A study on the effect of fungicide mancozeb and its metabolite ethylene thiourea (ETU) in modulating spheroid attachment and trophoblast invasion

Abstract

Mancozeb belongs to the ethylene-bis-dithiocarbamate (EBDC) fungicide family and is commonly used as a fungicide in agriculture. Mancozeb acts as an endocrine disrupting chemical (EDC) which interferes human endocrine and reproductive systems. Mancozeb has a short half-life of 1-3 days, but its main metabolite ethylene thiourea (ETU) persists for 5-10 weeks. Mancozeb and ETU were reported to have teratogenic, mutagenetic, and carcinogenetic effects. However, the possible roles of Mancozeb and ETU on human reproductive function, especially embryo implantation, remain largely unknown. Our laboratory established several in vitro models to study the effect of EDC on the trophoblast-endometrial interaction during embryo implantation, including the trophoblastic spheroid attachment, trophoblast invasion and migration, and in vitro endometrial decidualization. The human blastocyst surrogate trophoblastic spheroids (Jeg-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa) or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Different concentrations of Mancozeb and ETU were used to treat Ishikawa and Jeg-3 cells; it was found that Mancozeb and ETU treatment for 48 hours inhibited cell viability at 10μg/mL and 5000µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb and ETU significantly reduced spheroid attachment at 3μg/mL and 300μg/mL. Mancozeb at 3μg/mL but not ETU dramatically decreased the expression of endometrial ERβ, integrin β3, and Mucin1 transcripts. Moreover, microarray and real-time PCR confirmed that Mancozeb differentially regulated genes related to cell junction, transporter activity, and biological adhesion. Knockdown of ERβ, or integrin β3 but not Mucin1 by siRNA in Ishikawa cells suppressed spheroid attachment. Similarly, the addition of ERβ selective antagonist (PHTPP) to Ishikawa cells suppressed spheroid attachment; while overexpressing ERβ or integrin β3 proteins in the transfected Ishikawa cells rescued the suppressive effect of Mancozeb on spheroid attachment. Intriguingly, addition of ERβ agonist (DPN) with or without Mancozeb suppressed the attachment of spheroids. In vivo mouse study confirmed Mancozeb treatment at 32mg/kg BW/day from postnatal day (PD) 1 to 8 reduced the expression of ERβ in pregnant mouse uterus. Mancozeb also significantly inhibited invasion, migration, and outgrowth of Jeg-3 cells and spheroids in the transwell and wound healing assays. Mancozeb at 1 and 3μg/mL down-regulated the expression of matrix metalloproteinase-2 (MMP-2) transcript and protein in the trophoblastic Jeg-3 and BeWo cells, and also inhibited MMP-2 activity in the conditioned media of treated cells. However, similar changes were not found when the cells were treated with Mancozeb metabolites, ETU or ZnCl2. Moreover, Mancozeb at 3μg/mL induced ERK1/2 phosphorylation at the first hour in the Jeg-3 cells and then reduced to a low level until 48 hours, suggesting that suppression of MMP-2 by Mancozeb was through ERK1/2 signaling pathway. Interestingly, Mancozeb at 3μg/mL suppressed the morphological transformation of human primary endometrial stromal cells without changing the expression of decidual markers, PRL, and IGFBP1 in the in-vitro decidualization model. In conclusion, Mancozeb but not its metabolite ETU suppressed spheroid attachment, trophoblast invasion and disrupted stromal cell decidualization through modulating multiple genes expression profile and ERK pathway activation with the in vitro and in vivo implantation models.