Aquatic Toxicology 155 (2014) 327–336
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Proteomic response of mussels Mytilus galloprovincialis exposed to CuO NPs and Cu2+ : An exploratory biomarker discovery Tânia Gomes ∗ , Suze Chora, Catarina G. Pereira, Cátia Cardoso, Maria João Bebianno CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 13 February 2014 Received in revised form 11 June 2014 Accepted 13 July 2014 Available online 21 July 2014 Keywords: Mytilus galloprovincialis Proteomic analysis CuO NPs Two-dimensional gel electrophoresis MALDI–TOF–TOF
a b s t r a c t CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other commercial applications. Nevertheless, less is known about the mechanisms by which these NPs inflict toxicity in mussels and to what extent it differs from Cu2+ . The aim of this study was to investigate changes in protein expression profiles in mussels Mytilus galloprovincialis exposed for 15 days to CuO NPs and Cu2+ (10 g L−1 ) using a proteomic approach. Results demonstrate that CuO NPs and Cu2+ induced major changes in protein expression in mussels’ showing several tissue and metal-dependent responses. CuO NPs showed a higher tendency to up-regulate proteins in the gills and down-regulate in the digestive gland, while Cu2+ showed the opposite tendency. Distinctive sets of differentially expressed proteins were found, either common or specific to each Cu form and tissue, reflecting different mechanisms involved in their toxicity. Fifteen of the differentially expressed proteins from both tissues were identified by MALDI-TOF-TOF. Identified proteins indicate common response mechanisms induced by CuO NPs and Cu2+ , namely in cytoskeleton and cell structure (actin, ␣-tubulin, paramyosin), stress response (heat shock cognate 71, putative C1q domain containing protein), transcription regulation (zinc-finger BED domain-containing protein 1, nuclear receptor subfamily 1G) and energy metabolism (ATP synthase F0 subunit 6). CuO NPs alone also had a marked effect on other biological processes, namely oxidative stress (GST), proteolysis (cathepsin L) and apoptosis (caspase 3/7-1). On the other hand, Cu2+ affected a protein associated with adhesion and mobility, precollagen-D that is associated with the detoxification mechanism of Cu2+ . Protein identification clearly showed that the toxicity of CuO NPs is not solely due to Cu2+ dissolution and can result in mitochondrial and nucleus stress-induced cell signalling cascades that can lead to apoptosis. While the absence of the mussel genome precluded the identification of other proteins relevant to clarify the effects of CuO NPs in mussels’ tissues, proteomics analysis provided additional knowledge of their potential effects at the protein level that after confirmation and validation can be used as putative new biomarkers in nanotoxicology. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Copper is an essential metal with a known role as a co-factor in many enzyme systems and other proteins (e.g. cytochrome oxidase, superoxide dismutase), involved in several biological processes required for growth, development and maintenance of organisms. However, this metal can be extremely toxic if present in high concentrations or if organisms are exposed chronically to low levels in the environment (da Silva, 2001; Gaetke and Chow, 2003). Its inherent toxicity is a consequence of the propensity of free Cu ions
∗ Corresponding author at: Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norway. Tel.: +47 22185100; fax: +47 22 18 52 00. E-mail address: [email protected] (T. Gomes). http://dx.doi.org/10.1016/j.aquatox.2014.07.015 0166-445X/© 2014 Elsevier B.V. All rights reserved.
to participate in the formation of reactive oxygen species (ROS). Additionally, Cu has a high affinity for thiol groups being capable to bind cysteine and lead to protein inactivation and their adverse effects and bioaccumulation in aquatic organisms have been extensively investigated (e.g. Maria and Bebianno, 2011; Regoli and Principato, 1995). The unique and attractive properties of nanoparticles (NPs) have made nanotechnology a rapidly growing industry with applications in a variety of different areas such as electronics, medicine, consumer products, food packaging, water treatment technology, fuel cells, catalysts and biosensors. Although clear benefits are expected from the expansion of nanotechnology products, concern is growing about their impact in the environment and possible interactions with the aquatic biota. The physical and chemical characteristics of NPs (e.g. chemical composition, size, solubility, agglomeration, mobility, density, concentration and charge),
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T. Gomes et al. / Aquatic Toxicology 155 (2014) 327–336
behaviour in the environment, mechanisms of biological uptake and toxic effects in aquatic systems can differ considerably from those of corresponding ionic and bulk counterparts (Bhatt and Tripathi, 2011; Handy et al., 2008; Scown et al., 2010). Even though the number of studies concerning NP-induced toxicity on aquatic organisms under laboratory conditions continues to increase, the mode of action behind NP toxicity in invertebrate species needs further clarification (Canesi et al., 2012; Moore, 2006; Scown et al., 2010). Copper oxide nanoparticles (CuO NPs) are one of the most used metal nanomaterials with several industrial and commercial applications (e.g. air and liquid filtration, coatings of integrated circuits and batteries, wood preservation, inks, skin products and textiles) associated with their antimicrobial properties and elevated thermal and electrical conductivity (Buffet et al., 2011; Griffitt et al., 2009). These NPs can be accumulated in different tissues of filter-feeding bivalves but their toxic mechanisms are still poorly understood, as well as if they are associated with the dissolution of NPs to free Cu ions or the inherent NP properties (Buffet et al., 2011; Gomes et al., 2011, 2012). Previous studies have show that when mussels are exposed to CuO NPs for two weeks (10 g L−1 , 31 ± 10 nm), the antioxidant defence system fail, lipid peroxidation increase and metallothioneins are induced in both gills and digestive gland, as well as neurotoxic impairment in the gills and DNA damage in hemolymph cells (Gomes et al., 2011, 2012, 2013a). Conventional biomarkers proved to be sensitive indicators in assessing the toxic effects of NPs (e.g. antioxidant enzymes, lipid peroxidation, metallothionein, DNA damage), nevertheless, it is likely that there are other specific proteins that may be more effective in establishing a nano-specific biological response (Handy et al., 2012; Moore, 2006). Proteomics applied to nanotoxicology may help understanding the major toxic mechanisms and modes of action of different types of NPs in aquatic organisms and identify novel and unbiased biomarkers of NP exposure and effect. In the last years, this technology was applied to mussels for the screening of protein expression signatures (PESs) in response to conventional contaminants, including Cu (e.g. Apraiz et al., 2006; Shepard and Bradley, 2000) but also to silver nanoparticles (10 g L−1 ,
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Proteomic response of mussels Mytilus galloprovincialis exposed to CuO NPs and Cu2+ : An exploratory biomarker discovery Tânia Gomes ∗ , Suze Chora, Catarina G. Pereira, Cátia Cardoso, Maria João Bebianno CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 13 February 2014 Received in revised form 11 June 2014 Accepted 13 July 2014 Available online 21 July 2014 Keywords: Mytilus galloprovincialis Proteomic analysis CuO NPs Two-dimensional gel electrophoresis MALDI–TOF–TOF
a b s t r a c t CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other commercial applications. Nevertheless, less is known about the mechanisms by which these NPs inflict toxicity in mussels and to what extent it differs from Cu2+ . The aim of this study was to investigate changes in protein expression profiles in mussels Mytilus galloprovincialis exposed for 15 days to CuO NPs and Cu2+ (10 g L−1 ) using a proteomic approach. Results demonstrate that CuO NPs and Cu2+ induced major changes in protein expression in mussels’ showing several tissue and metal-dependent responses. CuO NPs showed a higher tendency to up-regulate proteins in the gills and down-regulate in the digestive gland, while Cu2+ showed the opposite tendency. Distinctive sets of differentially expressed proteins were found, either common or specific to each Cu form and tissue, reflecting different mechanisms involved in their toxicity. Fifteen of the differentially expressed proteins from both tissues were identified by MALDI-TOF-TOF. Identified proteins indicate common response mechanisms induced by CuO NPs and Cu2+ , namely in cytoskeleton and cell structure (actin, ␣-tubulin, paramyosin), stress response (heat shock cognate 71, putative C1q domain containing protein), transcription regulation (zinc-finger BED domain-containing protein 1, nuclear receptor subfamily 1G) and energy metabolism (ATP synthase F0 subunit 6). CuO NPs alone also had a marked effect on other biological processes, namely oxidative stress (GST), proteolysis (cathepsin L) and apoptosis (caspase 3/7-1). On the other hand, Cu2+ affected a protein associated with adhesion and mobility, precollagen-D that is associated with the detoxification mechanism of Cu2+ . Protein identification clearly showed that the toxicity of CuO NPs is not solely due to Cu2+ dissolution and can result in mitochondrial and nucleus stress-induced cell signalling cascades that can lead to apoptosis. While the absence of the mussel genome precluded the identification of other proteins relevant to clarify the effects of CuO NPs in mussels’ tissues, proteomics analysis provided additional knowledge of their potential effects at the protein level that after confirmation and validation can be used as putative new biomarkers in nanotoxicology. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Copper is an essential metal with a known role as a co-factor in many enzyme systems and other proteins (e.g. cytochrome oxidase, superoxide dismutase), involved in several biological processes required for growth, development and maintenance of organisms. However, this metal can be extremely toxic if present in high concentrations or if organisms are exposed chronically to low levels in the environment (da Silva, 2001; Gaetke and Chow, 2003). Its inherent toxicity is a consequence of the propensity of free Cu ions
∗ Corresponding author at: Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norway. Tel.: +47 22185100; fax: +47 22 18 52 00. E-mail address: [email protected] (T. Gomes). http://dx.doi.org/10.1016/j.aquatox.2014.07.015 0166-445X/© 2014 Elsevier B.V. All rights reserved.
to participate in the formation of reactive oxygen species (ROS). Additionally, Cu has a high affinity for thiol groups being capable to bind cysteine and lead to protein inactivation and their adverse effects and bioaccumulation in aquatic organisms have been extensively investigated (e.g. Maria and Bebianno, 2011; Regoli and Principato, 1995). The unique and attractive properties of nanoparticles (NPs) have made nanotechnology a rapidly growing industry with applications in a variety of different areas such as electronics, medicine, consumer products, food packaging, water treatment technology, fuel cells, catalysts and biosensors. Although clear benefits are expected from the expansion of nanotechnology products, concern is growing about their impact in the environment and possible interactions with the aquatic biota. The physical and chemical characteristics of NPs (e.g. chemical composition, size, solubility, agglomeration, mobility, density, concentration and charge),
328
T. Gomes et al. / Aquatic Toxicology 155 (2014) 327–336
behaviour in the environment, mechanisms of biological uptake and toxic effects in aquatic systems can differ considerably from those of corresponding ionic and bulk counterparts (Bhatt and Tripathi, 2011; Handy et al., 2008; Scown et al., 2010). Even though the number of studies concerning NP-induced toxicity on aquatic organisms under laboratory conditions continues to increase, the mode of action behind NP toxicity in invertebrate species needs further clarification (Canesi et al., 2012; Moore, 2006; Scown et al., 2010). Copper oxide nanoparticles (CuO NPs) are one of the most used metal nanomaterials with several industrial and commercial applications (e.g. air and liquid filtration, coatings of integrated circuits and batteries, wood preservation, inks, skin products and textiles) associated with their antimicrobial properties and elevated thermal and electrical conductivity (Buffet et al., 2011; Griffitt et al., 2009). These NPs can be accumulated in different tissues of filter-feeding bivalves but their toxic mechanisms are still poorly understood, as well as if they are associated with the dissolution of NPs to free Cu ions or the inherent NP properties (Buffet et al., 2011; Gomes et al., 2011, 2012). Previous studies have show that when mussels are exposed to CuO NPs for two weeks (10 g L−1 , 31 ± 10 nm), the antioxidant defence system fail, lipid peroxidation increase and metallothioneins are induced in both gills and digestive gland, as well as neurotoxic impairment in the gills and DNA damage in hemolymph cells (Gomes et al., 2011, 2012, 2013a). Conventional biomarkers proved to be sensitive indicators in assessing the toxic effects of NPs (e.g. antioxidant enzymes, lipid peroxidation, metallothionein, DNA damage), nevertheless, it is likely that there are other specific proteins that may be more effective in establishing a nano-specific biological response (Handy et al., 2012; Moore, 2006). Proteomics applied to nanotoxicology may help understanding the major toxic mechanisms and modes of action of different types of NPs in aquatic organisms and identify novel and unbiased biomarkers of NP exposure and effect. In the last years, this technology was applied to mussels for the screening of protein expression signatures (PESs) in response to conventional contaminants, including Cu (e.g. Apraiz et al., 2006; Shepard and Bradley, 2000) but also to silver nanoparticles (10 g L−1 ,
