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Original Article Sodium Benzoate Exposure Downregulates the Expression of Tyrosine Hydroxylase and Dopamine Transporter in Dopaminergic Neurons in Developing Zebrafish Qian Chen, 1 Nan-nan Huang, 1 Jin-tao Huang, 1 Shen Chen, 1 Jinjin Fan, 2 Chaohong Li, 1 and Fu-kang Xie 1 1 Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong Province, China 2 Department of Nephrology, Key Lab of Kidney Disease of Education Ministry, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong Province, China BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B) 86:85–91, 2009. r 2009 Wiley-Liss, Inc. Key words: Sodium benzoate; Zebrafish embryo; Dopaminergic neurons; Neurotoxicity INTRODUCTION Sodium benzoate (SB), the sodium salt of benzoic acid, has been widely used as preservative in the food and beverage industries. Much work addressing its safety has been done since it was first used in 1911 as an artificial food additive. Using the protozoan Tetrahymena pyrifor- mis as a toxicological model, SB was found to stimulate the mitotic process and caused a statistically significant increase in DNA content, which substantially altered the susceptibility of the organism to chemical carcinogenesis (Stefanidou et al., 2003). Mouse, rat, and medaka studies with SB treatment indicated that there were no detectable tumorigenic effects, but mean rat pup weights and lengths were decreased by in utero exposure to SB (Sodemoto and Enomoto, 1980; Hatanaka et al., 1982; Toth, 1984; Mowafy et al., 2001). However, Tsay et al. recently reported that zebrafish (Danio rerio) larvae with SB treatment displayed significant defects in motor axons and neuromuscular junctions, thereby suggesting Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bdrb.20187 Grant sponsor: China Medical Board scholar program; Grant number: 98-677; Grant sponsor: Doctoral Foundation of Chinese Ministry of Education; Grant number: 20060558002; Grant sponsor: National Nature Science Foundation of China; Grant numbers: 39970237; 30570762; Grant sponsor: Nature Science Foundation of Guangdong Province; Grant number: 8151008901000044. Correspondence to: Prof. Fu-kang Xie or and Prof. Chaohong Li, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong Province 510080, China. E-mail: [email protected] Received 20 November 2008; Revised 16 January 2009; Accepted 16 January 2009 Birth Defects Research (Part B) 86:85–91 (2009) & 2009 Wiley-Liss, Inc.

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Original Article

Sodium Benzoate Exposure Downregulatesthe Expression of Tyrosine Hydroxylase and

Dopamine Transporter in Dopaminergic Neuronsin Developing Zebrafish

Qian Chen,1 Nan-nan Huang,1 Jin-tao Huang,1 Shen Chen,1 Jinjin Fan,2 Chaohong Li,1�

and Fu-kang Xie1�

1Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou,Guangdong Province, China

2Department of Nephrology, Key Lab of Kidney Disease of Education Ministry, First Affiliated Hospital, Sun Yat-Sen University,Guangzhou, Guangdong Province, China

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significantdevelopmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SBcan be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafishbrain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, duringwhich the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) inthe neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and thelocomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with theincrease of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment withSB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-daypost-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decreasein locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest thatSB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependentmanner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon,which results in decreased locomotor activity of zebrafish larvae. This study may provide some important informationfor further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B)86:85–91, 2009. r 2009 Wiley-Liss, Inc.

Key words: Sodium benzoate; Zebrafish embryo; Dopaminergic neurons;Neurotoxicity

INTRODUCTION

Sodium benzoate (SB), the sodium salt of benzoic acid,has been widely used as preservative in the food andbeverage industries. Much work addressing its safety hasbeen done since it was first used in 1911 as an artificialfood additive. Using the protozoan Tetrahymena pyrifor-mis as a toxicological model, SB was found to stimulatethe mitotic process and caused a statistically significantincrease in DNA content, which substantially altered thesusceptibility of the organism to chemical carcinogenesis(Stefanidou et al., 2003). Mouse, rat, and medaka studieswith SB treatment indicated that there were no detectabletumorigenic effects, but mean rat pup weights andlengths were decreased by in utero exposure to SB(Sodemoto and Enomoto, 1980; Hatanaka et al., 1982;Toth, 1984; Mowafy et al., 2001). However, Tsay et al.

recently reported that zebrafish (Danio rerio) larvae withSB treatment displayed significant defects in motoraxons and neuromuscular junctions, thereby suggesting

Published online in Wiley InterScience (www.interscience.wiley.com)DOI: 10.1002/bdrb.20187

Grant sponsor: China Medical Board scholar program; Grant number:98-677; Grant sponsor: Doctoral Foundation of Chinese Ministry ofEducation; Grant number: 20060558002; Grant sponsor: National NatureScience Foundation of China; Grant numbers: 39970237; 30570762;Grant sponsor: Nature Science Foundation of Guangdong Province; Grantnumber: 8151008901000044.

�Correspondence to: Prof. Fu-kang Xie or and Prof. Chaohong Li,Department of Histology and Embryology, Zhongshan School ofMedicine, Sun Yat-Sen University, Guangzhou, Guangdong Province510080, China. E-mail: [email protected]

Received 20 November 2008; Revised 16 January 2009; Accepted 16January 2009

Birth Defects Research (Part B) 86:85–91 (2009)& 2009 Wiley-Liss, Inc.

SB neurotoxicity (Tsay et al., 2007). However, whether SBaffects the development of the dopaminergic (DA)neurons in the zebrafish brain is still not known.

Dopaminergic (DA) neurons, one of the most inten-sively studied brain neurons, have attracted considerableattention because of their functional and medicalimportance. They control postural reflexes, initiation ofmovement, reward associated behavior, and hormonalhomeostasis. Degeneration of substantia nigra DAneurons in humans is a hallmark of Parkinson’s disease(PD), and the malfunction of DA neurons in other brainregions is implicated in psychiatric disorders andneuroendocrine dysregulation (Guo et al., 1999a; Smidtet al., 2003; Jeong et al., 2006). Accumulating data fromneuropathologic studies and animal models have shownthat a range of environmental neurotoxicants, such as1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),6-hydroxydopamine (6-OHDA), rotenone, and paraquat,can induce progressive degeneration of DA neurons inthe substantia nigra in mammals leading to the devel-opment of movement disorders (e.g., resting tremor,rigidity, bradykinesia, and gait disturbance) (Jellinger,2001; Bove et al., 2005; Landrigan et al., 2005; Logroscino,2005). To help determine whether the early developmentprocesses of DA neurons are impacted by SB, it would behelpful to develop a model in which the processes ofneurodevelopment can be more directly analyzed.Zebrafish offer such a model.

In recent years, the zebrafish has been widely used tostudy developmental biology, molecular genetics, humandisease modeling, and drug and toxicant analysis (Eisen,1991; Key and Devine, 2003; Carney et al., 2006; Kariet al., 2007; Lieschke and Currie, 2007; McGrath and Li,2008; Scholz et al., 2008). The advantages of this animalmodel include a short generation time, external fertiliza-tion, fast development, transparent embryos, and a largeand growing biological database (Westerfield, 2000; Hillet al., 2005; Zon and Peterson, 2005; Kari et al., 2007;Lieschke and Currie, 2007). Recent studies on theneurobiology of the zebrafish indicate that this is a goodmodel organism, which is not only well suited for thegenetic analysis of DA system development but also forreplicating neurodegenerative diseases such as PDbecause of its genotypic conservation, and morphologicaland physiological similarity to mammals (Guo et al.,1999a; Ma, 2003; Panula et al., 2006). In the present study,we examined the effect of SB on the development of DAneurons in zebrafish embryos and larvae, and relateddefects in locomotor activity. Results indicated signifi-cantly reduced expression of tyrosine hydroxylase (TH)and dopamine transporter (DAT) in DA neurons anddecreased locomotor activity in zebrafish larvae aftertreatment with SB. Our findings provide a further insightinto the neurotoxic effects of SB on early embryonicdevelopment.

MATERIALS AND METHODS

Animal Care And Maintenance

The wild-type zebrafish (AB strain) were obtainedfrom Pearl River Fisheries Research Institute (PRFRI) ofthe Chinese Academy of Fishery Sciences and main-tained as described previously with minor modifications(Westerfield, 2000; Chen et al., 2007; Tsay et al., 2007).

Briefly, the adult zebrafish were maintained at 281C on a14-h light and 10-h dark cycle under standard proce-dures. Zebrafish embryos were obtained through naturalmating and synchronously raised in the egg water(3.5 g/L Instant Ocean Salt, 0.1 g/L CaCl2, and 0.05 g/LKCl) at 28.51C. Zebrafish were staged by hours postfertilization (hpf) and days post fertilization (dpf) aspreviously described (Westerfield, 2000).

Drug Administration

Drug exposure was performed as described (McKinleyet al., 2005; Tsay et al., 2007) with the followingmodifications. Sodium benzoate (SB; Sigma-Aldrich, St.Louis, MO) was dissolved in distilled water to make a10-mg/ml stock solution. Newly fertilized embryos werecollected and randomly allocated into 24-well cultureplates at a density of 12 embryos per well (1 ml solutionfor each well), and all the solutions were changed daily.In the survival rate tests, 2-hpf zebrafish embryos weretreated with either egg water alone (untreated control) orwith SB (40–800 mg/ml) for up to 6 dpf. Subsequently,survival rates were counted every 24 h. In the neurotoxi-city tests, 2-hpf zebrafish embryos were treated in eitheregg water alone (untreated control) or with SB (40 or100mg/ml) for up to 3 dpf. At the end of exposure,embryos were collected for in situ hybridization andimmunofluorescence. To investigate whether the zebra-fish embryos and larvae with SB treatment displayeddifferences in their locomotor activity, embryos weresubdivided into the following groups according to thedifferent SB exposure times: the untreated control group(normally incubated up to 6 dpf), the first 3-day group(treated with 20 or 40mg/ml SB from 2 hpf to 3 dpf, andthen incubated for additional 3 days under normalconditions) and the latter 3-day group (normally in-cubated up to 3 dpf and then treated with 20 or 40mg/mlSB from 3 dpf to 6 dpf). Thereafter, all larvae were usedfor locomotor activity assessments. In a positive control,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP;Sigma-Aldrich) was dissolved in distilled water to makea 10-mg/ml stock solution. Afterwards, 2 hpf zebrafishembryos were treated in the egg water with MPTP(45mg/ml) up to 3 dpf. At the end of the exposure,embryos were collected for survival rates, in situhybridization, and immunofluorescence assays.

Whole Mount In Situ Hybridization

The cDNA sequence of zebrafish tyrosine hydroxylase(TH) has been deposited in GenBank (accession number:XM_682702). To generate a probe for detecting THexpression, a 537-bp cDNA fragment was amplifiedfrom 3 dpf embryonic total RNA by RT-PCR(primers: 50-TCCACCATCTTGAAACCAGACCCA-30 and50-GCCTCAACTGAAATCCTGTGCGTT-30). The PCRproducts were TA-cloned into pCRsII Topos (Invitro-gen, Carlsbad, CA) and confirmed by sequencing. Theconstructs were subsequently linearized with the restric-tion enzyme EcoRV (Takara, Kyoto, Japan) and anantisense riboprobe was transcribed with Sp6 RNApolymerase according to the manufacturer’s protocol(Roche Applied Science, Indianapolis, IN). In orderto make the dopamine transporter (DAT) probe, a760-bp fragment was amplified using the forwardprimer 50-TACCTGTGCTACAAGAATGGCGGA-30 and

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the reverse primer 50-TGATGGCGTCTCTGTAGCAG-TTGT-30, based on the published DAT cDNA sequence(GenBank accession number: AF318177). In situ hybridi-zation was carried out according to Guo et al. (1999b).Briefly, embryos were fixed in 4% paraformaldehyde/PBS and then digested with proteinase K. After that, theembryos were prehybridized for 4 h and then hybridizedovernight at 651C with digoxigenin-labeled antisenseprobes. Digoxigenin was detected with an anti-DIG-APFab fragment antibody (Roche). Finally, the embryoswere developed with NBT/BCIP reagents (Roche).

Whole Mount Immunofluorescence

Whole mount immunofluorescence was performed asdescribed previously (McLean and Fetcho 2004) withminor modifications. Briefly, the zebrafish embryos werefixed in 4% paraformaldehyde overnight at 41C. Theywere then rinsed thoroughly in PBS before beingdehydrated in a graded methanol series (10, 30, 50, 70,90, and 100%), permeabilized in acetone for 5–10 minutesat �201C. Nonspecific blocking was performed with aphosphate buffered solution (PBS) containing 0.5%Triton X-100 (TXPBS) and 5% normal goat serum (NGS)overnight at 41C. Embryos were then incubated in theTXPBS with primary antiserum containing mouse anti-tyrosine hydroxylase (1:500; Chemicon, Temecula, CA)for 3 days at 41C. After a thorough wash with TXPBS, theembryos were incubated in the TXPBS with secondaryantiserum (in TXPBS) containing goat anti-mouse con-jugated to Texas Red (1:200; Santa Cruz Biotechnology,Santa Cruz, CA) for 2 days at 41C and washed again withTXPBS.

Locomotor Activity Assay

Locomotor activity assays were performed as de-scribed with minor modifications (Boehmler et al.,2007). Group assignment and drug administration aredescribed in Drug Administration in the Materials andMethods section. After drug treatments, zebrafish larvae(6 dpf) were transferred into a clear 12-well tissue culturedish (one fish per well) containing 2 ml egg waterat 28.51C. Each well measured 25 mm� 20 mm(diameter�depth). The bottom of each well was dividedwith gridlines into four equal portions. Larvae wereallowed to habituate to the new environment for 5 min.The locomotor activity of zebrafish larvae was recordedwith digital video microscopy using a stereomicroscopeequipped with a video camera. Afterwards, the videowas analyzed using the Image-Pro Plus software (MediaCybernetics, Houston, TX). The locomotor activity wasquantitative determination by counting the number oftimes each larva crossed the line within 1 min. Data of thelocomotor activity of zebrafish larvae were then statisti-cally analyzed. Each dosage group was subjected to 24independent trials.

Imaging

In situ hybridization images were captured using aLeica DMIRB microscope equipped with a Leica DC300digital camera. Quantification of the neuronal area in theventral diencephalon of zebrafish embryos by wholemount in situ hybridization was done as previouslydescribed (McKinley et al., 2005). Briefly, the periphery of

each neuronal group was outlined by manually tracingthe edge. The area of each enclosed region wasmeasured, and the subsections were summed to givethe total neuronal area. Fluorescence signals weredetected using a Zeiss LSM 510 META laser scanningconfocal microscope. Serial optical sections were taken,and 3-D images were reconstructed from the stackedconfocal images by the supplied software.

Statistical Analysis

The comparison of survival rates between the un-treated control and the SB-treated groups was analyzedby multivariate ANOVA. SB-treatment effects on dopa-minergic neurons and locomotor activity were firstrevealed via one-way ANOVA and subsequently LeastSignificant Difference (LSD) t-tests were conducted todetermine significant differences between individualtreatments. SPSS 13.0 package for Windows (SPSS Inc.,Chicago, IL) was used to conduct statistical analyses. Asignificance level of Po0.05 was used in all statisticalanalyses. Each experiment was repeated at least threetimes and was carried out in triplicate.

RESULTS

SB Decreased Survival Rates of ZebrafishEmbryos

The impact of sodium benzoate (SB) on the survival of2-dpf zebrafish embryos was reported recently (Tsayet al., 2007). In our study, the toxicity of SB during thedevelopment was examined in 2-hpf embryos that wereexposed to various concentrations of SB (0, 40, 100, 200,400, and 800 mg/ml) for different durations and thesurvival rates were recorded every 24 h as shown inFigure 1. Compared to untreated clutch mates (untreatedcontrols), 40mg/ml SB treatment did not significantlydecrease the survival rates of embryos at variouslyindicated stages (P40.05). However, the survival ratessignificantly decreased as the duration of exposure anddosage of SB (from 100 to 800 mg/ml) increased. Forinstance, after exposure to 100mg/ml SB, the survivalrates significantly decreased daily to 93.0675.19%,88.8976.42%, 87.5076.75%, 55.56710.14%, and30.5679.40% when embryos were treated for 24, 48, 72,96, and 120 hpf, respectively. After 144 hpf of exposure to100mg/ml SB, no living larvae remained. In addition,after 48 hpf of exposure, the survival rates significantly

Fig. 1. Effects of various concentrations of SB on the survival ofzebrafish embryos at different development stages. Datarepresent the mean7standard error of five separateexperiments.

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decreased from untreated controls (98.6172.39% to90.2876.05%, 88.8976.42%, 86.1177.06%, and44.44710.14% corresponding to treatments with 40,100, 200, and 400mg/ml of SB, respectively). No embryossurvived upon exposure to 800 mg/ml or more SB. Basedon these results, we concluded that SB-induced embryo-nic death is time and dose dependent.

SB Downregulated the Expression of TH andDAT in DA Neurons in Zebrafish Embryos

SB-induced defects in motor neuron axons andneuromuscular junctions in zebrafish larvae have beenpreviously reported, thereby implying SB’s neurotoxicity(Tsay et al., 2007). To explore whether SB affected thedevelopment of the dopaminergic (DA) neurons in thezebrafish brain, 2-hpf embryos were treated with SB (40and 100 mg/ml) and 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP, 45 mg/ml, positive control) up to3 dpf. The expression of tyrosine hydroxylase (TH) in the

DA neurons of the ventral diencephalon of zebrafishembryos was detected by in situ hybridization andimmunofluorescence assays. As shown in Figure 2,results from in situ hybridization demonstrated thatseveral clusters of TH-positive neurons were observed inthe ventral diencephalon of zebrafish embryos ofuntreated controls at 3 dpf. These cells could besubdivided into the following two groups: the anteriorgroup of the paraventricular organ (PVO) and theposterior group of tubercular nuclei (PTN), which wereseparated by a gap (Fig. 2A–a). However, embryosexposed to various dosages of SB (40 and 100mg/ml)up to 3 dpf significantly downregulated the expression ofTH gene in neurons in the ventral diencephaloncompared to untreated controls (10077.2% to51.4274.1% and 36.7874.59%, corresponding to treat-ments with 40 and 100mg/ml SB, respectively; po0.001,compared with control) (Fig. 2A–b, c, C). In addition,compared to the effect of SB 40 mg/ml, the effect of SB100mg/ml was significantly more severe (po0.001). In the

Fig. 2. Effects of SB on TH-expressing neurons in zebrafish embryos. A: Comparison of the lesion effects by SB with MPTP via whole-mount in situ hybridization detecting TH mRNA expression in zebrafish embryos at 3 dpf. a: Untreated control. Embryos treated with(b) 40mg/ml or (c) 100mg/ml SB. d: Embryos treated with 45mg/ml MPTP. a: Views of the expression of TH-mRNA in the ventraldiencephalon (circled). The TH-expressing neurons in the ventral diencephalon can be divided into the two following groups: theanterior group of the paraventricular organ (PVO) and the posterior group of posterior tubercular nuclei (PTN), and there is a gap (blackarrow) between them. b: The TH-expressing neurons in the diencephalon decreased after exposure to 40mg/ml of SB. c: The damage toTH-expressing neurons became more severe after exposure to 100mg/ml SB. d: The application of 45 mg/ml MPTP produced similarimpairments on TH-expressing neurons in the diencephalon similar to SB. All embryos are in the dorsal view, anterior to the top.Bar 5 50mm. B: Compare the lesion effects of SB with MPTP by whole-mount immunofluorescence detection of the TH proteinexpressed in zebrafish embryos at 3 dpf. a: Untreated control. Embryos treated with (b) 40 mg/ml or (c) 100mg/ml SB. d: Embryos treatedwith 45 mg/ml MPTP. Composite photos were made from stacked confocal images, focusing on the catecholaminergic neurons of theventral diencephalon. Bar 5 20 mm. C: Statistical data derived from A showing the area of TH-positive neurons in the ventraldiencephalon of 3-dpf zebrafish embryos treated with 40 or 100mg/ml SB, and 45mg/ml MPTP (n 5 16). Data are presented asmean7standard error. ��Po0.001 compared with control, Po0.001 compared with embryos treated with 100mg/ml SB.

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positive controls, MPTP (45mg/ml) induced significantlydownregulated the expression of TH gene in neurons inthe ventral diencephalon (po0.001, compared withcontrol) (Fig. 2A–d, C). Consistent with the expressionpattern of TH transcripts, SB caused significant decreasesin the TH protein within the diencephalic neurons asdetected by immunofluorescence staining (Fig. 2B).

It has been reported that TH expression is widespreadamong catecholaminergic (CA) neurons including dopa-minergic (DA), noradrenergic (NA), and adrenergicneurons (Holzschuh et al., 2001). In contrast, thedopamine transporter (DAT, a membrane transportprotein involved in dopamine reuptake) was found tobe exclusively expressed in dopaminergic terminals.Thus, DAT expression has been used to distinguish DAneurons from other CA neurons in the zebrafish(Holzschuh et al., 2001). To investigate whether theobserved SB-induced decrease of TH expression inneurons was due to decrease of TH expression in DAneurons, a DAT RNA probe was used to detect thepopulation of DA cells in the diencephalon of zebrafishembryos. As shown in Figure 3, an SB-induced reductionof DAT expression in the neuronal area of embryos wassignificantly observed (untreated control of 10078.8%vs. 56.4478.8%, 41.1373.9%, and 48.8377.3% corre-sponding to treatments with 40, 100 mg/ml SB, and45 mg/ml MPTP, respectively; po0.001, compared withcontrol). And the effect of SB 100 mg/ml was alsosignificantly more severe than that of SB 40 mg/ml(p 5 0.002). This result was consistent with observed SB-induced decreases in expression of TH, further implyingthat SB has effects on the neurotoxicity during thedevelopment of zebrafish embryos.

SB Decreased the Locomotor Activity ofZebrafish Larvae

Several methods have been established to detectzebrafish motor changes by monitoring swimming

behavior in response to a tactile stimuli (Lam et al.,2005) or the distance traveled in a given time period(Bretaud et al., 2004; Boehmler et al., 2007). To investigatewhether the zebrafish embryos and larvae subjected toSB treatment displayed differences in locomotor activity,embryos were subdivided into three groups as describedin Materials and Methods. SB was used at 20 and40mg/ml because less than 40mg/ml SB did not causeany survival abnormalities in the zebrafish larvae duringthe experimental period. As shown in Figure 4, SBtreatment caused a significant reduction of locomotoractivity in zebrafish larvae (po0.001, compared withcontrol). In addition, when the embryos were treated atthe same dosage of SB, the earlier treatment was moreeffective than the later (po0.001). These results indicatedthat SB treatment impaired locomotor activity in zebra-fish larvae and early embryos were more sensitive thanlarvae in their responses to decreased locomotor activityinduced by SB treatment.

DISCUSSION

In this study, we investigated the neurotoxic effects ofSB on early zebrafish embryonic development. Ourresults show that SB treatment induced significantdecreases in the survival rates of zebrafish embryos ina time- and dose-dependent manner. SB also resulted insignificant decreases in the expression of both TH andDAT in DA neurons in the embryonic ventral dience-phalon and locomotor activity in zebrafish larvae. Theseresults were in good agreement with what was pre-viously observed in MPTP-treated zebrafish (Bretaudet al., 2004; Lam et al., 2005; McKinley et al., 2005; Wenet al., 2008). To our knowledge, this is the first evidenceto show that SB can significantly decrease the expressionof TH and DAT in DA neurons in the ventral dience-phalon of zebrafish embryos.

Fig. 3. Effects of SB on DAT-expressing neurons in zebrafish embryos. (A) Comparison of the lesion effects of SB with MPTP by whole-mount in situ hybridization detecting DAT mRNA expression in the ventral diencephalon of 3 dpf zebrafish embryos. (a) Untreatedcontrol. Embryos treated with (b) 40 mg/ml or (c) 100mg/ml SB. (d) Embryos treated with 45 mg/ml MPTP. All embryos are in the dorsalview, anterior to the top. Bar 5 50mm. (B) Statistical data derived from panel A showing the area of DAT-positive neurons in the ventraldiencephalon of 3 dpf zebrafish embryos treated with 40 mg/ml or 100mg/ml SB, and 45mg/ml MPTP (n 5 17). Data are presented asmean 7 standard error. ��Po0.001 compared with control, Po0.01 compared with embryos treated with 100mg/ml SB.

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A growing number of reports have indicated that SBhas multiple applications for food chemical and biome-dical research. Thus, the biosafety of SB is an importantissue that should be discussed. Although the acceptabledaily intake of SB in adults was established by the WorldHealth Organization at 5 mg/kg/day (B5 ppm), the safedosages for embryonic development are still relativelyunknown, even in higher vertebrates. It has recently beennoted that the amount of SB preservative in the dietinfluences children’s behavior, subsequently leadingto increased hyperactivity in 3- and 8/9-year-oldchildren in the general population (Bateman et al., 2004;McCann et al., 2007). However, whether SB exposure caninduce pathophysiological changes in human embryos orchildren has not been fully elucidated. Some reports havedemonstrated that there were no significant increases infetal abnormalities in pregnant hamsters, mice, rats, andrabbits that were dosed with 20–250 mg/kg SB (Nair,2001; Andersen, 2006). However, the report by Tsay et al.indicated that SB could induce significant decreases inthe survival rate and increased teratogenicity of zebrafishembryos in a dose-dependent manner (Tsay et al., 2007).These paradoxical results may be due to the use ofdifferent model species where one group was receivingthe dose in the diet and the other was systemicallyexposed to a solution of the toxin. Another possibility isthe results from species differences in drug metabolism(Harris et al., 2008). In this study, we used zebrafishembryos as an animal model to study SB-inducedtoxicity and found that SB induced a significant decreasein the survival rates of zebrafish embryos, furthersupporting Tsay et al.’s (2007) findings (Fig. 1). Thesestudies indicated that SB has deleterious effects onembryonic development of zebrafish.

The developing brain is more sensitive to chemicaltoxicants than the adult brain, and exposure duringdevelopment has been implicated in neurological dis-eases and mental retardation (Anderson et al., 2000).Recent data have indicated that the development ofdiencephalic DA neurons in zebrafish was specificallydisrupted when exposed to MPTP or other environmen-tal toxicants (Bretaud et al., 2004; Lam et al., 2005;

McKinley et al., 2005; Ton et al., 2006; Wen et al., 2008).Tsay et al. reported that SB caused motor axon andneuromuscular junction defects during development ofzebrafish embryos, implying that SB has neurotoxiccharacteristics (Tsay et al., 2007). However, to date therehave been no reports showing whether SB influences thegene expression in DA neurons in zebrafish embryos. Inthe present study, we provided the first evidence that SB,like MPTP, can induce significant downregulation ofexpression of both TH and DAT in diencephalic DAneurons in zebrafish embryos. This downregulation ofexpression of neuronal genes was dose-dependent(Figs. 2, 3). These studies suggested that SB hasdeleterious effects on the development of embryonicDA neurons. Whether SB can affect development of otherneurons in zebrafish brain remains to be determined.

The mesencephalic DA neurons in mammals areinvolved in initiation of movement and behavioralresponses. Their degeneration is the leading cause ofthe debilitating motor symptoms in Parkinson’s disease(PD) (Guo et al., 1999a; Smidt et al., 2003; Jeong et al.,2006). Based on our current findings, we presumed thatSB-induced decreased gene expression in DA neuronsmay also impair the locomotor activity of zebrafishlarvae. To confirm the hypothesis, we chose zebrafishlarvae at 6 dpf to study the effects of SB on locomotoractivity. These 6-day-old larvae already possess manypatterns of behavior including free swimming, foodhunting, optomotor responses, and escape behaviors(Guo, 2004). Our results showed that locomotor activitywas significantly reduced in SB-treated zebrafish larvae,which was related to the exposure period. The larvaetreated with SB during the first 3 days showed a moreremarkable reduction in locomotor activity than thelarvae treated for the latter 3 days (Fig. 4). Suchdifferences may reflect the time window for neuraldevelopment and maturation. The nervous system inzebrafish develops rapidly after fertilization and the first3 days is a critical time frame for development. Five ofthe 7 ventral diencephalic TH cell populations seen at5 dpf are already present at 2 dpf (Rink and Wullimann,2002). By 3 dpf, the primary nervous system is welldeveloped. After hatching from the chorion, embryoshave acquired spontaneous swimming bouts and canrespond to tactile stimuli. Treatment with SB during thefirst 3 days appears to interfere with the critical period ofdevelopment for DA neurons. These results suggestthat SB caused a remarkable reduction in the locomotoractivity of zebrafish larvae, and younger zebrafish larvaewere more sensitive to SB exposure than older ones.

In conclusion, in this study we have shown that SB wasable to induce neurotoxicity in DA neurons of zebrafishembryos and younger zebrafish larvae were moresensitive to SB exposure than older ones. These datamay provide some important information for furtherelucidating the mechanisms underlying SB-induceddevelopment neurotoxicity.

ACKNOWLEDGMENTS

We thank Dr. Hui Chen (Weis Center for Research,Geisinger Health System, Danville, PA) for criticallyreading the manuscript and Yu-peng Feng for maintain-ing the zebrafish colony.

Fig. 4. Effects of SB on the locomotor activity of larval zebrafishat 6 dpf. The untreated control group: normally incubated up to6 dpf; the first 3-day group: treated with 20 or 40 mg/ml SB from2 hpf to 3 dpf, and then incubated for an additional 3 days (up to6 dpf) under normal conditions; and the latter 3-day group:normally incubated up to 3 dpf and then treated with 20 or40 mg/ml SB from 3 dpf to 6 dpf. Data are presented asmean7standard error of three separate experiments. ��Po0.001compared with control; #Po0.001 compared with larvae treatedwith 20mg/ml SB during the latter 3 days; Po0.001 comparedwith larvae treated with 40mg/ml SB during the latter 3 days.

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