Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants that have been widely used in consumer products, but that are problematic because of their environmental persistence and endocrine‐disrupting properties. To date, very little is known about PBDE degradation by aerobic microorganisms and the enzymes involved in PBDE transformation. Resting cells of the polychlorinated biphenyl‐degrading actinomycete, Rhodococcus jostii RHA1, depleted nine mono‐ through penta‐BDEs in separate assays. Extensive depletion of PBDEs occurred with cells grown on biphenyl, ethylbenzene, propane, or styrene, whereas very limited depletion occurred with cells grown on pyruvate or benzoate. In RHA1, expression of bphAa encoding biphenyl dioxygenase (BPDO) and etbAa1 and etbAc encoding ethylbenzene dioxygenase (EBDO) was induced 30‐ to 3,000‐fold during growth on the substrates that supported PBDE depletion. The BPDO and EBDO enzymes had gene expression profiles that matched the PBDE‐depletion profiles exhibited by RHA1 grown on different substrates. Using the non‐PBDE‐degrading bacterium Rhodococcus erythropolis as a host, two recombinant strains were developed by inserting the eth and bph genes of RHA1, respectively. The resultant EBDO extensively depleted mono‐ through penta‐BDEs, while the BPDO depleted only mono‐, di‐, and one tetra‐BDE. A dihydroxylated‐BDE was detected as the primary metabolite of 4‐bromodiphenyl ether in both recombinant strains. These results indicate that although both dioxygenases are capable of transforming PBDEs, EBDO more potently transforms the highly brominated congeners. The availability of substrates or inducing compounds can markedly affect total PBDE removal as well as patterns of removal of individual congeners. Biotechnol. Bioeng. 2011;108: 313–321. © 2010 Wiley Periodicals, Inc.     

Isolation of Acetobacterium sp. Strain AG,Which Reductively Debrominates Octa- and Pentabrominated Diphenyl Ether Technical Mixtures

Polybrominated diphenyl ethers (PBDEs) are a class of environmental pollutants that have been classified as persistent organic pollutants since 2009. In this study, a sediment-free enrichment culture (culture G) was found to reductively debrominate octa- and penta-BDE technical mixtures to less-brominated congeners (tetra-, tri-, and di-BDEs) via a para-dominant debromination pattern for the former and a strict para debromination pattern for the latter. Culture G could debrominate 96% of 280 nM PBDEs in an octa-BDE mixture to primarily tetra-BDEs in 21 weeks. Continuous transferring of culture G with octa-/penta-BDEs dissolved in n-nonane or trichloroethene (TCE) yielded two strains (Acetobacterium sp. strain AG and Dehalococcoides sp. strain DG) that retained debromination capabilities. In the presence of lactate but without TCE, strain AG could cometabolically debrominate 75% of 275 nM PBDEs in a penta-BDE mixture in 33 days. Strain AG shows 99% identity to its closest relative, Acetobacterium malicum. In contrast to strain AG, strain DG debrominated PBDEs only in the presence of TCE. In addition, 18 out of 19 unknown PBDE debromination products were successfully identified from octa- and penta-BDE mixtures and revealed, for the first time, a comprehensive microbial PBDE debromination pathway. As an acetogenic autotroph that rapidly debrominates octa- and penta-BDE technical mixtures, Acetobacterium sp. strain AG adds to the still-limited understanding of PBDE debromination by microorganisms.     

Polybrominated diphenyl ethers as endocrine disruptors of adipocyte metabolism

Objective: 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. Research Methods and Procedures: 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. Results: 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. Discussion: 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.     

Effects of electron donors on anaerobic microbial debromination of polybrominated diphenyl ethers (PBDEs)

Polybrominated diphenyl ethers (PBDEs) are a class of widely used flame retardants that have been highly accumulated in sediments. It is reported that microorganisms play an important role in the reductive debromination of PBDEs in anaerobic sediments. However, little is known about the effects of electron donors on the microbial community structure and their debromination capacity in PBDE transformation. In this study, alternate carbon substrates were used as electron donors to enrich the PBDE-debrominating microbial consortia to evaluate the effects of electron donors on PBDE microbial debromination. Decabromodiphenyl ether (BDE-209) was found to be the dominant (more than 50%) PBDEs congener in all consortia, and the percentage of BDE-209 was deceased by 12% (methanol), 11% (ethanol), 8% (acetate), 9% (lactate), 5% (pyruvate), and 11% (no electron donors), while the relative abundances of most lesser-brominated PBDEs increased after 90-day incubation compared to the initial profile of PBDEs. Substantial shifts in the microbial community structure among different amendments were observed based on denaturing gradient gel electrophoresis results. Pseudomonas spp. were identified to be the predominant organisms and the abundances of Band R, which was associated with Pseudomonas sp. SCSWA09, was well correlated with the biodegradation rate of BDE-209. Finally, the microbial community structure was highly correlated with the concentration of deca-BDE, octa-BDE and total nitrogen. These results provide insights into in situ bioremediation of environments contaminated by PBDEs and our understanding of microbial ecology associated with PBDE-debromination.     

Binding of hydroxylated polybrominated diphenyl ethers with human serum albumin: Spectroscopic characterization and molecular modeling

Three hydroxylated polybrominated diphenyl ethers (OH‐PBDEs), 3‐OH‐BDE‐47, 5‐OH‐BDE‐47, and 6‐OH‐BDE‐47, were selected to investigate the interactions between OH‐PBDEs with human serum albumin (HSA) under physiological conditions. The observed fluorescence quenching can be attributed to the formation of complexes between HSA and OH‐PBDEs. The thermodynamic parameters at different temperatures indicate that the binding was caused by hydrophobic forces and hydrogen bonds. Molecular modeling and three‐dimensional fluorescence spectrum showed conformational and microenvironmental changes in HSA. Circular dichroism analysis showed that the addition of OH‐PBDEs changed the conformation of HSA with a minor reduction in α‐helix content and increase in β‐sheet content. Furthermore, binding distance r between the donor (HSA) and acceptor (three OH‐PBDEs) calculated using Förster's nonradiative energy transfer theory was <7 nm; therefore, the quenching mechanisms for the binding between HSA and OH‐PBDEs involve static quenching and energy transfer. Combined with molecular dynamics simulations, the binding free energies (ΔG _bind ) were calculated using molecular mechanics/Poisson ? Boltzmann surface area method, and the crucial residues in HSA were identified.     

Reductive Debromination of Polybrominated Diphenyl Ethers by Anaerobic Bacteria from Soils and Sediments

Polybrominated diphenyl ethers (PBDEs) have attracted attention recently due to their proven adverse effects on animals and their increasing concentrations in various environmental media and biota. To gain insight into the fate of PBDEs, microcosms established with soils and sediments from 28 locations were investigated to determine their debromination potential with an octa-brominated diphenyl ether (octa-BDE) mixture consisting of hexa- to nona-BDEs. Debromination occurred in microcosms containing samples from 20 of the 28 locations when they were spiked with octa-BDE dissolved in the solvent trichloroethene (TCE), which is a potential cosubstrate for stimulating PBDE debromination, and in microcosms containing samples from 11 of the 28 locations when they were spiked with octa-BDE dissolved in nonane. Debromination products ranging from hexa- to mono-BDEs were generated within 2 months. Notably, the toxic tetra-BDEs accounted for 50% of the total product. In sediment-free culture C-N-7* amended with the octa-BDE mixture and nonane (containing 45 nM nona-BDE, 181 nM octa-BDEs, 294 nM hepta-BDE, and 19 nM hexa-BDE) there was extensive debromination of the parent compounds, which produced hexa-BDE (56 nM), penta-BDEs (124 nM), and tetra-BDEs (150 nM) within 42 days, possibly by a metabolic process. A 16S rRNA gene-based analysis revealed that Dehalococcoides species were present in 11 of 14 active microcosms. However, unknown debrominating species in some of the microcosms debrominated the octa-BDE mixture in the absence of other added halogenated electron acceptors (such as TCE). These findings provide information that is useful for assessing microbial reductive debromination of higher brominated PBDEs to less-brominated congeners, a possible source of the more toxic congeners (e.g., penta- and tetra-BDEs) detected in the environment.Since they were first developed in the 1960s, polybrominated diphenyl ethers (PBDEs) have been used as flame retardant additives in an array of common household and industrial appliances. As a result of their widespread use, PBDEs have become ubiquitous environmental contaminants, and increasing levels have been detected in the air, soil, and water (5, 12). In a recent study, Leung et al. reported the highest PBDE concentrations in soil samples (2.7 to ∼4.3 ppb) and combusted residues (33.0 to ∼97.4 ppb) that were collected in Guiyu, Guangdong Province, China (18). More worrisome is the fact that increasing concentrations of PBDEs have also been detected in marine mammals, birds, fishes, and human tissues (3, 14, 20, 30), and 63 ppm of PBDEs in bird eggs is the highest level ever found in biota (23). The PBDE concentrations in both environmental samples and biota have been increasing exponentially, with a doubling time of 4 to 6 years (5, 12). Although the PBDEs comprise 209 different congeners designated 1 to 209, the PBDE congeners most often detected in biota (e.g., human tissues) include tetra-brominated diphenyl ether (tetra-BDE) (congener 47), penta-BDEs (congeners 99 and 100), and hexa-BDEs (congeners 153 and 154), which may have originated directly from a commercially available penta-BDE technical mixture or indirectly via breakdown of an octa- or deca-BDE technical mixture (10, 12). PBDEs began to receive worldwide scientific and public attention when a temporal study performed from 1972 to 1997 revealed increasing concentrations of PBDEs in Swedish human breast milk (19). Toxicological studies of rodents using a commercial penta-BDE mixture (including tetra-, penta- and hexa-BDEs) and congeners in a commercial octa-BDE mixture (such as hepta-BDE [congener 183] and octa-BDE [congener 203]) revealed developmental neurotoxicity, reproductive toxicity, liver toxicity, and disruption of thyroid hormone levels (24, 26, 29).To date, studies of PBDEs have focused mainly on detection of these compounds in the environment and their potential adverse health effects; only a few studies have reported microbial debromination of PBDEs (7, 10, 22, 25). Recently, He et al. demonstrated debromination of a technical octa-BDE mixture by pure isolates of Dehalococcoides species, which generated hepta- to di-BDEs after 6 months of incubation (10). Additionally, microbes belonging to the genera Dehalobacter and Desulfitobacterium were also found to be able to debrominate individual PBDE congeners present in commercial octa-BDE mixtures (10, 22). However, the debromination of PBDEs in both studies required the presence of a primary electron acceptor (e.g., chloroethenes or chlorophenols); in other words, debromination occurred cometabolically.In addition to debromination of PBDEs by pure cultures, a previous study demonstrated that in anaerobic sludge 5% of added deca-BDE (congener 209) was debrominated to nona- and octa-BDEs (total amount of product, 0.5 nmol) after 238 days of incubation (7). Moreover, another study showed that deca-BDE was debrominated to products ranging from nona-BDEs to hexa-BDEs in 3.5 years with anaerobic sediments as the inocula (25). These findings suggest that microbial reductive debromination of highly brominated congeners, such as deca-, nona-, octa-, and hepta-BDEs, may contribute to formation of less-brominated PBDEs in the environment, which are potentially more toxic (e.g., tetra- and penta-BDEs). Additionally, debromination of less-brominated PBDE congeners, such as di-BDE, to mono-BDE and diphenyl ether was demonstrated in a fixed-film plug flow biological reactor (21). Besides microbial debromination, highly brominated PBDEs were also found to be transformed to lower congeners via photodegradation or in vivo metabolism in aquatic and terrestrial animals (1, 16).This study was initiated to obtain information about the distribution of microorganisms capable of debrominating highly brominated PBDE congeners to more toxic daughter products or the final product diphenyl ether by assessing microcosm samples collected from various locations. Debromination of an octa-BDE mixture was evaluated in the presence of the potential energy-generating cosubstrate trichloroethene (TCE) (PBDEs dissolved in TCE) or in the presence of the relatively inert solvent nonane (PBDEs dissolved in nonane). The latter experiment provided, for the first time, information about the possible microbes living on the energy generated from the debromination of an octa-BDE mixture in the absence of any cosubstrate, such as TCE or another primer compound. Initial insights into the key debrominating microbes were obtained by using genus-specific 16S rRNA gene-based techniques.     

2,2′,4,4′,5‐Pentabromodiphenyl ether induces lipid accumulation throughout differentiation in 3T3‐L1 and human preadipocytes in vitro

Flame retardants, specifically polybrominated diphenyl ethers (PBDEs), are chemical compounds widely used for industrial purposes and household materials. NHANES data indicate that nearly all Americans have trace amounts of PBDEs in serum, with even higher levels associated with occupational exposure. PBDEs are known to bioaccumulate in the environment due to their lipophilicity and stability, and more importantly, they have been detected in human adipose tissue. The present study examined whether the PBDE congener, BDE‐99 (2,2′,4,4′,5‐pentabromodiphenyl ether; 0.2‐20 μM), enhances the adipogenesis of mouse and human preadipocyte cell models in vitro via induced lipid accumulation. 3T3‐L1 mouse preadipocytes and human visceral preadipocytes demonstrated enhanced hormone‐induced lipid accumulation upon BDE‐99 treatment. In addition, BDE‐99 (20 μM) induced preadipocyte differentiation and lipid development in nondifferentiated human preadipocytes. BDE‐99, the second most abundant congener in human adipose tissue, increased total lipids in differentiating adipocytes and therefore showed a potential role in the regulation of adipogenesis. This warrants more research to further understand the impact of lipophilic persistent pollutants on adipose tissue homeostasis.     

Pleurozium schreberi as an ecological indicator of polybrominated diphenyl ethers (PBDEs) in a heavily industrialized urban area

Polybrominated diphenyl ethers (PBDEs) are toxic contaminants with a persistent character and adverse effects on humans and wildlife. Therefore, the deposition of these chemicals in vegetation must be carefully controlled. Our objective was to determine PBDE concentrations (BDEs 28, 47, 66, 85, 99, 100, 153, 154, 183 and 209) in Pleurozium schreberi collected in a heavily industrialized urban agglomeration. High PBDE concentrations in the moss confirm the presence of active sources of atmospheric pollution in an area tested. The distribution of these xenobiotics was related to the vegetation cover being lower in sites surrounded by forests which indicates that PBDEs may have a tendency to be trapped from the air by tree leaves and needles. Congener profiles in P. schreberi were dominated by BDE 209 which was for 79% (in case of the coke smelter) to 95% (in case of the chemical plant) part of the total PBDE burden in this moss. The principal component and classification analysis classifying the concentration of PBDEs in P. schreberi allowed us to distinguish the pattern of these compounds characteristic for the origin of pollution. P. schreberi may be used as a bioindicator for PBDEs in areas contaminated with these chemicals.     

The effects of marginal maternal vitamin A status on penta‐brominated diphenyl ether mixture‐induced alterations in maternal and conceptal vitamin A and fetal development in the Sprague Dawley rat

BACKGROUND: Polybrominated diphenyl ether (PBDE) toxicity in rodents can be associated with disruptions in endocrine signaling. We previously reported that the penta‐BDE mixture, DE‐71, disrupts thyroid hormones and vitamin A metabolism in rats during lactation, and that this disruption is amplified in animals fed diets marginal in vitamin A. The ability of the DE‐71 to disrupt vitamin A metabolism during the prenatal period has not been evaluated. While penta‐BDE mixtures are not strong teratogens in pregnant animals fed standard commercial laboratory diets, we hypothesized that they could be teratogenic under conditions of marginal vitamin A status. METHODS : rats were fed diets containing 0.4 retinyl equivalents (RE, marginal) or 4.0 RE (adequate) of vitamin A per gram of diet. Pregnant animals were exposed to DE‐71 (0, 6, 18, 60, or 120 mg/kg) from gestation days (GD) 6–11.5, or on GD 6–19.5. RESULTS : DE‐71 treatment resulted in dose‐responsive reductions in maternal thyroid hormone and markers of vitamin A metabolism, with the latter reduction amplified in marginal vitamin A dams. Fetuses from marginal vitamin A, DE‐71‐exposed dams exhibited a dose‐responsive increase in liver retinol binding protein levels. DE‐71 treatment did not result in gross malformations; however, consistent with our hypothesis, GD 20 fetal weights were lower, and skeletal ossification was less when DE‐71 exposure occurred concomitant with a marginal vitamin A status. For several endpoints, observable effects were evident at the lowest dose tested, consistent with a dose‐response trend. CONCLUSIONS : The results of this study support the concept that marginal vitamin A status enhances the disruptive effects of DE‐71 during prenatal development. Birth Defects Research (Part B) 86:48‐57, 2009. © 2009 Wiley‐Liss, Inc.     

Elevated Serum Polybrominated Diphenyl Ethers and Alteration of Thyroid Hormones in Children from Guiyu,China

Informal electronic waste (e-waste) recycling results in serious environmental pollution of polybrominated diphenyl ethers (PBDEs) and heavy metals. This study explored whether there is an association between PBDEs, heavy metal and key growth- and development-related hormones in children from Guiyu, an e-waste area in southern China. We quantified eight PBDE congeners using gas chromatographic mass spectrometry, lead and cadmium utilizing graphite furnace atomic absorption spectrometry, three thyroids with radioimmunoassay and two types of growth hormones by an enzyme-linked immune-sorbent assay (ELISA) in 162 children, 4 to 6 years old, from Guiyu. In blood, median total PBDE was 189.99 ng/g lipid. Lead and cadmium concentrations in blood averaged 14.53±4.85 µg dL⁻¹ and 0.77±0.35 µg L⁻¹, respectively. Spearman partial correlation analysis illustrated that lead was positively correlated with BDE153 and BDE183. Thyroid-stimulating hormone (TSH) was positively correlated with almost all PBDE congeners and negatively correlated with insulin-like growth factor binding protein-3 (IGFBP-3), whereas free triiodothyronine (FT3) and free thyroxine (FT4) were negatively correlated with BDE154. However, no correlation between the hormones and blood lead or cadmium levels was found in this study. Adjusted multiple linear regression analysis showed that total PBDEs was negatively associated with FT3 and positively associated with TSH. Notably, FT4 was positively correlated with FT3, house functions as a workshop, and father''s work involved in e-waste recycling and negatively correlated with vitamin consumptions. TSH was negatively related with FT4, paternal residence time in Guiyu, working hours of mother, and child bean products intake. IGFBP-3 was positively correlated with IGF-1 and house close to an e-waste dump. These results suggest that elevated PBDEs and heavy metals related to e-waste in Guiyu may be important risk factors for hormone alterations in children.     

Embryonic exposure to environmentally relevant concentrations of a brominated flame retardant reduces the size of song‐control nuclei in a songbird

Environmental contaminants have the potential to act as developmental stressors and impair development of song and the brain of songbirds, but they have been largely unstudied in this context. 2,2′,4,4′,5‐Pentabromodiphenyl ether (BDE‐99) is a brominated flame retardant congener that has demonstrated endocrine disrupting effects, and has pervaded the global environment. We assessed the effects of in ovo exposure to environmentally relevant levels of BDE‐99 on the neuroanatomy of the song‐control system in a model songbird species, the zebra finch (Taeniopygia guttata). Embryos were exposed via egg injection to a vehicle control (DMSO), 10, 100, or 1000 ng BDE‐99/g egg on the day the egg was laid. Chicks were raised to sexual maturity to investigate long‐term effects of BDE‐99 on the adult male brain. Three key song‐control nuclei (Area X, HVC, RA) all showed a dose‐dependent trend toward decreasing volume as BDE‐99 concentration increased, and birds exposed to 1000 ng/g in ovo BDE‐99 had significantly smaller song‐control nuclei volume compared to control birds. High environmental concentrations of BDE‐99 in avian tissues can be within that range and thus could affect development of the song‐control system in birds, and potentially other processes. We previously found that BDE‐99 exposure during the nestling period had no effect of on the song‐control system, although it did have significant effects on some behaviural endpoints. Taken together, these results suggest that exposure to polybrominated diphenyl ether (PBDEs) during critical developmental windows can significantly alter neurological development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

Biodegradation of 2,4,6-Tribromophenol by Ochrobactrum sp. Strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 μM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Aerobic debromination of deca-BDE: Isolation and characterization of an indigenous isolate from a PBDE contaminated sediment

The environmental safety of decabromodiphenyl ether (deca-BDE) has been the topic of controversial discussions during the recent years. Reductive debromination of deca-BDE in the environment was proved to be a significant source of lower-brominated Polybrominated diphenyl ethers (PBDEs) to the ecosystem. Currently, very little is known about the susceptibility of deca-BDE to aerobic biotransformation. Lysinibacillus fusiformis strain DB-1, an aerobic bacterium capable of debromination of deca-BDE, was isolated from sediments of LianjiangRiver, Guiyu in Guangdong of China. DB-1 can efficiently transform deca-BDE to lower brominated BDEs using carbon sources such as lactate, pyruvate and acetate, respectively. In liquid cultures, free bromide concentration accumulated to 1220 ??g L⁻¹ with 6 mg L⁻¹ of the nominal initial concentration of deca-BDE after 72 h aerobic incubation. The resting cell activity tests showed that debromination of deca-BDE by DB-1 was an aerobic process. This is the first report for biotransformation of deca-BDE by an indigenous bacterium isolated from PBDEs contaminated environment.     

Optimization of the determination of polybrominated diphenyl ethers in human serum using solid-phase extraction and gas chromatography-electron capture negative ionization mass spectrometry

A simple, rapid, sensitive and reproducible method based on solid-phase extraction (SPE) and acidified silica clean-up was developed for the measurement of 12 polybrominated diphenyl ethers (PBDEs), including BDE 209, and 2,2',4,4',5,5'-hexabromobiphenyl (BB 153) in human serum. Several solid-phase sorbents (Empore C(18), Isolute Phenyl, Isolute ENV+ and OASIS HLB) were tested and it was found that OASIStrade mark HLB (500 mg) gives the highest absolute recoveries (between 64% and 95%, R.S.D.<17%, n=3) for all tested analytes and internal standards. Removal of co-extracted biogenic materials was performed using a 6 ml disposable cartridge containing (from bottom to top) silica impregnated with sulphuric acid, activated silica and anhydrous sodium sulphate. PBDEs and BB 153 were quantified using a gas chromatograph coupled with a mass spectrometer (MS) operated in electron-capture negative ionization mode. The method limits of quantification (LOQ) ranged between 0.2 and 25 pg/ml serum (0.1 and 4 ng/g lipid weight). LOQs were dependent on the analyte levels in procedural blanks which resulted in the highest LOQs for PBDE congeners found in higher concentrations in blanks (e.g. BDE 47, 99 and 209). The use of OASIS HLB SPE cartridge allowed a good method repeatability (within- and between-day precision<12% for all congeners, except for BDE 209<17%, n=3). The method was applied to serum samples from a random Belgian population. The obtained results were within the range of PBDE levels in other non-exposed population from Europe.     

Identification and biodegradation efficiency of a newly isolated 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) aerobic degrading bacterial strain

Polybrominated diphenyl ethers (PBDEs) are bioaccumulative, toxic and persistent, globally distributed organic chemicals in environment. However, very little is known for their aerobic biodegradation. In this research, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was selected as a model congener of PBDEs to study its aerobic biodegradation. A new BDE-47 degrading strain BFR01 identified as Pseudomonas stutzeri was isolated from polluted soil in a former brominated flame retardant production corporation. Stain BFR01 could utilize BDE-47 as a sole source of carbon and energy, and transformed 97.94% of BDE-47 in two weeks; the biodegradation of BDE-47 fitted well with the first-order kinetics, with the first-order kinetics constant of 0.32 d⁻¹. The biodegradation efficiency of stain BFR01 was higher than other reported PBDEs aerobic degrading bacteria. The biodegradation efficiency achieved maximum at pH 7.0 and 40 °C. The presence of additional carbon sources could enhance the biodegradation efficiency of BDE-47 by 1–6%. Furthermore, no lower brominated diphenyl ethers or biphenyl were detected, suggesting that the pathway of BDE-47 biodegradation by strain BFR01 might not be debromination with lower brominated diphenyl ethers as products. This is the first report of aerobic degradation of BDE-47 by P. stutzeri.     

The LpxL acyltransferase is required for normal growth and penta‐acylation of lipid A in Burkholderia cenocepacia

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa‐acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra‐ and penta‐acylated lipid A. “Late” acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2′ position of lipid A resulting in penta‐acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β ‐hydroxymyristate (3‐OH‐C14:0) chain at the 3 position, leading to tetra‐acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta‐acylated lipid A, suggesting lipid A penta‐acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.     

Multi‐spectroscopic and molecular dynamics simulations investigation of the binding mechanism of polybrominated diphenyl ethers to hen egg white lysozyme

Three PBDEs (BDE25, BDE47, and BDE154) were selected to investigate the interactions between PBDEs and hen egg white lysozyme (HEWL) by molecular modeling, fluorescence spectroscopy, and FT‐IR spectra. The docking results showed that hydrogen bonds were formed between BDE25 and residue TRP63 and between BDE47 and TRP63 with bond lengths of 2.178 Å and 2.146 Å, respectively. The molecular dynamics simulations indicated that van der Waals forces played a predominant role in the binding of three PBDEs to HEWL. The observed fluorescence quenching can be attributed to the formation of complexes between HEWL and PBDEs, and the quenching mechanism is a static quenching. According to Förster's non‐radiative energy transfer theory, the binding distances r were < 7 nm, indicating a high probability of energy transfer from HEWL to the three PBDEs. The synchronous fluorescence showed that the emission maximum wavelength of tryptophan (TRP) residues emerged a red‐shift. FT‐IR spectra indicated that BDE25, BDE47 and BDE154 induced the α‐helix percentage of HEWL decreased from 32.70% ± 1.64% to 28.27% ± 1.41%, 27.50% ± 1.38% and 29.78% ± 1.49%, respectively, whereas the percentage of random coil increased from 26.67% ± 1.33% to 27.60% ± 1.38%, 29.18% ± 1.46% and 30.59% ± 1.53%, respectively.     

Biodegradation of diphenyl ether and transformation of selected brominated congeners by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. PH-07

Polybrominated diphenyl ethers (PBDEs) are common flame-retardant chemicals that are used in diverse commercial products such as textiles, circuit boards, and plastics. Because of the widespread production and improper disposal of materials that contain PBDEs, there has been an increasing accumulation of these compounds in the environment. The toxicity and bioavailability of PBDEs are variable for different congeners, with some congeners showing dioxin-like activities and estrogenicity. The diphenyl ether-utilizing bacterium Sphingomonas sp. PH-07 was enriched from activated sludge of a wastewater treatment plant. In liquid cultures, this strain mineralized 1 g of diphenyl ether per liter completely within 6 days. The metabolites detected and identified by gas chromatography/mass spectrometry (MS) and electrospray ionization/MS analysis corresponded with a feasible degradative pathway. However, the strain PH-07 even catabolized several brominated congeners such as mono-, di-, and tribrominated diphenyl ethers thereby producing the corresponding metabolites.     

Aberrant 5’-CpG Methylation of Cord Blood TNFα Associated with Maternal Exposure to Polybrominated Diphenyl Ethers

Growing evidence suggests that maternal exposures to endocrine disrupting chemicals during pregnancy may lead to poor pregnancy outcomes and increased fetal susceptibility to adult diseases. Polybrominated diphenyl ethers (PBDEs), which are ubiquitously used flame-retardants, could leach into the environment; and become persistent organic pollutants via bioaccumulation. In the United States, blood PBDE levels in adults range from 30–100 ng/g- lipid but the alarming health concern revolves around children who have reported blood PBDE levels 3 to 9-fold higher than adults. PBDEs disrupt endocrine, immune, reproductive and nervous systems. However, the mechanism underlying its adverse health effect is not fully understood. Epigenetics is a possible biological mechanism underlying maternal exposure-child health outcomes by regulating gene expression without changes in the DNA sequence. We sought to examine the relationship between maternal exposure to environmental PBDEs and promoter methylation of a proinflammatory gene, tumor necrosis factor alpha (TNFα). We measured the maternal blood PBDE levels and cord blood TNFα promoter methylation levels on 46 paired samples of maternal and cord blood from the Boston Birth Cohort (BBC). We showed that decreased cord blood TNFα methylation associated with high maternal PBDE47 exposure. CpG site-specific methylation showed significantly hypomethylation in the girl whose mother has a high blood PBDE47 level. Consistently, decreased TNFα methylation associated with an increase in TNFα protein level in cord blood. In conclusion, our finding provided evidence that in utero exposure to PBDEs may epigenetically reprogram the offspring’s immunological response through promoter methylation of a proinflammatory gene.     

Reductive dehalogenation of brominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S

Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S are anaerobes that can utilize tetrachloroethene (PCE) as an electron acceptor in their energy metabolism. The end‐product of PCE reduction for both organisms is cis‐1,2‐dichloroethene, which is formed via trichloroethene as the intermediate. The bacteria were able to dehalogenate cis‐ and trans‐1,2‐dibromoethene (cDBE and tDBE) in growing cultures and cell extracts. Dibromoethene supported growth of both organisms. The organisms debrominated cDBE and tDBE to vinyl bromide (VB); D. hafniense PCE‐S also produced ethene in addition to VB. The PCE reductive dehalogenases (PCE dehalogenases) of S. multivorans and D. hafniense PCE‐S mediated the debromination of tribromoethene (TBE) and both isomers of 1,2‐DBE, indicating that this enzyme was responsible for the reductive dehalogenation of brominated ethenes. cDBE, tDBE, 1,1‐DBE and VB were formed upon TBE debromination; VB was the major end‐product. The PCE dehalogenase of D. hafniense PCE‐S also formed ethene. With the purified enzymes from both organisms the kinetic properties of dehalogenation of brominated alkenes were studied and compared with those of their chlorinated analogues.