\u00a0\u00a0<\/p>\n
5-7 pages not including cover page and literature cited page Format 12 pt. Times New Roman font, double-spaced, 1\u201d margins with proper grammar & spelling Content Using own words to write cohesive review (see next page for specifics) Literature Cited Single format for bibliography & in-text citations using correct information with at least 4 references total including the two primary articles chosen. No quotes or paraphrasing, explained importance of topic and sufficient background to tie together articles. Briefly explained methods, results, conclusions. Briefly explained methods, results, conclusions. Tied together articles and suggested future directions for research in the topic.\u00a0<\/p>\n<\/div>\n<\/div>\n<\/div>\n
RESEARCH ARTICLE\n<\/p>\n
Impact of exposure time, particle size and uptake pathway on silver nanoparticle
\neffects on circulating immune cells in mytilus galloprovincialis\n<\/p>\n
Younes Bouallegui, Ridha Ben Younes, Faten Turki and Ridha Oueslati\n<\/p>\n
Research Unit for Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of Bizerte, University of Carthage, Bizerte, Tunisia\n<\/p>\n
ABSTRACT
\nNanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are
\nknown to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed,
\nassessments of hemocyte-related parameters are a valuable tool in the determination of potentials
\nfor nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanopar-
\nticles (AgNP: <50 nm and <100nm) on the frequency of hemocytes subpopulations as immunomodula-
\ntion biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after
\ninhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over dif-
\nferent durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant varia-
\ntions in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as
\nexposure duration progressed cell levels were subsequently differentially altered depending on particle
\nsize (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed\/varied after
\nblockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative
\ncorrelations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils
\nas a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and
\nduration of exposure dependent. This study highlighted how not only was NP size important, but that dif-
\nfering internalization mechanisms could be key factors impacting on the potential for NP in the environ-
\nment to induce immunomodulation in a model\/test sentinel host like M. galloprovincialis.\n<\/p>\n
ARTICLE HISTORY
\nReceived 13 February 2017
\nRevised 6 May 2017
\nAccepted 24 May 2017\n<\/p>\n
KEYWORDS
\nSilver nanoparticles;
\nendocytosis; hyalinocytes;
\ngranulocytes; Pappenheim
\npanoptical staining\n<\/p>\n
Introduction\n<\/p>\n
Nanoparticles (NP) are defined as materials with all dimensions
\nin nanoscale [1\u2013100 nm] (Luoma 2008). Silver nanoparticles
\n(AgNP) have become the fastest growing product category in
\nnanotechnology due to their thermoelectrical conductivity, cata-
\nlytic activity and nonlinear optical behavior and have great value
\nin the formulation of inks, microelectronic products and biomed-
\nical facilities (i.e. imaging devices) (Tiede et al. 2009; Katsumiti
\net al. 2015). Their exceptional broad-spectrum bactericidal prop-
\nerties and biocompatibility (i.e. as drug delivery agent) have also
\nmade AgNP extremely useful in a diverse range of consumer
\ngoods (Luoma 2008; Rainville et al. 2014; Cozzari et al. 2015;
\nKatsumiti et al. 2015; Marisa et al. 2016).\n<\/p>\n
Worldwide AgNP production is estimated at \ufffd 55 tonne\/yr
\n(Piccinno et al. 2012). However, release of AgNP into aquatic
\nenvirons can happen through wastewaters generated during
\nAgNP synthesis and\/or incorporation into goods and consumer
\nproducts (Canesi et al. 2012; Matranga & Corsi 2012; Katsumiti
\net al. 2015; Marisa et al. 2016). As such, AgNP have emerged as
\npotential stressors that might enter marine environment (Luoma
\n2008). A lack of appropriate tools to evaluate effective NP
\n(of AgNP in particular) levels in aquatic environments make
\nselection of appropriate testing levels a major problem in risk
\nassessment of engineered NP. As a result, predicted environmen-
\ntal concentrations for AgNP are often set at a level of
\n\ufffd 0.01 lg\/L (Tiede et al. 2009; Katsumiti et al. 2015). Even so,\n<\/p>\n
levels much lower than that have commonly been used in aquatic
\nspecies ecotoxicity tests (1\u2013100 lg\/L) (Tiede et al. 2009; Canesi &
\nCorsi 2016), including those with mollusk models.\n<\/p>\n
In the mussel Mytilus galloprovincialis (filter-feeding organ-
\nism), hemocytes are hemolymph cells responsible for immune
\ndefence and serve as a first line of defence against foreign substan-
\nces (Gosling 2003; Parisi et al. 2008; Giron-Perez 2010; Matozzo &
\nBailo 2015). Immune defences carried out by hemocytes constitute
\nimportant targets for potential NP toxicity (Canesi et al. 2012;
\nCanesi & Prochazkova 2013; Katsumiti et al. 2015).\n<\/p>\n
Several studies have shown that different NP types, that is, car-
\nbon black, C60 fullerenes, TiO2, SiO2, ZnO, CeO2, Cd-based, Au-
\nbased and Ag-based, are rapidly taken up by hemocytes.
\nInternalization of these NP subsequently impacted on morpho-
\nlogic\/functional characteristics including immune responses
\n(Canesi et al. 2008, 2010a, b, 2012; Katsumiti et al. 2015; Marisa
\net al. 2016). Various mussel hemocyte parameters, including total
\nhemocyte count (THC), differential hemocyte count (DHC),
\nhemocyte viability, phagocytic activity and lysosomal membrane
\nstability, have been used as a tool for screening of immunomodu-
\nlatory effects of differing NP (Matozzo et al. 2007; Parisi et al.
\n2008; Hoher et al. 2013; Matozzo & Bailo 2015; Canesi & Corsi,
\n2016; Marisa et al. 2016). Specifically, hyalinocytes and granulo-
\ncytes have been assessed for morphological changes among hemo-
\ncytes in Mytilus galloprovincialis (Pipe et al. 1997; Chang et al.
\n2005; Garcia-Garcia et al. 2008).\n<\/p>\n
CONTACT Younes Bouallegui [email\u00a0protected]<\/a> Research Unit of Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of JOURNAL OF IMMUNOTOXICOLOGY, 2017 While granulocytes are large ovoid-shaped cells with a small Cellular uptake by endocytosis (clathrin- or caveolae-mediated In this context, the present study aimed to record the vari- Material and methods\n<\/p>\n Silver nanoparticles (AgNP) source and characterization\n<\/p>\n Poly-vinyl-pyrrolidone (PVP)-coated AgNP of <100 nm (99.5% A stock solution of each AgNP size was suspended in artificial CaCl2; 1.65% KCl; 0.5% NaHCO3; 0.24% KBr; 0.07% H3BO3; Endocytotic internalization blockers\n<\/p>\n A stock solution of amantadine (3 mg\/mL; Sigma, Steinheim, Sampling and experimental design\n<\/p>\n Mature mussels (M. galloprovincialis) of average shell length 75 Mussels (n \u00bc 10\/group) were separately exposed to AgNP Pappenheim\u2019s panoptical staining (MGG) and differential At the completion of the given exposure period, hemolymph JOURNAL OF IMMUNOTOXICOLOGY 117<\/p>\n<\/p>\n<\/div>\n from the adductor muscles of each animal, using nn 18-G needle mesh sterile gauze into a 5-mL tube at 4 \ufffdC to avoid aggregation Statistical analysis\n<\/p>\n All results are expressed as percentages (\u00b1SD) of total hemocytes. Results\n<\/p>\n Source and characterization of AgNP\n<\/p>\n Purchased AgNP (<100 nm; AgNP100) were characterized; charac- The XRD pattern recorded from a representative batch of sil- Determination of hemocyte subpopulations\n<\/p>\n Evaluations based on cytoplasmic granules (presence or absence) circulating hemocytes from mussels exposed to AgNP suspen- No significant variations were noted in levels of hyalinocytes Effect of uptake pathway on circulating hemocytes\n<\/p>\n Clathrin-mediated endocytosis inhibition (amantadine [AMA])\n<\/p>\n Significant increases in basophils were seen [16.02 [\u00b11.62] % vs. Caveolae-mediated endocytosis inhibition\n<\/p>\n Effect of exposure to AgNP in presence of DMSO (Vehicle)\n<\/p>\n Percentages of circulating hemocytes in mussels exposed to 118 Y. BOUALLEGUI ET AL.<\/p>\n<\/p>\n<\/div>\n Effect of exposure to AgNP in presence of nystatin No significant changes in circulating hemocytes sub-populations presence of NYS (hyalinocytes: 13.91 [\u00b13.64]% AgNP50, 10.39 Figure 1. (A) TEM image of AgNP shows homogenous distribution in size (average size \ufffd 50 nm). (B) Histogram of size (diameter) distribution for AgNP <50 nm. JOURNAL OF IMMUNOTOXICOLOGY 119<\/p>\n<\/p>\n<\/div>\n Figure 2. Representative light micrograph of Mytilus galloprovincialis hemocyte sub-populations. May\u2013Gr\u20acunwald\u2013Giemsa (MGG) staining. AG: acidophilic granulocytes; Figure 3. Variations in circulating hemocyte sub-populations (%) as marker of immunomodulation from AgNP. (A) Exposure to only AgNP. (B) Exposure to AgNP and 120 Y. BOUALLEGUI ET AL.<\/p>\n<\/p>\n<\/div>\n [11.97 [\u00b13.64] % vs. NYS at 8.71 [\u00b13.37]%). Significant Correlation between variations in hemocyte sub-populations\n<\/p>\n The variations in hemocyte sub-population levels under the con- 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- JOURNAL OF IMMUNOTOXICOLOGY 121<\/p>\n<\/p>\n<\/div>\n (r \u00bc 0.703). In contrast, significant [negative] correlations were Discussion\n<\/p>\n The present in vivo study aimed to elucidate the ability of AgNP these studies, Canesi et al. asserted that effects from NP were less With regard to AgNP, several studies have reported cytotoxic Apart from any increased presence of \u201cNP-detoxifying acid- The results also indicated significant decreases in basophil lev- In the present study, the reasonable choice to have used Table 1. Correlations of percentage variations in hemocyte sub-populations Hyalinocytes Basophils Acidophils\n<\/p>\n Hyalinocytes 1.0000 \u2013 \u2013 Table 2. Correlations of percentage variations in hemocyte sub-populations Hyalinocytes Basophils Acidophils\n<\/p>\n Hyalinocytes 1.0000 \u2013 \u2013 Table 3. Correlations of percentage variations in hemocyte sub-populations Hyalinocytes Basophils Acidophils\n<\/p>\n Hyalinocytes 1.0000 \u2013 \u2013 122 Y. BOUALLEGUI ET AL.<\/p>\n<\/p>\n<\/div>\n in diameter. Thus, while effects on clathrin-mediated endocytosis Apparently in keeping with this assumption, an AgNP size- The present study also sought to clarify the role of varying Conclusions\n<\/p>\n Overall, the results here showed how silver nanoparticles (AgNP) Acknowledgements\n<\/p>\n This study is funded by the immunomicrobiology, environmental University of Carthage, Tunisia. The authors acknowledge Prof. Disclosure statement\n<\/p>\n No potential conflict of interest was reported by the authors.\n<\/p>\n Funding\n<\/p>\n This study is funded by the immunomicrobiology, environmental References\n<\/p>\n Angel B, Batley G, Jarolimek C, Rogers N. 2013. The impact of size on the Canesi L, Corsi I. 2016. Effects of nanomaterials on marine invertebrates. Sci Canesi L, Proch\ufffdazov\ufffda P. 2013. Invertebrate immune system as a model for Canesi L, Ciacci C, Betti M, Fabbri R, Canonico B, Fantinati A, Marcomini Canesi L, Ciacci C, Betti M, Vallotto D, Gallo G, Marcomini A, Pojana G. Canesi L, Ciacci C, Fabbri R, Marcomini A, Pojana G, Gallo G. 2012. Bivalve Canesi L, Fabbri R, Gallo G, Vallotto D, Marcomini A, Pojana G. 2010b. Carballal M, Lopez M, Azevedo C, Villalba A. 1997. Hemolymph cell types of Chang S, Tseng S, Chou H. 2005. Morphological characterization via light Cima F. 2010. Microscopy methods for morpho-functional characterization of Cozzari M, Elia A, Pacini N, Smith B, Boyle D, Rainbow P, Khan F. 2015. Dobias J, Bernier-Latmani R. 2013. Silver release from silver nanoparticles in Doherty G, McMahon H. 2009. Mechanisms of endocytosis. Annu Rev dos Santos T, Varela J, Lynch I, Salvati A, Dawson K. 2011. Effects of trans- Garcia-Garcia E, Prado-Alvarez M, Novoa B, Figueras A, Rosales C. 2008. Giron-Perez M. 2010. Relationships between innate immunity in bivalve mol- Gliga A, Skoglund S, Wallinder I, Fadeel B, Karlsson H. 2014. Size-dependent Gosling E, editor. 2003. Bivalves mollusks: Biology, ecology and culture. Haucke V. 2006. Cargo takes control of endocytosis. Cell. 127:35\u201337.\n<\/p>\n JOURNAL OF IMMUNOTOXICOLOGY 123<\/p>\n<\/p>\n<\/div>\n Hine P. 1999. The inter-relationships of bivalve haemocytes. Fish Shellfish H\u20acoher N, Regoli F, Dissanayake A, Nagel M, Kriews M, K\u20acohler A, Broeg K. Ivanov A, editor. 2008. Pharmacological inhibition of endocytic pathways: is Katsumiti A, Gilliland D, Arostegui I, Cajaraville M. 2015. Mechanisms of Khan F, Mirsa S, Bury N, Smith Brian D, Rainbow P, Luoma S, Valsami- Leopold N, Lendl B. 2003. A new method for fast preparation of highly sur- Levard C, Hotze E, Lowry G, Brown G. 2012. Environmental transformations Liu J, Wang Z, Liu F, Kane A, Hurt R. 2012. Chemical transformations of Luoma S. 2008. Silver nanotechnologies and the environment. Washington Marisa I, Matozzo V, Munari M, Binelli A, Parolini M, Martucci A, Matranga V, Corsi I. 2012. Toxic effects of engineered nanoparticles in the Matozzo V, Bailo L. 2015. A first insight into haemocytes of the smooth Matozzo V, Gagn\ufffde F. 2016. Immunotoxicology approaches in ecotoxicology: Matozzo V, Rova G, Marin M. 2007. Hamocytes of the cockle Cerastoderma Matozzo V. 2016. Aspects of eco-immunology in mollusks. Invert Survival J. Mezni A, Mlayah A, Serin V, Smiri L. 2014b. Synthesis of hybrid Au-ZnO Mezni A, Dammak T, Fkiri T, Mlayah A, Abid A, Smiri L. 2014a. Minetto D, Ghirardini V, Libralato G. 2016. Saltwater ecotoxicology of Ag, Moore M. 2006. Do nanoparticles present ecotoxicologic risks for the health Nichols B, Lippincott-Shwartz J. 2001. Endocytosis without clathrin coats. Ottaviani E, Malagoli D. 2009. Around the word stress: Its biological and Ottaviani E, Franchini A, Barbieri D, Kletsas D. 1998. Comparative and mor- Parisi M, Li H, Jouvet Lionel B, Dyrynda E, Parrinello N, Cammarata M, Park K, Tuttle G, Sinche F, Harper S. 2013. Stability of citrate-capped silver Piccinno F, Gottschalk F, Seeger S, Nowack B. 2012. Industrial production Pinsino A, Russo R, Bonaventura R, Brunelli A, Marcomini A, Matranga V. Pipe R, Coles J. 1995. Environmental contaminants influencing immune func- Pipe R, Farley S, Coles J. 1997. The separation and characterisation of haemo- Puthenveedu M, von Zastrow M. 2006. Cargo regulates clathrin-coated pit Rainville L, Carolan D, Varela A, Doyle H, Sheehan D. 2014. Proteomic Razani B, Lisanti M. 2002. Role of caveolae and the caveolins in mammalian Sandvig K, Pust S, Skotland T, van Deurs B. 2011. Clathrin-independent Tiede K, Hassell\u20acov M, Breitbarth E, Chaudhry Q, Boxall A. 2009. Yu S, Yin Y, Chao J, Shen M, Liu J. 2014. Highly dynamic PVP-coated silver 124 Y. BOUALLEGUI ET AL.<\/p>\n<\/p>\n<\/div>\n Copyright of Journal of Immunotoxicology is the property of Taylor & Francis Ltd and its Research Article Konstantinos Michalakis ,1,2,3 Athina Bakopoulou ,1 Eleni Papachristou,1\n<\/p>\n Dimitra Vasilaki ,1 Alexandros Tsouknidas ,4 Nikolaos Michailidis ,5\n<\/p>\n and Elaine Johnstone6\n<\/p>\n 1School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece Correspondence should be addressed to Konstantinos Michalakis; [email\u00a0protected]<\/a>\n<\/p>\n Received 12 September 2021; Revised 20 November 2021; Accepted 22 November 2021; Published 13 December 2021\n<\/p>\n Academic Editor: Aziz ur Rehman Aziz\n<\/p>\n Copyright \u00a9 2021 Konstantinos Michalakis et al. This is an open access article distributed under the Creative Commons Osteosarcoma is considered to be a highly malignant tumor affecting primarily long bones. It metastasizes widely, primarily to the 1. Introduction\n<\/p>\n Osteosarcoma is considered a relatively uncommon malig- coma is 60-70% [2\u20135]. The chemotherapy agents employed Hindawi
\nBizerte, Zarzouna 7021, Bizerte, Tunisia
\n\ufffd 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
\nThis 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
\nunrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.\n<\/p>\n
\nVOL. 14, NO. 1, 116\u2013124
\nhttps:\/\/doi.org\/10.1080\/1547691X.2017.1335810<\/p>\n<\/p>\n
\neccentric nucleus and granulated cytoplasm (low nucleus\/cyto-
\nplasm [N\/C] ratio) that are able to spread out and produce pseudo-
\npodia), hyalinocytes are small round cells with an agranular (zero-
\nfew granules) small cytoplasm surrounding a large nucleus (high
\nN\/C ratio) (Carballal et al. 1997; Parisi et al. 2008; Cima 2010;
\nMatozzo & Bailo 2015). Overall, hemocytes can be classified into
\ntwo types, granulocytes and hyalinocytes (so-called agranulocytes),
\nbased on morphological characteristics (the presence\/absence of
\ngranules in cytoplasm). Staining of the cytoplasm by certain dyes
\nallows for sub-distinguishing of acidophils from basophils among
\nthe granulocytes. Ultimately, the basophils of M. edulis appear as
\ngranulocytes with small granules, while acidophilic granulocytes
\ncontain large granules. In comparison to the granulocytes, hyalino-
\ncytes in bivalve have only basophilic properties. Thus, in earlier
\nstudies that described hemocyte subpopulations, the author indi-
\ncated that basophilic cells (hyalinocytes \u00fe basophils) made up
\nabout 40% of the total hemocyte pool in bivalves\/mussels while
\neosinophils accounted for the remaining \ufffd 60% of all hemocytes
\n(Chang et al. 2005; Garcia-Garcia et al. 2008).\n<\/p>\n
\nroutes) are crucial for a variety of cellular and physiological
\nactivities (i.e. nutrient uptake, immune defence) (Haucke 2006;
\nSandvig et al. 2011); each has also been identified as potential
\nmeans for NP entry into cells (Moore 2006; dos Santos et al.
\n2011; Khan et al. 2015). Clathrin-dependent endocytosis involves
\nformation of a clathrin (protein)-coated pit used in enzymatic
\ndestruction of internalized contents. Caveolae-dependent endo-
\ncytosis occurs via cell-surface flask-shaped invaginations enriched
\nwith caveolin (cholesterol-binding proteins) (Nichols &
\nLippincott-Shwartz 2001; Razani & Lisanti 2002) that permit sub-
\ncellular movements of ingested materials through a series of
\nendosomal compartments of increasing acidity allowing for
\nhydrolytic breakdown (Moore 2006; Puthenveedu & von Zastrow
\n2006; Doherty & McMahon 2009). Each route can be modified
\nwith inhibitors (Moore 2006; Ivanov 2008; dos Santos et al. 2011;
\nKhan et al. 2015). Clathrin-mediated endocytosis could be inhib-
\nited by the antiviral amantadine through disruption of the cla-
\nthrin coat, while antibiotic nystatin can impact on cholesterol-
\nrich microdomains of caveolae-mediated endocytosis (Ivanov
\n2008; Khan et al. 2015).\n<\/p>\n
\nation in the percentages of circulating subpopulations of hemo-
\ncytes, using as method differential hemocytes count [DHC] after
\nPappenheim\u2019s panoptical staining [MGG] to: (1) assess effects of
\nAgNP on circulating hemocyte sub-populations; (2) establish a
\nrelationship linking length of exposure to different size AgNP
\nand variations in sub-populations [DHC]; and (3) evaluate the
\nrole of uptake pathways (clathrin- and caveolae-dependent endo-
\ncytosis) \u2013 as well as changes in their function \u2013 in the effect of
\nNP on circulating hemocyte subpopulations.\n<\/p>\n
\npure) were purchased from Sigma (Steinheim, Germany). PVP-
\ncoated AgNP <50 nm were produced by a modified process
\nwherein AgNO3 (Sigma) was dissolved in ethylene glycol (EG)
\nsolvent (ACROS Organics, 98%, Geel, Belgium) in the presence
\nof PVP (K30, Sigma) as a capping agent (Mezni et al. 2014a,b).\n<\/p>\n
\nseawater (ASW; 58.5% NaCl; 26.5% MgCl2; 9.8% Na2SO4; 2.8%\n<\/p>\n
\n0.0095% SrCl2; 0.007% NaF (Pinsino et al. 2015)). Prior to use,
\neach AgNP stock was mixed several times and an aliquot removed
\nas a working solution that was sonicated 15 min in alternating
\ncycles (2 \ufffd 30 s) in an ultrasonic bath (VWR, Strasbourg, France).
\nPrimary physicochemical properties of each AgNP was confirmed
\nby transmission electron microscopy (TEM) coupled with a micro-
\nanalysis characterization (TECNAI G20, Ultra-Twin, FSB, Bizerte,
\nTunisia) and ultraviolet-visible (UV-Vis) spectroscopy (T60; PG-
\nInstruments, Leicestershire, UK). X-ray diffraction (XRD) charac-
\nterization was performed using a D8 Advance diffracto-meter
\n(Bruker, Bizerte), with analyses performed in Bragg\u2013Brentano con-
\nfiguration at 40 kV and 40 mA.\n<\/p>\n
\nGermany) was prepared in ultrapure water. Nystatin (Sigma)
\nstock solution (5 mg\/mL; Sigma) was prepared in dimethyl sulf-
\noxide (DMSO) vehicle (Sigma); the final concentration of DMSO
\nin all Nystatin exposures was 0.05% (v\/v). Exposures to vehicle
\nalone or in the presence of AgNP of differing sizes were con-
\nducted to assure effects were not caused by any carrier modula-
\ntion of NP behavior or by the carrier itself. Effective
\nconcentration ranges used were chosen based on previous study
\nby Khan et al. (2015).\n<\/p>\n
\n[\u00b15] mm were collected from Bizerte lagoon (Tunisia) and main-
\ntained in oxygenated ASW (35% salinity, pH 8.0; as for local nat-
\nural seawater) in static tanks under standard conditions
\n(aeration, 12\/12 h photoperiod, 16 \ufffdC). Animals used for exposure
\nexperiments were acclimated for 1\u20133 days (Canesi et al. 2010b)
\nand were not fed during either acclimation or exposure.
\nExposure in each tank was 1 mussel\/0.5 L ASW in all studies. As
\nonly predicted environmental concentrations (PEC) were avail-
\nable in literature, the chosen dose of 100 lg AgNP\/L was selected
\nas the test concentration; this dose is usually used in ecotoxicity
\ntests on aquatic species and would be effective in producing
\nadverse effects that could be correlated with outcomes of previ-
\nous in vitro studies (Katsumiti et al. 2015; Canesi & Corsi 2016).\n<\/p>\n
\n<50 nm (AgNP50) or AgNP <100 nm (AgNP100) for 3, 6 and
\n12 h with\/without initial treatment with the pharmaceutical
\ninhibitors. For inhibitor-treated groups, mussels were incubated
\nfor 3 h with 100 lM amantadine (AMA), then placed in AgNP
\nexposure solutions (without AMA) for the required times. For
\nnystatin (NYS), mussels were exposed with 50 lM NYS for 1 h
\nbefore and then continuing over into the AgNP exposure time-
\nframes (Ivanov 2008; Angel et al. 2013; Khan et al. 2015).
\nControl groups (n \u00bc 10) of mussels were maintained in oxygen-
\nated tanks of only ASW and\/or ASW with the inhibitors exactly
\nas above with the AgNP treatments. All exposures were done in
\ntriplicate.\n<\/p>\n
\nhemocyte counts (DHC)\n<\/p>\n
\nsamples were quickly withdrawn (to minimize stress inflicted)\n<\/p>\n
\nfitted onto a 3-mL syringe. All samples were collected at 16 \ufffdC.
\nFor each sample, hemolymph of all 10 individuals\/treatment regi-
\nmen was pooled; the material was then filtered through 1-mm2\n<\/p>\n
\n(Canesi et al. 2010a). After mixing, 40 lL aliquots were deposited
\nonto glass slides; after 15 min, the attached cells were fixed with
\nmethanol and then the hemocytes were stained with May-
\nGr\u20acunwald solution (Bio-optica, Milan, Italy). Slides were then
\ncounterstained with 5% Giemsa, air-dried and then mounted
\nusing a mounting medium (Entellan Neu, Merck, Darmstadt,
\nGermany) and cover slipped. Slides were then evaluated using a
\nGX-10 light microscope (Olympus, Tokyo, Japan); differential
\nhemocyte counts were made from counts of stained cells in 10
\ndifferent fields\/slide. A minimum of 350 cells\/slide was counted.
\nTen slides\/experimental condition were evaluated.\n<\/p>\n
\nNormal distribution and homogeneity of variance were tested
\nusing Shapiro\u2013Wilk and Bartlett tests prior to statistical analysis.
\nStatistical analysis of absolute percentages was performed using a
\none-way analysis of variance (ANOVA) with a Tukey\u2019s HSD post
\nhoc test. Modulation in the percentages of hemocyte subpopula-
\ntions were compared to those of controls (untreated mussels).
\nCorrelation tests were used to determine relationships among
\nmodulated hemocyte subpopulations. Significance overall and
\nwithin any correlation (confirmed by linear regression test) was
\naccepted at p < 0.05.\n<\/p>\n
\nterizations met the manufacturer supplied valued (99.5% trace
\nmetal basis). Representative TEM showed homogeneous spherical
\ncharacteristics with an approximate primary size of 90 nm (Figure
\n1(A)); size distribution histograms revealed a median size of 85.0
\n[\u00b132.6] nm (Figure 1(C)). Representative TEM of synthesized
\nAgNP (<50 nm; AgNP50) demonstrated homogeneous spherical
\ncharacteristics with an approximate size of 50 nm (Figure 1(B));
\nsize distribution histograms revealed a median size of 41.6 [\u00b118.8]
\nnm (Figure 1(D)). Analyses of each sample indicated that the level
\nof particles <50 nm within the AgNP100 mixture was \ufffd 1.38\/each
\n100 particles from AgNP mixture (i.e. <1.5%).\n<\/p>\n
\nver powder is shown in Figure 1(E). The crystalline nature of the
\nAgNP was demonstrated by diffraction peaks that matched the
\nface-centered cubic (fcc) phase of silver. The absorption max-
\nimum of the measured UV-vis spectrum of the colloidal solution
\nprovides information on the average particle size, whereas its full
\nwidth at half-maximum (fwhm) can be used to estimate particle
\ndispersion as demonstrated by Leopold and Lendl (2003).
\nAgglomeration status analyses performed prior to exposure was
\nconfirmed by absorbance spectra measures at kmax \u00bc 400 nm
\n(Figure 1(F)) that clearly indicated the AgNP had a homogenous
\ndispersion in aqueous solutions.\n<\/p>\n
\nand stained granule color (Figure 2) showed that levels of\n<\/p>\n
\nsions at the same dose (100 lg\/L) varied as a function of differing
\nparticle size. For example, when exposed to AgNP50 for only 3 h,
\nmussels evinced a significant increase in acidophilic granulocytes
\n(acidophils) (78.93 [\u00b16.29]%) compared to levels in controls
\n(60.28 [\u00b18.63]%); however, the AgNP100 at this timepoint
\nimparted no significant effect. Conversely, exposure to either size
\nAgNP led to a significant decrease in basophilic granulocyte
\n(basophils) levels in the same timeframes (i.e. 10.76 [\u00b12.78]% for
\nAgNP50 and 13.43 [\u00b10.90]% for AgNP100) vs. control (19.77
\n[\u00b12.89]%).\n<\/p>\n
\n(10.30 [\u00b13.68]% AgNP50, 10.37 [\u00b13.33]% AgNP100, 19.94
\n[\u00b15.77]% control). Conversely, when exposed to AgNP50 for 6 h,
\nmussel levels of hyalinocytes displayed a significant increase
\n(16.21 [\u00b13.69]%) versus control values (7.48 [\u00b13.43]%). No other
\nsignificant variations were recorded for basophils (16.24
\n[\u00b12.49]% AgNP50, 14.27 [\u00b1 1.97]% AgNP100, 15.32 [\u00b11.82]% con-
\ntrol) or acidophils (67.54 [\u00b16.07]% AgNP50, 77.49% [\u00b12.69]%
\nAgNP100, 77.19 [\u00b14.21]% control) in the same timeframe. For the
\n12-h exposure, no significant variations in hemocyte sub-popula-
\ntions were noted with either AgNP [hyalinocytes \u00bc16.63 [\u00b15.37]
\n% AgNP50, 18.02 [\u00b13.52]% AgNP100, 20.33 [\u00b11.44]% control;
\nbasophils \u00bc 24.11 [\u00b17.03]% AgNP50, 19.62 [\u00b12.33]% AgNP100,
\n17.58 [\u00b10.96]% control; acidophils \u00bc59.20 [\u00b112.30]% AgNP50,
\n62.35 [\u00b12.23]% AgNP100, 62.07 [\u00b10.52]% control) (Figure 3(A)).\n<\/p>\n
\nAMA at 12.00 [\u00b10.90] %) in hosts exposed to AgNP100 for 3 h
\nbut not to AgNP50 [15.03 [\u00b11.99] %). No significant variations
\nwere recorded with any 6-h exposures (hyalinocytes: 15.49
\n[\u00b10.93]% AMA, 13.8 [\u00b12.09]% AMA \u00fe AgNP50, 18.12 [\u00b11.10] %
\nAMA \u00fe AgNP100; basophils: 15.12 [\u00b10.95]% AMA, 15.62
\n[\u00b14.09]% AMA \u00fe AgNP50, 14.79 [\u00b12.11]% AMA \u00fe AgNP100;
\nacidophils: 69.37 [\u00b11.88] % [AMA], 70.57 [\u00b16.15] %
\nAMA \u00fe AgNP50, 67.07 [\u00b13.21] % AMA \u00fe AgNP100). At 12 h,
\nacidophil levels were significantly increased in hosts exposed to
\neither AgNP [74.23 [\u00b12.81] % AgNP50, 73.85 [\u00b10.77] % AgNP100,
\n68.28 [\u00b10.63] % AMA. Conversely, basophil levels were signifi-
\ncantly decreased in mussels exposed for 12 h to AgNP100 with
\nclathrin path blocking (14.51 [\u00b10.15]% vs. AMA at 19.29
\n[\u00b11.33]%) but not to AgNP50 (19.29 [\u00b11.33]%). Hyalinocyte lev-
\nels were also significantly reduced in mussels exposed for 12 h to
\nAgNP50 with clathrin path blocking (8.76 [\u00b10.12] % vs. AMA at
\n11.79 [\u00b11.03] %); AgNP100 imparted no significant effect (11.63
\n[\u00b10.76] %) (Figure 3(B)).\n<\/p>\n
\nDMSO (0.05%) alone for 3, 6 or 12 h were not significantly
\nchanged from levels in untreated mussels (control) (Figure 4(A)).
\nHowever, in the presence of AgNP50 or AgNP100, only a signifi-
\ncant decrease in basophil levels was noted at the 6-h timepoint
\n(13.46 [\u00b13.78]% and 12.07 [\u00b12.65]%, respectively) as compared
\nto in hosts exposed only to DMSO (18.25 [\u00b19.06]%). No other
\nsignificant changes due to either form of AgNP at all other time-
\npoints was noted (Figure 4(B)).\n<\/p>\n
\n(NYS; caveolae blocker)\n<\/p>\n
\nwere evident for either size AgNP with 3 h of exposure in the\n<\/p>\n
\n[\u00b12.31]% AgNP100, 10.13 [\u00b13.37]% NYS). In contrast, exposure
\nto AgNP50 for 6 h in the presence of NYS caused only a sig-
\nnificant decrease in acidophils [79.15 [\u00b11.02]% vs. NYS at
\n84.51 [\u00b12.14]%) and a significant increase in basophils\n<\/p>\n
\n(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.\n<\/p>\n
\nEndo: endoplasm (dense stained granules); Ect: ectoplasm (hyaline with thin pseudopodia); Hy: hyalinocytes; BG: basophilic granulocytes. Magnification \u00bc 400\ufffd.\n<\/p>\n
\nAmantadine. Data shown are percentages. Hyalinocytes (dark grey), basophilic granulocytes (light grey), acidophilic granulocytes (medium grey), Cont: untreated,
\nAman: amantadine, Ag50: AgNP < 50 nm, Ag100: AgNP< 100nm for 3, 6 or 12 h. N \u00bc 10\/group. Value significantly different from negative control [\ufffdp < 0.05].\n<\/p>\n
\nincreases in acidophils were evident only after 12 h of exposure
\nto AgNP100 in the presence of NYS [73.89 [\u00b10.56] % vs. NYS
\nat 63.62 [\u00b12.08] %); in contrast, a significant decrease in baso-
\nphils was noted with exposures to either size AgNP in the
\npresence of NYS in this same timeframe [20.09 [\u00b10.49]%
\nAgNP50, 15.74 [\u00b10.89]% AgNP100, 23.23 [\u00b11.08]% NYS]. For
\nhyalinocytes, a significant increase was only evident with
\nexposure to AgNP50 in the presence of NYS for 12 h [17.44
\n[\u00b11.96] % vs. NYS at 13.13 [\u00b11.01]%); no significant effects
\nwere induced with AgNP100 (10.35 [\u00b10.51] %) (Figure 4(C)).\n<\/p>\n
\nditions tested here were seen to be intercorrelated. Mussels
\nexposed under differing conditions for 3 h demonstrated signifi-
\ncant negative correlations between changes in levels hyalinocytes
\nand acidophils or in levels basophils and acidophils (r \u00bc \ufffd0773
\nand r \u00bc \ufffd0.900, respectively). No significant correlation was
\nfound between levels of hyalinocytes and basophils (r \u00bc 0.466)
\n(Table 1). With 6-h exposures, a significant [positive] correlation
\nwas seen between changes in levels of hyalinocytes and basophils\n<\/p>\n
\nsels (control). Control (black), DMSO (grey). N \u00bc 10\/group. Data shown are mean percentages\u00b1 SD. (B) Variations in circulating hemocyte sub-populations (%) due to
\nAgNP or (C) Nystatin for 3, 6 or 12 h. N \u00bc 10\/group. Hyalinocytes (dark grey), basophilic granulocytes (light grey), acidophilic granulocytes (medium grey), Cont:
\nuntreated, Nyst: Nystatin, Ag50: AgNP <50 nm, Ag100: AgNP <100nm. Value significantly different from negative control at \ufffdp < 0.05, \ufffd\ufffdp < 0.01.\n<\/p>\n
\nevident for variations in levels of hyalinocytes and acidophils and
\nbasophils and acidophils (r \u00bc \ufffd0.951 and r \u00bc \ufffd0.888, respect-
\nively) (Table 2). The 12-h exposure gave rise to significant nega-
\ntive correlations among the variations in levels of hyalinocytes
\nand acidophils and of basophils and acidophils (r \u00bc \ufffd0.824 and
\nr \u00bc \ufffd0.757, respectively). No significant correlations between
\nchanges in the levels of hyalinocytes and of basophils was noted
\n(r \u00bc 0.255) (Table 3).\n<\/p>\n
\nto enter into Mytilus galloprovincialis marine mussels and modu-
\nlate the percentages of their immune system cell sub-populations.
\nPrevious studies noted the ability of environmental pollutants,
\nsuch as mercury and cadmium, to significantly enhanced varia-
\ntions in hemocyte counts in mussels (Pipe & Coles 1995). In the
\nsame context, changes in immune functions of organisms often
\ncorrespond with a presence of environmental stressors (i.e. chem-
\nicals or toxins) and thus can be used as good indices of local
\nenvironmental health status (Parisi et al. 2008; Ottaviani &
\nMalagoli 2009; Canesi & Corsi 2016; Matozzo 2016; Matozzo &
\nGagn\ufffde 2016). The ability of various NP to be taken up by hemo-
\ncytes and affect immune functions (i.e. lysosomal function,
\nphagocytic activity, oxyradicals (ROS) production and induce
\npro-apoptotic processes) have been investigated in invertebrate
\nmodels, as with most invertebrates, mussels possess only innate
\nimmune mechanisms \u2013 including phagocytosis, production of
\nreactive oxygen species (ROS) and nitrogen radicals, etc. \u2013 as
\nmeans of host protection (Canesi & Prochazova 2013). Canesi
\net al. (2008) reported that mussel hemocytes exposed in vitro
\nfrom 0.5\u20134 h to carbon black NP (1\u201310 lg\/mL) displayed
\nincreases in release of lysosomal hydrolytic enzymes, oxidative
\nburst and NO. In contrast, with C60 fullerene, TiO2 and SiO2,
\nthere were no significant cytotoxic effects in mussel hemocytes
\neven though each NP-stimulated immune\/inflammatory parame-
\nters in the exposed hosts (Canesi et al. 2010a,b). Based on all\n<\/p>\n
\nlike dependent on the chemical nature of the materials but mor-
\neso on associated redox properties that could cause oxidative
\nstress.\n<\/p>\n
\neffects were closely related to increase in production of ROS.
\nKatsumiti et al. (2015) demonstrated ROS production in mussel
\nhemocytes reached a peak early (3 h) when exposed to malatose-
\nstabilized AgNP. Such results could help explain outcomes in the
\npresent study whereby a 3-h exposure to AgNP50 led to signifi-
\ncant increases in levels of acidophil percentages in mussels, while
\nno variations were recorded after 6 or 12 h. This short \u201ctoxicity
\ntimeframe\u201d may indicate any putative cytotoxic effect caused by
\nAgNP could potentially be neutralized by the increased presence
\nof acidophils; this is plausible in that other studies have described
\na prominent role for acidophils in host internal defense (Chang
\net al. 2005; Garcia-Garcia et al. 2008; Parisi et al. 2008; Matozzo
\n& Bailo 2015).\n<\/p>\n
\nophils,\u201d the current results showing that the effect of the AgNP
\nwas duration of exposure\u2013related effect could also be a result of
\nchanges in the bioavailability of these NP over time. As bioavail-
\nability of NP is a major factor in ultimate toxicity, surrounding
\nenvironment effects on particle size stability, shape, surface
\ncharge, etc. are key variables that will determine effects on
\nexposed hosts, including mollusks (Levard et al. 2012; Liu et al.
\n2012; Dobias & Bernier-Latmani 2013; Yu et al. 2014; Katsumiti
\net al. 2015; Minetto et al. 2016). Canesi and Corsi (2016)
\nhypothesize putative trans-formations of NP including how
\nextracellular proteins could be adsorbed onto a NP surface, form-
\ning a protein corona of naturally occurring colloids, particles and
\nmacromolecules in the water column. The protein corona could
\nthen impact how specific cellular receptors, cellular internaliza-
\ntion pathways, and ultimately in immune responses as well, see
\nand respond to the now-modified NP.\n<\/p>\n
\nels with host exposures for 3 h to either size AgNP (but no sig-
\nnificant variations with 6- and 12-h exposures) and a significant
\nincrease in hyalinocytes levels only with AgNP50 for 6 h. Here,
\nthe variations showed again that AgNP effects were duration-of-
\nexposure-dependent. In this same context, the recorded varia-
\ntions in the different sub-populations could be explained by an
\nability of other cell categories, apart from acidophils, to be acti-
\nvated as part of the immune response. This result was in agree-
\nment with outcomes of studies conducted with bacteria in
\nmussels by Parisi et al. (2008) showed that dramatically varied
\nproportions of the three cell categories clearly reflected how hya-
\nlinocytes participated in antibacterial responses despite being
\nreported as \u201cless active\u201d than granulocytes. It was thus concluded
\nthat more than one cell type had been involved in immune
\ndefense. Such activation of different cell types as immune effec-
\ntors corroborates the hypothesis of Ottaviani et al. (1998) that
\nsuggested that, in bivalve hemolymph (M. galloprovincialis), there
\nis only one hemocyte type \u2013 with two or more different matur-
\nation (aging)-related stages, that is, hyalinocytes in a proliferative
\nstage mature to become granulocytes (Ottaviani et al. 1998).\n<\/p>\n
\nAgNP with sizes of <50 and <100 nm was based on the litera-
\nture on potential uptake pathways for each size particle. Typical
\nclathrin-coated pits (vessels for clathrin-mediated endocytosis)
\nhave diameters in the range 120 nm; conversely, internalization
\nvia caveolae-mediated endocytosis is considered the predominant
\nmechanism of entry for structures of 40\u201350 nm (and below)\n<\/p>\n
\nfrom mussels exposed for 3 h.\n<\/p>\n
\nBasophils 0.4661 1.0000 \u2013
\nAcidophils \ufffd0.7738\ufffd\ufffd \ufffd0.9008\ufffd\ufffd 1.0000
\n\ufffd\ufffdValue significantly correlated at p < 0.01.\n<\/p>\n
\nfrom mussels exposed for 6 h.\n<\/p>\n
\nBasophils 0.7034\ufffd 1.0000 \u2013
\nAcidophils \ufffd0.9511\ufffd\ufffd \ufffd0.8886\ufffd\ufffd 1.0000
\nValue significantly correlated at \ufffdp < 0.05 or \ufffd\ufffdp < 0.01.\n<\/p>\n
\nfrom mussels exposed for 12 h.\n<\/p>\n
\nBasophils 0.2550 1.0000 \u2013
\nAcidophils \ufffd0.8243\ufffd\ufffd \ufffd0.7577\ufffd\ufffd 1.0000
\n\ufffd\ufffdValue significantly correlated at p < 0.01.\n<\/p>\n
\nwould reflect how the cells interacted with both size AgNP here,
\nany impact of exposure on caveolae-mediated endocytosis would
\nthen be more directly impactful upon the AgNP <50 nm only
\n(Moore 2006; Doherty & McMahon 2009; Khan et al. 2015). This
\nis an important distinction in that these studies did not segregate
\nout the relatively few particles <50 nm from the AgNP100 parent
\nsample so as to provide hypothetical data for AgNP50 versus
\nAgNP51\u2013100. While such analyses would be interesting and
\ninformative, the reality is that there is no way in the real world
\nto face such segregated selections from a parent mixture of par-
\nticles (any type) even if the original cutoff value was set at
\n100 nm. Further, as the AgNP100 samples only contained \ufffd1.4%
\nparticles <50 nm, their relative contribution to the observed out-
\ncomes for the AgNP100 would be expected to be nominal.\n<\/p>\n
\ndependent effect variation in the percentages of cell categories
\nwas in fact observed here. Other studies also reported size-
\ndependent toxicity of AgNP, that is, with maltose-stabilized
\nAgNP (Katsumiti et al. 2015). In that study, small NP (Ag20-Mal)
\nwere significantly more toxic than larger NP (Ag40-Mal and
\nAg100-Mal). Such outcomes were expected based on a concept
\nproposed by Hine (1999) that posited differences in phagocytosis
\nbetween granulocytes and hyalinocytes were related to character-
\nistics of the involved particles (i.e. differences in size properties
\nhere) rather than differences in immune cell ability to phagocyt-
\nize\/process the particles.\n<\/p>\n
\nuptake mechanisms for NP (here AgNP) in influencing effects on
\nthe frequency of immune cell types. The variations in the percen-
\ntages of different sub-populations seen here showed that when
\nclathrin- or caveolae-mediated endocytosis was inhibited, effects
\ncaused by either size AgNP were delayed. Such results might be
\ndue to a potential ability of either uptake route to initially
\n\u201cmitigate\u201d toxic effects of AgNP as each pathway enables any
\nearly-internalized particles to be broken-down\/digested. While
\nthis might reduce initial levels of intracellular AgNP, it con-
\nversely increases the presence of the AgNP externally (such as in
\nan actual water environment) to putatively serve as continuous
\nsource of Ag ions due to particle oxidation (involving dissolved
\nO2 and protons in aqueous system) (Dobias & Bernier-Latmani
\n2013; Gliga et al. 2014; Yu et al. 2014). Over time, the now
\nincreasingly present Ag\u00fe ions could then impart their own forms
\nof cytotoxicity as was demonstrated in studies by Park et al.
\n(2013) and Katsumiti et al. (2015).\n<\/p>\n
\nmay influence the frequency of different hemocyte sub-popula-
\ntions as biomarker of the immunomodulation of mussel hemo-
\ncytes by NP. It was clearly noted that nanotoxicity of AgNP was
\nsize and indirectly duration of exposure dependent. The internal-
\nization mechanism of NP most likely considered as major factor
\nunderlying NP effects in hemocytes of M. galloprovincialis.
\nLastly, it is highly recommended further research be undertaken
\nto clarify how specific uptake routes could be involved in deter-
\nmining NP toxicity.\n<\/p>\n
\nand cancerogesis IMEC Research Unit, Sciences Faculty of Bizerte,\n<\/p>\n
\nDavid Sheehan at the Proteomic Research Group in the School of
\nBiochemistry and Cell Biology at University College Cork (Ireland),
\nfor reviewing this paper.\n<\/p>\n
\nand cancerogesis IMEC Research Unit, Sciences Faculty of Bizerte,
\nUniversity of Carthage, Tunisia.\n<\/p>\n
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\ncontent may not be copied or emailed to multiple sites or posted to a listserv without the
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\narticles for individual use.<\/p>\n<\/p>\n<\/div>\n\n
\n
\n
\n
\n
\n
\nEvaluation of the Response of HOS and Saos-2 Osteosarcoma Cell
\nLines When Exposed to Different Sizes and Concentrations of
\nSilver Nanoparticles\n<\/p>\n
\n2Tufts University, Boston, MA, USA
\n3University of Oxford, Oxford, UK
\n4Laboratory for Biomaterials and Computational Mechanics, Department of Mechanical Engineering, University of
\nWestern Macedonia, Kozani, Greece
\n5Department of Mechanical Engineering, School of Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece
\n6Department of Oncology, University of Oxford, Oxford, UK\n<\/p>\n
\nAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
\nis properly cited.\n<\/p>\n
\nlungs, resulting in poor survival rates of between 19 and 30%. Standard treatment consists of surgical removal of the affected site,
\nwith neoadjuvant and adjuvant chemotherapy commonly used, with the usual side effects and complications. There is a need for
\nnew treatments in this area, and silver nanoparticles (AgNPs) are one potential avenue for exploration. AgNPs have been found to
\npossess antitumor and cytotoxic activity in vitro, by demonstrating decreased viability of cancer cells through cell cycle arrest and
\nsubsequent apoptosis. Integral to these pathways is tumor protein p53, a tumor suppressor which plays a critical role in
\nmaintaining genome stability by regulating cell division, after DNA damage. The purpose of this study was to determine if p53
\nmediates any difference in the response of the osteosarcoma cells in vitro when different sizes and concentrations of AgNPs are
\nadministered. Two cell lines were studied: p53-expressing HOS cells and p53-deficient Saos-2 cells. The results of this study
\nsuggest that the presence of protein p53 significantly affects the efficacy of AgNPs on osteosarcoma cells.\n<\/p>\n
\nnant disease. Nevertheless, it is the most common cancer
\narising from bone [1]. It usually affects adolescents and
\nyoung adults. In recent years, much advancement has been
\nmade in treating osteosarcoma, which combines surgery,
\nchemotherapy, and sometimes radiotherapy. Currently, the
\n5-year survival rate for patients diagnosed with osteosar-\n<\/p>\n
\ninclude cisplatin, doxorubicin, ifosfamide, and methotrexate.
\nOther cytotoxic agents such as etoposide and different com-
\nbinations have also been suggested in the literature [6]. Nev-
\nertheless, the use of these drugs has several side effects and
\ncomplications including neutropenia, mouth ulcers, fatigue,
\nsevere diarrhea, nausea, and vomiting. The side effects can
\nbe very serious and commonly require hospitalization. Car-
\ndiomyopathies and irreversible lung fibrosis have also been\n<\/p>\n
\nBioMed Research International
\nVolume 2021, Article ID 5013065, 16 pages
\nhttps:\/\/doi.org\/10.1155\/2021\/5013065<\/p>\n<\/p>\n