Differential Expression of CYP1A, 2B, and 3A Genes in the F344 Rat following Exposure to a Polybrominated Diphenyl Ether Mixture or Individu
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《毒物学科学杂志》
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina 27709
Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695
Battelle, Columbus, Ohio 43201
ABSTRACT
Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have been detected in the environment and in mammalian tissues and fluids. Evidence indicates that PBDE mixtures induce CYPs through aryl hydrocarbon receptor (AhR)-dependent and -independent pathways. The present work has investigated the effects of individual components of a commercial PBDE mixture (DE71) on expression of CYP1A1, a biomarker for activation of the AhR (dioxin-like), and CYP2B and CYP3A, biomarkers for activation of the constitutive androstane and pregnanexreceptors (CAR and PXR), respectively, in the rat. Male F344 rats were dosed orally on three consecutive days with either DE71, PBDE components, 2,2',4,4'-tetraBDE (BDE47), 2,2',4,4',5-pentaBDE (BDE99), 2,2',4,4',5,5'-hexaBDE (BDE153), representative polybrominated dibenzofurans (PBDFs) present in DE71, or reference PCBs. Differential expression of target genes was determined in liver 24 h after the last dose. Quantitative PCR analysis indicated up-regulation of CYP1A1 by DE71; however, the response was weak compared to that for dioxin-like PCB126. Individual PBDE components of DE71 up-regulated CYP1A1 only at the highest administered dose (100 μmol/kg/day). Representative PBDFs efficiently up-regulated CYP1A1; therefore, they, along with other PBDFs and polybrominated dibenzodioxins detected in DE71 and individual PBDE components, may be responsible for most, if not all, dioxin-like properties previously observed for PBDEs. Conversely, PBDEs appear capable of up-regulating CYP2B and CYP3A in rats at doses similar to that for non-dioxin-like PCB153. These results indicate that in vivo PBDE-mediated toxicity would be better categorized by AhR-independent mechanisms, rather than the well-characterized AhR-dependent mechanism associated with exposure to dioxin-like chemicals.
Key Words: polybrominated diphenyl ethers; polybrominated dibenzodioxins and dibenzofurans; polychlorinated biphenyls; persistent organic compounds; gene expression; quantitative RT-PCR.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have accumulated in the environment and have been detected in humans (de Wit, 2002). PBDE concentrations in human adipose tissue range from ca. 1–500 ng/g lipid (McDonald, 2002). Human intake of PBDEs in Sweden has been estimated at 51 ng/day, but may be higher in the United States (Darnerud et al., 2001; McDonald, 2002). 2,2',4,4'-TetraBDE (BDE47) and 2,2',4,4',5-pentaBDE (BDE99), major components of the commercial flame retardant Great Lakes DE-71TM (DE71), are prevalent PBDEs found in human tissues and fluids (Schecter et al., 2003; Sjdin et al., 2004). BDE47, BDE99, and other PBDE components of DE71, e.g., 2,2',4,4',5,5'-hexaBDE (BDE153) may be increasing over time in some human populations. (Sjdin et al., 2004).
Administration of 10 mg/kg of DE71 for 4 days induced UDP-glucuronosyl transferase (UDPGT) and decreased serum T4 in weanling female rats (Zhou et al., 2001). Disruption of thyroid homeostasis may be attributable to enzyme induction through aryl hydrocarbon receptor (AhR)-dependent and/or -independent pathways (McClain, 1989). PBDE mixtures appear to be mixed-type monoxygenase inducers in vivo. Repeated exposure to PBDE products similar to DE71 resulted in phenobarbital-like and/or 3-methylcholanthrene-like induction in rats (Carlson, 1980; von Meyerinck, 1990). Ethoxy- and pentoxy-resorufin-O-dealkylase activity increased (indicating respective induction of CYP1A1 and CYP2B) in liver microsomes of DE71-treated rats (Zhou et al., 2001). Induction of CYP2B was detected in livers of mice 5 days following a single dose of 100 mg/kg of BDE47 (Staskal et al., 2005).
Induction of CYP1A1 indicates activation of the AhR, and planarity of polyhalogenated chemicals such as dibenzo-p-dioxins is required for AhR binding (Birnbaum, 1994; Mimura and Fujii-Kuriyama, 2003; Nebert et al. 2000). However, this well-characterized structure–activity relationship does not explain apparent AhR activation by PBDEs observed in previous in vivo experiments. Diphenyl ethers, including PBDEs (Fig. 1), are non-coplanar (Hu et al., 1994; Wang et al., 2005); thus, PBDEs should be poor AhR agonists. The reference AhR agonist used in the present study was 3,3',4,4',5-pentachlorobiphenyl (PCB126). PCB126 is coplanar (Fig. 1), is one-tenth as potent as TCDD at inducing CYP1A1, and is capable of producing dioxin-like toxicity, including carcinogenicity in experimental animals (Giesy and Kannan, 1998; Kafafi et al., 1993; NTP, 2005; Okey, 1990; Safe, 1990).
Phenobarbital-like induction of CYP2B by PBDEs indicates shared mechanism(s) of actions with non-coplanar PCBs. PCB congeners with multiple ortho-chlorine substitutions, e.g., 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), are non-coplanar (Fig. 1) and are poor AhR agonists (non-dioxin-like). Non-coplanar PCBs can elicit neurotoxic, carcinogenic, and endocrinal effects through AhR-independent pathways (Giesy and Kannan, 1998). Non-coplanar PCBs presumably induce CYP2Bs and CYP3As through respective activation of the constitutive androstane and pregnane x receptors (CAR and PXR) (Waxman, 1999; Dean et al., 2002). Overlap of enzyme induction may occur due to "crosstalk" between the two receptors (Dussault and Forman, 2002; Waxman, 1999). At present, data are lacking for adequate characterization of risk to humans following exposure to non-coplanar PCBs (Fischer et al., 1998).
Mechanisms of toxicity of PBDE mixtures and their individual components are not fully characterized. Investigations of the influence of metabolism and disposition of BDE47, BDE99, and BDE153 on toxicity of these chemicals in rodents are ongoing in this laboratory (Lebetkin et al., 2004, 2005; Sanders et al., 2004). However, gene expression changes may be more sensitive indicators of potential adverse effects than measurements derived from metabolism and disposition studies (Heinloth et al., 2004). Therefore, differential expression of selected PCB-target genes has been determined in the present study in rats dosed with DE71 or individual components of DE71. Expression of CYP1A1 was investigated to assess dioxin-like properties of DE71 and individual PBDE components. Expression of CYP2B and CYP3A was investigated to assess non-dioxin-like PCB properties of DE71 and individual PBDE components. Finally, the DE71 used in these studies was analyzed for the presence and activity of potential AhR agonists other than PBDEs. Results of this work will be useful for characterizing risks involved as a consequence of human exposure to PBDEs in the environment.
MATERIALS AND METHODS
Chemicals and chemical analysis.
DE71 used in these studies was obtained from Great Lakes Chemical Corporation (West Lafayette, IN). DE71 samples of varying concentrations were diluted in chloroform and injected at 1 μl each (injector temperature = 260°C) into an Agilent (Palo Alto, CA) model 6890 GC equipped with a flame ionization detector. A Restek (Bellefonte, PA) RTX 30 m x 0.35 mm column was used. The carrier gas was helium, delivered at a flow rate of 3 ml/min. Oven temperature was initially 80°C for 1 min, increased to 200°C at 20°/min, increased to 280°C at 10°C/min, then held at that temperature for an additional 10 min (total run time = 25 min). Retention times of peaks in DE71 were compared with those of authentic PBDE standards obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). The identities of PBDE components of DE71 were further verified using an Agilent 5973 mass spectrometer coupled to a similar GC system, with the exception that the helium flow was reduced to 1 ml/min and the final temperature hold time increased to 20 min (total run time = 35 min). BDE47, BDE99, and BDE153 used in these studies were obtained from Cerilliant (Round Rock, TX). PBDE purities were determined using authentic standards, GC/MS systems described above, and proton and 13C NMR spectroscopy.
GC/MS was used to search for the presence of polyhalogenated dibenzodioxins and dibenzofurans in DE71, BDE47, BDE99, and BDE153. Polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) were determined using a method with conditions and acceptance criteria similar to EPA Method 1613. Limits of detection (LOD) were 0.04 and 0.02–0.4 pg/g for 2,3,7,8-TCDD and 16 other PCDD/F authentic standards, respectively. The system used for identifying polybrominated dibenzodioxins and dibenzofurans (PBDD/Fs) consisted of a VG (Manchester, UK) Autospec mass spectrometer coupled to a Hewlett-Packard (Palo Alto, CA) 5890 Series II GC. Direct on-column injection volumes were 1 μl. The carrier gas was helium at 140 kpa. A DB-5MS 30 m x 0.32 mm column from J & W Scientific (Folsom, CA) was used. Oven temperature was held at 130°C for 2.5 min, increased at 30°C/min to 210°C, increased at 3°C/min to 315°C, then held for 25 min (total run time = 65 min). Authentic standards of PBDD/Fs (including 2,3,7,8-tetraBDF, 1,2,3,7,8-pentaBDF, and 1,2,3,4,7,8-hexaBDF used in the gene expression experiments) were obtained from Cambridge Isotope Laboratories, Inc. Aliquots diluted in dibromomethane were spiked with four 13C-labeled 2,3,7,8 substituted PBDD/F internal standards and processed through acid/base silica and two subsequent Florisil columns. Following the second Florisil cleanup, the samples were spiked with a 13C12-octachlorodibenzodioxin recovery standard. Method blanks contained no PBDD/Fs above their LOD, except for 2,3,7,8-tetraBDD, detected at 110 pg/g. Internal standard recoveries ranged from 89 to 125% for DE71. Matrix spike recoveries were acceptable (93–129%) for PBDD/Fs except for octabromodibenzodioxin, which was not detectable, and heptabromodibenzofuran, which had a 52% recovery.
3,3',5,5'-TetraBDE (BDE80) used in these studies was provided by Dr. Kun Chae (NIEHS). The material was the by-product of a copper-catalyzed coupling of p-methoxyphenol and 3,5-dibromophenylboronic acid (unpublished). The proton NMR (300 MHz, CDCl3), 7.59 (d, J = 2 Hz) and 7.70 (t, J = 2 Hz), was consistent with a symmetrical, meta-substituted diphenyl ether. Purity was determined by HPLC analysis to be >98%. The HPLC system utilized a Phenomenex (Torrance, CA) C18 RP column, with an isocratic mobile phase of 10% water and 90% acetonitrile at a flow rate of 1 ml/min. BDE80, eluting at ca.16 min in the system, was monitored by UV at 254 nm.
Dosing solutions and animal procedures.
DE71, PCBs, and PBDEs (except BDE153) dissolved in acetone, PBDFs dissolved in nonane, or BDE153 dissolved in 1,4-dioxane, were added to corn oil. Acetone and nonane were evaporated from dosing solutions under a steady stream of N2. The final volume of each dosing solution was adjusted with corn oil to administer the dose in 5 ml/kg. Vehicle controls containing acetone, nonane, or 1,4-dioxane were similarly prepared. Male F344 rats of 200–260 g and 10–12 weeks old were dosed by gavage once daily with one of the test chemicals (n = 3/treatment group) for three consecutive days. Each rat was euthanized with CO2 24 h after receiving the last dose. Procedures involving animals were carried out in accordance with institutional guidelines. Doses of test compounds are shown in Table 1, with the exception of a prepared mixture listed as PBDE-mix in Table 4. PBDE-mix delivered ca. 110 μmol/kg each of BDE47 and BDE99 and ca. 10 μmol/kg of BDE153, approximating the amounts of each congener in DE71–300.
RNA isolation.
Immediately following sacrifice of a rat, a portion of the right medial lobe of the liver was excised, diced, frozen in liquid N2, and stored at –80°C. RNA was isolated from ca. 100 mg liver using Qiagen, Inc. (Valencia, CA) RNeasy Midi Kits according to the manufacturer's instructions. The concentration and purity of the isolated RNA was determined using a Nanodrop Technologies (Wilmington, DE) ND-1000 Spectrophotometer. Isolated RNA was determined to be of high integrity using an Agilent Technologies (Palo Alto, CA) model 2100 Bioanalyzer.
Reverse transcriptase reactions and real-time PCR.
Each RNA sample was diluted in water to 4 ng/ml and converted to cDNA using Applied Biosystem's (ABI) (Foster City, CA) High Capacity cDNA Archive Kit according to the manufacturer's instructions. Real-time PCR (45 ng cDNA/well) was performed on an ABI Prism 7700 Sequence Detector. PCR conditions were as follows: denaturation for 10 min at 95°C followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Detection of target gene amplification was performed using ABI Assays on DemandTM Gene Expression primers and TaqMan probes. Primer/probes: Set #1 for rat CYP1A1. Set #2 with primers straddling exons 1 and 2 and Set #3 with primers straddling exons 8 and 9 of the gene sequence producing the splice variant for rat CYP2B1 (GenBank accession #J00719). Set #4 with primers straddling exons 5 and 6 and Set #5 with primers straddling exons 6 and 7 of the gene sequence producing the splice variant for rat CYP3A1 (GenBank accession #X96721).
Data analysis.
Conformational analysis of the structures in Figure 1 was performed using Cambridge Soft (Cambridge, MA) Chem 3D software and MOPAC Module. The conformations depicted in Figure 1 represent energy minima. Gene expression data are presented in Table 2 as fold change in target gene normalized to an endogenous reference gene (-actin) and relative to vehicle-treated controls by the method described by Livak and Schmittghen, 2001. Data represent the mean ± SD for 1–4 observations for each primer set for each of three rats/treatment group. Vehicle controls containing possible residues of either acetone or nonane had no effect on target gene expression and are combined in Table 4.
Statistical analysis.
Statistical analyses were performed using one-way ANOVA followed by the Tukey-Kramer test for pair-wise comparisons (JMP Statistical Software, SAS Institute Inc., Cary NC). Values were considered to be significantly different at p < 0.05.
RESULTS
GC analysis indicated that the DE71 used in these studies contained 36% BDE47, 42% BDE99, and 3% BDE153. BDE47 was ca. 99% pure. The purity of either BDE99 or BDE153, was ca. 96%. Conformational analysis predicted non-coplanar configurations for the three PBDEs and non-dioxin-like PCB153, all containing ortho-halogens (Fig. 1). A stable conformation for non-ortho brominated 3,3',5,5'-tetraBDE (BDE80) was also non-coplanar. In contrast, a stable conformation for dioxin-like PCB126 was coplanar.
No signs of toxicity were observed in rats of any treatment group. CYP1A1 expression in liver of rats receiving DE71 increased at a threshold between 3–30 μmol/kg/day (Table 2). The fold increase in CYP1A1 expression was comparable for 0.01 μmol PCB126/kg/day and a 30,000-fold higher dose of DE71 (300 μmol/kg/day). CYP1A1 expression was weakly induced in liver of PCB153-treated rats. Up-regulation of CYP1A1 was observed in liver only after high dose (100 μmol/kg/day) treatment of BDE47, BDE99, or BDE153 to rats. Increased CYP1A1 expression correlated with increased bromine content of the three congeners. In a separate experiment (data not included in Table 2), the mean fold change (±SD) of CYP1A1 expression was slightly greater in liver of rats receiving 10 μmol/kg of BDE80 for 3 days (4.2 ± 3.2) than in rats receiving a similar dose of BDE47 (1.4 ± 0.9) (corn oil-treated rats = 1.0 ± 0.2).
The threshold for induction of CYP2B expression in liver of DE71-treated rats was between 3–30 μmol/kg/day, resulting in a fold increase within the same order of magnitude as that following administration of 10 μmol PCB153/kg/day (Table 2). Thresholds for induction of CYP2B expression by the PBDEs were between 1 and 10 μmol/kg/day. BDE153 up-regulated CYP2B to a greatest extent than did BDE47 and BDE99 at 10 μmol/kg/day, comparable to results for PCB153. Transcriptional activity was greatest for CYP2B than for CYP3A following administration of DE71, PBDE congeners, or PCB153. The threshold for induction of CYP3A expression in liver of DE71-treated rats was between 30 and 300 μmol/kg/day. Thresholds for CYP3A up-regulation by the PBDEs were between 10 and 100 μmol/kg/day, except for 1–10 μmol/kg/day for BDE153 using primer set #5. Fold increases in expression of either CYP2B or CYP3A were similar among PBDE high dose groups.
The commercial sample of DE71 was found to contain brominated chemicals other than PBDEs (Table 3). Several tetra- through hexaBDD/Fs were identified in DE71 and individual PBDE samples. By weight, DE71 contained more PBDFs than PBDDs. The amount of PBDD/Fs in the PBDE samples correlated with an increase in bromine number. Only one PBDD/F was detected in BDE47, while BDE99 contained 4–5 PBDD/Fs. The greatest number and concentrations of PBDFs were detected in BDE153. No PCDD/Fs were detected in DE71, BDE47, BDE99, or BDE153.
The relative contribution of DE71 components, 2,3,7,8-tetraBDF, 1,2,3,7,8-pentaBDF, or 1,2,3,4,7,8-hexaBDF, to observed CYP1A1 up-regulation in these studies was investigated. Results demonstrated that increased CYP1A1 expression in liver of treated rats correlated with decreased bromine content of the administered PBDFs (Table 4). The tetraBDF was the most potent furan, with the dioxin-like response similar to that for an equimolar dose of PCB126. A group of rats dosed with a combination of BDE47, BDE99, and BDE153 in amounts similar to those contained in DE71–300 was included in this experiment to investigate the possibility of greater-than-additive effects of the three congeners on CYP1A1 up-regulation in DE71-treated rats. Results indicated much lower expression of CYP1A1 in liver of rats dosed with the prepared mixture than in liver of rats dosed with the DE71 high dose (Table 4).
DISCUSSION
BDE47, BDE99, or BDE153 contributed little to the observed increase in CYP1A1 expression in liver of rats treated with DE71. CYP1A1 expression increased over that of controls only after high dose (100 μmol/kg/day) administration of each individual congener. Further, CYP1A1 expression was weak (23 ± 5 fold increase) in liver of rats dosed with a combination of BDE47, BDE99, and BDE153 in amounts similar to those in the DE71 high dose (ca. 80% bromine content), relative to the response (2600 ± 1200 fold increase) in livers of rats treated with the DE71 high dose. Clearly, other components are more responsible for dioxin-like properties of DE71. 3,3',5,5'-tetraBDE (BDE80) was used to test the hypothesis that a PBDE lacking ortho-bromines could assume a coplanar conformation and have appreciable affinity for the AhR. However, the increase in CYP1A1 expression was minimal in liver of BDE80-treated rats, indicating that the PBDE congener resists coplanarity at physiological conditions. The present work supports in vitro data suggesting PBDE congeners are poor AhR agonists (Chen et al., 2001; Peters et al., 2004). Given the weight of evidence, current levels of exposure to PBDEs such as BDE47 and BDE99 may pose little or no risk of dioxin-like toxicity to humans.
Peters et al. (2004) postulated that CYP1A1 induction observed in studies of PBDE mixtures was due to molecules other than PBDEs, putatively PBDD/Fs. PBDD/Fs can bind to the AhR and have been shown to exhibit TCDD-like toxicity in experimental animals (Birnbaum et al., 2003). PBDFs have previously been detected in some PBDE mixtures. For instance, an unspecified commercial product containing tetra- through hexaBDEs was found to contain up to 2–4 ppm each of various tetra-, penta-, and hexaBDFs (Hileman et al., 1989). However, to the best of our knowledge, the presence of PBDD/Fs in the DE71 product has never been reported. Analytical results presented here confirm the presence of PBDD/Fs in both DE71 and the individual PBDEs used in our investigations. It is probable that DE71 and some PBDE components used in previous studies were contaminated with PBDD/Fs.
In the present study, the BDE47 high dose contained little or no PBDD/Fs and had minimal ability to up-regulate CYP1A1. The BDE99 high dose was intermediate among the PDBE high doses in both PBDD/F content and ability to up-regulate CYP1A1. The BDE153 high dose contained the highest amounts of PBDD/Fs and up-regulated CYP1A1 to a greater extent than did the other PBDE doses. These results provide indirect evidence for PBDD/F involvement in CYP1A1 up-regulation following PBDE administration. Direct evidence was obtained by investigating induction of CYP1A1 expression in liver following administration of three PBDF components of DE71 to rats. All three furans (a tetra-, penta-, and hexaBDF) significantly increased CYP1A1 expression over that of controls. 2,3,7,8-TetraBDF was, by far, the most potent of the three furans in up-regulating CYP1A1 and the most comparable to PCB126 in activity. The doses of PBDFs used to survey for this dioxin-like response were higher than their concentrations in the DE71 dosing solutions. Assuming the dose-response curve is hyperbolic, linear extrapolation of high dose response to zero would underestimate CYP1A1 up-regulation by lower concentrations of PBDD/Fs in DE71. It must also be pointed out that the present analysis of DE71 was limited to the availability of specific PBDD/Fs authentic standards. Therefore, it is likely that unidentified PBDD/Fs are present in the mixture that would contribute to CYP1A1 expression in DE71-treated rats.
Low-molecular-weight PBDEs appear to share AhR-independent mechanism(s) of action with non-coplanar PCBs. PBDEs and PCB153 up-regulated CYP2B and CYP3A gene expression in livers of treated rats and, thus, are apparent CAR and PXR agonists. Biological effects observed in rodents exposed to high doses of PBDE may be rationalized by well-characterized effects of phenobarbital-type inducers. For instance, phenobarbital induction of UDPGTs in rodents is associated with disruption of thyroid homeostasis and thyroid neoplasia (McClain, 1989). PBDEs are capable of altering thyroid hormone levels in rodents, but the response appears to occur at doses higher than environmental exposures to humans (McDonald, 2002). Further, there is no apparent correlation with environmental exposure to phenobarbital-type inducers and thyroid neoplasia in humans (McClain, 1995). However, thyroid hormone is critical to normal brain development in mammals, and altered thyroid hormone levels may relate to neurodevelopmental toxicity observed in rodents following exposure to either non-coplanar PCBs or PBDEs (Eriksson et al., 2001, 2002; Fischer et al., 1998; Porterfield, 2000). Additionally, AhR-independent mechanisms involving disruption of cell signaling and/or intracellular calcium homeostasis may contribute to observed neurotoxicity in rodents treated with PBDEs or non-coplanar PCBs (Fischer et al., 1998; Kodavanti, 2003; Kodavanti and Derr-Yellin, 2002; Tilson and Kodavanti, 1998). The relevance of these data to humans, particularly following perinatal exposure, is uncertain.
BDE153 appeared to be the most potent phenobarbital-type inducer among the three DE71 PBDE components, up-regulating CYP2B and CYP3A to a greater extent than the other congeners at 10 μmol/kg/day. Increased expression of these genes by BDE153 may be due to the presence of more BDE153-derived material and less BDE47- or BDE99-derived material in liver following equivalent doses to rats. This is supported by dosimetry data (unpublished) from our laboratory showing more BDE153 (2.8 ± 0.5 nmol equivalents/g) than either BDE47 or BDE99 (0.7 ± 0.1 and 0.9 ± 0.1 nmol equivalents/g, respectively) in liver of rats receiving equimolar doses (three consecutive daily doses of 1 μmol/kg). There was little difference in either CYP2B or CYP3A expression between the PBDEs at the high dose (100 μmol/kg/day), perhaps indicating saturation of the induction response at or below that dose for all three congeners.
In conclusion, results presented here indicate that major PBDE components of DE71 are poor AhR agonists and should contribute little, if any, dioxin-like properties to DE71. Other components of DE71, specifically PBDD/Fs, may be responsible for up-regulation of CYP1A1 following administration of the mixture to rats. The presence of these minor components should be considered in future studies and in risk characterization of products containing PBDEs. Conversely, biological pathways affected by exposure to non-dioxin-like PCBs may be similarly affected by exposure to PBDEs in the environment. Work is needed to characterize AhR-independent effects on biological systems following exposure to PBDEs, particularly at doses relevant to human exposure.
ACKNOWLEDGMENTS
The authors thank Drs. J. Dunnick and S. S. Ferguson of NIEHS for review of this manuscript. Thanks also go to Dr. N. Walker of NIEHS, Dr. L. S. Birnbaum of U.S. EPA, and Drs. D. Shea, R. Rose, A. Wallace, and C. Hofelt of North Carolina State University, for helpful discussions concerning these studies. This research was supported by the Intermural Research Program.
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Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695
Battelle, Columbus, Ohio 43201
ABSTRACT
Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have been detected in the environment and in mammalian tissues and fluids. Evidence indicates that PBDE mixtures induce CYPs through aryl hydrocarbon receptor (AhR)-dependent and -independent pathways. The present work has investigated the effects of individual components of a commercial PBDE mixture (DE71) on expression of CYP1A1, a biomarker for activation of the AhR (dioxin-like), and CYP2B and CYP3A, biomarkers for activation of the constitutive androstane and pregnanexreceptors (CAR and PXR), respectively, in the rat. Male F344 rats were dosed orally on three consecutive days with either DE71, PBDE components, 2,2',4,4'-tetraBDE (BDE47), 2,2',4,4',5-pentaBDE (BDE99), 2,2',4,4',5,5'-hexaBDE (BDE153), representative polybrominated dibenzofurans (PBDFs) present in DE71, or reference PCBs. Differential expression of target genes was determined in liver 24 h after the last dose. Quantitative PCR analysis indicated up-regulation of CYP1A1 by DE71; however, the response was weak compared to that for dioxin-like PCB126. Individual PBDE components of DE71 up-regulated CYP1A1 only at the highest administered dose (100 μmol/kg/day). Representative PBDFs efficiently up-regulated CYP1A1; therefore, they, along with other PBDFs and polybrominated dibenzodioxins detected in DE71 and individual PBDE components, may be responsible for most, if not all, dioxin-like properties previously observed for PBDEs. Conversely, PBDEs appear capable of up-regulating CYP2B and CYP3A in rats at doses similar to that for non-dioxin-like PCB153. These results indicate that in vivo PBDE-mediated toxicity would be better categorized by AhR-independent mechanisms, rather than the well-characterized AhR-dependent mechanism associated with exposure to dioxin-like chemicals.
Key Words: polybrominated diphenyl ethers; polybrominated dibenzodioxins and dibenzofurans; polychlorinated biphenyls; persistent organic compounds; gene expression; quantitative RT-PCR.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have accumulated in the environment and have been detected in humans (de Wit, 2002). PBDE concentrations in human adipose tissue range from ca. 1–500 ng/g lipid (McDonald, 2002). Human intake of PBDEs in Sweden has been estimated at 51 ng/day, but may be higher in the United States (Darnerud et al., 2001; McDonald, 2002). 2,2',4,4'-TetraBDE (BDE47) and 2,2',4,4',5-pentaBDE (BDE99), major components of the commercial flame retardant Great Lakes DE-71TM (DE71), are prevalent PBDEs found in human tissues and fluids (Schecter et al., 2003; Sjdin et al., 2004). BDE47, BDE99, and other PBDE components of DE71, e.g., 2,2',4,4',5,5'-hexaBDE (BDE153) may be increasing over time in some human populations. (Sjdin et al., 2004).
Administration of 10 mg/kg of DE71 for 4 days induced UDP-glucuronosyl transferase (UDPGT) and decreased serum T4 in weanling female rats (Zhou et al., 2001). Disruption of thyroid homeostasis may be attributable to enzyme induction through aryl hydrocarbon receptor (AhR)-dependent and/or -independent pathways (McClain, 1989). PBDE mixtures appear to be mixed-type monoxygenase inducers in vivo. Repeated exposure to PBDE products similar to DE71 resulted in phenobarbital-like and/or 3-methylcholanthrene-like induction in rats (Carlson, 1980; von Meyerinck, 1990). Ethoxy- and pentoxy-resorufin-O-dealkylase activity increased (indicating respective induction of CYP1A1 and CYP2B) in liver microsomes of DE71-treated rats (Zhou et al., 2001). Induction of CYP2B was detected in livers of mice 5 days following a single dose of 100 mg/kg of BDE47 (Staskal et al., 2005).
Induction of CYP1A1 indicates activation of the AhR, and planarity of polyhalogenated chemicals such as dibenzo-p-dioxins is required for AhR binding (Birnbaum, 1994; Mimura and Fujii-Kuriyama, 2003; Nebert et al. 2000). However, this well-characterized structure–activity relationship does not explain apparent AhR activation by PBDEs observed in previous in vivo experiments. Diphenyl ethers, including PBDEs (Fig. 1), are non-coplanar (Hu et al., 1994; Wang et al., 2005); thus, PBDEs should be poor AhR agonists. The reference AhR agonist used in the present study was 3,3',4,4',5-pentachlorobiphenyl (PCB126). PCB126 is coplanar (Fig. 1), is one-tenth as potent as TCDD at inducing CYP1A1, and is capable of producing dioxin-like toxicity, including carcinogenicity in experimental animals (Giesy and Kannan, 1998; Kafafi et al., 1993; NTP, 2005; Okey, 1990; Safe, 1990).
Phenobarbital-like induction of CYP2B by PBDEs indicates shared mechanism(s) of actions with non-coplanar PCBs. PCB congeners with multiple ortho-chlorine substitutions, e.g., 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), are non-coplanar (Fig. 1) and are poor AhR agonists (non-dioxin-like). Non-coplanar PCBs can elicit neurotoxic, carcinogenic, and endocrinal effects through AhR-independent pathways (Giesy and Kannan, 1998). Non-coplanar PCBs presumably induce CYP2Bs and CYP3As through respective activation of the constitutive androstane and pregnane x receptors (CAR and PXR) (Waxman, 1999; Dean et al., 2002). Overlap of enzyme induction may occur due to "crosstalk" between the two receptors (Dussault and Forman, 2002; Waxman, 1999). At present, data are lacking for adequate characterization of risk to humans following exposure to non-coplanar PCBs (Fischer et al., 1998).
Mechanisms of toxicity of PBDE mixtures and their individual components are not fully characterized. Investigations of the influence of metabolism and disposition of BDE47, BDE99, and BDE153 on toxicity of these chemicals in rodents are ongoing in this laboratory (Lebetkin et al., 2004, 2005; Sanders et al., 2004). However, gene expression changes may be more sensitive indicators of potential adverse effects than measurements derived from metabolism and disposition studies (Heinloth et al., 2004). Therefore, differential expression of selected PCB-target genes has been determined in the present study in rats dosed with DE71 or individual components of DE71. Expression of CYP1A1 was investigated to assess dioxin-like properties of DE71 and individual PBDE components. Expression of CYP2B and CYP3A was investigated to assess non-dioxin-like PCB properties of DE71 and individual PBDE components. Finally, the DE71 used in these studies was analyzed for the presence and activity of potential AhR agonists other than PBDEs. Results of this work will be useful for characterizing risks involved as a consequence of human exposure to PBDEs in the environment.
MATERIALS AND METHODS
Chemicals and chemical analysis.
DE71 used in these studies was obtained from Great Lakes Chemical Corporation (West Lafayette, IN). DE71 samples of varying concentrations were diluted in chloroform and injected at 1 μl each (injector temperature = 260°C) into an Agilent (Palo Alto, CA) model 6890 GC equipped with a flame ionization detector. A Restek (Bellefonte, PA) RTX 30 m x 0.35 mm column was used. The carrier gas was helium, delivered at a flow rate of 3 ml/min. Oven temperature was initially 80°C for 1 min, increased to 200°C at 20°/min, increased to 280°C at 10°C/min, then held at that temperature for an additional 10 min (total run time = 25 min). Retention times of peaks in DE71 were compared with those of authentic PBDE standards obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). The identities of PBDE components of DE71 were further verified using an Agilent 5973 mass spectrometer coupled to a similar GC system, with the exception that the helium flow was reduced to 1 ml/min and the final temperature hold time increased to 20 min (total run time = 35 min). BDE47, BDE99, and BDE153 used in these studies were obtained from Cerilliant (Round Rock, TX). PBDE purities were determined using authentic standards, GC/MS systems described above, and proton and 13C NMR spectroscopy.
GC/MS was used to search for the presence of polyhalogenated dibenzodioxins and dibenzofurans in DE71, BDE47, BDE99, and BDE153. Polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) were determined using a method with conditions and acceptance criteria similar to EPA Method 1613. Limits of detection (LOD) were 0.04 and 0.02–0.4 pg/g for 2,3,7,8-TCDD and 16 other PCDD/F authentic standards, respectively. The system used for identifying polybrominated dibenzodioxins and dibenzofurans (PBDD/Fs) consisted of a VG (Manchester, UK) Autospec mass spectrometer coupled to a Hewlett-Packard (Palo Alto, CA) 5890 Series II GC. Direct on-column injection volumes were 1 μl. The carrier gas was helium at 140 kpa. A DB-5MS 30 m x 0.32 mm column from J & W Scientific (Folsom, CA) was used. Oven temperature was held at 130°C for 2.5 min, increased at 30°C/min to 210°C, increased at 3°C/min to 315°C, then held for 25 min (total run time = 65 min). Authentic standards of PBDD/Fs (including 2,3,7,8-tetraBDF, 1,2,3,7,8-pentaBDF, and 1,2,3,4,7,8-hexaBDF used in the gene expression experiments) were obtained from Cambridge Isotope Laboratories, Inc. Aliquots diluted in dibromomethane were spiked with four 13C-labeled 2,3,7,8 substituted PBDD/F internal standards and processed through acid/base silica and two subsequent Florisil columns. Following the second Florisil cleanup, the samples were spiked with a 13C12-octachlorodibenzodioxin recovery standard. Method blanks contained no PBDD/Fs above their LOD, except for 2,3,7,8-tetraBDD, detected at 110 pg/g. Internal standard recoveries ranged from 89 to 125% for DE71. Matrix spike recoveries were acceptable (93–129%) for PBDD/Fs except for octabromodibenzodioxin, which was not detectable, and heptabromodibenzofuran, which had a 52% recovery.
3,3',5,5'-TetraBDE (BDE80) used in these studies was provided by Dr. Kun Chae (NIEHS). The material was the by-product of a copper-catalyzed coupling of p-methoxyphenol and 3,5-dibromophenylboronic acid (unpublished). The proton NMR (300 MHz, CDCl3), 7.59 (d, J = 2 Hz) and 7.70 (t, J = 2 Hz), was consistent with a symmetrical, meta-substituted diphenyl ether. Purity was determined by HPLC analysis to be >98%. The HPLC system utilized a Phenomenex (Torrance, CA) C18 RP column, with an isocratic mobile phase of 10% water and 90% acetonitrile at a flow rate of 1 ml/min. BDE80, eluting at ca.16 min in the system, was monitored by UV at 254 nm.
Dosing solutions and animal procedures.
DE71, PCBs, and PBDEs (except BDE153) dissolved in acetone, PBDFs dissolved in nonane, or BDE153 dissolved in 1,4-dioxane, were added to corn oil. Acetone and nonane were evaporated from dosing solutions under a steady stream of N2. The final volume of each dosing solution was adjusted with corn oil to administer the dose in 5 ml/kg. Vehicle controls containing acetone, nonane, or 1,4-dioxane were similarly prepared. Male F344 rats of 200–260 g and 10–12 weeks old were dosed by gavage once daily with one of the test chemicals (n = 3/treatment group) for three consecutive days. Each rat was euthanized with CO2 24 h after receiving the last dose. Procedures involving animals were carried out in accordance with institutional guidelines. Doses of test compounds are shown in Table 1, with the exception of a prepared mixture listed as PBDE-mix in Table 4. PBDE-mix delivered ca. 110 μmol/kg each of BDE47 and BDE99 and ca. 10 μmol/kg of BDE153, approximating the amounts of each congener in DE71–300.
RNA isolation.
Immediately following sacrifice of a rat, a portion of the right medial lobe of the liver was excised, diced, frozen in liquid N2, and stored at –80°C. RNA was isolated from ca. 100 mg liver using Qiagen, Inc. (Valencia, CA) RNeasy Midi Kits according to the manufacturer's instructions. The concentration and purity of the isolated RNA was determined using a Nanodrop Technologies (Wilmington, DE) ND-1000 Spectrophotometer. Isolated RNA was determined to be of high integrity using an Agilent Technologies (Palo Alto, CA) model 2100 Bioanalyzer.
Reverse transcriptase reactions and real-time PCR.
Each RNA sample was diluted in water to 4 ng/ml and converted to cDNA using Applied Biosystem's (ABI) (Foster City, CA) High Capacity cDNA Archive Kit according to the manufacturer's instructions. Real-time PCR (45 ng cDNA/well) was performed on an ABI Prism 7700 Sequence Detector. PCR conditions were as follows: denaturation for 10 min at 95°C followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Detection of target gene amplification was performed using ABI Assays on DemandTM Gene Expression primers and TaqMan probes. Primer/probes: Set #1 for rat CYP1A1. Set #2 with primers straddling exons 1 and 2 and Set #3 with primers straddling exons 8 and 9 of the gene sequence producing the splice variant for rat CYP2B1 (GenBank accession #J00719). Set #4 with primers straddling exons 5 and 6 and Set #5 with primers straddling exons 6 and 7 of the gene sequence producing the splice variant for rat CYP3A1 (GenBank accession #X96721).
Data analysis.
Conformational analysis of the structures in Figure 1 was performed using Cambridge Soft (Cambridge, MA) Chem 3D software and MOPAC Module. The conformations depicted in Figure 1 represent energy minima. Gene expression data are presented in Table 2 as fold change in target gene normalized to an endogenous reference gene (-actin) and relative to vehicle-treated controls by the method described by Livak and Schmittghen, 2001. Data represent the mean ± SD for 1–4 observations for each primer set for each of three rats/treatment group. Vehicle controls containing possible residues of either acetone or nonane had no effect on target gene expression and are combined in Table 4.
Statistical analysis.
Statistical analyses were performed using one-way ANOVA followed by the Tukey-Kramer test for pair-wise comparisons (JMP Statistical Software, SAS Institute Inc., Cary NC). Values were considered to be significantly different at p < 0.05.
RESULTS
GC analysis indicated that the DE71 used in these studies contained 36% BDE47, 42% BDE99, and 3% BDE153. BDE47 was ca. 99% pure. The purity of either BDE99 or BDE153, was ca. 96%. Conformational analysis predicted non-coplanar configurations for the three PBDEs and non-dioxin-like PCB153, all containing ortho-halogens (Fig. 1). A stable conformation for non-ortho brominated 3,3',5,5'-tetraBDE (BDE80) was also non-coplanar. In contrast, a stable conformation for dioxin-like PCB126 was coplanar.
No signs of toxicity were observed in rats of any treatment group. CYP1A1 expression in liver of rats receiving DE71 increased at a threshold between 3–30 μmol/kg/day (Table 2). The fold increase in CYP1A1 expression was comparable for 0.01 μmol PCB126/kg/day and a 30,000-fold higher dose of DE71 (300 μmol/kg/day). CYP1A1 expression was weakly induced in liver of PCB153-treated rats. Up-regulation of CYP1A1 was observed in liver only after high dose (100 μmol/kg/day) treatment of BDE47, BDE99, or BDE153 to rats. Increased CYP1A1 expression correlated with increased bromine content of the three congeners. In a separate experiment (data not included in Table 2), the mean fold change (±SD) of CYP1A1 expression was slightly greater in liver of rats receiving 10 μmol/kg of BDE80 for 3 days (4.2 ± 3.2) than in rats receiving a similar dose of BDE47 (1.4 ± 0.9) (corn oil-treated rats = 1.0 ± 0.2).
The threshold for induction of CYP2B expression in liver of DE71-treated rats was between 3–30 μmol/kg/day, resulting in a fold increase within the same order of magnitude as that following administration of 10 μmol PCB153/kg/day (Table 2). Thresholds for induction of CYP2B expression by the PBDEs were between 1 and 10 μmol/kg/day. BDE153 up-regulated CYP2B to a greatest extent than did BDE47 and BDE99 at 10 μmol/kg/day, comparable to results for PCB153. Transcriptional activity was greatest for CYP2B than for CYP3A following administration of DE71, PBDE congeners, or PCB153. The threshold for induction of CYP3A expression in liver of DE71-treated rats was between 30 and 300 μmol/kg/day. Thresholds for CYP3A up-regulation by the PBDEs were between 10 and 100 μmol/kg/day, except for 1–10 μmol/kg/day for BDE153 using primer set #5. Fold increases in expression of either CYP2B or CYP3A were similar among PBDE high dose groups.
The commercial sample of DE71 was found to contain brominated chemicals other than PBDEs (Table 3). Several tetra- through hexaBDD/Fs were identified in DE71 and individual PBDE samples. By weight, DE71 contained more PBDFs than PBDDs. The amount of PBDD/Fs in the PBDE samples correlated with an increase in bromine number. Only one PBDD/F was detected in BDE47, while BDE99 contained 4–5 PBDD/Fs. The greatest number and concentrations of PBDFs were detected in BDE153. No PCDD/Fs were detected in DE71, BDE47, BDE99, or BDE153.
The relative contribution of DE71 components, 2,3,7,8-tetraBDF, 1,2,3,7,8-pentaBDF, or 1,2,3,4,7,8-hexaBDF, to observed CYP1A1 up-regulation in these studies was investigated. Results demonstrated that increased CYP1A1 expression in liver of treated rats correlated with decreased bromine content of the administered PBDFs (Table 4). The tetraBDF was the most potent furan, with the dioxin-like response similar to that for an equimolar dose of PCB126. A group of rats dosed with a combination of BDE47, BDE99, and BDE153 in amounts similar to those contained in DE71–300 was included in this experiment to investigate the possibility of greater-than-additive effects of the three congeners on CYP1A1 up-regulation in DE71-treated rats. Results indicated much lower expression of CYP1A1 in liver of rats dosed with the prepared mixture than in liver of rats dosed with the DE71 high dose (Table 4).
DISCUSSION
BDE47, BDE99, or BDE153 contributed little to the observed increase in CYP1A1 expression in liver of rats treated with DE71. CYP1A1 expression increased over that of controls only after high dose (100 μmol/kg/day) administration of each individual congener. Further, CYP1A1 expression was weak (23 ± 5 fold increase) in liver of rats dosed with a combination of BDE47, BDE99, and BDE153 in amounts similar to those in the DE71 high dose (ca. 80% bromine content), relative to the response (2600 ± 1200 fold increase) in livers of rats treated with the DE71 high dose. Clearly, other components are more responsible for dioxin-like properties of DE71. 3,3',5,5'-tetraBDE (BDE80) was used to test the hypothesis that a PBDE lacking ortho-bromines could assume a coplanar conformation and have appreciable affinity for the AhR. However, the increase in CYP1A1 expression was minimal in liver of BDE80-treated rats, indicating that the PBDE congener resists coplanarity at physiological conditions. The present work supports in vitro data suggesting PBDE congeners are poor AhR agonists (Chen et al., 2001; Peters et al., 2004). Given the weight of evidence, current levels of exposure to PBDEs such as BDE47 and BDE99 may pose little or no risk of dioxin-like toxicity to humans.
Peters et al. (2004) postulated that CYP1A1 induction observed in studies of PBDE mixtures was due to molecules other than PBDEs, putatively PBDD/Fs. PBDD/Fs can bind to the AhR and have been shown to exhibit TCDD-like toxicity in experimental animals (Birnbaum et al., 2003). PBDFs have previously been detected in some PBDE mixtures. For instance, an unspecified commercial product containing tetra- through hexaBDEs was found to contain up to 2–4 ppm each of various tetra-, penta-, and hexaBDFs (Hileman et al., 1989). However, to the best of our knowledge, the presence of PBDD/Fs in the DE71 product has never been reported. Analytical results presented here confirm the presence of PBDD/Fs in both DE71 and the individual PBDEs used in our investigations. It is probable that DE71 and some PBDE components used in previous studies were contaminated with PBDD/Fs.
In the present study, the BDE47 high dose contained little or no PBDD/Fs and had minimal ability to up-regulate CYP1A1. The BDE99 high dose was intermediate among the PDBE high doses in both PBDD/F content and ability to up-regulate CYP1A1. The BDE153 high dose contained the highest amounts of PBDD/Fs and up-regulated CYP1A1 to a greater extent than did the other PBDE doses. These results provide indirect evidence for PBDD/F involvement in CYP1A1 up-regulation following PBDE administration. Direct evidence was obtained by investigating induction of CYP1A1 expression in liver following administration of three PBDF components of DE71 to rats. All three furans (a tetra-, penta-, and hexaBDF) significantly increased CYP1A1 expression over that of controls. 2,3,7,8-TetraBDF was, by far, the most potent of the three furans in up-regulating CYP1A1 and the most comparable to PCB126 in activity. The doses of PBDFs used to survey for this dioxin-like response were higher than their concentrations in the DE71 dosing solutions. Assuming the dose-response curve is hyperbolic, linear extrapolation of high dose response to zero would underestimate CYP1A1 up-regulation by lower concentrations of PBDD/Fs in DE71. It must also be pointed out that the present analysis of DE71 was limited to the availability of specific PBDD/Fs authentic standards. Therefore, it is likely that unidentified PBDD/Fs are present in the mixture that would contribute to CYP1A1 expression in DE71-treated rats.
Low-molecular-weight PBDEs appear to share AhR-independent mechanism(s) of action with non-coplanar PCBs. PBDEs and PCB153 up-regulated CYP2B and CYP3A gene expression in livers of treated rats and, thus, are apparent CAR and PXR agonists. Biological effects observed in rodents exposed to high doses of PBDE may be rationalized by well-characterized effects of phenobarbital-type inducers. For instance, phenobarbital induction of UDPGTs in rodents is associated with disruption of thyroid homeostasis and thyroid neoplasia (McClain, 1989). PBDEs are capable of altering thyroid hormone levels in rodents, but the response appears to occur at doses higher than environmental exposures to humans (McDonald, 2002). Further, there is no apparent correlation with environmental exposure to phenobarbital-type inducers and thyroid neoplasia in humans (McClain, 1995). However, thyroid hormone is critical to normal brain development in mammals, and altered thyroid hormone levels may relate to neurodevelopmental toxicity observed in rodents following exposure to either non-coplanar PCBs or PBDEs (Eriksson et al., 2001, 2002; Fischer et al., 1998; Porterfield, 2000). Additionally, AhR-independent mechanisms involving disruption of cell signaling and/or intracellular calcium homeostasis may contribute to observed neurotoxicity in rodents treated with PBDEs or non-coplanar PCBs (Fischer et al., 1998; Kodavanti, 2003; Kodavanti and Derr-Yellin, 2002; Tilson and Kodavanti, 1998). The relevance of these data to humans, particularly following perinatal exposure, is uncertain.
BDE153 appeared to be the most potent phenobarbital-type inducer among the three DE71 PBDE components, up-regulating CYP2B and CYP3A to a greater extent than the other congeners at 10 μmol/kg/day. Increased expression of these genes by BDE153 may be due to the presence of more BDE153-derived material and less BDE47- or BDE99-derived material in liver following equivalent doses to rats. This is supported by dosimetry data (unpublished) from our laboratory showing more BDE153 (2.8 ± 0.5 nmol equivalents/g) than either BDE47 or BDE99 (0.7 ± 0.1 and 0.9 ± 0.1 nmol equivalents/g, respectively) in liver of rats receiving equimolar doses (three consecutive daily doses of 1 μmol/kg). There was little difference in either CYP2B or CYP3A expression between the PBDEs at the high dose (100 μmol/kg/day), perhaps indicating saturation of the induction response at or below that dose for all three congeners.
In conclusion, results presented here indicate that major PBDE components of DE71 are poor AhR agonists and should contribute little, if any, dioxin-like properties to DE71. Other components of DE71, specifically PBDD/Fs, may be responsible for up-regulation of CYP1A1 following administration of the mixture to rats. The presence of these minor components should be considered in future studies and in risk characterization of products containing PBDEs. Conversely, biological pathways affected by exposure to non-dioxin-like PCBs may be similarly affected by exposure to PBDEs in the environment. Work is needed to characterize AhR-independent effects on biological systems following exposure to PBDEs, particularly at doses relevant to human exposure.
ACKNOWLEDGMENTS
The authors thank Drs. J. Dunnick and S. S. Ferguson of NIEHS for review of this manuscript. Thanks also go to Dr. N. Walker of NIEHS, Dr. L. S. Birnbaum of U.S. EPA, and Drs. D. Shea, R. Rose, A. Wallace, and C. Hofelt of North Carolina State University, for helpful discussions concerning these studies. This research was supported by the Intermural Research Program.
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