Gestational Exposure to EHDPP Disrupts Pancreatic Endocrine Specification and Impairs Postnatal Glucose Homeostasis in Offspring

Background: Organophosphate flame retardants (OPFRs) are widely detected in pregnant women and have been associated with increased diabetes risk. However, whether gestational OPFR exposure disrupts embryonic pancreatic development, a critical determinant of glucose homeostasis, remains unclear. Objectives: We investigated the effects of gestational 2-ethylhexyl diphenyl phosphate (EHDPP) exposure on pancreatic developmental programming and offspring glucose homeostasis. Methods: Pregnant mice were exposed to EHDPP at 0.4 or 2 mg/kg/day by oral gavage. Transcriptional profiling of the E15.5 embryo pancreas was performed, with findings contextualized using published single-cell RNA-seq pseudotime trajectories. Key transcription factor expression and cell-type distribution were validated and quantified by immunofluorescence. Lineage deviation was assessed in vivo and in ES-E14TG2a embryonic stem cell differentiation. Islet maturation markers were evaluated at E18.5 and PND4, and offspring glucose homeostasis was assessed on PND21. Results: At PND21, EHDPP-exposed offspring exhibited disrupted glucose homeostasis in both sexes, as evidenced by altered glucose tolerance and reduced glucose-stimulated insulin secretion. Transcriptomic profiling of the E15.5 embryonic pancreas revealed coordinated suppression of pancreatic developmental and endocrine transcriptional programs at both doses, including multiple genes annotated to the maturity-onset diabetes of the young (MODY)-associated category. Single-cell pseudotime analysis indicated impaired progression from pancreatic progenitors toward endocrine lineages. Immunofluorescence confirmed reduced protein levels of FOXA2, PDX1, and NEUROD1, alongside a decreased proportion of NGN3+/PDX1+ and NEUROD1+/NGN3+ double-positive cells. At a higher dose, aberrant hepatic-like molecular features were observed at both mRNA and protein levels and were corroborated by ES-E14TG2a embryonic stem cell differentiation. These developmental defects converged to reduce the β-cell pool and impair the β-cell identity at E18.5 and PND4. Discussion: Gestational EHDPP exposure perturbs pancreatic endocrine development at both exposure doses, with additional hepatic-like molecular features observed at a higher dose. These developmental alterations were accompanied by impaired postnatal islet secretory function and glucose homeostasis. This study identifies the embryonic pancreas as a sensitive developmental endpoint of OPFR exposure and provides insight into how gestational exposure may influence later metabolic health.