Abstract
Methyl mercury (MeHg) is an organic highly toxic compound that is transported efficiently via the human placenta. Our previous data suggest that MeHg is taken up into placental cells by amino acid transporters while mercury export from placental cells mainly involves ATP binding cassette (ABC) transporters. We hypothesized that the ABC transporter multidrug resistance-associated protein (MRP)1 (ABCC1) plays an essential role in mercury export from the human placenta. Transwell transport studies with MRP1-overexpressing Madin-Darby Canine Kidney (MDCK)II cells confirmed the function of MRP1 in polarized mercury efflux. Consistent with this, siRNA-mediated MRP1 gene knockdown in the human placental cell line HTR-8/SVneo resulted in intracellular mercury accumulation, which was associated with reduced cell viability, accompanied by increased cytotoxicity, apoptosis, and oxidative stress as determined via the glutathione (GSH) status. In addition, the many sources claiming different localization of MRP1 in the placenta required a re-evaluation of its localization in placental tissue sections by immunofluorescence microscopy using an MRP1-specific antibody that was validated in-house. Taken together, our results show that (1) MRP1 preferentially mediates apical-to-basolateral mercury transport in epithelial cells, (2) MRP1 regulates the GSH status of placental cells, (3) MRP1 function has a decisive influence on the viability of placental cells exposed to low MeHg concentrations, and (4) the in situ localization of MRP1 corresponds to mercury transport from maternal circulation to the placenta and fetus. We conclude that MRP1 protects placental cells from MeHg-induced oxidative stress by exporting the toxic metal and by maintaining the placental cells' GSH status in equilibrium.
Highlights
The metal mercury (Hg) is among the most hazardous chemicals with high significance for public health (WHO 2017, ATSDR 2019)
Gene knockdown was confirmed by RT-qPCR. e The antiMRP1 antibody detected a protein of appropriate size (190 kDA) by western blotting in control cells, but hardly any in siMRP1 treated cells. f Relative human MRP1 gene expression levels of MDCKII cells constitutively expressing MRP1 and of MDCKII cells overexpressing MRP1 (MDCKII-MRP1) were analyzed by RT-qPCR. g Anti-MRP1 antibody detected a significant increase in protein expression in MDCKII-MRP1 cells. h In Immunofluorescence microscopy (IFM), the anti-MRP1 antibody produced a strong fluorescence signal in MDCKII-MRP1 cells, but not in MDCKII cells or the negative controls
We performed western blotting with total tissue lysates as in IFM/IHC the antibody is used on total tissue
Summary
The metal mercury (Hg) is among the most hazardous chemicals with high significance for public health (WHO 2017, ATSDR 2019). Mercury with its exceptionally high affinity to thiols is known to modify the redox state of its ligands. It affects the mitochondrial electron transfer chain leading to increased formation of reactive oxygen species (ROS), i.e. superoxide anion and hydrogen peroxide (Farina et al 2013). ◂Fig. 1 Structure of the human placenta and the placental barrier, validation of MRP1 expression levels in HTR-8/SVneo and MDCKII cells, and anti-MRP1 antibody specificity. D siRNA-mediated gene knockdown was performed in HTR-8/SVneo cells using MRP1-specific siRNA (siMRP1). G Anti-MRP1 antibody detected a significant increase in protein expression in MDCKII-MRP1 cells (a representative western blot is shown). The letters A-D denote homogeneous subgroups derived from one-way ANOVA and S–N-K posthoc test (P < 0.05)
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