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Repurposing Archival Samples for Investigating Toxicological Modes of Action
Citation:
Wehmas, L., S. Hester, AND C. Wood. Repurposing Archival Samples for Investigating Toxicological Modes of Action. GEM, rtp, Nc, September 27, 2017.
Impact/Purpose:
While archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold huge promise for retrospective mode of action studies, little is known about potential genomic artifacts induced by formalin fixation, which could affect toxicological and clinical studies being conducted in FFPE samples. Here we investigated whether direct contact of live tissue with formalin was initiating a transcriptomic response independent of tissue processing or RNA sequencing. We found that tissue directly fixed with formalin had many differentially expressed genes most of which were down-regulated compared to frozen. Comparative analysis of this formalin-induced gene set with two independent studies in Ingenuity Pathway Analysis identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways lending support to our formalin effect hypothesis. Direct fixation did not have a clear impact on chemical response. Upstream regulator analysis identified receptor activation consistent with our referent chemical indicating the formalin gene signature did not confound the results. These finding highlight distinct transcriptional effects of formalin fixation that could impact toxicological mode of action studies using FFPE samples. Our results will aid in advancing chemical safety through retrospective analysis of FFPE tissue samples for AOP discovery and development.
Description:
Little is known about formalin fixation induced genomic artifacts, limiting the use of formalin-fixed paraffin-embedded (FFPE) samples in toxicological and clinical studies. Previously, we identified a consistent shift in transcriptional profiles between paired frozen and FFPE samples, which we hypothesized was caused by fixation of fresh tissue formalin. To test this, liver samples were collected from male B6C3F1 mice treated with 600 ppm phenobarbital (PB) or vehicle control (Con) for 7 days. Samples were: 1) fresh-frozen (FR); 2) directly fixed in 10% buffered formalin for 18 hours and processed to FFPE (FIX); or 3) initially frozen (FR>FIX) and processed as FIX (n=6/group/condition). The FR>FIX group served as a technical control. Samples were sequenced on an Illumina Hi-seq 2500. Reads were aligned using Star (2.4) and analyzed in Partek Flow (6.0). FIX vs. FR resulted in 2946 differentially expressed genes (DEGs) (98% down-regulated). FR>FIX vs. FR resulted in 95% fewer DEGs, suggesting the formalin effect occurs at fixation. Comparative analysis of the formalin-induced DEGs with two independent studies in Ingenuity Pathway Analysis identified enrichment in oxidative stress, mitochondrial dysfunction and transcription elongation pathways. However, direct fixation did not clearly impact chemical response. PB treatment induced 180 DEGs within the FIX and 159 in FR>FIX of which 120 were shared. The DEGs were consistent with CAR/PXR activation and PB exposure, suggesting the formalin effect did not confound the chemical response. Our results could advance the use of FFPE samples for investigating chemical modes of action. This abstract does not reflect EPA policy.