Bisphenol A and its analogues induce Fibrosis in a 3D Human Uterine Fibroid Model through TGF‐beta Signaling
Authors: Liu Jingli , Castro Lysandra , Clayton Natasha P. , Bushel Pierre , Li Stephen , Yu Linda , Flagler Norris D. , Scappini Erica , Yan Yitang , Dixon Darlene 
Published: April 2020

BackgroundBisphenol A (BPA) and its analogues (BPS, BPAF) are ubiquitous environmental contaminants with some having estrogenic properties. Recently, BPA was found to induce fibrosis in the liver, lung, and heart, and both BPA and BPAF, reportedly, can stimulate fibrosis in uteri of mice (1, 2, 3). In women, uterine leiomyomas (fibroids) are highly prevalent estrogen‐responsive tumors that often have excessive accumulation of extracellular matrix (ECM) components. The role of environmental estrogens in fibrotic changes observed histologically in uterine fibroids is unknown. A deeper understanding of the mechanisms of fibrosis in fibroids requires model systems that closely mimic the in vivo tumors. A 3D human fibroid culture system is being explored as a possibly more realistic system to study the effects of environmental estrogens on fibrosis development.ObjectivesTo develop a human uterine leiomyoma (ht‐UtLM) 3D cell culture system to evaluate the role of environmental estrogens (BPA, BPS, and BPAF) on the induction and molecular mechanisms of fibrosis.MethodsHt‐UtLM 3D cell cultures (spheroids) were developed and assessed for morphologic characteristics. Cell proliferation was evaluated in 3D cultures after treatment with BPA, BPS, or BPAF (10−6 – 200 μM). Fibrosis was evaluated using a Masson’s Trichrome stain and western blotting targeting ECM components at 7 days. Differential gene expression was determined using RT2 Profiler™ PCR arrays of ECM and fibrosis genes. Network and pathway data analyses were done using Ingenuity Pathway Analysis (IPA). Activation of pathways was analyzed by TGF‐beta protein array. The profibrotic effect of environmental estrogens was also evaluated in uterine tissue sections from rats exposed to BPAF.ResultsThe 3D ht‐UtLM spheroids had a cell viability percentage of 84.17%. BPA, BPS, and BPAF all increased cell proliferation and gene expression including subtypes of collagen (COL1A1, COL15A1, and COL16A1) and regulators of fibrosis (TGFB, TGFBR, and SMAD). Compared to controls, fibrosis was more evident in spheroids treated with BPA, BPS, and BPAF for 7 days. ECM components (collagen, fibronectin and versican) were increased with all three treatments. BPA, BPS, and BPAF all upregulated expression of profibrotic genes, predicting the activation of the TGF‐beta pathway. Phosphorylation of TGFBR1 was increased significantly after treatment with BPA, BPS or BPAF at 3 hours. Both Smad and non‐Smad (Erk) pathway mediated by TGF‐beta were activated by all three compounds. Increased fibrosis was observed in the uteri of rats exposed to BPAF compared to controls.ConclusionsThe 3D fibroid spheroid culture system is an effective and valuable model for studying fibrosis in human uterine leiomyomas. BPA, BPS and BPAF, can promote fibrosis in uterine fibroids through TGF‐beta signaling, and may pose a health risk to women with uterine fibroids.