Associations between maternal phthalate exposure and cord sex hormones
in human infants
Lung-Cheng Lin
a, Shu-Li Wang
b,c, Yu-Chen Chang
a, Po-Chin Huang
b, Joan-Tin Cheng
a, Pen-Hua Su
d,e,
Pao-Chi Liao
a,f,⇑a
Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan 704, Taiwan
bDivision of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli 350, Taiwan c
Institute of Environmental Medicine, College of Public Health, China Medical University and Hospital, Taichung, Taiwan
d
Department of Pediatrics, Division of Genetics, Chung Shan Medical University Hospital, Taichung 402, Taiwan
e
School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan
f
Sustainable Environment Research Center, National Cheng Kung University, University Road, Tainan 701, Taiwan
a r t i c l e
i n f o
Article history: Received 15 July 2010
Received in revised form 27 December 2010 Accepted 27 December 2010
Available online 26 January 2011 Keywords:
Sex steroid hormones Maternal exposure Phthalate
Umbilical cord blood
a b s t r a c t
It has been speculated that maternal phthalate exposure may affect reproductive development in human newborns. However, the mechanism awaits further investigation. The aim is to evaluate the association between maternal phthalate exposure and cord sex steroid hormones in pregnant women and their new-borns from the general population. A total of 155 maternal and infant pair were recruited and analyzed. Levels of urinary phthalate metabolites and sex steroid hormones were determined using liquid chroma-tography/electrospray tandem mass spectrometry (LC–ESI-MS/MS) and radioimmunoassay (RIA), respec-tively. No significant correlation was found between each steroid hormones and phthalate metabolites for male newborns, except MMP was marginally significantly correlated with E2. After adjusting for
maternal age, estradiol (E2) levels in cord serum from male newborns were not correlated with maternal
urinary phthalate metabolites. In female newborns, the maternal urinary levels of mono-(2-ethylhexyl) phthalate (MEHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP) were negatively corre-lated with the free testosterone (fT) and fT/E2levels in cord serum with Pearson correlation coefficients
ranging between 0.24 and 0.29 (p < 0.05). Additionally, after gestational age was adjusted, the mater-nal urinary level of DEHP was negatively correlated with the free testosterone (fT) and fT/E2levels in cord
serum. We suggest that maternal exposure to phthalates may affect sex steroid hormones status in fetal and newborn stage.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Phthalates are widely used in plastics, building materials, and personal care products, and they are considered to be ubiquitous compounds to which humans are frequently exposed. In recent years, exposure to phthalates has drawn a lot of concern due to their adverse effects on reproductive system. Epidemiological studies have revealed that exposure to several commonly used phthalates such as di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-n-butyl phthalate (DBP), butyl-benzyl phthalate (BBP), and di-(2-ethylhexyl) phthalate (DEHP) can alter sex steroid
hor-mone levels in human subjects (Duty et al., 2005; Main et al.,
2006; Pan et al., 2006; Chou et al., 2009). It was recently hypothe-sized that the effects for prenatal exposure might be more
profound because of fasting differentiation and proliferation of go-nadal organs (Moore et al., 2001; Lottrup et al., 2006). The health effects of prenatal phthalate exposure and the later effects are of great concern.
Animal studies indicate that prenatal exposure to phthalates could be associated with in various reproductive effects in off-spring, especially in male newborns. Prenatal exposure to DBP and/or DEHP is associated with reduced androgen-dependent or-gan weights (Kai et al., 2005; Howdeshell et al., 2007), decreased fetal serum testosterone (T) levels (Thompson et al., 2004; Borch et al., 2006; Howdeshell et al., 2007; Mahood et al., 2007), in-creased reproductive malformation (Wilson et al., 2007), altera-tions of sexual behavior (Dalsenter et al., 2006), increased the number of ovarian atretic tertiary follicles (Grande et al., 2007), re-duced Leydig and Sertoli cell function (Mahood et al., 2007; Scott et al., 2007), and reduced anogenital distance (AGD) in exposed off-spring (Ema et al., 2000; Barlow et al., 2004; Borch et al., 2006). In addition, prenatal exposure to BBP could be related to reduced body weight, and litter size during lactation, retention of nipples,
0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.12.079
⇑Corresponding author at: Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 704, Taiwan. Tel.: +886 6 2353535x5566; fax: +886 6 2743748.
E-mail address:[email protected](P.-C. Liao).
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and an increased incidence of male reproductive system malforma-tions in offspring (Nagao et al., 2000; Ema and Miyawaki, 2002; Tyl et al., 2004).
Phthalate exposure has also been reported to be related to dis-torted levels of reproductive hormones such as free testosterone (fT), estradiol (E2), follicle stimulating hormone (FSH), and inhibin B (Duty et al., 2005; Pan et al., 2006; Meeker et al., 2009) in adult men. Additionally, a study reported that the DEHP exposure of fer-tile men is associated with minor alterations of markers of fT (Mendiola et al., 2010). So far, only a few studies have been
re-ported prenatal phthalate exposure in humans (Latini et al.,
2003; Swan et al., 2005; Huang et al., 2009). Prenatal exposure to
DEHP and/or its metabolite mono-(2-ethylhexyl) phthalate
(MEHP) was associated with shorter gestational duration (Latini et al., 2003). Reductions in the AGD in male newborns are corre-lated with increasing levels of the mono-ethyl phthalate (MEP), mono-butyl phthalate (MBP), and mono-benzyl phthalate (MBzP) corresponding metabolites DEP, DBP, and BBP in human urine sam-ples taken during pregnancy (Swan et al., 2005). Another study re-cently report that the levels of the phthalate metabolites MBP and MEHP in the amniotic fluid of female newborns were negatively correlated with the anogenital index (Huang et al., 2009).
Despite the fact that prenatal or neonatal phthalate exposure caused adverse effects for both male and female offspring, limited information is available regarding the effect of phthalate exposure on hormone levels. Our aim was to examine the association be-tween maternal phthalate exposure in the general population and sex steroid hormone levels in cord blood.
2. Material and methods 2.1. Subjects
The usage of plastic material and potential exposure of un-known toxicants from plastics in Taiwanese has been a long and lasting issue in Taiwan, and recent studies have provided certain evidence about the phthalate exposure level in Taiwanese (Huang et al., 2007, 2009, 2010; Chen et al., 2008). The subjects of current study comes from a sub-study of a long-term birth cohort study (Wang et al., 2004, 2005), which also included the exposure assess-ment of dioxin, PCBs and lead in the same population. Briefly, all pregnant women were invited to visit the local medical center to participate in the study during December 2000 to November 2001. Initially, the spot urine samples of 430 subjects were collected at the third trimester along with their personal data, including reproductive and medical histories and physical param-eters. All of the pregnant women were between the ages of 18 and 39, with a single pregnancy, and without clinical complication. Of the 430 subjects, 275 provided urine samples. The remaining 155 subjects with cord blood specimens and complete personal data were included in the present study. The study protocol was re-viewed and approved by the Human Ethical Committee of the Na-tional Health Research Institutes in Taiwan. This study followed the ethical standards formulated from the Helsinki Declarations of 1964 and revised in 2000 (World Medical Association, 2000). Each participant provided informed consent after receiving a de-tailed explanation of the study and its potential consequences. 2.2. Phthalate metabolite measurements in maternal urine samples
Maternal urine was collected from subjects during third trimes-ter of pregnancy (28–36 weeks) and spot urine samples were col-lected using glass beaker and immediately transferred into amber glass bottle at the hospital. Standards of phthalate metabo-lites including mono-methyl phthalate (MMP), MEP, MBP, MBzP,
MEHP, mono-(2-ethyl-5-oxo-hexyl) phthalate (5oxo-MEHP),
mono-(2-ethyl-5-hydroxy hexyl) phthalate (5OH-MEHP) and their
corresponding 13C4-labeled compounds were purchased from
Cambridge Isotope Laboratories (Andover, MA, USA). Formic acid (FA), acetic acid (AA), buffer salts, and b-Glucuronidase (Helix pomatia) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol (HPLC grade) was purchased from Merck (Darms-tadt, Germany). Deionized water was acquired from a Millipore system (Milford, MA,USA). b-Glucuronidase enzymatic deconjuga-tion was used to change the glucuronid-conjugated forms of seven phthalate metabolites into their free forms for quantification of the total amounts in urine samples. As previously reported, on-line so-lid phase extraction (SPE) was used to extract the seven urinary phthalate metabolites (Lin et al., 2004). Briefly, this analytical ap-proach involved the enzymatic deconjugation of phthalate metab-olites followed by on-line SPE and quantification using liquid chromatography/electrospray tandem mass spectrometry (LC– ESI-MS/MS). The analytical system consisted of two PE series 200 pumps (PerkinElmer, Norfolk, CT) and an API365 triple quadrupole MS equipped with a TurboIonSpray source (PE Sciex, Throhill, ON, Canada). A PE series 200 autosampler (PerkinElmer, Norfolk, CT) performed the sample introduction, on-line SPE, and chromato-graphic separation using an electric two-position switching valve (6-ports, Valco Europe, Schenkon, Switzerland), a C18 trap car-tridge (2.0 55-mm, 3-
l
m, Merck, Darmstadt, Germany) for SPE, and a Chromolith Flash RP-18e column (4.6 50 mm, Merck, Darmstadt, Germany). To deconjugate the samples, aliquots (1 mL) containing 750l
L urine, 50l
L of 2000 ppb 13C4-labledphthalate metabolites as internal standards (IS), 200
l
L of100 mM ammonium acetate buffer (pH 6.5), and 10 units of b-glu-curonidase were incubated at 37 °C for 90 min. The deconjugation reaction was stopped by the addition of 50
l
L of 20% acetic acid/ACN. The mixture was passed through a 0.2
l
m PVDF membranefilter (MSF-3, Advantec MFS, Inc., Pleasanton, CA, USA) and stored at 4 °C prior to loading onto the analytical system. The urine mix-ture was loaded onto the on-line SPE cartridge and washed with 2% formic acid/H2O at a flow rate of 600
l
L min 1for 10 min before the switching valve was triggered to start the LC gradient and ini-tiate chromatography on the Chromolith column. The gradient elu-tion started with the mobile phase from 0.001% formic acid/H2O at a flow rate of 600l
L min 1to 100% MeOH in 10 min. The LC eluent was diluted 1:20 before entering the mass spectrometer. After gra-dient analysis was completed, the Chromolith column was washed with MeOH for 5 min and re-equilibrated with 2% formic acid/H2O for 1 min before the next injection.The MS/MS data acquisition was initiated by triggering the switching valve. The metabolites and the13C4-compounds were ana-lyzed by monitoring their precursor to product ion transitions using the negative ion mode. The precursor to product ion transitions of MMP, MEP, MBP, MBzP, MEHP, 5OH-MEHP, 5oxo-MEHP, and their
corresponding13C4-labeled compounds were the same as those in
the previous report (Kato et al., 2005). Other instrumental parame-ters were optimized to generate the highest signal intensities.
The dynamic concentration range of the MMP, MEP, MBzP, 5OH-MEHP, and 5oxo-MEHP calibration curves was 0.67–1300 (ng mL 1), while the range of calibration curves for MBP and MEHP was 0.67–
670 (ng mL 1). We examined the intra-day precision, apparent
recovery, and method detection limit (MDL) of the analytical approach for these metabolites (Supp. Table 1A). The intra-day vari-ations of all seven urine phthalate metabolites were below 10%, with intra-day recoveries at 100 ± 20% at three different concentrations, 25%, 50% and 75%, of individual substance. The detection limits of MMP, MEP, MBP, MBzP, MEHP, 5OH-MEHP, and 5oxo-MEHP were 3.4, 2.2, 1.6, 0.99, 0.55, 0.23, and 0.26 ng mL 1, respectively.
The accuracy of the analytical approach was tested against two reference urine samples with different known phthalate
metabolite concentrations. The samples were received from the
laboratory intercomparison program (www.g-equas.de) in 2006.
These reference urinary samples contained MBP, MBzP, MEHP, 5OH-MEHP, and 5oxo-MEHP, but not MMP and MEP. In both con-centrations, the relative errors (RE) of these five urinary metabo-lites were below 16% (Supp. Table 1B). Our analytical approach can therefore accurately quantify these five urinary phthalate metabolites, but the accuracy for MMP and MEP was not accessed. Urinary 5OH-MEHP, and 5oxo-MEHP levels were used to repre-sent the whole OH-MEHP and oxo-MEHP levels in urine, respec-tively. The creatinine-adjusted urinary concentrations of MEHP, OH-MEHP, and oxo-MEHP were summed for assessment of the DEHP exposure.
2.3. Quantification of serum reproductive hormones
Cord blood samples were collected and immediately centri-fuged for 20 min at 4 °C. The serum was separated into aliquots
and stored at 80 °C before hormone quantitative analysis. Cord
blood levels of fT, and E2were quantified using radioimmunoassay in a clinical laboratory (Department of Nuclear Medicine, Kaoh-siung Medical University, KaohKaoh-siung, Taiwan). We carried out blind duplicates for every 10 samples. The analytical precision values, coefficient of variation (CV), from the duplicate analyses of samples were below 20%. The CV values of fT QC control samples at 3 differ-ent concdiffer-entrations, including 150, 400, 800 ng dL 1, were 7.1%, 5.3%, 7.0%, respectively. The CV values of E2 QC control samples at three different concentrations, including 80, 250, 350 ng dL 1, were 3.2%, 0.64%, 3.1%, respectively. The detection limits of fT, and E2 were 20 ng dL 1, and 20 pg mL 1, respectively. For each
cord blood sample, the fT was analyzed first and then E2 was.
Due to the limited volumes of cord blood samples, the number of analytical results for fT, and E2were 155, and 152, respectively. 2.4. Statistical analysis
To examine sexual differences at varied maternal phthalate metabolite levels, the phthalate metabolite levels were log-trans-formed and tested using a student T test for two independent sam-ples. When the levels of the urinary phthalate metabolites showed geometrical distributions, the data were log10-transformed for fur-ther statistical comparison and correlation analyses. If the levels of metabolites or hormones were below their detection limits (DL), they were assigned values equal to half of their DL. We calculated the ratio of fT to E2because fT is the precursor of E2through the aromatase in metabolism. The Mann–Whitney U test was used to examine whether hormone levels, fT/E2, or demographic data were
significantly different between male and female newborns. The Pearson correlation coefficients were used to show the association between the maternal phthalate metabolite level and the sex ste-roid hormones in cord serum. Potential confounding factors were assessed with a stepwise multivariate regression analysis of the independent variables, including maternal and newborn character-istics. Stepwise multiple regression analysis was completed to con-trol for confounders. A p-value of <0.05 was considered to be statistically significant. SPSS version 12 was employed for all sta-tistical tests and correlation analysis.
3. Results
3.1. Subject characteristics
The characteristics of our subjects and their newborn by gender
were shown inTable 1. Mean age and BMI of these women were
28.8 ± 3.6 years old and 26.1 ± 3.3. Less than 5% of them had smok-ing and drinksmok-ing habit dursmok-ing pregnancy. Only three subjects have ever worked in a chemical factory. The mean age and BMI of the un-followed subjects were 27.9 ± 4.6 years old and 25.4 ± 3.8, respectively. Their smoking and drinking rates were 3.4% and 4.0%, respectively. No significant differences of these variables be-tween participants and un-followed subjects were observed.
Mean gestational age of newborns were around 39 weeks. Birth weight and head circumference of the male newborns were signif-icantly greater than those of the female newborns (p < 0.05). In addition, there was a marginally significant difference in the birth length between male and female newborns (p < 0.1).
3.2. Phthalate metabolites in maternal urine samples
Levels and distribution of phthalate metabolites in maternal
ur-ine were shown inTable 2. Median levels of urinary MMP, MEP,
MBP, MBzP, MEHP, 5oxo-MEHP, 5OH-MEHP andRDEHP without
creatinine-adjusted were 34.6, 34.6, 65.5, 8.85, 11.7, 17.2, 11.8 and 43.9 ng mL 1, and those with creatinine-adjusted were 54.7, 56.0, 95.9, 15.6, 19.1, 25.6, 19.8 and 68.8
l
g g creatinine 1, respec-tively. For all of these metabolites, the frequencies of samples with phthalate metabolite levels above their MDLs were higher than 98.7%.3.3. Sex steroid hormones in the cord blood
Levels of cord blood fT, E2and the fT/E2in male and female new-borns are shown inTable 3. All samples had sex steroid hormone levels above the MDL. Median levels of fT in male newborns were
Table 1
Characteristics of the mothers and their newborns according to newborn sex.
Characteristics Male (n = 81) Female (n = 74) p valuea
Maternal
Age (mean ± SD, years) 28.8 ± 3.6 29.0 ± 4.5 0.449 BMI (mean ± SD, kg m2
) 26.1 ± 3.3 25.6 ± 3.7 0.266
Smoking habit during pregnancy, n (%) 0 (0%) 2 (2.7%) 0.138 Drinking habit during pregnancy, n (%) 3 (3.7%) 1 (1.4%) 0.358 Ever work history in chemical factories, n (%) 1 (1.2%) 2 (2.7%) 0.509 Newborn
Gestational age (mean ± SD, weeks) 38.8 ± 1.47 38.6 ± 1.64 0.491 Birth weight (mean ± SD, g) 3250 ± 393 3040 ± 414 0.001**
Birth length (mean ± SD, cm) 52 ± 2.4 51 ± 2.7 0.073 Birth head circumference (mean ± SD, cm) 34 ± 1.4 33 ± 1.6 0.039*
Chest girth (mean ± SD, cm) 33 ± 1.8 33 ± 1.5 0.109
a
Mann–Whitney U test or Fisher’s exact test.
*p < 0.05. ** p < 0.01.
significantly 1.2-fold higher than those in female newborns (p = 0.029). This difference was similar to a previous report (Dawood and Saxena, 1977). There were also significant differ-ences between sexes for the fT/E2in newborns (p = 0.019). There were no gender differences in the concentration of E2in cord blood (p = 0.679). Although all 155 samples were tested for fT, three samples were not tested for E2due to the limited volumes of the cord blood samples.
3.4. Associations between maternal phthalate exposure and cord sex steroid hormones levels
Correlations between maternal phthalate metabolite concentra-tions and the cord serum hormone levels of newborns are shown in
Table 4. fT concentration in cord blood was negatively correlated
with maternal MEP (r = 0.24, p < 0.05), MEHP (r = 0.32,
p < 0.01), 5OH-MEHP (r = 0.28, p < 0.05) and RDEHP (r = 0.38, p < 0.001) for female newborns. In addition, the fT/E2ratios were
negatively correlated with MEP (r = 0.29, p < 0.1), MEHP
(r = 0.27, p < 0.05), 5OH-MEHP (r = 0.30, p < 0.05) and RDEHP (r = 0.35, p < 0.01). Fig. 1showed clear negative linear correla-tions between fT, fT/E2andRDEHP in female newborns. However, no significant correlation was found between each steroid
hormones and phthalate metabolites for male newborns, except MMP was marginally significantly correlated with E2.
A stepwise multivariate regression model was used to examine the associations between cod sex hormones of newborns, and maternal phthalate metabolites (Table 5). Several potential con-founding factors, such as maternal age, BMI, smoking habit, gesta-tional age, pregnant times, and ever using contraceptive drug, which could be associated with phthalate exposure and/or endo-crine disruption were included for stepwise multivariate regres-sion analysis.
Gestational age were significantly positively associated with
cord blood fT, whereas RDEHP showed a negatively correlation
with fT (b: 0.23, p < 0.05) in female newborns. In addition, we
also found thatRDEHP showed a negatively association with fT/
E2(b: 0.22, p < 0.05). In male newborns, only E2level in cord
ser-um was negatively correlated with maternal age (b: 0.01,
p < 0.05). Additionally, fT/E2ratio of male newborns was negatively correlated with pregnant times.
4. Discussion
This is the first report on the association of maternal phthalate exposure and sex steroid hormones in cord serum samples. We
Table 2
Unadjusted and creatinine-adjusted concentrations of seven phthalate metabolites in pregnant women’s urine samples (n = 155). Phthalate metabolitesa
Percentile Median (range)
Minb 5th 25th 50th 75th 95th Max Taiwan USe Creatinine-unadjusted (ng mL1 ) 1st trimesterc 2nd trimesterd 3rd trimester MMP ND 1.71 18.6 34.6 60.1 127 461 7.1 (0.7–48.4) 4.3 (0.7–237.2) – MEP 10.1 12.6 22.3 34.6 61.3 241 397 22.8 (0.5–415) 27.7 (0.7–5466) – MBP 9.34 16.4 36.1 65.5 121 275 662 78.0 (8.9–541) 81.8 (13.2–580) – MBzP ND 3.09 5.90 8.85 15.1 40.3 68.0 3.0 (0.7–845) 0.9 (0.9–35.3) – MEHP 0.83 5.07 8.85 11.7 16.5 34.6 84.7 24.6 (0.5–1140) 20.6 (0.7–381) – 5oxo-MEHP ND 2.76 7.60 17.2 30.2 127 271 – – – 5OH-MEHP ND 2.63 7.33 11.8 22.2 66.1 186 – – – RDEHP 11.6 17.3 28.2 43.9 71.1 181 479 – – – Creatinine-adjusted (lg g creatinine 1 ) MMP ND 8.82 26.9 54.7 83.6 184 728 – 10.8 (0.4–363)) – MEP 9.33 18.9 34.5 56.0 106 346 863 – 68.0 (5.0–13 299) 236 (26.7–5520) MBP 11.3 29.9 58.4 95.9 169 507 926 – 195 (57.8–1901) 42.6 (21.3–105) MBzP ND 4.56 10.1 15.6 25.9 43.9 104 – 3.7 (0.5–69.9)) 12.1 (5.6–120) MEHP 3.73 4.91 10.4 19.1 33.7 100 193 – 60.8 (12.2–1251) 4.6 (1.8–449) 5oxo-MEHP ND 5.44 14.6 25.6 43.6 158 801 – – – 5OH-MEHP ND 4.17 11.7 19.7 30.7 109 407 – – – RDEHP 16.2 25.6 43.2 68.8 105.5 316 927 – – – a R
DEHP = MEHP + 5oxo-MEHP + 5OH-MEHP.
b
ND: not detected. Detection limits (LOD) of seven phthalate metabolites were: MMP, 3.4; MEP, 2.2; MBP, 1.6; MBzP, 0.99; MEHP, 0.55; 5oxo-MEHP, 0.26; 5OH-MEHP, 0.23 ng mL 1, respectively. ND was calculated as 1/2 LOD.
c Huang et al., 2009. Taiwanes pregnant women carried female fetus at first trimester (n = 31). d
Huang et al., 2007. Taiwanes pregnant women 33.6 ± 3.3 years old (n = 76).
e
Adibi et al., 2003. New York pregnant women 18–35 years old (n = 25).
Table 3
Levels of cord free testosterone (fT) and estrogen (E2) in male and female newborns.
Hormones Percentile p valueb
na Min 5th 25th 50th 75th 95th Max fT (ng dL1 ) Male 81 56 92 140 220 290 490 650 0.029* Female 74 34 70 120 167 260 370 482 E2(pg mL 1) Male 80 4100 4400 5800 6500 8300 10 000 11 000 0.679 Female 72 2500 3600 5900 6700 9500 7900 9800 fT/E2cMale 80 0.11 0.13 0.23 0.31 0.39 0.66 1.06 0.019* Female 72 0.10 0.12 0.20 0.26 0.36 0.48 0.66 a
One male and two female newborn do not have enough serum sample to measure estrogen.
b
Sex differences of hormone levels in cord blood were tested using the Mann–Whitney U test.
c
fT/E2was non-dimensional unit. *p < 0.05.
found that both the fT concentration and fT/E2ratio in the cord blood of female newborns were negatively correlated with two DEHP metabolites, MEHP and 5OH-MEHP, in maternal urine. Alter-ation of the reproductive hormone concentrAlter-ations in newborns might be associated with maternal, perinatal, and pregnancy fac-tors that are related to hormone-related diseases (Troisi et al., 2003). According to our data, prenatal phthalate exposure may al-ter sex sal-teroid hormone levels and proportions, which may be re-lated to the factors for subsequent cancer risk. fT and E2cord blood concentrations were consistent with those in an earlier report, which identified the fT and E2levels in a general population ( Sava-rese et al., 2007). The ratio of fT to E2is considered as an index of aromatase activity as fT is the metabolic precursor of E2through aromatase. Furthermore, the fT/E2ratio has been related to coro-nary artery disease (Kajinami et al., 2004; He et al., 2007), gyneco-mastia (Beck, 1981), and hepatocellular carcinoma (Tanaka et al., 2000). In the present study, male newborns had different fT/E2 ra-tios in cord blood than female newborns. In female newborns, the fT/E2level was associated with maternal DEP and DEHP exposure, indicating that prenatal phthalate exposure may alter the propor-tion of sex steroid hormones.
One study reported the levels of urinary phthalate metabolites at second trimester from pregnant women in southern Taiwan (Huang et al., 2007). Among the phthalate monoesters, median lev-els of MMP, and MBzP were 4-to-5 folds higher than those in Huang’s report, whereas those of MBP and MEHP were 2-to-4 folds lower (Table 2). Similar phthalate metabolite profile at first trimes-ter from pregnant women in southern Taiwan was observed in Huang’s another report (Huang et al., 2009). These different expo-sure profiles could be possibly caused by the difference expoexpo-sure sources of phthalates, like dietary habits, from subjects with differ-ence area. In our study, the levels of the urinary phthalate metab-olites MBP, MBzP, MEHP, 5OH-MEHP, and 5oxo-MEHP were validated using authentic samples from a continuous laboratory intercomparison program, which allowed us to provide validated reference values for urinary phthalate metabolite levels in preg-nant women in Taiwan.
Compared to the median levels of urinary MEP, MBP, MBzP, and MEHP in pregnant New York women (Adibi et al., 2003), median le-vel of MEP in this study was fold lower, and that of MEHP was
4-fold higher (Table 2). The median levels of MBP and MBzP were
similar between the two reports. Another previous report identical identified phthalate metabolite concentrations (ng mL 1) in
preg-nant women (Swan et al., 2005). For comparing their urinary
metabolite levels with our data, we assumed that the urinary cre-atinine levels of the previous report and our study were similar.
We used the 0.8 g L creatinine 1concentration identified by our
study to adjust the urinary metabolite levels in the previous report. With the exceptions of MMP, MBP, and MEHP, the metabolite medians were similar. Among DEHP-derived metabolites, the lev-els of the secondary metabolites 5oxo-MEHP and 5OH-MEHP were higher than the primary metabolite MEHP. This finding was consis-tent with an earlier report (Swan et al., 2005).
Various human and animal studies have demonstrated that phthalate exposure has anti-androgenic effects on reproductive systems, especially in males (Fisher, 2004; Swan et al., 2005; Latini et al., 2006). In a recent report, male workers exposed to DBP and DEHP had a decreased serum fT level (Pan et al., 2006). It has been suggested that phthalates affect the reproductive system by influ-encing Leydig cell function in the testis (Lottrup et al., 2006). In hu-man and animal models concerning male offspring, hu-many studies have focused on the effects of prenatal phthalate exposure on the reproductive system, especially DBP, BBP and DEHP (Ema et al., 2000; Ema and Miyawaki, 2002; Thompson et al., 2004; Barlow et al., 2004; Swan et al., 2005; Dalsenter et al., 2006; Foster, 2006; Howdeshell et al., 2007; Mahood et al., 2007; Scott et al., 2007; Wilson et al., 2007). However, we did not identify an associ-ation between fT concentrassoci-ations in the cord blood of male new-borns and prenatal phthalate exposure. Additionally, another report showed that maternal fT was correlated with the fT concen-trations in the cord blood of male, but not female, newborns (r = 0.34) (Troisi et al., 2003). Therefore, the effects of prenatal phthalate exposure on the fT levels in the cord blood of male new-borns may be veiled by the effect of maternal fT. Meanwhile, sev-eral studies have demonstrated that prenatal phthalate exposure also affects the reproductive system of female offspring. Female offspring exposed to BBP in utero and during lactation had an in-creased AGD, but the levels of sexual hormones were not
associ-ated with phthalate exposure (Nagao et al., 2000). Prenatal
exposure to BBP has been related to delayed puberty in female off-spring, although BBP exposure has more reproductive effects for male offspring (Tyl et al., 2004). In addition, an increase in the number of ovarian atretic tertiary follicles has been observed in fe-male offspring exposed to DEHP in utero and during lactation (Grande et al., 2007).
The phthalate exposure assessment was dependent upon the phthalate metabolite levels in spot urine samples from pregnant women in their third trimester. We assumed that the maternal phthalate exposure during pregnancy was consistent, as a number of earlier reports have supported the assumption that daily human
phthalate exposure was consistent (Hoppin et al., 2002; Hauser
et al., 2004). However, if this was not true, it is possible that mis-classification of exposure levels based on spot urine samples could minimize the prenatal exposure effects on reproductive hormones.
Table 4
Pearson correlation coefficients between levels of maternal phthalate metabolites and cord blood hormone levels. Phthalate metabolites
(lg g creatinine1
)a
Male newborns Female newborns fT (ng dL1 ) (n = 81) E2(pg mL1) (n = 80) fT/E2(n = 80) fT (ng dL1) (n = 74) E2(pg mL1) (n = 72) fT/E2(n = 72) MMP 0.11 0.21# 0.00 0.01 0.03 0.01 MEP 0.10 0.02 0.13 0.24* 0.01 0.29* MBP 0.11 0.05 0.15 0.07 0.07 0.06 MBzP 0.05 0.14 0.03 0.18 0.20# 0.10 MEHP 0.07 0.07 0.04 0.32** 0.19 0.27* 5oxo-MEHP 0.11 0.02 0.11 0.19 0.07 0.20 5OH-MEHP 0.16 0.07 0.15 0.28* 0.09 0.30* RDEHPb 0.06 0.00 0.07 0.38*** 0.17 0.35**
aPhthalate metabolites were all log-transformed. b RDEHP = MEHP + 5oxo-MEHP + 5OH-MEHP. #p value for Pearson correlation: p < 0.1. *p value for Pearson correlation: p < 0.05. ** p value for Pearson correlation: p < 0.01. ***p value for Pearson correlation: p < 0.001.
More significant effects of phthalate exposure on hormone levels should be observed when there is less exposure misclassification. Therefore, we believe that using spot urine samples for the assess-ment of phthalate exposure would not cause a false significant correlation.
In the present study, we found that prenatal phthalate exposure might be related to a reduction in the cord blood fT concentration of female offspring. Interestingly, pregnant women exposed to endocrine disruptors, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs), had reduced fT concentrations in the cord blood of female newborns (Cao et al., 2008) but not in male newborns. Such obser-vations indicate that endocrine disruptors affect the reproductive
systems of female offspring through different mechanisms than male offspring. As the enzyme aromatase is responsible for the conversion of T to E2, the endocrine disruption effect of phthalates may be a result of aromatase activity regulation, resulting in the distortion of T and E2concentrations (Lovekamp-Swan and Davis, 2003). For illustrating the mechanism of ovarian action of DEHP, they proposed a model which DEHP or/and its active metabolites cause decreased serum E2levels through the suppression of aroma-tase. However, the model is not consistent with our observations, which may be resulted from our observation based on the human and/or prenatal phthalate exposure. A previous report showed that brain aromatase activity is inhibited at low DEHP doses and in-creased at high DEHP doses in rat male offspring after in utero or
lactational treatment. In female offspring, the aromatase activity was increased regardless of the doses, which enhanced the conver-sion of T to E2(Andrade et al., 2006). The enhancement of the T to E2conversion in female offspring is similar to our observation of a decreasing fT/E2ratio with an increasing phthalate exposure level. Whether the endocrine disruption effect of phthalates offers a sim-ilar mechanism remains to be studied.
5. Conclusion
This is the first report associating prenatal phthalate exposure with reproductive hormone concentrations in human cord blood samples. The levels of DEHP metabolites in maternal urine were negatively correlated with the fT and fT/E2levels in the cord blood of the female newborns. A negative association was also observed between the level of DEHP and the fT/E2ratio in the cord blood of female newborns. Our results suggest that prenatal phthalate exposure may affect the hormone levels of newborns at the time of delivery. The long term adverse effects of prenatal phthalate exposure on the reproductive systems of female newborns should be of great concern.
Acknowledgments
This work was supported by Grants NSC94-2113-M006-06 and NSC97-2113-M-006-005-MY3 from National Science Council, Tai-wan, and Grants EO-097-PP-05 and EO-098-PP-03 from National Health Research Institutes, Taiwan.
Appendix A. Supplementary material
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Stepwise multivariate linear regression between sex steroid hormones and phthalate exposure in cord blood of newborns.
Newborn gender Male Female Hormone/Phthalate
metabolitesa
E2(pg mL 1) fT/E2 fT (ng dL1) fT/E2
Estimate Estimate Estimate Estimate Constant 4.11** 1.42** 0.99 1.17** Age 0.01** 0.03 0.03 0.05 BMI 0.04 0.01 0.19# 0.16 Gestational age 0.06 0.89 0.04* 1.86 Smoking – – 0.08 0.01# Times of pregnant 0.13 0.04* 0.00 0.08 Ever using contraceptive drug 0.07 0.17 0.21 0.23# MMP 0.15 0.03 0.06 0.06 MEP 0.02 0.17 0.02 0.02 MBP 0.02 0.22 0.01 0.01 MBzP 0.11 0.01 0.00 0.10 RDEHPb 0.05 0.01 0.23* 0.22** R2 0.12 0.07 0.26 0.16 p value 0.003 0.031 0.000 0.001 a Creatinine-adjusted. b R
DEHP = MEHP + 5oxo-MEHP + 5OH-MEHP.
#
p < 0.1.
*
p < 0.05.
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