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Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is the most common plasticizer used in polyvinyl chloride-based plastics. DEHP is not covalently bound to the plastics and is easily released to the environment, resulting in human exposure. In this study, the adult rats were exposed to DEHP and its effects on the uterus was evaluated. Healthy adult female rats were treated with DEHP orally (with dose level 0, 1, 10, and 100 mg/kg body weight/day) for 30 days. No significant changes in the body weight and wet uterine weight were observed. Ovarian hormones and their receptor levels in the uterus were increased. Histological studies exhibited the structural abnormalities such as decrease in diameter, thinning of the layers and disruption in the glandular epithelium.

Keywords

DEHP estrogen receptors progesterone receptors PPAR uterus

Introduction

Phthalates are alkyl diesters of phthalic acid, termed based on the lengths of the alkyl chains, they are used to impart flexibility in plastic or as a matrix in cosmetic products. Phthalates are not covalently bound to the plastic and thus leach into the environment over time where they become ubiquitous contaminants.¹ Phthalates, including the widely used plasticizer, di-(2-ethylhexyl) phthalate (DEHP), are the most abundant pollutants in our general environment. DEHP remains to be components of food wraps, medical devices, and many cosmetic products. The Agency for Toxic Substances and Disease Registry evaluated that the maximum daily exposure to DEHP for the general population is about 2 mg/day.² After ingestion in rodents, DEHP is hydrolyzed by lipases to mono 2-ethyl hexyl phthalate (MEHP) and 2-ethylhexanol in the digestive tract prior to absorption.³

In vivo studies demonstrated that ovary is the target site for DEHP and hence decrease the estradiol production. DEHP acts through peroxisome proliferator-activated receptor (PPAR) and other aryl hydrogen receptors. Decreased expression of aromatase as a result of PPARγ activation and increased 17β-HSD expression as a result of PPARα activation may be responsible for the change in steroidogenesis.³ A relative increase in estrone production and a decrease in estradiol production from mouse follicles were observed with MEHP treatment.⁴

DEHP (2 g/kg) treatment to Sprague-Dawley rats decreased serum estradiol levels, prolonged estrous cycles, and no ovulation.⁵ In vivo studies in rats showed that DEHP (300 and 600 mg/kg) significantly decreased the estradiol level and also the aromatase mRNA and protein levels.⁶ In mice, Antral follicles from adult mice cultured with DEHP and MEHP inhibited follicle growth, estradiol production and decreased aromatase mRNA levels. Interestingly, estradiol co-treatment prevented phthaltes-induced inhibition of follicle growth and estradiol production.⁷

The perinatal exposure to estradiol compounds or related endocrine disrupting chemicals (EDCs) produced lesions in adult uteri that including cystic endometrial hyperplasia, squamous metaplasia, adenomyosis, and myometrial and general uterine hypoplasia.⁸

There are reports documenting the interactions between PPARγ and estrogen signaling or uterine physiology. It has been shown that activation of PPARγ inhibits the growth of uterine leiomyoma, which is also an estrogen-dependent disease.⁹

The major target organ for ovarian hormones is uterus. When the ovary is the target for these EDCs, eventually it will affect the uterus in bringing some abnormal changes. The reports on DEHP affecting the uterus of adult female rats during the cycling condition are very scarce. DEHP effect on the uterus at a low concentration level is less clear, although it may cause uterine related impairments. Several studies proved DEHP-altered ovarian steroidogenesis and its action on its target organs. The current study focuses on the effects of DEHP on the uterus through ovarian hormones action. In the present study, DEHP doses were selected were within the range of normal to occupational exposure levels in humans. Our data suggests that the change in ovarian hormones levels in DEHP-treated groups also altered steroid hormone receptor expression in the uterus. Histoarchitecture of uterine showed disintegrated epithelial cells, which suggests that DEHP to affect the uterus through estrogenic action.

Materials and methods

Chemicals

DEHP or dioctyl phthalate with ≥99.5% purity (D201154; CAS no. 117-81-7; 500 ml) was purchased from Sigma-Aldrich (St Louis, Missouri, USA). All other chemicals and reagents used in this study were of analytical grade and were purchased from Sisco Research Laboratories (Mumbai, Maharashtra, India). Antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). β-Actin antibody was purchased from Sigma-Aldrich.

Animals

Animals were maintained as per the National Guidelines and Protocols approved by the Institutional Animal Ethical Committee (IAEC No. 01/01/10). Female Wistar adult rats, weighing around 150–200 g were used in this study. Animals were housed in polypropylene cages under specific humidity (65 ± 5%) and temperature (21 ± 2°C) with constant 12-h light/12-h dark schedule. They were fed with standard rat-pelleted diet (Lipton India, Mumbai, Maharashtra, India), and clean drinking water was made available ad libitum.

Dose selection and treatment

The adult female rats were administered with DEHP by gavage at different dose of 0 (olive oil), 1, 10, and 100 mg/kg body weight/day for 30 days. The dose range was selected based on the reference value close to the predictable, normal to occupational exposure to the human population. This is based on the formula that suggestively converse chemical exposure to various species by considering body surface area and metabolism.¹⁰ Fresh solutions were prepared daily according to the weight of rats. The dose volume was 0.2 ml in all groups. The rats in the vehicle control group received olive oil in equal volume as in experimental groups for 30 days. During the treatment period, body weight and estrous cycles were monitored. After treatment, the animals were weighed and killed; trunk blood was collected, centrifuged at 3000 g for 10 min at 4°C and stored at −80°C for the estimation of serum hormones. The uterus was excised immediately and adjacent fat tissues were removed, weighed, and immediately used for RNA and protein isolation. One uterine horn from each rat was fixed in 10% formalin for histopathological evaluation.

Serum progesterone and estradiol assay

Serum progesterone and estradiol concentrations were measured using Direct ELISA kits (Diagnostic Biochem Canada Inc., Ontorio, Canada), according to the manufacturer’s instructions. ELISA plates were read in BioTek plate reader (Winooski, Vermont, USA). Samples and standards were analyzed in duplicate. The sensitivity of progesterone and estradiol ELISA kits were 0.1 ng/ml and 10 pg/ml, respectively. The intra- and inter-assay coefficients of variation were 10.6% and 12.6, respectively, for progesterone and 9.3% and 10.1% for estradiol.

Histology of uterine structure

The uterine tissue was fixed in 10% formalin and with paraffin, sectioned at 7 mm, and stained with hematoxylin and eosin for microscopic examination. Sections were then captured using a Nikon eclipse 80i microscope and differentiated (Chiyoda, Tokyo, Japan).

Total RNA extraction and real-time RT-PCR

Total RNA was extracted from the uterus by a single-step technique using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The quantity and the integrity of total RNA was determined by ultraviolet spectrophotometer and agarose gel electrophoresis, respectively. Reverse transcription (RT) was done using M-MuLV Reverse Transcriptase enzyme (NEB, Ipswich, Massachusetts, USA). For cDNA synthesis, 2 μg of total RNA was reverse transcribed using random hexamer primers and MMLV-RT enzyme. The cDNA was subsequently used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the Mesa Green qPCR kit (Eurogentec, Fremont, California, USA) with gene-specific primers (Table 1). The real-time RT-PCR was performed on a CFX 96 Touch Real-Time PCR (Bio-Rad, Hercules, California, USA) with the PCR conditions as given in Table 2. The specificity of the amplification product was determined by melting curve analysis for each primer pairs. The data were analyzed by comparative threshold cycle (CT) method and the fold change was calculated by 2^−ΔΔCT method using CFX Manager Version 2.1 (Bio-Rad).

Table 1.

List of primers.

Name	Accession number	Primer sequence
β-actin	NM_031144.3	Forward 5′-CGTCCACCCGCGAGTACAACC-3′
β-actin	NM_031144.3	Reverse 5′-TCCATGGCGAACTGGTGGCG-3′
PR	NM_022847.1	Forward 5′-GGAAGCCTCGCGCCGAAGAA-3′
PR	NM_022847.1	Reverse 5′-GAGGGCGCCAACAGGGTGTC-3′
PPARγ	NM_013124.3	Forward 5′-GCATGGTGCCTTCGCTGATGC-3′
PPARγ	NM_013124.3	Reverse 5′-AGTTGGTGGGCCAGAATGGCA-3′
ERα	NM_012689.1	Forward 5′-ATCACACACCGCGCCACTCG-3′
ERα	NM_012689.1	Reverse 5′-AGCAGCGGATGAGCCACCCT-3′
ERβ	NM_012754.1	Forward 5′-CAGGCCTGCCGACTTCGCAA-3′
ERβ	NM_012754.1	Reverse 5′-TCAGGCAGTGTACCTGCTCGCT-3′

PR: progesterone receptor; PPAR: peroxisome proliferator-activated receptor; PCR: polymerase chain reaction.

Table 2.

Conditions used in quantitative PCR reactions.

Reaction	Temperature (°C)	Time
Initial denaturation	95	5 min
Denaturation	95	30 s
Annealing	56	30 s
Extension	72	30 s
Final extension	95	5 min
Melt curve	56–95°C	Increment 0.5°C 0:05 Plate read

Immunoblot analysis of ER and PR in the uterus

Protein from the uterus was extracted using RIPA buffer with proteinase inhibitors (Roche, Germany). Protein concentration in the tissue extract was determined in triplicate using Bio-Rad dye reagent (Bio-Rad). Total protein lysates were mixed 1:1 with 2× Laemmli sample buffer and boiled for 5 min and then centrifuged at 12,000 r/min for 2 min. An equal volume of the total protein was resolved on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membrane (Bio-Rad). After blocking in 5% non-fat milk protein in PBS/Tween, the membranes were incubated with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) such as ERα (1:500; cat. no. sc-542), ERβ (1:1000; cat. no. sc-6822), progesterone receptor (PR; 1:500; cat. no. sc-7208), PPARγ (1:500; cat. no. sc-7273), and β-actin (1:5000; cat. no. A1978; Sigma-Aldrich) for overnight at 4°C. After washing in PBS with Tween-20, the blots were incubated with respective secondary antibodies conjugated with HRP (1:10,000; GeNei, Bangalore, Karnataka, India) for 1 h at room temperature. The membranes were then washed four times in PBS-Tween buffer. Immunoactive antigen–antibody complexes were visualized with Enhanced Chemiluminescent reagent (ECL) (Thermo Scientific, Rockford, Illinois, USA), the signals were captured by the Chemi Doc XRS system (Bio-Rad), and the intensity of the bands were quantified by Quantity One software (Bio-Rad). The protein products of all the genes studied were normalized with β-actin.

Statistical analysis

All data were analyzed using GraphPad Prism 5.0 Software (GraphPad Software, La Jolla, California, USA) and expressed as mean ± standard error of mean. Differences between groups were analyzed by one-way analysis of variance followed by the Student–Newman–Keul’s test for multiple post hoc comparison tests and p < 0.05 was considered to be significant.

Results

Effect of DEHP on the body weight and uterus weight of adult rats

All animals were healthy and no evidence of diseases or death was observed during the treatment period. Body weight was recorded throughout the treatment period. No change in the body weight was observed in treated group when compared to control. The relative uterine wet weight in the treated group did not show significant difference when compared with control (Figure 1(a) and (b)).

Figure 1.

Impact of oral exposure to DEHP on the body weight and the uterus weight of adult female rats. (a) Body weight, (b) relative uterine wet weight, (c) estradiol, (d) progesterone, and (e) histoarchitecture of the uterus. Values are represented as mean ± SEM of six rats. p < 0.05 was considered statistically significant, (a′) compared with control, (b′) compared with 1 mg, and (c′) compared with 10 mg DEHP kg⁻¹ day⁻¹. DEHP: di-(2-ethylhexyl) phthalate; SEM: standard error of mean; P: perimetrium; M: myometrium; E: endometrium; EG: endometrial gland; GE: glandular epithelium.

Effect of DEHP exposure on the serum estradiol and progesterone levels in adult female rats

The effect of DEHP exposure on ovarian steroidogenesis was assessed by ELISA method. The serum levels of estradiol were not altered in 1 mg and 10 mg DEHP treatment group but marginally increased in 100 mg DEHP treatment group.

The serum level of progesterone was increased in 1 mg and 10 mg DEHP treatment groups compared to control. Whereas, in 100 mg DEHP-treated group, serum progesterone level was not altered (Figure 1(c) and (d)).

Effect of DEHP exposure on the structure of the uterus of adult Wistar rats

The histology of uterine tissues showed decrease in diameter of the uterus and numbers of uterine glands in DEHP-treated rats. The affected glandular epithelial cells were observed in 10 and 100 mg DEHP-treated rats. Myometrial and perimetrial layers disc disintegrated in 10 and 100 mg DEHP-treated rats (Figure 1(e)).

Effect of DEHP exposure on the levels of mRNA of ER_s, PR, and PPARγ in the uterus of adult rats

Although both ERα and ERβ are expressed in the uterus, ERα is highly expressed in the uterine epithelium and induces proliferation of epithelial cells. It has been reported that ERβ has antiuterotrophic effect. Decrease in the mRNA expression was observed in the expression of ERα in the 100 mg DEHP-treated group when compared to control, but there was no change in the ERβ (Figure 2(a) and (b)). A dose-dependent decrease in the PR mRNA was observed in the DEHP-treated groups as compared with the control group. Interestingly, DEHP treatment increased PPARγ expression in the 1 mg treated group, whereas 10 and 100 mg treatment shows decreased mRNA expression (Figure 2(c) and (d)).

Figure 2.

Impact of DEHP exposure on the gene expression of ERs, PR, and PPARγ in the uterus of adult rats. (a) ERα mRNA and protein, (b) ERβ mRNA and protein, (c) PR mRNA and protein, and (d) PPARγ mRNA and protein in the whole uterine lysate of adult rats. The mRNAs levels were analyzed by real-time RT-PCR and protein levels by western blotting. Target gene expression was normalized to β-actin mRNA and the results are expressed as fold change from control. For western blotting, β-actin was used as loading control. The immunoreactive bands were detected with ECL reagent in Chemi Doc XRS Imaging System, Bio-Rad. Each bar represents mean ± SEM of three observations. p < 0.05 was considered statistically significant, (a′) compared with control, (b′) compared with 1 mg, and (c′) compared with 10 mg DEHP kg⁻¹ day⁻¹. DEHP: di-(2-ethylhexyl) phthalate; ER: estrogen receptor; TRIR: total RNA isolation reagent; cDNA: complementary DNA; PR: progesterone receptor; PPAR: peroxisome proliferator-activated receptor; RT-PCR: reverse transcriptase polymerase chain reaction; SEM: standard error of mean.

Effect of DEHP exposure on protein levels of ER_s, PR, and PPARγ in the uterus of adult rats

ERs and PR levels were increased in the dose-dependent manner (Figure 2(a) to (c)). DEHP treatment also increased PPARγ protein level in the all DEHP treatment groups (Figure 2(d)). Data from both real-time RT-PCR and western blot analyses showed DEHP treatment upregulated both ERα and ERβ expression as compared to control.

Discussion

This study was designed to evaluate the impact of DEHP exposure on the uterus of adult Wistar rats. Body weight is an important non-specific indicator comprehensively reflecting the toxicity of substances and can be used to evaluate the effect of DEHP on the growth status of rats. It has been reported that DEHP can limit the body weight gain through interfering with the fat metabolism and synthesis.¹¹ In the present study, the animals exposed to DEHP did not show any significant change in the body weight and relative uterus weight. Earlier reports indicated that high DEHP doses (>1000 mg/kg/day) adversely affected body weight and organ weight of adult female rats.^5,12 It appears that low-dose DEHP did not exert obvious toxic effect on the animals as observed by unaltered body weight and uterine weight.

Sex steroids influence the growth, differentiation, and function of female reproductive organs such as the uterus. During the estrous cycle, fluctuating levels of estradiol and progesterone elicit profound effects on epithelial proliferation and cytodifferentiation of these organs.¹³ In the current study, DEHP treatment showed effects on the uterus epithelial cell morphology and on its proliferation. The uterus responds to ovarian steroids to produce a collective physiological response which is vital for a successful reproductive cycle. The increase in hormonal level of estradiol and progesterone is likely to increase the proliferation of uterine cells, which is the major target of ovarian hormones.

The ERα is highly expressed in the uterine epithelium and induces proliferation of epithelial cells. ERβ is also present in the uterus and the level changes during the ovarian cycle. It has been reported that ERβ has an anti uterotrophic effect and dampen the effects on ERα.¹⁴ In this study, the increase in estradiol level was also associated with the upregulated receptor expression in the uterus among DEHP-treated groups, which could have implicated in the development of inflammatory diseases in the uterus.

In the present study, an inverse relationship between ERα and ERβ protein expression observed in control and DEHP-treated group except in 1 mg DEHP treatment. The downregulation of ERβ may be responsible for the upregulation of ERα. The PR gene is a well-described target of estradiol-induced expression via the classic model of ERα action, especially in the uterus. Progesterone is involved in the differentiation of uterine epithelium and estradiol induces the expression of PR in uterine epithelium through ERα. A dose-dependent upregulation of PR expression in the DEHP-treated rat was observed, which may be attributed to the estrogenic activity of the DEHP.

It has been established that phthalates act as an agonist for nuclear receptors, PPARα and PPARγ. PPARs are expressed in uterine tissues and the action of PPARs on estradiol signaling in the uterus has also been reported in earlier study.⁸ In the present study, PPARγ was upregulated in DEHP-treated rats. Depending on the experimental system, PPAR ligands, such as DEHP, can be pro-apoptotic or anti-apoptotic.¹⁵ The increased ERα observed in the DEHP-treated animals might have a role in the induction of PPARγ because PPARγ is an estradiol responsive gene and the levels of estradiol are positively correlated with PPARγ. On the other hand, it has been reported that PPARγ acts as an anti-adhesive molecule on the uterine surface, thus preventing implantation.¹⁶

The uterus is composed of heterogeneous cell types (stroma, luminal epithelial, glandular epithelium, and smooth muscle) that undergo continuous synchronized changes of proliferation and differentiation in response to changes in levels of circulating estradiol and progesterone. The perinatal exposure to estradiol compounds or related EDCs produced lesions in adult uteri;⁸ similar effect of DEHP were observed in our study on the rat uterus. The histopathological abnormalities were observed in the uterus of DEHP-treated rats which showed decreased diameter of the uterine horn and endometrial glands. The thinning of the uterine layer as well as disruption in the uterine glands structure was observed in DEHP-treated animals. The present study suggests that uterine disorders can develop if there is exposure to DEHP for longer period or increasing dosage. In this regard, women with endometriosis showed significantly higher plasma DEHP concentration than controls.¹⁷

In summary, the present study demonstrates that DEHP exposure upregulated ovarian steroidogenesis and altered uterine architecture and steroid sex hormone receptor expression which may lead to any uterine related inflammatory diseases.

Footnotes

Authors’ note

DB Somasundaram and K Manokaran have equally contributed.

Acknowledgments

Financial support provided in the form of UGC-RFSMS to DBS is acknowledged. The authors acknowledged financial assistance to the Department of Endocrinology by UGC-SAP-DSA-I programme.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by DST-EMEQ project to RSB.

References

Wittassek

Koch

Angerer

. Assessing exposure to phthalates - the human biomonitoring approach. Mol Nutr Food Res 2011; 55: 7–31.

Albro

. Absorption, metabolism, and excretion of di(2-ethylhexyl) phthalate by rats and mice. Environ Health Perspect 1986; 65: 293–298.

Lovekamp-Swan

Davis

. Mechanisms of phthalate ester toxicity in the female reproductive system. Environ Health Perspect 2003; 111: 139–145.

Lenie

Smitz

. Steroidogenesis-disrupting compounds can be effectively studied for major fertility-related endpoints using in vitro cultured mouse follicles. Toxicol Lett 2009; 185: 143–152.

Davis

Maronpot

Heindel

. Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicol Appl Pharmacol 1994; 128: 216–223.

Chen

Qiu

. Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicol Lett 2010; 199: 323–332.

Gupta

Singh

Leslie

. Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro. Toxicol Appl Pharmacol 2010; 242: 224–230.

Houston

Copland

Broaddus

. Inhibition of proliferation and estrogen receptor signaling by peroxisome proliferator-activated receptor gamma ligands in uterine leiomyoma. Cancer Res 2003; 63: 1221–1227.

Goldsworthy

Popp

. Chlorinated hydrocarbon-induced peroxisomal enzyme activity in relation to species and organ carcinogenicity. Toxicol Appl Pharmacol 1987; 88: 225–233.

10.

Reagan-Shaw

Nihal

Ahmad

. Dose translation from animal to human studies revisited. FASEB J 2008; 22: 659–661.

11.

Itsuki-Yoneda

Kimoto

Tsuji

. Effect of a hypolipidemic drug, Di (2-ethylhexyl) phthalate, on mRNA-expression associated fatty acid and acetate metabolism in rat tissues. Biosci Biotechnol Biochem 2007; 71: 414–420.

12.

Takai

Hayashi

Kiyokawa

. Collaborative work on evaluation of ovarian toxicity. Two- or four-week repeated dose studies and fertility study of di-(2-ethylhexyl) phthalate (DEHP) in female rats. J Toxicol Sci 2009; 34 Suppl 1: SP111–SP119.

13.

Boutin

Cunha

. Estrogen-induced epithelial proliferation and cornification are uncoupled in sinus vaginal epithelium associated with uterine stroma. Differentiation 1997; 62: 171–178.

14.

Weihua

Saji

Makinen

. Estrogen receptor (ER) beta, a modulator of ER alpha in the uterus. Proc Natl Acad Sci USA 2000; 97: 5936–5941.

15.

Atarod

Kehrer

. Dissociation of oxidant production by peroxisome proliferator-activated receptor ligands from cell death in human cell lines. Free Radic Biol Med 2004; 37: 36–47.

16.

Shalom-Barak

Nicholas

Wang

. Peroxisome proliferator-activated receptor gamma controls Muc1 transcription in trophoblasts. Mol Cell Biol 2004; 24: 10661–10669.

17.

Cobellis

Latini

De Felice

. Endometriosis women showed significantly higher plasma DEHP concentration than controls. Human Reprod 2003; 18: 1512–1515.

Impact of di-(2-ethylhexyl) phthalate on the uterus of adult Wistar rats