Year of Publication

2019

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Toxicology and Cancer Biology

First Advisor

Dr. Olga V. Tsyusko

Abstract

There has been an increase in the incorporation of silver nanomaterials into consumer products due to their antimicrobial properties. Therefore there is potential for silver nanoparticles (Ag-NPs) to leach out into the environment during different life-cycle stages of these nanomaterial-containing products. Concern about the toxicity of Ag-NPs has led to investigations into their toxic effects on a variety of organisms mainly using acute and sub-chronic, single-generation exposures. The focus of this project was to understand the effects of long-term continuous multigenerational exposure to AgNO3 and Ag-NPs in both pristine and environmentally transformed forms, on the model organism, Caenorhabditis elegans, a soil nematode.

A previous multigenerational C. elegans study, showed increased sensitivity in terms of reproductive toxicity, in response to AgNO3 and Ag-NPs, but not sulfidized Ag-NPs (sAg-NPs), with increasing generations of exposure. The reproductive toxicity persisted in subsequently unexposed generations even after rescue from the exposure. We hypothesized that genomic mutations and/or epigenetic changes were possible mechanisms by which the reproductive toxicity was inherited. We investigated the potential for induction of germline mutations in C. elegans after exposures for ten generations to AgNO3, Ag-NPs, and sAg-NPs using whole genome DNA sequencing. Epigenetic changes at histone methylation markers, (H3K4me2 and H3K9me3), and DNA methylation at adenosine (N6-methyl-2’-deoxyadenosine) were investigated after multigenerational exposure as well as after rescue from the exposure using enzyme-linked immunosorbent assays (ELISA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), respectively. Expression levels of the genes of methyltransferases and demethylases, associated with the histone methylation markers and DNA methylation, were also examined.

Our results for germline mutations reveal no significant differences between the nematodes exposed to AgNO3 or pristine Ag-NPs when compared to controls. The significant increase in the number of transversion was observed only for sAg-NPs. However, a trend toward an increase in the total number of mutations was observed in all Ag treatments with some of those mutations having a predicted moderate or high impact. This potentially contributed towards reproductive as well as growth toxicity shown previously after ten generations of exposure in every treatment.. These results did not entirely support the multigenerational reproductive toxicity observed previously. Epigenetic responses at histone methylation markers revealed opposite patterns between pristine and transformed Ag-NPs with Ag-NPs causing a significant increase while exposure to sAg-NPs resulted in significant decrease in methylation at H3K4me2 mark. The increase in H3K4me2 levels was also inherited by subsequent unexposed generations rescued from Ag-NP exposure. Only sAg-NPs caused a significant decrease in methylation at H3K9me3 mark. Changes in mRNA levels for histone methyltransferases and demethylase corresponded with the histone methylation levels affected by Ag-NPs and sAg-NPs. For DNA methylation, a significant increase was observed only for AgNO3, which was not inherited after the rescue.

In conclusion, while germline mutations with a high or moderate impact may affect reproduction, our results do not support this as a mechanism for the heritable increase in C. elegans sensitivity to reproductive toxicity from AgNO3 and pristine Ag-NPs. The epigenetic changes, however, do show partial correlation with the observed reproductive toxicity. The reproductive multigenerational effects of AgNO3 can be attributed to changes in DNA methylation whereas that of Ag-NPs can be attributed to changes in histone methylation. Further studies, focused on the investigation of changes in histone and DNA methylation levels at specific loci using chromatin immunoprecipitation sequencing (ChIP-Seq) and methylated DNA immunoprecipitation sequencing (MeDIP-Seq), respectively, are warranted for a better understanding of the impact of such changes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2019.146

Funding Information

This work was supported by the U.S. EPA and NSF through the U.S. National Science Foundation (NSF) under EF-0830093, DBI-1266252, and CBET- 1530594. AW was also supported by the Lyman T. Johnson fellowship and the NIEHS through the 5T32ES007266-24 training.

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