Exposure to Wildfire Smoke Particulate Matter Induces Ovarian Hyperandrogenism by Activating Aryl Hydrocarbon Receptor

Kuhelika Mali^1,3; Delong Zhang^2,3; Lila Bazina^3,4; Elena Abramova^1,2,3; Tingjie Zhan^2,3; Andrew Gow^1,2,3; Audrey Gaskins⁵; Philip Demokritou^3,4; and Shuo Xiao^1,2,3  

1. Department of Animal Sciences, Endocrinology and Animal Biosciences Graduate Program, Rutgers University, New Brunswick, NJ 08901, USA

2. Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA

3. Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA

4. Department of Environmental and Occupational Health and Justice, School of Public Health, Rutgers University, Piscataway, NJ 08854, USA

5. Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA 33032, USA

Abstract Text:

Wildfires have become more frequent and intense over the past decades, a trend expected to continue. Recent wildfires in California and Canada have significantly affected the air quality across vast areas in the US. Wildfire-generated particulate matter 2.5 (WFPM_2.5), particularly its nano-sized fraction (PM_0.1) can be more toxic than urban background PM_2.5 due to a higher proportion of polar organic compounds, including polycyclic aromatic hydrocarbons (PAHs). WFPM has been related to higher risks of respiratory and cardiovascular diseases, yet the effects on female reproductive health remain largely unknown. Herein, we used a mouse model and a 3D in vitro ovarian follicle culture model to investigate the effects of nano-sized WFPM (WFPM_0.1) on ovarian functions and potential mechanisms involved.

We first exposed 8-week-old CD-1 female mice with 8.9 µg/µL lab-synthesized WFPM_0.1 (LS-WFPM_0.1) in 50 µl water once through intratracheal lung instillation. LS-WFPM_0.1 was generated by complete combustion of pine wood, the major wood in California and Canadian wildfires, using a wildfire smoke simulator (WiSS). Blood sera were collected on days 1, 3, and 7 post-instillation for measuring sex hormones using ELSIA. Late-stage antral follicles were isolated from ovaries, and RT-qPCR was performed to analyze the expression of genes involved in follicle development, steroidogenesis, and AhR signaling, given that PAHs are known AhR activators with endocrine-disrupting potential.

ELISA results revealed comparable circulating estradiol levels between LS-WFPM_0.1-treated and control mice across all three timepoints. However, a tendency of higher testosterone levels was observed on day 3 and 7, although the differences were not statistically significant. RT-qPCR data revealed that antral follicles from LS-WFPM_0.1-treated mice had significantly higher expression levels of androgen synthesis related genes (Star, Cyp11a1, Cyp17a1) and AhR target genes (Cyp1b1, Cyp1a2, Ahrr). These results suggest that in vivo exposure to LS-WFPM_0.1 through intratracheal lung instillation interferes with ovarian steroidogenesis and activates AhR in antral follicles.

PM_2.5 can infiltrate the lung alveoli, reach the bloodstream, and accumulate in other organs, including the ovary. We next used a 3D hydrogel encapsulated in vitro follicle growth (eIVFG) system to examine the direct ovarian impacts of LS-WFPM_0.1. Immature mouse follicles were cultured in eIVFG for 6 days and treated with LS-WFPM_0.1 at 0, 1, 10 and 100 µg/mL from days 4-6 during follicle maturation. Follicles were treated with human chorionic gonadotropin (hCG) on day 6 to induce ovulation. While LS-WFPM_0.1 treatment did not affect follicle growth, ovulation, or oocyte meiotic resumption, it concentration-dependently increased testosterone secretion and upregulated androgen synthesis and AhR target genes, consistent with our in-vivo findings. Single-follicle RNA sequencing (RNA-seq) analysis confirmed the RT-qPCR results and discovered new differentially expressed genes (DEGs) related to inflammation. Single oocyte RNA-seq analysis revealed changes in oocytes transcriptomics, with DEGs associated with mitochondrial respiratory chain and cell cycle.

The same concentration range of real world WFPM_0.1 collected from central New Jersey during 2022 Canadian wildfire were used for in-vitro exposure in eIVFG. Real world WFPM_0.1 treatment resulted in similar ovarian hyperandrogenism and AhR activation. Mechanistic studies using eIVFG revealed that co-treatment of 10 µM resveratrol, a selective AhR inhibitor, reversed LS-WFPM_0.1 induced ovarian hyperandrogenism and transcriptional induction of androgen synthesis and AhR target genes.
In summary, our study indicates that exposure to WFPM_0.1 interferes with ovarian functions by promoting testosterone secretion, inducing ovarian inflammation, and altering oocyte transcriptomics likely via AhR activation.