Targeted RNA-sequencing of testes from fetal rats exposed to dicyclohexyl phthalate informs potency and adverse outcome pathway development

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.

SourceEPA

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