Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis

Accepted Manuscript Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis Pamela Ruiz, Alberto Katsumiti, Jose A. Nieto, Jaume Bori, Alba Jimeno-Romero, Paul Reip, Inmaculada Arostegui, Amaia Orbea, Miren P. Cajaraville PII:

S0141-1136(15)30025-8

DOI:

10.1016/j.marenvres.2015.07.018

Reference:

MERE 4045

To appear in:

Marine Environmental Research

Received Date: 19 January 2015 Revised Date:

27 July 2015

Accepted Date: 28 July 2015

Please cite this article as: Ruiz, P., Katsumiti, A., Nieto, J.A., Bori, J., Jimeno-Romero, A., Reip, P., Arostegui, I., Orbea, A., Cajaraville, M.P., Short-term effects on antioxidant enzymes and longterm genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis, Marine Environmental Research (2015), doi: 10.1016/ j.marenvres.2015.07.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Short-term effects on antioxidant enzymes and long-term genotoxic and

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carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic

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copper in mussels Mytilus galloprovincialis

4 Pamela Ruiza, Alberto Katsumitia, Jose A. Nietoa, Jaume Boria, Alba Jimeno-Romeroa,

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Paul Reipb, Inmaculada Arosteguic, Amaia Orbeaa, Miren P. Cajaravillea*

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a

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and Technology and Research Centre for Experimental Marine Biology and

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Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country,

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Spain.

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b

Intrinsiq Materials Ltd, Cody Technology Park, Hampshire, UK.

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Department of Applied Mathematics, Statistics and Operations Research, Faculty of

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Science and Technology, University of the Basque Country UPV/EHU, Leioa, Spain.

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CBET Research Group, Dept. Zoology and Animal Cell Biology; Faculty of Science

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*Author for correspondence:

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Miren P. Cajaraville, CBET Research Group, Dept. Zoology and Animal Cell Biology,

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Science and Technology Faculty and Research Centre for Experimental Marine Biology

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and Biotechnology PIE, University of the Basque Country UPV/EHU. Basque Country,

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Spain.

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Tel.: + 34 94 6012697

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Fax: + 34 94 6013500

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e-mail address: [email protected]

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Abstract

26 The aim of this work was to study short-term effects on antioxidant enzyme activities

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and long-term genotoxic and carcinogenic potential of CuO nanoparticles (NPs) in

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comparison to bulk CuO and ionic copper in mussels Mytilus galloprovincialis after 21

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days exposure to 10 µg Cu L-1. Then, mussels were kept for up to 122 days in clean

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water. Cu accumulation depended on the form of the metal and on the exposure time.

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CuO NPs were localized in lysosomes of digestive cells, as confirmed by TEM and X

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ray microanalysis. CuO NPs, bulk CuO and ionic copper produced different effects on

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antioxidant enzyme activities in digestive glands, overall increasing antioxidant

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activities. CuO NPs significantly induced catalase and superoxide dismutase activities.

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Fewer effects were observed in gills. Micronuclei frequency increased significantly in

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mussels exposed to CuO NPs and one organism treated with CuO NPs showed

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disseminated neoplasia. However, transcription levels of cancer-related genes did not

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vary significantly. Thus, short-term exposure to CuO NPs provoked oxidative stress and

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genotoxicity, but further studies are needed to determine whether these early events can

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lead to cancer development in mussels.

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Key Words: copper oxide nanoparticles, Mytilus galloprovincialis, bioaccumulation

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and subcellular localization, long-term effects, micronuclei frequency, histopathology,

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transcription level of cancer-related genes p53, ras and gadd45α.

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Abbreviations

48 CAT, catalase

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dH2O, deionised water

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EDTA, ethylenediamine tetraacetic acid

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GADD45α, growth arrest- and DNA damage inducible 45 alpha

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GPx, glutathione peroxidase

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MN, micronuclei

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NM, nanomaterial

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NP, nanoparticle

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ROS, reactive oxygen species

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RQ, relative quantification

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SOD, superoxide dismutase

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1. Introduction

62 During recent years engineered nanoparticles (NPs) are emerging as a potential new

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type of environmental pollutant due to the extensive development in the field of

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nanotechnology. NPs are particles less than 100 nm in size in more than one dimension

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(EPA, 2007). The characteristic size of NPs gives special mechanical, catalytic and

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optical properties that make them suitable for developing applications in many areas

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including cosmetics, medicine, food and food packaging, bioremediation, paints,

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coatings, electronics, fuel catalysts and water treatment (Aitken et al., 2006; Chaudhry

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et al., 2008; Savage and Diallo, 2005). Those man-made NPs, commonly known as

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engineered NPs, already include a high number of substances like metals, metal oxides

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and alloys, carbon-based materials such as fullerenes, silicates and quantum dots as well

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as polymer composites (Aitken et al., 2006; Chaudhry et al., 2008). Although CuO NPs

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are currently not as commonly used as other metal or metal-bearing NPs, such as Ag or

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TiO2 NPs, they are industrially produced and commercially available in the market

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place. They show potential to replace noble metal catalysts for carbon monoxide

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oxidation (Zhou et al., 2006) and to be used as additives in lubricants, polymers/plastics

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and metallic coating inks.

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During past decades, marine invertebrates, and especially bivalve molluscs like mussels,

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have been extensively used as sentinel organisms for studying the biological effects of

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both organic and inorganic pollution (Cajaraville et al., 2000; Zorita et al., 2007).

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Recent studies have highlighted the utility of marine invertebrates as test organisms for

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NP ecotoxicity too. Since invertebrates represent about 95% of animal species, they

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play an important ecological role and participate in transfer of NPs through food chains

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(Baun et al., 2008). Filter-feeding invertebrates, especially bivalve molluscs, constitute

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ACCEPTED MANUSCRIPT an important target group for NP toxicity due to their highly developed processes for

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cellular internalisation of nano- and micro-scale particles by endocytosis and

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phagocytosis, respectively (Moore, 2006). As the final destination of filtered particles

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within the organism, digestive gland cells are useful to determine NPs fate and effects in

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mussels (Canesi et al., 2012). Haemocytes (haemolymph cells in charge of the innate

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immune response in bivalves) are also a major target of NPs (Canesi et al., 2012).

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During recent years several experiments of exposure to NPs have been carried out both

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in vitro (Canesi et al., 2010; Katsumiti et al., 2014a, 2014b) and in vivo (Buffet et al.,

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2011; 2012; Gagné et al., 2008; Gomes et al., 2011, 2012, 2013, 2014; Tedesco et al.,

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2008; 2010) using mussels and other bivalves.

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Toxicity mechanisms at the cellular level have not been yet completely elucidated for

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most NPs, but possible mechanisms include disruption of membranes or membrane

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potential, oxidation of proteins, genotoxicity, interruption of energy transduction,

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formation of reactive oxygen species (ROS) and release of toxic constituents (Klaine et

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al., 2008). Oxidative stress has been postulated in several in vivo and in vitro studies as

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a primary toxicity mechanism of NPs both in mammalian models (Park and Park, 2009;

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Ye et al., 2010) and in different aquatic species (reviewed by Klaine et al., 2008 and Fu

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et al., 2014) including freshwater fish such as zebrafish (Zhu et al., 2008), estuarine fish

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such as sticklebacks (Sanders et al., 2008) and mussels (Gagné et al., 2008; Tedesco et

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al., 2008; 2010). Observed toxic effects seem to be mediated through the formation of

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the very reactive hydroxyl radicals (Reeves et al., 2008). CuO NPs have been shown to

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be toxic to both vertebrates and invertebrates by increasing intracellular ROS

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production (Aruoja et al., 2009; Buffet et al., 2011; Chen et al., 2006; Karlsson et al.,

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2008; Meng et al., 2007).

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The capacity of CuO NPs to produce ROS may lead to activation or inhibition of

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ACCEPTED MANUSCRIPT antioxidant enzymes and consequently the alteration of the antioxidant capacity

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(Ahamed et al., 2010; Buffet et al., 2011; Gomes et al., 2011, 2012; Karlsson et al.,

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2008). In case antioxidant capacity is overwhelmed, ROS can damage DNA by

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production of strand breaks, cross links and adducts of nucleotide bases or sugars (Chen

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et al., 2006; Kang et al., 2012).

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In addition to NP induced indirect DNA damage through ROS, NPs can directly interact

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with DNA due to their small size and high surface area (Singh et al., 2009). Thus, both

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by direct or indirect mechanisms, NPs can cause DNA damage, as shown by Comet

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assays or micronuclei (MN) frequency tests. CuO NPs were found to induce DNA

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fragmentation and MN formation in N2A cells (Perreault et al., 2012). Similar results

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were observed in murine macrophages RAW 264.7 and in peripheral whole blood from

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healthy volunteers exposed in vitro to CuO NPs of different shapes (Di Bucchianico et

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al., 2013). In agreement, Gomes and co-workers (2013) and Rocha et al. (2014) showed

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that CuO NPs and CdTe quantum dots, respectively, were genotoxic to mussels’

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haemocytes after 1 and 2 weeks of exposure. Similar results have been reported for

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other bivalve species, such as the clam Scrobicularia plana after 21 days exposure to

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CuO NPs (Buffet et al., 2103; Mouneyrac et al., 2014).

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DNA damage caused by nanomaterials (NMs) can invoke various cellular responses

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such as cell cycle arrest, apoptosis and DNA repair. When DNA is damaged, a key

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effector molecule, P53, is activated. This tumour suppressor gene is responsible for

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arresting the cell cycle and activating transcription of genes that mediate DNA repair,

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thus preventing the conversion of damage to mutation (Harris and Levine, 2005).

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However, if the damage is extensive, apoptotic pathways are triggered and elicit cell

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death (Sancar et al., 2004). Treatment with CdTe quantum dots, TiO2 NPs and Ag NPs

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ACCEPTED MANUSCRIPT increased P53 expression in zebrafish liver, human lymphocytes and mouse embryonic

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stem cells and fibroblasts (Ahamed et al., 2008; Choi et al., 2008; Kang et al., 2008). In

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the case of CuO NPs, exposure of human hepatocellular carcinoma HepG2 cells and

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human lung epithelial (A549) cells induces the expression of P53 (Siddiqui et al., 2013;

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Wang et al., 2012). However, exposure of HaCaT human keratinocytes and mouse

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embryonic fibroblasts to CuO NPs induced decreases in P53 and p-P53 levels,

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indicating that P53 had not a prodeath function (Luo et al., 2014). In order to maintain

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genomic stability, DNA repair genes are activated after DNA damage (Hanahan and

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Weinberg, 2000). Thus, exposure of HepG2 human hepatoma cells to Ag NPs led to up-

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regulation of DNA repair specific genes, such as rad51 and gadd45 (Kawata et al.,

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2009). Similarly, Ag NPs up-regulated DNA damage repair protein RAD51 in mouse

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embryonic cells (Ahamed et al., 2008). ras oncogene plays a pivotal role in regulating

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cell growth, differentiation and survival (Patra, 2008) and it is oncogenically activated

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by mutations in over 25% of all human tumours (Bos, 1989). Mutations of ras gene

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locus were found in the lung of mice exposed to single-walled carbon nanotubes

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(Shvedova et al., 2008).

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Little is known about the potential mutagenic and carcinogenic effects of NMs in vivo.

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It is possible that NMs affect tumour formation through DNA damage, increasing cell

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proliferation associated with inflammation or by oxidative stress, which is considered as

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a main non-genotoxic mechanism of carcinogenesis (Klaine et al., 2008; Singh et al.,

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2009).

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Thus, the present work aimed to study the short-term effects on the antioxidant system

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and the long-term genotoxic and carcinogenic potential of CuO NPs in comparison to

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effects caused by bulk CuO or ionic copper in mussels. While there are many studies

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comparing the effects caused by NPs and their counterpart ionic forms, recently Duester

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ACCEPTED MANUSCRIPT et al. (2014) have highlighted the lack of comparative studies with bulk products in

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order to identify possible nano-specific effects and to assess the need for nano-specific

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regulations. In order to achieve this objective, an experiment was designed in which

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mussels were maintained unexposed or exposed to CuO NPs, bulk CuO and ionic

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copper for 21 days and then kept in clean water for up to 122 days in an attempt to

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allow possible initiation of tumour lesions. As cancer is a multistage, progressive

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disease, long-term experiments are needed for carcinogens to induce neoplasia in fish

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and mammals (Spitsbergen and Kent, 2003; Winslow and Jacks, 2008). In this study,

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bioaccumulation of copper in mussels exposed to the three copper forms was quantified

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by chemical analyses and subcellular localization of CuO NPs was addressed by TEM

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and X-ray microanalysis. Oxidative stress was assessed measuring the activity of the

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main antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx) and

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superoxide dismutase (SOD)), genotoxicity was studied by the MN frequency assay,

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and carcinogenic potential was evaluated through the measurement of the transcription

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level of cancer-related genes p53, ras and gadd45α and through histopathological

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analysis of mussel tissues. This is the first report on a long-term experiment designed to

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address potential effects of NPs on cancer-related genes and cancer development.

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2.1. Animals and experimental procedure

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Adult mussels, M. galloprovincialis, of 3.5-4.5 cm shell lengths were obtained from an

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aquaculture facility in Boiro, A Coruña (42º 39.00´N; 8º 53.00´O) (Spain) in late

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January 2010. After arrival to the laboratory, mussels were placed in a 600 L tank with

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running seawater. Seawater was obtained from Getaria (43º 18.00´N; 2º 12.00´O) 8

ACCEPTED MANUSCRIPT (Basque Country, Spain) and subsequently treated with ultraviolet light, filtered with

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active charcoal and passed through a mechanic filter (0.45 µm) before use (T = 16-18

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ºC; salinity = 33%; hardness = 7 dKH; conductivity = 41-45 KµS; pH = 7.6-7.8). Water

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quality (nitrate, nitrite and ammonia concentration) was checked every day using

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commercial tests (SERA GmbH, Heisenberg, Germany). Mussels were acclimatised for

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14 days before their transfer to 300 L exposure tanks containing 250 L seawater.

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Animals were not fed in the first 5 days of acclimatisation; for the next days mussels

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were fed with the microalgae Isochrysis galbana (4x106 algae mL-1) supplemented with

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the commercial marine invertebrate diet Coraliquid (Sera Marin, Heinsberg, Germany).

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One exposure tank containing 250 mussels was used for each experimental condition.

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Mussels were exposed for 21 days to 10 µg Cu L-1 in the form of CuO NPs, bulk CuO

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or ionic copper (from CuCl2) in a recirculating system with water aeration. The

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recirculating system was stopped for 30 min each day in order to feed animals with I.

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galbana (4x106 algae mL-1). Every 3 days, all water was removed and the tanks were

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cleaned to remove CuO aggregates deposited at the bottom of the tanks, as well as

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mussels and food debris. Clean seawater was added and CuO suspensions and ionic

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copper solution were redosed after each change up to a nominal concentration of 10 µg

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Cu L-1. The feeding and Cu redosification were always done separately, with an interval

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of 6 h between them. In parallel, an unexposed water control group was maintained in

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the same experimental conditions.

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CuO NPs were synthesised at Intrinsiq Materials Limited (Farnborough, UK) as powder

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and a 25 mg L-1 suspension of these NPs in deionized water was stable for

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approximately 1 month. NP characterisation has been reported previously (Buffet et al.,

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2011). Briefly, size distribution of CuO NPs in dH2O ranged from 40 to 500 nm with an

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average of 197 nm. When suspended in seawater, the NPs aggregated/agglomerated and

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ACCEPTED MANUSCRIPT the sample was not suitable for dynamic light scattering measurements. Zeta potential

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measurements showed that the particles were positively charged (26.3 mV) in dH2O,

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which corroborated the stability of the suspension, while the NPs appeared slightly

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negatively charged in seawater collected at t=0 and t=2 days (-8.69 and -7.72 mV,

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respectively), an indication of poor stability (Buffet et al., 2011). CuO NPs were almost

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non-soluble in seawater (Buffet et al., 2011), as confirmed by speciation simulation

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studies

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(http://www.kemi.kth.se/medusa/). According to the simulation, CuO particles form

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complexes such as CuCl+, CuCO3 and Cu2+ depending on the pH of the media. At the

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range of pH values of natural seawater (pH ≈ 8), only a minor fraction of Cu is released

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from CuO particles, the majority remaining as CuO.

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CuO NPs and bulk CuO (Sigma-Aldrich, St. Louis, Missouri, USA) stock solutions (25

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mg Cu L-1) were sonicated for 10 min in a sonication bath (50 Hz/ 220 V, Ultrasons-H,

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JP Selecta, Barcelona, Spain) and stirred overnight. The flasks containing the

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suspensions were sonicated again for 10 min before dosing. The bulk CuO suspension

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was unstable in seawater along time and precipitated in the stock solution. CuCl2

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(Probus, Barcelona, Spain) solution was prepared in the same way as the CuO

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suspensions except for the sonication steps, which were not necessary as the chloride

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form is soluble in water.

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Each experimental tank, including the control group, was equipped with a set of two

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water pumps (3000 L h-1) placed in the exterior of the tank that created two directional

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water currents inside the tank. This system design aimed to maintain bulk CuO and CuO

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NPs in suspension during the exposure period. After the 21 day exposure period,

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mussels were maintained for up to 122 days in clean seawater. Samples of mussels were

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collected after 1 day and 21 days of exposure and at 63 and 122 days post-exposure.

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2.2. Chemical analyses in mussel tissues

238 Mussels (n = 20 per experimental group) were de-shelled and soft tissues were placed in

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glass plates and dried in an oven at 130ºC for 24 h. Flesh dry weight was determined for

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each mussel. Soft tissues were pooled (5 pools of 4 mussels), placed into 25 mL

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Erlenmeyer’s, grinded to fine powder and digested in nitric acid (65%, Scharlau, extra

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pure quality). Upon full digestion the remnant liquid was left to evaporate in a hot plate

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(80ºC). Later on, 6 mL nitric acid (0.1 M) were added to each Erlenmeyer and the

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resulting contents were transferred to sealed test tubes and stored at 4ºC prior to

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chemical analysis. ICP-MS (Agilent, 7700. Agilent Technologies, Santa Clara, CA,

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USA) analysis was carried out for Cu content in mussel soft tissue by the Analytical

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Chemistry Service of the University of the Basque Country (SGiKER) following US-

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EPA 6020A directions. Certified reference material TMDA 54.4 LOT1107 fortified

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water from Lake Ontario (Environment Canada) was used as analytical reference.

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2.3. Subcellular localization of CuO NPs in mussel digestive gland

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A portion of the digestive gland of 3 mussels per treatment was processed for

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Transmission Electron Microscopy (TEM) using a standard procedure modified from

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Hayat (2000). Briefly, various pieces (<1 mm3 in size) of each digestive gland were

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fixed in filtered seawater containing 2.5% glutaraldehyde at 4ºC for 1 h. Then, samples

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were postfixed with osmium tetroxide and ferrocyanide (1:1), cleaned in filtered

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seawater, dehydrated in an ethanol series and embedded in EPON (Fluka; Sigma-

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Aldrich). Semithin (500 nm) and ultrathin (100 nm) sections were cut in a Reichert-

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Jung Ultracut E ultramicrotome (Leica Microsystems; Wetzlar, Germany) and mounted

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ACCEPTED MANUSCRIPT on Ni 100-mesh square grids. Sections were carbon-coated in an Edwards coating

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system (Edwards Auto 306, Edwards High Vacuum International, UK) and observed in

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a FEI-Tecnai T12 TEM (FEI Company, Hilsboro, USA) at 120 kV. X-ray microanalysis

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(EDX) was performed with the aid of an EDAX X-Ray detector (EDAX Inc. Mahwah,

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USA).

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2.4. Antioxidant enzymes in mussel digestive glands and gills

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Digestive glands and gills of 18 mussels per experimental group were dissected and

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stored at -80ºC until analysis. Tissues of three individuals were pooled and

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homogenised (3:10, w/v) in 10 mM Tris-HCl, pH 7.6, containing 0.15 M KCl and 0.5 M

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sucrose using a glass-Teflon® homogeniser in an ice water-cooled bath (Potter S

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Homogeniser, B. Braun, Melsungen, Germany). Homogenised samples were

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centrifuged at 500 g for 15 min in a Beckman Coulter Allegra 25R Centrifuge (Palo

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Alto, USA) and then centrifuged at 12,000 g for 45 min in an Optima L-90K Beckman

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Coulter ultracentrifuge (Pasadena, USA) in order to obtain the mitochondrial fraction.

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Supernatants were then centrifuged at 100,000 g for 90 min in the same ultracentrifuge

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in order to obtain the cytosolic fraction.

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Cytosolic and mitochondrial fractions were used for enzyme activity determinations.

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CAT activity was assessed in both mitochondrial and cytosolic fractions by measuring

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the disappearance of H2O2 at 240 nm (ext. coeff. 40 M−1 cm−1) in a Shimadzu

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spectrophotometer (Columbia, USA) using 50 mM H2O2 as substrate in 80 mM

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potassium phosphate buffer (pH 7) according to Aebi et al. (1974). CAT activity was

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calculated as the sum of the activity measured in both fractions and expressed in mmol

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min−1 mg protein−1. GPx activity was measured in the cytosolic fraction at 340 nm (ext.

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ACCEPTED MANUSCRIPT coeff. 6.2 mM−1 cm−1) in an assay mixture that contained 100 mM potassium phosphate

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buffer, pH 7, 2 mM glutathione, 1 mM sodium azide, 2 U mL−1glutathione reductase

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and 120 µM NADPH (Guntzer and Flohe, 1985). GPx activity was expressed in nmol

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min−1 mg protein−1. SOD activity was determined in the cytosolic fraction at 550 nm by

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measuring the inhibition of cytochrome c reduction by O2−• generated by the xanthine

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oxidase/hypoxanthine system in an assay mixture that contained 50 mM potassium

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phosphate buffer plus 0.1 mM EDTA (pH 7.8), 50 µM hypoxanthine, 1.87 mU mL−1

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xanthine oxidase and 10 µM cytochrome c (Porte, 1991). One SOD unit was defined as

295

the amount of enzyme that inhibits the rate of cytochrome c reduction by 50%. SOD

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activity was expressed in SOD unit mg protein−1. Protein concentration of each

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subcellular fraction was measured following the method of Lowry et al. (1951).

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2.5. Micronuclei (MN) frequency

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Haemolymph from each of 8 mussels per experimental group was withdrawn from the

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posterior adductor muscle through the shell hinge using a sterile, 21 gauge needle

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attached to a 2 mL syringe. Haemolymph from each mussel was transferred into an

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individual microtube and kept cold on crushed ice to prevent haemocyte aggregation. 30

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µL haemolymph was mixed with 120 µL of cold Alsever´s anti-aggregant solution

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(glucose: 0.11 M; sodium citrate: 37 mM; EDTA: 11 mM; NaCl: 0.38 M) and

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cytocentrifuged at 92 x g for 5 min using a Shandon Cytospin 4 cytocentrifuge

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(Thermo, Cheshire, UK). The haemolymph cells were fixed and stained with the kit

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Hemacolor® (Merck, Darmstadt, Germany) and mounted with DPX. 1000 agranular

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haemocytes per mussel were examined under an Olympus BX51 microscope (Tokyo,

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Japan) using a 100X objective. Micronucleated cells were classified following generally

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ACCEPTED MANUSCRIPT accepted criteria for mussels: well-preserved cell cytoplasm, MN not touching the main

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nucleus, similar or weaker staining than the main nucleus and size of MN ≤ 1/3 in

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comparison to the main nucleus (Venier et al., 1997). Results are reported in ‰

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frequencies.

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2.6. Quantitative real-time RT-PCR

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Digestive glands of 4-7 mussels per experimental group were dissected, immersed

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individually in RNA later® (Sigma-Aldrich), frozen in liquid nitrogen and stored at -

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80ºC.

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Transcription levels of p53 (DQ158079), ras (DQ305041) and gadd45α (AJ623737)

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were measured by real time quantitative PCR using custom TaqMan probes. About 50-

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100 mg of each individual mussel digestive gland were homogenised in TRIzol®. Total

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RNA was isolated and its purity was checked with a Biophotometer Spectrophotometer

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(Eppendorf, Hamburg, Germany). cDNA was obtained from 2 µg of total RNA by

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Super ScriptTM II reverse transcriptase PCR (Invitrogen, Leek, Netherlands) using

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random hexamers as primers and following the manufacturer’s recommendations in a

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conventional 2720 Thermal Cycler (Applied Biosystems Life Technologies, California,

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USA).

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The real time PCR was run in 25 µL reactions on a 7003 PCR machine (Applied

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Biosystems Life Technologies) using TaqMan Reverse Transcription Reagent (Applied

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Biosystems Life Technologies, New Jersey, USA). TaqMan probes and primers (Table

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1) from mussel specific sequences were designed using Primer Express 3.0 software

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(Applied Biosystems Life Technologies). Universal conditions were used in PCR for all

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genes: 1 cycle at 50ºC for 2 min, 1 cycle at 95ºC for 10 min, 40 cycles at 95ºC for 15 s

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ACCEPTED MANUSCRIPT and at 60ºC for 1 min. A control without template and RT-control reactions were run for

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quality assessment.

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Amplified fragments were visualised in ethidium bromide stained 1.5% agarose gels,

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cloned using TOPO-TA cloning reagents (Invitrogen, Carlsbad, California, USA) and

341

sequenced. 18S rRNA (L33452) and elongation factor 1 alpha (EF1-α, AB162021) were

342

used for normalisation of transcription levels of target genes (Table 1). Relative

343

transcription levels were calculated with the 2-∆∆ct method (Livak and Schmittgen, 2001)

344

relative to the mean of control animals sampled at day 1.

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2.7. Histopathological analysis

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For histopathological analysis, 20 mussels per experimental group were used. The

349

digestive gland, gonad and gills were dissected out, fixed in 10% neutral buffered

350

formalin and routinely processed for paraffin embedding in a Leica Tissue processor

351

ASP 3000 (Leica Instruments, Wetzlar, Germany). Histological sections (5 µm in

352

thickness) were cut in a Leica RM2255 microtome (Leica Instruments) and stained with

353

hematoxylin/eosin (Wilson and Gamble, 2002). One section per individual was

354

examined “blind” under an Olypmus BX61 motorised upright microscope (Tokyo,

355

Japan).

356

Prevalence of histopathological alterations such as presence of disseminated neoplasia,

357

infiltration of tissues by haemocytes, gonadal neoplasia, granulocytomas, aggregates of

358

brown cells, necrotic areas and parasites was determined in one section per organ. The

359

histopathological evaluation was based on the criteria established by Bignell et al.

360

(2008, 2012). Results are given in percentages.

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2.8. Statistical analysis 15

ACCEPTED MANUSCRIPT 363 Bootstrap resampling techniques (Efron and Tibshirani, 1993) were used to assess

365

differences between treatment groups and time settings. For each experiment, N = 2000

366

repetitions of the same size of the original sample were selected by bootstrap

367

resampling. After that, Bonferroni’s correction was used for multiple comparisons.

368

Significance level was globally stated at 5% for all the comparisons. Bootstrap analyses

369

were performed using the SAS 9.2 software (Cary, USA). For histopathological data the

370

Chi-square test was used (significance level p<0.05) using SPSS v. 21 (SPSS Inc.,

371

Microsoft Co., Redmond, WA).

373

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374 375

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3.1. Copper content in mussel tissue

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Cu accumulation depended on the form of the metal and on the exposure time. After 1

378

day of exposure Cu concentration was similar in mussels exposed to the three forms of

379

copper, ranging from 2.4 to 5 µg Cu g-1 dry weight with no significant differences

380

among treatments. After 21 days of exposure, mussels exposed to CuO NPs or ionic

381

copper showed significantly higher Cu level than the control group, being mussels

382

exposed to ionic copper the ones with the highest Cu concentration in soft tissues. Bulk

383

CuO was the less available metal form to mussels. Level of Cu in mussels exposed to

384

bulk CuO was significantly lower than after exposure to ionic copper and similar to that

385

registered in control mussels. As to time-related effects, Cu accumulation was higher

386

after 21 days of exposure than after 1 day for all the exposed groups. This increase was

387

significant in mussels exposed to CuO NPs and to ionic copper (Figure 1).

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ACCEPTED MANUSCRIPT 388 389

3.2. Subcellular localization of CuO NPs in mussel digestive gland

390 Control mussels lacked well-defined electron-dense structures in the lumen of the

392

digestive tubules or in the tubule epithelium (results not shown). Electrondense particles

393

were found inside the lumen of the stomach and of digestive diverticula, where they

394

were attached to organic matter, cell debris or inside residual bodies. Particles were also

395

found among microvilli of digestive cells and inside digestive cell lysosomes (Figure

396

2a). X-ray microanalysis confirmed the elemental composition of these particles (Figure

397

2b).

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398 399

3.3. Antioxidant enzymes in mussel digestive glands and gills

400

Exposure to the three forms of copper induced different responses of the antioxidant

402

enzyme activities in mussel digestive glands and gills. In digestive glands, CAT activity

403

increased significantly in mussels exposed for 1 and 21 days to CuO NPs and bulk CuO

404

with respect to control animals (Figure 3a). CAT activity increased along the time in

405

mussels treated with the three forms of copper (Figure 3a). SOD activity increased

406

significantly respect to control animals in mussels exposed for 1 day to bulk CuO and

407

ionic copper and in mussels treated with CuO NPs for 21 days (Figure 3b). SOD

408

activity increased along the time in control animals and animals treated with CuO NPs,

409

and decreased in animals exposed to bulk CuO (Figure 3b). GPx activity increased

410

significantly in animals treated with bulk CuO and ionic copper with respect to control

411

after 1 day of exposure. After 21 days, all treated mussels showed higher GPx activities

412

than control mussels, but these differences were not significant due to the high

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ACCEPTED MANUSCRIPT variability recorded in exposed animals. Only mussels exposed to ionic copper

414

displayed significantly higher GPx activity after 21 days than after 1 day of exposure

415

(Figure 3c).

416

In gills, fewer effects were found in the activity of the antioxidant enzymes. CAT

417

activity increased in mussels exposed for 1 day to bulk CuO with respect to control

418

animals (Figure 3d). After 21 days exposure, CAT activity increased in control animals

419

and animals treated with CuO NPs and decreased in animals exposed to bulk CuO

420

compared to animals sampled at day 1 (Figure 3d). SOD activity did not show

421

variations among treatments, but decreased along the time in animals treated with ionic

422

copper (Figure 3e). GPx activity did not vary among treatments, but increased along the

423

time in control animals and in animals treated with CuO NPs (Figure 3f).

424 425

3.4. Micronuclei (MN) frequency

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MN frequency was significantly higher in haemocytes of mussels exposed for 21 days

428

to CuO NPs with respect to control mussels (Figure 4). Temporal differences were

429

observed only in this treatment, where MN frequency was significantly higher at 21

430

days exposure compared to those in mussels at 122 days post exposure. At 63 and 122

431

days post-exposure, MN frequency did not show significant variation among treatments

432

(Figure 4).

433

In addition to MN (Figure 5a), other cellular alterations were infrequently noted in

434

individual cases in haemolymph preparations. Binucleated cells were seen in two

435

individuals, one exposed to ionic copper and sampled at 63 days post-exposure and the

436

other exposed to bulk CuO and sampled at 122 days post-exposure. Disseminated

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ACCEPTED MANUSCRIPT 437

neoplasia (Figure 5b) was found in haemolymph preparations of one individual exposed

438

to CuO NPs sampled at 63 days post-exposure.

439 440

3.5. Quantitative real-time RT-PCR

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The transcription level of 18S rRNA showed significant variation in all experimental

443

groups throughout the experiment. On the contrary, EF1-α did not show significant

444

variation either among treatments or among time periods and its transcription levels

445

remained almost constant in all conditions (data not shown). Therefore, levels of

446

transcription of EF1-α were used to normalise the transcription of genes of interest.

447

For p53, ras and gadd45α transcription levels did not show significant differences,

448

neither among treatments nor throughout time (Figures 6a-c).

449

451

3.6. Histopathological analysis

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In general, a higher prevalence of different histopathological responses was observed in

453

mussels sampled after 63 and 122 days post-exposure than in mussels exposed for 1 and

454

21 days. Both control and exposed mussels showed similar histopathological conditions,

455

consisting mostly of haemocytic infiltrations and brown cells aggregations found in the

456

different tissues. There were no significant differences between different treatments

457

(CuO NPs, bulk CuO and ionic copper) and controls at any time period. There were

458

significant differences within each treatment throughout time (Table 2).

459

Diffuse (Figure 7a, b) and focal (Figure 7c) infiltration appeared mainly in digestive

460

glands. Infiltration also occurred in the gonadal tissue (Figure 7d), but the prevalence

461

was lower than that found in digestive glands. The prevalence of brown cell aggregation

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ACCEPTED MANUSCRIPT was high in gills (Figure 7e) and in digestive glands. Granulocytomas (Figure 7f) were

463

also found mainly in digestive glands although their presence was rare. Neoplasias,

464

either disseminated or gonadal, were not found in any of these organs. Parasites

465

appeared only in one organism of the control group in the third sampling (63 days post-

466

exposure). Most of the organisms sampled during the post-exposure period showed part

467

of the digestive gland tissue occupied by a disorganized connective tissue with atrophy

468

of digestive tubules and a high degree of infiltration by haemocytes.

469

The presence of diffuse and focal haemocytic infiltration in control individuals showed

470

an upward trend from day 1 of the experiment until the third sampling, when diffuse

471

infiltration reached the maximum level with a significantly higher prevalence than in the

472

two previous time periods. After 63 days post-exposure, the prevalence dropped

473

although at the end of the experiment prevalence was still higher than at day 1.

474

Prevalence of brown cell aggregations in controls increased during the first 21 days of

475

the experiment followed by a decrease afterwards and finally increased significantly at

476

the end of the experiment.

477

In mussels treated with CuO NPs, the prevalence of diffuse haemocytic infiltration

478

decreased after 21 days of exposure compared to the first sampling while the prevalence

479

of focal infiltration rose slightly. After that sampling, the prevalence of both types of

480

infiltration increased significantly at 63 days post-exposure, followed by a non-

481

significant decrease after 122 days in clean water. The prevalence of brown cell

482

aggregations did not vary until 63 days post-exposure, significantly increasing after 122

483

days post-exposure, when almost all individuals presented this condition. In the case of

484

mussels treated with bulk CuO, the prevalence of both diffuse and focal haemocytic

485

infiltration showed the same pattern than that described for mussels treated with CuO

486

NPs. Regarding brown cell aggregations, the prevalence increased significantly after 21

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20

ACCEPTED MANUSCRIPT days of exposure and then kept at that level at 63 days post-exposure, finally reaching

488

the highest prevalence at 122 days post-exposure. Diffuse and focal infiltrations

489

exhibited similar tendencies in mussels treated with ionic copper as in mussels exposed

490

to NPs. Similar pattern was shown for brown cell aggregation in control individuals

491

though its increase was more constant throughout time.

492 493

4. Discussion

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Among metal-bearing NPs, CuO NPs are toxic to both vertebrates and invertebrates

496

(Aruoja et al., 2009; Buffet et al., 2011; Karlsson et al., 2008; Gomes et al., 2013;

497

Mouneyrac et al., 2014), but their toxicity mechanisms are not totally understood yet. It

498

is not clear whether their toxicity is attributable to soluble copper ions released from the

499

NPs and known to be toxic (Aruoja et al., 2009) or to the form of the NP itself

500

(Karlsson et al., 2008). Griffitt et al. (2007) reported that after exposure of zebrafish to

501

Cu NPs the toxicity observed was not adequately explained by dissolution of the NPs

502

alone and concluded that Cu NPs exert a toxic effect on zebrafish separate from the well

503

understood effects of soluble copper. Gomes et al. (2014) reached the same conclusion

504

after analyzing the proteomic response of mussels exposed to CuO NPs and Cu2+. In the

505

present work, CuO NPs together with bulk CuO and ionic copper at a concentration of

506

10 µg Cu L-1 were tested under laboratory conditions in order to determine copper

507

bioavailability, short-term effects on antioxidant enzymes and long-term genotoxic and

508

carcinogenic potential in sentinel mussels Mytilus galloprovincialis.

509

Exposure of mussels for 21 days to CuO NPs and to ionic copper resulted in the

510

incorporation of Cu in soft tissues of mussels. Accumulation was slightly higher in

511

mussels exposed to ionic copper than in those exposed to CuO NPs. CuO NPs used in

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ACCEPTED MANUSCRIPT the present work were almost non-soluble in seawater (Buffet et al., 2011), indicating

513

that bioavailability of Cu is similar for nano CuO than for soluble Cu. Cu

514

bioaccumulation was also demonstrated in the bivalve Scrobicularia plana after

515

exposure to the same concentration of the same CuO NPs during 16 days (Buffet et al.,

516

2011). Gomes and co-workers (2011; 2012; 2014) also observed accumulation of Cu in

517

digestive glands and in gills of mussels exposed to 10 µg L-1 of CuO NPs (<50 nm) as

518

well as to ionic copper for 1 and 2 weeks. However, in the present work copper content

519

in mussels exposed to bulk CuO was lower than for those exposed to CuO NPs,

520

reflecting the reduced availability of the bulk compared to the nano CuO. This apparent

521

selective incorporation of CuO NPs, even in an aggregated state, is described by Ward

522

and Kach (2009).

523

TEM and X-ray microanalysis confirmed incorporation and subcellular localization of

524

CuO NPs in mussel digestive cells. CuO NPs appeared to be internalized via endocytic

525

vesicles and consequently incorporated into lysosomes and excreted through residual

526

bodies into the lumen of digestive diverticula. This intracellular trafficking route

527

resembles that used for soluble metals (Marigómez et al., 2002). It is worth mentioning

528

that TEM images showed single particles inside the lysosomes, suggesting that the

529

aggregates present in seawater get somehow disassembled in the gut, and only single

530

particles or very small aggregates incorporate into the cells. This agrees with results

531

obtained for TiO2 NPs in mussels (Jimeno-Romero et al., submitted) and in the

532

polychaete Arenicola marina (Galloway et al., 2010).

533

The toxicity of ionic copper towards aquatic organisms is well known. As a transitional

534

metal, copper participates in Fenton and Haber–Weiss reactions, facilitating the

535

generation of ROS and leading to oxidative stress (Regoli and Principato, 1995). The

536

increase in ROS production and consequently oxidative stress are already recognized as

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22

ACCEPTED MANUSCRIPT main mechanisms of nanotoxicity (Fu et al., 2014). The ability of CuO NPs to cause

538

oxidative stress has been described (Buffet et al., 2011; Gomes et al., 2011; Hu et al.,

539

2014; Karlsson et al., 2008). In the present work, copper exposures increased

540

antioxidant enzyme activities in mussel digestive gland, reflecting the capacity of the

541

three forms of copper to produce ROS and the need of cells to cope with the resulting

542

oxidative stress in this organ. However, the effects were less marked in the gills,

543

consistent with lower activities of antioxidant enzymes in gills compared to digestive

544

gland in mussels (Power and Sheehan, 1996). Less marked responses of CAT, SOD and

545

GPx activities in the gills compared to those in digestive glands could be due to a higher

546

contribution of other antioxidant enzymes such as glutathione-S-transferase (GST) to

547

neutralize ROS in the gill tissue (Power and Sheehan, 1996). According to the work of

548

Gomes et al. (2014), alterations in antioxidant enzyme activities depend on the copper

549

form and on the tissue analyzed. Ionic copper exposure induced a prompt increase (at 1

550

day exposure) in SOD activity in digestive glands, but after 21 days only CuO NPs had

551

a significant effect. In S. plana, Buffet and co-workers (2011) also found stronger

552

effects on SOD activity (from whole mussel body tissue) at 16 days of CuO NP

553

exposure than those in ionic copper exposure. In gills of M. galloprovincialis, Gomes et

554

al. (2011) found that CuO NPs induced oxidative stress by overwhelming gill

555

antioxidant defense system, while for ionic copper enzymatic activities remained

556

unchanged or increased. In digestive glands of the same mussel species, Gomes et al.

557

(2012) found that ionic copper induced SOD activity more strongly than CuO NPs. Our

558

results agree with those of Gomes et al. (2011; 2012) indicating that antioxidant

559

enzymes of mussel digestive glands and gills respond differently to CuO NPs and ionic

560

copper.

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23

ACCEPTED MANUSCRIPT Studies reporting effects of NPs in comparison to those caused by bulk counterparts are

562

scarce (Duester et al., 2014). Exposure to bulk CuO provoked an increase in CAT

563

activity in the digestive gland at both time points, SOD and GPx only responded after 1

564

day treatment. In gills, bulk CuO was able to induce CAT activity after 1 day exposure,

565

whereas no effects of CuO NPs or ionic copper were found at any sampling time with

566

respect to the control. Thus, exposure to bulk CuO induced antioxidant enzyme

567

activities especially in the digestive gland, although Cu levels were not significantly

568

increased in mussels exposed to bulk CuO compared to controls. This apparent

569

contradiction may be explained by the fact that Cu levels were measured in whole soft

570

tissues and not in individual organs, as antioxidant enzyme activities.

571

Few studies have addressed the genotoxic effects of NPs in bivalves and different

572

results have been described depending on the NP type and exposure concentration and

573

time. Gagné et al. (2008) exposed freshwater mussels Elliptio complanata up to 1.6 mg

574

L-1 of CdTe quantum dots for 1 h and found reduced DNA damage in exposed animals

575

compared to control organisms. More prolonged exposure (14 days) of mussels M.

576

galloprovincialis to lower concentrations of CdTe quantum dots (10 µg L-1) resulted in

577

genotoxic damage (Rocha et al., 2014). Gomes et al. (2013) and Buffet et al. (2013)

578

showed that CuO NPs caused DNA damage in haemocytes of mussels and clams,

579

respectively, as determined by Comet assay. In the present study, mussels exposed to

580

CuO NPs for 21 days showed an increase in MN frequency compared to controls. This

581

increase was not found in animals at 63 and 122 days post exposure indicating that the

582

effect disappeared after removal of the contaminant. Nevertheless, MN frequencies

583

recorded were low when compared with those of mussels and oysters exposed to model

584

genotoxic compounds such as benzo(a)pyrene (Burgeot et al., 1995; Venier et al.,

585

1997).

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24

ACCEPTED MANUSCRIPT During the analysis of haemolymph samples for MN analysis, one mussel treated with

587

CuO NPs and sampled at day 63 post-exposure showed disseminated neoplasia. Cells in

588

mitotic division or binucleated cells, typical in cancer development, were seldom seen

589

in studied preparations. In the histopathological analysis of sections of digestive glands,

590

gonads and gills, no cases of neoplasia were found. Thus, although the appearance of

591

neoplasia in molluscs has been related to the exposure to different chemical carcinogens

592

(Gardner et al., 1991; 1992), a clear link between exposure to CuO NPs and

593

development of disseminated neoplasia can not be established in the present work.

594

Nonetheless, cancer development is known to be related to DNA damage, so it can not

595

be ruled out that the single case of disseminated neoplasia found in the present work is

596

related to DNA damage produced as a consequence of exposure to CuO NPs.

597

As mentioned before, NMs can produce DNA damage by promoting oxidative stress

598

and inflammatory responses (Singh et al., 2009), processes strongly associated with

599

carcinogenesis (Federico et al., 2007; Ohshima et al., 2005). DNA damage stimulates

600

the production of P53 protein, which can modulate the transcription of genes related

601

with DNA repair (e.g. gadd45) and cell cycle arrest (Hanahan and Weinberg, 2000). On

602

the other hand, the ras proto-oncogene is involved in the control of cell growth,

603

differentiation and apoptosis (Buday and Downward, 2008).

604

In vitro exposure of vertebrate cells to metal and metal-bearing NPs, such as Au, Ag,

605

CdTe, TiO2 and SiO2 has been shown to regulate the transcription level of genes

606

involved in these cellular processes (Ahamed et al., 2008; Cha et al., 2008; Choi et al.,

607

2008; Kang et al., 2008; Kawata et al., 2009; Li et al., 2008; Ye et al., 2010).

608

Specifically, CuO NPs induced P53 protein and DNA damage repair proteins RAD51

609

and MSH2 expression in human pulmonary epithelial cells (Ahamed et al., 2010). In the

610

present work, the transcription levels of p53, ras and gadd45α in mussel digestive

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25

ACCEPTED MANUSCRIPT glands were not regulated after CuO NP, bulk CuO and ionic copper exposure. Choi et

612

al. (2010) have shown that p53 was unchanged in zebrafish treated with Ag NPs.

613

Nevertheless, they observed that P53 protein was induced in a dose-dependent manner

614

after treatment with Ag NPs and consequently, p53-related pro-apoptotic genes bax,

615

noxa, and p21 were up-regulated after treatment with Ag NPs in zebrafish liver.

616

Similarly, exposure of ionic copper did not significantly affect p53 transcription levels

617

in HepG2 cells, whereas P53 protein levels increased in a dose-dependent manner (Song

618

et al., 2009; 2011). Thus, it has been suggested that P53 activation results from protein

619

stabilisation and post-transcriptional modifications rather than from changes in gene

620

transcription (Pluquet and Hainaut, 2001; Song and Freedman, 2011).

621

The finding that transcription levels of EF1-α did not vary with the different treatments

622

is in agreement with the above results. EF1-α takes part in protein synthesis (Kaziro et

623

al., 1991) and is also involved in several other cellular processes such as cell growth,

624

differentiation and apoptosis (Frum et al., 2007; Lamberti et al., 2004; Negrutskii and

625

El´Skaya, 1998). The control of EF1-α expression levels is of fundamental importance

626

for normal cell functions. Indeed, it has been demonstrated that up-regulation of EF1-α

627

expression is related to the increase of cell proliferation (Frum et al., 2007; Talapatra et

628

al., 2002), oncogenic transformation (Borradaile et al., 2006) and delayed cell

629

senescence (Lamberti et al., 2004). Our finding that EF1-α transcription did not vary

630

either among treatments or with time, and together with the other data of this study,

631

suggests that CuO NPs, bulk CuO and ionic copper did not induce cell proliferation and

632

consequently, they did not provoke cancer development under the present experimental

633

conditions.

634

A similar conclusion can be drawn from our histopathological analysis. Histopathology

635

of aquatic organisms is a valuable tool for providing health assessments of individuals

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26

ACCEPTED MANUSCRIPT and of populations (Bignell et al., 2008; 2012). In the present work, different

637

histopathological alterations were found, including atrophy of digestive tubules, mainly

638

in the post-exposure period, and several types of inflammatory responses. Prevalences

639

of these histopathological alterations did not show significant differences between

640

treatments. As a central organ for digestion and homeostasis maintenance (Moore and

641

Allen, 2002), changes in the morphology of digestive tubules are considered as non-

642

adaptative responses to pollutant exposure (Bignell et al., 2012; Cajaraville et al., 1992).

643

Many other environmental factors such as food availability as well as saline and thermal

644

stress may also produce changes in the morphology of the digestive gland structure,

645

which can result in the failure of an organism’s digestive and storage functions and the

646

impairment of individual physiology (Cajaraville et al., 1992; Kim and Powell, 2004).

647

Haemocytic infiltrations, both focal and diffuse, may constitute repair processes

648

following tissue damage (Lee et al., 2001). The prevalence of both types of haemocytic

649

infiltration increased along the experimental period until they reached their maximum

650

level at 63 days post-exposure. Brown cell aggregation prevalence also followed an

651

upward tendency during the experimental period in all groups, reaching the highest

652

values at 122 days post-exposure. As cells involved in metabolite accumulation and

653

detoxification (Zaroogian et al., 1993), brown cell aggregation prevalence has been

654

proposed to be a non-specific indicator of environmental pollution in mussels (Feist et

655

al., 2006). In this line, Hu and co-workers (2014) observed a deposition of brown cells

656

in mussels exposed to 1000 µg L-1 of CuO NPs after 1 h. Nevertheless, the increase of

657

recorded histopathological alterations throughout time in this study indicates a stress

658

situation that could be related to the conditions of long-time maintenance in the

659

laboratory.

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27

ACCEPTED MANUSCRIPT 661

5. Conclusions

662 Exposure to CuO NPs and to ionic copper, but not to bulk CuO, resulted in a significant

664

Cu bioaccumulation in mussels and CuO NPs were localized in lysosomes of digestive

665

cells. In accordance, the three forms of copper affected differently antioxidant enzyme

666

activities in digestive gland and gills and micronuclei frequency in haemocytes of

667

mussels. Exposure to CuO NPs increased the activity of antioxidant enzymes indicating

668

a situation of oxidative stress and produced genotoxic effects, which disappeared during

669

the post-exposure period. Although an individual exposed to CuO NPs presented

670

disseminated neoplasia, transcription levels of p53, ras, gadd45α and EF1-α remained

671

at an almost constant level after CuO NP exposure and no remarkable histopathological

672

alterations were observed as a consequence of this treatment. Thus, we conclude, based

673

on the available data, that an association between exposure to CuO NPs and cancer

674

development can not be established in mussels.

675

677

6. Acknowledgments

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This work was funded by EU 7th FP (project NanoReTox, ref CP-FP 214478-2),

679

Spanish Ministry of Science and Innovation (project NanoCancer CTM2009-13477 and

680

PhD grant to P. Ruiz), Basque Government (grant to consolidated research groups

681

IT810-13 and IT620-13) and the University of the Basque Country (UPV/EHU) through

682

the grant to the Unit of Formation and Research (UFI11/37) and the PhD grant to A.

683

Jimeno-Romero. TEM and X-ray microanalysis were carried out at the Centre for

684

Ultrastructural Imaging, King's College London. SGIker technical and human support

685

(UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged.

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ACCEPTED MANUSCRIPT 686 687

7. References

688 Aebi, H., 1974. Catalase methods of enzymatic analysis, II. AcademicPress, New York,

690

pp. 673-683.

691

Ahamed, M., Karns, M., Goodson, M., Rowe, J., Hussain, S., Schlager, J., Hong, Y.,

692

2008. DNA damage response to different surface chemistry of silver nanoparticles in

693

mammalian cells. Toxicol. Appl. Pharmacol. 233, 404-410.

694

Ahamed, M., Siddiqui, M.A., Akhtar, M.J., Ahmad, I., Pant, A.B., Alhadlq, H.A., 2000.

695

Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells.

696

Biochem. Biophys. Res. Commun. 396, 578-583.

697

Aitken, R.J., Chaudhry, M.Q., Boxall, A.B.A., Hull, M., 2006. Manufacture and use of

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nanomaterials: current status in the UK and global trends. Occup. Med. 56, 300-306.

699

Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of

700

nanoparticles of CuO, ZnO, and TiO2 to microalgae Pseudokirchneriella subcapitata.

701

Sci. Tot. Environ. 407, 1461-1468.

702

Baun, A., Hartmann, N.B., Grieger, K., Kusk, K.O., 2008. Ecotoxicity of engineered

703

nanoparticles to aquatic invertebrates: a brief review and recommendations for future

704

toxicity testing. Ecotoxicology 17, 387-395.

705

Bignell, J.P., Dodge, M.J., Feist, S.W., Lyons, B., Martin, P.D., Taylor, N.G.H., Stone,

706

D., Travalent, L., Stentiford, G.D., 2008. Mussel histopathology: effects of season,

707

disease and species. Aquat. Biol. 2, 1-15.

708

Bignell, J., Cajaraville, M.P., Marigómez, I., 2012. Background document:

709

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AC C

EP

TE D

M AN U

SC

980

41

ACCEPTED MANUSCRIPT Figure 1. Cu concentration (µg Cu g-1 dry weight) in soft tissues of mussels exposed to

982

10 µg Cu L-1 in the form of CuO NPs, bulk CuO and ionic copper for 1 and 21 days.

983

Results are given as means and standard deviations. Significant differences (p<0.05) are

984

based on the bootstrap analysis followed by Bonferroni’s correction. Significant

985

differences among mussels exposed to different treatments at 21 days are indicated with

986

asterisks in the upper triangular matrix and differences between time periods for each

987

treatment are indicated with #.

RI PT

981

SC

988

Figure 2. Transmission electron micrographs and X-ray microanalysis spectrum of the

990

digestive gland of mussels exposed to 10 µg Cu L-1 in the form of CuO NPs. (A) CuO

991

NPs (as electrondense particles) being incorporated in a digestive cell through an

992

endocytic vesicle. Inset: Detail of several particles found among the microvilli. L,

993

lumen of digestive tubule. (B) CuO particles inside a residual body in the lumen of a

994

digestive tubule. X-ray energy spectrum showing the elemental composition of the

995

particles found in (A). Cu peaks are indicated with arrows. Ni peaks correspond to Ni

996

grids. Scale bars: (A) 500 nm, Inset: 100 nm, (B) 100 nm.

TE D

EP

997

M AN U

989

Figure 3. Catalase, superoxide dismutase and glutathione peroxidase activities in

999

digestive glands (A, B and C) and in gills (D, E and F) of mussels exposed to CuO NPs,

1000

bulk CuO and ionic copper for 1 and 21 days. Results are given as means and standard

1001

deviations. Significant differences (p<0.05) are based on the bootstrap analysis followed

1002

by Bonferroni’s correction. Significant differences among mussels exposed to different

1003

treatments at 21 days are indicated with asterisks in the upper triangular matrix and

1004

differences between time periods for each treatment are indicated with #.

AC C

998

1005

42

ACCEPTED MANUSCRIPT Figure 4. Box-plot of the micronuclei frequency (‰) in mussels exposed for 21 days to

1007

different treatments and at 63 or 122 days post-exposure. Box-plot boxes represent the

1008

percentage data value in between the 25th and the 75th percentile, median indicated by a

1009

line in the middle of the box. Whiskers are the data values in up to the 5th percentile and

1010

95th percentile. Outliers are represented by circles. Significant differences (p<0.05)

1011

between time periods are indicated with #, according to the bootstrap analysis followed

1012

by Bonferroni’s correction.

RI PT

1006

SC

1013

Figure 5. Samples of haemolymph from experimental mussels. (A) Micronucleus

1015

(arrow) in a haemocyte of a mussel exposed to ionic copper and then kept for 63 days in

1016

clean water. (B) Micrographs of haemolymph from a mussel exposed to CuO NPs and

1017

then kept 63 days in clean water affected by disseminated neoplasia; neoplastic cells

1018

(large arrows) and normal haemocytes (small arrows). The inset shows a higher

1019

magnification of a neoplastic cell. Scale bars for A: 10 µm and for B: 200 µm, inset 20

1020

µm.

TE D

1021

M AN U

1014

Figure 6. Box-plots of the transcription levels of (A) p53, (B) ras and (C) gadd45α

1023

determined in digestive gland of mussels exposed for 1 and 21 days to different

1024

treatments and at 63 and 122 days post-exposure. Transcription level of each gene was

1025

normalised to EF1-α. Box-plot boxes represent the percentage data value in between the

1026

25th and the 75th percentile, median indicated by a line in the middle of the box.

1027

Whiskers are the data values in up to the 5th percentile and 95th percentile. Outliers are

1028

represented by circles. RQ: relative quantification.

AC C

EP

1022

1029

43

ACCEPTED MANUSCRIPT Figure 7. Micrographs of the digestive gland, gonad and gill tissue of mussels exposed

1031

to CuO NPs, bulk CuO or ionic copper showing different pathologies. (A) Diffuse

1032

haemocytic infiltration (asterisk) in digestive gland of a mussel sampled at 63 days post-

1033

exposure to ionic copper. (B) Atrophic digestive tubules (arrows) and haemocytic

1034

infiltration (asterisk) in the connective tissue of a mussel sampled at 63 post-exposure to

1035

CuO NPs. (C) Focal haemocytic infiltrations (asterisks) in digestive gland of a mussel

1036

previously exposed to CuO NPs after 63 days in clean water. (D) Haemocytic

1037

infiltration (asterisks) in gonad of a mussel exposed to bulk CuO for 1 day. (E)

1038

Aggregation of brown cells (arrows) in the gills of a mussel after 122 days in clean

1039

water following exposure to bulk CuO. (F) Granulocytomas (arrows) in digestive gland

1040

of a mussel after 63 days in clean water following exposure to CuO NPs. Scale bars for

1041

A, C, D and F: 500 µm, for B: 200 µm and for E: 100 µm.

M AN U

SC

RI PT

1030

AC C

EP

TE D

1042

44

ACCEPTED MANUSCRIPT 1043

Table 1. 5´-3´forward (Fw) primers, 5´-3´reverse (Rv) primers, and 5´-3´dual label

1044

probes (Probe) with indicated fluorophore reporter molecule (FAM) and the quencher

1045

NFQ dye used for TaqMan real time PCR of target and reference genes in mussels.

1046 Gene

FW: CAACAACTTGCCCAATCCGATTTAA p53 (DQ158079)

RV: GGTTCTTGGACATGTTCAGGTTTCA

FW:

SC

Probe: FAM-CAGGGATGTGTTATTCG-NFQ

RI PT

Product size (bp)

(GenBank Accession nº)

103

ACAGATCAAAAGAGTTAAAGATGCAGATGA ras (DQ305041)

77

M AN U

Rv: TCCGTGTCGGTAAATCCACTTT

Probe: FAM-TGCCAATGGTCTTGGTAGG-NFQ FW: AAGAAATGTGAACAACATAGGGTTTGC gadd45α (AJ623737)

RV: ACAACAATTCTGCCGTCTTCCT

75

Probe: FAM-GAAAAAGTTGCTAGTGAAG-NFQ FW: CTGAGATGGGAAAAGGCTCCTT EF1-α (AB162021)

RV: GACAAACTGAAGGCTGAGCG

61

TE D

Probe: FAM-CAAGTACGCCTGGGTTT-NFQ FW: CTGACCTACCTCCCGGTTTT

18S rRNA (L33452)

RV: GCCACCCGAGACACTCA

57

1048

AC C

1047

EP

Probe: FAM-TCGCCCTTGGTGCTCT-NFQ

45

ACCEPTED MANUSCRIPT Table 2. Prevalence of histopathological alterations found in control and exposed

1050

mussels. The total percentage of individuals showing a specific alteration, as well as

1051

the prevalence observed in each organ (DG: digestive gland; GO: gonad; and gill) is

1052

shown. Significant differences in total prevalence for each treatment according to the

1053

Chi-square test (p<0.05) throughout time: a Significant differences with respect to day

1054

1; b Significant differences with respect to day 21; c Significant differences with respect

1055

to day 63 post-exposure;

1056

exposure.

Significant differences with respect to day 122 post-

SC

d

RI PT

1049

AC C

EP

TE D

M AN U

1057

46

1059

1 day exposure

exposure

122 days post-

exposure

63 days post-

exposure

21 days

22.2

Ionic copper

70

Ionic copper

30

47.4

25

CuO NPs

Bulk CuO

Ionic copper

20

45

Bulk CuO

Control

55

CuO NPs

60

13.3

Bulk CuO

26.3

5

21

6.7

Control

5.16

0

GO

0

0

Gill

30

15.8

25

30

30

45

45

35

0

6.7

0

10.5

10

31.6

15

0

0

0

0

0

5

0

0

6.7

0

5.3

0

5.3

TE D

16.7

15

DG

CuO NPs

Control

Ionic copper

Bulk CuO

CuO NPs

Control

EP

Groups

AC C Diffuse haemocytic infiltration

15.8

15.8

25

45c

20

57.9

55b

5

50

80a b d

45

20

30

70a

70b

16.7

22.2c

45

20

26.7c

65a b

6.7

6.7c d

10

10.3

35

30

20

40

25

25

5.6

0

6.7

15.8

5

5.3

5.6

5

GO

10

0

10

5

5

5

20

0

0

0

0

0

5

10.5

0

0

Gill

M AN U

36.8

10

15c

5.6

22.2c

10.5

15

15c

42.1

DG

Total

Focal haemocytic infiltration

5

0

0

0

5.3

0

40

31.6

50a b

40

60a b

60a b

60a b

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Gill

0

5

5

5.6

0

0

5.3

0

0

5.6

0

Total

20

40

25

0

33.3

40

15.8

15

0

16.7

10

DG

0

0

0

0

0

0

0

0

0

0

35

47.4

40

45

35

RI PT

5.6

0

0

0

0

0

0

0

GO

SC

50

16.7c

20c

6.7c d

31.6

0

0

15.8c

15c

5.6

0

DG

5.6c d

20

Total

Granulocytomas

Non-specific inflammatory responses

10

0

20

20

0

20

5

5

0

0

6.67

5.3

0

0

0

0

GO

70

78.9

75

45

40

30

30

25

33.3

33.3

26.7

36.8

20

21.1

16.7

5

Gill

90a b c

78.9a

95a b c

75a

55d

55

45d

40

33.3d

60a

53.3d

47.4

25d

21.1b d

33.3d

15d

Total

Aggregations of brown cells

ACCEPTED MANUSCRIPT

1058

47

ACCEPTED MANUSCRIPT

C NP B

* * * 30

15

SC

10

5

EP

TE D

1 day

M AN U

0

AC C

µg Cu / g dry tissue

#

20

21 days

C NP B I

CuO NPs (NP) Bulk CuO (B) Ionic copper (I)

RI PT

#

25

Control (C)

I

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

A

EP AC C

B

TE D

L

Catalase ACCEPTED MANUSCRIPT C NP B

I

**

NP

mmol / min·mg prot

0.4

I

**

C

#

C

*

B

#

I

D C NP B

NP B

* *

0.4

I

0.3

# 0.2

0.1

0.3

1 day

B NP B

C

*

#

I

30

#

I NP B I

#

20

10

0

80

SOD units / mg prot

SOD units / mg prot

40

*

C

21 days

M AN U

** **

#

SC

E C NP B

#

0.1

Superoxide dismutase

I

*

B

#

0

60

#

40

20

0

1 day

21 days

TE D

1 day

21 days

Glutathione peroxidase

I

**

150

C NP B

#

I

100

50

0

1 day

Control (C)

F 150

nmol / min·mg prot

C NP B

AC C

nmol / min·mg prot

200

EP

C

#

50

0

CuO NPs (NP)

#

100

21 days

Bulk CuO (B)

I C

NP

I

21 days

C NP B

C NP B C

0.2

0

1 day

I

RI PT

C NP B

mmol / min·mg prot

A

1 day

21 days

Ionic copper (I)

NP

*

B I

ACCEPTED MANUSCRIPT 30

C NP B

* 25

Control (C)

I C

CuO NPs (NP)

NP B

Bulk CuO (B) Ionic copper (I)

# 20

10

5

0

63 days post-exposure

122 days post-exposure

EP

TE D

M AN U

21 days

SC

RI PT

15

AC C

MN frequency (‰)

I

ACCEPTED MANUSCRIPT

A

AC C

EP

TE D

M AN U

SC

RI PT

B

A

11

ACCEPTED MANUSCRIPT p53

10

Control

8

RQ

CuO NPs Bulk CuO Ionic copper

6

RI PT

4

2

21 days

1 day

B

63 days post-exposure

TE D

RQ

4

EP

2

0

122 days post-exposure

M AN U

ras

6

1 day

AC C

C

SC

0

10

21 days

63 days post-exposure

122 days post-exposure

gadd45α

8

RQ

6

4

2

0

1 day

21 days

63 days post-exposure

122 days post-exposure

ACCEPTED MANUSCRIPT

B

A

RI PT

*

D *

EP AC C

E

TE D

*

* *

M AN U

C

SC

*

F

*

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

-Copper accumulation occurred in mussels exposed to CuO NPs and ionic copper for 21 d -CuO NPs, bulk CuO and ionic copper produced different effects on antioxidant activities -Micronuclei frequency increased significantly in mussels exposed to CuO NPs -Transcription levels of cancer-related genes did not change after exposure to CuO NPs -Further studies are needed to determine genotoxic and carcinogenic potential of CuO NPs