The sublethal effects of nanosilver on thyroid hormone-dependent frog metamorphosis




Carew, Amanda

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Nanoparticles (NPs) are engineered in the nanoscale (<100nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver (nAg) is the most prevalent nanoparticle in consumer products due to its strong antimicrobial action and can be released to the environment during product manufacture, usage and disposal. The predicted environmental concentrations are within the North American guidelines for the protection of aquatic life and in drinking water. While nAg toxicity at high concentrations has been well described, the sublethal effects at environmentally-relevant concentrations are relatively unknown. Initial screening in our lab showed nAg was a potential endocrine disrupting chemical (EDC). Amphibian metamorphosis is mediated by thyroid hormone (TH), and nAg perturbed TH-dependent transcriptional responses in the tailfin of bullfrog (Rana catesbeiana) tadpoles. The primary objective of this thesis was to further investigate and characterize the effects of low, environmentally relevant concentrations of nAg on TH-dependent metamorphosis in R. catesbeiana and Xenopus laevis. Two chronic, 28 day in vivo exposures at 0.06 and 6µg/L nAg were conducted with premetamorphic R. catesbeiana tadpoles using TH to induce precocious metamorphosis. Ionic silver (iAg) was also examined to control for the complete dissolution of Ag. Analysis of metamorphic stage progression demonstrated nAg-induced acceleration of hindlimb growth and development. After 6 days of nAg exposure, analysis with quantitative real-time polymerase chain reaction (QPCR) demonstrated nAg-induced disruption of TH-responsive transcripts in a tissue-specific manner. Furthermore, the nAg effects could not be fully explained by iAg, indicating NP-specific disruption. Two chronic, 28 day exposures to 0.018-1.8 µg/L nAg were conducted on X. laevis premetamorphic and prometamorphic tadpoles. nAg was found to significantly bioaccumulate in tadpole tissue after 28 days. Furthermore, nAg increased the hindlimb length during early premetamorphosis and in post-metamorphic juvenile tadpoles. Using an in-house MAGEX microarray and QPCR transcriptional analysis, 7 biomarkers of nAg exposure were validated. Five of these targets showed disruption to their TH-response. Furthermore, the increased mRNA abundance of two peroxidases indicated that nAg generated reactive oxygen species (ROS) even at low, environmental concentrations. This thesis demonstrates that nAg has consistent EDC actions across two distinct amphibian species, and the data suggest that regulatory guidelines for silver may need revision. A X. laevis derived fibroblast-like TH-responsive cell line, XTC-2, was used in conjunction with the 7 biomarkers of nAg exposure to gain mechanistic insight into the role of ROS in TH signaling disruption. Monocultures were created and validated to increase the specificity of TH-response. While the monocultures were successfully created, the biomarkers were not responsive to nAg in this cell line. Additional investigations were made into the relationship between genetic sex and responsiveness to TH. Genetic sexing methods were used to investigate transcriptional differences between males and females during natural and TH-induced metamorphosis. The sexing protocol was optimized and validated successfully. The genetic sex was determined for premetamorphic and prometamorphic X. laevis tadpoles exposed to TH for 48 h. QPCR and microarray analysis were used to identify three markers that demonstrated transcriptional sex-bias during early gonadal differentiation stages.



endocrine disruption, thyroid hormone, silver nanoparticles, amphibian metamorphosis, toxicology, molecular biology