Biologically relevant reductions in fetal testosterone and Insl3 induced by in utero exposure to high levels of di-isononyl phthalate (DINP) in male rats

I Some phthalate esters alter male rat reproductive development during sexual differentiation by interfering with fetal testis maturation resulting in reduced Leydig Cell synthesis of testosterone and insulin-like 3 (Insl3) hormones. Gene transcripts associated with steroid hormone and cholesterol transport, and cholesterol synthesis and lipid metabolism also are reduced. These alterations cause permanent malformations of hormone-dependent tissues, sperm production and fertility in male offspring; effects known as the “Phthalate Syndrome.” We have shown that administration of a high dose of 750 mg diisononyl phthalate (750 mg/kg/d DINP) during sex differentiation reduced fetal testis testosterone production (T Prod), testis gene expression and induced a low incidence of reproductive malformations in male rat offspring. In the current study we administered DINP at even higher dose levels (1.0 and 1.5 g/kg/d) from gestational day (GD) 14 to postnatal (PND) 3 to determine if these effects were dose related and if the magnitude of the effects could be predicted from a statistical model of fetal testosterone production (T Prod) and Insl3 mRNA levels. These models were previously developed using dipentyl phthalate (DPeP) data from fetal T Prod and postnatal studies. We found that the severity of the demasculinizing effects on the androgen-dependent organs and gubernaculum by DINP were accurately predicted from the statistical models of fetal T prod and Insl3 mRNA, respectively. Taken together, our results indicate that reductions fetal T prod and Insl3 predict the severity of demasculinizing effects in utero exposure to the phthalates DINP and DPeP regardless of potency.

Impact/Purpose

In utero exposure to a mixture of the perfluroalkyl-isopropyl pesticide (PFAP) Pyrifluquinazon with a Phthalate cumulatively disrupts male rat reproductive development via different mechanisms of action. Background/Overview:             Humans carry residues of multiple synthetic chemicals at any given point in time.  Research has demonstrated that compounds with varying molecular mechanisms that disrupt common Key Events can act in concert to produce cumulative adverse effects.  As such, one of the most pressing issues in toxicology and risk assessment is the evaluation and cumulative assessment of chemical mixtures. For example, OPPT is currently considering approaches to estimating the risk of mixtures of PFAS and mixtures of phthalates.  In addition, several phthalates including the one used herein are on the TSCS high priority list for regulation.               Our research group has published several research studies on the combined effects of in utero exposure to chemicals that have converging Key Events within an androgen receptor signaling Adverse Outcome Pathway network.  This abstract describes a new studt that tests this hypothesis.  The study design is based on a binary combination of the pyrifluquinazon (PFQ), a pesticide with perfluoroalkyl isopropyl chain, known to disrupt androgen receptor function, and dibutyl phthalate (DBP), a phthalate on the TSCA high priority list that disrupts fetal testosterone synthesis. Since these two chemicals disrupt fetal androgen signaling, albeit via different mechanisms of toxicity, we hypothesized that this binary mixture would produce permanent, adverse male reproductive effects in cumulative, dose additive manner when the mixture was administered to the pregnant rat during the period of fetal male sexual differentiation. Relevance to EPA Program/Regional Research Needs/Priorities:  This paper contributes to  subproduct CSS.4.2.1.1 and is relevant to the needs of OPPT as they consider cumulative risk assessment approach for phthalates and PFAS chemicals, PFQ being a perfluoroalkyl pesticide (PFAP).  Further, this information adds to the growing body of scientific information that contributes to the methods and scientific rationale for addressing issues related to chemical mixtures.  In general, this body of literature indicates that dose addition should be the default model for assessing risk the risk of chemical mixtures that disrupt common signaling pathways even when they contain chemicals that disrupt development via different mechanisms of action Name(s) of Program Office Reviewer(s) of Earlier Drafts:  ORD/CCTE scientist Jason Lambert provided Technical Manuscript Reviews and OW/ECD scientist Dr. Colleen Flaherty also provided input on the abstract. Impact/Potential Implication of the Findings: This study demonstrates the importance of assessing the toxicity for multiple chemicals with overlapping Key Events in an AOP network.  Like the results of the current study, an emerging body of information from multiple laboratories supports the hypothesis that when individual chemicals that disrupt androgen receptor signaling via multiple MIEs are administered as a mixture to pregnant female rats during the critical window of sexual differentiation, permanent adverse effects occur in the offspring at doses lower than those at which the individual chemicals produce effects in individual chemical exposures.  Findings Advancing Existing Scientific Knowledge:  The present study suggests that male fetuses may be at increased risk due to cumulative exposure of the pregnant woman to multiple anti-androgenic chemicals.  Moving forward, cumulative risk assessment approaches should consider grouping chemicals based on a common biological signaling pathway and these pathways can be elucidated using the AOP framework.  Further, the dose addition continues to be the most accurate model for predicting mixture-based toxicological effects.     

Citation

Gray, L. Biologically relevant reductions in fetal testosterone and Insl3 induced by in utero exposure to high levels of di-isononyl phthalate (DINP) in male rats. Academic Press Incorporated, Orlando, FL, 465(116454):1, (2023). [DOI: 10.1016/j.taap.2023.116454]

DOI: Biologically relevant reductions in fetal testosterone and Insl3 induced by in utero exposure to high levels of di-isononyl phthalate (DINP) in male rats