Reproductive Toxicology 45 (2014) 8–13
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Estimation of bisphenol A (BPA) concentrations in pregnant women, fetuses and nonpregnant women in Eastern Townships of Canada Aziz Aris ∗ Department of Obstetrics and Gynecology, Clinical Research Centre of Sherbrooke University Hospital Centre, Sherbrooke, Quebec, Canada
a r t i c l e
i n f o
Article history: Received 17 March 2013 Received in revised form 30 November 2013 Accepted 11 December 2013 Available online 28 December 2013 Keywords: Bisphenol A Pregnant women Fetuses Nonpregnant women Blood Peritoneal fluid
a b s t r a c t We determined bisphenol A (BPA) concentrations of 61 pregnant women (PW), their fetuses and 26 nonpregnant women (NPW) in Eastern Townships of Canada; and evaluated potential correlations between maternal and fetal blood, and between peripheral blood and peritoneal fluid. In PW, BPA levels were ranged from non-detected to 4.46 ng/ml and from non-detected to 4.60 ng/ml for maternal and fetal serum, respectively. In NPW, BPA levels were ranged from 1.30 to 8.17 ng/ml and from 0.19 to 13.45 ng/ml for serum and peritoneal fluid, respectively. Positive correlation was found between maternal and fetal serum, and between serum and peritoneal fluid. In conclusion, our findings highlight a continuous distribution of BPA between the mother and its fetus and reveal a role of pregnancy in underestimating the actual levels of blood BPA. Our study also provides a temporal-spatial reference on BPA exposure, which is a useful tool in monitoring, comparing and correcting. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Xenoestrogens are molecules mimicking estrogen, including bisphenol A (BPA) which is widely used as a product of epoxy resins, polycarbonate plastics, and flame retardants. It is extensively used in a broad range of products including toys, water pipes, drinking glasses, baby bottles, food-storage containers, the lining of food beverage containers, medical equipment, tubing, consumer electronics and dental sealants [1]. At high temperatures, BPA can migrate from polymer to food [2,3]. There has been an exponential increase in the use of BPA during the last 30 years, increasing its potential effects [4]. Several studies indicate that humans are regularly in contact with BPA, through water, air, soil, environment, and food contamination [5,6]. Levels of BPA have been shown to range between 0.2 and 20 ng/ml in adult serum [1], suggesting a possible involvement of this synthetic estrogen in human diseases by triggering endocrine disruption, immune disorder, epigenetic modulation and oxidative stress [7]. Other studies have already reported the presence of BPA in human blood, fetal serum during pregnancy, amniotic fluid, follicular fluid, placental tissue, umbilical cord blood, and urine [1,8–16].
∗ Correspondence to: Department of Obstetrics and Gynecology, University of Sherbrooke Hospital Centre, 3001, 12e Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4. Tel.: +1 819 820 6868x12538; fax: +1 819 564 5302. E-mail address: [email protected] 0890-6238/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.12.006
Recently, maternal levels of unconjugated BPA in blood ranging between 0.5 and 22.3 ng/ml have been shown in southeastern Michigan mothers [17]. In Korea, BPA levels were ranged from nondetectable to 66.48 g/l in maternal blood of pregnant women, and from non-detectable to 8.86 g/l in cord blood [11]. Unconjugated BPA readily crosses the placenta but conjugated BPA would have to be un-conjugated to do so [18]. Other studies show that BPA undergoes a rapid transport of BPA across the placenta and passes into the cord blood of human fetuses [19]. Additionally, BPA has been detected in human breast milk indicating that exposure during lactation is also likely [18]. Levels of unconjugated BPA in human blood and other fluids are higher than levels needed to stimulate a number of molecular endpoints in cell culture in vitro, and appear to be within BPA levels in animal studies. A study conducted by the Centres for Disease Control and Prevention (CDCP) examined 2500 Americans and reported that BPA was found in 92.6% of urine samples, indicating that humans are routinely exposed to this chemical. Urine concentrations ranged from 0.4 to 149 g/l were significantly higher in children and adolescents compared to adults [1]. Since the basis of better health is prevention, one would hope that we can develop procedures to avoid environmentally induced disease in susceptible population such as pregnant women and their fetuses. The fetus is considered to be highly susceptible to the adverse effects of xenobiotics. This is because environmental agents could disrupt the biological events that are required to ensure normal growth and development [20,21]. The objective of this study
A. Aris / Reproductive Toxicology 45 (2014) 8–13 Table 1 Clinical characteristics of subjects.
Age (year, mean ± SD) BMI (kg/m2 , mean ± SD) Gestational age (week, mean ± SD) Birth weight (g, mean ± SD)
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2.3. Blood and peritoneal fluid sampling
Pregnant women (n = 61)
Nonpregnant women (n = 26)
P valuea
31.72 ± 3.66
33.12 ± 4.04
NS
24.8 ± 3.1
24.7 ± 3.2
NS
38.2 ± 2.1
N/A
N/A
3236 ± 324
N/A
N/A
BMI = body mass index, N/A: not applicable, data are expressed as mean ± SD. NS = not significant. a P values were determined by Mann–Whitney test.
was to evaluate human exposure of BPA in a specific period and geography, providing a reference for monitoring the evolution of measurable BPA.
2. Materials and methods 2.1. Chemicals and reagents Bisphenol A (BPA), 2.2-bis-(4-hydroxy-3-methylphenyl)propane (BPC) and N-methyl-N-(tert-butyldimethylsilyl) trifluoro(MTBSTFA) + 1% acetamide tert-buryldimethylchlorosilane (TBDMCS) were purchased from Sigma (St. Louis, MO, USA). Sep-Pak Plus PS-2 cartridges, from Waters Corporation (Milford, MA, USA). Ethyl acetate was purchased from VWR (Radnor, PA, USA). All other chemicals and reagents were of analytical grade (Sigma, MO, USA). 2.2. Study subjects At the Centre Hospitalier Universitaire de Sherbrooke (CHUS), we formed two groups of subjects: (1) a group of healthy pregnant women (n = 61), recruited at delivery; and (2) a group of healthy fertile nonpregnant women (n = 26), recruited during their tubal ligation of sterilization. As shown in Table 1 of clinical characteristics of subjects, eligible groups were matched for age and body mass index (BMI). Participants were not known for cigarette, illicit drug use, medical condition (i.e. diabetes, hypertension or metabolic disease) or for working in contact with BPA. The Eastern Townships population is mainly rural and there is no significant industrial activity nearby. The diet taken is typical of a middle class population of Western industrialized countries. A food market-basket, representative for the local population, contains various meat including margarine, canola oil, rice, corn, grain, peanuts, potatoes, fruits and vegetables, eggs, poultry, meat and fish. Beverages include milk, juice, tea, coffee, bottled water, soft drinks and beer. Most of these foods come mainly from the province of Quebec, then the rest of Canada and the United States of America. Our study did not quantify the exact levels of BPA in a market-basket study. Also, there is no data from public health agencies about the state of air or water pollution. Pregnant women had vaginal delivery and did not have any adverse pregnancy outcomes. The sex ratio was 1.03 (31 males to 30 females) and all neonates were of appropriate size for gestational age and without observable congenital malformations such as cryptorchidism, hypospadias and urogenital distance. The study was approved by the CHUS Ethics Human Research Committee on Clinical Research. All participants gave written consent.
For pregnant women, peripheral blood samples were obtained from the antecubital vein before delivery. Umbilical cord blood sampling was done after birth using the syringe method. Blood samples were collected in BD Vacutainer 10 ml glass serum tubes (Franklin Lakes, NJ, USA). To obtain serum, whole blood was centrifuged at 2000 × g for 15 min within 1 h of collection. For maternal samples, about 10 ml of blood was collected, resulting in 5–6.5 ml of serum. For cord blood samples, about 10 ml of blood was also collected by syringe, giving 3–4.5 ml of serum. Serum was stored at −20 ◦ C until assayed for BPA levels. For nonpregnant women, peripheral blood samples were obtained as described above before the induction of anesthesia prior the laparoscopic sterilization. Preparation for laparoscopy was routine, using umbilical puncture and pneumoperitoneum. Two ml of peritoneal fluid (PF) was collected through an abdominal port with a syringe before any operative manipulation to minimize blood contamination. All fluid present in the pouch Douglas was sucked up. Samples were centrifuged at 2000 rpm for 15 min within 1 h of collection. Glass syringes and glass tubes were used to avoid a possible contamination of BPA from plastic materials. Serum and PF were conserved in glass vials at −20 ◦ C until BPA assays were done. 2.4. BPA assessment Levels of free BPA were measured using gas chromatography– mass spectrometry (GC–MS). Calibration curve: adapted from Schönfelder et al. [9], BPA and BPC (used as internal standard) (1 mg/ml) were prepared in 50% ethanol and stored for a maximum of 3 months at 4 ◦ C. A 5 g/ml solution from previous components was made prior BPA extraction with 10% ethanol, and an additional 0.5 g/ml solution of BPA. These solutions were used as calibrators. Blank serum samples (0.2 ml) were spiked with 20 l of BPC (5 g/ml), 20 l, 4 l of BPA (5 g/ml), 20 l 4 l of 500 ng/ml solution, resulting in calibration samples containing 100 ng of internal standard (500 ng/ml), with 100 ng (500 ng/ml), 20 ng (100 ng/ml), 10 ng (50 ng/ml), 2 ng (10 ng/ml), 1 ng (5 ng/ml), 0.5 ng (2.5 ng/ml), 0.005 ng (0.025 ng/ml) of BPA. Extraction procedure: the control samples and calibration curve were extracted by employing a liquid-liquid extraction. Serum samples (200 l) were transferred into 5-ml glass tubes with 2 ml ethyl acetate containing the internal standard. The vials were sealed with Teflon-coated caps and agitated for 30 min. After a 2 min centrifugation at 1000 × g, 1.5 ml of the supernatant was transferred to 5-ml glass tubes, and the solvent was evaporated to dryness under nitrogen. The samples were reconstituted in 50 l each of MTBSTFA with 1% TBDMCS and ethyl acetate. The mixture was vortexed for 30 s every 5 min, 6 times. Samples of solution containing the derivatives were used directly for GC–MS (Agilent Technologies 6890N GC and 5973 Inert MS). GC–MS analysis: chromatographic conditions for these analyses were as followed: a 30 mm × 0.25 mm Zebron ZB-5MS fused-silica capillary column with a film thickness of 0.25 m from Phenomenex (Torrance, CA, USA) was used. Helium was used as a carrier gas at 1.1 ml/min. A 2 l extract was injected in a split mode at an injection temperature of 250 ◦ C. The oven temperature was programmed to increase from an initial temperature of 100 ◦ C (held for 3 min) to 300 ◦ C (held for 5 min) at 5 ◦ C/min. The temperatures of the quadrupole, ion source and mass-selective detector interface were respectively 150, 230, and 280 ◦ C. The MS was operated in the selected-ion monitoring (SIM) mode. The following ions were monitored (with quantitative ions in parentheses): BPA (441.0) 456.0; BPC (469.0) 484.0. The limit of quantification (LOQ) was 0.10 ng/ml,
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Table 2 Concentrations of BPA in maternal and fetal cord serum.
BPA Number of detection Range of detection (ng/ml) Mean ± SD (ng/ml)
Maternal (n = 61)
Fetal cord (n = 61)
P valuea
59/61 (97%) nd-4.46 1.36 ± 1.18
58/61 (95%) nd-4.60 1.23 ± 1.04
NS
BPA = bisphenol-A. Data are expressed as number (n, %) of detection, range and mean ± SD (ng/ml). NS = not significant, nd = non-detected (below the limit of detection = 0.010 ng/ml). a P values were determined by Wilcoxon matched pairs test.
whereas the limit of detection (LOD) was 0.01 ng/ml after determination as the lowest concentration giving a response three times the average baseline noise. Measures taken to prevent BPA contamination: potential crosscontamination of samples with BPA by external factors (tubes, pipette tips, collection kit, chemical reagents and the GC/MS itself) was evaluated with additional control experiments. The experiment was conducted by collecting distillate water into sterile BD Vacutainer 10 ml glass serum tubes (Franklin Lakes, NJ, USA) via a sterile 21 G gage Vacutainer needles instead of human blood. Afterwards, the extraction procedure and the GC/MS analysis were performed following the same procedures as human serum samples. There was no evidence of BPA contamination due to leaching from any material used during the experimentation. The calibration curves were constructed from a pool of human serum where no BPA was detected (blank serum) after addition of known amounts of BPA and internal standard (BPC). The standard samples were injected before and after serial sample analysis. The 0 ng/ml standard sample injected at the end of a serial sample analysis indicated that there was no carrying over between two samples. The other standard samples injected at the end of serial sample analysis indicated that there was no degradation in time of BPA during the GC/MS analysis. Levels of BPA in the samples were calculated based on the ratio of sample BPA to internal standard peak area response. The slope of the plot (peak-area ratio vs. amount of BPA added) indicated a linear dependence (r = 1.000). All the BPA assessments were based on a single measurement. 2.5. Statistical analysis BPA exposure was expressed as number, range and mean ± SD for each group. Characteristics of cases and controls and BPA exposure were compared using the Mann–Whitney U-test for continuous data and by Fisher’s exact test for categorical data. Wilcoxon matched pairs test compared two dependent groups. Other statistical analyses were performed using Spearman correlations. Analyses were realized with the software SPSS version 17.0. A value of P < 0.05 was considered as significant for every statistical analysis. 3. Results As shown in Table 1, pregnant women and nonpregnant women were similar in terms of age and body mass index. Pregnant women had normal deliveries and infants of normal birth weight (Table 1). BPA was detectable in maternal and fetal serum of pregnant women (Table 2, Fig. 1A), and in serum and peritoneal fluid of nonpregnant women (Table 3, Fig. 2A). BPA was [59/61 (97%), range (from non-detected to 4.46 ng/ml) and mean ± SD (1.36 ± 1.18)] in maternal serum and [58/61 (95%), range (from non-detected to 4.60 ng/ml) and mean ± SD (1.23 ± 1.04)] in fetal serum (Table 2, Fig. 1A). Of note, five samples (2 maternal serum samples and 3 fetal serum samples) had BPA
Fig. 1. Concentrations of BPA in maternal and fetal blood serum (A). Illustration of positive correlation between maternal and fetal exposure to BPA (B). Maternal and umbilical cord blood sampling were performed at delivery from sixty one pregnant women. BPA was assessed using GC–MS.
Table 3 Concentrations of BPA in peripheral blood and peritoneal fluid of nonpregnant women.
BPA Number of detection Range of detection (ng/ml) Mean ± SD (ng/ml)
Serum (n = 26)
Peritoneal fluid (n = 26)
P valuea
26/26 (100%) 1.30–8.17
26/26 (100%) 0.19–13.4
NS
3.83 ± 1.98
4.94 ± 3.71
BPA = bisphenol-A. Data are expressed as number (n, %) of detection, range and mean ± SD (ng/ml). NS = not significant. a P values were determined by Wilcoxon matched pairs test.
A. Aris / Reproductive Toxicology 45 (2014) 8–13
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Fig. 3. Concentrations of BPA in blood serum of pregnant women and nonpregnant women. Blood sampling were performed at delivery from sixty-one pregnant women and at tubal ligation for sterilization from twenty six nonpregnant women. BPA was assessed using GC–MS. Note the significant increase of BPA levels in nonpregnant women (P < 0.0001, determined by Mann Whitney test).
mean ± SD = 3.827 ± 1.98 vs. 1.36 ± 1.18, respectively, P < 0.0001 (Fig. 3). 4. Discussion
Fig. 2. Concentrations of BPA in blood serum and peritoneal fluid of nonpregnant women (A). Illustration of positive correlation between serum and peritoneal fluid exposure to BPA (B). Blood and peritoneal fluid sampling were performed at tubal ligation for sterilization from twenty six nonpregnant women. BPA was assessed using GC–MS.
levels below the limit of quantification (established at 0.10 ng/ml) and therefore were considered “non-detection”. BPA was detected in 26/26 (100%) peripheral serum and peritoneal fluid of NPW. Levels of BPA were ranged from 1.30 to 8.17 ng/ml (mean ± SD = 3.827 ± 1.98) and 0.19 to 13.45 ng/ml (mean ± SD = 4.94 ± 3.71) for serum and peritoneal fluid, respectively (Table 3, Fig. 2A). We also investigated a possible correlation between levels of BPA in maternal and fetal blood, and between peripheral blood and peritoneal fluid. Positive correlation was found between maternal and fetal serum: Spearman r = 0.823, P < 0.0001 (Fig. 1B), and between peripheral serum and peritoneal fluid: Spearman r = 0.461, P = 0.018 (Fig. 2B). On the other hand, levels of BPA in blood serum were higher in nonpregnant women than pregnant women:
Our results showed that BPA was detected in more than 95% of blood samples from healthy mothers and their fetuses. Obviously, this is not the first study showing the presence of BPA in the circulation of pregnant women or their fetuses, as others have already done that [9,11,22,23], but it is the first to compare between pregnant and nonpregnant women in Canada. Among the main findings of our study is the positive correlation between maternal and fetal blood BPA. The placenta does not seem to oppose or reduce the transfer of BPA, as its levels in both maternal and fetal sides are identical. This reflects the exposure of the fetus and suggests that maternal BPA may have a direct effect on fetal health. According to inclusion criteria of this study, all of the studied mothers and their babies were healthy and have no observable congenital diseases. It is important to note that “to be considered healthy” does not mean absence of BPA effects. Unfortunately, there are no studies on the long-term effects of low doses exposures and nonmonotonic dose–response curves (NMDRCs) of BPA on maternal–fetal health. The National Toxicology Program defined low-dose effects as any biological changes occurring in the range of typical human exposures or occurring at doses lower than those typically used in standard testing protocols, i.e. doses below those tested in traditional toxicology assessments [24]. Nonmonotonic dose–response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. In this regard, our previous study demonstrated that very low doses of BPA are able to induce necrosis, apoptosis and inflammatory response in human placental cytotrophoblasts, taken as an in vitro model for fetal toxicities, according to nonmonotonic dose-response curves [25]. This previous study suggested that exposure placental cells to low doses of BPA may cause detrimental effects, leading in vivo to adverse pregnancy outcomes which are associated with placental dysfunction (i.e. preeclampsia, intrauterine growth restriction, prematurity
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and pregnancy loss, among other). However, appropriate studies are needed to confirm or refute these hypotheses. It is important to note that although low dose exposures and NMDRCs are two major issues in the study of endocrine-disrupting chemicals, these concepts are interrelated, and NMDRCs are especially problematic for assessing potential impacts of exposure when nonmonotonicity is evident at levels of exposure below those that are typically used in toxicological assessments. A study of Vandenberg et al. [26] has investigated in depth the different aspects related to these concepts. Moreover, our findings showed that BPA was measurable in both peripheral blood and peritoneal fluid of healthy nonpregnant women. We also showed a positive correlation between levels of BPA in peripheral blood and peritoneal fluid, suggesting a continuum between these compartments. It is interesting to note that BPA levels tend to be higher in peritoneal fluid in comparison with peripheral blood, suggesting more presence and/or less elimination of BPA in peritoneal cavity. Large portion of female reproductive system bathes in peritoneal environment and it is conceivable to investigate possible links between accumulated BPA and increased risk of estrogen-dependent reproductive diseases, such as endometriosis, uterine leiomyoma and gynecological cancers, which are supposed to be sensitive to xenoestrogens as BPA [7]. For these reasons, peritoneal measurable BPA must be taken into consideration in our future studies. It is important to note that BPA levels reported in our study are higher than those reported by Teeguarden et al. [27], as well as those of many studies summarized in the review of Vandenberg et al. [28]. These differences cannot be explained by an effect related to anesthesia and/or the stress response related to surgery, because the blood collection was done before the administration of the anesthetic. Moreover, we have already observed that surgery does not affect the detection of BPA (unpublished data). Thus, the difference in the quantification of BPA levels observed between our study and that of Teeguarden et al. [27] may be explained by a higher exposure of our population or the sensitivity of the methods used, since the LOD in our study was 0.01 ng/ml, whereas it was 0.30 ng/ml (30 times higher) in the study of Teeguarden et al. [27]. On the other hand, a wide range of free BPA concentrations has been shown between our study and many studies reported by Vandenberg et al. [28]. It has been argued that contamination and/or deconjugation of BPA metabolites in collection, storage or analytical processes may be responsible for this inconsistency. Efforts to develop validated assays to quantify BPA in human blood are already ongoing [29]. The wide inter-individual variability as well as temporal variability should also be taken into account. From another angle, the higher levels of BPA in our population may be explained by dietary habits exposing to BPA, because there are at least two common practices that may contribute to the increase of measurable levels of BPA in Eastern Townships population: (1) eating canned foods, and (2) microwaving foods in plastic containers. These fairly common practices may be the result of socio-economic level of the region that is considered among the lowest in Canada. However, further studies are needed to confirm or refute these speculations. On the other hand, our study showed that BPA levels were higher in nonpregnant women than pregnant women. This is difficult to explain, but it may be the result of larger volume of distribution known in pregnancy, a process called hemodilution. This seems to suggest that pregnancy can affect the determination of BPA by underestimating its actual concentrations. 5. Conclusions This is the first study to assess simultaneously the levels of free bisphenol A in maternal and fetal blood and in peripheral blood and
peritoneal fluid of women. Our study highlights a continuum in the BPA distribution between the mother and the fetus and between peripheral blood and peritoneal cavity. It also suggests considering the potential role of pregnancy in the underestimation of blood BPA concentration. Furthermore, our study provides a temporal-spatial reference to the literature on BPA exposure, which is a valuable tool in monitoring, comparing and correcting. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported by the Grant No. 14441 from the FRQS (Fonds de recherche du Québec – Santé). This work was also supported by Fondation des Étoiles. The author wish to thank Dr. Youssef Ainmelk, Samuel Leblanc and Denis Cyr for their material and technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox. 2013.12.006. References [1] Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reproductive Toxicology 2007;24(2):139–77. [2] Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N. Xenoestrogens released from lacquer coatings in food cans. Environmental Health Perspectives 1995;103(6):608–12. [3] Biles JE, White KD, McNeal TP, Begley TH. Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods. Journal of Agricultural and Food Chemistry 1999;47(5):1965–9. [4] vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA, Eriksen M, et al. Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reproductive Toxicology 2007;24(2):131–8. [5] Kuo HW, Ding WH. Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography–mass spectrometry. Journal of Chromatography A 2004;1027(1–2):67–74. [6] Thomson BM, Grounds PR. Bisphenol A in canned foods in New Zealand: an exposure assessment. Food Additives and Contaminants 2005;22(1):65–72. [7] Aris A, Paris K. Hypothetical link between endometriosis and xenobioticsassociated genetically modified food. Gynecologie, Obstetrique & Fertilite 2010;38(12):747–53. [8] Brock JW, Yoshimura Y, Barr JR, Maggio VL, Graiser SR, Nakazawa H, et al. Measurement of bisphenol A levels in human urine. Journal of Exposure Analysis and Environmental Epidemiology 2001;11(4):323–8. [9] Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I. Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environmental Health Perspectives 2002;110(11):A703–7. [10] Yamada H, Furuta I, Kato EH, Kataoka S, Usuki Y, Kobashi G, et al. Maternal serum and amniotic fluid bisphenol A concentrations in the early second trimester. Reproductive Toxicology 2002;16(6):735–9. [11] Lee YJ, Ryu HY, Kim HK, Min CS, Lee JH, Kim E, et al. Maternal and fetal exposure to bisphenol A in Korea. Reproductive Toxicology 2008;25(4):413–9. [12] Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL. Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environmental Health Perspectives 2005;113(4):391–5. [13] Wan Y, Choi K, Kim S, Ji K, Chang H, Wiseman S, et al. Hydroxylated polybrominated diphenyl ethers and bisphenol A in pregnant women and their matching fetuses: placental transfer and potential risks. Environmental Science & Technology 2010;44(13):5233–9. [14] Chou WC, Chen JL, Lin CF, Chen YC, Shih FC, Chuang CY. Biomonitoring of bisphenol A concentrations in maternal and umbilical cord blood in regard to birth outcomes and adipokine expression: a birth cohort study in Taiwan. Environmental Health: A Global Access Science Source 2011;10:94. [15] Casas M, Valvi D, Luque N, Ballesteros-Gomez A, Carsin AE, Fernandez MF, et al. Dietary and sociodemographic determinants of bisphenol A urine concentrations in pregnant women and children. Environment International 2013;56:10–8. [16] Zhang T, Sun H, Kannan K. Blood and urinary bisphenol A concentrations in children, adults, and pregnant women from china: partitioning between blood and
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Estimation of bisphenol A (BPA) concentrations in pregnant women, fetuses and nonpregnant women in Eastern Townships of Canada Aziz Aris ∗ Department of Obstetrics and Gynecology, Clinical Research Centre of Sherbrooke University Hospital Centre, Sherbrooke, Quebec, Canada
a r t i c l e
i n f o
Article history: Received 17 March 2013 Received in revised form 30 November 2013 Accepted 11 December 2013 Available online 28 December 2013 Keywords: Bisphenol A Pregnant women Fetuses Nonpregnant women Blood Peritoneal fluid
a b s t r a c t We determined bisphenol A (BPA) concentrations of 61 pregnant women (PW), their fetuses and 26 nonpregnant women (NPW) in Eastern Townships of Canada; and evaluated potential correlations between maternal and fetal blood, and between peripheral blood and peritoneal fluid. In PW, BPA levels were ranged from non-detected to 4.46 ng/ml and from non-detected to 4.60 ng/ml for maternal and fetal serum, respectively. In NPW, BPA levels were ranged from 1.30 to 8.17 ng/ml and from 0.19 to 13.45 ng/ml for serum and peritoneal fluid, respectively. Positive correlation was found between maternal and fetal serum, and between serum and peritoneal fluid. In conclusion, our findings highlight a continuous distribution of BPA between the mother and its fetus and reveal a role of pregnancy in underestimating the actual levels of blood BPA. Our study also provides a temporal-spatial reference on BPA exposure, which is a useful tool in monitoring, comparing and correcting. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Xenoestrogens are molecules mimicking estrogen, including bisphenol A (BPA) which is widely used as a product of epoxy resins, polycarbonate plastics, and flame retardants. It is extensively used in a broad range of products including toys, water pipes, drinking glasses, baby bottles, food-storage containers, the lining of food beverage containers, medical equipment, tubing, consumer electronics and dental sealants [1]. At high temperatures, BPA can migrate from polymer to food [2,3]. There has been an exponential increase in the use of BPA during the last 30 years, increasing its potential effects [4]. Several studies indicate that humans are regularly in contact with BPA, through water, air, soil, environment, and food contamination [5,6]. Levels of BPA have been shown to range between 0.2 and 20 ng/ml in adult serum [1], suggesting a possible involvement of this synthetic estrogen in human diseases by triggering endocrine disruption, immune disorder, epigenetic modulation and oxidative stress [7]. Other studies have already reported the presence of BPA in human blood, fetal serum during pregnancy, amniotic fluid, follicular fluid, placental tissue, umbilical cord blood, and urine [1,8–16].
∗ Correspondence to: Department of Obstetrics and Gynecology, University of Sherbrooke Hospital Centre, 3001, 12e Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4. Tel.: +1 819 820 6868x12538; fax: +1 819 564 5302. E-mail address: [email protected] 0890-6238/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.12.006
Recently, maternal levels of unconjugated BPA in blood ranging between 0.5 and 22.3 ng/ml have been shown in southeastern Michigan mothers [17]. In Korea, BPA levels were ranged from nondetectable to 66.48 g/l in maternal blood of pregnant women, and from non-detectable to 8.86 g/l in cord blood [11]. Unconjugated BPA readily crosses the placenta but conjugated BPA would have to be un-conjugated to do so [18]. Other studies show that BPA undergoes a rapid transport of BPA across the placenta and passes into the cord blood of human fetuses [19]. Additionally, BPA has been detected in human breast milk indicating that exposure during lactation is also likely [18]. Levels of unconjugated BPA in human blood and other fluids are higher than levels needed to stimulate a number of molecular endpoints in cell culture in vitro, and appear to be within BPA levels in animal studies. A study conducted by the Centres for Disease Control and Prevention (CDCP) examined 2500 Americans and reported that BPA was found in 92.6% of urine samples, indicating that humans are routinely exposed to this chemical. Urine concentrations ranged from 0.4 to 149 g/l were significantly higher in children and adolescents compared to adults [1]. Since the basis of better health is prevention, one would hope that we can develop procedures to avoid environmentally induced disease in susceptible population such as pregnant women and their fetuses. The fetus is considered to be highly susceptible to the adverse effects of xenobiotics. This is because environmental agents could disrupt the biological events that are required to ensure normal growth and development [20,21]. The objective of this study
A. Aris / Reproductive Toxicology 45 (2014) 8–13 Table 1 Clinical characteristics of subjects.
Age (year, mean ± SD) BMI (kg/m2 , mean ± SD) Gestational age (week, mean ± SD) Birth weight (g, mean ± SD)
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2.3. Blood and peritoneal fluid sampling
Pregnant women (n = 61)
Nonpregnant women (n = 26)
P valuea
31.72 ± 3.66
33.12 ± 4.04
NS
24.8 ± 3.1
24.7 ± 3.2
NS
38.2 ± 2.1
N/A
N/A
3236 ± 324
N/A
N/A
BMI = body mass index, N/A: not applicable, data are expressed as mean ± SD. NS = not significant. a P values were determined by Mann–Whitney test.
was to evaluate human exposure of BPA in a specific period and geography, providing a reference for monitoring the evolution of measurable BPA.
2. Materials and methods 2.1. Chemicals and reagents Bisphenol A (BPA), 2.2-bis-(4-hydroxy-3-methylphenyl)propane (BPC) and N-methyl-N-(tert-butyldimethylsilyl) trifluoro(MTBSTFA) + 1% acetamide tert-buryldimethylchlorosilane (TBDMCS) were purchased from Sigma (St. Louis, MO, USA). Sep-Pak Plus PS-2 cartridges, from Waters Corporation (Milford, MA, USA). Ethyl acetate was purchased from VWR (Radnor, PA, USA). All other chemicals and reagents were of analytical grade (Sigma, MO, USA). 2.2. Study subjects At the Centre Hospitalier Universitaire de Sherbrooke (CHUS), we formed two groups of subjects: (1) a group of healthy pregnant women (n = 61), recruited at delivery; and (2) a group of healthy fertile nonpregnant women (n = 26), recruited during their tubal ligation of sterilization. As shown in Table 1 of clinical characteristics of subjects, eligible groups were matched for age and body mass index (BMI). Participants were not known for cigarette, illicit drug use, medical condition (i.e. diabetes, hypertension or metabolic disease) or for working in contact with BPA. The Eastern Townships population is mainly rural and there is no significant industrial activity nearby. The diet taken is typical of a middle class population of Western industrialized countries. A food market-basket, representative for the local population, contains various meat including margarine, canola oil, rice, corn, grain, peanuts, potatoes, fruits and vegetables, eggs, poultry, meat and fish. Beverages include milk, juice, tea, coffee, bottled water, soft drinks and beer. Most of these foods come mainly from the province of Quebec, then the rest of Canada and the United States of America. Our study did not quantify the exact levels of BPA in a market-basket study. Also, there is no data from public health agencies about the state of air or water pollution. Pregnant women had vaginal delivery and did not have any adverse pregnancy outcomes. The sex ratio was 1.03 (31 males to 30 females) and all neonates were of appropriate size for gestational age and without observable congenital malformations such as cryptorchidism, hypospadias and urogenital distance. The study was approved by the CHUS Ethics Human Research Committee on Clinical Research. All participants gave written consent.
For pregnant women, peripheral blood samples were obtained from the antecubital vein before delivery. Umbilical cord blood sampling was done after birth using the syringe method. Blood samples were collected in BD Vacutainer 10 ml glass serum tubes (Franklin Lakes, NJ, USA). To obtain serum, whole blood was centrifuged at 2000 × g for 15 min within 1 h of collection. For maternal samples, about 10 ml of blood was collected, resulting in 5–6.5 ml of serum. For cord blood samples, about 10 ml of blood was also collected by syringe, giving 3–4.5 ml of serum. Serum was stored at −20 ◦ C until assayed for BPA levels. For nonpregnant women, peripheral blood samples were obtained as described above before the induction of anesthesia prior the laparoscopic sterilization. Preparation for laparoscopy was routine, using umbilical puncture and pneumoperitoneum. Two ml of peritoneal fluid (PF) was collected through an abdominal port with a syringe before any operative manipulation to minimize blood contamination. All fluid present in the pouch Douglas was sucked up. Samples were centrifuged at 2000 rpm for 15 min within 1 h of collection. Glass syringes and glass tubes were used to avoid a possible contamination of BPA from plastic materials. Serum and PF were conserved in glass vials at −20 ◦ C until BPA assays were done. 2.4. BPA assessment Levels of free BPA were measured using gas chromatography– mass spectrometry (GC–MS). Calibration curve: adapted from Schönfelder et al. [9], BPA and BPC (used as internal standard) (1 mg/ml) were prepared in 50% ethanol and stored for a maximum of 3 months at 4 ◦ C. A 5 g/ml solution from previous components was made prior BPA extraction with 10% ethanol, and an additional 0.5 g/ml solution of BPA. These solutions were used as calibrators. Blank serum samples (0.2 ml) were spiked with 20 l of BPC (5 g/ml), 20 l, 4 l of BPA (5 g/ml), 20 l 4 l of 500 ng/ml solution, resulting in calibration samples containing 100 ng of internal standard (500 ng/ml), with 100 ng (500 ng/ml), 20 ng (100 ng/ml), 10 ng (50 ng/ml), 2 ng (10 ng/ml), 1 ng (5 ng/ml), 0.5 ng (2.5 ng/ml), 0.005 ng (0.025 ng/ml) of BPA. Extraction procedure: the control samples and calibration curve were extracted by employing a liquid-liquid extraction. Serum samples (200 l) were transferred into 5-ml glass tubes with 2 ml ethyl acetate containing the internal standard. The vials were sealed with Teflon-coated caps and agitated for 30 min. After a 2 min centrifugation at 1000 × g, 1.5 ml of the supernatant was transferred to 5-ml glass tubes, and the solvent was evaporated to dryness under nitrogen. The samples were reconstituted in 50 l each of MTBSTFA with 1% TBDMCS and ethyl acetate. The mixture was vortexed for 30 s every 5 min, 6 times. Samples of solution containing the derivatives were used directly for GC–MS (Agilent Technologies 6890N GC and 5973 Inert MS). GC–MS analysis: chromatographic conditions for these analyses were as followed: a 30 mm × 0.25 mm Zebron ZB-5MS fused-silica capillary column with a film thickness of 0.25 m from Phenomenex (Torrance, CA, USA) was used. Helium was used as a carrier gas at 1.1 ml/min. A 2 l extract was injected in a split mode at an injection temperature of 250 ◦ C. The oven temperature was programmed to increase from an initial temperature of 100 ◦ C (held for 3 min) to 300 ◦ C (held for 5 min) at 5 ◦ C/min. The temperatures of the quadrupole, ion source and mass-selective detector interface were respectively 150, 230, and 280 ◦ C. The MS was operated in the selected-ion monitoring (SIM) mode. The following ions were monitored (with quantitative ions in parentheses): BPA (441.0) 456.0; BPC (469.0) 484.0. The limit of quantification (LOQ) was 0.10 ng/ml,
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Table 2 Concentrations of BPA in maternal and fetal cord serum.
BPA Number of detection Range of detection (ng/ml) Mean ± SD (ng/ml)
Maternal (n = 61)
Fetal cord (n = 61)
P valuea
59/61 (97%) nd-4.46 1.36 ± 1.18
58/61 (95%) nd-4.60 1.23 ± 1.04
NS
BPA = bisphenol-A. Data are expressed as number (n, %) of detection, range and mean ± SD (ng/ml). NS = not significant, nd = non-detected (below the limit of detection = 0.010 ng/ml). a P values were determined by Wilcoxon matched pairs test.
whereas the limit of detection (LOD) was 0.01 ng/ml after determination as the lowest concentration giving a response three times the average baseline noise. Measures taken to prevent BPA contamination: potential crosscontamination of samples with BPA by external factors (tubes, pipette tips, collection kit, chemical reagents and the GC/MS itself) was evaluated with additional control experiments. The experiment was conducted by collecting distillate water into sterile BD Vacutainer 10 ml glass serum tubes (Franklin Lakes, NJ, USA) via a sterile 21 G gage Vacutainer needles instead of human blood. Afterwards, the extraction procedure and the GC/MS analysis were performed following the same procedures as human serum samples. There was no evidence of BPA contamination due to leaching from any material used during the experimentation. The calibration curves were constructed from a pool of human serum where no BPA was detected (blank serum) after addition of known amounts of BPA and internal standard (BPC). The standard samples were injected before and after serial sample analysis. The 0 ng/ml standard sample injected at the end of a serial sample analysis indicated that there was no carrying over between two samples. The other standard samples injected at the end of serial sample analysis indicated that there was no degradation in time of BPA during the GC/MS analysis. Levels of BPA in the samples were calculated based on the ratio of sample BPA to internal standard peak area response. The slope of the plot (peak-area ratio vs. amount of BPA added) indicated a linear dependence (r = 1.000). All the BPA assessments were based on a single measurement. 2.5. Statistical analysis BPA exposure was expressed as number, range and mean ± SD for each group. Characteristics of cases and controls and BPA exposure were compared using the Mann–Whitney U-test for continuous data and by Fisher’s exact test for categorical data. Wilcoxon matched pairs test compared two dependent groups. Other statistical analyses were performed using Spearman correlations. Analyses were realized with the software SPSS version 17.0. A value of P < 0.05 was considered as significant for every statistical analysis. 3. Results As shown in Table 1, pregnant women and nonpregnant women were similar in terms of age and body mass index. Pregnant women had normal deliveries and infants of normal birth weight (Table 1). BPA was detectable in maternal and fetal serum of pregnant women (Table 2, Fig. 1A), and in serum and peritoneal fluid of nonpregnant women (Table 3, Fig. 2A). BPA was [59/61 (97%), range (from non-detected to 4.46 ng/ml) and mean ± SD (1.36 ± 1.18)] in maternal serum and [58/61 (95%), range (from non-detected to 4.60 ng/ml) and mean ± SD (1.23 ± 1.04)] in fetal serum (Table 2, Fig. 1A). Of note, five samples (2 maternal serum samples and 3 fetal serum samples) had BPA
Fig. 1. Concentrations of BPA in maternal and fetal blood serum (A). Illustration of positive correlation between maternal and fetal exposure to BPA (B). Maternal and umbilical cord blood sampling were performed at delivery from sixty one pregnant women. BPA was assessed using GC–MS.
Table 3 Concentrations of BPA in peripheral blood and peritoneal fluid of nonpregnant women.
BPA Number of detection Range of detection (ng/ml) Mean ± SD (ng/ml)
Serum (n = 26)
Peritoneal fluid (n = 26)
P valuea
26/26 (100%) 1.30–8.17
26/26 (100%) 0.19–13.4
NS
3.83 ± 1.98
4.94 ± 3.71
BPA = bisphenol-A. Data are expressed as number (n, %) of detection, range and mean ± SD (ng/ml). NS = not significant. a P values were determined by Wilcoxon matched pairs test.
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Fig. 3. Concentrations of BPA in blood serum of pregnant women and nonpregnant women. Blood sampling were performed at delivery from sixty-one pregnant women and at tubal ligation for sterilization from twenty six nonpregnant women. BPA was assessed using GC–MS. Note the significant increase of BPA levels in nonpregnant women (P < 0.0001, determined by Mann Whitney test).
mean ± SD = 3.827 ± 1.98 vs. 1.36 ± 1.18, respectively, P < 0.0001 (Fig. 3). 4. Discussion
Fig. 2. Concentrations of BPA in blood serum and peritoneal fluid of nonpregnant women (A). Illustration of positive correlation between serum and peritoneal fluid exposure to BPA (B). Blood and peritoneal fluid sampling were performed at tubal ligation for sterilization from twenty six nonpregnant women. BPA was assessed using GC–MS.
levels below the limit of quantification (established at 0.10 ng/ml) and therefore were considered “non-detection”. BPA was detected in 26/26 (100%) peripheral serum and peritoneal fluid of NPW. Levels of BPA were ranged from 1.30 to 8.17 ng/ml (mean ± SD = 3.827 ± 1.98) and 0.19 to 13.45 ng/ml (mean ± SD = 4.94 ± 3.71) for serum and peritoneal fluid, respectively (Table 3, Fig. 2A). We also investigated a possible correlation between levels of BPA in maternal and fetal blood, and between peripheral blood and peritoneal fluid. Positive correlation was found between maternal and fetal serum: Spearman r = 0.823, P < 0.0001 (Fig. 1B), and between peripheral serum and peritoneal fluid: Spearman r = 0.461, P = 0.018 (Fig. 2B). On the other hand, levels of BPA in blood serum were higher in nonpregnant women than pregnant women:
Our results showed that BPA was detected in more than 95% of blood samples from healthy mothers and their fetuses. Obviously, this is not the first study showing the presence of BPA in the circulation of pregnant women or their fetuses, as others have already done that [9,11,22,23], but it is the first to compare between pregnant and nonpregnant women in Canada. Among the main findings of our study is the positive correlation between maternal and fetal blood BPA. The placenta does not seem to oppose or reduce the transfer of BPA, as its levels in both maternal and fetal sides are identical. This reflects the exposure of the fetus and suggests that maternal BPA may have a direct effect on fetal health. According to inclusion criteria of this study, all of the studied mothers and their babies were healthy and have no observable congenital diseases. It is important to note that “to be considered healthy” does not mean absence of BPA effects. Unfortunately, there are no studies on the long-term effects of low doses exposures and nonmonotonic dose–response curves (NMDRCs) of BPA on maternal–fetal health. The National Toxicology Program defined low-dose effects as any biological changes occurring in the range of typical human exposures or occurring at doses lower than those typically used in standard testing protocols, i.e. doses below those tested in traditional toxicology assessments [24]. Nonmonotonic dose–response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. In this regard, our previous study demonstrated that very low doses of BPA are able to induce necrosis, apoptosis and inflammatory response in human placental cytotrophoblasts, taken as an in vitro model for fetal toxicities, according to nonmonotonic dose-response curves [25]. This previous study suggested that exposure placental cells to low doses of BPA may cause detrimental effects, leading in vivo to adverse pregnancy outcomes which are associated with placental dysfunction (i.e. preeclampsia, intrauterine growth restriction, prematurity
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and pregnancy loss, among other). However, appropriate studies are needed to confirm or refute these hypotheses. It is important to note that although low dose exposures and NMDRCs are two major issues in the study of endocrine-disrupting chemicals, these concepts are interrelated, and NMDRCs are especially problematic for assessing potential impacts of exposure when nonmonotonicity is evident at levels of exposure below those that are typically used in toxicological assessments. A study of Vandenberg et al. [26] has investigated in depth the different aspects related to these concepts. Moreover, our findings showed that BPA was measurable in both peripheral blood and peritoneal fluid of healthy nonpregnant women. We also showed a positive correlation between levels of BPA in peripheral blood and peritoneal fluid, suggesting a continuum between these compartments. It is interesting to note that BPA levels tend to be higher in peritoneal fluid in comparison with peripheral blood, suggesting more presence and/or less elimination of BPA in peritoneal cavity. Large portion of female reproductive system bathes in peritoneal environment and it is conceivable to investigate possible links between accumulated BPA and increased risk of estrogen-dependent reproductive diseases, such as endometriosis, uterine leiomyoma and gynecological cancers, which are supposed to be sensitive to xenoestrogens as BPA [7]. For these reasons, peritoneal measurable BPA must be taken into consideration in our future studies. It is important to note that BPA levels reported in our study are higher than those reported by Teeguarden et al. [27], as well as those of many studies summarized in the review of Vandenberg et al. [28]. These differences cannot be explained by an effect related to anesthesia and/or the stress response related to surgery, because the blood collection was done before the administration of the anesthetic. Moreover, we have already observed that surgery does not affect the detection of BPA (unpublished data). Thus, the difference in the quantification of BPA levels observed between our study and that of Teeguarden et al. [27] may be explained by a higher exposure of our population or the sensitivity of the methods used, since the LOD in our study was 0.01 ng/ml, whereas it was 0.30 ng/ml (30 times higher) in the study of Teeguarden et al. [27]. On the other hand, a wide range of free BPA concentrations has been shown between our study and many studies reported by Vandenberg et al. [28]. It has been argued that contamination and/or deconjugation of BPA metabolites in collection, storage or analytical processes may be responsible for this inconsistency. Efforts to develop validated assays to quantify BPA in human blood are already ongoing [29]. The wide inter-individual variability as well as temporal variability should also be taken into account. From another angle, the higher levels of BPA in our population may be explained by dietary habits exposing to BPA, because there are at least two common practices that may contribute to the increase of measurable levels of BPA in Eastern Townships population: (1) eating canned foods, and (2) microwaving foods in plastic containers. These fairly common practices may be the result of socio-economic level of the region that is considered among the lowest in Canada. However, further studies are needed to confirm or refute these speculations. On the other hand, our study showed that BPA levels were higher in nonpregnant women than pregnant women. This is difficult to explain, but it may be the result of larger volume of distribution known in pregnancy, a process called hemodilution. This seems to suggest that pregnancy can affect the determination of BPA by underestimating its actual concentrations. 5. Conclusions This is the first study to assess simultaneously the levels of free bisphenol A in maternal and fetal blood and in peripheral blood and
peritoneal fluid of women. Our study highlights a continuum in the BPA distribution between the mother and the fetus and between peripheral blood and peritoneal cavity. It also suggests considering the potential role of pregnancy in the underestimation of blood BPA concentration. Furthermore, our study provides a temporal-spatial reference to the literature on BPA exposure, which is a valuable tool in monitoring, comparing and correcting. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported by the Grant No. 14441 from the FRQS (Fonds de recherche du Québec – Santé). This work was also supported by Fondation des Étoiles. The author wish to thank Dr. Youssef Ainmelk, Samuel Leblanc and Denis Cyr for their material and technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox. 2013.12.006. References [1] Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reproductive Toxicology 2007;24(2):139–77. [2] Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N. Xenoestrogens released from lacquer coatings in food cans. Environmental Health Perspectives 1995;103(6):608–12. [3] Biles JE, White KD, McNeal TP, Begley TH. Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods. Journal of Agricultural and Food Chemistry 1999;47(5):1965–9. [4] vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA, Eriksen M, et al. Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reproductive Toxicology 2007;24(2):131–8. [5] Kuo HW, Ding WH. Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography–mass spectrometry. Journal of Chromatography A 2004;1027(1–2):67–74. [6] Thomson BM, Grounds PR. Bisphenol A in canned foods in New Zealand: an exposure assessment. Food Additives and Contaminants 2005;22(1):65–72. [7] Aris A, Paris K. Hypothetical link between endometriosis and xenobioticsassociated genetically modified food. Gynecologie, Obstetrique & Fertilite 2010;38(12):747–53. [8] Brock JW, Yoshimura Y, Barr JR, Maggio VL, Graiser SR, Nakazawa H, et al. Measurement of bisphenol A levels in human urine. Journal of Exposure Analysis and Environmental Epidemiology 2001;11(4):323–8. [9] Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I. Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environmental Health Perspectives 2002;110(11):A703–7. [10] Yamada H, Furuta I, Kato EH, Kataoka S, Usuki Y, Kobashi G, et al. Maternal serum and amniotic fluid bisphenol A concentrations in the early second trimester. Reproductive Toxicology 2002;16(6):735–9. [11] Lee YJ, Ryu HY, Kim HK, Min CS, Lee JH, Kim E, et al. Maternal and fetal exposure to bisphenol A in Korea. Reproductive Toxicology 2008;25(4):413–9. [12] Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL. Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environmental Health Perspectives 2005;113(4):391–5. [13] Wan Y, Choi K, Kim S, Ji K, Chang H, Wiseman S, et al. Hydroxylated polybrominated diphenyl ethers and bisphenol A in pregnant women and their matching fetuses: placental transfer and potential risks. Environmental Science & Technology 2010;44(13):5233–9. [14] Chou WC, Chen JL, Lin CF, Chen YC, Shih FC, Chuang CY. Biomonitoring of bisphenol A concentrations in maternal and umbilical cord blood in regard to birth outcomes and adipokine expression: a birth cohort study in Taiwan. Environmental Health: A Global Access Science Source 2011;10:94. [15] Casas M, Valvi D, Luque N, Ballesteros-Gomez A, Carsin AE, Fernandez MF, et al. Dietary and sociodemographic determinants of bisphenol A urine concentrations in pregnant women and children. Environment International 2013;56:10–8. [16] Zhang T, Sun H, Kannan K. Blood and urinary bisphenol A concentrations in children, adults, and pregnant women from china: partitioning between blood and
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