WHAT’S KNOWN ON THIS SUBJECT: Being an androgenantagonist and a possible estrogen agonist, severalendocrinologic effects of DEHP have been demonstrated mostly invitro and in animals. A limited number of studies have linked theendocrinologic effects of DEHP and its metabolite, MEHP, inhumans.
WHAT THIS STUDY ADDS: To our knowledge, this is the firststudy to define a possible effect of DEHP and MEHP in pubertalgynecomastia. The etiology of pubertal gynecomastia is attributedeither to excessive estrogen, deficient androgen, increasedaromatase enzyme activity, or a combination.
abstractOBJECTIVE: Several untoward health effects of phthalates, which are agroup of industrial chemicals with many commercial uses includingpersonal-care products and plastic materials, have been defined. Themost commonly used, di-(2-ethylhexyl)-phthalate (DEHP), is known tohave antiandrogenic or estrogenic effects or both. Mono-(2-ethylhexyl)-phthalate (MEHP) is the main metabolite of DEHP. In this study, weaimed to determine the plasma DEHP and MEHP levels in pubertalgynecomastia cases.
PATIENTS AND METHODS: The study group comprised 40 newly diag-nosed pubertal gynecomastia cases who were admitted to HacettepeUniversity Ihsan Dogramacı Children’s Hospital. The control groupcomprised 21 age-matched children without gynecomastia or otherendocrinologic disorder. Plasma DEHP and MEHP levels were mea-sured by using high-performance liquid chromatography. Serum hor-mone levels were determined in some pubertal gynecomastia casesaccording to the physician’s evaluation.
RESULTS: Plasma DEHP and MEHP levels were found to be statisticallysignificantly higher in the pubertal gynecomastia group comparedwith the control group (P � .001) (DEHP, 4.66 � 1.58 and 3.09 �0.90 �g/mL, respectively [odds ratio: 2.77 (95% confidence interval:1.48–5.21)]; MEHP, 3.19 � 1.41 and 1.37 � 0.36 �g/mL [odds ratio:24.76 (95% confidence interval: 3.5–172.6)]). There was a statisticallysignificant correlation between plasma DEHP and MEHP levels (r : 0.58;P� .001). In the pubertal gynecomastia group, no correlation could bedetermined between plasma DEHP and MEHP levels and any of thehormone levels.
CONCLUSIONS: DEHP, which has antiandrogenic or estrogenic effects,may be an etiologic factor in pubertal gynecomastia. These resultsmaypioneer larger-scale studies on the etiologic role of DEHP in pubertalgynecomastia. Pediatrics 2010;125:e122–e129
AUTHORS: Erdem Durmaz, MD,a Elif N. Ozmert, MD, PhD,a
Pınar Erkekoglu, PhD,b Belma Giray, PhD,b Orhan Derman,MD,a Filiz Hıncal, PhD,b and Kadriye Yurdakok, MDa
Departments of aPediatrics and bToxicology, HacettepeUniversity, Ankara, Turkey
Address correspondence to Elif N. Ozmert, MD, PhD, HacettepeUniversity, Faculty of Medicine, Department of Pediatrics, SocialPediatrics Unit, 06100, Ankara, Turkey. E-mail: [email protected]
Pubertal gynecomastia refers to thebenign enlargement of male breast at-tributable to the proliferation of duc-tile elements. Pubertal gynecomastiais a common problem occurring in upto 65% of adolescent boys.1
Male breast tissue has estrogen andandrogen receptors. Estrogens sti-mulate and androgens inhibit breasttissue proliferation; pubertal gyneco-mastia is usually caused by an imbal-ance between these 2 factors, whichmay be attributable to excessive estro-gen activity, deficient androgen activ-ity, increased aromatase enzyme activ-ity, or a combination of these effectson breast tissue.2
Phthalates, esters of a-phthalic acid,are a group of industrial chemicalswith many commercial uses, includingpersonal-care products (eg, perfumes,lotions, cosmetics), paints, buildingmaterials, household furnishings,clothing, dentures, children’s toys,cleaning materials, insecticides, andmost commonly as plasticizers in thefood, certain medical devices, andpharmaceuticals.3–6 Di-(2-ethylhexyl)-phthalate (DEHP) is one of the mostwidespread phthalate plasticizers. Theannual production volume of DEHPalone has been estimated at 2 milliontons.7–9 Consumer products containingphthalates can result in human expo-sure through direct contact and use,indirectly through leaching into otherproducts, or general environmentalcontamination. Humans are exposedthrough ingestion, inhalation, and der-mal exposure.4–6,10 The Agency for ToxicSubstances and Disease Registry esti-mates that the maximum daily expo-sure to DEHP for the general popula-tion is �2 mg/d.10 It is also estimatedthat the total intake of DEHP is higherin all children younger than 19 yearsold than in adults.11 DEHP and itsmetabolites have been detected inblood and urine samples from a highpercentage of the people screened
for phthalates. Mono-(2-ethylhexyl)-phthalate (MEHP) is known as thefirst and the main metabolite ofDEHP.12
DEHP has been reported to be an an-drogen antagonist,13 an initiator ofliver and testicular cancer in rats,14,15
and to interfere with tamoxifen-induced apoptosis in human breastcancer cells because of its estrogenic-ity.16 Although DEHP is considered to bean estrogen agonist and a testoster-one antagonist, its mechanisms of tox-icity are still not well understood, butare thought to be a function of the dis-ruption of endocrine-regulated geneexpression by interaction with estro-gen receptor �.17
Although there are some in vivo stud-ies that show no estrogenic effect,18
several articles have been recentlypublished in which the authors re-ported a possible estrogenic effect ofDEHP in humans, such as a probablerole in premature thelarche,19 endo-metriosis,20 and precocious puberty.21
As far as we know there is no report inwhich the authors investigated the re-lationship between DEHP/MEHP levelsand pubertal gynecomastia, whichdevelops as a result of an imbalancebetween androgenic and estrogenicactivity. In this study, we aimed todetermine the plasma DEHP andMEHP levels in patients with pubertalgynecomastia.
MATERIALS AND METHODS
Subjects
The study group comprised 40 patientswith pubertal gynecomastia, 11 to 15years old (mean, 13.2 � 0.9 years),who were admitted to Hacettepe Uni-versity Ihsan Dogramacı Children’sHospital in Ankara between Octoberand December 2007. Twenty-onehealthy male children of comparableage with no history of gynecomastiaand any other endocrinologic disor-
der comprised the control group(11.5–14.5 years old; mean, 13.2� 1.1years; P� .05).
All patients were examined by thesame pediatrician. Diagnosis of gy-necomastia was made by standardapproach2; testis volume was esti-mated by “Prader orchidometry” andstretched penis length (SPL) was mea-sured. Patients who had normal medi-cal history and physical examinationfindings apart from gynecomastiawere classified as pubertal gynecom-astia. In both groups, testis volumeswere minimum, 8 mm, and the Tannersexual development stage was 3 andabove. The BMI was calculated. Fami-lies filled out a questionnaire for pos-sible DEHP exposure.
The study was approved by HacettepeUniversity’s ethical committee. Writteninformed consent was obtained fromthe parents and children beforeparticipation.
Blood Sampling
Venous blood samples, for DEHP mea-surement, were taken by a stainlesssteel needle from the left arm cubitalvein, and the sample was allowed todrop into heparinized glass test tubesdirectly. The tube openings and allaround were covered by clean alumi-num foil to protect the sample fromcontacts with the screw caps and sun-light. Centrifugation was performed at800g, plasma was separated, and allsamples were immediately aliquotedinto glass vials and stored in a freezerat�80°C until analysis. All glass equip-ment was heated in an oven at 400°Cfor 4 hours after the general cleaningprocedure.
Chemicals
DEHP and MEHP were purchasedfrom Fluka (St Louis, MO) and Cam-bridge Chemicals Laboratories (An-dover, MA), respectively. Acetonitrile(high-performance liquid chromatog-
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raphy [HPLC] grade) and all other ana-lytical grade reagents were obtainedfrom Sigma Co (St Louis, MO) andMerck Co (Darmstadt, Germany).
Commercial kits for luteinizing hormone(LH), follicle-stimulating hormone (FSH),estradiol, prolactin, thyrotropin, free tri-iodothyronine (FT3), and free thyroxine(FT4)werepurchased fromAbbott Archi-tect (Abbott Park, IL), and the kits for thesex hormone-binding globulin (SHBG)were purchased from Zentech (Angleur,Belgium).Serumdehydroepiandrosteronesulfate (DHEA-S) and total testosteronelevels were tested by using the kits fromBio DPC Immulite (Los Angeles, CA).
Measurement of DEHP and MEHPLevels in Plasma
Determination of DEHP and MEHP con-centrations was conducted by HPLCequipped with an auto sampler(Hewlett Packard Agilent 1100 Series,Vienna, Austria) using a UV detector(� � 230 nm). A Spherisorb C18 ODS2column was used (4.6 mm, insidediameter; length: 25 cm, 5-�m particlesize; Waters, Milford, MA). The mobilephase was orthophosphoric acid 0.1%(acetonitrile [90:10, vol/vol]), and theflow rate was 1 mL/min.22
Two hundred microliters of plasmawere extracted by 400�L of NaOH (1N),100 �L of H3PO4 (50%), and 600 �L ofacetonitrile. The extraction was re-peated again by using 600 �L of aceto-nitrile, and supernatants collected af-ter the extractions were evaporatedunder nitrogen stream. The samplewas dissolved in 400 �L of mobilephase and injected to HPLC. The injec-tion volume was 100 �L. The retentiontimes for DEHP andMEHPwere 9.8min-utes and 3.1 minutes, respectively.
Recovery studies were performed onblank samples of plasma spiked withlevels of 7.5 ppm of DEHP and 1.25 ppmof MEHP, and the average recoverieswere found to be (mean � SD) 89 �1.24% for DEHP and 98 � 1.12% for
MEHP on 20 occasions. Between-runprecisions were 7.43 � 0.14% coeffi-cient of variation (CV) for DEHP and10.07� 1.25% CV for MEHP. Within-dayprecisions were 9.88 � 0.51% CV forDEHP and 6.99� 0.19% for MEHP.
The concentrations of DEHP and MEHPin the samples were calculated by us-ing the calibration curve of peak areaprepared for DEHP and MEHP stan-dards (Fig 1). The detection limits weredetermined as 0.05 ppm for DEHP andas 1 ppm for MEHP.
Determination of Serum HormoneLevels
Serum LH, FSH, estradiol, prolactin,thyrotropin, FT3, and FT4 levels were
measured by using the 2-step chemilu-minescence microparticle immunoas-say method. SHBG levels were testedby immunoradiometric assay. SerumDHEA-S and total testosterone levelswere also determined by solid-phasechemiluminescence immunoassay.Serum-free testosterone levels werecalculated according to the formu-la23,24 by using albumin, total testoster-one, and SHBG levels.
Statistical Analysis
Statistical analysis was performed byusing SPSS 13.0 (SPSS Inc, Chicago, IL).Data with normal distribution were ex-pressed as mean � SD. The distribu-tion of DEHP and MEHP values were an-
y = 121.91x + 28.655 = 0.9977
0
1000
2000
3000
4000
5000
6000
7000
0 10 20 30 40 50 60DEHP, ppm
Peak
are
a
y = 158.25x + 9.1537R2
R2
= 0.9912
0
200
400
600
800
1000
1200
1400
1600
1800
0.00 2.00 4.00 6.00 8.00 10.00 12.00MEHP, ppm
Peak
are
a
A
B
FIGURE 1Calibration curves of DEHP (A) and MEHP (B) standards.
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alyzed by using the Shapiro-Wilk testand were found to have normal distri-bution. The comparison between 2parametric values was made by usingStudent’s t test. Comparisons of multi-ple groups were made by using one-way analysis of variance and Bon-ferroni correction. The correlationbetween parametric values was ana-lyzed by using Pearson’s correlation.Nonparametric values were comparedby using the �2 test. When evaluatingDHEA-S, multivariate logistic regres-sion was used for determining the in-dependent variables. A P value of�.05was accepted as significant.
RESULTS
Forty patients with pubertal gynecomas-tia and21control patientswere includedin the study. However, 1 patient from thegynecomastia group and 1 patient fromthe control group were excluded fromanalysis because the values were at theend points and changed the normal dis-tribution curve.
The clinical characteristics of the pa-tients with pubertal gynecomastia andthe control patients are displayedin Table 1. There was no statisticallysignificant difference between thegroups. Pubertal gynecomastia was bi-lateral in 30 (76.9%) patients. Severepain was present in 9 (23%) patients,and 18 patients had been admitted tothe hospital with the complaint ofgynecomastia.
DEHP levels were detectable in allplasma samples and MEHP levels weredetectable in all patients with pubertalgynecomastia and 19 of the 20 controlpatients. Plasma DEHP andMEHP levelswere statistically significantly higherin the pubertal gynecomastia groupcompared with the control group (P�.001) (DEHP, 4.66 � 1.58 and 3.09 �0.90 �g/mL, respectively [odds ratio:2.77 (95% confidence interval: 1.48–5.21)]; MEHP, 3.19 � 1.41 and 1.37 �0.36 �g/mL, respectively [odds ratio:
24.76 (95% confidence interval: 3.5–172.6)]). There was a statistically sig-nificant correlation between DEHP andMEHP values in the pubertal gynecom-astia group (r� 0.44, P� .005); how-ever, the correlation was not signifi-cant in the control group (r � 0.43,P� .065). When all of the patients withpubertal gynecomastia and the controlpatients were evaluated totally again, astatistically significant high correlationwas found between DEHP and MEHP val-ues (r� 0.58, P� .01). The destitution ofplasma MEHP and DEHP levels in puber-tal gynecomastia and control cases areshown in Figs 2 and 3.
There was no statistically significantrelationship between DEHP and
MEHP levels and height, weight, BMIpercentile for age, testis volume, andSPL in gynecomastia or controlcases. Also, no relationship could bedetected between the breast en-largement (disk size) and DEHP andMEHP levels in the pubertal gyneco-mastia group (Table 2). The only sta-tistically significant difference inDEHP and MEHP levels was found inthe gynecomastia group accordingto the presence of pain on admission(Table 2).
The hormone levels of the patientswith pubertal gynecomastia were de-termined according to the suggestedalgorithm for the management of gy-necomastia.2 The hormone levels were
0
1
2
3
4
5
6
7
8
9
10D
EHP,
µg/
mL
Pubertal gynecomastia Control
P < .001
FIGURE 2The distribution of DEHP levels in patients with pubertal gynecomastia and control patients.
TABLE 1 Clinical Characteristics of Patients With Pubertal Gynecomastia and Control Patients
Characteristic Patients With PubertalGynecomastia (n� 39)
Control Patients(n� 20)
Age, mo 158.2� 11.7 159.4� 13.1Height, cm 157.9� 8.4 158.2� 7.1Weight, kg 50.5� 14.6 51.9� 8.9BMI 19.9� 3.9 20.6� 3.1BMI-for-age percentile, n (%)1–24 9 (23.1) 2 (10)25–75 17 (43.6) 10 (50)76–100 13 (33.3) 8 (40)Family history of pubertal gynecomastia 12 (30.7) 4 (20)Left testis, mL 12.8� 4.2 13.1� 4.5Right testis, mL 12.8� 4.2 12.6� 4.3Average of testis volumes consideringthe bigger testis, mL
12.8� 4.1 13.2� 4.4
SPL, cm 9.3� 1.4 9.8� 1.9
Data are presented as mean� SD unless otherwise indicated. P� .05 for all comparisons.
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all in the normal range (Table 3). Therewas no correlation between plasmaDEHP/MEHP levels and FSH, LH, SHBG,thyrotropin, FT4, FT3, and total and freetestosterone levels (Table 4). Therewas only a statistically significant cor-relation between DEHP and DHEA-S lev-
els. However, when the effect of SHBGwas adjusted, no correlation wasfound (r� �0.268, P� .13). All levelsfor �-human chorionic gonadotropinwere found to be�1.2 mIU/mL; there-fore, no additional analysis for corre-lation was made.
DISCUSSION
DEHP, the most commonly used plasti-cizer, is a widespread ubiquitous envi-ronmental contaminant. There aremany reports showing its long-termtoxic effects and tissue accumulationin animals.15,25–27 The toxic effects de-pend on dose, the age of the person,and the duration of exposure.28 It hasbeen shown that the toxic effects aremuch more during infancy, puberty,and pregnancy periods, and long-termsmall amounts of exposure can causeevident toxic effects.18 Experimentalanimal models showed its affects ondevelopmental stages.25,29–31 AlthoughDEHP has been shown to have toxiceffects for many systems, the mostimportant effects after long-term
0
1
2
3
4
5
6
7
8
Pubertal gynecomastia Control
MEH
P, µ
g/m
L
P < .001
FIGURE 3The distribution of MEHP levels in patients with pubertal gynecomastia and control patients.
TABLE 2 Plasma DEHP and MEHP Levels According to Gynecomastia Characteristics andAnthropometric Values
In this study, the possible effect ofDEHP in the etiology of pubertal gy-necomastia was investigated. It wasfound that for every 1 �g/mL increasein DEHP levels, the risk of pubertal gy-necomastia increased nearly three-fold. Plasma MEHP levels were studiedto increase the reliability of the re-sults. A high correlation was found be-tween DEHP and MEHP levels. The in-crease in the risk of gynecomastiawithMEHP levels was found to bemuchhigher (�25-fold).
This is the first study in which the au-thors investigated the relationship be-tween pubertal gynecomastia andDEHP levels, making it impossible tocompare with other study results. Wehave tried to minimize the risk for en-vironmental contamination as outlinedin the Materials and Methods section.No plastic material was used in anystage of the study. Blood was taken tothe laboratory in 30 minutes. Plasmasamples were separated and stored at�80°C immediately. The analyses ofthe pubertal gynecomastia cases andcontrol blood samples were made si-multaneously so if any contaminationoccurred it would have affected bothgroups. The diurnal and inter-day re-peatability of the DEHP analysismethod was found to be very high, theaverage recovery was 89% and the de-tection limit was low (0.05 �g/mL).However, the number of the pubertalgynecomastia cases and controls inthe study is limited, and the study de-sign does not allow for a direct conclu-sion of cause-effect.
We could not encounter any studyabout the DEHP levels in pubertal boys.However, in several studies, the bloodDEHP levels were studied in specificrisk groups such as women with endo-metriosis20 or newborn infants afterblood transfusion.32 Although it is notpossible to define a blood range for
DEHP levels, the DEHP levels found inthese studies are comparable with ourresults.
A clue to the cause-effect relationshipof DEHP in pubertal gynecomastia wasthe higher DEHP levels in patients whowere admitted to the hospital withpain. These patients mentioned thatgynecomastia had started in the previ-ous 3 months and most were bilateral.It is known that pain is experienced inthe early florid stage of gynecomastia.Patients who present with symptomsof pain and tenderness generally havegynecomastia of more recent onset,and pathologic findings include hyper-plasia of the ductal epithelium, infiltra-tion of the periductal tissue withinflammatory cells, and increased sub-areolar fat.33 Still, these results have tobe confirmed in larger case series.
Mostly, antiandrogenic propertieshave been demonstrated in animals. Ina study where pregnant rats weregiven DEHP during their pregnancy, in-fant rats were found to have de-creased anogenital distance, femaletype areola, and genital malforma-tions.27 In another study, intrauterineDEHP exposure was found to cause adecrease in testis size and testoster-one levels.28 Recently, similar resultswere reported for human infants.34 Theantiandrogenic effects of DEHP wererelated to decreased testosterone lev-els and it is thought that DEHP does nothave a direct effect on androgenreceptors.35–37
In our study, no difference could befound for testis size among the groups.Also, therewas no correlation betweenDEHP/MEHP levels and testis size. How-ever, there was a reverse but nonsig-nificant correlation between plasmaDEHP levels and free and total testos-terone levels. Again, these results needconfirmation from larger case series.
One of the etiologic explanations forpubertal gynecomastia is increasedestrogenic sensitivity in breast tissue
or increased estrogen levels (dis-turbed estrogen/androgen ratio, rela-tive elevation of estrogen level), orboth. Animal and in vitro studies indi-cate negligible estrogenic activity forDEHP.38 However, recent studies in hu-mans indicate a possible estrogeniceffect.17,19,20
The first study demonstrating a possi-ble estrogenic effect for DEHP was re-ported by Colon et al19 in Puerto Rico.They have shown that DEHP levels in 45girls with premature thelarche werestatistically significantly higher com-pared with the 35 control girls. Unfor-tunately, these findings were criticizedfor its statistical methods being vague;the blood samples were stored for 2 to6 years and the DEHP levels were veryhigh compared with other studies,which may be because of contamina-tion of samples. In our study, all sam-ples were collected and analyzedwithin 6 months and our results werecomparable with the literature. How-ever, being the first study19 related toestrogenic and/or antiandrogenic ef-fects of DEHP still it is remarkable.
In another study, DEHP/MEHP levelswere found to be higher in patientswith endometriosis compared with thecontrol group. The etiopathogenesis ofendometriosis is not clear, however, itis thought that estrogen receptors inthe uterus also play a role. In the study,it was speculated that DEHPmay play arole in the development of endometri-osis through the estrogen receptorstimulation.20
In a recent study conducted in Chinesegirls with precocious puberty, DEHPlevels were found to be higher com-pared with the control group. Also, itwas noted that girls with high DEHP lev-els have larger ovarian and uterussize. The authors have concluded thatDEHP exposure during the period ofrapid development may cause unex-plainable estrogenic or antiandro-genic effects.21
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All these studies point to a possible es-trogenic effect of DEHP. However, thisis not simply via increasing the estro-gen levels but probably through recep-tors or gene regulation, or both.
It is known that estrogen or estrogen-like substances increase the cellularproliferation in breast cancer cell cul-ture (MCF-7). Bloom et al39 have dem-onstrated a dose-dependent relation-ship between DEHP and cellularproliferation in these cell cultures. Theauthors of a recent study have demon-strated that DEHP inhibits tamoxifen-induced apoptosis in breast cancercell cultures.16 Tamoxifen is an estro-gen receptor antagonist used inbreast cancer treatment. Tamoxifendecreases the BCL-2 gene expression,which is an antiapoptotic oncogeneand increases the expression of BAXgene, which increases apoptosis.However, adding DEHP to the culturereverses all these effects. The anti-apoptotic BCL-2 gene expression in-creases and apoptotic BAX gene ex-pression decreases. In this study, itwas concluded that DEHPmay alleviate
tamoxifen-induced apoptosis throughestrogen receptor interaction, and itwas recommended that women usingtamoxifen use less cosmetic material(because of its DEHP content).16 Tamox-ifen is also a drug of choice in pubertalgynecomastia. It is effective in de-creasing breast size and pain.40,41
In this study, plasma DEHP and MEHPlevels were found significantly higherin patients with pubertal gynecomas-tia. Although in this study it is not pos-sible to show a cause-effect relation-ship, it may be speculated that DEHPmay lead to pubertal gynecomastia viareceptor or intracellular gene regula-tion, rather than effecting hormonelevels, at a period of rapid growth orincreased sensitivity.
The hormonal evaluation was not oneof the objectives of the study. There-fore, plasma hormone level determina-tion was made only from some of thepubertal gynecomastia cases accord-ing the algorithms.2,33 All hormone lev-els were within the normal range andno relationship between DEHP or MEHPlevels and thyrotropin, LH, FSH, total
testosterone, free testosterone, FT3,FT4, and SHBG were found. A negativesignificant relationship between plasmaDEHP levels and DHEA-S was found. How-ever, after correction with SHBG levels,no relationship persisted.
CONCLUSIONS
Plasma DEHP and MEHP levels werefound to be significantly higher in pa-tients with pubertal gynecomastiacompared with control patients. Sev-eral studies that included humans asthe subjects have demonstrated a pos-sible estrogenic effect of DEHP besidesantiandrogenic effects. Also, the ef-fects of DEHP in cell cultures supportan estrogenic effect at the receptorlevel. Being a common problem, thissubject deserves additional studies inlarger case series to confirm our re-sults and explain the cause-effectrelationship.
ACKNOWLEDGMENTThis study was funded by HacettepeUniversity Scientific Research Unitgrant 07 D06 103 001.
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