A. Wamucho, O. Tsyusko, J. Unrine
University of Kentucky,
Keywords: multigenerational, silver nanoparticles
Summary:Studies in the nematode Caenorhabditis elegans have shown that exposure to as-synthesized and transformed (sulfidized) silver nanoparticles (sAg-NPs) can cause various toxic effects, but genomic effects that can be induced by prolonged exposure over multiple generations have not been investigated. A previous multigenerational study of continuous exposure of C. elegans to AgNO3, Ag-NPs and sAg-NPs showed that increased sensitivity, in terms of reproductive toxicity from Ag, occurred as early as the second generation for AgNO3 and Ag-NPs, but not sAg-NPs. This suggested that Ag-NPs may cause mutations or epi-mutations. In this study, we used wild type N2 C. elegans as a model organism to determine if mutations and/or epi-mutations contribute to the observed multigenerational effects. Exposure was carried out using sub-lethal concentrations (EC30 for reproduction) of AgNO3, Ag-NPs and sAg-NPs in simulated soil pore water for 10 generations with the parent generation (F0) unexposed for all groups. Four replicates were used per treatment, from which DNA was extracted from an entire brood of a single worm for whole genome DNA sequencing by NextSeq. Preliminary analysis of sequencing data has been carried out and exposure to pristine and sAg-NPs show greater number of mutations compared to controls and AgNO3 exposed worms, suggesting a NP-specific effect. Mutations were mostly contributed by SNPs, not deletions or insertions. This suggests that exposure to both pristine and transformed Ag-NPs, but not Ag ions, may introduce germline mutations that may be passed to subsequent generations. These results do not explain the increased reproductive sensitivity to Ag ions and pristine Ag-NPs observed in our previous multigenerational study. We hypothesized that epigenetic mechanisms are involved. Increased DNA methylation (6-methyladenine), an epigenetic marker, which was recently discovered in C. elegans, has been shown to negatively impact reproduction over multiple generations. We are currently investigating DNA methylation as a potential mechanism by which reproductive toxicity can be passed on to subsequent generations after exposure to Ag-NPs. Preliminary experiments using slot blots show that there is a potential increase in DNA methylation after 2 generations of exposure to pristine Ag-NPs. Interestingly, there seems to be a decrease in DNA methylation for AgNO3 exposed worms. In addition to confirming these results and testing sAg-NPs, we will also examine if the higher levels of DNA methylation persist in subsequent generations after cessation of exposure.