Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish
Introduction
With the development of nanoparticles (NPs), nanotechnology is moving toward larger scale production and increasing applications, hence, it is inevitable that NPs and respective byproducts will be released directly and indirectly into the aquatic environment. The unique properties of small size (one or more dimensions of the order of 100 nm or less), chemical composition, agglomeration, high persistence, worldwide distribution and biocompatibility of NPs have received great concern over their toxicity. Several studies have focused on the toxicity of various NPs in the early life stages of fish. For example, silver NPs induce developmental retardation and abnormalities in the early life stages of Japanese medaka (Oryzias latipes) (Wu et al., 2010). Silver NPs at 0.25 mM is more toxic than gold NPs at the same concentration in zebrafish embryos (Bar-Ilan et al., 2009). Zebrafish specimens loaded with functionalized multi-walled carbon nanotubes (MWCNTs) were found to have normal primordial germ cells at early stages and damaged reproduction potential (Cheng et al., 2009). Copper NPs at ≥0.1 mg/L induces gill injury to adult zebrafish (Griffitt et al., 2007). Exposure to TiO2 NPs causes several gill pathologies, including edema and thickening of the lamellae, and significantly increases the total glutathione levels in rainbow trout (Oncorhynchus mykiss) (Federici et al., 2007).
Nano-scale zinc oxide (nano-ZnO), due to their unique properties and ability to form diverse nanostructures, has been widely applied in optoelectronics, cosmetics, catalysts, ceramics, and pigments, etc. The increased use of nano-ZnO has inevitably led to elevated human and environmental exposures and induced the toxicological effects environmental organisms. On the other hand, nano-ZnO particles are easily bio-accumulated by aquatic organisms, wherein they elicit toxic effects. The limited information currently available on the toxic effects of nano-ZnO in fish revealed that nano-ZnO induced the development (Yu et al., 2011) and hatch inhibition (Xia et al., 2011) in zebrafish embryo. The possible toxic mechanisms of nano-ZnO in fish are quite complex. Zhu et al. (2009) suggested that nano-ZnO and Zn2+ elicited embryonic toxicity by increasing the reactive oxidative species (ROS) and/or compromising the cellular oxidative stress response. Moreover, nano-ZnO suspensions have other toxic mechanisms, such as mechanical damage to gill cells by direct contact with the particles (Yu et al., 2011).
Recent studies have investigated the relationship between the toxicity of nano-ZnO and the release of Zn2+ ions (Franklin et al., 2007, Bai et al., 2010, Xia et al., 2011, Fukui et al., 2012, Gilbert et al., 2012). However, some disputes emerged on the role of dissolved Zn2+ in the toxic mechanisms of nano-ZnO. For example, some studies suggested that the toxicity of nano-ZnO in freshwater microalga (Pseudokirchneriella subcapitata) and marine organisms was significantly influenced by the release of Zn2+ (Franklin et al., 2007, Wong et al., 2010). By contrast, other studies seldom ascribed the toxicity of nano-ZnO to dissolved Zn2+ (Bai et al., 2010, Xia et al., 2011). To date, the role of soluble Zn2+ in the toxicity of nano-ZnO in aquatic organisms and the mechanism of the interaction between them have not been fully elucidated (Bai et al., 2010, Wong et al., 2010, Xia et al., 2011).
It has been reported that reactive oxygen species (ROS) can be generated in living organisms exposed to environmental contaminants. The production of ROS has long been regarded as a possible mechanism of nanoparticles-induced toxicity as evidenced by triggered oxidative stress in recent study (Xiong et al., 2011). Moreover, the induction of the ROS results in oxidative damage to macromolecules such as proteins, DNA and lipids, finally leading to the damage of different cellular organelles (Sabatini et al., 2009). Additionally, DNA damage is mainly caused by the hydroxyl radical and superoxide anion radical and this damage is of particular concern because it can cause heritable effects and disease. Under normal conditions, in living organisms damaging effects of oxidative stress are counteracted by antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Malondialdehyde (MDA), an indicator of lipid peroxidation contents, has been considered as one of the molecular mechanisms involved in nanoparticles-induced toxicity (Ma et al., 2010) and its predictive importance as a biomarker for oxidative stress is indicated in different investigation (Xu et al., 2011). Ma et al. had demonstrated that adult mice abdominal cavity injected nanoparticulate anatase (5 nm) exhibited reduced level of SOD, CAT, APX and GSH-Px the activities of brain. Additionally, a recent study suggested that acute exposure 5 mg/L nano-ZnO to adult zebrafish induced oxidative stress in the gills and elevated MDA level the liver (Xiong et al., 2011). Moreover, silver NPs (1 and 25 μg/L) caused cellular and DNA damages as well as oxidative stress in medaka, consequently activating the genes related to metal detoxification/metabolism regulation and radical scavenging action (Chae et al., 2009). Furthermore, exposure of zebrafish to silver NPs at 30 mg/L to 120 mg/L increased the mRNA expression of metallothionein-2 (MT2) in the liver and decreased the activities of the oxyradical-scavenging enzymes catalase (Cat) and glutathione peroxidase 1a (Gpx1a) (Choi et al., 2010). Although the previous studies showed that the nanoparticles could induce oxidative stress and DNA damage, the researches about genes related to oxidative damage were so inadequate that they could not supply the comprehensive information at the molecular level to indicate how the regulation of antioxidant enzyme activities and other negative effects in vertebrates is affected by nanoparticles stress.
The zebrafish embryo is a useful research model because it is small in size and transparent, easy to maintain and rapid embryogenesis and continuous reproduction. In addition, the zebrafish genome has been sequenced and genetic information is rapidly accumulating, which places this freshwater fish in a privileged position for toxicological studies (Berry et al., 2007). Therefore, it is feasible to select toxicological endpoints to find the genes that may be involved in toxicant exposure. Previous studies have demonstrated that embryonic and larval zebrafish were useful for investigating chemical toxicity by using survivorship and development, as well as gene expression as endpoints. Based on the fact that zebrafish embryo represents a good model to assess the toxicity of nanoparticles (Lee et al., 2007), this study has utilized zebrafish embryo as a model to evaluate the developmental toxicity of nano-ZnO. Furthermore, to better understand the oxidative stress process induced by nano-ZnO and at the molecular level to indicate how the regulation of antioxidant enzyme activities in vertebrates is affected by nano-ZnO stress, the present study determined MDA contents, and the activities of SOD, CAT, and GPx, as well as the mRNA expression levels of genes encoding antioxidant proteins. Moreover, employing single cell gel electrophoresis (SCGE) to detect zebrafish embryo DNA damage after nano-ZnO treated. Finally, to determine whether the toxicity of nano-ZnO was attributed to the released Zn2+ from the nano-ZnO, we analyzed the level of Zn2+ ions released from nano-ZnO, and investigated any potential negative effects. The results reveal new insights into the mechanisms underlying the oxidative damages caused by nano-ZnO on zebrafish embryos.
Section snippets
Nanoparticle characterization
Nano-ZnO dispersion with a published particle size of less than 100 nm was purchased from Sigma (St. Louis, MO, USA). A series of exposure suspensions (1, 5, 10, 20, 50, and 100 mg/L) were prepared by stepwise dilution with zebrafish culture medium (consisting of 64.75 mg/L, NaHCO3, 5.75 mg/L, KCl, 123.25 mg/L, MgSO4·7H2O, and 294 mg/L CaCl2·2H2O). And then the suspending solutions containing nano-ZnO particles dispersed by an Ultrasonic processor JY92-IID 900W (Scientz, China) (frequency 25 kHz,
Nano-ZnO characterization
The SEM image of nano-ZnO powder (Fig. 1A) shows that most nano-ZnO powders were spherical and short-rod in shape, and not strongly aggregated. TEM images of nano-ZnO revealed that nano-ZnO particles in suspensions gathered into large aggregates of irregular shapes (Fig. 1B), which may be due to the aggregation of particles. The particle size distribution of nano-ZnO obtained by DLS showed aggregates characterized by single particles with sizes from 50 to 100 nm (Fig. 1C).
Embryonic toxicity of nano-ZnO aggregates and soluble Zn2+
We examined a range of
Discussion
This study presented evidence that nano-ZnO could result in developmental toxicity, induced oxidative stress, and altered the oxidant-associated gene expression in the early stages of zebrafish development. A suite of abnormalities including hyperaemia, pericardial oedema, tail deformity and spinal curvature could be induced in zebrafish embryos after exposure to nano-ZnO. The results also showed that up-regulation of SOD, CAT, and Gpx activities, and MDA content, repressed mRNA of Bcl-2,
Conclusion
In summary, this study demonstrated the occurrence of developmental toxicity in the embryo-larval stages of zebrafish exposed to nano-ZnO. The developmental toxicity tests revealed that that nano-ZnO significantly retarded the embryo hatching and increased malformation after the 96-hpf exposure. Acute nano-ZnO exposure induced DNA damage through excessive ROS, changed the activities of the defense enzymes (SOD, CAT, and GPx), and increased the MDA concentration in zebrafish larvae. These
Acknowledgements
The authors would like to acknowledge support from the “Fundamental Research Funds for the Central Universities” (Grant No. HIT NSRIF. 201025). The authors also would like to thank the generous support from the “State Key Lab of Urban Water Resource and Environment (HIT)” (2010TS01 and QA200812).
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