Marta Hoffmann

Disruption of 17β-estradiol secretion by persistent organic pollutants present in human follicular fluid is dependent on the potential of ovarian granulosa tumor cell lines to metabolize estrogen

Endocrine-disrupting chemicals (EDCs), such as perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyl 153 are persistent pollutants that are found in human follicular fluid (FF). These compounds may affect endocrine function, disrupt steroid secretion by granulosa cells, and play a role in granulosa cell tumor (GCT) development. GCTs demonstrate endocrine activity, expressing aromatase and secreting 17β-estradiol (E2). We aimed to determine the effects of a mixture of EDCs, similar to that found in human FF, on human granulosa tumor cell lines representing the juvenile (JGCT) and adult (AGCT) forms (COV434 and KGN cells, respectively). We found that all the individual compounds and mixtures tested altered granulosa tumor cell function by disrupting E2 secretion. In KGN cells, which possess significantly higher basal aromatase gene expression, and therefore secrete more E2 than JGCT cells, EDC mixtures activated estrogen receptors (ERs) and G protein-coupled receptor-30 signaling, thereby stimulating E2 secretion, without affecting aromatase expression. By contrast, in COV434 cells, which demonstrate higher CYP1A1 expression, a key mediator of estrogen metabolism, than KGN cells, EDC mixtures reduced E2 secretion in parallel with increases in the 2-hydroxyestrogen 1/E2 ratio and CYP1A1 expression, implying an upregulation of E2 metabolism. These results indicate that the EDC mixture present in FF disrupts E2 secretion in JGCT and AGCT cells according to the estrogen metabolic potential of the cell type, involving both classical and non-classical ER pathways.

Mixtures of persistent organic pollutants increase ovarian granulosa tumor cell line migration and spheroid invasion by upregulating MMP2 expression and activity via IGF1R

Granulosa cell tumors (GCT) of the ovary have a good prognosis. Recurrence tends to be late; however, > 66 % of patients with recurrent GCT die from the disease. Most recurrences are abdominopelvic, although distant metastases have been documented. Here, we tested the hypothesis that a mixture of persistent endocrine-disrupting chemicals (EDCs) stimulates the invasion of GCT cells. We selected perfluorooctanoate (PFOA, 2 ng/mL), perfluorooctanesulfonate (PFOS, 8 ng/mL), 2,2-dichlorodiphenyldichloroethylene (p,p'-DDE, 1 ng/mL), polychlorinated biphenyl 153 (PCB153, 100 pg/mL), and hexachlorobenzene (HCB, 50 pg/mL), which have the highest measured concentrations in follicular fluid of women undergoing treatment with assisted reproductive technology. The human GCT cell lines COV434 and KGN have been used as in vitro models of juvenile (JGCT) and adult (AGCT) GCT subtypes, respectively. Cells were treated with a mixture of the test compounds for 15 min prior to analysis of protein phosphorylation; for 4 h prior to analysis in a circular chemorepellent-induced defect assay; for 6 h prior to analysis of matrix metalloproteinase 2 (MMP2) activity; for 24 h prior to analysis of migration, invasion, and gene expression; and for 48 h prior to analysis of protein expression. First, we showed that KGN cells migrated and exhibited invasive behavior. By contrast, COV434 cells lacked migration and invasion potential. Moreover, expression of mesenchymal genes and the gene encoding MMP2 was higher in KGN cells, and that of epithelial genes lower, than that in COV434 cells. Treatment of KGN cells with the EDC mixture stimulated cell migration, invasion, and lymphatic dissemination. The results suggest that the role of the EDC mixture in AGCT invasion is not related to changes in expression of epithelial and mesenchymal genes; rather, it is related to increased expression and activity of MMP2. Additionally, silencing insulin-like growth factor 1 (IGF1R) in AGCT abolished the stimulatory effect of the EDC mixture on KGN spheroid invasion. These results demonstrate that the EDC mixture increased KGN spheroid invasion by stimulating expression and activity of MMP2 via IGF1R.