Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Multi-biomarker approach to assess the acute effects of cerium dioxide nanoparticles in gills, liver and kidney of Oncorhynchus mykiss
Graphical abstract
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.
References (86)
- et al.
Histopathological effects of waterborne copper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchus mykiss)
Aquat. Toxicol.
(2013) - et al.
Impacts of metal and metal oxide nanoparticles on marine organisms
Environ. Pollut.
(2014) - et al.
Microsomal lipid peroxidation
Methods Enzymol.
(1978) - et al.
Effects of the chronic exposure to cerium dioxide nanoparticles in Oncorhynchus mykiss: assessment of oxidative stress, neurotoxicity and histological alterations
Environ, Toxicol. Phar.
(2019) - et al.
Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: a weighted indices approach
Aquat. Toxicol.
(2009) - et al.
Toxicity of metal oxide nanoparticles in immune cells of the sea urchin
Mar. Environ. Res.
(2012) - et al.
Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects
Aquat. Toxicol.
(2007) - et al.
DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide – elucidation of organ-specificity and the role of oxidative stress
Mutat. Res.
(2012) - et al.
Glutathione-S-transferases - the first enzymatic step in mercapturic acid formation
J. Biol. Chem.
(1974) - et al.
The role of cerium redox state in the SOD mimetic activity of nanoceria
Biomaterials
(2008)
Histological and histochemical alterations in the liver induced by lead chronic toxicity
Saudi. J. Biol. Sci.
Genotoxicity and physiological effects of CeO2 NPs on a freshwater bivalve (Corbicula fluminea)
Aquat. Toxicol.
Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals
Mutat. Res.
Genotoxicity and ecotoxicity assays using the freshwater crustacean Daphnia magna and the larva of the aquatic midge Chironomus riparius to screen the ecological risks of nanoparticle exposure
Environ. Toxicol. Phar.
Toxicity of CeO2 nanoparticles - the effect of nanoparticle properties
J. Photochem. Photobiol. B Biol.
The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress
Mutat. Res.
Roundup® causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus
Chemosphere
Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water
Ecotoxicol. Environ. Saf.
Oxidative stress induced by cerium oxide nanoparticles in cultured BEAS-2B cells
Toxicol
Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells
Mutat. Res.
Acute and chronic effects of erythromycin exposure on oxidative stress and genotoxicity parameters of Oncorhynchus mykiss
Sci. Total Environ.
Histological alterations in gills and liver of rainbow trout (Oncorhynchus mykiss) after exposure to the antibiotic oxytetracycline
Environ. Toxicol. Pharmacol.
Cerium and yttrium oxide nanoparticles are neuroprotective
Biochem. Biop. Re. Comm
Morphological changes in the kidney of a fish living in an urban stream
Environ. Toxicol. Pharmacol.
Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants
Ecotox. Environ. Safe.
Effects of selected metal oxide nanoparticles on multiple biomarkers in Carassius auratus
Biomed. Environ. Sci.
Effect on catfish (Clarias lazera) composition of ingestion rearing water contaminated with lead or aluminum compounds
J Archiv für Tierernaehrung
Catalase in vitro
Methods Enzymol.
Synthesis, characterization, and ecotoxicity of CeO2 nanoparticles with differing properties
J. Nanopart. Res.
Gill damage in the freshwater fish Gnathonemus petersii (Family: Mormyridae) exposed to selected pollutants: an ultrastructural study
Environ. Technol.
The molecular mechanism of the catalase reaction
J. Am. Chem. Soc.
Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans
Arch Environ Con Tox
The comet assay: a sensitive and quantitative method for analysis of DNA damage
Encycl Anal Chem
Histopathology in fish: proposal for a protocol to assess aquatic pollution
J. Fish Dis.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding
Anal. Biochem.
Casarett and Doull’s Toxicology: The Basic Science of Poisons
Cerium oxide nanoparticles: a promise for applications in therapy
J. Exp. Ther. Oncol
Environmental release, fate and ecotoxicological effects of manufactured ceria nanomaterials
Environ. Sci. Nano.
The comet assay for DNA damage and repair
Mol. Biotechnol.
Environmental geochemistry of cerium: applications and toxicology of cerium oxide nanoparticles
Int. J. Env. Res. Pub. He.
Cerium oxide nanoparticles induce oxidative stress in the sediment-dwelling amphipod Corophium volutator
Nanotoxicology
Toxicology Review of Cerium Oxide and Cerium Compounds
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