ARTICLE pubs.acs.org/est
Polybrominated Diphenyl Ethers, Hydroxylated Polybrominated Diphenyl Ethers, and Measures of Thyroid Function in Second Trimester Pregnant Women in California Ami R. Zota,*,† June-Soo Park,‡ Yunzhu Wang,‡ Myrto Petreas,‡ R.Thomas Zoeller,§ and Tracey J. Woodruff † †
Program on Reproductive Health and the Environment, University of California, San Francisco, Oakland, California, United States Environmental Chemistry Lab, California Department of Toxic Substances Control, Berkeley, California, United States § Biology Department, Morrill Science Center, University of Massachusetts at Amherst, Amherst, Massachusetts, United States ‡
bS Supporting Information ABSTRACT: Prenatal exposure to polybrominated diphenyl ethers (PBDEs) may disrupt thyroid function and contribute to adverse neurodevelopmental outcomes. We conducted a pilot study to explore the relationship between serum concentrations of lower-brominated PBDEs (BDE-17 to -154), higher-brominated PBDEs (BDE183 to -209), and hydroxylated PBDE metabolites (OH-PBDEs) with measures of thyroid function in pregnant women. Concentrations of PBDEs, OH-PBDEs, thyroid-stimulating hormone (TSH), total thyroxine (T4), and free T4 were measured in serum samples collected between 2008 and 2009 from 25 second trimester pregnant women in California. Median concentrations of lower-brominated PBDEs and OH-PBDEs were the highest reported to date in pregnant women. Median concentrations of BDE-47 and the sum of lower-brominated PBDEs (ΣPBDE5) were 43.1 ng/g lipid and 85.8 ng/g lipid, respectively, and the sum of OH-PBDEs (ΣOH-PBDE4) was 0.084 ng/mL. We observed a positive association between the weighted sum of chemicals known to bind to transthyretin (ΣTTR binders) and TSH levels. We also found positive associations between TSH and ΣPBDE5, ΣOH-PBDE4, BDE-47, BDE-85, 5-OH-BDE47, and 40 -OH-BDE49, and an inverse association with BDE-207. Relationships with free and total T4 were weak and inconsistent. Our results indicate that PBDE exposures are elevated in pregnant women in California and suggest a relationship with thyroid function. Further investigation is warranted to characterize the risks of PBDE exposures during pregnancy.
’ INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals widely used as flame retardants in consumer products since the 1970s. The commercial mixture penta-BDE has been most often added to polyurethane foam used in furniture, while octa- and deca-BDE commercial mixtures have been used in electronics and other plastic products. Although PBDEs are being phased out, human exposure in the U.S. continues due to the slow replacement time of PBDE-containing products and ingestion of contaminated foods.1 Concentrations of lower-brominated PBDE congeners, characteristic of penta-BDE, measured in household dust, breast milk, and serum from North America are an order of magnitude higher than those measured in Europe and Asia.2 Few biomonitoring studies have measured higher-brominated PBDEs which are commonly found in octa- and deca-BDE. Serum penta-BDEs levels are approximately 2-fold higher in California residents compared to the rest of the U.S., likely a result of California’s unique furniture flammability standards.3 Higher serum PBDE levels are also correlated with lower socioeconomic status (SES).4 Thus, low-income California residents may encounter elevated exposures to PBDEs. PBDE exposures may exert adverse effects by interfering with the thyroid system. Thyroid hormones (TH) in serum are r 2011 American Chemical Society
regulated through negative feedback such that, when TH levels decline, the pituitary gland secretes thyroid-stimulating hormone (TSH), which stimulates the thyroid to increase secretion of TH. When TH levels increase above a “set point”, TSH secretion is effectively suppressed.5 In animal studies, a pentaBDE mixture, as well as individual PBDE congeners, reduces serum free and total thyroxine (T4) in developing and adult rodents probably by activating liver enzymes that increase TH clearance from serum.68 However, associations of penta-BDEs and TH in humans show more variability. Yuan et al.9 found higher serum TSH in highly exposed Chinese electronic-waste workers relative to controls, which would suggest lower serum T4 in the exposed population. In contrast, other studies among adults show null associations or positive correlations between PBDE exposure and T410,11 and T3.12 Few studies have focused on pregnant women. A recent study of 270 pregnant women found inverse associations between PBDE levels and TSH but not T4.13 Two smaller studies of pregnant women reported null associations Received: February 4, 2011 Accepted: August 10, 2011 Revised: July 27, 2011 Published: August 10, 2011 7896
dx.doi.org/10.1021/es200422b | Environ. Sci. Technol. 2011, 45, 7896–7905
Environmental Science & Technology
ARTICLE
Figure 1. Molecular structures of thyroid hormones (TH) and PBDEs.
between PBDE levels and T4,14,15 and a study of newborns found an inverse association between PBDE levels in umbilical cord blood and total T4 in the subset of babies delivered vaginally.16 These studies indicate that PBDEs may interfere with thyroid function in humans, and if so, pregnant women may be particularly vulnerable. Pregnancy represents a period of increased demand on the thyroid gland. Serum TH levels increase by almost 50% during the first trimester due to an increase in thyroxine-binding globulin (TBG) and the direct action of human chorionic gonadotropin on the thyroid gland.17 TH insufficiency during pregnancy can impair the health of mother and offspring.18 Overt and subclinical hypothyroidism are associated with increased risk of miscarriage and preterm birth.19 Even modest reductions in maternal T4 during early pregnancy are associated with long lasting developmental deficits in their children, such as reduced IQ.20 Thus, PBDE exposure may represent an added stressor on the thyroid gland of pregnant women that could have lifelong effects on the offspring. PBDEs can be metabolized via cytochrome P450 mediated oxidation to form OH-PBDEs;21 these hydroxylated forms may be more potent on the thyroid system. As halogenated diphenyl ethers, PBDEs are structurally similar to T4 (Figure 1) and hydroxylation improves their ability to interact with TH-binding proteins in serum and perhaps in cells.22 For example, 5-OH-BDE-47 has a three times stronger binding affinity to serum binding protein transthyretin (TTR) than T4 and 3 orders of magnitude stronger binding affinity than BDE-47.23 Additionally, some OH-PBDEs can bind to the thyroid hormone receptor.24,25 It is possible that both of these mechanisms may result in altered thyroid status. However, OHPBDEs have rarely been studied in pregnant women. Given the growing evidence of the role of PBDEs and their metabolites in TH functioning and the opportunity to study these effects in a population with relatively high exposures, we conducted a pilot study to measure exposures to a large suite of PBDE flame retardants in an ethnically diverse and predominately low-income population of pregnant women in California and examined associations of PBDEs and OH-PBDEs with measures of thyroid function.
’ MATERIALS AND METHODS Study Population. We recruited pregnant women (n = 25) prior to second trimester pregnancy termination procedures at
the San Francisco General Hospital Women’s Options Center (WOC) in San Francisco, California, between 2008 and 2009. The WOC is an outpatient clinic serving an ethnically diverse and predominantly low-income population from the San Francisco Bay area and other parts of Northern and Central California. We excluded participants if they were currently smoking, using illicit drugs, or taking thyroid medication or obtained an abortion because of fetal anomalies. Because TH fluctuate throughout pregnancy,26 we limited pregnancy gestation from 19 to 23 weeks. We also abstracted information on pregnancy and demographic characteristics from medical records. Study protocols were approved by the University of California, San Francisco Committee on Human Research. Data Collection and Thyroid Hormone Analysis. Blood samples were collected from each participant prior to medical procedures. Approximately, 35 mL of serum was isolated from these samples and stored in aliquots of 2 mL in polypropylene storage tubes at 70 °C until analysis. To prevent unnecessary thawing and freezing, one tube of serum per participant was sent to Quest Diagnostics (San Jose, CA, USA) where it was analyzed for TSH, free and total T4, and lipid content immediately upon arrival. TSH was measured by third-generation immunochemiluminometric assay, and total T4 was measured by solid-phase immunochemiluminometric assay. Free T4 was measured by direct equilibrium dialysis followed by radioimmunoassay at Quest Diagnostics Nichols Institute (San Juan Capistrano, CA, USA). Equilibrium dialysis is considered the preferred method for free T4 analysis since it increases the accuracy of measurements in samples with normal or high transport proteins.27 The coefficients of variation (CV) for the TH assays were 4%, 5%, and 11% for TSH, total T4, and free T4, respectively. Total lipid content was calculated on the basis of measurements of total cholesterol and triglycerides using Phillip’s formula.28 PBDEs and OH-PBDEs Analysis. We analyzed 37 PBDE analytes in serum at the Department of Toxic Substances Control (DTSC) (Berkeley, CA, USA). Our suite of analytes consisted of 19 PBDE congeners and 18 OH-PBDEs and included lowerbrominated (BDE-17 to -154) and higher-brominated PBDEs (BDE-183 to -209). These analytes were representative of commercial mixtures penta-BDE (e.g., BDE-47), octa-BDE (e.g., BDE183), and deca-BDE (e.g., BDE-209)29 (analyte list provided in Table S1, Supporting Information). The serum preparation and 7897
dx.doi.org/10.1021/es200422b |Environ. Sci. Technol. 2011, 45, 7896–7905
Environmental Science & Technology extraction method, phase separation technique, and analysis were adopted from our earlier methods to separate phenolic compounds (e.g., OH-PCBs, PCP) from maternal serum.30,31 13 C12 labeled PBDEs (13C12-BDE-28, -47, -99, -153, -154, -183, -197, and -207) and 13C12 6-OH-BDE-47 (Wellington Laboratory, Guelph, Ontario, Canada) were used as internal standards. 13C12PCB-209 (Cambridge Isotope Laboratory, Andover, MA, USA) was used as a recovery standard. Native PBDE standards were purchased from Wellington Laboratories (Guelph, Ontario, Canada). MeO-BDE standards: 60 -MeO-BDE17, 40 -MeO-BDE17, 20 -MeO-BDE28, 60 -MeO-BDE49, 20 -MeO-BDE68, 6-MeOBDE47, 3-MeO-BDE47, 5-MeO-BDE47, 40 -MeO-BDE49, 6-MeO-BDE99, 50 -MeO-BDE100, 6-MeO-BDE90, 50 -MeOBDE99, 40 -MeO-BDE101, 2-MeO-BDE123, 6-MeO-BDE85, 40 -MeO-BDE103, and 6-MeO-BDE137 were purchased from Wellington Laboratories or donated by Dr. Robert Letcher. Diazomethane was synthesized in hexane using N-nitroso-Nmethylurea (Sigma-Aldrich, USA) as described elsewhere.32 Other chemicals and solvents used for the gas chromatography (GC) analysis were of the highest quality available and included dichloromethane and hexane (trace analysis, Burdick and Jackson), methanol, methyl-tert butyl ether (MTBE), and water (HPLC grade, Fisher Scientific, USA), 2-propanol (99.9%, pesticide grade, Fisher Scientific, USA), hydrochloric acid, sulfuric acid (98%), potassium hydroxide, potassium chloride, sodium hydroxide, and ethyl alcohol (9496%, 200 proof) (Fisher Scientific, USA), silica (200400 mesh) (Sigma-Aldrich, USA), and Florisil (60100 mesh) (Mallinckrodt, USA). Serum preparation and extraction procedures were completed in the ultraclean laboratory of DTSC. Before extraction, we spiked surrogate internal standards: 13C12 labeled PBDEs (2.510 ng) and 13C12 6-OH-BDE47 (1 ng). Maternal serum (1 mL) was denatured with 1 mL of 6 M hydrochloric acid and 6 mL of 2-propanol and extracted using 6 mL of hexane/MTBE (1:1; re-extraction with 3 mL). After a potassium chloride (1%) wash, the extracts were phase-separated by adding 2 mL of potassium hydroxide solution (0.5 M). After the neutral fraction was extracted, the alkaline aqueous solutions (potassium hydroxide) were acidified with 2 M hydrochloric acid and extracted for phenolic compounds with 4 mL of hexane/MTBE (9:1; reextraction with 3 mL). Phenolic extracts and five calibration standards (
