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FutureTox IV, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2018. Building upon FutureTox I, II, and III, this conference focused on the latest science and technology for in vitro profiling and in silico modeling as it relates to predictive developmental and reproductive toxicity (DART). Publicly available high-throughput screening data sets are now available for broad in vitro profiling of bioactivities across large inventories of chemicals. Coupling this vast amount of mechanistic data with a deeper understanding of molecular embryology and post-natal development lays the groundwork for using new approach methodologies (NAMs) to evaluate chemical toxicity, drug efficacy, and safety assessment for embryo-fetal development. NAM is a term recently adopted in reference to any technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment to avoid the use of intact animals (U.S. Environmental Protection Agency [EPA], Strategic plan to promote the development and implementation of alternative test methods within the tsca program, 2018, https://www.epa.gov/sites/production/files/2018-06/documents/epa_alt_strat_plan_6-20-18_clean_final.pdf). There are challenges to implementing NAMs to evaluate chemicals for developmental toxicity compared with adult toxicity. This forum article reviews the 2018 workshop activities, highlighting challenges and opportunities for applying NAMs for adverse pregnancy outcomes (eg, preterm labor, malformations, low birth weight) as well as disorders manifesting postnatally (eg, neurodevelopmental impairment, breast cancer, cardiovascular disease, fertility). DART is an important concern for different regulatory statutes and test guidelines. Leveraging advancements in such approaches and the accompanying efficiencies to detecting potential hazards to human development are the unifying concepts toward implementing NAMs in DART testing. Although use of NAMs for higher level regulatory decision making is still on the horizon, the conference highlighted novel testing platforms and computational models that cover multiple levels of biological organization, with the unique temporal dynamics of embryonic development, and novel approaches for estimating toxicokinetic parameters essential in supporting in vitro to in vivo extrapolation.

The brominated flame retardant, hexabromocyclododecane (HBCD), is added—but not bound—to consumer products and is eventually found in the environment and human tissues. Commercial-grade HBCD mixtures contain three major stereoisomers, alpha (α), beta (β), and gamma (γ), that are typically at a ratio of 12%:6%:82%, respectively. Although HBCD is widely used, the toxicological effects from its exposure in humans are not clearly understood. Using a physiologically based pharmacokinetic (PBPK) model could help improve our understanding of the toxicity of HBCD. The aim of this work was to develop a PBPK model, consisting of five permeability limited compartments (i.e., brain, liver, adipose tissue, blood, and rest of the body), to evaluate the pharmacokinetics of γ-HBCD in C57BL/6 mice. Physiological parameters related to body size, organ weights, and blood flow were taken from the literature. All partition coefficients were calculated based on the log K(ow). The elimination in urine and feces was optimized to reflect the percent dose eliminated, as published in the literature. Compared with data from the literature for brain, liver, blood, and adipose tissue, the model simulations accurately described the mouse data set within 1.5-fold of the data points. Also, two examples showing the utility of the PBPK model supplement the information regarding the internal dose that caused the health effects observed during these studies. Although this version of the PBPK model expressly describes γ-HBCD, more efforts are needed to clarify and improve the model to discriminate between the α, β, and γ stereoisomers.

Tetrabromobisphenol A (TBBPA) has become a ubiquitous indoor contaminant due to its widespread use as an additive flame retardant in consumer products. Reported evidence of endocrine disruption and accumulation of TBBPA in brain tissue has raised concerns regarding its potential effects on neurodevelopment and behavior. The goal of the present study was to examine the impact of developmental TBBPA exposure, across a wide range of doses, on sexually dimorphic non-reproductive behaviors in male and female Wistar rats. We first ran a pilot study using a single TBBPA dose hypothesized to produce behavioral effects. Wistar rat dams were orally exposed using cookie treats to 0 or 0.1 mg TBBPA/kg bw daily from gestational day (GD) 9 to postnatal day (PND) 21 to assess offspring (both sexes) activity and anxiety-related behaviors. Significant effects were evident in females, with exposure increasing activity levels. Thus, this dose was used as the lowest TBBPA dose in a subsequent, larger study conducted as part of a comprehensive assessment of TBBPA toxicity. Animals were exposed to 0, 0.1, 25, or 250 mg TBBPA/kg bw daily by oral gavage starting on GD 6 through PND 90 (dosed dams GD 6 – PND 21, dosed offspring PND 22 – PND 90). Significant behavioral findings were observed for male offspring, with increased anxiety-like behavior as the primary phenotype. These findings demonstrate that exposure to environmental contaminants, like TBBPA, can have sex-specific effects on behavior highlighting the vulnerability of the developing brain.

Tetrabromobisphenol A (TBBPA, CAS No. 79-94-7) is a brominated flame retardant used in 90% of epoxy coated circuit boards. Exposures to TBBPA can induce neurotoxicity and disrupt MAPK, estrogen, thyroid, and PPAR-associated signaling pathways. Because these pathways also regulate transporters of the central nervous system barriers, we sought to determine the effect of TBBPA on the expression and activity of 3 ATP binding cassette (ABC) transporters of the blood-brain barrier (BBB). Using a confocal based assay, we measured the ex vivo and in vivo effects of TBBPA on P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) transport activity in rat brain capillaries. Our rationale for using a rat model was based on tissue availability, ease of handling, and availability of historical TBBPA toxicokinetic data. We found that TBBPA (1-1000 nM) exposure had no significant effect on multidrug resistance-associated protein 2 transport activity in either sex, suggesting TBBPA does not compromise the physical integrity of the BBB. However, low concentrations of TBBPA (1-100 nM) significantly decreased breast cancer resistant protein transport activity in both sexes. Additionally, TBBPA exposures (1-100 nM), elicited a sex-dependent response in P-gp transport: increasing transport activity in males and decreasing transport activity in females. All TBBPA dependent changes in transport activity were dose- and time-dependent. Inhibitors of either transcription or translation abolished the TBBPA dependent increases in male P-gp transport activity. Western blot and immunofluorescent assays confirmed the TBBPA dependent P-gp increases expression in males and decreases in females. Antagonizing PPAR-γ abolished the TBBPA dependent increases in males but not the decreases in females. However, the decreases in female P-gp transport were blocked by an ER-α antagonist. This work indicates that environmentally relevant concentrations of TBBPA (1-100 nM) alter ABC transporter function at the BBB. Moreover, permeability changes in the BBB can alter brain homeostasis, hinder central nervous system drug delivery, and increase the brain's exposure to harmful xenobiotic toxicants.

Tetrabromobisphenol A Bis(2,3-dibromopropyl) ether (TBBPA-BDBPE) is a high production volume brominated flame retardant (BFR) used in consumer products, resulting in ubiquitous human exposure. Although the major route of exposure for this chemical is believed to be via ingestion, dermal contact is likely via contaminated dust. Independent trials of a single dose of 100 nmol/cm(2) (~1 µCi [(14)C]/cm(2)) of [(14)C]-radiolabeled TBBPA-BDBPE was applied to whole rat skin (in vivo) or split-thickness human and rat skin (ex vivo) to estimate in vivo human percutaneous uptake. [(14)C]-radioactivity was quantified to determine dermal absorption (dose retained in dosed skin) and penetrance (dose recovered in receptor fluid [ex vivo] or tissues/excreta [in vivo]) over 24 h. In vivo absorption and penetration for rat skin was 26% and 1%, with a maximum flux of 44±9 pmol/cm(2)/h. In ex vivo rat skin, absorption and penetration and absorption values were 23% and 0.3% (flux = 26±8 pmol/cm(2)/h). In ex vivo human skin, 53% was absorbed and penetration was 0.2% with a maximal flux of 16±12 pmol/cm(2)/h. Computed maximal flux for in vivo human skin was 21±9 pmol/cm(2)/h with expected total absorption of ~80% and a penetration of

2,4,6-Tribromophenol (TBP, CAS no. 118-79-6) is a brominated chemical used as a precursor, flame retardant, and wood antifungal agent. TBP is detected in environmental matrices and biota, including human breast milk, placenta, and serum. To address reports of TBP accumulation in human placenta and breast milk, studies were conducted to characterize TBP disposition and toxicokinetics in timed-pregnant or nursing Sprague Dawley rats following a single oral dose to the dam. Animals were administered [14C]-TBP (10 μmol/kg, 25 µCi/kg, 4 ml/kg) by gavage on gestation day 12 and 20, or postnatal day 12 and serially euthanized between 15 min and 24 h for collection of blood and tissues from the dam and fetuses/pups. Observed plasma TBP Cmax (3 and 7 nmol/ml) occurred at 15 min in both GD12 and GD20 dams while Cmax (3 nmol/ml) was observed at 30 min for PND12 dams. Concentrations in tissues followed plasma concentrations, with kidneys containing the highest concentrations at 30 min. GD12 litters contained a sustained 0.2%–0.3% of the dose (5–9 nmol/litter) between 15 min and 6 h while GD20 fetuses (2%–3%) and placentas (0.3%–0.5%) had sustained levels between 30 min and 12 h. The stomach contents (approx. 1 nmol-eq/g, 6–12 h), livers (0.04–0.1 nmol-eq/g) and kidneys (0.1–0.2 nmol-eq/g) of PND12 pups increased over time, indicating sustained exposure via milk. Systemic exposure to TBP and its metabolites occurs in both the directly exposed mother and the indirectly exposed offspring and is rapid and persistent after a single dose in pregnant and nursing rats.

Telomere length (TL) can be affected by various factors, including age and oxidative stress. Changes in TL have been associated with chronic disease, including a higher risk for several types of cancer. Environmental exposure of humans to PCBs and dioxins has been associated with longer or shorter leukocyte TL. Relative telomere length (RTL) may serve as a biomarker associated with neoplastic and/or non-neoplastic responses observed with chronic exposures to TCDD and PCBs. RTL was measured in DNA isolated from archived frozen liver and lung tissues from the National Toxicology Program (NTP) studies conducted in female Harlan Sprague Dawley rats exposed for 13, 30, and 52 weeks to TCDD, dioxin-like (DL) PCB 126, non-DL PCB 153, and a mixture of PCB 126 and PCB 153. RTL was assessed by quantitative polymerase chain reaction (qPCR). Consistent with literature, decreased liver and lung RTL was seen with aging. Relative to time-matched vehicle controls, RTL was increased in both the liver and lung tissues of rats exposed to TCDD, PCB 126, PCB 153, and the mixture of PCB 126 and PCB 153, which is consistent with most epidemiological studies that found PCB exposures were associated with increased leukocyte RTL. Increased RTL was observed at doses and/or time points where little to no pathology was observed. In addition to serving as a biomarker of exposure to these compounds in rats and humans, increases in RTL may be an early indicator of neoplastic and non-neoplastic responses that occur following chronic exposure to TCDD and PCBs.

Background: Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) is a replacement for perfluorooctanoic acid in the production of fluoropolymers used in a variety of consumer products. GenX alters fetal development and antibody production and elicits toxic responses in the livers and kidneys of rodents. The GenX effect on the blood–brain barrier (BBB) is unknown. The BBB protects the brain from xenobiotic neurotoxicants and harmful endogenous metabolites. Objectives: We aimed to investigate the effects of GenX on the transport activity and expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance–associated protein 2 (MRP2) at the BBB. Methods: Transporter activities were measured in isolated rat brain capillaries by a confocal microscopy–based method. ATPase (enzymatic hydrolysis of adenosine triphosphate to inorganic phosphate) levels were measured in vitro. Western blotting determined P-gp and BCRP protein levels. Cell survival after GenX exposure was determined for two human cell lines. Results: Nanomolar levels of GenX inhibited P-gp and BCRP but not MRP2 transport activities in male and female rat brain capillaries. P-gp transport activity returned to control levels after GenX removal. GenX did not reduce P-gp- or BCRP-associated ATPase activity in an in vitro transport assay system. Reductions of P-gp but not BCRP transport activity were blocked by a peroxisome proliferator–activated receptor γ (PPARγ) antagonist. GenX reduced P-gp and BCRP transport activity in human cells. Conclusion: In rats, GenX at 0.1–100 nM rapidly (in 1–2 h) inhibited P-gp and BCRP transport activities at the BBB through different mechanisms. PPARγ was required for the GenX effects on P-gp but not BCRP transport activity. https://doi.org/10.1289/EHP5884

Trimethylamine N-oxide (TMAO) is a diet and gut microbiota-derived metabolite that has been linked to cardiovascular disease risk in human studies and animal models. TMAO levels show wide inter and intra individual variability in humans that can likely be accounted for by multiple factors including diet, the gut microbiota, levels of the TMAO generating liver enzyme Flavin-containing monooxygenase 3 (FMO3) and kidney function. We recently found that dioxin-like (DL) environmental pollutants increased FMO3 expression to elevate circulating diet-derived TMAO in mice, suggesting that exposure to this class of pollutants might also contribute to inter-individual variability in circulating TMAO levels in humans. To begin to explore this possibility we examined the relationship between body burden of DL pollutants (reported by serum lipid concentrations) and serum TMAO levels (n = 340) in the Anniston, AL cohort, which was highly exposed to polychlorinated biphenyls (PCBs). TMAO concentrations in archived serum samples from the Anniston Community Health Survey (ACHS-II) were measured, and associations of TMAO with 28 indices of pollutant body burden, including total dioxins toxic equivalent (TEQ), were quantified. Twenty-three (22 after adjustment for multiple comparisons) of the 28 indices were significantly positively associated with TMAO. Although the design of ACHS-II does not enable quantitative assessment of the contributions of previously known determinants of TMAO variability to this relationship, limited multivariate modeling revealed that total dioxins TEQ was significantly associated with TMAO among females (except at high BMIs) but not among males. Our results from this cross-sectional study indicate that exposure to DL pollutants may contribute to elevated serum TMAO levels. Prospective longitudinal studies will be required to assess the joint relationship between DL pollutant exposures, other determinants of TMAO, and health outcomes.

The effects of body fat mass on the elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in mice. When male C57BL/6J mice are fed a high-fat, simple carbohydrate diet (HFD) for 13 weeks, they develop an obese phenotype. In contrast, A/J mice fed an HFD do not become obese. After 13 weeks on a normal diet (ND) or HFD, male C57BL/6J and A/J mice received a single dose by gavage of 0.1 or 5.0 µg of 2,3,7,8-tetrachloro[1,6-3H] dibenzo-p-dioxin per kg body weight. Using classical pharmacokinetics, the blood elimination half-life of TCDD was approximately 10 and 2 times longer in the C57BL/6J on the HFD compared with the mice on the ND at 0.1 and 5.0 μg/kg doses, respectively. The diet did not increase the blood half-life of TCDD in the A/J mice, which did not get obese. Using a physiologically based pharmacokinetic model for TCDD that incorporated experimentally derived percent body fat mass and tissue partition coefficients, as well as data on hepatic sequestration, did not provide accurate predictions to the data and could not explain the increase in half-life of TCDD in the HFD groups. This work demonstrates that obesity influences the half-life of TCDD, but other undetermined factors are involved in its elimination because the increase in body fat mass, decreases in cytochrome P4501A2, and altered partition coefficients could not completely explain the prolonged half-life.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant used globally at high volumes, primarily in the epoxy resin of circuit boards. It has been detected in the environment and in humans. The National Toxicology Program found that chronic oral TBBPA treatment of 250 mg/kg and higher caused an increased incidence of uterine lesions in female Wistar Han rats. The present laboratory has previously reported changes in gene expression associated with estrogen homeostasis in liver and uterine tissue of adult female Wistar Han rats after five days of gavage with 250 mg/kg of TBBPA. Microarray analysis of tissue from these same TBBPA-treated rats was performed to detect additional pathways perturbed by TBBPA. Microarray analysis of uterine tissue detected downregulation of genes in pathways of the immune response following TBBPA treatment. These results, along with validation of associated gene expression changes using droplet digital PCR, are reported here. Our findings suggest mechanisms that may be related to estrogen-mediated immunosuppression.

2,4,6-tribromophenol (TBP, CAS No. 118-79-6) is widely used as a brominated flame retardant and wood antifungal agent. TBP is frequently detected in environmental matrices, biota, and humans. In female SD rats, systemically available TBP (10 µmol/kg, IV) was rapidly excreted primarily via urine, with approximately 61% of the dose recovered after 4 h, and 89%–94% in 24 h; 5% was recovered in feces; and 1%–2% in blood/tissues. TBP administered to female SD rats (0.1–1000 µmol/kg) by gavage was well absorbed, with approximately 25% eliminated via urine after 4 h and approximately 88% after 24 h. Approximately 11% of a single oral dose was recovered in bile. Male SD rats and B6C3F1/J mice of both sexes had similar disposition profiles when administered a single oral dose of TBP (10 µmol/kg). Following administration, fecal recoveries varied only slightly by dose, sex, or species. TBP readily passed unchanged through both human (ex vivo only) and rat skin with between 55% and 85% of a 100 nmol/cm2 passing into or through skin. Concentrations of TBP in blood fit a two-compartment model after IV-dosing and a one-compartment model after oral dosing. Urine contained a mixture of TBP, TBP-glucuronide, and TBP-sulfate. Fecal extracts contained only parent TBP whereas bile contained only TBP-glucuronide. TBP did not appear to bioaccumulate or alter its own metabolism after repeated administration. TBP was readily absorbed at all doses and routes tested with an oral bioavailability of 23%–27%; 49% of TBP is expected to be dermally bioavailable in humans. From these data, we conclude that humans are likely to have significant systemic exposure when TBP is ingested or dermal exposure occurs.

Mitochondria are intracellular organelles responsible for biological oxidation and energy production. These organelles are susceptible to damage from oxidative stress and compensate for damage by increasing the number of copies of their own genome, mitochondrial DNA (mtDNA). Cancer and environmental exposure to some pollutants have also been associated with altered mtDNA copy number. Since exposures to polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been shown to increase oxidative stress, we hypothesize that mtDNA copy number will be altered with exposure to these compounds. mtDNA copy number was measured in DNA from archived frozen liver and lung specimens from the National Toxicology Program (NTP) study of female Harlan Sprague Dawley rats exposed to TCDD (3, 10, or 100 ng/kg/day), dioxin-like (DL) PCB 126 (10, 100, or 1000 ng/kg/day), non-DL PCB 153 (10, 100, or 1000 μg/kg/day), and PCB 126 + PCB 153 (10 ng/kg/day + 10 μg/kg/day, 100 ng/kg/day + 100 μg/kg/day, or 1000 ng/kg/day + 1000 μg/kg/day, respectively) for 13 and 52 weeks. An increase in mtDNA copy number was observed in the liver and lung of rats exposed to TCDD and the lung of rats exposed to the mixture of PCB 126 and PCB 153. A statistically significant positive dose-dependent trend was also observed in the lung of rats exposed to PCB 126 and a mixture of PCB 153 and PCB 126, although in neither case was the control copy number significantly exceeded at any dose level. These exposures produced a range of pathological responses in these organs in the two-year NTP studies. Conversely, there was a significant decrease or no change in mtDNA copy number in the liver and lung of rats exposed to non-DL PCB 153. This is consistent with a general lack of PCB 153 mediated liver or lung injury in the NTP study, with the exception of liver hypertrophy. Together, the results suggest that an increase in mtDNA copy number may serve as a sensitive, early biomarker of mitochondrial injury and oxidative stress that contributes to the development of the toxicity of dioxin-like compounds.

2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis(2-ethylhexyl)tetrabromophthalate (BEH-TEBP) are novel brominated flame retardants used in consumer products. A parallelogram approach was used to predict human dermal absorption and flux for EH-TBB and BEH-TEBP. [14C]-EH-TBB or [14C]-BEH-TEBP was applied to human or rat skin at 100 nmol/cm2 using a flow-through system. Intact rats received analogous dermal doses. Treated skin was washed and tape-stripped to remove “unabsorbed” [14C]-radioactivity after continuous exposure (24h). “Absorbed” was quantified using dermally retained [14C]-radioactivity; “penetrated” was calculated based on [14C]-radioactivity in media (in vitro) or excreta+tissues (in vivo). Human skin absorbed EH-TBB (24±1%) while 0.2±0.1% penetrated skin. Rat skin absorbed more (51±10%) and was more permeable (2±0.5%) to EH-TBB in vitro; maximal EH-TBB flux was 11±7 and 102±24 pmol-eq/cm2/h for human and rat skin, respectively. In vivo, 27±5% was absorbed and 13% reached systemic circulation after 24 h (maximum flux was 464±65 pmol-eq/cm2/h). BEH-TEBP in vitro penetrance was minimal (

The disposition of decabromodiphenyl ethane (DBDPE) was investigated based on concerns over its structural similarities to decaBDE, high potential for environmental persistence & bioaccumulation, and high production volume.In the present study, female Sprague Dawley rats were administered a single dose of [14C]-DBDPE by oral, topical, or IV routes. Another set of rats were administered 10 daily oral doses of 14C]-DBDPE. Male B6C3F1/Tac mice were administered a single oral dose.DBDPE was poorly absorbed following oral dosing, with 95% of administered [14C]-radioactivity recovered in the feces, 1% recovered in the urine and less than 3% in the tissues at 72 h. DBDPE excretion was similar in male mice and female rats. Accumulation of [14C]-DBDPE was observed in liver and the adrenal gland after 10 daily oral doses.The dermis was a depot for dermally applied DBDPE; conservative estimates predict ~14±8% of DBDPE may be absorbed into human skin in vivo; ~7±4% of the parent chemical is expected to reach systemic circulation following continuous exposure (24 h).Following intravenous administration, 6% of the dose was recovered in urine and 28% in the feces, while ~70% of the dose remained in tissues after 72h, with the highest concentrations found in the liver (42%) and lung (17%).

2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB; MW 549.92 g/mol; CAS 183658-27-7) is a brominated component of flame retardant mixtures used as substitutes for some PBDEs. EH-TBB is added to various consumer products, including polyurethane foams, and has been detected in humans. The present study characterized the fate of EH-TBB in rodents. [14C]-labeled EH-TBB was absorbed, metabolized, and eliminated via the urine and feces following single administrations of 0.1–100 µmol/kg (∼0.05–55 mg/kg) or repeated administration (0.1 µmol/kg/day × 5–10 days) by gavage to female Hsd:Sprague DawleySD (SD) rats. Cumulative excretion via feces increased (39–60%) with dose (0.1–10 µmol/kg) with corresponding decreases in urinary excretion (54 to 37%) after 72 h. Delayed excretion of [14C]-radioactivity in urine and feces of a 100 µmol/kg oral dose was noted. Recovery was complete for all doses by 72 h. IV-injected rats excreted more of the 0.1 µmol/kg dose in urine and less in feces than did gavaged rats, indicating partial biliary elimination of systemically available compound. No tissue bioaccumulation was found for rats given 5 oral daily doses of EH-TBB. Parent molecule was not detected in urine whereas 2 metabolites, tetrabromobenzoic acid (TBBA), a TBBA-sulfate conjugate, and a TBBA-glycine conjugate were identified. EH-TBB and TBBA were identified in extracts from feces. Data from gavaged male B6C3F1/Tac mice indicated minimal sex- or species differences are likely for the disposition of EH-TBB. Approximately 85% of a 0.1 µmol/kg dose was absorbed from the gut. Overall absorption of EH-TBB is expected to be even greater at lower levels.

Chronic oral treatment of tetrabromobisphenol A (TBBPA) to female Wistar Han rats resulted in increased incidence of cell proliferation at 250 mg/kg and tumor formation in the uterus at higher doses. The present study was designed to test the hypothesis that disruption of estrogen homeostasis was a major mode-of-action for the observed effects. Biological changes were assessed in serum, liver, and the proximal (nearest the cervix) and distal (nearest the ovaries) sections of the uterine horn of Wistar Han rats 24 hours following administration of the last of five daily oral doses of 250 mg/kg. Expression of genes associated with receptors, biosynthesis, and metabolism of estrogen was altered in the liver and uterus. TBBPA treatment also resulted in changes in expression of genes associated with cell division and growth. Changes were also observed in the concentration of thyroxine in serum and in expression of genes in the liver and uterus associated with thyroid hormone receptors. Differential expression of some genes was tissue-dependent or specific to tissue location in the uterus. The biological responses observed in the present study support the hypothesis that perturbation of estrogen homeostasis is a major mode-of-action for TBBPA-mediated cell proliferation and tumorigenesis previously observed in the uterus of TBBPA-treated Wistar Han rats.

Tetrabromobisphenol A (TBBPA) is currently the world’s highest production volume brominated flame retardant. Humans are frequently exposed to TBBPA by the dermal route. In the present study, a parallelogram approach was used to make predictions of internal dose in exposed humans. Human and rat skin samples received 100 nmol of TBBPA/cm2 skin and absorption and penetrance were determined using a flow-through in vitro system. TBBPA-derived [14C]-radioactivity was determined at 6 hour intervals in the media and at 24 hours post-dosing in the skin. The human skin and media contained an average of 3.4% and 0.2% of the total dose at the terminal time point, respectively, while the rat skin and media contained 9.3% and 3.5%, respectively. In the intact rat, 14% of a dermally-administered dose of ~100 nmol/cm2 remained in the skin at the dosing site, with an additional 8% reaching systemic circulation by 24 hours post-dosing. Relative absorption and penetrance was less (10% total) at 24 hours following dermal administration of a ten-fold higher dose (~1,000 nmol/cm2) to rats. However, by 72 hours, 70% of this dose was either absorbed into the dosing-site skin or had reached systemic circulation. It is clear from these results that TBBPA can be absorbed by the skin and dermal contact with TBBPA may represent a small but important route of exposure. Together, these in vitro data in human and rat skin and in vivo data from rats may be used to predict TBBPA absorption in humans following dermal exposure. Based on this parallelogram calculation, up to 6% of dermally applied TBBPA may be bioavailable to humans exposed to TBBPA.

This review summarizes the report entitled: Breast Cancer and the Environment: Prioritizing Prevention, highlights research gaps and the importance of focusing on early life exposures for breast development and breast cancer risk.

Tetrabromobisphenol-A (TBBPA) is a brominated flame retardant (BFR) commonly used in electronics to meet fire safety standards and has the largest worldwide production of any BFR. TBBPA has been detected in human breast milk and maternal/cord serum, indicating exposure to mothers, fetuses, and breastfeeding newborns although exposure to fetuses and newborns is poorly understood. Pregnant or nursing Wistar Han IGS rats were administered [14C]-TBBPA in a single dose (25 mg/kg, 2.5 μCi/kg) and euthanized between 0.5&24 h post dose to determine disposition in pregnant and nursing rats and their pups. Systemic exposure was largely unchanged between 1&8 h post dose in pregnant rats; [14C]-radioactivity in blood varied only slightly between 0.5&8 h (2.6 ± 0.6 → 2.6 ± 0.8 nmol-eq/mL) but was below the limit of detection at 24 h with an absorption half-life of 16min and elimination half-life of 17 h. Cmax was observed at 30min in lactating rats and concentrations fell steadily through 8 h. Plasma from pregnant rats contained a mixture of TBBPA and TBBPA-conjugates at 30min but only metabolites in subsequent samples. TBBPA was not detected in lactating dam plasma in this study. Placental concentrations increased through 8 h while whole-fetus Cmax occurred at 2 h post dose. In lactating animals, liver, uterus, and mammary time-concentration curves lagged slightly behind blood-concentration curves. It was clear from these studies that TBBPA is available to both the developing fetus and nursing pup following maternal exposure, and nursing pups are continuously exposed via contaminated milk produced by their mother. This research was supported in part by the Intramural Research Program of NIH/NCI.

In June 2005, a World Health Organization (WHO)-International Programme on Chemical Safety expert meeting was held in Geneva during which the toxic equivalency factors (TEFs) for dioxin-like compounds, including some polychlorinated biphenyls (PCBs), were reevaluated. For this reevaluation process, the refined TEF database recently published by Haws et al. (2006, Toxicol. Sci. 89, 4-30) was used as a starting point. Decisions about a TEF value were made based on a combination of unweighted relative effect potency (REP) distributions from this database, expert judgment, and point estimates. Previous TEFs were assigned in increments of 0.01, 0.05, 0.1, etc., but for this reevaluation, it was decided to use half order of magnitude increments on a logarithmic scale of 0.03, 0.1, 0.3, etc. Changes were decided by the expert panel for 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) (TEF = 0.3), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF) (TEF = 0.03), octachlorodibenzo-p-dioxin and octachlorodibenzofuran (TEFs = 0.0003), 3,4,4',5-tetrachlorbiphenyl (PCB 81) (TEF = 0.0003), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) (TEF = 0.03), and a single TEF value (0.00003) for all relevant mono-ortho-substituted PCBs. Additivity, an important prerequisite of the TEF concept was again confirmed by results from recent in vivo mixture studies. Some experimental evidence shows that non-dioxin-like aryl hydrocarbon receptor agonists/antagonists are able to impact the overall toxic potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, and this needs to be investigated further. Certain individual and groups of compounds were identified for possible future inclusion in the TEF concept, including 3,4,4'-TCB (PCB 37), polybrominated dibenzo-p-dioxins and dibenzofurans, mixed polyhalogenated dibenzo-p-dioxins and dibenzofurans, polyhalogenated naphthalenes, and polybrominated biphenyls. Concern was expressed about direct application of the TEF/total toxic equivalency (TEQ) approach to abiotic matrices, such as soil, sediment, etc., for direct application in human risk assessment. This is problematic as the present TEF scheme and TEQ methodology are primarily intended for estimating exposure and risks via oral ingestion (e.g., by dietary intake). A number of future approaches to determine alternative or additional TEFs were also identified. These included the use of a probabilistic methodology to determine TEFs that better describe the associated levels of uncertainty and "systemic" TEFs for blood and adipose tissue and TEQ for body burden.

The use of brominated flame retardants and incineration of bromine-containing materials has lead to an increase in polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in the environment. Measurable amounts of PBDD/Fs have been detected in soil, seafood, and human breast milk and serum. Studies indicate that the relative potencies of some PBDD/Fs based on enzyme induction are equivalent to those of some polychlorinated dibenzo-p-dioxins and dibenzofurans. To assess the humoral immunity relative potencies of PBDD/Fs and compare them to their chlorinated analogs, female B6C3F1/N mice received a single oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), 1,2,3,7,8-pentachlorodibenzofuran (1PeCDF), 2,3,4,7,8-pentabromodibenzofuran (4PeBDF), 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (DBDCDD), or 2,3,7-tribromodibenzo-p-dioxin (TriBDD). Inhibition of the immunoglobulin M (IgM) antibody forming cell response was measured 4 days following immunization with sheep red blood cells. The data were fit to a Hill model to estimate the ED50 for inhibition. Expression of xenobiotic metabolizing enzyme (XME) and thyroxine transport protein (Ttr) genes in liver was measured by PCR to assess aryl hydrocarbon-mediated responses. TCDD, TBDF, TCDF, 1PeBDF, 4PeBDF, 4PeCDF, and DBDCDD suppressed the IgM antibody response and Ttr gene expression, and upregulated phase I XME genes. 1PeCDF suppressed the IgM antibody response but only upregulated phase I XME genes; TriBDD had no effect on antibody response. The rank order of potency (ED50) for these chemicals was TCDD>TBDF>4PeBDF>TCDF/4PeCDF/1PeBDF>1PeCDF. Whereas TCDD was the most potent compound tested, the brominated analogs were more potent than their chlorinated analogs, suggesting that these compounds should be considered in toxic equivalency factor evaluation and risk assessment.

Polybrominated diphenyl ethers (PBDEs) have been used in a wide variety of consumer applications as additive flame retardants. In North America, scientists have noted continuing increases in the levels of PBDE congeners measured in human serum. Some recent studies have found that PBDEs are associated with adverse health effects in humans, in experimental animals, and wildlife. This laboratory previously demonstrated that urinary elimination of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is saturable at high doses in mice; however, this dose-dependent urinary elimination has not been observed in adult rats or immature mice. Thus, the primary objective of this study was to examine the mechanism of urinary elimination of BDE-47 in adult mice using a physiologically based pharmacokinetic (PBPK) model. To support this objective, additional laboratory data were collected to evaluate the predictions of the PBPK model using novel information from adult multi-drug resistance 1a/b knockout mice. Using the PBPK model, the roles of mouse major urinary protein (a blood protein carrier) and P-glycoprotein (an apical membrane transporter in proximal tubule cells in the kidneys, brain, intestines, and liver) were investigated in BDE-47 elimination. The resulting model and new data supported the major role of m-MUP in excretion of BDE-47 in the urine of adult mice, and a lesser role of P-gp as a transporter of BDE-47 in mice. This work expands the knowledge of BDE-47 kinetics between species and provides information for determining the relevancy of these data for human risk assessment purposes.

Background: Few studies have evaluated predictors of childhood exposure to organochlorine pesticides (OCPs), a class of lipophilic persistent chemicals. Objectives: Our goal was to identify predictors of serum OCP concentrations—hexachlorobenzene (HCB), β-hexachlorocyclohexane (β-HCH), and p,p-dichlorodiphenyldichloroethylene (p,p´-DDE)—among boys in Chapaevsk, Russia. Methods: Between 2003 and 2005, 499 boys 8–9 years of age were recruited in a prospective cohort. The initial study visit included a physical examination; blood collection; health, lifestyle, and food-frequency questionnaires; and determination of residential distance from a local factory complex that produced HCB and β-HCH. Fasting serum samples were analyzed for OCPs at the U.S. Centers for Disease Control and Prevention. General linear regression models were used to identify predictors of the boys’ serum HCB, β-HCH, and p,p´-DDE concentrations. Results: Among 355 boys with OCP measurements, median serum HCB, β-HCH, and p,p´-DDE concentrations were 158, 167, and 284 ng/g lipid, respectively. Lower body mass index, longer breastfeeding duration, and local dairy consumption were associated with higher concentrations of OCPs. Boys who lived < 2 km from the factory complex had 64% (95% CI: 37, 96) and 57% (95% CI: 32, 87) higher mean HCB and β-HCH concentrations, respectively, than boys who lived ≥ 5 km away. Living > 3 years in Chapaevsk predicted higher β-HCH concentrations, and having parents who lacked a high school education predicted higher p,p´-DDE concentrations. Conclusions: Among this cohort of prepubertal Russian boys, predictors of serum OCPs included consumption of local dairy products, longer local residence, and residential proximity to the local factory complex. Citation: Lam T, Williams PL, Burns JS, Sergeyev O, Korrick SA, Lee MM, Birnbaum LS, Revich B, Altshul LM, Patterson DG Jr, Turner WE, Hauser R. 2013. Predictors of serum chlorinated pesticide concentrations among prepubertal Russian boys. Environ Health Perspect 121:1372–1377; http://dx.doi.org/10.1289/ehp.1306480

Within the past few decades, the concept of endocrine-disrupting chemicals (EDCs) has risen from a position of total obscurity to become a focus of dialogue, debate, and concern among scientists, physicians, regulators, and the public. The emergence and development of this field of study has not always followed a smooth path, and researchers continue to wrestle with questions about the low-dose effects and nonmonotonic dose responses seen with EDCs, their biological mechanisms of action, the true pervasiveness of these chemicals in our environment and in our bodies, and the extent of their effects on human and wildlife health. This review chronicles the development of the unique, multidisciplinary field of endocrine disruption, highlighting what we have learned about the threat of EDCs and lessons that could be relevant to other fields. It also offers perspectives on the future of the field and opportunities to better protect human health.

1. Bis(2-ethylhexyl)-tetrabromophthalate (BEH-TEBP; CAS No. 26040-51-7; PubChem CID: 117291; MW 706.15 g/mol, elsewhere: TeBrDEPH, TBPH, or BEHTBP) is used as an additive brominated flame retardant in consumer products. 2. Female Sprague Dawley rats eliminated 92-98% of [14C]-BEH-TEBP unchanged in feces after oral administration (0.1 or 10 μmol/kg). A minor amount of each dose (0.8-1%) was found in urine after 72 h. Disposition of orally administered BEH-TEBP in male B6C3F1/Tac mice was similar to female rats. 3. Bioaccumulation of [14C]-radioactivity was observed in liver and adrenals following 10 daily oral administrations (0.1 μmol/kg/day). These tissues contained 5- and 10-fold higher concentrations of [14C]-radioactivity, respectively, versus a single dose. 4. IV-administered [14C]-BEH-TEBP (0.1 μmol/kg) was slowly eliminated in feces, with >15% retained in tissues after 72 h. Bile and fecal extracts from these rats contained the metabolite mono-ethylhexyl tetrabromophthalate (TBMEHP). 5. BEH-TEBP was poorly absorbed, minimally metabolized and eliminated mostly by the fecal route after oral administration. Repeated exposure to BEH-TEBP led to accumulation in some tissues. The toxicological significance of this effect remains to be determined. This work was supported by the Intramural Research Program of the National Cancer Institute at the National Institutes of Health (Project ZIA BC 011476).

Summary Understanding and preventing adverse impacts from chemicals in the environment is fundamental to protecting public health, and chemical risk assessments are used to inform public health decisions in the United States and around the world. Traditional chemical risk assessments focus on health effects of environmental contaminants on a chemical-by-chemical basis, largely based on data from animal models using exposures that are typically higher than those experienced by humans. Results from environmental epidemiology studies sometimes show effects that are not observed in animal studies at human exposure levels that are lower than those used in animal studies. In addition, new approaches such as Toxicology in the 21st Century (Tox21) and exposure forecasting (ExpoCast) are generating mechanistic data that provide broad coverage of chemical space, chemical mixtures, and potential associated health outcomes, along with improved exposure estimates. It is becoming clear that risk assessments in the future will need to use the full range of available mechanistic, animal, and human data to integrate multiple types of data and to consider nontraditional health outcomes and end points. This perspective was developed at the “Strengthening the Scientific Basis of Chemical Safety Assessments” workshop, which was cosponsored by the U.S. Environmental Protection Agency and the National Institute of Environmental Health Sciences, where gaps between the emerging science and traditional chemical risk assessments were explored, and approaches for bridging the gaps were considered.

Almost 4 years ago, one of us (F.S.C.) wrote an Editorial ([ 1 ][1]) affirming the continued importance of basic research to the National Institutes of Health (NIH) mission. The Editorial emphasized that basic scientific discovery is the engine that powers the biomedical enterprise, and NIH

The metabolism of α- and γ-hexabromocyclododecane (HBCD) was investigated in adult C57BL/6 female mice. α- or γ-[(14)C]HBCD (3 mg/kg bw) was orally administered with subsequent urine and feces collection for 4 consecutive days; a separate group of mice was dosed and sacrificed 3 h postexposure to investigate tissue metabolite levels. Extractable and nonextractable HBCD metabolites were quantitated in liver, blood, fat, brain, bile, urine, and feces and characterized by LC/MS (ESI-). Metabolites identified were distinct between the two stereoisomers. In mice exposed to α-HBCD, four hydroxylated metabolites were detected in fecal extracts, and one of these metabolite isomers was consistently characterized in liver, brain, and adipose tissue extracts. In contrast, fecal extracts from mice exposed to γ-HBCD contained multiple isomers of monohydroxy-pentabromocyclododecene, dihydroxy-pentabromocyclododecene, and dihydroxy-pentabromocyclododecadiene, while in liver and adipose tissues extracts only a single monohydroxy-pentabromocyclododecane metabolite was observed. Both stereoisomers were transformed to metabolites which formed covalent bonds to proteins and/or lipids in the gut as suggested by high fecal nonextractables. The presence of tissue- and excreta-specific metabolic products after in vivo exposure to the two main HBCD stereoisomers supports previous toxicokinetic studies indicating that these two stereoisomers are biologically distinct. The distinct metabolic products identified in this study have the potential to aid in the identification of stereoisomer-specific HBCD exposures in future biomonitoring studies.

Background: Bisphenol A (BPA) is a high production volume chemical used to make polycarbonate plastic and is found in many consumer products. Some studies using animal models have suggested that BPA exposures may have adverse health effects. However, research gaps have precluded a full understanding of the effects of BPA in humans and engendered controversies surrounding the chemical’s potential toxicity. Objectives: The National Institute of Environmental Health Sciences (NIEHS) and National Toxicology Program (NTP) have developed an integrated, multipronged, consortium-based approach to optimize BPA-focused research investments to more effectively address data gaps and inform decision making. Discussion: NIEHS/NTP BPA research investments made over the past 4 years include extramural research grants, establishment of a BPA Grantee Consortium, intramural research activities on BPA’s mechanisms of action, the launch of two clinical studies and an occupational study, development of a round-robin experiment to validate BPA measurements in human serum, and, in collaboration with the Food and Drug Administration (FDA), formation of a consortium to design and execute a chronic toxicity study of BPA in rats. The NIEHS’s new consortium-based approach has led to more integrated, collaborative efforts and should improve our ability to resolve controversies over the potential human health effects of exposures to low levels of endocrine-active agents.

Hexabromocyclododecane (HBCD) is a mixture of three stereoisomers alpha (α), beta (β), and gamma (γ). γ-HBCD dominates the mixture (∼70%), and despite α-HBCD's minor contribution to global HBCD production and usage (∼10%), it is the dominant congener found in most biotic samples worldwide. Evidence of toxicity and lack of stereoisomer studies drives the importance of understanding HBCD toxicokinetics in potentially susceptible populations. The majority of public health concern has focused on hazardous effects resulting from exposure of infants and young children to HBCD due to reports on adverse developmental effects in rodent studies, in combination with human exposure estimates suggesting that nursing infants and young children have the highest exposure to HBCD. This study was designed to investigate differences in the disposition of both γ-HBCD and α-HBCD in infantile mice reported to be susceptible to the HBCD commercial mixture. The tissue distribution of α-[(14)C]HBCD- and γ-[(14)C]HBCD-derived radioactivity was monitored in C57BL/6 mice following a single oral dose of either compound (3 mg/kg) after direct gavage at postnatal day 10. Mice were held up to 7 days in shoebox cages after which pups were sacrificed, tissue collected, and internal dosimetry was measured. Developing mice exposed to α-HBCD had an overall higher body burden than γ-HBCD at every time point measured; at 4 days postexposure, they retained 22% of the α-HBCD administered dose, whereas pups exposed to γ-HBCD retained 10%. Total body burden in infantile mice after exposure to γ-HBCD was increased 10-fold as compared with adults. Similarly, after exposure to α-HBCD, infantile mice contained 2.5-fold higher levels than adult. These differences lead to higher concentrations of the HBCD diastereomers at target tissues during critical windows of development. The results indicate that the toxicokinetics of the two HBCD diastereomers differ between developing and adult mice; whereas distribution patterns are similar, concentrations of each HBCD diastereomer's-derived radioactivity are higher in the pup's liver, fat, kidney, brain, blood, muscle, and lungs than in the adult's. This study suggests that developmental stage may be a risk factor for the harmful effects of α-HBCD and γ-HBCD, when developing animals may be more sensitive to effects and have increased body burden.

Alpha-hexabromocyclododecane (α-HBCD) is an emerging persistent organic pollutant present in the hexabromocyclododecane (HBCD) commercial mixture. HBCD is used as an additive flame retardant in a wide variety of household consumer products. Three main stereoisomers, alpha (α), beta (β), and gamma (γ), comprise roughly 10, 10, and 80% of the mixture, respectively. Despite its small contribution to HBCD global production and usage, α-HBCD is the major stereoisomer found in wildlife and human tissues including breast milk and blood in North America, European Union, and Asia. No mammalian or human data are currently available regarding the toxicokinetics of α-HBCD. This study was conducted in an effort to fully characterize the absorption, distribution, metabolism, and elimination of α-HBCD following a single and repeated exposure with respect to dose, time, and route of administration in female C57BL/6 mice. Results indicate that ∼90% of the administered dose (3 mg/kg) was absorbed after oral exposure. Disposition was (1) dictated by lipophilicity, as adipose, liver, muscle, and skin were major depots and (2) was dose dependent with nonlinear accumulation at higher doses. Elimination, both whole-body and from individual tissues, was biphasic. α-HBCD-derived radioactivity was excreted in the feces as parent and metabolites, whereas urine only contained metabolites. Presence of polar metabolites in the blood and urine were a major factor in determining the rapid initial whole-body half-life after a single oral exposure. Initial half-lives were ∼1-3 days and much longer terminal half-lives of 17 days were observed, suggesting the potential for α-HBCD bioaccumulation. A 10-day repeated study supports α-HBCD bioaccumulation potential. Stereoisomerization previously observed after exposure to γ-HBCD was not seen after exposure of α-HBCD. The toxicokinetic behavior reported here has important implications for the extrapolation of toxicological studies of the commercial HBCD mixture to the assessment of risk of α-HBCD which is the major stereoisomer found in wildlife and people.

Toxicologists and pathologists worldwide will benefit from a new, website-based, and completely searchable Nonneoplastic Lesion Atlas just released by the U.S. National Toxicology Program (NTP). The atlas is a much-needed resource with thousands of high-quality, zoomable images and diagnostic guidelines for each rodent lesion. Liver, gallbladder, nervous system, bone marrow, lower urinary tract and skin lesion images, and diagnostic strategies are available now. More organ and biological systems will be added with a total of 22 chapters planned for the completed project. The atlas will be used by the NTP and its many pathology partners to standardize lesion diagnosis, terminology, and the way lesions are recorded. The goal is to improve our understanding of nonneoplastic lesions and the consistency and accuracy of their diagnosis between pathologists and laboratories. The atlas is also a useful training tool for pathology residents and can be used to bolster any organization’s own lesion databases. Researchers have free access to this online resource at www.ntp.niehs.nih.gov/nnl.

Developmental effects of polybrominated diphenyl ethers (PBDEs) have been suspected due to their structural similarities to polychlorinated biphenyls (PCBs). This study evaluated neurobehavioral, hormonal, and reproductive effects in rat offspring perinatally exposed to a widely used pentabrominated commercial mixture, DE-71. Pregnant Long-Evans rats were exposed to 0, 1.7, 10.2, or 30.6 mg/kg/day DE-71 in corn oil by oral gavage from gestational day 6 to weaning. DE-71 did not alter maternal or male offspring body weights. However, female offspring were smaller compared with controls from postnatal days (PNDs) 35-60. Although several neurobehavioral endpoints were assessed, the only statistically significant behavioral finding was a dose-by-age interaction in the number of rears in an open-field test. Developmental exposure to DE-71 caused severe hypothyroxinemia in the dams and early postnatal offspring. DE-71 also affected anogenital distance and preputial separation in male pups. Body weight gain over time, reproductive tissue weights, and serum testosterone concentrations at PND 60 were not altered. Mammary gland development of female offspring was significantly affected at PND 21. Congener-specific analysis of PBDEs indicated accumulation in all tissues examined. Highest PBDE concentrations were found in fat including milk, whereas blood had the lowest concentrations on a wet weight basis. PBDE concentrations were comparable among various brain regions. Thus, perinatal exposure to DE-71 leads to accumulation of PBDE congeners in various tissues crossing blood-placenta and blood-brain barriers, causing subtle changes in some parameters of neurobehavior and dramatic changes in circulating thyroid hormone levels, as well as changes in both male and female reproductive endpoints. Some of these effects are similar to those seen with PCBs, and the persistence of these changes requires further investigation.

Objectives The objectives of this study were to provide updated measurements of PBDEs in US food, to estimate possible difference in levels from differing geographical regions, and to provide an improved estimate of current dietary intake. Methods Thirty matched food samples for a total of 90 samples were collected from each of three cities (Los Angeles, California; Dallas, Texas; and Albany, New York) and were analyzed for 13 polybrominated diphenyl ether congeners (BDE 28, 47, 49, 66, 85, 99, 100, 138, 153, 154, 183, 203, and 209). Dietary intake of PBDEs was estimated by food type, age, and sex. Results In this pilot study, we did not note a statistically significant difference in total PBDE levels in food collected from the three locations. The median total PBDE levels (estimating non-detected values as half of the detection limit) in meat, dairy, eggs, and fish were 267 pg/g wet weight (ww) (range 102–3156 pg/g ww), 176 pg/g ww (range 41–954 pg/g ww), 637 pg/g ww (range 193–932 pg/g ww), and 243 pg/g ww (range 36–2161 pg/g ww). PBDE intake from food was estimated to range from 2.7 ng/kg/day for children 2 through 5 years of age to 0.8 ng/kg/day for women aged 60 years and older. This compares closely with our previous study where the intake estimate was 2.7 ng/kg/day for children 2 through 5 years of age and 0.9 ng/kg/day for women aged 60 years and older. Conclusion We did not find a decrease of PBDEs in food since our previous studies which we expected to find due to the declining use of PBDEs in the USA. These findings could be consistent with food contamination from depot sources of PBDEs. A larger, more representative sampling of the US food supply is indicated based on our findings.

Polybrominated diphenyl ethers (PBDEs) are used commercially as additive flame retardants and have been shown to transfer into environmental compartments, where they have the potential to bioaccumulate in wildlife and humans. Of the 209 possible PBDEs, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is usually the dominant congener found in human blood and milk samples. BDE-47 has been shown to have endocrine activity and produce developmental, reproductive, and neurotoxic effects. The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model for BDE-47 in male and female (pregnant and non-pregnant) adult rats to facilitate investigations of developmental exposure. This model consists of eight compartments: liver, brain, adipose tissue, kidney, placenta, fetus, blood, and the rest of the body. Concentrations of BDE-47 from the literature and from maternal-fetal pharmacokinetic studies conducted at RTI International were used to parameterize and evaluate the model. The results showed that the model simulated BDE-47 tissue concentrations in adult male, maternal, and fetal compartments within the standard deviations of the experimental data. The model's ability to estimate BDE-47 concentrations in the fetus after maternal exposure will be useful to design in utero exposure/effect studies. This PBPK model is the first one designed for any PBDE pharmaco/toxicokinetic description. The next steps will be to expand this model to simulate BDE-47 pharmacokinetics and distributions across species (mice), and then extrapolate it to humans. After mouse and human model development, additional PBDE congeners will be incorporated into the model and simulated as a mixture.

Background Results from previous studies have suggested that breast cancer risk correlates with total lifetime exposure to estrogens and that early-life 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or diets high in fat can also increase cancer risk. Objectives Because both TCDD and diet affect the estrogen pathway, we examined how TCDD and a high-fat diet (HFD) interact to alter breast cancer susceptibility. Methods We exposed pregnant female FVB/NJ mice (12.5 days postcoitus) to 1 μg/kg TCDD or vehicle; at parturition, the dams were randomly assigned to a low-fat diet (LFD) or a high-fat diet (HFD). Female offspring were maintained on the same diets after weaning and were exposed to 7,12-dimethylbenz[a]anthracene on postnatal days (PNDs) 35, 49, and 63 to initiate mammary tumors. A second cohort of females was treated identically until PND35 or PND49, when mammary gland morphology was examined, or PND50, when mammary gland mRNA was analyzed. Results We found that maternal TCDD exposure doubled mammary tumor incidence only in mice fed the HFD. Among HFD-fed mice, maternal TCDD exposure caused rapid mammary development with increased Cyp1b1 (cytochrome P450 1B1) expression and decreased Comt (catechol-O-methyltransferase) expression in mammary tissue. Maternal TCDD exposure also increased mammary tumor Cyp1b1 expression. Conclusions Our data suggest that the HFD increases sensitivity to maternal TCDD exposure, resulting in increased breast cancer incidence, by changing metabolism capability. These results provide a mechanism to explain epidemiological data linking early-life TCDD exposure and diets high in fat to increased risk for breast cancer in humans.

Objectives This study was designed to update previous U.S. market basket surveys of levels and polybrominated diphenyl ether (PBDE) dietary intake calculations. This study also quantifies hexabromocyclododecane (HBCD) levels in U.S.-purchased foods for the first time and estimates U.S. dietary intake of HBCD. This is part of a larger market basket study reported in two companion articles, of current levels of certain persistent organic pollutants (POPs) PBDEs, HBCD, perfluorinated compounds, polychlorinated biphenyls, and pesticides in composite food samples collected in 2008–2009. Methods In this study, we measured concentrations of 24 PBDE congeners and total HBCD in composite samples of 31 food types (310 samples). U.S. dietary intake of PBDEs and HBCD was estimated referencing the most current U.S. Department of Agriculture loss-adjusted food availability report. Results Total PBDE concentrations in food varied by food type, ranging from 12 pg/g wet weight (ww) in whole milk to 1,545 pg/g ww in canned sardines and 6,211 pg/g ww in butter. Total HBCD concentrations also varied substantially within and among food groups, ranging from 23 pg/g in canned beef chili to 593 pg/g in canned sardines. HBCD was not detected in any dairy samples. Dietary intake of all PBDE congeners measured was estimated to be 50 ng/day, mostly from dairy consumption but also from meat and fish. HBCD intake was estimated at 16 ng/day, primarily from meat consumption. Conclusion PBDEs and HBCDs currently contaminate some food purchased in the United States, although PBDE intake estimated in this study is lower than reported in our previous market basket surveys. HBCD is in food at higher levels than expected based on previously reported levels in milk and blood compared with PBDE levels and is comparable to European levels.

Background Increased fat intake is associated with obesity and may make obese individuals uniquely susceptible to the effects of lipophilic aryl hydrocarbon receptor (AHR) ligands. Objectives We investigated the consequences of high-fat diet (HFD) and AHR ligands on body composition, mammary development, and hepatic P450 expression. Methods Pregnant C57BL/6J (B6) and DBA/2J (D2) dams, respectively expressing high- or low-responsive AHR, were dosed at mid-gestation with TCDD. At parturition, mice were placed on an HFD or a low-fat diet (LFD). Body fat of progeny was measured before dosing with 7,12-dimethylbenz[a]anthracene (DMBA). Fasting blood glucose was measured, and liver and mammary glands were analyzed. Results Maternal TCDD exposure resulted in reduced litter size in D2 mice and, on HFD, reduced postpartum survival in B6 mice. In D2 mice, HFD increased body mass and fat in off-spring, induced precocious mammary gland development, and increased AHR expression compared with mice given an LFD. Maternal TCDD exposure increased hepatic Cyp1a1 and Cyp1b1 expression in offspring on both diets, but DMBA depressed Cyp1b1 expression only in mice fed an HFD. In D2 progeny, TCDD exposure decreased mammary terminal end bud size, and DMBA exposure decreased the number of terminal end buds. Only in D2 progeny fed HFD did perinatal TCDD increase blood glucose and the size of mammary fat pads, while decreasing both branch elongation and the number of terminal end buds. Conclusions We conclude that despite having a low-responsive AHR, D2 progeny fed a diet similar to that consumed by most people are susceptible to TCDD and DMBA exposure effects blood glucose levels, mammary differentiation, and hepatic Cyp1 expression.

Previous studies demonstrated that perinatal exposure to polybrominated diphenyl ethers (PBDEs), a major class of brominated flame retardants, may affect thyroid hormone (TH) concentrations by inducing hepatic uridinediphosphate-glucoronosyltransferases (UGTs). This study further examines effects of the commercial penta mixture, DE-71, on genes related to TH metabolism at different developmental time points in male rats. DE-71 is predominately composed of PBDE congeners 47, 99, 100, 153, 154 with low levels of brominated dioxin and dibenzofuran contaminants. Pregnant Long-Evans rats were orally administered 1.7 (low), 10.2 (mid), or 30.6 (high) mg/kg/day of DE-71 in corn oil from gestational day (GD) 6 to postnatal day (PND) 21. Serum and liver were collected from male pups at PND 4, 21, and 60. Total serum thyroxine (T(4)) decreased to 57% (mid) and 51% (high) on PND 4, and 46% (mid) dose and 25% (high) on PND 21. Cyp1a1, Cyp2b1/2, and Cyp3a1 enzyme and mRNA expression, regulated by aryl hydrocarbon receptor, constitutive androstane receptor, and pregnane xenobiotic receptor, respectively, increased in a dose-dependent manner. UGT-T(4) enzymatic activity significantly increased, whereas age and dose-dependent effects were observed for Ugt1a6, 1a7, and 2b mRNA. Sult1b1 mRNA expression increased, whereas that of transthyretin (Ttr) decreased as did both the deiodinase I (D1) enzyme activity and mRNA expression. Hepatic efflux transporters Mdr1 (multidrug resistance), Mrp2 (multidrug resistance-associated protein), and Mrp3 and influx transporter Oatp1a4 mRNA expression increased. In this study the most sensitive responses to PBDEs following DE-71 exposure were CYP2B and D1 activities and Cyb2b1/2, d1, Mdr1, Mrp2, and Mrp3 gene expression. All responses were reversible by PND 60. In conclusion, deiodination, active transport, and sulfation, in addition to glucuronidation, may be involved in disruption of TH homeostasis due to perinatal exposure to DE-71 in male rat offspring.

Oxidative DNA damage is one of the key events thought to be involved in mutation and cancer. The present study examined the accumulation of M1dG, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, DNA adducts after single dose or 1-year exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to evaluate the potential role of oxidative DNA damage in PHAH toxicity and carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in polychlorinated biphenyl (PCB)-induced toxicity and carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153, at doses up to 3000 microg/kg/day, had no significant effect on the number of M1dG adducts in liver and brain tissues from the exposed rats compared to controls. However, 1000 ng/kg/day of PCB 126 resulted in M1dG adduct accumulation in the liver. More importantly, coadministration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adduct accumulation in the liver from 300 to 1000 ng/kg/day of PCB 126 with 300-1000 microg/kg/day of PCB 153. Interestingly, the coadministration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adduct accumulation with higher doses of PCB 153. These results are consistent with the results from cancer bioassays that demonstrated a synergistic effect between PCB 126 and PCB 153 on toxicity and tumor development. In summary, the results from the present study support the hypothesis that oxidative DNA damage plays a key role in toxicity and carcinogenicity following long-term PCB exposure.

Polybrominated diphenyl ethers (PBDEs) are a class of widely used flame retardants which have been found to persist, bioaccumulate, and potentially affect development in animals. Exposure to PBDEs can be through both diet and the environment and is generally estimated by measuring PBDEs in blood, adipose tissue, muscle, or milk samples. Using rats as a model, we investigated tissue distribution of PBDEs after oral administration and evaluated a suitable matrix for body burden estimation. Male rats were administered dust or corn oil containing 8 or 6 microg PBDEs kg(-1) body wt, respectively, in the diet for 21 days (N=4 rats per treatment), and the concentration of 15 PBDEs were measured in various tissues, plasma, and feces. PBDEs were found in all tissues, including the brain, and showed no difference in distribution patterns between treatments for most PBDEs. Tri- to hexa-BDEs comprised80% of the total PBDEs in the adipose, brain, kidney, lung, and residual carcass, but40% in the liver and plasma. The ratio of the lipid-weight concentration of tri- to hexa-BDEs in adipose tissue, residual carcass, and plasma was 1:1:2. For the hepta- to nona-BDEs, lipid-weight concentrations increased from adipose tissue to residual carcass to plasma in the ratio 0.3:1:4. BDE-209 was the dominant congener in the liver and plasma, but was not detected in the adipose tissue or carcass. In summary, the lower brominated congeners tended to distribute equally into lipids implying both adipose tissue and plasma would be suitable matrices for biomonitoring. Plasma was the best matrix for detection of the higher brominated congeners (especially BDE-209), although on a lipid-weight basis tended to overestimate the total body burdens.

The human body burden of polychlorinated biphenyls (PCBs) sharply declined after production was banned in the US in 1979. For the 10% of the US population that remains most exposed to PCBs, fish consumption is the primary source. National Health and Nutrition Examination Survey (NHANES) data indicates that the highest remaining PCB levels exist in a non-Hispanic black subpopulation. Our review suggests that catfish consumption may be a significant PCB source for the one million non-Hispanic black anglers who fish for catfish. In comparison to non-Hispanic white anglers, non-Hispanic black anglers consume more catfish, are more likely to eat the whole fish rather than just the fillets that contain less PCBs, and are more likely to fish in watersheds with high PCB contamination. Efforts to diminish potential racial disparities in PCB exposure are challenged by geographic, economic, cultural, and educational barriers. In response, we propose that a fish consumption survey be performed that identifies the extent of subsistence fishing by non-Hispanic black anglers for catfish in watersheds with PCB contamination, the type and quantity of catfish subsistence fishing provides, and what actions would help moderate PCB exposure due to subsistence fishing for catfish in such areas. Understanding the contamination and consumption factors that contribute to higher PCB body burdens will help identify and offer solutions to racial disparities in exposure to PCBs due to subsistence fishing while providing a model to prevent similar disparities in exposure to toxics ranging from mercury to polybrominated diphenyl ethers.

The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo- p -dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo- p -dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3′,4,4′-tetrachlorobiphenyl (IUPAC No. 77), 3,3′,4,4′,5-pentachlorobiphenyl (126), 3,3′,4,4′,5,5 N -hexachlorobiphenyl (169), 2,3,3′,4,4′-pentachlorobiphenyl (105), 2,3′,4,4′,5-pentachlorobiphenyl (118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose–response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197–208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose–response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157–172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured, and antibody response to SRBCs was observed. Exposure to TCDD, MIX-1, and MIX-2 suppressed the antibody response in a dose-dependent manner. Two-way ANOVA indicated no differences in the response between TCDD and the mixtures for body weight, spleen/body weight and decreased antibody responses. The results support the use of the TEF methodology and suggest that immune suppression by dioxin-like chemicals may be of concern at or near background human exposures.

Household dust has been implicated as a major source of polybrominated diphenyl ether (PBDE) exposure in humans. This finding has important implications for young children, who tend to ingest more dust than adults and may be more susceptible to some of the putative developmental effects of PBDEs. Absorption parameters of PBDEs from ingested dust are unknown; therefore, the objectives of this study were to determine and to compare the uptake of PBDEs from either household dust (NIST Standard Reference Material 2585) or a corn oil solution. Male rats were administered dust or corn oil doses at 1 or 6 microg of PBDEs kg(-1) body wt in the diet for 21 days (n = 4 rats per group). The concentrations of 15 PBDEs were measured in adipose tissue and liver from each treatment group and showed that bioconcentration was congener dependent, but for the majority of congeners, the concentrations did not differ with either dose level or dose vehicle. Hepatic Cyp2b1 and 2b2 mRNA expression increased in rats receiving the higher PBDE doses, suggesting potential effects on metabolic activity. Retention of PBDEs in tissues ranged from5% of the dose for BDE-209 to 70% for BDEs-47, 100, and 153 but generally did not differ between the high dust and high oil treatment groups. Excretion via the feces was significantly lower in the high oil dosed rats suggesting differences in absorption, excretion, and/or metabolism. The present study shows that PBDEs in dust are readily bioavailable and are biologically active, as indicated by increased transcription of hepatic enzymes.

Co-incident with the introduction of polybrominated diphenyl ethers (PBDEs) into household materials nearly 30 years ago, feline hyperthyroidism (FH) has increased dramatically. Risk of developing FH is associated with indoor living and consumption of canned catfood. We hypothesized that increases in FH were, in part, related to increased PBDE exposure, with key routes of exposure being diet and ingestion of house dust. This study was designed to determine whether body burdens of PBDEs in hyperthyroid (HT) cats were greater than that of young or sick non-HT cats. Serum samples and clinical information were collected from 23 cats. Serum and dry and canned cat food were analyzed for PBDEs. A spectrum of BDE congeners was detected in all cats, with BDE-47, 99, 207, and 209 predominating. Mean +/- standard error (and median) cumulative sigma PBDE serum concentrations of young, old non-HT, and HT cats were 4.3 +/- 1.5 (3.5), 10.5 +/- 3.5 (5.9), and 12.7 +/- 3.9 (6.2) ng/mL, respectively. Due to high variability within each group, no association was detected between HT cats and sigma PBDE levels. Indicative of age- or disease-dependent changes in PBDE metabolism, BDE-47/99 ratios were inversely correlated with age, and 47/99 and 100/ 99 ratios in HT cats were significantly lower than those in the other cats. Overall, sigma PBDE levels in cats were 20- to 100-fold greater than median levels in U.S. adults. Our results support the hypothesis that cats are highly exposed to PBDEs; hence, pet cats may serve as sentinels to better assess human exposure and adverse health outcomes related to low-level but chronic PBDE exposure.

Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/kg/day, in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10 to 1,000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low-dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long-lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.

Background: Hexabromocyclododecane (HBCD) is a high production volume brominated flame retardant added to building insulation foams, electronics, and textiles. HBCD is a commercial mixture (CM-HBCD) composed of three main stereoisomers: α-HBCD (10%), β-HBCD (10%), and γ-HBCD (80%). A shift from the dominant stereoisomer γ-HBCD to α-HBCD is detected in humans and wildlife. Objectives: Considering CM-HBCD has been implicated in neurodevelopment and endocrine disruption, with expected metabolism perturbations, we performed metabolomics on mice serum obtained during a window-of-developmental neurotoxicity to draw correlations between early-life exposures and developmental outcomes and to predict health risks. Methods: Six female C57BL/6 mice at postnatal day (PND) 10 were administered a single gavage dose of α-, γ-, or CM-HBCD at 3, 10, and 30 mg/kg. Nuclear magnetic resonance metabolomics was used to analyze 60 μL serum aliquots of blood collected 4 days post-oral exposure. Results: Infantile mice exposed to α-, γ-, or CM-HBCD demonstrated differences in endogenous metabolites by treatment and dose groups, including metabolites involved in glycolysis, gluconeogenesis, lipid metabolism, citric acid cycle, and neurodevelopment. Ketone bodies, 3-hydroxybutyrate, and acetoacetate, were nonstatistically elevated, when compared with mean control levels, in all treatment and dose groups, while glucose, pyruvate, and alanine varied. Acetoacetate was significantly increased in the 10 mg/kg α-HBCD and was nonsignificantly decreased with CM-HBCD. A third ketone body, acetone, was significantly lower in the 30 mg/kg α-HBCD group with significant increases in pyruvate at the same treatment and dose group. Metabolites significant in differentiating treatment and dose groups were also identified, including decreases in amino acids glutamate (excitatory neurotransmitter in learning and memory) and phenylalanine (neurotransmitter precursor) after α-HBCD and γ-HBCD exposure, respectively. Conclusions: We demonstrated that 4 days following a single neonatal oral exposure to α-, γ-, and CM-HBCD resulted in different serum metabolomic profiles, indicating stereoisomer- and mixture-specific effects and possible mechanisms of action. Citation: Szabo DT, Pathmasiri W, Sumner S, Birnbaum LS. 2017. Serum metabolomic profiles in neonatal mice following oral brominated flame retardant exposures to hexabromocyclododecane (HBCD) alpha, gamma, and commercial mixture. Environ Health Perspect 125:651–659; http://dx.doi.org/10.1289/EHP242

There are now robust data supporting the Developmental Origins of Health and Disease (DOHaD) paradigm. This includes human and animal data focusing on nutrition or environmental chemicals during development. However, the term DOHaD has not been generally accepted as the official term to be used when one is concerned with understanding the pathophysiological basis for how environmental influences acting during early development influence the risk of later noncommunicable diseases. Similarly, there is no global research or public health program built around the DOHaD paradigm that encompasses all aspects of environment. To better inform the global health efforts aimed at addressing the growing epidemic of chronic noncommunicable diseases of environmental origin, we propose a two-pronged approach: first, to make it clear that the current concept of DOHaD comprehensively includes a range of environmental factors and their relevance to disease occurrence not just throughout the life span but potentially across several generations; and second, to initiate the discussion of how adoption of DOHaD can promote a more realistic, accurate, and integrative approach to understanding environmental disruption of developmental programming and better inform clinical and policy interventions. (Endocrinology 156: 3416–3421, 2015)

The developmental origin of health and disease hypothesis posits that early-life exposures, including prenatal, can influence disease outcomes throughout the entire lifespan of an organism. Over the past 30 years, scientific researchers have compiled robust epidemiological and mechanistic data showing the effects of early-life nutrition, chemical exposures, and stress on prenatal programing and toxicity. Using novel techniques in genomics and epigenetics, science is now establishing strong links between low-level early-life environmental exposures and the later development of noncommunicable diseases, such as cardiovascular disease, obesity, diabetes, neurodevelopmental and neurodegenerative disease, reproductive effects, immune system function and cancer. Now scientists must engage with communities, industry, policy makers, and clinicians to leverage our newfound understanding of prenatal programing and toxicity into better health outcomes across the lifespan.