Proteomic and metabolomic analysis of earthworm Eisenia fetida exposed to different concentrations of 2,2′,4,4′-tetrabromodiphenyl ether

    Proteomic and metabolomic analysis of earthworm Eisenia fetida exposed to different concentrations of 2,2’,4,4’-tetrabromodiphenyl ether Chenglong Ji, Huifeng Wu, Lei Wei, Jianmin Zhao, Hongjian Lu, Junbao Yu PII: DOI: Reference:

S1874-3919(13)00442-9 doi: 10.1016/j.jprot.2013.08.004 JPROT 1532

To appear in:

Journal of Proteomics

Received date: Accepted date:

10 May 2013 3 August 2013

Please cite this article as: Ji Chenglong, Wu Huifeng, Wei Lei, Zhao Jianmin, Lu Hongjian, Yu Junbao, Proteomic and metabolomic analysis of earthworm Eisenia fetida exposed to different concentrations of 2,2’,4,4’-tetrabromodiphenyl ether, Journal of Proteomics (2013), doi: 10.1016/j.jprot.2013.08.004

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ACCEPTED MANUSCRIPT Proteomic and metabolomic analysis of earthworm Eisenia fetida exposed

to

different

concentrations

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2,2’,4,4’-tetrabromodiphenyl ether

of

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Chenglong Jia,b, Huifeng Wua,*, Lei Weia,b, Jianmin Zhaoa, Hongjian

a

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Luc, Junbao Yua

Key Laboratory of Coastal Zone Environmental Processes, Yantai Institute of

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Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS); Shandong Provincial Key Laboratory of Coastal Zone Environmental Processes, YICCAS,

c

The Graduate School of Chinese Academy of Sciences, Beijing 100049, P. R. China

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b

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Yantai 264003, P. R. China

Institute of Chemistry and BioMedical Sciences (ICBMS), Nanjing University,

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Nanjing 210046, P. R. China

*

To whom correspondence should be addressed. Huifeng Wu: Fax: +86-535-2109000. Tel:

+86-535-2109190. E-mail: [email protected]. 1

ACCEPTED MANUSCRIPT ABSTRACT As a class of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs), are widely used in industrial products. PBDEs have been detected in local terrestrial biota from the Laizhou Bay in China. They can induce

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various toxicities, such as hepatotoxicity, neurotoxicity, cytotoxicity, genotoxicity and

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endocrine disrupting effects in animals. In this work, we characterized the dose-responsive effects of 2,2’,4,4’-tetrabromodiphenyl ether (BDE 47) in earthworm

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Eisenia fetida using an integrated proteomic and metabolomic approach. Metabolic responses indicated that BDE 47 mainly caused disturbance in osmotic regulation and

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energy metabolism marked by differentially altered betaine, amino acids, ATP, glucose, maltose and succinate in E. fedita. Proteomic responses revealed that BDE

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47 induced cell apoptosis (or injury), oxidative stress, disturbance in protein biosynthesis and energy metabolism in E. fedita in terms of differential proteomic

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biomarkers. Especially, the increased ATP was confirmed by up-regulated nucleoside

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diphosphate kinase A and ATP synthase in 1 and 100 µg/L of BDE 47-treated groups, respectively. In addition, several metabolic biomarkers including betaine, glycine and

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2-hexyl-5-ethyl-3-furansulfonate were relatively stable in all BDE 47-exposed groups. This work demonstrated that proteomics and metabolomics could partially validate one another and their combination could better understand toxicological effects of environmental pollutants.

Keywords: Proteomics; Metabolomics; Eisenia fetida; BDE 47

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ACCEPTED MANUSCRIPT 1. Introduction In the Laizhou Bay along the Bohai Sea, there are abundant resources of seawater and underground brine, and therefore this region has the largest industrial of

brominated

flame-retardants

(BFRs)

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manufacturing base

in

Asia

[1].

Polybrominated diphenyl ethers (PBDEs) are the main products in the BFR output

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and have been extensively used in a variety of industrial products, such as plastics,

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electronic equipment, textiles and upholstery foam, to reduce the risk of fire [2]. PBDE pollutions have been found in both soil and aquatic environments due to the

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release from PBDE products and discharge of industrial wastewaters [2, 3]. Although a few PBDE chemicals, including penta-BDE and octa-BDE, have been banned,

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PBDEs have been increasingly detected in the environments and organisms because of the increasing usage of other congeners, such as Deca-PBDE mixtures [4]. Environmental PBDEs can be broken down to lower brominated PBDEs that have

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higher persistence and toxicity in organisms and consequently higher ecotoxicological

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risk [5]. In the environments, 2,2’,4,4’-tetrabromodiphenylether (BDE 47) and

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2,2’,4,4’,5-pentabromodiphenyl ether (BDE 99) are the main congeners of PBDEs [6]. However, BDE 47 is one of the most toxic congeners to organisms [7]. PBDEs

have

diverse

toxicities

including

hepatotoxicity,

neurotoxicity,

cytotoxicity, genotoxicity, carcinogenecity and immunotoxicity [1, 8-11]. These compounds can also exert effects in the sex steroid and thyroid endocrine systems in humans and wildlife together with their metabolites [12-14]. Previous studies revealed that PBDEs could induce disruption in the thyroid system by binding to the receptors of thyroid hormone, accelerating clearance rates of thyroid hormones and competing with thyroxine for transport protein, transthyretin [15]. Traditional toxicological studies usually focus on the measurement of specific responses such as the anti-oxidant activities (e.g., superoxide dismutase and catalase) to test for oxidative stress or specifically expressed genes and proteins to test for certain toxicities [16, 17]. However, there is a lack of broad toxicological biomarkers to characterize the toxicological effects of contaminants in bioindicators. To achieve a comprehensive understanding on toxicological effects and mechanisms, a global analysis on the 3

ACCEPTED MANUSCRIPT biological responses and corresponding biomarkers should be performed at molecular levels (gene, protein and metabolite) in bioindicators to contaminant exposures. Recent development in analytical chemistry has greatly improved the efficiency

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of toxicological biomarker discovery using the global biomarker approaches including

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genomics, proteomics, transcriptomics and metabolomics [18-21]. These –omic approaches are capable to discover broader ranges of biomarkers at molecular levels

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[18, 21-22]. In proteomics, the two-dimensional electrophoresis (2-DE)-based proteomics remains widely useful to characterize complex biologically functional

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protein networks [22, 23]. Therefore, a comparison of protein spots resolved in 2-DE gels can detect the alterations in the protein profiles between contaminant-exposed

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and control conditions, which allow us to interpret the contaminant-induced toxicological effects and mechanisms [24]. Metabolomics usually focuses on the

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small molecular weight metabolites that are the end products of all metabolisms in

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diverse biological systems, such as tissue, cell or fluid [25]. The characterization of the metabolome can provide an insight into the toxicological mechanisms of

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contaminants on metabolism in organisms. Obviously, an integration of proteomics and metabolomics can simultaneously characterize the perturbations in metabolism and interactive enzymes [19]. It therefore has a capability to better understand toxicological mechanisms of contaminants [26]. In the terrestrial system, earthworms are excellent model organisms and have been widely used as preferable bioindicators in soil ecotoxicology [27, 28]. Some recent studies have shown that both NMR-based metabolomics and 2-DE-based proteomics on earthworm Eisenia fetida have great potential to elucidate the toxicological effects and mechanisms induced by environmental contaminants, such as heavy metals (e.g., cadmium) and organic chemicals (e.g., phenanthrene) [29, 30]. To our knowledge, however, no combined proteomic and metabolomic investigation has been carried out in ecotoxicology using E. fetida as a bioindicator. In the present study, an integrated metabolomics and proteomic approach was used to elucidate the toxicological effects of BDE 47 at different concentrations in E. fetida. 4

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Experimental design The adult earthworms Eisenia fetida were purchased from a farming factory in

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Haiyang, Yantai. The earthworms were cultured in cattle manure in a climate

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controlled room with a relative humidity of 65%, in darkness at 25 oC for 1 month. Before exposure experiment, animals with similar sizes (approximately 400 mg) were

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transferred to wet filter paper to empty their intestinal tract. Meanwhile, the test solution used for exposure experiment was prepared by adding different volumes of

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stock solutions of Ca(NO3)2, MgSO4, NaNO3, and KNO3 to deionized water [31]. These chemicals were analytical reagent grade or higher and purchased from Guoyao

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Co., Ltd (Shanghai, China). After removal of faeces in the intestinal tract, all earthworms were transferred to clean glass containers with the test solution (1 liter)

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and acclimatized for 3 days. Then these earthworms were randomly divided into six

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groups, including control, solvent (dimethyl sulfoxide (DMSO), 0.002% v/v) control, 0.1, 1, 10 and 100 μg/L BDE 47 (99.5% purity, Chem Service, West Chester, PA,

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USA). It should be noted that these concentrations of BDE 47 were not environmentally relevant, since this work basically focused on the acute effects of BDE 47 in E. fetida. Each group had two replicate exposures, which meant there were two glass containers containing six earthworms in each group. The concentration of BDE 47 stock solutions was 5 mg/mL in DMSO and diluted to appropriate ratios with DMSO for BDE 47 exposures to ensure the same DMSO concentrations to that of solvent control group. The actual concentrations of BDE 47 in the exposed groups were at the range of 88% to 97% of the nominal concentrations, which were determined using the published method [32]. During the acclimation and exposure periods, animals were kept at 25 oC in darkness and test solution was changed daily. After exposure for 96 hours, all the earthworms were flash-frozen in liquid nitrogen and stored at -80 oC before metabolite, protein and RNA extraction and enzyme assay. For further procedures, each earthworm was ground homogeneously in liquid nitrogen and divided into four glass vials. Thus these glass vials contained the identical biological samples from an individual earthworm. Each treatment consisted of 6 and 3 5

ACCEPTED MANUSCRIPT (2 pooled into 1) biological replicates for metabolomic and proteomic analysis, respectively. 2.2. Metabolite and protein extraction

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Metabolite and protein extraction protocols and procedures are described in the

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Supporting Information. 2.3. Two-dimensional gel electrophoresis

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The first dimension (IEF) was performed using Immobiline Drystrip (24 cm, pH 3-10, linear). One hundred and forty microgram of proteins to a final volume of 450 μL

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were loaded. Isoelectric focusing gel solution containing 7 M urea, 2 M thiourea, 4% m/v CHAPS, 65 mM DTT, 0.001% m/v Bromophenol blue and 0.2% W/V Bio-lyte

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buffer. IEF was conducted at 20 oC with an Etan IPGphor3 system for a total of 85858 Vh (Active rehydration was carried out at 30 V for 12 h, followed by 100 V for 5 h,

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500 V for 1 h, 1000 V for 1 h, and a linear increase of voltage to 8000 V for 11 h).

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After the first dimension, strips were placed in equilibration buffer (0.05 M Tris-HCl, pH 8.8; 6 M urea; 30% glycerol; 2% w/v SDS; containing 1% w/v DTT)

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and were slowly shaken for 15 min. The strips were then incubated for another 15 min in the equilibration buffer with 2.5% (w/v) iodoacetamide without DTT [33]. The second dimension was conducted on 12.5% SDS-PAGE gels using the Ettan DALTsix system. After electrophoresis, the gels were silver stained by following the method of Mortz et al., (2001) and Gharahdaghi et al., (1999) [33, 34]. Images were captured by ImageScanner ൗ and spots were quantitatively analyzed by using the software ImageMaster 2D Platinum 7.0. The abundance of each protein spot was calculated by the percentage volume (% vol). Only those spots with significant changes (> 1.5 folds and P < 0.05) were considered to be differentially expressed proteins. 2.4. In gel digestion and MS analysis The details of in gel digestion are described in the Supporting Information. After being completely dried, the samples were re-suspended with 5 µL 0.1% TFA

followed

by

mixing

in

1:1

ratio

with

a

saturated

solution

of 6

ACCEPTED MANUSCRIPT α-cyano-4-hydroxy-trans-cinnamic acid in 50% acetonitrile [35]. One microliter of mixture was analyzed by an ABI 4800 MALDI-TOF/TOF Plus mass spectrometer (AB Sciex Inc., Foster City, USA), data were acquired in a positive MS reflector

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using a CalMix5 standard to calibrate the instrument (ABI4800 Calibration Mixture).

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Both the MS and MS/MS data were integrated and processed by using the GPS Explorer V3.6 software (AB Sciex Inc., Foster City, USA) with default parameters.

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Proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.4 search engine (Matrix Science Ltd., London, U.K.).

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The following parameters were used in the search: NCBInr Metazoa (Animals) (2861494 sequences) database; trypsin as the digestion enzyme; one missed cleavage

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site; partial modifications of cysteine carbamidomethylation and methionine oxidization; no fixed modifications; 0.15 Da for precursor ion tolerance and 0.25 Da

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for fragment ion tolerance. Individual ions scores >40 indicate identity or extensive

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homology (P < 0.05). For the protein spots with multiple identifications, only the proteins with the highest scores within each sample are reported.

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2.5. RNA extraction and quantitation of gene expressions The details of RNA extraction, determination of internal reference gene and qRT-PCR are described in the Supporting Information. The information of primer sequences for the determination of housekeeping genes and tested genes was listed in Table S1 and Table S2, respectively.

2.6. Antioxidant enzyme activities and lipid peroxidation Six ground samples of E. Fetida from each group were subjected to antioxidant enzyme activity assays. Measurement of superoxide dismutase (SOD, EC 1.15.1.1), glutathione S-transferases (GST, EC 2.5.1.18) and catalase (CAT, EC 1.11.1.6) were performed by using commercial enzyme kits (Nanjing Jiancheng Bioengineering Institute, China). Lipid peroxidation was determined by measuring the generation of thiobarbituric acid reactive substances (TBARS) and expressed in terms of malondialdehyde (MDA) content. Protein concentration was determined with Bradford method by using bovine serum albumin as standard [36]. The enzyme 7

ACCEPTED MANUSCRIPT activities and MDA content were expressed as U/mg protein and nmol/mg protein, respectively. 2.7. 1H NMR spectroscopy, spectral pre-processing and pattern recognition analysis

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Metabolite extracts of homogenized earthworms were analyzed on a Bruker AV 500 NMR spectrometer performed at 500.18 MHz (at 25 oC) as described previously [37].

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All 1H NMR spectra were phased, baseline-corrected, and calibrated (internal

manually using TopSpin (version 2.1, Bruker).

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reference: 2,2,3,3-d(4)-3-(trimethylsilyl)propionic acid sodium salt (TSP) at 0.0 ppm)

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All one dimensional 1H NMR spectra were converted to a data matrix using the custom-written ProMetab software in Matlab version 7.1 (The MathsWorks, Natick,

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MA, USA) [38]. Each spectrum was segmented into bins with a width of 0.005 ppm between 0.2 and 10.0 ppm. The bins of residual water peak between 4.70 and 5.20

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ppm were excluded from all the NMR spectra. The total spectral area of the remaining

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bins was normalized to unity to facilitate the comparison between the spectra. All the NMR spectra were generalized log transformed (glog) with a transformation

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parameter λ = 1.0 × 10-8 to stabilize the variance across the spectral bins and to increase the weightings of the less intense peaks [38]. Pattern recognition analysis was performed with the software SIMCA-P+ (V11.0, Umetric, Sweden). The unsupervised pattern recognition method, principal component analysis (PCA) was used to reduce the dimensionality of the data and summarize the similarities and differences between multiple NMR spectra [39]. The algorithm of PCA calculates the highest amount of correlated variation along PC1, with subsequent PCs containing correspondingly smaller amounts of variance. One-way analysis of variance (ANOVA) was conducted on the PC scores from each group to test the statistical significance (P < 0.05) of separations. Furthermore, the supervised multivariate data analysis methods, partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (O-PLS-DA), were sequentially carried out to uncover and extract the statistically significant metabolite variations related to BDE 47 exposures. The results were visualized in terms of scores plots to show the classifications and corresponding 8

ACCEPTED MANUSCRIPT loadings plots to show the NMR spectral variables contributing to the classifications. The model coefficients were calculated from the coefficients incorporating the weight of the variables in order to enhance interpretability of the model. Then metabolic

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differences responsible for the classifications between control and BDE-exposed

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groups could be detected in the coefficient-coded loadings plots. The coefficient plots were generated by using MATLAB (V7.0, the Mathworks Inc., Natwick, USA) with

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an in-house developed program and were color-coded with absolute value of coefficients (r). A hot color (i.e., red) corresponds to the metabolites being highly

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positive/negative significant in discriminating between groups, while a cool color (i. e. blue) corresponds to no significance. The correlation coefficient was determined

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according to the test for the significance of the Pearson’s product-moment correlation coefficient. The validation of the model was conducted using 10-fold cross validation

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and the cross-validation parameter Q2 was calculated, and an additional validation

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method, permutation test (permutation number = 200), was also conducted in order to evaluate the validity of the PLS-DA models. The R2 in the permutated plot described

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how well the data fit the derived model, whereas Q2 describes the predictive ability of the derived model and provides a measure of the model quality. If the maximum value of Q2 max from the permutation test was smaller than or equal to the Q2 of the real model, the model was regarded as a predictable model. Similarly, the R2 value and difference between the R2 and Q2 were used to evaluate the possibility of over-fitted models [26]. Metabolites were assigned following the tabulated chemical shifts [40], based on the Human Metabolome Database (HMDB, http://www.hmdb.ca/) database and by using the software, Chenomx (Evaluation Version, Chenomx Inc., Edmonton, Alberta, Canada). 2.8. Statistical analysis All biochemical indices including SOD, CAT, GST and MDA were expressed as means ± standard deviation. One way ANOVA with Tukey’s test was conducted on the values from both control and solvent (DMSO) control blank groups to test possible differences induced by DMSO in earthworms. Furthermore, one-way ANOVA combined with Tukey’s test was performed on the biochemical indices 9

ACCEPTED MANUSCRIPT between solvent control and BDE 47-exposed groups, respectively. A P value less than 0.05 was considered statistically significant. The Minitab software (Version 15,

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Minitab Inc., USA) was used for the statistical analysis.

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3. Results and discussion

3.1. Effects of BDE 47 on antioxidant enzyme activities and MDA in E. fetida

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Several biochemical indices (SOD, CAT, GST activities and MDA content) involved in anti-oxidative defense were measured to examine oxidative stress induced by

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different concentrations of BDE 47. Results indicated that both SOD and GST activities were significantly (P < 0.05) increased in 100 µg/L BDE 47-exposed group.

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In addition, the activity of SOD was increased in 10 µg/L BDE 47-exposed group as well (Table 1). Neither CAT activity nor MDA was significantly altered in all BDE

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47-exposed groups. The increased SOD activity might suggest the enhanced

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dismutation of superoxide into oxygen and hydrogen peroxide that can be catalyzed into hydrogen peroxide to water and oxygen. However, one CAT molecule can

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convert millions of molecules of hydrogen peroxide into water and oxygen [41]. Therefore, CAT activity was not significantly elevated in BDE 47-exposed groups. The unchanged levels of MDA meant that BDE 47 did not induced detectable lipid peroxidation in E. fetida. These findings suggested that the higher doses (10 and 100 µg/L) of BDE 47 could induce oxidative stress in E. fetida. 3.2. Effects of BDE 47 on the metabolome of E. fetida Representative 1H NMR spectra of earthworm extracts from solvent (DMSO) control and BDE 47-exposed groups were shown in Fig. S1. All the detectable metabolites in 1

H NMR spectra were labeled in Fig. S1 and listed in Table S3. Several classes of

metabolites were identified including amino acids (valine, leucine, isoleucine, alanine, arginine, glutamate, glycine, etc.), organic acids (lactate, succinate, fumarate, etc.), organic osmolytes (betaine, dimethylglycine and dimethylamine) and energy storage compounds (ATP, glucose, glycogen and maltose). PCA was initially performed on the control and solvent control groups. However, no significant separation was found between these two groups with a P value at 0.96 10

ACCEPTED MANUSCRIPT (data not shown). Therefore only solvent control was used as control for further analysis. PCA resulted in significant (P < 0.05) separations between solvent control and BDE 47-exposed groups along either PC1 axis or PC2 axis (Fig. S2), which

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implied that these BDE 47 exposures induced significant metabolic differences in

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earthworms. In further analysis of O-PLS-DA, scores plots and corresponding loading plots for the pair-wise discrimination between control and BDE 47-exposed groups

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were shown in Fig. 1.

The O-PLS-DA coefficients of the lowest concentration (0.1 µg/L) of BDE 47

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revealed that betaine and glycine were higher than those in solvent control group. Meanwhile, the level of 2-hexyl-5-ethyl-3-furansulfonate (HEFS) was lower than that

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in control group (Fig. 1E). At the concentration of 1 µg/L, BDE 47 induced higher levels of glucose and ATP in earthworm E. fetida. Other metabolites including HEFS,

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betaine and glycine were changed similarly to those in 0.1 µg/L BDE 47-treated group

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(Fig. 1F). For both 10 and 100 µg/L BDE 47-treated groups (Fig. 1G and 1H), the metabolic changes, including increased betaine, alanine, glucose, and glycine and

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decreased HEFS and maltose were commonly found. The intermediate in Krebs cycle, succinate, was depleted in 10 µg/L BDE 47-treated earthworms (Fig. 1G), which was not observed in 100 µg/L BDE 47-treated group. HEFS was identified in diverse earthworm species including Eisenia veneta, Lumbricus rubellus, Aporrectodea caliginosa and Eisenia fetida [27, 28, 42, 43]. Although the function of HEFS in earthworms was not fully understood, it was suggested that HEFS might play a role in membrane stabilization due to its amphiphilic property [27, 28]. The decrease of HEFS could mean that BDE 47 induced disturbance in membrane stabilization in E. fetida at concentrations ranged from 0.1 to 100 µg/L. Betaine plays an important role in osmotic regulation in invertebrates. Therefore the elevation of betaine meant the osmotic stress caused by BDE 47 exposures in E. fetida. Except the lowest concentration (0.1 µg/L) of BDE 47-treated samples, glucose was elevated in all the other BDE 47-exposed groups. It might indicate the disturbance in energy metabolism. In addition, ATP, another compound of energy storage was increased in 1 and 100 µg/L BDE 47-treated groups, 11

ACCEPTED MANUSCRIPT which was consistent with increased glucose. Maltose can be broken down to two glucose molecules that are the substrates for glycolysis [44]. Therefore the decrease of maltose in 10 and 100 µg/L BDE 47-treated groups correlated with the increase of

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glucose, which could imply the increasing energy requirement for BDE 47-exposed

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earthworms. The similar result was observed in phenanthrene-treated E. fetida [29]. Previous studies showed that hydrophobic organic contaminants could result in an

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enhanced energy metabolism marked by decreased maltose and increased amino acids due to protein breakdown [28, 45]. In our case, the elevated amino acids including

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glycine and alanine were observed in both 10 and 100 µg/L BDE 47-treated groups. Especially, the increased alanine was related to gluconeogenesis since alanine is the

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major substrate [46, 47]. As an intermediate in Krebs cycle, succinate was decreased in 10 µg/L BDE 47-treated group, it probably meant the disturbance in energy

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metabolism combined with other metabolic biomarkers, such as glucose, alanine,

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maltose.

3.3. Effects of BDE 47 on the proteome of E. fetida

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The representative 2-DE gels of control and BDE 47-exposed earthworm samples are shown in Fig. 2. The quantitative comparisons of protein spots (~1000) were conducted by ImageMaster 2D Platinum 7.0 software. The comparison showed no significant difference between control and solvent control groups. To detect the protein biomarkers induced by BDE 47 in earthworms, we compared the 2-DE gels from solvent control and BDE 47-treated groups. A total of twenty eight protein spots in BDE 47-exposed groups were significantly altered in abundance (> 1.5 folds; P < 0.05), and twenty four of them were identified by MALDI-TOF-MS/MS (Table 1). These proteins were found to be involved in energy and primary metabolism, homeostasis and protein synthesis, defense system, cell growth/division and cytoskeleton and signal transduction. Fig. 3 summarized the proteomic and metabolomic responses involved in the metabolic pathways. For the lowest concentration (0.1 µg/L) of BDE 47-exposed group, five proteins (extracellular globin-4, fibrinolytic protease 1, alpha-1 tubulin, troponin 1 and calmodulin-like protein) were significantly altered. Tubulin family has two common 12

ACCEPTED MANUSCRIPT members, alpha tubulin and beta tubulin that make up microtubules of cytoskeleton. The down-regulation of alpha tubulin might be the marker of cellular injury induced by BDE 47 at 0.1 µg/L. Extracellular globin-4 is related to oxygen transporters [48]. It

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might be involved in anti-oxidative defense. Fibrinolytic enzymes are fibrin-specific

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proteases that have high activity in the presence or absence of plasminogen [30]. Due to the potent activity and stability, they have therapeutic benefits for the treatment of

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thromboembolic symptoms [30]. Wang et al. (2010) have found that cadmium exposure resulted in the down-regulation of fibrinolytic enzymes. It was similarly

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observed in BDE 47-exposed E. fetida, which may indicate that fibrinolytic enzymes are stress-responsive [28]. Troponins are the mediators of Ca2+-dependent regulation

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for striated muscle contraction [49]. They are linked to actin filaments and functions combined with tropomyosin [49]. Calmodulin is an acidic Ca2+-binding protein which

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is involved in numerous biological processes including muscle contraction, cellular

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metabolism, cell proliferation, differentiation and apoptosis [50]. Therefore the down-regulation of troponin 1 and calmodulin-like protein implied cellular injury

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together with alpha tubulin in 0.1 µg/L of BDE 47-treated earthworms. Eight

proteins

including

glyceraldehyde-3-phosphate

nucleoside

dehydrogenase

diphosphate (GPD),

kinase

fibrinolytic

A

1-like,

protease

1,

multifunctional chaperone, heat shock protein 70 (Hsp70), actin A3, SCBP3 protein and calmodulin-like protein were identified in 1 µg/L BDE 47-treated group. Nucleoside diphosphate kinases (NDKs) are enzymes catalyzing the exchange of phosphate groups between various nucleoside diphosphates. For example, NDKs can convert guanosine triphosphate (GTP) to ATP in citrate cycle [51]. Hereby the up-regulation of NDK resulted in an energy increase including elevated ATP, which was observed in corresponding metabolic profiles (Fig. 3). Ellington reported that GPD could be up-regulated in apoptotic cells to 3-fold higher than that in non-apoptotic cells [52]. In a previous study, the exposure of Escherichia coli induced an obvious up-regulation of GPD in E. fetida [48]. In our case, therefore, the up-regulation of GPD could be the indicator of cell apoptosis induced by 1 µg/L BDE 47 in E. fetida. Fibrinolytic proteases are the enzymes involved in proteolysis. They 13

ACCEPTED MANUSCRIPT are important chemotherapeutic agents and can dissolve blood fibrin clots [30]. Down-regulation of fibrinolytic protease 1 could mean the disturbance in innate immunity, which was also found in Cd-exposed E. fetida [30]. Heat shock proteins are

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ubiquitous molecular chaperones that are involved in the inhibition of cell apoptosis

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caused by stressors. Actins are the abundant cytoskeletal proteins that are composed of microfilaments in cells. The alteration in actins has been reported in response to

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cellular injury or apoptosis combined with down-regulated calmodulin-like protein. The function of SCBP proteins is not fully characterized, however, they probably play

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a similar role as soluble relaxing factors in fast muscle [53]. In 10 µg/L BDE 47-treated group, nine differentially expressed proteins were

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found, including lombricine kinase, hemoglobin chain d1, elongation factor 1 alpha, indoleamine 2,3-dioxygenase 2-like protein, protein disulfide isomerase 2,

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fructose-bisphosphate aldolase, smoothelin-like protein, myosin regulatory light chain

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and actin A3. Lombricine kinase plays an important role in the coupling of energy production in animals [48]. The down-regulation of lombricine kinase meant

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disturbance in energy metabolism, which was also observed in E. coli-treated E. fetida [48]. Hemoglobin is involved in anti-oxidative defense [54]. Elongation factors are a class of proteins that are involved in protein synthesis in the cell. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic heme-containing enzyme catalyzing the first step in tryptophan catabolism [55]. The enzyme can deplete tryptophan, resulting in reduced proteins biosynthesis. Both elongation factor 1 alpha and IDO were significantly down-regulated, which suggested the disturbance in protein biosynthesis. Protein disulfide isomerase (PDI) is a metabolic enzyme catalyzing the formation and breakage of disulfide bonds between sulfides (S-) in cysteine residues of folding proteins [56]. As a member of the superfamily of thioredoxin, PDI has been found in response to oxidative stress [56]. The down-regulation of PDI could imply that BDE 47 (10 µg/L) affected the antioxidant system by induction of oxidative stress in E. fetida, which was indicated by elevated SOD activity. Myosin regulatory light chains are regulators in the myosin contractile activity that is related to muscle contraction [57]. Fructose-bisphosphate aldolase (FBPA) is a ubiquitous enzyme that is essential 14

ACCEPTED MANUSCRIPT for glycolysis and gluconeogenesis [58]. The alteration of FBPA probably suggested the disturbance in energy metabolism induced by BDE 47 (10 and 100 µg/L). Smoothelins are actin-binding proteins playing roles for smoothelins in smooth

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muscle contraction [59]. A down-regulation of smoothelin-like protein was likely

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related to cellular injury together with altered actin A3 in BDE 47 (10 µg/L)-treated E. fetida.

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In the highest dose (100 µg/L) of BDE 47-exposed group, ATP synthase beta subunit, extracellular hemoglobin linker L2 precursor, FBPA, heat shock cognate 70,

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actin A3, intermediate filament protein and calmodulin-like were significantly altered. An up-regulation of ATP synthase implied an energy increase in E. fetida exposed to

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100 µg/L of BDE 47, which was confirmed by the elevation of ATP in corresponding metabolomes (Fig. 3). Extracellular hemoglobin linker L2 precursor could be related

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to anti-oxidative defense [54]. As the constituents of deformable cellular latticework,

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down-regulated intermediate filament proteins (IFP) demonstrated the cellular injury by BDE 47 (100 µg/L) in E. fetida combined with actin A3. As mentioned above,

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BDE 47 (100 µg/L) induced oxidative stress indicated by increased SOD and GST activities in E. fetida. Oxidative stress can damage cells in organisms. Therefore the cellular injury marked by actin and IFP could be related to BDE 47-induced oxidative stress in E. fetida. Heat shock cognate 70 (HSC70) usually functions as a molecular chaperon and exerts important roles in folding of newly synthesized polypeptides, membrane translocation and degradation of misfolded proteins [60]. The down-regulation of HSC70 probably meant the disruption in protein stability. Three proteins including fructose-bisphosphate aldolase, actin A3 and calmodulin-like protein were altered in more than one specific concentration of the BDE 47 treatments. Interestingly, all these three proteins were down-regulated in the lower concentrations of BDE 47-treated groups, however, they were up-regulated in the highest concentration (100 μg/L) of BDE-treated group. The contrary alterations of these proteins probably meant a BDE 47-induced hormesis effect which is defined as the dose-response relationship phenomenon characterized by low-dose stimulation and high-dose inhibition. This dose-responsive effect has been observed in 15

ACCEPTED MANUSCRIPT toxicological studies on the organic toxic chemicals (e.g., bisphenol A) [61]. 3.4. Correlation between mRNA expressions and protein abundances The expressions of seven genes corresponding to (NDK, GPD, ATP synthase,

PT

fibrinolytic protease, HSP70, IFP and calmodulin-like protein) in E. fetida were

RI

quantified to explore the correlation between protein abundances and mRNA expression levels (Table S2). However, the results indicated that mRNA expressions

SC

did not correlate well with the protein abundances, except for NDK and GPD; the remaining five altered independently (Fig. 4). The disparity between mRNA

NU

expressions and corresponding proteins was not supervising [48], since mRNA expression means the tendency of the corresponding encoded protein which, however,

MA

does not always happen due to the posttranscriptional and posttranslational

D

modifications, differential degradation rates between mRNA and protein [48].

TE

4. Conclusion

In this work, an integrated metabolomic and proteomic approach was used to

AC CE P

characterize the dose-dependent responses of BDE 47 in earthworm Eisenia fetida. Both metabolic and proteomic biomarkers demonstrated the differentiation in toxicological effects induced by various concentrations (0.1, 1, 10 and 100 µg/L) of BDE 47 in E. fetida. Basically, metabolic biomarkers indicated the disturbance in osmotic regulation and energy metabolism marked by differentially altered betaine, amino acids, ATP, glucose, maltose and succinate in E. fedita exposed to BDE 47. The differentially expressed proteins induced by BDE 47 were involved in cell apoptosis (or injury), oxidative stress, disturbance in protein biosynthesis and energy metabolism in E. fedita in terms of the differential proteomic biomarkers. Especially, the increased ATP was confirmed by up-regulated nucleoside diphosphate kinase A and ATP synthase in 1 and 100 µg/L of BDE 47-treated groups, respectively (Fig. 3). This work demonstrated that a combination of proteomics and metabolomics could better understand toxicological effects of environmental pollutants.

Acknowledgment 16

ACCEPTED MANUSCRIPT This research was supported by The 100 Talents Program of the Chinese Academy of Sciences and Key Deployment Program of Chinese Academy of Sciences

AC CE P

TE

D

MA

NU

SC

RI

PT

(KZZD-EW-14-03). We thank Prof. Mark Viant for the use of ProMetab software.

17

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in

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Cancer

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24

ACCEPTED MANUSCRIPT Table 1 The biochemical indices including antioxidant enzyme activities (SOD, CAT and GST) and MDA contents in E. fetida exposed to various concentrations of BDE 47. Biochemical

Exposures b

indices

SC

0.1 μg/L

1 μg/L

10 μg/L

PT

a

100 μg/L *

15.17 ± 2.07*

12.02 ± 1.52

14.02 ± 1.58

14.18 ± 2.56

15.65 ± 3.25

CAT

11.05 ± 2.49

12.38 ± 3.00

11.42 ± 2.54

12.26 ± 6.32

10.67 ± 2.99

GST

26.53 ± 4.10

26.49 ± 3.81

27.21 ± 3.16

26.80 ± 4.84

32.88 ± 4.97*

MDA

0.65 ± 0.07

0.61 ± 0.06

0.62 ± 0.05

0.68 ± 0.14

0.64 ± 0.11

SC

RI

SOD

The enzyme activities and MDA content were expressed as U mg-1 protein and nmol mg-1 protein, respectively.

b

SC means solvent control.

*

Statistical significances between solvent control and BDE 47-exposed groups were less than 0.05.

AC CE P

TE

D

MA

NU

a

25

protein name

ATP synthase beta subunit, partial

Fibrinolytic enzyme

Elongation factor 1 alpha

582

1555

Multifunctional chaperone

Defense and chaperones

Fibrinolytic protease 1

Glossina morsitans

rotundatus

58396588

283765337

16660643

Eisenia fetida Calathus

89520725

121230

51846266

51846257

71370834

29.4/4.7

27.8/6.6

20.2/4.6

25.3/4.7

17.5/6.0

32.0/5.7

17.9/5.9

46.1/5.1

Exp.

28.5/7.8

45.7/9.3

32.2/4.5

25.2/5.4

18.5/6.1

16.9/5.5

76

447

185

792

142

Score g

Protein

58

135

110

168

156

323

5

2

7

2

9

4

PN h

1

3

2

2

3

7

13

14

16

15

26

9

23

5

24

30

SC i

fold

1.67b

-1.90c

-1.68a

-1.55b

-1.93a

-1.53d

-1.64c

1.68d

2.28b

-1.94c

2.04b

changes j

PT

RI

SC

NU 15.5/5.7

53.8/5.2

42.1/5.7

45.3/8.3

12.8/3.9

MW/pI

MA

36.0/6.7

41.8/7.7

D

TE

239730800

3183058

193505769

Eisenia fetida

terrestris

Lumbricus

terrestris

terrestris

Lumbricus

lacteus

Cerebratulus

Salmo salar

Eisenia fetida

precursor

369

463

Amphimedon

queenslandica

Lumbricus

Extracellular globin-4

Number f

17.1/8.3

Theor. MW/pI

Accession

AC CE P

species

Extracellular hemoglobin linker L2

Hemoglobin chain d1

185

156

50

Homeostasis and protein synthesis

1572

dehydrogenase

Glyceraldehyde-3-phosphate

Lombricine kinase

916

935

Nucleoside diphosphate kinase A

36

Energy and primary metabolism

Spot ID e

Table 2 List of protein spots differentially expressed in earthworm E. fetida exposed to different concentrations of BDE 47.

26

ACCEPTED MANUSCRIPT

Fructose-bisphosphate aldolase

Heat shock protein 70

Protein disulfide isomerase-2

Heat shock cognate 70

895

1569

1581

1640

Smoothelin-like protein 1

Intermediate filament protein

Alpha-1 tubulin

730

1144

1131

146

SCBP3 protein

Actin A3

583

Signal transduction

Myosin regulatory light chain, LC25

229

Cell growth/division and cytoskeleton

Indoleamine 2,3-dioxygenase 2-like

864

157424658

terrestris

Lumbricus

Hirudo medicinalis

terrestris

209529123

1527170

633240

49511054

Crassostrea gigas Lumbricus

5751

157423849

13.2/4.5

50.8/4.9

68.9/5.6

24.9/5.9

42.2/5.5

21.9/4.9

16.7/9.0

62.6/4.9

66.7/6.0

35.5/7.5

31.8/4.5

20.7/4.9

124

239

125

46

447

55

483

49

499

187

133

2

6

2

17

19

5

PT

19

2

RI

14

12

15

24

20

14

20

2

6

2

3

2

2

6

SC

NU

75.2/5.3

52.8/9.5

77.1/5.7

40.3/7.4

41.0/6.6

MA

73.0/5.3

57.3/4.9

73.4/8.9

21.5/5.2

45.2/6.4

D

TE

124013459

148717315

319433534

34995386

Bombyx mori

terrestris

Lumbricus

interpunctella

Plodia

longicornis

Haemaphysalis

Eisenia fetida

MR-2009

Oligochaeta sp.

AC CE P

Anolis carolinensis

morsitans

2.17b

-1.87a

-2.40d

-1.76c

2.25d

-1.56c

-1.86b

3.71c

2.07d

-2.70c

-2.72b

2.46d

-1.78c

-3.06c

27

ACCEPTED MANUSCRIPT

37190777

Sus scrofa

37191551

AC CE P

japonicum

Schistosoma

16.7/4.2

29.1/6.4 42.1/8.4

23.3/9.6 96

94 3

5 22

30

2.92d

-1.77b

-1.73a

-1.53a

D

with significant changes (> 1.5 folds and p < 0.05) were calculated using ImageMaster 2D Platinum 7.0.

TE

PT

RI

SC

NU

MA

28

spot ID as indicated in Fig. 2. f Database GI numbers after searching against the NCBInr database. g Mascot score reported. h Number of peptide sequences. i Sequence coverage. j Fold changes

Identification of diơerentially expressed proteins from the comparisons between control and 0.1 µg/L, 1 µg/L, 10 µg/L, 100 µg/L of BDE-47-exposed groups, respectively. e Assigned

Calmodulin-like

1549

a, b, c, d,

Troponin I

283

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Figure legends Figure 1. O-PLS-DA scores derived from 1H NMR spectra of earthworm tissue extracts from solvent control (♦) and BDE 47-exposed groups (■), (A) 0.1 µg/L BDE 47, (B) 1 µg/L BDE 47, (C) 10 µg/L BDE 47 and (D) 100 µg/L BDE 47 and corresponding coefficient plots (E), (F), (G) and

PT

(H). The color map shows the significance of metabolite variations between the two classes (solvent control and BDE 47 treatment). Peaks in the positive direction indicate metabolites that

RI

are more abundant in the BDE 47-exposed groups. Consequently, metabolites that are more abundant in the control group are presented as peaks in the negative direction. Metabolites were

SC

assigned and labeled in Fig. S1 and Table S3. Abbreviations: ATP, Adenosine triphosphate;

NU

HEFS, 2-hexyl-5-ethyl-3-furansulfonate.

Figure 2. Representative 2-DE images of proteins from Eisenia fetida. Proteins were submitted to isoelectric focusing on 3-10 IPG strips (24 cm) followed by electrophoresis on 12.5% SDS-PAGE.

MA

Gels were silver-stained. Gels (A, B, C, D, E and F) are from (A) control, (B) solvent control, (C) 0.1 µg/L BDE 47, (D) 1 µg/L BDE 47, (E) 10 µg/L BDE 47 and (F) 100 µg/L BDE 47, respectively. The proteins spots observed in all five biological replicates were analyzed by

TE

D

MALDI-TOF/TOF mass spectrometry.

Figure 3. A schematic presentation of pathways indicated by altered metabolites and proteins. The identified metabolites and proteins involved in different pathways were marked in red (increased)

AC CE P

and blue (decreased). Abbreviations: ADP, Adenosine diphosphate; ATP, Adenosine triphosphate; CaM, calmodulin; CaMK, calmodulin-dependent kinase; EF1, Elongation factor 1 alpha; FBPA, Fructose-bisphosphate aldolase; FE: Fibrinolytic enzyme; GPD, Glyceraldehyde-3-phosphate dehydrogenase; HSC70: Heat shock cognate 70; HSP70, Heat shock protein 70; IDO, Indoleamine 2,3-dioxygenase 2-like; LK, Lombricine kinase; MC: Multifunctional chaperone; NDK, Nucleoside diphosphate kinase; PDI, Protein disulfide isomerase.

Figure 4. Comparison of seven genes at the mRNA and protein levels in the earthworm E. fetida exposed to different concentrations of BDE 47. The mRNA and protein values of the ratio of exposed group to the control are plotted. Abbreviations: GPD, Glyceraldehyde-3-phosphate dehydrogenase; Heat shock protein 70; IFP, Intermediate filament protein; NDK, Nucleoside diphosphate kinase.

29

ACCEPTED MANUSCRIPT Control

0.1 μg/L 0.5

A

8

0.4

Loading value

6 4

t[2]O

2 0 -2

-4

1

E 0.8

0.3

Betaine

0.2

0 -0.1

-6











-0.3 10



9

8

2X=84.0%, 1 μg/LQ2=0.69 RControl

15

F Loading value

t[2]O

-5

0.1

ATP







 t[1]p





10 μg/L R2Control X=52.9%, Q2=0.67



3

2

1

0

Glycine

0.8

Betaine

0.6

ATP

0.4

-0.2 10



9

8

0.2

HEFS Unknown 1 7

6

5

4

3

2

1

0

TE

-2

-12



8

t[2]O

3

-2

AC CE P

-7





  t[1]p Control 100 μg/L R2X=41.6%, Q2=0.61

0.6

-0.1

HEFS -0.2

0.4

Maltose

-0.3

0.2







-0.5 10

9

8

7

 t[1]p

6

5

4

3

2

1

0

0.3

1

H





0

Chemical shift (ppm) Betaine

Glycine Alanine

0.2

Glucose 0.1

0.8

0.6

ATP

ATP

0

0.4

-0.1



0.8

Glucose

0

D

 

Alanine Glycine

Betaine

Succinate

-7

-12

1

G

-0.4

Loading value

t[2]O

3

Loading value

D

8

0.1

0

Chemical shift (ppm)

0.2

C

0

1

0

MA



4

Glucose

-0.1

-10 -15

0.2

NU

0

5

SC

0.3

B

5

6

0.2

Chemical shift (ppm)

t[1]p

10

7

RI

-10

0.4

Unknown 1 HEFS

HEFS

-0.2

-8

0.6

Glycine

0.1

PT

10

-0.2 10

HEFS Maltose 9

8

7

6

5

4

Unknown 1 3

2

0.2

1

0

0

Chemical shift (ppm)

R2X=60.1%, Q2=0.57

Figure 1

30

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 2

31

ACCEPTED MANUSCRIPT

IDO

Glycolysis

FBPA

Maltose

β-D-Fructose-1,6 P2

LK

RI

ATP

Pyruvate

SC

D-lombricine

Anthranilate

Serine

Glycine

Succinate

TE

transcription translation

AC CE P

EF1

signal

Ca2+

PP P P

ribosome

protein

CaM

Krebs cycle

Citrate

ATP

Betaine

D

N-PhosphoD-lombricine

Acetyl CoA

Oxaloacetate

MA

ADP

N-Formylkynurenine

Glucose

NU

ATP synthase

Tryptophan

Glycerate-1,3 P2

GPD

PT

Glyceraldehyde-3P

ADP Succinyl CoA Su NDK

MC HS 0 HSC70 HSP70 PDI

Stress response

CaMK

Figure 3

32

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 4

33

ACCEPTED MANUSCRIPT Significance: As a class of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs), are widely used in industrial products and have been detected in local

PT

terrestrial biota from the Laizhou Bay in China. Therefore a study on PBDE-induced toxicological effects is necessary. The earthworm Eisenia fetida, a terrestrial sentinel

RI

animal, is the most frequently used bioindicator for terrestrial environmental contaminants. To our knowledge, however, very few studies have focused on the

SC

dose-dependent responses induced by PBDEs, in terrestrial sentinel animal at protein and metabolite levels. In the present study, an integrated metabolomic and proteomic

NU

approach was used to elucidate the dose-dependent toxicological effects of BDE 47 in

AC CE P

TE

D

MA

E. fetida.

34

ACCEPTED MANUSCRIPT

IDO

Glycolysis

FBPA

Maltose

β-D-Fructose-1,6 P2

LK

RI

ATP

Pyruvate

SC

D-lombricine

Anthranilate

Serine

Glycine

Succinate

Krebs cycle

Betaine

TE

transcription translation

AC CE P

EF1

signal

Ca2+

PP P P

ribosome

CaM

Citrate

ATP

D

N-PhosphoD-lombricine

Acetyl CoA

Oxaloacetate

MA

ADP

N-Formylkynurenine

Glucose

NU

ATP synthase

Tryptophan

Glycerate-1,3 P2

GPD

PT

Glyceraldehyde-3P

protein

ADP Succinyl CoA Su NDK

MC HS 0 HSC70 HSP70 PDI

Stress response

CaMK

Graphical abstract

35

ACCEPTED MANUSCRIPT Research Highlights: BDE 47 induced dose-dependent effects in E. fetida.

l

Some protein biomarkers were validated by metabolite biomarkers in same

PT

l

pathways.

RI

Proteomics and metabolomics provided a broader view into pollutant-induced

TE

D

MA

NU

SC

effects.

AC CE P

l

36