ARTICLE
pubs.acs.org/est
Toxicogenomic Mechanisms of 6-HO-BDE-47, 6-MeO-BDE-47,
and BDE-47 in E. coli
Guanyong Su,† Xiaowei Zhang,†,* Hongling Liu,† John P. Giesy,†,‡,§,|| Michael H. W. Lam,§ Paul K. S. Lam,§
Maqsood A. Siddiqui,|| Javed Musarrat,|| Abdulaziz Al-Khedhairy,|| and Hongxia Yu†,*
†
)
State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing,
People's Republic of China
‡
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3,
Canada
§
State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue,
Kowloon, Hong Kong SAR, China
Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
bS Supporting Information
ABSTRACT: Cytotoxicity of 6-HO-BDE-47 and its two analogues, BDE-47
and 6-MeO-BDE-47, and the associated molecular mechanisms were assessed
by use of a live cell reporter assay system which contains a library of 1820
modified green fluorescent protein (GFP) expressing promoter reporter
vectors constructed from E. coli K12 strains. 6-HO-BDE-47 inhibited growth
of E. coli with a 4 h median effect concentration (EC50) of 22.52 ( 2.20 mg/L,
but neither BDE-47 nor 6-MeO-BDE-47 were cytotoxic. Thus, 6-HOBDE-47 might serve as an antibiotic in some living organisms. Exposure to
6-HO-BDE-47 resulted in 65 (fold change >2) or 129 (fold change >1.5)
genes being differentially expressed. The no observed transcriptional effect
concentration (NOTEC) and median transcriptional effect concentration
(TEC50) based on transcriptional end points, of 6-HO-BDE-47 were 0.0438
and 0.580 mg/L, respectively. The transcriptional responses were 514- and
39-fold more sensitive than the acute EC50 to inhibit cell growth. Most of the
genes that were differentially expressed in response to 6-HO-BDE-47 were
not modulated by BDE-47 or 6-MeO-BDE-47. These results suggest that
cytotoxicity of 6-HO-BDE-47 to E. coli was via a mechanism that was different
from that of either BDE-47 or 6-MeO-BDE-47. Gene expression associated with metabolic pathways was more responsive to 6-HOBDE-47, which suggests that this pathway might be the primary target of this compound.
’ INTRODUCTION
Polybrominated diphenyl ethers (PBDEs) have been widely
used for many years as flame retardants in various commercial
products, such as furniture, textiles, plastics, paints, and electronic appliances due to their low cost and high-performance.1,2
Because of their persistence and bioaccumulation potential,3
PBDEs have been reported in various environmental matrices
and their environmental concentrations have continuously
increased.4 HO-PBDEs and MeO-PBDEs were identified as
potential transformation products of PBDEs, which have also
been detected in humans,5,6 especially 6-HO-BDE-47, 5-HOBDE-47 and 50 -HO-BDE-99. This raised concern about the
potential toxicity of these transformation products of PBDE
and their modes of molecular toxicity. Some early studies of HOPBDEs found that for some end points they were more potent
than the postulated precursor PBDEs and corresponding MeOPBDEs.7 Recently, it has been demonstrated that HO-PBDEs,
r 2011 American Chemical Society
especially 6-HO-BDE-47, are not produced from synthetic
PBDEs, but rather from naturally occurring MeO-PBDEs.810
Specifically, demethylation of 6-MeO-BDE-47 was the primary
transformation pathway leading to formation of 6-HO-BDE-47
in medaka, while the previously hypothesized formation of HOPBDEs from synthetic BDE-47 did not occur.8 The fact that HOPBDEs, including 6-HO-BDE-47, were detected in human
blood,5 led to interest in the potential of MeO-PBDEs and
HO-BDEs to modulate gene expression and lead to toxicity.
Assessment of PBDEs and their analogues have focused on
their nuclear hormone receptor activity, 7,1113 reproductive
effects,14 or neurotoxicity. 15 However, the general toxicity
Received: September 13, 2011
Accepted: November 23, 2011
Revised:
November 14, 2011
Published: November 23, 2011
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mechanisms of these brominated compounds have scarcely been
investigated.
Genome-wide transcriptional investigations, using whole cell
arrays, is a powerful toxicogenomic approach for evaluating the
toxicity of chemicals.16,17 The microbial, whole cell array consists
of an assortment of genetically engineered microorganisms
tailored to respond to the activation of specific promoters. The
fusion of stress promoters to reporter genes, such as fluorescence
protein; GFP, provides the basic concept of cellular signals
detection.18 Compared with microarray technology, the use of
whole cell arrays avoids complex protocols of pretreatment, highcost experimental materials, less interference and lack of temporal
resolution, but can achieve comparable results.19 Furthermore,
their short testing time makes these arrays a rapid, portable and
economical, high-throughput biosensor system for detecting
environmental toxicities. Previous work in our laboratory 16,17
has shown that these reporter fusions allow accurate measurements
of the input-output relationship of gene circuits in living cells,
which might not be bacteria-specific and can represent responses
of systems that are conserved in multiple organisms, including
metazoans.20
Despite extensive information on mechanisms of toxicity of
PBDEs, MeO-PBDEs, and HO-PBDEs, less information was
available for 6-HO-BDE-47, which occurs at relatively great
concentrations in the environment and has recently been found
in human blood. Therefore, mechanisms of modulation of gene
expression by 6-HO-BDE-47 were assessed by use of a live cell
reporter assay system with library of 1820 modified GFP
expressing promoter reporter vectors constructed from E. coli
K12 strains. For comparative purposes, mechanisms of toxicity of
BDE-47 and 6-MeO-BDE-47 were assessed at the same time.
The No Observed Transcriptional Effect Concentration
(NOTEC) was used as one measurement end point in interpreting potency of individual compounds. Pathway analysis and
transcriptional network were also conducted to determine the
possible mechanisms of toxicity of 6-HO-BDE-47.
’ MATERIALS AND METHODS
Chemicals and Reagents. 6-HO-BDE-47 and 6-MeO-BDE47 were synthesized in the Department of Biology and Chemistry of City University of Hong Kong following previously
published methods.21 Purities of the synthesized compounds
were >98%. The synthesis procedure did not generate any
brominated dioxin and/or furan contaminants based on proton
NMR and electrospray-MS identity analysis of the intermediates
and end products.12 BDE-47 (>98.5% purity) was from Chem
Service (Lot 41421B, West Chester, PA).
Microbial Live Cell Array. Assessment of effects of chemicals
by use of microbial live cell array has already been described in
detail in our previous publications.16 The library of 1900 promoter strains in the entire genome of E. coli K12 was produced by
researchers at the Weizmann Institute of Science (Rehovot,
Israel).20 Each of the reporter strains is coupled with a bright,
fast-folding green fluorescent protein (GFP) fused to a fulllength copy of an E. coli promoter in a low-copy plasmid and
contains a kanamycin resistance gene. Because of this, genomewide transcriptional investigations have a decisive competitive
advantage, which enables measurement of gene expression within minutes with high accuracy and reproducibility. It is also
suitable for rapid screening of numerous chemicals, which is now
in progress in our laboratory.
ARTICLE
Cytotoxicity. Stock solutions of test chemicals (2000 mg/mL)
were prepared in dimethyl sulfoxide (DMSO; Tedia, U.S.) and
other stock solutions were made by serial dilution with DMSO.
Nine different concentrations of 6-HO-BDE-47 (210, 100, 25,
6.4, 1.6, 0.39, 0.098, 0.024, 0.006 mg/L, respectively) (n = 3)
were used in the E. coli cytotoxicity test. Because of their small
solubility in water, 25 mg/L was selected as the maximal
concentration for BDE-47 and 6-MeO-BDE-47. After 4 or 24 h
of incubation at 37 °C, growth and division of E. coli was
determined by measurement of OD at 600 nm, by use of a
Synergy H4 hybrid microplate reader (BioTek Instruments
Inc., Winooski, VT). In parallel, 10 μL Alamar blue (Beijing
CellChip Biotechnology Inc., Beijing, BJ) was added to 150 μL
LB medium for each well to assess cell viability after 3 h
incubation. Alamar blue was known to be nontoxic to cells.
After dyeing for 1 h with Alamar blue, the blue-red fluorescence
was detected by a Synergy H4 hybrid microplate reader (excitation/
emission: 545 nm/590 nm) (BioTek Instruments Inc., Winooski,
VT).
Chemical Exposure. Exposure was done with a slight modification of our previously described methods.16 Strains of E. coli
were inoculated into a fresh 96-well plate from a 96-well stock
plate by use of disposable replicators (Genetix, San Jose, CA, U.S.).
Cells were incubated at 37 °C for 3.0 h in 96-well plate and then
transferred into 384-well plates. Finally, 3.79 μL of DMSO
(solvent control) or chemical stock solutions were added into
individual wells on the 384-well plates to make a final concentration of 0, 0.09, 0.9, and 9 mg chemical/L. GFP intensity of each
well was consecutively monitored every 10 min for 4 h by a
Synergy H4 hybrid microplate reader (excitation/emission:
485 nm/528 nm) (BioTek Instruments Inc., Winooski, VT).
Data Analyses. A linear regression model was applied to select
the promoter reporters of which expression was significantly
different after exposure to the chemicals. The response measured
as GFP fluorescence was fitted to a function of time for each
promoter reporter strain. Through the data analyses process, a
p value less than 0.001 was considered significant. All of the data
analysis procedures have been previously described.16,17
Determination of Transcriptional End Points. The No
Observed Transcriptional Effect Concentration (NOTEC) was
calculated based on the number of 1820 promoter strains that
were significantly altered by 6-HO-BDE-47. First, the 4 h EC20
based on inhibition of cell growth determined by optical density
at a wavelength of 600 nm (OD600) was selected as the maximal
concentration of each chemical to be used in the exposure study
for gene expression analysis. The percentage of genes differentially expressed at each concentrations compared to the number
of differentially altered genes at the EC20 of 6-HO-BDE-47 was
calculated. Finally, a linear model was fitted to assess the concentration-dependent response curve of the percentage of the
differentially expressed genes. The NOTEC was calculated as the
maximum concentration of 6-HO-BDE-47 at which less than 5%
of the genes are differentially expressed in when exposed to
6-HO-BDE-47 relative to that of the control.17,22 The median
transcriptional effect concentration (TEC50) was calculated as
the maximum concentration of 6-HO-BDE-47 at which fewer
than 50% of the genes were differentially expressed compared to
the EC20 of 6-HO-BDE-47.
Pathway Analysis. All of procedures used for analysis of
the data have been previously described.16 Gene lists were
developed for further analysis based on statistical significance
and use of a 1.5 or 2.0 fold-change cutoff. Annotations of
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Figure 1. Inhibition of cell division in E. coli by concentrations of 6-HOBDE-47 and its analogues after 4 h exposure. Values are shown with
mean. “A” means absorbance (600 nm); “AB” means alamar blue
accumulation.
differentially expressed genes were obtained from genes databases (www.ecogene.org, www.geneontology.org, and www.ecoliwiki.net) (See Table S1 of the Supporting Information, SI).
Pathway analysis was conducted by use of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The transcriptional
network was constructed by use of Cytoscape, which is an open
source bioinformatics software platform.23,24
’ RESULTS AND DISCUSSION
E. coli Cytotoxicity Test. After a 4 h exposure, no inhibition of
cell division was observed for 6-MeO-BDE-47 or BDE-47 at
concentrations ranging from 0 to 25 mg/L. However, 6-HOBDE-47 caused significantly less cell division of E. coli cells in a
concentration-dependent manner (Figure 1). By using both
absorbance as a measure of the total number of cells and Alamar
blue as a measure of viability of cells, median effect concentration
(EC50) of 6-HO-BDE-47 were 22.52 ( 2.20 and 12.13 ( 1.12 mg/L,
respectively. These results suggested that Alamar blue was the
more sensitive end point and cells treated by 6-HO-BDE-47 were
unable to produce enough energy to proliferate 25 before the
absorbance showed its down-trend. Most of the ECx after 24 h
exposure were less than that after 4 h exposure, which means
that 6-HO-BDE-47 could be toxic at even lesser concentrations if
the exposure time was longer (Table S1 of the SI). Finally, 0.09,
0.9, and 9 mg 6-HO-BDE-47/L were then selected to assess the
transcriptional expression profiles of E. coli.
Gene Expression Profiles by 6-HO-BDE-47. Expression of
genes in the microbial reporter stains was modulated by 6-HOBDE-47 in a time- and concentration-dependent manner. Exposure to 6-HO-BDE-47 resulted in fewer up-regulated genes
than down-regulated genes within the 4 h exposure for either
1.5-fold cutoff (see Figure S1 of the SI) or 2-fold cutoff (Figure 2)
genes. Of the 65 genes selected using a 2-fold cutoff, exposure to
6-HO-BDE-47 resulted in up-regulation of 11 and downregulation
ARTICLE
of 54 genes. For the 129 genes selected by 1.5-fold cutoff, 32 and 97
genes were up- and down-regulated, respectively.
The numbers of genes altered by exposure to 6-HO-BDE-47
were concentration-dependent. Using a 2.0-fold change as a
cutoff, exposure to 0.09, 0.9, and 9 mg/L of 6-HO-BDE-47
significantly altered the activity of 11, 17, and 60 promoters,
respectively. And 5 strains were responsive to all three concentrations (Figure 3A). A total of 129 genes were differentially
expressed with a maximum absolute fold change of at least 1.5,
where 40, 55, and 116 genes were differentially expressed after
exposure to 0.09, 0.9, and 9 mg/L of 6-HO-BDE-47, respectively,
and 24 strains were responsive to all three concentrations
(Figure 3B). At a concentration of 0.09 mg 6-HO-BDE-47/L,
three toxin-induced genes, yccF, ygbA, and yddM, had completely
different expression profiles from these in cells exposed to 0.9 or
9 mg/L. This demonstrates that expression of genes was a
concentration-dependent phenomenon.22 Using either a 1.5-fold
or a 2.0-fold cutoff, the number of genes altered by 6-HO-BDE47 was proportional to concentration of 6-HO-BDE-47 (see
Figure S2 of the SI).16
On the basis of the number of differentially expressed genes,
the transcriptional end points, NOTEC and TEC50, for effects of
6-HO-BDE-47 in E. coli were 0.0438 and 0.580 mg/L (Figure S2
of the SI). These end points were 514- and 39-fold less than the
4 h median effect concentration (EC50) on E. coli cell growth
inhibition, and 274- and 21-fold more sensitive than the 24 h
EC50 on E. coli cell growth inhibition. This is consistent with
transcriptional end points representing a sensitive end point to
assess chemical toxicity and be more protective of other species.
The NOTEC is an emerging measurement end point in chemical
toxicity assessment. Since it reflects sublethal, molecular responses to a toxicant, the NOTEC has been observed to be less
than concentrations associated with conventional end points.
Thus, the NOTEC has recently been proposed to be a potentially
useful end point and regulatory bench mark for chemical screening and effluent toxicity testing.17,22
Toxicity Pathway by 6-HO-BDE-47. On the basis of the
Kyoto Encyclopedia of Genes and Genomes (KEGG), multiple
pathways were identified as being most responsive to 6-HOBDE-47 during the 4 h exposure. These pathways included the
metabolic pathway, phosphoenolpyruvate (PEP)-dependent
phosphotransferase system (PTS), and aminoacyl-tRNAs, and
stress responsive pathways. Using a 2-fold change cutoff, 10
differentially expressed genes were observed to be in the metabolic pathway. Of these, fdhF and katG were up-regualted, and
dcd dgkA lacZ lpxC manX moaA pepD were down-regulated. In
biochemistry, chemical reactions of metabolism are organized
into metabolic pathways, in which one chemical is transformed
through a series of steps into another chemical, by a sequence of
enzymes. 6-HO-BDE-47 modulated expression of these metabolic
pathways might be due to it being difficult to be transformed into
other corresponding products.8 Exposure to 6-HO-BDE-47 might
disrupt the metabolic balance. Metabolic pathways are a series of
chemical reactions occurring within a cell and important to the
maintenance of homeostasis.26 The PEP-dependent PTS altered
by 6-HO-BDE-47 is a mechanism used by bacteria for uptake of
carbohydrates, particularly hexoses, hexitols, and disaccharides.27
After exposure to 6-HO-BDE-47 for 4 h some proteins associated with the PTS, including mannose-specific enzyme IIA component (manX), glucitol/sorbitol-specific IIB component (srlA)
and trehalose-specificenzyme IIBC component (treB) were downregulated. Aminoacyl-tRNAs is another pathway modulated by
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ARTICLE
Figure 2. Real-time, quantitative determination of mRNA abundances as measures of differentially expressed genes in E. coli. Clustering of the timedependent expression of the 6-HO-BDE-47 altered genes selected by 2-fold change cutoff. Exposures to 0.09, 0.9, and 9 mg 6-HO-BDE-47/L were
represented by the lower, middle, and upper bands in each gene column. Classification and visualization of the gene expression were derived by use of
ToxClust.36 The dissimilarity between genes was calculated by the Manhattan distance between the gene expressions at all the concentration vs time
combinations. The fold change of gene expression is indicated by color gradient, and the time course of expression changes is indicated from left to right.
6-HO-BDE-47, which is important for translation and pivotal in
determining how the genetic code is interpreted as amino acids.
Aminoacyl-tRNAs precisely match amino acids with tRNAs containing the corresponding anticodon. Three genes, ileX, lysU, and
serU, involved in this pathway were down-regulated more than
2-fold after exposure to 6-HO-BDE-47. These genes encode
isoleucine tRNA 2, lysine tRNA synthetase and serine tRNA 2.
Down-regulation of these genes might inhibit the delivery of
aminoacids to the ribosome where it will be incorporated into
the polypeptide chain that is being produced. Expression of four
stress responsive genes, bolA, katG, ybgI and yhjX, was altered more
than 2-fold by 6-HO-BDE-47. These genes can be divided into four
categories: general stress (bolA), redox stress (katG), general
function (ybgI) and drug resistance/sensitivity (yhjX).19 bolA is
related to disturbance of the biochemical and biophysical homeostasis of the cell. katG can increase concentrations of superoxides
and peroxides, and other conditions which alter redox potential
of the cell. Specifically, uspA has been suggested to be related to
chemical-induced stress.
Transcriptional Network by 6-HO-BDE-47. A total of 298
genes were significantly modulated by 6-HO-BDE-47 (p <
0.001). Genes modulated by exposure to 6-HO-BDE-47 were
predominately regulated through several transcriptional factors,
including ada, alsR, bolA, crp, cspA, cytr, deoR, envY, evgA, fur,
galR, galS, lsrR, torR, and yacH. Of these 298 modulated genes, 35
can be directly regulated by transcriptional factor cAMP receptor
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protein (crp) and 5 was directly regulated by another transcriptional factor (fur) (Figure S3 of the SI). Using a 2-fold cutoff, 13
and 7 genes were down- and up-regulated by crp, and the 3 genes
were up-regulated by fur. Transcriptional regulation of crpdependent genes requires the binding of cAMP and crp protein
Figure 3. Concentration-dependent expression of genes of E. coli
exposed to 6-HO-BDE-47. (Venn diagram displayed the differentially
expressed genes with the maximum fold change over 1.5- or 2.0-fold at
three different 6-HO-BDE-47 concentrations (0.09, 0.9, and 9 mg/L),
which were marked with red, green, and blue, respectively).
ARTICLE
to DNA, which causes a conformational change to allow the
protein to bind tightly to a specific DNA sequence in the promoters of the genes it controls.28 Genes activated by cAMP-crp
can be grouped into two groups. The first group of crp-dependent genes require one cAMP-crp for activation, while genes in
the second group require multiple activator molecules in which
two or more CAP dimersor one crp dimer and additional
activator proteins synergistically activate transcription.29 Furthermore, transcriptional factors fur, which can be directly
regulated by crp, also showed a down-regulation after exposure
of 6-HO-BDE-47. As a ferric iron uptake global transcriptional
repressor, fur could be activated by Fe2+ and was regard as zinc
metalloprotein, and its down-regulation might be related to upregulation of 3 different genes (sodB, entD, and katG) involved in
formation of three enzymes, including superoxide dismutase,
enterochelin synthetase, catalase, which also indicated that fur
could directly or indirectly regulates transcription of these genes
(sodB katG) encoding antioxidant enzymes.30
Comparison among BDE-47, 6-MeO-BDE-47, and 6-HOBDE-47. Expressions of the 54 modulated genes (see Table S2,
Supporting Information) by 6-HO-BDE-47 were also observed
after exposure to 0.09, 0.9 or 9 mg/L of 6-MeO-BDE-47 or
BDE-47, respectively (Figure 4). When exposed to BDE-47, four
Figure 4. Comparison of gene expression after exposure to BDE-47, 6-MeO-BDE-47, and 6-HO-BDE-47.
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genes, mipA, deoR, lacZ, and ucpA, were modulated significantly
from 1.5-fold cutoff, and two genes, deoR and mipA, had over
2-fold expression changes. All of these genes were up-regulated
by BDE-47. However, three of them, except for deoR, were downregulated after exposure to 6-HO-BDE-47. When exposed to
6-MeO-BDE-47, only pepD and mscS had over 1.5-fold significant changes, but none of these changes was greater than 2-fold.
Both of them were up-regulated by 6-MeO-BDE-47. However,
pepD showed an up-regulation profile after exposure of 6-HOBDE-47.
The pattern of differential gene expression caused by 6-HOBDE-47 was different than that caused by BDE-47 or 6-MeOBDE-47. Thus, different pathways were affected by the three
chemicals and their modes of toxic action are different. Among
these four genes, expression of which was up-regulated by BDE47, mipA deoR lacZ and ucpA, only lacZ is in the metabolic
pathway, which was identified as being most responsive to 6-HOBDE-47. Alternatively, expressions of these genes were downregulated by 6-HO-BDE-47. β-galactosidase (lacZ) is a hydrolase
enzyme that catalyzes the hydrolysis of β-galactosides into
monosaccharides. Since lacZ is an enzyme associated with
senescence, its down-regulation is consistent with E. coli cells
being senescent after exposure to BDE-47.31 The other three
genes encoded scaffolding protein for murein-synthesizing holoenzyme (mipA), transcriptional repressor for nucleotide catabolism (deoR) and putative acetoin dehydrogenase (ucpA), but
their actual mechanism is still unknown. Two genes, pepD and
mscS, were altered less than 2-fold by 6-MeO-BDE-47. PepD
encodes peptidase D, which functions as a dimer of pepD
monomers. Transcription of pepD increased after exposure to
6-MeO-BDE-47,32 which means that this chemical might cause
phosphate starvation. mscS was one of four classes of E. coli
mechanosensitive channels, which indicated that 6-MeO-BDE47 might cause disturbance to cross-linking of the C-terminus or
adding Ni2+ to C-terminally, hexahistidine tagged proteins.33
Natural Occurrence. Both HO-PBDEs and MeO-PBDEs
were mostly produced by marine organisms,10,34 as are a number
of other brominated compounds. One question is why concentrations of HO-PBDEs were lower in marine organisms.35 In our
study, that 6-HO-BDE-47 could cause cytotoxicity to bacteria
more easily and alter more genes expression rather than BDE-47
or 6-MeO-BDE-47. The metabolic pathway was identified as being
most responsive to 6-HO-BDE-47. Therefore, it was speculated
that up-regulation of the metabolic pathway might result in HOPBDEs’ lesser concentrations in organisms. The results of this study
also suggest that 6-MeO-BDE-47 and the subsequently produced
6-HO-BDE-47 might function as an antibiotic in some marine
organisms.
The other question is how are these compounds synthesized
by marine organisms? Synthesis of chemicals requires energy and
it can be postulated that the synthesis of any compounds should
impart some survival fitness to organisms that synthesize them.
Currently, it is unknown why 6-MeO-BDE-47 is synthesized by
marine organisms. In fact, it is not known exactly which organisms are responsible for the synthesis of these compounds. It is
known that concentrations tend to be greater in some red algae
and in some marine sponges, but it is still not known whether the
6-MeO-BDE-47 is synthesized by bacteria or the algae and or
sponge cells with which it is associated. If HO-PBDEs or MeOPBDEs could be synthesized by bacteria, then one or several of
these selected genes might act an important role in synthesis of
PBDEs analogs.
ARTICLE
’ ASSOCIATED CONTENT
bS
Supporting Information. This material is available free
of charge via the Internet at http://pubs.acs.org. Table S1 Acute
Toxicity End Point of 6-HO-BDE-47. Table S2 Altered Genes’
Functions by 6-HO-BDE-47 using 2-Fold Cut-off. Figure S1
Clustering of the time-dependent expression of the altered genes
selected by 1.5-fold change cutoff after exposure to 9 mg 6-HOBDE-47/L. (The dissimilarity between genes was calculated by
the Manhattan distance between the gene expressions at all the
concentration vs time combinations. The fold change of gene
expression is indicated by color gradient, and the time course of
expression changes is indicated from left to right.) Figure S2
Concentration-dependent transcriptional response curve of
6-HO-BDE-47 in E.coli. (Responses were based on the portion
of differentially expressed genes after exposure to three 6-HOBDE-47concentrations. NOTEC and TEC 50 were marked
with green and red, respectively.) Figure S3 Active functional
modules of a transcriptional network of patters of gene response
in E. coli exposed to 6-HO-BDE-47. Genes are displayed by
circular node, and transcriptional factor (TF)-target gene
interaction is indicated by arrow edge. Fold change of gene
expression in cells exposed to 9 mg 6-HO-BDE-47/L is indicated by
different color.
’ AUTHOR INFORMATION
Corresponding Author
* Phone: 86-25-83593649 Fax: 86-25-83707304 E-mail: yuhx@
nju.edu.cn (H.Y.); howard50003250@yahoo.com (X.Z.).
’ ACKNOWLEDGMENT
The research was supported by a grant from National Natural
Science Foundation of China (Grant Nos. 21007025, 20977047
and 20737001), Major State Basic Research Development Program (Grant No.2008CB418102), and National Science and
Technology Major Project (No. 2008ZX08526-003). This work
was also supported in part by grants from the Discovery Grant
program of the National Science and Engineering Research
Council of Canada (Project No. 326415-07) and the National
Plan for Science and Technology (10-ENV1314-02), King Saud
University. J.P.G. was supported by the Canada Research Chair
program, an at large Chair Professorship at the Department of
Biology and Chemistry and State Key Laboratory in Marine
Pollution, City University of Hong Kong, the Einstein Professor
Program of the Chinese Academy of Sciences and the Visiting
Professor Program of King Saud University.
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