Mechanism of TiO2 Nanoparticle-Induced Neurotoxicity in Zebrafish (Danio rerio) Lei Sheng,1* Ling Wang,2* Mingyu Su,1,3 Xiaoyang Zhao,1* Renping Hu,1 Xiaohong Yu,1 Jie Hong,1 Dong Liu,1 Bingqing Xu,1 Yunting Zhu,1 Han Wang,1 Fashui Hong1* 1
Medical College of Soochow University, Suzhou 215123, China
2
Libary of Soochow University, Suzhou 215021, China
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Suzhou Environmental Monitor Center, Suzhou 215004, China
Received 7 June 2014; revised 7 July 2014; accepted 13 July 2014 ABSTRACT: Zebrafish (Danio rerio) has been used historically for evaluating the toxicity of environmental and aqueous toxicants, and there is an emerging literature reporting toxic effects of manufactured nanoparticles (NPs) in zebrafish embryos. Few researches, however, are focused on the neurotoxicity on adult zebrafish after subchronic exposure to TiO2 NPs. This study was designed to evaluate the morphological changes, alterations of neurochemical contents, and expressions of memory behavior-related genes in zebrafish brains caused by exposures to 5, 10, 20, and 40 lg/L TiO2 NPs for 45 consecutive days. Our data indicated that spatial recognition memory and levels of norepinephrine, dopamine, and 5hydroxytryptamine were significantly decreased and NO levels were markedly elevated, and over proliferation of glial cells, neuron apoptosis, and TiO2 NP aggregation were observed after low dose exposures of TiO2 NPs. Furthermore, the low dose exposures of TiO2 NPs significantly activated expressions of C-fos, C-jun, and BDNF genes, and suppressed expressions of p38, NGF, CREB, NR1, NR2ab, and GluR2 genes. These findings imply that low dose exposures of TiO2 NPs may result in the brain damages in zebrafish, provide a developmental basis for evaluating the neurotoxicity of subchronic exposure, and raise the cauC 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. tion of aquatic application of TiO2 NPs. V Keywords: TiO2 nanoparticles; zebrafish; brain injury; neurotransmitters; gene expression
INTRODUCTION The use of nanoscale materials is growing exponentially, and metal-containing engineered titanium nanoparticles Correspondence to: Fashui Hong; e-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: National Natural Science Foundation of China. Contract grant number: 81273036, 30901218 Contract grant sponsor: Priority Academic Program Development of Jiangsu Higher Education Institutions, National Bringing New Ideas Foundation of Student of Soochow University Contract. Contract grant number: 201310285036 *These authors contributed equally to this work. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22031
(TiO2 NPs) are an important group of these new materials. For their tiny size and many specific features such as high surface area to particle mass ratio and high reactivity (Gurra et al., 2005), they are widely used in paint, food, sunscreen, cosmetics, building materials, pesticide degradation, air clearance, environmental cleanup of waste, and mould coating for domestic baths (Colvin, 2003; Kevin, 2004; Yu et al., 2008). The widespread production and use of TiO2 NPs will undoubtedly result in direct and indirect release into aquatic environments via bathing (Gao et al., 2009), industrial sewage effluent (Ternes et al., 2004; Handy et al., 2007) and other engineering applications (Obserdorster, 2004; Johnson et al., 2011; Wang et al., 2011; Xiong et al., 2011). The environmental concentrations from 0.01 to 16 lg/L in European surface waters were predicted (Mueller and Nowack, 2008; Gottschalk et al., 2009; Johnson et al., 2011). Accordingly,
C 2014 Wiley Periodicals, Inc. V
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much public concerns of toxicological effects of TiO2 NPs have been in aquatic organisms (Boyle et al., 2013a). Zebrafish (Danio rerio) is a tropical freshwater fish and acts as a useful model organism in scientific research. Their enduring popularity can be attributed to their playful disposition, as well as their rapid breeding, cheap price, and broad availability (Gerhard and Cheng, 2002; Parichy, 2006). Previous studies in zebrafish demonstrated that NPs, after different exposure conditions and durations, lead to various pathological and gene expression changes. Griffitt et al. observed an increase of gill filaments and different gene expression profiles after a 48 h of zebrafish to 0–10 mg/L Cu-NPs when comparing Cu-NPs and dissolved Cu (Griffitt et al., 2008). Ispas et al. (2009) observed the aggregation of Ni, thinner intestinal epithelium and separation of skeletal muscle fiber when applying 10–1000 mg/L Ni-NP to zebrafish embryos. TiO2 NPs were demonstrated accumulated in the kidney and gill tissue, but had minimal effects on kidney, spleen, or other tissues functions for 96 h (Boyle et al., 2013a) or 14 days (Scown et al., 2009) and locomotor behavior in rainbow trout for 14 days (Boyle et al., 2013b). Exposures to 100 and 200 mg/L TiO2 NPs for 8 days resulted in reductions of superoxide dismutase, catalase, and peroxidase activities, and an increase of lipid peroxidation level in liver, gill, and brain tissues of carp (Hao et al., 2009). Zhu et al. proved that after 14 days dietary exposure of TiO2 NPs on zebrafish resulted in higher body Ti burden than seen in aqueous exposed ones in complementary experiments (Zhu et al., 2009). When juvenile zebrafish were exposed to contaminated food for 14 days, Fouqueray et al. demonstrated that TiO2 residues of nanoparticles have a negative effect on digestive enzyme activity (Fouqueray et al., 2013). Ramsden et al. found that after the 14-d exposure, TiO2 NPs showed limited toxicity, but affected reproduction to zebrafish (Ramsden et al., 2013). The brain is critical to survival of the zebrafish upon injury owing to its pivotal role in many vital processes, for instance, control of movement and complex behaviors and the regulation of the endocrine system (Kizil et al., 2012). Because of its high sensitivity to drugs and convenient operation, zebrafish has been used to investigate behavioral and neurochemical changes, provides an attractive tool to assess molecular process of environmental toxicity (Bernardos et al., 2007; Patricia and Li, 2008; Poss, 2010). It has already been demonstrated that short-term exposure of TiO2 NPs could cause neurotoxicity in zebrafish, for example, Palaniappan et al. observed lipid degradation, carbonyl formation and overproduction of ROS after 14 days exposure of 10 ppm TiO2 NPs in zebrafish brains (Palaniappan and Pramod, 2011). Chen et al. demonstrated that the behavioral endpoints were more sensitive than the others (e.g., hatchability and survival) to detect toxicity of TiO2 NPs on developing zebrafish for 120 h-post-fertilization (Chen et al., 2011). The emerging studies on the toxicity of TiO2 NPs have so far
Environmental Toxicology DOI 10.1002/tox
shown that this kind of material can be harmful to zebrafish brain in the mg/L range and in short-term exposure. The molecular mechanisms of zebrafish neurotoxicity following low dose and subchronic exposure to TiO2 NPs, however, are still unclear. For example, whether some of the effects of TiO2 NPs on neuronal activity and behavior of zebrafish are due to its alteration of memory-related gene expression in zebrafish brain is not well understood. As suggested, memory-related factors, including N-methyl-D-aspartate (NMDA) receptor subunits (NR1, and NR2ab) and metabotropic glutamate receptor 2 (GluR2), cyclic-AMP responsive element binding protein (CREB), C-fos, C-jun, brain derived neurotrophic factor (BDNF), p38, and nerve growth factor (NGF), are associated with behavioral responses (Platenik et al., 2000; Yu et al., 2006; Liu et al., 2007). In addition, norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) have been proved to play a role in behavioral functions, including tonic and repetitive behavioral patterns, food intake, aggression, sexual behavior, learning, sensorimotor reactivity, circadian rhythm entrainment, sleep-wake cycles, the maturation of locomotor networks, swimming abilities, and in development such as weight gain in animals (Nakajima et al., 1998; Holschneider et al., 2001). We speculated that impairment of behavioral responses in zebrafish caused by low dose and subchronic exposure to TiO2 NPs may be associated with alterations of neurochemicals and memory behavior-related gene expressions in adult zebrafish. To gain new insights into the mechanisms underlying the potential neurotoxicity, here we assessed the brain pathological changes, and examined alterations of neurochemicals and memory behavior-related gene expressions in zebrafish caused by TiO2 NP aqueous exposure. Novelty of this article compared to previous work resides in both concentration (mg/L compared with mg/L in previous work) and duration (45 days versus 5–14 days). Our findings will provide an important developmental basis for evaluating the neurotoxicity of low dose and subchronic TiO2 NP exposures on aquatic organisms.
MATERIALS AND METHODS Characterization of TiO2 NPs The preparation, characteristics of TiO2 NPs particles have been described in our previously work (Yang et al., 2002; Hu et al., 2011). TiO2 powder was dispersed onto the zebrafish culture medium (consisting with zebrafish culture medium (consisting with 64.75 mg/L, NaHCO3, 5.75 mg/L, KCl, 123.25 mg/L, MgSO47H2O, and 294 mg/L, CaCl22H2O), and then the suspending solutions containing TiO2 particles were treated ultrasonically for 15–20 min and mechanically vibrated for 2 or 3 min. The anatase structure, size, surface area, mean hydrodynamic diameter, and f potential have been described in Supporting Information.
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
Animals and Treatment TiO2 powder was dispersed onto the surface of zebrafish culture medium to get 0, 5, 10, 20, and 40 lg/L TiO2 NP suspensions, and then treated ultrasonically for 15–20 min and mechanically vibrated for 2 or 3 min. Adult male and female zebrafish (Danio rerio) with a mean age of 120 d, mean length of 2.00 6 0.30 cm, and mean weight of 0.15 6 0.02 g were obtained from the Center for Circadian Clocks (Soochow University, China). The control group was provided with zebrafish culture medium without TiO2 NPs. In the experiment, zebrafish were randomly divided into five groups (N 5 40 each), including a control group (zebrafish culture medium without TiO2 NPs) and four experimental groups (zebrafish culture medium containing 5, 10, 20, and 40 lg/L TiO2 NPs, respectively, individual treatment replicates 5 3), and housed in separate but identical aquariums (60 3 30 3 40 cm). Prior to experimentation, zebrafish were acclimatized in deionized and single-distilled water for at least 7 d in the laboratory, with a natural light-dark cycle (12 h light /12 h dark), water temperature maintained at 28 6 2 C and pH of 6.7–7.2. Zebrafish were fed twice daily with newly hatched brine shrimp (Artemia). During the exposure term of consecutive 45 days, the dissolved oxygen was supplied by oxygen increasing pump. The number of dead zebrafish was recorded every 12 h, and they were removed immediately to avoid contamination of the exposure solutions. During this period, no zebrafish died. To ensure a constant concentration, all the test solutions were changed every 48 h.
Behavioral Tests During the exposure term of 45 days of TiO2 NPs, the acquisition of spatial recognition memory in zebrafish was detected by Y-maze (N 5 20 each). The Y-maze was made of black–white painted timber, consisted of three arms with an angle of 120 between each two arms and filled with dechlorinated tap water. Each arm was 8 cm 3 30 cm 3 15 cm (width 3 length 3 height). The three identical arms were randomly designated: Start arm, in which the zebrafish started to explore (always open), Novel arm, which was blocked during the 1st trial, but open during the 2nd trial, and Other arm (always open). The maze was placed in a sound attenuated room with dim illumination. The water of the maze was changed after each individual group in order to eliminate zebrafish olfactory stimuli. To assess spatial recognition memory, the Y-maze test consisted of two trials separated by an inter-trial interval (ITI) which described previously. During the first trial (5 min duration), the zebrafish was allowed to freely explore only two arms (start arm and other arm) of the maze, and the third arm (novel arm) of the Y-maze was blocked. After 30 min ITI, the second trial (retention) was conducted where all three arms were accessible, and novelty vs. familiarity was analyzed by comparing the behavior in all three arms. For the second trial, the zebrafish was placed
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back in the maze in the same starting arm with free access to all three arms for 5 min. Using a ceiling-mounted CCD camera, all trials were recorded on a VCR. The video recordings were later analyzed to determine the number of entries and time spent in each arm. The number of arms visited was taken as an indicator of locomotor and exploratory activity.
Preparation of Organs At the end of behavioral testing of Y-maze, zebrafish were quickly removed and placed on ice. Each set of experimental zebrafish was weighed and placed under the microscope (Leica polarizing microscope, Germany) to dissect heart, liver, and whole brain. The tissues were excised and rinsed in phosphate buffered saline (PBS) after weighed, and quickly taken and frozen at 280 C until sample preparation for further studies. After weighing the body and organs, the coefficients of organ to body weight were calculated as the ratio of organ (wet weight, mg) to body weight (g). The surgical protocols were approved by the Soochow Committee.
Titanium Content Analysis The frozen liver, heart and brain tissues were thawed and the samples were weighed, digested, and analyzed for titanium content. Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7; Thermo Electron, Waltham, MA) was used to determine the titanium concentration in the samples. Indium (20 ng/mL) was chosen as an internal standard element. The detection limit of titanium was 0.089 ng/mL.
Histopathological Evaluation of Brain For pathologic studies, all histopathologic examinations were performed using standard laboratory procedures. The brains (n 5 5 each) were embedded in paraffin blocks, then sliced (5 lm thickness) and placed onto glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (Nikon U-III Multi-point Sensor System, Japan). In the brain, cell morphous is pyramidal or polygonal to be judged to a neuron under optical microscope.
Observation of Brain Ultrastructure Brains (n 5 5 each) were fixed in a fresh solution of 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2-h fixation period at 4 C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol (75, 85, 95, and 100%), and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed
Environmental Toxicology DOI 10.1002/tox
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TABLE I. The increases of net weight and coefficient of organs in zebrafish caused by TiO2 NP exposure for 45 consecutive days TiO2 NPs (lg/L) Index Net increase of BW(mg) Heart indices (mg/g) Liver indices (mg/g) Brain indices (mg/g)
0
5
10
20
40
60.1 6 3.0 2.1 6 0.1 2.3 6 0.1 2.9 6 0.2
54.2 6 2.7 2.4 6 0.1 2.6 6 0.1 2.6 6 0.1
47.4 6 2.4* 2.5 6 0.1* 2.8 6 0.2* 2.3 6 0.1*
45.9 6 2.3* 2.8 6 0.2** 3.1 6 0.2** 2.2 6 0.1*
42.1 6 2.1* 2.9 6 0.2** 3.4 6 0.2** 2.0 6 0.1**
*p < 0.05, and **p < 0.01. Values represent means 6 SD (n 5 20).
with a HITACHI H600 TEM (HITACHI, Japan). Brain apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). In the brain, cell morphous is pyramidal or polygonal to be judged to a neuron under TEM.
(NR1, and NR2ab), and metabotropic glutamate receptor 2 (GluR2) were designed by the manufacturer and purchased from Shinegene Company (Shanghai, China). The RT-qPCR data were processed with the sequence detection software version 1.3.1 following the method of Schefe et al. (2006).
Neurotransmitters
Statistical Analysis
The homogenate of brains was centrifuged at 12,000 3 g for 20 min at 4 C and their supernatants were collected. The 20 lL supernatants were applied to an HPLC-ECD (Shimadzu, Kyoto, Japan) to determine the concentrations of monoamine neurotransmitter and their metabolites, including norepinephrine (NE), dopamine (DA) and 5hydroxytryptamine (5-HT). The 0.1 M sodium acetate/0.01 M citric acid buffer (pH 4.8) containing 0.25 M disodium edetate, 0.4 M dibutylamine, 1 M sodium octyl sulfate, and 82% methanol (v/v) was used as elution solution. ELISA was performed using commercial kits which were selective for nitric oxide (NO) concentration assay in the brain, following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of NO were calculated from a standard curve for each sample.
All results are expressed as means 6 SD. One-way analysis of variance (ANOVA) was carried out to compare the differences of means among the multi-group data using SPSS 11.9 software (SPSS, Chicago, IL). Dunnett’s test was performed when each dataset was compared with the solvent control data. Statistical significance for all tests was judged probability level of 0.05 (p < 0.05).
Brain Genes Expression Assay Total RNA was extracted from individual brains using from the homogenates was isolated using Tripure Isolation Reagent (Roche, USA) according to the manufacturer’s instructions. Probes and cycling condition were optimized in accordance with MIQE guidelines for PCR (Bustin et al., 2009). Synthesized cDNA was used for the real-time PCR by employing primers designed using Primer Express Software according to the software guidelines. PCR primers used in the gene expression analysis are listed in Supporting Information Table I. Gene expression levels were calculated as a ratio to the expression of the reference gene, actins and data were analyzed using the DDCt method. The probes for C-fos, C-jun, brain derived neurotrophic factor (BDNF), p38, cyclic-AMP responsive element binding protein (CREB), nerve growth factor (NGF), N-methyl-D-aspartate (NMDA) receptor subunits
Environmental Toxicology DOI 10.1002/tox
RESULTS Spatial Recognition Memory Under TiO2 NP Exposure Figure 1 exhibited the effects of TiO2 NPs on the spatial recognition memory of zebrafish. It can be seen that the percentage duration in the novel arm in the control was significantly higher than that in the start and other arms (p < 0.05), while the percentage duration in the novel arm in 5 lg/L, 10, 20 lg/L, and 40 lg/L TiO2 NP-treated zebrafish was lower than the start and other arms (p < 0.05), and lower than the control (p < 0.05 or 0.01). These results suggest that low dose and subchronic exposures of TiO2 NPs impaired the spatial recognition memory of zebrafish. This memory impairment might be related to brain injuries, which confirmed by morphological examination.
Locomotor Activity Under TiO2 NP Exposure To further measure spatial recognition memory, the number of entries and time spent in each arm of the maze by each zebrafish was recorded and novelty versus familiarity was analyzed by comparing behavior in all three arms. The effect of TiO2 NPs on arm visits in zebrafish is presented in Figure 2. The results indicate that TiO2 NPs decreased the number of arm entries compared to the control. Measurement of total
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
Fig. 1. The percentage of duration for zebrafish visiting the novel, start, and other arms in the Y-maze after TiO2 NPs exposure for consecutive 45 days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 5 20).
number of arm entries during the second trial revealed a significant difference between the three arms in each group after 30 min ITI.
Body Weight, Organ Indices, and Titanium Content Under TiO2 NP Exposure Table I shows the coefficients of the tissue to body weight (BW), which were expressed as milligrams (wet weight of tissues)/grams (BW). Significant differences were found in the net increase of body weight of five groups, indicating that zebrafish body weight was markedly reduced by TiO2 NP exposure (p < 0.05 or 0.01). With increased concentrations, the coefficients of the heart and liver to body weight were increased gradually, and those of various TiO2 NP-
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Fig. 3. Titanium contents in zebrafish organs caused by TiO2 NP exposure for 45 consecutive days. ***p < 0.001. Values represent mean 6 SD (n 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
exposed groups were significantly higher than that of the control (p < 0.05 or 0.01). On the contrary, the coefficients of the brain to body weight decreased gradually with increased TiO2 NP concentrations (Table I, p < 0.05 or 0.01). A differential accumulation of TiO2 NP was observed with an increasing concentration of NP in brain, heart, and liver (Fig. 3, p < 0.05).
Histopathological Observation Under TiO2 NP Exposure The histopathological changes in zebrafish brains are presented in Figure 4. Compared with the control (normal hippocampal architecture), hipocampal tissues of 5 lg/L, 10, and 20 lg/L TiO2 NP exposed groups exhibited slight glial cell proliferation (Fig. 4). In the 40 lg/L TiO2 NP exposure group, over proliferation of glial cells could be significantly observed in the hippocampus (Fig. 4), indicating that the CNS was injured by exposure to TiO2 NPs.
Observation of Neuron Cell Ultrastructure Under TiO2 NP Exposure
Fig. 2. Effects of acute TiO2 NPs locomotor activity of zebrafish in the Y-maze test following TiO2 NPs exposure for consecutive 45 days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 5 20).
Changes to deltoid or polygonal neuron ultrastructure in zebrafish brain samples are presented in Figure 5. As shown, brain cells of the control group contained elliptical nuclei with homogeneous chromatin (Fig. 5); however, ultrastructure of neuron cells in the brain tissue treated with TiO2 NPs indicated a typical apoptosis, including significant mitochondrial swelling and cristae disappearance, nuclear membrane collapse and chromatin marginalization (Fig. 5). In addition, we also significantly observed black particles agglomerates in cytoplasm and/or nucleus in the TiO2 NP-exposed brain
Environmental Toxicology DOI 10.1002/tox
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Fig. 4. Histopathology of zebrafish brains after TiO2 NP exposure for 45 consecutive days (n 5 5). Green circle indicates glial cell proliferation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
tissues (Fig. 5), further confirming that TiO2 NPs were possibly deposited in zebrafish brain.
Reduction of Neurotransmitters Under TiO2 NP Exposure Figure 6 reveals the contents of monoamine transmitters in zebrafish brain after TiO2 NP exposure for 45 consecutive days. Compared with the control, the levels of NE, DA, and 5-HT in
Environmental Toxicology DOI 10.1002/tox
the brain were decreased gradually with increased TiO2 NP concentrations (p < 0.05 or 0.01). The results are in accordance with our study on histopathological changes of zebrafish brain.
Increase of NO Level Under TiO2 NP Exposure The changes of NO concentrations in zebrafish brain are presented in Figure 7. It can be seen that the generation of NO
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
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Fig. 5. Ultrastructure of neuron cells in zebrafish brain caused by TiO2 NP exposure for 45 consecutive days and evidence of TiO2 NP translocation through the blood brain barrier and entering the neuron cells (n 5 5). (a) Control (unexposed group); (b) 5 lg/L TiO2 NP exposure group; (c) 10 lg/L TiO2 NP exposure group; (d) 20 lg/L TiO2 NP exposure group; (e) 40 lg/L TiO2 NP exposure group. Red circle indicates TiO2 NP aggregation in brain cell. Yellow arrow indicates mitochondrial swelling, green arrow indicates nuclear membrane collapse and chromatin marginalization. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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Fig. 6. Contents of monoamine transmitters in the zebrafish brain caused by TiO2 NP exposure for 45 consecutive days. **p < 0.01, and ***p < 0.001. Values represent mean 6 SD (n 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
in the brain was increased with increased TiO2 NP concentrations (p < 0.05 or 0.01).
Gene Expression Under TiO2 NP Exposure To further verify molecular mechanisms of neurotoxicity, expressions of learning and memory behavior-related genes were evaluated and are listed in Table II. Three genes including C-fos, C-jun, and BDNF were significantly upregulated, whereas 5 genes including p38, NGF, CREB, NR1, NR2ab. and GluR2 in zebrafish brain were greatly down-regulated by exposures to 10, 20, and 40 mg/L TiO2 NPs (p < 0.05 or 0.01), respectively.
DISCUSSION The results of this study indicated that exposures to 5, 10, 20, and 40 lg/L TiO2 NPs caused significant reductions in spatial recognition memory. According to Ohnishi, the twotrial Y-maze task is a specific and sensitive test of spatial recognition memory in goldfish (Ohnishi, 1997). Our data also supported this view by showing that there were always significant arm effects on percentage measures of total duration of visits and number of visits during the retention test in zebrafish. Following exposure to increased TiO2 NP dose, the time spent in the unfamiliar novel arm, and or the frequency with which zebrafish entered this arm, were lower than other arms after the 30 min ITI. However, in unexposed zebrafish, the time spent in the unfamiliar novel arm, and or the frequency with which zebrafish entered these arms, were higher than those for the familiar start and other arms. This suggested that zebrafish were highly sensitive to their spatial and contextual environment. Moreover, our data were consistent with previous findings in zebrafish which
Environmental Toxicology DOI 10.1002/tox
demonstrated a very high level of novelty exploration. In the retention test, zebrafish had to make a choice between the novel arm (unfamiliar) and the other arm (familiar) when they were released from the start arm in the Y-maze. Zebrafish exposed to low dose TiO2 NPs showed a lower score in discrimination memory than unexposed zebrafish. The findings demonstrated that low dose TiO2 NPs may impair spatial recognition memory in zebrafish in the Y-maze. Locomotor activity is a function of the level of excitability of the central nervous system (Masur et al. 1971). We found that low dose TiO2 NPs also reduced locomotor activity in zebrafish. Our data showed that low dose and suchronic exposures to TiO2 NPs caused significant reductions in body weight, and brain indices. Furthermore, titanium accumulation or TiO2 NP aggregation in the brain, over proliferation of glial cells and cell apoptosis were observed in the low dose TiO2 NPexposed zebrafish. The TiO2 NP concentrations (lg/L) are lower than previous reports (mg/L), while exposure duration (45 days) is longer than previous reports (5–14 days) (Hao et al., 2009; Scown et al., 2009; Zhu et al., 2009; Boyle et al., 2013a, b; Fouqueray et al., 2013; Ramsden et al., 2013), suggesting that TiO2 NP exposure of low concentration and long duration could lead to neurotoxicity in the TiO2 NP-exposed zebrafish. Our previous studies observed that TiO2 NP exposure resulted in titanium accumulation, over proliferation of glial cells and cell apoptosis in mouse brain, resulting in reduction of spatial recognition (Barakat et al., 2005; Ma et al., 2010; Hu et al., 2010; Hu et al., 2011; Ze et al., 2013, 2014a, b). TiO2 NP deposition in the brain or neuron cell may be via gill (Zhu et al., 2009; Palaniappan and Pramod, 2011; Wang et al., 2011; Boyle et al., 2013a; Fouqueray et al., 2013; Ramsden et al., 2013) or olfactory bulb route (Thurn et al., 2010), and endocytosis or pinocytosis route (Handy et al., 2008; Iavicoli et al., 2011), and would decrease behavior responses of zebrafish. TiO2 NP accumulation in the liver and heart of zebrafish may be circulation system
Fig. 7. NO concentrations in zebrafish brain caused by TiO2 NP exposure for 45 consecutive days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent mean 6 SD (n 5 5).
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TABLE II. mRNA expressions of genes in zebrafish brain following exposure to TiO2 NPs for 45 consecutive days TiO2 NPs (lg/L) Gene expression (Fold) C-fos/action C-jun/action BDNF/action p38/action NR1/action NGF/action CREB/action GluR2/action NR2ab/action
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5
10
20
40
3.19 6 0.16 5.51 6 0.28 0.13 6 0.01 3.88 6 0.19 0.22 6 0.01 2.98 6 0.15 0.50 6 0.03 0.25 6 0.01 0.30 6 0.02
3.25 6 0.16 5.84 6 0.29 0.12 6 0.01 3.39 6 0.17 0.15 6 0.01 2.79 6 0.14 0.48 6 0.02 0.24 6 0.01 0.28 60.01
3.51 6 0.189 7.49 6 0.38** 0.16 6 0.01* 2.83 6 0.14* 0.13 6 0.01* 2.20 6 0.11* 0.356 0.02* 0.22 6 0.01 0.19 6 0.01*
5.52 6 0.28** 8.31 6 0.42** 0.21 6 0.01** 2.58 6 0.13* 0.12 6 0.01** 1.70 6 0.091** 0.15 6 0.01** 0.13 6 0.01* 0.16 6 0.01**
6.11 6 0.31*** 9.11 6 0.46*** 0.25 6 0.01** 1.24 60.06*** 0.10 6 0.01*** 1.16 6 0.06*** 0.08 6 0.01*** 0.09 60.01** 0.12 6 0.01**
*p < 0.05, **p
Medical College of Soochow University, Suzhou 215123, China
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Libary of Soochow University, Suzhou 215021, China
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Suzhou Environmental Monitor Center, Suzhou 215004, China
Received 7 June 2014; revised 7 July 2014; accepted 13 July 2014 ABSTRACT: Zebrafish (Danio rerio) has been used historically for evaluating the toxicity of environmental and aqueous toxicants, and there is an emerging literature reporting toxic effects of manufactured nanoparticles (NPs) in zebrafish embryos. Few researches, however, are focused on the neurotoxicity on adult zebrafish after subchronic exposure to TiO2 NPs. This study was designed to evaluate the morphological changes, alterations of neurochemical contents, and expressions of memory behavior-related genes in zebrafish brains caused by exposures to 5, 10, 20, and 40 lg/L TiO2 NPs for 45 consecutive days. Our data indicated that spatial recognition memory and levels of norepinephrine, dopamine, and 5hydroxytryptamine were significantly decreased and NO levels were markedly elevated, and over proliferation of glial cells, neuron apoptosis, and TiO2 NP aggregation were observed after low dose exposures of TiO2 NPs. Furthermore, the low dose exposures of TiO2 NPs significantly activated expressions of C-fos, C-jun, and BDNF genes, and suppressed expressions of p38, NGF, CREB, NR1, NR2ab, and GluR2 genes. These findings imply that low dose exposures of TiO2 NPs may result in the brain damages in zebrafish, provide a developmental basis for evaluating the neurotoxicity of subchronic exposure, and raise the cauC 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. tion of aquatic application of TiO2 NPs. V Keywords: TiO2 nanoparticles; zebrafish; brain injury; neurotransmitters; gene expression
INTRODUCTION The use of nanoscale materials is growing exponentially, and metal-containing engineered titanium nanoparticles Correspondence to: Fashui Hong; e-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: National Natural Science Foundation of China. Contract grant number: 81273036, 30901218 Contract grant sponsor: Priority Academic Program Development of Jiangsu Higher Education Institutions, National Bringing New Ideas Foundation of Student of Soochow University Contract. Contract grant number: 201310285036 *These authors contributed equally to this work. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22031
(TiO2 NPs) are an important group of these new materials. For their tiny size and many specific features such as high surface area to particle mass ratio and high reactivity (Gurra et al., 2005), they are widely used in paint, food, sunscreen, cosmetics, building materials, pesticide degradation, air clearance, environmental cleanup of waste, and mould coating for domestic baths (Colvin, 2003; Kevin, 2004; Yu et al., 2008). The widespread production and use of TiO2 NPs will undoubtedly result in direct and indirect release into aquatic environments via bathing (Gao et al., 2009), industrial sewage effluent (Ternes et al., 2004; Handy et al., 2007) and other engineering applications (Obserdorster, 2004; Johnson et al., 2011; Wang et al., 2011; Xiong et al., 2011). The environmental concentrations from 0.01 to 16 lg/L in European surface waters were predicted (Mueller and Nowack, 2008; Gottschalk et al., 2009; Johnson et al., 2011). Accordingly,
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much public concerns of toxicological effects of TiO2 NPs have been in aquatic organisms (Boyle et al., 2013a). Zebrafish (Danio rerio) is a tropical freshwater fish and acts as a useful model organism in scientific research. Their enduring popularity can be attributed to their playful disposition, as well as their rapid breeding, cheap price, and broad availability (Gerhard and Cheng, 2002; Parichy, 2006). Previous studies in zebrafish demonstrated that NPs, after different exposure conditions and durations, lead to various pathological and gene expression changes. Griffitt et al. observed an increase of gill filaments and different gene expression profiles after a 48 h of zebrafish to 0–10 mg/L Cu-NPs when comparing Cu-NPs and dissolved Cu (Griffitt et al., 2008). Ispas et al. (2009) observed the aggregation of Ni, thinner intestinal epithelium and separation of skeletal muscle fiber when applying 10–1000 mg/L Ni-NP to zebrafish embryos. TiO2 NPs were demonstrated accumulated in the kidney and gill tissue, but had minimal effects on kidney, spleen, or other tissues functions for 96 h (Boyle et al., 2013a) or 14 days (Scown et al., 2009) and locomotor behavior in rainbow trout for 14 days (Boyle et al., 2013b). Exposures to 100 and 200 mg/L TiO2 NPs for 8 days resulted in reductions of superoxide dismutase, catalase, and peroxidase activities, and an increase of lipid peroxidation level in liver, gill, and brain tissues of carp (Hao et al., 2009). Zhu et al. proved that after 14 days dietary exposure of TiO2 NPs on zebrafish resulted in higher body Ti burden than seen in aqueous exposed ones in complementary experiments (Zhu et al., 2009). When juvenile zebrafish were exposed to contaminated food for 14 days, Fouqueray et al. demonstrated that TiO2 residues of nanoparticles have a negative effect on digestive enzyme activity (Fouqueray et al., 2013). Ramsden et al. found that after the 14-d exposure, TiO2 NPs showed limited toxicity, but affected reproduction to zebrafish (Ramsden et al., 2013). The brain is critical to survival of the zebrafish upon injury owing to its pivotal role in many vital processes, for instance, control of movement and complex behaviors and the regulation of the endocrine system (Kizil et al., 2012). Because of its high sensitivity to drugs and convenient operation, zebrafish has been used to investigate behavioral and neurochemical changes, provides an attractive tool to assess molecular process of environmental toxicity (Bernardos et al., 2007; Patricia and Li, 2008; Poss, 2010). It has already been demonstrated that short-term exposure of TiO2 NPs could cause neurotoxicity in zebrafish, for example, Palaniappan et al. observed lipid degradation, carbonyl formation and overproduction of ROS after 14 days exposure of 10 ppm TiO2 NPs in zebrafish brains (Palaniappan and Pramod, 2011). Chen et al. demonstrated that the behavioral endpoints were more sensitive than the others (e.g., hatchability and survival) to detect toxicity of TiO2 NPs on developing zebrafish for 120 h-post-fertilization (Chen et al., 2011). The emerging studies on the toxicity of TiO2 NPs have so far
Environmental Toxicology DOI 10.1002/tox
shown that this kind of material can be harmful to zebrafish brain in the mg/L range and in short-term exposure. The molecular mechanisms of zebrafish neurotoxicity following low dose and subchronic exposure to TiO2 NPs, however, are still unclear. For example, whether some of the effects of TiO2 NPs on neuronal activity and behavior of zebrafish are due to its alteration of memory-related gene expression in zebrafish brain is not well understood. As suggested, memory-related factors, including N-methyl-D-aspartate (NMDA) receptor subunits (NR1, and NR2ab) and metabotropic glutamate receptor 2 (GluR2), cyclic-AMP responsive element binding protein (CREB), C-fos, C-jun, brain derived neurotrophic factor (BDNF), p38, and nerve growth factor (NGF), are associated with behavioral responses (Platenik et al., 2000; Yu et al., 2006; Liu et al., 2007). In addition, norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) have been proved to play a role in behavioral functions, including tonic and repetitive behavioral patterns, food intake, aggression, sexual behavior, learning, sensorimotor reactivity, circadian rhythm entrainment, sleep-wake cycles, the maturation of locomotor networks, swimming abilities, and in development such as weight gain in animals (Nakajima et al., 1998; Holschneider et al., 2001). We speculated that impairment of behavioral responses in zebrafish caused by low dose and subchronic exposure to TiO2 NPs may be associated with alterations of neurochemicals and memory behavior-related gene expressions in adult zebrafish. To gain new insights into the mechanisms underlying the potential neurotoxicity, here we assessed the brain pathological changes, and examined alterations of neurochemicals and memory behavior-related gene expressions in zebrafish caused by TiO2 NP aqueous exposure. Novelty of this article compared to previous work resides in both concentration (mg/L compared with mg/L in previous work) and duration (45 days versus 5–14 days). Our findings will provide an important developmental basis for evaluating the neurotoxicity of low dose and subchronic TiO2 NP exposures on aquatic organisms.
MATERIALS AND METHODS Characterization of TiO2 NPs The preparation, characteristics of TiO2 NPs particles have been described in our previously work (Yang et al., 2002; Hu et al., 2011). TiO2 powder was dispersed onto the zebrafish culture medium (consisting with zebrafish culture medium (consisting with 64.75 mg/L, NaHCO3, 5.75 mg/L, KCl, 123.25 mg/L, MgSO47H2O, and 294 mg/L, CaCl22H2O), and then the suspending solutions containing TiO2 particles were treated ultrasonically for 15–20 min and mechanically vibrated for 2 or 3 min. The anatase structure, size, surface area, mean hydrodynamic diameter, and f potential have been described in Supporting Information.
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
Animals and Treatment TiO2 powder was dispersed onto the surface of zebrafish culture medium to get 0, 5, 10, 20, and 40 lg/L TiO2 NP suspensions, and then treated ultrasonically for 15–20 min and mechanically vibrated for 2 or 3 min. Adult male and female zebrafish (Danio rerio) with a mean age of 120 d, mean length of 2.00 6 0.30 cm, and mean weight of 0.15 6 0.02 g were obtained from the Center for Circadian Clocks (Soochow University, China). The control group was provided with zebrafish culture medium without TiO2 NPs. In the experiment, zebrafish were randomly divided into five groups (N 5 40 each), including a control group (zebrafish culture medium without TiO2 NPs) and four experimental groups (zebrafish culture medium containing 5, 10, 20, and 40 lg/L TiO2 NPs, respectively, individual treatment replicates 5 3), and housed in separate but identical aquariums (60 3 30 3 40 cm). Prior to experimentation, zebrafish were acclimatized in deionized and single-distilled water for at least 7 d in the laboratory, with a natural light-dark cycle (12 h light /12 h dark), water temperature maintained at 28 6 2 C and pH of 6.7–7.2. Zebrafish were fed twice daily with newly hatched brine shrimp (Artemia). During the exposure term of consecutive 45 days, the dissolved oxygen was supplied by oxygen increasing pump. The number of dead zebrafish was recorded every 12 h, and they were removed immediately to avoid contamination of the exposure solutions. During this period, no zebrafish died. To ensure a constant concentration, all the test solutions were changed every 48 h.
Behavioral Tests During the exposure term of 45 days of TiO2 NPs, the acquisition of spatial recognition memory in zebrafish was detected by Y-maze (N 5 20 each). The Y-maze was made of black–white painted timber, consisted of three arms with an angle of 120 between each two arms and filled with dechlorinated tap water. Each arm was 8 cm 3 30 cm 3 15 cm (width 3 length 3 height). The three identical arms were randomly designated: Start arm, in which the zebrafish started to explore (always open), Novel arm, which was blocked during the 1st trial, but open during the 2nd trial, and Other arm (always open). The maze was placed in a sound attenuated room with dim illumination. The water of the maze was changed after each individual group in order to eliminate zebrafish olfactory stimuli. To assess spatial recognition memory, the Y-maze test consisted of two trials separated by an inter-trial interval (ITI) which described previously. During the first trial (5 min duration), the zebrafish was allowed to freely explore only two arms (start arm and other arm) of the maze, and the third arm (novel arm) of the Y-maze was blocked. After 30 min ITI, the second trial (retention) was conducted where all three arms were accessible, and novelty vs. familiarity was analyzed by comparing the behavior in all three arms. For the second trial, the zebrafish was placed
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back in the maze in the same starting arm with free access to all three arms for 5 min. Using a ceiling-mounted CCD camera, all trials were recorded on a VCR. The video recordings were later analyzed to determine the number of entries and time spent in each arm. The number of arms visited was taken as an indicator of locomotor and exploratory activity.
Preparation of Organs At the end of behavioral testing of Y-maze, zebrafish were quickly removed and placed on ice. Each set of experimental zebrafish was weighed and placed under the microscope (Leica polarizing microscope, Germany) to dissect heart, liver, and whole brain. The tissues were excised and rinsed in phosphate buffered saline (PBS) after weighed, and quickly taken and frozen at 280 C until sample preparation for further studies. After weighing the body and organs, the coefficients of organ to body weight were calculated as the ratio of organ (wet weight, mg) to body weight (g). The surgical protocols were approved by the Soochow Committee.
Titanium Content Analysis The frozen liver, heart and brain tissues were thawed and the samples were weighed, digested, and analyzed for titanium content. Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7; Thermo Electron, Waltham, MA) was used to determine the titanium concentration in the samples. Indium (20 ng/mL) was chosen as an internal standard element. The detection limit of titanium was 0.089 ng/mL.
Histopathological Evaluation of Brain For pathologic studies, all histopathologic examinations were performed using standard laboratory procedures. The brains (n 5 5 each) were embedded in paraffin blocks, then sliced (5 lm thickness) and placed onto glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (Nikon U-III Multi-point Sensor System, Japan). In the brain, cell morphous is pyramidal or polygonal to be judged to a neuron under optical microscope.
Observation of Brain Ultrastructure Brains (n 5 5 each) were fixed in a fresh solution of 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2-h fixation period at 4 C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol (75, 85, 95, and 100%), and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed
Environmental Toxicology DOI 10.1002/tox
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TABLE I. The increases of net weight and coefficient of organs in zebrafish caused by TiO2 NP exposure for 45 consecutive days TiO2 NPs (lg/L) Index Net increase of BW(mg) Heart indices (mg/g) Liver indices (mg/g) Brain indices (mg/g)
0
5
10
20
40
60.1 6 3.0 2.1 6 0.1 2.3 6 0.1 2.9 6 0.2
54.2 6 2.7 2.4 6 0.1 2.6 6 0.1 2.6 6 0.1
47.4 6 2.4* 2.5 6 0.1* 2.8 6 0.2* 2.3 6 0.1*
45.9 6 2.3* 2.8 6 0.2** 3.1 6 0.2** 2.2 6 0.1*
42.1 6 2.1* 2.9 6 0.2** 3.4 6 0.2** 2.0 6 0.1**
*p < 0.05, and **p < 0.01. Values represent means 6 SD (n 5 20).
with a HITACHI H600 TEM (HITACHI, Japan). Brain apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). In the brain, cell morphous is pyramidal or polygonal to be judged to a neuron under TEM.
(NR1, and NR2ab), and metabotropic glutamate receptor 2 (GluR2) were designed by the manufacturer and purchased from Shinegene Company (Shanghai, China). The RT-qPCR data were processed with the sequence detection software version 1.3.1 following the method of Schefe et al. (2006).
Neurotransmitters
Statistical Analysis
The homogenate of brains was centrifuged at 12,000 3 g for 20 min at 4 C and their supernatants were collected. The 20 lL supernatants were applied to an HPLC-ECD (Shimadzu, Kyoto, Japan) to determine the concentrations of monoamine neurotransmitter and their metabolites, including norepinephrine (NE), dopamine (DA) and 5hydroxytryptamine (5-HT). The 0.1 M sodium acetate/0.01 M citric acid buffer (pH 4.8) containing 0.25 M disodium edetate, 0.4 M dibutylamine, 1 M sodium octyl sulfate, and 82% methanol (v/v) was used as elution solution. ELISA was performed using commercial kits which were selective for nitric oxide (NO) concentration assay in the brain, following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of NO were calculated from a standard curve for each sample.
All results are expressed as means 6 SD. One-way analysis of variance (ANOVA) was carried out to compare the differences of means among the multi-group data using SPSS 11.9 software (SPSS, Chicago, IL). Dunnett’s test was performed when each dataset was compared with the solvent control data. Statistical significance for all tests was judged probability level of 0.05 (p < 0.05).
Brain Genes Expression Assay Total RNA was extracted from individual brains using from the homogenates was isolated using Tripure Isolation Reagent (Roche, USA) according to the manufacturer’s instructions. Probes and cycling condition were optimized in accordance with MIQE guidelines for PCR (Bustin et al., 2009). Synthesized cDNA was used for the real-time PCR by employing primers designed using Primer Express Software according to the software guidelines. PCR primers used in the gene expression analysis are listed in Supporting Information Table I. Gene expression levels were calculated as a ratio to the expression of the reference gene, actins and data were analyzed using the DDCt method. The probes for C-fos, C-jun, brain derived neurotrophic factor (BDNF), p38, cyclic-AMP responsive element binding protein (CREB), nerve growth factor (NGF), N-methyl-D-aspartate (NMDA) receptor subunits
Environmental Toxicology DOI 10.1002/tox
RESULTS Spatial Recognition Memory Under TiO2 NP Exposure Figure 1 exhibited the effects of TiO2 NPs on the spatial recognition memory of zebrafish. It can be seen that the percentage duration in the novel arm in the control was significantly higher than that in the start and other arms (p < 0.05), while the percentage duration in the novel arm in 5 lg/L, 10, 20 lg/L, and 40 lg/L TiO2 NP-treated zebrafish was lower than the start and other arms (p < 0.05), and lower than the control (p < 0.05 or 0.01). These results suggest that low dose and subchronic exposures of TiO2 NPs impaired the spatial recognition memory of zebrafish. This memory impairment might be related to brain injuries, which confirmed by morphological examination.
Locomotor Activity Under TiO2 NP Exposure To further measure spatial recognition memory, the number of entries and time spent in each arm of the maze by each zebrafish was recorded and novelty versus familiarity was analyzed by comparing behavior in all three arms. The effect of TiO2 NPs on arm visits in zebrafish is presented in Figure 2. The results indicate that TiO2 NPs decreased the number of arm entries compared to the control. Measurement of total
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
Fig. 1. The percentage of duration for zebrafish visiting the novel, start, and other arms in the Y-maze after TiO2 NPs exposure for consecutive 45 days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 5 20).
number of arm entries during the second trial revealed a significant difference between the three arms in each group after 30 min ITI.
Body Weight, Organ Indices, and Titanium Content Under TiO2 NP Exposure Table I shows the coefficients of the tissue to body weight (BW), which were expressed as milligrams (wet weight of tissues)/grams (BW). Significant differences were found in the net increase of body weight of five groups, indicating that zebrafish body weight was markedly reduced by TiO2 NP exposure (p < 0.05 or 0.01). With increased concentrations, the coefficients of the heart and liver to body weight were increased gradually, and those of various TiO2 NP-
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Fig. 3. Titanium contents in zebrafish organs caused by TiO2 NP exposure for 45 consecutive days. ***p < 0.001. Values represent mean 6 SD (n 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
exposed groups were significantly higher than that of the control (p < 0.05 or 0.01). On the contrary, the coefficients of the brain to body weight decreased gradually with increased TiO2 NP concentrations (Table I, p < 0.05 or 0.01). A differential accumulation of TiO2 NP was observed with an increasing concentration of NP in brain, heart, and liver (Fig. 3, p < 0.05).
Histopathological Observation Under TiO2 NP Exposure The histopathological changes in zebrafish brains are presented in Figure 4. Compared with the control (normal hippocampal architecture), hipocampal tissues of 5 lg/L, 10, and 20 lg/L TiO2 NP exposed groups exhibited slight glial cell proliferation (Fig. 4). In the 40 lg/L TiO2 NP exposure group, over proliferation of glial cells could be significantly observed in the hippocampus (Fig. 4), indicating that the CNS was injured by exposure to TiO2 NPs.
Observation of Neuron Cell Ultrastructure Under TiO2 NP Exposure
Fig. 2. Effects of acute TiO2 NPs locomotor activity of zebrafish in the Y-maze test following TiO2 NPs exposure for consecutive 45 days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 5 20).
Changes to deltoid or polygonal neuron ultrastructure in zebrafish brain samples are presented in Figure 5. As shown, brain cells of the control group contained elliptical nuclei with homogeneous chromatin (Fig. 5); however, ultrastructure of neuron cells in the brain tissue treated with TiO2 NPs indicated a typical apoptosis, including significant mitochondrial swelling and cristae disappearance, nuclear membrane collapse and chromatin marginalization (Fig. 5). In addition, we also significantly observed black particles agglomerates in cytoplasm and/or nucleus in the TiO2 NP-exposed brain
Environmental Toxicology DOI 10.1002/tox
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Fig. 4. Histopathology of zebrafish brains after TiO2 NP exposure for 45 consecutive days (n 5 5). Green circle indicates glial cell proliferation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
tissues (Fig. 5), further confirming that TiO2 NPs were possibly deposited in zebrafish brain.
Reduction of Neurotransmitters Under TiO2 NP Exposure Figure 6 reveals the contents of monoamine transmitters in zebrafish brain after TiO2 NP exposure for 45 consecutive days. Compared with the control, the levels of NE, DA, and 5-HT in
Environmental Toxicology DOI 10.1002/tox
the brain were decreased gradually with increased TiO2 NP concentrations (p < 0.05 or 0.01). The results are in accordance with our study on histopathological changes of zebrafish brain.
Increase of NO Level Under TiO2 NP Exposure The changes of NO concentrations in zebrafish brain are presented in Figure 7. It can be seen that the generation of NO
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
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Fig. 5. Ultrastructure of neuron cells in zebrafish brain caused by TiO2 NP exposure for 45 consecutive days and evidence of TiO2 NP translocation through the blood brain barrier and entering the neuron cells (n 5 5). (a) Control (unexposed group); (b) 5 lg/L TiO2 NP exposure group; (c) 10 lg/L TiO2 NP exposure group; (d) 20 lg/L TiO2 NP exposure group; (e) 40 lg/L TiO2 NP exposure group. Red circle indicates TiO2 NP aggregation in brain cell. Yellow arrow indicates mitochondrial swelling, green arrow indicates nuclear membrane collapse and chromatin marginalization. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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Fig. 6. Contents of monoamine transmitters in the zebrafish brain caused by TiO2 NP exposure for 45 consecutive days. **p < 0.01, and ***p < 0.001. Values represent mean 6 SD (n 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
in the brain was increased with increased TiO2 NP concentrations (p < 0.05 or 0.01).
Gene Expression Under TiO2 NP Exposure To further verify molecular mechanisms of neurotoxicity, expressions of learning and memory behavior-related genes were evaluated and are listed in Table II. Three genes including C-fos, C-jun, and BDNF were significantly upregulated, whereas 5 genes including p38, NGF, CREB, NR1, NR2ab. and GluR2 in zebrafish brain were greatly down-regulated by exposures to 10, 20, and 40 mg/L TiO2 NPs (p < 0.05 or 0.01), respectively.
DISCUSSION The results of this study indicated that exposures to 5, 10, 20, and 40 lg/L TiO2 NPs caused significant reductions in spatial recognition memory. According to Ohnishi, the twotrial Y-maze task is a specific and sensitive test of spatial recognition memory in goldfish (Ohnishi, 1997). Our data also supported this view by showing that there were always significant arm effects on percentage measures of total duration of visits and number of visits during the retention test in zebrafish. Following exposure to increased TiO2 NP dose, the time spent in the unfamiliar novel arm, and or the frequency with which zebrafish entered this arm, were lower than other arms after the 30 min ITI. However, in unexposed zebrafish, the time spent in the unfamiliar novel arm, and or the frequency with which zebrafish entered these arms, were higher than those for the familiar start and other arms. This suggested that zebrafish were highly sensitive to their spatial and contextual environment. Moreover, our data were consistent with previous findings in zebrafish which
Environmental Toxicology DOI 10.1002/tox
demonstrated a very high level of novelty exploration. In the retention test, zebrafish had to make a choice between the novel arm (unfamiliar) and the other arm (familiar) when they were released from the start arm in the Y-maze. Zebrafish exposed to low dose TiO2 NPs showed a lower score in discrimination memory than unexposed zebrafish. The findings demonstrated that low dose TiO2 NPs may impair spatial recognition memory in zebrafish in the Y-maze. Locomotor activity is a function of the level of excitability of the central nervous system (Masur et al. 1971). We found that low dose TiO2 NPs also reduced locomotor activity in zebrafish. Our data showed that low dose and suchronic exposures to TiO2 NPs caused significant reductions in body weight, and brain indices. Furthermore, titanium accumulation or TiO2 NP aggregation in the brain, over proliferation of glial cells and cell apoptosis were observed in the low dose TiO2 NPexposed zebrafish. The TiO2 NP concentrations (lg/L) are lower than previous reports (mg/L), while exposure duration (45 days) is longer than previous reports (5–14 days) (Hao et al., 2009; Scown et al., 2009; Zhu et al., 2009; Boyle et al., 2013a, b; Fouqueray et al., 2013; Ramsden et al., 2013), suggesting that TiO2 NP exposure of low concentration and long duration could lead to neurotoxicity in the TiO2 NP-exposed zebrafish. Our previous studies observed that TiO2 NP exposure resulted in titanium accumulation, over proliferation of glial cells and cell apoptosis in mouse brain, resulting in reduction of spatial recognition (Barakat et al., 2005; Ma et al., 2010; Hu et al., 2010; Hu et al., 2011; Ze et al., 2013, 2014a, b). TiO2 NP deposition in the brain or neuron cell may be via gill (Zhu et al., 2009; Palaniappan and Pramod, 2011; Wang et al., 2011; Boyle et al., 2013a; Fouqueray et al., 2013; Ramsden et al., 2013) or olfactory bulb route (Thurn et al., 2010), and endocytosis or pinocytosis route (Handy et al., 2008; Iavicoli et al., 2011), and would decrease behavior responses of zebrafish. TiO2 NP accumulation in the liver and heart of zebrafish may be circulation system
Fig. 7. NO concentrations in zebrafish brain caused by TiO2 NP exposure for 45 consecutive days. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent mean 6 SD (n 5 5).
TIO2-NANOPARTICLE INDUCED BRAIN INJURY IN ZEBRAFISH
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TABLE II. mRNA expressions of genes in zebrafish brain following exposure to TiO2 NPs for 45 consecutive days TiO2 NPs (lg/L) Gene expression (Fold) C-fos/action C-jun/action BDNF/action p38/action NR1/action NGF/action CREB/action GluR2/action NR2ab/action
0
5
10
20
40
3.19 6 0.16 5.51 6 0.28 0.13 6 0.01 3.88 6 0.19 0.22 6 0.01 2.98 6 0.15 0.50 6 0.03 0.25 6 0.01 0.30 6 0.02
3.25 6 0.16 5.84 6 0.29 0.12 6 0.01 3.39 6 0.17 0.15 6 0.01 2.79 6 0.14 0.48 6 0.02 0.24 6 0.01 0.28 60.01
3.51 6 0.189 7.49 6 0.38** 0.16 6 0.01* 2.83 6 0.14* 0.13 6 0.01* 2.20 6 0.11* 0.356 0.02* 0.22 6 0.01 0.19 6 0.01*
5.52 6 0.28** 8.31 6 0.42** 0.21 6 0.01** 2.58 6 0.13* 0.12 6 0.01** 1.70 6 0.091** 0.15 6 0.01** 0.13 6 0.01* 0.16 6 0.01**
6.11 6 0.31*** 9.11 6 0.46*** 0.25 6 0.01** 1.24 60.06*** 0.10 6 0.01*** 1.16 6 0.06*** 0.08 6 0.01*** 0.09 60.01** 0.12 6 0.01**
*p < 0.05, **p
