Zinc oxide nanoparticles in predicted environmentally ...

Science of the Total Environment 613–614 (2018) 653–662

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Zinc oxide nanoparticles in predicted environmentally relevant concentrations leading to behavioral impairments in male swiss mice Joyce Moreira de Souza a,b, Bruna de Oliveira Mendes a,b, Abraão Tiago Batista Guimarães a,b, Aline Sueli de Lima Rodrigues a,b, Thales Quintão Chagas b, Thiago Lopes Rocha c, Guilherme Malafaia a,c,d,e,⁎ a

Post-Graduation Program in Conservation of Cerrado Natural Resources, Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil Biological Research Laboratory, Goiano Federal Institute – Urutaí Campos, GO, Brazil Institute of Tropical Pathology and Public Health, Federal University of Goiás, GO, Brazil d Biological Sciences Department, Post-Graduation Program in Conservation of Cerrado Natural Resources, Goiano Federal Institute – Urutaí Campus, GO, Brazil e Post-Graduation Program in Animal Biodiversity, Federal University of Goiás – Samambaia Campus, Goiânia, GO, Brazil b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Predicted environmentally relevant concentration of ZnO NPs cause behavioral change. • ZnO NPs causes anxiogenic effect in male Swiss mice. • ZnO NPs are able to overcome the blood-brain barrier in mice.

a r t i c l e

i n f o

Article history: Received 17 June 2017 Received in revised form 19 August 2017 Accepted 6 September 2017 Available online xxxx Editor: Henner Hollert Keywords: Experimental model Nanotoxicology Nanomaterials Anxiety Neurotoxicity

a b s t r a c t Although the potential neurotoxic effects from the exposure to zinc oxide nanoparticles (ZnO NPs) on humans and on experimental models have been reported in previous studies, the effects from the exposure to environmentally relevant concentrations of them remain unclear. Thus, the aim of the present study is to investigate the effects from the exposure to environmentally relevant concentrations of ZnO NPs on the behavior of male Swiss mice. The animals were daily exposed to environmentally relevant concentrations of ZnO NPs (5.625 × 10−5 mg kg−1) at toxic level (300 mg kg−1) through intraperitoneal injection for five days; a control group was set for comparison purposes. Positive control groups (clonazepam and fluoxetine) and a baseline group were included in the experimental design to help analyzing the behavioral tests (open field, elevated plus maze and forced swim tests). Although we did not observe any behavioral change in the animals subjected to the elevated plus maze and forced swim tests, our data evidence the anxiogenic behavior of animals exposed to the two herein tested ZnO NPs concentrations in the open field test. The animals stayed in the central part of the apparatus and presented lower locomotion ratio in the central quadrants/total of locomotion during this test. It indicates that the anxiogenic behavior was induced by ZnO NP exposure, because it leads to Zn accumulation in the brain. Thus, the current study is the first to demonstrate that the predicted environmentally relevant

⁎ Corresponding author at: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campos, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO CEP: 75790-000, Brazil. E-mail address: [email protected] (G. Malafaia).

http://dx.doi.org/10.1016/j.scitotenv.2017.09.051 0048-9697/© 2017 Elsevier B.V. All rights reserved.

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J.M. de Souza et al. / Science of the Total Environment 613–614 (2018) 653–662

ZnO NPs concentration induces behavioral changes in mammalian experimental models. Our results corroborate previous studies that have indicated the biological risks related to the water surface contamination by metalbased nanomaterials. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Nanoscience and nanotechnology involve processes applied to different segments such as food production, electronics, pharmacy, biotechnology, cosmetics, nanomedicine, agriculture and national security, as well as are related to materials and products ranging from 1 to 100 nm (Hu et al., 2016). There is no doubt nanotechnology is one of the science fields facing great development in the last decade due to high investments in research; the United States is the greatest investor in this technology, followed by Germany and Japan (Sant'Anna et al., 2013). The estimated annual production of nanomaterials (NMs) jumped from 1000 tons in 2004 to 5000 tons in 2010; the estimate for the next decade is of approximately 100,000 tons (Paschoalino et al., 2010). This growing production indicates the inevitable exposure of the environment to NMs, and it means environmental risk depending on these materials' nanospecific properties such as size, surface area, hydrodynamic diameter, agglomeration/dispersion capacity and sedimentation rate. Such properties can facilitate NMs translocation to organic systems and lead to different toxic effects (Rocha et al., 2015, Rocha et al., 2017). Currently, zinc oxide NPs (ZnO NPs) are some of the most widely used nanoparticles (McCall, 2011). They compose commercial products such as toothpastes, cosmetics, sunscreens, textile material, wall paints and building materials (Xia et al., 2008; Zvyagin et al., 2008; Smijs & Pavel, 2011; Vanderiel & Jong, 2012). Previous studies have already shown that ZnO NPs lead to different toxic effects on mammal systems (mainly in mice and rats). Therefore, ZnO NPs promote toxic effects at different biological organization levels due to their cyto- and genotoxicity (Han et al., 2017; Pati et al., 2016), neurotoxicity (Tian et al., 2015), reproductive toxicity (Jo et al., 2013; Talebi et al., 2013), immunotoxicity (Kim et al., 2014). Moreover, they cause behavioral (Xie et al., 2012; Torabi et al., 2013; Xiaoli et al., 2017; Sheida et al., 2017) histopathological (Yan et al., 2015; Almansour et al., 2017), metabolic and biochemical changes (Amara et al., 2014; Wang et al., 2016, 2017). However, most of these studies worked with ZnO NPs doses or concentrations from 1 mg kg−1 to 300 mg kg−1 (Vandebriel & Jong, 2012). These concentrations are higher than the ones often found in the environment (water surface: 1.5 ng L− 1 to 360 ng L− 1) (Dumont et al., 2015), fact that implies lack of realistic results about the ecotoxicological impact from these NPs. According to Fabrega et al. (2012), ZnO NPs can enter the water surface from multiple non-point sources. The aquatic or land animals feed on the contaminated water and accumulate ZnO NPs in their bodies, fact that reinforces the environmental and health concerns. Previous studies have shown that several microscopic, spectroscopic and separation techniques have been employed to detect NMs such as gold, silver, zinc oxide NPs and quantum dots (Segets et al., 2009; Howard, 2010; Weinberg et al., 2011; Silva et al., 2011; Majedi et al., 2012). However, these methodologies are insufficient to monitor the current environmental concentrations of engineered NMs (Farré et al., 2011). Thus, unfortunately, we are far from having methods to collect data about occurrence levels, fate and engineered NMs transportation in the environment. Before developing a solid analytical approach, it is worth fully understanding the NMs domain, but such understanding requires assessing the material-source matrices, these materials' transformation in the natural aquatic environment, and their specific physical/ chemical behavior in water medium (Weinberg et al., 2011).

Accordingly, some studies have used spatially and temporally explicit methods to model/estimate ZnO NPs concentrations in the environment (Boxall et al., 2007; Gottschalk et al., 2009; Gottschalk et al., 2011; Sun et al., 2014; Dumont et al., 2015; Markus et al., 2016). According to Hassellov et al. (2008), it is obvious that the experimental validation of these model/estimate is highly desirable, although it is still impossible assessing and quantifying the nano-sized fraction of a certain material in the environment at trace concentrations. Nevertheless, the results shown in these studies provide ways to develop analytical methods able to improve estimates on predicted environmental concentrations. A method to be successful must provide important knowledge concerning on-going investigations about the possible effects from the environmentally relevant NMs concentrations on different organisms. Despite their important contributions, the focus of many of the aforementioned studies on ZnO NPs toxicity has definitely not taken into account these materials' potential effects when they are available at environmentally relevant concentrations (Vanderiel & Jong, 2012; Saptarshi et al., 2015). Accordingly, the aim of the present study was to analyze the effects of the exposure to ZnO NPs at environmentally relevant (5.625 × 10−5 mg kg−1) and high concentrations (300 ng L−1) on the behavior of male Swiss mice, as well as to set the Zn concentration in their brain tissue. Mice exposed to ZnO NPs are expected to suffer the neurotoxic effects possibly resulting in neurobehavioral disorders, if one considers the evidence of ZnO NPs accumulation in the central nervous system (CNS) of experimental mammalian models (Feng et al., 2015). Thus, the present study emerges as an incremental step within a series of studies focused on the toxicity of ZnO NP. 2. Materials and methods 2.1. Characterizing the ZnO NPs The ZnO NPs were purchased at Sigma Aldrich (Saint Louis, MO, USA; CAS number 544906). The morphology and individual diameter distributions were measured through transmission electron microscopy (TEM). The NPs were suspended in 100 mg L−1 distilled water, stirred and then sonicated for 1 min; 3-mL aliquots were pipetted and deposited on formvar-coated 200 mesh copper grids immediately after the last procedure, then the water excess was gently blotted on filter paper. The grids were directly inserted into a Jeol-JEM1220 TEM operating at 100 kV after they were dried. The images were taken at 50 k magnification in a dedicated CCD camera. Approximately 180 NPs were measured and the mean diameter (±SD) of each isolated particle was determined. The purity of the ZnO NPs was also analyzed through image analysis in the Scandium software of the Olympus Soft Imaging Solutions GmbH and ImageJ (National Institute of Health, USA). The stock solution of ZnO NPs (18 mg L−1) was prepared in distilled water and dispersed for 10 min in a sonicator to prevent aggregation. It was kept at 4 °C and used in the experiment after 5 days. The stock solution was sonicated for 20 min before each experiment and subsequently diluted in distilled water. 2.2. Animals and experimental design Sixty-five (65) adult male Swiss mice (2.5–3 months old – nulliparous) were kept in the bioterium of the Biological Research Laboratory at Instituto Federal Goiano – Campus Urutaí (Urutaí, Goiás State, Brazil).


The mice were kept in polypropylene boxes (30.3 × 19.3 × 12.6 cm) under 12/12 h light/dark cycle on a ventilated shelf, under controlled temperature and humidity conditions (22–25 °C and 55–60% humidity); each box hosted 5 animals. All the procedures were approved by the Ethics Committee on Animal Use of Federal Goiano Institute (Comissão de Ética no Uso de Animais do Instituto Federal Goiano) (GO, Brazil – protocol n. 5678.2016). Meticulous efforts were made to assure that the animals were subjected to the least suffering possible and to reduced external stress, pain and discomfort sources. The current study did not exceed the number of animals necessary to produce trustworthy scientific data. The animals were counter-balanced according to the co-variables ‘age and body mass’, so that the mean age and mean body mass of the experimental groups were statistically equal. Next, the animals were divided into three experimental groups: control group (not exposed to NPs) (n = 10); group exposed to ZnO NPs at environmentally relevant concentration (5.625 × 10−5 mg kg−1) (n = 10) and the group of animals exposed to high ZnO NPs concentrations (300 mg kg−1) (n = 10). The animals were exposed to intraperitoneally injected ZnO NPs (5.625 × 10−5 mg kg−1 and 300 mg kg−1) diluted in distilled water for five days. Each animal was daily weighed so that the drug dosage could be accurately adjusted. The positive control groups and the baseline group were added to the experimental design to support the analysis applied to the behavioral tests. The positive control groups were composed of mice treated with intraperitoneal clonazepam (0.5 mg kg−1) (n = 10) and fluoxetine (30 mg kg−1) (n = 10) injections 30 min before the behavioral test, according to Guimarães et al. (2017). These positive groups were used to validate the sensitivity of the elevated plus maze (EPM) and forced swim tests. The baseline group (n = 15) was composed of treatmentfree animals who were minimally manipulated during the experimental period and 30 days before the experiment. Except for the baseline group and the positive control groups, all other groups were treated with intraperitoneal injections of phosphate buffered saline - PBS before the behavioral tests. The predicted environmentally relevant ZnO NPs concentration was defined according to Dumont et al. (2015), who used the Global Water Availability Assessment (GWAVA) model to simulate exposures resulting from the current ZnO NPs production levels in Europe. This simulation was based on the representative period of 31 years of monthly meteorological data. The concentration 300 ng L−1 was chosen in the present study among the concentrations estimated by Dumont et al. (2015), because it can be found in the water surface of rivers located close to the major European cities and further in the downstream of river systems. This concentration was modeled for a “conservationist” scenario without the transformation/deposition or removal of the ZnO nanoparticles from the rivers. The mice were daily exposed to a number of ZnO nanoparticles corresponding to the number of NPS the animals would ingest daily in case they would exclusively drink water from a source contaminated with ZnO nanoparticles at concentration 300 ng L−1. In order to do so, the animals' biomass (32.02 ± 1.22 g) and the mean daily volume of water ingested by the mice (6.04 mL ± 0.3/day – volume measured 30 days before the experiment) were taken into account to determine the exposure doses. Based on these data, each mouse was daily exposed to the dose 5.625 × 10− 5 mg kg− 1. On the other hand, the high ZnO NPs (300 mg kg− 1) concentration was chosen according to the previous study by Talebi et al. (2013), who showed the negative effects of this ZnO NPs concentration on the spermatogenesis of adult NMR mice males after 35 days of exposure. 2.3. Biometry The animals' total body mass, their relative brain mass, and their water consumption in a daily ratio were measured as parameters indicating systemic toxicity. The brain mass was normalized to the body

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weight through the following formula: brain mass (g)/body weight (g). The daily water and feed consumption was calculated by subtracting the leftovers from the total amount of feed offered per day. 2.4. Zn concentration in the brain tissues of the mice The animals were anesthetized with an intraperitoneal injection of 40 mg kg−1 pentobarbital followed by cervical dislocation after the behavioral tests. Next, the brain was collected to quantify the Zn amount in its tissues and to assess the possible link between Zn concentration and behavior impairments. The Zn concentration was quantified through inductively-coupled plasma-mass spectrometry (ICP-MS) (Element, Finnigan MAT, Germany), according to the method by Li et al. (2012), with modifications. Briefly, the brain was macerated and digested in a screw cap sample beaker containing 2 mL of wet-digest solution (75% nitric acid (HNO3): 70% perchloric acid (HClO4) = 1:1 v/v). The samples were placed on a hot plate for 6 h at 100 °C. After the sample's digest was clear, the cover was removed and the heating process continued at 80 °C until total drying. Five percent (5%) HNO3 was added in order to dissolve the sample digest residue; thus, the final volume dropped to 5 mL. The resulting solution was then diluted in 5% HNO3 and the total dilution corresponded to 5000 times the original weight of the sample. The solution was then analyzed through ICP-MS. We used the calibration standards 0.1, 1.0 and 10 mg kg−1 Zn to validate the method. All lines were observed based on a plasma-axial viewpoint in order to increase sensitivity. All samples were re-diluted and analyzed in duplicates to assure reproducibility. The Zn concentrations in the brain were expressed in μg/g of dry weight tissue. 2.5. Behavioral tests The animals were subjected to different behavioral tests one day after the last drug administration in order to assess the locomotor activity and the predictive anxiety and depression behaviors. Accordingly, the “triple tests” model described below was adopted based on the method by Ramos et al. (2008), with modifications. The animals were sequentially subjected to the open field, elevated plus maze and forced swim tests. 2.5.1. Open-field test The open-field arena (60 cm × 40 cm) comprised a white floor divided into 24 squares (15 cm × 15 cm) enclosed by continuous 40-cm-high walls. Each mouse was placed in the center of the open field in this test; the animal did not know the field before. The number of peripheral (adjacent to the walls) and central (away from the walls) squares touched by all four paws of the mouse were recorded for 5 min, according to the method by Souza et al. (2017). The locomotion ratio within the central quadrants/total of locomotion was calculated. The frequency of crossings through the quadrants (central and peripheral) was used to measure the locomotor activity during the open-field test. The arena was carefully cleaned with 10% ethanol solution after each test session. 2.5.2. Elevated plus maze (EPM) test The EPM device consisted of two opposing open arms (30 × 5 × 25 cm) and two opposing closed arms (30 × 5 × 25 cm) extending from a common central platform (5 × 5 cm). The adopted apparatus was made of wood and elevated 45 cm from ground level. The edges (0.25 cm) of the open arms were tested to prevent the mice from falling. The behavior rehearsal room was soundproof and the light intensity was kept at 100 lx. Subsequently, each animal was individually placed in the center of the EPM device with its face turned to one of the open arms. The animal was allowed to freely explore the apparatus for 5 min; all mice were tested once. The EPM device was cleaned with 10% ethanol before each test. The anxiety index was calculated as follows: Anxiety index = 1 − [([time the animal stayed in the open

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arms, in seconds/test duration in seconds (300 s)] + [input frequency in the open arms/total number of entries])/2]. The animals' locomotor activity in this test was accounted as the total number of entries, which were defined as the sum of the number of times the animal entered the opened and closed arms. 2.5.3. Forced swim test The forced swim test consisted of individually placing the mice in a cylindrical tank (height 39 cm, diameter 20 cm) containing water at 25 °C (20 cm depth) for 6 min. Subsequently, the animals were removed from the water and left to dry under light heating. Next, they were taken back to their crates. All test sessions were video-recorded in a video camera located 30 cm above the tank. This procedure was adopted to allow further assessing the time the mice stayed immobile. The immobility behavior is commonly used as depression predictor in the forced swim test (David et al., 2003; Petit-Demouliere et al., 2005, Can et al., 2012; Costa et al., 2013). Immobility was defined as the total absence of movement in the whole body, i.e., when the mouse stopped struggling and kept motionless, floating on the water, or when the animal was only doing the necessary movements to keep its head above the water.

comparisons of mean ranks used to compare p-values. One-way ANOVA, followed by the Tukey's post hoc analysis, was applied to the normally-distributed data. The body mass data were analyzed through factorial and repeated ANOVA measurements. The “time” (initial and end) and “treatment” (control, environmental concentration and toxic concentration) correlation analysis was performed through the Spearman's method. In addition, the regression analysis was performed when significant differences were detected between different treatments. Differences in the distributions of the groups were significant at pvalues lower than 0.05. The recordings from all the behavioral tests were watched by two trained observers. The same recording was analyzed twice, thus resulting in 85% intra-observer compliance. The analysis applied to the behavioral parameters assessed through the EPM test was performed in the PlusMZ software; the OpenFLD software was used in the open field test. Behavioral measures were later scored continuously from the videotapes by an experimenter blind to the experimental condition of each animal. All the statistical analyses and graphing were carried out in the Prism6® software (GraphPad Software, Inc., La Jolla, CA, USA). 3. Results and discussion

2.6. Data analysis 3.1. ZnO NP characterization Initially, the residual normality of all data was verified through the Shapiro-Wilk test. The Bartlett's test was used to check the homoscedasticity of the collected data. Nonparametric tests consisting of the Kruskal-Wallis ANOVA and median tests, applied to the data sets that were not normally distributed, were used to set the significance level of the differences found in their distributions, as well as in multiple

The ZnO NPs (purity N99.99%) used in the current study were nanocrystals without any surface modification (uncoated NPs). The TEM analysis showed crystalline and polygonal particles (Fig. 1A–F) of individual diameter 68.96 ± 33.71 nm (Fig. 2A), because this diameter is consistent with that reported by the manufacturer (b 100 nm). The

Fig. 1. Transmission Electron Microscopic (TEM) image of the ZnO NPs in different magnifications.


crystal structure of the ZnO NPs was characterized through XRD using Cu Kα radiation (λ = 0.15418 nm). Fig. 2B shows the XRD patterns of the ZnO NPs. Peaks at 2θ = 31.67°, 34.31°, 36.14°, 47.40°, 56.52°, 62.73°, 66.28°, 67.91°, 69.03° and 72.48° were assigned to (100), (002), (101), (102), (110), (103), (200), (112), (201), and (004) of ZnO NPs, respectively. These results indicate that the samples were formed by a polycrystalline wurtzite structure (Zincite, JCPDS 36–1451). No peaks of characteristic impurity were identified, fact that suggests the high quality of the ZnO NPs. The fourier transform– infrared (FTIR) spectra of the samples were recorded using the AlphaT spectrometer (Perkin Elmer Lambda 1050) (Fig. 2C).

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reduced the respective parameters, fact that indicated the anxiogenic effects (Fig. 4B–C) without any sedation sign in the animals. Therefore, it is surprising observing that the 5-day mice exposure to the relevant environmental concentration of ZnO NPs resulted in similar behavior in the open field test, when it was compared to the group of animals subjected to higher NP concentration. Regarding the EPM test, the results indicated the absence of anxiogenic or anxiolytic effects on the animals exposed to both ZnO NPs concentrations in comparison to animals in the control and baseline groups (Fig. 5A). The anxiety index was statistically lower in the animals exposed to clonazepam (pharmacological control) in comparison to the

3.2. Biometry The results showed significant interaction between the factors “exposure time” and “treatment” and the animals' body mass (F(2,53) = 8.779; p = 0.0006), fact that evidences that animals exposed to high ZnO NPs (300 mg kg− 1) concentrations presented significant total body mass reduction at the end of the experiment (Fig. 3A). These data can be explained by the significant decrease in daily water intake (F(2,12) = 16.93; p = 0.0003) (Fig. 3B) and food (F(2,12) = 80.61; p b 0.0001) (Fig. 3C) presented by the animals exposed to 300 mg kg−1 of ZnO NPs. These results may also explain the differences between the relative brain masses of animals after they were exposed to high ZnO NPs concentrations (F(2.27) = 14.66; p b 0.0001) (Fig. 3C). We observed a positive and statistically significant correlation between the variables “body mass (g) vs. daily feed consumption (g) per mice” (r = 0.7013; p = 0.0036) and “body mass (g) vs. daily water consumption (mL) per mice” (r = 0.5238; p = 0.0450), and the regression model best fitting the data of the simple linear regression model (R2 = 0.4918; y = 0.3968x − 6.757; R2 = 0.2744; y = 0.2115x − 1.432, respectively). It is worth highlighting that we did not observe accumulative effect of ZnO NP, i.e., animals slowly reduced the feed and water intake throughout the exposure period. On the other hand, the systemic toxicity of ZnO NPs is dose dependent; ZnO NPs at 300 mg kg−1 has high toxicity in male Swiss mice after 5 days of exposure, whereas no significant effects were observed on the biometric parameters (no changes in the body weight, daily water and food consumption and relative brain weight) of animals exposed to the environmentally relevant concentration of ZnO NPs (5.625 × 10−5 mg kg−1) (p N 0.05; Fig. 3A–D). According to Júnior et al. (2012), the body weight variation is one of the most commonly used parameters in toxicological assessments conducted to indicate the toxic effects of a given substance on the health status of animals. Therefore, different studies have assessed the body mass of animals, as well as the relative mass of their organs in research aiming at studying the effect of certain substances on the body in the medical, food production or environmental toxicology fields (Bhaskar & Mohanty, 2014; Lu et al., 2014; Mukerji et al., 2015; Das et al., 2015; Olson et al., 2015; Malafaia et al., 2015). 3.3. Behavioral impairments The ZnO NPs led to non-significant changes in the total number of quadrant crossings (H = 0.911; p = 0.923) by the mice in the open field test in comparison to the control group (Fig. 4A), thus indicating that the animals did not show hyper- or hypoactivity. On the other hand, both ZnO NPs concentrations decreased the permanence time in the central part (F(4.50) = 0.877; p b 0.0001) (Fig. 4B), as well as the locomotion ratio in the central quadrants/total locomotion assessed during the open field test (F(4,50) = 8.324; p = 0.0001) (Fig. 4C). As it was expected for animals treated with clonazepam (positive control group), the results were consistent with an anxiolytic effect (Fig. 4B– C), since the longer time spent in the central part, as well as the ratio central/total locomotion, indicate anxiolysis, according to Prut & Belzung (2003). On the other hand, both ZnO NPs concentrations

Fig. 2. Characterization of the ZnO NPs: (A) Distribution of the individual ZnO NP diameters; (B) X-ray diffraction patterns of the crystal quality of the ZnO NPs, and (C) Fourier transform–infrared transmission spectra.

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ones in the other groups (F(4,50) = 7.891; p b 0.0001). This result validates the EPM test sensitivity to identify anxiety-related behaviors. In addition, no changes in the locomotor activity of the mice were observed (H = 1.182; p = 0.881), and it indicated that the treatments did not sedate the animals during the EPM test (Fig. 5B). It is known that both the open field and EPM tests are widely used to predict anxiety behaviors in murine models (Carola et al., 2002). These two tests are based on the natural conflict between the drive to explore a new environment and the trend to avoid a potentially dangerous area (Ramos, 2008). Thus, they represent the tests of choice to assess the genetic basis of anxiety behaviors in mice, including the genetically modified animal models such as transgenic and knock-out mice (Carola et al., 2002). However, the present study evidenced a discrepancy between the anxiety indices of the animals exposed to ZnO NPs in the open field and EPM tests, as already reported in previous studies (Trullas & Skolnick, 1993; Rogers et al., 1999; Anchan et al., 2014). Although these studies present designs and objectives very different from those defined in the present study, they converge to the interpretation that the discrepant results recorded in both tests can have several explanations. One of the explanations refers to the nature of the behavioral tests used as anxiety predictors (open field and PEM tests). According to Ramos (2008), numerous studies have shown that the two main emotionality indices (ambulation and defecation) in the open field test are often unrelated, fact that supports the concept that emotionality is multidimensional, and, therefore, that it may manifest differently in distinct tests. Such concept has been further supported by factor analyses

involving multiple tests. For example, Trullas & Skolnick (1993) described that the behavioral performance of inbred mouse strains was assessed in animal models with anxiety symptoms as a way to find the potential contribution of genetic factors to fear-motivated behaviors. These authors showed that emotionality-related behaviors during the open field and EPM tests were based on distinct factors, thus they had different reflex on the behavioral dimensions. Similar results were reported by Ramos et al. (1998), who found that EPM and open field test variables do not generate usual anxiety-related factors in mice. Assumingly, the animals exposed to ZnO NPs may have presented greater emotionality in the open field test, and it reflects the observed behavioral changes. The effects of undergoing the open field test before the EMP test cannot be ruled out. It is possible that the animals had different intrinsic anxiety indices at the time of the EPM test was performed, if one considers that the open field test was the first to be conducted (Ramos, 2008). Holmes and Rodgers (1998) assessed the nature of the behavioral changes in male Swiss mice prior to the plus-maze test. They found that the primary anxiety (and activity) indices in naïve, and in subsequent trials based on independent factors, helped further emphasizing the impact of the prior experience on the emotional responses to the behavioral strategy applied to future plus-maze test encounters. Some studies, such as the review conducted by Walf & Frye, (2007), address that the exposure to a new environment immediately before the elevated plus maze test improves the motor activity during the test session by increasing the likelihood of the mice to enter the open arms of the maze. Although no changes were observed in the locomotor activity of the

Fig. 3. Initial and final body weight (A), average daily water (B) and food (C) consumption and relative brain weight (D) of male Swiss mice in the control group and of the ones exposed to ZnO NPs at environmentally relevant concentration (5.625 × 10−5 mg kg−1) and toxic levels (300 mg kg−1). The bars indicate the mean + standard deviation (SD). In “A”, the body mass data were analyzed through the factorial and repeated ANOVA measurements of “time” (initial and end) and “treatment” (control, environmental concentration and toxic concentration). In case of significant F value (p b 0.05), the Tukey post-test was applied at 5% probability. The mean values + SD with the same lower-case letter in the figures did not differ in the Tukey post-test, at 5% probability. In “B–D”, the data were subjected to analysis of variance (one-way ANOVA) and Tukey post-test at 5% probability. Different letters indicate significant differences among the experimental groups. C: control group (n = 10); EC: environmental concentration group (n = 10), and TC: toxic concentration group (n = 10).


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have shown that based on the EPM (Pellow et al., 1985) and open field (Prut & Belzung, 2003) tests it is possible finding the responses of mice pharmacologically treated with benzodiazepine anxiolytics, different parameters linked to anxiety were observed in rodents subjected to these two tests (Trullas & Skonick, 1993; Rogers et al., 1999; Anchan et al., 2014). Therefore, these tests can measure different emotionality aspects of the mice, fact that explains the discrepancies in the results recorded in our study. On the other hand, the animals' physical condition (indicated by the body mass, for example) may be an important factor influencing their behavior during the open field test. The low feed and water consumption, together with the reduced body mass of animals exposed to higher ZnO NPs concentrations, can explain the anxiogenic condition shown by the animals during the open field test. This hypothesis complies with Heiderstadt et al. (2000), who showed that animals fed with enough feed to achieve 20% initial body weight reduction presented increased anxiety in an open field test, as well as increased serum corticosterone levels 37 days after the experimental period had started. On the other hand, we cannot deny that the anxiogenic effect, especially on the animals exposed to higher ZnO NPs concentrations, can be

Fig. 4. (A) Frequency of crossings in the quadrants (central and peripheral), (B) time spent in the central part, and (C) locomotion ratio in the central quadrants/total locomotion of male Swiss mice in the control group and of the ones exposed to ZnO NPs at environmentally relevant concentrations (5.625 × 10−5 mg kg−1) and toxic levels (300 mg kg−1) assessed during the open field test (C). The bars indicate the mean + standard deviation (SD). The data were analyzed through the nonparametric KruskalWallis test in “A”; the probability b0.05 was considered statistically significant. The data were subjected to analysis of variance (one-way ANOVA) and Tukey post-test at 5% probability in “B–C”. Different letters indicate significant differences among the experimental groups. B: baseline group (n = 15); C: control group (n = 10); PC: positive control (clonazepam); EC: environmental concentration group (n = 10), and TC: toxic concentration group (n = 10).

animals during the EPM test (Fig. 5B), the aforementioned studies reinforce the hypothesis about the possible influence of the open field test on the animals' behaviors in the EPM test; however, further studies focused on this issue are needed. A third explanation may be related to the fact that the open field and EPM tests measure distinct anxiety aspects. Although previous studies

Fig. 5. (A) Anxiety index and (B) total number of crossings assessed during the elevated plus maze test applied to male Swiss mice in the control group and to animals exposed to ZnO NPs at environmentally relevant concentrations (5.625 × 10−5 mg kg−1) and toxic levels (300 mg kg−1). The bars indicate the mean + standard deviation (SD). The data were subjected to analysis of variance (one-way ANOVA) and Tukey post-test at 5% probability in “A”. Different letters indicate significant differences among the experimental groups. The data were analyzed through the nonparametric KruskalWallis test in “B”. The same lower-case letter means that there was not statically significant difference at 5% probability. B: baseline group (n = 15); C: control group (n = 10); PC: positive control (clonazepam) (n = 10); EC: environmental concentration group (n = 10), and TC: toxic concentration group (n = 10).

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Fig. 6. Zn concentration (μg/g) in the brain tissue of male Swiss mice from the control group and from the ones exposed to ZnO NPs at environmentally relevant concentrations (5.625 × 10−5 mg kg−1) and toxic levels (300 mg kg−1). The bars indicate the mean + standard deviation (SD). Data were subjected to analysis of variance (one-way ANOVA) and Tukey post-test at 5% probability. Different letters indicate significant differences among the experimental groups. C: control group (n = 10); EC: environmental concentration group (n = 10), and TC: toxic concentration group (n = 10).

related to both the physical damages observed in the animals (hair loss, small rashes, increased urination and defecation were effects recorded after the intraperitoneal applications) and the action of the NPs in the central nervous system of the animals. Similar behavior was observed in the animals exposed to the lowest ZnO NPs concentration, although without food, water and body mass reduction (Fig. 3). Thus, an important point to be noticed in the present study refers to the absence of a dose-response relation in the open field test. The anxiogenic effects in the open field test were equally observed in animals exposed to different ZnO NPs concentrations. Assumingly, the presence of ZnO NPs may have caused the effects, rather than the amount of NPs, although we have observed increased Zn accumulation in the brain tissues of animals exposed to NPs (Fig. 6) (F(2,27) = 0.877; p b 0.0001). Thus, the results suggest that even at small concentrations (i.e. b 5 million times the toxic concentration), the ZnO NPs are able to overcome the blood-brain barrier and to reach the central nervous system of these animals. We

Fig. 7. Immobility time (s) during the forced swim test applied to male Swiss mice from the control group and to the animals exposed to ZnO NPs at environmentally relevant concentration (5.625 × 10−5 mg kg−1) and toxic levels (300 mg kg−1). The bars indicate the mean + standard deviation (SD). Data were subjected to analysis of variance (one-way ANOVA) and Tukey post-test at 5% probability. Different letters indicate significant differences among the experimental groups. B: baseline group (n = 15); C: control group (n = 10); PC: positive control (fluoxetine) (n = 10); EC: environmental concentration group (n = 10), and TC: toxic concentration group (n = 10).

found a negative and statistically significant correlation between the variables “zinc concentration (μg/g) brain vs. ratio locomotion in the central quadrants/total locomotion” (r = −0.646; p = 0.0037), “body mass (g) vs. zinc concentration (μg/g) brain” (r = − 0.7610; p = 0.0002) and the regression model best fitting the data of the simple linear regression model (R2 = 0.4179; y = − 8.674x + 18.35; R2 = 0.5791; y = −2.154x + 67.46, respectively). Obviously, the Zn concentrations identified in the animals' brains are lower than the concentrations the animals were exposed to. Thus, it is possible that part of these NPs has accumulated in other organs such as liver, spleen and kidneys, or maybe they were simply excreted. Further investigations may better elucidate the metabolic route taken by these ZnO NPs in animals exposed to relevant environmental concentrations. Previous studies have presented different results regarding the anxiety condition in rodents exposed to ZnO NPs. Torabi et al. (2013) investigated the effects of ZnO NPs on the anxiety-like behaviors in adult male Wistar rats, and found that the anxiolytic effect of ZnO NPs (5, 10, and 20 mg kg−1) is much higher than its conventional form. On the other hand, Zahra et al. (2017) have recently observed that the applied ZnO NPs (50, 300 and 600 mg/mL solvent/kg body weight for 4 days) doses did not affect the exploratory and anxiolytic behaviors and the object recognition capability of adult male albino mice. Furthermore, Amara et al. (2015) assessed whether the exposure to ZnO NPs (20–30 nm; 25 mg kg−1, by i.p. injection every day for 10 days) leads to changes in the emotional behavior and trace elements homeostasis in the brain of rats. These authors showed that the treatment with ZnO NPs slightly modulated the exploratory behaviors of these animals. However, no significant differences were observed in the anxious index between ZnO NP-treated rats and the control group. Therefore, these studies indicated that the effects of the exposure to ZnO NPs are dependent on the studied rodent species, on the route and time of exposure, on the nano-specific properties of ZnO NPs, as well as on the used concentrations/doses. Based on the present study, it is tempting speculating that the anxiogenic effect observed during the open field may be related to the small size of the ZnO NPs (sufficient to bioaccumulate in the central nervous system) (Figs. 4 and 6) and to the ability of these NPs to impair the functioning of anxiety-related neurological circuits. Several neurotransmission systems are involved in the anxiety neurobiological regulatory mechanisms (Nutt, David J., 2001). Assumingly, ZnO NPs may have influenced the anxiogenic systems, if one considers that the existing cholecystokinin, excitatory amino acids, serotonin and noradrenaline compose these systems (Kalueff, 2007). In addition, changes in the GABA neurotransmitters, GABAergic neutrotransmission or the GABAreceptor complex production may have happened because these systems are closely related to anxiety disorders (Wong & Snead, 2001). Some distinctive properties typical of ZnO NPs, including the ability to generate reactive oxygen species (ROS) and membrane damage possibly caused by the generation of ROS (Reddy et al., 2007), are aspects that give us an idea about the possible mode of action associated with the anxiogenic-like effects observed in animals after the exposure to ZnO NPs. Finally, regarding the forced swim test, which is a validated depression predictive test applied to laboratory animals (Porsolt et al., 1977), we did not observe changes in the immobility of animals exposed to ZnO NPs when they were compared to animals in the control or baseline groups. Immobility reduction was observed in animals treated with the pharmacological treatment (fluoxetine) (F(4,50) = 3.066; p = 0.0004), only; these results meet those in previous studies involving rodent models (Contreras et al., 2001; Mezadri et al., 2011; Can et al., 2012; Costa et al., 2013). Thus, in opposition to the results described by Xie et al. (2012), although we observed anxiogenic behavior in mice exposed to ZnO NPs during the open field test, we can suggest that the animals' exposure to both NPs concentrations did not cause depressionlike behavior in male Swiss mice. Xie et al. (2012)showed that male


Swiss mice exposed to ZnO NPs (5.6 mg kg−1, via i.p.) were immobile in the tail suspension test and forced swim test for a shorter time, thus suggesting an antidepressant-like behavioral effect. Given the paucity of studies about the effects of ZnO NPs exposure and about the possible effects linked to depression, further investigations should be conducted (see Fig. 7). According to Guimarães et al. (2016), it is important emphasizing that the lack of a clear distinction between depression and anxiety poses a major issue to the interpretation of behavioral tests. Depression, by definition, is considered a pathological mental condition (Leonardo & Hen, 2008), whereas anxiety is a normal state of cognitive and behavioral preparedness mobilized by the body in response to future or distant potential threats (Leonardo & Hen, 2008). Krishnan et al. (2007) and Wallace et al. (2009) argue that anxiety can often emerge as part of a depressive syndrome, but this is not the rule for all patients and certainly not for all animal models, as it was observed in our study. It is also important highlighting that the neural circuits connected to depression and anxiety are found in different axes (Nestler and Hyman, 2010), and it would explain the anxiogenic effect on the animals exposed to ZnO NPs who, however, did not show to suffer from any depressive effect. 4. Conclusion The present results support the hypothesis that the exposure to predicted environmentally relevant concentration of ZnO NPs (5.625 × 10−5 mg kg−1), even for a short period of time (5 days), cause behavioral changes related to anxiety in male Swiss mice. Our study is pioneer in evidencing the anxiogenic effect on animals subjected to the open field test presenting no behavioral changes related to depression. Therefore, more investigations are required in order to better understand the neuro-effect of ZnO NPs exposure and its underlying mechanisms. Acknowledgements The authors acknowledge the “Centro Regional para o Desenvolvimento Tecnológico e Inovação (CRTI/UFG, Brazil)”, “Laboratório Multiusuário de Microscopia de Alta Resolução (LabMicUFG, Brazil)” and “Central Analítica (IQ-UFG, Brazil)” for their collaboration in the characterization of ZnO NPs. References Almansour, M.I., Alferah, M.A., Shraideh, Z.A., Jarrar, B.M., 2017 Apr. Zinc oxide nanoparticles hepatotoxicity: histological and histochemical study. Environ. Toxicol. Pharmacol. 51:124–130. http://dx.doi.org/10.1016/j.etap.2017.02.015 (Epub 2017 Feb 16). Amara, S., Slama, I.B., Mrad, I., Rihane, N., Khemissi, W., El Mir, L., Rhouma, K.B., Abdelmelek, H., Sakly, M., 2014 Nov. Effects of zinc oxide nanoparticles and/or zinc chloride on biochemical parameters and mineral levels in rat liver and kidney. Hum. Exp. Toxicol. 33 (11):1150–1157. http://dx.doi.org/10.1177/ 0960327113510327 (Epub 2014 Feb 5). Amara, S., Slama, I.B., Omri, K., El Ghoul, J., El Mir, L., Rhouma, K.B., Abdelmelek, H., Sakly, M., 2015 Dec. Effects of nanoparticle zinc oxide on emotional behavior and trace elements homeostasis in rat brain. Toxicol. Ind. Health 31 (12):1202–1209. http:// dx.doi.org/10.1177/0748233713491802 (Epub 2013 Jun 6). Anchan, D., Clark, S., Pollard, K., Vasudevan, N., 2014. GPR30 Activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice. Brain Behav. 4 (1), 51–59. Bhaskar, R., Mohanty, B., 2014. Pesticides in mixture disrupt metabolic regulation: in silico and in vivo analysis of cumulative toxicity of mancozeb and imidacloprid on body weight of mice. Gen. Comp. Endocrinol. 205, 2226–2234. Boxall, A.B.A., Chaudhry, Q., Sinclair, C., Jones, A., Aitken, R., Jefferson, B., Watts, C., 2007. Current and Future Predicted Environmental Exposure to Engineered Nanoparticles. Central Science Laboratory, York. Can, A., Dao, D.T., Arad, M., Terrillion, C.E., Piantadosi, S.C., Gould, T.D., 2012. The mouse forced swim test. J. Vis. Exp. 59, 3638. Carola, V., D'Olimpio, F., Brunamonti, E., Mangia, F., Renzi, P., 2002. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav. Brain Res. 134 (1–2), 49–57 Aug 21. Contreras, C.M., Rodriguez-Landa, J.F., Gutiérrez-García, A.G., Bernal-Morales, B., 2001 Dec. The lowest effective dose of fluoxetine in the forced swim test significantly affects the firing rate of lateral septal nucleus neurones in the rat. J. Psychopharmacol. 15 (4), 231–236.

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