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The glucagon-like peptide-1 receptor (GLP-1R) is found in a variety of tissues outside of the pancreas. For example, GLP-1R is expressed in the lung, where it has been implicated in the regulation of the lipid fraction of surfactants, suggesting it fulfills an important role in lung function. Here, we show that GLP-1R expression is strongly up-regulated immediately after birth in neonatal rats, particular in male offspring. Moreover, administering long half-life GLP-1R agonists to the mother from gestational day 14 to birth (exendin-4 or liraglutide) increased surfactant protein (SP)-A and SP-B mRNA expression and the amount of SPs in the amniotic fluid at the end of pregnancy. These effects were similar or more potent to those induced by the glucocorticoid dexamethasone, which also increased GLP-1R expression in fetuses just before delivery. Lir increased fetal SP-A and GLP-1R expression in control rats and in a nitrofen-induced model of lung hypoplasia. Moreover, lung size increased in controls after Lir administration, which also prevented the decrease in lung weight and the poor neonatal survival of the offspring from nitrofen-treated dams, effects that were not produced by dexamethasone. Taken together, our results demonstrate the importance of the GLP-1 system in regulating SP production and lung development.

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca(2+)](i)) levels using flow cytometry and observed an increase in the intracellular Ca(2+) concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 microM) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of [Ca(2+)](i) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Among proposed uses for microarrays in environmental toxiciology is the identification of key contributors to toxicity within a mixture. However, it remains uncertain whether the transcriptomic profiles resulting from exposure to a mixture have patterns of altered gene expression that contain identifiable contributions from each toxicant component. We exposed isogenic rainbow trout Onchorynchus mykiss, to sublethal levels of ethynylestradiol, 2,2,4,4-tetrabromodiphenyl ether, and chromium VI or to a mixture of all three toxicants Fluorescently labeled complementary DNA (cDNA) were generated and hybridized against a commercially available Salmonid array spotted with 16,000 cDNAs. Data were analyzed using analysis of variance (p<0.05) with a Benjamani-Hochberg multiple test correction (Genespring [Agilent] software package) to identify up and downregulated genes. Gene clustering patterns that can be used as "expression signatures" were determined using hierarchical cluster analysis. The gene ontology terms associated with significantly altered genes were also used to identify functional groups that were associated with toxicant exposure. Cross-ontological analytics approach was used to assign functional annotations to genes with "unknown" function. Our analysis indicates that transcriptomic profiles resulting from the mixture exposure resemble those of the individual contaminant exposures, but are not a simple additive list. However, patterns of altered genes representative of each component of the mixture are clearly discernible, and the functional classes of genes altered represent the individual components of the mixture. These findings indicate that the use of microarrays to identify transcriptomic profiles may aid in the identification of key stressors within a chemical mixture, ultimately improving environmental assessment.

Polybrominated diphenyl ethers (PBDEs) are increasing in biota. Here, captive adult American kestrels were exposed daily by diet to safflower oil (controls), or one of two levels of a pentaBDE technical formulation, DE-71 (0.3 or 1.6 ppm), for approximately 75 days, commencing 21 days before breeding. This exposure resulted in eggs having PBDE concentrations similar (low exposure) or within the same order of magnitude (high exposure) reported for wild American kestrels and gulls in the Great Lakes. Compared to controls, kestrels in both exposure groups copulated less, spent less time in their nest boxes, and participated in fewer pair-bonding behaviors. Furthermore, the timing of these behaviors, which is important to creating and maintaining the pair-bond, also differed significantly from the controls. The females in the low-exposure group made fewer compatible trilling calls and ate less frequently. These behavioral changes were compounded by increasing exposure to DE-71 during the 9-day courtship period immediately preceding egg laying, a standard measure of the kestrel courtship period. The birds in the high-exposure group made more food transfers, excited "klee" calls, and copulations, the latter only when compared to the low-exposure birds, whereas the low-exposure males performed fewer pair-bonding behaviors. This study demonstrates that the exposure of kestrels to environmentally relevant levels of DE-71 modifies the quality of the pair-bond, affects the reproductive behavior of both sexes, and occurs when birds are exposed for a short period as adults. In addition, these behavioral effects are consistent with the observed reproductive changes in these birds.

Epidemiological studies indicate that exposure to environmental pollutants during early human development can have deleterious effects on cognitive development. The interaction between environmental pollutants is suggested as one reason for the observed defective neurological development in children from the Faeroe Islands as compared to children from the Seychelles. We have previously seen in mice that polychlorinated biphenyls (PCBs) can interact together with methyl mercury (MeHg), as well as PCB together with polybrominated diphenyl ether (PBDE 99) to exacerbate developmental neurotoxic effects when present during a critical period of neonatal brain development. PBDEs are a new class of global environmental contaminants. The present study shows that neonatal coexposure to PBDE 99 (0.8 mg/kg body weight) and MeHg (0.4 or 4.0 mg/kg body weight) can exacerbate developmental neurotoxic effects. These effects are manifested as disrupted spontaneous behavior, reduced habituation, and impaired learning/memory abilities. This is seen in the low dose range, where the sole compounds do no give rise to developmental neurotoxic effects. The effects seen are more than just additive. Furthermore, a significant effect of interaction was seen on the cholinergic nicotinic receptors in the cerebral cortex and hippocampus. This suggests that a mechanism for the observed cognitive defects is via the cholinergic system. Furthermore, PBDE can interact with MeHg causing developmental neurotoxic effects similar to those we previously have observed between PCB 153 + MeHg and PCB 52 + PBDE 99. This is of vital importance, as the levels of PBDEs are increasing in mother's milk and in the environment generally.

The neuropeptide, vasopressin (VP) is synthesized in magnocellular neuroendocrine cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the mammalian hypothalamus. VP has multiple functions including maintenance of body fluid homeostasis, cardiovascular control, learning and memory, and nervous system development. Polybrominated diphenyl ethers (PBDEs), used as additive flame retardants, have been shown to interfere with hormone metabolism and function. Previously, we demonstrated that the technical polychlorinated biphenyl (PCB) mixture, Aroclor 1254, inhibits somatodendritic VP release from the SON of osmotically stimulated rats. The objectives of the current study were to test whether PBDEs affect central VP release in a similar manner and to determine the potency of several PCB and PBDE congeners in order to identify a common mode of action for these persistent chemicals. The current work shows that the commercial PBDE mixture (DE-71) significantly decreased somatodendritic VP release from rat SON punches in a strain-independent manner. In addition, the specific congeners PBDE 47 and PCB 47 (15 and 5 microM) were also neuroactive in this system. To explore structure/activity relationships, we compared the effects of PBDE 77 with PCB 77. PBDE 77, but not PCB 77 significantly reduced VP release. These results show that like PCBs, PBDEs perturb signaling mechanisms responsible for hormone release, and that environmentally relevant PBDE congeners are more neuroactive than the commercial mixtures with noncoplanarity of these compounds playing a role in promoting neuroactivity.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and are universally present in the environment. An exponential increase in PBDE concentrations in the U.S. population have been reported over the last 3 decades. PBDEs 47 (tetraBDE) and 99 (pentaBDE) are the most commonly detected PBDE congeners in the environment and in human samples. PBDE209 (decaBDE) is the only remaining PBDE flame retardant commercially manufactured in the United States. Several PBDEs are known to induce cyp3a in rats, but the mechanism of induction remains unclear. The goal of this study was to clarify the mechanism by which PBDE congeners induce cyp3a. Treatment of C57BL6 mice with PBDEs 47, 99, and 209 induced gene expressions of cyp3a11 and 2b10, but not cyp1a1/2. Because the first two genes are known target genes of pregnane X receptor (PXR), a ligand-activated transcription factor in the nuclear hormone receptor superfamily, we hypothesized that PBDE congeners are PXR activators. Using reporter gene luciferase assays, the present data show that PBDEs 47, 99, and 209 activated PXR and its human counterpart, steroid X receptor, but not aryl hydrocarbon receptor. Furthermore, induction of cyp3a11 and 2b10 by PBDEs 47, 99, and 209 was markedly suppressed in PXR-knockout mice, indicating that PBDE congeners activate PXR in vivo. In summary, our study provides the first evidence that PBDEs are activators for xenobiotic nuclear receptor.

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are recognized as global environmental contaminants and a potential health risk. They have been shown to elicit neurodevelopmental toxicity through disruption of the cholinergic neurotransmitter system in rodent models, but the effects of environmentally relevant exposures in wildlife species are unknown. The objective of this study was to assess the effects of the commercial pentabrominated diphenyl ether mixture DE-71 on cholinergic parameters in ranch mink (Mustela vison) following dietary exposure of adult females and in utero, lactational, and dietary exposure of their offspring. Adult females were fed diets containing 0, 0.1, 0.5, or 2.5 microg DE-71/g feed from four weeks prior to breeding through weaning of their kits at six weeks of age. A portion of the weaned kits were maintained on their respective diets through 27 weeks of age. Cholinergic parameters, including muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) binding, cholinesterase (ChE) activity, and acetylcholine (ACh) concentration, were assayed in the cerebral cortex, and ChE activity was measured in the plasma. In the cerebral cortex, results indicated a significant exposure-dependent increase in PBDE concentrations, but no significant effects of DE-71 on cholinergic parameters. There was a threefold increase in ChE activity in the plasma of adult females in the 2.5 microg DE-71/g feed group, but was likely due to effects on liver function. This study demonstrated that environmentally relevant exposures to DE-71 did not affect key parameters of the cholinergic neurotransmitter system in the brain of ranch mink.

The present study shows that polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) can interact and enhance developmental neurobehavioral defects when the exposure occurs during a critical stage of neonatal brain development. PBDEs are used in large quantities as flame-retardant additives in polymers, especially in the manufacture of a great variety of electrical appliances, and textiles. In contrast to the well-known persistent compounds PCBs and DDT, the PBDEs have been found to increase in the environment and in human mother's milk. We have previously shown that low-dose exposure to environmental toxic agents such as PCB can cause developmental neurotoxic effects when present during a critical stage of neonatal brain development. Epidemiological studies indicate the adverse neurobehavioral impact of PCBs. Recently, we reported that neonatal exposure to PBDEs causes developmental neurotoxic effects. In the present study, 10-day-old Naval Medical Research Institute male mice were given one single oral dose of PCB 52 (1.4 micromol/kg body weight [bw]) + PBDE 99 (1.4 micromol), PCB 52 (1.4 micromol or 14 micromol), or PBDE 99 (1.4 micromol or 14 micromol). Controls received a vehicle (20% fat emulsion). Animals exposed to the combined dose of PCB 52 (1.4 micromol) + PBDE 99 (1.4 micromol) and the high dose of PCB 52 (14 micromol) or PBDE 99 (14 micromol) showed significantly impaired spontaneous motor behavior and habituation capability at the age of 4 and 6 months. The neurobehavioral defects were also seen to worsen with age in mice neonatally exposed to PCB 52 + PBDE 99.

The congener profiles of polybrominated diphenyl ethers (PBDEs) in human and wildlife samples are dominated by brominated diphenyl ether (BDE) congeners 47, 99, 100, 153, and 154, all of which are components of the commercial pentaBDE mixtures commonly used in a variety of flammable consumer products. Very little information is available on the toxicokinetics of these congeners and no studies are available directly comparing these BDE congeners in mice. Therefore, the objective of this study was to compare the distribution, metabolism, and excretion of BDEs 47, 99, 100 and 153. Female C57BL/6 mice were administered a single dose of BDE (1 mg/kg: 2.1, 1.9, 1.9, and 1.8 mumol/kg, respectively) intravenously. Excretion was monitored daily, and terminal tissue disposition was examined 5 days following exposure. All BDE congeners in this study distribute with similar patterns into lipophilic tissues; however, tissue concentrations 5 days following exposure were much higher for BDE-153 than for 100, 99, and 47, respectively. Excretion rates were inversely related to tissue concentrations as BDE-47 was the most rapidly excreted congener, followed by BDE-99, -100, and -153. Differences in tissue concentrations were largely driven by congener-specific urinary elimination rates which were associated with protein binding in the urine. While the overall rate of metabolism appeared to be low, analysis of metabolites in daily feces samples revealed that BDE-99 was the most rapidly metabolized congener in this study. The results of this study demonstrate that congener substitution plays a role in the distribution, metabolism, and excretion of PBDEs in mice and it is therefore important to consider the differential toxicokinetic parameters associated with each congener when assessing the risk to human health from these PBDE congeners.

Polybrominated diphenyl ethers (PBDEs) are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate i.e. in human biota. We examined the micronucleus-forming activities of low-dose PBDEs (congeners 47, 99, 153, 183 or 209) in MCF-7 cells along with their ability to modulate growth, cell biochemistry [by infrared (IR) microspectroscopy], clonogenic survival or quantitative expression of cytochrome P450 isoenzymes (CYP1A1, CYP1A2 and CYP1B1), cyclin-dependent kinase inhibitor 1A [CDKN1A (P21(WAF1/CIP1))], B-cell leukaemia/lymphoma-2 (BCL-2) and Bcl-2-associated X (BAX). Elevations in micronucleus formation were observed following treatment with 10(-12) to 10(-9) M PBDE concentrations despite the fact that less than one-fourth of the concentration of each test agent administered partitioned out of the media and into the incubating cells. However, low-dose treatment levels remained within the range of reported concentrations measured in UK serum samples collected in 2003. Clonogenic survival and gene expression was unaltered following 10(-12) to 10(-9) M PBDE treatment but significant (P < 0.05) elevations in growth kinetics were observed. Significant alterations in IR cell spectra were associated with treatments, and plotted clusters following principal component analysis highlighted these changes. Whether such in vitro effects point to an underlying ability of PBDEs to initiate and drive target-cell alterations in vivo now needs to be addressed.

Polybrominated diphenyl ethers (PBDEs), a class of additive flame retardants, are temporally increasing in wildlife tissues and capable of disrupting normal endocrine function. We determined whether in ovo and post-hatch exposure of captive American kestrels (Falco sparverius) to environmentally relevant PBDEs alter thyroid, retinol, and oxidative stress measures. Control eggs were injected with safflower oil and subsequent nestlings fed the same vehicle; dosed eggs received PBDE congeners (BDE-47, -99, -100, -153), which mainly comprise the Penta-BDE commercial mixture, dissolved in safflower oil at concentrations (1500 ng/g total [Sigma] PBDEs) approximating those in Great Lakes gull eggs. Nestlings hatching from dosed eggs were orally exposed for 29 days to variable SigmaPBDE concentrations that are similar to levels reported in tissues of Great Lakes trout (100 ng/g). Treatment kestrels had lower plasma thyroxine (T(4)), plasma retinol, and hepatic retinol and retinyl palmitate concentrations, but unaltered triiodothyronine (T(3)) concentrations and thyroid glandular structure. BDE-47, -100, and -99 were negatively associated with plasma T(4), plasma retinol (BDE-100, -99) and hepatic retinol (BDE-47). Despite an antioxidant-rich diet, PBDE exposure induced hepatic oxidative stress, particularly in females, with an increased hepatic GSSG:GSH ratio, a marginal increase in lipid peroxidation, and increased oxidized glutathione. Positive associations were found between concentrations of BDE-183 and thiols and, in males, between BDE-99 and reduced GSH, but a negative association occurred between BDE-99 and TBARS. Subsequently, concentrations of PBDE congeners in wild birds may alter thyroid hormone and vitamin A concentrations, glutathione metabolism and oxidative stress.

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 micromol/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.

Polybrominated diphenyl ethers (PBDEs), widely used as flame-retardants, are now recognized as globally distributed pollutants, and are detected in most environmental and biological samples, including human blood, adipose tissue, and breast milk. Due to their wide use in commercial products and their persistent nature, long-term exposure to PBDEs may pose a human health risk, especially to children. Our previous reports showed that the commercial PBDE mixture, DE-71, affected protein kinase C (PKC) and calcium homeostasis in a similar way to those of a structurally-related polychlorinated biphenyl (PCB) mixture. These intracellular signaling events are associated with neuronal development and learning and memory function. The objectives of the present study were to test whether environmentally relevant PBDE congeners, with different position and number of bromines, affected PKC translocation in cerebellar granule neuronal cultures and compare the potency and efficacy of PBDE congeners with their 14C-accumulation. All the tested PBDE congeners increased 3H-phorbol ester (PDBu) binding, and a significant effect was seen as low as 10 microM. Among the congeners tested, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent than other congeners. These effects were seen at concentrations and exposure times where no cytotoxicity was observed. The efficacy of PBDE congeners varied with their structural composition, and the effects seen on 3H-PDBu binding with some PBDE congeners are similar to those of PCB congeners. Cerebellar granule neurons accumulated all three PBDE congeners (PBDEs 47, 99, and 153) following exposure. At the lowest concentration (0.67 microM), about 13-18% of the total dose of 14C-PBDE congeners was accumulated by these neurons. There were distinct differences in the pattern of 14C-PBDE accumulation among the PBDE congeners. The 14C-PBDE accumulation, either represented as percent basis or nanomole basis, was much lower for the 30.69 microM PBDE 99 and 10.69-30.69 microM PBDE 153 than at the lower concentrations, which may be due to low solubility of these congeners. The accumulation pattern with PBDE 47 did not vary with concentration. On a nanomole accumulation basis, PBDEs 47, 99, and 153 accumulation was linear with time. While the nanomole accumulation was linear with concentration for PBDE 47, it is nonlinear for PBDEs 99 and 153. The pattern of PBDE accumulation seems to correlate with the effects on PKC translocation, with regression values of 0.773-0.991. These results indicate that PBDEs affected PKC translocation in neurons in a similar way to those of other organohalogens, some PBDE congeners are equally efficacious as the respective PCB congeners, and PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals.

Polybrominated diphenyl ethers (PBDEs), chemicals commonly used as flame retardants, are ubiquitous in the environment and bioaccumulate in humans and wildlife. However, little is known about their potential toxicological properties. In the present study, male Long-Evans rats orally administered the commercial PBDE mixture DE-71 or corn oil for 1 week, beginning at postnatal day (PND) 6, were tested on a visual discrimination task and two sustained attention tasks. After completion of these tasks, the rats were administered a drug challenge with the muscarinic antagonist scopolamine (0, 0.01, 0.03, 0.05 mg/kg), which was injected subcutaneously 30 min prior to testing on the second sustained attention task. The DE-71-exposed rats demonstrated deficits in learning but not in sustained attention when compared to controls. Scopolamine impaired the animals' ability to detect the brief visual cues in controls, as evidenced by decreases in accuracy and increases in omission errors. However, the DE-71-exposed rats were subsensitive to the effects of scopolamine on omission errors, particularly on trials in which a long delay preceded the cue, suggesting alterations in the cholinergic modulation of sustained attention. For the DE-71-exposed rats, the lack of sustained attention deficits in the absence of the drug, coupled with the subsensitivity to scopolamine's effects on sustained attention, suggest that although this PBDE mixture produced lasting alterations in cholinergic functioning, either (1) these alterations were not of sufficient magnitude to be behaviorally relevant, or (2) behavioral deficits resulting from these alterations were overcome by the development of compensatory neural mechanisms or response strategies in adulthood.

Polybrominated diphenyl ethers (PBDEs) are widely used brominated flame retardants (BFRs), which have become ubiquitous in the environment. This study investigates the effects of the pentabrominated diphenyl ether mixture, DE-71, on human neutrophil granulocytes in vitro. DE-71 enhanced production of reactive oxygen species (ROS) in a concentration-dependent manner measured as lucigenin-amplified chemiluminescence. Octabrominated diphenyl ether (OBDE), decabrominated diphenyl ether (DBDE), and the non-brominated diphenyl ether did not induce ROS formation at the concentrations tested. DPI (4 microM), an inhibitor of the NADPH oxidase completely inhibited DE-71 induced ROS formation, highlighting a role for NADPH oxidase activation. The protein kinase C inhibitor BIM (0.25 microM) and the selective chelator of intracellular calcium, BAPTA-AM (5 microM), also inhibited NADPH oxidase activation, indicating a calcium-dependent activation of PKC. ROS formation was also inhibited by the tyrosine kinase inhibitor tyrphostin (1 microM), the phospholipase C inhibitor ET-18-OCH3 (5 microM), and the phosphatidylinositol-3 kinase inhibitor LY294002 (25 microM). Alterations in intracellular calcium were measured using fura-2/AM, and a significant increase was measured after exposure to DE-71 both with and without extracellular calcium. The tetra brominated compound BDE-47 also enhanced ROS formation in a concentration dependent manner. The combination of DE-71 with the bacteria-derived N-formyl peptide fMLP and PCB153 induced an additive effect in the lucigenin assay. We suggest that tyrosine kinase mediated activation of PI3K could result in enhanced activation of calcium-dependent PKC by enhanced PLC activity, followed by intracellular calcium release leading to ROS formation in neutrophil granulocytes.

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 approximately 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.

In this paper the benchmark dose (BMD) method was introduced for spontaneous behavior data observed in 2-, 5-, and 8-month-old male and female C57Bl mice exposed orally on postnatal day 10 to different doses of 2,2',4,4',5-pentabromodiphenyl ether (PBDE 99). Spontaneous behavior (locomotion, rearing, and total activity) was in the present work quantified in terms of a fractional response defined as the cumulative response after 20 min divided by the cumulative response produced over the whole 1-h test period. The fractional response contains information about the time-response profile (which differs between the treatment groups) and has appropriate statistical characteristics. In the analysis, male and female mice could be characterized by a common dose-response model (i.e., they responded equally to the exposure to PBDE 99). As a primary approach, the BMD was defined as the dose producing a 5 or 10% change in the mean fractional response. According to the Hill model, considering a 10% change the lower bound of the BMD for rearing, locomotion, and total activity was 1.2, 0.85, and 0.31 mg PBDE 99/kg body weight, respectively. A probability-based procedure for BMD modeling was also considered. Using this methodology, the BMD was defined as corresponding to an excess risk of 5 or 10% of falling below cutoff points representing adverse levels of fractional response.

Polybrominated diphenyl ethers (PBDEs), one class of flame retardants used to suppress or inhibit the risk of fire, are regularly found in the environment and in human milk. The present study shows that neonatal exposure to a widely, environmentally found PBDE, 2,2',4,4',5-pentaBDE (PBDE 99), can induce developmental neurotoxic effects, such as changes in spontaneous behavior (hyperactivity), effects that are dose-response related and worsen with age. These changes are seen in C57/Bl mice of both sexes. Neonatal C57/Bl male and female mice were orally exposed on day 10 to 0.4, 0.8, 4.0, 8.0, or 16 mg PBDE 99/kg body weight. Spontaneous behavior (locomotion, rearing, and total activity) was observed in two-, five-, and eight-month-old mice. The behavior tests showed that the effects were dose-response and time-response related for both male and female mice. The observed developmental neurotoxic effects seen for PBDE 99, in C57/Bl mice, are similar to effects seen for 2,2',4,4'-tetraBDE (PBDE 47), PBDE 99, 2,2',4,4',5,5'- hexaBDE (PBDE 153), 2,2',3,3',4,4',5,5',6,6'-decaBDE (PBDE 209) and for certain PCBs, in male NMRI mice. Furthermore, the effects of PBDEs appear to be as potent in female mice as in male mice, and as potent in C57/Bl mice as in NMRI mice, concerning developmental neurotoxicity.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

Polybrominated diphenyl ethers (PBDEs), which are widely used as flame-retardants, have been increasing in environmental and human tissue samples during the past 20-30 years, while other structurally related, persistent organic pollutants such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (on a TEQ basis), have decreased. PBDEs have been detected in human blood, adipose tissue, and breast milk, and developmental and long-term exposure to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that PCBs, which cause neurotoxic effects, including changes in learning and memory, stimulated the release of [(3)H]arachidonic acid ([(3)H]AA) by a cPLA(2)/iPLA(2)-dependent mechanism. PLA(2)(phospholipase A(2)) activity has been associated with learning and memory, and AA has been identified as a second messenger involved in synaptic plasticity. The objective of the present study was to test whether PBDE mixtures (DE-71 and DE-79), like other organohalogen mixtures, have a similar action on [(3)H]AA release in an in vitro neuronal culture model. Cerebellar granule cells at 7 days in culture were labeled with [(3)H]AA for 16-20 h and then exposed in vitro to PBDEs. DE-71, a mostly pentabromodiphenyl ether mixture, significantly stimulated [(3)H]AA release at concentrations as low as 10 microg/ml, while DE-79, a mostly octabromodiphenyl ether mixture, did not stimulate [(3)H]AA release, even at 50 microg/ml. The release of [(3)H]AA by DE-71 is time-dependent, and a significant increase was seen after only 5-10 min of exposure. The removal and chelation of calcium from the exposure buffer, using 0.3 mM EGTA, significantly attenuated the DE-71-stimulated [(3)H]AA release; however, only an 18% inhibition of the release was demonstrated for the calcium replete conditions at 30 microg/ml DE-71. Methyl arachidonylfluorophosphonate (5 microM), an inhibitor of cPLA(2)/iPLA(2), completely attenuated the DE-71-stimulated [(3)H]AA release. Further studies focused on comparing the effects of DE-71 with PCB mixtures such as Aroclors 1016 and 1254. Both PCB mixtures stimulated [(3)H]AA release in a concentration-dependent manner; however, the effect for PCBs was about two times greater than that of the PBDEs on a weight basis, but was comparable on a molar basis. These results indicate that PBDEs stimulated the release of [(3)H]AA by activating PLA(2), which is similar to the effect of other organohalogen mixtures.

Polybrominated diphenyl ethers (PBDEs) are used as flame-retardants and have recently been shown to be increasing in the environment and in human mother's milk. We have recently reported that neonatal exposure to 2,2',4,4',5-pentaBDE (PBDE 99) can induce persistent aberrations in spontaneous behavior and also affect learning and memory functions in the adult animal. The present study indicates that the cholinergic system, in its developing stage, may be a target of and sensitive to PBDEs. Neonatal exposure of male NMRI mice on postnatal day 10, to 2,2',4,4',5-pentaBDE (8 mg/kg bw) was shown to alter the response to a cholinergic agent, nicotine, at an adult age. The nicotine-induced behavior test revealed a hypoactive response to nicotine in PBDE 99-treated animals, whereas the response of controls was an increased activity. These findings show similarities to observations made from neonatal exposure to PCBs and nicotine, compounds shown to affect cholinergic nicotinic receptors. This indicates that PBDE 99 can affect the cholinergic system and might thereby interact with other environmental toxicants.

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as flame retardant additives. In a recent study, we have seen that neonatal exposure to some brominated flame retardants can cause permanent aberrations in spontaneous motor behavior that seem to worsen with age. In view of an increasing amount of PBDEs in mother's milk and in the environment, the present study was undertaken to investigate whether there is a critical and limited phase, during neonatal life, for induction of persistent neurotoxic effects of 2,2',4,4',5-pentaBDE (PBDE 99). Neonatal NMRI male mice were exposed on day 3, 10, or 19 to 8 mg 2,2',4,4',5-pentaBDE/kg body weight. Uptake and retention of 2,2',4,4',5-penta[(14)C]BDE were studied in the mouse brain after exposure to 1.5 M becquerel (Bq) 2,2',4,4',5-penta[(14)C]BDE /kg body weight (bw) on postnatal day 3, 10, or 19. Spontaneous motor behavior was observed in 4-month-old mice. Mice exposed to 2,2',4,4',5-pentaBDE on day 3 or 10 showed significantly impaired spontaneous motor behavior, whereas no effect was seen in mice exposed on day 19. Neonatal mice exposed to 2,2',4,4',5-penta[(14)C]BDE 99 on postnatal day 3, 10, or 19 were sacrificed 24 h or 7 days posttreatment. The amount of radioactivity, given as per mille ( per thousand) of total amount administered, was between 3.7 and 5.1 per thousand in the three different age categories at 24 h after administration. Seven days after the administration, 2,2',4,4',5-penta[(14)C]BDE or its metabolites could still be detected in the brain. The amount of radioactivity in the brain was not higher in mice exposed on day 3 or 10 when compared to exposure on day 19. Thus, the behavioral disturbances observed in adult mice following neonatal exposure to 2,2',4,4',5-pentaBDE are induced during a defined critical period of neonatal brain development.

The objective of the current study was to characterize the effects of DE-71 (a commercial polybrominated diphenyl ether mixture containing mostly tetra- and penta-bromodiphenyl ethers) on thyroid hormones and hepatic enzyme activity in offspring, following perinatal maternal exposure. Primiparous Long-Evans rats were orally administered DE-71 (0, 1, 10, and 30 mg/kg/day) in corn oil from gestation day (GD) 6 to postnatal day (PND) 21. Serum and liver samples obtained from dams (GD 20 and PND 22), fetuses (GD 20), and offspring (PNDs 4, 14, 36, and 90) were analyzed for circulating total serum thyroxine (T(4)) and triiodothyronine (T(3)), or hepatic microsomal ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD), and uridine diphosphoglucuronosyl transferase (UDPGT) activity. There were no significant effects of treatment on maternal body weight gain, litter size, or sex ratio, nor were there any effects on any measures of offspring viability or growth. Serum T(4) was reduced in a dose-dependent manner in fetuses on GD 20 (at least 15%) and offspring on PND 4 and PND 14 (50 and 64% maximal in the 10 and 30 mg/kg/day groups, respectively), but recovered to control levels by PND 36. Reduction in serum T(4) was also noted in GD 20 dams (48% at highest dose), as well as PND 22 dams (44% at highest dose). There was no significant effect of DE 71 on T(3) concentrations at any time in the dams or the offspring. Increased liver to body weight ratios in offspring were consistent with induction of EROD (maximal 95-fold), PROD (maximal 26-fold) or UDPGT (maximal 4.7-fold). Induction of PROD was similar in both dams and offspring; however, EROD and UDPGT induction were much greater in offspring compared to dams (EROD = 3.8-fold; UDPGT = 0.5-fold). These data support the conclusion that DE-71 is an endocrine disrupter in rats during development.