Bisphenol A exposure leads to specific microRNA alterations in placental cells

https://doi.org/10.1016/j.reprotox.2010.04.004Get rights and content

Abstract

Exposure to bisphenol A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity.

Introduction

Bisphenol A (BPA), an industrial plasticizer, is a key monomer in the production of polycarbonate plastic, a fungicide, a flame retardant, and a component of epoxy resins used as food-contact surface coating for a variety of objects including cans and metal jar lids [1]. Because of the increased use of both polycarbonate plastics and epoxy resins in the coatings of food cans and plastic water bottles, the frequency of human exposure to BPA has steadily increased in recent years [2].

Exposure of the developing fetus to BPA is of particular concern as the compound readily crosses the placental barrier and accumulates both in the placenta and in the fetus [3], [4]. The placenta, comprised of both fetal tissue derived from the chorionic sac and maternal tissue from the endometrium, is the first complex mammalian organ to form in pregnancy and is required for the early development of all viviparous species [5]. It not only allows the transport of nutrients and waste products between the mother and the fetus, but also provides a barrier protecting the fetus against harmful substances in the mother's circulation. Importantly, the placenta also serves as an endocrine organ, providing peptide and steroid hormones, which influence placental, fetal, and maternal metabolism and development.

BPA's toxicity has been linked to interference with endogenous estrogens and thyroid hormone, and to the disruption of reproductive, immune, and central nervous systems [6], [7], [8], [9], [10]. These functions may be mediated through BPA's binding to the estrogen receptor, though they may also be related to nuclear-receptor independent activation of cell signaling systems [11]. Evidence is weaker regarding BPA's potential as a carcinogen, although literature on the association of BPA with increased cancer risk in adult life through fetal exposures or across generations suggests that BPA may elicit epigenetic effects [12], [13]. Further, experiments in mice have demonstrated that BPA exposure can cause altered DNA methylation patterns in cell signaling genes, suggesting that BPA can exert its effects through epigenetic mechanisms [13], [14], [15].

MicroRNAs (miRNAs) are approximately 22 nucleotide long non-coding RNAs capable of regulating gene expression post-transcriptionally through imperfect complementarity with a target mRNA, and are considered a mode of epigenetic regulation [16]. As they potentially target up to one third of human mRNAs, miRNAs regulate myriad cellular processes, playing pivotal roles in developmental timing and patterning [16], [17]. Our group and others have demonstrated that miRNAs are susceptible to cellular stresses and environmental exposures, such as folate deficiency, exposure to arsenic and nicotine, ionizing radiation, and diesel exhaust particles [18], [19], [20], [21]. Several studies have suggested that miRNA expression modulation may be a mechanism through which toxicants can exert both developmental and carcinogenic effects [22], [23].

The effect of BPA on miRNA expression has yet to be studied and the high prevalence of fetal exposure to BPA from maternal sources makes the placenta a particularly important model. The specific risks associated with fetal exposures and the key role of the placenta in mediating the potential toxic effects of BPA make the study of molecular alterations at different stages of fetal development particularly important. Here we have used an in vitro cell culture system to examine the effect of BPA exposure on alterations in miRNA expression, and our results suggest that changes in miRNA expression may be critical mediators of BPA toxicity.

Section snippets

Cell lines and BPA exposure conditions

Human SV40 transformed placental cell lines 3A, TCL-1, and HTR-8 were cultured in phenol-free RPMI 1640 (Invitrogen, Inc., Gaithersburg, MD). Phenol-free growth medium was supplemented with 10% charcoal-stripped fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin for both 3A and TCL-1 cell lines. HTR-8 cell line growth medium was supplemented with 5% charcoal-stripped fetal bovine serum (FBS; Invitrogen) with no penicillin–streptomycin. For the microarray experiment, 3A, TCL-1,

miRNA expression profiles of placental cell lines following bisphenol A exposure

To examine the effects of BPA exposure on miRNA expression in placental cell lines, 3A, TCL-1, and HTR-8 cells were treated over a 6-day period with 25 ng/μL of BPA and a microarray platform was used to determine microRNA expression of all the known miRNA sequences based on the Sanger Institutes microRNA database Release 10.0 as well novel proprietary miRNAs in multiple species. Data from the microarray analysis were visualized using heat maps generated using unsupervised hierarchical clustering

Discussion

The commercial use of BPA is increasing in frequency and research has shown that exposure to BPA, for example through plastic beverage containers, can significantly increase urinary BPA concentrations [29], [30]. The increase in maternal exposure during critical windows of fetal development is of particular concern as even low-dose exposure to BPA can lead to adverse effects in laboratory animals including delayed developmental outcomes in offspring of exposed mothers [31], [32], [33].

Funding information

This work was supported by [National Institutes of Health NCRR P20 RR018728, P20 RR016457]; [National Institutes of Health–National Institute of Environmental Health Sciences P42 ES013660] and [National Science Foundation-The Experimental Program to Stimulate Competitive Research 0554548].

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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