliQ water. Narrow peak confirms the size of the particles.

JOURNAL OF IMMUNOTOXICOLOGY 119

Figure 2. Representative light micrograph of Mytilus galloprovincialis hemocyte sub-populations. May–Gr€unwald–Giemsa (MGG) staining. AG: acidophilic granulocytes; Endo: endoplasm (dense stained granules); Ect: ectoplasm (hyaline with thin pseudopodia); Hy: hyalinocytes; BG: basophilic granulocytes. Magnification ¼ 400�.

Figure 3. Variations in circulating hemocyte sub-populations (%) as marker of immunomodulation from AgNP. (A) Exposure to only AgNP. (B) Exposure to AgNP and Amantadine. Data shown are percentages. Hyalinocytes (dark grey), basophilic granulocytes (light grey), acidophilic granulocytes (medium grey), Cont: untreated, Aman: amantadine, Ag50: AgNP < 50 nm, Ag100: AgNP< 100nm for 3, 6 or 12 h. N ¼ 10/group. Value significantly different from negative control [�p < 0.05].

120 Y. BOUALLEGUI ET AL.

[11.97 [±3.64] % vs. NYS at 8.71 [±3.37]%). Significant increases in acidophils were evident only after 12 h of exposure to AgNP100 in the presence of NYS [73.89 [±0.56] % vs. NYS at 63.62 [±2.08] %); in contrast, a significant decrease in baso- phils was noted with exposures to either size AgNP in the presence of NYS in this same timeframe [20.09 [±0.49]% AgNP50, 15.74 [±0.89]% AgNP100, 23.23 [±1.08]% NYS]. For hyalinocytes, a significant increase was only evident with exposure to AgNP50 in the presence of NYS for 12 h [17.44 [±1.96] % vs. NYS at 13.13 [±1.01]%); no significant effects were induced with AgNP100 (10.35 [±0.51] %) (Figure 4(C)).

Correlation between variations in hemocyte sub-populations

The variations in hemocyte sub-population levels under the con- ditions tested here were seen to be intercorrelated. Mussels exposed under differing conditions for 3 h demonstrated signifi- cant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils (r ¼ �0773 and r ¼ �0.900, respectively). No significant correlation was found between levels of hyalinocytes and basophils (r ¼ 0.466) (Table 1). With 6-h exposures, a significant [positive] correlation was seen between changes in levels of hyalinocytes and basophils

Figure 4. (A) Percentages (%) circulating hemocyte sub-populations of mussels exposed to DMSO (0.05%) vehicle, alone for 3, 6 or 12 h compared to untreated mus- sels (control). Control (black), DMSO (grey). N ¼ 10/group. Data shown are mean percentages± SD. (B) Variations in circulating hemocyte sub-populations (%) due to AgNP or (C) Nystatin for 3, 6 or 12 h. N ¼ 10/group. Hyalinocytes (dark grey), basophilic granulocytes (light grey), acidophilic granulocytes (medium grey), Cont: untreated, Nyst: Nystatin, Ag50: AgNP <50 nm, Ag100: AgNP <100nm. Value significantly different from negative control at �p < 0.05, ��p < 0.01.

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(r ¼ 0.703). In contrast, significant [negative] correlations were evident for variations in levels of hyalinocytes and acidophils and basophils and acidophils (r ¼ �0.951 and r ¼ �0.888, respect- ively) (Table 2). The 12-h exposure gave rise to significant nega- tive correlations among the variations in levels of hyalinocytes and acidophils and of basophils and acidophils (r ¼ �0.824 and r ¼ �0.757, respectively). No significant correlations between changes in the levels of hyalinocytes and of basophils was noted (r ¼ 0.255) (Table 3).

Discussion

The present in vivo study aimed to elucidate the ability of AgNP to enter into Mytilus galloprovincialis marine mussels and modu- late the percentages of their immune system cell sub-populations. Previous studies noted the ability of environmental pollutants, such as mercury and cadmium, to significantly enhanced varia- tions in hemocyte counts in mussels (Pipe & Coles 1995). In the same context, changes in immune functions of organisms often correspond with a presence of environmental stressors (i.e. chem- icals or toxins) and thus can be used as good indices of local environmental health status (Parisi et al. 2008; Ottaviani & Malagoli 2009; Canesi & Corsi 2016; Matozzo 2016; Matozzo & Gagn�e 2016). The ability of various NP to be taken up by hemo- cytes and affect immune functions (i.e. lysosomal function, phagocytic activity, oxyradicals (ROS) production and induce pro-apoptotic processes) have been investigated in invertebrate models, as with most invertebrates, mussels possess only innate immune mechanisms – including phagocytosis, production of reactive oxygen species (ROS) and nitrogen radicals, etc. – as means of host protection (Canesi & Prochazova 2013). Canesi et al. (2008) reported that mussel hemocytes exposed in vitro from 0.5–4 h to carbon black NP (1–10 lg/mL) displayed increases in release of lysosomal hydrolytic enzymes, oxidative burst and NO. In contrast, with C60 fullerene, TiO2 and SiO2, there were no significant cytotoxic effects in mussel hemocytes even though each NP-stimulated immune/inflammatory parame- ters in the exposed hosts (Canesi et al. 2010a,b). Based on all

these studies, Canesi et al. asserted that effects from NP were less like dependent on the chemical nature of the materials but mor- eso on associated redox properties that could cause oxidative stress.

With regard to AgNP, several studies have reported cytotoxic effects were closely related to increase in production of ROS. Katsumiti et al. (2015) demonstrated ROS production in mussel hemocytes reached a peak early (3 h) when exposed to malatose- stabilized AgNP. Such results could help explain outcomes in the present study whereby a 3-h exposure to AgNP50 led to signifi- cant increases in levels of acidophil percentages in mussels, while no variations were recorded after 6 or 12 h. This short “toxicity timeframe” may indicate any putative cytotoxic effect caused by AgNP could potentially be neutralized by the increased presence of acidophils; this is plausible in that other studies have described a prominent role for acidophils in host internal defense (Chang et al. 2005; Garcia-Garcia et al. 2008; Parisi et al. 2008; Matozzo & Bailo 2015).

Apart from any increased presence of “NP-detoxifying acid- ophils,” the current results showing that the effect of the AgNP was duration of exposure–related effect could also be a result of changes in the bioavailability of these NP over time. As bioavail- ability of NP is a major factor in ultimate toxicity, surrounding environment effects on particle size stability, shape, surface charge, etc. are key variables that will determine effects on exposed hosts, including mollusks (Levard et al. 2012; Liu et al. 2012; Dobias & Bernier-Latmani 2013; Yu et al. 2014; Katsumiti et al. 2015; Minetto et al. 2016). Canesi and Corsi (2016) hypothesize putative trans-formations of NP including how extracellular proteins could be adsorbed onto a NP surface, form- ing a protein corona of naturally occurring colloids, particles and macromolecules in the water column. The protein corona could then impact how specific cellular receptors, cellular internaliza- tion pathways, and ultimately in immune responses as well, see and respond to the now-modified NP.

The results also indicated significant decreases in basophil lev- els with host exposures for 3 h to either size AgNP (but no sig- nificant variations with 6- and 12-h exposures) and a significant increase in hyalinocytes levels only with AgNP50 for 6 h. Here, the variations showed again that AgNP effects were duration-of- exposure-dependent. In this same context, the recorded varia- tions in the different sub-populations could be explained by an ability of other cell categories, apart from acidophils, to be acti- vated as part of the immune response. This result was in agree- ment with outcomes of studies conducted with bacteria in mussels by Parisi et al. (2008) showed that dramatically varied proportions of the three cell categories clearly reflected how hya- linocytes participated in antibacterial responses despite being reported as “less active” than granulocytes. It was thus concluded that more than one cell type had been involved in immune defense. Such activation of different cell types as immune effec- tors corroborates the hypothesis of Ottaviani et al. (1998) that suggested that, in bivalve hemolymph (M. galloprovincialis), there is only one hemocyte type – with two or more different matur- ation (aging)-related stages, that is, hyalinocytes in a proliferative stage mature to become granulocytes (Ottaviani et al. 1998).

In the present study, the reasonable choice to have used AgNP with sizes of <50 and <100 nm was based on the litera- ture on potential uptake pathways for each size particle. Typical clathrin-coated pits (vessels for clathrin-mediated endocytosis) have diameters in the range 120 nm; conversely, internalization via caveolae-mediated endocytosis is considered the predominant mechanism of entry for structures of 40–50 nm (and below)

Table 1. Correlations of percentage variations in hemocyte sub-populations from mussels exposed for 3 h.

Hyalinocytes Basophils Acidophils

Hyalinocytes 1.0000 – – Basophils 0.4661 1.0000 – Acidophils �0.7738�� �0.9008�� 1.0000 ��Value significantly correlated at p < 0.01.

Table 2. Correlations of percentage variations in hemocyte sub-populations from mussels exposed for 6 h.

Hyalinocytes Basophils Acidophils

Hyalinocytes 1.0000 – – Basophils 0.7034� 1.0000 – Acidophils �0.9511�� �0.8886�� 1.0000 Value significantly correlated at �p < 0.05 or �&

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