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Chemosphere 144 (2016) 2150e2157
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Chemosphere
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Classification and toxicity mechanisms of novel flame retardants(NFRs) based on whole genome expression profiling
Miao Guan a, Guanyong Su a, John P. Giesy a, b, c, d, e, Xiaowei Zhang a, *
a State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, Chinab Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canadac Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USAd School of Biological Sciences, University of Hong Kong, Hong Kong, Chinae Department of Biology, Hong Kong Baptist University, Hong Kong, China
h i g h l i g h t s
� Assess mechanisms of toxic modes of action of six NFRs in genome-wide level.� NFRs were clustered based on expression of multiple genes that responded.� Clustering by molecular descriptors was consistent with that by gene profiles.
a r t i c l e i n f o
Article history:Received 8 August 2015Received in revised form25 October 2015Accepted 26 October 2015Available online xxx
Keywords:NFRsE coliGFP reporterGenomicsToxic mechanismClustering
* Corresponding author. School of the Environment210046, China.
E-mail addresses: [email protected], h(X. Zhang).
http://dx.doi.org/10.1016/j.chemosphere.2015.10.1140045-6535/© 2015 Elsevier Ltd. All rights reserved.
a b s t r a c t
Recently some novel alternative flame retardants (NFRs), which have been widely applied to meet de-mands for mandated flame retardation of products, have been detected in various matrices of theenvironment. However, knowledge on toxic effects and associated molecular mechanisms of thesechemicals was limited. Here, toxic mechanisms of action of six NFRs, bis (2-ethylhexyl) phosphate(BEHP), chlorendic acid (Het acid), 2,2-bis (bromomethyl)-1,3-propanediol (BMP), tris (2-butoxyethyl)phosphate (TBEP), triethyl phosphate (TEP), tributyl phosphate (TBP) were investigated by use of a li-brary containing ~1820 modified green fluorescent protein (GFP) expressing promoter reporter vectorsconstructed from Escherichia coli K12(E.coli). BEHP, Het acid, BMP, TBEP, TEP, TBP inhibited growth ofE. coli with 4 h 10%-inhibition concentrations of 53.0e3102.3 mM. A total of 119, 44, 26, 131, 62, 103 genesout of 336 genes selected during preliminary screening were significantly altered with fold-changesgreater than 1.5 by BEHP, Het acid, BMP, TBEP, TEP and TBP, respectively. GO analyses of responsivegenes suggested that RNA and primary metabolism process were involved in molecular mechanisms oftoxicity. Chemical clustering based on expression of 62 multi-responsive genes showed that BEHP, TBPand TBEP were grouped together, which is consistent with similarity of their chemical structures,especially for BEHP and TBP. Clustering by molecular descriptors and molecular activity by use of themultivariate classification system ToxCast was consistent with that by profiles of multi-responsive genes.The results of this study demonstrated the utility of the E. coli, whole-cell assay for determiningmechanisms of toxic action of chemicals.
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Flame retardants are ubiquitous in the environment due to their
, Nanjing University, Nanjing,
use to inhibit or resist the spread of fire, in thermoplastics, ther-mosets, foams, textiles, electronics and coatings. Since the globalbanishing of some brominated flame retardants that were usedhistorically, such as polybrominated diphenyl ethers (PBDEs),because of their high-performance and low-cost, production ofsome novel flame retardants (NFRs) like organophosphate flameretardants have been increased to meet demand required byvarious jurisdictions (van der Veen and de Boer, 2012). Moreover,some alternative flame retardants have been widely detected in
M. Guan et al. / Chemosphere 144 (2016) 2150e2157 2151
indoor air (Marklund et al., 2003; Hartmann et al., 2004), waters(Andresen et al., 2004; Teo et al., 2015) and soils (Ingram et al.,1996). This has raised concerns about potential toxicity of novelalternative flame retardants to wildlife and humans.
Previously, toxicity data for NFRs have been based primarily onin vivo tests with animals. Bis (2-ethylhexyl) phosphate (BEHP)induced oxidative stress by proliferation of both peroxisomes andmitochondria in rat liver (Lundgren and DePierre, 1987). Chlorendicacid (Het acid) was determined to be a clastogen by use of thein vitro mouse lymphoma assay and mutagenic in the L5178Y/TK ± mouse lymphoma assay in the absence of S9 activation(McGregor et al., 1988; Sofuni et al., 1996). 2,2-Bis (bromomethyl)-1,3-propanediol (BMP) can damage DNA in-vivo and might beassociated with oxidative stress (Kong et al., 2011). Tris (2-butoxyethyl) phosphate (TBEP) caused developmental toxicity byinhibiting degradation and utilization of nutrients and inducingapoptosis in zebrafish (Han et al., 2014). Triethyl phosphate (TEP)increased activity of reductase in rat liver microsomal preparations(Noboru et al., 1987). Triethyl phosphate (TBP) induced lung dam-age probably via the depression of key antioxidant enzymes andelevation of lipid peroxidation (Salovsky et al., 1998). However,information on molecular mechanisms of toxicity for these NFRswas limited, especially genome-wide information.
Sequencing of the complete genome of Escherichia coli K-12 andfusion of stress promoters to fluorescent transcriptional reportersprompted development of a useful toxicogenic approach to char-acterize toxic modes of action of chemicals or samples (Su et al.,2012; Zhang et al., 2011; Fu et al., 2013; Su et al., 2013; Hug et al.,2015). For each strain, fusion of stress promoters to the GFP pro-tein gene provides a mechanism for detection of modulation ofcellular signaling, which makes analyses of differential expressionof genes easier and more accurate (Elad et al., 2010). Comparedwith microarray technology, live cell arrays avoid complex pro-tocols of pre-treatment and high-costs of experimental materialshave fewer interferences and can provide temporal resolution(Onnis-Hayden et al., 2009). Furthermore, the short time requiredto complete a test makes use of live cell arrays rapid, economical,high-throughput biosensor systems for detecting toxicity anddetermining effects on specific signaling pathways.
Profiles of expression of genes can reveal mechanisms of toxicactions of chemicals, which are correlated to both the structure ofchemicals (molecular descriptors) and structure of the target of testorganism, which can be assessed by use of E. coli reporter genes asdemonstrated by the results presented here. Chemicals with similarmechanisms of toxic action produced similar profiles of transcrip-tional expression (Waring et al., 2001), which was useful for clus-tering of compounds or samples based on changes in patterns ofexpression of genes caused by each chemical (Su et al., 2014; Huget al., 2015).
In this study the E.coli, microbial reporter gene assay was usedto: 1) assess mechanisms of toxic actions of six NFRs; 2) identifymulti-responsive genes which were responsive to multiple chem-icals and based on expression of these multi-responsive genes tocluster six NFRs based on their effects on expression of genes inmultiple pathways.
2. Materials and methods
2.1. Chemicals
Bis (2-ethylhexyl) phosphate (BEHP), chlorendic acid (Het acid),2,2-bis (bromomethyl)-1,3-propanediol (BMP), tris (2-butoxyethyl)phosphate (TBEP), triethyl phosphate (TEP), tributyl phosphate(TBP) were obtained from Sigma Aldrich (St. Louis, MO, USA). Stocksolutions of six chemicals were prepared in dimethyl sulfoxide
(DMSO), and other test concentrations were made by serial dilutionwith DMSO. Structures and potencies for cytotoxicity informationare given (Table 1 and Fig. 1).
2.2. Microbial live cell array
The collection of microbial promoters developed by the Weiz-mann Institute of Science, which includes most of the genome ofE. coli K12 strain MG1655 (1820/2500) was used to assess dynamicexpression of genes (Zaslaver et al., 2006). Each of the reporterstrains is coupled with a bright, fast-folding green fluorescentprotein (GFP) fused to a full-length copy of an E. coli promoter in alow-copy plasmid. This makes quantification of expression of geneseasier and faster without the need to extract DNA/RNA. All cloneswere grown separately using 96-well plate (Corning, NY, USA) at37 �C in LB-Lennox media plus 25 mg/L kanamycin.
2.3. Cytotoxicity
For each of the NFRs studied, six concentrations (n ¼ 3) wereselected for use in tests of cytotoxicity to E. coli in 96-well plate, andthe maximum concentration which was determined by chemicalcytotoxicity and maximum solubility is listed in Table 1. Here, thevital stain, alamar blue was used as an indicator of cytotoxicity.Alamar blue is not toxic to cells and is a more sensitive measurethan OD600 (Su et al., 2012). After 3 h incubation at 37 �C, alamarblue was used to assess whether cells had sufficient capacity toproliferate. After dyeing for 1 h with alamar blue, blue-red fluo-rescence was quantified by use of a Synergy H4 hybrid microplatereader (excitation at 545 nm and emission at 590 nm) (Bio TekInstruments Inc., Winooski, VT).
2.4. High throughput screening
Strains of E. coliwere inoculated into a fresh 96-well plate from a96-well stock plate by use of disposable replicators (Genetix, SanJose, CA, USA). Cells were incubated at 37 �C for 3 h in 96-well plateand then transferred into 384-well plate (NUNC, Rochester, NY,USA). Finally, 3.75 mL of DMSO (solvent control) or chemical solu-tions were added into individual wells on the 384-well plate tomake a final concentration of 10% inhibition concentration (IC10).Intensity of fluorescence of GFP in each well was consecutivelymonitored every 10 min for 4 h by use of a Synergy H4 hybridmicroplate reader (excitation/emission: 485 nm/528 nm). Differ-ential expressions of genes of 1820 E. coli reporter strains exposedto the six NFRs, which were BEHP, Het acid, BMP, TBEP, TEP and TBP,were obtained in two batches, where each batch contained 21 96-well plates. The response measured as fluorescence of GFP wasfitted to a function of time for each promoter reporter strain with ap value less than 0.001. Genes that changed in response to exposureto the six NFRs with maximum fold changes greater than 2-foldwere selected to be monitored in a series of concentrations ofeach of the individual NFRs.
For validation, all selected E. coli reporter strains were exposedto each of three concentrations representing 0.01*IC10, 0.1*IC10and IC10. To select the final promoter reporter genes that weresignificantly differentially expressed in response to NFRs, a linearregression model was applied. Changes in expressions of geneswhich exhibited a significant correlation between magnitude ofresponse and time and also concentrations with p values less than0.001 and a maximum fold change greater than 1.5 or 2 wereconsidered to be significant. Details of the analyses applied to thedata have been previously described (Su et al., 2012; Zhang et al.,2011; Gou et al., 2010). Lists of genes which derived from threeseries concentrations validation test were developed for analysis of
Table 1Cytotoxicity of six novel flame retardants (NFRs) to E. coli.
No. Chemicals Abbreviation CAS Test concentration range (mM) IC10b (mM) IC50c (mM)
1 Bis (2-ethylhexyl) phosphate BEHP 298-07-7 0e17114.3 145.8 5867.82 Chlorendic acid Het acid 115-28-6 0e8381.3 53.0 2484.33 2,2-Bis(bromomethyl)-1,3-propanediol BMP 3296-90-0 0e19874.8 871.6 6721.84 Tris(2-butoxyethyl) phosphate TBEP 78-51-3 0e15511.8 549.9 NAa
5 Triethyl phosphate TEP 78-40-0 0e39224.9 3102.3 NAa
6 Tributyl phosphate TBP 126-73-8 0e36678.5 375.1 NAa
a NA means not achieved within the test concentration.b IC10 means 10% inhibitory concentration of a NFR after a 4-hr exposure.c IC50 means median inhibition concentration of a NFR after a 4-hr exposure.
Fig. 1. Structures of six novel flame retardants (NFRs).
M. Guan et al. / Chemosphere 144 (2016) 2150e21572152
mechanisms of toxic action based on a cutoff of 1.5 fold-changes.Assessment of mechanisms of toxic action was conducted by useof GO gene set enrichment analysis by use of the R package clus-terProfiler (Yu et al., 2012). P-values which were adjusted formultiple comparisons less than 0.01 and q-values less than 0.05were also calculated as cutoff for FDR control (Storey, 2003). An-notations of responsive genes were obtained from website (www.ecogene.org).
2.5. Clustering of NFRs
Chemicals with similar patterns of expression of genes wereclustered together. To avoid inclusion of unaltered genes that didnot contribute to categorization of NFRs, only those altered by atleast three NFRs (p value < 0.001& fold change > 1.5) in the vali-dation test were used to classify NFRs. Classification of NFRs basedon similarities of differentially expressed genes was accomplishedby use of ToxClust (Zhang et al., 2009). Dissimilarities among geneswere calculated by use of Manhattan distances between expres-sions among genes across three concentrations and 25 time points.
Other data on molecular toxicity for the six NFRs by U.S. Envi-ronmental Protection Agency (EPA) were obtained from ToxCast(Dix et al., 2007) (http://actor.epa.gov/dashboard/). There were 174,382, 384, 170, 167 and 404 tested assays and 42, 1, 1, 15, 1 and 16
active assays for BEHP, Het acid, BMP, TBEP, TEP and TBP, respec-tively. Of 431 assays conducted, 61which were active by at least oneNFR were chosen for toxicity mechanism comparison. NFRs withmore overlap active assays were considered having more similartoxicity mechanism.
Molecular descriptors for the six NFRs were calculated by use ofE-Dragon (Mauri et al., 2006), which provides more than 1600molecular descriptors that are divided into 20 logical blocks (http://www.vcclab.org). Molecular descriptors of constant expressionamong the six NFRs were removed. The final dataset consisted of1492 descriptors which contained 31 constitutional indices, 10 ringdescriptors, 72 topological indices, 42 walk and path counts, 37connectivity indices, 48 information indices, 492 2-D matrix-baseddescriptors, 211 2-D autocorrelations, 91 burden eigenvalues, 33 P-VSA-like descriptors, 23 ETA indices, 303 edge adjacency indices, 10atom-centered fragments, 10 atom-type E-state indices, 14 CATS2D, 37 2-D atom pairs, 20 molecular properties and 8 drug-likeindices (http://www.talete.mi.it/products/dragon_molecular_descriptor_list.pdf). Molecular descriptors were normalized byrange standardization (X-Xmin)/(Xmax-Xmin). The Cluster AffinitySearch Technique (CAST) was applied to 1492 descriptors and 81clusters with an inclusion threshold of 0.8 identified (Ben-Dor et al.,1999). The median profile of each cluster of molecular descriptors(MCP) was chosen to represent each cluster. Hierarchical clustering
Fig. 2. Inhibition of growth of E. coli growth inhibition profiles by novel flame re-tardants (NFRs) at different concentrations (Data points represent mean and standarderror).
M. Guan et al. / Chemosphere 144 (2016) 2150e2157 2153
of the six NFRs by use of MCPs was conducted by use of Spearmandistances.
3. Results and discussion
3.1. Cytotoxicity
After a 4-h exposure of E. coli reporter strains to NFRs, differentprofiles of term were observed (Fig. 2, Table 1). Three NFRs, BEHP,Het acid and BMP, were cytotoxic and inhibited E. coli cells in aconcentration-dependent manner, with maximum inhibitions of100%, 95% and 81%, respectively. For TBEP, TEP and TBP, slight in-hibitions were observed, with maximum inhibitions 33% 33%, and43%, respectively. After exposure to BEHP, Het acid, BMP, TBEP, TEPor TBP, IC10 values based on probit model analyses, were 145.8,
Fig. 3. Biological processes (BP) GO of responsive genes (fold change > 1.5, IC10) modulatedphosphate (TBEP), triethyl phosphate (TEP) and tributyl phosphate (TBP) (A) ~ (E). The Ven
53.0, 871.6, 549.9, 3102.3 and 375.1 mM (Hamilton et al., 1977). IC50values could be calculated for only BEHP, Het acid and BMP andwere 5867.8, 2484.3 and 6721.8 mM, respectively. This result sug-gested that these NFRs can cause toxicity to E. coli but only at veryhigh concentrations. The IC10 was selected as the test concentra-tion to be used in studies of expression of genes for use in classi-fication of the six NFRs.
3.2. Profiles of expression of genes
After exposure to BEHP, Het acid, BMP, TBEP, TEP or TBP, 336genes were modulated by at least 2-fold by at least one of the NFRs.Of these 336 genes, 119, 44, 26, 131, 62, 103 were significantlyaltered by 1.5-fold or greater by BEHP, Het acid, BMP, TBEP, TEP andTBP, respectively (Table S2eS7). Furthermore, 62, 24, 9, 75, 37, 56genes were significantly altered by 2-fold or greater for the sixNFRs, respectively. More genes were up-regulated than down-regulated due to 4 h exposure to the six NFRs with cutoffs ofeither 1.5 or 2 (Fig. S1eS12). The number of genes altered byexposure to NFRs with maximum fold change greater than 1.5 wasproportional to concentrations of the individual NFRs. Strains thatresponded to lesser or moderate concentrations were responsive togreater concentrations as well (Fig. S13). Genes that were respon-sive to at least one concentration were selected for use in deter-mining toxicity mechanisms of toxic actions. Genes modulated bythe greatest concentration, which represented the most compre-hensive potential toxicity mechanism, were chosen for furtherclustering NFRs. Meanwhile, 62 genes which were modulated by atleast three NFRs were selected as multi-responsive genes forfurther clustering NFRs.
3.3. Mechanisms of toxic action
Gene ontology (GO) biological processes were inferred and usedto understand biochemical pathways that were modulated byexposure to each of the six NFRs. GO biological processes modu-lated by the five NFRs (except BMP) included primary metabolic
by bis (2-ethylhexyl) phosphate (BEHP), chlorendic acid (Het acid), tris(2-butoxyethyl)n diagram displays the overlap of five NFRs' BP GO terms (F).
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process, cellular metabolic process, organic substance metabolicprocess, cellular process, metabolic process and biological processby responsive genes of NFRs (p < 0.01, q < 0.05). There was nosignificant GO pathway enriched with BMP. The common pathwaysaffected by the five NFRs were primary processes occurring in celland important for maintaining homeostasis (Maughan andNicholson, 2011). Modification of RNA; tRNA metabolism process,tRNA aminoacylation for protein translation, ncRNA metabolicprocess, amino acid activation, tRNA aminoacylation and organo-nitrogen compound metabolic process; small molecular meta-bolism process; macromolecular biosynthetic process and cellularmacromolecule biosynthetic process were the pathways onlymodulated by BEHP, TBP, TBEP and Het acid, respectively. Gene
Fig. 4. Clustering of time-dependent expression of six novel flame retardants (NFRs) basedexpression were derived by use of ToxClust (Zhang et al., 2009). Color gradient represent folform left to right. Bis (2-ethylhexyl) phosphate (BEHP), tributyl phosphate (TBP) and tris(chlorendic acid (Het acid), triethyl phosphate (TEP) and 2,2-bis(bromomethyl)-1,3-propanepretation of the references to color in this figure legend, the reader is referred to the web
expression was the pathway modulated by Het acid and BEHP.Translation, cellular amino acid metabolic process, protein meta-bolic process, cellular biosynthetic process and cellular proteinmetabolic process were modulated by BEHP and TBP. Nucleobase-containing compound metabolic process, cellular aromatic com-pound metabolic process, RNA metabolic process, cellular nitrogencompound metabolic process, heterocycle metabolic process,nucleic acid metabolic process and organic cyclic compoundmetabolic process were enriched by BEHP, Het acid and TBP; BEHP,Het acid, TBEP and TBP and common pathway of nitrogen com-pound metabolic process. BEHP, Het acid, TEP and TBP have com-mon pathways of macromolecule metabolic process and cellularmacromolecule metabolic process. 21 common GO pathways of
on profiles of 62 multi-responsive genes. Classification and visualization of the gened change of gene expression and the time course of expression changes were indicated2-butoxyethyl) phosphate (TBEP) were classified together, and the other three NFRs,diol (BMP), were different from others and classified into unique clusters. (For inter-version of this article.)
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BEHP (23 pathways) and TBP (27 pathways) indicated that themechanisms of toxicity of the two OPFRs were more similar to theother four NFRs; Six GO pathways of TBEP (8 pathways) and TEP (8pathways) indicated that mechanisms toxicity of the two OPFRswere more similar to the other four NFRs based on the GOenrichment analysis (Fig. 3). Potential mechanisms of toxicity ofBEHP, TEP and Het acid were primary processes of metabolismthose that influence transcription of RNA. Potential key mecha-nisms of toxic actions of TBEP and TEP were primary metabolismprocesses.
3.4. Clustering of NFRs
Based on differential expression of genes, a chemical classifi-cation was conducted. A total of 62 genes which were significantlyaltered by at least three NFRs (p < 0.001 & fold change > 1.5) wereselected and data from the greatest concentration was used for
Fig. 5. Parallel heat map of six novel flame retardants (NFRs) based on the activity of ToxCassays and gray color represented assays not tested with this chemical. Bis (2-ethylhexyl)have similar active assays, while the other three NFRs, triethyl phosphate (TEP), 2,2-bis(bdifferent active assay. (For interpretation of the references to color in this figure legend, th
clustering of the six NFRs (Fig. 4). BEHP, TBP and TBEP, which havesimilar chemical structures, were classified together (Fig. 1). Espe-cially for BEHP and TBP, patterns of modulations of genes caused bythese two chemicals were quite similar, while the patterns ofexpression caused by the other three NFRs, Het acid, TEP and BMP,were different from the others.
Multi-responsive genes that were altered by exposure to at leastthree of the NFRs and thus might provide superior power ofdiscrimination are listed. These 62 genes were classified into fourgroups: enzymes or putative enzymes; regulatory proteins or pu-tative regulator proteins; structural proteins and those of unspec-ified function, accounted for 48.4%, 32.3%, 14.5%, and 4.8% of the 62modulated genes, respectively (Table S1). Among these genes, iscR,pspB, ycaC and yncC were differentially expressed due to exposureamong five NFRs. Genes expression of dksA, ecfG, efp, galU, hisS, slyA,stpA, tolB, trmA, xseA, yadF, ycfQ, yciA, ydiV, yeaT, yhcO, yjjK and znuAwere altered by exposure to four NFRs. The other 40 genes were
ast test assays. Yellow color represented active assays, blue color represented inactivephosphate (BEHP), tributyl phosphate (TBP) and tris(2-butoxyethyl) phosphate (TBEP)romomethyl)-1,3-propanediol (BMP) and chlorendic acid (Het acid), which has verye reader is referred to the web version of this article.)
M. Guan et al. / Chemosphere 144 (2016) 2150e21572156
altered by exposure to three NFRs. iscR the transcript of which is aprotein that is a repressor of DNA-binding transcription, whichregulates transcription of several operons and genes involved inbiogenesis of FeeS clusters was modulated by exposure to BEHP,Het acid, TBEP, TEP and TBP. PspB, psp operon transcription co-activator was responsive for BEHP, TBEP, TEP, TBP and Het acid.YcaC, putative isochorismatase family hydrolase was responsive forBEHP, Het acid, BMP, TBEP and TBP. YncC, coding colonic acid andbiofilm gene transcriptional regulator was responsive for BEHP, Hetacid, BMP, TBEP and TEP. Of these 62 genes, 11 genes which werestpA, yncC, iscR, allR, yjeB, slyA, yahB, ycfQ, yeaT, ygfI and yegEwere inthe term for regulation of transcription, regulation of RNA meta-bolic process. 12 genes which were deaD, yncC, iscR, allR, yjeB, papB,lsrR, slyA, yahB, ycfQ, yeaT and ygfIwere in the term for transcriptionbased on gene ontology biological process analysis. That mightimplied that these six NFRs could be distinguished in the expres-sion of genes coding transcription.
Sixty one molecular toxicity assays from ToxCast also indicatedthat BEHP, TBP and TBEP have similar toxicity activity. Three assayswhich were ATG_PPARg_TRANS_up (target gene: PPARG),ATG_PXRE_CIS_up (target gene: NR1I2) and ATG_VDRE_CIS_up(target gene: VDR) were active for BEHP, TBEP and TBP. Four assayswhich were Tox21_ARE_BLA_agonist_ratio (target gene: NFE2L2),Tox21_Aromatase_Inhibition (target gene: CYP19A1), Tox21_TR_-LUC_GH3_Antagonist (target gene: THRB) and ACEA_T47D_80hr_-Negative were active for BEHP and TBEP. Three assays which wereNVS_MP_hPBR (target gene: TSPO), NVS_MP_rPBR (target gene:Tspo) and OT_FXR_FXRSRC1_0480 (target gene: NR1H4) wereactive for TBEP and TBP. One assay which was ATG_PXR_TRANS_up(target gene: NR1I2) was active for BEHP and TBP. While the otherthree NFRs, which were TEP (only active by ACEA_T47D_80hr_-Positive assay, ESR1), BMP (only active by NVS_ENZ_oCOX2 assay,
Fig. 6. Clustering of six novel flame retardants (NFRs) based on median cluster profiles of mtris(2-butoxyethyl) phosphate (TBEP) were classified together, while the other three NFRspropanediol (BMP), which were clustered separately (cutoff was represented by the red lreferred to the web version of this article.)
PTGS2) and Het acid (only active by NVS_GPCR_gLTB4 assay, Ltb4r),have very different active assays (Fig. 5). The grouping of six NFRsby molecular toxicity data from ToxCast was consistent with thatobtained by use of profiles of differential expression of genes.
Molecular descriptors of NFRs can be used to gain insights intopotential modes of toxic action of NFRs. Based on the 81 mediancluster profiles of molecular descriptors, the six NFRs were clus-tered into four clusters: Cluster 1 contained BEHP, TBP and TBEPwhile Cluster 2 contained: Het acid and TEP and BMP were clus-tered alone in Clusters 3 and 4 (Fig. 6). Clustering by use of mo-lecular descriptors was also consistent with that obtained by use ofprofiles of differential expression of genes.
4. Conclusions
Cytotoxicity of six NFRs, BEHP, Het acid, BMP, TBEP, TEP and TBP,expressed as the IC10, were 145.8, 53.0, 871.6, 549.9, 3102.3 and375.1 mM, respectively. Of the 336 genes identified in the initialscreening, expression of 119, 44, 26, 131, 62, 103 genes weremodulated by a factor of 1.5 by BEHP, Het acid, BMP, TBEP, TEP andTBP, respectively. Analysis of biological processes based on GOhelped elucidate potential mechanisms of toxic actions of the sixNFRs. BEHP, TBP and TBEP were clustered together based on bothgene expression of multi-responsive genes and molecular de-scriptors. Het acid, BMP and TEP separated into separate clusters.
Flame retardants with similar mode of action trend to havesimilar gene expression profiles. In the future, we can investigatemode of action of some other novel flame retardants by clusteringthem with flame retardants with known mode of action by geneexpression profiles to predict mode of action of novel flameretardants.
olecular descriptors. Bis (2-ethylhexyl) phosphate (BEHP), tributyl phosphate (TBP) and, chlorendic acid (Het acid), triethyl phosphate (TEP) and 2,2-bis(bromomethyl)-1,3-ine). (For interpretation of the references to color in this figure legend, the reader is
M. Guan et al. / Chemosphere 144 (2016) 2150e2157 2157
Acknowledgments
We thank the National Science Foundation of China for theExcellent Yong Scientist Grant (21322704) and Research Fund forthe Doctoral Program of Higher Education of China (Grant:20120091110034). This work is also supported by the SeventhFramework Programme (the SOLUTIONS Project, FP7-ENV-2013) ofthe European Union under grant agreement no. 603437. Prof. Giesywas supported by the program of 2012 “High Level Foreign Experts”(#GDT20143200016) funded by the State Administration of ForeignExperts Affairs, the P.R. China to Nanjing University and the EinsteinProfessor Program of the Chinese Academy of Sciences. He was alsosupported by the Canada Research Chair program.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2015.10.114.
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