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Mono-(2-ethylhexyl) Phthalate Increases Oxidative Stress Responsive miRNAs in 1st trimester Placental Cell Line HTR8/SVneo Sunitha Meruvu, Jian Zhang, and Mahua Choudhury Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00038 • Publication Date (Web): 12 Feb 2016 Downloaded from http://pubs.acs.org on February 17, 2016
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Chemical Research in Toxicology
Mono-(2-ethylhexyl) Phthalate Increases Oxidative Stress Responsive miRNAs in 1st trimester Placental Cell Line HTR8/SVneo Sunitha Meruvu, Jian Zhang, Mahua Choudhury* Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, TX USA ABSTRACT: Phthalates, an endocrine disruptor group, cause oxidative stress (OS) in the placenta. However, no studies have reported OS related miRNAs induced by phthalates. In the present study, we demonstrate that mono-(2-ethylhexyl) phthalate (MEHP) induces OS responsive miR-17-5p, miR-155-5p, and miR-126-3p in HTR8/SVneo in a dose and timedependent manner. Furthermore, MEHP altered the expression of phosphoinositide-3-kinase regulatory subunit 1α α, phosphatase and tensin homolog, CDKN2A interacting protein, superoxide dismutase 2, and 3β-hydroxysterol-D24 reductase which are involved in OS and predicted to be regulated by these miRNAs. Our results suggest that placental exposure to MEHP may result in an aberrant miRNAs expression leading to pregnancy complications.
1640 with L-glutamine and FBS were from Corning Cellgro (Manassas, VA). Cell Culture HTR-8/SVneo cell line was a gift from Dr. Peeyush Lala (University of Western Ontario, Ontario, Canada). These are the human first-trimester trophoblast cells immortalized with SV40 T antigen. They were cultured in RPMI 1640 with Lglutamine with 10% FBS, 1% penicillin/streptomycin. Cell Treatment HTR8/SVneo cells were treated with MEHP for the times and concentrations specified in the result section. MEHP was diluted in DMSO, and the final concentration of DMSO was maintained at 0.1% for both control and treatments. The MEHP concentrations selected for the treatments were shown to induce oxidative stress in HTR8/SVneo cells.5 MEHP has been detected in human placenta, amniotic fluid, and umbilical cord blood.11, 12 Latini et al. reported an average concentration of 0.52 µg/mL or 1.8 µM in the umbilical cord serum.13 H2O2 was added in complete DMEM medium for the times and concentrations specified in the result section. The H2O2 concentrations selected for the treatment were based on the previous OS studies in villous 3A first trimester trophoblast cells and in HTR8/SVneo cells.7, 14 The cell viability assay with H2O2 in HTR8/SVneo cells did not show any significant cell death from 0 to 25 µM of H2O2 concentrations (Supplementary section, Fig. S1). Cell Viability Assay and ROS Measurement The effect of MEHP on cell viability was measured using CellTiter-Glo® 2.0 Assay kit from Promega (Madison, WI) following manufacturer’s instructions. Cells plated in a 96well plate was exposed to 25, 50, 100 and 180 µM MEHP for 24 and 48 h. CellTiter-Glo® 2.0 Reagent was added directly to cells and the luminescent signal generated is proportional to the amount of ATP present. Luminescent signal was quantified using a microplate reader (Infinite M100 PRO, TECAN, Morrisville, NC). The percentage of viable cells was determined by comparing the luminescent signal of MEHP treated cells to DMSO treatment. Intracellular reactive oxygen species (ROS) levels were measured by DCFDA assay. Cells plated in a 96- well plate
INTRODUCTION Oxidative stress (OS) is implicated in several pregnancy complications including preeclampsia, preterm births, and gestational diabetes. Excessive reactive oxygen species (ROS) and OS can lead to abnormal placental development1. Phthalates, primarily used to improve the flexibility of plastic, are known endocrine disruptors. Di-(2-ethylhexyl) phthalate, one of the most commonly used phthalates, is hydrolyzed in the body to a biologically active metabolite, mono-(2ethylhexyl) phthalate (MEHP).2 In vitro and in vivo studies demonstrated that MEHP adversely affects the developmental and reproductive functions in both sexes by inducing oxidative stress.3 Epidemiological studies also showed higher levels of maternal urine phthalates in preterm birth, low birth weight, and pregnancy loss.4 Tetz et al. showed that MEHP increased ROS generation, oxidative DNA damage, and apoptosis in HTR8/SVneo cells.5 Emerging evidence suggests that miRNA dysregulation during early pregnancy might lead to pregnancy complications.6 In this study, we investigated if MEHP can regulate OS responsive miRNAs and their targets in a human 1st-trimester placental cell line, HTR8/SVneo. To examine if MEHP can elicit the induction of OS related miRNAs, we analyzed miR-17-5p, miR-126-3p, and miR-1555p expression in HTR8/SVneo cells under MEHP exposure. As these miRNAs are reported to be involved in OS response, there is a possibility of them being implicated in the onset and progression of preeclampsia.7-10 We also validated our study by investigating the expression profile of these OS responsive miRNAs while exposed to hydrogen peroxide (H2O2), a wellknown OS inducer. This is the first report showing that MEHP induces OS responsive placental miRNAs in vitro which may lead to pregnancy complications including preeclampsia. MATERIALS AND METHODS Chemicals MEHP (purity 97% purity, CAS Number 4376-20-9), H2O2, dimethyl sulfoxide (DMSO) and 6-carboxydichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI
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RESULTS Effect of MEHP on cell viability and ROS generation in HTR-8/SVneo cells To evaluate the effect of MEHP on cell viability, HTR8/SVneo cells were exposed to various doses of MEHP for 24 and 48 h. MEHP did not show any significant cell death till 100 µM (Fig. 1 (i) A) while 180 µM MEHP treatment showed 11 % decrease in cell viability (p
