Silver Nanocolloids Disrupt Medaka Embryogenesis through Vital

Silver nanomaterials are the major components of healthcare products largely because of their antimicrobial effects. However, their unintended toxicit...
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Silver Nanocolloids Disrupt Medaka Embryogenesis through Vital Gene Expressions Shosaku Kashiwada,†,‡,§,∥,* Maria E. Ariza,† Tomohiro Kawaguchi,†,⊥ Yuya Nakagame,§ B. Sumith Jayasinghe,† Karin Gar̈ tner,† Hiroshi Nakamura,# Yoshihiro, Kagami,# Tara Sabo-Attwood,†,‡ P. Lee Ferguson,‡,▽ and G. Thomas Chandler*,†,‡ †

Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina 29208, United States ‡ USC Nano Center, University of South Carolina, Columbia, South Carolina 29208, United States § Department of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Gunma 374-0193, Japan ∥ Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan ⊥ Department of Biology, Division of Mathematics and Natural sciences, Allen University, Columbia, South Carolina 29209, United States # Ecogenomics, Inc., 1-1 Hyakunenkouen, Kurume, Fukuoka 839-0864, Japan ▽ Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States S Supporting Information *

ABSTRACT: Silver nanomaterials are the major components of healthcare products largely because of their antimicrobial effects. However, their unintended toxicity to biological organisms and its mechanism are not well understood. Using medaka fish embryo model, the toxic effects and corresponding mechanisms of silver nanocolloids (SNC, particle size 3.8 ± 1.0-diameter nm) were investigated. SNC caused morphological changes in embryos including cardiovascular malformations, ischemia, underdeveloped central nervous system and eyes, and kyphosis at exposures of 0.5 mg/L. Interestingly, SNC were observed inside the eggs at a level of 786.1 ± 32.5 pg/mg egg weight, and TEM analysis showed that SNC adhered to the surface and inside of the chorion. Meanwhile, medaka oligo DNA microarray and qRT-PCR were used for gene expression analysis in the embryos exposed to 0.05 mg/L SNC for 48 h. As a result, expressions of six of the oxidative stress-, embryogenesis- and morphogenesis-related genes, ctsL, tpm1, rbp, mt, atp2a1, and hox6b6, were affected by the SNC exposure, and these genes’ involvement in those malformations was implied. Thus, SNC could potentially cause malformations in the cardiovascular and central nervous systems in developing medaka embryo through SNC-induced differential expression of the genes related to oxidative stress, embryonic cellular proliferation, and morphological development.

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INTRODUCTION The use of nanotechnology is considerably increasing in both high-tech industries and medicine. It is estimated that nanotechnology would support $1.5 trillion world market by 2015.1 While such technology offers numerous benefits, there are mounting concerns regarding the potential environmental and human health risks associated with exposure to nanomaterials. In particular, silver nanomaterials incluing nanocolloids and nanoparticles are the most widely used for their disinfectant properties,2 and have high likelihood of emission and impacting aquatic environments3 through municipal and other wastewater inputs. Recently, there are increasing number of researches using fish embryos to investigate developmental toxicity of nanomaterials. For example, Li et al. reported that nanoiron decreased © 2012 American Chemical Society

superoxide dismutase (SOD) and increased malondialdehyde (MDA) in medaka embryos.4 Blickley and McClellan-Green studied tocixity of aqueous fullerene in embryos of mummichog, but there was little mortality and no influence to its embryonic developments or to hatching success.5 Furthermore, Bai et al. reported that toxicity of copper nanoparticles in zebrafish embryos delayed their hatching and caused morphological malformations in the larvae.6 Last but not least, George et al. screened developmental hazards of engineered nanomaterilas using zebrafish embryos, and reported that ZnO and CdSe/ZnS Received: Revised: Accepted: Published: 6278

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Then, fertilized eggs were selected, and they were placed in ERM (embryo-rearing medium; 1.0 g NaCl, 0.03 g KCl, 0.04 g CaCl2·2H2O, 0.163 g MgSO4·7H2O in 1 L of ultrapure water, adjusted pH to 7.2 with 1.25% NaHCO3) and incubated at 28 ± 0.1 °C in an incubator. The embryos at developmental stages 11 (blastula), 21 (brain regionalization and otic vesicle formation) and 30 (blood vessel development 20) were subjected to this study, and they received different concentrations of SNC exposures. Silver Nanocolloids. Purified silver nanocolloids (SNC, 10 mg/L, 99.99% purity, nominal size 3.6 nm of diameter, dissolved in distilled water) were purchased from Synergenesis, Inc. (Pengilly, MN). Diluted SNC solutions for exposure tests were prepared with ultrapure water. Purity of SNC was validated by ICP-OES (inductively coupled plasma-optical emission spectrometer) analysis (See Supporting Information for details). SNC diameter was certified using a JEOL 200CX operated at 120 kV, and also measured as 3.8 ± 1.0 nm by Delsa Nano Zeta Potential and Submicrometer Particle Size Analyzer (Beckman Coulter, Inc., Fullerton, CA) by courtesy of Dr. Pei-Jen Chen in National Taiwan University. Ag+ concentration in 10 mg/L SNC solution was measured with a silver ion selective electrode (Thermo Fisher Scientific, Beverly, MA), and AgNO3 was used as a reference. Concentration of Ag+ released from SNC in aqueous suspension was dependent on pH of the solution. Therefore, Ag+ concentrations of 10 mg/L SNC were measured in triplicates in the pH range of 5.03− 7.52. pH of the 10 mg/L SNC solution was adjusted with minimal drop additions of 0.1 N HNO3 and 0.1 N NaOH solutions as required (See Figure S1, Supporting Information). All of the SNC concentrations used for the exposure tests were nominal concentration values. Age-Dependent Toxic Effects of SNC to Medaka Embryos. Fifteen medaka embryos at developmental stages of 11, 21, and 30 were exposed in triplicates to 5 mL each of 0, 0.5, and 1.0 mg/L SNC solutions in the wells of 6-well polystyrene plate at 28 ± 0.1 °C in the dark until hatch or up to 14 days. During the exposure period, the embryo’s heart rate was measured at day 6, and other phenotypic deformities (Table 1) were observed until day 7 (since the unexposed embryos hatched at day 7. The hatching time and hatching rate of the exposed embryos were measured up until day 14. Embryos that did not hatch within 14 days were defined as dead. pH of the SNC solutions remained stable in the 5.81− 5.90 range throughout the exposure. The exposed embryos were observed for morphological abnormalities under the dissection microscope (Nikon SMZ 1500, Nikon Instruments Inc.) at 24 h intervals. Test solutions were renewed every 24 h. Epifluorescence Microscopy Observations for Effects of SNC on Embryonic Vertebra. In the SNC-exposure test to measure age-dependent toxic effects, embryonic vertebral defects were observed and evaluated by immersing the freshly hatched embryos in 0.02% calcein (3,3-bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein, Sigma-Aldrich, Inc.) solution for 10 min, followed by 30-min rinsing with fresh ERM.17 Calcein stained embryos were anesthetized with 0.05% MS-222 (Finquel, Argent Chemical Laboratories, Redmond, WA), and then observed for vertebral structure abnormalities under the epifluorescence microscope (Nikon SMZ 1500, Nikon Instruments Inc.) equipped with a green fluorescent protein filter (excitation at 480 nm and emission at 510 nm). Fluorescence and regular light images were captured for 10 s and 10 ms, respectively, with a CCD-cooled digital camera

were the most hazardous nanomaterials followed by Ag, CdSe, and Pt, but no toxic effect was observed with Au, Al2O3 and SiO2.7 Although silver is rare in the natural environment, its environmental concentrations at large have been elevated due to anthropogenic activities. According to a report,8 the highest silver concentration in fresh water system was 260 μg/L in Genesee River, New York, U.S., which was a recipient of postphotoprocessing effluent, and the postphotoprocessing effluent was one of the major anthropogenic pollutant sources of silver. The background concentrations of silver were in fact 0.01 μg/L in unpolluted areas, and the concentrations were measured at 0.01−0.1 μg/L in urban and industrialized areas.9 In the environment, silver can be present in four different oxidation states: Ag0, Ag+, Ag2+, and Ag3+ with the former two being stable and most abundant, and the latter two being unstable and much less abundant in aquatic systems.10 Thus, silver is typically found as a monovalent ion together with sulfide, bicarbonate or sulfate, or complexed with chlorides and sulfates adsorbed onto particulate matter.9 Despite the widespread use of silver and free silver ions (Ag+) in industry and medicine/healthcare as well as the increasing use of nanoscale silver as an antimicrobial agent in personal care products, there remains limited information on silver toxicity in aquatic environments. Nevertheless, existing environmental and human studies previously demonstrated that free silver ions (Ag+) were typically more toxic than other forms of silver, which lead to the following hypothesis: Toxic effcts of silver substances are proportional to the rate of release of f ree Ag+.3 Various adverse effects of silver nanoparticles in algae,11 daphnia,12 and zebrafish13,14 were reported. Lee et al. reported phenotypic abnormalities including spinal deformity, cardiac malformation, yolk sac edema, head edema, and eye malformation in zebrafish embryos that were exposed to 0.38 nM (41 ng/L) silver nanoparticles (5−46 nm in diameter).14 Asharani et al. reported that silver nanoparticles (5−20 nm in diameter) capped with starch or bovine serum albumin (BSA) induced similar abnormalities in zebrafish embryos but at much higher concentration (100 mg/L).13 To be able to compare these studies mechanistically, effective measurement of released Ag+ from the various nano-Ag forms seems to be the key for understanding the toxic mechanisms of silver nanomaterials in their various sizes and forms. In this study, we used Japanese medaka (Oryzias latipes) to investigate vertebrate developmental toxicity15−19 by silver nanocolloids (SNC) themselves and by the Ag+ ion released from the SNC. We present novel results revealing that SNC were capable of traversing medaka egg chorion and reaching embryonic mass. The localization of SNC might have caused oxidative stress and severe gross malformations, potentially linked to down-regulation of the genes involved in growth regulation, cell proliferation and differentiation, namely, cathepsin L and retinolbinding protein.

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MATERIALS AND METHODS Medaka Embryos. Medaka (Oryzias latipes) cultured for this study were obtained from Nagoya University (National BioResource Project) in Japan, and they were fed Artemia salina nauplii once a day and Otohime larval β-1 (Marubeni Nissin Feed Co. Ltd., Tokyo, Japan) twice a day. 16-h:8-h light:dark cycle at 26 ± 0.5 °C condition was maintained. After female medaka spawned eggs, external egg clusters were removed. 6279

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Environmental Science & Technology (0.0) (0.0) (1.2) (23.6)b (0.1)b (0.5)b (0.0) (0.0) (2.3) (0.0) (0.0) (0.5)b 0.0 0.0 30.2 100 3.3 8.4 (0.0) (0.0) (2.1) (0.0) (0.0) (0.6) 0.0 0.0 31.7 100 0.0 7.6 13.3 (0.0)b 0.0 (0.0) NA 2.2 (3.8)b NA NA 0.0 (0.0) 10.0b (0.0) 30 (1.9) 56.7 (0.0)b 23.3 (0.2)b 8.9 (0.3)b (0.0) (0.0) (3.6) (0.0) (0.0) (0.0)

NA: not available. Standard errors are in parentheses. bAnalysis of variance versus control: p < 0.05.

0.0 0.0 29.5 100 0.0 8.0

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RESULTS AND DISCUSSION pH-dependent Ag+ Concentration in SNC Solution. As expected, silver ion (Ag+) release was increased with acidic pH levels in SNC solution (Supporting Information Figure S1). Colloidal silver clusters should reach equilibrium with ionic Ag+

a

0.6 (1.4) 0.0 (0.0) NA 0 (0.0)b NA NA (0.0) (0.0) (1.7) (0.0)b (0.0) (0.5)b 0.0 0.0 29.8 70 3.3 8.9 (0.0) (0.0) (3.2) (0.0) (0.0) (0.3) 0.0 0.0 29.1 93.3 0.0 7.9

0.0

less-development of brain and eyes, short spinal cord, vascular defects and ischemia blood clots (%) pericardiovascular edema and tubular heart (%) heart rate per 15s at day 6 hatch rate (%) spinal deformity (kyphosis, %) time to hatch (days)

SNC (mg/L)

(MicroPublisher 5.0, QImaging Inc., Surrey, BC, Canada). The captured fluorescence was pseudocolored yellow-green using QCapture Suite software (QImaging Inc.) to aid visualization. TEM (Transmission Electron Microscope) Analysis of Medaka Embryos Exposed to SNC. Fifteen medaka embryos at stage-21 were exposed to 5 mL of 0.5 mg/L SNC in the wells of 6-well polystyrene plate at 28 ± 0.1 °C in the dark for 6 days. The exposed embryos were rinsed in ultrapure water and fixed in 72.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for 2 h, postfixed in 1% osmium tetroxide at 4 °C for 2 h, dehydrated in ethanol and embedded in Spurr’s medium via acetone. A series of ultrathin sections were cut using a Leica UCT ultramicrotome, double stained with uranyl acetone and lead citrate and viewed on a JEOL 200CX TEM operated at 120 kV.21 Measurements of GSH, MDA, CAT, and SOD. Seventy medaka embryos at stage-21 were exposed to each of the 5 mL SNC solutions (0, 0.005, 0.05, and 0.5 mg/L) separately in the wells of 6-well polystyrene plate at 28 ± 0.1 °C in the dark for 6 days. For each of the exposure conditions triplicate samples were prepared. Their initial pH values were 5.81, 5.81, 5.86, and 5.90 for 0, 0.005, 0.05, and 0.5 mg/L SNC, respectively. Ten exposed embryos were harvested at every 24th hr, immediately rinsed in ultrapure water, and snap-frozen in liquid nitrogen and kept at −80 °C for further analyses. In order to detect GSH (glutathione) and MDA (malondialdehyde) levels and CAT (catalase) and SOD (superoxide dismutase) activities, Glutathione Assay Kit, TBARS (thiobarbituric acid reactive substances) Assay Kit, Catalase Assay Kit, and Superoxide Dismutase Assay Kit, respectively (all kits were purchased from Cayman Chemical Company, Ann Arbor, MI), were used. Rescue assays. Fifteen medaka embryos at stage-21 were pretreated either 0.5 mM GSH (Acros Organics, Morris Plains NJ) or 0.05 mM NAC (N-acetyl-L-cysteine, Sigma-Aldrich, Inc.) for 30 min, then after confirming no lethality, 0.5 mg/L SNC was added to the GSH/NAC-treated embryos and incubated further for 96 h. The tests were carried out in triplicates, and survival of the embryos was determined. DNA Microarray Analysis and Microarray Data Validation/Transcript Quantification by qRT-PCR. Three medaka embryos at stage-21 were separately exposed to each of the 5 mL SNC solutions (0 and 0.05 mg/L) in triplicates in the wells of 6-well polystyrene plate at 28 ± 0.1 °C in the dark for 48 h. Since differential gene expression analysis was more sensitive method than phenotypic analysis in terms of detection of xenobiotics’ toxic influences, 0.05 mg/L (10 times lower concentration than the concentration to observe morphological alterations) was chosen for this DNA microarray analysis. Furthermore, changes in the transcriptional expression level of the differentially expressed genes that were involved in embryogenesis and/or morphogenesis were quantified using qRT-PCR (quantitative reverse transcription-polymerase chain reaction) analysis. See Supporting Information for details. Statistical Analyses. Data other than the DNA microarray results were analyzed using analysis of variance (ANOVA) and Dunnett’s a posteriori test to evaluate the impact of SNC at each concentration compared with the control.

0.0 3.3 30.3 43.3 26.7 9.6

1.0 + −

0.5 0.0

− ++

1.0 0.5 0.0

+ − −

0.5

+

1.0

−

stage 30 stage 21 stage 11 embryo stages

Table 1. Age-Dependent Toxic Effects of SNC to Medaka Embryosa

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Figure 1. Morphological toxic effects of SNC to medaka egg embryos. Medaka embryos at stage-21 were exposed to 10 mg/L SNC for 5.5 h to investigate acute toxicities (a−f). Other groups of embryos were exposed to 0 (control) and 0.5 mg/L SNC for 6 days to investigate morphological toxicities (g−k) and hatched embryos were stained with a 0.02% calcein solution to examine the effects on osteogenesis (l−q). a, 0 h, the germ ring was well-defined from chorion membrane and it was clear between germ and chorion membrane, this stage had well-defined brain and the neural fold seen as a median line. b, 3 h, embryo moved weakly and rhythmically undulating. c, 4 h, the rhythmical undulation stopped. d, 5 h, regionalized brain and neural fold started disappearing. e, 5.25 h, damaged embryonic body was seen on the shrunk yolk sack, yellowish yolk leaked from the yolk sack. f, 5.5 h, the embryonic body was completely damaged and organs could no longer be identified. g, control embryo at day 6 (short arrow; heart ventricle, long arrow; heart atrium, small arrowhead; oil droplet, white large arrowhead; right duct of Cuvier, black large arrowhead; left duct of Cuvier, tail rises up over a head of embryo). h, exposed embryo at day 6 (short arrow; heart ventricle, long arrow; heart atrium, white large arrowhead; a blood clot on right duct of Cuvier, black large arrowhead; enfeebled left duct of Cuvier, red large arrowhead; pericardiovascular edema, a tail did not developed well). i, exposed embryo at day 6 (short arrow; heart ventricle, long arrow; tubular atrium of the heart, red large arrowhead; pericardiovascular edema, a tail did not developed well). j, exposed embryo at day 6 (two blue arrowheads; significant eye defects). k, exposed embryo at day 6 (a back image of j, two yellow arrowheads; artery defect and vein defect on the vertebral column. l, dechorionated healthy control embryo. m, dechorionated embryo with spinal deformities and shortened tails after exposure. n, a hatched healthy embryo control. o, calcein-stained image of n. p, a hatched damaged embryo. q, calcein-stained image of p.

in aqueous solution. This equilibrium is shifted to the ionic form with decreasing pH. Released Ag+ concentrations in SNC solutions used in this study were estimated as 0.00126, 0.0123, 0.121 mg/L in SNC 0.005 (pH5.82), 0.05 (pH5.86) and 0.5 (pH5.90) mg/L, respectively. Hence, ca. 25% of total silver existed as Ag+ ion in the SNC solution at ca. pH5.8. Acute Toxicities, Malformations, And Embryonic Distributions of SNC. First, we conducted acute toxicity tests by exposing stage-21 medaka embryos to 10 mg/L SNC. Results from this study indicated that exposure to SNC caused termination of rhythmical undulation of whole embryo at 3 h, followed by disappearance of regionalized brain and neural fold at 5 h, shrinkage/burst of yolk sack with yolk leakage at 5.25 h, and final destruction of embryonic body at 5.5 h after treatment (Figure 1a−f). See Supporting Information for details.

Second, LC50 of SNC by 96 h exposure test was calculated for the developmental stages 11, 21, and 3020 because developmental stage-dependent sensitivity was reported by Gonzalez-Doncel et al.22 However, there was no difference in 96-h LC50 (1.39 ± 0.02 mg/L) among these tested developmental stages (Supporting Information Table S1). The SNC solution’s pH was 5.8, and therefore determination of the silver ion concentration in 10 mg/L SNC solution at pH 5.8 from the calibration curve (Supporting Information Figure S1) was done as in 262.89 × 5.8−2.642 = 2.53 (mg/mL). Since the ratio of silver ion to SNC should be 2.53/10 = 0.253, the silver ion concentration in 1.39 mg/L SNC solution at pH5.8 (the acute LC50) was calculated as in 0.253 × 1.39 = 0.352 mg/L and thus a practical LC50. See Supporting Information for details. 6281

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Figure 2. Transmission electron microscope (TEM) images of SNC and their distributions in medaka egg chorion. a, SNC (arrows). b, a section image of medaka egg embryo in control (×37 000). Proteins of inner chorionic layer started broken down by hatching enzymes that were secreted from an embryo and chorion lysate, and looked like smear. Only the outer edge layer of chorion remained (large arrowhead). A double arrowhead indicates original thickness of chorion. c, a magnified image of a square in image b (×330 000). A double arrowhead indicates thickness of the outer edge layer of the chorion. d, a section image of an egg embryo exposed to SNC (×37 000). Chain-like formations of SNC were seen on the egg surface (arrows). High electron density layers were seen on the surface of chorion, which might penetrate SNC, were seen. A double arrowhead indicates original thickness of chorion. e, a magnified image of the square in image d (×330 000). Arrows indicate chain-like formations of SNC. Arrowheads indicate clusters with high electron densities, which were assumed to be penetrating SNC.

There was no such high electron density material in any untreated chorions (Figure 2b and 2c). Fish embryonic chorion is well-known to play a protective role against chemical exposure.24 Under normal culturing conditions the chorion of untreated embryos is lysed by hatching enzymes after 5 days of SNC exposure (i.e., on day 6, as seen in this study) (Figure 2b). However, in SNC-treated eggs, the chorionic layer remained intact on day 6 (i.e., enzymatic digestion was perhaps inhibited in the presence of SNC) (Figure 2d), and delayed hatching was observed in the embryos (Table 1). Additionally, the outermost edge of the chorionic layer in untreated eggs showed a regionalized microlayered structure (Figure 2b and 2c), but in contrast, the SNC-treated eggs lacked this layered structure (Figure 2d and 2e). Thus, it suggested that physical interference of SNC on and in the chorionic layer could be a cause of SNC-mediated alteration of chorionic breakdown at hatching (Figure 2d, e). Markers of Oxidative Stress. Exposure to silvercontaining compounds is associated with the production of oxidative stress in multiple aquatic species. GSH and MDA levels and CAT and SOD activities were known markers of oxidative stress induced by activated oxygen species, which are catalyzed by metal ions,25 in many organisms including fish.26,27 Consequently, we tried to determine if these markers were suitable indicators of SNC-induced oxidative stress by exposing stage-21 medaka embryos to 0.005, 0.05, and 0.5 mg/L SNC for 6 days. As a result, only the GSH level was significantly (p < 0.001) reduced among these four markers, and this was observed only by 0.5 mg/L SNC exposure and not by the lower SNC concentrations (Figure 3a-d). On the contrary, MDA was in fact significantly (p < 0.001) elevated by 0.5 mg/L SNC but on day 6 only, and no significant MDA up-regulation was observed at the lower SNC concentrations (Figure 3d).

Third, we observed the age-dependent morphological malformations in medaka embryos using 0.5 and 1.0 mg/L SNC solutions, which were below 96 h LC50 (1.39 ± 0.02 mg/L). The medaka embryos at stage-21 showed the most increased sensitivity to SNC by underdeveloped brain and eyes, short deformed spinal cord, tubular heart, vascular defects, blood clots, ischemia, and pericardiovascular edema (Table 1 and Figure 1g-m). In contrast, medaka embryos at stage 11 and 30 showed higher hatch rates, lower ratios of spinal deformities in comparison with medaka embryos at stage 21. Although similar malformations were reported for zebrafish embryo exposed to silver nanoparticles,14 our results revealed that there was strong stage-dependent developmental sensitivity to SNC. Since similar developmental deformities were described in response to conventional exposures to some pesticides17 and dioxins,23 we examined by epifluorescence microscopy whether the spinal deformities observed in developing embryo were caused by interference with osteogenesis. However, no perturbation of osteogenesis was observed in any spine-deformed embryos (Figure 1n−o), suggesting that some other mechanism were driving the deformation under SNC exposure. Lastly, since stage-21 medaka embryos showed the most increased sensitivity to SNC in the study of age-dependent SNC-toxicity, analysis of the distribution of SNC-derived silver nanoparticles was carried out using stage-21 medaka embryos exposed at 0.5 mg/L SNC for 6 days. The silver nanoparticles were detected in medaka eggs at a level of 786.1 ± 32.5 pg/mg eggs (wet weight of single medaka egg embryo was 0.937 ± 0.00 mg). In addition, 16.6 ± 9.3 pg and 720 ± 29 pg of silver were detected in “single embryo” and “chorion + embryonic fluid (solution filled inside the egg)”, respectively. Furthermore, TEM analyses showed high electron density materials: resembling chains of SNC, adhered onto the surface of the chorion and within the chorionic layer (Figure 2d and 2e). 6282

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Figure 3. Biomarkers measured in embryos exposed to SNC. a, GSH levels; b, CAT activity; c, SOD activity; d, MDA level in medaka embryos exposed to various SNC concentrations (0, 0.005, 0.05, and/or 0.5 mg/L) for 6 days (stages 21 to 38 in control). *Significantly different from control (two-way ANOVA, p < 0.001).

oxidative stress was one of the key mechanisms involved in the SNC toxicity. DNA Microarray and qRT-PCR Analyses. From our DNA microarray analysis, 118 genes were up-regulated (>2.0 fold) and 117 genes were down-regulated (