Multi-biomarker approach to assess the acute effects of cerium dioxide nanoparticles in gills, liver and kidney of Oncorhynchus mykiss

https://doi.org/10.1016/j.cbpc.2020.108842Get rights and content

Highlights

  • Ecotoxicological effects on the aquatic biota of Cerium oxide nanoparticles (CeO2-NP) is scarce.

  • Results showed that the CeO2-NP promotes biochemical, genotoxic and histopathological damages in fish.

  • The mechanisms underlying the occurrence of such effects require further investigation.

Abstract

Cerium oxide nanoparticles (CeO2-NP) have already been detected in the aquatic compartment, however, the evaluation of potential ecotoxicological effects on biota are scarce. The present study aimed to assess the toxic effects of CeO2-NP in Oncorhynchus mykiss in different organs/tissues (gills, liver and kidney) after acute exposure (96 h) to three concentrations: 0.25, 2.5 and 25 mg/L. Oxidative stress response (catalase - CAT; glutathione S-transferases - GSTs), lipid peroxidation (thiobarbituric acid reactive substances - TBARS), Na+/K+-ATPase activity, genotoxicity (genetic damage index - GDI) and histopathology (organ's pathological indices) were evaluated. CAT activity was increased in gills and decreased in liver of fish exposed to the highest CeO2–NPs concentration tested. However, GSTs and Na+/K+-ATPase activities and TBARS levels were not significantly altered in analysed organs. CeO2–NP caused marked changes in the gills (aneurysms, blood capillary congestion, lamellar hypertrophy and hyperplasia, secondary lamella fusion and epithelial lifting), in liver (pyknotic nucleus, hyperemia, enlargement of sinusoids and leucocyte infiltration) and kidney (shrinkage of the glomeruli, enlargement of the Bowman space, tubular degeneration and nuclear hypertrophy). Moreover, a semi-quantitative histopathological scoring system (pathological index) confirmed significant alterations in the three organs of all exposed fish. Furthermore, a significant increase of GDI indices observed in gills and liver, for all tested concentrations, indicated a dose-dependent effect. The present study suggests that the release of CeO2-NP into the aquatic environment promotes biochemical, genotoxic and histopathological damages in fish. However, the mechanisms underlying the occurrence of such effects require further investigation.

Introduction

Nanoparticles (NP) are defined as particles with size ranging from 1 to 100 nm, which present new and improved characteristics (e.g. improved chemical reactivity, optical behavior and large surface area) compared to larger particles of the same material (Felix et al., 2013; Xia et al., 2013). Currently, cerium dioxide nanoparticles (CeO2-NP) are among the most produced NP in the world, around 10,000 tons/year (Keller et al., 2013). CeO2-NP can be found in an increasing number of applications in commercial products and industrial processes, such as abrasives in chemical mechanical planarization slurries, in the semiconductor manufacturing industry and as a catalyst in automobiles, but they are also used for several medical and therapeutic purposes (Tarnuzzer et al., 2005; Leung et al., 2015; Alam et al., 2016). In general, NP metal oxides can interact with the aquatic ecosystems leading to environment and human health concerns (Xia et al., 2013). Because of their world-wide use, CeO2-NP are released into the environment, namely in the aquatic compartment, where their fate and potential impacts are poorly studied. Hence, the CeO2-NP are on the list of the substances that the Organization for Economic Co-operation and Development consider to be priority to test (OECD, 2010).

Although scarce, previous studies on the effects of CeO2-NP exposure on biological systems have shown both beneficial as well as ecotoxicological effects (Tarnuzzer et al., 2005; Schubert et al., 2006; Heckert et al., 2008; E.J. Park et al., 2008; B. Park et al., 2008; Nalabotu et al., 2011; Arnold et al., 2013; Xia et al., 2013). Prior literature suggests that their protective effects are a result of general radical scavenging capabilities. Among the positive effects, researchers enhance their antioxidant capacity providing protection against free radicals and reactive oxygen species (ROS) (Heckert et al., 2008). In addition, CeO2-NP also demonstrated protective capacity against oxidative stress in nerve cells culture exposed to this compound (Schubert et al., 2006). Furthermore, in anticancer therapy, CeO2-NP protect normal cells against damage due to the radiation (Tarnuzzer et al., 2005). Moreover, some researches also reported toxic effects in human lung epithelial cells (E.J. Park et al., 2008) and liver damage of rats (Nalabotu et al., 2011) following exposure to CeO2-NP.

The information regarding the ecotoxicity of CeO2-NP for aquatic organisms is also limited. Arnold et al. (2013) exposed Caenorhabditis elegans and Danio rerio during 3 days to concentrations ranging from 2.5 to 93.75 mg/L of CeO2-NP and observed growth inhibition in C. elegans. Van Hoecke et al. (2009) and Felix et al. (2013) suggested the LC50 is higher than 200 mg/L to CeO2-NP for embryos of Danio rerio. Furthermore, Gaiser et al. (2009) reported no mortality on Daphnia magna after acute exposure to concentrations of 0–10 μg/mL or no cytotoxicity in trout hepatocytes after exposure to concentrations ranging 0–1000 μg/mL. Lee et al. (2009) exposed D. magna and Chironomus riparius to 1 mg/L of CeO2-NP of 15 and 30 nm and observed genotoxic effects in both species. Xia et al. (2013) exposed Carassius auratus to CeO2-NP for 4 days at concentrations ranging from 20 to 320 mg/L, and reported enzymatic changes in brain (acetylcholinesterase) and liver (antioxidant enzymes as catalase and superoxide dismutase). Dogra et al. (2016) reported that CeO2-NP also elicited pro-oxidant and genotoxic responses in Corophium volutator. Gagnon et al. (2018) observed a reduction of the immunotoxic potential and increased mortality in O. mykiss. Correia et al. (2019) recently reported alterations in antioxidant enzymes activities and histological damages in O. mykiss following a chronic exposure to CeO2-NP (0.1, 0.01, and 0.001 μg/L).

Information about the metabolism and excretion of metallic NP is limited, although a hepatic route and excretion into the bile is suggested as the most important pathways (Handy et al., 2008). It is also proposed that the production of reactive oxygen species (ROS) and free radicals is a primary mechanism of NP toxicity (Dahle and Arai, 2015). Despite these indications of mechanistic nature and toxicological, it is necessary to evaluate early warning signals or biomarkers that reflect adverse biological responses towards NPs that are likely to occur via environment. Xenobiotics, in general, are not exempt of toxicity, and some compounds can be bioactivated or involved in redox cycles. As such, this can result in deleterious effects to the organism in different metabolic/biochemical pathways and/or still cause genotoxic and tissue damages, compromising physiological functions. Organisms are able to develop adaptive responses such as an increase of antioxidant defenses and repair mechanisms, but severe oxidative damage can lead to cellular death (Limón-Pacheco and Gonsebatt, 2009). The antioxidant defense system involve enzymes such as catalase (CAT), present in peroxisomes, which degrades hydrogen peroxide into water and molecular oxygen; glutathione S-transferases (GSTs), for instance, catalyses the conjugation of a variety of compounds (such as xenobiotic metabolites and lipid peroxidation products) with electrophilic centers with glutathione (GSH) facilitating their excretion (Modesto and Martinez, 2010). Despite the efficiency of the antioxidant defense system, the peroxidative damage can occur. Lipid peroxidation (LPO) extent can be accessed by the amount of malondialdehyde (MDA), product of oxidative degradation of membrane lipids (Nunes et al., 2014). The spread of lipid peroxidation induced by oxidative stress severely affects Na+/K+-ATPase, a transmembrane protein, composed of α and β subunits, responsible for the active transport of Na+ and K+ across the plasma membranes (Kaplan, 2002; Maiti et al., 2010). In teleost fish, gills and kidney are used as a control of ion transport mechanism (e.g. Na+/K+-ATPase) to guarantee a successful osmoregulation (Tipsmark and Madsen, 2003). Na+/K+-ATPase is a transmembrane enzyme that catalyses the active Na+ and K+ transport in fish gills and kidney for osmoregulation purposes (Marshall and Grosell, 2006). Genotoxicity can be assessed using the comet assay, a fast and very sensitive method to measure DNA damage in individual cells (Lee and Steinert, 2003). Furthermore, response to stressors can be assessed by the appearance of histological changes in the tissues and organs, which can be used as a biomarker of intermediate level of biological organization (Bernet et al., 1999).

This work intended to evaluate the acute effects of CeO2-NP toxicity on biochemical, genotoxic and histological biomarkers of the freshwater fish species Oncorhynchus mykiss. Gills and liver were evaluated in terms of the oxidative stress response [catalase (CAT) and glutathione S-transferase (GST)], lipid peroxidation [thiobarbituric acid reactive substances (TBARS levels)] and genotoxicity [genetic damage index (measured by comet assay)]. Additionally, gills and kidney Na+/K+-ATPase activities were evaluated. Gills, liver and kidney were also assessed in terms of histological damage [pathological indices (PI)]. Thus, the integration of different biomarkers of NP effects, coupling different scales of organization should be a good approach to understand their potential toxicity. Thus, monitoring both histopathology, genotoxicity and other cellular responses to environmental stressors should allow a broad and complementary view of CeO2-NP effects.

Section snippets

Chemicals

Cerium (IV) oxide nanopowder (CeO2-NP) used in this study was acquired from Sigma-Aldrich (Schnelldorf, Germany) with a degree of purity of 99.95% and size under 50 nm (CeO2-NP; CAS: 1306-38-3; Reference Number: 700290). Exposure media were prepared by successive dispersion of the stock solution in dechlorinated tap water. The Bradford test reagent was purchased from Bio-Rad UK. All other chemicals (for media, buffers preparation and for biomarkers assays) were obtained either from

Biomarkers

Exposure to CeO2-NP revealed significant differences in CAT activities (Fig. 1A). A significant increase of CAT activity in gills was observed for the highest concentration tested (25.0 mg/L; F[3, 56] = 30.673; p < 0.001), while a significant inhibition on CAT activity was observed in liver in the same CeO2-NPs concentration tested (25 mg/L) (F[3, 56] = 4.479; p = 0.007).

No significant differences in GSTs activity were observed among experimental groups for gills and liver (F[3, 56] = 4.064; p

Biochemical analyses

Considering that aquatic ecosystems are the final sink for many environmental contaminants (Reeves et al., 2008), it becomes urgent to understand the potential harmful of a recent xenobiotic, such as metallic nanoparticles, on these ecosystems. To date, few studies on NPs ecotoxicity have been performed, especially with evaluation of effects of CeO2-NP on aquatic organisms (Gaiser et al., 2009; Lee et al., 2009; Van Hoecke et al., 2009; Arnold et al., 2013; Felix et al., 2013; Xia et al., 2013;

Conclusions

This work demonstrated acute effects of CeO2-NP on several biochemical, genotoxic, physiological and pathological parameters in rainbow trout. Our finding of unchanged GSTs activity suggests that metabolic responses to CeO2-NP exposure do not involve the glutathione pathway, at least at the exposure concentrations and durations evaluated. GSTs also did not appear to be involved in the antioxidant defense. In contrast, the activity of CAT was significantly affected in animals exposed to the

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgments

Sara Antunes was hired through the Regulamento do Emprego Científico e Tecnológico – RJEC from the FCT program (CEECIND/01756/2017). This study was also supported by national funds through FCT - Foundation for Science and Technology of Portugal within the scope of UIDB/04423/2020 and UIDP/04423/2020.

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