Supporting Information. New Insights into the Cytotoxic Mechanism of. Hexabromocyclododecane from a Metabolomic Approach

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1 Supporting Information New Insights into the Cytotoxic Mechanism of Hexabromocyclododecane from a Metabolomic Approach Feidi Wang,, Haijun Zhang, *, Ningbo Geng,, Baoqin Zhang, Xiaoqian Ren,, Jiping Chen *, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China University of Chinese Academy of Sciences, Beijing , China Number of pages: 28 Number of Tables: 2 Number of Figures: 11. Corresponding Authors * Phone: Fax: hjzhang@dicp.ac.cn. * Phone/Fax: chenjp@dicp.ac.cn. S1

2 1. Supplementary description on cell viability assay HepG2 cells were seeded in a 96-well plate at a density of , , and cells/well respectively for 24, 48, and 72 h exposure, and then HBCD dissolved in DMSO were incorporated into the cell culture medium with varying concentrations (0.05, 0.5, 1, 5, and 10 mg/l). Ten replicates were performed for the control and each dose treatment. The final DMSO content in culture medium was 0.05% (v/v), and background control wells were treated with only 0.05% DMSO. Then cell viability was tested using the MTT assay after HBCD exposure for different time (24 h, 48 h, or 72 h). When the HBCD exposure experiment was finished, 20 μl of MTT (5 mg/ml, in PBS) was added directly to the culture medium, and then an additional 4-h incubation at 37 C and 5% CO 2 was conducted. Violet crystals generated by viable cells were directly solved by 150 μl DMSO and well-mixed for analysis. The optical density of the sample was quantified at a wavelength of 492 nm with a multiwell microplate reader (Tecan Infinite F50). The cell viability was calculated by setting the viability of the control cells as 100%, and the result is shown in Figure S1. Figure S1 Viability of HepG2 cells exposed to HBCD at various concentrations of 0.05, 0.5, 1, 5, 10 mg/l for 24 h, 48 h, and 72 h. Significant differences were indicated in comparison of the control by T-test. *, P < 0.05; **, P < S2

3 2. Supplementary description on instrumental Analysis UHPLC/Q-TOF MS for Untargeted Tandem MS For untargeted tandem MS, the auto MS/MS function of the Q-TOF MS system with data-dependent acquisition was performed in positive ion mode and negative ion mode, respectively. For positive ion mode, 5 μl of extract containing metabolites was injected into the UHPLC/Q-TOF MS system with an ACQUITY UPLC BEH C8 column (2.1 mm 100 mm, 1.7 μm, Waters, USA) maintained at 50 C. Water and acetonitrile both containing 0.1% (v/v) formic acid were used as mobile phases A and B, respectively. The flow rate was 0.35 ml/min, and the gradient elution was as follows (time, %B): 0 min, 10%; 3 min, 40%; 15 min, 100%, and maintained for 5 min; 20.1 min, 10%, and re-equilibrated for 2.9 min. The mass spectrometer was operated with a capillary voltage of 4000 V, fragmentor voltage of 175 V, skimmer voltage of 65 V, nebulizer gas (N 2 ) pressure at 45 psi, drying gas (N 2 ) flow rate of 9 L/min, and a temperature of 350 C. Five most intense precursors were chosen within one full scan cycle (0.25 s) with a precursor ion scan range of m/z and a tandem mass scan range of m/z The collision energies were set at 10, 20, 30, and 40 ev, and all samples were analyzed to obtain abundant and complementary product ion information. For negative ion mode, 5 μl of extract containing metabolites was injected into the UHPLC/Q-TOF MS system with an ACQUITY UPLC HSS T3 column (2.1 mm 100 mm, 1.8 μm, Waters, USA) maintained at 50 C. Water and methanol both containing 5 mmol/l ammonium bicarbonate were used as mobile phases A and B, respectively. The flow rate was also 0.35 ml/min, and the gradient elution was as follows (time, %B): 0 min, 2%; 3 min, 42%; 12 min, 100%, and maintained for 4 min; 16.1 min, 2%, and re-equilibrated for 3.9 min. The mass spectrometer was operated with a capillary voltage of 3500 V, fragmentor voltage of 175 V, skimmer voltage of 65 V, nebulizer gas (N 2 ) pressure at 45 psi, drying gas (N 2 ) flow rate of 9 L/min, and a temperature of 350 C. Five most intense precursors were chosen within one full scan cycle (0.25 s) with a precursor ion scan range of m/z and a tandem mass scan range of m/z The collision energies were set at 10, 20, 30, and 40 ev, and all samples were analyzed to obtain abundant and complementary product ion information. After data acquisition, the Find by Auto MS/MS function of MassHunter Qualitative Analysis software was used to automatically extract ion pair information for subsequent MRM detection. The retention time window was set to 0.15 min; the MS/MS threshold S3

4 was set to 100, and the mass match tolerance was set to 0.02 Da. The single mass expansion was set to symmetric 100 ppm, and the persistent background ions, such as reference mass ions, were excluded. After execution, 241 detected ion pairs and 59 detected ion pairs respectively for positive ion mode and negative ion mode with information about the precursor ion, product ions, retention time, and collision energy were exported to a spreadsheet. Ion pairs were selected on the basis of the following rules: different precursor ions eluted in the neighboring time range were scrutinized to exclude the isotopic, fragmentation, adduct, and dimer ions; and the product ion that appeared with the most applied collision energy and with the highest intensity was selected as the characteristic product ion. UHPLC/Q-Trap MRM MS for Pseudo-targeted Metabolomic Analysis A Waters Acquity Ultra Prerformance liquid chromatography system (UHPLC) coupled online to an ABI Q-Trap 5500 (AB SCIEX, USA) via an electrospray ionization (ESI) interface was adopted for pseudo-targeted metabolomic analysis using the spreadsheet produced from the analysis of UHPLC/Q-TOF MS. The same chromatographic condition, including chromatographic column, mobile phases, and gradient elution procedure, was performed on both UHPLC/Q-TOF MS system and UHPLC/Q-Trap MS system. For positive ion mode, The MS instrumental parameters were set as those for the following: source temperature, 550 C; gas I, 40 arbitrary units; gas II, 40 arbitrary units; curtain gas, 35 arbitrary units; ion spray voltage, 5500 V. Four internal standards containing 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, 1,2-diheptadecanoyl-snglycero-3-phosphoethanolamine, octanoyl (8,8,8-D3)-L-carnitine, and L-phenylalanine-d5 were used for normalizing the peak areas of all other analytes. For negative ion mode, The MS instrumental parameters were set as follows: source temperature, 550 C; gas I, 40 arbitrary units; gas II, 40 arbitrary units; curtain gas, 35 arbitrary units; ion spray voltage, 4500 V. Hendecanoic acid and nonadecanoic acid served as internal standards to normalize the peak areas of all other analytes. S4

5 3. Supplementary description on the repeatability of metabolic profiling analysis using UHPLC/Q-Trap MRM MS To ensure data quality for metabolic profiling, pooled quality control (QC) samples were prepared by mixing 20 μl Metabolites Extraction from all of the samples. 7 replicates of QC samples inserting into the analytical sequence were measured using UHPLC/Q-Trap MS system. For ESI + mode, the relative standard deviation (RSD) for the peak areas of 241 peaks detected from QC samples was calculated after each peak area was normalized to the sum peak area of the 241 peaks, and the results are shown in Figure S2A. 65% of the 241 peaks had an RSD of less than 10%, and 92% of the 241 peaks had an RSD of less than 20%. Furthermore, the score plot of principal component analysis (PCA) (Figure S2C) revealed that the scores of all QC samples along the first component distributed within the confidence interval corresponding to two standard deviations (SD) (R 2 X = 0.348), indicating that the sample analysis sequence had a satisfactory stability and repeatability. For ESI mode, 59 peaks had been detected from QC samples, and the RSD for the peak areas of these 59 peaks was also calculated after each peak area was normalized to the total peak area of the 59 peaks, and the results are shown in Figure S2B. 55% of the 59 peaks had an RSD of less than 10%, and 92% of the 59 peaks had an RSD of less than 20%. In addition, the score plot of PCA (Figure S2D) also indicated that the scores of all QC samples along the first component distributed within the confidence interval corresponding to two SD (R 2 X = 0.398). The statistical results for QC samples pointed out that UHPLC/Q-Trap MS based platform had favorable repeatability for the pseudo-targeted metabolomic analysis. S5

6 Figure S2. Distribution of % RSD and score plots of PCA for QC samples. RSD: the relative standard deviation; QC: quality control; PCA: principal component analysis. S6

7 4. Supplementary description on the measurement of ROS generation Reactive oxygen species (ROS) in HepG2 cells was measured with the specific assay kit (Nanjing Jiancheng Bioengineering Institute, China). Six replicates were performed for the control and each dose treatment (0.05, 1, and 10 mg/l) using a 6-well plate. After HBCD exposure experiment, the culture medium was removed. Cells were rinsed with PBS for three times, trypsinized, and then terminated digestion with FBS. The cell suspension of each well was transferred into an Eppendorf tube, adding 1 ml PBS, and followed by centrifugation for 5 min at 1500 g, 8 C, and then the supernatant was discarded. Soon after, 1 ml PBS was added into the Eppendorf tube to resuspend cells, and 0.5 ml of the cell suspension was used for the determination of ROS, while the other half was used for total protein quantitative assay. Next the cells were stained with 10 μmol/l DCFH-DA for 60 min at 37 C. Otherwise two positive control experiments were set to verify the determination method of ROS generation was efficient. One positive control experiment was conducted by adding 100 μmol/l H 2 O 2 into cell suspension before staining with 10 μmol/l DCFH-DA for 60 min at 37 C. The other one was conducted by adding 100 μmol/l H 2 O 2 into PBS solution without cells before staining with 10 μmol/l DCFH-DA for 60 min at 37 C. DCFH-DA was cleaved by nonspecific esterases inside cells to form DCFH, a non-fluorescent compound, and subsequently it was oxidized to the fluorescent compound DCF by intracellular ROS. After 1-h incubation with DCFH-DA and centrifugation for 5 min at 1500 g, 8 C, discarding the supernatant, the cells were re-suspended with 0.25 ml PBS. The conversion of DCFH to the fluorescent product DCF was measured using a TECAN GENios Microplate Reader (Tecan, Switzerland) with excitation/emission at 485/525 nm. Simultaneously Background fluorescence (conversion of DCFH to DCF in the absence of cells) was corrected by the inclusion of parallel blanks. ROS level was expressed in arbitrary units (intensity/mg protein). Then ROS level was calculated by setting the level of the control cells as 100%, and the result is shown in Figure S3. S7

8 ROS production (% of control) * * * 0 control HBCD dose (mg/l) Figure S3. Changes of ROS in HepG2 cells exposed to HBCD at various concentrations of 0.05 mg/l, 1 mg/l, 10 mg/l for 24 h. ROS: reactive oxygen species. Significant differences were indicated in comparison of the control by T-test. *, P < 0.05; **, P < Supplementary description on the determination of oxidative stress markers, relevant metabolic enzyme activities, and total protein HBCD exposure was performed with six replicates for the control and each dose treatment 0.05, 1, and 10 mg/l) using a 6-well plate for 24 h. Then HepG2 cells were rinsed with PBS for three times, trypsinized, and then terminated digestion with FBS. The cell suspension of each well was transferred into an Eppendorf tube, adding 1mL PBS, and followed by centrifugation for 5 min at 1500 g, 8 C, and then the supernatant was discarded. Soon after, 1 ml PBS was added into the Eppendorf tube to wash the cells, and centrifugation for 5 min at 1500 g, 8 C was also done and then the supernatant was discarded. After repeating this washing procedure twice, the cells were resuspended in 0.5 ml PBS for ultrasonic cell disruption using a SCIENTZ JY 96-IIN ULTRASONIC HOMOGENIZER (Ningbo Scientz Biotechnology Company, China) in an ice bath. Soon afterwards, the specific assay kits (Nanjing Jiancheng Bioengineering Institute, China) were adopted to determine the level of reduced glutathione (GSH), malondialdehyde (MDA), and total protein (TP), the activities of superoxide dismutase (SOD), catalase (CAT), and lactate dehydrogenase (LDH) using the suspension containing cell debris directly. But for determination of the activities of other metabolic enzymes, the suspension was first centrifuged for 5 min at 6000 g, 8 C, and the supernatant was collected. Then the activities of other metabolic enzymes, phosphofructokinase (PFK), hexokinase (HK), S8

9 Activity (U/mg protein) Activity (U/mg protein) long-chain acyl-coa dehydrogenase (LCACD), long-chain acyl-coa synthetase (LCACS), adenosine-triphosphate (ATP), Ca 2+ -ATPase, Na + /K + -ATPase, phopholipase A1 (PLA1), and phopholipase A2 (PLA2) were measured using the enzyme-linked immune sorbent assay (ELISA). 4 CAT ** 160 SOD 3 ** ** 120 * Concentration (umol/g protein) Control GSH ** Control HBCD dose(mg/l) Concentration (umol/g protein) 0 Control MDA ** 0.8 ** 0.6 ** Control HBCD dose(mg/l) Figure S4. Changes of several oxidative stress markers in HepG2 cells exposed to HBCD at various concentrations of 0.05 mg/l, 1 mg/l, 10 mg/l for 24 h. CAT: catalase; SOD: superoxide dismutase; GSH: reduced glutathione; MDA: malondialdehyde. Significant differences were indicated in comparison of the control by T-test. *, P < 0.05; **, P < S9

10 Figure S5. Effects of HBCD exposure on the level of total protein and the activities of some crucial metabolic enzymes regulating glycolytic pathway, transport across cellular membranes, β-oxidation of fatty acid and phospholipid metabolism. LCACS: long-chain acyl-coa synthetase; LCACD: long-chain acyl-coa dehydrogenase; PLA1: phopholipase A1; PLA2: phopholipase A2; PFK: phosphofructokinase; HK: hexokinase; LDH: lactate dehydrogenase; ATP: adenosine-triphosphate. * P < 0.05; ** P < S10

11 Table S1. PLS-DA and ANOVA results for differential metabolites (exposure group vs. control group) ESI mode Metabolites Parent Ion Product Ion VIP P FDR + SM(d18:0/12:0) E E-08 + Behenic acid E E-06 - L-Aspartic acid E E-08 + SM(d18:1/15:0) E E-08 + Methyl stearate E E-07 + L-alanine E E-09 + SM(d18:1/20:1) E E-09 + L-Proline E E-07 + Sarcosine E E-09 - Palmitoleic acid E E-08 + L-Cysteine E E-08 + Triene-3-nonadecanoic acid ester E E-05 + SM(d18:1/16:1) E E-08 + Acetone E E-06 + Pyroglutamic acid E E-11 + LysoPC(16:0/0:0) E E-06 + Glycine E E-08 + L-Lactic acid E E-06 + PC(16:0/14:1) E E-06 + phenylalanylphenylalanine E E-04 + Choline E E-04 + PC(24:1/15:0) E E-06 + L-Threonine E E-11 + L-Lysine E E-09 - Myristic acid E E-03 + LysoPC(18:1/0:0) E E-04 + Anandamide E E-03 + L-Serine E E-08 + PC(14:0/14:0) E E-04 + L-glutamine E E-09 + SM(d18:1/14:0) E E-07 + LysoPC(18:0/0:0) E E-07 - Tetracosapentaenoic acid (24:5n-3) E E-05 + PC(20:4/14:0) E E-09 + SM(d18:1/18:1) E E-05 S11

12 + Creatinine E E-05 + LysoPC(O-16:0/0:0) E E-07 + Propionylcarnitine E E-09 + PC(18:2/15:0) E E-07 + L-Isoleucine E E-08 + LysoPC(O-18:0/0:0) E E-09 + L-Leucine E E-08 - Pentadecanoic Acid E E-06 - Nonadecanoic acid E E-03 + LysoPC(15:0/0:0) E E-08 + Linoleyl carnitine E E-02 + LysoPE(22:4/0:0) E E-06 + Taurine E E-03 + L-Valine E E-07 + LysoPC(18:3/0:0) E E-05 + Indole-3-carbinol E E-02 + LysoPC(19:0) E E-07 + PC(18:1/15:0) E E-06 + LysoSM(d18:1) E E-06 + L-Alpha-aminobutyric acid E E-05 + LysoPC(P-18:0) E E-07 + LysoPC(20:3/0:0) E E-04 + Docosahexaenoic acid E E-06 + SM(d18:1/16:0)(OH) E E-08 + SM(d18:0/18:1) E E-06 + Butyrylcarnitine E E-05 + L-Histidine E E-08 + L-Tyrosine E E-07 + L-Asparagine E E-07 - LysoPE(18:1/0:0) E E-02 + L-Phenylalanine E E-07 + L-Octanoylcarnitine E E-02 + LysoPC(14:0/0:0) E E-04 - Arachidonic acid E E-02 + PE(20:0/14:0) E E-02 - Docosapentaenoic acid E E-04 - LysoPE(18:0/0:0) E E-05 + Ornithine E E-08 + PC(16:1/16:1) E E-04 + PC(20:4/18:3) E E-05 - LysoPE(16:0/0:0) E E-04 S12

13 + PC(20:4/16:0) E E-03 - Docosaheptaenoic acid E E-02-8,11,14-Eicosatrienoic acid E E-07 + L-Tryptophan E E-05 + Citrulline E E-03 + L-Methionine E E-07 + Serotonin E E-03 + Hypoxanthine E E-02 - Eicosapentaenoic acid E E-04 - LysoPE(18:2/0:0) E E-02 - Nonadeca-10(Z)-enoic acid E E-03 + L-Acetylcarnitine E E-09 - Uridine E E-05 + LysoPC(22:6/0:0) E E-03 + Valerylcarnitine E E-03 - Erucic acid E E-02 + L-Kynurenine E E-03 + LysoPC(18:2/0:0) E E-02 + LysoPC(20:5/0:0) E E-02 + LysoPE(22:6/0:0) E E-03 * VIP values were obtained from Partial least squares discriminate analysis (PLS-DA), P values were obtained from comparing dose groups with the control group (one-way ANOVA), and the positive false discovery rate (FDR) from the P values of multiple-hypothesis testing was estimated. S13

14 6. Supplementary description on hierarchical clustering analysis We used the MeV software package to arrange 96 metabolites with most significant change, and the heat map was shown in figure S6. The metabolites were clustered according to their Euclidean distance coefficients, and similar dose-response relationship were clearly clustered in the left tree with five dose-response trajectories being generated (Figure S6). In group A, B, and C, part of phospholipids, fatty acids, carnitine, and other kinds of metabolites displayed a significant increase in low-, middle-, and high-dose groups compared with the control group. However, group A, B, and C exhibited different tendencies from low-dose to high-dose exposure group. Metabolites clustered in group A significantly increased with the increase of HBCD doses. In group B, metabolites showed a fall from low-dose to middle-dose, followed by a increase in the high-dose group. In contrast, metabolites of group C exhibited a rising trend from low-dose to middle-dose group and then decreased in the high-dose group. The remaining phospholipids and fatty acids were classified into group D, with an obvious elevation in low- and middle- groups but a significant decrease in the high-dose group compared with the control. It is worth noting that most of amino acids were classified into group E, showing a significant decrease in all three exposure groups compared with the control. In addition, glutamine, lactic acid and serotonin were also classified into group E. Based on the results of hierarchical clustering analysis, significant disturbance of metabolites in HepG2 cells induced by HBCD exposure could be verified. S14

15 Figure S6. Heat map of 96 metabolites with most significant change (PLS-DA, VIP > 0.76 and one-way ANOVA, P < 0.05) among the different groups. PLS-DA: partial least squares discriminate analysis; ANOVA: a one-way analysis of variance. S15

16 Table S2. The involved pathways of each differential metabolite No. Metabolites Pathway Class Alanine, aspartate, and glutamate metabolism Glycine, serine, and threonine 1 L-Aspartic acid metabolism Cysteine and methionine metabolism Lysine biosynthesis Arginine and proline metabolism Histidine metabolism Carbon fixation in photosynthetic organisms Energy metabolism 2 L-Threonine Glycine, serine, and threonine metabolism Valine, leucine, and isoleucine biosynthesis 3 L-Lysine Lysine biosynthesis Lysine degradation Glycine, serine, and threonine 4 Sarcosine metabolism Arginine and proline metabolism Glycine, serine, and threonine metabolism Cysteine and methionine metabolism 5 L-Cysteine Taurine and hypotaurine metabolism Metabolism of other amino acids Glutathione metabolism Metabolism of other amino acids Sulfur metabolism Energy metabolism 6 L-Proline Arginine and proline metabolism 7 Glycine Primary bile acid biosynthesis Purine metabolism Glycine, serine, and threonine metabolism Lysine degradation Glutathione metabolism Synaptic vesicle cycle Nucleotide metabolism Metabolism of other amino acids Organismal Systems; 8 L-alanine Alanine, aspartate, and glutamate metabolism Cysteine and methionine metabolism Taurine and hypotaurine metabolism Metabolism of other amino acids S16

17 Carbon fixation in photosynthetic organisms Energy metabolism Glycine, serine, and threonine metabolism Cysteine and methionine metabolism 9 L-Serine Environmental Information Sphingolipid metabolism Sulfur metabolism Energy metabolism Sphingolipid signaling pathway Processing; Signal transduction Purine metabolism Pyrimidine metabolism Alanine, aspartate, and glutamate Nucleotide metabolism Nucleotide metabolism 10 L-glutamine metabolism Arginine and proline metabolism Glutamatergic synapse GABAergic synapse Organismal Systems; Organismal Systems; 11 L-Isoleucine Valine, leucine, and isoleucine degradation Valine, leucine, and isoleucine biosynthesis 12 L-Leucine Valine, leucine, and isoleucine degradation Valine, leucine, and isoleucine biosynthesis 13 Creatinine Arginine and proline metabolism 14 Taurine Primary bile acid biosynthesis Taurine and hypotaurine metabolism Sulfur metabolism Neuroactive ligand-receptor interaction Metabolism of other amino acids Energy metabolism Environmental Information Processing; Signaling molecules and interaction 15 L-Histidine Histidine metabolism 16 L-Tyrosine Tyrosine metabolism Phenylalanine metabolism Dopaminergic synapse Prolactin signaling pathway Phenylalanine, tyrosine, and tryptophan Organismal Systems; Organismal Systems; Endocrine system S17

18 biosynthesis 17 L-Asparagine Alanine, aspartate, and glutamate metabolism 18 Ornithine Arginine and proline metabolism Glutathione metabolism Metabolism of other amino acids 19 L-Phenylalanine Phenylalanine metabolism Phenylalanine, tyrosine, and tryptophan biosynthesis 20 L-Tryptophan Glycine, serine, and threonine metabolism Tryptophan metabolism Phenylalanine, tyrosine and tryptophan biosynthesis 21 Citrulline Arginine and proline metabolism 22 L-Valine Valine, leucine, and isoleucine degradation Valine, leucine, and isoleucine biosynthesis 23 L-Methionine Cysteine and methionine metabolism 24 L-Kynurenine Tryptophan metabolism Tryptophan metabolism camp signaling pathway Gap junction Environmental Information Processing; Signal transduction Cellular Processes; Cellular 25 Serotonin community Synaptic vesicle cycle Organismal Systems; Serotonergic synapse Inflammatory mediator regulation of TRP channels Neuroactive ligand-receptor interaction Organismal Systems; Sensory system Organismal Systems; Digestive system 26 Hypoxanthine Purine metabolism Nucleotide metabolism 27 phenylalanylphenylalanine Unavailable 28 SM(d18: 1/16: 1) Sphingolipid metabolism Sphingolipid signaling pathway Environmental Information Processing; Signal S18

19 transduction Sphingolipid metabolism 29 SM(d18: 1/15: 0) Sphingolipid signaling pathway Environmental Information Processing; Signal transduction Sphingolipid metabolism 30 SM(d18: 1/20: 1) Environmental Information Sphingolipid signaling pathway Processing; Signal transduction Sphingolipid metabolism 31 SM(d18: 1/18: 1) Environmental Information Sphingolipid signaling pathway Processing; Signal transduction Sphingolipid metabolism 32 SM(d18: 0/18: 1) Environmental Information Sphingolipid signaling pathway Processing; Signal transduction Sphingolipid metabolism 33 SM(d18: 1/14: 0) Environmental Sphingolipid signaling pathway Information Processing; Signal transduction Sphingolipid metabolism 34 SM(d18: 0/12: 0) Environmental Information Sphingolipid signaling pathway Processing; Signal transduction Sphingolipid metabolism 35 SM(d18: 1/16: 0)(OH) Environmental Information Sphingolipid signaling pathway Processing; Signal transduction 36 LysoSM(d18: 1) Sphingolipid metabolism 37 Methyl stearate Unavailable 38 Triene-3-nonadecanoic acid ester Unavailable 39 LysoPC(18: 1/0: 0) Glycerophospholipid metabolism 40 LysoPC(18: 3/0: 0) Glycerophospholipid metabolism 41 LysoPC(19: 0) Glycerophospholipid metabolism 42 LysoPC(20: 3/0: 0) Glycerophospholipid metabolism 43 LysoPC(14: 0) Glycerophospholipid metabolism 44 LysoPC(18: 0) Glycerophospholipid metabolism 45 LysoPC(16: 0) Glycerophospholipid metabolism 46 LysoPC(22: 6/0: 0) Glycerophospholipid metabolism 47 LysoPC(18: 2/0: 0) Glycerophospholipid metabolism 48 LysoPC(20: 5/0 :0) Glycerophospholipid metabolism S19

20 49 LysoPC(15: 0) Glycerophospholipid metabolism 50 LysoPC(O-18:0) Ether lipid metabolism 51 LysoPC(O-16:0) Ether lipid metabolism 52 LysoPC(P-18:0) Glycerophospholipid metabolism 53 Choline Glycine, serine, and threonine metabolism Glycerophospholipid metabolism Bile secretion Organismal Systems; Digestive system Glycerophospholipid metabolism Arachidonic acid metabolism 54 PC(20: 4/16: 0) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 55 PC(16: 0/14: 1) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 56 PC(18: 1/15: 0) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 57 PC(16: 1/16: 1) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 58 PC(20: 4/18: 3) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; 59 PC(18: 2/15: 0) Glycerophospholipid metabolism Arachidonic acid metabolism Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; S20

21 Glycerophospholipid metabolism Arachidonic acid metabolism 60 PC(24: 1/15: 0) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 61 PC(20: 4/14: 0) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; Glycerophospholipid metabolism Arachidonic acid metabolism 62 PC(14: 0/14: 0) Linoleic acid metabolism alpha-linolenic acid metabolism Retrograde endocannabinoid signaling Organismal Systems; 63 LysoPE(18: 2/0: 0) Glycerophospholipid metabolism 64 LysoPE(16: 0/0: 0) Glycerophospholipid metabolism 65 LysoPE(22: 4/0: 0) Glycerophospholipid metabolism 66 LysoPE(18: 0/0: 0) Glycerophospholipid metabolism 67 LysoPE(18: 1/0: 0) Glycerophospholipid metabolism 68 LysoPE(22: 6/0: 0) Glycerophospholipid metabolism Glycerophospholipid metabolism Retrograde endocannabinoid signaling Organismal Systems; 69 PE(20: 0/14: 0) Glycosylphosphatidylinositol(GPI)-anch or biosynthesis Glycan biosynthesis and metabolism Retrograde endocannabinoid signaling Organismal Systems; 70 anandamide Organismal Systems; Inflammatory mediator regulation of Sensory system TRP channels Environmental Information Neuroactive ligand-receptor interaction Processing; Signaling molecules and interaction 71 Valerylcarnitine Unavailable 72 L-Acetylcarnitine Beta Oxidation of Very Long-Chain Fatty Acids Oxidation of Branched Chain Fatty Acids S21

22 73 Propionylcarnitine Lipid catabolism, Fatty acid transport Oxidation of Branched Chain Fatty Acids 74 Linoleyl carnitine Lipid catabolism, Fatty acid transport 75 Indole-3-carbinol Unavailable 76 Butyrylcarnitine Lipid catabolism, Fatty acid transport Membrane integrity/stability 77 L-Octanoylcarnitine Lipid catabolism, Fatty acid transport Mitochondrial Beta-Oxidation of Short Chain Saturated Fatty Acids 78 Pyroglutamic acid Glutathione metabolism Metabolism of other amino acids 79 L-Lactic acid Glycolysis / Gluconeogenesis Fructose and mannose metabolism Pyruvate metabolism Propanoate metabolism Glucagon signaling pathway Styrene degradation camp signaling pathway Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism Endocrine system Xenobiotics biodegradation and metabolism Environmental Information Processing; Signal transduction 80 Palmitoleic acid Fatty acid biosynthesis 81 Behenic acid Biosynthesis of unsaturated fatty acids Cutin, suberine and wax biosynthesis Arachidonic acid metabolism Linoleic acid metabolism Biosynthesis of unsaturated fatty acids 82 Arachidonic acid Retrograde endocannabinoid signaling Organismal Systems; Serotonergic synapse Inflammatory mediator regulation of TRP channels Organismal Systems; Sensory system 83 Myristic acid Fatty acid biosynthesis 84 Pentadecanoic Acid Membrane integrity/stability 85 Docosaheptaenoic acid Unavailable 86 8,11,14-Eicosatrienoic acid Linoleic acid metabolism Biosynthesis of unsaturated fatty acids 87 Nonadecanoic acid Enzyme cofactor Membrane integrity/stability 88 Tetracosapentaenoic acid (24: Alpha Linolenic Acid and Linoleic Acid 5n-3) Metabolism 89 Docosahexaenoic acid Biosynthesis of unsaturated fatty acids Alpha Linolenic Acid and Linoleic Acid S22

23 Metabolism Biosynthesis of unsaturated fatty acids 90 Docosapentaenoic acid Alpha Linolenic Acid and Linoleic Acid Metabolism Biosynthesis of unsaturated fatty acids 91 Eicosapentaenoic acid Alpha Linolenic Acid and Linoleic Acid Metabolism 92 Erucic acid Biosynthesis of unsaturated fatty acids 93 Nonadeca-10(Z)-enoic acid Unavailable 94 L-Alpha-aminobutyric acid Protein synthesis, amino acid biosynthesis 95 Acetone Synthesis and degradation of ketone bodies Propanoate metabolism Carbohydrate metabolism 96 Uridine Pyrimidine metabolism Nucleotide metabolism S23

24 7. Supplementary description on pathway analysis Pathway analysis was conducted to reveal the most relevant pathways influenced by different exposure doses. There were 88 metabolites with identified HMDB ( ID and changed significantly upon HBCD intervention, considered to be highly responsible for the perturbation of alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; arginine and proline metabolism; glycerol phospholipid metabolism; arachidonic acid metabolism; taurine and hypotaurine metabolism; histidine metabolism; phenylalanine metabolism; pyruvate metabolism; cysteine and methionine metabolism; lysine biosynthesis; lysine degradation; tryptophan metabolism. Figure S7. The most relevant pathways influenced by HBCD based on the pathway analysis. S24

25 Figure S8. The most relevant metabolite sets perturbed by HBCD based on the pathway enrichment analysis. S25

26 Figure S9. The disturbed pathways of amino acid metabolism by HBCD. Significant differences were indicated in comparison of the control by T-test. *, P < 0.05; **, P < S26

27 Figure S10. HBCD-induced perturbation of extracellular nutrients. * P < 0.05; ** P < S27

28 Figure S11. HBCD-induced perturbation of intracellular glucose and amino acid nutrients. * P < 0.05; ** P < S28