Pentabrominated Diphenyl Ether Research Articles

Determination of organic priority pollutants in sewage treatment plant effluents by gas chromatography high-resolution mass spectrometry

In this work, we report the development and validation of an analytical method for the trace level determination of 14 selected (EU-directive) priority organic pollutants (namely, 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene, 1,3,5-trichlorobenzene, hexachloro-1,3-butadiene, pentachlorobenzene, hexachlorobenzene, alachlor, α-hexachloro-cyclohexane (α-HCH), β-HCH, γ-HCH (lindane), δ-HCH, tetra-brominated diphenyl ether (tetra-BDE), penta-brominated diphenyl ether and hepta-brominated diphenyl ether) in wastewater samples from 5 different sewage treatment plants (STPs) located in Spain. The proposed methodology is based on liquid–liquid extraction with n-hexane followed by identification and confirmation of the selected pollutants by gas chromatography high-resolution mass spectrometry in selected ion recording acquisition mode. Recovery studies performed with spiked wastewater samples at two different concentration levels (0.1 and 1 μg L −1) gave mean recoveries in the range 80–120% (except for trichlorobenzenes, ca. with 50%) with RSD values below 10% in most cases, thus confirming the usefulness of the proposed methodology for the analyses of this kind of complex samples. The obtained detection limits in effluent wastewater matrices were in the low nanogram per liter range, with values as low as 0.09 ng L −1 for tetra-BDE and 0.3 ng L −1 for hexachlorobenzene. Finally, the proposed methodology was successfully applied to a monitoring study intended to characterize wastewater effluents of 5 different sewage treatment plants with different major activities (Industrial, Coastal, Urban). Most of the compounds targeted were detected in the ng L −1 range at concentrations ranging from 0.19 ng L −1 to 135 ng L −1 (hexachlorobenzene).

Molecular mechanisms underlying pentabrominated diphenyl ether-induced proliferation in breast cancer MCF-7 cells

The effects of pentabrominated diphenyl ethers (BDE-99) were examined on mRNA and protein expressions of c-Myc and p53 in breast cancer MCF-7 cells. Human breast cancer MCF-7 cells were cultured in Dulbecco's modified Eagle's medium. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was employed to investigate cell proliferation. Real time-polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to detect mRNA and protein expressions of c-Myc and p53. At the concentration of 1 × (10−4–10−8) mol L−1, BDE-99 stimulated proliferation of MCF-7 cells in a concentration- and time-dependent manner. RT-PCR and immunohistochemistry analyses revealed that treatment with 1 × (10−4–10−7) mol L−1 for 72 h resulted in significantly increased mRNA and protein expressions of c-Myc and inhibited p53. It is possible that BDE-99 exerted estrogenic actions and might markedly stimulate proliferation in estrogen responsive breast cancer MCF-7 cells. BDE-99 similar to estrogen promoted c-Myc mRNA and protein, and inhibited p53 mRNA and protein expression in MCF-7 cell.

Absorption and biotransformation of polybrominated diphenyl ethers DE-71 and DE-79 in chicken ( Gallus gallus), mallard ( Anas platyrhynchos), American kestrel ( Falco sparverius) and black-crowned night-heron ( Nycticorax nycticorax) eggs

We recently reported that air cell administration of penta-brominated diphenyl ether (penta-BDE; DE-71) evokes biochemical and immunologic effects in chicken ( Gallus gallus) embryos at very low doses, and impairs pipping (i.e., stage immediately prior to hatching) and hatching success at 1.8 μg g −1 egg (actual dose absorbed) in American kestrels ( Falco sparverius). In the present study, absorption of polybrominated diphenyl ether (PBDE) congeners was measured following air cell administration of a penta-BDE mixture (11.1 μg DE-71 g −1 egg) or an octa-brominated diphenyl ether mixture (octa-BDE; DE-79; 15.4 μg DE-79 g −1 egg). Uptake of PBDE congeners was measured at 24 h post-injection, midway through incubation, and at pipping in chicken, mallard ( Anas platyrhynchos), and American kestrel egg contents, and at the end of incubation in black-crowned night-heron ( Nycticorax nycticorax) egg contents. Absorption of penta-BDE and octa-BDE from the air cell into egg contents occurred throughout incubation; at pipping, up to 29.6% of penta-BDE was absorbed, but only 1.40–6.48% of octa-BDE was absorbed. Higher brominated congeners appeared to be absorbed more slowly than lower brominated congeners, and uptake rate was inversely proportional to the log K ow of predominant BDE congeners. Six congeners or co-eluting pairs of congeners were detected in penta-BDE-treated eggs that were not found in the dosing solution suggesting debromination in the developing embryo, extraembryonic membranes, and possibly even in the air cell membrane. This study demonstrates the importance of determining the fraction of xenobiotic absorbed into the egg following air cell administration for estimation of the lowest-observed-effect level.

Polybrominated Diphenyl Ethers Orally Administration to Mice Were Tansferred to Offspring during Gestation and Lactation with Disruptions on the Immune System.

BackgroundThe present study was undertaken to examine the immunological effects of pentabrominated diphenyl ether (penta-BDE) and decabrominated diphenyl ether (deca-BDE) on the immune system of the dams and the developmental immune system of the offsprings.MethodsIn this study, mated female C57BL/6J mice were orally administered penta-BDE, deca-BDE or corn oil for 5 weeks, from gestational day 6 to lactational day 21.ResultsThe body weight of PND21 exposed to penta-BDE was significantly decreased relative to control mice, but that of post-natal day 63 (PND63) were recovered. Orally dosed dams with penta-BDE had significantly smaller absolute and relative spleen masses than control mice. Absolute and relative spleen and thymus masses of PND21 exposed to penta-BDE were significantly decreased over control. The exposure of dams and PND21 with penta-BDE reduced the number of splenocytes and thymocytes. As results of hematologic analysis, percentage WBC and percentage neutrophils increased in dams with deca-BDE. Splenic T cell proliferation in dams and PND21 exposed to penta-BDE was increased, and there were no significant difference in splenic B cell proliferation in all treatment groups. As results of flow cytometric analysis of splenocyte, percentage total T cell, Th cell and Tc cell in PND21 exposed to penta-BDE was slightly increased, and percentage macrophage in dams and PND21 exposed to deca-BDE was decreased. The ELISA results of antibody production show no significant difference in all treatment groups relative to controls.ConclusionThese results imply that PBDEs given to the dam were transferred to the offspring during gestation and lactation, and PBDEs transferred from the dam affect immune system of offspring.

Technical pentabromodiphenyl ether and hexabromocyclododecane as activators of the pregnane-X-receptor (PXR)

Technical pentabrominated diphenyl ether (pentaBDE mix) is a mixture of polybrominated diphenyl ethers (PBDEs) which has been widely used as a flame retardant. Since its ban in several countries it has been replaced by other brominated flame retardants such as hexabromocyclododecane (HBCD). Both certain PBDE congeners and HBCD are present in environmental and human samples reflecting their persistent and bioaccumulative properties. PentaBDE mix and HBCD have recently been found to induce cytochrome P450 (CYP) 3 enzymes in rat liver. In this study we tested both technical pentaBDE mix and HBCD for their potency to induce CYP3A enzymes in rat hepatocytes in primary culture, and in rat H4IIE and human HepG2 hepatoma cells. In rat hepatocytes, HBCD was a more effective CYP3A1 inducer than pentaBDE mix, being less effective, however, than the prototype inducer dexamethasone. In human HepG2 cells, both compounds and the prototype inducer rifampicin were about equally effective. In contrast, in HepG2 cells, HBCD failed to induce luciferin-PFBE dealkylase, a common catalytic activity of a number of CYP3A enzymes, possibly reflecting enzyme inhibition. A significant induction of catalytic activity was observed in rat hepatocytes with both compounds. Analysis of a XREM-driven reporter gene activity in transfected cells confirmed that both compounds act as agonists of the human and rat pregnane-X-receptor, which was detectable in all cell types used.

Molecular mechanisms underlying proliferation and apoptosis in breast cancer MCF-7 cells induced by pentabrominated diphenyl ethers

The molecular mechanisms underlying proliferation and apoptosis induced by pentabrominated diphenyl ethers (5-BDE) through regulation of mRNA and protein expression of Bcl-2 and Bax was investigated using breast cancer MCF-7 cells. Human breast cancer MCF-7 cells were grown in DMEM medium, MTT assay was employed to investigate cell proliferation. RT-PCR and immunohistochemistry was performed to determine mRNA and protein expression of Bcl-2 and Bax. Within the range of 1 × 10−8–10−4mol L−1, polybrominated diphenyl ethers (PBDE) stimulated proliferation in MCF-7 cells in a concentration- and time-dependent manner. RT-PCR and immunohistochemistry analyses revealed that treatment with PBDE for 72 h resulted in increased mRNA and protein expression of Bcl-2 and inhibition of Bax. Evidence indicates that 5-BDE exerted estrogenic actions and stimulated proliferation in estrogen responsive breast cancer MCF-7 cells via up-regulation of Bcl-2 mRNA and protein expression.

Reproductive and Developmental Toxicity of a Pentabrominated Diphenyl Ether Mixture, DE-71, to Ranch Mink (Mustela vison) and Hazard Assessment for Wild Mink in the Great Lakes Region

Polybrominated diphenyl ethers (PBDEs) are widespread persistent and bioaccumulative environmental contaminants. Recent scientific attention has focused on the developmental toxicity of PBDE commercial mixtures following perinatal exposure of rodents; however, these studies do not necessarily predict toxicity to highly exposed top predators, such as mink (Mustela vison). Here we assessed the effects of environmentally relevant doses (0, 0.1, 0.5, and 2.5 ppm [wt/wt] in feed) of a technical pentabrominated diphenyl ether mixture, DE-71, on reproductive performance of mink and on development of offspring exposed perinatally and post-weaning until 33 weeks. A dietary concentration that causes no effects on reproduction in rodents, 2.5-ppm DE-71, resulted in complete reproductive failure in these mink, while whelping rates were not affected at all lower does. Developmental effects in offspring were evident in 33-week-old juveniles, which were more sensitive to effects than their respective dams. Juvenile thyroid hormone homeostasis was also much more sensitive compared to rodents, and at 0.5-ppm DE-71, total triiodothyronine (T3) was significantly decreased in all males and females, even despite a compensatory increase of total thyroxine (T4) in females. T4-outer-ring deiodinase activity, mainly contributed by type II deiodinase, was not affected at any dose for any life stage, but thyroid follicular epithelium cell height was elevated in the 0.5-ppm-treated juveniles (p = 0.057). Ethoxyresorufin O-deethylase activity was significantly induced in all offspring at 33 weeks, most likely as a consequence of polybrominated dioxin, furan, or biphenyl impurities in DE-71. Biomonitoring of wild mink in the Great Lakes region indicated that most populations had lower concentrations than what are expected to affect thyroid hormone homeostasis, but margins of safety are small and mink around Hamilton Harbour exceeded the no observed adverse effect level for T3 disruption.

Toxicity of polybrominated diphenyl ethers (de‐71) in chicken (Gallus gallus), mallard (Anas platyrhynchos), and American kestrel (Falco sparverius) embryos and hatchlings

Embryonic survival, pipping and hatching success, and sublethal biochemical, endocrine, and histological endpoints were examined in hatchling chickens (Gallus gallus), mallards (Anas platyrhynchos), and American kestrels (Falco sparverius) following air cell administration of a pentabrominated diphenyl ether (penta-BDE; DE-71) mixture (0.01-20 microg/g egg) or polychlorinated biphenyl (PCB) congener 126 (3,3',4,4',5-pentachlorobiphenyl; 0.002 microg/g egg). The penta-BDE decreased pipping and hatching success at concentrations of 10 and 20 microg/g egg in kestrels but had no effect on survival endpoints in chickens or mallards. Sublethal effects in hatchling chickens included ethoxyresorufin-O-dealkylase (EROD) induction and histological changes in the bursa, but these responses were not observed in other species. Polychlorinated biphenyl congener 126 (positive control) reduced survival endpoints in chicken and kestrel embryos and caused sublethal effects (EROD induction, reduced bursal mass and follicle size) in chickens. Mallards were clearly less sensitive than the other species to administered penta-BDE and PCB 126. In a second experiment, the absorption of penta-BDE (11.1 microg/g egg, air cell administered during early development) into the contents of chicken and kestrel eggs was determined at various intervals (24 h postinjection, midincubation, and pipping). By pipping, 29% of the penta-BDE administered dose was present in the egg contents in chickens, and 18% of the administered dose was present in kestrel egg contents. Based on uptake in kestrels, the lowest-observed-effect level on pipping and hatching success may be as low as 1.8 microg total penta-BDE/g egg, which approaches concentrations detected in eggs of free-ranging birds. Because some penta-BDE congeners are still increasing in the environment, the toxic effects observed in the present study are cause for concern in wildlife.

Dose-dependent effects of pentabrominated diphenyl ethers on thyroid histological structure and function in rats

The aim of this study was to determine the dose-dependent effects of pentabrominated diphenyl ether (PBED-99), a flame retardant, on thyroid histological structure and function in rats. Four-week-old Sprague-Dawley rats were divided into four groups randomly, with five animals of each gender per group. Rats in treatment groups were orally gavaged with (PBED-99) at the dose of 30, 60 or 120 mg kg−1, while controls received an equal volume of corn oil. The levels of serum hormone were measured as well as change in histopathology, histochemistry and follicular cells ultramicroscopic structure 15 days following exposure to PBED-99. Serum levels of thyroxine (FT4) and triiodothyronine (FT3) decreased gradually dose-dependently at 15 days of exposure to PBED-99. However, thyroid-stimulating hormone levels increased. The thyroid follicular epithelium hyperplasia was aggravated gradually as dose increased. Proliferating cell nuclear antigen positive cells rose as dose increased. The activities of thyroperoxidase and succinate dehydrogenase in all treatment groups was significantly elevated compared to controls. The rough endoplasmic reticulum of the thyroid follicular cells appeared expanded with saccate using electron microscopy, whereas nucleolus, cell membrane and mitochondria were not damaged. BDE-99 may disturb thyroxine histological structure and function in a dose-dependent manner.

Persistent Organic Pollutant Residues in Human Fetal Liver and Placenta from Greater Montreal, Quebec: A Longitudinal Study from 1998 through 2006

BackgroundThere is general concern that persistent organic pollutants (POPs) found in the environment, wildlife, food, water, house dust, human tissues, and fluids may alter normal human physiologic activities (e.g., fetal development, immune and endocrine systems). Although the levels of some POPs [polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs)] in these matrices have decreased after their ban, others [polybrominated diphenyl ethers (PBDEs)] have increased in recent years.ObjectiveTo determine the longitudinal trend of specific POPs in human fetal tissues for risk assessment purposes.MethodsWe analyzed early to mid-gestation fetal liver (n = 52) and placental (n = 60) tissues, obtained after elective abortions during 1998–2006, for selected PBDEs, PCBs, and OCs using gas chromatography–mass spectroscopy.ResultsTotal PBDEs in fetal liver increased over time (mean ± SE: 1998, 284.4 ± 229.8 ng/g lipid; 2006, 1,607.7 ± 605.9; p < 0.03), whereas placental levels were generally lower, with no clear trend. Low levels of PCBs and OCs varied yearly, with no evident trend. The major analytes in 1998 were OCs (liver, 49%; placenta, 71%), whereas the major analytes in 2006 were PBDEs (liver, 89%; placenta, 98%). The 1998–2006 tissue PBDE congener profile is similar to that of DE-71, a commercial primarily pentabrominated diphenyl ether mixture manufactured in North America.ConclusionsAlthough commercial production of penta- and octa-brominated diphenyl ethers in North America was halted in 2004, their concentrations in fetal liver and placenta are now greater than the tissue burdens for the analyzed OCs and PCBs. Our findings also demonstrate that PBDEs accumulate within the fetal compartment at a very early stage in gestation.

Development and validation of a congener‐specific photodegradation model for polybrominated diphenyl ethers

With the phaseout of the manufacture of some polybrominated diphenyl ether (PBDE) formulations, namely penta-brominated diphenyl ether (BDE) and octa-BDE, and the continued use of the deca-BDE formulation, it is important to be able to predict the photodegradation of the more highly brominated congeners. A model was developed and validated to predict the products and their relative concentrations from the photodegradation of PBDEs. The enthalpies of formation of the 209 PBDE congeners were calculated, and the relative reaction rate constants were obtained. The predicted reaction rate constants for PBDEs show linear correlation with previous experimental results. Because of their large volume use, their presence in the environment, and/or importance in the photodegradation of the deca-BDE formulation, BDE-209, BDE-184, BDE-100, and BDE-99 were chosen for further ultraviolet photodegradation experiments in isooctane. The photodegradation model successfully predicted the products of the photochemical reactions of PBDEs in experimental studies. A gas chromatography retention time model for PBDEs was developed using a multiple linear regression analysis and, together with the photodegradation model and additional PBDE standards, provided a way to identify unknown products from PBDE photodegradation experiments. Based on the results of the photodegradation experiments, as well as the model predictions, it appears that the photodegradation of PBDEs is a first-order reaction and, further, that the rate-determining step is the stepwise loss of bromine. Our results suggest that, based on photodegradation, over time, BDE-99 will remain the most abundant penta-BDE, while BDE-49 and BDE-66 will increase greatly and will be comparable in abundance to BDE-47.

Interaction of polybrominated diphenyl ethers (PBDEs) with anaerobic mixed bacterial cultures isolated from river sediment

The degradation of flame retardant polybrominated diphenyl ethers (PBDE), including tetra-brominated diphenyl ether (BDE-47), penta-brominated diphenyl ether (BDE-99 and -100), and hexa-brominated diphenyl ether (BDE-153 and -154), by anaerobic bacterial mixed cultures isolated from river sediment was investigated. The effects of PBDEs on changes of anaerobic bacterial community in sediment culture were also studied. Sediments were collected from Er-Jen River and Nan-Kan River basins, which were both heavily polluted rivers in Taiwan, and bacteria from the sediment samples were enriched before the experiment was conducted. Into the anaerobic bacterial mixed cultures, 0.1 μg/mL of PBDEs was added followed by incubation under 30 °C for 70 days. Residues of PBDE were determined by gas chromatography with electron capture detector (GC-ECD), and the changes of bacterial community were analyzed by denaturing gradient gel electrophoresis (DGGE). Less than 20% of PBDEs were degraded after 70 days of incubation in all samples except for BDE-47 from the Nan-Kan River sediment. In that culture, BDE-47 was found to have notably degraded. In particular, after 42 days of incubation; BDE-47 was degraded, suddenly and sharply, to a negligible level on Day 70, and the result was confirmed by a repeated experiment. An interesting result was that although BDE-47 was degraded fast in the Nan-Kan River sediment, the bacterial communities did not shift significantly as we had speculated at Day 70. From UPGMA dendrograms, PBDEs changed the composition of bacterial communities, and the extents varied with the variety of PBDE congeners. By the amendment with BDE-153 or -154, bacterial communities would be changed immediately and irreversibly throughout the rest of the incubation period. No significant difference in degradation of PBDEs was observed between sediment bacteria from Er-Jen River and Nan-Kan River. However, the results verified the persistence of PBDEs in the environment.

Effects of Hexabromocyclododecane and Polybrominated Diphenyl Ethers on mRNA Expression in Chicken (Gallus domesticus) Hepatocytes

Hexabromocyclododecane (HBCD) and polybrominated diphenyl ethers (PBDEs) are additive flame retardants used in a wide range of consumer products. Both compounds have been detected in free-living avian species, but toxicological and molecular end points of exposure are limited. An in vitro approach was used to compare concentration-dependent effects of HBCD and the commercial penta-brominated diphenyl ether mixture DE-71 on cytotoxicity and mRNA expression in cultured hepatocytes derived from embryonic chickens. Neither HBCD-alpha, HBCD-technical mixture (TM), nor DE-71 effected hepatocyte viability at the highest concentrations assessed (30-100 microM). Real-time RT-PCR assays were developed to quantify changes in mRNA abundance of genes associated with chicken xenobiotic-sensing orphan nuclear receptor activation, the thyroid hormone (TH) pathway, and lipid regulation. Exposure to >or= 1 microM HBCD-alpha and HBCD-TM resulted in significant upregulation of cytochrome P450 (CYP) 2H1 (fourfold to sevenfold) and CYP3A37 (5- to 30-fold) at 24 and 36 h. In contrast, 30 microM DE-71 caused a twofold increase of CYP2H1 only. UGT1A9 expression was only upregulated by HBCD-alpha to a maximum of fourfold at >or= 1 microM. Transthyretin, thyroid hormone-responsive spot 14-alpha, and liver fatty acid-binding protein were all significantly downregulated (up to sevenfold) for cells exposed to >or= 1 microM HBCD-alpha and HBCD-TM. DE-71 also downregulated these three target genes twofold to fivefold at concentrations >or= 3 microM. Taken together, our results indicate that xenobiotic-metabolizing enzymes and genes associated with the TH pathway and lipid regulation are vulnerable to HBCD and DE-71 administration in cultured avian hepatocytes and might be useful molecular markers of exposure.

Metabolism of Polybrominated Diphenyl Ethers (PBDEs) by Human Hepatocytes in Vitro

BackgroundPolybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that accumulate in human tissues and are potential toxicants. Concentrations of PBDEs in human tissues have increased recently, and body burdens in the U.S. and Canadian populations are higher than in any other region.ObjectivesAlthough metabolism in animal laboratory studies has been examined, no studies have explored the metabolism of these contaminants in human tissues. We undertook this study to determine whether PBDEs could be metabolized by human liver cells in vitro and to identify what types of metabolites are formed.MethodsWe exposed hepatocytes from three different donors (two cryopreserved batches and one fresh batch) to solutions containing 10 μM of either of two environmentally relevant and prominent PBDE congeners—BDE-99 or BDE-209—for periods of 24–72 hr. We also conducted gene expression analysis to provide information on potential induction of xenobiotic metabolizing enzymes.ResultsExposing hepatocytes to BDE-99 resulted in the formation of 2,4,5-tribromo phenol, two monohydroxylated pentabrominated diphenyl ether metabolites, and a yet unidentified tetrabrominated metabolite. No hydroxylated or debrominated metabolites were observed in the cells exposed to BDE-209. This suggests that BDE-209 was not metabolized, that nonextractable, covalently protein-bound metabolites were formed, or that the exposure time was not long enough for BDE-209 to diffuse into the cell to be metabolized. However, we observed up-regulation of genes encoding for cytochrome P450 monooxygenase (CYP) 1A2, CYP3A4, deiodinase type 1, and glutathione S-transferase M1 in hepatocyes exposed to both BDE-99 and BDE-209.ConclusionsOur in vitro results suggest that the human liver will likely metabolize some BDE congeners (e.g., BDE-99) in vivo. These metabolites have been shown to elicit greater toxicity than the parent BDE congeners in laboratory bioassays; thus, more research on body burdens and human health effects from these metabolites are warranted.

Time-dependent effects of pentabrominated diphenyl ether (PBDE) on thyroid hormones and histology in rats

In order to examine the time-dependent effects of pentabrominated diphenyl ether (BDE-99) on thyroid hormones levels and histological structure in rats, 4-week-old SD rats were divided into 10 groups randomly, according to their body weight. Rats in treatment groups were orally gavaged with BDE-99 at the dose of 60 mg kg−1 for 10, 15, 20, 30 or 40 days, while rats in control group received equal volume of corn oil. Serum levels of thyroxine (FT4), triiodothyronine (FT3), and thyroid stimulating hormone (TSH) were measured by radioimmunoassay. Sections of thyroids were dyed with hematoxylin-eosin (HE) to detect any pathologically alterations. In BDE-99 treated groups, on day 10, changes in serum levels of FT4, FT3 and TSH were not found. On days 15 and 20, serum levels of FT4 and FT3 decreased, accompanied by increased levels of TSH. On days 30 and 40, serum levels of these hormones returned to control values. As for alterations in histology, on day 15, follicular epithelium of the thyroid were found to proliferate into two layers in BDE-99 treated rats. On day 20, in focal areas the two-layer follicular epithelia were observed to continuously proliferate into 3–4 layers of follicular epithelium, called hyperplasia plaques. On days 30 and 40, solid buds and secondary follicles to solid buds were detected. The earliest alteration of thyroid hormones in treated SD rats occurred at the duration of 10–15 days, with corresponding histopathologic changes. As the exposure time increased, the serum level of hormones and histopathology altered accordingly in a time-dependent manner.

DE-71-Induced Apoptosis Involving Intracellular Calcium and the Bax-Mitochondria-Caspase Protease Pathway in Human Neuroblastoma Cells In Vitro

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.

Dietary accumulation, disposition, and metabolism of technical pentabrominated diphenyl ether (DE‐71) in pregnant mink (Mustela vison) and their offspring

Concentrations of polybrominated diphenyl ethers (PBDEs) in humans and wildlife suggest significant bioaccumulation potential in mammals, but no quantitative biomagnification data from controlled experiments are currently available. As part of a larger study examining the effects of PBDEs on mink (Mustela vison) reproduction and development, we examined congener-specific tissue distribution, maternal transfer, biotransformation, and biomagnification of the technical penta-BDE mixture, DE-71, in farmed mink. Adult female mink were fed one of four concentrations of DE-71 (0-2.5 microg/g) in the diet from breeding through gestation and until weaning at 6 weeks postparturition. Parent PBDEs were measured in tissues and excreta of adult mink, kits, and juveniles, whereas hydroxylated PBDEs (OH-PBDEs) were measured in juveniles only. Similar lipid-normalized concentrations of PBDEs were detected in most tissues of adult mink with the exception of brain, in which concentrations were significantly lower. Kits, however, had a higher proportion of PBDEs in brain compared with adults, presumably because of incomplete development of the blood-brain barrier. Maternal transfer favored lower-brominated PBDE congeners, and the bulk of the body burden in kits at weaning resulted from lactational rather than transplacental transfer. Lipid-normalized, whole-body biomagnification factors ranged from 0.5 to 5.2 for the major congeners and were highest for BDEs 47 and 153. Metabolism clearly limited the biomagnification of some PBDEs, and OH-PBDEs were detectable in plasma, liver, and feces. On a mass basis, OH-PBDEs accounted for 28 to 32% of the excreted fraction, indicating metabolism was an important elimination pathway. Further studies are required to understand the mechanisms of PBDE biotransformation.

Polybrominated Diphenyl Ethers as Endocrine Disruptors of Adipocyte Metabolism

Obesity is thought to result from poor diet and insufficient exercise. An additional factor may be endocrine-disrupting environmental chemicals that contaminate the air, water, and food supply. We tested the hypothesis that a class of lipid-soluble flame retardant chemicals known to accumulate in adipose tissue, polybrominated diphenyl ethers (PBDEs), disrupts insulin and isoproterenol sensitivity of isolated rat adipocytes. Six-week-old Sprague-Dawley rats were gavaged daily with 14 mg/kg body weight (BW) pentabrominated diphenyl ether (penta-BDE) in corn oil (n = 24) or corn oil alone (n = 24). At 2 and 4 weeks of treatment, epididymal fat pad adipocytes were isolated, and isoproterenol-stimulated lipolysis, insulin-stimulated glucose oxidation, and adipocyte size were measured. There was no alteration in adipocyte metabolism after 2 weeks of in vivo penta-BDE treatment, but after 4 weeks of treatment, adipocytes averaged a 30% increase in isoproterenol-stimulated lipolysis and a 59% decrease in insulin-stimulated glucose oxidation, compared with control. There were no differences in average rat BW and adipocyte size between treated and control rats, but plasma total thyroxine level in 2- and 4-week treated rats was 30% of control. Daily exposure of rats to 14 mg/kg BW penta-BDE for 4 weeks has no effect on animal or adipocyte size but significantly alters insulin and isoproterenol-stimulated metabolism of isolated adipocytes. These alterations, hallmark features of metabolic obesity, suggest the need for further research on the contribution of lipid-soluble, endocrine-disrupting environmental chemicals to the obesity epidemic.

Determination of polybrominated diphenyl ethers in environmental standard reference materials

Standard reference materials (SRMs) are valuable tools in developing and validating analytical methods to improve quality assurance standards. The National Institute of Standards and Technology (NIST) has a long history of providing environmental SRMs with certified concentrations of organic and inorganic contaminants. Here we report on new certified and reference concentrations for 27 polybrominated diphenyl ether (PBDE) congeners in seven different SRMs: cod-liver oil, whale blubber, fish tissue (two materials), mussel tissue and sediment (two materials). PBDEs were measured in these SRMs, with the lowest concentrations measured in mussel tissue (SRM 1974b) and the highest in sediment collected from the New York/New Jersey Waterway (SRM 1944). Comparing the relative PBDE congener concentrations within the samples, we found the biota SRMs contained primarily tetrabrominated and pentabrominated diphenyl ethers, whereas the sediment SRMs contained primarily decabromodiphenyl ether (BDE 209). The cod-liver oil (SRM 1588b) and whale blubber (SRM 1945) materials were also found to contain measurable concentrations of two methoxylated PBDEs (MeO-BDEs). Certified and reference concentrations are reported for 12 PBDE congeners measured in the biota SRMs and reference values are available for two MeO-BDEs. Results from a sediment interlaboratory comparison PBDE exercise are available for the two sediment SRMs (1941b and 1944).

Dietary and In Utero Exposure to a Pentabrominated Diphenyl Ether Mixture Did Not Affect Cholinergic Parameters in the Cerebral Cortex of Ranch Mink (Mustela vison)

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.