Histochem Cell Biol DOI 10.1007/s00418-015-1348-9

ORIGINAL PAPER

Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse Lan Li1 · Jing‑Cai Liu2,3 · Yong Zhao2,4 · Fang‑Nong Lai2,3 · Fan Yang2,3 · Wei Ge2 · Cheng‑Li Dou2 · Wei Shen2 · Xi‑Feng Zhang5 · Hong Chen1 

Accepted: 30 June 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5–3.5 days post-coitum had increased proliferation, growth rate and Electronic supplementary material  The online version of this article (doi:10.1007/s00418-015-1348-9) contains supplementary material, which is available to authorized users. * Xi‑Feng Zhang [email protected] * Hong Chen [email protected] Wei Shen [email protected] 1

College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, Shaanxi, China

2

Institute of Reproductive Sciences, Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China

3

College of Life Science, Qingdao Agricultural University, Qingdao 266109, China

4

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China

5

College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China







number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice. Keywords  DEHP · Mammary gland · Gene expression pattern · Microarray

Introduction Plastics are widely used in the world. More than 10 million tons of di(2-ethylhexyl) phthalate (diethylhexyl phthalate, DEHP), an estrogen-like unbound chemical, are used worldwide every year to increase the softness of plastics or liquefy materials (Zhang et al. 2013a, b, c). Particularly, softer plastic products contain more DEHP. One of the most widely used plastic materials, polyvinyl chloride (PVC) cling film, contains large amount of DEHP. DEHP is not covalently bound to plastic matrix; therefore, it can be released from plastic products and pollute the external environment. DEHP is also present in medical devices and common household items such as food containers, packaging, toys and clothing, posing a potential risk to human health (Zhang et al. 2013a, b, c). In the general population, most people intake 2 mg of DEHP via food and water daily. Thus, DEHP exposure poses health risks to human and is a growing concern, because it mimics the actions of endogenous hormones, which can potentially disrupt endocrine functions in humans or other animals (Pocar et al. 2012). Many studies involving fetal and perinatal DEHP exposure have shown persistent alterations of the structures and functions of estrogen target tissues. DEHP acts analogously to estrogen by inducing polyovular follicles (POF), negatively modulating oocyte growth, DNA

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methylation of imprint genes and meiotic maturation, as well as impairing ovarian functions that lead to loss of fertility (Lovekamp and Davis 2001; Dalman et al. 2008; Ambruosi et al. 2009; Zhang et al. 2013a, b, c; Li et al. 2014). Particularly, epidemiological studies have suggested that increased levels of endocrine-disrupting compounds (EDCs) during pregnancy increase the risk of breast cancer of offspring (Potischman and Troisi 1999). During the past 10 years, many people have studied the potential relationship between breast carcinogenesis and EDC exposure (Potischman and Troisi 1999; Vandenberg et al. 2007, 2008; Lamartiniere et al. 2011). Bisphenol A (BPA), a known EDC like DEHP, has been intensively investigated for its role in the development of breast carcinogenesis. After exposure to BPA, fat pad differentiation accelerates and the collagen localization in the mesenchyme changes in the mammary glands of 18-dpc CD1 mouse fetuses. As a consequence, cell size reduces, lumen formation delays, and the area of duct epithelium increases (Vandenberg et al. 2007). Mice or rats exposed to BPA prenatally have delayed ductal invasion of the mammary stroma at puberty, induced lateral branching and terminal ends, and increased heightened response to estradiol at adulthood (Munoz de Toro et al. 2005; Markey et al. 2001; Murray et al. 2007). The development of in situ preneoplastic lesions or carcinoma, i.e., intraductal hyperplasias, is observed (Vandenberg et al. 2008). Similarly, prenatal exposure to BPA results in the development of intraductal hyperplasia in rat (Murray et al. 2007). It has been shown that BPA [fed 400 μg of BPA per kg of body weight (BW) daily from gestational day 100 to term] alters the development of fetal mammary glands in rhesus monkeys (Tharp et al. 2012). Dairkee et al. have developed a system for culturing human non-cancerous high-risk donor breast epithelial cells (HRBECs) and found that BPA affects the expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis (Dairkee et al. 2013). Bhan et al. have demonstrated that BPA and DES induce gene expressions in both cultured human breast cancer cells (MCF7) and the mammary glands of rats, and alter the epigenetic programming of the HOTAIR promoter, leading to endocrine disruption in vitro and in vivo (Bhan et al. 2014). Soto’s group has also demonstrated that BPA affects the transcriptomes of fetal mammary gland and may act directly on stromal cells, which in turn affect gene expression in epithelial cells (Wadia et al. 2013). Furthermore, they have found that BPA exposure impacts the epigenome of postnatal and adult mammary gland and alters gene expression in rat fetus (Dhimolea et al. 2014). However, the effects of DEHP exposure on gene expression of breast development during early pregnancy have not been studied. In this study, we used female mouse as a model to investigate the effects of DEHP on gene expression of mammary

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Histochem Cell Biol

gland cells in vivo and in vitro during early pregnancy. We asked whether low-dose exposure to DEHP is sufficient to alter gene expression during mammary gland development and impact the proliferation of mammary gland cells during pregnancy.

Materials and methods Animals and experimental design CD1 mice (Vital River, Beijing, China) were housed under temperature-controlled (21–22 °C) and light-controlled (12-h light and 12-h dark cycle) conditions. Natural mating was conducted by housing female mice with males together overnight and then checking for vaginal plugs the next morning (0.5 dpc) (Zhang et al. 2012, 2013a, b, c; Chao et al. 2012). All procedures were reviewed and approved by the Ethical Committee of Qingdao Agricultural University. Experimental DEHP dose was lower than the “safe” dosage suggested by the USA Food and Drug Administration (FDA). A total of 81 mice were used in this study, and each group included at least nine mice. Each group received a different dosage of DEHP treatment from 0.5 to 3.5 dpc (vehicle only, 20, 40, 80 or 160 µg/kg BW/day). DEHP was dissolved in 0.1 % DMSO and further diluted into drinking water. DEHP treatment was carried out by oral administration (Zhang et al. 2013a, b, c). Mammary gland tissue collection and preparation The fourth inguinal mammary glands of pregnant female mice (3.5 dpc) were isolated with the aid of a stereomicroscope (Nikon, SMZ1500, Japan) as described previously (Zhao et al. 2010). The glands were spread onto porous specimen collection bags and then placed in vials containing RNAlater (RNA-EZ Reagents RNA-Be-Locker A, Sangon, B644171-0025, China). The tissues were then fixed in 4 % paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4 °C overnight for histological analyses. After washed with deionized water, the fixed tissues were placed in carmine solution and aluminum potassium sulfate solution (1 g carmine (Sigma, C-1022) and 2.5 g aluminum potassium sulfate (Sigma, A-7167) in 500 mL ddH2O) at 4 °C overnight, followed by dehydration in 70 % alcohol for 15 min, 95 % alcohol for 15 min, 100 % alcohol for 15 min, and then cleaned with toluene for 15 min. The mammary gland tissues were kept in salicylic acid methyl ester for preservation. The paraformaldehyde-fixed tissues were embedded in paraffin and sectioned (5 μm in thickness) on a microtome (Nikon, SMZ1500, Japan).

Histochem Cell Biol

In vitro culturing of mouse mammary gland cells The fourth inguinal mammary glands of pregnant mice were isolated with the aid of a stereomicroscope (Nikon, SMZ1500, Japan). Then mammary gland tissue samples other than breast nodules were washed in Hank’s balanced salt solution (HBSS) for eight times, cut into 1-mm2 pieces and digested in 0.25 % trypsin–EDTA for 25 min at 37 °C. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and the tissues were digested in 2–3 mL 1.5 mg/mL collagenase I for 25 min at 37 °C. Cells were washed and passed through 150-μm nylon cell strainers (BD, USA), centrifuged and suspended in 3 mL DMEM/F12 (Gibco, C1130500BT, China) containing 10 % fatal bovine serum (FBS) (Gibco, 10099-141, China), 1 % penicillin/streptomycin, epidermal growth factor (EGF) (10 ng/mL, Sigma, E4127, USA), 5 µg/ mL insulin and 5 µg/mL hydrocortisone. Cells were cultured in 60-mm dishes at 37 °C in a humidified incubator with 5 % CO2. Media were changed every other day. Primary mammary gland cell cultures were grown to 80 % confluence and then washed two times in the D-Hank’s medium. The cells were treated with 0.25 % trypsin–EDTA for 2 min at 37 °C and collected for subculture in 24-well plates. To study the impact of DEHP on mammary gland cells, in vitro culture cells were exposed to 10 or 100 μM DEHP in 0.1 % DMSO, or 0.1 % DMSO alone as the vehicle control. After being exposed to DEHP for 3 days, the cells were collected and rinsed once with precooled PBS and kept at −20 °C for further examination. Cell viability analysis Cell viability was measured using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide staining (MTT) (Sigma, M5655, USA). Briefly, mammary gland cells were seeded in a 96-well plate at 2000 cells/well after primary culture. Cells were then divided into three treatment groups, each composed of five repeats. DEHP was diluted in the culture media at 0 (vehicle control only), 10 and 100 µM, and cells were cultured for 72 h after DEHP exposure. The supernatant was aspirated, and cells were incubated in 0.5 mg/mL MTT solution at 37 °C for 4 h. Then formazan crystals were dissolved by the addition of 150 µL DMSO to each well and incubation for 1 h. Absorbance was measured with a multiwell microplate reader at a wavelength of 570 nm. Data were presented as the ratios of the optical density (OD) values of the experiment group to the control group. BrdU assay To detect the degree of proliferation, BrdU labeling and anti-BrdU antibody (Sigma, B2531, USA) were used. The mammary gland cells of pregnant mouse were incubated

with 10 mM BrdU for 48 h with or without DEHP treatment (0, 10 and 100 µM). Cells were then fixed in 4 % paraformaldehyde for 30 min and incubated with the anti-BrdU antibody (Chen and Chien 2014). Images were obtained using a fluorescence microscope (Olympus BX51, Japan). Positive signal was scored in more than 500 cells. Cell cycle analysis using flow cytometry To analyze cell cycle, mammary gland cells were resuspended after 0.25 % trypsin treatment, fixed in 70 % cold ethanol and incubated for a minimum of 20 min. Then cells were pelleted (1000 rpm for 5–7 min). The tubes were carefully inverted to decant the ethanol, and 0.5 mL PI-RNAse solution (final concentrations: 50 µg/mL PI + 100 µg/ mL RNAse Type I-A in PBS) was added into the tubes and incubated at 37 °C in the dark for a minimum of 20 min before flow cytometry analysis (BD FACSCalibur, FACS101, USA). Forty thousand events were acquired per sample. Quantification analysis was completed using the ModFit software. Flow cytometry analysis of intracellular reactive oxygen species (ROS) The cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) (Beyotime, S0033, China) was used as an indicator of intracellular reactive oxygen species (ROS) via fluorescent staining. After incubation for 24 h, the mammary gland cells of pregnant mice were trypsinized and collected and washed three times in PBS and then incubated in 10 µM DCFH-DA at 37 °C for 20 min according to the manufacturer’s instructions. 2′,7′-Dichlorofluorescein (DCF) fluorescence was detected at the emission wavelength of 525 nm by flow cytometry (Becton–Dickinson) upon excitation at 488 nm. Dihydroethidium (DHE) fluorescent probe (Beyotime, S0063, China) was used for detecting superoxide anion (O2−) in the gland cells. The cells were incubated with 10 μM DHE for 30 min at 37 °C according to the manufacturer’s instructions. Measurement of fluorescence was taken on flow cytometry at excitation wavelength of 535 nm and emission wavelength of 610 nm. Western blot analysis Total proteins were released from mammary gland cell samples in RIPA lysis solution (Beyotime, P0013C, China) for 30 min on ice with frequent vortexing. Then 16 µL protein extract was mixed with 40 µL of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer and boiled for 5 min. Lysates were collected by centrifugation (14,000 rpm for 5 min). Proteins

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were separated by SDS-PAGE with a 4 % stacking gel and a 10 % separating gel for 2 h at 100 V and then transferred onto polyvinylidene fluoride membrane (18 min, 15 V) by electrophoresis. The membrane was blocked in TBST (Tris-buffered saline with Tween-20) with 10 % BSA at 4 °C for 4 h and incubated with the mouse anti-PCNA (proliferating cell nuclear antigen) antibody (Zhongshan Goldenbridge Biotechnology, ZM-0213, China) at 1:1000 dilution in TBST buffer containing 5 % bovine serum albumin (BSA) (Solarbio, A8020, China) overnight at 4 °C. Then membranes were washed three times in TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime, A0216, China) at 1:2000 dilution in TBST. The BeyoECL plus kit (Beyotime, P0018, China) was used for signal development. Beta-actin was used as the loading control. The intensity of the signal was analyzed using the AlphaView SA software. Immunofluorescence Cultured mammary gland cells were washed three times with PBS and fixed in 4 % paraformaldehyde for 20 min at 4 °C. After being washed three times in PBS, cells were permeabilized in PBS solution supplemented with 0.5 % Triton X-100 for 10 min. Next, cells were washed one time in PBS and blocked for 45 min with 10 % normal goat serum (Boster, AR0009, China) and 0.5 % Triton X-100 in PBS, followed by incubation with the primary PCNA antibody overnight at 4 °C. After being washed three times in PBS with 1 % BSA, cells were incubated with Cy3-conjugated goat anti-mouse IgG secondary antibody (Beyotime, A0521, China) for 2 h at 37 °C. Vectashield mounting media containing Hoechst 33342 was used to stain the nuclei. RNA extraction and RNA expression profiling Total RNAs were extracted from mammary gland tissues using the Trizol reagent and eluted in RNase-free water. The integrity of total RNAs was analyzed by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and no significant RNA degradation was shown. Total RNA concentration was measured using the Nanodrop spectrophotometer. A total of 6 μg Cy5-labeled RNA targets were hybridized to a mouse oligo microarray (Phalanx Mouse Whole Genome OneArray™; Phalanx Biotech Group, Palo Alto, CA, USA) according to the manufacturer’s protocol. Each array contained 26,423 mouse DNA oligonucleotide probes, and each probe was a 60-mer designed in the sense direction. Among the probes, 872 control probes were on the array, and 25,551 probes were designed according to the RefSeq release 42 and Ensembl release 59 database. The data were analyzed according to the manufacturer’s protocol. After hybridization, the

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Histochem Cell Biol

fluorescent signals on the array were scanned by an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA, USA). Two replicates from two independent mice were used. Microarray signal intensity of each spot was analyzed by the GenePix 4.1 software (Molecular Devices, Sunnyvale, CA, USA). Each signal value was normalized by the R program in the Limma package. The Limma pipeline included linear modeling to analyze complex experiments for testing differential expression, and we used it for signal value quantile normalization (Limma powers differential expression analyses for RNA sequencing and microarray studies). Bioinformatics analysis of mRNA expression profile The expressions of mRNAs with log2 |fold change| >1 (absolute |fold change| >2) and P