Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early Online: 1–6 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2015.1011803
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The effect of silver nanoparticles on zebrafish embryonic development and toxicology Guangqing Xia1, Tiantian Liu2, Zhenwei Wang2, Yi Hou2, Lihong Dong1, Junyi Zhu1 & Jie Qi2 1Department of Life Science, College of Life Science, Tonghua Normal University, Tonghua, P. R. China and 2Key Laboratory of
Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, P. R. China
the last decade, nano-Ag have attracted much interest due to the interesting optical, electrical and magnetic properties that lead to applications in sensing, chemical catalysis, medical fields and personal healthcare (He et al. 2013). Currently, nano-Ag have become the most common group of metallic engineered nano-particles in commercial products, and are widely applied in agricultural sciences, medical products (Chen and Schluesener 2008), and food packaging (Gottesman et al. 2011). The increasing uses of nano-Ag in these fields have increased their release into the environment, and meanwhile, resulted in their exposure to living tissues. This would thus promote special concerns of environmental risk and human health posed by these particles. Earlier reports (Braydich-Stolle et al. 2005, Hussain et al. 2005, Skebo et al. 2007) proved that nano-Ag are more lethal to cell-based in vitro systems than other metal nanoparticles screened. Some evaluations also had been carried out to demonstrate the plausible deleterious effects of nano-Ag in cell lines and/or isolated primary human cells (Braydich-Stolle et al. 2005, Hussain et al. 2005, Skebo et al. 2007, Rizzo et al. 2013). However, this nanotoxicity testing, owing to its over-simplified systems and setups, moderately informative and conclusive results, and limited translational value, cannot be properly addressed using in vitro experimental setups. In the present study, zebrafish embryos, which are relatively well-established to assess the acute toxic effects from exposure to environmental chemicals and nanoparticles, were chosen as model systems for testing the toxicity of the nano-Ag. Unravelling the genetic networks in vertebrates that guide the formation of the three germ layers-ectoderm, mesoderm and endoderm. The embryonic ectoderm is fated to become either neural or non-neural (epidermal), depending on the patterning process that occurs before and during gastrulation (Bakkers et al. 2002). During gastrulation, cells expressing gsc move anteriorly, while cells expressing no tail (ntl) remain more posterior (Schulte-Merker et al. 1994). By mid-gastrula, otx2 RNAs are restricted to the anterior dorsal epiblast (superficial layers) and mesoderm (hypoblast) (Li
Abstract The unique physical and chemical characteristics of nanomaterials, such as the effects of their small size, surface effects, very high rates of reaction, and quantum tunnel effect, have aroused great interest among scholars. However, improper usage has led to an increasing number of nanomaterials entering the environment through various channels, greatly threatening the security of the ecological environment and human health. The urgent need for a scientific assessment of their biosafety can enable nanomaterials to truly benefit humanity. However, the current research in this field is extremely limited with regard to safety standards and waste disposal. In this study, we used silver nanoparticles (nano-Ag) and zebrafish embryos as experimental subjects, and we have reported the deleterious effect on zebrafish embryos treated with different concentrations of nano-Ag, with respect to morphological features (mortality, deformity rate, and heartbeat) and the analysis of expression of relevant genes (sox17, gsc, ntl, otx2); we found a dose-dependent increase in mortality and hatching delay. The results of in situ hybridization indicated that nano-Ag causes a dose-dependent toxicity in embryonic development, and would affect their development and lead to deformity, delayed development, and even death. The safety limit for the concentration of nano-Ag was found to be less than 5 mg/L. Keywords: deformity, heartbeat, malformation, silver nanoparticles, zebrafish embryonic development
Introduction The development of nanotechnology has redesigned the current scenario of science and technology. Many scientists have considered nanotechnology as the next logical roadmap in some areas, such as drug delivery, diagnostics, tissue engineering, and environmental remediation (Alexiou et al. 2005, Abarrategi et al. 2008, Navarro et al. 2008, Meyer et al. 2009). Silver nanoparticles (nano-Ag), as common nano-materials, have taken on wonderful and unique chemo-physical virtues and biological properties. During
Correspondence: Jie Qi, Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, #5 Yushan Road, Qingdao 266003, Shandong, P. R. China. Tel: ⫹ 86-0532-82031986. E-mail: [email protected] (Received 3 December 2014; accepted 21 January 2015)
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et al. 1994). The Sox17 homolog is expressed during gastrulation exclusively in the endoderm, with a restricted pattern of sox17expression and its postulated importance for endoderm formation (Alexander and Stainier 1999). Herein, a green, cost effective pathway was designed for synthesizing nano-Ag with a size of 10–20 nm, by adopting DL-α-aminopropanoic acid as a reductant and capping agent. To enable simple, efficient, and high-throughput toxicity testing in systems significantly more complex than cultured cells, the nano-Ag particles were used as prepared, to evaluate their plausible deleterious effects on zebrafish embryos. Based on the fact that the zebrafish represents a bridge between in vitro cell culture and in vivo mammalian models (Fako and Furgeson 2009), the objective of this study was to evaluate the acute toxicity effects of nano-Ag on zebrafish embryos, especially concerning the toxicity of nano-Ag to some relative gene expressions during the early stages of zebrafish embryonic development by in situ hybridization.
Materials and methods Preparation and characterization of particle suspensions All the chemicals were used as received, and all aqueous solutions were prepared with deionized water. The preparation process was green and simple. In a typical procedure, 0.2 g of PVP was dissolved in 20 mL of 0.1 mol/L AgNO3 solution at an ambient temperature of 15°C. Then 20 mL of 0.1 mol/L DL-α-aminopropanoic acid solution was added into the AgNO3 solution and the mixture was left to settle undisturbed. About 10 min later, the system took on a dark, brownish color, with optical transparency, which indicates the formation of small-sized nano-Ag particles. The solution was centrifuged immediately at 10,000 rpm for 10 min, to separate the nano-Ag from the mother liquid. After centrifugation, the clear supernatant was allowed to stand for another 30 min, to allow the formation of the next batch of nano-Ag. Next, the nanoparticles were separated again by centrifugation. Such a process could be repeated five times, to obtain smaller nano-Ag particles without wastage of raw materials, until the supernatant did not turn dark brown during placement. It should be noted that the settling time should be prolonged with the increasing number of repetitions, and the actual reaction time was also affected by the ambient temperature. The particle size and shape were determined via transmission electron microscope (TEM, Hitachi H-7500) studies.
a different concentration of nano-Ag, was repeated three times, and the standard deviation was calculated. Treated and untreated embryos were anesthetized with 0.2 mg/ml tricaine (3-amino benzoic acid ethyl ester; Sigma-Aldrich, St. Louis, MO, USA), and embedded in methylcellulose.
The whole mount in situ hybridization and immunohistochemistry RNA probes of the germ layer marker gene were prepared from cDNA of the zebrafish otx2 (Li et al. 1994), sox17 (Alexander and Stainier 1999), ntl (Shestopalov et al. 2012), gsc (Slater and Birney 2005) (The plasmid of ntl and gsc were gifted by Dr. Michael REBAGLIATI). The primer sequences were given as the following: otx2: Forward: 5′-CCTGCCATCCTTCCAATA ACA-3′, Reverse: 5′-CACTCGCACACATCCTCTCTA-3′; sox17: Forward: 5′-ATGAGCA GTCCCGATGCG-3′, Reverse: 5′-TCAAGAATTATTATAGC CGC-3′. Their PCR products were cloned into the pEASY-T5 vector and sequenced. RNA probes containing digoxigenin11-UTP were synthesized from the linearized plasmid DNA by PstI for pEASY-T5-otx2,SpeI for pEASY-T5-sox17, SalI for ntl, and NotI for gsc, respectively, using T7 RNA polymerase. Whole mount in situ hybridization was performed principally as described previously (Thisse and Thisse 2008). Embryos of shield period (6 hpf ) and tail bud (10 hpf ) were fixed in 4% paraformaldehyde (PFA, rinsed in PBST, dehydrated in methanol, and stored at ⫺ 20°C until used.
Results and discussions Morphology of nano-Ag The morphology of the silver product as obtained in the typical experiment was investigated by TEM (Figure 1). It could be seen that the product was composed of nano-particles in the range of 10–20 nm. No other morphology was found, which indicated high purity of the product.
Deformity, heartbeat, and malformation Embryos treated with nano-Ag showed dose-dependent toxicity (Figure 2). They showed less deformity under a low concentration of nano-Ag, compared with the control. The embryonic lethality rate showed dose-dependency from 5% to 76.7%, at the concentration levels ranging from 0 to 100 mg/L at 4 hpf, but 100% of the embryos died at 50 and
Collection and exposure of the embryos to nano-Ag Zebrafish were maintained in standard fish facility conditions with a 14:10 h light/dark cycle and fed with living brine shrimp twice per day. The water temperature was maintained at 28°C. For toxicity studies, 20 healthy embryos were transferred to the wells of a 24-well plate, and incubated in different concentrations of nano-Ag (5, 10, 25, 50 and 100 mg/L) at 28°C. Lethality and deformity of the embryos was noted at 4 h post-fertilization (hpf ), 24 hpf, and 48 hpf. The rate of heartbeat was recorded using a stopwatch at 24 hpf by direct microscopic observation. Each treatment, with
Figure 1. Characterization of nanoparticles - TEM image of nano-Ag.
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Silver nanoparticles on zebrafish embryonic development 3
Figure 2. Deformity rate of zebrafish embryos after treatment with different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag at 4 hpf, 24 hpf, and 48 hpf.
100 mg/L, at 24 and 48 hpf (even 8 hpf, data not shown). For development of the embryonic heart, the number of heartbeats was calculated for all samples treated by the serial concentrations of nano-Ag (Figure 3). No significant difference for the heartbeat count was seen at concentrations between 0 and 25 mg/L, while no embryo survived at 50 and 100 mg/L. The morphological development of zebrafish embryos also showed differences at different concentrations of nano-Ag. At 4 hpf, compared to control, the embryos appeared abnormal, regardless of whether the concentration of nano-Ag was 5 mg/L or 100 mg/L, although the percentage was low at the concentration of 5 mg/L (Figure 4A), while at a concentration of 10 mg/L, there was an irregular bulge at the top of the fertilized eggs, and with an increase in nano-Ag concentration, the surface of
Figure 3. Heartbeat of zebrafish embryos after treatment with different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag. No embryo survived to the stage of heartbeat period under 50 and 100 mg/L.
the fertilized eggs became rough and condensed, and the yolk sac of the embryos appeared distorted. After 12 hpf, when the concentration of nano-Ag was higher than 10 mg/L, most of the zebrafish eggs could not finish the transition from the gastrula period to the segmentation period (Figure 4B). At the pharyngula period (24 hpf ), at a lower concentration (5 mg/L) of nano-Ag, the tail of the zebrafish embryos separated from the yolk, while at a concentration of 10 mg/L, there was curved tail separated from the yolk (Figure 4C). When the concentration was higher than 25 mg/L, the tail could not be separated from the yolk (Figure 4C). Above a concentration of 10 mg/L, 60–90% of the surviving embryos showed body malformations after 48 hpf, and the embryos exhibited severe phenotypic changes characterized by bent and twisted notochord, pericardial edema, and degeneration of body parts (Figure 4D).
Figure 4. The effect of different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on zebrafish embryonic development during the cleavage period (A: 4 hpf, B: 12 hpf, C: 24 hpf, and D: 48 hpf ).
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Figure 5. The effect of different concentrations (A–F: 0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on the expression of the sox17 gene.
Expression of zebrafish sox17, gsc, ntl and otx2 During the development of zebrafish embryos, sox17 is expressed in the mesodermal and forerunner cells at 6 hpf, while gsc is expressed in precursor cells at 10 hpf. The Ntl gene is related with the formation of tail, and expresses in precursor cells and chorda-mesoderm at the stage of tail bud (10 hpf ). At the stage of the tail bud (10 hpf ) and 6 somites (12 hpf ), the otx2 gene is expressed in the pituitary gland and the hippocampus of developed head. In order to be more comprehensive in detecting the effect of nano-Ag on the early embryonic development, four reproductive stages of marker gene sox17, gsc, ntl and otx2 were selected for whole mount in situ hybridization, based on the analysis of the four gene maps. We observed different trends with treatment at differ-
ent concentrations of nano-Ag. The expression of sox17 did not show significant difference from 0 to 10 mg/L at the 50% epiboly stage, while the signal of hybridization was darker than at 0 mg/L, with the nano-Ag concentration increased (25–100 mg/L) (Figure 5).To the gsc gene, its transcription levels started to express more from a concentration up to 10 mg/L of nano-Ag (Figure 6). During the stage of tail bud, nano-Ag showed obvious effect on the expression of ntl, at the concentration ranging from 25 to 100 mg/L (Figure 7), and the expression of otx2, at the concentration ranging from 5 to 25 mg/L (Figure 8). All evidence showed that nano-Ag have more or less effect on zebrafish embryonic development, and the effect might be more serious when the concentration is increased up to10 mg/L or higher.
Figure 6. The effect of different concentrations (A–F: 0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on the expression of the gsc gene.
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Silver nanoparticles on zebrafish embryonic development 5
Figure 7. The effect of different concentrations (A–D: 0, 5, 10, and 25 mg/L) of nano-Ag on the expression of the ntl gene. No picture at 50 and 100 mg/L is available, for the reason that the embryos had all died at a really early developmental stage.
Over 120 years ago, Lea (1889) reported the synthesis of a citrate-stabilized silver colloid, which was the first report of nano-silvers. Since then, silver nanomaterials have been used widely in many fields. Nano-Ag are employed in detergents and as antimicrobial agents that could end up in the environment or inside the organs of animals. We have demonstrated here the toxicity of nano-Ag using zebrafish embryos as experimental materials, and the assay could be used to validate and complement findings obtained in vitro. Compared to control, the results suggested that long-term exposure to a relative dose of nano-Ag would cause varying degrees of deformity, such as fin membrane damage and tail bend, and the greater the concentration, the higher the impact. Moreover, nano-Ag could induce death when the concentration increases up to some extent, such as 50 mg/L or 100 mg/L. Our results also indicated that the nano-Ag not only had the function on the surface of the cells, but also could enter the cells through the fragile skin of embryos, which might
be the consequence of local injury. An earlier report on zinc oxide nanoparticles elucidated the passage through rat and rabbit skins (Kapur et al. 1974, Hallmans and Liden 1979). Unlike teratogenesis, we found that nano-Ag had almost no effect on heartbeat in our experiment. Therefore, we concluded that low concentration of nano-Ag would not affect the development and function of the heart. However, the effect on heart of high dose of nano-Ag was unclear, for the reason that the embryos had died before the heart appeared. At the same time, we could find that the embryonic mortality most likely occurred before 8 hpf. A study by Herrmann had showed that the zebrafish embryos were vulnerable to exogenous chemical influence at the beginning of embryonic development, and that the egg shell was not rigid (Herrmann 1993). The high sensitivity of zebrafish embryo to toxic materials also supported the view. In addition, the study had reported that nano-Ag could not only have an effect on the surface of cells, but also penetrate biofilm
Figure 8. The effect of different concentrations (A–D: 0, 5, 10 and 25 mg/L) of nano-Ag on the expression of the otx2 gene. No picture at 50 and 100 mg/L is available, for the reason that the embryos had all died at a really early developmental stage.
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barriers into cells, which could inevitably affect the embryonic development (Morones et al. 2005). All the results illustrated the potential nanotoxicity of nano-Ag for zebrafish embryos, when they reach a certain concentration. Of course, nano-Ag was relatively safe at a low dose, lower than 5 mg/L. Earlier reports (Braydich-Stolle et al. 2005, Morones et al. 2005, Panacek et al. 2006) have shown that nano-Ag was translocated to various cellular organelles from the site of entry during the early embryonic stage, and the deposition of nanoparticles inside the nucleus of the cells led to toxicity, observed through various mechanisms. In zebrafish, sox17 is expressed in the mesodermal and forerunner cells at 6 hpf (Kanai-Azuma et al. 2002, Wang et al. 2003). Gsc and ntl are expressed in the precursor cells at 10 hpf, which regulate the early development of embryos (Slater and Birney 2005) and tail respectively. The Otx2 gene is expressed at the stage of somites in the pituitary gland and the hippocampus of the developed head, and as a marker gene to detect the ectoderm (Acampora et al. 1995). As indicated by evidence from the in situ hybridization, the nano-Ag invades the cells during the early embryonic stages and bears effect at different levels of expression on embryonic development. Only a low concentration of nano-Ag (5 mg/L or less than 5 mg/L) could have no effect on the normal development on the embryos. Higher concentrations of nano-Ag result in significant effects on mesodermal and ectodermal development, which could be due to delay or inhibition of cell division.
Conclusions From the present study, it is clear that nano-Ag have the potential to pose health and toxicity risks in a dosedependent manner. The nano-Ag-treated embryos exhibited phenotypic defects, altered physiological functions, and degradation of body parts. These results have important implications of potential discrepancies for facilitating the translation of nanomedicine materials into clinical traits.
Acknowledgement This study was financially supported by grants from the Science and Technology Department of Jilin Province (20130102034jc).
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. Development. 121: 3279–3290. Alexander J, Stainier DY. 1999. A molecular pathway leading to endoderm formation in zebrafish. Curr Biol. 9:1147–1157. Alexiou C, Jurgons R, Schmid R, Hilpert A , Bergemann C, Parak F, Iro H. 2005. In vitro and in vivo investigations of targeted chemotherapy with magnetic nanoparticles. J Magn Magn Mater. 293: 389–393. Bakkers J, Hild M, Kramer C, Furutani-Seiki M, Hammerschmidt M. 2002. Zebrafish DeltaNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm. Dev Cell. 2:617–627. Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann MC. 2005. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol Sci. 88:412–419. Chen X, Schluesener HJ. 2008. Nanosilver: a nanoproduct in medical application. Toxicol Lett. 176:1–12. Fako VE, Furgeson DY. 2009. Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity. Adv Drug Deliv Rev. 61:478–486. Gottesman R, Shukla S, Perkas N, Solovyov LA , Nitzan Y, Gedanken A . 2011. Sonochemical coating of paper by microbiocidal silver nanoparticles. Langmuir. 27:720–726. Hallmans G, Liden S. 1979. Penetration of 65Zn through the skin of rats. Acta Derm Venereol. 59:105–112. He S, Chen H, Guo Z, Wang B, Tang C, Feng Y. 2013. High-concentration silver colloid stabilized by a cationic gemini surfactant. Colloids Surf A Physicochem Eng Aspects. 429:98–105. Herrmann K . 1993. Effects of the anticonvulsant drug valproic acid and related substances on the early development of the zebrafish (Brachydanio rerio). “http://www.sciencedirect.com/science/ journal/08872333” Toxicology in Vitro. 7:41–54. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. 2005. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 19:975–983. Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, Hayashi Y. 2002. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 129:2367–2379. Kapur SP, Bhussry BR, Rao S, Harmuth-Hoene E. 1974. Percutaneous uptake of zinc in rabbit skin. Proc Soc Exp Biol Med. 145:932–937. Lea MC. 1889. On allotropic forms of silver. Am J Sci. 37:476–491. Li Y, Allende ML, Finkelstein R, Weinberg ES. 1994. Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Mech Dev. 48:229–244. Meyer DE, Curran MA , Gonzalez MA . 2009. An examination of existing data for the industrial manufacture and use of nanocomponents and their role in the life cycle impact of nanoproducts. Environ Sci Technol. 43:1256–1263. Morones JR, Elechiguerra JL, Camacho A , Holt K, Kouri JB, Ramirez JT, Yacaman MJ. 2005. The bactericidal effect of silver nanoparticles. Nanotechnology. 16:2346–2353. Navarro E, Baun A , Behra R, Hartmann NB, Filser J, Miao AJ, Sigg L. 2008. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology. 17:372–386. Panacek A , Kvitek L, Prucek R, Kolar M, Vecerova R, Pizurova N, Zboril R. 2006. Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. J Phys Chem B. 110:16248–16253. Rizzo LY, Golombek SK, Mertens ME, Pan Y, Laaf D, Broda J, Lammers T. 2013. In Vivo nanotoxicity testing using the zebrafish embryo assay. J Mater Chem B Mater Biol Med. 1:3918–3925. Schulte-Merker S, Hammerschmidt M, Beuchle D, Cho KW, De Robertis EM, Nusslein-Volhard C. 1994. Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos. Development. 120:843–852. Shestopalov IA , Pitt CL, Chen JK. 2012. Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development. Nat Chem Biol. 8:270–276. Skebo JE, Grabinski CM, Schrand AM, Schlager JJ, Hussain SM. 2007. Assessment of metal nanoparticle agglomeration, uptake, and interaction using high-illuminating system. Int J Toxicol. 26:135–141. Slater GS, Birney E. 2005. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 6:31. Thisse C, Thisse B. 2008. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 3:59–69. Wang R, Cheng H, Xia L, Guo Y, Huang X, Zhou R. 2003. Molecular cloning and expression of Sox17 in gonads during sex reversal in the rice field eel, a teleost fish with a characteristic of natural sex transformation. Biochem Biophys Res Commun. 303:452–457.
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The effect of silver nanoparticles on zebrafish embryonic development and toxicology Guangqing Xia1, Tiantian Liu2, Zhenwei Wang2, Yi Hou2, Lihong Dong1, Junyi Zhu1 & Jie Qi2 1Department of Life Science, College of Life Science, Tonghua Normal University, Tonghua, P. R. China and 2Key Laboratory of
Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, P. R. China
the last decade, nano-Ag have attracted much interest due to the interesting optical, electrical and magnetic properties that lead to applications in sensing, chemical catalysis, medical fields and personal healthcare (He et al. 2013). Currently, nano-Ag have become the most common group of metallic engineered nano-particles in commercial products, and are widely applied in agricultural sciences, medical products (Chen and Schluesener 2008), and food packaging (Gottesman et al. 2011). The increasing uses of nano-Ag in these fields have increased their release into the environment, and meanwhile, resulted in their exposure to living tissues. This would thus promote special concerns of environmental risk and human health posed by these particles. Earlier reports (Braydich-Stolle et al. 2005, Hussain et al. 2005, Skebo et al. 2007) proved that nano-Ag are more lethal to cell-based in vitro systems than other metal nanoparticles screened. Some evaluations also had been carried out to demonstrate the plausible deleterious effects of nano-Ag in cell lines and/or isolated primary human cells (Braydich-Stolle et al. 2005, Hussain et al. 2005, Skebo et al. 2007, Rizzo et al. 2013). However, this nanotoxicity testing, owing to its over-simplified systems and setups, moderately informative and conclusive results, and limited translational value, cannot be properly addressed using in vitro experimental setups. In the present study, zebrafish embryos, which are relatively well-established to assess the acute toxic effects from exposure to environmental chemicals and nanoparticles, were chosen as model systems for testing the toxicity of the nano-Ag. Unravelling the genetic networks in vertebrates that guide the formation of the three germ layers-ectoderm, mesoderm and endoderm. The embryonic ectoderm is fated to become either neural or non-neural (epidermal), depending on the patterning process that occurs before and during gastrulation (Bakkers et al. 2002). During gastrulation, cells expressing gsc move anteriorly, while cells expressing no tail (ntl) remain more posterior (Schulte-Merker et al. 1994). By mid-gastrula, otx2 RNAs are restricted to the anterior dorsal epiblast (superficial layers) and mesoderm (hypoblast) (Li
Abstract The unique physical and chemical characteristics of nanomaterials, such as the effects of their small size, surface effects, very high rates of reaction, and quantum tunnel effect, have aroused great interest among scholars. However, improper usage has led to an increasing number of nanomaterials entering the environment through various channels, greatly threatening the security of the ecological environment and human health. The urgent need for a scientific assessment of their biosafety can enable nanomaterials to truly benefit humanity. However, the current research in this field is extremely limited with regard to safety standards and waste disposal. In this study, we used silver nanoparticles (nano-Ag) and zebrafish embryos as experimental subjects, and we have reported the deleterious effect on zebrafish embryos treated with different concentrations of nano-Ag, with respect to morphological features (mortality, deformity rate, and heartbeat) and the analysis of expression of relevant genes (sox17, gsc, ntl, otx2); we found a dose-dependent increase in mortality and hatching delay. The results of in situ hybridization indicated that nano-Ag causes a dose-dependent toxicity in embryonic development, and would affect their development and lead to deformity, delayed development, and even death. The safety limit for the concentration of nano-Ag was found to be less than 5 mg/L. Keywords: deformity, heartbeat, malformation, silver nanoparticles, zebrafish embryonic development
Introduction The development of nanotechnology has redesigned the current scenario of science and technology. Many scientists have considered nanotechnology as the next logical roadmap in some areas, such as drug delivery, diagnostics, tissue engineering, and environmental remediation (Alexiou et al. 2005, Abarrategi et al. 2008, Navarro et al. 2008, Meyer et al. 2009). Silver nanoparticles (nano-Ag), as common nano-materials, have taken on wonderful and unique chemo-physical virtues and biological properties. During
Correspondence: Jie Qi, Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, #5 Yushan Road, Qingdao 266003, Shandong, P. R. China. Tel: ⫹ 86-0532-82031986. E-mail: [email protected] (Received 3 December 2014; accepted 21 January 2015)
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et al. 1994). The Sox17 homolog is expressed during gastrulation exclusively in the endoderm, with a restricted pattern of sox17expression and its postulated importance for endoderm formation (Alexander and Stainier 1999). Herein, a green, cost effective pathway was designed for synthesizing nano-Ag with a size of 10–20 nm, by adopting DL-α-aminopropanoic acid as a reductant and capping agent. To enable simple, efficient, and high-throughput toxicity testing in systems significantly more complex than cultured cells, the nano-Ag particles were used as prepared, to evaluate their plausible deleterious effects on zebrafish embryos. Based on the fact that the zebrafish represents a bridge between in vitro cell culture and in vivo mammalian models (Fako and Furgeson 2009), the objective of this study was to evaluate the acute toxicity effects of nano-Ag on zebrafish embryos, especially concerning the toxicity of nano-Ag to some relative gene expressions during the early stages of zebrafish embryonic development by in situ hybridization.
Materials and methods Preparation and characterization of particle suspensions All the chemicals were used as received, and all aqueous solutions were prepared with deionized water. The preparation process was green and simple. In a typical procedure, 0.2 g of PVP was dissolved in 20 mL of 0.1 mol/L AgNO3 solution at an ambient temperature of 15°C. Then 20 mL of 0.1 mol/L DL-α-aminopropanoic acid solution was added into the AgNO3 solution and the mixture was left to settle undisturbed. About 10 min later, the system took on a dark, brownish color, with optical transparency, which indicates the formation of small-sized nano-Ag particles. The solution was centrifuged immediately at 10,000 rpm for 10 min, to separate the nano-Ag from the mother liquid. After centrifugation, the clear supernatant was allowed to stand for another 30 min, to allow the formation of the next batch of nano-Ag. Next, the nanoparticles were separated again by centrifugation. Such a process could be repeated five times, to obtain smaller nano-Ag particles without wastage of raw materials, until the supernatant did not turn dark brown during placement. It should be noted that the settling time should be prolonged with the increasing number of repetitions, and the actual reaction time was also affected by the ambient temperature. The particle size and shape were determined via transmission electron microscope (TEM, Hitachi H-7500) studies.
a different concentration of nano-Ag, was repeated three times, and the standard deviation was calculated. Treated and untreated embryos were anesthetized with 0.2 mg/ml tricaine (3-amino benzoic acid ethyl ester; Sigma-Aldrich, St. Louis, MO, USA), and embedded in methylcellulose.
The whole mount in situ hybridization and immunohistochemistry RNA probes of the germ layer marker gene were prepared from cDNA of the zebrafish otx2 (Li et al. 1994), sox17 (Alexander and Stainier 1999), ntl (Shestopalov et al. 2012), gsc (Slater and Birney 2005) (The plasmid of ntl and gsc were gifted by Dr. Michael REBAGLIATI). The primer sequences were given as the following: otx2: Forward: 5′-CCTGCCATCCTTCCAATA ACA-3′, Reverse: 5′-CACTCGCACACATCCTCTCTA-3′; sox17: Forward: 5′-ATGAGCA GTCCCGATGCG-3′, Reverse: 5′-TCAAGAATTATTATAGC CGC-3′. Their PCR products were cloned into the pEASY-T5 vector and sequenced. RNA probes containing digoxigenin11-UTP were synthesized from the linearized plasmid DNA by PstI for pEASY-T5-otx2,SpeI for pEASY-T5-sox17, SalI for ntl, and NotI for gsc, respectively, using T7 RNA polymerase. Whole mount in situ hybridization was performed principally as described previously (Thisse and Thisse 2008). Embryos of shield period (6 hpf ) and tail bud (10 hpf ) were fixed in 4% paraformaldehyde (PFA, rinsed in PBST, dehydrated in methanol, and stored at ⫺ 20°C until used.
Results and discussions Morphology of nano-Ag The morphology of the silver product as obtained in the typical experiment was investigated by TEM (Figure 1). It could be seen that the product was composed of nano-particles in the range of 10–20 nm. No other morphology was found, which indicated high purity of the product.
Deformity, heartbeat, and malformation Embryos treated with nano-Ag showed dose-dependent toxicity (Figure 2). They showed less deformity under a low concentration of nano-Ag, compared with the control. The embryonic lethality rate showed dose-dependency from 5% to 76.7%, at the concentration levels ranging from 0 to 100 mg/L at 4 hpf, but 100% of the embryos died at 50 and
Collection and exposure of the embryos to nano-Ag Zebrafish were maintained in standard fish facility conditions with a 14:10 h light/dark cycle and fed with living brine shrimp twice per day. The water temperature was maintained at 28°C. For toxicity studies, 20 healthy embryos were transferred to the wells of a 24-well plate, and incubated in different concentrations of nano-Ag (5, 10, 25, 50 and 100 mg/L) at 28°C. Lethality and deformity of the embryos was noted at 4 h post-fertilization (hpf ), 24 hpf, and 48 hpf. The rate of heartbeat was recorded using a stopwatch at 24 hpf by direct microscopic observation. Each treatment, with
Figure 1. Characterization of nanoparticles - TEM image of nano-Ag.
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Silver nanoparticles on zebrafish embryonic development 3
Figure 2. Deformity rate of zebrafish embryos after treatment with different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag at 4 hpf, 24 hpf, and 48 hpf.
100 mg/L, at 24 and 48 hpf (even 8 hpf, data not shown). For development of the embryonic heart, the number of heartbeats was calculated for all samples treated by the serial concentrations of nano-Ag (Figure 3). No significant difference for the heartbeat count was seen at concentrations between 0 and 25 mg/L, while no embryo survived at 50 and 100 mg/L. The morphological development of zebrafish embryos also showed differences at different concentrations of nano-Ag. At 4 hpf, compared to control, the embryos appeared abnormal, regardless of whether the concentration of nano-Ag was 5 mg/L or 100 mg/L, although the percentage was low at the concentration of 5 mg/L (Figure 4A), while at a concentration of 10 mg/L, there was an irregular bulge at the top of the fertilized eggs, and with an increase in nano-Ag concentration, the surface of
Figure 3. Heartbeat of zebrafish embryos after treatment with different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag. No embryo survived to the stage of heartbeat period under 50 and 100 mg/L.
the fertilized eggs became rough and condensed, and the yolk sac of the embryos appeared distorted. After 12 hpf, when the concentration of nano-Ag was higher than 10 mg/L, most of the zebrafish eggs could not finish the transition from the gastrula period to the segmentation period (Figure 4B). At the pharyngula period (24 hpf ), at a lower concentration (5 mg/L) of nano-Ag, the tail of the zebrafish embryos separated from the yolk, while at a concentration of 10 mg/L, there was curved tail separated from the yolk (Figure 4C). When the concentration was higher than 25 mg/L, the tail could not be separated from the yolk (Figure 4C). Above a concentration of 10 mg/L, 60–90% of the surviving embryos showed body malformations after 48 hpf, and the embryos exhibited severe phenotypic changes characterized by bent and twisted notochord, pericardial edema, and degeneration of body parts (Figure 4D).
Figure 4. The effect of different concentrations (0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on zebrafish embryonic development during the cleavage period (A: 4 hpf, B: 12 hpf, C: 24 hpf, and D: 48 hpf ).
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Figure 5. The effect of different concentrations (A–F: 0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on the expression of the sox17 gene.
Expression of zebrafish sox17, gsc, ntl and otx2 During the development of zebrafish embryos, sox17 is expressed in the mesodermal and forerunner cells at 6 hpf, while gsc is expressed in precursor cells at 10 hpf. The Ntl gene is related with the formation of tail, and expresses in precursor cells and chorda-mesoderm at the stage of tail bud (10 hpf ). At the stage of the tail bud (10 hpf ) and 6 somites (12 hpf ), the otx2 gene is expressed in the pituitary gland and the hippocampus of developed head. In order to be more comprehensive in detecting the effect of nano-Ag on the early embryonic development, four reproductive stages of marker gene sox17, gsc, ntl and otx2 were selected for whole mount in situ hybridization, based on the analysis of the four gene maps. We observed different trends with treatment at differ-
ent concentrations of nano-Ag. The expression of sox17 did not show significant difference from 0 to 10 mg/L at the 50% epiboly stage, while the signal of hybridization was darker than at 0 mg/L, with the nano-Ag concentration increased (25–100 mg/L) (Figure 5).To the gsc gene, its transcription levels started to express more from a concentration up to 10 mg/L of nano-Ag (Figure 6). During the stage of tail bud, nano-Ag showed obvious effect on the expression of ntl, at the concentration ranging from 25 to 100 mg/L (Figure 7), and the expression of otx2, at the concentration ranging from 5 to 25 mg/L (Figure 8). All evidence showed that nano-Ag have more or less effect on zebrafish embryonic development, and the effect might be more serious when the concentration is increased up to10 mg/L or higher.
Figure 6. The effect of different concentrations (A–F: 0, 5, 10, 25, 50, and 100 mg/L) of nano-Ag on the expression of the gsc gene.
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Silver nanoparticles on zebrafish embryonic development 5
Figure 7. The effect of different concentrations (A–D: 0, 5, 10, and 25 mg/L) of nano-Ag on the expression of the ntl gene. No picture at 50 and 100 mg/L is available, for the reason that the embryos had all died at a really early developmental stage.
Over 120 years ago, Lea (1889) reported the synthesis of a citrate-stabilized silver colloid, which was the first report of nano-silvers. Since then, silver nanomaterials have been used widely in many fields. Nano-Ag are employed in detergents and as antimicrobial agents that could end up in the environment or inside the organs of animals. We have demonstrated here the toxicity of nano-Ag using zebrafish embryos as experimental materials, and the assay could be used to validate and complement findings obtained in vitro. Compared to control, the results suggested that long-term exposure to a relative dose of nano-Ag would cause varying degrees of deformity, such as fin membrane damage and tail bend, and the greater the concentration, the higher the impact. Moreover, nano-Ag could induce death when the concentration increases up to some extent, such as 50 mg/L or 100 mg/L. Our results also indicated that the nano-Ag not only had the function on the surface of the cells, but also could enter the cells through the fragile skin of embryos, which might
be the consequence of local injury. An earlier report on zinc oxide nanoparticles elucidated the passage through rat and rabbit skins (Kapur et al. 1974, Hallmans and Liden 1979). Unlike teratogenesis, we found that nano-Ag had almost no effect on heartbeat in our experiment. Therefore, we concluded that low concentration of nano-Ag would not affect the development and function of the heart. However, the effect on heart of high dose of nano-Ag was unclear, for the reason that the embryos had died before the heart appeared. At the same time, we could find that the embryonic mortality most likely occurred before 8 hpf. A study by Herrmann had showed that the zebrafish embryos were vulnerable to exogenous chemical influence at the beginning of embryonic development, and that the egg shell was not rigid (Herrmann 1993). The high sensitivity of zebrafish embryo to toxic materials also supported the view. In addition, the study had reported that nano-Ag could not only have an effect on the surface of cells, but also penetrate biofilm
Figure 8. The effect of different concentrations (A–D: 0, 5, 10 and 25 mg/L) of nano-Ag on the expression of the otx2 gene. No picture at 50 and 100 mg/L is available, for the reason that the embryos had all died at a really early developmental stage.
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barriers into cells, which could inevitably affect the embryonic development (Morones et al. 2005). All the results illustrated the potential nanotoxicity of nano-Ag for zebrafish embryos, when they reach a certain concentration. Of course, nano-Ag was relatively safe at a low dose, lower than 5 mg/L. Earlier reports (Braydich-Stolle et al. 2005, Morones et al. 2005, Panacek et al. 2006) have shown that nano-Ag was translocated to various cellular organelles from the site of entry during the early embryonic stage, and the deposition of nanoparticles inside the nucleus of the cells led to toxicity, observed through various mechanisms. In zebrafish, sox17 is expressed in the mesodermal and forerunner cells at 6 hpf (Kanai-Azuma et al. 2002, Wang et al. 2003). Gsc and ntl are expressed in the precursor cells at 10 hpf, which regulate the early development of embryos (Slater and Birney 2005) and tail respectively. The Otx2 gene is expressed at the stage of somites in the pituitary gland and the hippocampus of the developed head, and as a marker gene to detect the ectoderm (Acampora et al. 1995). As indicated by evidence from the in situ hybridization, the nano-Ag invades the cells during the early embryonic stages and bears effect at different levels of expression on embryonic development. Only a low concentration of nano-Ag (5 mg/L or less than 5 mg/L) could have no effect on the normal development on the embryos. Higher concentrations of nano-Ag result in significant effects on mesodermal and ectodermal development, which could be due to delay or inhibition of cell division.
Conclusions From the present study, it is clear that nano-Ag have the potential to pose health and toxicity risks in a dosedependent manner. The nano-Ag-treated embryos exhibited phenotypic defects, altered physiological functions, and degradation of body parts. These results have important implications of potential discrepancies for facilitating the translation of nanomedicine materials into clinical traits.
Acknowledgement This study was financially supported by grants from the Science and Technology Department of Jilin Province (20130102034jc).
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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