Mono-2-ethylhexyl phthalate inhibits human extravillous trophoblast invasion via the PPARγ pathway

Highlights

• MEHP inhibits HTR-8/SVneo cell invasion.

• MEHP exposure imbalanced the expression of MMP-9/TIMP-1 in HTR-8/SVneo cells.

• This effect of MEHP is mediated via the PPARγ pathway.

Abstract

Concerns over the adverse reproductive outcomes in human have been raised, more evidence including the underlying mechanism are required. Since extravillous trophoblast (EVT) invasion is an important physiological step during early development, the effects of mono-2-ethylhexyl phthalate (MEHP), the bioactive metabolite of DEHP, on EVT invasion were investigated using Matrigel-coated transwell chambers and cell line HTR-8/SVneo. In the transwell-based invasive assay, MEHP exposure inhibited EVT invasion as judged by decreased invasion index. Further analysis showed that MEHP exposure significantly inhibited the activity of matrix metalloproteinase-9 (MMP-9), which is an important positive regulator of EVT invasion. Meanwhile, the protein levels of tissue inhibitor matrix metalloproteinase-1 (TIMP-1), one key negative regulator of EVT invasion, were upregulated by MEHP treatment. Finally, inactivation of PPARγ pathway by either PPARγ inhibitors or PPARγ shRNA knockdown rescued the MEHP-induced inhibited invasion of HTR-8/SVneo cells, which is accompanied by the recovery of inhibited MMP-9 expression. The present study provides the evidence that MEHP exposure inhibits trophoblast invasion via PPARγ at concentrations comparable to those found in humans, which provides an insight in understanding the mechanisms of DEHP-associated early pregnancy loss.

Introduction

Early pregnancy loss or first-trimester miscarriage is one of the most common complications of female reproduction, with its incidence increasing worldwide in recent decades (Lang and Nuevo-Chiquero, 2012). Although many factors affecting early pregnancy loss have been identified in recent years, the causes for approximately 40% of early pregnancy loss remains unclear (Hempstock et al., 2003). Notably, there is increasing evidence showing that exposure to endocrine disrupting chemicals (EDCs) elicits adverse effects on female reproductive health. Human epidemiological studies and animal exposure experiments have shown that perinatal exposure to several EDCs, such as dichlorodiphenyltrichloroethane, arsenic, lead, bisphenol A and phthalates, is associated with an increased risk of miscarriage (Fay et al., 1999, Brown, 2008, Balabanic et al., 2011, Toft et al., 2012).

Among the EDCs showing reproductive toxicity, phthalate acid esters (PAEs), commonly referred to as, phthalates, are of particular concern. PAEs are a diverse group of chemicals widely used in the manufacture of industrial products such as paint, glue, lubricants, solvents, medical product plastics including polyvinyl chloride, residential building materials and personal products such as cosmetics (Fay et al., 1999). Maternal exposure to PAEs is associated with impaired gonadal development and fertility in human males (Hauser et al., 2007, Pant et al., 2008). Due to concerns over the potential health effects of PAEs, the U.S. has banned six PAEs (di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), diisononyl phthalate (DINP), diisodecyl phthalate (DIDP), and di-n-octyl phthalate (DNOP)) from use in certain products made for young children (Braun et al., 2013). However, many PAEs are still used as ingredients in paints, medical tubes, vinyl flooring, soaps and shampoos, and therefore humans are exposed to PAEs via various pathways including ingestion, inhalation, and dermal absorption (Guo et al., 2011). PAEs can be rapidly metabolized to mono-phthalates and have been frequently detected in human urine (Latini, 2005, Hogberg et al., 2008, Mu et al., 2015), and mono-2-ethylhexyl phthalate (MEHP) is often detected as a major metabolite of PAEs since DEHP is the most commonly used plasticizer for polyvinyl chloride, with production reaching 230,000 tons in the USA in 2006 (Zolfaghari et al., 2014). There is increasing evidence showing that MEHP is the bioactive chemical of DEHP, which plays an important role in the induction of adverse effects of DEHP (Albro, 1986, Huber et al., 1996, Lovekamp-Swan and Davis, 2003, Shea and Comm Environm, 2003). One recent epidemiological study has shown that high urinary concentration of MEHP has been associated with increased risk of early pregnancy loss (Toft et al., 2012). Although another critical review of epidemiological study has been unable to show similar linkages (Kay et al., 2013), there is evidence that DEHP exposure can induce abortion of mouse (Schmidt et al., 2012). Thus, the study on the underlying mechanism will be helpful to understand the potential adverse effects of DEHP on early pregnancy loss.

Early embryonic development involves complicated interactions between the maternal body and the conceptus, and dysfunction of either the maternal endocrine environment or embryonic development can induce pregnancy loss. Extravillous trophoblast (EVT) invasion is a key event in embryonic development (Jauniaux and Burton, 2005). Invasion of EVT into uterine tissue and vessels is an essential process for normal placentation and successful maintenance of human pregnancy (Red-Horse et al., 2004), with two-thirds of early pregnancy failures attributed to the inhibition of EVT invasion (Hustin et al., 1990). In the process of EVT invasion, matrix metalloproteinases (MMPs), a family of zinc-dependent proteolytic enzymes, are key proteinases collectively capable of degrading all components of the extracellular matrix (ECM) (Cohen et al., 2006). Accumulating evidence suggest that MMP-9 expression/activation is a prerequisite to EVT invasion and may play a more important role in EVT invasion during pregnancy (Ferretti et al., 2007). On the other hand, peroxisome proliferator-activated receptor γ (PPARγ), is a nuclear receptor, is specially expressed in human placenta and serves as an essential regulator of EVT invasion of human placenta which plays a vital role in EVT invasion (Fournier et al., 2008). Considering that MEHP can elicit PPARγ binding activity with an effective concentration for half-maximal response (EC₅₀) of 6.2 μΜ (Hurst and Waxman, 2003), we hypothesized that MEHP might inhibit EVT invasion by down-regulating MMPs via the PPARγ pathway.

To test this hypothesis, we used the transwell-based invasion assay to investigate the effects of MEHP exposure on the invasion of HTR-8/SVneo cells, which is a model for EVT invasion in the first trimester of human pregnancy. The protein and RNA expression of MMP-9 and their inhibitors (TIMP-1) involved in cell invasion were also analyzed to elucidate MEHP-mediated trophoblast invasion. Furthermore, the PPARγ-mediated mechanism was clarified by both pharmacological inhibitors and shRNA knockdown of PPARγ. This study provides evidence that MEHP can inhibit human EVT cell invasion via the PPARγ pathway, implicating female reproductive toxicity.