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

There is growing interest in introducing efficiencies in chemical safety assessment by reducing reliance on conventional chronic toxicity tests. One approach involves benchmark dose (BMD) analysis of gene expression data from short-term adult animal studies to assess chronic toxicity. Whether this approach applies to chemicals that cause developmental and reproductive toxicity (DART), such as dicyclohexyl phthalate (DCHP), is unknown. The present study aimed to (i) investigate how well BMD analysis of gene expression data performed at indicating DART potency using an in utero rat exposure (gestational days 14 to 18 at 0, 100, 300, 600, and 900 mg DCHP/kg-day), and (ii) inform new key events in phthalate syndrome through use of targeted RNA-sequencing. A sub-aim evaluated the consistency of existing polymerase chain reaction array vs. targeted RNA-sequencing data, which were relatively similar. BMD analysis identified gene set points-of-departure (PODs) of 10.4 and 24.7 mg/kg-day, which were similar to some of the lowest PODs for DCHP DART endpoints at 10 mg/kg-day. Further analysis of targeted RNA-sequencing results identified Testin (found in Sertoli cell junctions) as one of a few significantly upregulated genes. Upstream regulator analysis predicted inhibition of SREBPs and gonadotropins, consistent with downregulation of steroidogenesis genes and testosterone production. These results show that transcriptomics can quickly identify a gene set POD comparable to that of DART PODs while also discovering upregulation of Testin as a putative mediator of rodent phthalate syndrome. These data present an important first step to evaluating a transcriptomic approach as an efficient and cost-effective means to assess chemical impacts related to DART.