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Toxicogenomic analysis of >25-year-old archival tissue samples indicate metal sulfate mode of action and potency
Citation:
Wehmas, L. AND S. Hester. Toxicogenomic analysis of >25-year-old archival tissue samples indicate metal sulfate mode of action and potency. SOT, Nashville, TN, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.22630594
Impact/Purpose:
Presentation to the Society of Toxicology 62nd Annual Meeting and ToxExpo March 2023
Description:
Archival tissues provide an invaluable resource for retrospective molecular analyses; yet widespread use for toxicogenomics has been limited by formalin-fixation related damage to nucleic acids. Advances in targeted RNA-sequencing technologies have helped overcome this challenge by focusing reads on smaller expressed regions of the transcriptome; thereby opening archives for new studies into chemical modes of action and retrospective proof of principle research. In the present effort, formalin-fixed paraffin-embedded (FFPE) rat lung or liver tissue (>25- years-old) was obtained from early timepoints (15-16 days) of two chronic toxicity studies (nickel sulfate: NiSO4 by inhalation or cupric sulfate: CuSO4 by ingestion) archived by the National Toxicology Program (NTP). Each study tested multiple dose levels (3.5 – 30 mg/m3 NiSO4 or 1000-16000 ppm CuSO4 plus concurrent controls) in both male and female rats. Tissue type was selected based on the 2-year pathology reports and collected from the NTP archive for targeted, whole transcriptome sequencing by BioSpyder Technologies (n=5/sex/treatment). We analyzed each chemical for significant differentially expressed genes (DEGs, FDR < 0.05, fold change ± 2) across treatment groups and looked for consistencies in molecular pathway response using comparison analysis in Ingenuity (Qiagen). Dose responsive genes underwent benchmark dose (BMD) analysis in BMDExpress (v.2) to compare short-term transcriptomic measures of chemical potency with traditional measures from the 2-year NTP pathology data. We observed a dose dependent increase in significant genes for the NiSO4 exposure from 54 – 106 DEGs at 3.5 – 15 mg/m3 with a slight drop at the highest treatment group whereas for CuSO4, there was an increase in DEGs across the upper three treatments, which identified 31 – 458 DEGs at 4000-16000 ppm. A total of 26 and 8 DEGs were shared across treatment groups for NiSO4 and CuSO4, respectively representing consistency in response that was not likely a result of fixation related damage. When mapped to pathways, NiSO4 DEGs indicated upregulation in inflammation and immune response, which has been linked to metal exposures while pathway analysis of CuSO4 DEGs suggested disruption in sterol synthesis as the top impacted pathway across treatments, which is known to be modified by copper levels. Dose response modeling of NiSO4 genes resulted in median BMDs of 0.91 mg/m3 and 0.99 mg/m3 for female and male rats, respectively, when mapped to the most sensitive gene ontology biologic process. This was 8-fold higher than the most sensitive lung-related BMD (hyperplasia) at 2 years. For CuSO4, the median gene set BMD values (2558 ppm for female and 4260 ppm for male rats) were also ~8-fold higher than the most sensitive 2-year BMD values of adverse health outcomes (liver inflammation and forestomach hyperplasia, respectively). These results show promise in obtaining potentially useful transcriptomic data from decades old archival tissue samples and suggest utility in using gene response to model chemical potency. The views presented in the abstract do not necessarily represent the US EPA.
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DOI: Toxicogenomic analysis of >25-year-old archival tissue samples indicate metal sulfate mode of action and potency
20230306_WEHMAS_SOT_POSTER_V6_CLEAN.PDF (PDF, NA pp, 1786.559 KB, about PDF)