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Establishing methods to assess epigenetic signatures in archived study tissues
Citation:
Malloy, M., G. Carswell, B. Bennet, P. Bushel, L. Wehmas, S. Hester, AND B. Chorley. Establishing methods to assess epigenetic signatures in archived study tissues. 2020 SOT Annual Meeting, Research Triangle Park, NC, March 15 - 19, 2020. https://doi.org/10.23645/epacomptox.14340239
Impact/Purpose:
Poster presented to the Society of Toxicology annual meeting March 2020. Biorepositories contain millions of preserved tissue samples from human, rodent, and alternative models. These samples often come from informative studies covering hundreds of compounds with data on study methods, animal condition, and results. Revisiting these experiments using new methods on preserved samples will aid in a molecular understanding of adverse chemical effects. These advances will also help in translating chemical effects on the animal to those that occur in cell culture without the need for new and costly experiments. This reduces animal use, helps prioritize hazard, and increases risk assessment efficiency - all of which is useful to ORD and other organizations (such as chemical/pesticide registrants). We propose to establish methods to measure DNA methylation and hydroxymethylation in preserved formalin-fixed paraffin-embedded (FFPE) mouse liver tissues. DNA methylation and hydroxymethylation (the methylome) are known to respond to environmental exposures and these changes can persist over a lifetime, or even across generations, and may contribute to disease. By measuring changes in the methylome using archived tissues, we can link pre-existing animal data to molecular targets involved in disease susceptibility resulting from environmental chemical exposures. Long-term, these methods will improve and refine early key events in adverse outcome pathways while providing a bridge between animal studies and cell-based assays.
Description:
Revisiting archived studies using new methods on preserved samples will aid in a molecular understanding of adverse chemical effects, thereby reducing animal use, enhancing chemical hazard prioritization, and providing weight of evidence for regulatory decision making. Alterations in DNA methylation can occur with environmental chemical exposures, persist over time, and result in adverse health outcomes. In this pilot work, we tested the ability of Reduced Representation Bisulfite Sequencing (RRBS) to measure DNA methylation in formalin fixed paraffin embedded (FFPE) tissue samples relative to paired frozen controls. The 5 yr old liver tissue samples were obtained from male mice treated with 600 ppm phenobarbital (PB), a known mediator of DNA methylation changes and potential tumorigen, or vehicle control in drinking water for 28 d. Sequencing results indicate that FFPE samples contained significantly higher CpG sites relative to frozen samples (average 8.5 and 4.4 million reads, respectively with p=0.010; Kruskal-Wallis rank sum test); however, with higher read depth criteria (≥ 5X CpG site coverage), the remaining read counts for each sample type were statistically similar. In addition, read count variability in FFPE samples was significantly higher (p-value = 1.767e-11; Kruskal-Wallis rank sum test) than in frozen samples, overall indicating poorer quality sequencing data associated with FFPE. Only a few differentially methylated regions (DMRs) due to PB exposure were quantified in either sample type (4 and 1 DMRs, FDR p1%), 644 and 963 DMRs were identified for frozen and FFPE samples, respectively; however, only 30 DMRs were shared between sample types. In addition, genes linked to DMR regions overlapped less than 2% of previously published DMR genes due to short-term PB exposure in mouse liver, regardless of sample source. This indicates the study sample size (n=3 per condition) was of insufficient power to determine true DMRs. Follow-up studies in progress will address this issue. With sufficient power, we estimate that these FFPE-sourced data will accurately represent data from frozen/fresh tissue, thereby unlocking epigenetic-based data from archived tissue to refine mechanistic understanding of susceptibility to chemical-mediated toxicity. This abstract does not necessarily reflect the policy of the US EPA.
URLs/Downloads:
DOI: Establishing methods to assess epigenetic signatures in archived study tissues
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