Toxicity and Transcriptome Sequencing (RNA-seq) Analyses of Adult Zebrafish in Response to Exposure to Stabilized FeS and Fe3O4 Nanoparticles
Type of DegreePhD Dissertation
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Iron-based nanoparticles (NPs) have been widely studied for potential applications in many fields, particularly in environmental remediation and biomedical areas. For example, iron sulfide (FeS) nanoparticles, have been frequently tested for removal and immobilization of pollutants in soil and groundwater because of their reducing and adsorbing properties. Magnetite (Fe3O4) nanoparticles have been applied to various biomedical areas such as cell tracking and drug delivery, as well as various environmental remediation settings. This is not only because of their small particle size and high surface-area-to-volume ratio, but also their magnetism. However, bare NPs have a strong tendency to form large aggregates, impeding their delivery and performance. To prevent particle aggregation, various stabilizers are often employed. For instance, polysaccharide stabilizers, carboxymethyl cellulose (CMC) and starch have been used to successfully stabilize FeS and Fe3O4 NPs. While such surface modifications can greatly facilitate their applications, there is limited information available regarding the toxicity and potential environmental impacts of stabilized NPs. As the applications of stabilized NPs continue to expand, it is imperative to understand and assess the associated toxicity to key ecosystem organisms. The zebrafish (Danio rerio) has been widely used as a model organism in eco-toxicological testing, particularly for assessing the risk of chemicals and nanoparticles. Two representative iron based nanoparticles, CMC-stabilized FeS and starch-stabilized Fe3O4 NPs, will be evaluated. The overall goal of this research is to investigate the stress response of adult zebrafish to stabilized FeS and Fe3O4 NPs through the state of the art of transcriptome sequencing (RNA-seq) technique together with tissue burdens and histological alternations assessment. Adult zebrafish were exposed to 10 mg/L bare and CMC stabilized FeS NPs for 96 hours, demonstrating striking differences in gene expression profiles in the liver. This exposure caused significant alterations in gene expression related to immune and inflammatory responses, detoxification, oxidative stress and DNA damage/repair. The Kyoto encyclopedia of genes and genomes (KEGG) pathways related to immune system response and complement and coagulation cascades were found to be significantly up-regulated. A quantitative real-time polymerase chain reaction (RT-qPCR) of candidate genes commonly regulated in the liver confirmed the RNA-seq results. Hepatic inflammation was further confirmed by histological observation of pyknotic nuclei, as well as vacuole formation upon exposure. Additionally, tissue accumulation tests showed a 2.2-times higher iron concentration in the fish tissue upon exposure. Further, when CMC-FeS NPs toxicity was compared with bare FeS NPs, we discovered that CMC coating can alleviate the toxicity caused by FeS NPs. This study provides preliminary mechanistic insights into potential toxic effects of organic matter stabilized FeS NPs, which will improve our understanding of the genotoxicity caused by stabilized NPs. In an effort to understand the impact of coating on NPs induced toxicity, we used RNA-seq to characterize gill and liver transcriptomes from adult zebrafish exposed to Fe3O4 NPs and starch-Fe3O4 NPs for 7 days. Striking differences in gene expression profiles were observed in both tissues. Surface coating dependent toxicity was revealed on both the gill and liver. Fe3O4 NPs exerted greater toxicity than starch-Fe3O4 NPs in the gill. In contrast, starch-Fe3O4 NPs triggered more severe damage on the liver, but likely shared a similar regulatory mechanism with Fe3O4 NPs. The RNA-seq results were verified through RT-qPCR using six genes each for two tissues. Surface coating plays an important role in determining the nanoparticle toxicity, which in turn modulates cell uptake and biological responses. Consequently, surface coating impacts the potential safety and efficacy of nanomaterials. Our findings will aid with the evaluation of risks associated with fate, transport and toxicity of NPs. Additionally, these findings guide the application of NPs and their coatings for optimal utility and decrease the potential for deleterious environmental impacts.
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