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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biochem Mol Toxicol. Author manuscript; available in PMC 2013 October 2.
Published in final edited form as:
PMCID: PMC3788594
NIHMSID: NIHMS517066

Accumulation and Endocrine Disrupting Effects of the Flame Retardant Mixture Firemaster® 550 in Rats: An Exploratory Assessment

Abstract

Firemaster® 550 (FM 550), a fire-retardant mixture used in foam-based products, was recently identified as a common contaminant in household dust. The chemical structures of its principle components suggest they have endocrine disrupting activity, but nothing is known about their physiological effects at environmentally relevant exposure levels. The goal of this exploratory study was to evaluate accumulation, metabolism and endocrine disrupting effects of FM 550 in rats exposed to 100 or 1000 μg/day across gestation and lactation. FM 550 components accumulated in tissues of exposed dams and offspring and induced phenotypic hallmarks associated with metabolic syndrome in the offspring. Effects included increased serum thyroxine levels and reduced hepatic carboxylesterease activity in dams, and advanced female puberty, weight gain, male cardiac hypertrophy, and altered exploratory behaviors in offspring. Results of this study are the first to implicate FM 550 as an endocrine disruptor and an obesogen at environmentally relevant levels.

Keywords: Anxiety, Cardiac Hypertrophy, Endocrine Disruptor, Flame Retardants, Metabolic Syndrome, Metabolism, Obesity, Obesogen, Polybrominated Diphenyl Ethers, Rodents

INTRODUCTION

Flame retardants (FRs) are chemicals or treatments applied to materials to reduce their flammability or delay their combustion. Over the past few decades, it has become increasingly common to treat polymers and resins used in commercial furniture, electronics, and construction materials with FR chemical additives. Polybrominated diphenyl ethers (PBDEs) were, at one time, the prevailing choice for this purpose in furniture foam produced in the United States, but the majority of PBDEs (e.g., PentaPBDE and OctaBDE) are persistent, bioaccumulate, and potentially toxic [1]. Consequently, several classes of PBDEs were recently added to Annex A of the Stockholm Convention [2], an international treaty that aims to eliminate or restrict the use of persistent organic pollutants. This prompted the introduction of new FRs to meet furniture flammability standards, like California’s Technical Bulletin 117 (TB 117).

In 2005, to maintain compliance with the TB 117 standard, the U.S. Environmental Protection Agency (EPA) Furniture Flame Retardancy Partnership identified 14 FR formulations as potential replacements [3]. Many of these formulations are proprietary; however, several recent research studies have identified their chemical components [4, 5]. A study by Stapleton et al. [4] identified components of a proprietary FR mixture known as Firemaster® 550 (FM 550) marketed by Chemtura (West Lafayette, IN). It contains four different components: (1) triphenyl phosphate (TPP); (2) a mixture of isopropylated triphenylphosphate isomers (ITPs); (3) 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB); and (4) bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) (Figure 1). After screening 102 different foam samples collected from baby products, Stapleton et al. [5] found that FM 550 is the second most commonly detected FR in polyurethane foam used and sold in the United States following the 2005 PentaBDE phaseout. Studies measuring FR levels in the environment have now detected both TBB and TBPH in indoor dust [4], outdoor air [6], marine mammal tissues [7], and wastewater sewage sludge [8,9], suggesting that, like some of their PentaPBDE predecessors, these chemicals are leaching from treated products and entering the environment. Relatively little is known regarding the potential for these compounds to bioaccumulate or their ability to induce adverse health effects.

FIGURE 1
Chemical structures of the four FM 550 components.

Owing to concerns about the potential toxicity of FM 550 components, the EPA entered into a modified consent order with Chemtura and required further testing of FM 550 [10]. Specifically, they required a two generation reproductive study (MPI Research Study 1038–008; “CN-2065: An Oral Two-Generation Reproduction and Fertility Study in Rats”) and a developmental toxicity study (MPI Research Study 1038–006; CN-2065: Prenatal Developmental Toxicity Study in Rats”). Exposures in both toxicity studies were conducted at doses ranging from 15 to 300 mg/kg/day using oral gavage. Numerous effects were observed but those that did not increase with dose or were observed only at an intermediate dose, were interpreted as “spurious” and unrelated to exposure. Observations included decreased litter size in the 15 and 50 mg/kg/day exposure groups and fused cervical vertebral neural arches in fetuses of exposed dams, an effect attributed to exposure at the 300 mg/kg dose but considered “incidental” at the 100 mg/kg dose. In both studies, the no observable adverse effects level (NOAEL) was set at 50 mg/kg/ day.

Since FM 550 is a common FR used in polyurethane foam found in both baby products and residential furniture, and because it is commonly detected in house dust, it is important to understand potential health effects associated with exposure in a human-relevant range. Children in particular are likely receiving chronic exposure to FM 550 components similar to their exposure to PBDEs [11]. Based on the observations of potentially adverse effects at the lower doses reported in both the two generation reproductive and developmental toxicity studies, we sought to conduct an exploratory study using oral exposure levels that more closely approximate human-relevant exposures.

Our primary objectives were to characterize effects of low-dose perinatal FM 550 exposure on growth, developmental, behavioral, and metabolic parameters in perinatally exposed Wistar rats. In addition, because many of the brominated FRs, including PBDEs, are known to disrupt thyroid hormone signaling (due to their structural similarity to thyroid hormones), we also investigated the effects of FM 550 exposure on circulating levels of thyroid hormones in both the dams and the pups. Finally, we sought to examine the accumulation potential of the brominated components of FM 550 and the primary metabolites of the two brominated components [12], in both exposed dams and potential transfer to developing pups. Collectively, this is the first study to test the hypothesis that FM 550 is endocrine disrupting at human-relevant exposure levels.

MATERIALS AND METHODS

Materials

Individual authentic standards of TBB and TBPH were purchased from Wellington Laboratories (Guelph, Ontario, Canada). The internal standard used for TBB and TBPH analysis, 4-fluoro-2,3,4,6-tetrabromodiphenylether (FBDE 69), was purchased from Chiron (Trondheim, Norway). Tetrabromobenzoic acid (TBBA), the primary metabolite of TBB, was synthesized as described in our previous study [12]. Two chemical standards, 2,3,5 triiodobenzoic acid (TIBA) and 2,3,4,5 tetrachloromonohexyl phthalate (TCMHP), were purchased from Sigma Aldrich (St. Louis, MI) and used as an internal quantification standards for TBBA and mono(2-ethylhexyl) tetrabromophthalate (TBMEHP; the primary metabolite of TBPH), respectively. All solvents used throughout this study were HPLC grade.

Two doses of FM 550 were selected for this study (100 and 1000 μg per day; referred to as low dose and high dose, respectively) in addition to a vehicle control. These doses represent potential high-range human exposures from household dust, as levels of TBB and TBPH combined have been measured as high as 124 μg/g (H. Stapleton, unpublished data). A commercial standard of FM 550 was supplied by Great Lakes Chemical (West Lafayette, IN), a company owned by Chemtura (Philadelphia, PA). FM550 was first accurately weighed and then diluted into 100% ethanol with stirring for 6 h, to yield a 53.82 mg/mL high-dose solution. A 300-μL aliquot of the high-dose solution was then diluted with 2.7 mL of 100% ethanol to prepare the low-dose solution (5.38 mg/mL). Ethanol diluent was used as the vehicle control. Aliquots (5 μL) of each dosing solution were transferred to autosampler vials, diluted to 1 mL with hexane, and analyzed by gas chromatography mass spectrometry to confirm dosing levels. It was assumed that the sum total of TBB and TBPH in FM 550 was 50% based on the range reported in the Material Safety Data Sheet (40%–60%) supplied by Great Lakes Chemical. The sum measurement of TBB and TBPH in the solutions ranged from 45.4% to 46.8% of the total mass of FM 550 in each solution. Therefore, the concentrations of FM 550 in each dose were considered accurate. Each preparation was coded for all dosing and analyses.

Animal Husbandry

Animal care and maintenance were conducted in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services’ Guide for the Care and Use of Laboratory Animals and approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee. All animals were obtained from our existing colony of Wistar rats maintained in an environment that minimizes exogenous exposure to endocrine disrupting compounds (EDCs) at the NCSU Biological Resource Facility. Study animals were kept in a climate controlled room at 25°C and 45%–60% average relative humidity. To minimize exposure to phytoestrogens, and other EDCs, the animals were fed a standardized, phytoestrogen free diet (Teklad 2020, Harlan) ad libitum, and housed in thoroughly washed polysulfone cages with glass water bottles (rubber stoppers and metal sippers) and woodchip bedding [13,14]. The light cycle was reversed and lengthened over time to account for season. Animals were initially maintained on a 10-h light cycle (lights on from 12:00 to 22:00), then a 12-h light cycle 2 months later (lights on from 12:00 to 24:00) and finally a 14-h light cycle 6-weeks after that for the remainder of the project (lights on from 12:00 to 3:00). After a change in light cycle, animals were allowed a minimum of 1 week to acclimate prior to testing. The first behavioral test was completed in the 12-h light cycle, and glucose challenge and the second behavioral assay occurred during the 14-h light cycle.

Adult female Wistar rats (n = 9) from our existing colony were paired with males until a sperm plug was detected (defined as gestational day zero (GD 0) by vaginal lavage), at which point the male was removed. All dosing was done using the coded solutions (described above) with all investigators blinded to the three exposure groups (vehicle control, 100 μg FM 550 and 1000 μg FM 550 daily) for the duration of the study. Daily oral exposure to the dams (n = 3 per exposure group) spanned GD 8 through weaning by dispensing 20 μL of solution onto ¼ of a soy-free food treat pellet (apple- or chocolate-flavored AIN-76A Rodent Diet Test Tabs, Test Diet, Richmond, IN). Dams were habituated to the treat prior to exposure, and all readily consumed the treat with no sign of taste aversion for any exposure group. This exposure method was selected because (1) oral administration is considered to be human relevant and (2) compared to gavage, it eliminates confounding due to maternal stress, which can have lifelong impacts on the offspring [1517]. All dams became pregnant, but one in the high-dose group failed to litter. Dosing for that individual continued, and she was sacrificed with the others on the day their litters were weaned. Thus, in the high-dose group, there were three dams but two litters. Day of birth was designated postnatal day zero (PND 0). Sex ratio and body weights of each litter were obtained on PNDs 1, 10, and 21. On PND 21, pups were weaned into same sex littermate groups of up to four and housed in the same conditions as the dams. Remaining animals were pair housed after PND 50.

Tissue Collection

On PND 21, a subset of animals and all dams were sacrificed (90–330 min postexposure) by CO2 asphyxiation and rapid decapitation. Animal weight was recorded, and blood, muscle, liver, and gonadal adipose tissue were collected. Liver was removed immediately, wet weighed, and snap frozen via liquid nitrogen. In dams, gonadal adipose tissue was additionally wet weighed. All other tissue was directly frozen on dry ice. For PND 21 animals, mixed sex pools of blood, muscle, and adipose were collected from pups of the same dam. Trunk blood was rapidly collected on ice, spun at 4°C for 10 min at 13,000 rpm and the plasma extracted and transferred to a clean microcentrifuge tube with disposable glass transfer pipets, then stored at –80°C. All tissue samples remained frozen and were stored at –80°C until processing. The remaining offspring were sacrificed as adults (approximately PND 220) and the same tissues collected as described for the juveniles (above), plus the hearts. In addition, heart, testis, ovaries, and uteri were collected and weighed after being cleaned of excess connective tissue and fat and blotted free of excess fluids.

Chemical Analysis

TBB, and TBPH were analyzed in adipose, muscle, and liver tissue collected from dams on PND 21 and pooled (three to six individuals for each mixed sex pool) adipose tissue collected from pups on PND 21 and PND 210. TBBA and TBMEHP were also analyzed in liver and adipose tissue from the dams on PND 21. Approximately 0.1–0.3 g of tissue was ground with sodium sulfate, spiked with appropriate internal standards (see the Materials section) and extracted using pressurized fluid extraction (ASE 300; Dionex Inc., Sunnyvale, CA) following our previous method [4] and using a 1:1 mixture of dichloromethane (DCM) and ethyl acetate. Gravimetric analysis of a subsample of the extract was used to measure total lipid content of the tissue. The remaining extract was purified using gel permeation chromatography to remove lipid residues following our previously published method [18]. The extracts of all the solid tissue types were reduced in volume to near dryness, reconstituted in 1.0 mL of DCM, and purified through Supelclean ENVI-Florisil extraction cartridges (6 mL, 1.0 g; Supelco, Bellefonte, PA). TBB and TBPH were eluted from the Florisil cartridges with 3 mL of DCM and 3 mL of ethyl acetate added sequentially to the column, and TBBA and TBMEHP were eluted from the column with 3 mL of methanol containing 10 mM acetic acid. The fraction containing TBB and TBPH was reduced in volume under nitrogen to near dryness and reconstituted in DCM (0.5 mL). The fraction containing TBBA and TBMEHP was evaporated to near dryness and reconstituted in a 1:1 mixture of methanol and water.

Quantification was performed using the following internal standards: 4′-fluoro-2,3′,4,6-terabromodiphenyl ether (FBDE-69) for TBB and TBPH; TIBA for TBBA; and TCMHP for TBMEHP. TBB and TBPH were quantified using our previously published GC/ECNI-MS method [4], and TBBA and TBMEHP were quantified following our previously published LC/MS/MS method [12]. Recovery of F-BDE 69 in the tissue extracts averaged 73 ± 12%. Recovery of TBB and TBPH through the ENVI-Florisil extraction cartridges was evaluated using matrix spikes (n = 3) and averaged 109 ± 16 and 82 ± 21%, respectively. In some cases, minor background levels of TBB or TBPH were detected in lab blanks and ranged from <IDL to 1.9 ng for TBB and IDL to 8.5 ng for TBPH. All sample values were blank corrected using the average blank level. Method detection limits were calculated using three times the standard deviation of the blanks normalized to average tissue mass extracted.

Thyroid Hormone Assays

Total (free and protein bound) thyroxine (T4) and total triiodothyronine (T3) were measured in serum collected from individual dams and pups (pooled) on PND 21 and in individual pups at 7 months of age. T4 and T3 were measured with previously described methods that were modified to improve the precipitation of serum proteins by the addition of 10% trichloroacetic acid to the acetone used in the serum deproteination procedure [12, 19]. 13C-labeled T4 and T3 were added to serum samples prior to extraction for use as internal standards. Recovery of the 13C T4 and T3 in serum extracts averaged 65 ± 22 and 67 ± 31%, respectively.

Thyroid hormone deiodinase activity (DI) was measured in liver and brain microsomal subcellular fractions using our previously reported method [20]. Hepatic DI activity was measured using rT3 as a substrate to evaluate outer ring DI; whereas T4 was used a substrate in the brain microsomal samples to evaluate inner ring DI. For each individual microsomal sample, DI activity was measured in triplicate and the average value used for statistical analyses. The relative standard deviation for triplicate DI measurements ranged from 0.9% to 7.0%.

Hepatic Enzyme Activities in the Dams

Measurement of CYP1A1 activity was measured in liver microsomal fractions using a standard ethoxyresorufin-o-deethylase assay. Carboxylesterase activity was evaluated by measuring the hydrolysis of p-nitrophenyl acetate (2.5 mM) to p-nitrophenol in assays containing hepatic microsomal protein at a concentration of approximately 5 μg mL−1 in KPO4 buffer (0.1 M; pH 7.4) [21]. Our previous research has shown that carboxylesterases can metabolize both TBB and TBPH [12].

Assessment of Body Weight and Female Reproductive Maturation

Offspring were weighed on PNDs 1, 10, 21, 120, 180, and the day of sacrifice (PND 220). Beginning on PND 25, the females were checked daily for vaginal opening (VO), a hallmark of puberty in the rat [22] and weighed on the day of opening to determine whether pubertal onset was associated with elevated body weight.

Behavioral Assessment of Activity and Anxiety

Exploratory and anxiety-related behavior were assessed with the zero maze (ZM) and the elevated plus maze (EPM) using well-established procedures described in detail elsewhere by us and others [2326]. Briefly, both mazes test for anxiety and general activity by quantifying exploration of the enclosed arms compared to unprotected open, and thus more aversive, arms. Testing duration was 5 min, and the apparatus was thoroughly cleaned between trials. All testing was done in the first 4 h of the dark cycle under red light (65 lux) when the animals are naturally more active. To eliminate transport stress, testing took place in an alcove of the room in which the animals are housed. All females were tested in estrous (established via vaginal lavage) [27] to eliminate cycle effects. ZM testing commenced at 13 weeks (approximately PND 90), and was completed over 9 days. EPM testing began at 19 weeks (approximately PND 135) and took place over 8 days. All trials were videotaped from a video camera suspended above the maze and scored live using behavioral analysis software (Stopwatch, courtesy of David A. Brown, Center for Behavioral Neuroscience, Emory University) by an observer blind to the treatment groups. All behavioral measures were then validated by a second observer from the videotape. For both mazes, the number of open arm entries and latency to enter an open arm were analyzed as a measure of anxiety and closed arm activity was analyzed as a measure of general activity.

Glucose Challenge Test

The glucose challenge test was performed at 17 weeks of age (approximately PND 120), 2 weeks prior to the onset of EPM testing. Animals were fasted for 18 h prior to testing in clean cages to minimize coprophagia. To ensure that the β-form, which is better transported, was predominant, the glucose solution was prepared the night before testing by dissolving 5 g D-glucose (Sigma) in 10 mL purified water and incubated overnight at 37°C. All animals were weighed at the time of testing, tested for baseline blood glucose (time zero) from a tail nick using glucometer (One-Touch Ultra; Lifescan, Milpitas, CA), intraperitoneally injected with 2 g/kg bw glucose solution and returned to the home cage. Subsequent glucose measures (10, 30, 60, and 120 min) were obtained from the tail nick while the animal was in the home cage to minimize handling stress. For each animal, the area under the curve (AUC) was calculated with SYSTAT 13 (Systat Software, Inc., Chicago, IL), using the trapezoidal rule.

Cardiac Histopathology

Hearts from the adult offspring were flushed with 0.9% NaCl followed by 4% paraformaldehyde in phosphate buffered saline (KPBS) and postfixed fixed in 4% paraformaldehyde for 72 h, then repeatedly washed in fresh 70% EtOH. Following automated tissue processing for 40–45 min each in seven changes of graded alcohols followed by embedding with three changes in paraffin at 58°C with applied vacuum (Histocenter 3; Thermo-Shandon Kalamazoo, MI), concentric sections of 1 mm were embedded at the level of the papillary muscle and microtome sections were cut at 5 μm from blocks at 4°C and placed on positively charged slides. Standard H&E (Richard-Allan, Kalamazoo, MI) staining was performed using a standard H&E protocol to examine tissue structure and morphology. 1× images were acquired using a Nikon Eclipse 55i microscope and a DS-Fi1 CCD camera controlled with Digital Sight software to examine tissue structure and measure left ventricular (LV) free wall thickness. Using Photoshop version CS2, five evenly spaced lines spanning the width of the LV free wall were measured, and the average LV free wall thickness was calculated from a single section at the level of the papillary muscle for each animal.

Statistical Analysis

Statistical analyses of the thyroid hormone, carboxylesterase, and DI activity measurements in the dams (n = 3/treatment) were performed using a one-way ANOVA and a Dunnett’s post hoc when appropriate. While one dam did not litter, she did continue to receive exposure to FM 550 and her tissues were used for analysis. Statistical significance was set at α = 0.05.

For the remaining endpoints measured in pups, due to the small scale of this exploratory study, there was not sufficient power to analyze the data by litter. Thus, to test for possible litter effects, the data were first approached by ANCOVA with litter as the covariate. No significant main effects or significant interactions with litter were identified for any endpoint. The subsequent statistical approach maximized power while simultaneously considering variance within the litters. Statistical analysis started with an examination of the univariate distribution (means, medians, standard deviations [SD], and range) of the various outcome measurements. Data were analyzed using a linear mixed model with a random effect for dam. This accounted for the within-litter correlation of outcomes among littermates without losing information by summarizing outcomes within-dam. [28, 29]. We tested for exposure effects using a solution for fixed effects. Body weight was included as a covariate in regression analyses performed for age at VO, and all cardiac measures to determine whether these endpoints were confounded by body weight. Body weight was only significantly associated with VO and heart weight, thus concurrent body weight was controlled for as a covariate in analyses of VO, and heart weight but no other measure. We examined the effect of treatment on the ZM, EPM, glucose challenge, body weight, and cardiac outcomes separately by sex since these traits are sexually dimorphic, and the effect of treatment may be sex specific. When a main effect was identified, post hoc tests were conducted using t tests to compare each exposure group to the control group. For LV thickness, ANOVA was used, followed by a Dunnett’s test. All analyses were conducted in SAS version 9.2 or Graph Pad version 5.

RESULTS

Gestational Data and Litter Composition Preweaning

Gestation length, litter size, and litter composition did not significantly differ between groups (Table 1). In the male offspring, significant group differences in body weight became apparent by PND 10 (F(2,33) = 5.58; p ≤ 0.01) and persisted through weaning on PND 21 (F(2,32) = 6.44; p ≤ 0.005) (and into adulthood, as shown below) with the males in the high-dose group weighing significantly more than controls (p ≤ 0.003). Among the female offspring, group differences in body weight were significant at PND 21 (F(2,33) = 3.65; p ≤ 0.04 with high-dose group females weighing significantly more than controls (p ≤ 0.03; Table 1). No significant differences in dam weight at sacrifice were observed.

TABLE 1
Preweaning Outcomes

Measurement of FM 550 in Dam and PND 21 Offspring Tissues

Adipose, liver, and muscle tissue samples were collected from each dam on PND 21 and analyzed for TBB and TBPH. Results are presented on a wet weight (ww) basis and not a lipid weight basis due to low signal-to-noise responses for lipid measurements in muscle, liver, and brain tissues. In the dams, TBB significantly accumulated in all three tissues with the highest concentrations measured in adipose tissues (Figure 2), measuring 768 ± 135 ng/g ww in the high-dose group, 29.6 ± 12.8 ng/g ww in the low-dose group, and <7.0 ng/g ww in the controls. Lipid content of dam adipose tissue on PND 21 averaged 74 ± 7%. Concentrations of TBB in liver tissue were 101 ± 40 ng/g ww in the high-dose group and <7 ng/g in the low-dose and control groups. TBB in muscle tissue was 13.4 ± 7.3 ng/g ww in the high-dose group and <0.9 ng/g in the controls. One of the three replicates from the low-dose group was lost during sample processing; however, TBB was detected in the two muscle tissue extracts from the low-dose group and averaged 14.3 ± 13.1 ng/g ww. TBPH was only detected in dam liver tissues. Concentrations of TBPH were 596 ± 189 ng/g ww in the high-dose group, 80.6 ± 27.6 ng/g ww in the low-dose group, and <18 ng/g ww in the control group.

FIGURE 2
Adipose levels of the FM 550 component TBB in dam (black bars) and pup (gray bars) adipose tissue on the day of weaning (PND 21). Samples were collected 60–330 min after exposure (n = 3/treatment; high dose = 1000 μg per day; low dose ...

TBB was detected in pooled PND 21 pup adipose tissue (Figure 2). Concentrations were 174 ± 27.5 ng/g ww in the high-dose group, 28.5 ± 19.9 ng/g ww in the low-dose group, and <18 ng/g ww in the control group. Lipid content of the pup adipose tissue on PND 21 averaged 49 ± 10%. TBPH was not detected in any pup adipose tissues. Neither TBB nor TBPH was detected in pooled adipose tissues from pups collected on PND 220.

The two main metabolites of TBB and TBPH, TBBA, and TBMEHP, respectively, were measured in dam liver tissue on PND 21. TBBA measured 1025 ± 342 ng/g ww in the high-dose group, 57.4 ± 34.0 ng/g ww in the low-dose group, and <7.0 ng/g ww in the control group. TBMEHP was not detected in any tissues (<15.4 ng/g ww).

Thyroid Hormone Levels in Dam and PND 21 Offspring Serum

Total serum thyroxine (T4) levels were significantly (65%) higher (p ≤ 0.05) in the high-dose exposed dams compared to control dams, averaging 26.3 ± 3.98 ng/mL in controls and 43.4 ± 1.88 ng/mL in the high-dose exposure group (Figure 3). T4 levels in dams from the low-dose group averaged 32.2 ± 2.94 ng/mL but were not significantly higher compared to controls. There was no statistically significant main effect of the experimental group on total triiodothyronine (T3) levels in dam serum; however, T3 levels in the low-and high-dose exposed dams averaged 0.272 ± 0.034 and 0.286 ± 0.023 ng/mL, respectively, compared to 0.371 ± 0.090 ng/mL in the control dams. There were also no statistically significant differences in T4 or T3 levels in pup serum on PND 21. There was a suggestive (p = 0.09) decrease, however, in T4 levels in exposed pups. T4 levels averaged 61.4 ± 6.7 ng/mL in control group pups, compared to 47.2 ± 11.0 and 44.5 ± 4.8 ng/mL in the low- and high-dose exposure groups, respectively.

FIGURE 3
Total thyroxine levels measured in dam serum collected on PND 21. Samples were collected 60–330 min after exposure (n = 3/treatment; bars depict means ± SEM; *p ≤ 0.05; high dose = 1000 μg per day; low dose = 100 μg/day ...

Enzyme Activity in Exposed Dams

No significant differences were observed in hepatic dam outer ring DI or brain inner ring DI among the three experimental groups (data not shown). Since halogenated contaminants are known to upregulate cytochrome P450 enzyme systems, we investigated potential effects of FM 550 exposure on CYP1A1. CYP1A1 activity was also not significantly different among groups on PND 21, with measurements ranging from 0.119 ± 0.014 μmol/min/mg protein in controls to 0.0893 ± 0.0129 μmol/min/mg protein in the high-dose group. Because our previous research has shown that carboxylesterases can metabolize both TBB and TBPH [12], we also investigated hepatic esterase activity. In contrast to the other enzyme activities investigated, hepatic carboxylesterase activity was significantly reduced in the high-dose group (0.658 ± 0.117 μmol/min/mg protein), compared to controls (1.75 ± 0.347 μmol/min/mg protein; p ≤ 0.05) (Figure 4).

FIGURE 4
Hepatic carboxylesterase activity measured in dams on PND 21. Samples were collected 60–330 min after exposure (n = 3/treatment; bars depict means ± SEM. *p ≤ 0.05; high dose = 1000 μg per day; low dose = 100 μg/day ...

Postweaning Reproductive Maturation and Body Weight

Age, but not body weight, at VO was significantly impacted by exposure (Table 2). Females exposed to the high dose displayed VO significantly earlier than unexposed controls (p ≤ 0.006). In both sexes, a significant main effect of exposure on body weight was retained into adulthood (p ≤ 0.03 for all cases; Table 2). At PND 120, males (p ≤ 0.02) and females (p ≤ 0.001) in the high-dose exposure group weighed significantly more than same sex controls and this effect persisted through PND 180 to PND 220, with males in the high-dose group weighing 32% more and females weighing 22% more than same sex controls at the time of sacrifice (p ≤ 0.001 for both sexes).

TABLE 2
Reproductive Maturation and Weight Gain

Anxiety and Exploratory Behavior

On the ZM, as expected, the number of open arm entries was higher in control females compared to control males (p ≤ 0.03; Figure 5A). Latency to enter an open arm also trended toward the expected sex difference (p = 0.09). Within females, the number of open arm entries was significantly impacted by exposure group (F(2,17) = 3.87; p ≤ 0.04) with females in both the low-dose (p ≤ 0.05) and high-dose (p ≤ 0.02) groups making significantly fewer entries. Consistent with a high anxiety phenotype, latency to enter an open arm was also impacted by FM 550. Although the main effect did not reach statistical significance in females (p = 0.2; Figure 5B), follow-up t tests revealed that the high-dose group took significantly longer to enter an open arm than controls (p ≤ 0.05). Among males, a main effect of exposure was also found for ZM behaviors, at the low dose only. The number of open arm entries made by the males significantly differed across experimental groups (F(2,16) = 5.49; p ≤ 0.02), with the low-dose exposure group making significantly more entries (p ≤ 0.01) than same sex controls. A trend for an exposure effect on latency to enter an open arm (p = 0.07) was also noted with low-dose exposed males showing enhanced exploration of the open arms compared to same sex controls; a reversal of the typical sex difference in maze performance.

FIGURE 5
Offspring performance on the ZM (A, B) and EPM (C, D) differed by sex and exposure. As expected, performance among controls was sexually dimorphic, with females showing less anxiety than males, although latency to enter an open arm in the ZM did not reach ...

On the EPM, the expected sex differences in open arm entry number (p ≤ 0.04) and latency to enter an open arm (p ≤ 0.01) were observed in the unexposed controls. Among the FM 550-exposed animals, none of the animals exposed to the high dose, regardless of sex, ventured out on the open arms compared to 55% (5/11) of the low-dose females and 11% (1/9) of the low-dose males. Among females, there was a dose-dependent decrease in the number of open arm entries (p ≤ 0.05) and, correspondingly, a dose-dependent increase in latency to enter an open arm (p ≤ 0.001). FM 550 effects on male EPM performance were inconclusive, primarily because overall activity was low.

Glucose Challenge

Baseline blood glucose levels were significantly altered by exposure in males; F(1,12) = 5.53; p ≤ 0.02) but not females, with elevated levels in the high-dose group (p ≤ 0.006) compared to same sex controls. Significant main effects on blood glucose levels during the glucose challenge test were only observed in females (Figure 6). At 30 min, the high-dose females had significantly higher blood glucose levels compared to controls (p ≤ 0.03), an effect which was no longer significant at 60 min. AUC was higher in the high-dose females compared to controls, but the effect did not reach statistical significance (p = 0.2).

FIGURE 6
Female offspring in the high-dose (1000 μg/day; n = 4) group had significantly higher blood glucose levels 30 min after glucose administration (A) compared to unexposed controls (n = 6). No significant effect of low-dose exposure (100 μg/day; ...

Cardiac Pathology

No significant differences were observed in female heart weight and LV thickness, nor in male heart weight. In males, LV wall thickness was significantly increased (F(3,18) = 4.754; p ≤ 0.024), with a significantly increased LV wall thickness in males exposed to the high dose (p ≤ 0.05) (Figure 7).

FIGURE 7
Impact of FM500 on LV wall thickness in the male offspring. The mean thickness of the LV wall was determined for controls (n = 9) and each exposure group. (A) Mean wall thickness for the control group was 2.58 mm (±0.06), 2.52 (±0.08), ...

DISCUSSION

This exploratory study reveals, for the first time, the potential for perinatal FM 550 exposure to have adverse effects indicative of endocrine disruption, at levels much lower than the NOAEL reported by the manufacturer. These findings are significant because FM 550 appears to be one of most commonly used replacements for PBDEs in foam and is prevalent in house dust [4,5]. Collectively, the data from this exploratory study identify FM 550 as a potential obesogen and contributor to metabolic syndrome, a collective term for a set of comorbid risk factors (including obesity, elevated fasting glucose, and impaired glucose tolerance) that together increase the risk for coronary artery disease, stroke, and type 2 diabetes [30,31]. In both sexes, the most distinctive physiological outcome was markedly elevated body weight at the high exposure dose. This effect became evident prior to adolescence and persisted into adulthood. In females, this increased mass contributed to accelerated pubertal onset and was accompanied by glucose intolerance, reduced activity, and elevated anxiety. In males, hallmarks of metabolic syndrome were also evident but the overall phenotype was distinct from the females and included higher baseline blood glucose levels and a marked increase in LV wall thickening, suggestive of poor cardiovascular performance. Maze performance suggested altered exploratory activity consistent with an obese phenotype and suggestive of altered anxiety. Future work exploring other metabolic parameters including blood pressure, serum lipid levels, and insulin levels will be needed to establish whether FM 550 exposure contributes to metabolic syndrome as has been suggested for other FRs including the PBDEs it was designed to replace [32, 33].

The FM 550 component TBB accumulated in tissues from both the exposed dams and their offspring. Levels of TBB in adipose tissue were comparable, demonstrating clear transplacental and/or lactational transfer of TBB from mother to offspring. However, TBPH was only detected in dam hepatic tissues. FM 550 also contains a mixture of isopropylated triaryl phosphate isomers and triphenyl phosphate, which are known to be well metabolized and were not investigated in this study.

In the female pups, pubertal onset was significantly advanced in the high-dose group, an effect that was associated with elevated body weight. Early pubertal onset girls are well documented in the United States and elsewhere [3438], and the reason for this remain to be elucidated. Increased bodyweight is contributing factor [3941], but hormonally active compounds may also play a contributing role. FM550 is a concern because it appears to be both obesogenic and endocrine disrupting, suggesting that it may be contributing to early puberty via multiple mechanisms.

Disruption of the thyroid hormone axis was observed in the dams. T4 was significantly elevated in exposed dams, whereas there was a suggestive decrease in T4 in pups. In contrast to animal studies investigating effects of PBDEs on thyroid hormone levels [42,43], we observed a positive association between T4 and exposure to FM 550. It is unclear why T4 levels increased with exposure. No effects on hepatic DI were observed, suggesting the increase in T4 was not due to inhibition of outer ring deiodination. Like PBDEs and their metabolites, it may be possible that TBB or TBPH is capable of inhibiting the activity of thyroid hormone conjugating systems that are responsible for clearing T4 from the body. In humans, maternal hyperthyroidism is associated with an increased risk of miscarriage, stillbirth, preeclampsia, and low birth weight [4447]. In severe cases, unmanaged hyperthyroidism can induce fetal musculo-skeletal malformations [48,49] similar to those reported in the guideline study conducted by Chemtura. Moreover, emerging evidence from animal studies suggests that offspring born to hyperthyroid dams may be more vulnerable to stress [50], resulting in a more anxious phenotype consistent with what was found in the present study. Thus, maternal hyperthyroidism may contribute to the offspring effects reported here, a possibility that should be followed up in future studies.

Activity of the phase 1 metabolic enzyme CYP1A1 was not influenced by exposure to FM550. However, hepatic carboxylesterase activity was decreased in the high-dose exposure group. Because carboxylesterases are implicated in the direct metabolism of TBB and TBPH, the biologic half-life of TBB or TBPH may be increased [12]. Because it remains unclear whether or not pregnancy itself heightens carboxylesterase activity, an alternative possibility is that FM 550 may blunt this induction, thereby resulting in lower levels compared to unexposed controls. Exposure testing of nonpregnant animals would be needed to explore this possibility. In addition, based on the present findings, relationships between carboxylesterase activity and lipid accumulation and adipogenesis following exposure to FM 550 should also be investigated in future studies to gain further insight as to the obesogenic potential of FM 550. Notably, a recent study reported higher expression of carboxylesterase 1 in obese individuals compared to lean individuals; however, they did not observe any effect on lipolytic activity [51].

The behavioral testing data suggest sex-specific effects on exploratory and anxiety-related behavior. Sex-specific effects were anticipated based on prior work with EDCs [17, 25, 52, 53]. In females, anxiety increased with increasing exposure. In males, the outcome was more complex with low-dose effects on anxiety-related behavior in the ZM but ambiguous effects in the EPM. Several factors may account for the inconsistent outcomes on the two mazes. Although exploratory drive naturally declines with age and thus reduced activity was not unexpected, ultimately, none of the animals in the high-dose exposure group ventured onto the EPM open arms. These animals were also markedly heavier, especially the males, thus the behavioral outcomes could be interpreted as a symptom of morbid obesity. Further study will be needed to delineate whether elevated anxiety or reduced activity accounts for the observed reduction in exploratory drive with age.

In summary, this is the first study to test for health effects of perinatal FM 550 exposure in a dose range that is potentially relevant to human exposure from dust. These results suggest that FM 550 may impact growth and neurodevelopmental endpoints. Given these findings and the increasing and widespread use of FM 550, additional research is warranted to validate and replicate these findings. Specifically, future research should further examine the endocrine-disrupting properties of FM 550 and elucidate the mechanisms responsible for the observed changes in weight, anxiety, and insulin resistance. Importantly, additional exposure assessment and biomonitoring studies are needed to determine the current levels of human exposure. While the FM 550 mixture is a relatively new FR (introduced around 2003), the organophosphate components of FM550 have been used individually as plasticizers and FRs for several decades, thus exposure may be ubiquitous and increasing.

Acknowledgments

The authors are grateful to Karina Todd, Sandra Losa-Ward, and Jinyan Cao for assisting with the tissue collection; Meghan Radford, Alana Sullivan, and Nicole Russ for their help with the behavioral testing; and the staff of the NCSU Biological Resource Facility for overseeing animal care and husbandry. In addition we thank Eric Kendig for his work on the cardiac assessments. This study was supported by research grants to HMS (NIEHS R01 ES016099), HBP (NIEHS, R01 ES016001), and SMB (NIEHS R01 ES015145, RC2 ES018765). FM 550 was provided by Chemtura.

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