Toxicokinetics of BDE 47 in Female Mice: Effect of Dose, Route of Exposure, and Time
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《毒物学科学杂志》
UNC Curriculum in Toxicology, US EPA, MD B143–01, Research Triangle Park, NC 27711
US EPA, ORD, NHEERL, ETD, US EPA, MD B143–01, Research Triangle Park, NC 27711
ABSTRACT
2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) is present in commercial mixtures of polybrominated diphenyl ethers (PBDEs), which are used as flame retardants in a wide variety of consumer products. Despite its small contribution to PBDE global production and usage, BDE 47 is the major congener found in environmental samples and human tissue. No human data are currently available regarding the toxicokinetics of BDE 47 either as an individual congener or in the commercial mixture. Because previous studies have suggested potential toxicokinetic differences between rodent species, this study was conducted in an effort to fully characterize absorption, distribution, and excretion parameters following a single dose with respect to dose, time, and route of exposure in female C57BL/6 mice. Over 80% of the administered dose was absorbed after oral or intratracheal administration, whereas 62% was absorbed when the dose was applied dermally. Disposition was dictated by lipophilicity as adipose and skin were major depot tissues. BDE 47 was rapidly excreted in the urine and feces. Of particular interest was the amount of parent compound found in the urine, which was a major factor in determining an initial whole-body half life of 1.5 days after a single oral exposure. Elimination, both whole-body and from individual tissues, was biphasic. Initial half-lives were 1–3 days, whereas terminal half-lives were much longer, suggesting the potential for bioaccumulation. This toxicokinetic behavior has important implications for extrapolation of toxicological studies to the assessment of health risk in humans.
Key Words: BFRs; PBDEs; BDE 47; toxicokinetics.
INTRODUCTION
Worldwide regulations require that flame retardants be added to a wide variety of consumer goods in an effort to reduce flammability. Generally, flame retardants are divided into classes which include halogenated organic, phosphorus-containing, nitrogen-containing, and inorganic flame retardants. Brominated flame retardants (BFRs) are the largest market group because of their low cost and high performance efficiency. Over 75 BFRs are recognized commercially; however, five BFRs constitute the overwhelming majority of BFR production: tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), decabromodiphenyl ether (DecaBDE), octabromodiphenyl ether (OctaBDE), and pentabromodiphenyl ether (PentaBDE). Recently, concern for PBDEs has risen after their detection in the environment and in humans at steadily increasing concentrations (Betts, 2002; Birnbaum and Staskal, 2004; de Wit, 2002; Law et al., 2003). The polybrominated diphenyl ethers potentially involve 209 different congeners, varying in both number and position of bromination. Each commercial product is a mixture of brominated diphenyl ether congeners.
The commercial pentaBDE product, commonly known as DE-71, is composed of 50–60% pentaBDEs, 24–38% tetraBDEs, and 4–8% hexaBDEs (World Health Organization, 1994). The commercial pentaBDE product is used in a number of highly flammable consumer applications including flexible polyurethane foam often found in upholstered furniture, mattresses, and bedding and carpet underlay (BSEF 2004). 2,2',4,4'-tetrabromodiphenyl ether (BDE 47, Fig. 1) is a specific PBDE congener found only in the pentaBDE commercial mixture. Despite its small contribution to PBDE global production and usage, BDE 47 is the major congener found in environmental samples and human tissue (Birnbaum and Staskal, 2004; de Wit, 2002; Hale et al., 2001; Law et al., 2003; Petreas et al., 2003; Schecter et al., 2003).
Concentrations of PBDEs in breast milk and serum from U.S. women are the highest reported in humans and are often tens to hundreds of times higher than human tissue samples from European countries. Petreas et al. (2003) measured BDE 47 in serum and adipose from contemporary California women in the late 1990s; concentrations of this congener ranged from 5 to 510 ng/g lipid (median 16.5 ng/g). Comparative serum samples from women residing in the same area in the 1960s revealed no detectable BDE 47. More recently, Schecter et al. (2003) reported high concentrations of PBDEs in U.S. breast milk collected in 2002. BDE 47 specifically had a range of 3 to 272 ng/g lipid with a median value of 18.4 (median PBDEs 34 ng/g). A recent review by Hites et al. (2004) highlights the ratio of BDE 47 as compared to the sum of all other PBDEs measured: BDE 47 accounts for approximately 55% of the total PBDE body burden despite the date or location of sampling.
Results from multiple studies make it clear that PBDEs are ubiquitous pollutants with increasing concentrations in people in North America; however, the mechanism(s) by which humans are exposed is not clear. Several researchers have found high concentrations of PBDEs in outdoor, indoor, and occupational air sampling (Butt et al., 2004; Jakobsson et al., 2002; Sinkkonen et al., 2003; Sjodin et al., 1999) as well as in house dust samples (Knoth et al., 2002; Stapleton et al., 2004). Correlations with other persistent, bioaccumulative, and toxic chemicals suggest that the primary pathway of exposure is through the diet (Bocio et al., 2003; Ohta et al., 2002). Recently, multiple investigators have reported that PBDEs are present at high levels in meat, fish, and dairy products (Huwe, 2004; Schecter et al., 2004). Because of these recent findings of high levels of PBDEs in the human diet, air, and house dust, it is essential to understand the relevance of each potential route of exposure.
In a human health risk assessment, the dose/response relationship of a chemical must be well characterized in order to accurately describe the associated risk. One of the major components of this is establishing a basic knowledge of absorption, distribution, metabolism, and excretion (ADME) of BDE 47. At present, studies on the toxicokinetics of PBDEs are very limited. Because of high octanol/water partition coefficients, the PBDEs are expected to distribute into the fat. There are no data in the scientific literature comparing uptake from oral, pulmonary, and dermal routes after PBDE exposure. By analogy to other persistent organic pollutants (POPs), it is likely that relative absorption may decrease at high concentrations; that the higher the degree of bromination, the poorer the absorption; that oral absorption will be similar to pulmonary absorption; and that dermal absorption will be quite limited.
A single disposition study of BDE 47 was conducted by Orn and Klasson-Wehler (1998), who examined the terminal tissue concentrations in both rats and mice after administration of an oral dose. The distribution and excretion were surprisingly different between rats and mice, as 14% and 20% were excreted in the feces, and <0.5% and 33% via the urine, respectively, after 5 days. Of the remaining dose, BDE 47 distributed preferentially to adipose tissue, followed by liver, lung, kidney, and brain. While the focus of the current study is not on metabolism, Orn and Klasson-Wehler (1998) performed extensive analyses of tissues and excreta for the presence and identification of metabolites. Their results indicate that mice have a limited capacity to metabolize BDE 47. The percent of metabolites found increased with time; however, in all tissues at all time points, the majority of chemical was parent compound.
The objective of this three-part study was to determine basic absorption, distribution, and excretion parameters of BDE 47 following a single dose with respect to dose, time, and route of exposure. Basic toxicokinetic characteristics are reported in this article; further analyses of these and other data will be included in the development of a physiologically based pharmacokinetic (PBPK) model. Because this congener has been implicated as a developmental neurotoxicant, reproductive toxicant, enzyme inducer, and endocrine disruptor (Eriksson et al., 2001; Hallgren et al., 2001; Stoker et al., 2004), these studies were conducted in female mice.
MATERIALS AND METHODS
Chemicals. 14C-2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) (26.7 mCi/mmol) was donated by Great Lakes Chemical Corporation (Indianapolis, IN) with a purity of >97% as determined by reverse-phase high-performance liquid chromatography (HPLC) (System Gold, Beckman Instruments, Inc., Fullerton, CA) using an Ultrasphere ODS column (5 μm, 25 x 4.6 cm, Beckman Instruments, Inc., Fullerton, CA) and a gradient elution of 50:50 methanol:water over 30 min to 100% methanol at a flow rate of 1.5 ml/min. A radioactive flow detector (Beckman Model 171, Beckman Instruments), with 1 ml/min Flo Scint III (Packard Instrument Co., Meriden, CT) was used to monitor radioactivity. Unlabeled BDE 47 was also provided by Great Lakes Chemical (>98% purity). All other chemicals used were of the highest grade commercially available.
Dosing solutions. Doses were selected based on environmental relevance, human tissue concentrations, and various toxicity studies (Birnbaum and Staskal, 2004; de Wit, 2002; Schecter et al., 2003). A stock solution of 14C-BDE 47 was made by sonicating 63.6 mg of 14C-BDE 47 (55 μCi/mg) in toluene (1 ml) until dissolved. Aliquots were used directly from this solution for all dosing regimens. All solutions were subjected to pre-dosing and post-dosing radioactivity examination to ensure proper delivered dose. All solutions were designed to deliver <2μCi to each mouse; cold BDE 47 was added to the 14C-BDE 47 to achieve desired mass.
Oral (po). Unlabeled BDE 47 was added directly to the dosing solution vial and dissolved in acetone. Corn oil was then added to the vials by weight, followed by the addition of labeled BDE 47. Volatile solvents were evaporated under vacuum (Speed Vac, Savant Instruments, Inc. Farmingdale, NY).
Intravenous (iv) and Intraperitoneal (ip). 10 μl of stock 14C-BDE 47 was allowed to evaporate in an amber vial, and the BDE 47 was resuspended in 95% ethanol followed by Emulphor. Water was slowly added to a final volume of 2.55 ml with an ethanol: Emulphor:water ratio of 1:1:8.
Dermal. Solution was prepared fresh immediately prior to dosing: 10 μl of stock 14C-BDE 47 in toluene was air-dried and resuspended in 0.64 ml acetone.
Intratracheal (itr). 10 μl of stock 14C-BDE 47 in toluene was air-dried and resuspended in 1.59 ml Hanks balanced salt solution and sonicated for 3 h immediately prior to dosing.
Animals. Female C57BL/6J mice were obtained from Charles River Breeding Laboratories (Raleigh, NC) and Jackson Laboratories (Bar Harbor, ME). Animals were maintained on a 12 h light/dark cycle at ambient temperature (22°C) and relative humidity (55 ± 5%), and were provided with Purina 5001 Rodent Chow (Ralston Purina Co., St. Louis, MO) and tap water ad libitum. Prior to the commencement of the study, mice were adapted (3 mice/cage) for 1 week in Nalge metabolism cages (Nalgene, Rochester, NY). Mice were then randomly assigned to treatment groups (n = 4 or 6) and housed individually for the remainder of the study. All mice were 100 days old at time of treatment.
Treatment. The treatment groups included intratracheal instillation, oral by gavage, intravenous injection, intraperitoneal injection, and dermal. Because intravenous dosing eliminates the absorption process, i.v. administration serves as a standard for evaluating the absorption and bioavailability of a compound when compared to other routes. In this study, itr via involuntary aspiration was used as a surrogate for inhalation exposure. All animals
Intratracheal treatment (n = 6). Mice were given a single dose of 14C-BDE 47 via involuntary aspiration. Full methodology is described by Ward et al. (1998). Briefly, mice were anaesthetized with isoflurane and suspended by their incisors on a vertical support by a wire loop. The esophagus was closed by gently pulling the tongue around the teeth. Dosing solution was injected into the oropharynx and the mouse's nose was covered, forcing inspiration of the solution.
Oral treatment (n = 6). A single dose (0.0, 0.1, 1.0, 10, or 100 mg/kg) was administered directly by oral gavage into the stomach of each mouse using a curved ball-tipped animal feeding needle.
Intravenous treatment (n = 4). A single dose (1 mg/kg) was administered intravenously via the tail vein at a dosing volume of 4 ml/kg.
Intraperitoneal treatment (n = 4). A single dose (1 mg/kg) was administered into the abdominal cavity at a dosing volume of 4 ml/kg.
Dermal treatment (n = 4). Mice were administered a single dose of 14C-BDE 47 using methods previously described (Banks and Birnbaum, 1991). Briefly, hair was removed from the circular application site (2 cm2) the day prior to dosing. Mice were lightly anesthetized using ketamine/xylazine (95 mg/kg/5 mg/kg b.w., final concentration 20 mg/ml i.p.). The dermal solution (1 mg/kg) was applied to the center of the site and the vehicle was allowed to volatilize. The application site was immediately covered with a stainless-steel perforated cap (Lippshaw, Detroit, MI) and attached to the skin with cyanolacrylate adhesive (Krazy Glue, Itasca, IL) to prevent ingestion. After euthanasia, the cap was removed and swabbed three times with alcohol swabs. The application site was swabbed three times with non-antibacterial soap solution and collected separately from other tissues for determination of radioactivity remaining at the site of application in the epidermis 5 days after dosing.
Sample analysis. Radioactivity in the tissues was determined by combustion (Packard 306B Biological Oxidizer, Downers Grove, IL) of triplicate samples when available (100 mg/sample) followed by liquid scintillation spectrometry (LSS; Beckman Scintillation Counter, Beckman Instruments, Fullerton, CA). All data is reported using wet weight values. Feces were air dried following collection, weighed and analyzed for radioactivity by combustion and LSS. Daily urine volume was recorded, and then 100 μl alliquots (triplicate) were analyzed by direct addition into scintillant for radioactivity determination by LSS. Select urine samples were analyzed by HPLC for the presence of metabolites. Individual urine samples were combined with equal amounts of solvent (1:1 methanol:water) and filtered using a 0.2 μm Nylon membrane filter (Rainin Instrument Co, Woburn, MA). Filtrate was injected manually onto an Ultrasphere ODS column (5 μm, 25 x 4.6 cm, Beckman Instruments, Inc., Fullerton, CA). Initial mobile phase was 50% methanol and 50% water, gradient was run over 30 minutes up to 100% methanol.
EROD and PROD assays. Microsomal fractions were prepared from liver according to the method of DeVito et al. (1993). Protein concentrations were calculated photometrically using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. Activities of ethoxyresorufin O-deethylase (EROD), a marker of CYP1A1, and pentoxyresorufin O-deethylase (PROD), a marker for CYP2B, were determined using a spectrofluorimetrically based assay. All samples were run in triplicate as described by Abbott et al. (2003).
Data analysis. Body composition estimates for blood, fat, skin, and muscle were 8, 8, 12, and 35%, respectively (ILSI, 1994). In the route of exposure study, the ‘oral’ tissue disposition data refers to the mean of all data collected from the effect of dose and time phases in which a) animals were exposed orally with a 1 mg/kg dose, and b) 5 day time points were available. Intergroup comparisons were performed by a two-way ANOVA followed by Bonferroni post tests (Graphpad Prizm 4.0). Differences between treatment groups were considered significant when p < 0.05. All data are presented as mean ± standard deviation. PK Solutions 2.0: Noncompartmental Pharmacokinetics Data Analysis Software (Montrose, CA) was used for half life calculations.
RESULTS
The present study was carried out in three phases to examine the effects of dose, route of exposure, and time on the absorption, distribution, and excretion of BDE 47 in C57BL/6 J female mice.
Phase 1: Effect of Dose
Female C57BL/6 J mice were administered a single oral dose (0.0, 0.1, 1.0, 10, or 100 mg/kg) of [14C] BDE 47. Excreta were collected daily and tissue distribution analyzed five days after the administration of BDE 47. All tissues examined had measurable BDE 47 (Table 1) five days after dosing. Tissue distribution was primarily dictated by lipophilicity; adipose, skin and liver had the highest concentrations, followed by muscle, lung, kidney, blood, and brain. The majority of the BDE 47 remaining in the animal was found in the adipose tissue (8–14%). Approximately 1% of the dose was found in the liver and 2–3% in the skin and muscle. The brain, kidneys, and lungs had less than 0.05% of the administered BDE 47 in all dose groups five days after exposure.
Approximately 65–81% of the administered dose had been excreted in the urine and feces, collectively, by the fifth day (Figure 2). In the 0.1 and 1.0 mg/kg dose groups, 18% of the dose was excreted in the urine on the first day whereas 12% and 3% were excreted in the 10 and 100 mg/kg dose groups, respectively. By the end of the collection period (five days), up to 40% was excreted in the urine (0.1 mg/kg). Expressed as percent dose, the data demonstrate a dose dependency in excretion via this route of elimination as 38%, 33%, and 14% of the dose was cumulatively excreted in the urine in the 1.0, 10, and 100 mg/kg dose groups, respectively. Because of the large amounts of BDE 47 being unexpectedly excreted in the urine, samples were further analyzed for the presence of metabolites. HPLC analysis consistently revealed one major peak which corresponded to parent compound (data not shown).
Excretion of BDE 47 into feces was also rapid. The dose-dependency pattern observed in the urinary excretion was not observed in fecal elimination of BDE 47. The percent of the dose excreted in feces was relatively consistent between dose groups on all days. In all dose groups, approximately 28% was excreted on the first day (Figure 2), a portion of which was most likely unabsorbed material. Significantly less was excreted on the second day, averaging 6% of the dose across all dose groups. By day five, 34, 41, 48 and 51% cumulatively had been excreted in the feces, respectively.
Several studies have demonstrated that these chemicals have the potential to induce hepatic enzymes (Chen et al., 2001; Zhou et al., 2001). The ability of BDE 47 to induce CYP1A1 and CYP2B was examined (Figure 3). Specifically, some studies suggest that PBDEs have structure-activity relationships similar to dioxins and therefore may act through the Ah receptor (Chen et al. 2001). EROD was used as a specific substrate for CYP1A1, a marker for the AhR pathway. BDE 47 did not induce EROD at any dose. CYP2B induction was also measured, as it is an indicator for a PXR or CAR-activated mechanism. Induction of PROD, a CYP2B marker, was only observed at the highest dose (100 mg/kg).
Phase 2: Route of Exposure
From the results of the effect of dose study, a single dose was chosen (1 mg/kg) for the remaining studies based on its environmental relevance and because its behavior appeared to be in the linear range. Mice were given a single dose of [14C] BDE 47 (1 mg/kg) by intratracheal, oral, intravenous, intraperitoneal, or dermal administration and kept in metabolism cages for five days. BDE 47 was found in all tissues examined from all routes of administration at the time of collection. Tissue distribution (Table 1) was similar for all routes of exposure; the proportion of the dose reaching each tissue was dependent on lipid content of the tissue, which is consistent with Phase 1. Adipose had the highest concentration of BDE 47 (8–14% of the dose). Muscle, skin, and liver all contained 1–3% of the administered dose, whereas brain and kidney consistently had 0.05% of the administered BDE 47 five days following administration. Relative to other tissues, high concentrations of BDE 47 were observed in the lung of the itr-dosed animals, which can most likely be attributed to unabsorbed dose. Also, the concentrations in muscle and fat of the ip-dosed animals were high, which may be due to the sites of collection (abdominal fat and muscle) if the dose had not dispersed entirely.
Figure 4 compares the percent of dose in the urine and feces over five days. Excreta profiles are consistent between iv, oral, and itr routes of administration assuming that the difference in the percent dose in the feces on day one represents the unabsorbed materials. Nine percent of the dose was excreted in the feces of the iv-administered mice on the first day whereas 18–27% was excreted in the oral and itr-dosed animals. After five days, 38% of the dose was excreted in the feces following oral exposure, 30% following itr and ip, 17% following iv, and 30% following dermal exposure.
The amount of BDE 47 in the urine excreted on the first day is relatively consistent (17–20% of the dose) for the iv, oral and itr administrations indicating that BDE 47 was rapidly absorbed and excreted after exposure by these routes. Cumulatively, 32–40% of the dose was excreted in the urine following exposure through these routes in five days. The excretion pattern in the dermally-exposed mice demonstrates that BDE 47 has a slower uptake through this route. A significantly lower amount of BDE 47 was excreted in the urine on the first day (3%), yet 21% of the dose was found to be in the urine by day five. In contrast to the other routes, the peak urine concentrations were observed on the second and third days (6%) following dermal exposure.
Based on excretion patterns, comparison between the iv route and all other routes of exposure indicate that BDE 47 is well absorbed (>80%) orally and intratracheally. Approximately 62% of the BDE 47 administered through a dermal application had been absorbed after five days. While it was not included in the calculations for percent dose in the skin, approximately 15% of the administered dose remained at the site of application five days following application.
Phase 3: Effect of Time
Disposition and excretion were examined 1, 3, 8 hrs, 1, 2, 3, 7, 10, 14, and 21 days following a single, oral dose of [14C] BDE 47 (1 mg/kg). BDE 47 was found in all tissues at all time points measured (Table 1). None of the tissues examined appear to have a selective retention or sequestration of BDE 47. Twenty-one days after exposure, approximately 80% of the administered dose was accounted for in the excreta, 5% in the cage wash, with the remaining radioactivity primarily distributed in the adipose tissue, skin, and muscle. Terminal tissue concentrations accounted for approximately 3% of the administered dose and averaged 121.5 ng/g in adipose, 29.0 ng/g in skin, and 7.1 ng/g in muscle. Although only a small amount remained, BDE 47 was detectable in brain, kidney, blood, and lung tissue 21 days following a single administration, with concentrations in these tissues of 2.0, 4.5, 4.6, and 10.8 ng/g, respectively.
Peak tissue concentrations were dependent on blood flow and partitioning. Following a single oral dose (1 mg/kg), rapidly perfuse tissues such as kidney, liver, and lung had peak concentrations three hours (the second sampling interval) following the administration of BDE 47 (306 ± 60, 1662 ± 219, 408 ± 92 ng/g, respectively). These peak tissue concentrations parallel the peak concentrations found in blood. The brain, which is a rapidly perfused tissue, peaked at eight hours (the third sampling interval) with a concentration of 104 ± 15 ng/g suggesting a diffusion limitation in this tissue. Muscle also peaked at eight hours (214 ± 21 ng/g). Both skin and fat maintained high concentrations on days 1–3, with the highest at day 2 (303 ± 147 and 1785 ± 696 ng/g). For the tissues with concentrations peaking at three hours, the elimination is more rapid from 8 hrs – 2 days as compared to the 7 – 21 day time points (Table 1).
The majority of the administered BDE 47 was rapidly excreted in the urine and feces (Figure 5). On the first day of collection, 15% of the oral dose was found in the urine and 20% in the feces. The trends between the two modes of excretion were very similar; however, the percent in urine is always greater than the feces except on day one. Following the large amount excreted on the first day by each route, the excretion is approximately 5–7% on the second day, 3–4% on the third day, 2–3% on the fourth day and fifth day, and 1–2% on days six and seven. After this time, the percent excreted decreases by small amounts days 8–21. At the end of the collection period, 37% of the dose had been excreted in the feces and 42% in the urine.
The data generated in this phase of the study provided data for initial calculations on whole-body and tissue specific half lives of BDE 47 (Table 2). This congener was eliminated in a biphasic pattern; during the first phase of elimination (), the majority of the BDE 47 is eliminated from the body (67%), leaving only a small percentage of the dose to be eliminated during the second, terminal phase (). For the whole body, it was assumed that all of the BDE 47 was absorbed and in the distribution process by the end of day one. Days 2–6 were considered the initial elimination phase (t1/2). Only 33% of the dose remained to be eliminated during the terminal elimination phase (t1/2), which was considered to be days 7–21 in this study. Tissue half lives were based on peak tissue concentrations; the absorption and distribution phase was assumed to be the time period prior to the peak tissue concentration. All time points following the peak concentration were used in the calculations for t1/2 and t1/2.
Data from this study suggest that the initial whole-body t1/2 in female mice following a single, oral dose in mice is 1.5 days. By the end of the first phase of elimination, 67% of the dose was excreted. By the seventh day, the excretion rate becomes slower, producing a terminal half life (t1/2) of approximately 23 days. Elimination rates from specific tissues are presented in Table 2. The t1/2 in blood is approximately one day and the terminal t1/2 appears to be 13 days. The study design does not support the determination of terminal half life calculations from skin and fat as the duration of the study was too short and too few data were collected at the later time points; however, t1/2 values appear to be six and five days, respectively.
DISCUSSION
The presence and rapid increase in environmental and human concentrations of polybrominated diphenyl ethers has heightened interest in toxicological consequences of these chemicals. Because some studies have shown that both the chemical mixtures and the individual congeners have the potential to be toxic, it is essential to understand the factors involved in human health risk calculations. BDE 47 accounts for a very small proportion of global production and usage, yet is consistently the dominant congener found in human and wildlife tissue. Furthermore, there is a large variation in observed human body burden concentrations (McDonald, 2004) which are not explained by high levels of exposure. The literature does not describe the reasons for the dominating presence of BDE 47 or the small portion of the population with particularly high levels of BDE 47 in relation to the general population. Some hypotheses include breakdown of higher-brominated congeners, selective uptake or retention of BDE 47, intraspecies differences in elimination, and differential metabolism of other PBDEs to BDE 47.
No human data is currently available regarding the kinetics of BDE 47 either as an individual congener or in the commercial mixture.
The present paper was focused on determining toxicokinetic parameters related to a single dose of BDE 47 in female mice. In this three part study, the effects of dose, route of exposure, and time on the distribution and excretion of BDE 47 were examined. This study design allows for characterization of a number of parameters essential for extrapolation: relative absorption following various routes of exposure, half life calculations, potential changes of disposition as a result of a high dose exposure, as well as data needed to establish parameters of linearity.
As predicted, BDE 47 was well absorbed and tissue distribution and dosimetry were dictated by lipophilicity. In the effect of dose phase, the concentrations found in the tissues five days after exposure were linear with respect to dose in almost all cases. Only the adipose tissue exhibits significant non-linear distribution; the 0.1 and 1.0 dose groups have approximately 9% of the BDE 47 contained in the adipose tissue whereas the 10 and 100 mg/kg dose groups have 14%. These results demonstrate a lack of tissue-specific sequestration as seen with dioxin and dioxin-like chemicals.
Tissue distribution trends in the effect of route phase were similar between all routes examined. Slightly more BDE 47 was found in the adipose and muscle of the ip-dosed animals. The high concentrations are most likely an effect of the slow and unequal distribution following an ip dosing regimen as abdominal fat and abdominal muscle were collected for analysis. High concentrations of BDE 47 in the lung following itr exposure are also likely due to the dosing regimen; the difference in the BDE 47 lung concentrations from itr and iv exposure suggest that a small portion of the dose still remained (unabsorbed and/or out of solution) in the lungs five days following exposure.
Despite small variations in the disposition of BDE 47, the results of this study have shown that this PBDE congener is well absorbed. Calculations based on the percent dose excreted in the feces on day one from each route of exposure as compared to iv administration (100% absorption) demonstrate the oral absorption was approximately 82%, 91% following intratracheal exposure, and 62% following dermal exposure. In general, a higher percentage of the dose is found in all tissues following i.v. exposure when compared to other routes of exposure. However, when differential absorption rates are considered, the data normalize to the levels found in the i.v. exposure. These results are of interest when correlated to recent studies reporting high levels of PBDEs in food, air samples, and house dust (Butt et al., 2004; Schecter et al., in press; Stapleton et al., 2004; Wilford et al., 2004). Dietary intake is the dominating source of exposure to several other halogenated environmental contaminants; while it is most likely a major source of intake of PBDEs, the results of this study support the theory that inhalation and dermal contact may also play a role in determining PBDE exposure.
The time course analyses of BDE 47 tissue concentrations more fully characterize the disposition patterns between tissues. The distribution of BDE 47 to the kidneys, liver, and brain is governed by a flow limited process, whereas adipose, muscle, and skin distribution is dependent on a diffusion limited process. Peak tissue concentrations are dependent on absorption and blood flow; highly perfused tissues achieved peak BDE 47 concentrations 3 hours after exposure. In other tissues, such as adipose and skin, peak concentrations were not seen until 2–3 days following the single exposure.
BDE 47 elimination follows a trend similar to the distribution; the chemical is eliminated quickly from the flow-limited tissues, and more slowly from the more lipophilic, diffusion-limited tissues. In addition, tissue and whole-body elimination of BDE 47 appears to be biphasic. A large majority of the chemical is excreted within the first few days of exposure, after which a slower elimination rate ensues. Although the whole body t1/2 is only 1.5 days, the terminal half life is much longer (23 days), which is indicative of the potential for bioaccumulation.
The half lives found in this study can be compared to calculations from other studies examining other species. Geyer et al. (2004) estimated that BDE 47 has a terminal half life of 664 days in humans using daily intake and total-body burden data. In rats, this same group of investigators reports an estimated whole body terminal half life of 31 days in rats and 1795 days in humans when using data from rat half life data. Looking just at adipose tissue, they calculated a 28-day terminal half life in rats. Although the current study was not extended past 21 days, the mice data suggest a t1/2 in adipose tissue of approximately 30–40 days.
The short initial half life (t1/2) of BDE 47 in mice is partly due to its rapid excretion in urine. Cumulatively, 40% of the dose was excreted in the urine over five days in the 0.1 mg/kg group; 38%, 33%, and 14% of the dose were excreted in the other dose groups (1.0, 10, 100 mg/kg respectively). This was a surprising result considering the high molecular weight and lipid solubility of BDE 47. The mass amount of chemical excreted in the urine would not be as surprising if it were metabolized; however, the majority of BDE 47 excreted in urine and feces of mice appears to be parent compound. This result is in agreement with the studies of Orn and Klasson-Wehler (1998).
In summary, the congener profiles found in humans do not match those of commercial PBDE mixtures. The overwhelming presence of BDE 47 is consistent in almost all biota; it is not clear whether the variability in human body burden is due to differences in exposure or pharmacokinetics. Recent studies have shown BDE 47 to be slowly eliminated in rats (Hakk et al., 2002; Orn and Klasson-Wehler, 1998) where mice undergo a much more rapid excretion of this congener. Basic renal clearance calculations from data generated in this study suggest the presence of active renal elimination; a hypothesis which is currently being investigated in our laboratory. If the rapid excretion of unmetabolized BDE 47 in urine and feces is due to an active transport mechanism, this may play a major role in understanding the species differences in elimination and further contribute to our understanding of the congener profiles found in humans. While we do not discount that there is most likely a difference in the metabolic capabilities between the species, the dominating presence of parent compound in the urine is suggestive of active transport. This data would support the hypothesis that pharmacokinetics, in addition to differential exposure, play a role in observed congener profiles; thus the species difference between rats and mice in elimination of BDE 47 raises the question of which species is a better model for human risk assessment.
ACKNOWLEDGMENTS
Frances McQuaid, Brenda Edwards, David Ross, Vicki Richardson, Steve Godin, Mike Hughes, and Marsha Ward deserve special recognition for their assistance in these studies. Partial funding was provided by the NHEERL-DESE Training in Environmental Sciences Research, EPA CT 826513. The information in this document has been subjected to review by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The research presented in this document was funded in part by the U.S. Environmental Protection Agency.
NOTES
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US EPA, ORD, NHEERL, ETD, US EPA, MD B143–01, Research Triangle Park, NC 27711
ABSTRACT
2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) is present in commercial mixtures of polybrominated diphenyl ethers (PBDEs), which are used as flame retardants in a wide variety of consumer products. Despite its small contribution to PBDE global production and usage, BDE 47 is the major congener found in environmental samples and human tissue. No human data are currently available regarding the toxicokinetics of BDE 47 either as an individual congener or in the commercial mixture. Because previous studies have suggested potential toxicokinetic differences between rodent species, this study was conducted in an effort to fully characterize absorption, distribution, and excretion parameters following a single dose with respect to dose, time, and route of exposure in female C57BL/6 mice. Over 80% of the administered dose was absorbed after oral or intratracheal administration, whereas 62% was absorbed when the dose was applied dermally. Disposition was dictated by lipophilicity as adipose and skin were major depot tissues. BDE 47 was rapidly excreted in the urine and feces. Of particular interest was the amount of parent compound found in the urine, which was a major factor in determining an initial whole-body half life of 1.5 days after a single oral exposure. Elimination, both whole-body and from individual tissues, was biphasic. Initial half-lives were 1–3 days, whereas terminal half-lives were much longer, suggesting the potential for bioaccumulation. This toxicokinetic behavior has important implications for extrapolation of toxicological studies to the assessment of health risk in humans.
Key Words: BFRs; PBDEs; BDE 47; toxicokinetics.
INTRODUCTION
Worldwide regulations require that flame retardants be added to a wide variety of consumer goods in an effort to reduce flammability. Generally, flame retardants are divided into classes which include halogenated organic, phosphorus-containing, nitrogen-containing, and inorganic flame retardants. Brominated flame retardants (BFRs) are the largest market group because of their low cost and high performance efficiency. Over 75 BFRs are recognized commercially; however, five BFRs constitute the overwhelming majority of BFR production: tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), decabromodiphenyl ether (DecaBDE), octabromodiphenyl ether (OctaBDE), and pentabromodiphenyl ether (PentaBDE). Recently, concern for PBDEs has risen after their detection in the environment and in humans at steadily increasing concentrations (Betts, 2002; Birnbaum and Staskal, 2004; de Wit, 2002; Law et al., 2003). The polybrominated diphenyl ethers potentially involve 209 different congeners, varying in both number and position of bromination. Each commercial product is a mixture of brominated diphenyl ether congeners.
The commercial pentaBDE product, commonly known as DE-71, is composed of 50–60% pentaBDEs, 24–38% tetraBDEs, and 4–8% hexaBDEs (World Health Organization, 1994). The commercial pentaBDE product is used in a number of highly flammable consumer applications including flexible polyurethane foam often found in upholstered furniture, mattresses, and bedding and carpet underlay (BSEF 2004). 2,2',4,4'-tetrabromodiphenyl ether (BDE 47, Fig. 1) is a specific PBDE congener found only in the pentaBDE commercial mixture. Despite its small contribution to PBDE global production and usage, BDE 47 is the major congener found in environmental samples and human tissue (Birnbaum and Staskal, 2004; de Wit, 2002; Hale et al., 2001; Law et al., 2003; Petreas et al., 2003; Schecter et al., 2003).
Concentrations of PBDEs in breast milk and serum from U.S. women are the highest reported in humans and are often tens to hundreds of times higher than human tissue samples from European countries. Petreas et al. (2003) measured BDE 47 in serum and adipose from contemporary California women in the late 1990s; concentrations of this congener ranged from 5 to 510 ng/g lipid (median 16.5 ng/g). Comparative serum samples from women residing in the same area in the 1960s revealed no detectable BDE 47. More recently, Schecter et al. (2003) reported high concentrations of PBDEs in U.S. breast milk collected in 2002. BDE 47 specifically had a range of 3 to 272 ng/g lipid with a median value of 18.4 (median PBDEs 34 ng/g). A recent review by Hites et al. (2004) highlights the ratio of BDE 47 as compared to the sum of all other PBDEs measured: BDE 47 accounts for approximately 55% of the total PBDE body burden despite the date or location of sampling.
Results from multiple studies make it clear that PBDEs are ubiquitous pollutants with increasing concentrations in people in North America; however, the mechanism(s) by which humans are exposed is not clear. Several researchers have found high concentrations of PBDEs in outdoor, indoor, and occupational air sampling (Butt et al., 2004; Jakobsson et al., 2002; Sinkkonen et al., 2003; Sjodin et al., 1999) as well as in house dust samples (Knoth et al., 2002; Stapleton et al., 2004). Correlations with other persistent, bioaccumulative, and toxic chemicals suggest that the primary pathway of exposure is through the diet (Bocio et al., 2003; Ohta et al., 2002). Recently, multiple investigators have reported that PBDEs are present at high levels in meat, fish, and dairy products (Huwe, 2004; Schecter et al., 2004). Because of these recent findings of high levels of PBDEs in the human diet, air, and house dust, it is essential to understand the relevance of each potential route of exposure.
In a human health risk assessment, the dose/response relationship of a chemical must be well characterized in order to accurately describe the associated risk. One of the major components of this is establishing a basic knowledge of absorption, distribution, metabolism, and excretion (ADME) of BDE 47. At present, studies on the toxicokinetics of PBDEs are very limited. Because of high octanol/water partition coefficients, the PBDEs are expected to distribute into the fat. There are no data in the scientific literature comparing uptake from oral, pulmonary, and dermal routes after PBDE exposure. By analogy to other persistent organic pollutants (POPs), it is likely that relative absorption may decrease at high concentrations; that the higher the degree of bromination, the poorer the absorption; that oral absorption will be similar to pulmonary absorption; and that dermal absorption will be quite limited.
A single disposition study of BDE 47 was conducted by Orn and Klasson-Wehler (1998), who examined the terminal tissue concentrations in both rats and mice after administration of an oral dose. The distribution and excretion were surprisingly different between rats and mice, as 14% and 20% were excreted in the feces, and <0.5% and 33% via the urine, respectively, after 5 days. Of the remaining dose, BDE 47 distributed preferentially to adipose tissue, followed by liver, lung, kidney, and brain. While the focus of the current study is not on metabolism, Orn and Klasson-Wehler (1998) performed extensive analyses of tissues and excreta for the presence and identification of metabolites. Their results indicate that mice have a limited capacity to metabolize BDE 47. The percent of metabolites found increased with time; however, in all tissues at all time points, the majority of chemical was parent compound.
The objective of this three-part study was to determine basic absorption, distribution, and excretion parameters of BDE 47 following a single dose with respect to dose, time, and route of exposure. Basic toxicokinetic characteristics are reported in this article; further analyses of these and other data will be included in the development of a physiologically based pharmacokinetic (PBPK) model. Because this congener has been implicated as a developmental neurotoxicant, reproductive toxicant, enzyme inducer, and endocrine disruptor (Eriksson et al., 2001; Hallgren et al., 2001; Stoker et al., 2004), these studies were conducted in female mice.
MATERIALS AND METHODS
Chemicals. 14C-2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) (26.7 mCi/mmol) was donated by Great Lakes Chemical Corporation (Indianapolis, IN) with a purity of >97% as determined by reverse-phase high-performance liquid chromatography (HPLC) (System Gold, Beckman Instruments, Inc., Fullerton, CA) using an Ultrasphere ODS column (5 μm, 25 x 4.6 cm, Beckman Instruments, Inc., Fullerton, CA) and a gradient elution of 50:50 methanol:water over 30 min to 100% methanol at a flow rate of 1.5 ml/min. A radioactive flow detector (Beckman Model 171, Beckman Instruments), with 1 ml/min Flo Scint III (Packard Instrument Co., Meriden, CT) was used to monitor radioactivity. Unlabeled BDE 47 was also provided by Great Lakes Chemical (>98% purity). All other chemicals used were of the highest grade commercially available.
Dosing solutions. Doses were selected based on environmental relevance, human tissue concentrations, and various toxicity studies (Birnbaum and Staskal, 2004; de Wit, 2002; Schecter et al., 2003). A stock solution of 14C-BDE 47 was made by sonicating 63.6 mg of 14C-BDE 47 (55 μCi/mg) in toluene (1 ml) until dissolved. Aliquots were used directly from this solution for all dosing regimens. All solutions were subjected to pre-dosing and post-dosing radioactivity examination to ensure proper delivered dose. All solutions were designed to deliver <2μCi to each mouse; cold BDE 47 was added to the 14C-BDE 47 to achieve desired mass.
Oral (po). Unlabeled BDE 47 was added directly to the dosing solution vial and dissolved in acetone. Corn oil was then added to the vials by weight, followed by the addition of labeled BDE 47. Volatile solvents were evaporated under vacuum (Speed Vac, Savant Instruments, Inc. Farmingdale, NY).
Intravenous (iv) and Intraperitoneal (ip). 10 μl of stock 14C-BDE 47 was allowed to evaporate in an amber vial, and the BDE 47 was resuspended in 95% ethanol followed by Emulphor. Water was slowly added to a final volume of 2.55 ml with an ethanol: Emulphor:water ratio of 1:1:8.
Dermal. Solution was prepared fresh immediately prior to dosing: 10 μl of stock 14C-BDE 47 in toluene was air-dried and resuspended in 0.64 ml acetone.
Intratracheal (itr). 10 μl of stock 14C-BDE 47 in toluene was air-dried and resuspended in 1.59 ml Hanks balanced salt solution and sonicated for 3 h immediately prior to dosing.
Animals. Female C57BL/6J mice were obtained from Charles River Breeding Laboratories (Raleigh, NC) and Jackson Laboratories (Bar Harbor, ME). Animals were maintained on a 12 h light/dark cycle at ambient temperature (22°C) and relative humidity (55 ± 5%), and were provided with Purina 5001 Rodent Chow (Ralston Purina Co., St. Louis, MO) and tap water ad libitum. Prior to the commencement of the study, mice were adapted (3 mice/cage) for 1 week in Nalge metabolism cages (Nalgene, Rochester, NY). Mice were then randomly assigned to treatment groups (n = 4 or 6) and housed individually for the remainder of the study. All mice were 100 days old at time of treatment.
Treatment. The treatment groups included intratracheal instillation, oral by gavage, intravenous injection, intraperitoneal injection, and dermal. Because intravenous dosing eliminates the absorption process, i.v. administration serves as a standard for evaluating the absorption and bioavailability of a compound when compared to other routes. In this study, itr via involuntary aspiration was used as a surrogate for inhalation exposure. All animals
Intratracheal treatment (n = 6). Mice were given a single dose of 14C-BDE 47 via involuntary aspiration. Full methodology is described by Ward et al. (1998). Briefly, mice were anaesthetized with isoflurane and suspended by their incisors on a vertical support by a wire loop. The esophagus was closed by gently pulling the tongue around the teeth. Dosing solution was injected into the oropharynx and the mouse's nose was covered, forcing inspiration of the solution.
Oral treatment (n = 6). A single dose (0.0, 0.1, 1.0, 10, or 100 mg/kg) was administered directly by oral gavage into the stomach of each mouse using a curved ball-tipped animal feeding needle.
Intravenous treatment (n = 4). A single dose (1 mg/kg) was administered intravenously via the tail vein at a dosing volume of 4 ml/kg.
Intraperitoneal treatment (n = 4). A single dose (1 mg/kg) was administered into the abdominal cavity at a dosing volume of 4 ml/kg.
Dermal treatment (n = 4). Mice were administered a single dose of 14C-BDE 47 using methods previously described (Banks and Birnbaum, 1991). Briefly, hair was removed from the circular application site (2 cm2) the day prior to dosing. Mice were lightly anesthetized using ketamine/xylazine (95 mg/kg/5 mg/kg b.w., final concentration 20 mg/ml i.p.). The dermal solution (1 mg/kg) was applied to the center of the site and the vehicle was allowed to volatilize. The application site was immediately covered with a stainless-steel perforated cap (Lippshaw, Detroit, MI) and attached to the skin with cyanolacrylate adhesive (Krazy Glue, Itasca, IL) to prevent ingestion. After euthanasia, the cap was removed and swabbed three times with alcohol swabs. The application site was swabbed three times with non-antibacterial soap solution and collected separately from other tissues for determination of radioactivity remaining at the site of application in the epidermis 5 days after dosing.
Sample analysis. Radioactivity in the tissues was determined by combustion (Packard 306B Biological Oxidizer, Downers Grove, IL) of triplicate samples when available (100 mg/sample) followed by liquid scintillation spectrometry (LSS; Beckman Scintillation Counter, Beckman Instruments, Fullerton, CA). All data is reported using wet weight values. Feces were air dried following collection, weighed and analyzed for radioactivity by combustion and LSS. Daily urine volume was recorded, and then 100 μl alliquots (triplicate) were analyzed by direct addition into scintillant for radioactivity determination by LSS. Select urine samples were analyzed by HPLC for the presence of metabolites. Individual urine samples were combined with equal amounts of solvent (1:1 methanol:water) and filtered using a 0.2 μm Nylon membrane filter (Rainin Instrument Co, Woburn, MA). Filtrate was injected manually onto an Ultrasphere ODS column (5 μm, 25 x 4.6 cm, Beckman Instruments, Inc., Fullerton, CA). Initial mobile phase was 50% methanol and 50% water, gradient was run over 30 minutes up to 100% methanol.
EROD and PROD assays. Microsomal fractions were prepared from liver according to the method of DeVito et al. (1993). Protein concentrations were calculated photometrically using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. Activities of ethoxyresorufin O-deethylase (EROD), a marker of CYP1A1, and pentoxyresorufin O-deethylase (PROD), a marker for CYP2B, were determined using a spectrofluorimetrically based assay. All samples were run in triplicate as described by Abbott et al. (2003).
Data analysis. Body composition estimates for blood, fat, skin, and muscle were 8, 8, 12, and 35%, respectively (ILSI, 1994). In the route of exposure study, the ‘oral’ tissue disposition data refers to the mean of all data collected from the effect of dose and time phases in which a) animals were exposed orally with a 1 mg/kg dose, and b) 5 day time points were available. Intergroup comparisons were performed by a two-way ANOVA followed by Bonferroni post tests (Graphpad Prizm 4.0). Differences between treatment groups were considered significant when p < 0.05. All data are presented as mean ± standard deviation. PK Solutions 2.0: Noncompartmental Pharmacokinetics Data Analysis Software (Montrose, CA) was used for half life calculations.
RESULTS
The present study was carried out in three phases to examine the effects of dose, route of exposure, and time on the absorption, distribution, and excretion of BDE 47 in C57BL/6 J female mice.
Phase 1: Effect of Dose
Female C57BL/6 J mice were administered a single oral dose (0.0, 0.1, 1.0, 10, or 100 mg/kg) of [14C] BDE 47. Excreta were collected daily and tissue distribution analyzed five days after the administration of BDE 47. All tissues examined had measurable BDE 47 (Table 1) five days after dosing. Tissue distribution was primarily dictated by lipophilicity; adipose, skin and liver had the highest concentrations, followed by muscle, lung, kidney, blood, and brain. The majority of the BDE 47 remaining in the animal was found in the adipose tissue (8–14%). Approximately 1% of the dose was found in the liver and 2–3% in the skin and muscle. The brain, kidneys, and lungs had less than 0.05% of the administered BDE 47 in all dose groups five days after exposure.
Approximately 65–81% of the administered dose had been excreted in the urine and feces, collectively, by the fifth day (Figure 2). In the 0.1 and 1.0 mg/kg dose groups, 18% of the dose was excreted in the urine on the first day whereas 12% and 3% were excreted in the 10 and 100 mg/kg dose groups, respectively. By the end of the collection period (five days), up to 40% was excreted in the urine (0.1 mg/kg). Expressed as percent dose, the data demonstrate a dose dependency in excretion via this route of elimination as 38%, 33%, and 14% of the dose was cumulatively excreted in the urine in the 1.0, 10, and 100 mg/kg dose groups, respectively. Because of the large amounts of BDE 47 being unexpectedly excreted in the urine, samples were further analyzed for the presence of metabolites. HPLC analysis consistently revealed one major peak which corresponded to parent compound (data not shown).
Excretion of BDE 47 into feces was also rapid. The dose-dependency pattern observed in the urinary excretion was not observed in fecal elimination of BDE 47. The percent of the dose excreted in feces was relatively consistent between dose groups on all days. In all dose groups, approximately 28% was excreted on the first day (Figure 2), a portion of which was most likely unabsorbed material. Significantly less was excreted on the second day, averaging 6% of the dose across all dose groups. By day five, 34, 41, 48 and 51% cumulatively had been excreted in the feces, respectively.
Several studies have demonstrated that these chemicals have the potential to induce hepatic enzymes (Chen et al., 2001; Zhou et al., 2001). The ability of BDE 47 to induce CYP1A1 and CYP2B was examined (Figure 3). Specifically, some studies suggest that PBDEs have structure-activity relationships similar to dioxins and therefore may act through the Ah receptor (Chen et al. 2001). EROD was used as a specific substrate for CYP1A1, a marker for the AhR pathway. BDE 47 did not induce EROD at any dose. CYP2B induction was also measured, as it is an indicator for a PXR or CAR-activated mechanism. Induction of PROD, a CYP2B marker, was only observed at the highest dose (100 mg/kg).
Phase 2: Route of Exposure
From the results of the effect of dose study, a single dose was chosen (1 mg/kg) for the remaining studies based on its environmental relevance and because its behavior appeared to be in the linear range. Mice were given a single dose of [14C] BDE 47 (1 mg/kg) by intratracheal, oral, intravenous, intraperitoneal, or dermal administration and kept in metabolism cages for five days. BDE 47 was found in all tissues examined from all routes of administration at the time of collection. Tissue distribution (Table 1) was similar for all routes of exposure; the proportion of the dose reaching each tissue was dependent on lipid content of the tissue, which is consistent with Phase 1. Adipose had the highest concentration of BDE 47 (8–14% of the dose). Muscle, skin, and liver all contained 1–3% of the administered dose, whereas brain and kidney consistently had 0.05% of the administered BDE 47 five days following administration. Relative to other tissues, high concentrations of BDE 47 were observed in the lung of the itr-dosed animals, which can most likely be attributed to unabsorbed dose. Also, the concentrations in muscle and fat of the ip-dosed animals were high, which may be due to the sites of collection (abdominal fat and muscle) if the dose had not dispersed entirely.
Figure 4 compares the percent of dose in the urine and feces over five days. Excreta profiles are consistent between iv, oral, and itr routes of administration assuming that the difference in the percent dose in the feces on day one represents the unabsorbed materials. Nine percent of the dose was excreted in the feces of the iv-administered mice on the first day whereas 18–27% was excreted in the oral and itr-dosed animals. After five days, 38% of the dose was excreted in the feces following oral exposure, 30% following itr and ip, 17% following iv, and 30% following dermal exposure.
The amount of BDE 47 in the urine excreted on the first day is relatively consistent (17–20% of the dose) for the iv, oral and itr administrations indicating that BDE 47 was rapidly absorbed and excreted after exposure by these routes. Cumulatively, 32–40% of the dose was excreted in the urine following exposure through these routes in five days. The excretion pattern in the dermally-exposed mice demonstrates that BDE 47 has a slower uptake through this route. A significantly lower amount of BDE 47 was excreted in the urine on the first day (3%), yet 21% of the dose was found to be in the urine by day five. In contrast to the other routes, the peak urine concentrations were observed on the second and third days (6%) following dermal exposure.
Based on excretion patterns, comparison between the iv route and all other routes of exposure indicate that BDE 47 is well absorbed (>80%) orally and intratracheally. Approximately 62% of the BDE 47 administered through a dermal application had been absorbed after five days. While it was not included in the calculations for percent dose in the skin, approximately 15% of the administered dose remained at the site of application five days following application.
Phase 3: Effect of Time
Disposition and excretion were examined 1, 3, 8 hrs, 1, 2, 3, 7, 10, 14, and 21 days following a single, oral dose of [14C] BDE 47 (1 mg/kg). BDE 47 was found in all tissues at all time points measured (Table 1). None of the tissues examined appear to have a selective retention or sequestration of BDE 47. Twenty-one days after exposure, approximately 80% of the administered dose was accounted for in the excreta, 5% in the cage wash, with the remaining radioactivity primarily distributed in the adipose tissue, skin, and muscle. Terminal tissue concentrations accounted for approximately 3% of the administered dose and averaged 121.5 ng/g in adipose, 29.0 ng/g in skin, and 7.1 ng/g in muscle. Although only a small amount remained, BDE 47 was detectable in brain, kidney, blood, and lung tissue 21 days following a single administration, with concentrations in these tissues of 2.0, 4.5, 4.6, and 10.8 ng/g, respectively.
Peak tissue concentrations were dependent on blood flow and partitioning. Following a single oral dose (1 mg/kg), rapidly perfuse tissues such as kidney, liver, and lung had peak concentrations three hours (the second sampling interval) following the administration of BDE 47 (306 ± 60, 1662 ± 219, 408 ± 92 ng/g, respectively). These peak tissue concentrations parallel the peak concentrations found in blood. The brain, which is a rapidly perfused tissue, peaked at eight hours (the third sampling interval) with a concentration of 104 ± 15 ng/g suggesting a diffusion limitation in this tissue. Muscle also peaked at eight hours (214 ± 21 ng/g). Both skin and fat maintained high concentrations on days 1–3, with the highest at day 2 (303 ± 147 and 1785 ± 696 ng/g). For the tissues with concentrations peaking at three hours, the elimination is more rapid from 8 hrs – 2 days as compared to the 7 – 21 day time points (Table 1).
The majority of the administered BDE 47 was rapidly excreted in the urine and feces (Figure 5). On the first day of collection, 15% of the oral dose was found in the urine and 20% in the feces. The trends between the two modes of excretion were very similar; however, the percent in urine is always greater than the feces except on day one. Following the large amount excreted on the first day by each route, the excretion is approximately 5–7% on the second day, 3–4% on the third day, 2–3% on the fourth day and fifth day, and 1–2% on days six and seven. After this time, the percent excreted decreases by small amounts days 8–21. At the end of the collection period, 37% of the dose had been excreted in the feces and 42% in the urine.
The data generated in this phase of the study provided data for initial calculations on whole-body and tissue specific half lives of BDE 47 (Table 2). This congener was eliminated in a biphasic pattern; during the first phase of elimination (), the majority of the BDE 47 is eliminated from the body (67%), leaving only a small percentage of the dose to be eliminated during the second, terminal phase (). For the whole body, it was assumed that all of the BDE 47 was absorbed and in the distribution process by the end of day one. Days 2–6 were considered the initial elimination phase (t1/2). Only 33% of the dose remained to be eliminated during the terminal elimination phase (t1/2), which was considered to be days 7–21 in this study. Tissue half lives were based on peak tissue concentrations; the absorption and distribution phase was assumed to be the time period prior to the peak tissue concentration. All time points following the peak concentration were used in the calculations for t1/2 and t1/2.
Data from this study suggest that the initial whole-body t1/2 in female mice following a single, oral dose in mice is 1.5 days. By the end of the first phase of elimination, 67% of the dose was excreted. By the seventh day, the excretion rate becomes slower, producing a terminal half life (t1/2) of approximately 23 days. Elimination rates from specific tissues are presented in Table 2. The t1/2 in blood is approximately one day and the terminal t1/2 appears to be 13 days. The study design does not support the determination of terminal half life calculations from skin and fat as the duration of the study was too short and too few data were collected at the later time points; however, t1/2 values appear to be six and five days, respectively.
DISCUSSION
The presence and rapid increase in environmental and human concentrations of polybrominated diphenyl ethers has heightened interest in toxicological consequences of these chemicals. Because some studies have shown that both the chemical mixtures and the individual congeners have the potential to be toxic, it is essential to understand the factors involved in human health risk calculations. BDE 47 accounts for a very small proportion of global production and usage, yet is consistently the dominant congener found in human and wildlife tissue. Furthermore, there is a large variation in observed human body burden concentrations (McDonald, 2004) which are not explained by high levels of exposure. The literature does not describe the reasons for the dominating presence of BDE 47 or the small portion of the population with particularly high levels of BDE 47 in relation to the general population. Some hypotheses include breakdown of higher-brominated congeners, selective uptake or retention of BDE 47, intraspecies differences in elimination, and differential metabolism of other PBDEs to BDE 47.
No human data is currently available regarding the kinetics of BDE 47 either as an individual congener or in the commercial mixture.
The present paper was focused on determining toxicokinetic parameters related to a single dose of BDE 47 in female mice. In this three part study, the effects of dose, route of exposure, and time on the distribution and excretion of BDE 47 were examined. This study design allows for characterization of a number of parameters essential for extrapolation: relative absorption following various routes of exposure, half life calculations, potential changes of disposition as a result of a high dose exposure, as well as data needed to establish parameters of linearity.
As predicted, BDE 47 was well absorbed and tissue distribution and dosimetry were dictated by lipophilicity. In the effect of dose phase, the concentrations found in the tissues five days after exposure were linear with respect to dose in almost all cases. Only the adipose tissue exhibits significant non-linear distribution; the 0.1 and 1.0 dose groups have approximately 9% of the BDE 47 contained in the adipose tissue whereas the 10 and 100 mg/kg dose groups have 14%. These results demonstrate a lack of tissue-specific sequestration as seen with dioxin and dioxin-like chemicals.
Tissue distribution trends in the effect of route phase were similar between all routes examined. Slightly more BDE 47 was found in the adipose and muscle of the ip-dosed animals. The high concentrations are most likely an effect of the slow and unequal distribution following an ip dosing regimen as abdominal fat and abdominal muscle were collected for analysis. High concentrations of BDE 47 in the lung following itr exposure are also likely due to the dosing regimen; the difference in the BDE 47 lung concentrations from itr and iv exposure suggest that a small portion of the dose still remained (unabsorbed and/or out of solution) in the lungs five days following exposure.
Despite small variations in the disposition of BDE 47, the results of this study have shown that this PBDE congener is well absorbed. Calculations based on the percent dose excreted in the feces on day one from each route of exposure as compared to iv administration (100% absorption) demonstrate the oral absorption was approximately 82%, 91% following intratracheal exposure, and 62% following dermal exposure. In general, a higher percentage of the dose is found in all tissues following i.v. exposure when compared to other routes of exposure. However, when differential absorption rates are considered, the data normalize to the levels found in the i.v. exposure. These results are of interest when correlated to recent studies reporting high levels of PBDEs in food, air samples, and house dust (Butt et al., 2004; Schecter et al., in press; Stapleton et al., 2004; Wilford et al., 2004). Dietary intake is the dominating source of exposure to several other halogenated environmental contaminants; while it is most likely a major source of intake of PBDEs, the results of this study support the theory that inhalation and dermal contact may also play a role in determining PBDE exposure.
The time course analyses of BDE 47 tissue concentrations more fully characterize the disposition patterns between tissues. The distribution of BDE 47 to the kidneys, liver, and brain is governed by a flow limited process, whereas adipose, muscle, and skin distribution is dependent on a diffusion limited process. Peak tissue concentrations are dependent on absorption and blood flow; highly perfused tissues achieved peak BDE 47 concentrations 3 hours after exposure. In other tissues, such as adipose and skin, peak concentrations were not seen until 2–3 days following the single exposure.
BDE 47 elimination follows a trend similar to the distribution; the chemical is eliminated quickly from the flow-limited tissues, and more slowly from the more lipophilic, diffusion-limited tissues. In addition, tissue and whole-body elimination of BDE 47 appears to be biphasic. A large majority of the chemical is excreted within the first few days of exposure, after which a slower elimination rate ensues. Although the whole body t1/2 is only 1.5 days, the terminal half life is much longer (23 days), which is indicative of the potential for bioaccumulation.
The half lives found in this study can be compared to calculations from other studies examining other species. Geyer et al. (2004) estimated that BDE 47 has a terminal half life of 664 days in humans using daily intake and total-body burden data. In rats, this same group of investigators reports an estimated whole body terminal half life of 31 days in rats and 1795 days in humans when using data from rat half life data. Looking just at adipose tissue, they calculated a 28-day terminal half life in rats. Although the current study was not extended past 21 days, the mice data suggest a t1/2 in adipose tissue of approximately 30–40 days.
The short initial half life (t1/2) of BDE 47 in mice is partly due to its rapid excretion in urine. Cumulatively, 40% of the dose was excreted in the urine over five days in the 0.1 mg/kg group; 38%, 33%, and 14% of the dose were excreted in the other dose groups (1.0, 10, 100 mg/kg respectively). This was a surprising result considering the high molecular weight and lipid solubility of BDE 47. The mass amount of chemical excreted in the urine would not be as surprising if it were metabolized; however, the majority of BDE 47 excreted in urine and feces of mice appears to be parent compound. This result is in agreement with the studies of Orn and Klasson-Wehler (1998).
In summary, the congener profiles found in humans do not match those of commercial PBDE mixtures. The overwhelming presence of BDE 47 is consistent in almost all biota; it is not clear whether the variability in human body burden is due to differences in exposure or pharmacokinetics. Recent studies have shown BDE 47 to be slowly eliminated in rats (Hakk et al., 2002; Orn and Klasson-Wehler, 1998) where mice undergo a much more rapid excretion of this congener. Basic renal clearance calculations from data generated in this study suggest the presence of active renal elimination; a hypothesis which is currently being investigated in our laboratory. If the rapid excretion of unmetabolized BDE 47 in urine and feces is due to an active transport mechanism, this may play a major role in understanding the species differences in elimination and further contribute to our understanding of the congener profiles found in humans. While we do not discount that there is most likely a difference in the metabolic capabilities between the species, the dominating presence of parent compound in the urine is suggestive of active transport. This data would support the hypothesis that pharmacokinetics, in addition to differential exposure, play a role in observed congener profiles; thus the species difference between rats and mice in elimination of BDE 47 raises the question of which species is a better model for human risk assessment.
ACKNOWLEDGMENTS
Frances McQuaid, Brenda Edwards, David Ross, Vicki Richardson, Steve Godin, Mike Hughes, and Marsha Ward deserve special recognition for their assistance in these studies. Partial funding was provided by the NHEERL-DESE Training in Environmental Sciences Research, EPA CT 826513. The information in this document has been subjected to review by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The research presented in this document was funded in part by the U.S. Environmental Protection Agency.
NOTES
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