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Targeted RNA-sequencing of testes from fetal rats exposed to dicyclohexyl phthalate informs potency and adverse outcome pathway development (SOT 24)
Citation:
Waterbury, C., J. Conley, L. Gray, AND L. Wehmas. Targeted RNA-sequencing of testes from fetal rats exposed to dicyclohexyl phthalate informs potency and adverse outcome pathway development (SOT 24). Society of Toxicology (SOT) 63rd Annual Meeting and ToxExpo, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25475944
Impact/Purpose:
Presentation to the Society of Toxicology (SOT) 63rd Annual Meeting and ToxExpo March 2024. Dicyclohexyl phthalate (DCHP) is a high production volume phthalate with reproductive toxicity concerns. Previous research identified that DCHP reduced testosterone production and expression of 14 genes important to testis development following short-term exposure during pregnancy. This is consistent with other phthalates, yet questions remain as to how these effects start at the molecular level. The present work used whole genome, targeted RNA-sequencing (BioSpyder Technologies) to measure changes in gene expression related to DCHP exposure and discover molecular events involved in fetal male testes toxicity. Pregnant Sprague Dawley rats received 0 (vehicle), 100, 300, 600 (n=3/dose-level), or 900 (n=2) mg/kg-day DCHP orally during pregnancy from day 14 to 18. At day 18, messenger RNA was extracted and sequenced from testes pooled by litter. Reads were aligned using STAR, normalized using count per million mapped reads, and analyzed for differential gene expression in Partek Flow (filtered counts ≤ 25; pseudocount = 0.5). Dose response modeling of gene expression data to estimate chemical potency was completed with BMDExpress2 (best fit p-value > 0.1; genes passed filter ≥ 3) while molecular pathway analysis was completed with Ingenuity Pathway Analysis (p-value ≤ 0.05; fold change ± 1.50). Gene expression analysis identified 40 genes that were shared across DCHP treatments. Comparisons with previous gene expression measures identified 72 that were shared and 54 new genes including Testin, which is involved with blood-testis barrier disruption. Benchmark dose (BMD) analysis identified a gene expression-based potency estimate for DCHP of 40.9 mg/kg-day, which was slightly more sensitive than the rat developmental and reproductive potency estimate of 68 mg/kg-day. As for molecular pathway analysis, four of the five most significantly impacted pathways were downregulated and involved in cholesterol biosynthesis processes. These results fit with how DCHP decreases testosterone production in fetal male rats. Upstream regulator analysis predicted activation of NR0B1 and inhibition of NR5A1 as potential mediators of the gene expression results. Both regulators are important in testis development and may be significant in DCHP's adverse effects . Our results show that targeted RNA-sequencing data was able to identify new molecular effects consistent with DCHP developmental and reproductive toxicity. This abstract does not necessarily represent U.S. EPA policy.
Description:
Background and purpose Dicyclohexyl phthalate (DCHP) is a high production volume phthalate ester with reproductive toxicity concerns. Previous research identified that DCHP reduced overall testosterone production and expression of 14 genes vital to reproductive development in a dose-dependent manner in fetal rat testis following short-term in utero exposure. This is consistent with other phthalate esters, yet questions remain as to how these effects are initiated at the molecular level. Gene expression analysis using RNA-sequencing can provide a cost-effective means of answering this question while providing information on chemical hazard and potency. Therefore, the present work applied whole genome, targeted RNA-sequencing (BioSpyder Technologies) to in utero DCHP exposed fetal rat testis to expand on measures of gene expression previously limited to qRT-PCR arrays. The goal of this study was to use benchmark dose modeling of gene expression changes to indicate DCHP potency while identifying putative molecular events involved in male reproductive tract malformations. Methods and Materials Pregnant Sprague Dawley rats received 0 (vehicle), 100, 300, 600 (n=3/dose-level), or 900 (n=2) mg/kg-day DCHP by oral gavage from gestation day (GD) 14 to 18. Messenger RNA, extracted from testes pooled by litter at GD 18, underwent targeted RNA-sequencing. Reads were aligned using STAR, normalized using count per million mapped reads, and analyzed for differential gene expression in Partek Flow (filtered counts ≤ 25; pseudocount = 0.5). Gene expression-based potency estimates were calculated in BMDExpress2 (best fit p-value > 0.1; genes passed filter ≥ 3) while molecular pathway enrichment was performed using Ingenuity Pathway Analysis (p-value ≤ 0.05; fold change ± 1.50). Results After STAR alignment, between 93.0% and 95.9% of the reads were aligned in the samples, and, after filtering and normalization, 11,352 genes remained. Differential gene expression analysis identified 40 genes that were shared between all four treatment groups. Comparisons with previous custom qRT-PCR measures identified 53 genes that were not consistent with the custom arrays, 72 shared biomarker genes, and 54 new potential biomarker genes. Amongst the 72 shared genes, several demonstrated consistencies in dose-dependent changes between the two technologies, including Insl3, which is integral to male reproductive development. Conversely, targeted RNA-sequencing resulted in the identification of several new potential biomarkers like Testin, a Sertoli cell factor associated with disruption of the blood-testis barrier which was not included in the qRT-PCR array. Benchmark dose (BMD) analysis identified a gene expression-based potency estimate of BMD = 40.9 mg/kg-day with a BMDL = 29.9 mg/kg-day, which was about 2-fold more sensitive than the apical developmental and reproductive potency of BMDL10 = 68 mg/kg-day. The most sensitive gene ontology (GO) pathway was steroid hormone biosynthetic process, and the most downregulated canonical pathway was superpathway of cholesterol biosynthesis. In fact, four of the five most significantly impacted canonical pathways were downregulated and involved in some part of cholesterol biosynthesis. These results coincide with the known effects of DCHP on testosterone production in fetal male rats. Upstream regulator analysis predicted activation of NR0B1 and inhibition of NR5A1 as potential mediators of the gene expression results. Both regulators are important in testis development and may be significant in DCHP mechanism of action. Conclusion Our results show that targeted RNA-sequencing data was able to identify molecular effects consistent with DCHP developmental and reproductive toxicity. Additionally, several new, putative biomarker genes were identified, which require confirmation......... This abstract does not necessarily represent U.S. EPA policy.
URLs/Downloads:
DOI: Targeted RNA-sequencing of testes from fetal rats exposed to dicyclohexyl phthalate informs potency and adverse outcome pathway development (SOT 24)
20230908_WATERBURY_POSTER_V5_CLEAN.PDF (PDF, NA pp, 1785.113 KB, about PDF)