Citation:

Douglas, G., T. Thirkill, P. Kumar, M. Loi, AND E Hilborn. Effect of Microcystin-LR on human trophoblast differentiation in vitro. Presented at International Toxic Cyanobacteria Conference, Johannesburg, SOUTH AFRICA, August 11 - 16, 2013.

Impact/Purpose:

Microcystin is a cyanobacteria toxin listed on the third Contaminant Candidate List. It is a widely recognized liver toxin, but other health effects are poorly characterized. We hypothesized that microcystin would interrupt cellular differentiation, implantation and placental function in early pregnancy. Cultures of human trophoblasts harvested from term placentae were exposed to microcystin. We found that low doses of microcystin did not disrupt cellular integrity, but did increase production of human chorionic gonadotropin, a hormonal peptide essential to the maintenance of proper placentation and blood flow to the fetus during early gestation.

Description:

Background: Microcystin LR is a potent protein phosphatase 2a (PP2a) inhibitor and generates reactive oxygen species (ROS) believed to be an essential component of a toxic effect. Toxicological studies have demonstrated microcystin (MCYST) disruption of cytoskeletal function and cellular integrity. Our goal was to investigate the effect of MCYST on human placental trophoblast differentiation which is required for normal placental function and successful pregnancy outcome. Methods: Villous cytotrophoblasts were harvested from human term placentae. Cells were plated onto Hams/Weymouth medium for maintenance as mononucleated cytotrophblasts or onto keratinocyte growth medium to promote differentiation into multinucleated syncytiotrophoblast-like colonies. Both types of cultures were exposed to MCYST-LR at 0.1-50 µM with and without L-buthionine sulphoximine (BSO), an inhibitor of gamma glutamyl cysteine synthetase and hence glutathione-based ROS defense. Microcystin-LR exposed and control cell preparations were evaluated for viability, attachment, differentiation, apoptosis and human chorionic gonadotropin (CG) production. The expression of organic anion transporters (OATP) previously identified as essential for cellular uptake of microcystin was assessed. Results: MCYST-LR had no effect on cell morphology, attachment, differentiation or apoptosis below the cytotoxic dose of 25 µM. Trophoblasts expressed OATP1B3 mRNA and protein and lower amounts of OATB1A2. The production of CG was increased in a dose-dependent manner between MCYST-LR exposures from 0.1 – 10 µM; the effect was enhanced by BSO. CG is a trophoblast-derived peptide hormone which is essential for normal implantation and maintenance of pregnancy. It is also understood to provide protection from free oxygen radicals as the placenta transitions from a low oxygen environment to a more perfused state during weeks 10 – 12 of gestation. Since MCYST exposure is known to increase ROS production, these results suggest that toxic effects on trophoblasts might be attenuated by the simultaneous MCYST-dependent increase in CG production and this CG increase is further enhanced by the reduction of glutathione-based ROS protection. This abstract is of a proposed presentation and does not necessarily reflect EPA policy.

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