Bisphenol S Increases Cell Number and Stimulates Migration of Endometrial Epithelial Cells

Abstract

Objective: To determine whether bisphenol S (BPS), a common substitute for bisphenol A (BPA), induces cell proliferation and migration in human endometrial epithelial cells (Ishikawa) and adult mouse uterine tissues.

Methodology: Human endometrial Ishikawa cells were exposed to low doses of BPS (1 nM and 100 nM) for 72 hours. Cell proliferation was assessed through the viability assays MTT and CellTiter-Glo^®. Wound healing assays were also used to evaluate the migration potential of the cell line. The expression of genes related to proliferation and migration was also determined. Similarly, adult mice were exposed to BPS at a dose of 30 mg/kg body weight/day for 21 days, after which, the uterus was sent for histopathologic assessment.

Results: BPS increased cell number and stimulated migration in Ishikawa cells, in association with the upregulation of estrogen receptor beta (ESR2) and vimentin (VIM). In addition, mice exposed to BPS showed a significantly higher mean number of endometrial glands within the endometrium.

Conclusion: Overall, in vitro and in vivo results obtained in this study showed that BPS could significantly promote endometrial epithelial cell proliferation and migration, a phenotype also observed with BPA exposure. Hence, the use of BPS in BPA-free products must be reassessed, as it may pose adverse reproductive health effects to humans.

Keywords: BPS; Ishikawa cells; endocrine-disrupting chemicals; hyperplasia; uterus.

Figure 1

BPS increases the mean relative viability of human endometrial Ishikawa cells at low but not at high serum. Ishikawa cells were cultured in steroid-free media and then exposed to 1 nM and 100 nM (A, B) BPS and (C, D) BPA with 0% or 2% CS-FBS for 72 hrs before assessment of proliferation using (A, C) MTT and (B, D) CellTiter-Glo^® assay. Numerical data were analysed as mean relative viability ± SEM (n=3 and n=6 per treatment, respectively). Different letters denote significant differences at p<0.05 using two-way ANOVA followed by Tukey’s post hoc test.

Figure 2

A low-dose of BPS promotes migration in human endometrial Ishikawa cells. (A) Representative photomicrographs of Ishikawa cells exposed to vehicle, 1 nM, or 100 nM BPS or BPA in steroid-depleted medium supplemented with 2% FBS taken at 0 hr and 72 hrs post induction of gap. Cells were exposed to 10 μg/mL MMC before treatment with BPS. (B) Percent (%) gap closure of Ishikawa cells after 72 hr exposure to BPS or vehicle. Data were analyzed as mean % closure ± SEM (n=9). Different letters denote significant differences at p<0.05 using one-way ANOVA followed by Tukey’s post hoc test.

Figure 3

BPS significantly upregulates the expression of ESR2 and VIM genes. Relative log2 mRNA expression (-ΔΔCt) of (A) ESR and cell proliferation-related genes and (B) migration-associated genes in Ishikawa cells treated with BPS (1 nM, 100 nM) or vehicle (DMSO) for 72 hrs using qRT-PCR. All data were expressed as mean ± SEM (n=3 per treatment). Asterisks denote significant differences at p<0.05 relative to vehicle control using Kruskal–Wallis one-way ANOVA with Tukey post hoc test.

Figure 4

BPS increases the number of endometrial glands in the mouse uterus. (A) Representative photomicrographs of H&E-stained uteri from mice exposed orally to 30 mg/kg body weight BPS or vehicle for 21 days. (B) Quantification of the number of endometrial glands. Numerical data were analyzed as the mean number of glands ± SEM (n=5). Differences in letters denote significance at p<0.05 using Student’s t-test.

Figure 5

BPS promotes the migration of endometrial glands and stroma in the uterus of mice. (A) Representative photomicrographs of uteri from mice exposed orally to 30 mg/kg body weight per day BPS for 21 days showed the presence of endometrial mucosa and stroma (encircled) within the myometrium (M). (B) High power view of the myometrial layer showing hypertrophic smooth muscles. (C) High-power view of endometrial glands lined by normal-appearing epithelia within the myometrium. Histiocytes (yellow arrow) and neutrophils (white arrow) were seen in the ectopic endometrial tissues. (D) The thickness of uterine walls (i.e., perimetrium, myometrium, and endometrium) in mice treated with BPS or vehicle (n=5 per group). Numerical data were analyzed as the mean thickness of uterine walls ± SEM (n=5 per group). Differences in letters denote significance at p<0.05 using Student’s t-test.

Appendix 1

BPS increases absorbance and luminescence readings in low but not at high serum. Ishikawa cells were cultured in steroid-free media and then exposed to 1 nM and 100 nM (A, B) BPS and (C, D) BPA with 0% or 2% CS-FBS for 72 hrs before assessment of viability using (A, C) MTT and (B, D) CellTiter-Glo^® assay. Numerical data were analyzed as mean absorbance ± SEM (n=3 and n=6 per treatment, respectively). Different letters denote significant differences at p<0.05 using two-way ANOVA followed by Tukey’s post hoc test.