Effects of the chronic exposure to cerium dioxide nanoparticles in Oncorhynchus mykiss : assessment of oxidative stress, neurotoxicity and histological alterations

Cerium dioxide nanoparticles (CeO 2 -NPs) have a variety of uses, especially in the production of solar panels, oxygen pumps, gas sensors, computer chips and catalytic converters. Despite their worldwide use, the few published studies demonstrate that metallic nanoparticles, in general, are still not properly characterized in terms of their potencial ecotoxicological effects. CeO 2 -NPs, in particular, have demonstrated extreme antioxidant activity, but their in vivo toxicity is still unknown. This work intended to characterize the chronic toxicity (28 days) of three different ecologically relevant concentrations (0.1, 0.01, and 0.001 µg/L) of CeO 2 -NPs in the rainbow trout ( Oncorhynchus mykiss ), in terms of biomarkers of oxidative stress [activity of the enzymes glutathione S-transferases (GSTs) and catalase (CAT)] and neurotoxicity [activity of the enzyme acetylcholinesterase (AChE)], as well as histological alterations in liver and gills. In the hereby study, GSTs activity was increased in gills of fish exposed to the highest CeO 2 –NPs level. Moreover, a potential anti-oxidant response was also reported, with a significant increase of CAT activity observed in livers of the same fish. AChE, however, was not significantly altered in fish eyes. Individuals exposed to CeO 2 - NPs also presented marked changes in the gills (e.g. epithelial lifting, intercellular edema, lamellar hypertrophy and hyperplasia, secondary lamella fusion and aneurysms) and liver (e.g. hepatocyte vacuolization, pyknotic nucleus, enlargement of sinusoids and hyperemia). The semi-quantitative analysis (organs pathological index) also showed the

Nanotechnology is a growing science based on the application and production of equipment and/or material at a nanometer scale ranging from 1 µm to 1 nm (Park et al., 2007). Nanoparticles (NPs) are chemical compounds with small dimensions, usually less than 100 nanometers (Ju-Nam & Lead, 2008;Felix et al., 2013). Nanoparticles, due to their small size, have improved physicochemical properties compared to larger particles of the same substance. Among these improved properties, once can identify among others, better optical behavior and enhanced chemical reactivity (Ju-Nam & Lead, 2008;Gaiser et al., 2012;Xia et al., 2013). Given their versatility, NPs are currently used in domestic products, foods, sunscreens, medicine, optical equipment, cosmetics, textiles, bioremediation processes, paints and electronics production (Handy & Shaw, 2007;Gaiser et al., 2009;Dahle & Arai, 2015). One of the most common NPs in use at present is composed of cerium dioxide (CeO2-NPs). Also called nanoceria, CeO2-NPs are used as a fuel additive, as a catalyst in petroleum refining, as a semiconductor, and as an absorber of UV radiation in sun lotion Cassee et al., 2011;Dahle & Arai, 2015). CeO2-NPs, like many others, are also used for pharmaceutical purposes (Dahle & Arai, 2015). Cerium (Ce) is the most abundant of rare-earth metals found in the Earth's crust (Dahle & Arai, 2015). Thus, it is one of the most viable and valuable NPs that exists today (Hedrick, 2004;Wang et al., 2008;Xia et al., 2013). Because of their widespread use, CeO2-NPs can be dispersed into the environment, especially in the aquatic compartment, causing putative changes in aquatic organisms (Gaiser et al., 2009).
industry has led to an increasing concern about the potential impact of NPs on human health and in the environment (Dreher, 2004;Xia et al., 2013). However, despite their extensive use, some critical aspects of NPs are still largely unknown. Some studies in the areas of human toxicology and ecotoxicology of NPs have been developed. Even so, this amount of data is still insufficient to understand the long-term effects of NPs in organisms, mostly aquatic. Among the effects caused by NPs, the alterations in the structure and function of cells and tissues, and in the activity of specific key enzymes, should be further investigated and discussed. The existent literature suggests that CeO2-NPs can elicit antagonistic responses in biological systems. In some case, it can have benefit but also harmful effects in biota (Tarnuzzer et al., 2005;Schubert et al., 2006;Heckert et al., 2008;Nalabotu et al., 2011;Arnold et al., 2013;Xia et al., 2013). One of the beneficial effects of NPs is related with their antioxidant activity, which can act against reactive oxygen species and free radicals (Heckert et al., 2008).
NPs can also protect normal cells against the injury caused by radiation, in case of anticancer treatments (Tarnuzzer et al., 2005). On the contrary, other studies showed harmful effects in human lung epithelial cells, and liver damage in a chronical exposure in rats . It has been proposed that the metabolism of NPs is probably accomplished by a hepatic route, by excretion into the bile, which seems to be a more likely mechanism instead of renal or branchial excretion (Handy et al., 2008;Dahle & Arai, 2015). Despite these indications of mechanistic nature, only a few studies with aquatic organisms exposed to CeO2-NPs have been published so far. Among these, CeO2-NPs elicited genotoxic effects in Daphnia magna (Lee et al., 2009), caused growth inhibitory effects in Pseudokirchneriella subcapitata (Hoecke et al., 2009), significant increases in single-strand DNA breaks, lipid peroxidation and superoxide dismutase A C C E P T E D M A N U S C R I P T activity in Corophium volutator (Dogra et al., 2016), and simultaneously reduced the immunotoxic potential and increased mortality in Oncorhynchus mykiss (Gagnon et al., 2018).
Some metal oxides NPs can disrupt the biochemical balance of living organisms, leading to adaptive responses, measurable by biomarkers that are based on compromised biochemical processes due to xenobiotic exposure. These biomarkers can represent the health status of the biota and early-warning signs of environmental threats (Xia et al., 2013). Large varieties of substances that cause oxidative stress in aquatic species were already found in water (Valavanidis et al., 2006;Ray et al., 2012). Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) during the oxidative metabolism in mitochondria and cellular antioxidant defenses (Betteridge, 2000). It is related with the increase in the production of oxidant species, or with the significant decrease in the efficiency of the antioxidant defenses (Schafer & Buettner, 2001). Some examples of biomarkers involved in the antioxidant response are glutathione S-transferases (GSTs) and catalase (CAT) (Depledge & Fossi, 1994;Timbrell, 1998).
GSTs are a set of enzymes that lead to the conjugation of glutathione with diverse compounds with electrophilic centers, helping their excretion (Modesto & Martinez, 2010). CAT is also important for the defense of the organism against oxidative damage (Rahman, 2007). This enzyme is part of the antioxidant defense system that exists in peroxisomes (Modesto & Martinez, 2010), and its primary function is the reduction of hydrogen peroxide, produced from the metabolism of fatty acids (Xia et al., 2013), into water and molecular oxygen (Aebi, 1984). Among all cholinesterases, AChE is a biomarker of neurotoxicity, since it hydrolyses the neurotransmitter acetylcholine into choline and acetate (Tripathi & Srivastava, 2010), mainly at the central nervous system of living organisms. Thus, the proper function of the nervous systems depends on the activity of this enzyme, which is concentrated at the neuromuscular and cholinergic synapses (Chung & Bieber, 1993;Bajgar & Herick, 1997;Tripathi & Srivastava, 2010).
Furthermore, a severe oxidative stress can cause tissue alterations, trigger apoptosis and induce injured cell death (necrosis) (Lennon et al., 1991;Limón-Pacheco & Gonsebatt, 2009;Rodrigues et al., 2017). Tissue alterations are usually observed in two main fish organs: the gills, because of their extensive surface and superficial location, being in constant contact with the pollutant agents, and the liver which is responsible for the excretion and metabolism of the contaminants (Bucher & Hofer, 1993;Jobling & Sumpter, 1993;Camargo & Martinez, 2007). To face such environmental stressors, including NPs, fish show adaptive changes, through impairment of biochemical and structural traits in cells and tissues, which may be monitored by using a biomarker approach ( Van der Oost et al., 2003).
Based on previous studies, nanoparticles are able to interact with living systems leading to deleterious conditions and effects, including oxidative stress (Manke et al., 2013). Moreover it has recently shown that CeO2-NPs could be involved in the exertion of oxidative stress scenarios (Dogra et al., 2016). In addition, physical interference resulting in inhibition of AChE activity was already reported to occur after exposure to several types of nanoparticles (Wang et al., 2009). Furthermore, histopathological effects were already reported in fish cells and tissues as results of exposure and up-take of waterborne metallic NPs (Gaiser et al., 2009;Al-Bairuty et al., 2013).
The main goal of this work was to evaluate the toxic effects in rainbow trout resulting from a chronic exposure (28 days) to ecologically relevant amounts of CeO2-NPs by measuring the oxidative stress response (determination of the activity of the enzymes GSTs and CAT in the liver and gills), neurotoxicity (determination of the activity of AChE in the eyes) and histopathological (gills and liver tissue alterations) biomarkers. According to the existent knowledge, the here-proposed toxicological parameters seem suitable to diagnose the exposure of fish to environmentally relevant levels of NPs, namely CeO2-NPs. 9007-83-4) were purchased from Sigma-Aldrich. Bradford reagent was purchased from Bio-Rad UK. All other chemicals (for media and buffers preparation and for enzymatic assays) were obtained either from Sigma-Aldrich or Merck-Millipore.

Test organisms
Rainbow trout (Oncorhynchus mykiss) juveniles (10.6±0.2 cm and 12.2±0.5 g of total length and mass, respectively) were acquired at an aquaculture facility, Posto Aquícola do Torno, in Marão, north of Portugal. Rainbow trout is a freshwater fish species used for human consumption, easy to maintain in laboratory, highly abundant and it is a standard species in toxicology assays (Talbot, 2014). Fish were collected using hand nets and then transported in plastic bags with continuous air supply on cold water until the arrival at the laboratory. Fish were not fed during the first two days after the transport.
These specimens were kept in freshwater during an acclimation/quarantine period of fifteen days in 500 L tanks under optimal rearing controlled-conditions (continuous aeration, water temperature of 15±1°C and photoperiod of 12h L: 12h D) (Brown and Giral, 2000;Martínez, 2009). Every two days, nitrites (NO2), ammonium (NH4), dissolved oxygen (DO) and temperature were recorded. The water (dechlorinated tap water: OECD, 1992), was changed when the values of nitrites and ammonium were altered, and every day the specimens were fed ad libitium with commercial pellets (Aquagold 3mm, Aquasoja, Sorgal SA, Portugal). During this period, the fish that were diseased or died were discarded immediately. Only healthy fish were kept for exposure.

Chronic exposure
One hundred and twenty healthy specimens were selected from the quarantine tanks, and were transferred into the exposure aquariums (50 L each). Ten fish were assigned per aquarium, three replicates for each concentration, including a control group (unexposed fish). The aquaria were randomly distributed in the exposure room. The levels of CeO2-NPs used in this study were 0.1, 0.01 and 0.001 µg/L; these levels were defined based on the minimal and maximal values of 90% confidence intervals for mean predicted environmental concentrations modelled at a regional level (Ireland) in surface waters (O'Brien & Cummins, 2010;Gottschalk et al., 2013).
The period of exposure lasted twenty-eight days (chronic exposure) according to the guidelines (test nº. 204) of OECD (1984). Every two days, fish were fed ad libitium with commercial pellets (Aquagold 3mm, Aquasoja, Sorgal SA, Portugal), and water was partially renewed (by 80% according to a semi-continuous exposure regime).
Immediately after the water renewal, CeO2-NPs were added. Aquaria water pumps with a flow of 700 L/h (Trixie, model 86120) were used for the continuous resuspension of nanoparticles because, giving its nature, CeO2-NPs have a tendency to sediment. For test validation purposes, water quality during the exposures assays was monitored every 48 h (OECD 1992(OECD , 2000: guidelines nº 203 and 215). NO2 (0.02±0.00 mg/L) and NH3 (0.71± 0.05 mg/L) were recorded using a photometer (YSI, 9300 Photometer) and water test tablets (Palintest: Nitricol and Ammonia); and DO (9.56±0.08 mg/L) and temperature (16.2±0.1 °C) with a multi-probe (YSI, 556 MPS). During exposures, no mortality was recorded complying with the OECD requirements (mortality < 10% in the control group). (OECD 1992(OECD , 2000 Fish Sacrifice After the twenty-eight days of exposure, five specimens from each aquarium were euthanized by immersion into equal amounts of water and ice (water temperature under 4°C) until there was no observable opercular movement and fish could not swim (Wilson et al., 2009). Experiments took into consideration the AVMA Guidelines for the euthanasia of animals, the Portuguese animal welfare law (Decreto-Lei 113/2013) and has been previously authorized by the ethical committee of the host institution (CIIMAR-ORBEA).

Sample Processing
Fish were dissected on cold ice phosphate buffer, and liver, eyes and gills were removed, and divided into portions that were allocated into Eppendorf microtubes. These samples were immersed in liquid nitrogen, and then kept at -80°C, until the biochemical biomarker analyses. For the histopathological procedures, a portion of liver and gills tissue were hold in plastic tissue cassettes, followed by an immersion into Bouin solution for 24h.
For the biomarker analysis (GSTs and CAT) the hepatic and branchial tissues were homogenized in 1 mL of phosphate buffer (50 mM, pH=7.0, with Triton X-100 0.1%).
For AChE activity determination, eyes were homogenized in 1 mL of phosphate buffer (0.1M, pH=7.2). With a small scissors, the tissues were cut, especially the eyes and gills, to facilitate the homogenization by sonication (Branson S-250A), on ice, for 30 seconds.
Samples for the analysis of GSTs and CAT activities, were centrifuged at 15.000 g during 10 min at 4°C, and the ones that were intended to be analyzed for AChE activity were centrifuged at 3300 g during 3 min at 4°C. After centrifugation, the supernatants were then collected, being stored in the freezer at -80°C, and the remaining pellets were discarded.
Briefly, the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione is catalyzed by glutathione S-transferase, which leads to the formation of a thioether (ε= 9.6 mM -1 cm -1 ). This formation may be spectrophotometrically followed at  = 340 nm, by the increase of the absorbance. The enzymatic activity was then calculated in µmol min -1 mg -1 of protein.

Catalase Activity Quantification
CAT activity measurement was made according the method described by Aebi (1984). The decomposition of hydrogen peroxide (H2O2) was catalyzed by catalase. This decomposition was spectrophotometrically followed at  = 240 nm, by the decrease of the absorbance. The enzymatic activity was then calculated in µmol min -1 mg -1 of protein.
AChE activity was measured using a standard colorimetric method described (Ellman et al., 1961). The degradation of acetylthiocholine into acetate and thiocholine is catalyzed by acetylcholinesterase; thiocholine in turn reacts with dithionitrobenzoic acid (DTNB), leading to the development of a yellow complex. The formation of this yellow complex can be spectrophotometrically followed at  = 412 nm, by the increase in absorbance. The enzymatic activity was then calculated in µmol min -1 mg -1 of protein.
AChE (EC 3.1.1.7, nomenclature of the International Union of Biochemistry and Molecular Biology) is typically the predominant ChE enzyme present in muscle and brain of most fish species including rainbow trout (Sandahl and Jenkins, 2002;Sturm et al., 2007).

Protein Determination
The quantification of protein of all tissues was based on the method described by Bradford (1976). The conjugation of the Bradford reagent with the total protein leads to the formation of a colored and stable complex, whose absorbance was spectrophotometrically measured at  = 595 nm. Protein standards were prepared with bovine serum albumin (BSA), in a concentration of 1 mg/mL.

Histological Assessment
The second lamellar arch of each gill, and a medial portion of the liver, after chemically fixed in Bouin, were decalcified (12 h, only for gills), dehydrated through an increasing series of alcohols (70, 80, 90 and 100 %: one hour each), cleared with xylene (2 h), impregnated in paraffin wax (56 to 58 °C), and sectioned (5 to 7 µm) using a microtome (Reichert-Jung 2030). These sections were stained with hematoxylin-eosin, cover slips mounted with DPX, and analyzed at x200, x400, x1000 magnification by light microscopy (Olympus CX41). Micrographs were taken using a digital USB camera (Olympus SC30).
The tissue alterations were identified based on atlas of fish histology (Takashima and Hibiya, 1995;Genten et al., 2009) and other related papers (e.g. Federici et al., 2007;Al-Bairuty et al., 2013;Rodrigues et al., 2017). Histopathological condition indices were determined for liver and gills using a standard methodology (Bernet et al., 1999). Briefly, pathological findings were classified in five categories: circulatory, regressive, progressive, inflammatory, and neoplastic alterations. To each observed individual lesions an importance factor of 1, 2 or 3 corresponding to minimal, moderate and severe pathological importance was attributed. A score value was also attributed to each lesion according with to the extension of the pathological alteration: 0, 2, 4 or 6, corresponding to no observed alterations, mild occurrence, moderate occurrence and severe occurrence, respectively. The histopathological condition indices for both organs (gills and liver) were then determined by the sum of the multiplied importance factors and score values of all alterations found in each analyzed organ, allowing the use of appropriate statistics (Bernet et al., 1999).

Statistical Analyses
The data were tested for normality (Shapiro-Wil test) and equal variance (Levene test) prior to the statistical analyses and transformed (log) if necessary. Variables (biomarkers) were compared by a One-Way Analysis of Variance (One-way ANOVA) followed (if needed, p<0.05) by a Dunnett multi-comparison test to identify significant differences between the treatments and the control group. The adopted level of significance was 0.05.
Data are presented as the mean and standard error. Statistics were performed using the software Sigma Plot 13.0.

Acetylcholinesterase
Although there was a trend for the decline of AChE activity in the exposed groups, no statistically significant differences were observed (One-Way ANOVA: F3,56=1.932, p=0.136) (Fig. 3).
Individuals exposed to CeO2-NPs evidenced circulatory and regressive alterations in liver tissues. Among the reported circulatory changes, enlargement of sinusoids, hyperemia and intercellular edema were the most common. In terms of regressive alterations, cytoplasmic vacuolization and pyknotic nucleus in hepatocytes were observed (Fig. 4). However, progressive, inflammatory and neoplastic changes were not found. The hepatic pathological index determined from the histological semi-quantitative analysis showed significant statistical differences (One-Way ANOVA: F3,56=4.235, p=0.009), namely for fish exposed to the higher level of CeO2-NPs (Dunnett's test, p<0.05) (Fig.   5A).
Concerning the gills, circulatory, regressive, progressive and inflammatory alterations were observed in all exposed groups. Among the circulatory changes, hemorrhage and aneurisms were the most common. In terms of the regressive alterations, epithelial lifting, lamellar fusion and intercellular edema were recorded. Some of the proliferative changes found were hypertrophy and hyperplasia of gill epithelium, and proliferation of mucus cell. The most observed inflammatory alteration was leucocytes infiltration. (Fig. 6). No neoplastic changes were observed. The gills pathological index showed a dose-dependent incidence of alterations, with significant differences between fish exposed to CeO2-NP and those from the control group (One-Way ANOVA: F3,56=26,659, P<0.001; Dunnett test, p<0.05) (Fig. 5B).

Discussion
This study was conducted with the primary goal of understanding the potential impacts of chronic exposure of CeO2-NPs on the freshwater fish rainbow trout. The number of studies about the toxicity of CeO2-NPs in aquatic species is scarce, and none was so far performed with rainbow trout, as far as it is known. Given the potential toxicity of such NPs and the absence of toxicity data for freshwater fish species, it seems important that overviews of long-terms, ecologically realistic assessments are performed, especially focusing on likely pathways of contamination. CeO2-NPs can aggregate (Baker et al., 2016) and sediment (Quik et al., 2014) in the aquatic environment, as they have low water solubility (Batley et al., 2013), a factor that can affect their incorporation into fish tissues (Collin et al., 2014). However, the primary pathway of incorporation of CeO2-NPs by living organisms is by ingestion (Collin et al., 2014), as evidenced in zebrafish (Johnston et al., 2010), being unlikely that these NPs enter the organism through the gills of aquatic species, as opposed to many dissolved compounds (Baker et al., 2014).
Furthermore, it has also been shown that NPs have the capacity to cross biological membranes, from the gastrointestinal tract into blood vessels and other parts of the body, such as the brain (Ragnaill et al., 2011). The surface charge of NPs may alter their capacity to be transported through the blood-brain barrier and change their permeability to cells, but systemic effects are likely to occur (Saraiva et al., 2016).
The analysis of biochemical biomarkers such as GSTs and CAT activities intended to determine if CeO2-NPs could increase phase II detoxifying metabolism response, and to generate systemic oxidative stress, and thereby triggering an antioxidant response. GSTs are a set of enzymes that lead to the conjugation of glutathione with diverse compounds with electrophilic centers, helping their excretion (Modesto & Martinez, 2010), and it is vital against oxidative stress (Rahman, 2007). No significant differences in GSTs activity in liver were found. As previously suggested, the absence of significant effects in terms of GSTs activity may be due to the capacity of the liver tissue (the main metabolic organ involved in metabolism of toxicants and also antioxidant defense) to use alternative conjugation and antioxidant mechanisms to buffer some of the ROS generated by NPs (Federici et al., 2007). The catalytic properties of CeO2-NPs are A C C E P T E D M A N U S C R I P T not yet fully understood, but its role as scavenger for -NO is already known. However, the mechanism by which Ce02-NPs scavenges -NO is not totally elucidated, leading to the question if -NO is of a nucleophilic or electrophilic nature (Dowding, 2012). Because there were no significant differences in the activity of these isoenzymes in the liver, it may be suggested hereby that the conjugation of glutathione with these centers (putatively present in CeO2-NPs) did not occur; in addition, we can also hypothesize about the absence of oxidative stress. On the contrary, the results of GSTs activity in the gills may be due to a protective (possibly also antioxidant) mechanism that exists in specific tissues, such as gills, which are in close contact with waterborne contaminants. In fact, the conjugation capacity of gills seems to be common to a large number of aquatic species, and prevents the internalization of toxicants from the water (Xing et al., 2012;Carneiro et al., 2015).
Considering the results obtained for CAT activity determined in the liver, a dosedependent increase in the activity of the enzyme was observed, but significant differences were only observed for the higher concentration. As already reported, CeO2-NPs have redox properties that allow the unexpected transition from Ce 4+ to Ce 3+ or Ce 5+ , a condition that may be followed by an increase in reactive oxygen species in the cell (Collin et al., 2014). In fact, a previous study reported an increase of the amount of H2O2 following exposure to these specific NPs, in relation to a larger Ce 3+ /Ce 4+ ratio (Celardo et al., 2011). In particular, it displayed more competition with cytochrome C in efficiently reducing superoxides (Korsvik et al., 2007). The results of this assay may be related to this Ce 3+ /Ce 4+ ratio. If there were higher amounts of Ce 3+ than Ce 4+ , this competition with cytochrome C for the reduction of superoxides could have caused an increase in the hydrogen peroxide level, which in turn caused an increase of the activity of liver catalase, enzyme whose main function is the reduction of hydrogen peroxide into water and A C C E P T E D M A N U S C R I P T molecular oxygen (Alfonso-Prieto et al., 2009). An inhibition of CAT activity has been already reported after exposing the freshwater fish Carassius auratus to CeO2 in levels of ≥ 160 mg/L for 4 days (Xia et al., 2013). This inhibition of CAT activity may be due to a direct damage to the structure of the enzyme, which may result in a partial loss of its catalytic activity, or even to its inactivation (Modesto & Martinez, 2010;Loro et al., 2012). However, in the hereby study, although catalase activity in gills was decreased for the lower level of CeO2-NPs, it was statistically meaningless. Because these NPs have antioxidant properties, at least when in low levels, the differences observed for both tissues of fish exposed to the lower NPs levels may be due to this antioxidant effect; after surpassing a given amount or physiological barrier (e.g. gills), these NPs are metabolized, becoming pro-oxidant when in the remaining concentrations (Gambardella et al., 2014).
Coating of NPs influences homo and heteroaggregation, a factor that can facilitate the interactions between NPs and cells, frequently resulting in oxidative stress (Auffan et al., 2009;Collin et al., 2014). The primary mechanism of toxicity of NPs might be the production of ROS as free radicals (Heckert et al., 2008), inducing severe injury in cells and the deactivation of enzymes. It has been demonstrated that these NPs can induce and catalyze the formation of ROS in numerous biological systems (Hoshino et al., 2004;. However, it has also been reported that these NPs can protect cells against damage caused by free radicals and ROS (Falugi et al., 2012). As mentioned before, these nanoparticles have redox properties that allow the unexpected transition between the forms of Ce 3+ (that can generate ROS) and Ce 5+ , being affected by pH and reduction potential, becoming unstable, and then causing oxidative stress (Collin et al., 2014). The literature presents conflicting results concerning the behavior of nanoceria, i.e. some studies reported that CeO2-NPs can protect cells against damage caused by free radicals and ROS, while others found that these NPs can induce severe injury in cells, the deactivation of enzymes and the production of ROS. This distinct toxicological profile may be due to the presence of Ce, which may occur in both +3 and +4 valence states, which may vary among forms of these NPs (Zhang et al., 2011;Dunnick et al., 2015).
Although there was a visible pattern of reduction of AChE activity with increasing amounts of CeO2-NPs, no statistically significant differences were observed between experimental groups for the used concentrations. Metal oxide nanoparticles can inhibit the activity of cholinesterases (Wang et al., 2009), but this pattern of inhibition was not reported here. The mechanism which can explain the effects of NPs in eyes of exposed organisms is yet to be known. Chen et al. (2006) used CeO2-NPs to alleviate oxidative stress caused by light induced retinal degeneration, what may suggest that maybe these NPs can cross the blood-retina barrier, reaching the eyes. Because AChE maintains its proper function (Chung & Bieber, 1993;Tripathi & Srivastava, 2010), it is possible to suggest that no significant neurotoxicity occurred hereby.
Tissue and cell alterations may appear in sub-lethal doses, which could be a consequence of exposure to some environmental stressors, being identified via histological assessments (Johnson et al., 2002). ). In the hereby study the most significant differentiated group corresponds to the fish exposed to the higher concentration of CeO2-NPs (0.1 µg/L). The absence of statistical changes obtained in the hepatic pathological index in the lower and medium concentrations may be due to an early stage of the detoxification process conducted by the liver, which can imply that individuals exposed to the higher concentration, were not able to complete the process effectively, giving the high amount of CeO2-NPs.
In this assay, the liver of fish exposed to CeO2-NPs exhibited some circulatory and regressive changes. The observed hyperemia, i.e. a congestion of blood in an organ caused by venous as well as arterial processes, can act as a regulatory response, allowing change in blood supply to various tissues through vasodilation (Bernet et al., 1999). The vasodilation is often caused by the presence of by-products of the metabolism of glucose and fatty acids (Kumar et al., 2005). The hereby observed hyperemia, coincident with the increase in the activity of CAT in the liver, suggests that the NPs may have increased the metabolism of fatty acids, which result in the overproduction of H2O2. The by-products of this metabolism (fatty acids) lead to a vasodilation, which could have caused hyperemia. Intercellular edema is described as stagnant tissue fluid that has leaked from capillaries into tissue (Bernet et al., 1999). The quantity of intercellular edema is determined by the balance of fluid homeostasis, which can be caused by the increase of secretion of fluid into the interstitium. The increase in hydrostatic pressure is often due to the retention of water and sodium by the liver (Kumar et al., 2005). The obtained results can indicate that exposure to the CeO2-NPs may alter the ionic balance in the liver causing tissue damage. Pyknotic nuclei is known as the irreversible condensation of the chromatin, which can lead to necrosis or apoptosis (Jarrar & Taib, 2012). Pyknotic nuclei was also observed in rainbow trout exposed to Cu-NPs (Al-Bairuty et al., 2013). The presence of pyknotic nucleus may be due to an increase in cellular activity and nuclear interruption in the mechanism of detoxification of the NPs (Jarrar & Taib, 2012). Liver vacuolization can be a result of a metabolic imbalance by exposure to contaminants (Camargo & Martinez, 2007). Fish hepatocytes tend to be vacuolated due to the high content in glycogen and/or lipid (Gingerich, 1982;Ferguson, 1989). This vacuolation is often aparent in the liver of captive fish, which may be due to the artificial feeding and housing conditions (Wolf & Wolfe, 2005). It has been reported that rainbow trout liver stores mainly glycogen (Hinton et al., 2001). The results obtained on this assay may suggest that the NPs have a tendency to generate more glycogen and/or triglycerides, increasing the degree of hepatic vacuolization.

A C C E P T E D M A N U S C R I P T
It is well known that CeO2-NPs tends to aggregate in freshwater and to be adsorbed on gills (Gagné et al., 2018). Despite being absorbed in some extent, NPs can become also attached to the gills mucus, as a feedback response to the irritation induced by NPs, forming complexes that can affect the efficacy of respiratory mechanisms, and also impacting the ion transport (Baker et al., 2014). Both factors may be the basis of the here-observed proliferation of mucus cell. As previously mentioned, the intercellular edema is determined by the balance of fluid homeostasis, which can be caused by the increase of secretion of fluid into the interstitium. Some studies revealed that interstitial edema is one of the more frequent lesions observed in gill epithelium of fish exposed to metals (Mallatt, 2011), and the metallic core of NPs (namely with Ce) could have similar effet. Aneurisms occur by an increase in the blood in the lamellae, which leads to damage in pillar cells, and consequently a loss of vascular integrity (Poleksic & Mitrovic-Tutundzic 1994;Rosety-Rodrıǵuez et al., 2002). Both tissue damages are considered as a specific reaction to toxic compounds (Temmink et al., 1983). Epithelial lifting was probably induced by the incidence of severe edema (Arellano et al., 1999;Pane et al., 2004;Schwaiger et al., 2004). The reduction in surface area, caused by the epithelium lifting, reduces the entrance of the toxicant (NPs in our case), and combined with the hypertrophy of the epithelium, could result in an increased distance between water and blood, and eventually compromising the organ function (Rodrigues et al., 2017).
Although fish can increase their rate of respiration to compensate for this loss of oxygen uptake (Fernandes & Mazon 2003), these gill structural alterations typically result in an ionic imbalance in blood parameters and red cells of fish (Wood & Soivio 1991;Poleksic & Mitrovic-Tutundzic 1994). Almost all the tissue lesions reported on this assay are considered non-specific, reversible (if the toxicant compound is removed from the media) and establish an adaptive barrier mechanism to avoid or minimize the xenobiotic uptake A C C E P T E D M A N U S C R I P T (Poleksic & Mitrovic-Tutundzic 1994;Fernandes & Mazon 2003). In general, alterations in the gills typically lead to respiratory failure, hypoxia and ionic and acid-base imbalances (Hawkins et al., 1984;Alazemi et al., 2010;Yasser & Naser, 2011). Moreover the organisms become more susceptible to secondary infections and death (Hawkins et al. 1984). Moreover, it was recently shown that CeO2-NPs induced mortality at initial concentration of 10 µg/L in rainbow trout (Gagnon et al., 2018). Although the primary pathway of incorporation of CeO2-NPs by aquatic organisms is thought to be ingestion, the significant differences found between all the exposed groups and the control may evidence that the NPs do affect the gills, even more severely than the liver. This may be due to the direct exposure to the NPs in the media and the production of complexes mucus-NPs that was evidenced before. However, these changes are unspecific and can occur after the exposure of fish to many xenobiotics (Depledge & Fossi, 1994).

Conclusions
This study provided the first evidences of the long-term effects of CeO2-NPs, in terms of pathological and physiological effects in rainbow trout. Fish subjected to chronical exposure to these NPs evidenced alterations of minimal or moderate pathological importance in the liver and also of marked pathological importance in the gills. The analyses of the enzymatic activity of GSTs identified a significant metabolic response in gills of fish exposed to the higher tested level of NPs. The absence of alterations in the activity of GSTs in liver may be due to the capacity of this tissue to use alternative metabolic routes, and may suggest that these NPs metabolism does not occur via the glutathione pathway. However, the activity of catalase was significantly increased (in liver of animals subjected to the higher levels of NPs, showing the involvement of CeO2-NPs in an pro-oxidative pathway that required an anti-oxidant compensatory biological response. The data concerning the activity of the AChE, which was not significantly different between the exposed groups and the control, may suggest that the CeO2-NPs did not cause neurotoxicity. This work also evidenced tissue damages of the gills and liver of the exposed fish. The histopathological findings reported in this study can be an adaptive mechanism to the direct exposure to waterborne  and/or result of a physiological disorder produced by them (e.g. liver). Further studies about the ecotoxicological effects of the CeO2-NPs have yet to be conducted, considering their properties, as the aggregation chemistry and the ratio of its redox state, which may affect their availability to the organism and their toxicity in the environment and biota.  Brown, L., Giral, I. (2000). Acuicultura para veterinarios: producción y clínica de peces.