Neurotoxicology and Teratology 48 (2015) 9–17

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Embryonic exposure to cadmium (II) and chromium (VI) induce behavioral alterations, oxidative stress and immunotoxicity in zebrafish (Danio rerio) Yuanxiang Jin 1, Zhenzhen Liu 1, Fang Liu, Yang Ye, Tao Peng, Zhengwei Fu ⁎ College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China

a r t i c l e

i n f o

Article history: Received 24 October 2014 Received in revised form 4 January 2015 Accepted 12 January 2015 Available online 16 January 2015 Keywords: Cadmium Chromium Oxidative stress Immunotoxicity Zebrafish

a b s t r a c t Cadmium (Cd) and chromium (Cr) are considered as the main environmental contaminants which have serious risks for health. Firstly, we observed that the hatchability was significantly decreased by exposure to 10 μM Cd for 60 and 96 h post fertilization (hpf). And some abnormalities in embryos and larvae were observed especially in the 10 μM Cd treated group. Moreover, the free swimming activities and the swimming behaviors of the larval zebrafish in response to the stimulation of light-to-dark photoperiod transition were significantly influenced by both Cd and Cr treatments. Secondly, Cd and Cr exposure induced the changes in oxidative stress of the larval zebrafish. The malondialdehyde (MDA) levels increased and the glutathione (GSH) contents decreased significantly after the exposure to Cd or Cr for 96 hpf. Cd or Cr affected not only the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione S-transferase (GST), but also the transcriptional levels of their respective genes. Thirdly, with regard to the immune response, the mRNA levels of the main cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1 β (IL-1β) in the larvae increased significantly after the exposure to Cd and Cr for 96 hpf. Our results suggested that Cd and Cr have the potential to cause behavior alterations, oxidative stress and immunotoxicity in the larval zebrafish. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Health concerns regarding the potential interference of heavy metals in wildlife and humans have increased in recent years (Valko et al, 2005; Jomova and Valko, 2011; Ng et al, 2013; Notarachille et al, 2014). Cd and Cr are frequently used in many industrial branches such as batteries, metal plating, pigments, cast iron and metal finishes (Bagchi et al, 2000; Alghasham et al, 2013), and they are released into the environment through different pathways. Indeed, high levels of Cd and Cr were observed in the waterborne and sediment especially in developing countries. The dissolved Cd and Cr levels in freshwater generally ranged from 10 to 500 ng/L, but their levels exceeding 1 mg/L also have been recorded in some industrialized areas (Jones et al, 2001; Ma et al, 2008). In the sediment and soil, the levels of Cd and Cr reached much more than 1 mg/kg in seriously polluted areas (Smith et al, 1996; Zhang et al, 2014). More importantly, the environmental heavy metals could be bioaccumulated in zebrafish (Matz et al, 2007). As observed by Burnison et al. (2006), who reported that over a 5-h exposure, zebrafish eggs showed a steady increase in Cd-accumulation, and the ⁎ Corresponding author. Tel./fax: +86 571 8832 0599. E-mail address: [email protected] (Z. Fu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ntt.2015.01.002 0892-0362/© 2015 Elsevier Inc. All rights reserved.

accumulation was affected by Cd concentration, exposure time, presence of dissolved organic material, different origins of dissolved organic material and different parts of fish eggs. In a number of previous studies, Cd and Cr were reported to be associated with a wide range of toxic effects including nephrotoxicity, hepatotoxicity, oxidative stress, as well as disrupting effects on the endocrine systems in some aquatic organisms. For example, Woo et al. (2009) reported that exposure to Cd, Cr and other heavy metals for 24 h would change the transcriptional levels of eight genes related to oxidative stress in the liver of Japanese medaka. Li et al. (2014) observed that the exposure to 0.5 and 2.5 mg/L of Cd for 4 days caused significant changes in the expression profiles of genes related to the hypothalamic– pituitary–thyroid (HPT) axis in Chinese rare minnow (Gobiocypris rarus), resulting in the significant decrease of the thyroid hormone (TH) levels in the whole-body of fish. Recently, the toxicities of Cd and Cr have also been studied in zebrafish. For example, Wu et al. (2013) reported that maternal Cd exposure influenced reproduction ability in females and appeared to cause developmental delay and abnormality in embryos. More recently, Hussainzada et al. (2014) observed that the metals including nickel chloride (NiCl2), cobalt chloride (CoCl2), sodium dichromate (Na2Cr2O7) acute exposure efficiently activated biological processes associated with ribosome biogenesis, proteosomal degradation, and p53 signaling cascades in adult male zebrafish analyzed by

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microarray. However, a risk assessment for such heavy metals on the motor behavior and immune system in fish species has only received limited concern. Zebrafish is reported as a powerful vertebrate model for in vivo studies of aquatic toxicology (Yoder et al, 2002; Fraysse et al, 2006; Craig et al, 2007; Lahnsteiner, 2008; Jin et al, 2010a; Sawle et al, 2010). In the present study, to better understand the potential of Cd and Cr to induce oxidative stress and immunotoxicity in the early developmental stage of zebrafish, their effects on behavior, the oxidative stress responses, as well as the innate immune system indicated by the expression changes of some representative immunological genes, such as tumor necrosis factor α (TNFα), interleukin-1 β (IL-1β) and interleukin-6 (IL-6), were examined. Our results bring some insights into the toxicity mechanisms of heavy metals in aquatic ecosystem. 2. Materials and methods 2.1. Experimental fish and chemicals Healthy five-month-old adult fish were selected and acclimatized separately in glass tanks at ambient temperature (27 ± 1 °C) with 14-hour light/10-hour dark cycles. The fish were fed two times a day with brine shrimp. A total of forty male and forty female fish were maintained. Embryos were collected and staged using standard procedures as outlined by Westerfield (1993). Cadmium chloride (CdCl2, Purity N 99%) and potassium dichromate (K2Cr2O7, Purity N 99%) were purchased from Sigma-Aldrich. Stock solutions for both metals were prepared by dissolving in pure water. 2.2. Exposure experiments and sample collection To determine the hatchability affected by Cd or Cr, forty embryos were selected and exposed to 100 mL of 1, 3, 10 μM Cd and 3, 10, 30 μM Cr, respectively, in glass beakers (size: 250 mL), with four replicates for each Cd or Cr treated concentration (n = 4). At 60 and 96 h post fertilization (hpf), the hatched larvae were counted in each treated group, and the hatching rate was analyzed by the ratio of hatched numbers/total exposed numbers × 100. The abnormal and dead zebrafish embryos and larval were monitored at the points of 96 hpf exposure to Cd or Cr. To determine the locomotion affected by Cd or Cr, fertilized eggs were exposed to 1, 3, 10 μM Cd and 3, 10, 30 μM Cr in a 96-well plate (1 egg per well), respectively, for 96 h. Ten eggs were designed as each Cd or Cr treated group, with four replicates for each concentration. Before locomotion analysis, the exposure solutions were changed to clean water. To determine the antioxidant enzyme activities and their related gene expressions induced by Cd and Cr, zebrafish embryos were exposed to 1, 3, 10 μM Cd or 3, 10, 30 μM Cr for 96 hpf. Briefly, sixty fertilized eggs were selected and exposed to 200 mL of each of the above solutions in glass beakers (size: 500 mL), with four replicates for each Cd and Cr concentration (n = 4). After the exposure, about 30 larval zebrafish from each treatment group were collected as one sample for determining malondialdehyde (MDA) and glutathione (GSH) contents, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione S-transferase (GST) enzyme activities, and about 15 larval zebrafish from each beaker were collected as one sample for qRT-PCR analysis (n = 4). In all experiments, water was dechlorinated and filtered through activated carbon prior to use. The eggs were transferred to various exposure chambers containing Cd or Cr with indicated concentrations at least 60 min after the initial spawning. Non-fertilized eggs were separated from the fertilized ones using a pipette. Control embryos were exposed to water without any addition of Cd or Cr. In all experiments, the exposure solutions were changed daily. Incubation was carried out

at ambient temperature (28 ± 1 °C) with 14 h light/10 h dark cycles in a constant temperature–light incubator (Laifu, Ningbo, China). 2.3. Locomotion analysis Embryos were exposed to Cd or Cr in a 96-well (1 fish per well) plate for 96 hpf and then exposure water was changed with clean fresh water. Free swimming activities during 30 min visible light and larval swimming in response to a 10 min light-to-dark photoperiod stimulation were recorded after exposure of the larvae to various concentrations of Cd or Cr for 96 hpf. The tested larval fish were monitored with the Zebralab Video-Track system (ViewPoint Life Science, France) equipped with a PointGray IEEE-1394 camera (Model GRAS-03K2M-C, 30 fps) and an infrared filter. The entire record hardware is linked to the computer control program and kept isolated from lab environment in a sealed opaque plastic box (ViewPoint Life Science, France). 2.4. Determination of MDA, GSH contents and the activities of SOD, CAT, GPX and GST The 30 larvae fish from each beaker were defrosted and homogenized on ice with 180 μL ice-cold physiological saline. The homogenate was centrifuged at 4000 ×g at 4 °C for 15 min to obtain the supernatant. The supernatant was used to assay for MDA and GSH contents, in addition to SOD, CAT, GPX and GST activities, using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to previous studies (Liu et al, 2008; Jin et al., 2010b, 2011). Briefly, the SOD activity assay was based on its ability to inhibit the oxidation of oxymine by O− 2 produced by the xanthine–xanthine oxidase system. The red product (nitrite) produced by the oxidation of oxymine had absorbance at 550 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of the absorbance at 550 nm in 1 min per mg protein in the fish. GPX activity was determined by quantifying the oxidizing rate of reduced GSH to oxidized glutathione (GSSG) by using the H2O2 catalyzed by GPX. A yellow product, which had absorbance at 412 nm, could be formed as GSH reacted with dithiobisnitrobenzoic acid. One unit of GPX was defined as the amount that reduced the GSH level by 1 μmol/L in 1 min per mg protein in the fish. One unit of CAT activity was defined as the amount of enzyme required to consume 1 μmol H2O2 in 1 s and was expressed as U·mg/protein. GST activity was measured spectrophotometrically by the standard substrate, 1-chloro2,4-dinitrobenzene (CDNB) conjugated with GSH. The lipid peroxidation products (measured as MDA) were quantified using the thiobarbituric acid (TBARS) method, and the MDA concentration was expressed as nmol·mg/protein. Protein concentrations were determined using bicinchoninic acid (BCA) as a detection reagent for Cu+ following the reduction of Cu2+ by protein in an alkaline environment (BCA protein kit, Sangon company, China). Measurements were made on a microplate reader according to the manufacturer's instructions (Power wave XS, Bio-TEK, USA). 2.5. Gene expression analysis Total RNA was isolated from the 15 zebrafish larvae using TRIzol reagent (Takara Biochemicals, Dalian, China) according to the manufacturer's protocol. Subsequently, cDNA was synthesized using the M-MLV reverse transcriptase kit (Toyobo, Tokyo, Japan). Oligonucleotide primers were used to detect the gene expression of β-actin, Sod1, Sod2, Cat, Gpx, TNFα, IL-1β and IL6 using the SYBR green system (Toyobo, Tokyo, Japan); detailed information on the primers used can be read in previous reports (Jin et al., 2010a,b). As a housekeeping gene, β-actin transcript was used to standardize the results by eliminating variations in mRNA and cDNA quantity and quality. The qPCR was preformed with Eppendorf MasterCycler® ep realPlex2 (WesselingBerzdorf, Germany). The main protocols were described as follows: denaturation for 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and

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1 min at 60 °C. The relative quantification of gene expression among the different groups was analyzed according to previous reports (Livak and Schmittgen, 2001). 2.6. Data analysis The data were checked for normality and homogeneity of variance using the Kolmogorov–Smirnov one-sample test and Levene's test, respectively. Intergroup differences among the concentration levels were assessed by a one-way analysis of variance analysis of variance (ANOVA) followed by Dunnett's post hoc test. The F and P values were provided in each figure. The data of behavior were further evaluated by Tukey's multiple-comparison testing. The data analysis was performed using SPSS 19.0 (SPSS, Chicago, IL, USA). Values were considered statistically significant when p was less than 0.05 or 0.01. 3. Results 3.1. Effect of Cd and Cr on the hatchability The hatchability was significantly decreased by the exposure to 10 μM Cd for both 60 and 96 h (Fig. 1A and C). No significant difference in the hatchability was observed between any Cr treated group and the control group when exposed for both 60 and 96 h (Fig. 1B and D). Moreover, some abnormalities such as crooked bodies were also observed in the 10 μM Cd treated group (Fig. 1E). 3.2. Effect of Cd and Cr on behavior of larvae As shown in Fig. 2A, the free swimming speeds were not affected by Cd treatment. The free swimming speeds in Cr treated groups decreased, with a decrease by 14.1%, 22.2% and 41.5% after the treatment

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of 3, 10 and 30 μM Cr groups when compared with that of the control group, respectively (Fig. 2 B). The free swimming speeds in 30 μM Cr treated group was also significant lower than that in 3 μM Cr treated group (Fig. 2 B). Locomotion activities of larvae after exposure to Cd or Cr for 96 h were further assessed by alternating dark–light photoperiod stimulation. Both average swimming speed and distance of normal larvae in the tested 10 min could be significantly reduced or increased by a rapid transition from dark to light or by a reverse transition from light to dark, respectively. These behavioral changes in response to the dark–light stimulation disappeared after the treatment with Cd, and the average swimming speed and distance in the dark period were much lower than that in the control group (Fig. 2C and E). On the contrary, Cr treatment did not influence the behavioral response to the dark–light stimulation, except that the average swimming speed and distance of the larvae in 30 μM Cr treated group were lower than that of the control group during the 10 minute light period (Fig. 2D and F). The continuous changes in the swimming distance also indicated that Cd exposure blocked the behavioral response of the larvae to the dark–light shift (Fig. 2G) and the treatment with the high concentration Cr decreased the activity of the larvae in the light period (Fig. 2 H). 3.3. Effect of Cd and Cr on lipid peroxidation and GSH content The results in Fig. 3 illustrated that the MDA levels in the larval zebrafish increased significantly after the exposure to various concentrations of Cd or Cr for 96 h. The MDA levels increased significantly in all the 1, 3 and 10 μM Cd treatments, being about 1.5-, 1.8- and 2.5fold higher than that of the control fish, respectively (Fig. 3A). The MDA contents in the larvae also increased significantly by 1.6-, 2.1and 2.2-fold after the treatment with 3, 10 and 30 μM Cr for 96 h, respectively, as compared to that of the control group (Fig. 3 B).

Fig. 1. The hatchability in larval zebrafish after exposure to various concentrations of Cd and Cr for 60 and 96 hpf (A–D) and some of the abnormalities in larval zebrafish exposed to 10 μM Cd for 96 hpf (E). Data are expressed as the mean ± SEM of four replicates (40 eggs per replicate). The asterisk represents a statistically significant difference when compared with the controls; * at p b 0.05 level and ** at p b 0.01 level.

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Fig. 2. Locomotor behavior of the larval zebrafish after exposure to various concentrations of Cd and Cr for 96 hpf. (A and B) Free swimming speed during 30 min visible light; (C, D, E and F) Average swimming speeds and distances of 10-min intervals for each light state (dark or light). Data are expressed as the mean ± SEM of four replicates (10 larvae per replicate). Different letters above the adjacent bars indicate a significant difference (p b 0.05) among the different groups, whereas the same letter indicates no significant difference. Continuous locomotor traces in swimming distance of 10-min intervals for each light state (dark or light) (n = 40).

Correspondingly, the GSH contents decreased significantly in the larvae after the exposure to various concentrations of Cd for 96 h, being about 27.4%, 39.5% and 53.1% lower in the 1, 3 and 10 μM Cd treated groups

than that of the control, respectively (Fig. 3 C). The GSH content in the 30 μM Cr treated group also decreased significantly when compared to that of the control group (Fig. 3D).

Fig. 3. Effects of the Cd and Cr exposure on MDA (A and B) and GSH (C and D) contents in the larval zebrafish. Embryonic zebrafish was exposed to various concentrations of Cd or Cr for 96 hpf. Data are expressed as the mean ± SEM of four replicates (about 30 larvae per replicate). The asterisk represents a statistically significant difference when compared with the controls; * at p b 0.05 level and ** at p b 0.01 level.

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3.4. Effect of Cd and Cr on antioxidant enzymes The activities of the antioxidant enzymes including SOD, CAT, GPX and GST affected by Cd or Cr exposure in the larvae zebrafish were shown in Fig. 4. The SOD activities increased significantly in 1 and 3 μM Cd treated groups, while no significant induction was observed in the 10 μM Cd treated group (Fig. 4A). On the contrary, the SOD activities with the Cr treatment increased, being 1.7- and 2.6-fold higher in 10 and 30 μM Cr treated groups than that of the control group (Fig. 4B). As for the activities of CAT, no significant increase was observed both in Cd and Cr treated group (Fig. 4C and D). The activities of GPX decreased significantly in the larvae after the treatment with both Cd and Cr for 96 h (Fig. 4E and F), being significant lower in the 1, 3, 10 μM Cd and 10, 30 μM Cr treated groups, as compared to that of the control group. As for the GST activity, it increased significantly only in 10 μM Cd treated group, being 37-fold higher than the control group (Fig. 4G). While in the Cr treated groups, a significant increase in the GST activity was observed between the 10 μM treated group and the control group (Fig. 4 H). 3.5. Effect of Cd and Cr on the transcription of genes related to oxidative stress Transcriptional changes of oxidative stress related genes including Cu/Zn-Sod, Mn-Sod, Cat and Gpx were investigated in the larval zebrafish after the exposure to various concentrations of Cd and Cr for 96 h (Fig. 5). Generally, the mRNA levels of Cu/Zn-Sod and Mn-Sod increased, being induced significantly in all Cd and Cr concentration-treated

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groups (Fig. 5A, B, C and D). The mRNA levels of Cat in the larval zebrafish in all Cd and Cr treated groups were higher than that in the control group, while the level in 10 μM Cd or 30 μM Cr treated group was lower than those in two relative lower concentration-Cd or Cr treated groups, respectively (Fig. 5E and F). With respect to the mRNA levels of Gpx, significant increases were observed in all the Cd and Cr treated groups (Fig. 5G and H).

3.6. Effect of Cd and Cr on the transcription of genes related to the innate immune system As shown in Fig. 6, the effects of Cd and Cr on the transcription status of TNFα, IL6 and IL1β in the larval zebrafish were dependent on the exposure concentrations. Generally, the expression levels of these cytokines in the larvae increased significantly after the exposure to Cd or Cr, and they were significantly lower in the high concentration-Cd and Cr treated groups than those in the low and middle concentrations. For example, the mRNA levels of TNFα, IL6 and IL1β in the larvae increased significantly after the treatment with 1, 3, 10 μM Cd as compared with that of the control, while they decreased significantly among three Cd treated groups (Fig. 6A, C and E). The transcriptional changes of TNFα, IL6 and IL1β in the larval zebrafish in response to Cr challenge were similar to those in response to Cd (Fig. 6B, D and F). The mRNA levels of TNFα and IL6 increased significantly after the exposure to 3 and 10 μM Cr, while the corresponding levels in 30 μM Cr treated group had no significant difference in comparison with the control group (Fig. 6B and D).

Fig. 4. Effects of the Cd and Cr exposure on the activities of SOD (A and B), CAT (C and D), GPX (E and F) and GST (G and H) in the larval zebrafish. Embryonic zebrafish was exposed to various concentrations of Cd or Cr for 96 hpf. Data are expressed as the mean ± SEM of four replicates (about 30 larvae per replicate). The asterisk represents a statistically significant difference when compared with the controls; * at p b 0.05 level and ** at p b 0.01 level.

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Fig. 5. Expression of Cu/Zn-Sod (A and B), Mn-Sod (C and D), Cat (E and F) and Gpx (G and H) in larval zebrafish exposed to various concentrations of Cd or Cr for 96 hpf. Values were normalized against β-actin (used as a housekeeping gene) and represent the mean mRNA expression value ± SEM (n = 4) relative to those of the controls (about 15 larvae per replicate). The asterisk represents a statistically significant difference when compared with the controls; * at p b 0.05 level and ** at p b 0.01 level.

4 . Discussion Heavy metals have the potential to induce toxicological effects, as having been suggested by both in vivo and in vitro tests in a number of previous reports. Cd and Cr had been reported to exert deleterious effects in terms of nephrotoxic-, genotoxic-, immunotoxic- and even carcinogenic-effects (Risso-de-faverney et al, 2001; Riggio et al, 2003; Kumar and Singh, 2010; Jomova and Valko, 2011; Zhang et al, 2012; Abreu et al, 2014). Because the metabolic rate and elimination of heavy metals are very slow in fishes, the outcomes induced by Cd and Cr may be permanent for zebrafish (Kusch et al., 2007). In this study, we utilized zebrafish as a model system to investigate the toxic effects of Cd and Cr in early developmental stage. In fact, the embryo and developmental toxicity have been observed in zebrafish embryos exposed to Cd or Cr (Blechinger et al, 2002; Chan and Cheng, 2003; Chow et al, 2009; Domingues et al, 2010). In previous studies, the observed concentrations that caused developmental toxicity in zebrafish ranged form 0.5 to more than 10 mg/L for Cd and more than 7.5 mg/L for Cr at different endpoints (Dave et al., 1987; Hallare et al., 2005). In accordance with previous studies, our findings demonstrated that the hatching rates decreased significantly by exposure to 10 μM Cd (about 1.12 mg/L) for 60 and 96 hpf, and increased significantly by exposure to Cr for 60 hpf (Fig. 1A). And some abnormal dead embryos and larvae were also observed especially in the 10 μM Cd treated group (Fig. 1B). In addition, the locomotor behaviors changed significantly in the larval zebrafish

after the exposure to Cd and Cr (Fig. 2). Moreover, the main parameters related to the status of the oxidative stress and the innate immune system were also affected significantly by Cd and Cr exposure in early developmental stage of zebrafish (Figs. 3–6). All these results indicated that Cd and Cr not only affected the behavior but also induced serious oxidative stress and immunotoxicity in the larval fish. The changes in the spontaneous movement and swimming speed by the dark–light stimulation in the normal zebrafish were observed in this study, being as similar as those of previous reports (Eddins et al, 2010; Chen et al., 2012a,b). In fact, the behavioral alterations influenced by Cd, Cr or other heavy metals had been reported previously in other different adult fish species (Olojo et al, 2005; Begum et al, 2006; Mishra and Mohanty, 2008). To our knowledge, this is the first time to compare the abnormal behavior induced by Cd and Cr in larval zebrafish. In this study, the different endpoints in the behavior of the larval zebrafish were obviously influenced by Cd and Cr (Fig. 2). In fact, a number of studies indicated that the neurotoxicity induced by environmental chemicals was tightly linked to the behavioral alterations in the early developmental stage of zebrafish (Chen et al., 2012a,b). Moreover, the neurotoxicity and behavior alteration induced by Cd had been reported in rat (Desi et al, 1998; Kusch et al, 2007) and zebrafish (Chow et al, 2008). As for Cr, a previous study also indicated that the activities of AChE presented a dose-dependent inhibition in larval zebrafish when exposed to 21, 49, 75 and 116 mg/L Cr (VI) for 96 h, and the percentages of larvae with behavioral anomalies were significantly increased at all Cr

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Fig. 6. Expression of TNFα (A and B), IL6 (C and D) and IL1β (E and F) in larval zebrafish exposed to various concentrations of Cd or Cr for 96 hpf. Values were normalized against β-actin (used as a housekeeping gene) and represent the mean mRNA expression value ± SEM (n = 4) relative to those of the controls (about 15 larvae per replicate). The asterisk represents a statistically significant difference when compared with the controls; * at p b 0.05 level and ** at p b 0.01 level.

treated concentrations (Domingues et al, 2010). As for our results, it was possible that the differences in the behavioral alterations between Cdand Cr treatments might represent their different potentials in inducing the neurotoxicity, although no direct evidence is available in this study. In addition, a previous study reported that zebrafish embryonic Cd exposure led to abnormal somite patterning of the muscle fibers and defects in axonogenesis (Chow and Cheng, 2003). Oxidative stress has become an important subject in aquatic toxicity (Atli et al, 2006; Lushchak, 2011), and environmental chemicals, including heavy metals, may be directly involved in this process. Some published data proved that heavy metals could induce oxidative stress in zebrafish and other aquatic test models. For example, Blechinger et al. (2007) reported that acute Cd exposures (from 80 to 83 hpf to 0.2, 5, or 125 μM) lead to the activation of cell death pathways in the larval olfactory epithelium and more permanent disturbances in olfactory function in juvenile fish at 50 dpf. Lushchak et al. (2009) evaluated the effects of Cr3 + exposure at 1, 25, 5 or 10 mg/L for 96 h on oxidative stress and antioxidant defense of goldfish liver and kidney, and observed that Cr3+ exposure decreased total GSH levels in the liver and kidney. Kubrak et al. (2010) reported that the activities of SOD, CAT, GST, and glutathione reductase (GR) activities were altered in the brain, liver, kidney and gills of goldfish after the exposure to 10 mg/L Cr6+ and Cr3+ over 24, 48 and 96 h. Wang et al. (2011) found that in testis of freshwater crabs exposed to 725, 145, 29, 58 and 116 mg/L Cd for 7 days, the activities of SOD, GPX and CAT initially increased and subsequently decreased with increasing Cd concentrations, which was accompanied with the increase in MDA and H2O2 contents in a concentration dependent manner. Hsu et al. (2013) reported that the MDA contents increased significantly in zebrafish embryos after the treatment with 1, 3, 5 and 9 μM CdCl2 for 9 h. In agreement with these data, in the present study, we found that Cd- and Cr-induced oxidative stress affected several processes in the larval zebrafish. The MDA levels increased and the GSH contents decreased significantly in the larvae zebrafish exposed to various concentrations of Cd or Cr for 96 hpf, indicating that Cd and Cr induced serious oxidative damage in the larval

zebrafish. In addition, the treatment with Cd or Cr affected not only the activities of SOD, CAT, GPX and GST, but also the transcriptional levels of their respective genes. Fish have unique systems for protecting themselves against the oxidative damaging effects by evaluating the antioxidant enzymes (Valavanidis et al, 2006; Jin et al, 2010b). They can combat the elevated oxidative stress in their systems with antioxidant enzymes such as SOD and CAT, which convert superoxide anions (O2•−) into H2O2 and then into H2O and O2. Thus, the increases of SOD and CAT could be explained by the self-protection mechanism to antioxidative stress in fish (Valavanidis et al, 2006). In addition, according to a previous study, GSTs are also modulated in response to Cd and Cr exposure in river pufferfish (Kim et al, 2010) and zebrafish (Domingues et al, 2010). A surprising result was that the activities of GPX decreased, while its mRNA level increased significantly in all the Cd and Cr treated larvae. The GPX mainly catalyzes the reduction of H2O2, but at the expense of GSH (Liu et al, 2008). We thought that the decrease in the activity of GPX may be due to GSH depletion induced by Cd and Cr (Dudley and Klassen, 1984). Thus, it is possible that the increase of the transcriptional levels of Gpx is to compensate the decrease of GPX in the Cd or Cr treated larval fish. Taken together, these changed parameters in the larval zebrafish are closely associated with the oxidative stress induced by Cd and Cr. The main cytokines such as TNFα, ILs secreted by immune cells are vital in modulating the amplitude of an immune response. A number of previous reports proved that bacterial, viral or some environmental chemicals could induce or inhibit the mRNA expression of the innate immune-related cytokines in different fish species (Jin et al, 2010a; Phelan et al, 2004; Eder et al, 2008). For example, a previous study indicated that a chronic exposure (30 days) to 0.5 μM As2O3 led to significant increase in arsenic content in the head kidney accompanied by a reduction in head kidney macrophage numbers in a fish, Clarias batrachus. And the production of IL-1β from the head kidney macrophage was suppressed by the chronic exposure to arsenic (Datta et al, 2009). Although increasing numbers of researches have focused on the immunotoxicity arising from heavy metals, the reports on fish

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were still very limited. Sanchez-Dardon et al. (1999) reported that the parameters of the nonspecific cellular immune response (phagocytosis, respiratory burst, and lymphoblastic proliferation) and of the nonspecific humoral immune response (lysozyme activity and the level of immunoglobulin) were affected in the rainbow trout exposed to CdCl2 and HgCl2 for 30 days at environmentally relevant concentrations. Notch et al. (2011) found that cyanobacterial lipopolysaccharides (LPS) potentiated Cd toxicity in zebrafish embryos. Recently, Leite et al. (2013) observed that the mRNA levels of TNFα, IL1β and COX-2 increased significantly in the larval zebrafish after exposure to 10 μM copper for 4 and 24 h. In the present study, we discovered that the mRNA levels of TNFα, IL6 and IL1β were elevated in the larval zebrafish after the exposure to various concentrations of Cd and Cr, reflecting that Cd and Cr exhibited their effects on the immune system response in the early development of zebrafish. However, in the highest concentration of Cd and Cr treated groups, the corresponding mRNA levels of TNFα, IL6 and IL1β were much lower than those in the low and middle concentrations (Fig. 6). Thus, we thought that the innate immune function in early development stage of zebrafish was lightly linked to the exposure concentrations to Cd or Cr. Given the widespread use of Cd and Cr in the world, coupled with their generally water-soluble properties, there is need for further research to elucidate their effects and underlying mechanisms on aquatic species. Thus, a comprehensive investigation of their effects on the different endpoints is likely to be a key to understand the mechanisms of overall metals toxicity in fish. In the present study, we observed that the main parameters related to the behavior, oxidative stress and the innate immune system in the larval zebrafish were significantly influenced by Cd and Cr, suggesting that Cd and Cr induced multi-toxic effects on zebrafish. All the information presented in this study will help elucidate the possible mechanism of metal-induced developmental neurotoxicity in fish. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgments This work was supported by the National Natural Science Foundation of China (21277128) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13096). References Abreu PL, Ferreira LMR, Alpoim MC, Urbano AM. Impact of hexavalent chromium on mammalian cell bioenergetics: phenotypic changes, molecular basis and potential relevance to chromate-induced lung cancer. Biometals 2014;27:409–43. Alghasham A, Salem TA, Meki AM. Effect of cadmium-polluted water on plasma levels of tumor necrosis factor-α, interleukin-6 and oxidative status biomarkers in rats: Protective effect of curcumin. Food Chem Toxicol 2013;59:160–4. Atli G, Alptekin Ö, Tükel S, Canli M. Response of catalase activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus. Comp Biochem Physiol C Toxicol Pharmacol 2006;143:218–24. Bagchi D, Joshi SS, Bagchi M, Balmoori J, Benner EJ, Kuszynski CA, et al. Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. J Biochem Mol Toxicol 2000;14:33–41. Begum G, Venkateswara RJ, Srikanth K. Oxidative stress and changes in locomotor behavior and gill morphology of Gambusia affinis exposed to chromium. Toxicol Environ Chem 2006;88:355–65. Blechinger SR, Warren Jr JT, Kuwada JY, Krone PH. Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environ Health Perspect 2002; 110:1041–6. Blechinger SR, Kusch RC, Haugo K, Matz C, Chivers DP, Krone PH. Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish. Toxicol Appl Pharmacol 2007;224:72–80.

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