Placental Cell Responses to Trichloroethylene Metabolite S-(1, 2-dichlorovinyl)-l-cysteine Exposure
Elkin, Elana
2019
Abstract
The placenta provides the critical interface between mother and fetus, regulating fetal in utero development through the exchange of hormones nutrients, waste, and oxygen. Given its critical role, abnormalities of the placenta substantially increase the risk of adverse birth outcomes. Although exposure to environmental contaminants is increasingly thought to elevate risk for adverse birth outcomes, the mechanism(s) by which toxicants adversely impact pregnancy remain largely unknown. Recent epidemiology studies found that maternal exposure to trichloroethylene (TCE), an industrial solvent and pervasive environmental contaminant, was associated with increased risk for low birth weight and preterm birth. This thesis investigated the effects of the TCE metabolite S-(1, 2-dichlorovinyl)-L-cysteine on extravillous trophoblasts, specific cells that play a pivotal role in tissue and maternal spiral artery remodeling during placental development. DCVC effects on placental cells were studied using the HTR-8/SVneo immortalized extravillous trophoblast cell line. Following exposure to 5-20 µM DCVC for 6 or 12 h, targeted metabolomics detected changes in intracellular concentrations of metabolites from different energy metabolism pathways. Although no change in ATP levels were observed, glucose metabolism perturbations were detected that included a time-dependent accumulation of glucose-6-phosphate+frutose-6-phosphate (G6P+F6P), as well as independent shunting of glucose intermediates. Furthermore, DCVC treatment stimulated compensatory utilization of glycerol, lipid and amino acid amino metabolism to provide intermediate substrates entering downstream glycolysis or the tricarboxylic cycle. In addition, transcriptomics analysis revealed hundreds of differentially expressed genes with exposure to 20 µM DCVC for 12 h (FDR< 0.1 and fold-change < -1.5 or >1.5). Furthermore, gene set enrichment analyses demonstrated that the most substantially altered molecular signaling pathway was the EIF2α/ATF4 Integrated Stress Response (ISR), accompanied by ¬¬decreased global protein synthesis. However, no changes in cell cycle progression or proliferation were detected, suggesting that a generally successful adaptive process occurred after 12 h of DCVC treatment. In order to further investigate DCVC effects, HTR-8/SVneo cells were treated with 10-20 µM DCVC for mitochondrial bioenergentics experiments, or 10-100 µM DCVC for apoptosis experiments. Using the Seahorse XF Analyzer allowed evaluation of key aspects of mitochondrial function with concurrent real-time monitoring of oxygen consumption rates and extracellular acidification rates. Elevated basal oxygen consumption rate, mitochondrial proton leak and sustained energy coupling deficiency occurred after 6 h of exposure to 20 µM DCVC, whereas 12 h of exposure to DCVC decreased mitochondrial-dependent basal, ATP-linked and maximum oxygen consumption rates. Moreover, mitochondrial membrane potential dissipated after 12 h of exposure to 20 µM DCVC, as detected with the TMRE fluorochrome. The antioxidant (±)-α-tocopherol attenuated DCVC-stimulated mitochondrial membrane depolarization and caspase 3+7 activity but failed to rescue oxygen consumption perturbations. Furthermore, evaluation of DCVC-stimulated apoptosis revealed that DCVC simultaneously activated the mitochondrial-mediated and cell surface receptor-mediated apoptotic pathways, most likely via co-regulatory p53 signaling. Finally, mitochondria-centered crosstalk between the two activated pathways appeared to amplify the effect on the overall apoptotic response. Taken together, these findings showed progressive defects in mitochondrial function and suggest mitochondria as the primary intracellular targets for DCVC-mediated cytotoxicity in placental cells. Collectively, the series of studies presented in this thesis detail responses to the TCE metabolite DCVC in placental cells, contributing to our understanding of the toxicological mechanism of action of TCE. These new insights support epidemiology studies by providing plausible biological explanations for observed association with pregnancy complications and adverse birth outcomes.Subjects
Trichloroethylene S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) placenta extravillous trophoblasts reproductive toxicology
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