(346) Ovarian disrupting effects of real-world surface river water organic mixtures

Samantha L. Cheron^1,2,3, Tingjie Zhan^1,2,3, Jiyang Zhang^1,2,3, Congcong Zhang^1,2,3, Qiang Zhang⁴, Daniel R. Millemann⁵, Josephine A. Bonventre⁵, Brian Buckley^2,3, and Shuo Xiao^1,2,3
1Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA
²Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA
³Center for Environmental Exposures and Disease (CEED), Rutgers University, Piscataway, NJ 08854, USA 
⁴Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322
⁵New Jersey Department of Environmental Protection, Division of Science and Research, Trenton, NJ 08625

Abstract Text:

Rivers play a vital role in supporting diverse ecosystems and meeting various crucial human needs. There is now a growing health concern regarding various organic contaminants in the rivers, including dioxins, polychlorinated biphenyls (PCBs), per- and polyfluorinated substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), and phthalates. Many of these chemicals have been established as endocrine-disrupting chemicals (EDCs). Here, we aimed to study the effects of real-world river water organic mixtures on female ovarian functions and mechanisms involved.

Surface river waters were collected from the Raritan River, a major river in the central New Jersey (NJ) area that flows through several NJ townships hosting ~ 221,000 residents. Organic matters were extracted using the solid-phase extraction method, with double-distilled lab water as control. Targeted mass spectrometry was applied to identify and measure 72 organic chemicals. A 3D hydrogel encapsulated in vitro ovarian follicle growth (eIVFG) system and other approaches were used to investigate the effects of river water organic mixtures on key ovarian functions at morphological, hormonal, and molecular levels.

The results of targeted mass spectrometry and historical data detected PFAS, phthalates, PAHs, dioxins, and alkylphenols in the Raritan River. Follicles treated with extracted organic mixtures at concentrations of 1, 5, and 10-fold higher than the original river waters exhibited comparable outcomes of preovulatory estradiol secretion, ovulation, and oocyte meiotic resumption to the control. However, they dose-dependently inhibited follicle development, increased preovulatory testosterone secretion, and decreased postovulatory progesterone secretion. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed that follicles treated with river water organic mixtures had a decreasing trend of follicle maturation genes (Lhcgr & Pappa) and significantly increased expression of genes related to androgen synthesis (Star and Cyp11a1), oxidative stress (Sod1), apoptosis (Bax), and several aryl hydrocarbon receptor (AhR) target genes (Cyp1a1 & Cyp1b1). Single-follicle RNA sequencing analysis confirmed the RT-qPCR data and identified newly altered gene regulatory pathways related to cell-to-cell signaling and receptor binding. For instance, several genes related to the AhR signaling including Cyp1a1, Cyp1b1, and Ahrr were among top-ranked up-regulated genes, suggesting a potential mechanistic role of AhR in observed ovarian defects. Inflammatory marker genes, including Cxcl2, Il6, and Ccl2, were prominently upregulated, indicating an inflammatory microenvironment in follicles treated with river water organic mixtures.

Oocytes were collected before and after ovulation for single-oocyte RNA sequencing. The bioinformatic analysis revealed that post- but not pre-ovulatory oocyte transcriptomic profiling was altered, which may impair oocyte quality. The top-ranked differentially expressed genes (DEGs) include the up-regulation of Mars2, Acta2, and Itgbl1 and the downregulation of Hoxd8 Plpp4, and Fthl17f. The KEGG analysis showed that these oogenic DEGs are related to metabolism, cAMP signaling, AMPK signaling, and oocyte meiosis.

In summary, we use an ex vivo ovarian follicle culture system to demonstrate that organic mixtures extracted from the real world river surface waters interferes with essential follicle and oocyte health endpoints. In future studies, we will verify the in vitro findings with in vivo oral exposure model and elucidate the molecular toxic mechanisms involved.