Targeted RNA-sequencing of testes from fetal rats exposed to dicyclohexyl phthalate informs potency and adverse outcome pathway development
The US EPA is refining long-term animal testing to increase the pace of chemical risk assessments. One approach involves using benchmark dose (BMD) response modeling of gene expression data from short term adult animal studies to indicate longer term chemical risk. Whether this approach works with developmental and reproductive toxicants is less certain. Dicyclohexyl phthalate (DCHP) is a high production volume phthalate ester with known effects on testes. Despite knowledge on several key events in phthalate testicular dysgenesis, the molecular initiating event is still unknown. The present study aims to assess how well gene expression-based BMD analysis of fetal testes from an in utero exposure performs at predicting developmental and reproductive toxicity endpoints for DCHP while applying, whole transcriptome, targeted RNA-Sequencing (RNA-Seq) to  inform new molecular targets involved in phthalate-induced testicular dysgenesis. BMD analysis of targetd RNA-Seq data identified gene-set based BMD values that were 4 and 6-fold lower than development-related decreased anogenital distance and reproductive-related testis atrophy measures (101 and 144 mg/kg-day, respectively). Further analysis of top differentially expressed genes from targeted RNA-Seq identified Testin, Svs5, Lrrc39, etc. that may warrant further investigation in DCHP-induced testicular dysgenesis. Canonical pathway comparisons (Ingenuity Pathway Analysis) indicated suppression of steroid/cholesterol biosynthesis processes while biological function comparisons identified disrupted reproductive and genitourinary development processes across dose groups consistent with known DCHP effects. These results show promise in using targeted RNA-Seq from short-term in utero exposures to rapidly and quantitatively indicate developmental and reproductive effects, thereby helping EPA more quickly assess chemical impacts across a range of adverse effects. This abstract does not represent US EPA policy and mention of products does not constitute endorsement. Overall design: Pregnant dams were exposed by oral gavage to 0 (corn oil vehicle), 100, 300, 600, or 900 mg/kg-day dicyclohexyl phthalate from gestational day 14 to 18. On gestational day 18, fetal rats were necropsied with testes collected and pooled by litter. There were 3 pooled testes samples per treatment except 900 mg/kg-day which had 2 pooled testes.