Grantee Research Project Results
2005 Progress Report: Pre-natal Exposures of Children to Polybrominated Diphenyl Ethers: The Collection of Animal and Human Data along with the Development and Validation of a PBPK Model
EPA Grant Number: R830756Title: Pre-natal Exposures of Children to Polybrominated Diphenyl Ethers: The Collection of Animal and Human Data along with the Development and Validation of a PBPK Model
Investigators: Raymer, James H. , Licata, Amy C. , Mathews, J. M. , Birnbaum, Linda , Studabaker, W.
Current Investigators: Raymer, James H. , Garner, C Edwin , Emond, C. , Birnbaum, Linda , Studabaker, W.
Institution: Desert Research Institute
Current Institution: RTI International
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2003 through December 31, 2006 (Extended to December 31, 2007)
Project Period Covered by this Report: January 1, 2005 through December 31, 2006
Project Amount: $749,654
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
The objectives of this research project are to: (1) develop a physiologically based pharmacokinetic (PBPK) animal model for the polybrominated diphenyl ethers (PBDEs) and 2,2',4,4'-tetrabromomodiphenyl ether (BDE-47) that can be used to estimate fetal exposures to PBDEs in humans and measure the parameters necessary to develop the model for PBDEs; and (2) develop, install, and apply analytical methods for PBDEs in human blood and meconium to samples collected during this project, both to estimate the utility of the model and to determine if chemical analysis of cord blood and meconium are appropriate media for measurement of cumulative exposures of newborn babies to PBDEs.
Specific hypotheses to be tested include:
- A rodent PBPK model for PBDEs can be scaled to be applicable to humans.
- The PBDE concentrations in cord blood and meconium from newborns are proportional.
- Mother’s blood concentrations of PBDEs are predictive of the cord blood and/or meconium concentrations in newborn babies.
- Meconium is a useful medium for assessing cumulative dose of the developing fetus.
Progress Summary:
A 1-year, no-cost extension was requested and approved. In addition, Dr. Linda Birnbaum became involved as a collaborator, therefore, the grant was converted to a cooperative agreement. Dr. Birnbaum provided mouse data to be used for initial model development, as described below.
Analytical Method
During the reporting period, method development work for the rat matrices was completed. Results and initial application of the method were presented at the International Society of Exposure Analysis meeting in October 2005. An overview of the method is presented below.
Sample analyses were performed using procedures developed and validated for the matrix type. Methods are generally based on existing RTI International procedures for the analysis of polychlorinated biphenyls in biological matrices. These methods include homogenization (tissues), hexane extraction, florisil cleanup (tissues), and analysis by gas chromatography-electron capture detector.
Spike-Recovery Studies. BDE-47 in methanol was spiked into blood samples immediately prior to extraction. For tissue samples, spikes were made directly into the tissue (~1 g samples) and allowed to permeate for 10 minutes. Tissues evaluated included liver, adipose, abdominal muscle, skin, brain, and fetuses. BDE-47 was spiked into tissues at 0, 2.5, 25, and 250 ng/mL. BDE-47 is ubiquitous in the laboratory environment. We observed a consistent background of 0.2 -2 ng/mL in laboratory method blanks that we were unable to eliminate despite screening of plastics, baking and triple hexane rinsing of glassware, and careful cleaning of work areas. BDE-47 recoveries are reported in Table 1. Results for different tissue matrices are pooled; for the most part they were similar except for adipose, which yielded low recoveries (20%-60%). Recoveries of surrogate from both matrices were more variable than for BDE-47 and strongly tissue dependent (blood, 98% ± 18%; adipose, 59% ± 19%; muscle, 113% ± 18%; fetuses, 82% ± 7%).
Table 1. Recoveries of BDE-47 Spiked Into Rat Blood and Rat Tissues
Matrix |
Spike Level |
n |
Mean Recovery |
% Recovery |
SD |
RSD (%) |
Blood |
0.1 ng/mL |
4 |
0.313 ng/mL |
313 |
0.113 |
36.1 |
2.5 |
10 |
3.04 |
122 |
0.160 |
5.3 |
|
25 |
6 |
27.5 |
110 |
1.61 |
5.9 |
|
250 |
8 |
255 |
102 |
23.1 |
9.1 |
|
Tissue (composite results) |
0 (Reagent) |
13 |
1.04 ng |
n/aa |
0.44 |
42.3 |
0 (Tissue) |
24 |
1.97 |
n/a |
0.99 |
50.3 |
|
2.5 ng |
30 |
3.80 |
152 |
1.04 |
27.4 |
|
25 |
17 |
25.6 |
102 |
4.06 |
15.9 |
|
250 |
15 |
246 |
98.4 |
32.5 |
13.2 |
|
a Not applicable |
Discussion. Although recovery of BDE-47 from spiked blood samples essentially is quantitative and reproducible, the persistence of a laboratory background confounds the analysis of samples containing low initial concentrations (<10 ng/mL). These observations are consistent with previously reported results for reagent controls. For most of the experimental samples from dosed animals that have been tested so far, actual BDE-47 levels are one or more orders of magnitude higher, and the effect of background may be diluted out of those samples. Background contamination continues to be a concern, however, for the analysis of human samples. Similarly, recoveries from a variety of spiked tissues are centered in the vicinity of quantitative. Matrix components persist in the extract even following Florisil cleanup, however, particularly for those derived from tissues high in fat. There is an increasing effect of matrix on baseline noise, an effect which frequently persists on dilution. As a result, the range of recoveries is much wider, with imprecision increasing to 15 percent + RSD. For both matrices, high concentrations of BDE-47 resulted in masking of the surrogate; dilution pushed it near or below the limits of quantitation.
Application of the Method to Samples
The developed method was applied to the analysis of BDE-47 in samples collected from the rat dosing studies. Extraction and analysis of blood and tissue samples is ongoing. Muscle samples were pooled from abdominal and leg muscle from each animal; all samples of muscle have been analyzed. Approximately one-third of the blood samples have been analyzed. Preliminary data for these two matrices are presented in Table 2.
Table 2. Preliminary Ranges for BDE-47 in Blood and Muscle (ng/g) Overall Dosing Regimens
|
n |
Minimum |
Mean |
Median |
Maximum |
Blanks |
21 |
0.00 |
0.72 |
0.00 |
6.50 |
Controls |
16 |
24.1 |
35.3 |
36.6 |
42.3 |
(Corrected recovery, %) |
(80.8%) |
(119%) |
(123%) |
(142%) |
|
Blood |
192 |
0.8 |
1833 |
1477 |
8049 |
Muscle |
37 |
12 |
3909 |
3945 |
10840 |
Representative blood data from a single animal are plotted in Figure 1. In this case, the animal received a single oral dose of 20 mg/kg. Blood concentrations of BDE-47 increase approximately linearly for the first 6 hours then increase more slowly to about the 48-hour mark.
Conclusions for Methods and Initial Application
The methods described yield BDE-47 recoveries of 90-110 percent from blood in the range of 25-250 g/mL and tissues in the range of 25 ng/g. Precision in blood samples (< 10% RSD) is consistent with previously reported results for reagent controls; precision in tissue samples is impacted by matrix interferences. A persistent low-level contamination will require resolution before analysis of human samples, especially with BDE-47 concentration less than 10 ng/mL.
Initial Development of PBPK Model
An objective of this project is to describe early PBPK model development based on data generated from the studies described. PBPK modeling efforts are an ongoing collaboration with Dr. Linda Birnbaum of the U.S. Environmental Protection Agency (EPA) and Dr. Danielle Staskal of Chem Risk (formerly of EPA). Tissue partition coefficients were estimated, and the overall model was constructed, based on data generated by Dr. Birnbaum’s laboratory. As indicated earlier, work with the mouse data will help in the generation of a model that can be used in the rats when data are available. Discussions among members of the team (primarily Garner, Birnbaum, and Staskal) led to its refinementinto the version discussed below.
Figure 1. Time Course of BDE-57 in Blood Following an Oral Dose
The model structure for BDE-47 was established based on analysis of the distribution of BDE-47 in C57BL6 mice. The model structure is based on a diffusion limited PBPK model developed by Wang, et al. (1997). It consists of seven compartments (blood, brain, kidney, skin, fat, liver, muscle, and gastrointestinal tract). An intravenous model was constructed initially (data not shown) based on data from Dr. Birnbaum’s laboratory; adult, female C57BL6 mice were given a single, intravenous dose (1 mg/kg) of [14C]BDE-47. Excretion was monitored daily, and tissue distribution was assessed on the fifth day following exposure. Model parameters initially fit to IV data then were applied to an oral absorption model. Model schematics presented here are based on the single oral dose tissue and excreta time course data. Briefly, adult, female C57BL6 mice were administered a singe, oral dose (1 mg/kg) of [14C] BDE-47. Disposition and excretion were monitored for 21 days following the exposure.
The model assumes that BDE-47 is delivered to the blood circulation via the lymphatic system and the portal circulation from the GI tract (Lakshmanan, et al., 1986; Roth, et al., 1993). The model furthers assumes enterohepatic circulation. Fraction of dose absorbed (absolute bioavailability) was limited to 0.8-0.95. First order kinetics were assumed for metabolism of BDE-47 in the liver as well as excretion of BDE-47 parent and metabolite into the bile. The excretion of BDE-47 into the urine was described as a first order process by inserting a kidney constant (KK) that accounts for all filtration, secretion, and absorption processes in this preliminary model. Further work to characterize the nature of mouse renal clearance will lead to refinement of this aspect of the model. Model simulations were conducted using Berkeley Madonna (v 8.0, 2001) software.
A conceptual representation of the PBPK model for BDE-47 in mice is shown in Figure 2. Tissue partitioning was examined using predictions from the model based on area under the curve (AUC) and compared actual tissue to blood ratios (Table 3). The preliminary model closely describes the experimental data with respect to tissue partitioning; BDE-47 has a high affinity for adipose and lipid-rich tissues as demonstrated by partition coefficients of approximately 70 into adipose tissue and approximately 10 into the liver. BDE-47 is excreted rapidly relative to xenobiotics of similar structure (Staskal, et al., 2005). The preliminary model presented here is able to predict urinary and fecal excretion reasonably well using first order absorption and elimination kinetics following a single oral administration (Figure 3). The current model, when refined and validated, offers a construct for exploring the biological determinants of BDE-47 disposition across dose, exposure conditions, and species. As additional toxicity data become available, this model also will aid in the characterization between dose and early biochemical responses involved in the hypothesized mode-of-action. Perhaps the greatest value of this model will be its ability to contribute to dose metric selection in the evaluation of acceptable exposure levels based on extrapolation across dose, route, and species in human health risk assessment.
Human Study
A subcontract with Duke University Medical Center, Maternal and Fetal Medicine, was established for the recruitment and sample collection from 25 pregnant women. The Institutional Review Board protocol, including questionnaires and consent forms, was prepared and approved by both RTI and Duke University Medical Center. Documentation of approval was supplied to EPA; EPA also approved the protocol.
Figure 2. Conceptual Model Representation for BDE-47 in Mice (Berkley Madonna, version 8.0)
Table 3. Tissue Blood Partition Ratios (TBR) for Mice Following a Single Oral Dose (1 mg/kg)
|
Liver |
Fat |
Muscle |
Skin |
Lung |
Kidney |
Brain |
AUC-based TBR |
9.3 |
70.1 |
4.0 |
8.9 |
5.4 |
2.0 |
0.7 |
Average TBRa |
11.6 |
70.4 |
4.2 |
8.9 |
6.4 |
2.3 |
0.8 |
a Based on individual time points |
(A) Urine |
(B) Feces |
Figure 3. Measured Versus Predicted (A) Urine and (B) Fecal Excretion of BDE-47 Following a 1 mg/kg Oral Administration of BDE-47 in Female Mice
Future Activities:
The remaining samples from the rat studies will be analyzed. The data will be subjected to quality assurance review. Model development will be completed and applied to the rat data, into which a pregnancy compartment is added. Participant recruitment via Duke will be initiated, and samples will be subjected to analysis. The model development will be completed, scaled to humans, and resulting predictions for human meconium compared to the BDE-47 concentrations measured.
References:
Lakshmanan MR, Campbell BS, Chirtel SLJ, Ekarhita N, Ezekiel M. Studies on the mechanism of absorption and distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Journal of Pharmacology and Experimental Therapeutics 1986;239:673-677.
Roth WL, Freeman RA, Wilson AGE. A physiologically based model for gastrointestinal absorption and excretion of chemical carried by lipids. Risk Analysis 1993;13:531-543.
Staskal DF, Diliberto JJ, DeVito MJ, Birnbaum LS. Toxicokinetics of BDE 47 in female mice: effect of dose, route of exposure, and time. Toxicological Sciences 2005;83:215-223.
Wang X, Santostefano MJ, Evans MV, Richardson VM, Diliberto JJ, Birnbaum LS. Determination of parameters responsible for pharmacokinetic behavior of TCDD in female Sprague-Dawley rats. Toxicology Applied Pharmacology 1997;147:151-168.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
sensitive populations, human health, mathematics, measurement methods, bioavailability, metabolism, vulnerability, infant,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Toxicology, Genetics, Health Risk Assessment, Risk Assessments, Environmental Microbiology, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Physical Processes, Children's Health, genetic susceptability, Risk Assessment, health effects, pharmacodynamic model, sensitive populations, biomarkers, age-related differences, PBDE, gene-environment interaction, exposure, developmental effects, children, pharmacokinetic models, toxicity, genetic polymorphisms, insecticides, human exposure, pharmacokinetc model, biological markers, risk based model, exposure assessment, polybrominated diphenyl ethers, biochemical research, environmental hazard exposures, toxicsRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.